Dhiraj Kumar, Chengliang Gong-Trends in Insect Molecular Biology and Biotechnology-Springer International Publishing (2018) PDF
Dhiraj Kumar, Chengliang Gong-Trends in Insect Molecular Biology and Biotechnology-Springer International Publishing (2018) PDF
Dhiraj Kumar, Chengliang Gong-Trends in Insect Molecular Biology and Biotechnology-Springer International Publishing (2018) PDF
Trends in Insect
Molecular Biology
and Biotechnology
Trends in Insect Molecular Biology
and Biotechnology
Dhiraj Kumar • Chengliang Gong
Editors
Trends in Insect
Molecular Biology
and Biotechnology
Editors
Dhiraj Kumar Chengliang Gong
School of Biology and School of Biology and
Basic Medical Sciences Basic Medical Sciences
Soochow University Soochow University
Suzhou Suzhou
China China
This Springer imprint is published by the registered company Springer International Publishing AG part
of Springer Nature
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface
Insects are one of the versatile groups of the animal kingdom with a large popula-
tion and are long since attracting researchers to disclose their molecular biology and
use them for the benefit of humankind. Several traditional insects such as silkworms,
honey bee, lac, Drosophila, termites, etc. are genetically and economically impor-
tant and are the best invertebrate animal models. In the modern era of genetic engi-
neering, these insects open a new horizon in the molecular biology with a
multidisciplinary approach. Additionally, insect-derived products are widely used
in biomedical and biotechnology industries. In this book, we made an effort to club
together various recent aspects of insect molecular biology, including insect genom-
ics, proteomics, virology, noncoding RNA, nano-biotechnology, recombinant insect
products, and their applications in modern research. Therefore, this volume will
certainly help academics and scientists to better understand and carry out research
on insect genomics, proteomics, and transgenics and their utilization. The first sec-
tion of this book comprises topics on insect molecular marker-assisted selection in
breeding, molecular mechanism of communication, monocyclic aromatic hydrocar-
bons (MAHs)-induced toxicity, molecular studies of evolution, long noncoding
RNA discovery, and pathogen-driven proteomics of various insects. The second part
describes the role of viral lytic polysaccharide monooxygenase, antiviral mecha-
nism, and RNAi as a novel tool for crop protection. The third section deals with the
application of recombinant insect products and chitinolytic enzymes, the role of
insects in forensic science, and genome research on Cordycep, including informa-
tion on nanotechnology application in insect molecular biology. The content of the
book will also provide a common platform for the molecular entomologist and bio-
technologist to develop novel, significant, and accessible approach for mankind
across the world.
v
Acknowledgements
vii
Contents
ix
x Contents
xi
Part I
Insect Genomics and Proteomics
Molecular Marker-Assisted Selection
Breeding in Silkworm, Bombyx mori 1
Rajendra Mundkur and E. Muniraju
Abstract
Silkworm is the lepidopteran molecular model. Many biotechnologists have con-
structed large database of DNA sequences and have tried to correlate with the
traditional linkage maps. Molecular marker-assisted selection (MAS) has been
considered as a means to improve the efficiency, accuracy, and speed of the
breeding process. Markers are the specialized breeding tools which aid selection
for target genes that are not easily visible morphologically in individuals, mini-
mize the drag around the target gene, and reduce the number of generations
required to achieve the required result. In this hope, conventional breeders in all
the disciplines of life sciences, including silkworm, are turning into biotechnolo-
gists. Like in any field of computer science, before a technology in the field of
DNA marker is understood and put into practice, another simpler, more efficient,
economical technology emerges. In this context, to keep up the pace with the
technology, it has become necessary for the breeder to be familiar with the
advancement in the field of DNA-marker technology.
1.1 Introduction
The silkworm, Bombyx mori, known to mankind since more than 4700 years is a
highly domesticated and well-characterized genetic tool (Tazima 1962) next to the
fruit fly, Drosophila. After the advent of recombinant DNA technology in breeding,
the silkworm has become the lepidopteran molecular model system (Goldsmith
1995). In Japan, the information about improved races are available (Yokoyama 1973)
since as early as 1680. Till and even after the Mendelian era, the breeding was often
referred to as an art because a new breed developed based on the experience and
ingenuity of the breeder in correlating phenotypic expression with the genetic
makeup of the individual. Later, systematic breeding started using the principles of
traditional genetics. Though the knowledge of genes governing phenotypic expres-
sions was prevailing, there were no tools to have access to the genes. Characterization
of the silkworm genome was fast developed because of its importance as lepi-
dopteran model for breeding and genetic studies, for isolating valuable genes and
promoters, and for comparative genomics (Goldsmith et al. 2005; Goldsmith 2006).
This included molecular linkage maps, BAC libraries, large EST databases, and
whole-genome shotgun sequences (Goldsmith 2006). In contrast to the traditional
genetics, where individual organisms are used for crossing and genetic studies,
modern-day genomic studies involve test tubes, micropipettes, gels, and liquid
media. Along with the liquid genetics, the computational biology and bioinformat-
ics provide insight into the genetic molecules and their expression.
The comparative increase in silk yield over 90 years of conventional breeding in
Japan (Kuribayashi 1992) (Table 1.1) indicates that the cocoon production/50 dfls
has increased by 386.5% and raw silk % by 291.3%.
Thereafter, the increase in quantitative traits has become plateau. Employing bio-
technological tools becomes a necessity to achieve marked quantum jump beyond
this level. The recent DNA molecular architecture of silkworm, Bombyx mori (Gage
1974; Yasukochi 1998; Wu et al. 1999; Wang et al. 2005), is indicated in Table 1.2.
Breeders are contemplating on designing the breeding program using the molec-
ular information. This involves many terminologies and processes routinely used in
molecular biotechnology. The terminologies and processes that are related to the
DNA marker-assisted selection (MAS) breeding are discussed in this article.
A quantitative trait locus (Beavis et al. 1991) is a region of DNA that is associated
with a particular phenotypic trait. These QTLs are often found on different chromo-
somes. Knowing the number of QTLs that explains variation in the phenotypic trait
indicates the genetic architecture of a trait. For example, it may suggest that plant
height is controlled by many genes of small effect or by a few genes of large effect.
Another use of QTLs is to identify candidate genes underlying a trait. Once a
region of DNA is identified as contributing to a phenotype, it can be sequenced. The
DNA sequence of any genes in this region can then be compared to a database of
DNA for genes whose function is already known. Advancement in quantitative
genetics and work on QTL facilitated linking of certain markers to the gene of
focus, thereby increasing the accuracy of breeding values.
In a recent development, classical QTL analyses are combined with gene expres-
sion profiling, i.e., by DNA microarrays. Such expression QTLs (e-QTLs) describes
cis- and trans-controlling elements for the expression of often disease-associated
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 5
Table 1.1 Increase in quantitative productivity over 90 years in Japan through conventional
breeding
Character 1890–1904 1987–1991 % increase
Cocoon yield/50 dfls (kg) 8.9 34.40 386.5
Raw silk (%) 7.3 21.27 291.3
Genome annotation (Ghedin et al. 2004) is the process of marking the genes and
other biological features in a DNA sequence by genome annotation software system
(White 1995). The software system is used to find the genes (places in the DNA
sequence that encode a protein), the transfer RNA, and other features and to make
initial assignments of function to those genes. The advanced genome annotation
software systems work similarly, but the programs available for analysis of genomic
DNA are constantly changing and improving.
Both plasmids and chromosomal DNA molecules can be isolated from cells. In
both cases, the cell membrane is solubilized by the application of detergent. The
resultant lysate is then enzyme/heat treated to remove various contaminants from
the desired DNA. The DNA molecules are collected by precipitating them into
ethanol.
The restriction enzyme, found in bacterial cells, functions to protect those cells
from infection by bacteriophage particles. They carry out this function by cleav-
ing the invading phage DNA. DNA of the host cell remains protected by attaching
methyl group to some of its bases. The restriction enzymes bind to specific
sequences of bases known as recognition sequences. Each enzyme has a single
specific recognition sequence, e.g., EcoRI (pronounced as eco-r-one) (restriction
enzyme I of E. coli.) restriction enzyme cuts DNA whenever the base sequence
GAATTC is found.
Once the enzyme has bound to the specific base sequence, it cleaves the DNA
backbone, thus breaking the molecule into fragments. The most useful enzymes,
known as Type II restriction endonucleases, cleave the DNA molecule at a predict-
able site within the recognition sequence itself.
Each restriction enzyme, while cleaving, leaves two characteristic ends (termi-
nals) on the fragments: (1) blunt ends, in which there is no overhanging single-strand
tail, and (2) staggered ends, in which a single-strand tail overhangs, either in 3′ or 5′
direction.
The staggered ends sometimes complement one another, in which case the stag-
gered ends tend to hydrogen bond to one another and are thus called “sticky ends.”
Sticky ends make it possible to join two DNA fragments together, regardless of the
source of DNA. The enzyme DNA ligase functions to complete the sugar-phosphate
backbone between the newly joined (ligated) fragments. When the fragments are
hybridized from at least two different sources, the resultant ligated molecule is
called recombinant DNA (rDNA).
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 7
Fig. 1.1 Electropherogram printout from automated sequencer showing part of a DNA sequence
(Source: Wikimedia 2003)
The base sequence of DNA molecules can be determined using a variety of tech-
niques (Fig. 1.1).
The Maxam-Gilbert technique (Maxam and Gilbert 1977) depends on the selective
degradation of specific bases within the molecule to be sequenced. The degradation
results in the production of large population of DNA fragments which are separated
by using PAGE. The resulting band pattern determined by autoradiography is used
to determine the base sequence. Based on this protocol, more simple and advanced
methods have been developed.
The Sanger dideoxy DNA sequencing technique (Sanger et al. 1977) relies on the
interruption of DNA synthesis. By adding a carefully calculated amount of m odified
dideoxynucleotides to the reaction mixture of DNA subunits, it is possible to halt
DNA chain elongation at various base sites, resulting in a wide variety of fragments
of DNA. This approach is also known as “dye-primer sequencing.”
8 R. Mundkur and E. Muniraju
1.7.3 Pyrosequencing
Shotgun sequencing (Anderson 1981) is used for sequencing long DNA strands.
Since the chain termination method of DNA sequencing can only be used for fairly
short strands, it is necessary to divide longer sequences up and then assemble the
results to give the overall sequence. In chromosome walking, this division is done
by progressing through the entire strand, piece by piece; shotgun sequencing uses a
faster but more complex process to assemble random pieces of the sequence.
In shotgun sequencing, DNA is broken up randomly into numerous small segments,
which are sequenced using the chain termination method to obtain reads. Multiple
overlapping reads for the target DNA are obtained by performing several rounds of this
fragmentation and sequencing. Computer programs then use the overlapping ends of
different reads to assemble them into a contiguous sequence (Table 1.3).
For example, consider the following two rounds of shotgun reads:
Table 1.3 A simplified example of two rounds of shotgun sequencing (Anderson 1981)
Shotgun stages Sequenced nucleotides
Relation to original strand
Original strand AGCATGCTGCAGTCATGCTTAGGCTA
First round of AGCATGCTGCAG AGCATGCTGCAGTCATGCTTAGGCTA
shotgun reads TCATGCTTAGGCTA AGCATGCTGCAGTCATGCTTAGGCTA
Second round TTAGGCTA AGCATGCTGCAGTCATGCTTAGGCTA
of shotgun AGCATGCTGCAGTCATGC AGCATGCTGCAGTCATGCTTAGGCTA
reads
In this extremely simplified example, the four reads can be assembled into the
original sequence using the overlap of their ends to align and order them. However,
assembly of complex genomes is additionally complicated by the abundance of
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 9
repetitive sequence, meaning similar short reads could come from completely differ-
ent parts of the sequence. Many overlapping reads for each segment of the original
DNA are necessary to overcome these difficulties and accurately assemble the
sequence.
Gene libraries store genetic information. These libraries are composed of fragments
of donor DNA which are protected by insertion into cloning vectors. Recombinant
cloning vectors from a library, inserted into host cells, allow replication of the donor
10 R. Mundkur and E. Muniraju
genetic material. Donor genetic information comes primarily from two sources: (1)
Genomic DNA: It is the entire complement of DNA in a donor cell. Eukaryotic
genes composed of genomic DNA consist of noncoding introns interspersed among
coding exons. (2) cDNA (complementary DNA): Genes composed of cDNA are
created using reverse transcriptase to synthesize a DNA copy of an mRNA template.
Since mRNA template has already undergone posttranscriptional modifications, it
consists solely of coding exons. Therefore, cDNA differs from genomic DNA in
that it is composed of exons alone.
cDNA reduces the effective size of the eukaryotic cells since it represents only
the exon portion of the gene. The lack of introns in eukaryotic cDNA allows its
successful translation by bacterial cells, as these cells are unable to remove
noncoding introns from the mRNA which is transcribed from a genomic DNA
fragment.
The use of cDNA makes it easier to identify a particular target. By isolating
mRNA from appropriate cells (e.g., mRNA complementary to fibroin gene from
silk gland cells), the probability of locating a target molecule increases.
mRNA is isolated from a host cell by passing a preparation of nucleic acid over
a cellulose column, which has been treated with short lengths of thymine deoxyri-
bonucleotides. This type of column is called oligo-dT cellulose column. The poly(A)
tail which characterizes the mRNA molecules binds to the complementary thymine
nucleotides attached to the cellulose of the column. In this way the mRNA mole-
cules remain within the column, while others containing nucleic acids pass out
through the column. The bound mRNA molecules can be chemically removed from
the cellulose. cDNA is synthesized from mRNA templates using the retroviral
enzyme called reverse transcriptase.
1.10 D
NA Fingerprinting (or Genetic Fingerprinting or DNA
Testing or DNA Typing or DNA Profiling)
organism will not probably survive, effectively removing that altered gene from the
population. For this reason, random variations crop up in the noncoding (junk) DNA
sequences as often as once in every 200 DNA letters.
The base sequences of the chromosomes of different individuals of the same spe-
cies closely resemble one another. However, some differences, called polymor-
phisms, do exist. Each individual has enough polymorphic sites to make its DNA
unique. The analysis of various polymorphisms in any one organism will provide a
unique profile which can be used to identify it. This is called DNA fingerprinting
which was described by Sir Alec Jeffreys in 1985 (Jeffreys et al. 1991).
Donor genetic sequences, either genomic or cDNA, must be protected from degra-
dation and transported into appropriate host cells by cloning vectors. Cloning vec-
tors are lengths of DNA which generally have three properties: (1) unique recognition
sequences, (2) selectable visible markers, and (3) can replicate. The three types of
cloning vectors are (1) bacterial plasmids, (2) bacteriophage chromosomes, and
(3) cosmids:
1. Plasmid vectors often contain genes for antibiotic resistance and genes that gov-
ern their transmissions from cell to cell during the process of conjugation. Most
plasmids contain a polylinker or multiple cloning site (MCS), which is a short
region containing several commonly used restriction sites allowing the easy
insertion of DNA fragments at this location. The replication of plasmids can
either be linked to the replication of host chromosome “stringent plasmid” or
independent of host chromosome “relaxed plasmids.” The number of relaxed
plasmids per host cell increased by the technique known as amplification.
2. Bacteriophage (or phage) vector is a virus that can infect bacteria. Bacteriophage
vectors have linear double-stranded DNA molecules, which are flanked by com-
plementary single-stranded sequences, of bases known as “cos sites.” The cos
sites can bind to one another thus making the phage chromosome circular. Phage
particles inject their DNA into host bacterial cells. The phage DNA immediately
directs the host bacterial cells to synthesize new phage particles in the lytic pro-
cess or becomes relatively inert through incorporation into the host chromosome,
in the lysogenic process.
3. Shuttle vectors have both yeast and bacterial origins of replication and can there-
fore be maintained in both cell types. This property has allowed the identification
of genes within the yeast cell itself. In this system, a yeast DNA library is made
and propagated in E. coli cells. When sufficient plasmid DNA is available, those
plasmids are then isolated from the E. coli cells and introduced into yeast cells
with known mutations. Donor DNA fragments of about 4000–20,000 base pairs
can be incorporated into plasmid or phage vector, respectively.
4. Cosmid vectors represent hybrid vectors consisting of the phage cos site incor-
porated into a plasmid molecule. Cosmid vectors can accept donor DNA frag-
12 R. Mundkur and E. Muniraju
BACs are often used to sequence the genetic code of organisms in genome proj-
ects, for example, the Human Genome Project. A short piece of the organism’s
DNA is amplified as an insert in BACs and then sequenced. Finally, the sequenced
parts are rearranged, resulting in the genomic sequence of the organism.
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 13
Chromosomal DNA
Plasmid
Bacteria
Chromosomal DNA
with integrated
Plasmid
Fig. 1.2 (Above) Bacterial cell with chromosomal DNA of bacteria and plasmid DNA. (Below)
Plasmid DNA integrated with the chromosomal DNA
BACs can carry both the gene and various promoter sequences which can often
show the genes’ true expression level. They are transferred over to the organisms by
electroporation/transformation or transfection with a suitable virus or microinjec-
tion. BACs can also be utilized to detect genes or large sequences of interest and
then used to map them onto the human chromosome using BAC arrays.
1.12 Contig
1.14 Transposons
Transposons are sequences of DNA that can move around to different positions
within the genome of a single cell, a process called transposition. In the process,
they can cause mutations and change the amount of DNA in the genome. Transposons
are also called “jumping genes” or “mobile genetic elements.” There are a variety of
mobile genetic elements, and they can be grouped based on their mechanism of
transposition. Class I mobile genetic elements, or retrotransposons, move in the
genome by being transcribed to RNA and then back to DNA by reverse transcrip-
tase, while class II mobile genetic elements move directly from one position to
another within the genome using a transposase to “cut and paste” them within the
genome. Transposons are very useful to researchers as a means to alter DNA inside
of a living organism. Transposons make up a large fraction of genome sizes which
is evident through the C-values of eukaryotic species. For example, about 45% of
the human genome is composed of transposons and their defunct remnants.
1.15 Cloning
Donor genetic sequences of cDNA are cloned to get recombinant vectors. Once a
recombinant vector has been synthesized, it must be inserted into the target host cell
(e.g., bacterial cell) in order to allow replication of the exogenous genetic material
(Fig. 1.3) Watson JD (2007).
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 15
STOP
PRIMER CODON
EXON INTRON EXON INTRON EXON
5’UTR 3’UTR
CDS
5’ 3’ mRNA
3’ 5’
cDNA
EST
Transformed cells can often be identified with the use of a selective media which
interacts in some way with a selectable marker located on the cloning vector.
Typically the cloning vector carries gene which confers drug resistance on a trans-
formed cell, thus allowing only transformed cells to grow in selective media con-
taining that particular drug. Non-transformed cells cannot grow in the selective
medium.
16 R. Mundkur and E. Muniraju
5’ 3’
TCAGTTCCGACTGACTAGCTGAAAGGTC
AGTCAAGGCTGACTGATCGACTTTCCAG
3’ 5’
DNA FRAGMENT
FREE NUCLEOTIDES
DNA
DNA POLYMERASE
POL RADIOACTIVE
NUCLEOTIDES
NICK
A C A G T
T C A C
G A GC G
TA T C
5’ 3’
TCAGTTCCGACTGACTAGCTGAAAGGTC
AGTCAAGGCTGACTGATCGACTTTCCAG
3’ 5’
G T G C A T
A C T
T G C G A
DNA POLYMERASE
T
A C A G
T C A C
G G G A C
A T T C
5’ 3’
TCAGTTCCGACTGACTAGCTGAAAGGTC
CCGACTG
CGACTG
AGTCAAGGCTGACTGATCGACTTTCCAG
CTG
3’ 5’
G T G C A T
A C T
T G C G A
RADIOACTIVE
NUCLEOTIDE
A A G T
T A
G A C G G
A
T T C
5’ 3’
TCAGTTCCGA CTGACTAGCTGAAAGGTC
GTC
G
AGTCAAGGCTGACTGATCGACTTTCCAG
CAG
AG
3’ 5’
G T G A
T
A C T
T G C G A
RADIOACTIVE
NUCLEOTIDES
ACTGACTAGCTGAAAGGTC
The presence of an inserted donor gene in the cloning vector can sometimes be
determined by insertional inactivation. This is the loss of a gene product due to
insertion of foreign DNA in a recognition sequence located within a particular gene.
The basic screening techniques include the transfer of the host cells or the phage
vector to a solid substrate, usually nitrocellulose membrane (or nylon or PVDF)
through blotting, subsequent treatment of the sheet with the labeled probe which is
complementary to the target sequence. Probes can be cDNA, genomic DNA, RNA,
or antibody molecules. Specific probes are chosen based on the information known
about the target sequence. Probes will hybridize to complementary molecules on the
substrate. Hybridized probe/target complexes are visualized with autoradiography.
Alternatively, mRNA probes can be removed from the target molecule and used to
direct protein synthesis in order to identify the original gene sequence.
1.17 Blotting
Southern blotting is the analysis of DNA sequences with either a DNA or RNA
probe. Northern blotting is the analysis of RNA sequences with DNA or RNA
probe. Western blotting is the analysis of proteins with an antibody probe.
Transformed host cells can be maintained for a long period of time by storage at
very cold temperatures, making gene libraries reusable. Frozen cells thawed many
years after their original storage behave as if they are freshly prepared. Thus, gene
libraries can be created, conserved, probed, regenerated, and re-probed many times.
18 R. Mundkur and E. Muniraju
The Southern blot is a method of enhancing the result of an agarose gel electropho-
resis by marking specific DNA sequences. The method is named after its inventor,
the British biologist Southern, Edwin M (Southern 1975). This became a conven-
tion to other blot methods to be named similarly to indicate variants in blot technol-
ogy, e.g., Northern blot, Western blot, and Southwestern blot (Peters 1993).
DNA strands are broken into fragments by restriction endonucleases. The frag-
ments are then electrophoresed on a gel to separate cut DNA based on size. If DNA
is larger than 15 kb, prior to blotting, the gel may be treated with a dilute acid, such
as dilute HCl, which acts to depurinate the DNA fragments. This breaks the DNA
into smaller pieces that will be able to complete the transfer more efficiently than
larger fragments.
DNA bands are transferred to nylon sheet: The gel from the DNA electrophoresis
is treated with an alkaline solution (typically containing sodium hydroxide) to cause
the double-stranded DNA to denature, separating it into single strands. Denaturation
is necessary so that the DNA will stick to the membrane and be hybridized by the
probe. Since the gel is brittle, it cannot withstand further process. Therefore, the
bands are transferred to a firm supporting sheet (substrate or membrane) (Towbin
et al. 1979). A sheet of nitrocellulose (or nylon, polyvinylidene fluoride (PVDF, it is
also known as KYNAR®)) membrane is placed on top of the gel. Pressure is applied
evenly to the gel (either using suction or by placing a stack of paper towels and a
weight on top of the membrane and gel). This causes the DNA to move from the gel
onto the membrane by capillary action, where it sticks. The membrane is then baked
(in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to perma-
nently cross-link the DNA to the membrane.
The sheet is then treated with a hybridization probe—an isolated DNA molecule
with a specific sequence that pairs with the appropriate sequence (the appropriate
sequence is the complementary sequence of what the restriction enzyme recog-
nized). The probe DNA is labeled so that it can be detected, usually by incorporat-
ing radioactivity or tagging the molecule with a fluorescent or chromogenic dye. In
some cases, the hybridization probe may be made from RNA, rather than DNA.
After hybridization, excess probe is washed from the membrane, and the pattern
of hybridization is visualized on X-ray film by autoradiography in the case of a
radioactive or fluorescent probe or by development of color on the membrane itself
if a chromogenic detection is used. When making use of hybridization in the labora-
tory, DNA must first be denatured, usually by using heat or chemicals. Denaturing
is a process by which the hydrogen bonds of the original double-stranded DNA are
broken, leaving a single strand of DNA whose bases are available for hydrogen
bonding.
Once the DNA has been denatured, a single-stranded radioactive probe can be
used to see if the denatured DNA contains a sequence similar to that on the probe.
The denatured DNA is put into a plastic bag along with the probe and some saline
liquid; the bag is then shaken to allow sloshing. If the probe finds a fit, it will bind
to the DNA (Fig. 1.5).
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 19
The fit of the probe to the DNA does not have to be exact. Sequences of varying
homology (Fig. 1.6) can stick to the DNA even if the fit is poor; the poorer the fit,
the fewer the hydrogen bonds between the probe and the denatured DNA. The abil-
ity of low-homology probes to still bind to DNA can be manipulated through vary-
ing the temperature of the hybridization reaction environment or by varying the
amount of salt in the sloshing mixture.
The Northern blot (Alwine et al. 1977) is a technique to study gene expression. It is
similar to the Southern blot procedure, with the fundamental difference that RNA,
rather than DNA, is the substance being analyzed by electrophoresis and detection
with a hybridization probe. A notable difference in the procedure (as compared with
the Southern blot) is the addition of formaldehyde in the agarose gel, which acts as
a denaturant. As in the Southern blot, the hybridization probe may be made from
DNA or RNA. A variant of the procedure known as the reverse Northern blot was
occasionally used. In this procedure, the substrate nucleic acid (i.e., affixed to the
membrane) is a collection of isolated DNA fragments, and the probe is RNA
extracted from a tissue and radioactively labeled.
The use of DNA microarrays that have come into widespread use in the early
2000s is similar to the reverse procedure, in that they involve the use of isolated
DNA fragments affixed to a substrate and hybridization with a probe made from
cellular RNA. Thus, the reverse procedure enabled the one-at-a-time study of gene
expression using Northern analysis to evolve into gene expression profiling, in
which many of the genes in an organism may have their expression monitored.
Fig. 1.6 Varying degree of homology between the DNA strand and the probe
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 21
“noise” in the final product of the Western blot, leading to clearer results, and elimi-
nates false positives.
During the detection process, the membrane is “probed” for the protein of inter-
est with antibodies and links them to a reporter enzyme, which drives a colorimetric
or photometric signal. This process takes place in a two-step method (now one-step
method is also available for certain applications).
approximations are taken by comparing the stained bands to that of the marker or
ladder loaded during electrophoresis. The process is repeated for a structural pro-
tein, such as actin or tubulin, that should not change between samples. The amount
of target protein is indexed to the structural protein to control between groups. This
practice ensures correction for the amount of total protein on the membrane in case
of errors or incomplete transfers.
1.17.3.7 Chemiluminescence
Chemiluminescent detection methods depend on incubation of the Western blot
with a substrate that will luminesce when exposed to the reporter on the secondary
antibody. The light is then detected by photographic film and more recently by CCD
cameras which capture a digital image of the Western blot. The image is analyzed
by densitometry, which evaluates the relative amount of protein staining and quanti-
fies the results in terms of optical density. Newer software allows further data analy-
sis such as molecular weight analysis if appropriate standards are used. The
“enhanced chemiluminescent” (ECL) detection is considered to be among the most
sensitive detection methods for blotting analysis.
Markers are DNA sequences that can be identified by a simple assay, allowing the
presence or absence of neighboring stretches of genome to be inferred. Markers
may be short, such as single base pair change (single nucleotide polymorphism), or
long such as DNA fragment generated by restriction digestion. DNA markers can be
identified directly, e.g., by DNA sequencing or indirectly as in case of allozymes.
1. Non-PCR-based markers
a. RFLP
2. PCR-based markers
a. RAPD
b. AFLP
c. Minisatellites
d. Microsatellites (SSR, STR, SSLPs)
e. SNP
f. Sequence-tagged sites (SCARs, CAPS, ISSRs)
g. Diversity arrays
h. LCN-DNA
1.19.1.1 RFLP
DNA polymorphism can be identified by the ability of various restriction enzymes
to cleave DNA in the vicinity of the polymorphism into variable sizes of DNA frag-
ments. This is called Restriction Fragment Length Polymorphisms (RFLPs). When
DNA fingerprinting first began, RFLP (Zabeau and Vos 1993) analysis was used.
Now it has been almost completely replaced with newer PCR-based techniques.
RFLP analysis is performed by using a restriction enzyme to cut the DNA into frag-
ments which are separated into bands by agarose gel electrophoresis. These bands
of DNA are transferred by Southern blotting from the agarose gel to a nylon mem-
brane. This is treated with a radioactively labeled DNA probe which binds to certain
specific DNA sequences on the membrane. The excess DNA probe is then washed
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 25
off. An X-ray film placed next to the nylon membrane detects the radioactive pat-
tern. This film is then developed to make a visible pattern of bands called a DNA
fingerprint. By using multiple probes targeting various polymorphisms in succes-
sive X-ray images, a fairly high degree of discrimination was possible.
Advantages of RFLPs are as follows: RFLPs have higher level of polymorphism
than isozymes, larger number of loci can be identified, produce semidominat mark-
ers, allowing determination of homozygosity or heterozygosity, and have selective
neutrality. They are stable and reproducible.
Disadvantages of RFLPs are the exact sizes of the bands are unknown and com-
parison to a molecular weight ladder is done in a purely qualitative manner. RFLP
is a very time-consuming method which requires relatively high quantity of good-
quality DNA. One has to work with radioisotopes. Too many polymorphisms for a
short probe.
RAPD
RAPD (Random Amplification of Polymorphic DNA) is a type of PCR reaction, but
the segments of DNA that are amplified are random. The scientist performing
RAPD creates several arbitrary, short primers (8–12 nucleotides) and then proceeds
with the PCR using a large template of genomic DNA, hoping that fragments will
amplify. By resolving the resulting patterns, a semi-unique profile can be generated
from a RAPD reaction.
No knowledge of the DNA sequence for the targeted gene is required, as the
primers will bind somewhere in the sequence, but it is not certain exactly where.
This makes the method popular for comparing the DNA of biological systems that
have not had the attention of the scientific community or in a system in which rela-
tively few DNA sequences are compared (it is not suitable for forming a DNA data
bank). Due to the fact that it relies on a large, intact DNA template sequence, it has
some limitations in the use of degraded DNA samples. Its resolving power is much
lower than targeted, species-specific DNA comparison methods, such as Short
Tandem Repeats.
Advantages of RAPDs are they are more polymorphic than RFLPs, simple, and
quick and have selective neutrality. Disadvantages are they are dominant and do not
permit the scoring of heterozygous individuals. Reproducibility is limited.
26 R. Mundkur and E. Muniraju
AFLP
AFLP-PCR, amplified fragment length polymorphism-polymerase chain reaction
(Vos et al. 1995), is a highly sensitive method for detecting polymorphisms in
DNA. The procedure of this technique is divided into three steps: (1) digestion of
total cellular DNA with one or more restriction enzymes and ligation of restriction
half-site-specific adaptors to all restriction fragments, (2) selective amplification of
some of these fragments with two PCR primers that have corresponding adaptor and
restriction site-specific sequences, and (3) electrophoretic separation of amplicons
on a gel matrix, followed by visualization of the band pattern.
AFLP is relied on variable number tandem repeat (VNTR) polymorphisms to
distinguish various alleles, which were separated on a polyacrylamide gel using an
allelic ladder (as opposed to a molecular weight ladder). Bands could be visualized
by silver staining the gel. As with all PCR-based methods, highly degraded DNA or
very small amounts of DNA may cause allelic dropout (causing a mistake in think-
ing a heterozygote is a homozygote) or other stochastic effects. In addition, because
the analysis is done on a gel, very high number repeats may bunch together at the
top of the gel, making it difficult to resolve. AFLP analysis can be highly automated
and allows for easy creation of phylogenetic trees based on comparing individual
samples of DNA. A variation of AFLP is TE Display, used to detect transposable
element mobility.
Advantages: No sequence information required, reliable, highly sensitive and
very large number of polymorphisms per reaction, highly reproducible (repeatable),
selective neutrality. Disadvantages: Null allele not detected, proprietary
technology.
Minisatellite
Minisatellite is a section of DNA that consists of a short series of bases 10–100 bp;
these occur at more than 1000 locations in the genome. This series usually contains
the same central sequence of letters “GGGCAGGAXG” (where X can be any one
of A, T, G, C letters). This sequence encourages chromosomes to swap DNA. When
this happens, frequent mistakes are made; this causes minisatellites at over 1000
locations in the genome to have slightly different numbers of repeats, thereby mak-
ing them unique. Due to their high level of polymorphism, minisatellites were
extensively used for DNA fingerprinting as well as for genetic markers. Minisatellites
have also been implicated as regulators of gene expression (e.g., at levels of tran-
scription, alternative splicing, or imprint control) or as part of bona fide open read-
ing frames. Minisatellites have also been associated with chromosome fragile sites
and are proximal to a number of recurrent translocation breakpoints.
RAPD markers are used. This technique converts a band which is prone to difficul-
ties in interpretation and/or reproducibility into a very reliable marker.
Advantages: Simpler pattern than RAPDs, robust and reproducible, Mendelian
inheritance, sometimes convertible to codominant markers. Disadvantages: Require
a small degree of sequence knowledge, require effort and expense in designing spe-
cific primers for each locus.
the genome. Polymorphic clones in the library are identified by arraying insert from
a random set of clones and hybridizing the array to different samples. The inserts
from polymorphic clones are immobilized on a chip. (2) Genotyping a sample:
Label the representation (DNA) of the sample with fluorescence and hybridize
against the array. Scan the array and measure for each spot the amount of hybridiza-
tion signal. By using multiple labels, contrast a representation from one sample with
the other or with control probe.
Advantages: Do not require sequence information, high output, fast data acquisi-
tion and analysis, detects single base changes as well as insertions and/or deletions,
detects differences in DNA methylation, depending on the enzyme used to generate
the fragments, small DNA sample is enough, good transferability of markers among
breeding populations, full automation possible. Disadvantages: Dominance of
markers, technically demanding, low polymorphism in genomic library.
linkage groups which had been determined previously were analyzed, out of which
189 were unambiguously placed on the linkage map by repeated three-point analy-
sis. The majority of the mapped clones corresponded to single loci dispersed on
every 28 chromosomes. The map covers about 66% of the silkworm genome.
A RAPD linkage map of Bombyx mori was constructed by Li et al. (2000) with
Dazao/C108 and their F2 generation. The map consists of 182 RAPD loci, of which
103 loci come from Dazao and from the first 23 linkage groups and the other 79 loci
come from C108 and from the second 16 linkage groups. This map covered a total
genetic distance of over 1148.3 cM.
Mita et al. (2004) established draft sequence of silkworm Bombyx mori by three-
fold whole-genome shotgun (WGS) sequencing and assembled into 49,345 scaf-
folds that span a total length of 514 mb including gaps and 387 mb without gaps.
Because the genome size of the silkworm is estimated to be 530 mb, almost 97% of
the genome has been organized in scaffolds, of which 75% has been sequenced.
Yamamoto et al. (2006) have developed a linkage map for the silkworm Bombyx
mori based on single nucleotide polymorphisms (SNPs) between strains p50T and
C108T initially found on regions corresponding to the end sequences of bacterial
artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a
p50T female × F1 (p50T × C108T) male, they analyzed segregation patterns of
534 SNPs, detected among 3840 PCR amplicons, each associated with a p50T
BAC end sequence. They have constructed a linkage map composed of 534 SNP
markers spanning 1305 cM in total length distributed over the expected 28 linkage
groups.
Nagaraja and Nagaraju (1995) studied DNA profiling of 13 silkworm genotypes
using the RAPD technique. Two hundred sixteen amplified products were generated
using 40 random primers. Amplification products specific to diapausing genotypes
were identified.
Nagaraja et al. (2005) also constructed a genetic map of RAPD, SSR, and
FISSR markers for the Z chromosome using a backcross mapping population.
Sixteen Z-linked markers were identified, characterized, and mapped using od,
a recessive trait for translucent skin as an anchor marker yielding a total recom-
bination map of 334.5 cM distributed throughout the Z chromosome. Four
RAPD and four SSR markers that were linked to W chromosome were also
identified.
Nagaraju et al. (2002) showed that the FISSR-PCR markers are inherited and
segregated in Mendelian fashion as demonstrated on a panel of 99 F2 offspring
derived from a cross of two divergent silkworm strains.
SilkDB 2017 (Silkworm Knowledgebase from China) (http://silkworm.genom-
ics.org.cn) (Xia et al. 2004), SilkSatDB (SilkSatDB 2017) (a microsatellite database
of silkworm from CDFD, India) (www.cdfd.org.in/silksatdb), Silkbase (Silkbase
2017) (EST database and BAC library from Japan) (www.ab.a.u-tokyo.ac.jp/silk-
base) (Mita et al. 2004; Mita et al. 2003), and many other websites provide updated
information about genome sequence assembly, cDNAs, ESTs, SNPs, and functional
annotations of genes of silkworm.
32 R. Mundkur and E. Muniraju
References
Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by
transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad
Sci U S A 74(12):5350–5354
Anderson S (1981) Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic
Acids Res 9(13):3015–3027
Babak G, Jonas E, Nader N, Tommy N, Nyren P (2004) Improvements in pyrosequencing technol-
ogy by employing sequence polymerase. Anal Biochem 330(2):272–280
Beavis WD, Grant D, Albertsen MC, Fincher RR (1991) Quantitative trait loci for plant height in
four maize populations and their associations with qualitative genetic loci. Theor Appl Genet
83:141–145
Bornet B, Branchard M (2001) Nonanchored inter simple sequence repeat (ISSR) markers: repro-
ducible and specific tools for genome fingerprinting. Plant Mol Biol Report 19:209–215
Burnette WN (1981) Western blotting: electrophoretic transfer of proteins from sodium dodecyl
sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with
antibody and radioiodinated protein A. Ann Biochem 112(2):195–203
Chatterjee SN, Mohandas TP (2003) Identification of ISSR markers associated with productivity
traits in silkworm, Bombyx mori L. Genome 46(3):438–447
Edwards A, Caskey T (1991) Closure strategies for random DNA sequencing. Methods:
Companion Methods Enzymol 3(1):41–47
Edwards A, Voss H, Rice P, Civitello A, Stegemann J, Schwager C, Zimmerman J, Erfle H, Caskey
T, Ansorge W (1990) Automated DNA sequencing of the human HPRT locus. Genomics
6:593–608
Findlay I, Taylor A, Quirke P, Frazier R, Urquhart A (1997) DNA fingerprinting from single cells.
Nature 389(6651):555–556
Fleischmann RD (1995) Whole-genome random sequencing and assembly of Haemophilus influ-
enza. Science 269(5223):496–512
Gage GL (1974) Polyploidization of the silk gland of Bombyx mori. Chromosoma 45:27–42
Ghedin E, Bringaud F, Peterson J, Myler P, Berriman M, Ivens A, Andersson B, Bontempi E, Eisen
J, Angiuoli S, Wanless D, Von Arx A, Murphy L, Lennard N, Salzberg S, Adams MD, White
O, Hall N, Stuart K, Fraser CM, El-Sayed NM (2004) Gene synteny and evolution of genome
architecture in trypanosomatids. Mol Biochem Parasitol 134(2):183–191
Goldsmith MR (1995) In: Goldsmith MR, Wilkins AS (eds) Molecular model system in the
Lepidoptera. Cambridge University Press, New York, pp 21–76
Goldsmith MR (2006) What should we do with our molecular linkage maps? A position paper
on positional cloning targets for the silkworm, Bombyx mori. In: Abstracts of Int. Symp. on
Genetics and Genomics, CDFD, Hyderabad, India 9–11 Jan 2006
Goldsmith MR, Shimada T, Abe H (2005) The genetics and genomics of the silkworm, Bombyx
mori. Annu Rev Entomol 50:71–100
Gupta M, Chyi Y-S, Romero SJ, Owen JL (1994) Amplification of DNA markers from evolu-
tionarily diverse genomes using single primers of simple-sequence repeats. Theor Appl Genet
89:998–1006
Jeffreys AJ, McLead A, Tamaki K, Neil DL, Monckton DG (1991) Miniature repeat coding as a
digital approach to DNA typing. Nature 354:204–209
Kadono-Okuda K, Kosegawa E, Mase K, Hara W (2002) Linkage analysis of maternal EST, cDNA
clones covering all twenty-eight chromosomes in the silkworm, Bombyx mori. Insect Mol Biol
11(5):443–451
Kuribayashi S (1992) Sericulture technology. Farming Jpn 26(5):18–24
Li B, Lu C, Zhou ZY, Xiang ZH (2000) Construction of silkworm RAPD molecular linkage map.
Yichuan Xuebao 27(2):27–32
Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A
74(2):560–564
1 Molecular Marker-Assisted Selection Breeding in Silkworm, Bombyx mori 33
Abstract
Insects are the largest group of invertebrates having unique modalities of com-
munication among members of the same species. Conspecific communication
among insect species occurs mainly through visual, tactile, chemical, and behav-
ioral changes. A number of studies on different insect models have been con-
ducted by several researchers to understand the molecular, neuronal, and
behavioral mechanism underlying communication among conspecifics. Though
huge volume of research has been done to understand the mechanistic details of
insect communication, there are a number of answered questions which require
special attention. Understanding mechanisms of communication among insects
has a number of potential applications in devising appropriate and sustainable
control and/or management of insect population in the crop field. Pheromones
are being used to effectively manage insect population since long before. Genetic
basis of odor detections and interpretation of different odorants by insect species
that carry message for different purposes involves several signaling receptors
including G-protein-coupled receptor (GPCR) and second messenger signaling.
Neuronal firing pattern following exposure to a pheromonal compound explains
partially the mechanism of conspecific message delivery conspecific. However,
how limited number of odorant-binding proteins that detect large spectrum of
odorant species and differentiate as a different signal is not yet understood.
I. Baitharu (*)
Department Environmental Sciences, Sambalpur University,
Jyoti Vihar, Burla 768019, Odisha, India
e-mail: iswarbaitharu@suniv.ac.in
S. Shroff
School of Chemistry, Sambalpur University, Jyoti Vihar, Burla 768019, Odisha, India
J.K. Sahu
Department of Life Science, National Institute of Technology, Rourkela, Odisha, India
2.1 Introduction
There are a number of interesting social insects which lead a group life with distinct
division of labor among them. Close coordination among the members of such
insect species is essential at different level for various purposes such as reproduc-
tion, search for food sources. It is well known that members of an insect species
communicate frequently with organisms of the same species which is referred to as
intraspecific communication. Sometimes direct or indirect communication occurs
between members of one species with organisms of other species for different pur-
poses which is referred to as interspecific communication. There are a number of
reasons for communication among insect species which are enumerated as
follows:
with a series of antennal taps by the male on each side of the female’s body and
male gets reciprocation from the female partner by lifting of wing covers and allow-
ing to clump on the back.
Certain tree hoppers belonging to membracidae family produce vibrations in the
tissue of their host plant which can be felt by all other tree hoppers residing on the
same plant. Communication among bees exhibits a unique behavior similar to
dance. Bees perform various types of dance to communicate the distance and direc-
tion of food sources as well as nest sites. Running in a circle popularly known as
round dance is performed to indicate close sites and transitional or sickle dance for
sites at an intermediate distance from the hive. This dance involves running in a
semicircular or moon shape. The most complex of the dance types performed by
honeybees is the waggle dance which generally performed by honey bee Apis mel-
lifera to communicate the locations of food sources. The dance language of honey
bee consists of different patterns that convey information about distance of food
source from the bee hive. The number of interactions of the dance that bee performs
conveys information about distance while the liveliness of dance indicates the qual-
ity of the food source. The angle of the dance provides information about the direc-
tion of the food source to other insects. Sometimes bees stop dancing and provide a
food sample to other bees in the hive upon their request. Sound produced by bee
during dance generally plays important role in getting attention of other bees and to
keep their attention.
Many insects have ability to produce sound though they possess no vocal chords.
Insects use various other ways to produce sounds. Ways of producing sound include
rubbing of body parts together. Sounds are caused by vibrations that can pass
through air, water, and solid structures which insect use as a modality to convey
various messages to the members of the same species or different species. Crickets
sing by rubbing one wing over the other wing. Some other insects rub their legs,
scratch their bodies, or rub their jaws together to make audible sound. Buzzing
sound is produced by grasshoppers by rubbing the hind legs against the wings.
Sound of different frequencies is produced by mosquito’s resonation of antennal
hairs. Special organs are also found in different insect species to produce sound.
Male cicadas have special organs to produce sound called tymbals. Membranes
present inside the tymbal can vibrate to produce a “singing” sound. A tympanic
membrane in the abdomen (e.g., grasshoppers and moths) or in the tibiae of the
front legs (e.g., crickets and katydids) is mostly used to detect sound. Though sound
produced by most of the insects is clearly audible to human being such as that crick-
ets’ song, many insects make supersonic sounds that are above the human range of
hearing. These supersonic sounds produced by insects have more than 20,000 vibra-
tions each second. Some grasshoppers and moths have been known to produce
ultrasonic sounds of 80,000 Hz.
2 Molecular, Neuronal, and Behavioral Mechanism of Communication Among 39
One of the most common way of communication among insect species is the use
of odor or smell. Special scent glands are present in insects that release small vola-
tile odorant molecule from their body. These odors are popularly called as phero-
mones. The female insects can produce specific odorant molecules to attract
partners of its own species for mating and such molecules are known as sex phero-
mones. Some insect species have extraordinary sensitivity toward the sex phero-
mone which they can perceive even at long distance. Male moths can perceive the
pheromones of female moths over distances of many kilometers. Ants use odorant
molecule to mark a trail, so that other ants can use the trail to get back to the nest
or to find food. The special scent released by ants enables them to know the other
members of their colony. Some insects use smell to notify about the danger to each
other. Sense of taste or smell is sometimes exploited various insect species to
detect the presence of odors. However, most insects possess specialized receptors
in their feet, antennae, and ovipositors for perception of odorant signals. One of the
most important organs for detecting odors in the insect species is the antennae. In
species where the female produces an odor, the males often have extra big antennae
which help them to find the female and the vice versa. These chemicals are divided
into two groups:
in restricting water loss and prevent a lethal rate of desiccation (Nelson and
Blomquist 1995). It is challenging for all terrestrial animals with high surface area
to volume ratio such as insects to conserve water in their bodies. The cuticular
waxes function as anti-desiccation agent and play crucial role in meeting the need
of water conservation and thus cuticular lipid is the focused target for insect
control.
chemosensory systems in almost all organisms and can vary from complete termi-
nation of signaling to graded attenuation of agonist potency (Dohlman et al. 1991).
Desensitization of GPCR-mediated signal transduction is carried out mainly through
the combined activity of two classes of proteins: G-protein-coupled serine/threo-
nine receptor kinases (GRKs) and arrestins (Freedman and Lefkowitz 1996). Second
messenger-induced kinases such as cAMP-dependent protein kinase A (PKA) and
protein kinase C (PKC) cause phosphorylation of specific intracellular residues on
GPCRs resulting in slow desensitization, GRKs phosphorylate only the agonist-
bound (activated) form of GPCRs and are responsible for rapid receptor-specific
desensitization (Inglese et al. 1993). Phosphorylation by GRKs serves to promote
the binding of arrestin proteins, which further uncouple GPCRs from the G-protein-
based signaling cascade (Pippig et al. 1993).
Furthermore, GRKs and arrestins are also intimately involved in GPCR internal-
ization, an integral component of GPCR resensitization (Ferguson et al. 1996).
Recent studies show that visual arrestins also function in olfactory signal transduc-
tion pathways in D. melanogaster and Anopheles gambiae (Merrill et al. 2002),
while huge number of ORs and OBPs are present in both these insects, only three
genes encode dual-functional arrestins which make them an attractive target for
reducing the olfactory sensitivity of insects of medical and economic importance.
GPCRs contain seven transmembrane spanning regions of 20–25 amino acids and
are most prevalent superfamily of proteins currently known and are having more
than 5000 members (Gether 2000; Strader et al. 1994). These proteins link ligands
and downstream effectors by transmitting, amplifying, and integrating other cellular
signals (Dohlman et al. 1991). ORs being a member of the GPCR superfamily are
hypothesized to function through a signal transduction pathway similar to other
GPCRs and with specific components unique to olfactory tissue, such as Golf (a
Gs-like protein), adenylate cyclase III, and cAMP-gated channel (Pilpel et al. 1998).
Due to the small body size of insects, their ability to produce and perceive auditory
and visual signals over large distances is limited (Greenfield 2002). Social commu-
nication in insects largely depends on chemo sensation through chemicals involved
in communication known as semiochemicals. Semiochemicals can be grouped into
two classes: allelochemicals and pheromones. While allelochemicals are chemicals
produced and secreted by one species of organism that elicit a behavioral or physi-
ological response in a member of the other species, pheromones are those that elicit
a response in a member of a same species (Wyatt 2014). Understanding the mecha-
nisms behind chemical communications in insects using recent advances in insect
genomics, molecular genetics, and neuroanatomical techniques has been a major
focus because of its potent and high impact application in disease control and
agriculture.
Diverse classes of chemicals such as ketones, aldehydes, and fatty acids have
been co-opted by several insect species to serve as pheromones over time through
2 Molecular, Neuronal, and Behavioral Mechanism of Communication Among 43
evolution (Yew and Chung 2015). The original function of cuticular hydrocarbons
(CHCs) was as anti-desiccants but now it serves a dual role in pheromone signaling
(Chung and Carroll 2015). Intrinsic properties of pheromones such as volatility vary
depending upon its chemical nature. Some pheromones are volatile compounds
while some are nonvolatile such as cuticular hydrocarbons. To coup with such vari-
able volatility of pheromones, insects have evolved sophisticated pheromone-
sensing organs for volatile and nonvolatile chemicals. While olfactory receptors
present in the antennae and maxillary palps detect volatile pheromones like ketones,
contact chemosensory receptors distributed across the body of the insect are impli-
cated in the detection of low-volatile or nonvolatile pheromones, such as long chain
CHCs (Ferveur 2005; Aquiloni et al. 2015).
Pheromones have been widely investigated as a sex attractant to drive behaviors
associated with mating. Diverse classes of insect mating pheromones have been
identified in numerous insect species which are secreted and perceived species spe-
cifically. Lepidopteran (butterflies and moths) are known to release volatile phero-
mones primarily for long-distance sexual advertisement (Greenfield 2002). On the
other hand, fruit flies exploit both high-volatile and low-volatile CHCs pheromones
for complex courtship behaviors (Haberer et al. 2014). Insects exhibiting dual paren-
tal care secrete pheromones to recognize mating partners (Müller et al. 2003). Beetle
females are the best example that recognize their mate via nonvolatile CHC phero-
mones using contact chemo sensation mechanism (Wang and Anderson 2010; Carde
2014). Male–male interactions like aggression are also regulated by pheromonal sig-
naling in many insect species. For example, a male-specific volatile pheromone
11-cis-vaccenyl acetate (cVA) is secreted by D. melanogaster that pleiotropically
suppresses male–male courtship and aggression (Wertheim et al. 2006).
Apart from mating and sexual behaviors, pheromones are also used as a signal to
induce the formation of groups of conspecifics and designated as aggregation pher-
omones (Imen et al. 2015). Aggregation pheromones are typically volatile long dis-
tance signal and are perceived by the olfactory system (van Zweden and d’Ettorre
2010). However, the cockroach, Periplaneta americana, uses both high-volatile and
low-volatile CHCs as a signal for aggregation at diurnal resting site (Suh et al.
2014). Additionally, pheromone-driven social behaviors such as nest mate recogni-
tion and nest defense are independent of mating and are prevalent in social insects.
Volatile alarm pheromones are mostly used for recruiting conspecifics to attack
intruders (Wyatt 2014; Sakurai et al. 2014).
Insect species come across large numbers of volatile organic compounds of natu-
ral as well as anthropogenic origin. Thus it is imperative for insects to differentiate a
myriad of physiologically irrelevant chemical compounds in the environment from
essential semiochemical signals such as sex pheromones. The ability of pheromones
in conveying message to the conspecific insects is dependent on chemical structure
of the molecule and even tiny change in the pheromone molecules renders them
completely inactive (Kaissling 1987). The extraordinary selectivity of the olfactory
system (i.e., its ability to discriminate) is coupled with an inordinate sensitivity. To
advertise their readiness to mate for reproduction, females secrete very minute quan-
tity of sex pheromones and thus avoid being noticeable. On the other hand, detectors
44 I. Baitharu et al.
in males display remarkable sensitivity and perceive such small amounts of phero-
mone in a way that the signal-to-noise ratio of the system approaches the theoretical
limit. Furthermore a dynamic process of signal inactivation is a prerequisite in case
of odor oriented navigation. Males encounter pheromone molecules as flashing sig-
nals consisting of diminutive burst of high flux estranged by periods during which
the flux is zero while flying toward a pheromone releasing female. The average dura-
tion of spikes within puffs of pheromones is on the millisecond scale, and it declines
as the moth approaches the source of pheromone (Murlis et al. 2000). Thus, a male
moth has to perceive selectively minute quantity of pheromones and reset the phero-
mone detectors on a millisecond timescale.
Olfactory receptors (ORs) and odorant binding proteins (OBPs) have been studied
extensively to understand their role in odor sensitivity and discrimination. OBPs
have got special attention as regulator of dynamics of olfaction system in insects as
well as in higher vertebrates which has been two strong line of evidence as below:
First, expression of a Drosophila odorant receptor in Xenopus oocytes provided
direct evidence for its function, its activation was slower requiring timescale of
second than normally observed millisecond timescale in in vivo function. This
extreme slow response of ORs could be because of lack of OBPs in the heterologous
system of xenopus olfaction process.
Second, kinetic studies demonstrated that the pH-dependent conformational
change in BmPBP requires less than 4 ms. Studies on structural biology aspect of
the molecules indicate that conformational change in BmPBP is an intramolecular
mechanism to facilitate binding and release of pheromones by pheromone-binding
proteins. Whether the remarkable selectivity of the insect’s olfactory system
(Kaissling 1987) is achieved by the specificity of pheromone-binding proteins or the
olfactory receptors is still unclear. When tested with a limited number of candidate
ligands, OBPs bind to candidate ligands specifically (Du and Prestwich 1995;
Maïbèche-Coisné et al. 1997; Maida et al. 2000; Plettner et al. 2000; Wojtasek et al.
1999). However, the number of OBPs is significantly less than the number of com-
pounds that insects can smell. Even in the case of Drosophila, a species which has
been extensively studied, only a few number of OBPs have been identified (Graham
and Davies 2002). How limited number of OBPs detect unlimited numbers of dif-
ferent odorant species is still a matter of research. Evidences show that a Drosophila
olfactory receptors are not specific to a single ligand (Wetzel et al. 2001). It can be
stimulated by compounds with remarkably different chemical structures, such as
cyclohexanol and cyclohexanone, benzaldehyde, and benzyl alcohol. The extraordi-
nary specificity of insect olfactory system has been extensively explored using pio-
neering electroantennogram (Schneider 1957) and single sensillum recordings
(Schneider and Boeckh 1962) at the Max Planck Institute. Even the generalist
detectors for plant compounds have now been demonstrated to have inordinate
specificity (Hansson and Christensen 1999; Nikonov et al. 2001; Nikonov et al.
2 Molecular, Neuronal, and Behavioral Mechanism of Communication Among 45
2002). The mechanism of such specificity of a receptor could be based on the con-
cept of “layers of filters” of participating OBPs that operate step by step. OBPs
transport only small subset of the ligands to reach the pore tubule where each OR
can be stimulated by a small number of ligands out of which only few of them reach
the dendrite. Thus though neither the OBPs nor the ORs are extremely specific, the
whole machinery can show remarkable selectivity by acting as two step filter.
The antennae and maxillary palps are the primary sensory organs in insects that
detect volatile ligands. A huge array of anatomically and functionally diverse spe-
cialized structure called sensilla cover these organs. Inside the sensilla, olfactory
receptor neurons (ORNs) are found in large numbers that are responsible for the
detection of various chemicals (Suh et al. 2014). For example, four different types of
sensilla are found on the antennae of silkworm Bombyx mori, out of which three are
found to detect general, non-pheromone chemicals while the other one a long tricho-
dea is uniquely tuned for detection of the sex pheromones such as bombykol and
bombykal (Sakurai et al. 2014). D. melanogaster possess a trichoid sensilla which
can specifically detect volatile pheromones like cVA and methyl laurate (ML)
(Dweck et al. 2015). Chemical and molecular identities of diverse compound acting
as pheromones are well characterized, however, the receptors responsible for specifi-
cally detecting such diverse pheromones in insect species are still unexplored.
Though the recent advancement in Drosophila molecular genetics and in some
insects has largely filled this gap, neurophysiological processes and behavioral alter-
ation involved in pheromonal signaling require further research in numerous other
insect species. It is now known that two different families of olfactory receptors
(ORs) seem to detect the majority of insect volatile pheromones Kaissling (1986).
The members of the olfactory receptor family were identified first of all as volatile
pheromone receptors (Vosshall et al. 2000; Clyne et al. 1999). cVA, a known phero-
mone, was shown to activate and inhibit innate behavioral programs via the activa-
tion of Or67d expressing and Or65a-expressing neurons using neuronal and
behavioral approaches (Datta et al. 2008; Liu et al. 2011). Furthermore, these neuro-
nal and genetic architectures have been known to be evolutionarily conserved across
the Drosophila species group (Dweck et al. 2015; Dekker et al. 2015; Lebreton et al.
2014). Pheromone receptor neurons synapse with central projection neurons in dis-
crete glomeruli within the antennal lobe similar to olfactory receptor neurons.
2.10 B
ehavioral Mechanism of Communication Among
Insect Species
Most of the insects live a solitary life except few conspecific contacts. Temporary
aggregations among the insect species is often associated with the abundance of
food materials as in case of grasshoppers and the encounter of conspecific males
46 I. Baitharu et al.
and females prior to copulation during breeding season. Social insects are character-
ized by the communities where they live in permanent association with their nest
mates. In this regard, bees, bumblebees, wasps, ants, and termites have fascinated
human beings due to their well-organized and impressive colonies. The social life-
style of insects goes along with the foreseeable development of a communication
system which allows the individual members of the colony to exchange informa-
tion. This mode of communication occurs through various sensory channels, using
visual, acoustic, tactile, sometimes magnetic, and especially chemical signals.
drastically decrease the fidelity of the reopened route (Rosengren and Pamilo 1978).
However, some insect species possess no visual system such as eye and hence visual
cues play no role in their case. For example, some ants and termite species because of
total absence of eyes cannot use visual cues. Winged social insects use acoustic com-
munication by producing buzzing sound through high-frequency wing movements.
As in the case of honey bee, sound produced through rapid wing movement at high
frequency and movement of thoracic muscle during waggle dance helps to attract
attention and provide information about distance and quality of food sources to the
nest mates (Nieh 2004). The queen’s tooting and quacking signals give acoustic com-
munication about newly enclosing queens to make contact with each other (Michelsen
et al. 1986). Sounds produced by knocking body parts onto the substrate called as
drumming in wingless termites (Röhrig et al. 1999) and in some ants provide acoustic
signals and bring about behavioral responses (Hölldobler 1999).
Stridulation behavior in some ant species such as rapid movement of the scraper
situated at the posterior dorsal margin against parallel ridges of first gastral tergite
plays important role in nest mate selection. Atta ants stridulate while cutting leaf
fragments in order to recruit nest mates (Roces and Hölldobler 1996; Eibl-Eibesfeldt
and Eibl-Eibesfeldt 1967). Stridulation activities appear to regulate ant’s species in
maneuvering the leaf fragment into a carrying position (Roces and Hölldobler
1995). However, there are a number of controversial reports regarding the transmis-
sion of ant stridulatory signals through air (Hickling and Brown (2000). It is still
unknown whether the ants are deaf and hence detection of sound occur through
substrate-borne vibrations and not by sound produced (Roces and Tautz 2001).
Magnetic orientation among few insect species such as ants with respect to earth’s
magnetic field has been reported to be used as communication modalities. Several
reports suggest that the magnetic nanoparticles present in the body of the insect
detect the geomagnetic (Acosta-Avalos et al. 1999). In the absence of sunlight cues,
leaf-cutting ants appear to be responding to the geomagnetic field during its forag-
ing journey (Banks and Srygley 2003). The ability to perceive the earth’s magnetic
field has also been demonstrated in a number of insect species such as the fire ant
Solenopsis invicta (Anderson and Vander Meer 1993), bees (Gould 1980), and bum-
ble bees (Chittka et al. 1998). However, orientation along the earth’s magnetic field
is not a true mode of communication among the insect species.
Conclusion
Communication among insect species involves complex process of exchange of
information encoded in semiochemicals like pheromones, sex attractants, acous-
tic exchange of messages through production of unique sound, complex and
peculiar behavior, and highly sensitive and selective reception of signals. In spite
of many years of research into the role of pheromones and other related factors
regulating the behavior of insects, our understanding of the mechanisms and
48 I. Baitharu et al.
evolutionary processes that support these complex signals is still in their infancy.
Although studies in the fruit fly D. melanogaster are paving the way for under-
standing the sensory, neuroethological, and genetic principles of pheromonal
communication, the current lack of comparable genetic tool for other insect spe-
cies hinders progress in the field. A number of studies have been undertaken in
the recent past to understand the neuronal, molecular, and behavioral basis of
insect communication in few insect models. Insects are the members of largest
phylum arthropoda with huge numbers of insect species and unique communica-
tion modalities. Insect pest is the major threat to modern crop system that
includes numerous hybrid varieties with reduced pest resistance. Modern agri-
cultural practice in recent years has introduced large numbers of dangerous per-
sistent pesticides to the environment which has resulted in incidence of number
of diseases in the human system. Understanding the mechanism of insect com-
munication would help in managing pest species without polluting the environ-
ment. For that purpose, identification of receptors and cells responsible for
pheromonal communication in diverse insect species will enable the field to take
advantage of the wealth of existing behavioral and physiological data from these
species. Furthermore, as a number of insect species act as pest or as disease vec-
tors, understanding the mechanism of pheromonal signaling in regulating behav-
ior of these insect species can be implicated for the development of more
sustainable and specific environment-friendly control methods.
References
Acosta-Avalos D, Wajnberg E, Oliveira PS, Leal I, Farina M, Esquivel DMS (1999) Isolation of
magnetic nanoparticles from Pachycondyla marginata ants. J Exp Biol 202:2687–2692
Anderson JB, Vander Meer RK (1993) Magnetic orientation in the fire ant, Solenopsis invicta.
Naturwissenschaften 80:568–570
Anton S (1996) Central olfactory pathways in mosquitoes and other insects. Ciba Found Symp
200:184–192. discussion 192–196, 226–232
Aquiloni L, Tricarico E, SpringerLink (Online service) Springer International Publishing (2015)
In: Aquiloni L, Tricarico E (eds) Social recognition in invertebrates the knowns and the
unknowns. Springer International Publishing, Berlin
Banks AN, Srygley RB (2003) Orientation by magnetic field in leaf-cutter ants, Atta Colombica
(hymenoptera: Formicidae). Ethology 109:835–846
Blaustein DN, Simmons RB, Burgess MF, Derby CD, Nishikawa M, Olson KS (1993)
Ultrastructural localization of 5’AMP odorant receptor sites on the dendrites of olfactory
receptor neurons of the spiny lobster. J Neurosci 13:2821–2828
Blomquist GJ, Tillman JA, Mpuru S, Seybold SJ (1998) The cuticle and cuticular hydrocarbons
of insects: structure, function and biochemistry. In: Vander Meer RK, Breed M, Winston M,
Espelie C (eds) Pheromone communication in social insects. Westview Press, Boulder, CO,
pp 34–54
Carde RT (2014) Defining attraction and aggregation pheromones: teleological versus functional
perspectives. J Chem Ecol 40:519–520
Chittka L, Williams NM, Rasmussen H, Thomson JD (1998) Navigation without vision: bumble-
bee orientation in complete darkness. Proc R Soc Lond B 266:45–50
2 Molecular, Neuronal, and Behavioral Mechanism of Communication Among 49
Chung H, Carroll SB (2015) Wax, sex and the origin of species: dual roles of insect cuticular
hydrocarbons in adaptation and mating. BioEssays 37:822–830
Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim J, Carlson JR (1999) A novel family of divergent
seven-transmembrane proteins: candidate odorant receptors in drosophila. Neuron 22:327–338
Datta SR, Vasconcelos ML, Ruta V, Luo S, Wong A, Demir E, Flores J, Balonze K, Dickson BJ,
Axel R (2008) The drosophila pheromone cVA activates a sexually dimorphic neural circuit.
Nature 452:473–477
Dear TN, Boehm T, Keverne EB, Rabbitts TH (1991) Novel genes for potential ligand-binding
proteins in subregions of the olfactory mucosa. EMBO J 10:2813–2819
Dekker T, Revadi S, Mansourian S, Ramasamy S, Lebreton S, Becher PG, Angeli S, Rota-Stabelli
O, Anfora G (2015) Loss of drosophila pheromone reverses its role in sexual communication
in Drosophila suzukii. Proc Biol Sci 282:20143018
Dickens JC, Callahan FE, Wergin WP, Murphy CA, Vogt RG (1998) Odorant binding proteins of
true bugs. Generic specificity, sexual dimorphism, and association with subsets of chemosen-
sory sensilla. Ann N Y Acad Sci 855:306–310
Dohlman HG, Thorner J, Caron MG, Lefkowitz RJ (1991) Model systems for the study of seven-
transmembrane-segment receptors. Annu Rev Biochem 60:653–688
Du G, Prestwich GD (1995) Protein structure encodes the ligand binding specificity in pheromone
binding proteins. Biochemistry 34:8726–8732
Dweck HK, Ebrahim SA, Thoma M, Mohamed AA, Keesey IW, Trona F, Lavista-Llanos S, Svatos
A, Sachse S, Knaden M et al (2015) Pheromones mediating copulation and attraction in dro-
sophila. Proc Natl Acad Sci U S A 112:E2829–E2835
Eibl-Eibesfeldt I, Eibl-Eibesfeldt E (1967) Das Parasitenabwehren der Minima- Arbeiterinnen der
Blattschneider-Ameise (Atta cephalotes). Z Tierpsychol 24:278–281
Ferguson SS, Downey WER, Colapietro AM, Barak LS, Menard L, Caron MG (1996) Role of
beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization.
Science 271:363–366
Ferveur JF (2005) Cuticular hydrocarbons: their evolution and roles in drosophila pheromonal
communication. Behav Genet 35:279–295
Freedman NJ, Lefkowitz RJ (1996) Desensitization of G protein-coupled receptors. Recent Prog
Horm Res 51:319–351. discussion 352–353
Frisch K (1954) The dancing bees: an account of the life and senses of the honey bee, 1st edn.
Springer-Verlag, Wien, pp XIV–183
Frisch K, Wenner AM, Johnson DL (1967) Honeybees: do they use direction and distance informa-
tion provided by their dancers? Science 158:1072–1077
Gether U (2000) Uncovering molecular mechanisms involved in activation of G protein coupled
receptors. Endocr Rev 21:90–113
Gould JL (1980) The case for magnetic sensitivity in birds and bees (such as it is): surprising
concentrations of magnetite in the tissues of some animals may explain their sensitivity to the
earth’s magnetic field. Am Sci 68:256–267
Graham LA, Davies PL (2002) The odorant-binding proteins of Drosophila melanogaster.
Annotation and characterization of a divergent gene family. Gene 292:43–55
Greenfield MD (2002) Signalers and receivers: mechanisms and evolution of arthropod communi-
cation. Oxford University Press, Oxford
Haberer W, Steiger S, Müller JK (2014) Dynamic changes in volatile emissions of breeding bury-
ing beetles. Physiol Entomol 39:153–164
Hansson BS, Christensen TA (1999) Functional characteristics of the antennal lobe. In: Hansson
BS (ed) Insect olfaction. Springer, Berlin, pp 125–161
Hickling R, Brown RL (2000) Analysis of acoustic communication by ants. J Acoust Soc Am
108:1920–1929
Hildebrand JG, Shepherd GM (1997) Mechanisms of olfactory discrimination: converging evi-
dence for common principles across phyla. Annu Rev Neurosci 20:595–631
Hölldobler B (1999) Multimodal signals in ant communication. J Comp Physiol 184A:129–141
50 I. Baitharu et al.
Pippig S, Andexinger S, Daniel K, Puzicha M, Caron MG, Lefkowitz RJ, Lohse MJ (1993)
Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization
of beta 2-adrenergic receptors. J Biol Chem 268:3201–3208
Plettner E, Lazar J, Prestwich EG, Prestwich GD (2000) Discrimination of pheromone enantio-
mers by two pheromone binding proteins from the gypsy moth Lymantria dispar. Biochemistry
39:8953–8962
Roces F, Hölldobler B (1995) Vibrational communication between hitchhikers and foragers in leaf-
cutting ants (Atta cephalotes). Behav Ecol Sociobiol 37:297–302
Roces F, Hölldobler B (1996) Use of stridulation in foraging leaf-cutting ants: mechanical support
during cutting or short-range recruitment signal? Behav Ecol Sociobiol 39:293–299
Roces F, Tautz J (2001) Ants are deaf. J Acoust Soc Am 109:3080–3082
Röhrig A, Kirchner WH, Leuthold RH (1999) Vibrational alarm communication in the African
fungus-growing termite genus Macrotermes (Isoptera, Termitidae). Insect Soc 46:71–77
Rosengren R, Pamilo P (1978) Effect of winter timber felling on behaviour of foraging wood ants
(Formica rufagroup) in early spring. Memorabilia Zool 29:143–155
Sakurai T, Namiki S, Kanzaki R (2014) Molecular and neural mechanisms of sex pheromone
reception and processing in the silkmoth Bombyx mori. Front Physiol 5:125
Schneider D (1957) Elektrophysiologische untersuchungen von chemo- und mechanorezeptoren
der antenne des seidenspinners Bombyx mori L. Z Vergl Physiol 40:8–41
Schneider D, Boeckh J (1962) Rezeptorpotential und nervenimpulse einzelner olfaktorischer sen-
sillen der insektenantenne. Z Vergl Physiol 45:405–412
Stocker RF (1994) The organization of the chemosensory system in Drosophila melanogaster: a
review. Cell Tissue Res 275:3–26
Strader CD, Fong TM, Tota MR, Underwood D (1994) Structure and function of G protein-coupled
receptors. Annu Rev Biochem 63:101–132
Suh E, Bohbot JD, Zwiebel LJ (2014) Peripheral olfactory signaling in insects. Curr Opin Insect
Sci 6:86–92
van Zweden JS, d’Ettorre P (2010) Nestmate recognition in social insects and the role of hydrocar-
bons. In: Insect hydrocarbons: biology, biochemistry and chemical ecology, vol 11. Cambridge
University Press, Cambridge, pp 222–243
Vogt RG (1987) The molecular basis of pheromone reception: its influence on behavior. In:
Prestwich GD, Blomquis GJ (eds) Pheromone biochemistry. Academic Press, New York,
pp 385–431
Vogt RG, Rybczynski R, Lerner MR (1991) Molecular cloning and sequencing of general odorant-
binding proteins GOBP1 and GOBP2 from the tobacco hawk moth Manduca sexta: compari-
son with other insect OBPs and their signal peptides. J Neurosci 11:2972–2984
Vosshall LB, Wong AM, Axel R (2000) An olfactory sensory map in the fly brain. Cell 102:147–159
Wang L, Anderson DJ (2010) Identification of an aggression-promoting pheromone and its recep-
tor neurons in drosophila. Nature 463:227–231
Wehner R (2003) Desert ant navigation: how miniature brains solve complex tasks. J Comp
Physiol 189:579–588
Wertheim B, Allemand R, Vet LEM, Dicke M (2006) Effects of aggregation pheromone on individ-
ual behaviour and food web interactions: a field study on drosophila. Ecol Entomol 31:216–226
Wetzel CH, Behrendt H-J, Gisselmann G, Störtkuhl KF, Hovemann B, Hatt H (2001) Functional
expression and characterization of a drosophila odorant receptor in a heterologous cell system.
Proc Natl Acad Sci 98:9377–9380
Wojtasek H, Picimbon JF, Leal WS (1999) Identification and cloning of odorant binding proteins
from the scarab beetle Phyllopertha diversa. Biochem Biophys Res Commun 263:832–837
Wyatt TD (2014) Pheromones and animal behavior: chemical signals and signatures, 2nd edn.
Cambridge University Press, Cambridge
Yew JY, Chung H (2015) Insect pheromones: an overview of function, form, and discovery. Prog
Lipid Res 59:88–105
Monocyclic Aromatic Hydrocarbons
(MAHs) Induced Toxicity in Drosophila: 3
How Close How Far?
Mahendra P. Singh and Ranjana Himalian
Abstract
Monocyclic aromatic hydrocarbons (MAHs) are being used as individual chem-
icals or as in mixtures of two or more chemicals in several industrial and house-
hold processes across the world. Among MAHs, the most common chemicals
are benzene, toluene and xylene, and they are also known as volatile organic
compounds (VOCs), among them benzene categorised as highly toxic chemical
and also listed as human carcinogen. Benzene, toluene and xylene cause cyto-
toxicity to a nontarget organism-like Drosophila melanogaster as an individual
(benzene or toluene or xylene)/in mixture (benzene-toluene-xylene or benzene-
toluene or benzene-xylene). In this chapter, several cellular, biochemical and
molecular approaches were used to evaluate cellular toxicity due to MAHs like
benzene, toluene and xylene using Drosophila melanogaster as an alternative to
animal. We also judged variable cytotoxicity patterns of MAHs when they are
exposed individually or in a mixture of two/three chemicals. An antagonistic
effect of xylene and toluene on benzene toxicity and additive/synergistic effect
of xylene on toluene-induced toxicity were evident in Drosophila. This study
shows that co-exposure of benzene-toluene-xylene causes reduced cellular and
organismal toxicity as compared to individual test chemical on Drosophila
melanogaster.
3.1 Introduction
Monocyclic aromatic hydrocarbons have diverse ring and side chain structure that
could independently affect their absorption and toxicity effectively (Chou et al.
2003). Unleaded petrol contains significant amount of benzene, toluene and xylene,
collectively called as BTX. A number of chemicals are used in chemical and phar-
maceutical industries. Among the major monocyclic aromatic hydrocarbons
(MAHs) are styrene, benzene, toluene and isomers o-xylene, m-xylene and
p-xylene. These chemicals are constituted in gasoline up to 15% and also signifi-
cantly present in paints, plastics, detergents, dyes, adhesives, pesticides, rubber
products and several other products (Derwent et al. 2000; Kim and Kim 2002;
Chang et al. 2007). They are one of the major pollutants of the outdoor and indoor
environment (Lee et al. 2002; Srivastava and Devotta 2007). Benzene is causally
linked to leukaemia (IARC) (US-EPA 1996), whereas currently toluene and xylene
are not listed as carcinogens (Gallegos et al. 2007). BTX is a special lipophilic
substance that has capability to penetrate the skin or subcutaneous fat (Adami et al.
2006a, b and also used as an individual or as a mixture compound. It is shown that
benzene is more toxic as compared to toluene and xylene (Singh et al. 2009, 2010,
2011). Earlier studies reported the presence of benzene and benzene mixtures in
different occupational environments, like leather, electronics, machinery and sports
equipment industries (Wong and Raabe 1989; Dosemeci et al. 1994; Kuang and
Liang 2005; Wang et al. 2006). Exposure to these nonoxygenated aromatic hydro-
carbons is of great concern, even at low concentrations because of their toxicity
and carcinogenicity. An increasing number of young people inhale these volatile
substances for recreational purposes (Greer 1984; Kozel et al. 1995; Spiller and
Krenzelok 1997).
Toluene has a protective effect on male mice against benzene toxicity as toluene
is a competitive inhibitor of benzene metabolism (Andrew et al. 1977). Prior or co-
exposure to other chemicals is also likely to modulate the benzene metabolism,
suggesting synergistic or antagonistic effect of one or the other chemicals (Medinsky
et al. 1994). Benzene, toluene and xylene toxicity mechanism depends on their
metabolites (Croute et al. 2002). Benzene-induced toxicity is related to some types
of cancer and blood disorders, including bone marrow depression (Wan and Winn
2004; Wetmore et al. 2008). BTX induces genotoxicity and apoptosis in in vitro and
in vivo models (Smith 1996; Ross 2000; Snyder 2000; Nakai et al. 2003; Al-Ghamdi
et al. 2004; Wan and Winn 2004; Wetmore et al. 2008).
3.2 Benzene
Benzene has a diverse ring and is the smallest and is the most stable aromatic
hydrocarbon with no side chain. Benzene is classified as a class I carcinogen that
causes DNA damage both in vitro and in vivo (Sul et al. 2005; Weaver et al. 2007;
Weaver and Liu 2008). Benzene toxicity is the result of synergistic interaction
among metabolites (Smith et al. 1989; Irons et al. 1992; Kolachana et al. 1993;
3 Monocyclic Aromatic Hydrocarbons (MAHs) Induced Toxicity in Drosophila 55
Chapman et al. 1994), like phenol, hydroquinone and catechol (Medinsky et al.
1996; Snyder 2007). There is significant increase in the chromosomal breaks in
K-562 cells, haematopoietic stem cells (HSC), sister chromosomal exchanges and
clastogenicity when exposed to benzene and its metabolites (Singh and Winn 2008;
Faiola et al. 2004; Erexson et al. 1986; Zhang et al. 2002). Applying comet assay,
benzene and its metabolites showed increased comet parameters in the human lym-
phocytes and HeLa cells (Chen et al. 2008; Galvan et al. 2008). Benzene occupa-
tional and environmental hazard is associated with increased risk of leukaemia.
Benzene is recognised as a hematotoxin and human carcinogen. (Irons et al. 2013;
Lagorio et al. 2013; Li and Yin 2006; Snyder 2012) and is an important pollutant
in indoor air (Rappaport et al. 2013; Weisel 2010; Xing et al. 2013). Benzene toxic-
ity is recognised by oxidative stress, which mediates change in DNA methylation
(Ahmed et al. 2009; Adami et al. 2006a, b; Ayalogu et al. 2001; Lippmann et al.
2011; Revilla et al. 2007; Brautbar et al. 2006; Saadat and Ansari-Lari 2005; Dogru
et al. 2007). 1,4-Benzoquinone (1,4-BQ) is the benzene’s most toxic metabolite
and is used to evaluate the benzene-induced toxicity (Das et al. 2010; Hu et al.
2014; Stokes and Winn 2014; Tian et al. 2012; Yang and Zhou 2010). Aberrant
DNA methylation helps in the early diagnosis of the disease (Deng and Liu 2010).
Previous reports documented benzene metabolism-induced reactive oxygen spe-
cies (ROS) (Badham and Winn 2010), which increases free radicals and attacks
DNA molecule, resulting in DNA strand break and oxidative damage (Atkinson
2009; Barreto et al. 2009).
These are aromatic hydrocarbons with diverse side chain. These including benzene
are the most dangerous component of petrol (Perigo and Prado 2005). Risk of acute
or chronic toxicity is associated with production distribution and use of petrol
(Bruckner and Warren 2001). Their exposure leads to hepatotoxicity and nephrotox-
icity (Benson et al. 2011).
bg a b c d
pv
mg
hg
Fig. 3.1 Trypan blue staining in the internal tissues isolated from third instar larvae of D. melano-
gaster (Oregon R+) control (a) and in third instar larvae exposed to 100.0 mM benzene (b), toluene
(c) and xylene (d) after 48 h. bg brain ganglia, pv proventriculus, mg midgut, hg hind gut. Bar
represents 100 μm (taken from Singh et al. 2011)
xylene and toluene. A maximum depletion in GSH content was observed after 48 h
at the highest dietary concentration of the benzene, toluene and xylene. Larvae
exposed to the highest concentration of test chemicals exhibited a significantly
enhanced enzyme activity after 6 h in benzene and 12 h in both xylene and toluene.
BTX induced cellular and organismal toxicity in Drosophila by the expression of
stress, oxidative stress and organismal assays.
Flies were exposed to either individual test chemical or in a mixture of two or three
chemicals, i.e. benzene-toluene, benzene-xylene or benzene-toluene and xylene
(Singh et al. 2010. The magnitude of toxicity of a benzene-toluene (BT) or benzene-
xylene (BX) or benzene-toluene-xylene mixture (BTX) was statistically signifi-
cantly lower in flies than in the individual chemical cases. When BT is induced, it’s
seen it has protective effect on mice as toluene is a competitive inhibitor of benzene
metabolism. Benzene metabolism is modulated by co-exposure or prior exposure to
other chemicals (Medinsky et al. 1994). Combination of toluene and xylene expo-
sure results in additive effect (Chen et al. 1994). There is a reduction in benzene-
induced cellular and genetic toxicity when exposed to BT or BX mixture
(Gad-El-Karim et al. 1984; Plappert et al. 1994).
We did not observe any mortality in any of the mixture concentration throughout
the exposure duration (Singh et al. 2010). In mixture-treated groups, they observed
the delay in the emergence of the flies by 1 day. BT, BX and BTX mixture-treated
groups also show significantly lower β-galactosidase activity. Levels of hsp70 in
mixture-treated groups also increased significantly after 48 h as compared to that
after 24 h (Fig. 3.2). ROS generation and CAT activity were significantly lower in
mixture-treated group as compared to the individual tested groups. They showed
that benzene, toluene and xylene either individually or in mixture are toxic to the
exposed organism, but the mixture is less toxic than the individual chemicals. The
highest concentrations of all the components of mixtures cause 100% larval lethal-
ity after 2 h indicating a very severe additive effect of the mixture. This indicates
that after threshold limit, these may work synergistically or additively with benzene.
The exposed organism was rescued means organismal toxicity was less severe when
larvae were exposed to BT, BX or BTX mixture. Mixed chemicals affect the hsp70
expression only after 48 h as compared to individual chemical exposure which is
after 24 h. This indicates lesser generation of damaging signals in cells when treated
by mixed chemicals. BTX, BT and BX mixture decreased the activities of CAT,
MDA and PC content and lesser depletion of GSH content in the exposed
organism.
Exposure of organisms to the pollutants, which includes monocyclic aromatic
hydrocarbons, shows genotoxicity and apoptosis. Genotoxicity is defined as a
destructive effect on a cells genetic material that is DNA and RNA, affecting its
integrity. Apoptosis is the death of cells which occurs as a normal and controlled
58 M.P. Singh and R. Himalian
a
hsp 70
9 Control
8 * * B25
T * *
7
Fold change (normalized
T25
*
6 * * T* * * * X25
with GAPDH)
# # *T*
* *
5 T * S S T *
* S * T T* T* B50
T
4 * * *
T50
3
X50
2
B50+T50
1
B50+X50
0
24 48 B50+T25+X25
Exposure time (h)
b 9
Control
8 * *
B25
7
Fold change (normalized
6 T25
with GAPDH)
* *
5 X25
** ** * *
4 * * S S S
B50
* * *
*
3 * * * * T50
*
2
X50
1
B50+T50
0
24 48 B50+X50
Exposure time (h)
B50+T25+X25
Fig. 3.2 Quantitative real-time PCR (qRT-PCR) analysis of hsp70 (a), hsp83 (b), hsp60 (c) and
hsp26 (d) mRNA in control, individual and mixtures exposed third instar larvae of D. melanogas-
ter (Oregon R+) for 24 and 48 h (taken from Singh et al. 2010)
3 Monocyclic Aromatic Hydrocarbons (MAHs) Induced Toxicity in Drosophila 59
c 9
Control
8
* *
B25
7
Fold change (normalized
T25
6
with GAPDH)
** X25
5
* * S SS B50
4 * * *
* * * T50
3 * * * * X50
* *
2 *
* B50+T50
1 B50+X50
0 B50+T25+X25
24 48
Exposure time (h)
d 9
Control
8
B25
7
Fold change (normalized
T25
* * **
6 ** # # # X25
with GAPDH)
5 ** S SS
* * * B50
4 * * * *
* T50
* * *
3 * *
X50
2 B50+T50
1
B50+X50
0 B50+T25+X25
24 48
Fig. 3.2 (continued)
part of an organism’s development and growth. Comet assay parameters show dam-
age only after 24 h in benzene and 48 h in toluene and xylene. There is significantly
increase in the AV-positive cells when exposed to low concentration of benzene
after 24 h and after 48 h in xylene and toluene. At the highest concentration, this
increase is after 12 h and 24 h, respectively. For caspase activity change in mito-
chondrial membrane potential is required (Hay and Guo 2006). Larvae when
exposed to low concentration of benzene exhibit significant change in membrane
potential after 24 h and XT showed after 48 h. BTX showed this effect at high con-
centration after 24 h.
60 M.P. Singh and R. Himalian
a
10
Control
9
* DMSO
Fold increase in ROS generation
8 B100
(mean fluorescent intensity)
7 T100
X100
6
* QC
5 B100+QC
s * * T100+QC
4 * s
* * * s s s s s X100+QC
* * s
3 * * * * * * * *
CUR
2 B100+CUR
*
T100+CUR
1
X100+CUR
0
24 48
b Control c QC Control d Cur Control
200 200 200
11% 10.6%
8%
Count
Count
50 50 50
0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
DCF-DA DCF-DA DCF-DA
e B100 f B100+QC
g B100+Cur
200 200 200
68% 24% 23.9%
Count
50 50 50
0 0 0
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104
DCF-DA DCF-DA DCF-DA
Fig. 3.3 ROS generation in D. melanogaster (Oregon R+) in control, DMSO and benzene, tolu-
ene or xylene alone or in combinations with QC or CUR treatments for 24 and 48 h. Histogram (a)
depicts ROS generation in test chemical exposed organisms, and flow cytometric panels show the
ROS generation in (b) control, (c) QC control, (d) CUR control, (e) B100, (f) B100 + QC and (g)
B100 + CUR exposed organisms after 48 h. Data represent mean ± SD of three identical experi-
ments made in triplicates, and significance is ascribed as *P < 0.01 vs. control; $P < 0.01, reduction
vs. individual chemical (B100 or T100 or X100) (taken from Singh et al. 2011)
3 Monocyclic Aromatic Hydrocarbons (MAHs) Induced Toxicity in Drosophila 61
Larvae when exposed to curcumin and quercetin mixed with chemical mixtures
show significant diminution in the GST activity and reduction in ROS generation,
SOD, CAT activity and MDA content after 48 h. There is also significant reduction
in DNA damage and DNA migration after 24 and 48 h when exposed to QC and
CUR (Fig. 3.3).
References
Adami G, Larese F, Venier M, Barbieri P, LoCoco F, Reisenhofer E (2006a) Penetration of ben-
zene, toluene and xylenes contained in gasoline’s through human abdominal skin in vitro.
Toxicol In Vitro 20:1321e30
Adami G, Larese F, Venier M, Barbieri P, Lo Coco F, Reisenhofer E (2006b) Penetration of ben-
zene, toluene and xylenes contained in gasolines through human abdominal skin in vitro.
Toxicol In Vitro 20:1321–1330
Ahmed HH, Metwally FM, Rashad HM (2009) Toxicity of solvents exposure on the neuroendo-
crine system in rats: role of amino acids supplementation. Toxic Solvents Rep Opin 1:66e83
Al-Ghamdi SS, Raftery MJ, Yaqoob MM (2004) Toluene and p-xylene induced LLCPK1 apopto-
sis. Drug Chem Toxicol 27:425–432
Andrew LS, Lee EW, Witmer CM, Kocsis JJ, Snyder R (1977) Effects of toluene on the metabo-
lism, disposition and hemopoietic toxicity of [3H] benzene. Biochem Pharmacol 26:293–300
Atkinson TJ (2009) A review of the role of benzene metabolites and mechanisms in malignant
transformation: summative evidence for a lack of research in nonmyelogenous cancer types.
Int J Hyg Environ Health 212:1–10
62 M.P. Singh and R. Himalian
Ayalogu OE, Igboh NM, Dede EB (2001) Biochemical changes in the serum and liver of albino
rats exposed to petroleum samples (gasoline, kerosene, and crude petroleum). J Appl Sci
Environ Manage 5:97e100
Badham HJ, Winn LM (2010) In utero exposure to benzene disrupts fetal hematopoietic progenitor
cell growth via reactive oxygen species. Toxicol Sci 113:207–215
Barreto G, Madureira D, Capani F, Aon-Bertolino L, Saraceno E, Alvarez-Giraldez LD (2009) The
role of catechols and free radicals in benzene toxicity: an oxidative DNA damage pathway.
Environ Mol Mutagen 50:771–780
Benson JM, Gigliotti AP, March TH, Barr EB, Tibbetts BM, Skipper BJ, Clark CR, Twerdok L
(2011) Chronic carcinogenicity of gasoline vapour condensate (GVC) and GVC containing
methyl tertiary-butyl ether in f344 rats. J Toxicol Environ Health A 74:638e57
Brautbar N, Wu MP, Gabel E, Regev L (2006) Occupational kidney cancer exposure to industrial
solvents. Ann N Y Acad Sci 1076:753e64
Bruckner JV, Warren DA (2001) Toxic effects of solvents and vapors. In: Klaassen CD (ed)
Casarette and Doulls toxicology the basic science of poisons, 6th edn. McGraw-Hill Medical,
New York, p 869e944
Chang FK, Chen ML, Cheng SF, Shih TS, Mao IF (2007) Dermal absorption of solvents as a major
source of exposure among shipyard spray painters. J Occup Environ Med 49:430–436
Chapman DE, Namkung MJ, Juchau MR (1994) Benzene and benzene metabolites as embryotoxic
agents: effects on cultured rat embryos. Toxicol Appl Pharmacol 128:129–137
Chen Z, Liu SJ, Cai SX, Yao YM, Yin H, Ukai H, Uchida Y, Nakatsuka H, Watanabe T, Ikeda M
(1994) Exposure of workers to a mixture of toluene and xylenes. II. Effects. Occup Environ
Med 51:47–59
Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB (2008) Oxidative stress induces autoph-
agic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ
15:171–182
Chou CC, Riviere JE, Monteiro-Riviere NA (2003) The cytotoxicity of jet fuel aromatic hydrocar-
bons and dose-related interleukin-8 release from human epidermal keratinocytes. Arch Toxicol
77:384–391
Croute F, Poinsot J, Gaubin Y, Beau B, Simon V, Murat JC, Soleilhavoup JP (2002) Volatile organic
compounds cytotoxicity and expression of HSP72, HSP90 and GRP78 stress proteins in cul-
tured human cells. Biochim Biophys Acta 1591:147–155
Das S, Chakrabarty D, Choudhury GC (2010) 1,4-benzoquinone (PBQ) induced toxicity in lung
epithelial cells is mediated by the disruption of the microtubule network and activation of
caspase-3. Chem Res Toxicol 23:1054–1066
Deng Z, Liu YD (2010) Epigenetic alterations as cancer diagnostic, prognostic, and predictive
biomarkers. Adv Genet 71:125–176
Derwent RG, Davies TJ, Delaney M, Dollard GJ, Field RA, Dumitrean P, Nason PD, Jones BMR,
Pepler SA (2000) Analysis and interpretation of the continuous hourly monitoring data for 26
C2–C8 hydrocarbons at 12 United Kingdom sites during 1996. Atmos Environ 34:297–312
Dogru O, Celkan T, Demir T (2007) Hematological and biochemical changes in volatile substance
abusing street children in Istanbul. Turk J Hematol 24:52e6
Dosemeci M, Li GL, Hayes RB, Yin SN, Linet M, Chow WH, Wang YZ, Jiang ZL, Dai TR, Zhang
WU (1994) Cohort study among workers exposed to benzene in China: II. Exposure assess-
ment. Am J Ind Med 26:401–411
Erexson GL, Wilmer JL, Steinhagen WH, Kligerman AD (1986) Induction of cytogenetic damage
in rodents after short-term inhalation of benzene. Environ Mutagen 8:29–40
Faiola B, Fuller ES, Wong VA, Pluta L, Abernethy DJ, Rose J, Recio L (2004) Exposure of hema-
topoietic stem cells to benzene or 1,4-benzoquinone induces gender-specific gene expression.
Stem Cells 22:750–758
Gad-El-Karim MM, Harper BL, Legator MS (1984) Modifications in the myeloclastogenic effect
of benzene in mice with toluene, phenobarbital, 3- methylcholanthrene, Aroclor 1254, SKF-
525A. Mutat Res 135:225–243
3 Monocyclic Aromatic Hydrocarbons (MAHs) Induced Toxicity in Drosophila 63
Gallegos P, Lutz J, Markwiese J, Ryti R, Mirenda R (2007) Wildlife ecological screening levels for
inhalation of volatile organic chemicals. Environ Toxicol Chem 26:1299–1303
Galvan N, Lim S, Zmugg S, Smith MT, Zhang L (2008) Depletion of WRN enhances DNA dam-
age in HeLa cells exposed to the benzene metabolite, hydroquinone. Mutat Res 649:54–61
Gayathri MV, Krishnamurthy NB (1981) Studies on the toxicity of mercurial fungicide Agallol3 in
Drosophila melanogaster. Environ Res 24:89–95
Greer JE (1984) Adolescent abuse of typewriter correction fluid. South Med J 77:297–298
Hay BA, Guo M (2006) Caspase-dependent cell death in Drosophila. Annu Rev Cell Dev Biol
22:623–650
Hu J, Ma H, Zhang W, Yu Z, Fu J (2014) Effects of benzene and its metabolites on global DNA
methylation in human normal hepatic L02 cells. Environ Toxicol 29:108–116
Irons RD, Stillman WS, Colagiovanni DB, Henry VA (1992) Synergistic action of benzene metab-
olite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-
stimulating in vitro. Proc Natl Acad Sci U S A 89:3691–3695
Irons RD, Chen Y, Wang X, Ryder J, Kerzic PJ (2013) Acute myeloid leukemia following exposure
to benzene more closely resembles de novo than therapy related-disease. Genes Chromosomes
Cancer 52:887–894
Kim KH, Kim MY (2002) The distributions of BTEX compounds in the ambient atmosphere of the
Nan-Ji-Do abandoned landfill site in Seoul. Atmos Environ 36:2433–2446
Kolachana P, Subrahmanyam VV, Meyer K, Zhang L, Smith M (1993) Benzene and its phenolic
metabolites produce oxidative DNA damage in HL-60 cells in vitro and in the bone marrow
in vivo. Cancer Res 53:1023–1026
Kozel N, Sloboda Z, De La Rosa M (eds) (1995) Epidemiology of inhalant abuse: an international
perspective. US Department of Health and Human Services, Washington, DC. NIDA Res. Mon. 14
Kuang S, Liang W (2005) Clinical analysis of 43 cases of chronic benzene poisoning. Chem Biol
Interact 30:129–135
Lagorio S, Ferrante D, Ranucci A, Negri S, Sacco P, Rondelli R, Cannizzaro S, Torregrossa MV,
Cocco P, Forastiere F, Miligi L, Bisanti L, Magnani C (2013) Exposure to benzene and child-
hood leukaemia: a pilot case-control study. BMJ Open 3:e002275
Lee SC, Chiu MY, Ho KF, Zou SC, Wang X (2002) Volatile organic compounds (VOCs) in urban
atmosphere of Hong Kong. Chemosphere 48:375–382
Li G, Yin S (2006) Progress of epidemiological and molecular epidemiological studies on benzene
in China. Ann N Y Acad Sci 1076:800–809
Lippmann SJ, Richardson DB, Chen JCM (2011) Elevated serum liver enzymes and fatty liver
changes associated with long driving among taxi drivers. Am J Ind Med 54:618e27
Medinsky MA, Schlosser PM, Bond JA (1994) Critical issues in benzene toxicity and metabolism:
the effect of interactions with other organic chemicals on risk assessment. Environ Health
Perspect 102:119–124
Medinsky MA, Kenyon EM, Seaton MJ, Schlosser PM (1996) Mechanistic considerations in ben-
zene physiological model development. Environ Health Perspect 104:1399–1404
Nakai N, Murata M, Nagahama M, Hirase T, Tanaka M, Fujikawa T, Nakao N, Nakashima K,
Kawanishi S (2003) Oxidative DNA damage induced by toluene is involved in its male repro-
ductive toxicity. Free Radic Res 37:69–76
Perigo JF, Prado C (2005) Evolution of occupational exposure to environmental levels of aromatic
hydrocarbons in service stations. Ann Occup Hyg 49(233e):40
Plappert U, Barthel E, Seidel HJ (1994) Reduction of benzene toxicity by toluene. Environ Mol
Mutagen 24:283–292
Rappaport SM, Kim S, Thomas R, Johnson BA, Bois FY, Kupper LL (2013) Low-dose metabolism
of benzene in humans: science and obfuscation. Carcinogenesis 34:2–9
Revilla AS, Pestana CR, Pardo-Andreu GL, Santos AC, Uyemura SA, Gonzales ME (2007) Potential
toxicity of toluene and xylene evoked by mitochondrial uncoupling. Toxicol In Vitro 21:782e8
Ross D (2000) The role of metabolism and specific metabolites in benzene-induced toxicity: evi-
dence and issues. J Toxicol Environ Health A 61:357–372
64 M.P. Singh and R. Himalian
Saadat M, Ansari-Lari M (2005) Alterations of liver function test indices of filling station workers
with respect of genetic polymorphisms of GSTM1 and GSTT1. Cancer Lett 227:163e7
Siddique HR, Kar Chowdhuri D, Saxena DK, Dhawan A (2005) Validation of Drosophila melano-
gaster as an in vivo model for genotoxicity assessment using modified alkaline Comet assay.
Mutagenesis 4:285–290
Singh R, Winn LM (2008) The effects of 1,4-benzoquinone on c-Myb and topoisomerase II in
K-562 cells. Mutat Res 645:33–38
Singh MP, Reddy MM, Mathur N, Saxena DK, Chowdhuri DK (2009) Induction of hsp70,
hsp60, hsp83 and hsp26 and oxidative stress markers in benzene, toluene and xylene exposed
Drosophila melanogaster: role of ROS generation. Toxicol Appl Pharmacol 235(2):226–243
Singh MP, Ram KR, Mishra M, Shrivastava M, Saxena DK, Chowdhuri DK (2010) Effects of co-
exposure of benzene, toluene and xylene to Drosophila melanogaster: alteration in hsp70, hsp60,
hsp83, hsp26, ROS generation and oxidative stress markers. Chemosphere 79(5):577–587
Singh MP, Mishra M, Sharma A, Shukla AK, Mudiam MK, Patel DK, Ram KR, Chowdhuri DK
(2011) Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene
and xylene: attenuation by quercetin and curcumin. Toxicol Appl Pharmacol 253(1):14–30
Smith MT (1996) Mechanistic studies of benzene toxicity—implications for risk assessment. Adv
Exp Med Biol 387:259–266
Smith MT, Yager JW, Steinmetz KL, Eastmond DA (1989) Peroxidase-dependent metabolism of
benzene’s phenolic metabolites and its potential role in benzene toxicity and carcinogenicity.
Environ Health Perspect 82:23–29
Snyder R (2000) Overview of the toxicology of benzene. J Toxicol Environ Health A 61:339–346
Snyder R (2007) Benzene’s toxicity: a consolidated short review of human and animal studies by
HA Khan. Hum Exp Toxicol 26:687–696
Snyder R (2012) Leukemia and benzene. Int J Environ Res Public Health 9:2875–2893
Spiller HA, Krenzelok EP (1997) Epidemiology of inhalant abuse reported to two regional poison
centers. J Toxicol Clin Toxicol 35:167–173
Srivastava A, Devotta S (2007) Indoor air quality of public places in Mumbai, India in terms of
volatile organic compounds. Environ Monit Assess 133:127–138
Stokes SE, Winn LM (2014) NF-kappaB signaling is increased in hd3 cells following exposure to
1,4-benzoquinone: role of reactive oxygen species and p38-MAPK. Toxicol Sci 137:303–310
Sul D, Lee E, Lee MY, Oh E, Im H, Lee J, Jung WW, Won N, Kang HS, Kim EM, Kang SK (2005)
DNA damage in lymphocytes of benzene exposed workers correlates with trans, trans-muconic
acids and breath benzene levels. Mutat Res 582:61–70
Tian JF, Peng CH, Yu XY, Yang XJ, Yan HT (2012) Expression and methylation analysis of p15 and
p16 in mouse bone marrow cells exposed to 1,4-benzoquinone. Hum Exp Toxicol 31:718–725
US EPA 1996 Priority pollutants, code of federal regulations. Title 40; U.S. Environmental Protection
Agency. U.S. Government Printing Office, Washington, DC. Part 423, App. A (chapter 1)
Wan J, Winn LM (2004) The effects of benzene and the metabolites phenol and catechol on c-Myb
and Pim-1 signaling in HD3 cells. Toxicol Appl Pharmacol 201:194–201
Wang L, Zhou Y, Liang Y, Wong O, Armstrong T, Schnatter AR, Wu Q, Fang J, Ye X, Fu H,
Irons RD (2006) Benzene exposure in the shoemaking industry in China, a literature survey,
1978–2004. Regul Toxicol Pharmacol 46:149–156
Weaver CV, Liu SP (2008) Differentially expressed pro- and anti-apoptogenic genes in response to
benzene exposure: immunohistochemical localization of p53, Bag, Bad, Bax, Bcl-2, and Bcl-w
in lung epithelia. Exp Toxicol Pathol 59:265–272
Weaver CV, Liu SP, Lu JF, Lin BS (2007) The effects of benzene exposure on apoptosisin epithelial
lung cells: localization by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL) and the immunocytochemical localization of apoptosis-related gene
products. Cell Biol Toxicol 23:201–220
Weisel CP (2010) Benzene exposure: an overview of monitoring methods and their findings. Chem
Biol Interact 184:58–66
3 Monocyclic Aromatic Hydrocarbons (MAHs) Induced Toxicity in Drosophila 65
Wetmore BA, Struve MF, Gao P, Sharma S, Allison N, Roberts KC, Letinski DJ, Nicolich MJ, Bird
MG, Dorman DC (2008) Genotoxicity of intermittent co-exposure to benzene and toluene in
male CD-1 mice. Chem Biol Interact 173:166–178
Wong O, Raabe GK (1989) Critical review of cancer epidemiology in petroleum industry employ-
ees, with a quantitative meta-analysis by cancer site. Am J Ind Med 15:283–310
Xing Q, Chen G, Li L, Zhang M, Zheng Z, Zou L, Hou QF, Wang X, Liu XG (2013) Microsomal
epoxide hydrolase (EPHX1) polymorphisms are associated with aberrant promoter meth-
ylation of ERCC3 and hematotoxicity in benzene-exposed workers. Environ Mol Mutagen
54:397–405
Yang F, Zhou JH (2010) Cytotoxicity and DNA damage induced by 1,4-benzoquinone in v79
Chinese hamster lung cells. J Toxicol Environ Health A 73:483–489
Zhang L, Eastmond DA, Smith MT (2002) The nature of chromosomal aberrations detected in
humans exposed to benzene. Crit Rev Toxicol 32:1–42
Tracing of Evolution in Silkworm,
Bombyx mori L., on the Basis 4
of Molecular Studies
E. Muniraju and Rajendra Mundkur
Abstract
Pure Mysore and Nistari are known oldest races of India. Both are multivoltine
races which produce colored cocoons. The origin of these races is obscure. Pure
Mysore was supposed to have originated from the race that Mysore King Tippu
Sultan brought from China in 1875 and established in Karnataka State. Nistari is
the well-known race also believed to have brought from China and established in
West Bengal area. However, the wild sericigenous species of Bombyx, Theophila,
and Ocinara are naturally distributed in the Himalayan ranges of Indo-China
range and also in the Andaman Islands in India, besides Jawa, Sumatra, Borneo,
and Malay Peninsula (Barlow, An introduction to the moths of South East Asia,
1982). Apart from these, there are wild relatives of silkworm, B. mandarina,
which have been collected from Kedarnath. It is believed that the silkworm,
Bombyx mori L., has evolved from B. mandarina in China and spread across the
globe. There are many theories about the pattern of silkworm evolution and
spread. In this article, the theories are discussed on the molecular basis.
4.1 Introduction
Pure Mysore and Nistari are known oldest races of India. Both are Multivoltine
races which produce colored cocoons. The origin of these races is obscure. Pure
Mysore was supposed to have originated from the race that Mysore King Tippu
Sultan brought from China in 1875 and established in Karnataka State. Nistari is
the well-known race also believed to have brought from China and established in
Fig. 4.1 Geographical location of Kedarnath where Ancestor of Silkworm, Bombyx mandarina is
also available in the wild
West Bengal area. However, the wild sericigenous species of Bombyx, Theophila,
and Ocinara are naturally distributed in the Himalayan ranges of Indo-China
range and also in Andaman Islands in India, besides Jawa, Sumatra, Borneo, and
Malaya Peninsular (Barlow 1982). In addition to these species, wild relatives of
silkworm, B. mandarina, has been collected from Kedarnath (Fig. 4.1). It is
believed that the Silkworm, Bombyx mori L. has evolved from B. mandarina in
China and spread across the globe. There are many theories about the pattern of
silkworm evolution and spread. In this article, the theories are discussed on the
molecular basis.
4.2 Voltinism
Voltinism is one of the important factors for discussing about the evolution and
adaptation of the silkworm. The silkworm, Bombyx mori is classified based on the
geographical regions, as Temperate and Tropical races. They are classified on the
basis of voltinism as Univoltines, Bivoltines, and Multivoltines (or Polyvoltines);
depending upon the number of generations, they undergo in a year. Generally,
tropical silkworms are polyvoltines and the polyvoltine eggs do not undergo hiber-
nation, which means that embryos develop continuously and hatch in about
10–11 days after egg laying. In univoltines and bivoltines, the embryos “pause”
development and enter hibernation at blastoderm stage which is about 20–22 h
after oviposition. Univoltines are temperate silkworm races, show facultative type
of diapause wherein the eggs exhibit diapause phenomenon irrespective of envi-
ronmental factors. Bivoltines are also temperate races which show obligatory type
of diapause wherein diapause phenomenon is modified by environmental factors
(Fig. 4.2).
4 Tracing of Evolution in Silkworm, Bombyx mori L. 69
Fig. 4.3 Bivoltine to multivoltine theory of evolution (Gamo and Ohtsuka 1980)
Fitness involves the ability of organisms (or populations or species) to survive and
reproduce in the environment in which they find themselves (Allen Orr 2009).
Natural selection is the process by which the best adaptations survive long enough
to reproduce. Natural selection is the process by which the traits that are useful for
the survival will continue on to the next generation and what does not work will be
eliminated. The most important factor influencing the fitness is the environment. Let
us take the example of Pure Mysore, with only two parameters, survival and cocoon
weight. About 25 years ago, during the 1990s, the average cocoon weight of Pure
Mysore cocoon was 1.00 g, and the survival (ERR) was 95%. Today, with improved
4 Tracing of Evolution in Silkworm, Bombyx mori L. 73
inputs, the selection could be made up to 1.4 g cocoon weight with the same
ERR. However, there is a limit. We cannot match the productivity of temperate
bivoltines in temperate conditions (2.0 g cocoon weight with 95% ERR) in Pure
Mysore. Let us relocate these two breeds, Pure Mysore to temperate congenial envi-
ronment and temperate bivoltine to the tropical unfavorable environment. Pure
Mysore in temperate conditions will gradually turn into diapausing type and show
little higher cocoon weight (1.6 g) than in tropical conditions. It will never match
the temperate bivoltine in productivity even if it is placed in the same environment
as of temperate bivoltine breed. It looks like it simply does not have the genetic
machinery for producing higher cocoon weight. The relocated bivoltine in tropical
conditions lose its productivity to retain its survivability (as in case of C-Nichi). The
net result is that there is no improvement in survivability, but the productivity suf-
fered. Therefore, the environment plays a major role in deciding the quality and
productivity parameters.
In Japan, silkworm eggs hatch after completion of hibernation during spring (about
middle of May), in warmer climate with about 20°C temperature. They turn into
moths around later half of June and lay eggs. The deposited eggs are nonpigmented
pale yellow in color and nonhibernating. They hatch and grow during early July,
74
Fig. 4.6 Seesaw balance of quality versus resistance. Yield attributes are linked to the biochemical
parameters (Chatterjee et al. 1993)
Hibernation
DEC JAN
NOV FEB
OCT MAR
SEP APR
AUG MAY
JUL JUN
I Generation
II Generation
SPRING
SUMMER
Fig. 4.7 Behavior of bivoltines in temperate regions. They show two successive crops in spring
and following summer. Spring crop lays non-diapausing eggs, while summer crop lays diapausing
eggs
with the temperature of 25–30°C. They become moths during later half of August
and deposit eggs. The laid eggs are brown colored and hibernating (Tazima 1986)
(Fig. 4.7).
The freshly laid diapausing eggs also do not have pigments, just like nonhiber-
nating eggs, but after 10–20 days in Japan, eggs become pigmented and turn to
purple black. The pigmented eggs stop their development at the age of blastoderm
formation and become dormant. Eggs pass through summer, and once they
76 E. Muniraju and R. Mundkur
encounter cold weather of winter for 3–4 months, they continue their development.
Hence, “bivoltine” in Japan is alternation of diapausing and non-diapausing genera-
tions (Mundkur et al. 2004).
Extensive studies on diapause mechanism are undertaken in temperate condi-
tions, but under tropical conditions, the studies are scanty. In Indian tropical condi-
tions, bivoltines do not behave as they behave in Japanese conditions. They undergo
hibernation in every generation. During summer season, sporadic incidences of
bivoltines laying non-diapausing eggs are recorded. Conversely, during winter sea-
son, sporadic incidences of polyvoltine silkworm races laying diapausing eggs are
also recorded. Generally, polyvoltine females lay nondiapausing eggs irrespective
of whether they pair with polyvoltine or bivoltine males. Similarly, bivoltine females
lay diapausing eggs irrespective of whether they pair with bivoltine or polyvoltine
males. Therefore, diapause is a maternally inherited character, and males do not
have any role in determining the voltinism of the offspring. However, an exception
to this well-established phenomenon was recorded where bivoltine females (which
are destined to lay hibernating eggs) lay nonhibernating eggs when they are mated
with a special character race, KS-10 males (Mundkur et al. 2004, 2006, 2009,
2010b, c, d). This kind of paternal influence on diapause has opened up many views
on the diapause phenomenon in silkworm Bombyx mori, from point of view of its
expression under tropical conditions.
Generally there are many differences between diapause and non-diapause type of
silkworm eggs (Table 4.4).
4.8 Heritability
There are many theories of breeding methodology to make the breed best “fit” to the
given environment.
Falconer (1952) advocates that “the character required is best selected for under
environmental conditions which favour its fullest expression. Once developed, other
characters specially required for new environment also will be present in such ani-
mals.” Many silkworm breeders subscribe to this theory (Toyama 1906a, b, 1913).
during F10 to the different Regional Research Stations so that they are exposed to
various climates. Then they are brought back to the main breeding laboratory for
applying appropriate selection pressure.
Discovery of pnd and npnd genes and their behavior in tropical environment lead to
this special branch of breeding (Chatterjee 1993; Murakami 1986, 1988, 1989;
Murakami and Ohtsuki 1989; Subramanya and Murakami 1994). Murakami (1986)
showed that npnd (nonpigmented non-diapausing) is a sex-linked gene and respon-
sible for multivoltinism exhibiting maternal inheritance. He also revealed that npnd
is epistatic to pnd (pigmented non-diapausing) which was described by Katsumata
(1968).
The tropical bivoltine and multivoltine race exhibit distinct economic advantages.
Survival is generally attributed to multivoltines and productivity to bivoltines.
Voltinism remains a barrier and bottleneck in balancing and harvesting these two
critical attributes. Discovery of new dominant voltinism gene “Id” has become a
new tool in silkworm breeding. The presence of Id gene overcomes the effect of
voltinism. It can be transduced to the desired race thereby making it a “voltine-less”
race. A new terminology is coined for such character, “avoltinism,” which means
voltinism-less or “without voltinism.” This tool holds the promise for breaking the
inverse relationship and balancing the polyvoltine survival traits with bivoltine
quantitative and qualitative traits (Mundkur et al. 2004, 2010a, b, c, d, 2011, 2012).
Lepidoptera-specific genes, provide evidence for horizontal gene transfer, and con-
struct microarrays. Physical maps using large-fragment bacterial artificial chromo-
some libraries have been constructed, and whole-genome shotgun sequencing is
underway. Germline transformation and transient expression systems are well
established and available for functional studies, high-level protein expression, and
gene silencing via RNA interference.
4.11 U
nderstanding Tolerance and Resistance in Silkworm
Breeds
Conclusion
Silkworm breeders of Indian tropical conditions are trying very best for the sub-
stantial improvement in the cocoon yield, silk recovery, and quality in the silk-
worm breeds they develop, through the integration of all the available approaches
which include conventional as well as molecular biology tools. Foregoing dis-
cussions clearly indicate that the environment is the critical limiting factor, which
include biotic and abiotic factors, which determine the final performance of the
developed breeds. Southern zone of India is characterized by semiarid areas by
tropical conditions, often experiencing deficit rainfall and high temperature, with
higher biotic as well as abiotic stress. Natural selection for this area is the poly-
voltine breed with moderate production with higher survival rates. Balanced
increase in productivity, quality, and survivability is possible by considering all
the relevant factors in breeding.
82 E. Muniraju and R. Mundkur
References
Aizawa K (1962) Antiviral substance in the gut juice of the silkworm, Bombyx mori (L). J Insect
Pathol 4:72–76
Aizawa K (1991) Defense reactions of the silkworm, Bombyx mori (L), against the nuclear poly-
hedrosis. Sericologia 31:201–203
Allen Orr A (2009) Fitness and its role in evolutionary genetics. Nat Rev Genet 10(8):531–539
Andrewartha MG (1952) Diapause in relation to the ecology of insects. Bio Rev 27:50–107
Barlow HS (1982) An introduction to the moths of South East Asia. Malayan Nature Society,
Kaulalumpur, pp 1–305
Chatterjee SN (1993) Silkworm breeding in India. Sericologia 33(3):427–447
Chatterjee SN, Datta RK (1992) Hierarchical clustering of 54 races and strains of the mul-
berry silkworm, Bombyx mori L: significance of biochemical parameters. Theor Appl Genet
85(4):394–402
Chatterjee SN, Rao CGP, Chatterjee GK, Ashwath SK, Patnaik AK (1993) Correlation between
yield and biochemical parameters in the mulberry silkworm, Bombyx mori L. Theor Appl
Genet 87(3):385–391
Falconer DS (1952) The problem of environment and selection. Am Nat 86:293–298
Falconer DS (1960) Introduction to quantitative genetics. Oliver & Boyd, Edinburgh
Falconer DS (1981) Introduction to quantitative genetics, 2nd edn. Longman, London
Felix Horns F, Hood Michael E (2012) The evolution of disease resistance and tolerance in spa-
tially structured populations. Ecol Evol 2(7):1705–1711
Gamo T, Hirabayashi T (1983) Genetic analysis of growth rate, pupation rate and some quantita-
tive characters by diallel cross in the silkworm, Bombyx mori. Jpn J Breed 33:178–190
Gamo T, Ohtsuka Y (1980) Phylogenetic studies on the racial differences of the silkworm, Bombyx
mori, on the basis of polymorphic genes in haemolymph proteins. Bull Sericult Exp Sta
28:15–50
Goldsmith MR, Shimada T, Abe H (2005) The genetics and genomics of the silkworm, Bombyx
mori. Annu Rev Entomol 50:71–100
Gowda BLV, Narayanaswamy TK, Munirajappa R (1988) Impact of pupal weight on growth and
development of the following generation in the silkworm Indian race NB7 (Bombyx mori).
Sericologia 29:481–489
Gowda BLV, Sannaveerappanayar VT, Shivayogeshwar B (1989) Fecundity and hatchability in
mulberry silkworm, Bombyx mori (L.) as influenced by pupal weight. International congress on
tropical sericultural practices, part VI, Bangalore, 18–23 February 1989, pp 21–24
Grekov D, Petkov N (1990) Correlation between some traits and possibilities for prognostication
heterosis of F1 crosses on hybridization of Bombyx mori L. Zhivotnov’s Nauki 27:80
Hayashita K, Nishida J, Matsubara F (1968) Inactivation of nuclear polyhedrosis virus in the
digestive juice of silkworm, Bombyx mori (L). I. Comparison of antiviral activities in the diges-
tive juice of larvae reared on natural and artificial diets. Jpn J Appl Entomol Zool 12:189–193
HoZoo L (1997) Principles and techniques of silkworm breeding. ESCAP, United Nations,
New York
Jayaswal KP, Singh T, Sen SK (1990) Correlation between some economic parameters and their
application in silkworm breeding. Indian Silk 29:25–27
Jayaswal KP, Singh T, Subba RG (1991) Effect of female pupal weight on fecundity of mulberry
silkworm Bombyx mori. Indian J Seric 30:141–143
Jonaka N (1986) Some parameters of basic quantitative breeding characters in silkworm, Bombyx
mori. II. Correlations between the basic breeding characters. Genet Sel 19:144–150
Katsumata F (1968) Non maternal inheritance in Voltinism observed in the crossing experiments
between Indonesian polyvoltine and Japanese bivoltine races of silkworm, Bombyx mori. J
Seric Sci Jpn 37:453–461
Krishnaswami S (1978) New technology of silkworm rearing. Bulletin No. 2. Central Sericultural
Research and Training Institute, Mysore, Central Silk Board, India, pp 1–23
4 Tracing of Evolution in Silkworm, Bombyx mori L. 83
Liu Q-X, He S-M (1991) Studies on the efficiency of selection for improving technological char-
acters of cocoon filament of Bombyx mori L. Canye Kaxue 17:75–79
Long NV, Petkov N (1987) Breeding—genetic studies in some silkworm (Bombyx mori) breeds.
I. Variability and correlations of quantitative characters. Genet Sel 20:58–62
Miyahara T (1978) Selection of long filament length basic variety. I. Effect of selection in the later
generations. Acta Sericologia Sinica 106:73–78
Morohoshi S (2000) Development physiology of silkworms. In: Translation of second Japanese
edition of Kaiko no Hatsuiku Seiri by Morohoshi S, (1979). Oxford and IBH Pub.Co.Pvt.Ltd,
New Delhi
Mundkur R, Murthy M, Raghuraman MR, Bongale UD (2004) Prevention of diapause in bivoltine
eggs of the silkworm, Bombyx mori L., through a cross with the race KS-10 as male parent. Int
J Ind Entomol 9(1):107–109
Mundkur R, Murthy M, Rao SK, Raghuraman R, Bongale UD (2006) Paternal inheritance of
voltinism: a new tool to understand the gene regulation in the silkworm, Bombyx mori. In:
Proceedings of international symposium on insect genetics and genome, CDFD, Hyderabad,
9–11 Jan 2006
Mundkur R, Murthy M, Latha R, Krishna Rao S, Sekharappa BM (2009) Indication of expression
of inhibitor of diapauses gene in silkworm, Bombyx mori. In: National conference on recent
trends in animal physiology, Manasa Gangotri, Mysore, 29–30 Oct 2009
Mundkur R, Katti SR, Chandrakala MV, Sekharappa BM (2010a) Diapause in silkworm, Bombyx
mori L with special reference to the paternal influence: a review. Sericologia 50(2):145–170
Mundkur R, Murthy M, Latha R, Krishna Rao S, Sekharappa BM (2010b) Avoltinism breeding:
a new concept in silkworm breeding. In: Silkworm breeder’s meet, RSRS Coonoor, 2–3 Dec
2010
Mundkur R, Murthy M, Latha R, Krishna R, Sekharappa BM (2010c) “Id” inhibitor of diapauses,
a new gene as diapauses switch in silkworm, Bombyx mori. In: Nattional seminar on emerging
trends in animal biotchnology, Kakatiya University, Warangal, 26–27 Mar 2010
Mundkur R, Murthy M, Latha R, Rajanna GS, Krishna Rao S (2010d) Reporting of a new gene
inhibitor of diapause(Id) in the silkworm, Bombyx mori L. Sericologia 50(3):359–364
Mundkur R, Murthy M, Krishna Rao S (2011) Modification of diapause induced by inhibitor of
diapause gene (Id), after fertilization in silkworm, Bombyx mori. In: International conference
on life science research for rural and agricultural development, CPRS, Patna, Bihar, 27–29
Dec 2011
Mundkur R, Murthy M, Krishna Rao S (2012) Termination of diapause by Inhibitor of diapause
gene (Id) after fertilization in silkworm, Bombyx mori. In: National seminar on advances in
zoology and life processes, Goa University, 9–11 Feb 2012
Murakami A (1986) Changes in embryonic diapause in the tropical multivoltine silkworm stock
Cambodge. In: Abstract: 34th Annual meetings of the Tokyo district Sericul. Sci Soc 27
Murakami A (1988) Ecogenetical studies on tropical mulberry Bombyx mori. In: Proceedings of
the international congress on tropical sericulture practices, Bangalore, India, pp 11–23
Murakami A (1989) Genetic studies on tropical races of silkworm (Bombyx mori) with special
reference to cross breeding strategy between tropical and temperate races. 2. Multivoltine silk-
worm strains in Japan and their origin. JARQ 23:123–127
Murakami A, Ohtsuki Y (1989) Genetic studies on tropical races of silkworm, Bombyx mori, with
special reference to cross breeding strategy between tropical and temperate races. 1. Genetic
nature of the tropical multivoltine strain, Cambodge. JARQ 23:37–45
Nagaraju J (1998) Silk yield attributes and complexities. In: Reddy GS (ed) Silkworm breeding.
Oxford Press, New Delhi, pp 168–185
Nagaraju J (1999) Biotechnology: a novel concept for silkworm improvement. In: Devaiah
MC, Narayanaswamy KC, Maribashetty VG (eds) Advances in mulberry sericulture.
C.V.G. Publications, Bangalore, pp 208–242
Nagaraju J, Klimenko V, Couble P (2001) The silkworm, Bombyx mori: a model genetic system. In:
Reeve ECR (ed) Encyclopaedia of genetics. Fitzroy Dearborn Publishers, London, pp 219–239
84 E. Muniraju and R. Mundkur
Ozdzenska B, Kremky J (1987) Estimation of heritability and genotypic, phenotypic and environ-
mental correlations in out-bred populations of mulberry silkworm, Bombyx mori L. Sericologia
27:633–638
Petkov N (1981a) The possibility of forecasting the effectiveness of selecting lines of the silkworm,
Bombyx Mori based on weight and silk content of cocoons. Selskotopanska Nauka 19:92–96
Petkov N (1981b) Variability and correlations between some characteristic features of silkworm
(Bombyx mori L). Zhivotnov’d Nauki 18:83–86
Petkov N (1981c) Phenotypic correlations and regressions between some silkworm (Bombyx mori)
breeding characters. Genet Sel 14:386–390
Rajanna GS, Reddy GS (1990a) Studies on the variability and interrelationship between some
quantitative characters in different breeds of silkworm, Bombyx mori L. Sericologia 30:67–74
Rajanna GS, Reddy GS (1990b) New dimensions in tropical silkworm race breeding. Indian Silk
28:8–12
Rajanna GS, Reddy GS (1998) New approaches to silkworm improvement aimed at narrowing
yield gaps. In: Reddy GS (ed) Silkworm breeding. Oxford Press, New Delhi, pp 168–185
Satenahalli SB, Govindan R, Goud JV, Magdum SB (1990) Genetic parameters and correlation
coefficient analysis in silkworm, Bombyx mori. Mysore J Agric Sci 24:491–495
Singh T (1994) Correlation between pupal weight and fecundity in Bombyx mori (L). Ann Entomol
12:5–7
Singh T, Singh K (1993) Heritability and correlations between some economic characters in
Bombyx mori (L). Sci Cult 59:51–52
Singh T, Jayaswal KP, Subba RG (1992a) Correlation studies between some breeding parameters
of silkworm, Bombyx mori (L). J Zool Res 5:47–50
Singh T, Singh K, Das M (1992b) Astounding effect of correlated parameters in silk industry.
Indian Text J 102:26–30
Singh T, Jayaswal KP, Subba RG (1992c) Correlation studies between some breeding parameters
of silkworm, Bombyx mori (L). J Zool Res 5:47–50
Singh T, Chandrashekharaiah A, Samson MV (1994) Selection strategies in relation to correlation
and heritability in the silkworm, Bombyx mori L. Bull Seric Res 5:37–41
Singh T, Bhat MM, Khan MA (2011) Critical analysis of correlation and heritability phenom-
enon in the silkworm, Bombyx mori (Lepidoptera: bombycidae). Adv Biosci Biotechnol
2(5):347–353
Sonobe H, Odake H (1986) Studies on embryonic diapause in the pnd mutant of the silkworm,
Bombyx mori; V. Identification of a pnd+ gene-specific protein. Rouxs Arch Dev Biol
195:229–235
Subramanya G, Murakami A (1994) Climatic differential phenotypic expression of voltine genes
in Bombyx mori L. Indian J Seric 33(2):103–109
Tazima Y (1986) The genetics of the silkworm. Logos Press, Academic Press, London
Toyama K (1906a) Mendel’s law of heredity as applied to the silkworm crosses. Biol Zentralbl
26:321–334
Toyama K (1906b) Breeding methods of silkworm. Sangyo Shimpo 158:282–286
Toyama K (1913) Maternal inheritance and mendelism. J Genet 2:351–405
Yan LL (1983) The estimates of heritability of pupal weight, cocoon weight and number of eggs
laid in the silkworm (Bombyx mori) and its genetic correlation between these characters and
path coefficient analysis. J Seric Sci China 9:149–155
Yoshitake N (1968) Phylogenetic aspects on the origin of Japanese race of the ilkworm, Bombyx
mori L. J Sericol Sci Jpn 37:83–87
Long Noncoding RNA: Disclosing
New Horizon in the Molecular 5
World of Insects
Abstract
Long noncoding RNAs (lncRNAs) are the most versatile group of nonprotein-
coding RNAs consisting of nucleotides of length more than 200 bp. Similar to
mammal’s genome phenomenon, thousands of lncRNAs have been discovered in
the insect’s genome through RNA sequencing technology and computational
methods, which contributes to the diverse biological processes including dis-
eases to regulate the gene expression, dosage compensation, and epigenetic
imprinting of entire chromosome. In fruit fly, lncRNAs exposed noteworthy
functions in the behavioral processes, sex, and neural development. However, in
the silkworm, lncRNAs were linked with silk synthesis and affect the apoptosis;
additionally, other baculoviral lncRNAs contributed to establishing the complex
regulation of viral gene expression in baculovirus-infected BmN cells. In dia-
mondback moth, lncRNA gene expression study revealed the insecticidal resis-
tant activity, whereas caste differentiation and behavior mechanism in honeybee
were also significantly investigated. Therefore, lncRNAs exist in various insect’s
genomes, opening a new horizon for biotechnologist to identify, study, and dis-
close the gene expression, regulatory and biological functions of lncRNAs in
insects.
5.1 Introduction
In the past, RNA fit cleanly into the central dogma as a messenger between DNA
and protein. RNA molecules have frequently emerged as vibrant and resourceful
regulators of the genome. However, several scientists have identified a different
kind of RNA which does not participate in the protein synthesis, the so-called dark
matter of genome (Kapranov and Laurent 2012). In eukaryotes, despite the fact that
noncoding DNA has been frequently named as “junk DNA” with no evolutionary
imperative, it encounters natural selection (Ponting et al. 2009). A little information
is accessible about the origin and advancement of lncRNAs compared with the
protein-coding gene. LncRNAs display a low conservation of sequence and rapid
evolution among mammals (Kutter et al. 2012). In the last decades, bioinformatics
and RNA sequencing technologies have been proven as influential and significant
tools to explore whole genome sequencing including lncRNA. In human and mouse
transcriptome analysis, about 58,000 and 8000 expressed genes were, respectively,
classified as lncRNAs (Iyer et al. 2015; Sun et al. 2013) and present at a specific
time-point in a particular cell type or tissue (Wang et al. 2009; Djebali et al. 2012).
Just a few years back, specific consideration has been paid to the class of long non-
coding RNAs (lncRNAs) as they have been associated with different systems, for
example, cis- and trans-control of interpretation, dosage remuneration, engraving
and contending endogenous RNA (Ulitsky and Bartel 2013; Fatica and Bozzoni
2013; Gardini and Shiekhattar 2014; Bonasio and Shiekhattar 2004), and their
diverse roles in a variety of critical biological processes in higher organisms (Eddy
2001). Long noncoding RNA (lncRNA), which is the most versatile group of RNA
consisting of more than 200 bp without a coding ORF (Kapranov et al. 2007),
emerges as potent regulator of the gene expression and dramatically altered our
understanding of the cell biology in diseases. The majority of the known miRNAs
are exceptionally rationed crosswise over different life-forms from insect to human
(Lim et al. 2003a, b). Similar to mammal genome phenomenon, in recent years,
through computational methods and RNA sequencing technology, thousands of
lncRNAs have been discovered in the insect’s genome. The main function of
lncRNA in regulation is to control the expression of distinct genes. As lncRNAs are
less conserved in the nucleotide sequence across phylogenetically related species,
therefore it is challenging to identify lncRNAs by general sequence searching
(Necsulea et al. 2014). LncRNAs are homologous to mRNAs in terms of posttran-
scriptional modifications such as splicing, polyadenylation, and capping (Derrien
et al. 2012). Nevertheless, lncRNAs are less conserved comparatively in human and
other vertebrates. Thousands of lncRNAs have been discovered in the genome of
Drosophila melanogaster, Anopheles gambiae, Apis mellifera, and Bombyx mori
(Li et al. 2014; Zhang et al. 2014; Pauli et al. 2012; Wang et al. 2015; Hao et al.
2015; Young et al. 2012; Padron et al. 2014; Jayakodi et al. 2015; Jenkins et al.
2015; Liu et al. 2012; Wu et al. 2016). Initially, Wahlestedt (Wahlestedt 2013)
described in details about the formation of lncRNA (Fig. 5.1) including various
linked processes such as translation and transcription. He also explained about how
lncRNA developed and played a significant role in various biological functions.
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 87
DNA
• Chromatin modifications
Transcription • PolII activity regulation Transcription
• Transcriptional interference
IncRNAs
ncRNA
miRNAs
and IncRNAs
• Splicing
Translation • Editing
• mRNA stability
• Translation initiation
Protein
The fruit fly has been one of the globally exploited and first-choice research
model organism for genetics research. The fast extension of genome sequence data
of Drosophila has led to the emergence of comparative genomics protocols to inves-
tigate the genome-wide alignment. There are about 40,000 introns with sequence
length, accounting for 22% of the entire D. melanogaster genome, which is a little
bit larger than 19% of the total coding sequences (Adams et al. 2000; Holt et al.
2002).
Hartfelder 2011). Various noncoding RNAs’ biological functions have been well
studied in B. mori such as PiWi-interacting RNAs (Kawaoka et al. 2011), microR-
NAs (Jia et al. 2015), and snoRNA (Li et al. 2014). However, lncRNAs function-
ally remain poorly classified in the silkworms except for lncRNA Fben-1 (Taguchi
et al. 2011) which is expressed in the female brain. In the last few years, the role of
various lncRNAs (lncRNA-p21, lncRNA-GAS5, lncRNA UCA1) in apoptosis was
also examined in humans (Tran et al. 2015; Shi et al. 2015; Wu et al. 2013). In the
case of invertebrates, two Bcl-2 family proteins homologous to Bok (a mammalian
pro-apoptotic member) in D. melanogaster were successfully recognized (Tatsushi
and Masayuki 2004). After identification of Bcl-2 gene, functional analysis of
numerous lncRNAs has been discovered in D. melanogaster (Smith et al. 2001).
Afterward, Pan (Pan et al. 2010) studied that BmBuffy played a significant role in
hydroxycamptothecin-induced apoptosis in BmN-SWU1 cells of B. mori and pos-
sesses potential apoptosis-related genes which give an important evidence to inves-
tigate the role of lncRNA in apoptosis. Systematic identification of lncRNAs (Wu
et al. 2016) in the few tissues of B. mori was disclosed in the presence of long
nonprotein-coding RNAs in the genome of the silkworm; nonetheless, studies
related to gene expression and biological function including apoptosis are still
scanty and need further investigation. Our research group also identified a number
of lncRNAs in the silkworm genome and is studying the lncRNAs’ gene functions
in the silkworm.
According to an important report of Kowalczyk and Higgs (Kowalczyk et al. 2012), they
made an effort to the insight of various researcher findings and revealed that nonprotein-
coding gene has been divided into noncoding RNA and long noncoding RNA. They also
explained about the number of known transcripts and transcript length. On that basis,
ncRNA (miRNA, snoRNA, snRNA, piRNA, tRNA) and lncRNAs (antisense ncRNA,
enhancer ncRNA (eRNA), enhancer ncRNA (meRNA) ΙΙ, intergenic ncRNA, pseudo-
gene ncRNA) have been categorized (Fig. 5.2 and Table 5.1).
Generally, a little extent of lncRNAs has so far been explored, and despite the fact,
this is predominantly because it doesn’t look like protein-coding gene, whose suc-
cession themes and sequence motifs are symbolic of their function; lncRNA
sequences are not usually conserved and they don’t tend to contain conserved motif.
The characteristics of lncRNA and mRNA are explained in Table 5.2.
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 89
miRNA
siRNA snRNA
Non-Coding
piRNA RNAs snoRNA
tRNA scRNA
IncRNA
Table 5.1 Types of noncoding and long noncoding RNAs and their transcripts
No. of known
ncRNA transcripts Transcript lengths (nucleotides: nt)
Precursors to short RNAs
snRNA 1944 1000
miRNA 1756 >1000
snoRNA 1521 >100
tRNA 497 >100
piRNA 89 Unknown
Long ncRNAs
Intergenic ncRNA 6742 102–105
Antisense ncRNA 5446 100–>1000
Enhancer ncRNA (eRNA) >2000 >1000
3′ UTR ncRNA 12 >100
Pseudogene ncRNA 680 102–104
LncRNAs can be classified into the following locus biotypes based on their
genome location with respect to protein-coding genes (Ponting et al. 2009; Mercer
et al. 2009) (Figs. 5.3 and 5.4).
Intergenic lncRNA: Intergenic lncRNAs are transcribed intergenetically from
both strands.
90 D. Kumar et al.
Table 5.2 Gray and cyan between lncRNA and mRNA (courtesy: Exon)
lncRNA mRNA
Tissue-specific expression Tissue-specific expression
Form secondary structure Form secondary structure
Undergo posttranscriptional processing, i.e., Undergo posttranscriptional processing, i.e.,
5′ cap, polyadenylation, splicing 5′ cap, polyadenylation, splicing
Important roles in diseases and development Important roles in diseases and development
Nonprotein-coding, regulatory functions Protein-coding transcript
Poorly conserved between species Well conserved between species
Many predominantly nuclear, others nuclear Present in both nucleus and cytoplasm
and/or cytoplasmic
Currently ~30,000 lncRNA transcripts, Total 20–24,000 mRNAs
predicted 3–100-fold of mRNA in number
Expression level: very low to moderate Expression level: low to high
Table 5.3 LncRNAs functions in insect model D. melanogaster and A. mellifera (Fabrice and
Thomas 2015)
Gene Species Function Mode
Gene regulation hsr-v D. melanogaster Long noncoding RNAs produced Trans
by the hsw-v gene are actively
expressed in nuclei, forming
spots called perinuclear omega
speckles, in response to heat
shock stress. These speckles are
involved in the redistribution and
sequestration of multiple
processing proteins, in particular,
heterogeneous nuclear
ribonucleoproteins (hnRNPs),
HP1, or polII, which strongly
affect multiple cellular networks
subsequent to a stress (Lakhotia
et al. 2012; Lakhotia 2011)
Epigenetics control D. melanogaster In X0 male Drosophila, the Trans
of gene regulation transcription of genes located on
rox1/rox2 the X chromosome is increased
relative to the level of XX
females by a mechanism known
as dosage compensation. This
mechanism is connected to the
acetylation of histone H4 at
lysine 16 induced by a protein
complex which involves two long
noncoding RNAs roX1 and roX2
(Deng and Meller 2006; Smith
et al. 2001)
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 91
Table 5.3 (continued)
Gene Species Function Mode
Development bithorax D. melanogaster The bithorax complex (BX-C) Cis
plays a key role in Drosophila
development and covers a region
larger than 300 kb that includes
only four protein-coding genes.
Noncoding genes have already
been shown to be concomitantly
expressed from the BX-C domain
to regulate in cis the BX-C
proteins in specific abdominal
segments (Lipshitz et al. 1987;
Pease et al. 2013)
Behavior yar D. melanogaster Yar is a long noncoding RNA, Cis
highly expressed during
embryogenesis and located in a
neural gene cluster between
yellow (y) and achaete (ac)
(Soshnev et al. 2008). With the
help of mutants, the function of
this long noncoding RNA has
been recently refined as a
regulator of y and ac
transcription, affecting as well
the sleep behavior of the
Drosophila (Soshnev et al. 2011)
Sphinx D. melanogaster Sphinx is a lncRNA involved in Unknown
the regulation of male courtship
behavior. Sequence variations
between close Drosophila species
advocate for a functional role and
a rapid adaptation. The
mutagenesis of sphinx in
Drosophila reveals the
emergence of male-male
courtship behavior, probably by
the disruption of some sensory
circuits, which is supported by its
specific expression in
chemosensory organs (Chen et al.
2011)
Nb-1 A. mellifera Nb-1 is a 700 nt transcript, whose Unknown
longest ORF encodes a putative
32 amino acid without any
sequence conservation. This
lncRNA is expressed in the
honeybee brain with variation
according to the age of the
colony workers testifying to its
putative role in polyethism (Tran
et al. 2015)
(continued)
92 D. Kumar et al.
Table 5.3 (continued)
Gene Species Function Mode
Neural expression A. mellifera Sawata et al. identified a 17knt Unknown
Ks-1 transcript that is expressed
restrictively in the mushroom
body of Kenyon cells in the
honeybee brain and which
accumulates in the nucleus. The
transcript exhibits seven putative
ORFs longer than 67 amino acids
without any conservation in a
related species (Apis cerana) nor
similarity with known proteins
(Sawata et al. 2002)
AncR-1 A. mellifera AncR-1 is preferentially Unknown
expressed in the brain, sexual
tissues, and some secretory
organs and accumulates in nuclei.
It contains multiple alternate
isoforms, which are derived from
a 6.9 kbp genomic locus (Sawata
et al. 2004)
Kakusei A. mellifera The kakusei is a 7000 nt long Unknown
noncoding RNA with multiple
constitutive and inducible
variants, the expression of which
is transiently upregulated by
neural activity. It is localized
exclusively in neural nuclei in
discrete nuclear compartments.
This gene may play specific roles
in RNA metabolism in the
honeybee brains, irrespective of
behavioral experience (Kiya et al.
2008b)
Lnccov1/lnccov2 A. mellifera These two transcripts lack Unknown
evidence of functional ORFs and
are differentially expressed in
queen and worker ovariole
transcriptomes at the embryonic
stage. Temporal expression
shows that lnccov1 might be
involved in the autophagic cell
death of ovarioles during worker
embryogenesis, and fluorescent
in situ hybridization (FISH)
indicates perinuclear localization
in omega speckle-like structures
(Humann and Hartfelder 2011)
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 93
Coding Regions
Non-coding Regions
Chromosomal Rearrangement
Duplications
short ncRNA long ncRNA
Transposon Insertion
Antisense
Bidirectional
<1000 bp
Intronic
Intergenic
probably reflecting more relaxed chromatin structure (Brown et al. 2014). In addi-
tion, many lncRNAs are expressed antisense to protein-coding genes which they
often regulate (He et al. 2008; Magistri et al. 2012; Johnsson et al. 2013). It is thus
recommended to favor stranded RNA-Seq protocols or directional transcriptome
sequencing that keeps track of the strand of origin of the transcript (Ponting et al.
2009), if the purpose of the study includes the identification of antisense lncRNAs.
For example, using these stranded protocols, the modENCODE project recently
discovered 402 lncRNA loci (21% of all lncRNA loci) located antisense to mRNA
transcripts of protein-coding genes in D. melanogaster (Brown et al. 2014), while
this proportion is slightly lower (15%) in the human genome (Derrien et al. 2012).
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 95
Mapping genome
Library preparation
TopHat v2.0.13
Reference assembly
NO
IncRNA transcript set
4,481 IncRNAs 375 IncRNAs
4,856 IncRNAs
et al. 2010). The second was the silkworm unigenes. The unigene transcripts were
combined from expressed sequence tag, and some of the lncRNAs were addition-
ally contained in the unigene transcripts (Zhou et al. 2014). Thus, the transcripts
assembled from RNA-Seq data and unigenes were used to identify lncRNAs in his
study (Zhou et al. 2016).
60°C,
5min
Construction of recombinant plasmid
Convert to cDNA
Fig. 5.6 Pipeline of cloning of lncRNAs and gene expression study in insects
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 97
I. Signaling
II. Decoys
The molecular decoy type of activity takes place when specific lncRNAs are
transcribed and then bind to and titrate away protein factors. Decoy lncRNAs
can “sponge” protein factors such as transcription factors and chromatin mod-
ifiers. This leads to broad changes in the transcriptome of the cell (Guenther
et al. 2007; Bernard et al. 2010).
III. Guides
IV. Scaffolds
I. Signaling
IncRNA
Gene Activation
II. Decoy
III. Guides
Promote chromatin
modification
IncRNA
IV. Scaffolds
Act on chromatin
structure
A C
Conclusion
After discovery of lncRNAs, the diversity of nonprotein-coding genes and their
molecular functions has been widely investigated in various important insect
research models from tissue to cell level. LncRNAs disclosed the various hidden
and key gene functions and also performing significant role in the various dis-
eases of insects. However, its beginning, in the coming future, lncRNAs investi-
gation will uncover the numerous conceals of the other genetically important
insects.
References
Adams MD, Celniker SE, Holt RA et al (2000) The genome sequence of Drosophila melanogas-
ter. Science 287:2185–2195
Bernard D, Prasanth KV, Tripathi V, Colasse S, Nakamura T et al (2010) A long nuclear-
retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO
J 29:3082–3093
Bonasio R, Shiekhattar R (2004) Regulation of transcription by long noncoding RNAs. Annu Rev
Genet 48:433–455
Bonasio R, Tu S, Reinberg D (2010) Molecular signals of epigenetic states. Science 330:612–616
Brown JB, Boley N, Eisman R, May GE, Stoiber MH et al (2014) Diversity and dynamics of the
Drosophila transcriptome. Nature 512:393–399
Chen Y, Dai H, Chen S, Zhang L, Long M (2011) Highly tissue specific expression of sphinx sup-
ports its male courtship related role in Drosophila melanogaster. PLoS One 6:e18853
Cheng T, Fu B, Wu Y, Long R, Liu C et al (2015) Transcriptome sequencing and positive selected
genes analysis of Bombyx Mandarina. PLoS One 10:e0122837
Deng X, Meller VH (2006) roX RNAs are required for increased expression of X-linked genes in
Drosophila melanogaster males. Genetics 174:1859–1866
Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S et al (2012) The GENCODE v7 catalog
of human long noncoding RNAs: analysis of their gene structure, evolution, and expression.
Genome Res 22:1775–1789
Djebali S, Davis CA, Merkel A, Dobin A, Lassmann T et al (2012) Landscape of transcription in
human cells. Nature 488:101–108
Eddy SR (2001) Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2:919–929
Etebari K, Michael J, Furlong S (2015) Intergenic non-coding RNAs in diamondback moth
(Plutella xylostella) and their expression in insecticide resistant strains. Sci Rep 5:14642
Fabrice L, Thomas D (2015) Identification of long non-coding RNAs in insects genomes. Curr
Opin Insect Sci 7:1–8 (Elsevier: 4054001009011)
Fang SM, Hu BL, Zhou QZ, Yu QY, Zhang Z (2015) Comparative analysis of the silk gland tran-
scriptomes between the domestic and wild silkworms. BMC Genomics 16:60
Fatica A, Bozzoni I (2013) Long non-coding RNAs: new players in cell differentiation and devel-
opment. Nat Rev Genet 15:7–21
Gardini A, Shiekhattar R (2014) The many faces of long noncoding RNAs. FEBS J 282(9):1647–
1657. doi:10.1111/febs.13101
Gong L, Chen X, Liu C, Jin F, Hu Q (2014) Gene expression profile of Bombyx mori hemocyte
under the stress of destruxin a. PLoS One 9:e96170
Good MC, Zalatan JG, Lim WA (2011) Scaffold proteins: hubs for controlling the flow of cellular
information. Science 332:680–686
Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA (2007) A chromatin landmark and
transcription initiation at most promoters in human cells. Cell 130:77–88
Guo R, Guangli C, Yahong L, Renyu X, Dhiraj K et al (2016) Exogenous gene can be integrated
into Nosema bombycis genome by mediating with a non-transposon vector. Parasitol Res
115:3093–3098
100 D. Kumar et al.
Guttman M, Amit I, Garber M, French C, Lin MF (2009) Chromatin signature reveals over a thou-
sand highly conserved large non-coding RNAs in mammals. Nature 458:223–227
Hao Z, Fan C, Cheng T, Su Y, Wei Q (2015) Genome-wide identification, characterization and evo-
lutionary analysis of long intergenic noncoding RNAs in cucumber. PLoS One 10:e0121800
He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW (2008) The antisense transcrip-
tomes of human cells. Science 322:1855–1857
Holt RA, Subramanian GM, Halpern A et al (2002) The genome sequence of the malaria mosquito
Anopheles gambiae. Science 298:12–149
Humann FC, Hartfelder K (2011) Representational difference analysis (RDA) reveals differential
expression of conserved as well as novel genes during caste-specific development of the honey
bee (Apis mellifera L.) ovary. Insect Biochem Mol Biol 41:602–612
Ilott NE, Ponting CP (2013) Predicting long non-coding RNAs using RNA sequencing. Methods
63:50–59
Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A et al (2015) The landscape of long noncoding
RNAs in the human transcriptome. Nat Genet 47:199–208
Jacobson A, Baran I, Popovi C, Sorkine O (2011) Bounded biharmonic weights for real-time
deformation. ACM Trans Graph 30:78:1–78:8
Jayakodi M, Jung JW, Park D, Ahn YJ, Lee SC et al (2015) Genome-wide characterization of long
intergenic non-coding RNAs (lincRNAs) provides new insight into viral diseases in honey bees
Apis Cerana and Apis Mellifera. BMC Genomics 16:680
Jenkins AM, Waterhouse RM, Muskavitch MA (2015) Long non-coding RNA discovery across
the genus anopheles reveals conserved secondary structures within and beyond the Gambiae
complex. BMC Genomics 16:337
Jia L, Dayan Z, Zhonghuai X, Ningjia H (2015) Nonfunctional ingestion of plant miRNAs in silk-
worm revealed by digital droplet PCR and transcriptome analysis. Sci Rep 5:12290
Johnsson P, Ackley A, Vidarsdottir L, Lui WO, Corcoran M et al (2013) A pseudogene long-
noncoding-RNA network regulates PTEN transcription and translation in human cells. Nat
Struct Mol Biol 20:440–446
Kapranov P, Laurent G (2012) Dark matter RNA: existence, function and controversy. Front Genet
3:60
Kapranov P, Cheng J, Dike S, Nix DA, Duttagupta R et al (2007) RNA maps reveal new RNA
classes and a possible function for pervasive transcription. Science 316:1484–1488
Kawaoka S, Kadota K, Arai Y, Suzuki Y, Fujii T et al (2011) The silkworm W chromosome is a
source of female-enriched piRNAs. RNA 17:2144–2151
Kiuchi T, Koga H, Kawamoto M, Shoji K, Sakai H et al (2014) A single female-specific piRNA is
the primary determiner of sex in the silkworm. Nature 509(7502):633–636
Kiya T, Kunieda T, Kubo T (2008a) Inducible- and constitutive-type transcript variants of kakusei,
a novel non-coding immediate early gene, in the honeybee brain. Insect Mol Biol 17:531–536
Kiya T, Kunieda T, Kubo T (2008b) Inducible and constitutive-type transcript variants of
kakusei, a novel noncoding immediate early gene, in the honeybee brain. Insect Mol Bio
17:531–536
Kiya T, Ugajin A, Kunieda T, Kubo T (2012) Identification of kakusei, a nuclear non-coding
RNA. Molecular Sci 13:15496–15509
Kowalczyk MS, Higgs DR, Gingeras TR (2012) Molecular biology: RNA discrimination. Nature
482:310–311
Kutter C, Watt S, Stefflova K, Wilson MD, Goncalves A et al (2012) Rapid turnover of long non-
coding RNAs and the evolution of gene expression. PLoS Genet 8:e1002841
Lakhotia SC (2011) Forty years of the 93D puff of Drosophila melanogaster. J Biosci 36:399–423
Lakhotia SC, Mallik M, Singh AK, Ray M (2012) The large noncoding hsrv-n transcripts are
essential for thermotolerance and remobilization of hnRNPs, HP1 and RNA polymerase II dur-
ing recovery from heat shock in Drosophila. Chromosoma 121:49–70
Lee JT (2009) Lessons from X-chromosome inactivation: long ncRNA as guides and tethers to the
epigenome. Genes Dev 23:1831–1842
5 Long Noncoding RNA: Disclosing New Horizon in the Molecular World of Insects 101
Legeai F, Derrien T (2015) Identification of long non-coding RNAs in insects genomes. Curr Opin
Insect Sci 7:37–44
Li M, Wen S, Guo X, Bai B, Gong Z et al (2012) The novel long non-coding RNA CRG regulates
Drosophila locomotor behavior. Nucleic Acids Res 40:11714–11727
Li L, Eichten SR, Shimizu R, Petsch K, Yeh CT et al (2014) Genome-wide discovery and charac-
terization of maize long non-coding RNAs. Genome Bio 5:40
Liao Q, Shen J, Liu J, Sun X, Zhao G et al (2014) Genome-wide identification and functional
annotation of plasmodium falciparum long noncoding RNAs from RNA-seq data. Parasitol
Res 113:1269–1281
Lim LP, Glasner ME, Yekta S et al (2003a) Vertebrate microRNA genes. Science 299:1540
Lim LP, Lau NC, Weinstein EG et al (2003b) The microRNAs of Caenorhabditis elegans. Genes
Dev 17:991–1008
Lipshitz HD, Peattie DA, Hogness DS (1987) Novel transcripts from the Ultrabithorax domain of
the bithorax complex. Genes Dev 1:307–322
Liu J, Jung C, Xu J, Wang H, Deng S et al (2012) Genome-wide analysis uncovers regulation of
long intergenic noncoding RNAs in Arabidopsis. Plant Cell 24:4333–4345
Ma L, Ma Q, Li X, Cheng L, Li K (2014) Transcriptomic analysis of differentially expressed genes
in the Ras1(CA)-overexpressed and wildtype posterior silk glands. BMC Genomics 15:182
Magistri M, Faghihi MA, St Laurent G, Wahlestedt C (2012) Regulation of chromatin structure
by long noncoding RNAs: focus on natural antisense transcripts. Trends Genet 28:389–396
Mattick JS, Rinn JL (2015) Discovery and annotation of long noncoding RNAs. Nat Struct Mol
Bio 22:5–7
Mercer TR, Dinger ME, Mattick JS (2009) Long non-coding RNAs: insights into functions. Nat
Rev Genetics 10:155–159
Nam JW, Bartel DP (2012) Long noncoding RNAs in C. elegans. Genome Res 22:2529–2540
Necsulea A, Soumillon M, Warnefors M, Liechti A, Daish T et al (2014) The evolution of lncRNA
repertoires and expression patterns in tetrapods. Nature 505:635–640
Nie H, Liu C, Cheng T, Li Q, Wu Y et al (2014) Transcriptome analysis of integument differentially
expressed genes in the pigment mutant (quail) during molting of silkworm, Bombyx mori.
PLoS One 9:e94185
Nishida KM, Iwasaki YW, Murota Y, Nagao A, Mannen T et al (2015) Respective functions of two
distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ
cells. Cell Rep 10:193–203
Padron A, Molina-Cruz A, Quinones M, Ribeiro JM, Ramphul U et al (2014) In depth annotation
of the Anopheles gambiae Mosquito midgut transcriptome. BMC Genomics 15:636
Pan MH, Cai XJ, Liu M (2010) Establishment and characterization of an ovaria cell line of the
silkworm, Bombyx mori. Tissue Cell 42:42–46
Pauli A, Valen E, Lin MF, Garber M, Vastenhouw NL et al (2012) Systematic identification of long
noncoding RNAs expressed during zebrafish embryogenesis. Genome Res 22:577–591
Pease B, Borges AC, Bender W (2013) Noncoding RNAs of the ultrabithorax domain of the
Drosophila bithorax complex. Genetics 195:1253–1264
Ponting CP, Oliver PL, Reik W (2009) Evolution and functions of long noncoding RNAs. Cell
139:629–641
Sawata M, Daisuke Y, Takeuchi H, Kamikouchi A, Kazuaki O, Kubo T (2002) Identification and
punctate nuclear localization of a novel noncoding identification and punctate nuclear localiza-
tion of a novel noncoding RNA, Ks-1, from the honeybee brain. RNA 8(6):772–785
Sawata M, Takeuchi H, Kubo T (2004) Identification and analysis of the minimal promoter activity
of a novel noncoding nuclear RNA gene, AncR-1, from the honeybee (Apis mellifera L.) RNA
10(7):1047–1058. doi:10.1261/rna.5231504.use
Shao W, Zhao QY, Wang XY, Xu XY, Tang Q et al (2012) Alternative splicing and trans-splicing
events revealed by analysis of the Bombyx mori transcriptome. RNA 18(7):1395–1407
Shi X, Ming S, Hongbing L, Yanwen Y, Rong K (2015) A critical role for the long non-coding RNA
GAS5 in proliferation and apoptosis in non-small-cell lung cancer. Mol Carcino 54(Suppl 1):E1–E12
102 D. Kumar et al.
Shoji K, Hara K, Kawamoto M, Kiuchi T, Kawaoka S et al (2014) Silkworm HP1a transcription-
ally enhances highly expressed euchromatic genes via association with their transcription start
sites. Nucleic Acids Res 42:11462–11471
Smith ER, Allis CD, Lucchesi JC (2001) Linking global histone acetylation to the transcription
enhancement of X-chromosomal genes in Drosophila males. J Biol Chem 276:31483–31486
Soshnev AA, Li X, Wehling MD, Geyer PK (2008) Context differences reveal insulator and activa-
tor functions of a Su(Hw) binding region. PLoS Genet 4:e1000159
Soshnev AA, Ishimoto H, McAllister BF, Li X, Wehling MD (2011) A conserved long noncoding
RNA affects sleep behavior in Drosophila. Genetics 189:455–468
Spitale RC, Tsai MC, Chang HY (2011) RNA templating the epigenome: long noncoding RNAs
as molecular scaffolds. Epigenetics 6:539–543
Sun J, Lin Y, Wu J (2013) Long non-coding RNA expression profiling of mouse testis during post-
natal development. PLoS One 8:e75750
Tadano H, Yamazaki Y, Takeuchi H, Kubo T (2009) Age and division-of-labour-dependent dif-
ferential expression of a novel non-coding RNA, Nb-1, in the brain of worker honeybees, Apis
mellifera L. Insect Mol Biol 18:715–726
Taguchi S, Iwami M, Kiya T (2011) Identification and characterization of a novel nuclear noncod-
ing RNA, Fben-1, which is preferentially expressed in the higher brain center of the female
silkworm moth, Bombyx mori. Neurosci Lett 496:176–180
Tatsushi I, Masayuki M (2004) Role of Bcl-2 family members in invertebrates, Biochim. Biophys
Acta 1644:73–81
Tran UM, Rajarajacholan U, Soh J, Kim T-S (2015) LincRNA-p21 acts as a mediator of ING1b-
induced apoptosis. Cell Death Dis 6:e1668. doi:10.1038/cddis
Ulitsky I, Bartel DP (2013) LincRNAs: genomics, evolution, and mechanisms. Cell 154:26–46
Wahlestedt C (2013) Targeting long non-coding RNA to therapeutically upregulate gene expres-
sion. Nat Rev Drug Dis 12:433–446
Wang KC, Chang HY (2011) Molecular mechanisms of long noncoding RNAs. Mol Cell
43:904–914
Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat
Rev Genet 10:57–63
Wang M, Yuan D, Tu L, Gao W, He Y et al (2015) Long noncoding RNAs and their proposed func-
tions in fibre development of cotton (Gossypium spp.) New Phytol 207:1181–1197
Wu W, Zhang S, Li X, Xue M, Cao S et al (2013) Ets-2 regulates cell apoptosis via the Akt path-
way, through the regulation of urothelial cancer associated 1, a long non-coding RNA, in blad-
der cancer cells. PLoS One 8:e73920
Wu Y, Cheng T, Liu C, Liu D, Zhang Q et al (2016) Systematic identification and characterization
of long non-coding RNAs in the silkworm, Bombyx mori. PLoS One 11:e0147147
Xue J, Qiao N, Zhang W, Cheng RL, Zhang XQ et al (2012) Dynamic interactions between
Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. J
Virol 86:7345–7359
Young RS, Marques AC, Tibbit C, Haerty W, Bassett AR et al (2012) Identification and properties
of 1,119 candidate lincRNA loci in the Drosophila melanogaster genome. Genome Bio Evol
4:427–442
Zemach A, McDaniel IE, Silva P, Zilberman D (2010) Genome-wide evolutionary analysis of
eukaryotic DNA methylation. Science 328:916–919
Zhang YC, Liao JY, Li ZY, Yu Y, Zhang JP et al (2014) Genome-wide screening and functional
analysis identify a large number of long noncoding RNAs involved in the sexual reproduction
of rice. Genome Bio 15:512
Zhou ZY, Li AM, Adeola AC, Liu YH, Irwin DM et al (2014) Genome-wide identification of long
intergenic noncoding RNA genes and their potential association with domestication in pigs.
Genome Biol Evol 6:1387–1392
Zhou QZ, Bindan Z, Quan-You Y, Ze Z (2016) BmncRNAdb: a comprehensive database of non-
coding RNAs in the silkworm, Bombyx mori. BMC Bioinformatics 17:370
Pathogen-Driven Proteomic Changes
in Hemolymph of Nuclear Polyhedrosis 6
Virus-Infected Silkworm Bombyx mori L.
M. Sayed Iqbal Ahamad, Neetha N. Kari,
and Shyam Kumar Vootla
Abstract
Viral infections are distinct in the capacity of the viruses to overtake the host’s
protein synthesis machinery and regulate it for the viral replication. Interaction
and infection process of BmNPV in its host Bombyx mori is an important step to
understand host-pathogen interaction studies. In present research work, we have
screened the BmNPV isolate from the grasserie-infected silkworms from rearing
fields of Central Karnataka, India. Isolation, purification, and characterization of
the BmNPV virus were done by sucrose density gradient centrifugation, scan-
ning electron microscopy, and SDS-PAGE of the occlusion bodies (OBs).
Comparative proteomic analysis revealed drastic up- and downregulation of sev-
eral proteins in control and infected silkworms. The role of various proteins in
comparison with reported proteins responsible for disease infection was
elucidated.
6.1 Introduction
The infectivity of baculoviruses to the specific host insect is of two modes, viz.,
horizontal transmission and vertical transmission. Baculoviruses replicate by ODVs
embedded in OBs that are produced in the final stage of the replication cycle and are
released upon the death and disintegration of the insect. The alkaline microenviron-
ment of insect midgut with pH 8–11 dissolves the OBs, within few seconds ODVs
are released. The released virions infect midgut epithelial cells through peritrophic
membrane (PM). The metalloproteinases and enhancins are concentrated in occlu-
sion bodies, digest mucin on the midgut, and allow virus to access the epithelial cell
surface (Wang and Granados 1997). Then virion envelope fuses with the microvillar
membrane, allowing the nucleocapsids to enter the microvilli; these cells produce
the BVs. BVs are responsible for systemic vertical infection to the surrounding
healthy cells. They enter other cell through receptor-mediated endocytosis. After the
penetration of the plasma membrane, the nucleocapsid uncoats and releases viral
DNA in the nucleus to initiate viral DNA replication. Finally, along with hemo-
lymph, BVs circulate throughout the body and infect other tissues of the host.
6 Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear Polyhedrosis 105
After 12 h of postinfection, mature ODVs embedded in the envelope get pack-
aged into the OBs due to the overexpression of polyhedrin (polh), a structural pro-
tein. At the last stage of infection, larvae stop feeding and exhibit melanization of
cuticle, intersegmental swelling, flaccid musculature, and wandering movements;
ultimately death and disintegration of larvae takes place. Larval disintegration and
contamination of fecal matter of infected larvae by OBs lead to horizontal transmis-
sion of the disease to other healthy larvae.
Immobilization of virus within the crystalline protein lattice of the occlusion body
allows virions to remain viable indefinitely from extremes of heat and UV
radiations.
P10 (BmNPV orf 114) Although p10 does not appear to be a major occlusion
body protein, it colonizes with the PE protein and appears to be required for the
formation of the polyhedron envelope (Gross et al. 1994).
protein that forms about 5% of the mass of OBs. Enhancin is thought to facilitate
baculovirus infection by digesting the peritrophic membrane (Lepore et al. 1996).
Ac145 and Ac150 These two genes encode small proteins ∼9 and 11 kDa, respec-
tively, that are related to one another. Ac145 and Ac150 were found to be associated
with both BV and ODV. Evidence suggested that the mutant had a reduction in its
ability to establish primary infections in midgut cells (Zhang 2005).
6 Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear Polyhedrosis 107
VLF-1 (Ac77) It influences the hyperexpression of the very late genes. VLF-1
appears to be a structural protein present in both BVs and ODVs and is clearly
required for the production of nucleocapsids (Yang and Miller 1998).
VP39 (BmNPV orf 72) VP39 is thought to be the major capsid protein with
39 kDa.
GP41 Tegument Protein (BmNPV orf 66) GP41 homologs are present in all bac-
ulovirus genomes; it is 41 kDa protein required for the release of nucleocapsids
from the nucleus.
Ac98 (38K) Ac98 is encoded by all baculoviruses and is associated with both BV
and ODV nucleocapsids. When deleted, tubelike structures devoid of DNA are pro-
duced (Wu 2006).
p49 (Ac142) p49 is associated with both BV and ODV virions. Deletion of Ac142
affects nucleocapsid formation but does not affect DNA synthesis (Vanarsdall et al.
2007).
The silkworm Bombyx mori has open circulatory system filled with hemolymph.
The hemolymph plays a very important role in transporting nutrients, oxygen,
enzymes, and hormones. It is a very good reservoir of nutrition and energy. The
larval stage of silkworm is the feeding stage, wherein it derives nutrition for its life
processes. This stage is a very crucial period for enhanced metabolic activities and
synthesis of immune proteins during infections. The fifth instar is the intermediate
phase to produce silk proteins and metamorphosis from larva to pupa. The activity
of transferase, phosphatase, and other metabolic enzymes for carbohydrate and lipid
metabolism drastically changes at this stage.
Recent proteomic studies on fifth instar silkworm hemolymph proteins revealed
variations in 30 and 80 kDa proteins. These two proteins are predicted to be the stor-
age proteins. In this feeding and energy accumulating stage, essential enzymes for
metabolism like aldose reductase, glyoxylate reductase, hydroxypyruvate isomer-
ase, and aminoacylase are upregulated. For the metamorphosis from larva to pupa,
proteins like beta-N-acetyl glucose aminidase, chitinolytic enzymes, juvenile
hormone-binding protein, and imaginal disk growth factor overexpressed. Proteins
for the biosynthesis of silk and metamorphosis dehydrogenase, hypothetical pro-
teins, alcohol dehydrogenase II (fragment), transcriptional regulator, and HAD-type
hydrolase/phosphatase related to fatty acid biosynthesis are identified. The upregu-
lation of immune proteins like hemolin, prophenoloxidase, serine proteases, para-
lytic peptide-binding protein, and trypsin inhibitors is also reported (Li et al. 2006;
Hou et al. 2010).
infection and bud out from the nucleus with the envelope from the nuclear mem-
brane (Granados and Lawler 1981).
A remarkable feature of NPV infection is that in some instances the insects can
grow and continue feeding right until pupation. They appear healthy yet when
examined are heavily infected with high concentrations of occlusion bodies in their
cells and hemolymph. A feature of the final stage of baculovirus replication that
takes place after DNA replication has occurred is the hyperexpression of very late
genes resulting in the production of high levels of polyhedrin and p10. Polyhedrin
accumulates in nuclei and at some point crystallizes into a lattice that surrounds
virions.
The dispersal of virus takes place by the OBs from the infected larvae. The
infected insects migrate to a higher elevation on the branch of the tree. This facili-
tates easy dispersal of the OBs. A gene (Ac1) encoding an RNA processing enzyme
(RNA 5′-triphosphatase) has implicated characteristic terminal movement of
infected insects. Late in infection after the wandering stage, the insects undergo
disintegration or liquefaction at later stage.
increased levels of PKR activity are known to induce the apoptosis in response to viral
infections. But the baculovirus has developed countermeasures to combat the antiviral
defense mechanism of the host by synthesizing antiapoptotic proteins like p35 and
inhibitors of apoptosis (IAP), so as to prevent cell death induced by the insect cell
apoptotic mechanism (Prudhomme and Couble 2002). The feces of silkworm larvae
also possess an antiviral substance L4-1 that shows antiviral activity by damaging
viral proteins by producing reactive oxygen species (Lim et al. 2002).
6.2.1 P
urification of Occlusion Bodies (OBs) by Sucrose Density
Gradient Centrifugation
The collected hemolymph was centrifuged at 8000 rpm for 10 min at room tempera-
ture. The pelleted OBs were further washed in Milli-Q water by centrifugation at
5000 rpm for 10 min. The washing process was repeated and then OBs were col-
lected as a white pellet.
The partially purified OBs were mixed in 1 mL of 0.2% Triton X-100. This solu-
tion was layered on 35–65% sucrose gradients and centrifuged at 20,000 rpm for 1 h
at 4 °C in SW55Ti rotor using the Beckman Coulter Optima LE-80K Ultracentrifuge.
The purified OBs were suspended in 0.5% SDS and observed in Olympus SZX16
stereo zoom microscope at 20× and 40× magnifications.
The ODVs were isolated and purified as described by Braunagel and Summers
(2007); accordingly, the OB suspension was incubated in polyhedra lysis buffer
(0.1 M Na2CO3; 0.166 M NaCl; 0.01 M EDTA, pH 10.5) at 37 °C for 2 h, and the
suspension was neutralized by using 0.5 M Tris-HCl (pH 7.5). The alkali-treated
OB suspension was layered onto the 35–65% (w/v) sucrose gradients and centri-
fuged at 28,000 rpm for 1 h. The purified band of ODVs was collected and stored at
−20 °C.
The purity of the OBs was confirmed by microscopic observation in Olympus
SZX16 stereo zoom microscope at 40× magnification. The density of the OB sus-
pension was adjusted to 5 × 107 OBs/mL using Blankenburg Neubauer chamber.
112 M. Sayed Iqbal Ahamad et al.
6.2.3 S
canning Electron Microscopy of Occlusion Bodies (OBs)
of BmNPV
The purified OBs were smeared on stubs on previously covered carbon tape and
allowed to air-dry till all the moisture content was removed and gold coated by
Polaron, SEM Coating System to 3A° for 1.3 min and observed in LEO 435VP
Scanning Electron Microscope at 2000× and 30, 000× magnification.
The purified OB suspension with 5 × 107 OBs/mL was mixed with sample buffer
(125 mM Tris-HCl, pH 6.7; 4% SDS; 30% glycerol; 0.002% bromophenol blue and
10% β-mercaptoethanol) and boiled for 5 min at 95 °C and separated on 12% resolv-
ing gel at 150 V for 1 h.
6.2.5 S
DS-PAGE Profile of BmNPV-Infected Hemolymph
Proteins
6.2.6 T
wo-Dimensional Electrophoresis of BmNPV-Infected
Silkworm Hemolymph Proteins
The IEF gel was pre-run with a constant voltage of 200 V for 15 min, 300 V for
30 min, and 500 V for 30 min. After pre-run, pre-estimated solubilized hemolymph
protein samples of 100 μg were loaded in each tube. On the protein samples, GOS
and upper tank buffer were filled. Electrophoresis was performed with constant
voltage of 600 V for 12 h and 700 V for 1 h at room temperature.
The gels after the first dimension electrophoresis were retrieved from the glass
tubes. Then the second dimension SDS-PAGE was performed in 12% polyacryl-
amide gel at 150 V. Protein spots on the gels after electrophoresis were visualized
by silver staining.
Fig. 6.1 Grasserie-
infected and
healthy silkworms
Fig. 6.2 BmNPV-infected
silkworm showing fragile
skin
The major protein of the NPV occlusion body is polyhedrin, which is highly con-
served of all baculovirus proteins. Initiation and formation of polyhedron-shaped
occlusion bodies are believed to require specific protein-protein interactions between
polyhedrin and proteins in the virion envelope (Russell and Rohrmann 1990). The
OBs of BmNPV are commonly polyhedral in shape, but mutations in polyhedrin gene
and some other genes may affect OBs morphogenesis. The shape of OBs may be tet-
rahedral, hexahedral, or octahedral in BmNPV are reported (Bilimoria 1991). The
genetic difference is responsible for variations in the formation of OBs of BmNPV. A
recent study confirmed the mutation in the genome of BmNPV leads to the formation
of cubic OBs, in addition to the single point mutation in polyhedrin gene (Lin et al.
2000); baculovirus repeated ORFs (bro) and homologous repeat (HR) regions showed
the major differences in genome size of the normal and cubic strains of BmNPV
(Cheng et al. 2012) (Fig. 6.7).
For the proteomic studies of OBs, sucrose density gradient purified OBs and
SDS-PAGE analysis revealed 13 predominant bands on 12% polyacrylamide gel
(Fig. 6.8). The molecular weight of all the bands was predicted by comparing with
medium range protein marker with 98–29 kDa range, and our results are also in
confirmation with previous studies (Perera et al. 2007; Braunagel and Summers
6 Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear Polyhedrosis 115
a b
Fig. 6.3 Anatomy of (a) healthy silkworm showing mulberry leaves the in gut. (b) BmNPV-
infected silkworm showing empty gut
20 X Magnification 40 X Magnification
Fig. 6.5 Microscopic image of occlusion bodies (OBs) in 20× and 40× magnifications
116 M. Sayed Iqbal Ahamad et al.
a b
Immature OBs
Mature OBs
Fig. 6.6 Sucrose density gradient purification of (a) BmNPV OBs and (b) ODVs
a b
Fig. 6.7 Scanning electron micrographs of OBs (a) 2000× magnification and (b) 3000×
magnification
Fig. 6.8 SDS-PAGE M
profiling of OBs, M
medium range protein kDa
marker
98
1
68 2
3
4
5
6
43 7
8
9
10
29 11 Polyhedrin
12
13
Table 6.1 Structural proteins of OBs and ODVs on 12% polyacrylamide gel of SDS-PAGE
Predicted size Number
Band from SDS- Protein of amino
no. PAGE (kDa) name acids (aa) BmNPV ORF Function
1 79.9 VP80 692 BmNPV orf88 Capsid protein
2 74 P74 645 BmNPV orf115 Oral infectivity
3 44.9 GP41 403 BmNPV orf66 Tegument protein
4 79.2 ODV-E66 702 BmNPV orf37 ODV envelope
5 59.8 PIF-1 527 BmNPV orf97 per os infectivity of
NPVs, ODV envelope
protein
6 55.5 P49 476 BmNPV orf118 ODV associated protein
7 41.6 BV/ 362 BmNPV orf85 Regulating the cell cycle
ODV-C42 and enhancing viral
infectivity
8 45 ODV-EC43 391 BmNPV orf92 ODV protein of envelope
and nucleocapsids, having
role during infection
9 41.3 ODV-E56 375 BmNPV orf124 ODV envelope protein
10 44.9 GP41 403 BmNPV orf66 Tegument protein
11 28.8 Polyhedrin 245 BmNPV orf1 OB structural protein
12 25.6 ODV-E25 228 BmNPV orf77 ODV envelope
13 8.1 P6.9 65 BmNPV orf84 DNA binding protein
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
kDa
185 53kDa
116.25kDa
108.20kDa
98 106.81kDa
102.85kDa
68
42.16kDa
41.48kDa
43 40.13kDa
29 54kDa
29 28.48kDa
26.17kDa
Fig. 6.9 SDS-PAGE of BmNPV-infected silkworm hemolymph proteins. M medium range pro-
tein marker. Lanes 1, 3, 5, 7, 9, 11, 13, 15, 17: control hemolymph of fifth instar first to ninth days
postinfection. Lanes 2, 4, 6, 8, 10, 12, 14, 16, 18: BmNPV-infected hemolymph of fifth instar day
1 to day 9
But the intensity of expression was more in sixth dpi and suddenly reduced in sev-
enth and eighth dpi (Fig. 6.7. White arrows). On the grounds of these observations,
we predicted that it may be an antiviral protein band but failed to overexpress to take
over BmNPV infection due to the lack of host insect physiological state to support
its expression. But further analysis is required to confirm this hypothesis.
We predicted 36 kDa protein of the aldo-keto reductase from day 1 to day 9 of con-
trol hemolymph, whereas it is progressively diffused in fifth to sixth dpi and sharply
disappeared in seventh dpi of BmNPV-infected hemolymph (Fig. 6.7, Lanes 10, 12, and
14). This enzyme is actively involved in the aldo-keta reductase activity in carbohydrate
metabolism of silkworm. The underexpression or suppression of aldo-keto reductase
may be responsible for the interruption in normal bioenergetics of carbohydrates, fail-
ure of appetite, and other physiological activities in BmNPV-infected silkworms.
The temporal fluctuations in expression observed in ∼260–270 kDa host iso-
forms of hemolymph proteins. In fifth dpi, these bands abruptly disappeared and
mildly expressed in sixth dpi and again disappeared in seventh dpi but clearly
expressed in 8th and 9th dpi. The highly expressed hemolymph protein band of
∼210–230 kDa is underexpressed in sixth dpi but failed to express in seventh dpi
(Fig. 6.7, Lanes 12 and 14) and normally expressed in 8th and 9th dpi. The protein
expression pattern in case of ∼185 kDa protein band is underexpression at sixth dpi
but not expressed in 7th dpi and again poorly expressed in 8th dpi and normally
expressed in 9th dpi of BmNPV-infected hemolymph (Fig. 6.7, Lanes 16 and 18).
The silkworm has an open circulatory system, and hemolymph is the main circu-
lating fluid in the hemocoel of the silkworm. It bathes all tissues and organs in the
insect body and transports nutrients, hormones, and metabolic wastes (Gilbert and
Chino 1974). The hemolymph proteins represent the products of gene expression.
Their levels show generic-specific, tissue-specific, and instar-specific variations
during insect metamorphosis (Hou et al. 2010). The hemolymph of silkworm acts as
a reservoir for many proteins that includes juvenile hormone-binding proteins,
RNA-binding proteins, paralytic peptide-binding protein, aldose reductase, low
molecular weight lipoproteins, carboxylesterases, zinc finger proteins, imaginal
disk growth factor, gelsolin, glyoxylate reductase, hydroxypyruvate isomerase, ami-
noacylase, trypsin inhibitor, transferrin protein, serine proteases, chymotrypsin
inhibitor, hemolin, prophenoloxidase, 30 kDa lipoproteins, instar-specific proteins,
N-acetylglucosaminidase, and many other unidentified proteins (Hou et al. 2010).
These proteins and enzymes are associated with silk formation, hemocyte pro-
duction, ecdysis, eclosion, metabolism, metamorphosis, immunity, locomotion,
cocoon spinning, digestion, respiration, tissue degeneration, organ growth, and
from heat shock control to gene expression (Li et al. 2006; Choi et al. 2008;
Nakahara et al. 2009). The hemolymph proteins represent 93 silk gland proteins,
177 fat body proteins, and 278 skeletal muscle proteins (Takasu et al. 2005; Kyung
et al. 2006; Zhang et al. 2007).
The electrophoretic analysis of hemolymph proteins of NPV-infected silkworm
shows the reduction in all the protein fractions (Watanabe 1986). The level of amino
acid transferases and parasitic polypeptides increases in NPV-infected insects and
decreases in host cellular polypeptides (Kobayashi et al. 1990). BmNPV infection
120 M. Sayed Iqbal Ahamad et al.
Table 6.2 2-DE number of protein spots observed in control and infected hemolymph proteins in
1st to 8th dpi of BmNPV infection
Days of post infection in 5th Protein spots in control Protein spots in infected
instar larvae hemolymph hemolymph
1st day 66 63
2nd day 49 38
3rd day 60 50
4th day 37 30
5th day 48 21
6th day 30 11
7th day 29 17
8th day 33 29
6 Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear Polyhedrosis 121
5th instar 1st day control 5th instar 1st day BmNPV infected
5th instar 2nd day control 5th instar 2nd day BmNPV infected
5th instar 3rd day control 5th instar 3rd day BmNPV infected
5th instar 4th day control 5th instar 4th day BmNPV infected
Fig. 6.10 2-DE profile of BmNPV-infected silkworm hemolymph proteins of fifth instar first day
to fourth days postinfection
5th instar 5th day control 5th instar 5th day BmNPV infected
5th instar 6th day control 5th instar 6th day BmNPV infected
5th instar 7th day control 5th instar7th day BmNPV infected
Fig. 6.11 2-DE profile of BmNPV-infected silkworm hemolymph proteins at fifth instar fifth day
to seventh days postinfection
intensely with different decoration and three new protein spots were overexpressed
(arrow mark). The enzyme alcohol dehydrogenase II is related to the fat biosynthe-
sis at fifth instar stage in preparation for pupation, and transcriptional factor is
essential for transcription. These two proteins are underexpressed in BmNPV-
infected hemolymph which adversely affects fat biosynthesis and transcription in
BmNPV-infected silkworms.
Conclusion
Silkworms have emerged as new tools for biotechnological studies as model ani-
mal and bioreactor. In addition to genomics, proteomics, and metabolomics stud-
ies on silkworms, basic studies like infectious diseases of silkworms, mode of
infections, disease establishment, host-pathogen interactions, and resistance of
the host toward the infection of entomopathogens are scarce. All these studies are
incomplete without authentic knowledge of relevant proteins which are the ulti-
mate biomolecules to decide most of the biological functions. Though a lot of
information on crystal structure, catalytic activity, and the functions of proteins
have been elucidated, the role of several proteins still remains unresolved.
The biological functioning of all the organisms not only depends on static
genome but the dynamic population of proteins determined by an interplay of
gene and protein regulation with extracellular influences. By using genome
sequence data, active domains of functional proteins, posttranslational modifica-
tions, cannot be predicted. To know the functional feature of proteins, posttrans-
lational modifications like phosphorylation, glycosylation, ubiquitination, and
methylation are very important. There is increasing interest in the field of pro-
teomics, the large-scale study of proteins as a complement to genomics, and
functional genomics is emerging.
The silkworm host and baculoviral interaction studies by using SDS-PAGE in
fifth instar day 1 to day 9 in BmNPV-infected silkworms with reference to
healthy silkworm hemolymph revealed the downregulation of housekeeping,
host-specific hemolymph proteins, and upregulation of BmNPV-specific struc-
tural proteins, specifically polyhedrin. In contrast to this, a new protein band of
∼70–90 kDa is identified in sixth, seventh, and eighth dpi of BmNPV predicted
to be an antiviral protein. Two-dimensional gel electrophoresis of BmNPV-
infected fifth instar day 1 to day 7 in contrast to healthy hemolymph proteins
dictates downregulation of host-specific proteins which are responsible for all
the physiological activities of the host and overexpression of pathogen-derived
and pathogen-induced proteins in the late phase of infection. We observed nearly
70 discrete protein spots and predicted their names and role in host-pathogen
interactions by comparing with the available protein data base of hemolymph
proteins.
These research findings are clearly evident in confirming the changes of host
protein profiles which mostly favor the disease course and give the pathogen a
very proven strategy. Our understanding from the present study will help us to
elucidate much more detailed approach to host-pathogen interactions in
silkworms.
124 M. Sayed Iqbal Ahamad et al.
References
Belyavskyi M, Braunagel SC, Summers MD (1998) The structural protein ODV-EC27 of
Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin. Proc Natl Acad
Sci U S A 95(19):11205–11210
Bilimoria SL (1991) The biology of nuclear polyhedrosis viruses. In: Kurstak E (ed) Viruses of
invertebrates. Marcel Dekker, New York, pp 1–72. 360p
Braunagel SC, Elton DM, Ma H, Summers MD (1996) Identification and analysis of an Autographa
californica nuclear polyhedrosis virus structural protein of the occlusion-derived virus enve-
lope: ODV-E56. Virology 217:97–110
Braunagel SC, Summers MD (2007) Molecular biology of the Baculovirus occlusion-derived virus
envelope. Curr Drug Targets 8:1084–1095
Cheng R-L, Xu Y-P, Zhang C-X (2012) Genome sequence of a Bombyx mori nucleopolyhedrovirus
strain with cubic occlusion bodies. J Virol 86(18):10245
Choi SS, Rhee WJ, Park TH (2008) Inhibition of human cell apoptosis by silkworm hemolymph.
Biotechnol Prog 18:874–878
Clemens MJ, Elia A (1997) The double stranded RNA-dependent protein kinase PKR: structure
and function. J Interf Cytokine Res 17(9):503–524
Deng F, Wang R, Fang M, Jiang Y, Xu X, Wang H, Chen X, Arif BM, Lin G, Wang H, Zhihong H
(2007) Proteomics analysis of Helicoverpa armigera single nucleocapsid nucleopolyhedrovi-
rus identified two new occlusion-derived virus-associated proteins, HA44 and HA100. J Virol
81(17):9377–9385
Du X, Thiem SM (1997) Characterization of host range factor 1(hrf-1) expression in Lymantria
dispar Multinucleopolyhedrovirus and recombinant Autographa californica infected IPLB-
Ld652Y cells. Virology 227:420–430
Engelhard EK (1994) The insect tracheal system: a conduit for the systemic spread of Autographa
californica multinuclear polyhedrosis virus. Proc Natl Acad Sci U S A 91:3224–3227
Fang M, Dai X, Theilmann DA (2007) Autographa californica multiple nucleopolyhedrovirus
EXON0 (ORF141) is required for efficient egress of nucleocapsids from the nucleus. J Virol
81(18):9859–9869
Gilbert LI, Chino H (1974) Transport of lipids in insects. J Lipid Res 15:439–456
Goley ED (2006) Dynamic nuclear actin assembly by Arp2/3 complex and a baculovirus WASP-
like protein. Science 314(5798):464–467
Granados RR, Lawler KA (1981) In vivo pathway of Autographa californica baculovirus invasion
and infection. Virology 108:297–308
Gross CH, Russell RLQ, Rohrmann GF (1994) The Orgyia pseudotsugata baculovirus p10 and
polyhedron envelope protein genes: analysis of their relative expression levels and role in poly-
hedron structure. J Gen Virol 75:1115–1123
Gururaj CS, Sekharappa BM, Sarangi SK (1999) Effect of BmNPV infection on the digestive
enzyme activity in the silkworm, Bombyx mori L. Indian J Seric 38(2):102–106
Hou Y, Zou Y, Wang F, Gong J, Zhong X, Xia Q, Zhao P (2010) Comparative analysis of pro-
teome maps of silkworm hemolymph during different developmental stages. Proteome Sci
8(45):1–10
Kajiwara H, Imamaki A, Nakamura M, Mita K, Xia Q, Ishizaka M (2009) Proteome analysis of
silkworm 2. Hemolymph. J Electrophor 53:27–31
Kishimoto A, Nakato H, Izumi S, Tomino S (1999) Biosynthesis of major plasma proteins in
the primary culture of fat body cells from the silkworm, Bombyx mori. Cell Tissue Res 297:
329–335
Kobayashi M, Kotake M, Sugimori H, Nagamine T, Kajiura Z (1990) Identification of virus spe-
cific polypeptides and translatable mRNAs in the isolated pupal abdomens of the silkworm,
Bombyx mori infected with nuclear polyhedrosis. J Invertebr Pathol 55(1):52–60
Kyung HS, Su JJ, Young RS, Seok WK, Sung SH (2006) Identification of up-regulated proteins
in the haemolymph of immunized Bombyx mori larvae. Comp Biochem Physiol D1:260–266
6 Pathogen-Driven Proteomic Changes in Hemolymph of Nuclear Polyhedrosis 125
Lehane MJ, Aksoy S, Levashina (2004) Blood immune responses and parasite transmission in
insects. Trends Parasitol 29(9):433–439
Lepore LS, Roelvink PR, Granados RR (1996) Enhancin, the granulosis virus protein that
facilitates nucleopolyhedrovirus (NPV) infections, is a metalloprotease. J Invertebr Pathol
68(2):131–140
Li X-h, Wu X-f, Yue W-f, Liu J-m, Li G-l, Miao Y-g (2006) Proteomic analysis of the silkworm
(Bombyx mori L.) Hemolymph during developmental stage. J Proteome Res 5(10):2809–2814
Lim DS, Ko SH, Kim SJ, Park YJ, Park JH, Lee WY (2002) Photoinactivation of vesicular stomati-
tis virus by a photodynamic agent, chlorophyll derivatives from silkworm excreta. J Photochem
Photobiol B 67(3):149–156
Lin GY, Zhong J, Wang XZ (2000) Abnormal formation of polyhedra resulting from a single
mutation in the polyhedrin gene of Autographa californica multicapsid nucleopolyhedrovirus.
J Invertebr Pathol 76(1):13–19
Liu X, Chen K, Cai K, Yao Q (2008) Determination of protein composition and host-derived pro-
teins of Bombyx mori Nucleopolyhedrovirus by 2-dimensional electrophoresis and mass spec-
trometry. Intervirology 51:369–376
McCarthy CB, Theilmann DA (2008) AcMNPV ac143 (odv-e18) is essential for mediating budded
virus production and is the 30th baculovirus core gene. Virology 375:277–291
Murphy FA, Fauquet CM, Bishop DHL, Ghabrial SA, Jarvis AW, Martelli GP, Mayo MA, Summers
MD (1995) Virus taxonomy. Classification and nomenclature of viruses. Sixth Report of the
International Committee on Taxonomy of Viruses. Springer, Wien New York, Arch Virol, 10
Nagata M, Kobayashi M (1990) Quantitative changes in storage proteins during larval develop-
ment of the silkworm, Bombyx mori. J Seric Sci Jpn 59:461–468
Nakahara Y, Shimura S, Ueno C, Kanamori Y, Mita K, Kiuchi T, Kamimura M (2009) Purification
and characterization of silkworm hemocytes by flow cytometry. Dev Comp Immunol 33:439–448
Nakazawa H, Tsuneishi E, Ponnuvel KM, Furukawa S, Asaoka A, Tanaka H, Ishibashi J, Yamakawa
M (2004) Antiviral activity of a serine protease from the digestive juice of B. mori larvae
against NPV. Virology 321(1):154–162
Nie Y, Fang M, Theilmann DA (2009) AcMNPV AC16 (DA26, BV/ODV-E26) regulates the levels
of IE0 and IE1 and binds to both proteins via a domain located within the acidic transcriptional
activation domain. Virology 385(2):484–495
Ohkawa T, Volkman LE, Welch MD (2010) Actin-based motility drives baculovirus transit to the
nucleus and cell surface. J Cell Biol 190(2):187–195
O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. Journal of bio-
logical chemistry 250(10):4007–4021
Palhan VB, Gopinathan KP (1996) Characterization of a local isolate of Bombyx mori nuclear
polyhedrosis virus. Curr Sci 70(2):147–153
Perera O, Green T B, Stevens S M, White S, Becnel JJ (2007) Proteins associated with Culex nigri-
palpus nucleopolyhedrovirus occluded virions. Journal of virology 81(9):4585–4590
Ponnuvel KM, Nakazawa H, Furukawa S, Asaoka A, Ishibashi J, Tanaka H, Yamakawa M (2003) A
lipase isolated from the silkworm shows antiviral activity against NPV. J Virol 77(19):10725–10729
Prudhomme JC, Couble P (2002) Perspectives in silkworm transgenesis. Curr Sci 83(4):432–438
Rohrmann GF (2011) Baculovirus Molecular Biology, 2nd edn. National Center for Biotechnology
Information (US), Bethesda
Russell RLQ, Rohrmann GF (1993) A 25 kilo dalton protein is associated with the envelopes of
occluded baculovirus virions. Virology 195:532–540
Russell RLQ, Funk CJ, Rohrmann GF (1997) Association of a baculovirus encoded protein with
the capsid basal region. Virology 227:142–152
Russell RLQ, Rohrmann GF (1990) A baculovirus polyhedron envelope protein: immunogold
localization in infected cells and mature polyhedra. Virology 174:177–184
Takasu Y, Yamada H, Saito H, Tsubouchi K (2005) Characterization of Bombyx mori sericins by
the partial amino acid sequences. J Insect Biotechnol Sericol 74:103–109
Terenius O (2004) Anti-parasitic and anti-viral immune responses in insects. Ph. D Thesis.
Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm
126 M. Sayed Iqbal Ahamad et al.
Ujita M, Katsuno Y, Kawachi I, Ueno Y, Banno Y, Fujii H, Hara A (2005) Glucan-binding activity
of silkworm 30-kDa apolipoprotein and its involvement in defense against fungal infection.
Biosci Biotech Bioch 69:1178–1185
Vanarsdall AL, Pearson MN, Rohrmann GF (2007) Characterization of baculovirus constructs
lacking either the Ac 101, Ac 142, or the Ac 144 open reading frame. Virology 367:187–195
Vigdorovich V, Miller AD, Strong RK (2007) Ability of hyaluronidase 2 to degrade extracellular
hyaluronan is not required for its function as a receptor for jaagsiekte sheep retrovirus. J Virol
8(7):3124–3129
Wang P, Granados RR (1997) An intestinal mucin is the target substrate for a baculovirus enhancin.
Proc Natl Acad Sci U S A 94(13):6977–6982
Wang R (2010) Proteomics of the Autographa californica Nucleopolyhedrovirus budded Virions.
J Virol 84:7233–7242
Wang Y (1995) Interaction of p53 with its consensus DNA-binding site. Mol Cell Biol
15(4):2157–2165
Watanabe H (1986) Resistance of the silkworm, Bombyx mori to viral infections. Agric Ecosyst
Environ 15(2–3):131–139
Wu W (2006) Autographa californica multiple nucleopolyhedrovirus nucleocapsid assembly is
interrupted upon deletion of the 38K gene. J Virol 80(23):11475–11485
Xu H (2008) Bombyx mori nucleopolyhedrovirus ORF56 encodes an occlusion-derived virus pro-
tein and is not essential for budded virus production. J Gen Virol 89:1212–1219
Yang S, Miller LK (1998) Expression and mutational analysis of the baculovirus very late factor 1
(vlf-1) gene. Virology 245(1):99–109
Zhang JH (2005) Effects of Ac150 on virulence and pathogenesis of Autographa californica mul-
tiple nucleopolyhedrovirus in noctuid hosts. J Gen Virol 86(6):1619–1627
Zhang P, Aso Y, Jikuya H, Kusakabe T, Lee JM, Kawaguchi Y, Yamamoto K, Banno Y, Fujii H
(2007) Proteomic profiling of silkworm skeletal proteins during larval-pupal metamorphosis.
J Proteome Res 6:2295–2303
Part II
Molecular Based Studies of Insect Pathology
Analysis of the Viral Lytic Polysaccharide
Monooxygenase Fusolin and Its 7
Potential Application to Pest Control
Wataru Mitsuhashi
Abstract
Although microbial insecticides are generally safe for vertebrates, plants and the
environment, their use has been very limited, mainly because their cost of pest
control is much higher than that of chemical insecticides. To expand the use of
microbial insecticides, their ability to kill pests needs to be strengthened.
Increased activity will reduce the amount applied per unit area and the cost of
pest control. The protein fusolin that is produced by the insect viruses entomo-
poxviruses and baculoviruses strongly synergistically increases the infectivity of
insect viruses. Recent studies further elucidated the synergistic effect of the pro-
tein on the insecticidal activity of major entomopathogenic bacterium Bacillus
thuringiensis. Furthermore studies have revealed that fusolin is a lytic chitin
monooxygenase, and thus the mechanism of increase in the infectivity and insec-
ticidal activity by fusolin has been elucidated in detail. These advances have
expanded the possible practical applications of this protein to pest control and
suggest its potential for use in a new field, namely, the development of technolo-
gies for efficient biofuel production from biomass such as chitin.
7.1 Introduction
Wide use of microbial insecticides in agriculture and forestry has been anticipated,
because they are considered safe for vertebrates (including humans and livestock),
plants and the environment. However, the sales of microbial insecticides constitute
W. Mitsuhashi
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization,
Tsukuba, Ibaraki 305-8634, Japan
e-mail: mitsuhas@affrc.go.jp
only a very small fraction of the total insecticide market, especially in developed
countries. The main reasons for their limited use are as follows:
However, if a drastic reduction in the cost of pest control can be achieved, this
may lead to expansion of the use of microbial insecticides. Therefore, attempts have
been made to strengthen the ability of microbes or the toxic proteins produced by
some entomopathogenic bacteria such as Bacillus thuringiensis (Bt) to kill pests. A
major approach to achieving this goal is the use of synergistic agents that can
enhance the peroral infectivity of microbes or insecticidal activity of toxins, thus
reducing the amount of insecticides needed per unit area and lowering costs. More
than ten synergists (synthetic compounds and proteins of insect viruses) have been
found (Mitsuhashi 2009). Natural synergists are likely to be less harmful than syn-
thetic compounds to vertebrates and the environment.
This review focuses on one of these synergists, fusolin, a lytic polysaccharide
monooxygenase produced by the insect viruses entomopoxviruses (EVs) and bacu-
loviruses. Fusolin may also potentially be used for the development of the methods
of efficient conversion of biomass to chitin.
EVs include all viruses of the subfamily Entomopoxvirinae in the family Poxviridae.
This subfamily is divided into the following three genera: Alphaentomopoxvirus, which
infect Coleoptera; Betaentomopoxvirus, which infect Lepidoptera and Orthoptera; and
Gammaentomopoxvirus of Diptera. EV virions are large, ovoid or brickshaped, and
they contain a large linear double-stranded DNA (225–380 kbp) with characteristic
terminal hairpin loop structures (Mitsuhashi et al. 2014b). Two types of hairpin loop
sequences that are in relationship as inverted repeat are found per one EV species, and
one loop links at each end of each genome molecule (Mitsuhashi et al. 2014b). EVs
replicate mainly in the fat bodies of hosts.
Baculoviruses include all viruses of the family Baculoviridae. This family is com-
posed of four genera: Alphabaculovirus which infects Lepidoptera; Betabaculovirus,
7 Analysis of the Viral Lytic Polysaccharide Monooxygenase Fusolin 131
7.3 Fusolin
Baculoviral fusolins are usually called GP37s, but in this review I refer to GP37s as
fusolins. Fusolins contain 220–390 amino acid residues (aa). Their N-termini (15–
20 aa) are signal peptides that are cleaved from the protein; the C-terminal regions
of fusolins are poorly conserved and in general are shorter in baculoviruses than in
EVs. The smallest fusolin reported so far is that of Epinotia aporema GV (Salvador
et al. 2012).
Many baculoviruses and some EVs (and apparently metazoans) lack fusolin
genes. Therefore, EVs and baculoviruses may have acquired these genes by hori-
zontal transfer from amoebae and bacteria (Thézé et al. 2015). As EVs and baculo-
viruses are phylogenetically unrelated, they seem to have acquired these genes
independently (Thézé et al. 2015).
7.4 Spindles
BmNPV (both the wild type and polyhedrin-negative recombinant) become highly
infectious perorally to B. mori larvae when administered with ACEV spindles. This
finding suggests that spindles, together with recombinant viruses, could be a power-
ful tool for mass production of proteins in biofactories. Peroral inoculation of these
recombinant viruses is much less labour consuming than the conventional inocula-
tion method (injection of viruses into the host haemocoel one by one).
Baculoviral fusolin enhances the insecticidal activity of the Bt bacterium (Liu
et al. 2011). EV spindles also enhance the insecticidal activity of Bt toxin alone and
a commercial complete Bt formulation (Mitsuhashi et al. 2014a).
EV spindles (fusolins) may have a wide activity spectrum. For instance, ACEV
spindles strongly enhance the infectivity of Spilosoma imparilis NPV (SiNPV) and
BmNPV (Mitsuhashi et al. 1998; Mitsuhashi and Sato 2000), even though these
NPVs are not very closely related taxonomically and ACEV does not infect the
hosts of both NPVs. In contrast, the GV protein enhancin does not seem to have a
wide spectrum of enhancement of baculovirus infection (Hukuhara et al. 1987;
Derksen and Granados 1988; Goto 1990; Wang et al. 1994).
The ability of ACEV spindles to enhance NPV infectivity is highly stable against
various abiotic factors, including high temperature, ultraviolet radiation and ethyl
alcohol (Mitsuhashi et al. 2008). For example, ACEV spindles retain high activity
even after heating at 95 °C for 30 min, whereas Pseudaletia unipuncta GV granules
(occlusion bodies containing enhancin) are inactivated by heating at 85 °C for
10 min (Tanada 1959). Spindle stability is necessary for the use of spindles as syner-
gists of microbial insecticides.
Fig. 7.2 Peritrophic
matrix (PM) of Bombyx
mori larvae. Reproduced
from Mitsuhashi (2013).
Upper, control PM. Lower,
disrupted PM by action of
spindles. Bar indicates
5 mm
7 Analysis of the Viral Lytic Polysaccharide Monooxygenase Fusolin 135
By assaying the activity of various ACEV fusolin mutants in a bioassay system that
included BmNPV and B. mori larvae, our group identified the regions of fusolin that are
involved in the enhancement of viral infectivity (Takemoto et al. 2008; Fig. 7.3). We
found that the N-terminal region (ca. 250 aa) containing a signal peptide is essential for
activity and N-glycosylation is important; the rest of the fusolin molecule is non-essen-
tial. We also found that the N-terminal region binds to chitin in vitro and that glycosyl-
ation is important for chitin binding (Takemoto et al. 2008). This essential region
contains regions constituting a chitin-binding domain that are found in fusolins and pro-
teins produced by many bacteria but very few eukaryotes. Indeed, Spodoptera litura
NPV fusolin binds chitin in vitro (Li et al. 2003). The non-essential region is easily
eliminated by serine protease(s) in insect digestive juice, whereas the essential region is
more stable, and glycosylation is required for its stability (Takemoto et al. 2008).
Our group observed that a certain degree of disruption of the PM occurred imme-
diately after the feeding of spindles to the larvae was completed and it was heavily
disrupted several hours after the observation (our unpublished data; Mitsuhashi and
Miyamoto 2003). However, the PM regenerated (except for its most posterior
region) the next day (our unpublished data; Mitsuhashi and Miyamoto 2003).
Recently, the atomic structures of the fusolins and spindles of three EVs were
determined by X-ray microcrystallography; for these studies, the in vivo spindles of
Melolontha melolontha EV (genus Alphaentomopoxvirus), ACEV, and an EV from
Wiseana spp. (genus Betaentomopoxvirus) that were purified from insects were
used (Chiu et al. 2015) (Fig. 7.4). The structures were very similar to each other, and
fusolins were found to be members of the auxiliary activity (AA) 10 family of lytic
polysaccharide monooxygenases (LPMOs) on the basis of the features of the struc-
tures (Levasseur et al. 2013). Therefore it was elucidated that fusolin acts as a lytic
chitin monooxygenase on the chitin of the PM, thereby disrupting it. AA10 LPMOs
specifically hydrolyse crystalline polysaccharides (Aachmann et al. 2012), and it is
speculated that more than 2000 organisms, including viruses, produce AA10
LPMOs (The Carbohydrate-Active enZYmes Database).
The N-terminal histidine (His1) of mature fusolin and a histidine located about
145 aa from the N-terminus form a histidine brace, a characteristic feature of LPMOs
Glycosylation site
190-192
1 17 ca. 250 373 aa
Fig. 7.3 Functional regions of Anomala cuprea EV fusolin. Functional regions were determined
by using fusolin mutants. The region essential for activity can bind chitin; glycosylation enhances
activity (N-glycosylation site; aa190–192). The region dispensable for activity is easily digested by
insect midgut juice. aa, amino acid
136 W. Mitsuhashi
a
b
Fig. 7.4 The 3D structure of fusolin from Melolontha melolontha EV. Reproduced from Chiu
et al. (2015). (a) Structure is represented by a ribbon diagram coloured in a blue-red gradient from
the N- to the C-terminus. Cysteine residues are shown as spheres. (b) Fusolin forms a domain-
swapped dimer shown as a ribbon diagram in a semi-transparent molecular surface. Inset: sche-
matic representation of the dimer
b
a g
c
e
d f
c d
Fig. 7.6 Structures of the unit cell of spindle of Melolontha melolontha EV. Spindles are crystal-
line polymers of fusolin dimers cross-linked by a 3D network of disulphide bonds. The figures
were cited from Chiu et al. (2015). (a) Scanning electron micrograph of spindles. A blue box rep-
resents a spindle unit cell (not to scale).(b) A unit cell viewed along a twofold crystallographic
axis. Cysteines involved in inter-dimer cross-links are shown as magenta spheres. The central
dimer is represented as a blue-red molecular surface; two crowns of four dimers are shown in green
and cyan. The two capping dimers are shown in brown as a semi-transparent molecular surface.
(c) Unit cell assembly. Inter-dimer disulphide bonds that involve the central dimer are indicated by
links between the interacting cysteines. (d) The full unit cell is shown in the same orientation as in
(c) and in an orthogonal view. (e) Representation of 18 unit cells with each dimer shown as a large
sphere and cysteines as small spheres. Inter-sphere links highlight the 3D network of covalent
bonds cross-linking spindles
Spindles are single crystals that diffract to high resolution (Chiu et al. 2015),
although they are generally described as paracrystalline. In spindles, fusolin forms
a dimer in which the disulphide bonds of the C-terminal extension mediate LPMO
domain swapping within dimers (Chiu et al. 2015; Fig. 7.4). Spindles are formed by
an intricate assembly of dimeric building blocks. The structure of a spindle unit cell
is as follows. Fusolin dimers assemble to form two crowns of four dimers that
encase a central dimer (Chiu et al. 2015; Fig. 7.6). Each crown is capped by a dimer
from neighbouring cells that projects its C-terminal extensions through the central
opening of the crown towards the central dimer. The central dimer is connected to
both capping dimers and two dimers of each crown by disulphide bonds between
the respective C-terminal extensions (Chiu et al. 2015; Fig. 7.6).
Spindles are inactive because the metal-binding site of fusolin is capped by an
amino acid and neighbouring molecules occlude the planar platform containing the
active site (Chiu et al. 2015). However, fusolin is released from the spindles by the
combined action of the alkaline pH of midgut juice and degradation by midgut ser-
ine proteases (Takemoto et al. 2008; Chiu et al. 2015), and thus the active site is
exposed. In addition to the intermolecular disulphide bonds stabilising spindles, the
C-terminal region contains an unusually high proportion of tyrosines, which are
located strategically at the dimer interface within helix H5 and around inter-dimer
crystal contacts next to the stabilising disulphide bonds (Chiu et al. 2015). Thus,
138 W. Mitsuhashi
Conclusion
Recent progress in research on fusolin has expanded its practical applicability.
Strengthening of Bt insecticidal activity (shortening survival times and increas-
ing the mortality rates of treated pests) remains desirable when such activity is
insufficient. Recent research strongly suggests that fusolin spindles may be use-
ful as a synergistic agent for Bt formulations and that expression of the fusolin
gene in crops may allow us to reduce the amounts of microbial insecticides
sprayed. Furthermore co-expressing fusolin with Bt toxin in crops may increase
their resistance to insects (Liu et al. 2011; Mitsuhashi et al. 2014a).
A characteristic feature of fusolin crystals is their stabilisation by a 3D net-
work of disulphide bonds that results in a fully cross-linked matrix (Chiu et al.
2015). This unique mechanism of fusolin stabilisation provides novel informa-
tion to develop more stable LPMOs, which are important for boosting biofuel
production from abundant recalcitrant biomass such as chitin and cellulose
(Vaaje-Kolstad et al. 2010; Gelfand et al. 2013).
7 Analysis of the Viral Lytic Polysaccharide Monooxygenase Fusolin 139
References
Aachmann FL, Sørlie M, Skjå-Bræk G, Eijsink VGH, Vaaje-Kolstad G (2012) NMR structure of a
lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics,
and substrate interactions. Proc Natl Acad Sci U S A 109:18779–18784
Adams JR, Wilcox TA (1968) Histopathology of the almond moth, Cadra cautella, infected with
a nuclear-polyhedrosis virus. J Invertebr Pathol 12:269–274
Afonso CL, Tulman ER, Lu Z, Oma E, Kutish GF, Rock DL (1999) The genome of Melanoplus
sanguinipes entomopoxvirus. J Virol 73:533–552
Bawden AL, Glassberg KJ, Diggans J, Shaw R, Farmerie W, Moyer RW (2000) Complete genomic
sequence of the Amsacta moorei entomopoxvirus: analysis and comparison with other poxvi-
ruses. Virology 274:120–139
Chakraborty M, Narayanan K, Sivaprakash MK (2004) In vivo enhancement of nucleopolyhedro-
virus of oriental armyworm, Mythimna separata using spindles from Helicoverpa armigera
entomopoxvirus. Indian J Exp Biol 42:121–123
Chakraborty M, Narayanan K, Suryanarayana VVS, Sivaprakash MK (2005) Enhancement of
nucleopolyhedrovirus of oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae)
using diamond shaped inclusion bodies of Galleria mellonella NPV (Lepidoptera: Pyralidae).
Entomon 30:343–346
Chiu E, Coulibaly F, Metcalf P (2012) Insect virus polyhedra, infectious protein crystals that con-
tain virus particles. Curr Opin Struct Biol 22:234–240
Chiu E, Hijnen M, Bunker R, Boudes M, Rajendran C, Aizel K, Olieric V, Schulze-Briese C,
Mitsuhashi W, Young V, Ward VK, Bergoin M, Metcalf P, Coulibaly F (2015) Structural basis
for the enhancement of virulence by viral spindles and their in vivo crystallization. Proc Natl
Acad Sci U S A 112:3973–3978
Derksen ACG, Granados RR (1988) Alteration of a lepidopteran peritrophic membrane by baculo-
viruses and enhancement of viral infectivity. Virology 167:242–250
Din N, Gilkes NR, Tekant B, Miller RC, Warren AJ, Kilburn DG (1991) Non-hydrolytic disruption
of cellulose fibres by the binding domain of a bacterial cellulose. Nat Biotechnol 9:1096–1099
Din N, Damude HG, Gilkes NR, Miller RC Jr, Warren RAJ, Kilburn DG (1994) C1-Cx revisited:
intramolecular synergism in a cellulase. Proc Natl Acad Sci U S A 91:11383–11387
Frederiksen RF, Paspaliari DK, Larsen T, Storgaard BG, Larsen MH, Ingmer H, Palcic MM, Leisner
JJ (2013) Bacterial chitinases and chitin-binding proteins as virulence factors. Microbiology
159:833–847
140 W. Mitsuhashi
Abstract
During the last two decades, insects have had the self-ability to develop a vigor-
ous and potent immune system that contests a huge diversity of pathogens and
lead them to grow into the most distinct and efficient organisms in the world.
Immune reactions against pathogens are basically characterized by invasion of
their cellular and humoral response. In the present era of challenging environ-
mental conditions, there is an urgent need for the prevention and control of viral
diseases. Serine proteases (SPs) are a vast group of proteolytic enzymes that play
an enormous role in anatomical systems (cell signaling, defenses, and move-
ment, etc.); thus, they are crucial to the antiviral mechanism (hemolymph coagu-
lation, activation of antimicrobial peptide, and melanin synthesis). They
participate in various biochemical and physiochemical pathways and act as cata-
lysts that break down the peptide bond in the protein. SPs are vital to numerous
microorganisms and contribute to several structural and biochemical concerns,
including a conserved catalytic triad (Ser, Asp, and His) that enacts the funda-
mental principle for the classification of a protein. SPs have diverse functions
and play a vital role in cellular differentiation, digestion, complement activation,
the immune response, and hemostasis. Recently, immunological responses in
many insects such as Bombyx mori, Drosophila, Anopheles, etc., are maintained
by circulatory hemocytes and performed a significant role in innate immune sys-
tem, namely, the synthesis of antimicrobial proteins, encapsulation, and pheno-
loxidase. Most of the antimicrobial proteins such as cecropins, attacins, lebocin,
moricin, gloverins, lysozyme, defensins, hemolin, etc., are effectively engaged in
defense reactions against invading pathogens. For antiviral mechanisms, molec-
ular and cell target-based analysis are valuable studies for identifying the genome
J. Verma
Department of Biotechnology, Babasaheb Bhimrao Ambedker University,
Lucknow, UP, India
e-mail: jyoti.verma873@gmail.com
and expression analysis of SPs, and their homologs in the silkworm, Drosophila
melanogaster, Apis mellifera, and Anopheles gambiae, are generally considered
to be model organisms for providing the relevant information regarding such
biological functions. In this chapter, we devote our endeavors to the antiviral
mechanism of SPs in various insects and critique the recent data on visualizing
the role of antiviral pathways. Furthermore, the antiviral pathways may encoun-
ter the infectious virus towards the systemic and specific level.
8.1 Introduction
Insect resistance plays a relevant role in the synergism within the host–pathogen
relationship, as a part of a survival strategy, along with physical impediments such as
epithelial barriers, cuticle and peritrophic matrix, protease cascades leading to coag-
ulation and melanization, and fundamental cellular responses (Choo et al. 2010;
Feng et al. 2013; Wang et al. 2016; Lavin and Strand 2002; Lehane et al. 2004;
Kotani et al. 1999). For many decades, the defense systems of insects, in contrast to
various pathogens such as bacteria, fungus, and protozoa, have been well recognized,
but articles on antiviral mechanism are scarce and surprising owing to a lack of
understanding of the mechanism of virus invasion and host–virus response (Popham
et al. 2004). In an earlier report, insect baculoviruses were accomplished in recombi-
nant protein expression systems, but there are virtually no studies including the anti-
viral mechanisms against viruses. Thus, there is specific consideration of Bombyx
mori, which is attracted to the area to regulate the gene and protein and to balance
expression in genetically modified cell lines (Popham et al. 2004). Serine proteases
(SPs) act as hydrolytic enzymes that are incorporated into the conserved catalogue of
triad residue (His, Ser, and Asp) and are routinely integrated as idle zymogen with
propeptide, which must be evacuated for their activation (Ross et al. 2003). Most
studies have delineated that insect SPs perform a fundamental aspect of dietary pro-
tein digestion (Herrero et al. 2005; Soares et al. 2011), molting (Wei et al. 2007; Liu
et al. 2009; He et al. 2009), metamorphosis (Danielli et al. 2000; Kaji et al. 2009),
and the immune response (An et al. 2009), whereas SP homologs(SPHs) are identi-
cal to SPs in amino acid sequences; nevertheless, there is a scarcity in amidase activ-
ity because of mutation (Romualdi et al. 2003). In various reports, Anopheles
gambiae, Drosophila melanogaster and Bombyx mori, and Bombus ignitus contain
305, 206, and 143 SP or SPH genes, 1,720 bp respectively (Choi et al. 2006; Romualdi
et al. 2003; Zdobnov et al. 2002; Zhao et al. 2010) and are categorized into different
families (Table 8.1). Insects have the ability to develop mechanisms that resist vari-
ous pathogens, including viruses (Qin et al. 2012). Choo et al. (2010) investigated the
bee venom SPs (Bi-VSPs), which promote the arthropods prophenoloxidase (proPO)-
activating factors (PPAFs) via a melanization process and illustrated fibrin(ogen)
8 Antiviral Mechanism of Serine Protease in Various Insects 145
Table 8.1 List of gene number of serine proteases and serine protease homolog gene in different
insects (adopted from Zhao et al. 2010)
Family D. melanogaster A. gambiae A. mellifera B. mori
SP_fam1 65 49 28 24
SP_fam2 12 37 4 9
SP_fam3 18 22 2 6
SP_fam4 17 19 4 5
SP_fam5 2 29 – 4
SP_fam6 9 9 1 2
SP_fam7 3 4 2 1
SP_fam8 – 1 0 5
SP_fam9 1 1 1 2
SP_fam10 1 1 1 1
SP_fam11 1 1 1 1
SP_fam12 – 49 28 15
SP_fam13 – 37 4 7
SP_fam14 – 22 2 6
SP_fam15 – 19 4 5
SP_fam16 – 29 – 3
SP_fam17 – 9 1 3
Occlusion
body
Occlusion Dispersion
derived virus
Occlusion Occlusion
Budded virus Bodies
derived
virus
Ingestion
Budded virus
Occlusion
derived virus
Nucleocapsid
Budding process
Envelop
glycoprotein
Capsid
Polyhedrin network
Capsid base
Fig. 8.2 Detailed structure of budded virus (BV) and occlusion-derived virus (ODV)
8 Antiviral Mechanism of Serine Protease in Various Insects 147
Proteases are the largest group of enzymes, ubiquitous in nature, that hydrolyze
proteins by adjoining water across peptide bonds (Saleem et al. 2012) and catalyze
peptide construction in an organic solvent with a low water content (Soares et al.
2011). According to Verma et al. (2011) proteases are subdivided into diverse groups
based on their catalytic activity with reference to reaction medium and can be iden-
tified as acidic, alkaline, neutral, and in active site groups. Proteases are also classi-
fied according to three major criteria: (1) the type of reaction catalyzed, (2) the
chemical nature of the catalytic site, and (3) the evolutionary relationship with
regard to structure (Barett 1994). Proteases are also categorized into exo- and endo-
peptidases depending on their action at or away from termini respectively and sub-
divided based on the nature of their functional active site groups, i.e., SPs, aspartic
proteases, cysteine proteases, and metalloproteases (Hartley 1960). Of these func-
tions, SPs act as mediators among the immune systems of different insects and
determine the defense mechanisms of various pathogens via antimicrobial peptide
synthesis, hemolymph coagulation, and melanization of pathogen surfaces (Gorman
and Paskewitz 2001). Previously, a protein was demonstrated to have a significant
antiviral activity against BmNPV from the digestive juice of Bombyx mori and was
termed B. mori SP (BMSP-2) (Nakazawa et al. 2004). According to Kotani et al.
(1999), BmSp-2 exhibited 94% amino acid sequence identity with SP. Recently, Lin
et al. (2017) reported that SP inhibitors (SPIs) were present in all living animals and
performed a vital role in development, digestion, and innate immunity. They studied
the genome-wide characterization and expression profiling of the SPI gene in
Plutella xylostella and noted that the SPI gene was categorized into serpins, canoni-
cal inhibitors, and alpha-2-macroglobulins. Of these, serpins demonstrated an asso-
ciation with the regulation of innate immunity of insects, whereas according Zhao
et al. (2010), the upregulation and downregulation of different SP inhibitor genes
may be participating in the combat with pathogenic microorganisms such as
Escherichia coli, Bacillus bombysepticus, Beauveria bassiana, or BmNPV.
In earlier studies, a diverse group of proteins and SPs played a very significant role
in insect immunity for the mechanism of antiviral infection and were referred to as
“serpocidins” (Kim et al. 2009). Of the various proteases, SPs have a zymogen-like
structure that comprises the catalytic domain of the C-terminal and the regulatory
domain of the N-terminal, are generally activated by His/Asp/Ser from the catalytic
site, and participate in the regulatory mechanism of melanization, coagulation, and
antimicrobial peptide production, through protease cascades (Kim et al. 2009;
Gabay 1994; Gorman and Paskewitz 2001). In addition, SP homologs possess the
catalytic triad and play a vital role in the antibacterial mechanism of human azuro-
cidin (Gabay and Almeid 1993) and horseshoe crab factor D (Iwanaga 2002); cell
148 J. Verma
The process of catalysis and specificity are not quietly inhibited by a scant residue,
nevertheless effects on the whole protein frameworks disciplined via the rationing of
the hydrogen bond charges, as it may be linking of domain drift to the chemical con-
version (Hedstrom 2002). The specificity of SP is basically deliberated the physiog-
raphy of the substrate binding sites which can adjoin the catalytic site of cleft (Polgar
2005). However, in an earlier report, SP specificity has produced a lot of information
regarding biological function and the establishment of efforts (Perona and Craik
1995). Of the industrial enzymes, about 75% of microbial proteas belong to SPs and
serve the nucleophilic Ser residue at their functioning site (Rao et al. 1998). SPs are
classified as the catalytic triad with the presence of Asp, His, and Ser, and these are
designated as catalytic machinery, which is subdivided into four separate groups. In
an earlier report, these four groups of SPs are described as chymotrypsin, subtilisin,
carboxypeptidase Y, and Clp protease (Rawlings and Barrett 2000).
Feng et al. (2013) reported that insects get participated in different ways to defend
themselves against different pathogen such as fungi, bacteria, nematodes but scanty
knowledge about the insect immune response against viruses. Among them, one of the
most important viruses is Autographa californica multiple nuclear polyhedrosis virus
(AcMNPV), which spreads almost 30 lepidopteron species, whereas baculoviruses
have a very narrow host range that contains a circular double-stranded genome ranging
from 80 to 180 kbp and moderated in the form of larvae of Bombyx mori (An et al.
2009; Rahman and Gopinathan 2004). The life cycle of Bombyx mori contains two
150 J. Verma
specific forms of virus, occlusion-derived virus (ODV) and the budded virus (BV).
Both forms play a different role in the interim pathogenesis in Fig. 8.1 (Katsuma et al.
2006). Antiviral immunity in other insects is well known, but there is not much more
information on the silkworm, B. mori. In addition, other factors such as hemolin, recep-
tors in midgut epithelial cells, phenoloxidase, and apoptosis also played a significant
role in the antiviral mechanism (Ponnuvel et al. 2012). Recently, a protein RNAse III
(dicer) was identified that showed the antiviral mechanism against infectious flu virus
(IFV) (Ponnuvel et al. 2008; Ponnuvel et al. 2012). Nakazawa et al. (2004) reported
that Bombyx mori possesses the two forms of virus particles, BmNPV and AcMNPV,
which showed that they are phenotypically different, but genetically identical, and
complied with their life cycle at the time of pathogenesis. The two forms play different
roles. During this process, infection begins and the ODV fuses with the microvillar
membrane (Monsma et al. 1996). This virus particle is enclosed with proteinaceous
occlusions, which are discharged into the midgut of the larvae with the combination of
alkaline gut pH and protease (Engelhard and Volkman 1995). The cell is infected and
produces many primary single nucleocapsids through the basal membrane. This BV
gained an envelope studded with glycoprotein GP-64, which infected the neighboring
host and tissues (Monsma et al. 1996). The detailed structure of budded virus (BV) and
occlusion-derived virus (ODV) is summarized in Fig. 8.2.
Various researchers reported that SP played a significant role in antiviral activity
against BmNPV, which is designated as BmSP-2 and analyses their gene expression
in the midgut of Bombyx mori (Nakazawa et al. 2004; Ponnuvel et al. 2008), whereas
Zhao et al. (2010) presumed that potential SPI genes based on the genome sequences
of the silkworm are susceptible to the antiviral mechanism. He reported that these
SPI genes may be responsible for defenses to pathogenic microorganisms through
microarray and qRT-PCR assay. This report highlights the upregulation and down-
regulation of several SPI genes subsequently infected by Escherichia coli, Bacillus
bombysepticus, Beauveria bassiana, or BmNPV. Recently, Liu et al. (2016) investi-
gated the roles of SP for antiviral mechanisms and identified SP gene BmSP36,
which has a 292-residue protein and is cloned. Liu et al. (2016) also defined that the
BmSP36 consists of an intact catalytic triad (H57, D102, and S195) and a conserved
substrate binding site (G189, H216, and G226), which is responsible for
chymotrypsin-like specificity. According to their reports, BmSP36 plays a signifi-
cant role in the midgut of B. mori and they analyzed the transcriptional and transla-
tional expression using western blotting, immunofluorescence, and liquid
chromatography-tandem mass spectrometry assay.
Recently, the mode of infection and mechanism of antiviral immunity were well
documented through the Toll and IMD pathways (De Gregorio et al. 2002).
According to An et al. (2013) melanization regulated the innate immunity via bind-
ing, eradicated the interfering organisms, and intervened using a SP cascade that is
regulated by serpins in a similar manner. Zhao et al. (2012) demonstrated that ser-
pins may be associated with the management of the innate immunity of different
152 J. Verma
For the study of genome-wide identification and expression analysis, there is a cur-
rent need to understand the genetics and regulatory mechanisms of immune
responses of insects that have developed the effective biological control system
(You et al. 2013). Recently, Xia et al. (2015) investigated the genome-wide identi-
fication, characterization, and expression analysis in Plutella xylostella by using
Toll, IMD, and JAK-STAT signaling pathways. Previously, genome-wide identifi-
cation and expression were reported by Zhao et al. (2010) in Bombyx mori who
compared the expression analysis of SPs and their homologs (Table 8.1). SP homo-
logs (SPHs) are similar to SPs in amino acid sequences, but they have no protease
activity because of the loss of one or more of the catalytic residues (Zou et al.
2006). In insects, SPHs participate in the innate immune response to regulate the
mechanism insect immunity (Dimopoulos et al. 1997; Kim et al. 2008). Genome-
wide characterization was also performed in SP and SP homologs of Drosophila
melanogaster (Ross et al. 2003). Besides these insects, Anopheles gambiae and
8 Antiviral Mechanism of Serine Protease in Various Insects 153
Table 8.2 List of serine proteases and serine protease inhibitors which upregulated and down-
regulated the transcripts region and their gene expression analysis (adopted from Riddell et al.
2014)
Upregulated Downregulated
Serine proteases
cSp3 BTT35293_1 BTT10579_1,
BTT10912_1, BTT25711_1
Sp18 BTT20808_1 BTT20808_1
Sp27 BTT40251_1 BTT40251_1
Sp28 BTT20637_1
Sp35 BTT05300_1 BTT10155_1
Sp40 BTT15256_1
Sp23 BTT01709_1, BTT05886_1, BTT09081_1,
BTT20661_1, BTT20725_1, BTT24359_1,
BTT25071_1
Serine protease homologs
cSPH39 BTT21868_1
Sph54 BTT27769_1
Sph56 BTT17814_1
Serine protease inhibitors
Kunitz ser- BTT14993_1
protease
inhibitor
Necrotic (nec) BTT35742_1
Spn 4 BTT04130_1 BTT04130_1
SRPN10 BTT02607_1, BTT4508_1,
BTT20259_1
Apis mellifera also had immunity-related SPs and SPH characteristics (Christophides
et al. 2002; Zou et al. 2006). There is little information about these proteins in
Bombyx mori against SPs and SPHs, but it emphasizes the biological functions
such as digestion, immune response, development (Nakazawa et al. 2004).
Furthermore, Tanaka et al. (2008) identified the potential immunity of SPs and
SPH-related geneswhile modifications to the mRNA level that were considered to
be involved in the antiviral mechanism. According to (Zhao et al. 2010; Xia et al.
2007; and Riddell et al. 2014) the SPs and SPH genes were quietly used in upregu-
lation and downregulation after pathogen induction by using microarray and real-
time quantitative experiments (Table 8.2).
In recent years, Chang et al. (2011) studied the comparative analysis of gene expres-
sion techniques and determined that cDNA microarray and two-dimensional gel
electrophoresis have become part of routine in checking the changes in gene
154 J. Verma
8.8.3 P
olyclonal Antibody-Based Preparation and Western Blot
Analysis
In the present scenario, multiple gene expression techniques used on the basis of
three potential criteria:
In recent years, there has been a need to analyze gene efficiency by comparing the
transcriptome of two strains, which is used to improve the efficiency and gene dis-
covery of host–pathogen responses. A new technique was developed by scientists
and used for comparative analysis of two strains with a transcriptome (Wang et al.
2016). According to their hypothesis, they reported that molecular changes in B.
mori occurred during BmNPV infection and were examined by transcriptome
sequencing in the isogenic line BC9 and the recurrent parent P50. These genes were
related to transport, virus replication, intracellular innate immune, and apoptosis
(Wang et al. 2016). After normalizing gene expression levels, DEGs were obtained
by pair-wise comparison of the four transcriptome libraries using IDEG6 software
(Romualdi et al. 2003). Genes participating in innate immunity pathways were
identified and analyzed with regard to their potential role in BmNPV infection in
silkworm, which could be classified into the Toll pathway, the IMD pathway, the
PPO pathway, the pattern recognition receptor, and the antimicrobial peptide. Wang
et al. (2016) further reported that the SP inhibitor performed a significant role in the
PPO pathway and it appears that there was downregulation in the gene of 47% and
57% respectively, whereas 30% and 20% were upregulated after infection with
156 J. Verma
BmNPV, and 57% were downregulated and 20% were upregulated in P50 after
BmNPV infection. By this comparative analysis, a total of 869 DEGs were obtained,
which included many genes potentially related to BmNPV resistance. After that,
Wang et al. (2016) predicted that reliable evidence may be produced to clarify the
molecular mechanism of the silkworm. Hu et al. (2014) also reported analysis of
several differentially expressed genes (DEGS), which is involved in metabolism,
immunity, and inflammatory responses in Microtus fortis following infection with
Schistosoma japonicum based on comparative transcriptome analysis.
Conclusion
In the current scenario, wonderful progress has been made in the antiviral mecha-
nism of SP against viral infection. The synergism of host and pathogen played a
pivotal role in viral infections, and various mechanisms such as genome-wide iden-
tification and expression, microarray-based expression, recombinant-based expres-
sion, polyclonal antibody-based expression, multiple gene-based expression, etc.,
were performed using the SP pathway. The discovery of Molecular and cellular
analysis - Genome-Wide Identification and Expression, cDNA Microarray-based
Assay, Polyclonal Antibody-Based Preparation and Western Blot, Multigene
Expression, Differentially expressed genes analysis of anti-protein detection plays
a vital role in fighting viral infection, but recently, antimicrobial target-based path-
ways, immensely used, such as siRNA, Toll, JAK-STAT, IMD, etc., is used to
determine the host–pathogen-specific immune response to the fascinating antiviral
mechanism. In this study, a new technology was developed for understanding the
fundamental and applied aspects of insect immunity against viral infection in the
functional genomic era, and potentially improving the antiviral mechanism.
References
Alto BW, Bettinardi D (2013) Temperature and dengue virus infection in mosquitoes: independent
effects on the immature and adult stages. Am J Trop Med Hyg 88:497–505
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped
BLAST and PSIBLAST: a new generation of protein database search programs. Nucl Acids
Res 25:3389–3402
An C, Ishibashi J, Ragan EJ, Jiang H, Kanost MR (2009) Functions of Manduca sexta hemolymph
proteinases HP6 and HP8 in two innate immune pathways. J Biol Chem 284:19716–19726
An C, Zhang M, Chu Y, Zhangwu Zhao Z (2013) Serine protease MP2 activates prophenoloxidase
in the melanization immune response of drosophila. PLoS One 8(11):1–10
Ashida M, Brey PT (1997) Recent advances in research on the insect prophenoloxidase cas-
cade. In: Brey PT, Hultmark D (eds) Molecular mechanisms of immune responses in insects.
Chapman and Hall, London, pp 135–172
Ashida M, Brey PT (1998) In: Brey PT, Hultmark D (eds) Molecular mechanisms of immune
responses in insects. Chapman & Hall, London, pp 135–172
Bao YY, Tang XD, Lv ZY, Wang XY, Tian CH, YP X, Zhang CX (2009) Gene expression profil-
ing of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated
variations in host gene transcript levels. Genomics 94(2):138–145
8 Antiviral Mechanism of Serine Protease in Various Insects 157
Barett AJ (1994) Proteolytic enzymes: serine and cysteine peptidases. Methods Enzymol 244:1–15
Beier JC (1998) Malaria parasite development in mosquitoes. Annu Rev Entomol 43:519–543
Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL et al (2013) The global distri-
bution and burden of dengue. Nature 496:504–507
Cerenius L, Lee BL, Soderhall K (2008) The proPO-system: pros and cons for its role in inverte-
brate immunity. Trends Immunol 29:263
Chang C, Liu X, Chen K (2011) Molecular cloning, expression and characterization of a novel
geneβ-N-acetylglucosaminidase from Bombyx mori. Adv Biosci Biotechnol 2(2):123–127
Chen J, X-F W, Zhang YZ (2006) Expression, purification and characterization of human GM-CSF
using silkworm pupae (Bombyx morie) as a bioreactor. J Biotechnol 123:236–247
Cheng Y, Wang X-Y, Hao H, Killiny N, Jia-Ping X (2014) A hypothetical model of crossing
Bombyx mori nucleopolyhedrovirus through Its host midgut physical barrier. PLoS One
9(12):e115032
Choi YS, Lee KS, Yoon HJ, Kim I, Sohn HD, Jin BR (2006) A bumblebee thioredoxin-like protein
gene that is upregulated by a temperature stimulus and lipopolysaccharide injection. Eur J
Entomol 103:291–296
Choo YM, Lee KS, Yoon HJ, Lee SB, Kim JH, Sohn HD, Jin BR (2007) A serine protease from
the midgut of the bumblebee, Bombus ignitus (hymenoptera: apidae): cDNA cloning, gene
structure, expression and enzyme activity. Eur J Entomol 104:1–7
Choo YM, Lee KS, Yoon HJ, Kim BY, Sohn MR, Roh JY, Je YH, Kim NJ, Kim I, Woo SD, Sohn
HD, Jin BR (2010) Dual function of a bee venom serine protease: prophenoloxidase-activating
factor in arthropods and fibrin(ogen)olytic enzyme in mammals. PLoS One 5(5):1–10
Christophides GK, Zdobnov E, Barillas-Mury C, Birney E, Blandin S, Blass C, Brey PT, Collins
FH, Danielli A, Dimopoulos G (2002) Immunity-related genes and gene families in Anopheles
gambiae. Science 298(5591):159–165
Danielli A, Loukeris TG, Lagueux M, Muller HM, Richman A, Kafatos FC (2000) A modular
chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles
gambiae. Proc Natl Acad Sci U S A 97:7136–7141
De Gregorio E, Han SJ, Lee WJ, Baek MJ, Osaki T et al (2002) An immune-responsive Serpin
regulates the melanization cascade in Drosophila. Dev Cell 3:581
Dimopoulos G, Richman A, Mullar HM, Kafatos FC (1997) Molecular immune responses of the
mosquito Anopheles gambiae to bacteria and malaria parasites. Proc Natl Acad Sci U S A
94:11508–11513
Engelhard EK, Volkman LE (1995) Developmental resistance in fourth instar Trichoplusia ni orally
inoculated with Autographa californica M nuclear polyhedrosis virus. Virology 209:384–389
Feng Fan, Hu Ping and Chen Keping (2013) Progress of antiviral mechanisms in the mulberry
silkworm: A review 7(14):1173–1178
Felfoldi G, Eleftherianos I, Ffrench-Constant RH, Venekei I (2011) A serine proteinase homolog,
SPH-3, plays a central role in insect immunity. J Immunol 186:4828–4834
Fragkoudis R, Chi Y, Siu RW, Barry G, Attarzadeh-Yazdi G, Merits A, Nash AA, Fazakerley JK,
Kohl A (2008) Semliki forest virus strongly reduces mosquito host defense signaling. Insect
Mol Biol 17:647–656
Fragkoudis R, Attarzadeh-Yazdi G, Nash AA, Fazakerley JK, Alain Kohl A (2009) Advances in
dissecting mosquito innate immune responses to arbovirus infection. J Gen Virol 90:2061–2072
Gabay JE (1994) Antimicrobial proteins with homology to serine protease. Ciba Found Symp
186:237–247
Gabay JE, Almeid RP (1993) Antibiotic peptides and serine protease homologs in human polymor-
phonuclear leukocytes: defensins and azurocidin. Curr Opin Immunol 5:97–102
Gettins PG (2002) Serpin structure, mechanism, and function. Chem Rev 102:4751
Gorman MJ, Paskewitz SM (2001) Serine proteases as mediators of mosquito immune responses.
Insect Mol Biol 31:257–262
Gorman MJ, Andreeva OV, Paskewitz SM (2000a) Sp22D: a multidomain serine protease with a
putative role in insect immunity. Gene 251:9–17
158 J. Verma
Gorman MJ, Olga V, Andreeva OV, Susan M, Paskewitz SM (2000b) Molecular characterization
of five serine protease genes cloned from Anopheles gambiae hemolymph. Insect Biochem
Mol Biol 30:35–46
Hartley BS (1960) Proteolytic enzymes. Annu Rev Biochem 29:45–72
He WY, Zheng YP, Tang L, Zheng SC, Beliveau C, Doucet D, Cusson M, Feng QL (2009)
Cloning, expression and localization of a trypsin-like serine protease in the spruce budworm,
Choristoneura fumiferana. Insect Sci 16:455–464
Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:4501–4524
Herrero S, Combes E, Oers V, Vlak MM, DeMaagd RA, Beekwilder J (2005) Identification and
recombinant expression of a novel chymotrypsin from Spodoptera exigua. Insect Biochem Mol
Biol 35:1073–1082
Holt RA, Subramanian GM, Halpern A, Sutton GG, Charlab R, Nusskern DR, Wincker P, Clark
AG, Ribeiro JM, authors o (2002) The genome sequence of the malaria mosquito Anopheles
gambiae. Science 298:129–149
Hu Y, YX X, WY L, Yuan ZY, Quan H, Shen YJ et al (2014) De novo assembly and transcrip-
tome characterization: novel insights into the natural resistance mechanisms of Microtus fortis
against Schistosoma japonicum. BMC Genomics 15(1):1–13
Huang HS, Wang H, Lee SY, Johansson WM, Soderhall K, Cerenius L (2000) A cell adhesion
protein from the crayfish Pacifastacus leniusculus, a serine protease homologue similar to dro-
sophila masquerade. J Biol Chem 275:9996–10001
Iwanaga S (2002) The molecular basis of innate immunity in the horseshoe crab. Curr Opin
Immunol 14:87–95
Jiang R, Kim EH, Gong JH, Kwon HM, Kim CH et al (2009) Three pairs of protease-serpin
complexes cooperatively regulate the insect innate immune responses. J Biol Chem 284:35652
Jupatanakul N, Shuzhen Sim S, Angleroa-Rodroaguez YI, Souza-Neto J, Das S, Poti KE, Rossi
SL, Bergren N, Vasilakis N, Dimopoulos G (2017) Engineered Aedes aegypti JAK/STAT
pathway-mediated immunity to dengue virus. PLoS Negl Trop Dis 1:24
Kaji K, Tomino S, Asano T (2009) A serine protease in the midgut of the silkworm, Bombyx mori:
protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form
and activation during midgut remodeling. Insect Biochem Mol Biol 39:207–217
Kanost MR, Gorman MJ (2008) Phenoloxidases in insect immunity. In: Beckage NE (ed) Insect
immunology. Elsevier/Academic, Manhattan, pp 69–96
Katsuma S, Daimon T, Horie S, Kobayashi M, Shimada T (2006) N-linked glycans of Bombyx
mori nucleopolyhedrovirus fibroblast growth factor are crucial for its secretion. Biochem
Biophys Res Commun 350(4):1069–1075
Kim CH, Kim SJ, Kan H, Kwon HM, Roh KB et al (2008) A three-step proteolytic cascade
mediates the activation of the peptidoglycan-induced toll pathway in an insect. J Biol Chem
283:7599–7607
Kim SY, Jeong EJ, Song J, Park KS (2009) Molecular cloning and characterization of a serine
protease-like protein from silkworm (Bombyx Morie). Gene Genom 31(5):387–395
Kotani E, Niwa T, Tokizane M, Suga K, Sugimura Y, Oda K, Mori M, Furusawa T, (1999) Cloning
and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm,
Bombyx mori. Insect Mol. Biol. 8:299–304
Kwon TH, Kim MS, Choi HW, Joo CH, Cho MY, Lee BL (2000) A masquerade-like serine pro-
tease homologue necessary for phenoloxidase activity in the coleopteran insect Holotrichia
diomphalia larvae. Eur J Biochem 267:6188–6196
Lavine MD, Strand MR (2002). Insect hemocytes and their role in immunity. Insect Biochem. Mol.
Biol. 32(10):1295–1309
Lehane MJ, Aksoy S, Levashina E (2004) Immune responses and parasite transmission in blood-
feeding insects. Trends Parasitol 20(9):433–439
Lemaitre B, Hoffmann J (2007) The host defense of Drosophila melanogaster. Annu Rev Immunol
25:697–743
Lin H, Lin X, Zhu J, XO Y, Xia X, Yao F, Guang Yang G, You M (2017) Characterization and
expression profiling of serine protease inhibitors in the diamondback moth, Plutella xylostella
(Lepidoptera: Plutellidae). BMC Genomics 18:162
8 Antiviral Mechanism of Serine Protease in Various Insects 159
Zdobnov EM, Von Mering C, Letunic I, Torrents D, Suyama M, Copley RR, Christophides GK,
Thomasova D, Holt RA, Subramanian GM, Mueller HM, Dimopoulos G, Law JH, Wells MA,
Birney E, Charlab R, Halpern AL, Kokoza E, Kraft CL, Lai Z, Lewis S, Louis C, Barillas-Mury
C, Nusskern D, Rubin GM, Salzberg SL, Sutton GG, Topalis P, Wides R, Wincker P, Yandell
M, Collins FH, Ribeiro J, Gelbart WM, Kafatos FC, Bork P (2002) Comparative genome and
proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298:149–159
Zhao P, Wang GH, Dong ZM, Duan J, PZ X, Cheng TC, Xiang ZH, Xia QY (2010) Genome-
wide identification and expression analysis of serine proteases and homologs in the silkworm
Bombyx mori. BMC Genomics 11:405
Zhao P, Dong ZM, Duan J, Wang GH, Wang LY, Li YS, Xiang ZH, Xia QY (2012) Genome-wide
identification and immune response analysis of serine protease inhibitor genes in the silkworm,
Bombyx mori. PLoS One 7(2):e31168
Zou Z, Lopez DL, Kanost MR, Evans JD, Jiang H (2006) Comparative analysis of serine protease-
related genes in the honey bee genome: possible involvement in embryonic development and
innate immunity. Insect Mol Biol 15(5):603–614
Preventive, Diagnostic and Therapeutic
Applications of Baculovirus Expression 9
Vector System
Abstract
Different strategies are being worked out for engineering the original baculovirus
expression vector (BEV) system to produce cost-effective clinical biologics at
commercial scale. To date, thousands of highly variable molecules in the form of
heterologous proteins, virus-like particles, surface display proteins/antigen carri-
ers, heterologous viral vectors and gene delivery vehicles have been produced
using this system. These products are being used in vaccine production, tissue
engineering, stem cell transduction, viral vector production, gene therapy, cancer
treatment and development of biosensors. Recombinant proteins that are
expressed and post-translationally modified using this system are also suitable
for functional, crystallographic studies, microarray and drug discovery-based
applications. Till now, four BEV-based commercial products (Cervarix®,
Provenge®, Glybera® and Flublok®) have been approved for humans, and myriad
of others are in different stages of preclinical or clinical trials. Five products
(Porcilis® Pesti, BAYOVAC CSF E2®, Circumvent® PCV, Ingelvac CircoFLEX®
and Porcilis® PCV) got approval for veterinary use, and many more are in the
pipeline. In the present chapter, we have emphasized on both approved and other
baculovirus-based products produced in insect cells or larvae that are important
from clinical perspective and are being developed as preventive, diagnostic or
therapeutic agents. Further, the potential of recombinant adeno-associated virus
(rAAV) as gene delivery vector has been described. This system, due to its rela-
tively extended gene expression, lack of pathogenicity and the ability to t ransduce
a wide variety of cells, gained extensive popularity just after the approval of first
AAV-based gene therapy drug alipogene tiparvovec (Glybera®). Numerous prod-
ucts based on AAV which are presently in different clinical trials have also been
highlighted.
9.1 Introduction
Baculovirus (family: Baculoviridae) derived its name from the Latin word “bacu-
lum” meaning “stick”. They are rod-shaped (30–60 × 250–300 nm) large enveloped
viruses with circular, supercoiled double-stranded DNA genomes, approximately
80–180 kb in size. While most of the baculoviruses infect their natural host, i.e.,
butterflies and moths (Lepidoptera), few are also known to infect sawflies
(Hymenoptera) and mosquitoes (Diptera) (King et al. 2011). They have not been
linked with any disease in any organism outside the phylum Arthropoda (Kost and
Condreay 2002). Baculoviruses are well known for their role as biopesticides and
are efficient tools for heterogeneous protein production in insect cells (Summers
2006). Morphologically, these enveloped viruses have been classified into two phe-
notypes: occlusion-derived viruses (ODVs) that are embedded in paracrystalline
matrix forming polyhedral occlusion bodies (OBs) which are responsible for hori-
zontal transmission between insects and the budded viruses (BuVs) present in the
haemolymph which spreads infection from cell to cell (Luckow and Summers
1988). Occlusion body morphology was initially used to define two major groups of
baculoviruses: nucleopolyhedroviruses (NPVs) and the granuloviruses (GVs).
NPVs obtain their envelop from host nuclear membrane and are occluded within
main occlusion protein polyhedrin forming large (1–15 μm) polyhedral inclusion
bodies, while GVs obtain their envelop from cell membrane to make oval-shaped
single virion structure called granule or capsule with diameter in the range of 0.2–
0.4 μm (King et al. 2011). NPVs are further distinguished as single nucleopolyhe-
drovirus or multiple nucleopolyhedrovirus based on the number of nucleocapsids in
a polyhedral inclusion body (O’Reilly et al. 1994). OBs allow virions to remain
infectious for long period due to their highly resistant and stable structure.
Baculovirus-infected insect cell expression system has been used for the routine
production of recombinant proteins, including several proteins of therapeutic nature
over the last three decades. The establishment of this system begins from the produc-
tion of human beta interferon (INF-β), the protein normally not produced in the cul-
tured human cells. It was produced with a recombinant Autographa californica multiple
nucleopolyhedrovirus (AcMNV) by exploiting its polyhedrin promoter (Smith et al.
1983). In this system, the protein coding sequence of human interferon gene was linked
to the AcNPV polyhedrin gene promoter. The interferon gene was inserted at different
positions relative to the AcNPV polyhedrin transcriptional and translational signals.
The interferon-polyhedrin hybrid plasmid was then transferred to infectious AcNPV
expression vectors by recombination within S. frugiperda insect cells, where more than
95% of biologically active glycosylated interferon was produced in the secreted form.
9 Preventive, Diagnostic and Therapeutic Applications 165
recombinant proteins, the Spodoptera frugiperda Sf21 and its subclone Sf9 and
High Five cell lines are being used. These insect cells exhibit several properties like
rapid growth, stress resistance and robust expression of recombinant proteins that
make them suitable for the production of clinical biologics and commercial
products.
Initially, insect-derived baculovirus expression vector (BEV) was recognized as
a safe system for routine production of recombinant proteins both in insect and
mammalian cells. During the last three decades, it has emerged as an effective tool
for research as well as various applications in the field of biotechnology. It has
shown tremendous potential as preventive, diagnostic and therapeutic agent against
a myriad of diseases in the form of vaccination, tissue engineering, stem cell trans-
duction, viral vector production and gene therapy (Airenne et al. 2013). It has been
extensively used for functional studies, crystallography, biosensors, protein micro-
array and drug discovery. All these applications are based on different baculovirus-
derived products such as heterologous proteins, protein/antigen displayed on
baculovirus particle surface, heterologous viral vectors and gene delivery vehicles
for mammalian cells (van Oers et al. 2015). In this chapter, we have presented the
application of these products from a clinical point of view in three main categories,
viz. preventive, diagnostic and therapeutic agents. Most of the approved biomole-
cules produced by using baculovirus expression system in insect cells have been
discussed. As thousands of other products are being developed by BEVs, it seems
ineffectual to include the entire list under the ambit of the present chapter; however
few among them have been mentioned to have an understanding about the scope of
this powerful expression system in the near future.
BEV exhibits many characteristics that make it suitable for the production of heter-
ologous proteins in insect cells. It can be easily handled in the BSL1/2 laboratories
due to its harmless nature to nontarget organisms. These viruses are environmen-
tally safe due to their instability outside the laboratory. It is used to produce high
level of proteins in insect cells or larvae where the eukaryotic environment provides
the appropriate post-translational modifications. BEVs host insect cells are mostly
free of human pathogens and do not require controlled oxygenic environment for
their growth. Insect cells can be grown into serum-free medium, and the heterolo-
gous protein production can be enhanced to the level of pilot plant or larger bioreac-
tors. Therefore, the proteins obtained by the BEVs can be used as vaccines either in
the form of heterologous subunit proteins or virus-like particles (VLPs) formed by
subunit proteins of virus.
Subunit vaccines are relatively safe as they are devoid of virus genetic material
but exhibit poor immunogenicity that might be due to incorrect folding of the target
protein. Structural proteins of viruses such as capsid and envelop proteins assemble
into particulate structure similar to the naturally occurring virus or subviral parti-
cles. Therefore, virus-like particles (VLPs) that are non-infectious and
9 Preventive, Diagnostic and Therapeutic Applications 167
non-replicating due to the absence of viral genetic material can be produced in het-
erologous system (Yamaji 2014). VLPs are highly effective in eliciting both humoral
and cellular immune response because of their densely repeated display of viral
antigens in right conformation (Roy and Noad 2008). VLPs comparatively exhibit
wide spectrum of clinical applications such as prevention of disease as vaccines,
diagnostics as antigens for the detection of antibodies and therapeutics in the form
of therapeutic vaccines and delivery agents. The use of heterologous proteins and
VLPs as preventive agents in the form of vaccines against different diseases is being
described (Table 9.1).
A decade back, only two veterinary products were manufactured using BEVs to
prevent classical swine fever in pigs. Now, five more new vaccines have been
approved, two of which are for humans, and many more products are in the develop-
ment phase. Here, approved vaccines as well as development of other vaccines in
preclinical stages have been highlighted.
(c) VLP-based vaccine for porcine parvovirus (PPV): PPV, a non-enveloped DNA
virus, causes major reproductive failure in swine. Its viral capsid is made up of
50–60 molecules of VP2, the major structural protein that are being targeted for
vaccine development. VP2 gene was expressed under the control of late p10
promoter of baculovirus and the LacZ gene under the control of Drosophila hsp
70 promoter. The recombinant baculovirus AcAs3-PPV was used to infect Sp21
insect cell line to express VP2 that leads to self-assembled empty PPV VLPs in
serum-free medium for safety point of view (Maranga et al. 2002). Earlier, it
was also produced in Sf9 cells in the presence of serum proteins. However, its
commercialization at large scale still needs more developmental efforts.
(d) VLP-based vaccine for sheep bluetongue virus (BTV): Bluetongue primarily
causes disease in ruminants due to infection by BTV double-stranded RNA
virus. Sheep generally shows more severe clinical signs than other cattle.
Recombinant baculovirus expression system in Sf9 insect cell lines shows great
potential to develop VLP-based vaccine against BTV (de Diego et al. 2011).
Monovalent and bivalent VLP vaccines are being developed for two serotypes 1
and 4 of BTV. BTV-1 exhibits more protection to virulent BTV live strain as
compared to BTV-4. Earlier, VLP expressing capsid proteins VP2 and VP5
were developed by co-transfection of dual transfer vector DNA (pAcVC3/BTV-
10-2/BTV-10-5) with wild-type AcNPV DNA in insect cells (French et al.
1990). Strong developmental efforts and further research are needed to com-
mercialize robust and effective BTV vaccine.
Table 9.2 (continued)
Targeted/used
for Expressed product Used to detect Test type
Humans Glutamic acid Insulin-dependent Immunoassay
decarboxylase diabetes mellitus
(GAD65 and
GAD67)
Humans Nucleocapsid Hantavirus Indirect immunofluorescence
protein of strain antibody (IFA)
SR-11
Humans E2 protein Human papillomavirus ELISA
(HPV)
Humans Hou/90 capsid Human calicivirus Immunoprecipitation and
(HuCV) EIA
Humans Fragment of gG Herpes simplex virus Indirect ELISA
comprising (HSV)
residues 321–580
of HSV-2
Humans Capsid proteins Human caliciviruses EIA
(HuCVs)
Humans C-terminus TBE complex virus ELISA and immunoblot
truncated form of assay
protein (Etr)
Humans Recombinant Fel Cat allergen Radioimmunoassay (RIA)
dl (rFel dl and ELISA
Ch1 + Ch2)
Humans Recombinant Autoantigen ELISA
human tissue TG transglutaminase (TG)
(hu-tTG)
Humans Envelop Herpes B virus (HBV) ELISA
glycoproteins gB,
gD, gC, gE and gG
nucleocapsid protein of strain SR-11 (rNP-SR-Sf9) was used as antigen for the indi-
rect immunofluorescence antibody (IFA) diagnostic test that detects three serotypes
(hantan 76-118, SR-11 and Puumala) of hantavirus (Yoshimatsu et al. 1993). Purified
human papillomavirus (HPV) E2 protein was used to develop ELISA to detect IgG
and IgA responses in cervical neoplasia patients (Rocha-Zavaleta et al. 1997).
Houston/90 (Hou/90) is a human calicivirus (HuCV) strain in one of the three clades
of Sapporo-like HuCVs that cause acute gastroenteritis in children. The viral capsid
gene of Hou/90 capsid was used as antigen for immunoprecipitation and EIA (Jiang
et al. 1998). Herpes simplex virus (HSV) infection is caused by two viruses HSV-1
and HSV-2. Diagnostic test that can distinguish between two strains has been devel-
oped that utilizes both type-specific and type-common HSV antigens in a single-step
assay format to perform accurate diagnosis (Burke 1999; Wald and Ashley-Morrow
2002; Liu et al. 2015). Eight different strains of human caliciviruses (HuCVs) capsid
proteins have been used to develop antigen-antibody detection assay by EIAs that are
highly specific (Jiang et al. 2000). Causative agent of tick-borne encephalitis (TBE),
C-terminus truncated form of protein E (Etr) of TBE complex virus tagged with his-
tidine was used to develop sensitive and specific ELISA as well as immunoblot assay
to detect the TBE virus-specific antibodies in infected individuals (Marx et al. 2001).
Fel dl, the major allergen from cats, consists of two polypeptide chains, chain 1 (ch1)
and chain 2 (ch2), which are usually linked with a disulphide bond. Recombinant Fel
dl (rFel dl Ch1 + Ch2) protein construct in which two chains are linked together with
glycine/serine linker was used as more potent antigen than bacterial-derived proteins
for the detection of IgE and IgG antibodies by radioimmunoassay (RIA) and ELISA
(Guyre et al. 2002). Coeliac disease (CD) is characterized by the presence of autoan-
tigen transglutaminase (TG). Recombinant human tissue TG (hu-tTG) expressed
with baculovirus system was used as antigen for ELISA that showed a sensitivity of
100% and a specificity of 98.6% (Osman et al. 2002). The envelope glycoproteins:
gB, gD, gC, gE and gG are thought to be the primary targets of IgG antibody response
in patients with Herpes B virus (HBV) infection. Therefore, ELISA test was devel-
oped by using the cocktail of these recombinant glycoproteins along with other cap-
sid proteins with high sensitivity and specificity (Perelygina et al. 2005). Similarly,
the recombinant proteins in single or multiple subunits for the diagnosis of different
types of viral infections in humans have been developed with baculovirus expression
system in insect cells.
BEVs express products like growth factors, cytokines, chemokines, enzymes, hor-
mones and monoclonal antibodies that can be used for human therapeutic purposes.
More recently, BEV has also been exploited as effective tool for gene therapy. For
simplicity, the applications of these products have been divided into two major
groups: biological drug therapy and gene therapy. Over thousands of such biomol-
ecules have been developed till now in this system, few among them are discussed
here (Table 9.3).
Table 9.3 BEVs produced biomolecules as disease therapeutic agents
176
Worcester
Enzyme-gene AAVrh.10halpha1AT Phase I Adverum
therapy Biotechnologies, Inc.
Enzyme-gene Aromatic amino acid Transgene AAV2-hAADC Aromatic L-amino Phase I/II National Taiwan
therapy decarboxylase acid decarboxylase University Hospital
deficiency
Protein gene Choroideremia Transgene rAAV2.REP1 Rab-escort protein Phase I University of
therapy 1 Oxford
(continued)
179
Table 9.3 (continued)
180
Human prepro (beta) nerve growth factor that has been suggested as a therapeu-
tic agent for the treatment of Alzheimer’s disease was produced in insect cells as
recombinant virus, mature human beta nerve growth factor (rhNGF). It was found
to be biologically active in cholinergic cell survival (Barnett et al. 1990). Similarly,
different strategies are being worked out with BEVs in insect cells or larvae for
biologically active, cost-effective, therapeutic and commercial scale production of
numerous highly variable molecules.
propensity for tissue-specific infection and infection kinetics (Zincarelli et al. 2008).
The major limitation of low production quantity was addressed recently by optimiz-
ing the BEVs platforms and adjusting different parameters such as multiplicity of
infection, cell density and fermentation mode that produced up to 104 vector
genomes per litre (Mena et al. 2010).
The strategy for rAAV production requires the production of three AAV capsid
proteins, VP1, VP2 and VP3. These capsid proteins assemble within BEV-transduced
insect cells to produce icosahedral VLPs (Aucoin et al. 2007). More efficient rAAVs
require co-infection of insect cells with three different kinds of baculoviral vectors.
The first one is Bac-Rep, expressing the major AAV replication enzymes Rep 78
and Rep 52 essential for AAV genomic replication and packaging, respectively.
Second is Bac-Cap, expressing the AAV virion capsid proteins (VP1, VP2 and
VP3), and third is Bac-GOI, expressing the gene of interest flanked by AAV inverted
terminal repeat elements required for the rescue, replication and packaging of the
heterologous gene. Co-infection with these three vectors in insect cells produces
efficiently replicated and encapsulated single-stranded AAV vector genome (Weyer
and Possee 1991). Further enhancement of AAV in terms of stability, robustness,
scalability and high-titre production involves both Rep and Cap protein expression
from a single baculovirus (Bac-Rep Cap), i.e. expression of both Rep 78 and Rep 52
transcription from a single mRNA and genetic modifications of the original Bac-
Rep and Bac-Cap constructs (Virag et al. 2009). The development of such robust
gene delivery vehicles was based on the fact that AAV genome is efficiently repli-
cated in Sf9 and Sf21 insect cell lines in a Rep-dependent fashion. Some of the dis-
eases that are being targeted by gene therapy using rAAV are discussed below:
(a) Gene therapy against lipoprotein lipase deficiency (LPLD): It is a rare autoso-
mal recessive genetic and metabolic disorder in which inactivation of familial
lipoprotein lipase enzyme occurs due to mutation in gene LPL. Functional
lipase is required for plasma triglyceride hydrolysis under normal condition.
Inactivated enzyme results into hypertriglyceridemia characterized by frequent
abdominal pain and fatty deposits in the skin and retina that in severe cases can
lead to fatal pancreatitis, diabetes and onset of cardiovascular diseases. Earlier
therapies targeted to lower the plasma triglycerides have not been proved much
effective. Alipogene tiparvovec (also known as AAV1-LPLS447X in the early
phases of clinical trial) is the first adeno-associated virus (AAV)-mediated gene
therapy manufactured by UniQure that got market authorization and govern-
ment approval in Europe. It is an AAV1 (serotype 1) vector expressing naturally
occurring variants of LPL transgene, LPLS447X linked with improved lipid pro-
file and is commercialized by the name of Glybera (Gaudet et al. 2010). It is
injected through intramuscular route in the patients that results in natural gain
of function of LPL gene variants to muscle tissues. Glybera use significantly
lowers plasma triglycerides by increasing the lipoprotein lipase enzyme
activity.
The major concern for using such vector-based gene therapy is to prevent
both humoral and cell-mediated immune response elicited against viral capsid
9 Preventive, Diagnostic and Therapeutic Applications 185
proteins that may impact the efficacy and safety of these drugs. Intramuscular
injections of Glybera has been proved clinically safe and efficient drug that
does not elicit any additional systemic and local immune response harmful for
humans. This approach was found to be relevant and promising for the treat-
ment of thousands of single gene disorders. Similar strategies are being investi-
gated in diverse range of therapeutic areas, and many products for the treatment
of human diseases are in different stages of clinical development. These AAV
gene therapy drugs at different clinical development phases are being discussed
here.
(b) Haemophilia: It is a blood clotting disorder caused by the mutation in the clot-
ting factor IX gene. Presently, four clinical trials are going on that involve rAAV
serotype 2 or 8, designed to express factor IX.
Haemophilia A, the most common severe inherited bleeding disorder caused
by mutation in factor VIII gene, is significantly more problematic for this treat-
ment because of a larger size of cDNA that prevents in achieving the adequate
level of transgene expression and elicits the anti-factor VIII immune response
(High et al. 2014).
(c) Retinal degeneration: Recombinant AAV has been used to treat a number of
animal models but is limited by carrying capacity, slow onset of expression and
limited ability to transduce some of the retinal cell types from the vitreous.
Next-generation AAVs have been produced to address these issues by creation
of self-complementary AAV vectors for faster onset of expression and specific
mutations of self-exposed residues to increase transduction. Such vectors were
further improved for broader applicability and advantageous characteristics by
directed evolution through an iterative process of selection (Day et al. 2014).
Age-related macular degeneration (AMD) that leads to the central vision loss in
elderly individuals due to choroidal neovascularization is marked by prolifera-
tion of blood vessels and retinal pigment epithelial (RPE) cells. It leads to pho-
toreceptor death and fibrous disciform scar formation. Treatment of AMD
patients requires neutralization of vascular endothelial growth factor (VEGF)
for which expression of modified soluble Flt1 receptor was designed and
expressed in AAV2-sFLT01 vector. Presently, this study is in Phase 1 trial
(MacLachlan et al. 2011). Leber congenital amaurosis (LCA) is an autosomal
recessive blinding disease that occurs due to mutations in RPE65 gene. Sub-
retinal administration of AAV2-hRPE65v2 has been reported both safe and effi-
cient for at least 1.5 years after injection. Currently six clinical trials, either in
stage 1 or 2, are going on to treat this retinal disease (Simonelli et al. 2010).
( d) Neurological diseases: rAAV has been used as an effective gene delivery sys-
tem for the treatment of central or peripheral nervous system with almost no
adverse effects in many clinical trials. First time, its clinical use in the human
brain has been used to treat Canavan disease, a childhood leukodystrophy also
known as Van Bogaert-Bertrand disease caused by the deficiency of enzyme
aspartoacylase (ASAP). It involves neurosurgical administration of approxi-
mately 10 billion infectious particles of recombinant adeno-associated virus
(AAV) containing the aspartoacylase gene (ASPA) directly to the affected
186 N. Kumar et al.
regions of the brain (Janson et al. 2002). To treat Alzheimer’s disease, transfer
of gene encoding nerve growth factor (NGF), which is essential for healthier
nerve cells, is transduced by an adeno-associated nerve growth factor (CERE-
110) (Bakay et al. 2007). Transduction of glutamic acid decarboxylase (GAD)
and trophic factor neurturin was assessed successfully in different Phase 1 and
2 clinical trials for the treatment of Parkinson’s disease (Marks et al. 2010;
Kaplitt et al. 2007).
(e) Duchenne muscular dystrophy (DMD): DMD is a severe recessive X-linked
muscle disorder caused by mutations in gene encoding dystrophin. Gene ther-
apy to treat DMD is a challenge due to the large size of DMD gene. However,
alternative gene delivery strategies like exon skipping, trans-splicing, micro-
and mini-dystrophin in Phase II/III clinical trials have been found to be promis-
ing (Jarmin et al. 2014).
A number of Phase I/II/III clinical trials are underway for the treatment of numer-
ous diseases such as acute intermittent porphyria, alpha 1-antitrypsin deficiency,
aromatic amino acid decarboxylase deficiency, Becker muscular dystrophy, choroi-
deremia, chronic heart failure, gastric cancer, HIV, inflammatory arthritis, late
infantile neuronal ceroid lipofuscinosis, Leber’s hereditary optic neuropathy, limb
girdle muscular dystrophy, macular degeneration, Pompe disease, spinal muscular
atrophy, etc. (Felberbaum 2015).
The future prospectives of baculovirus gene delivery applications in stem cell
transduction, cancer gene therapy and cartilage and bone tissue engineering are
also quite optimistic. Great interest in regenerative medicine begins with the
advancement in identification, isolation and derivation of human stem cells, spe-
cifically the generation of human-induced pluripotent stem cells. Prolonged expres-
sion of transgenes has been demonstrated in multiple multipotent stem cells such
as mesenchymal, neural, umbilical cord, bone marrow, adipose tissue, human
embryonic stem cells (hESCs) and pluripotent stem cells. These baculoviruses
have also been customized for stable gene expression in stem cells by genomic
integration for downstream therapeutic applications, for example, deriving unlim-
ited numbers of genetically corrected functional adult cells for cell replacement
therapy (Kotin et al. 1991).
De-differentiated chondrocytes transduced with baculovirus vector (Bac-CB)
expressing bone morphogenetic protein-2 (BMP-2) result into sustained expression
of BMP-2 with passaged chondrocytes in vitro. It was further improved by co-
expression of transforming growth factor beta with baculovirus vectors (Chen et al.
2008). These chondrocytes were further used to grow cartilage-like tissues in rotat-
ing shaft bioreactors that demonstrated the potential of baculovirus in cartilage tis-
sue engineering, but their clinical utility in humans is yet to be proved.
Bac-CB-based BMP-2 transduction into human bone marrow-derived mesen-
chymal stem cells (BMSCs) is also demonstrated to directing ontogenies of naïve
BMSCs. Implantation of these transduced cells induced ectopic bone formation in
nude mice and promoted calvarial bone repair in immunocompetent rats (Chuang
et al. 2009). For massive repairing of bone, sustained expression of genes promoting
9 Preventive, Diagnostic and Therapeutic Applications 187
References
Airenne KJ et al (2013) Baculovirus: an insect-derived vector for diverse gene transfer applica-
tions. Mol Ther 21(4):739–749
Ang WX et al (2016) Local immune stimulation by intravesical instillation of baculovirus to
enable bladder cancer therapy. Sci Rep 6:27455
Atmar RL et al (2011) Norovirus vaccine against experimental human Norwalk Virus illness. N
Engl J Med 365(23):2178–2187
Aucoin MG et al (2007) Virus-like particle and viral vector production using the baculovi-
rus expression vector system/insect cell system. In: Baculovirus and Insect Cell Expression
Protocols. Humana, New York, pp 281–296
Bakay RA et al (2007) Analyses of a phase 1 clinical trial of adeno-associated virus-nerve growth
factor (CERE-110) gene therapy in Alzheimer’s disease: 866. Neurosurgery 61(1):216
Ball JM et al (1999) Recombinant Norwalk virus–like particles given orally to volunteers: phase I
study. Gastroenterology 117(1):40–48
Barber GN, Clegg J, Lloyd G (1990) Expression of the Lassa virus nucleocapsid protein in insect
cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus
infection. J Gen Virol 71(1):19–28
Barnett J et al (1990) Human β nerve growth factor obtained from a baculovirus expression system
has potent in vitro and in vivo neurotrophic activity. Exp Neurol 110(1):11–24
Burke RL (1999) Herpes simplex virus diagnostics. Google Patents
Chen H-C et al (2008) Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor
culture synergistically enhances cartilage formation. Gene Ther 15(4):309–317
Choi K-S et al (2014) Baculovirus expression of the avian paramyxovirus 2 HN gene for diagnos-
tic applications. J Virol Methods 198:12–17
Chuang C-K et al (2009) Xenotransplantation of human mesenchymal stem cells into immuno-
competent rats for calvarial bone repair. Tissue Eng A 16(2):479–488
188 N. Kumar et al.
Chung C-Y et al (2010) Enterovirus 71 virus-like particle vaccine: improved production conditions
for enhanced yield. Vaccine 28(43):6951–6957
Cox MM, Hashimoto Y (2011) A fast track influenza virus vaccine produced in insect cells. J
Invertebr Pathol 107:S31–S41
Day TP et al (2014) Advances in AAV vector development for gene therapy in the retina. In:
Retinal degenerative diseases. Springer, New York, pp 687–693
de Diego ACP et al (2011) Characterization of protection afforded by a bivalent virus-like particle
vaccine against bluetongue virus serotypes 1 and 4 in sheep. PLoS One 6(10):e26666
Desrosiers R et al (2009) Use of a one-dose subunit vaccine to prevent losses associated with por-
cine circovirus type 2. J Swine Health Prod 17(3):148–154
dos Santos CND et al (1992) Trypanosoma cruzi flagellar repetitive antigen expression by recombi-
nant baculovirus: towards an improved diagnostic reagent for Chagas’ disease. Biotechnology
10:1474–1477
Dudognon B et al (2014) Production of functional active human growth factors in insects used as
living biofactories. J Biotechnol 184:229–239
Eshaghi M et al (2004) Nipah virus glycoprotein: production in baculovirus and application in
diagnosis. Virus Res 106(1):71–76
Fauquet CM et al (2005) Virus taxonomy: VIIIth report of the International Committee on
Taxonomy of Viruses. Academic, London
Felberbaum RS (2015) The baculovirus expression vector system: A commercial manufacturing
platform for viral vaccines and gene therapy vectors. Biotechnol J 10(5):702–714
French T, Marshall J, Roy P (1990) Assembly of double-shelled, viruslike particles of bluetongue
virus by the simultaneous expression of four structural proteins. J Virol 64(12):5695–5700
Frey S, Treanor JJ, Atmar RL, Topman D, Chen WH, Ferreira J (2011) Phase 1 dosage escalation,
safety and immunogenicity study of a bivalent norovirus VLP vaccine by the intramuscular
route. In Annual meeting of the infectious diseases society of America, Boston, 2011
Gaudet D et al (2010) Review of the clinical development of alipogene tiparvovec gene therapy for
lipoprotein lipase deficiency. Atheroscler Suppl 11(1):55–60
Gil F et al (2011) Targeting antigens to an invariant epitope of the MHC Class II DR molecule
potentiates the immune response to subunit vaccines. Virus Res 155(1):55–60
Goldman B, DeFrancesco L (2009) The cancer vaccine roller coaster. Nat Biotechnol 27(2):129–139
Gómez-Sebastián S et al (2012) Rotavirus A-specific single-domain antibodies produced in
baculovirus-infected insect larvae are protective in vivo. BMC Biotechnol 12(1):59
Guyre P et al (2002) Recombinant cat allergen, Fel dI, expressed in baculovirus for diagnosis and
treatment of cat allergy. Google Patents
High K et al (2014) Current status of haemophilia gene therapy. Haemophilia 20(s4):43–49
Hu YC, Bentley WE (2001) Effect of MOI ratio on the composition and yield of chimeric infec-
tious bursal disease virus-like particles by baculovirus co-infection: Deterministic predictions
and experimental results. Biotechnol Bioeng 75(1):104–119
Janson C et al (2002) Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of
aspartoacylase gene (ASPA) to the human brain. Hum Gene Ther 13(11):1391–1412
Jarmin S et al (2014) New developments in the use of gene therapy to treat Duchenne muscular
dystrophy. Expert Opin Biol Ther 14(2):209–230
Jiang X et al (1992) Expression, self-assembly, and antigenicity of the Norwalk virus capsid pro-
tein. J Virol 66(11):6527–6532
Jiang X et al (1998) Baculovirus-expressed Sapporo-like calicivirus capsid self-assembles into
virus-like particles (VLPs) and can be used to detect antibody in children† 854. Pediatr Res
43:148–148
Jiang X et al (2000) Diagnosis of human caliciviruses by use of enzyme immunoassays. J Infect
Dis 181(Supplement 2):S349–S359
Kanesashi S-N et al (2003) Simian virus 40 VP1 capsid protein forms polymorphic assemblies
in vitro. J Gen Virol 84(7):1899–1905
Kantoff PW et al (2010) Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N
Engl J Med 363(5):411–422
9 Preventive, Diagnostic and Therapeutic Applications 189
Kaplitt MG et al (2007) Safety and tolerability of gene therapy with an adeno-associated
virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet
369(9579):2097–2105
King AM et al (2011) Virus taxonomy: ninth report of the International Committee on Taxonomy
of Viruses. Elsevier, London
Ko Y-J et al (2005) Noninfectious virus-like particle antigen for detection of swine vesicular dis-
ease virus antibodies in pigs by enzyme-linked immunosorbent assay. Clin Diagn Lab Immunol
12(8):922–929
Ko Y-J et al (2010) Use of a baculovirus-expressed structural protein for the detection of antibod-
ies to foot-and-mouth disease virus type A by a blocking enzyme-linked immunosorbent assay.
Clin Vaccine Immunol 17(1):194–198
Kong XG et al (1997) Application of equine infectious anemia virus core proteins produced in a
baculovirus expression system to serological diagnosis. Microbiol Immunol 41(12):975–980
Kost TA, Condreay JP (2002) Innovations—biotechnology: baculovirus vectors as gene transfer
vectors for mammalian cells: biosafety considerations. Appl Biosaf 7(3):167–169
Kotin RM et al (1991) Mapping and direct visualization of a region-specific viral DNA integration
site on chromosome 19q13-qter. Genomics 10(3):831–834
Krammer F et al (2010) Trichoplusia ni cells (High FiveTM) are highly efficient for the production
of influenza A virus-like particles: a comparison of two insect cell lines as production platforms
for influenza vaccines. Mol Biotechnol 45(3):226–234
Laurent S et al (1994) Recombinant rabbit hemorrhagic disease virus capsid protein expressed
in baculovirus self-assembles into viruslike particles and induces protection. J Virol
68(10):6794–6798
Lee JH et al (2014) Expression of recombinant anti-breast cancer immunotherapeutic monoclonal
antibody in baculovirus–insect cell system. Entomol Res 44(5):207–214
Liu L-J et al (2008) Efficient production of type 2 porcine circovirus-like particles by a recombi-
nant baculovirus. Arch Virol 153(12):2291–2295
Liu T et al (2015) Production of a fragment of glycoprotein G of herpes simplex virus type 2 and
evaluation of its diagnostic potential. Singap Med J 56(6):346
Long G et al (2006) Functional entry of baculovirus into insect and mammalian cells is dependent
on clathrin-mediated endocytosis. J Virol 80(17):8830–8833
López-Macías C et al (2011) Safety and immunogenicity of a virus-like particle pandemic influ-
enza A (H1N1) 2009 vaccine in a blinded, randomized, placebo-controlled trial of adults in
Mexico. Vaccine 29(44):7826–7834
Luckow VA, Summers MD (1988) Trends in the development of baculovirus expression vectors.
Nat Biotechnol 6(1):47–55
Luo W et al (2011) Baculovirus vectors for antiangiogenesis-based cancer gene therapy. Cancer
Gene Ther 18(9):637–645
Luo W et al (2012) Development of the hybrid Sleeping Beauty-baculovirus vector for sustained
gene expression and cancer therapy. Gene Ther 19(8):844–851
MacLachlan TK et al (2011) Preclinical safety evaluation of AAV2-sFLT01—a gene therapy for
age-related macular degeneration. Mol Ther 19(2):326–334
Maranga L et al (2002) Large scale production and downstream processing of a recombinant por-
cine parvovirus vaccine. Appl Microbiol Biotechnol 59(1):45–50
Marks WJ et al (2010) Gene delivery of AAV2-neurturin for Parkinson’s disease: a double-blind,
randomised, controlled trial. Lancet Neurol 9(12):1164–1172
Marx F et al (2001) Diagnostic immunoassays for tick-borne encephalitis virus based on recombi-
nant baculovirus protein expression. J Virol Methods 91(1):75–84
Mathavan S et al (1995) High-level production of human parathyroid hormone in Bombyx mori
larvae and BmN cells using recombinant baculovirus. Gene 167(1):33–39
Mauch L et al (1993) Baculovirus-mediated expression of human 65 kDa and 67 kDa glutamic
acid decarboxylases in SF9 insect cells and their relevance in diagnosis of insulin-dependent
diabetes mellitus. J Biochem 113(6):699–704
190 N. Kumar et al.
Mazur NI et al (2015) Lower respiratory tract infection caused by respiratory syncytial virus: cur-
rent management and new therapeutics. Lancet Respir Med 3(11):888–900
Mecham J, Wilson W (2004) Antigen capture competitive enzyme-linked immunosorbent assays
using baculovirus-expressed antigens for diagnosis of bluetongue virus and epizootic hemor-
rhagic disease virus. J Clin Microbiol 42(2):518–523
Mena JA et al (2010) Improving adeno-associated vector yield in high density insect cell cultures.
J Gene Med 12(2):157–167
Monie A et al (2008) Cervarix™: a vaccine for the prevention of HPV 16, 18-associated cervical
cancer. Biol Targets Ther 2(1):107–113
Montross L et al (1991) Nuclear assembly of polyomavirus capsids in insect cells expressing the
major capsid protein VP1. J Virol 65(9):4991–4998
Moormann RJ et al (2000) Development of a classical swine fever subunit marker vaccine and
companion diagnostic test. Vet Microbiol 73(2):209–219
Mortola E, Roy P (2004) Efficient assembly and release of SARS coronavirus-like particles by a
heterologous expression system. FEBS Lett 576(1-2):174–178
Neuzil KM (2016) Progress toward a respiratory syncytial virus vaccine. Clin Vaccine Immunol
23(3):186–188
O’Reilly DR, Miller LK, Luckow VA (1994) Baculovirus expression vectors: a laboratory manual.
Oxford University Press on Demand, New York
Osman AA et al (2002) Production of recombinant human tissue transglutaminase using the bacu-
lovirus expression system, and its application for serological diagnosis of coeliac disease. Eur
J Gastroenterol Hepatol 14(11):1217–1223
Park D-Y et al (2011) Optimization of expression conditions for production of anti-colorectal cancer
monoclonal antibody CO17-1A in baculovirus-insect cell system. Hybridoma 30(5):419–426
Pastey MK, Samal SK (1998) Baculovirus expression of the fusion protein gene of bovine respira-
tory syncytial virus and utility of the recombinant protein in a diagnostic enzyme immunoas-
say. J Clin Microbiol 36(4):1105–1108
Paton D et al (1991) An ELISA detecting antibody to conserved pestivirus epitopes. J Virol
Methods 31(2-3):315–324
Perelygina L et al (2005) Production of herpes B virus recombinant glycoproteins and evaluation
of their diagnostic potential. J Clin Microbiol 43(2):620–628
Pham M-Q et al (1999) Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects
and partial control of proteolysis. Biotechnol Bioeng 62(2):175–182
Pillay S et al (2009) Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-
insect cell expression system. Biotechnol Prog 25(4):1153–1160
Prel A, Le Gall-Recule G, Jestin V (2008) Achievement of avian influenza virus-like particles that
could be used as a subunit vaccine against low-pathogenic avian influenza strains in ducks.
Avian Pathol 37(5):513–520
Reed JC et al (1992) A strategy for generating monoclonal antibodies against recombi-
nant baculovirus-produced proteins: application to the Bcl-2 oncoprotein. Anal Biochem
205(1):70–76
Rocha-Zavaleta L et al (1997) Differences in serological IgA responses to recombinant baculovirus-
derived human papillomavirus E2 protein in the natural history of cervical neoplasia. Br J
Cancer 75(8):1144
Roldão A et al (2010) Virus-like particles in vaccine development. Expert Rev Vaccines
9(10):1149–1176
Roy P, Noad R (2008) Virus-like particles as a vaccine delivery system: Myths and facts. Hum
Vaccin 4(1):5–12
Saijo M et al (2007) Development of recombinant nucleoprotein-based diagnostic systems for
Lassa fever. Clin Vaccine Immunol 14(9):1182–1189
Seppänen H et al (1991) Diagnostic potential of baculovirus-expressed rubella virus envelope
proteins. J Clin Microbiol 29(9):1877–1882
Sguazza GH et al (2013) Expression of the hemagglutinin HA1 subunit of the equine influenza
virus using a baculovirus expression system. Rev Argent Microbiol 45(4):222–228
9 Preventive, Diagnostic and Therapeutic Applications 191
Abstract
Protecting crops against insect pests is a major focus area in crop protection.
Over the past two decades, biotechnological interventions, especially Bt pro-
teins, have been successfully implemented across the world and have had major
impacts on reducing chemical pesticide applications. As insects continue to
adapt to insecticides, both chemical and protein-based, new methods, molecules,
and modes of action are necessary to provide sustainable solutions. RNA inter-
ference (RNAi) has emerged as a significant tool to knock down or alter gene
expression profiles in a species-specific manner. In the past decade, there has
been intense research on RNAi applications in crop protection. This chapter
looks at the current state of knowledge in the field and outlines the methodology,
delivery methods, and precautions required in designing targets. Assessing the
targeting of specific gene expression is also an important part of a successful
RNAi strategy. The current literature on the use of RNAi in major orders of
insect pests is reviewed, along with a perspective on the regulatory aspects of the
approach. Risk assessment of RNAi would focus on molecular characterization,
food/feed risk assessment, and environmental risk assessment. As more RNAi-
based products come through regulatory systems, either via direct application or
plant expression based, the impact of this approach on crop protection will
become clearer.
10.1 Introduction
Crop protection strategies need continuous improvement and innovation since the
ability of the insect herbivores to adapt to any pest control intervention is well docu-
mented (Georghiou and Lagunes-Tejeda 1991; Storer et al. 2010). The increased
strain on agricultural output due to global challenges such as population growth and
climate change in conjunction with the escalating costs of chemical pest control,
insecticide resistance, and rising environmental and health concerns creates a need
for developing new technologies to close yield gaps and minimize environmental
impacts (Pradhan et al. 2015). Sustainable intensification of crop production by
using the best of conventional plant breeding (adapted germplasm with native resis-
tance) and the best of biotechnology is a theme that is being widely advocated by
the global scientific community to meet the daunting challenge of feeding 9.7 bil-
lion in 2050 (Smith 2013). New biotechnological techniques developed during the
last two decades have helped agriculture to cope with different challenges like pest
resistance, disease, herbicide and stress tolerance, and improved yield and product
quality characteristics. Transgenic crops expressing Bacillus thuringiensis (Bt) pro-
teins have provided excellent yield protection from insect pest damage, and the
success of this technology is evident by the fact that the global planting of crops
genetically engineered to express Bt proteins increased to 78 million hectares in
2014 which is a significant fraction of the >170 M ha of transgenic crops cultivated
worldwide (James 2014; Baum and Roberts 2014). Although Bt sprays and Bt crops
have provided substantial economic and environmental benefits, insect adaptation
resulting from the strong selective pressures imposed, has reduced their effective-
ness (Tabashnik et al. 2013; Carrière et al. 2015). Therefore, as in the case for syn-
thetic and biological insecticides, alternative modes of action (MOAs) for
insect-protected crops are needed, either because some insect species are refractory
to Bt proteins or because some have evolved field resistance to the Bt Proteins. To
that end, RNA interference (RNAi) which targets and knocks down the expression
of genes in a species-specific manner provides significant opportunities for crop
protection by managing pest populations and reducing the spread of vector-borne
diseases (Price and Gatehouse 2008; Lundgren and Duan 2013). Therefore, there is
a growing interest in using dsRNA for insect control, both as a traditional RNAi-
based pesticide and RNAi-based genetically modified crop plants.
RNA interference (RNAi) refers to a collection of biological processes, by which
exogenously applied and endogenously expressed double-stranded RNAs (dsRNA)
target specific endogenous messenger RNAs (mRNAs) for degradation, thereby
silencing their expression by making use of conserved cellular machinery (Zamore
2001). Shortly following its discovery in the nematode, Caenorhabditis elegans
(Fire et al. 1998), RNAi has been observed in a wide range of eukaryotic organisms
and has proved itself to be a powerful tool for investigating gene function (Dykxhoorn
and Lieberman 2005). In plants, the dsRNA-triggered sequence-specific RNA deg-
radation pathway has been termed post-transcriptional gene silencing (PTGS). The
RNAi pathway is a major antiviral system in plants (Szittya and Burgyan 2013) and
nematodes (Sarkies and Miska 2013) and serves as a broadly acting (Kemp et al.
2013) and robust antiviral pathway in insects (Nayak et al. 2013; Bronkhorst and
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 195
van Rij 2014; Vijayendran et al. 2013). Not only has effective RNAi been demon-
strated in many insect species, but it has also been performed in insects in a variety
of developmental stages. The evidence for functional RNAi has been reported in a
wide range of insect species encompassing different taxonomic groups that include
the Coleoptera (Arakane et al. 2004; Suzuki et al. 2008), Diptera (Lum et al. 2003;
Dietzl et al. 2007), Dictyoptera, Hemiptera, Hymenoptera (Schluns and Crozier
2007; Antonio et al. 2008), Isoptera, Lepidoptera (Chen et al. 2008; YuQ et al. 2008;
Tian et al. 2009; Terenius et al. 2011), Neuroptera, and Orthoptera. Recent studies
have shown the potential applications of this tool in fundamental and applied
research and more specifically for crop protection against insect pests. The inges-
tion of double-stranded RNAs targeting essential insect genes by insects can trigger
RNAi and lead to growth inhibition, developmental aberrations, reduced fecundity,
and mortality. RNAi can therefore be considered as one of a suite of tools for crop
improvement and insect protection.
The range of potential applications of RNAi in agriculture is remarkable, and the
technology is being evaluated to introduce novel plant traits, increase crop yield,
and improve product quality. RNAi is being touted as the “game changer” in agri-
culture because it has provided a highly specific, non-chemical solution for pest and
pathogen control (Baum et al. 2007; Price and Gatehouse 2008; Huvenne and
Smagghe 2010). The remarkable systemic nature of this mechanism in insects
makes RNAi-based insecticides an exciting new IPM alternative for agricultural
pest control (Price and Gatehouse 2008). Proof-of-concept studies clearly illustrate
the efficacy of this technology for crop protection. RNAi-based transgenic plants
intended for market release can be designed to either induce silencing of target
genes in planta or in insect pests (Koch and Kogel 2014). RNAi-based GM crops
have been developed in the laboratory for three major crops: corn (Baum et al.
2007), cotton (Mao et al. 2007; Mao et al. 2011), and rice (Zha et al. 2011). Although
RNAi was perceived to provide greater specificity in pest control and have little or
no off-target effects, many studies have shown that unintentional off-target gene
silencing in target cells and gene silencing in non-target organisms occur more com-
monly than expected (Baum et al. 2007; Qiu et al. 2005; Mohr and Perrimon 2012).
An overall picture of the field risks, the environmental fate of dsRNA and RNAi
effects in variation trials (differential effects of RNAi treatments), is emerging (Chu
et al. 2014; Palli 2014; Lundgren and Duan 2013). Our review focuses on the cur-
rent knowledge of RNAi mechanism in general and highlights the scientific data
generated in insects with respect to mechanism, dsRNA uptake, and how RNAi can
complement the existing technologies for crop protection for achieving optimum
crop productivity.
Important insights have been gained in elucidating the detailed mechanism of RNAi,
since its initial discovery, and definitions have been proposed to differentiate the vari-
ous aspects of RNAi in plants and animals. Whangbo and Hunter (2008) categorized
the RNAi response into the following three types: cell autonomous, environmental,
196 L. Alamalakala et al.
and systemic. Environmental and systemic RNAi are together referred to as non-cell
autonomous RNAi. Cell autonomous RNAi refers to the silencing effect that is
encompassed within the cells where dsRNA is constitutively expressed or exoge-
nously introduced. In the non-cell autonomous RNAi, the silencing signal is directly
picked up by cells from the environment, viz., gut or hemocoel. The phenomena in
which the silencing signal (siRNA and/or dsRNA) spreads to neighboring cells or
remote tissues from an epicenter of cells is called systemic RNAi, whereas in envi-
ronmental RNAi, RNAi pathway is triggered by environmental exposure (either by
soaking or feeding), and this may or may not be followed by systemic movement of
the silencing signal (Baum and Roberts 2014). The presence of non-cell autonomous
RNAi in arthropods and its specificity are two important factors that paved the way
for using this technology in pest control. However, for the successful application of
the technology as a crop protection agent, environmental RNAi must first be evalu-
ated, and a suitable delivery system for dsRNA has to be identified.
The RNAi machinery can be categorized into two functional groups: (1) the
intracellular machinery, consisting of the Dicer and Argonaute proteins, and (2) the
“systemic machinery,” composed of factors that amplify the dsRNA trigger and
allow it to spread to other tissues within the animal or even to the next generation
(Siomi and Siomi 2009; Swevers 2012). The RNAi pathway is initiated upon recog-
nition of the long dsRNA precursor molecule that varies in length and origin and
can be introduced into the cell through microinjection, transfection, or expression
from endogenous genes (Huvenne and Smagghe 2010; Whangbo and Hunter 2008).
dsRNA can also move into the cells through the transmembrane transporters SID1/2
or the endocytosis machinery (Feinberg and Hunter 2003; Jose and Hunter 2007).
The dsRNA precursors are processed by Dicer-2, a ribonuclease III (RNase III)
family dsRNA endonuclease, into ~ 19–25 nt long siRNA duplexes with character-
istic 2 nucleotide (nt) 3′ overhangs. The dsRNA-binding motif proteins (dsRBMs)
facilitate the assembly of the siRNAs with the RNase H enzyme Argonaute-2
(Ago2), to form a multi-protein RNA-induced silencing complex (RISC) (Hammond
et al. 2001), where one of the siRNA strands (the passenger) is degraded in a process
dependent upon Ago2 and the endoribonuclease C3PO (component 3 promoter of
RISC) (Liu et al. 2009). The other strand (the guide) is retained and remains associ-
ated with Ago2 and is 2′-0-methylated on its 3′ terminal nt by the Hen1 methyl-
transferase, thus creating a mature RISC (Horwich et al. 2007; Saito et al. 2007).
Base pairing of the guide strand to a complementary target single-stranded RNA
(including mRNAs) leads to Ago2-mediated degradation of the target (Meister and
Tuschl 2004). The siRNA mechanism of the RNAi pathways is harnessed as an
experimental tool to target and degrade specific mRNAs with sequence homology
to the administered/incorporated dsRNA molecules.
The fundamental components of the RNAi machinery are evolutionarily con-
served among insects and are readily identified in insect species whose genomes
have been sequenced (Zhu et al. 2014). The ribonuclease III enzyme Dicer, one of
the key enzymes involved in RNAi pathways, is encoded by variable number of
genes and presents distinct functions among organisms. While mammals and nema-
todes have a single Dicer responsible for functions in siRNA and miRNA pathways
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 197
(Ghildiyal and Zamore 2009), insects have two Dicer proteins, Dcr-1 and Dcr-2 that
are assigned to the miRNA and siRNA pathways (Lee et al. 2004). Dcr-1 preferen-
tially processes the pre-miRNA to miRNA, whereas Dcr-2 is in charge of process-
ing long dsRNA into siRNAs (Tomoyasu et al. 2008; Aronstein et al. 2011; Asgari
2013). miRNAs are processed from endogenous genes and function in the regula-
tion of gene expression, while the siRNAs are derived from dsRNA molecules and
provide defense against invading viruses. The argonaute family proteins (AGO) are
the central protein components of the silencing complexes (RISC) that act in medi-
ating target recognition and silencing (Peters and Meister 2007) and have been
observed in different insect taxonomic groups (Aronstein et al. 2011; Swevers et al.
2013). The Ago proteins with a proven role in determining RNAi efficiency were
found to be duplicated in the Tribolium castaneum genome (Tc-Ago-2a and
Tc-Ago-2b), whereas Drosophila carries only one copy of the AGO-2 gene, thereby
suggesting a relationship between number of copies of AGO gene and insect RNAi
response (Tomoyasu et al. 2008).
In animals, the uptake of dsRNA is facilitated by two machineries, viz., the trans-
membrane channel-mediated uptake machinery based on SID-1(systemic interfer-
ence defective-1) and SID-2 proteins (Jose and Hunter 2007) and the
endocytosis-mediated uptake machinery (Saleh et al. 2006). In C. elegans, the
SID-1 protein is inferred to function as a dsRNA channel (Winston et al. 2002), and
the SID-2 has been implicated in dsRNA uptake by gut cells and probably functions
in environmental RNAi (Jose and Hunter 2007). SID-1 homologues are detected in
the genomes of insects belonging to Coleoptera, Lepidoptera, Hymenoptera, and
Hemiptera but not Diptera (Gordon and Waterhouse 2007). Although putative insect
orthologs of the C. elegans sid genes have been described in various insect species,
their involvement in RNAi is still not known (Xu and Han 2008; Huvenne and
Smagghe 2010). The sid-1-like genes of insects show greater sequence homology
with the C. elegans gene chup-1, a cholesterol transporter that has no involvement
in RNAi, than to sid-1 (Valdes et al. 2012; Luo et al. 2012). In Drosophila melano-
gaster, which lack a SID gene ortholog, dsRNA uptake occurs by receptor-mediated
endocytosis (Saleh et al. 2006). In the nematode C. elegans and in many plants,
there exists a host-derived RNA-dependent RNA polymerase (RdRp), for amplify-
ing the silencing signals by generating “secondary siRNAs” that sustain the RNAi
response (Carthew and Sontheimer 2009). There is no evidence of such RdRp
homologue in any insect genome sequenced to date (Tomoyasu et al. 2008). There
is considerable ambiguity on how the RNAi triggered by the acquisition of dsRNA
molecules is sustained in insect cells, and it is speculated that some unknown mech-
anism may be responsible for the systemic RNAi response (Barnard et al. 2012).
Although RNAi is a highly conserved cellular mechanism and its use for the control
of insect herbivores seems to be very straightforward, RNAi application and effi-
cacy remain variable between genes, life stages, and organisms. Factors that
198 L. Alamalakala et al.
determine the success of RNAi experiments in different insect species include the
uptake of dsRNA (environmental RNAi and/or systemic RNAi), presence/absence
of the core RNAi machinery, cellular uptake and propagation of signal (Roignant
et al. 2003; Miller et al. 2008), and dsRNA degrading enzymes (Arimatsu et al.
2007), as well as other differences in genetic backgrounds (Kitzmann et al. 2013;
Scott et al. 2013). These biological variables have been experimentally studied in
different insect species. A detailed description of different factors influencing the
success of RNAi technology in insects is presented below.
For successful RNAi, the choice of the essential genes that can trigger a lethal RNAi
response in the insect pest requires careful consideration. Target gene selection is
crucial yet challenging especially for those pest species that are usually difficult to
rear in the lab or those that lack the required genomic and genetic tools for a whole
animal-high-throughput-screen. In such cases, data from appropriate insect model
systems can be subjected to large scale unbiased RNAi screens for target gene iden-
tification (Ulrich et al. 2015). The abundance of target gene transcript and the rate
of protein turnover are two important factors that influence the outcome of an RNAi
experiment. An mRNA pool with high turnover that codes for a protein with a short
half-life is considered an ideal gene target for RNAi (Scott et al. 2013). Phenotypic
evaluation of gene function using RNAi may not be easy for a stable protein with a
long half-life. Another limitation is that for majority of genes, mRNA turnover and
protein half-life are not known. Potential target genes which will function under
field conditions can be identified by performing bioassays that closely mimic the
conditions in the field. A precise choice of the target region from the target gene for
dsRNA synthesis will ensure the specificity of RNAi and concurrently limits the
off-target effects. Identification of essential targets is possible by extended literature
search, analyses of available DNA/RNA sequence databases, and gene screening
mediated by second-generation sequencing (Andrade and Hunter 2016; Wang et al.
2011). Insect genomics research initiatives have steadily increased in the last two
decades due to the availability of cost-effective, high-throughput DNA sequencing
platforms that have contributed to the sequencing of genomes of agriculturally
important organisms. These efforts have broad implications in insect functional
genomics studies, enhancing the throughput of RNAi target identification and
development of insect management technologies.
(Scott et al. 2013; Andrade and Hunter 2016). The length of dsRNA is an important
parameter for successful RNAi as different efficacies by different sizes of dsRNA
were reported by Whyard et al. (2009) and Saleh et al. (2006). Although the mini-
mal required length to achieve an optimum RNAi effect varies among insect species
(Bolognesi et al. 2012), greater success with insect RNAi has been achieved with
dsRNA molecules of ≥50–200 bp in length (Huvenne and Smagghe 2010). Huvenne
and Smagghe (2010) provide a comprehensive survey of the length range of dsR-
NAs used in early studies: from 134 to 1842 bp, with most studies using 300–
520 bp. The advantages of using longer >200 bp dsRNA for RNAi strategies in pest
management are the production of many siRNAs against the targeted mRNA tran-
script, potentially maximizing the RNAi response. Occasionally, designing of RNAi
molecules, shorter in length than ideal, may achieve desired specificity. Studies
have shown the effectiveness of chemically synthesized siRNAs (obtained by dicing
the dsRNA in vitro before delivery to the insect) in the suppression of target gene
expression.
Another important aspect in the design of dsRNA sequences is the stringency to
be adopted in order to achieve the desired specificity and avoid/minimize off-target
effects. This property facilitates the designing of species-specific sequences that
mediate insect lethality and is a prerequisite for taking the technology from the
laboratory to the field. As reported by Zhang et al. (2010), two genes with high
sequence similarities can both be silenced by the same dsRNA. This has important
implications in off-target effects. Therefore the target gene and target region should
be carefully determined in order for adequate and specific RNAi. The gene regions
(e.g., 5′ or 3′ end of the gene) to which RNAi molecules are designed have also
yielded variable results, thus emphasizing the importance of screening multiple
RNAi sequences for a gene of interest (Mao and Zeng 2012; Pridgeon et al. 2008;
Loy et al. 2012).
The RNAi molecule design process can be aided by software tools, algorithms,
and databases that evaluate the genome sequence and RNA folding kinetics to opti-
mize effectiveness. Following are some of the online software tools that are avail-
able to minimize off-target effects and achieve specific and precise silencing effect:
the NEXT-RNAi software enables the design and evaluation of siRNAs and long
ds-RNAs and can be used for the design and evaluation of genome-wide RNAi
libraries in an organism-independent manner for all sequenced and annotated
genomes. The input data for the analysis are the desired target sequences and an
off-target database. The Next-RNAi software was deployed to design novel genome-
wide RNAi libraries of long dsRNA for the following insects, viz., D. melanogaster,
T. castaneum, and Anopheles gambiae, and to design multiple RNAi for a specific
gene to study associated phenotype (Horn et al. 2010). The web-based E-RNAi tool
initially developed for RNAi experiments in C. elegans and Drosophila (Zeynep
et al. 2005) provides siRNA and long dsRNA design suggestions suitable for RNAi
experiments in a variety of other species and insects that include Apis mellifera, T.
castaneum, Acyrthosiphon pisum, A. gambiae, and Aedes aegypti (Horn and Boutros
2010). It can calculate off-target impacts that may affect the phenotypic results. The
dsRNA sequences are evaluated for their specificity and efficiency. The dicer
200 L. Alamalakala et al.
enzyme in the RNAi machinery cleaves long dsRNA into small 19–22 nucleotides
long siRNAs. dsCheck is a software that investigates individual 19 nucleotide frag-
ments of long dsRNA and produces a list of potential off-target gene candidates
based on its novel algorithm. This tool provides off-target search to verify previ-
ously designed dsRNA sequences and also presents “off-target minimized” dsRNA
design (Naito et al. 2005).
The effective introduction of the RNAi trigger into an organism and its subsequent
entry into the RNAi pathway is the most limiting factor of the RNAi experiment.
There are many methods of dsRNA delivery reported and applied in RNAi experi-
ments, including microinjection (Tan et al. 2008; Martin et al. 2006), feeding (in
vitro synthesized dsRNA) (Zhou et al. 2008; Zhu et al. 2011), transgenic plants
expressing dsRNA(Baum et al. 2007; Mao et al. 2007), nanoparticle RNAi (Zhang
et al. 2010), soaking (Terenius et al. 2011; Ulvila et al. 2006), and topical applica-
tion (Pridgeon et al. 2008). Intracellular RNAi results from the expression of hairpin
RNAs as transgenes or during the introduction of dsRNA into cells by electropora-
tion or transfection or by direct delivery into a cell. Extracellular RNA, which
requires the uptake of dsRNA molecules by the cells, is achieved by soaking, feed-
ing, or injection into the hemocoel (Yu et al. 2013). From the studies it is evident
that most insect RNAi studies relied on the delivery of specific dsRNA triggers
through either microinjections (Fire et al. 1998; Adams et al. 2000) or ingestion
through feeding (Ulvila et al. 2006; Rangasamy and Siegfried 2012). Each of these
methods has its own advantages and limitations which are discussed further.
have been published for the two model insects, Tribolium and Drosophila, and these
protocols provide a quick reference and standard for similar experiments in other
arthropod species. Successful delivery of dsRNA by injection has also been demon-
strated in Lepidoptera; however, the method has shown great variation in effective-
ness between species and is not as simple as shown in other taxa (Terenius et al.
2011; Kolliopoulou and Swevers 2014). Nonetheless, quite a number of RNAi
microinjection experiments have been performed in species from the order
Lepidoptera with most notable success achieved with Bombyx mori and Manduca
sexta, and the members of the Saturniidae family were found to be quite sensitive to
RNAi using hemocoel injection as the dsRNA delivery method, compared to other
species within the order (Yu et al. 2013). In the moth species, RNAi based on dsRNA
microinjection has been applied to all life stages, viz., egg (Osanai-Futahashi et al.
2016; Fabrick et al. 2004), larvae (Mohammed et al. 2015; Sun et al. 2016; Zhao
et al. 2013), pupa (Choi et al. 2012; Qian et al. 2015), and adults (Abrieux et al.
2013; Hassanien et al. 2014). Injection of dsRNA has been proven successful to
cause a knockdown effect in the economically important model insect, the A. mel-
lifera (Farooqui et al. 2003; Gatehouse et al. 2004; Aronstein and Saldivar 2005).
Microinjection has also been used to deliver dsRNA or siRNA for RNAi in the
agriculturally important hemipteran herbivores, the pea aphid, A. pisum (Jaubert-
Possamai et al. 2007; Mutti et al. 2006), whitefly, Bemisia tabaci (Ghanim et al.
2007), and nymphs and adults of the small brown plant hopper, Laodelphax striatel-
lus (Liu et al. 2010). Microinjection of long dsRNA into the body cavity of B. tabaci
caused downregulation of genes uniquely expressed in the midgut and salivary
glands, and injection of dsRNA targeting the whitefly Drosophila chickadee homo-
logue caused phenotypic effects in the ovaries of B. tabaci. The disruption of gene
expression in the hemipteran herbivores opens the door to new strategies aimed at
curbing down the deleterious effects of these insect pests to agriculture (Ghanim
et al. 2007).
Microinjection protocols are currently available for various taxa, including
Lepidoptera, Diptera, Hymenoptera, the Orthopterans, and Cockroaches (Terenius
et al. 2011; Blandin et al. 2002; Martin et al. 2006; Belles 2010; Huang and Lee
2011; Nakamura et al. 2008). Microinjection has been applied to all life stages in
hemi- and holometabolous insects, and a large variation in the success rates of these
experiments has been observed between different species, genera, and taxa (Yu
et al. 2013). In the case of larvae, injections are usually carried out dorsally or
between segments, whereas in adults the tissue under the wings is the easiest loca-
tion to inject the organism. Microinjection has both its advantages and disadvan-
tages compared to the other methods of dsRNA delivery. This technique allows
researchers to get the dsRNA directly and effectively into the tissue of choice or into
the hemolymph without being hindered by barriers such as the integument or the gut
epithelium, in addition, to providing the flexibility to deliver the precise amount of
dsRNA. However, an important shortcoming of this technique in insects is the
mechanical damage during the injection, which is quite significant when targeting
embryos and neonatal larvae and pupae (Scott et al. 2013; Yu et al. 2013). The
mechanical damage may also have undesirable effects or even obscure the targeted
202 L. Alamalakala et al.
effects especially when studying the function of genes relation to behavior and sur-
vival using RNAi. Furthermore, this method is time-consuming and labor-intensive,
requires expertise, can only be used in the laboratory, and is not suitable for RNAi-
based pest control (Xu et al. 2016).
dsRNAs targeting specific genes from insects to increase their resistance to herbivo-
rous insects (Baum et al. 2007; Mao et al. 2007). Silencing of genes in target insects
of Lepidoptera, Coleoptera, and Hemiptera was evaluated effectively by delivery of
dsRNAs through transgenic plants (Baum et al. 2007; Pitino et al. 2011; Zha et al.
2011). Model plants such as thale cress (Arabidopsis thaliana) (Zha et al. 2011; Liu
et al. 2015), tobacco (Nicotiana tabacum) (Mao et al. 2007), rice (Oryza sativa)
(Zha et al. 2011), tomato (Solanum lycopersicum) (Mamta and Rajam 2016), and
cotton (Gossypium hirsutum) (Mao et al. 2011) were transformed to express dsRNA
against target herbivores.
Oral delivery of dsRNA into insects for RNAi can thus be performed by any of
the following approaches, viz., artificial diet, detached plant parts, or intact plants,
and provides several advantages. Not only is this technique easy to perform, it is a
labor-saving, cost-effective, and comparatively less invasive method with the
potential for high-throughput screening of target genes and potential for field
application (Tian et al. 2009; Kamath et al. 2000). This method may be the most
suitable method for developing RNAi pesticides since it allows RNAi through pest
insect feeding on sprayed dsRNA-based pesticide or transgenic plant and bacteria
that express dsRNA (Xu et al. 2016). The limitations of oral delivery of dsRNA
include the limited or no efficiency of dsRNA ingestion in inducing RNAi in some
insect species, thereby suggesting that the technique may not be suitable for all
species. In S. litura, ingested dsRNA targeting a gut-specific aminopeptidase N
failed to induce RNAi (Rajagopal et al. 2002). The gut environment of the target
insect species and the final effective dosage/concentration delivered or needed for
RNAi are difficult to determine and optimize, which could compromise the inves-
tigations (Turner et al. 2006; Surakasi et al. 2011).
when compared to soaking (Beck and Strand 2005; Valdes et al. 2003). Soaking
appears to work with similar efficiency as feeding in C. elegans; however, it is not
as effective as microinjection (Tabara et al. 1998). The method is easy to use and is
suitable for conducting high-throughput RNAi screens (Perrimon and Mathey-
Prevot 2007) and genome-wide analysis in the study of phenotypes characterization
(Sugimoto 2004).
impact the efficacy of RNAi. Furthermore, the mode of uptake, the ability to process
RNAi molecules, and the ability to spread the signal are other important factors that
influence the essential dose required to induce an RNAi response.
The desired result of an RNAi experiment varies with the objective of the study
(gene function analysis or insect control). For research attempting to develop novel
RNAi-based product for insect control, a successful outcome would be to obtain
high insect mortality, whereas for gene function analysis, physiological indices of
predicted function should be central to the analysis. Therefore, defining and inte-
grating the appropriate physiological and fitness assays in the experimental design
are critical (Scott et al. 2013). Furthermore, in order to expedite the process of
development of a viable pest control product using RNAi strategy, identifying the
best delivery mechanism (i.e., topical sprays, baits, or transgenic plants) early in the
product development phase is of utmost importance (Andrade and Hunter 2016).
10.3.5.1 Bioassays
RNAi bioassays for insects are optimized taking into consideration the feeding
behaviors of insects, and the in vitro experiments are designed to mimic conditions
the insects will encounter in the field. A range of concentrations of dsRNA are tested
to select an optimal concentration for effective RNAi. For insects with piercing-
sucking mouthparts, artificial feeding bioassays are being used widely for RNAi.
However, major drawbacks associated with liquid feeding bioassays (dsRNAs mixed
in a liquid diet or a sucrose solution) include the high mortality levels observed in the
controls, the significantly higher dsRNA concentrations required to achieve mortal-
ity, and the increased degradation of dsRNA in the liquid diet due to bacterial or
fungal contaminations (Upadhyay et al. 2011). Also, it has been reported that con-
centrations of up to 1 μg/μL cannot be reproduced inside plant vascular tissues
(Borgio 2010; Katoch et al. 2013; Tomizawa and Noda 2013), which is an important
parameter to consider when developing an effective RNAi control strategy against
the plant sap feeders (Andrade and Hunter 2016). Successful oral uptake of dsRNA
delivered via host plants treated with dsRNA either as a foliar spray or root drench
was demonstrated in two hemipteran insects, the xylem-feeding leafhopper
(Homalodisca vitripennis) and the phloem-feeding Asian citrus psyllid (Diaphorina
citri) (Hunter et al. 2012). Cost-effective feeding bioassays for screening large num-
ber of dsRNA molecules against the hemipteran pests can be developed using leaf
disks, whole leaf, new growth leaves and stem, or root cuttings (Andrade and Hunter
2016). These bioassays can be terminated after 8–10 days of observations for mortal-
ity and may be extended further to record observations on insect oviposition, egg
viability, or nymph development, since the plant material was found to remain viable
for up to 40 days on an average. These bioassays provide the flexibility to screen for
the synergistic effects of multiple dsRNAs in addition to screening single dsRNA
against multiple insect herbivores of a specific host plant (Andrade and Hunter 2016).
206 L. Alamalakala et al.
For bioassays in chewing insects, viz., the Lepidoptera and Coleoptera, which are
foliage feeders, topical foliar spray is a suitable method for delivery of dsRNA. Host
plant leaves sprayed with dsRNA solution are fed to the insect, and the RNAi effects
are evaluated. The effectiveness of this procedure was reported with the Coleopteran
insects such as western corn rootworm (WCR) (Bolognesi et al. 2012), Colorado
potato beetle (CPB) (Miguel and Scott 2016), the diaprepes root weevil (DRW), and
Diaprepes abbreviatus L. (Andrade and Hunter 2016) and in several other species as
well. In WCR, the dsRNA delivered as a foliar spray silenced genes in tissues far
from the gut epithelium, and in CPB, the actin dsRNA conferred protection against
insect damage for at least 28 days under greenhouse conditions, and the dsRNA was
found to be quite stable. Since chewing insects tend to consume a lot of leaf material
each day, a low-dose spray may be able to deliver a significant amount of RNAi
trigger.
10.3.5.2 Controls
When conducting RNAi experiments, depending on the type of assay/treatment,
including a negative control, viz., empty vector, empty cassette, buffer only, or a non-
specific control (such as dsGFP (green fluorescent protein) gene region), is essential
as this will aid in discriminating specific gene silencing from the simple induction of
siRNA processing machinery by exposure to a dsRNA. The reporter dsRNA used as
a negative control is selected on the basis of having no off-target effects; hence, it
should not show sequence similarity to any known insect mRNA transcript.
The potential of insecticidal RNAi for crop protection and management of insect
herbivores and beneficial insects is widely recognized, and the following options,
viz., transgenic plants expressing the insecticidal RNAi trait (plant-mediated RNAi)
and using dsRNA as a traditionally applied insecticide, are being pursued by indus-
try and academia for product development (Xue et al. 2012; Lundgren and Duan
2013). RNAi offers exquisite specificity and flexibility that cannot be matched by
other crop protection interventions such as chemical insecticides, biological con-
trol, or protein-coding transgenes (Scott et al. 2013). The breakthrough in applying
insecticidal RNAi strategy for controlling agricultural pests via transgenic plants
expressing hairpin dsRNA to target specific gene regions of the insect pests came
from studies conducted on the western corn rootworm, D. v. virgifera (WCR) (Baum
et al. 2007), and cotton bollworm, Helicoverpa armigera (CBW) (Mao et al. 2007).
Baum et al. (2007) screened 290 gene targets for evaluating RNAi response in larval
WCR, from which they observed rootworm mortality or stunting in approximately
2/5 of the targets screened at concentrations as low as ~50 ng/cm2 in surface overlay
diet bioassays (Baum et al. 2007; Baum et al. 2011). A distinct phenotypic response
was not observed on suppression of certain gene targets (Baum and Roberts 2014).
A gossypol-induced cytochrome P450 gene, CYP6AE14, was targeted for RNAi
analysis in CBW by Mao et al. (2007). The gene is expressed in the larval midgut
and permits the bollworm to tolerate inhibitory concentration of the cotton second-
ary metabolite, gossypol. When CBW larvae were fed A. thaliana or N. tabacum
leaves expressing CYP6AE14 dsRNA, lower expression levels of this transcript was
observed in the midgut, and larval growth was retarded, and both effects were more
dramatic in the presence of gossypol. The demonstration of plant resistance to
insects mediated by the RNAi-based trait has not only added a new tool to the crop
protection toolkit but has exemplified the following key issues for successful envi-
ronmental RNAi in crop pests: a large number of specific gene targets are available
that can be screened, the choice of the target sequence(s), the size of the RNAi trig-
ger, and the mode of delivery of the RNAi trigger (Baum et al. 2007; Mao et al.
2007; Bolognesi et al. 2012; Khajuria et al. 2013).
Due to the long product development timelines, slow regulatory approval of plant
delivered RNAi, and the recalcitrant nature of species in some taxa toward environ-
mental RNAi, the future of the RNAi-based insect pest management strategies may
depend on non-transformative RNAi strategies and development of topical formula-
tions (Hunter et al. 2010; Baum and Roberts 2014). Alternative approaches are being
developed for using RNAi strategy as a conventional topically applied pesticide. The
use of topical sprays relies on the penetration or adsorption of the RNAi trigger
through the insect cuticle, thereby bypassing the insect gut (Wang et al. 2011). For
achieving commercial success with the RNAi strategy as a conventional topically
applied pesticide, efficient methods for production, delivery, and increased stability
of dsRNA have to be developed. Nanoparticle-mediated RNAi was found to be an
effective delivery method and provided a better stability of dsRNA in some insects
(Yu et al. 2013; Zhang et al. 2010; Palli 2014). The delivery of dsRNA using
208 L. Alamalakala et al.
10.4.1 Coleoptera
RNAi is quite effective in insects that belong to order Coleoptera (Tomoyasu et al.
2008), and this fact has been corroborated through studies conducted in different
species from the order. The utility of RNAi in both basic and applied science has
been demonstrated in the beetle species, and they appear to be the first target group
to be controlled by the new generation of RNAi transgenics (Palli 2012; Palli 2014;
Rodrigues and Figueira 2016). Small quantities of ingested dsRNA appears to be
sufficient to initiate RNAi response in beetles, such as the western corn rootworm,
Colorado potato beetle, southern corn rootworm, Diabrotica undecimpunctata how-
ardi, and the canola flea beetle, Phyllotreta striolata (Baum et al. 2007; Bolognesi
et al. 2012; Zhao et al. 2008; Baum and Roberts 2014), which was evident by low
LC50 values (1–10 ppb). This level of sensitivity to ingested dsRNA was not observed
in insect species outside the order Coleoptera, and the sensitivity was exhibited by
both the larval and adult stages (Rangasamy and Siegfried 2012; Zhao et al. 2008),
although the in vivo amplification of dsRNA/siRNA has not been shown in beetles.
While systemic RNAi is functional in most beetle species studied, variable RNAi
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 209
responses have been documented across the species, notably to orally delivered
dsRNA. RNAi does not seem to work uniformly in all beetles, as illustrated in stud-
ies with red flour beetle, T. castaneum, and cotton boll weevil, Anthonomus grandis
(Baum et al. 2007; Whyard et al. 2009). The western corn rootworm is one of the
most important agricultural pests in which plant-mediated RNAi was successfully
demonstrated. Significant larval stunting and mortality were observed in the WCR
feeding on maize roots that express a hairpin version of the housekeeping gene
vacuolar ATPase (vATPase). The maize roots showed less injury as well (Baum
et al. 2007). Comprehensive studies of RNAi in corn rootworm by Baum et al.
(2007) provided important insights into the parameters for successful RNAi, for
example, their study showed that screening a large number of gene targets through
simple surface overlay diet bioassays at relatively low concentration of ~50 ng/cm2
was effective in identifying suitable targets causing lethal phenotypes for successful
environmental RNAi in corn rootworm. At least 2/5 of the total 290 gene targets
screened were found to cause rootworm mortality or stunting (Baum et al. 2007;
Baum et al. 2011). Specifically, the study by Baum et al. (2007) reported no signifi-
cant difference in efficacy of six ~300 bp dsRNAs corresponding to the V-ATPase
region in target gene knockdown in WCR suggesting that a single dsRNA of this
size is optimum for RNAi in rootworms. Growth inhibition and mortality were
observed in adults fed with vATPase dsRNA-treated artificial diet containing the
feeding stimulant cucurbitacin as bait (Rangasamy and Siegfried 2012). In this
study, mRNA levels were found to decrease within 24 h of ingestion of dsRNA;
however, decrease in protein levels was observed only after 3 days of feeding. The
RNAi effect resulted in mortality, although complete suppression of protein was not
achieved. A detailed study of the corn rootworm Snf7 ortholog (DvSnf7), which
encodes an essential protein involved in intracellular trafficking showed that
dsRNAs of greater than or equal to approximately 60 base pairs (bp) are required for
the initiation of biological activity in artificial diet bioassays (Bolognesi et al. 2012).
Additionally, 21bps short interfering (si) RNAs are not taken up by the midgut cells
and therefore failed to trigger the silencing of Snf7 gene, supporting the size versus
activity relationship observed in diet bioassays.
Parental RNAi (pRNAi) is an RNA interference response where the gene knock-
down phenotype is observed in the progeny of the treated organism (Vélez et al.
2017). In this type of RNAi, the uptake of dsRNA that targets genes regulating
embryonic development by adults results in reduced egg hatch rates or complete
absence of viable larvae, with the adults remaining unaffected (Khajuria et al. 2015;
Fishilevich et al. 2016). A recent study by Vélez et al. (2017) probed the parameters
for successful parental RNAi in WCR for two target genes the chromatin remodel-
ing gene brahma (brm) and the gap gene hunchback (hb). The parameters investi-
gated included the concentration, duration, and timing of exposure, with respect to
the mating status in WCR females, and the effects of brm and hb dsRNA on male
sperm viability and fecundity were also evaluated. Results from this study demon-
strate that all parameters studied affect the strength of pRNAi phenotype in females
and very subtle effects on sperm count were observed in males. These diet-based
pRNAi studies thus provide a framework for developing the technology for field
210 L. Alamalakala et al.
level testing of plant-based pRNAi. Hu et al. (2016) reported the discovery of two
new gene targets, the dvssj1 and dvssj2, in WCR that are orthologs of Drosophila
genes snakeskin (ssk) and mesh, respectively. Oral delivery of dsRNA targeting
dvssj1 and dvssj2 through diet-based insect feeding assays demonstrated target gene
suppression, larval growth inhibition, and mortality. Transgenic plants expressing
dsRNA of dvssj1 were protected from WCR damage and showed insecticidal
activity.
Since the leading insect model organism, D. melanogaster, lacks a robust sys-
temic RNAi response, Tomoyasu et al. (2008) analyzed the genes involved in RNA-
mediated gene silencing and the systemic RNAi response in T. castaneum. These
studies showed that T. castaneum contains a relatively larger inventory of core com-
ponent genes than D. melanogaster that probably is responsible for the observed
sensitivity of this coleopteran species to dsRNA. Functional analysis of three
Tribolium homologues of C. elegans sid-1 genes suggested that T. castaneum sid-
like genes are not required for systemic RNAi. Target genes having clear RNAi
phenotypes in the model insect T. castaneum were studied further in D. v. virgifera
larvae, to test the efficacy of RNAi for target-site screening. Delivery of dsRNA of
D. v. virgifera orthologs of laccase 2 (DvvLac2) and chitin synthase 2 (DvvCHS2)
by injection resulted in prevention of post-molt cuticular tanning and reduced chitin
levels in midguts, respectively, thus providing a tool for identifying potential insec-
ticidal target in western corn rootworm (Alves et al. 2010).
Colorado potato beetle (CPB), a notorious insect pest on solanaceous vegeta-
bles potatoes, tomatoes, and eggplants, has not only developed resistance against
insecticides but also has an exceptional ability to detoxify plant chemicals.
Transgenic potato plants expressing dsRNA targeting CPB genes demonstrated
limited success. Feeding dsRNA expressed in bacteria was found to work very well
in killing CPB (Palli 2014). In insects species where long dsRNA is more effective
than siRNA for effective environmental RNAi, plant-mediated RNAi may not be
effective against insect herbivory if there are low levels of dsRNA in the tissue due
to the presence of the endogenous plant RNAi pathways that processes dsRNAs
into short interfering RNAs. This bottleneck was addressed by expressing the
dsRNA in chloroplasts (which lack an RNAi machinery), thereby improving the
levels of dsRNA needed to provide protection (Zhang et al. 2015). Transplastomic
potato plants expressing hairpin versions of B-actin and Shrub genes in the chloro-
plasts conferred complete plant protection from insect herbivory; a dramatic
increase in the dsRNA levels that induced up to 100% mortality of the CPB in only
5 days was also observed in these plants.
10.4.2 Lepidoptera
The order Lepidoptera (moths, butterflies, and skippers) represents not only the
second largest order in the class Insecta, but also includes major pests of agricultural
importance (Xu et al. 2016). The lepidopteran insect herbivores were successfully
managed by the first-generation insecticidal plants expressing the Bt proteins for
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 211
Plutella xylostella, which reduced the larval resistance to permethrin (Bautista et al.
2009); the knockdown (63–64% reduction of transcript level) of a gut-specific chitin-
ase gene (OnCht) in the European corn borer (ECB), Ostrinia nubilalis larvae, which
facilitated an understanding of the regulation of chitin content in the peritrophic
matrix (PM) of ECB (Khajuria et al. 2010); the suppression of β1 subunit integrin
(βSe1) gene expression from the beet armyworm, S. exigua, to study its role in cel-
lular immune response and larval development (Surakasi et al. 2011); and a 5 μL
drop of 3μg SfT6 dsRNA was used in feeding assays to demonstrate the role of a
serine protease gene in the processing of the B. thuringiensis Cry1Ca1 insecticidal
protein in the fall armyworm (Rodriguez et al. 2010). High concentrations of dsRNA
(50–2500 ppm) were used in all cases, and a concentration-dependent mortality was
reported upon silencing of the βSe1 subunit gene in the beet armyworm (Surakasi
et al. 2011) and the vacuolar ATPase E subunit gene in M. sexta (Whyard et al. 2009).
A vacuolar ATPase-A gene and an arginine kinase gene were targeted in the tomato
leafminer, Tuta absoluta, an invasive lepidopteran insect pest that is a major threat to
commercial tomato production worldwide causing yield losses of up to 100% in vari-
ous regions (Desneux et al. 2011; Camargo Barbosa et al. 2016). The uptake of
dsRNA by the moth larvae from tomato leaflets treated with in vitro synthesized
dsRNA resulted in approximately 60% reduction in transcript accumulation in the
larvae for both the targets selected, increased larval mortality and protection against
insect herbivory (Camargo Barbosa et al. 2016).
Several studies in Lepidoptera also used chemically synthesized siRNA to sup-
press target gene expression because of the difficulties faced in delivering sufficient
dsRNA to the Lepidopteran gut epithelial cells (Gong et al. 2011). Feeding siRNAs
specific to acetylcholine esterase AChE to H. armigera larvae at ~0.35 ppm along
with the artificial diet resulted in a 15% increase in insect mortality followed by
other phenotypes which include growth inhibition of larvae, pupal weight reduc-
tion, malformation, and lower fecundity as compared to the control larvae (Kumar
et al. 2009). Similar results were reported when acetylcholine esterase genes AChE1
and AChE2 genes were targeted in P. xylostella using chemically synthesized and
modified siRNAs (Gong et al. 2013). The siRNAs were modified by addition of a
dTdT overhang in the 3′end, 2′-methyl-nucleotides, and 5′ polyethylene glycol
(PEG), and this sodium salt formulation contained chitosan. In this study it was
found that silencing of PxAChE2 caused higher mortality compared to PxAChE1,
thus confirming the importance of PxAChE2 in P. xylostella. In the laboratory cab-
bage leaf bioassays, one siRNA, Si-ace2_001, at a concentration of 3 μg cm−2 dis-
played the best insecticidal activity causing 89.0% mortality and exhibited LC50 and
LC90 values of 53.7 μg/mL and 759.71 μg/mL, respectively (Gong et al. 2013). In
the field evaluation, P. xylostella larvae feeding on Brassica oleracea and B. albo-
glabra treated with different siRNA doses had no negative effects on plant morphol-
ogy, color, and growth of vein; however, Si-ace2_001 in the dose of 200 ppm was
moderately harmful to the larvae with a mortality of 58.8% 5 days after exposure
(Gong et al. 2013). These studies suggest that siRNA can be readily taken up by
insect larvae with their diet, and there might not be a strict dsRNA size dependency
to the environmental RNAi in lepidopterans (Baum and Roberts 2014).
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 213
The uptake of large dsRNA expressed in Escherichia coli has also been reported
to impact the growth and survival of the lepidopteran larvae. In S. exigua, silencing
a chitin synthase A by feeding the larvae with bacterial culture expressing dsRNA
caused larval mortality to increase by 14%, 21%, 26%, and 18% in the first-instar
larvae, fourth and fifth larval instars, the prepupae, and pupae, respectively (Tian
et al. 2009). Silencing the CYP6B6 gene by feeding larvae with bacteria expressing
dsRNA caused a 27% increase in larval mortality in H. armigera (Zhang et al.
2013). Targeting the expression of arginine kinase (AK), an important regulation
factor of energy metabolism in invertebrates, by delivering the dsRNA to larvae
through diet containing bacteria expressing arginine kinase dsRNA caused larval
mortality in H. armigera to increase by 2–11% (Qi et al. 2015). A major limitation
in these studies was that neither the concentration of dsRNA in diet nor the effect of
dsRNA alone was reported (Baum and Roberts 2014). Yang and Han (2014) reported
the evaluation of different dsRNA delivery methods in H. armigera and concluded
that continuous ingestion of the bacteria expressing dsRNA was detrimental to
insect development and survival than naked dsRNA and the naked dsRNA degraded
much faster in the midgut than in hemolymph. Feeding-based RNAi mediated by
dsRNA expressed in bacteria or synthesized in vitro, of a molt-regulating transcrip-
tion factor CiHR3 in Sugarcane stem borer, Chilo infuscatellus Snellen, caused sig-
nificant abnormalities and weight loss in insects within 7 days of treatment (Zhang
et al. 2012). However, silencing a juvenile hormone esterase-related gene via bacte-
rial delivery of dsRNA did not result in a phenotype in the corn stalk borer, Sesamia
nonagrioides (Kontogiannatos et al. 2013).
Transgenic plants expressing insect-specific dsRNAs have been considered as a
promising strategy for improving pest resistance to insect herbivory in crops, and
therefore studies were undertaken in Lepidoptera to demonstrate the suppression of
gene expression via plant-mediated dsRNA delivery. Most studies reported so far
that used transgenic plants have targeted genes in H. armigera to suppress the devel-
opment and survival of the moth pest (Xu et al. 2016). Significant suppression of the
ecdysone receptor (EcR) gene expression was observed in H. armigera larvae that
fed on tobacco plants expressing EcR dsRNA, and it resulted in significantly higher
lethality (40%) compared to the gfp control group (10%). Moreover, the growth of
the larvae fed on leaves of transgenic tobacco plants expressing HaEcR dsRNA was
significantly delayed, their body sizes reduced, and the larvae died with significant
molting defects (Zhu et al. 2012). Elevated mortality and developmental aberrations
were reported in the larvae of the beet armyworm when fed on the same transgenic
tobacco tissue, probably because of the shared sequence similarity of the EcR target
sequences in these two species (Zhu et al. 2012).
Xiong et al. (2013) reported larval mortality of 22–30% and >50% mass reduc-
tion in H. armigera that fed on transgenic tobacco leaf disks expressing the dsRNA
of a molt-regulating transcription factor (HaHR3). Transgenic cotton plants express-
ing a dsRNA derived from the H. armigera gossypol-inducible cytochrome P450
CYP6AE14 did not cause mortality in H. armigera larvae that fed on the transgenic
tissue; however the plants showed increased tolerance to insect herbivory (Mao
et al. 2011). An increase in larval stunting was achieved by co-delivering a cysteine
214 L. Alamalakala et al.
proteinase to damage the larval peritrophic matrix that led to higher gossypol accu-
mulation (Mao et al. 2013). This study suggests that plant damage by insect her-
bivory can be mitigated by targeting detoxification mechanism in the insect midgut,
since it appears to not require a systemic RNAi response in the insect (Baum and
Roberts 2014). A recent study used transgenic tobacco (Nicotiana tabacum var.
Xanthi) and tomato (Solanum lycopersicum Mill cv. Pusa early dwarf) plants
expressing H. armigera chitinase dsRNA (Mamta and Rajam 2016) to show that
RNAi-induced mortality in H. armigera larvae that fed on transgenic tissue increased
by up to 45%. Transient expression of the dsRNA of the vacuolar ATPase-A gene
and an arginine kinase gene of T. absoluta, in the tomato plants by infiltration of
Agrobacterium cells carrying binary plasmids expressing the target gene hairpin
constructs, and uptake of dsRNA by the larvae by feeding on this tissue conferred
plant protection against insect feeding damage and reduced target transcript accu-
mulation in the larvae and associated lethality. This study provides evidence that
RNAi could be a promising alternative approach for the control of T. absoluta
(Camargo Barbosa et al. 2016).
The delivery of dsRNA targeting larval stage-specific transcripts, as a topical
application at 50 ppm, was found to cause significant gene silencing and larval mor-
tality at 5 days post-spray in the Asian corn borer, Ostrinia furnacalis (Wang et al.
2011). From this study it was inferred that sprayed dsRNA could have either directly
penetrated the body wall and reached the target site via the hemolymph or reached
the site of action via the tracheoles to produce RNAi effect. It was also reported that
significant RNAi-induced lethality was observed in many of the treatments despite
the absence of significant gene silencing at day 3; this raises pertinent questions
about the role of a non-RNAi mechanism in the effects observed or the sensitivity
of the method used for measuring transcript knockdown (Wang et al. 2011).
10.4.3 Hemiptera
were aimed at studying gene function and not insect mortality or pest control
(Belles 2010; Paim et al. 2013). These experiments revealed that the oral delivery
of dsRNA for gene silencing is an attractive alternative to microinjection in
Hemiptera because of the relatively small size and fragile nature of the immature
stages (Baum and Roberts 2014). Furthermore, for using RNAi trait in crop protec-
tion, oral uptake is a preferred route for dsRNA delivery to insect body, although
microinjection was the typical mode of delivery in many of the successful experi-
ments. Difficulties have also been reported in achieving optimum RNAi in some
Hemiptera, including A. pisum and the tarnished plant bug, Lygus lineolaris (Allen
and Walker 2012), due to the limited persistency of the RNAi trigger in the insect
body. Across all RNAi experimental data available for Hemiptera to date, it has
been observed that there is substantial amount of variability in the dietary concen-
trations of dsRNA required for knocking down gene expression levels and/or
obtaining lethal phenotypic effects, and response ranging from very low to com-
plete knockdown of the transcripts was reported (Baum and Roberts 2014;
Christiaens and Smagghe 2014). This variation is seen not only between different
species within the order but even between experiments conducted within the same
organism. It has been observed that the Hemiptera require a much higher dietary
concentrations of dsRNA, viz., at least three orders of magnitude higher than the
effective concentrations used with the coleopteran species (Baum and Roberts
2014). These studies also provide valuable insights on the best approaches and
targets for using RNAi as a pest control strategy.
Gene silencing following ingestion of dsRNA delivered via artificial diets and
transgenic plants has been reported for several hemipteran insects. The v-ATPase
subunit E gene knockdown in A. pisum and associated mortality (Whyard et al.
2009), suppression of gene expression by 41–48% in N. lugens (Li et al. 2011), the
silencing of aquaporin 1 (ApAQP1) gene leading to elevated hemolymph osmotic
pressure in A. pisum (Shakesby et al. 2009) and in the same species, depletion in the
expression of gap gene hunchback (Aphb), a key regulator in the antero-posterior
patterning causing the expression of a lethal phenotype (Mao and Zeng 2012),
silencing of the gene trehalose phosphate synthase (NlTPS) gene in N. lugens caus-
ing suppression of transcript expression, disturbed development and lethality in the
planthoppers (Chen et al. 2010), and the downregulation of the Ecdysone receptor
(SaEcR) and ultraspiracle protein (SaUSP) genes of the grain aphid Sitobion avenae
F. that impacted aphid survival and fecundity (Yan et al. 2016) are some examples
of gene silencing and/or associated phenotypes following ingestion of dsRNA in
Hemiptera. Upadhyay et al. (2011) reported silencing of the ribosomal protein L9
(RPL 9) and vacuolar ATPase subunit A in the whitefly, Bemisia tabaci, upon inges-
tion of dsRNA/siRNA, with the LC50 values of 11.21 and 3.08 μg/mL, respectively.
More than 80% mortality was observed when the target gene expression was
knocked down in whiteflies, and the insects showed remarkably higher sensitivity to
siRNA. Wuriyanghan et al. (2011) demonstrated the induction of specific RNAi
effects in the potato/tomato psyllid (B. cockerelli) by using a modified artificial
feeding system containing 15% sucrose, food coloring, and Cy™3-labeled
dsRNA. Their study reported that significant RNAi effects were observed when
216 L. Alamalakala et al.
observed. Injection of Coo2 siRNA into pea aphid adults (A. pisum) resulted in a
dramatic depletion of the target salivary gland transcript, and the aphids injected
with siCoo2-RNA died well before the control aphids injected with green fluores-
cent protein (Mutti et al. 2006), suggesting the greater efficiency of microinjection
over plant-mediated dsRNA uptake for controlling this species. A similar outcome,
viz., reduced fecundity but no mortality, was observed when M. persicae fed on A.
thaliana expressing dsRNA of a serine protease gene (Bhatia et al. 2012) and
tobacco plants expressing dsRNA targeting the hunchback (hb) gene (Mao and
Zeng 2014).
Three target genes (Rak1, MpCoo2, and MpPIntO2) with different functions in
aphids were selected to study the persistence and trans-generational effects of plant-
mediated RNAi in the green peach aphid (Coleman et al. 2015). This study demon-
strated that for the three genes examined RNAi-mediated downregulation and
persistence levels in the aphids were not influenced either by the gene sequence or
the function; however, a continuous supply of dsRNA was required to maintain the
RNAi effect since insects lack genes encoding an RNA-dependent RNA polymerase
(RdRP), the enzyme necessary for the siRNA amplification step that leads to persis-
tent RNAi effects (Sijen et al. 2001). The finding that the RNAi effect is transferred
to the next generation in aphids revealed by the downregulation of target genes in
nymphs born from mothers exposed to dsRNA-producing transgenic plants renders
plant-mediated RNAi as a powerful tool for aphid control (Coleman et al. 2015).
More recently, it was shown that transgenic tobacco lines expressing long dsRNA
precursors of v-ATPaseA provided resistance to whiteflies by delivering sufficient
siRNA to knockdown the whitefly v-ATPase gene expression. A significant silenc-
ing response leading to whitefly mortality was recorded in whiteflies feeding on
transgenic plants (Thakur et al. 2014). A comprehensive review of the plant-
mediated RNAi studies reveals that the dsRNA produced by the plants is processed
into short siRNA molecules by the plants own RNAi machinery. The presence of
long dsRNA in the plant phloem was mentioned in only one report. These data sug-
gest that the sap-sucking insects are mainly taking up siRNA rather than longer
dsRNA, although it has been suggested that the RNAi machinery in insects mainly
responds to dsRNA (Christiaens and Smagghe 2014).
facilitate to understand associated risks. Off-target gene silencing may occur both in
the RNAi-based GM plant and also in the organisms feeding on the plant. These
organisms include target pests and nontarget organisms (NTOs). It should be noted
that not all off-target silencing events would lead to significant reduction in gene
expression and/or result in detectable phenotypic changes (Casacuberta et al. 2014).
Non-availability of genome data of plants/varieties being used for introduction of
RNAi-based transformation event and genome data of nontarget organisms limits
the use of bioinformatics-based approaches for NTO risk assessment.
The food/feed risk assessment process follows comparative approach to identify
intended and unintended changes that may occur in a GM plant. The comparative
assessment includes proximate analysis, analysis of, compositional characteristics,
toxicity, allergenicity, and nutritional characteristics of the GM plant. This strategy
for evaluating potential food/feed risks of RNAi-based GM plants is accepted to be
appropriate by various regulatory agencies worldwide (FSANZ 2013; US EPA
2014). The choice of which component and characteristics to be chosen for compo-
sitional and agronomic evaluation is determined during the hazard identification
step of problem formulation process and is partly based on their ability to predict
harm. It should be noted that unintended effects are by nature not expected naturally
and so are difficult to test for them directly (Ladics et al. 2015; Schnell et al. 2015;
Devos et al. 2015).
The environmental and ecological risk assessment has been discussed by various
authors (Auer and Frederick 2009; Lundgren and Duan 2013; Ramon et al. 2014;
Vélez et al. 2017; Roberts et al. 2015). This primarily includes the adverse effects
on nontarget organisms, environmental fate, and risk of resistance evolution in tar-
get pests. The potential adverse effects on nontarget organisms can be studied fol-
lowing tier-based approach (US EPA 2007; Rose 2007; EFSA 2010a, b; ILSI-CERA
2011; Romeis et al. 2011, 2013). The tier-based assessment involves controlled
laboratory studies in lower tiers to field-based studies in highest tier. The early-tier
studies assess the adverse effects due to direct exposure to the insecticidal protein at
concentrations that are several folds higher than the environmental exposure con-
centrations (US EPA 2007; Raybould 2011; Romeis et al. 2013). The potential for
adverse ecological effects of MON 87411 maize, which expresses DvSnf7 RNA,
was studied and reported by Bachman et al. (2016). An assessment plan with the
routes and levels of exposure and testing representative functional taxa was devel-
oped, and the potential for toxicity of DvSnf7 RNA was evaluated. The test nontar-
get organisms (NTOs) included predators, parasitoids, pollinators, and soil biota
besides aquatic and terrestrial vertebrate species. Endpoint observations recorded
included survival, growth, development, and reproduction, and results of their study
demonstrated no adverse effects with any species tested at, or above, the maximum
expected environmental concentration (MEEC). All margins of exposure for NTOs
were >tenfold the MEEC. They concluded that exposure to DvSnf7 RNA, both
directly and indirectly, is safe for NTOs at the expected field exposure levels.
Surrogate species or model species are used to conduct early-tier studies (Romeis
et al. 2008). In general the surrogate species are selected based on exposure path-
way, knowledge on activity and mode of action, amenability of test system, and
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 219
availability of test organism. However, when risk assessment of RNAi products are
being considered, the surrogate species should be selected based on phylogenetic
relationship to the target organism, as the surrogate would likely be susceptible due
to sequence homology/similarity (Romeis et al. 2013). This necessitates evaluating
additional or different surrogate species from those tested for Bt crops (Vélez et al.
2017). Additionally, the susceptibility or unresponsiveness of a model organism to
the dsRNA in environment may help in selecting correct surrogate species for NTO
studies (Roberts et al. 2015). Among arthropod orders, coleopterans are more sensi-
tive to dsRNA (Belles 2010), and lepidopterans require high concentrations of
dsRNA to elicit a response as compared to coleopterans (Ivashuta et al. 2015). The
observed differences in RNAi efficiency in insects make it even more difficult and
complex in choosing surrogate or model organisms in the risk assessment process.
In most of the NTO studies, the measurable endpoint has been mortality, and
limited information exists on effects other than mortality (Vélez et al. 2017).
Considering that the effects of RNAi are not completely understood, endpoints
other than mortality need to be considered through standardized methods (Auer and
Frederick 2009; Vélez et al. 2017). Recently, the risks of RNAi-based GE crops on
a nontarget soil micro-arthropod, Sinella curviseta, a decomposer, were tested
through RNAi dietary toxicity assay, and the endpoint measurements included gene
expression profiles, survival, and life history traits (Pan et al. 2016). S. curviseta
larvae developed significantly faster under the treatments of dsDVV and dsSC than
the vehicle control, and results of this study suggest that the impacts of ingested
arthropod-active dsRNAs on this representative soil decomposer are negligible. The
selection and use of reference genes for RT-qPCR analysis in Coccinella septem-
punctata to assess unintended effects of RNAi GM plants was studied and reported
by Yang et al. (2016). This study will be a critical step toward the development of an
in vivo dietary RNAi toxicity assay for assessing the risks associated with RNAi
transgenic plants.
Higher-tier studies in semi-field, greenhouse, or open field conditions are under-
taken only when an adverse effect is detected in lower-tier studies. Long-term field
assessment of effects of Bt cotton and Bt corn showed minor or negligible risks to
nontarget species in these ecosystems (Daly and Buntin 2005; Head et al. 2005;
Lawo et al. 2009; Li and Romeis 2010; Naranjo 2005; Torres and Ruberson 2005,
2007). In a recent study reported by Ahmad et al. (2016), potential impact of GM
corn MON 87411 (expresses insecticidal dsRNA transcript and Cry3Bb1 protein
besides CP4EPSPS protein) on nontarget arthropods (NTAs) was evaluated in the
field. They evaluated NTA abundance and damage among GM corn and compara-
tors. Twenty taxa met minimum abundance criteria, out of which nine were consid-
ered to be representative of corn ecosystems. They conclude that there is no adverse
environmental impact of MON87411 on NTAs compared to conventional corn and
demonstrate utility of relevant transportable data for risk assessment in other corn
regions. The higher-tier studies may not always find adverse impacts on NTOs due
to the complexity of ecosystems and effects thereof.
Lundgren and Duan (2013) identified other reputed risks to NTOs based on the
pharmaceutical literature such as immune stimulation and over-saturation of the
220 L. Alamalakala et al.
RNAi machinery. However, as discussed by Bachman et al. (2016), the diets of
NTOs consist of plant or animal sources which naturally contain dsRNAs, and there
exists a long history of safe consumption of these endogenous dsRNA across
eukaryotes. With constant oral exposure to environmental dsRNA endogenously
present in natural food sources, unintended effects in nontarget organisms from
immune stimulation and RNA machinery saturation are extremely unlikely to result
from relatively low exposures to dsRNA resulting from cultivation of MON 87411.
The environmental fate of dsRNA can be addressed in terms of stability and
persistence in the environment (Heinemann et al. 2011). Recent studies reported by
Dubelman et al. (2014) indicate that the biological activity of Snf7 dsRNA is lost
within 2 days after application to different types of soil. Also, up to 90% degrada-
tion of the applied dsRNA in soil was observed within 35 h. The other potential
route of exposure is through food webs and risks associated can be addressed
through experiments with primary consumers and RNAi consumption by the same
(Roberts et al. 2015). Resistance evolution in insects to RNAi has not been addressed
and documented yet. It is anticipated that insects that carry viruses with RNAi sup-
pressors would be at a selective advantage on RNAi-protected crops, and RNAi-
based prophylactics for honey bee colonies would select for viral pathogens with
RNAi suppression (Scott et al. 2013). These authors also discuss how genetic vari-
ability within and among insect populations, mismatch between dsRNA and target
transcript, and single-nucleotide polymorphisms (SNPs) could provide selective
advantage for resistance evolution.
Although, the specificity and robustness of RNAi have triggered an immense
interest in using RNAi as a tool for creating insect-resistant crops, commercializa-
tion is likely to be fraught with challenges. Beyond safety issues, a major impedi-
ment includes the lack of a comprehensive federal regulatory framework for
estimating the environmental and ecological risks posed by these technologies.
Technology evaluation is ongoing, and the development of a standardized risk
assessment paradigm is being developed concurrently. However, a number of criti-
cal gaps remain including off-target effects, environmental fate, and importantly,
the risk of resistance evolution in target pests. Concurrent with limitations such as
off-target effects, toxicity, and unsafe delivery methods that have to be overcome
before RNAi can be considered for widespread commercial applications in agricul-
ture, it is crucial that a risk assessment paradigm that can proactively anticipate
potential nontarget effects be developed for pesticidal RNAs prior to the lifting of
deregulation of this technology. Studies reported by Bachman et al. (2016), Ahmad
et al. (2016), Pan et al. (2016), and Yang et al. (2016) can potentially form a basis
for risk assessment of RNAi-based GM plants or products, within the existing regu-
latory frameworks.
Conclusions
While significant advances in RNAi methods and applications in agriculture
have occurred recently, especially against viruses, the efficacy of the approach
against insect pests in the field is yet to be fully established. The discovery of
RNAi and the subsequent research on RNAi in insects have demonstrated the
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 221
profound impact that this technology can have not only in understanding gene
regulation in insects but developing pest management solutions for protecting
plants from insect herbivory. The applications of RNAi have been studied in
several target insect pests belonging to orders such as Coleoptera, Lepidoptera,
and Hemiptera. These studies paved the way to a better understanding of using
RNAi as a pest management tool, while concurrently highlighting the caveats of
using this tool for sustainable management of crop pests. The experimental data
generated in the laboratory on several targets shows that with the exception of
few studies, the phenotypes observed were mostly sublethal, and field-efficacy
data is lacking for many targets and species. It is now evident that the effects of
dsRNA; both target and off-target; are species-dependent and target gene-depen-
dent. RNAi provides a mode of action unique among insecticidal agents through
the mechanism of gene suppression and therefore can complement the current
methods deployed for pest control. Although the current regulatory system
allows following the existing methods, further refinement may be required in
terms of measurement of target and off-target effects. Therefore, the future
course of action for deploying this technology on a commercial scale depends on
how these challenges are addressed. Also, the technologies that enable effective
and efficient RNAi sprays such as BioClay, based on nanoparticles, should be
further explored.
References
Abrieux A, Debernard S, Maria A, Gaertner C, Anton S, Gadenne C, Duportets L (2013)
Involvement of the G-protein-coupled dopamine/ecdysteroid receptor DopEcR in the
behavioral response to sex pheromone in an insect. PLoS One 8:88. doi:10.1371/journal.
pone.0072785
Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD et al (2000) The genome sequence of
Drosophila melanogaster. Science 287:2185–2195
Agrawal N, Malhotra P, Bhatnagar RK (2004) siRNA-directed silencing of transgene expressed in
cultured insect cells. Biochem Biophys Res Commun 320:428–434
Ahmad A, Negri I, Oliveira W, Brown C, Asiimwe P, Sammons B, Horak M, Jiang C, Carson D
(2016) Transportable data from non-target arthropod field studies for the environmental risk
assessment of genetically modified maize expressing an insecticidal double-stranded RNA.
Transgenic Res 25:1–17
Allen ML, Walker WB (2012) Saliva of Lygus lineolaris digests double stranded ribonucleic acids.
J Insect Physiol 58:391–396. doi:10.1016/j.jinsphys.2011.12.014
Alves AP, Lorenzen MD, Beeman RW, Foster JE, Siegfried BD (2010) RNA interference as a
method for target-site screening in the western corn rootworm, Diabrotica virgifera virgifera.
J Insect Sci 10:162. doi:10.1673/031.010.14122
Andrade CE, Hunter WB (2016) RNA interference – natural gene-based technology for highly spe-
cific pest control (HiSPeC). In: Abdurakhmonov IY (ed) RNA interference. InTech, Croatia,
pp 391–409
Antonio DSM, Guidugli-Lazzarini KR, Do Nascimento AM, ZLP S, Hartfelder K (2008) RNAi-
mediated silencing of vitellogenin gene function turns honeybee (Apis mellifera) work-
ers into extremely precocious foragers. Naturwissenschaften 95:953–961. doi:10.1007/
s00114-008-0413-9
Arakane Y, Hogenkamp DG, Zhu YC, Kramer KJ, Specht CA, Beeman RW, Kanost MR,
Muthukrishnan S (2004) Characterization of two chitin synthase genes of the red flour beetle,
222 L. Alamalakala et al.
Tribolium castaneum, and alternate exon usage in one of the genes during development. Insect
Biochem Mol Biol 34:291–304
Arimatsu Y, Kotani E, Sugimura Y, Furusawa T (2007) Molecular characterization of a cDNA
encoding extracellular dsRNase and its expression in the silkworm, Bombyx mori. Insect
Biochem Mol Biol 37:176–183
Aronstein K, Saldivar E (2005) Characterization of a honey bee Toll related receptor gene Am18w
and its potential involvement in antimicrobial immune defense. Apidologie 36:3–14
Aronstein K, Oppert B, Lorenzen M (2011) RNAi in agriculturally-important arthropods. In:
Grabowski PP (ed) RNA processing. In Tech, Shanghai, pp 157–180
Asgari S (2013) MicroRNA functions in insect. Insect Biochem Mol Biol 43:388–397
Auer C, Frederick R (2009) Crop improvement using small RNAs: applications and predictive eco-
logical risk assessments. Trends Biotechnol 27:644–651. doi:10.1016/j.tibtech.2009.08.005
Bachman PM, Huizinga KM, Jensen PD, Mueller G, Tan J, Uffman JP, Levine SL (2016) Ecological
risk assessment for DvSnf7 RNA: a plant-incorporated protectant with targeted activity against
western corn rootworm. Regul Toxicol Pharmacol 81:77–88. doi:10.1016/j.yrtph.2016.08.001
Barnard A-C, Nijhof AM, Gaspar ARM, Neitz AWH, Jongejan F, Maritz-Olivier C (2012)
Expression profiling, gene silencing and transcriptional networking of metzincin metallo-
proteases in the cattle tick, Rhipicephalus (Boophilus) microplus. Vet Parasitol 186:403–414.
doi:10.1016/j.vetpar.2011.11.026
Baum JA, Roberts JK (2014) Progress towards RNAi-mediated insect pest management. In:
Dhadialla TS, Gill SS (eds) Insect midgut and insecticidal proteins, Advances in insect physi-
ology, vol 47. Academic, London, pp 249–295
Baum JA, Bogaert T, Clinton W, Heck GR, Feldmann P, Ilagan O et al (2007) Control of coleopter
an insect pests through RNA interference. Nat Biotechnol 25:1322–1326. doi:10.1038/nbt1359
Baum JA, Cajacob CA, Feldmann P, Heck GR, Nooren I, Plaetinck G, Maddelein W, Vaughn T
(2011) Methods for control of insect infestation in plants and compositions thereof. US Patent
No 7, 943,819
Bautista MAM, Miyata T, Miura K, Tanaka T (2009) RNA interference-mediated knockdown of a
cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval
resistance to permethrin. Insect Biochem Mol Biol 39:38–46
Beck M, Strand MR (2005) Glc1.8 from Microplitis demolitor bracovirus induces a loss of adhe-
sion and phagocytosis in insect high five and S2 cells. J Virol 79:1861–1870
Belles X (2010) Beyond Drosophila: RNAi in vivo and functional genomics in insects. Annu Rev
Entomol 55:111–128
Bettencourt R, Terenius O, Faye I (2002) Hemolin gene silencing by ds-RNA injected into
Cecropia pupae is lethal to next generation embryos. Insect Mol Biol 11:267–271
Bhatia V, Bhattacharya R, Uniyal PL, Singh R, Niranjan RS (2012) Host generated siRNAs attenu-
ate expression of serine protease gene in Myzus persicae. PLoS One 7:e46343. doi:10.1371/
journal.pone.0046343
Blandin S, Moita LF, Köcher T, Wilm M, Kafatos FC, Levashina EA (2002) Reverse genetics in the
mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep 3:852–856
Bolognesi R, Ramaseshadri P, Anderson J, Bachman P, Clinton W, Flannagan R et al (2012)
Characterizing the mechanism of action of double-stranded RNA activity against western corn
rootworm (Diabrotica virgifera virgifera LeConte). PLoS One 7:e47534. doi:10.1371/journal.
pone.0047534
Borgio JF (2010) RNAi mediated gene knockdown in sucking and chewing insect pests. J
Biopesticides 36:153–161
Bronkhorst AW, van Rij RP (2014) The long and short of antiviral defense: small RNA-based
immunity in insects. Curr Opin Virol 7:19–28. doi:10.1016/j.coviro.2014.03.010
Camargo Barbosa GO, Possignolo IP et al (2016) RNA interference as a gene silencing tool to control
Tuta absoluta in tomato (Solanum lycopersicum). Peer J 4:e2673. doi:10.7717/peerj.2673
Caplen NJ, Fleenor J, Fire A, Morgan RA (2000) dsRNA-mediated gene silencing in cultured
Drosophila cells, a tissue culture model for the analysis of RNA interference. Gene 252:95–105
Carrière Y, Crickmore N, Tabashnik BE (2015) Optimizing pyramided transgenic Bt crops for
sustainable pest management. Nat Biotechnol 33:161–168
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 223
Carthew RW, Sontheimer EJ (2009) Origins and mechanisms of miRNAs and siRNAs. Cell
136(4):642–655. doi:10.1016/j.cell.2009.01.035
Casacuberta JM, Devos Y, du Jardin P, Ramon M, Vaucheret H, Nogué F (2014) Biotechnological
uses of RNAi in plants: risk assessment considerations. Trends Biotechnol 33:145–147.
doi:10.1016/j.tibtech.2014.12.003
Chen J, Zhang D, Yao Q, Zhang J, Dong X, Tian H et al (2010) Feeding-based RNA interference
of a trehalose phosphate synthase gene in the brown planthopper, Nilaparvata lugens. Insect
Mol Biol 19(6):777–786. doi:10.1111/j.1365-2583.2010.01038.x
Chen X, Tian H, Zou L, Tang B, Hu J, Zhang W (2008) Disruption of Spodoptera exigua larval
development by silencing chitin synthase gene A with RNA interference. Bull Entomol Res
98(06):613–619
Choi H, Glatter T, Gstaiger M, Nesvizhskii AI (2012) SAINT-MS1: protein-protein interaction
scoring using label-free intensity data in affinity purification-mass spectrometry experiments.
J Proteome Res 11:2619–2624
Christiaens O, Smagghe G (2014) The challenge of RNAi-mediated control of hemipterans. Curr
Opin Insect Sci 6:15–21. doi:10.1016/j.cois.2014.09.012
Christiaens O, Sweveres L, Smagghe G (2014) DsRNA degradation in the pea aphid (Acyrthosiphon
pisum) associated with lack of response in RNAi feeding and injection assay. Peptides 53:307–
314. doi:10.1016/j.peptides.2013.12.014
Chu CC, Sun W, Spencer JL, Pittendrigh BR, Seufferheld MJ (2014) Differential effects of
RNAi treatments on field populations of the western corn rootworm. Pestic Biochem Physiol
110:1–6
Clemens JC, Worby CA, Simonson-Leff N, Muda M, Maehama T, Hemmings BA, Dixon JE
(2000) Use of double-stranded RNA interference in Drosophila cell lines to dissect signal
transduction pathways. Proc Natl Acad Sci U S A 97:6499–6503
Coleman AD, Wouters RH, Mugford ST, Hogenhout SA (2015) Persistence and transgenerational
effect of plant-mediated RNAi in aphids. J Exp Bot 66:541–548
Daly T, Buntin GD (2005) Effects of Bacillus thuringiensis transgenic corn for lepidopteran con-
trol on non-target arthropods. Environ Entomol 34:1292–1301. doi:10.1603/0046-225X(2005
)034[1292:EOBTTC]2.0.CO;2
Dass CR, Choong PF (2008) Chitosan-mediated orally delivered nucleic acids: a gutful of gene
therapy. J Drug Target 16:257–261
Desneux N, Luna MG, Guillemaud T, Urbaneja A (2011) The invasive South American tomato
pinworm, Tuta absoluta, continues to spread in Afro-Eurasia and beyond: the new threat to
tomato world production. J Pest Sci 84:403–408
Devos Y, Álvarez-Alfageme F, Gennaro A, Mestdagh S (2015) Assessment of unanticipated
unintended effects of genetically modified plants on non-target organisms: a controversy
worthy of pursuit? J Appl Entomol 140:1–10. doi:10.1111/jen.12248. [Epub ahead of
print]
Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S,
Scheiblauer S, Couto A, Marra V, Keleman K, Dickson B (2007) A genome-wide transgenic
RNAi library for conditional gene inactivation in Drosophila. Nature 448:151–156
Dubelman S, Fischer J, Zapata F, Huizinga K, Jiang C, Uffman J et al (2014) Environmental
fate of double-stranded RNA in agricultural soils. PLoS One 9:e93155. doi:10.1371/journal.
pone.0093155
Dykxhoorn DM, Lieberman J (2005) The silent revolution: RNA interference as basic biology,
research tool, and therapeutic. Annu Rev Med 56:401–423
Dzitoyeva S, Dimitrijevic N, Manev H (2001) Intra-abdominal injection of double-stranded RNA
into anesthetized adult Drosophila triggers RNA interference in the central nervous system.
Mol Psychiatry 6(6):665–670
EFSA (European Food Safety Authority) (2010a) EFSA panel on plant protection products and
their residues (PPR); scientific opinion on the development of specific protection goal options
for environmental risk assessment of pesticides, in particular in relation to the revision of
the guidance documents on aquatic and terrestrial ecotoxicology (SANCO/3268/2001 and
SANCO/10329/2002). EFSA J 8(10):1821. doi:10.2903/j.efsa.2010.1821. (55 pp)
224 L. Alamalakala et al.
EFSA (European Food Safety Authority) (2010b) EFSA panel on genetically modified organ-
isms (GMO); scientific opinion on the assessment of potential impacts of genetically modified
plants on non-target organisms. EFSA J 8(11):1877. doi:10.2903/j.efsa.2010.1877. (72 pp)
Fabrick JA, Kanost MR, Baker JE (2004) RNAi-induced silencing of embryonic tryptophan oxy-
genase in the Pyralid moth, Plodia interpunctella. J Insect Sci 4(15):9
Farooqui T, Robinson K, Vaessin H, Smith BH (2003) Modulation of early olfactory processing by
an octopaminergic reinforcement pathway in the honeybee. J Neurosci 23:5370–5380
Feinberg EH, Hunter CP (2003) Transport of dsRNA into cells by the transmembrane protein SID-
1. Science 301:1545–1547
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific
genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Fishilevich E, Velez AM, Storer NP, Li HR, Bowling AJ, Rangasamy M, Worden SE, Narva KE,
Siegfried BD (2016) RNAi as a management tool for the western corn rootworm, Diabrotica
virgifera virgifera. Pest Manag Sci 72:1652–1663. doi:10.1002/ps.4324
FSANZ (2013) Response to Heinemann et al. on the regulation of GM crops and foods devel-
oped using gene silencing. http://www.foodstandards.govt.nz/consumer/gmfood/Documents/
Heinemann%20Response%20210513.pdf
Garbutt JS, Bellés X, Richards EH, Reynolds SE (2013) Persistence of double-stranded RNA
in insect hemolymph as a potential determiner of RNA interference success: evidence from
Manduca sexta and Blattella germanica. J Insect Physiol 59:171–178
Gatehouse HS, Gatehouse LN, Malone LA, Hodges S, Tregidga E, Todd J (2004) Amylase activity
in honey bee hypopharyngeal glands reduced by RNA interference. J Apic Res 43:9–13
Georghiou GP, Lagunes-Tejeda A (1991) The occurrence of resistance to pesticides in arthropods:
an index of cases reported through 1989. Food and Agriculture Organization of the United
Nations, Rome
Ghanim M, Kontsedalov S, Czosnek H (2007) Tissue-specific gene silencing by RNA interfer-
ence in the whitefly, Bemisia tabaci (Gennadius). Insect Biochem Mol Biol 37:732–738.
doi:10.1016/j.ibmb.2007.04.006
Ghildiyal M, Zamore PD (2009) Small silencing RNAs: an expanding universe. Nat Rev Genet
10:94–108
Gong L, Chen Y, Hu Z, Hu MY (2013) Testing insecticidal activity of novel chemically synthe-
sized siRNA against Plutella xylostella under laboratory and field conditions. PLoS One 8:88.
doi:10.1371/journal.pone.0062990
Gong LA, Yang XQ, Zhang BL, Zhong GH, Hu MY (2011) Silencing of Rieske iron-sulfur protein
using chemically synthesised siRNA as a potential biopesticide against Plutella xylostella. Pest
Manag Sci 67:514–520. doi:10.1002/ps.2086
Gordon KH, Waterhouse PM (2007) RNAi for insect-proof plants. Nat Biotechnol 25:1231–1232
Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ (2001) Argonaute2, a link
between genetic and biochemical analyses of RNAi. Science 293:1146–1150
Hassanien ITE, Meyering-Vos M, Hoffmann KH (2014) RNA interference reveals allatotro-
pin functioning in larvae and adults of Spodoptera frugiperda (Lepidoptera, Noctuidae).
Entomologia 2:56–64. doi:10.4081/entomologia.2014.169
He B, Chu Y, Yin M, Müllen K, An C, Shen J (2013) Fluorescent nanoparticle delivered dsRNA
toward genetic control of insect pests. Adv Mater Weinheim 25:4580–4584. doi:10.1002/
adma.201301201
Head G, Moar M, Eubanks M, Freeman B, Ruberson J, Hagerty A, Turnipseed S (2005) A multi-
year, large-scale comparison of arthropod populations on commercially managed Bt and non-
Bt cotton fields. Environ Entomol 34:1257–1266
Heinemann JA, Kurenbach B, Quist D (2011) Molecular profiling – a tool for addressing emerging
gaps in the comparative risk assessment of GMOs. Environ Int 37:1285–1293
Horn T, Boutros M (2010) E-RNAi: a web application for the multi-species design of RNAi
reagents – 2010 update. Nucleic Acids Res 38:W332–W339
Horn T, Sandmann T, Boutros M (2010) Design and evaluation of genome-wide libraries for RNA
interference screens. Genome Biol 11:R61
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 225
Horwich MD, Li C, Matranga C, Vagin V, Farley G, Wang P, Zamore PD (2007) The Drosophila
RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in
RISC. Curr Biol 17:1265–1272
Hu X, Richtman NM, Zhao J-Z, Duncan KE, Niu X, Procyk LA, Oneal MA, Kernodle BM,
Steimel JP, Crane VC, Sandahl G, Ritland JL, Howard RJ, Presnail JK, Lu AL, Wu G (2016)
Discovery of midgut genes for the RNA interference control of corn rootworm. Sci Rep
6:30542. doi:10.1038/srep30542
Huang JH, Lee HJ (2011) RNA interference unveils functions of the hypertrehalosemic hormone
on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blatella
germanica. J Insect Physiol 57:858–864
Hunter W, Ellis J, Vanengelsdorp D, Hayes J, Westervelt D, Glick E, Williams M, Sela I, Maori
E, Pettis J et al (2010) Large-scale field application of RNAi technology reducing Israeli acute
paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae). PLoS Pathog
6:e1001160
Hunter WB, Glick E, Paldi N, Bextine BR (2012) Advances in RNA interference: dsRNA treat-
ment in trees and grapevines for insect pest suppression. Southwest Entomol 37:85–87.
doi:10.3958/059.037.0110
Huvenne H, Smagghe G (2010) Mechanisms of dsRNA uptake in insects and poten-
tial of RNAi for pest control: a review. J Insect Physiol 56(3):227–235. doi:10.1016/j.
jinsphys.2009.10.004
ILSI-CERA (2011) Problem formulation for the environmental risk assessment of RNAi plants.
International Life Sciences Institute, Center for Environmental Risk Assessment, Washington,
D.C.
Ivashuta S, Zhang Y, Wiggins BE, Ramaseshadri P, Segers GC, Johnson S et al (2015) Environmental
RNAi in herbivorous insects. RNA 21:840–850. doi:10.1261/rna.048116.114
James C (2014) Global status of commercialized biotech/GM Crops: 2014. ISAAA Brief No. 49.
ISAAA, Ithaca, NY
Jaubert-Possamai S, Le T, Bonhomme G, Christophides J, Rispe GK, Tagu D (2007) Gene knock-
down by RNAi in the pea aphid Acyrthosiphon pisum. BMC Biotechnol 7:63
Joga MR, Zotti MJ, Smagghe G, Christiaens O (2016) RNAi efficiency, systemic properties, and
novel delivery methods for pest insect control: what we know so far. Front Physiol 7:553.
doi:10.3389/fphys.2016.00553
Jose AM, Hunter CP (2007) Transport of sequence-specific RNA interference information between
cells. Annu Rev Genet 41:305–330
Kamath RS, Martinex-Campos M, Zipperlen P, Frasher AG, Ahringer J (2000) Effectiveness of
specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis
elegans. Genome Biol 2:research0002
Katoch R, Sethi A, Thakur N, Murdock LL (2013) RNAi for insect control: current perspective and
future challenges. Appl Biochem Biotechnol 171(4):847–873
Kemp C, Mueller S, Goto A, Barbier V, Paro S, Bonnay F, Dostert C, Troxler L, Hetru C, Meignin
C, Pfeffer S, Hoffmann JA, Imler JL (2013) Broad RNA interference-mediated antiviral immu-
nity and virus-specific inducible responses in Drosophila. J Immunol 190:650–658
Kennerdell JR, Carthew RW (1998) Use of dsRNA-mediated genetic interference to demonstrate
that frizzled and frizzled 2 act in the wingless pathway. Cell 95:1017–1026
Khajuria C, Buschman LL, Chen MS, Muthukrishnan S, Zhu KY (2010) A gut-specific chitinase
gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia
nubilalis larvae. Insect Biochem Mol Biol 40:621–629. doi:10.1016/j.ibmb.2010.06.003
Khajuria C, Li H, Narva K, Rangasamy M, Siegfried B (2013) Effectiveness of dsRNA versus
siRNA in RNAi mediated gene knock-down in western corn rootworm (Diabrotica virgifera
virgifera). In: Program and abstracts, 46th annual meeting of the Society for Invertebrate
Pathology Conference on Invertebrate Pathology and Microbial Control. Society for
Invertebrate Pathology. Pittsburgh, PA
Khajuria C, Vélez AM, Rangasamy M, Wang H, Fishilevich E, Frey MLF, Carneiro N, Premchand
G, Narva KE, Siegfried BD (2015) Parental RNA interference of genes involved in embryonic
226 L. Alamalakala et al.
development of the western corn rootworm, Diabrotica virgifera virgifera LeConte. Insect
Biochem Mol Biol 63:54–62
Kitzmann P, Schwirz J, Schmitt-Engel C, Bucher G (2013) RNAi phenotypes are influenced by the
genetic background of the injected strain. BMC Genomics 14(1):5. doi:10.1186/1471-2164-14-5
Koch A, Kogel KH (2014) New wind in the sails: improving the agronomic value of crop plants
through RNAi-mediated gene silencing. Plant Biotechnol J 12:821–831. doi:10.1111/pbi.12226
Kolliopoulou A, Swevers L (2014) Science direct recent progress in RNAi research in lepidop-
tera?: intracellular machinery, antiviral immune response and prospects for insect pest control.
Curr Opin Insect Sci:1–7. doi:10.1016/j.cois.2014.09.019
Kontogiannatos D, Swevers L, Maenaka K, Park EY, Iatrou K, Kourti A (2013) A functional char-
acterization of a Juvenile Hormone esterase related gene in the moth Sesamia nonagrioides
through RNA interference. PLoS One 8:88. doi:10.1371/journal.pone.0073834
Kumar M, Gupta GP, Rajam MV (2009) Silencing of acetylcholinesterase gene of Helicoverpa
armigera by siRNA affects larval growth and its life cycle. J Insect Physiol 55:273–278.
doi:10.1016/j.jinsphys.2008.12.005
Ladics GS, Bartholomaeus A, Bregitzer P, Doerrer NG, Gray A, Holzhauser T et al (2015) Genetic
basis and detection of unintended effects in genetically modified crop plants. Transgenic Res
24:587–603. doi:10.1007/s11248-015-9867-7
Lawo NC, Wackers FL, Romeis J (2009) Indian Bt cotton varieties do not affect the performance
of cotton aphids. PLoS One 4:e4804
Lee YS, Nakahara K, Pham JW, Kim K, He Z, Sontheimer EJ, Carthew RW (2004) Distinct roles
for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117:69–81
Li X, Zhang M, Zhang H (2011) RNA interference of four genes in adult Bactrocera dorsalis by
feeding their dsRNAs. PLoS One 6(3):e17788. doi:10.1371/journal.pone
Li Y, Romeis J (2010) Bt maize expressing Cry3Bb1 does not harm the spider mite, Tetranychus
urticae, or its ladybird beetle predator, Stethorus punctillum. Biol Control 53:337–344.
doi:10.1016/j.biocontrol.2009.12.003
Liu F, Wang X, Zhao Y, Li Y, Liu Y, Sun J (2015) Silencing the HaAK gene by transgenic plant-
mediated RNAi impairs larval growth of Helicoverpa armigera. Int J Biol Sci 11:67–74.
doi:10.7150/ijbs.10468
Liu S, Ding Z, Zhang C, Yang B, Liu Z (2010) Gene knockdown by introthoracic injection of
double-stranded RNA in the brown planthopper, Nilaparvata lugens. Insect Biochem Mol Biol
40:666–671
Liu Y, Ye X, Jiang F, Liang C, Chen D, Peng J, Kinch LN, Grishin NV, Liu Q (2009) C3PO, an
endoribonuclease that promotes RNAi by facilitating RISC activation. Science 325:750–753
Loy DL, Mogler MA, Loy DS, Janke B, Kamrud K, Scura ED, Harris DLH, Bartholomay LC
(2012) Double-stranded RNA provides sequence dependent protection against infectious myo-
necrosis virus in Litopenaeus vannamei. J Gen Virol 93:880–888
Lum L, Yao S, Mozer B, Rovescalli A, Von Kessler D, Nirenberg M, Beachy PA (2003) Science
299:2039–2045
Lundgren JG, Duan JJ (2013) RNAi-based insecticidal crops. Bioscience 63:657–665. doi:10.1525/
bio.2013.63.8.8
Luo Y, Wang X, Yu D, Kang L (2012) The SID-1 double-stranded RNA transporter is not required
for systemic RNAi in the migratory locust. RNA Biol 9:663–671
Mamta RKR, Rajam MV (2016) Targeting chitinase gene of Helicoverpa armigera by host-
induced RNA interference confers insect resistance in tobacco and tomato. Plant Mol Biol
90:281–292. doi:10.1007/s11103-015-0414-y
Mao J, Zeng F (2012) Feeding-based RNA interference of a gap gene is lethal to the pea aphid,
Acyrthosiphon pisum. PLoS One 7:e48718
Mao J, Zeng F (2014) Plant-mediated RNAi of a gap gene-enhanced tobacco tolerance against the
Myzus persicae. Transgenic Res 23:145–152. doi:10.1007/s11248-013-9739-y
Mao YB, Cai WJ, Wang JW, Hong GJ, Tao XY, Wang LJ, Huang YP, Chen XY (2007) Silencing a
cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance
of gossypol. Nat Biotechnol 25(11):1307–1313
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 227
Mao YB, Tao XY, Xue XY, Wang LJ, Chen XY (2011) Cotton plants expressing CYP6AE14
double-stranded RNA show enhanced resistance to bollworms. Transgenic Res 20:665–673.
doi:10.1007/s11248-010-9450-1
Mao YB, Xue XY, Tao XY, Yang CQ, Wang LJ, Chen XY (2013) Cysteine protease enhances
plant-mediated bollworm RNA interference. Plant Mol Biol 83:119–129. doi:10.1007/
s11103-013-0030-7
Martin D, Maestro O, Cruz J, Mane-Padros D, Belles X (2006) RNAi studies reveal a conserved
role for RXR in molting in the cockroach Blattella germanica. J Insect Physiol 52:410–416
Meister G, Tuschl T (2004) Mechanisms of gene silencing by double-stranded RNA. Nature
431(7006):343–349
Miguel SK, Scott JG (2016) The next generation of insecticides: dsRNA is stable as a foliar-
applied insecticide. Pest Manag Sci 72(4):801–809. doi:10.1002/ps.4056
Miller SC, Brown SJ, Tomoyasu Y (2008) Larval RNAi in Drosophila? Dev Genes Evol 218:505–510
Mitter N, Elizabeth AW, Karl ER, Li P, Jain RG et al (2017) Clay nanosheets for topical delivery
of RNAi for sustained protection against plant viruses. Nature Plants 3:16207. doi:10.1038/
nplants.2016.207
Mohammed A, Diab MR, Abd-Alla SMM, Hussien EHA (2015) RNA interference – mediated
knockdown of vacuolar ATPase genes in pink bollworm, Pectinophora gossypiella. Int J Biol
Pharm Allied Sci 4:2641–2660
Mohr S, Perrimon N (2012) RNAi screening: new approaches, understandings and organisms.
Wiley Interdiscip Rev RNA 2:145–158
Mutti NS, Park Y, Reese JC, Reeck GR (2006) RNAi knockdown of a salivary transcript leading to
lethality in the pea aphid, Acyrthosiphon pisum. J Insect Sci 6:1–7. doi:10.1673/031.006.3801
Mutti NS, Louis J, Pappan LK, Pappan K, Begum K, Chen MS, Park Y, Dittmer N, Marshall J,
Reese JC, Reeck GR (2008) A protein from the salivary glands of the pea aphid, Acyrthosiphon
pisum, is essential in feeding on a host plant. Proc Natl Acad Sci U S A 105:9965–9969
Mysore K, Andrews E, Li P, Duman-Scheel M (2014) Chitosan/siRNA nanoparticle targeting dem-
onstrates a requirement for single-minded during larval and pupal olfactory system develop-
ment of the vector mosquito Aedes aegypti. BMC Dev Biol 14:1. doi:10.1186/1471-213X-14-9
Naito Y, Yamuda T, Mastumiya T, Kumiko UT, Saigo K, Morishita S (2005) dsCheck: highly sen-
sitiveoff-target search software for double-stranded RNA-mediated RNA interference. Nucleic
Acids Res 33:W589–W591
Nakamura T, Mito T, Miyawaki K, Ohuchi H, Noji S (2008) EGFR signaling is required for re-
establishing the proximodistal axis during distal leg regeneration in the cricket Gryllus bimacu-
latus nymph. Dev Biol 319:46–55
Naranjo SE (2005) Long-term assessment of the effects of transgenic Bt cotton on the abundance
of nontarget arthropod natural enemies. Environ Entomol 34:1193–1210
Nayak A, Tassetto M, Kunitomi M, Andino R (2013) RNA interference-mediated intrinsic antiviral
immunity in invertebrates. Curr Top Microbiol Immunol 371:183–200
Osanai-Futahashi M, Tatematsu K-I, Futahashi R, Narukawa J, Takasu Y, Kayukawa T, Shinoda T,
Ishige T, Yajima S, Tamura T, Yamamoto K, Sezutsu H (2016) Positional cloning of a Bombyx
pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis.
Heredity 116:135–145. doi:10.1038/hdy.2015.74
Paim RM, Araujo RN, Lehane MJ, Gontijo NF, Pereira MH (2013) Long-term effects and parental
RNAi in the blood feeder, Rhodnius prolixus (Hemiptera; Reduviidae). Insect Biochem Mol
Biol 43:1015–1020. doi:10.1016/j.ibmb.2013.08.008
Palli SR (2012) RNAi methods for management of insects and their pathogens. CAB Rev 7:1–10
Palli SR (2014) RNA interference in Colorado potato beetle: steps toward development of dsRNA
as a commercial insecticide. Curr Opin Insect Sci 6:1–8. doi:10.1016/j.cois.2014.09.011
Pan H, Xu L, Noland JE, Li H, Siegfried BD, Zhou X (2016) Assessment of potential risks of
dietary RNAi to a soil micro-arthropod, Sinella curviseta Brook (Collembola: Entomobryidae).
Front Plant Sci 7:1028. doi:10.3389/fpls.2016.01028
Perrimon N, Mathey-Prevot B (2007) Applications of high-throughput RNA interference screens
to problems in cell and developmental biology. Genetics 175:7–16
228 L. Alamalakala et al.
Peters L, Meister G (2007) Argonaute proteins: mediators of RNA silencing. Mol Cell
26(5):611–623
Pitino M, Coleman AD, Maffei ME, Ridout CJ, Hogenhout SA (2011) Silencing of aphid genes by
dsRNA feeding from plants. PLoS One 6:e25709
Pradhan SK, Nayak DK, Mohanty S, Behera L, Barik SR, Pandit E, Lenka S, Anandan A (2015)
Pyramiding of three bacterial blight resistance genes for broad-spectrum resistance in deepwa-
ter rice variety, Jalmagna. Rice 8:19. doi:10.1186/s12284-015-0051-8
Price DRG, Gatehouse JA (2008) RNAi-mediated crop protection against insects. Trends
Biotechnol 26(7):393–400
Pridgeon JW, Zhao L, Becnel JJ, Strickman DA, Clark GG, Linthicum KJ (2008) Topically
applied AaeIAP1 double-stranded RNA kills female adults of Aedes aegypti. J Med Entomol
45:414–420
Qi XL, Su XF, Lu GQ, Liu CX, Liang GM, Cheng HM (2015) The effect of silencing arginine
kinase by RNAi on the larval development of Helicoverpa armigera. Bull Entomol Res
105:555–565. doi:10.1017/S0007485315000450
Qian D, Tian L, Qu L (2015) Proteomic analysis of endoplasmic reticulum stress responses in rice
seeds. Sci Rep 5:14255. doi:10.1038/srep14255
Qiu S, Adema CM, Lane T (2005) A computational study of off-target effects of RNA interference.
Nucleic Acids Res 33:1834–1847
Quan GX, Kanda T, Tamura T (2002) Induction of the white egg 3 mutant phenotype by injection
of the double-stranded RNA of the silkworm white gene. Insect Mol Biol 11:217–222
Rajagopal S, Sivakumar N, Agrawal P, Malhotra P, Bhatnagar RK (2002) Silencing of midgut
aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus
thuringiensis toxin receptor. J Biol Chem 277:46849–46851
Ramon M, Devos Y, Lanzoni A, Liu Y, Gomes A, Gennaro A et al (2014) RNAi-based GM plants:
food for thought for risk assessors. Plant Biotechnol J 12:1271–1273. doi:10.1111/pbi.12305
Rangasamy M, Siegfried BD (2012) Validation of RNA interference in western corn rootworm
Diabrotica virgifera virgifera LeConte (Coleoptera, Chrysomelidae) adults. Pest Manag Sci
68:587–591. doi:10.1002/ps.2301
Ratzka C, Gross R, Feldhaa H (2013) Systemic gene knockdown in Camponotus floridanus work-
ers by feeding of dsRNA. Insect Soc 60(4):475–484. doi:10.1007/s00040-013-0314-6
Raybould A (2011) The bucket and the searchlight: formulating and testing risk hypotheses about
the weediness and invasiveness potential of transgenic crops. Environ Biosaf Res 9:123–133
Rinkevich FD, Scott JG (2013) Limitations of RNAi of a6 nicotinic acetylcholine receptor sub-
units for assessing the in vivo sensitivity to spinosad. Insect Sci 20:101–108
Roberts AF, Devos Y, Lemgo GNY, Zhou X (2015) Biosafety research for non-target organ-
ism risk assessment of RNAi-based GE plants. Front Plant Sci 6:958. doi:10.3389/
fpls.2015.00958
Rodrigues TB, Figueira A (2016) Management of insect pest by RNAi – a new tool for crop pro-
tection. In: Abdurakhmonov IY (ed) RNA interference. InTech, Croatia. doi:10.5772/61807
Rodriguez CL, Trujillo BD, Borras HO, Wright DJ, Ayra-Pardo C (2010) RNAi-mediated
knockdown of a Spodoptera frugiperda trypsin-like serine-protease gene reduces suscep-
tibility to a Bacillus thuringiensis Cry1Ca1 protoxin. Environ Microbiol 12:2894–2903.
doi:10.1111/j.1462-2920.2010.02259.x
Roignant JY, Carré C, Mugat B, Szymczak D, Lepesant JA, Antoniewski C (2003) Absence of
transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila.
RNA 9:299–230
Romeis J, Bartsch D, Bigler F, Candolfi MP, Gielkens MMC et al (2008) Assessment of risk
of insect-resistant transgenic crops to nontarget arthropods. Nat Biotechnol 26(2):203–208.
doi:10.1038/nbt1381
Romeis J, Hellmich RL, Candolfi MP, Carstens K, De Schrijver A et al (2011) Recommendations
for the design of laboratory studies on non-target arthropods for risk assessment of genetically
engineered plants. Transgenic Res 20:1–22. doi:10.1007/s11248-010-9446-x
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 229
Romeis J, Raybould A, Bigler F, Candolfi MP, Hellmich RL, Huesing JE, Shelton AM (2013)
Deriving criteria to select arthropod species for laboratory tests to assess the ecological risks from
cultivating arthropod-resistant genetically engineered crops. Chemosphere 90(2013):901–909
Rose RI (ed) (2007) White paper on tier-based testing for the effects of proteinaceous insecti-
cidal plant-incorporated protectants on non-target invertebrates for regulatory risk assessment.
USDA-APHIS and US Environmental Protection Agency, Washington, DC. http://www.epa.
gov/pesticides/biopesticides/pips/non-target-arthropods.pdf
Sadeghi A, van Damme EJM, Smagghe G (2009) Evaluation of the susceptibility of the pea aphid,
Acyrthosiphon pisum, to a selection of novel biorational insecticides via artificial diet. J Insect
Sci 9:65. http://insectscience.org/9.65
Saito K, Sakaguchi Y, Suzuki T, Suzuki T, Siomi H, Siomi MC (2007) Pimet, the Drosophila
homolog of HEN1, mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends.
Genes Dev 21:1603–1608
Saleh MC, van Rij RP, Hekele A, Gillis A, Foley E, O’Farrell PH et al (2006) The endocytic
pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol 8:793–802.
doi:10.1038/ncbl439
Sarkies P, Miska EA (2013) Is there social RNA? Science 341(6145):467–468. doi:10.1126/
science.1243175
Schluns H, Crozier RH (2007) Relish regulates expression of antimicrobial peptide genes in the
honeybee, Apis mellifera, shown by RNA interference. Insect Mol Biol 16:753–759
Schnell J, Steele M, Bean J, Neuspiel M, Girard C, Dormann N et al (2015) A comparative analysis
of insertional effects in genetically engineered plants: considerations for pre-market assess-
ments. Transgenic Res 24:1–17. doi:10.1007/s11248-014-9843-7
Scott JG, Michel K, Bartholomay LC, Siegfried BD, Hunter WB, Smagghe G, Zhu KY, Douglas
AE (2013) Towards the elements of successful insect RNAi. J Insect Physiol 59:1212–1221.
doi:10.1016/j.jinsphys.2013.08.014
Shah C, Förstemann K (2008) Monitoring miRNA mediated silencing in Drosophila melanogaster
S2-cells. Biochim Biophys Acta 1779:766–772
Shakesby A, Wallace I, Isaacs H, Pritchard J, Roberts D, Douglas A (2009) A water-specific aqua-
porin involved in aphid osmoregulation. Insect Biochem Mol Biol 39:1–10. doi:10.1016/j.
ibmb.2008.08.008
Shukla JN, Kalsi M, Sethi A, Narva KE, Fishilevich E, Singh S et al (2016) Reduced stability and
intracellular transport of dsRNA contribute to poor RNAi response in lepidopteran insects.
RNA Biol 13(7):656–669. doi:10.1080/15476286.2016.1191728
Sijen T, Fleenor J, Simmer F, Thijssen KL, Parrish S, Timmons L, Plasterk RH, Fire A (2001) On
the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476
Singh AD, Wong S, Ryan CP, Whyard S (2013) Oral delivery of double stranded RNA in larvae of the
yellow fever mosquito, Aedes aegypti: implications for pest mosquito control. J Insect Sci 13:1–18
Siomi H, Siomi MC (2009) On the road to reading the RNA-interference code. Nature 457:396–404
Sivakumar S, Rajagopal R, Venkatesh GR, Srivastava A, Bhatnagar RK (2007) Knockdown of
aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA
interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein
Cry1Ac. J Biol Chem 282:7312–7319
Smith P (2013) Delivering food security without increasing pressure on land. Glob Food Sec
2(1):18–23
Soares CAG, Lima CMR, Dolan MC, Piesman J, Beard CB, Zeidner NS (2005) Capillary feeding
of specific dsRNA induces silencing of the isac gene in nymphal Ixodes scapularis ticks. Insect
Mol Biol 14:443–452
Storer NP, Babcock JM, Schlenz M, Meade T, Thompson GD, Bing JW, Huckaba RM (2010)
Discovery and characterization of field resistance to Bt maize: Spodoptera frugiperda
(Lepidoptera: Noctuidae) in Puerto Rico. J Econ Entomol 103(4):1031–1038
Sugimoto A (2004) High-throughput RNAi in Caenorhabditis elegans, genome-wide screens and
functional genomics. Differentiation 72:81–91
230 L. Alamalakala et al.
Sun K, Wolters AMA, Loonen AEHM, Huibers RP, van der Vlugt R, Goverse A, Jacobsen E,
Visser RGF, Bai Y (2016) Down-regulation of Arabidopsis DND1 orthologs in potato and
tomato leads to broad-spectrum resistance to late blight and powdery mildew. Transgenic Res
25:123–138. doi:10.1007/s11248-015-9921-5
Surakasi VP, Mohamed AAM, Kim Y (2011) RNA interference of beta 1 integrin subunit impairs
development and immune responses of the beet armyworm, Spodoptera exigua. J Insect
Physiol 57:1537–1544
Suzuki Y, Truman JW, Riddiford LM (2008) The role of Broad in the development of Tribolium
castaneum: implications for the evolution of the holometabolous insect pupa. Development
135:569–577. doi:10.1242/dev.015263
Swevers L, Smagghe G (2012 ) Use of RNAi for control of insect crop pests. In: Smagghe G, Diaz
I, editors. Arthropod-plant interactions: novel insights and approaches for IPM. Dordrecht:
Springer; pp. 177–197.
Swevers L, Broeck JV, Smagghe G (2013) The possible impact of persistent virus infection on the
function of the RNAi machinery in insects: a hypothesis. Front Physiol 4:319. doi:10.3389/
fphys.2013.00319
Szittya G, Burgyan J (2013) RNA interference-mediated intrinsic antiviral immunity in plants.
Curr Top Microbiol Immunol 371:153–181
Tabara H, Grishok A, Mello CC (1998) RNAi in C. elegans: soaking in the genome sequence.
Science 282:430–431
Tabashnik BE, Thierry B, Yves C (2013) Insect resistance to Bt crops: lessons from the first billion
acres. Nat Biotechnol 31:510–521
Tan EL, Tan TM, Chow VTK, Poh CL (2008) Inhibition of enterovirus 71 in virus-infected mice
by RNA interference. Mol Ther 15:1931–1938
Terenius O, Papanicolaou A, Garbutt JS, Eleftherianos I, Huvenne H, Kanginakudru S et al
(2011) RNA interference in Lepidoptera: an overview of successful and unsuccessful stud-
ies and implications for experimental design. J Insect Physiol 57(2):231–245. doi:10.1016/j.
jinsphys.2010.11.006
Thakur N, Upadhyay SK, Verma PC, Chandrashekar K, Tuli R, Singh PK (2014) Enhanced white-
fly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase a
gene. PLoS One 9:88. doi:10.1371/journal.pone.0087235
Tian H, Peng H, Yao Q, Chen H, Xie Q, Tang B et al (2009) Developmental control of a lepi-
dopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut
gene. PLoS One 4(7):e6225. doi:10.1371/journal.pone.0006225
Timmon L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395:854
Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce spe-
cific and potent genetic interference in Caenorhabditis elegans. Gene 263:103–112
Tomizawa M, Noda H (2013) High mortality caused by high dose of dsRNA in the green
rice leafhopper, Nephotettix cincticeps (Hemiptera: Cicadellidae). Appl Entomol Zool
48:553–559
Tomoyasu Y, Denell RE (2004) Larval RNAi in Tribolium (Coleoptera) for analyzing adult devel-
opment. Dev Genes Evol 214:575–578
Tomoyasu Y, Miller SC, Tomita S, Schoppmeier M, Grossmann D et al (2008) Exploring systemic
RNA interference in insects: a genome-wide survey for RNAi genes in Tribolium. Genome
Biol 9:R10. doi:10.1186/gb-2008-9-1-r10
Torres JB, Ruberson JR (2005) Canopy- and ground- dwelling predatory arthropods in commercial
Bt and non-Bt cotton fields: patterns and mechanisms. Environ Entomol 34:1242–1256
Torres JB, Ruberson JR (2007) Abundance and diversity of ground-dwelling arthropods of
pest management importance in commercial Bt and non-Bt cotton fields. Ann Appl Biol
150:27–39
Turner CT, Davy MW, MacDiarmid RM, Plummer KM, Birch NP, Newcomb RD (2006) RNA
interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-
stranded RNA feeding. Insect Mol Biol 15:383–391
10 Insect RNAi: Integrating a New Tool in the Crop Protection Toolkit 231
Ulrich J, Dao VA, Majumdar U, Schmitt-Engel C, Schwirz J, Schultheis D et al (2015) Large scale
RNAi screen in Tribolium reveals novel target genes for pest control and the proteasome as
prime target. BMC Genomics 16:674. doi:10.1186/s12864-015-1880-y
Ulvila J, Parikka M, Kleino A, Sormunen R, Ezekowitz RA, Kocks C et al (2006) Double-stranded
RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J Biol
Chem 281:14370–14375. doi:10.1074/jbc.M513868200
Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Borgio JF, Singh PK et al (2011) RNA
interference for the control of whiteflies (Bemisia tabaci) by oral route. J Biosci 36:153–161.
doi:10.1007/s12038-011-9009-1
US EPA (2007) White paper on tier-based testing for the effects of proteinaceous insecticidal
plant-incorporated protectants on non-target arthropods for regulatory risk assessments. United
States Environmental Protection Agency, Washington, DC
US EPA (2014) Scientific advisory panel minutes No. 2014-02 (Arlington, VA), 1–77. Available
online at: http://www.epa.gov/scipoly/sap/meetings/2014/january/012814minutes.pdf
Valdes VJ, Sampieri A, Sepulveda J, Vaca L (2003) Using double-stranded RNA to prevent in vitro
and in vivo viral infections by recombinant baculovirus. J Biol Chem 278:19317–19324
Valdes VJ, Athie A, Salinas LS, Navarro RE, Vaca L (2012) Correction: CUP-1 is a novel protein
involved in dietary cholesterol uptake in Caenorhabditis elegans. PLoS One 7(8). doi:10.1371/
annotation/5a203055-6c15-43b0-96ad-0fbd5eb9b810
Vélez AM, Fishilevich E, Matz N, Storer NP, Narva KE, Siegfried BD (2017) Parameters for suc-
cessful parental RNAi as an insect pest management tool in western corn rootworm, Diabrotica
virgifera virgifera. Genes 8:7. doi:10.3390/genes8010007
Vijayendran D, Airs PM, Dolezal K, Bonning BC (2013) Arthropod viruses and small RNAs. J
Invertebr Pathol 114(2):186–195
Walker WB, Allen ML (2011) RNA interference-mediated knockdown of IAP in Lygus line-
olaris induces mortality in adult and preadult life stages. Entomol Exp Appl 138:83–92.
doi:10.1111/j.1570-7458.2010.01078.x
Wang Y, Zhang H, Li H, Miao X (2011) Second-generation sequencing supply an effective way
to screen RNAi targets in large scale for potential application in pest insect control. PLoS One
6:e18644
Whangbo JS, Hunter CP (2008) Environmental RNA interference. Trends Genet 24(6):297–305.
doi:10.1016/j.tig.2008.03.007
Whitten MMA, Facey PD, Del Sol R, Fernández-Martínez LT, Evans MC, Mitchell JJ et al (2016)
Symbiont-mediated RNA interference in insects. Proc Biol Sci 283:20160042. doi:10.1098/
rspb.2016.0042
Whyard S, Singh AD, Wong S (2009) Ingested double-stranded RNAs can act as species-specific
insecticides. Insect Biochem Mol Biol 39:824–832. doi:10.1016/j.ibmb.2009.09.007
Winston WM, Molodowitch C, Hunter CP (2002) Systemic RNAi in C. elegans requires the putative
transmembrane protein SID-1. Science 295:2456–2459. doi:10.1126/science.1068836
Wu F, Wang PY, Zhao QL, Kang LQ, Xia DG, Qiu ZY, Tang SM, Li MW, Shen XJ, Zhang GZ
(2016) Mutation of a cuticle protein gene, BmCPG10, is responsible for silkworm non-moult-
ing in the 2nd instar mutant. PLoS One 11:88. doi:10.1371/journal.pone.0153549
Wuriyanghan H, Rosa C, Falk BW (2011) Oral delivery of double-stranded RNAs and siRNAs
induces RNAi effects in the potato/tomato psyllid, Bactericerca cockerelli. PLoS One 6:e27736
Xiong Y, Zeng H, Zhang Y, Xu D, Qiu D (2013) Silencing the HaHR3 Gene by transgenic plant-
mediated RNAi to disrupt Helicoverpa armigera development. Int J Biol Sci 9:370–381.
doi:10.7150/ijbs.5929
Xu J, Wang X-F, Chen P, Liu FT, Zheng SC, Ye H, Mo MH (2016) RNA interference in moths:
mechanisms, applications and progress. Genes 7:88. doi:10.3390/genes7100088
Xu W, Han Z (2008) Cloning and phylogenetic analysis of sid-1-like genes from aphids. J Insect
Sci 8:30. doi:10.1673/031.008.3001
Xue XY, Mao YB, Tao XY, Huang YP, Chen XY (2012) New approaches to agricultural insect pest
control based on RNA interference. Adv Insect Physiol 42:73–117
232 L. Alamalakala et al.
Yan T, Chen H, Sun Y, Yu X, Xia L (2016) RNA interference of the ecdysone receptor genes EcR
and USP in grain aphid (Sitobion avenae F.) affects its survival and fecundity upon feeding on
wheat plants. Davies TGE, ed. Int J Mol Sci 17(12):2098. doi:10.3390/ijms17122098
Yang C, Preisse EL, Zhang H, Liu Y, Dai L, Pan H, Pan H, Zhou X (2016) Selection of reference
genes for RT-qPCR analysis in Coccinella septempunctata to assess un-intended effects of
RNAi transgenic plants. Front Plant Sci 7:1672. doi:10.3389/fpls.2016.01672
Yang J, Han ZJ (2014) Efficiency of different methods for dsRNA delivery in cotton bollworm
(Helicoverpa armigera). J Integr Agric 13:115–123. doi:10.1016/S2095-3119(13)60511-0
Yao J, Rotenberg D, Afsharifar A, Barandoc AK, Whitfield AE (2013) Development of RNAi
methods for Peregrinus maidis, the corn planthopper. PLoS One 8:e70243
Yu N, Christiaens O, Liu JM, Niu J, Cappelle K, Caccia S, Huvenne H, Smagghe G (2013)
Delivery of dsRNA for RNAi in insects: an overview and future directions. Insect Sci 20:4–14
YuQ LT, Feng G, Yang K, Pang Y (2008) Functional analysis of the putative antiapoptotic genes,
p49 and iap4, of Spodoptera litura nucleopolyhedrovirus with RNAi. J Gen Virol 89:1873–1880
Zamore PD (2001) RNA interference: listening to the sound of silence. Nat Struct Mol Biol
8(9):746–750
Zeynep A, Horn T, Boutros M (2005) E-RNAi: a web application to design optimized RNAi con-
structs. Nucleic Acids Res 33:W582–W588. doi:10.1093/nar/gki468. Web Server issue
Zha W, Peng X, Chen R, Du B, Zhu L, He G (2011) Knockdown of midgut genes by dsRNA-trans-
genic plant-mediated RNA interference in the hemipteran insect Nilaparvata lugens. PLoS
One 6(5):e20504. doi:10.1371/journal.pone.0020504
Zhang H, Li FL, Cheng C, Jiao DX, Zhou Z, Cheng LG (2013) The identification and characteri-
sation of a new deltamethrin resistance-associated gene, UBL40, in the diamondback moth,
Plutella xylostella (L). Gene 530:51–56. doi:10.1016/j.gene.2013.07.075
Zhang X, Zhang J, Zhu KY (2010) Chitosan/double-stranded RNA nanoparticle-mediated RNA
interference to silence chitin synthase genes through larval feeding in the African malaria mos-
quito (Anopheles gambiae). Insect Mol Biol 19:683–693
Zhang X, Mysore K, Flannery E, Michel K, Severson DW, Zhu KY et al (2015) Chitosan/interfer-
ing RNA nanoparticle mediated gene silencing in disease vector mosquito larvae. J Vis Exp
97:52523. doi:10.3791/52523
Zhang Y, Wiggins B, Lawrence C, Petrick J, Ivashuta S, Heck G (2012) Analysis of plant derived
miRNAs in animal small RNA datasets. BMC Genomics 13:381
Zhao HM, Yi X, Hu Z, Chen SH, Dong XL, Gong L (2013) RNAi-mediated knockdown of cata-
lase causes cell cycle ar rest in SL-1 cells and results in low survival rate of Spodoptera litura
(Fabricius). PLoS One 8:e59527
Zhao YY, Yang G, Wang-PG YMS (2008) Phyllotreta striolata (Coleoptera: Chrysomelidae): argi-
nine kinase cloning and RNAi-based pest control. Eur J Entomol 105:815–822
Zhou XG, Wheeler MM, Oi FM, Scharf ME (2008) RNA interference in the termite Reticulitermes
flavipes through ingestion of double-stranded RNA. Insect Biochem Mol Biol 38:805–815.
doi:10.1016/j.ibmb.2008.05.005
Zhu F, Xu J, Palli R, Ferguson J, Palli SR (2011) Ingested RNA interference for managing the
populations of the Colorado potato beetle, Leptinotarsa decemlineata. Pest Manag Sci 67:175–
182. doi:10.1002/ps.2048
Zhu F, Cui Y, Walsh DB, Lavine LC (2014) Application of RNA interference toward insecticide
resistance management. In: Chandrasekar R, Tyagi BK, Gui ZZ, Reeck GR (eds) Short views
on insect biochemistry and molecular biology, vol 2. International Book Mission, Academic,
Manhattan, pp 595–619. Chapter-27
Zhu J-Q, Liu S, Ma Y, Zhang J-Q, Qi H-S, Wei Z-J et al (2012) Improvement of pest resistance
in transgenic tobacco plants expressing dsRNA of an insect-associated gene EcR. PLoS One
7:e38572. doi:10.1371/journal.pone.0038572
Egg-Laying Behaviour of Caryedon
serratus (Olivier) on the Essential Oils 11
of Skimmia anquetilia
Manjul Gondwal, Bhanu Pratap Singh Gautam,
and Navneet Kishore
Abstract
Nature has been a good source of medicinal agents for thousands of years, and
a vast number of modern drugs have been isolated from natural sources based
on the information about their uses in traditional medicine. The genus Skimmia
contains essential oils, coumarins and alkaloids. The principal constituent of
essential oil, ‘linalyl acetate’ is used in manufacture of cosmetics, perfumery
and flavouring. Egg-laying behaviour/antifeedant activity of Caryedon serra-
tus (Olivier) on the essential oils of flower and leaf of Skimmia anquetilia was
studied by choice experiment and was observed that number of eggs decreased
as the concentration of oil increased. The maximum number of eggs was
observed on solvent control. This showed egg-laying deterrent activity in
flowers as well as leaves with essential oil of Skimmia anquetilia at 1.5%
concentrations.
M. Gondwal
Department of Chemistry, Hemwati Nandan Bahuguna Garhwal University,
Srinagar 246174, India
e-mail: m.gondwal@gmail.com
B.P.S. Gautam
Department of Chemistry, Banaras Hindu University, Varanasi 221005, India
e-mail: gautambps@gmail.com
N. Kishore (*)
Department of Plant Science, University of Pretoria, Pretoria 0002, South Africa
e-mail: kishore.navneet6@gmail.com
11.1 Introduction
In many parts of the tropics, mainly in semiarid areas, groundnut (Arachis hypo-
gaea L.) that belongs to the family Leguminosae (Fabaceae) (Beghmin et al. 2003)
is an important commercial crop, also known as peanut earthnut, goobers, pinders,
etc. Other members of this family include cowpea, soybeans, tamarind, melon, etc.
(Ashle 1993). After the sixteenth century, Portuguese introduced groundnut into
Nigeria although groundnut originated from South America (Brazil) (Adeyemi
1968). Groundnut production in Africa has been estimated at 4.6 metric tons, with
Nigeria being the major producers in Africa (Ashle 1993). According to Nyilra,
Nigeria’s production of unshelled nut is about 2.6 metric tons annually from a land
area of approximately 2.5 million hectares (Nyilra 1988). Groundnut thrives best
on a well-drained sandy-loam soil; this type of soil facilitates easy penetration of
pegs and their development, hence their harvesting (Yayock 1984). Weiss (2000)
suggested that temperature range of 25–30 °C, rainfall of 500–1000 mm and a PH
range of 6.0–6.5 are considered optimum for groundnut production (Weiss 2000).
Groundnut is a major cash crop which serves as a foreign exchange earner prior to
the petroleum boom in Nigeria (Adeyemi 1968). According to Aribisala, the crop
is a good source of protein, fats and oil, vitamins, etc. Shelled groundnuts are fried,
roasted and salted which is eaten as snacks (Aribisala 1993). The crop serves as
raw materials for some food industries and also as feed concentrate for livestock
(Oaya et al. 2012).
Groundnuts are very important source of nutrition in human diet, are often con-
sumed either directly or as oil and are affected mainly by insects and pathogens.
Bruchid beetle, Caryedon serratus (Oliver), is one of the major storage pests affect-
ing the groundnut produce causing damage up to 70–80% in stored groundnuts
(Harish et al. 2012). It seems that in storage bruchids, besides causing direct dam-
age to groundnut, increase the contamination of aflatoxin in the stored groundnuts.
Aflatoxin shows considerable significance due to its deleterious effects on human
being, poultry and livestock (Abbas 2005; Chaytor et al. 2011; Diaz et al. 2010;
Hifnawy et al. 2004; Iheshiulor et al. 2011; Taranu et al. 2010; Vijayasamundeeswari
et al. 2009; Williams et al. 2010). It is a potent carcinogenic, mutagenic and immu-
nosuppressive agent, produced as secondary metabolites by the fungi, Aspergillus
flavus, A. parasiticus and A. nomius, on a variety of food commodities (Essono et al.
2009; Kurtzman et al. 1987).
Among the insect pests attacking the stored groundnuts, the groundnut seed bee-
tle (bruchid), Caryedon serratus (Olivier), is the only insect species known to infest
kernels and intact pods and is thus potentially the most important pest of unshelled
groundnut. The bruchid, Caryedon serratus (Olivier), is a serious pest of legumes in
West Africa, besides other hosts such as Acacia arabica, Tamarindus indica and
Bauhinia rufescens (Hall 1954; Diaollo and Huignard 1993). In Sudan, it attacks
groundnuts in the Rahad Scheme and also other parts of the country. The literature
on the biology of C. serratus is scanty and is only available for the other bruchids,
Callosobruchus maculatus, and other related species, C. chinensis. There is much
confusion in the past regarding the identity of the groundnut borer, Caryedon
11 Egg-Laying Behaviour of Caryedon serratus (Olivier) on the Essential Oils 235
serratus. More than 20 names were used before the name C. serratus was adopted
(Murkerji and Chatterjee 1957). C. serratus was given several common names, e.g.
tamarind beetle, groundnut bruchid, groundnut seed beetle and groundnut borer
(Davey 1958; Southgate and Pope 1957; Green 1960).
Olivier C. serratus was described as having a prognathous head type and distinct
serrate antennae (Singh 1977). Its body length varied from 6 to 7 mm and has dark
reddish brown spots with smudgy black spots on the wings. It has large prominent
compound eyes and is distinguished by the presence of broad-hind femur with con-
spicuous comb of spines. The femur has a strong spike in the middle followed by
10–14 smaller spikes. Davey studied the gender characteristics of C. serratus and
found that the abdominal segments of the female are wholly covered by the elytron,
while that of the male is partly covered, i.e. the elytron doesn’t reach the last abdom-
inal segment (Davey 1958b). Sexual dimorphism was distinguished by observing
the pygidium. In the case of males, pygidium projected downwards so that in dorsal
view it was hidden by the elytra, whereas in females pygidium projected beyond the
elytra and dorsally visible. The observations indicated that the female bruchid lived
longer than males (Figs. 11.1 and 11.2).
Fig. 11.1 Larva, adult male and female of C. serratus (Issoufou et al. 2016)
236 M. Gondwal et al.
Fig. 11.2 Life cycle and damage of C. serratus (Ouedraogo et al. 2016)
11.2 E
gg-Laying Behaviour/Antifeedant Activity Against
Caryedon serratus (Olivier)
Groundnut (Arachis hypogea L.) is an important legume cash crop for the tropical
farmers and its seeds contain high amount of edible oil (43–55%) and protein (25–
28%) (Reddy et al. 2003). Groundnuts are susceptible to the attack of many insect
pests when stored. Among these, the groundnut seed beetle (bruchid), Caryedon
serratus (Olivier), is the only insect species known to infest kernels and intact pods
and is thus potentially the most important pest of unshelled groundnut (Devi and
Rao 2005).
Antifeedant activity of extracts of Skimmia anquetelia and Aegle marmelos
(Rutaceae family) against the forest pest Plecoptera reflexa, Popular defoliator,
Clostera reflexa, Bamboo leafroller and Crysiptya coclesalis was reported (Negi
et al. 2006). Prates reported that some essential oils have acute toxicity, repellency,
feeding inhibition or harmful effects on the reproductive systems of insects. Leaf
powder, seed kernel powder and oil extracted from the seeds of Azadirachta indica
and leaf powder and oil extracted from the leaves of Eucalyptus camaldulensis and
benzene hexachloride (BHC) were tested at 1, 3 and 5% against Caryedon serratus
(Olivier) (Prates et al. 1998). Eucalyptus leaf oil and neem oil at 3 and 5% were as
efficient as BHC and significantly (P = 0.0001) reduced egg laying by C. serratus,
whereas Eucalyptus leaf powder had no significant effect (Atta and Ahmed 2002).
11 Egg-Laying Behaviour of Caryedon serratus (Olivier) on the Essential Oils 237
Some products from plant origin such as neem seed kernel powder, neem leaf
powder and Lantana camara leaf powder at 25 g/kg groundnuts pods and two aro-
matic oils (Citronella and Palmarosa) at 15 mL/kg pods against the groundnut bru-
chid, Caryedon serratus. It was found that Citronella oil and Palmarosa oil gave
total protection to groundnut pods by inhibition of oviposition by the bruchid for 6
months with an efficacy equal to that of malathion dust (malathion 5D). Among the
plant powders, L. camara had a good oviposition deterrent activity but lost effec-
tiveness gradually after 1 month (Kumari et al. 1998).
From thousands of years, nature has been a source of medicinal agents, and a num-
ber of modern drugs have been isolated from natural sources based on the informa-
tion about their use in traditional medicine (Cragg and Newman 2001). The herbal
medicines have been derived from rich traditions of ancient civilizations and scien-
tific heritage because the evidences of the use of traditional medicaments in Indian,
Chinese, Egyptian, Greek, Roman and Syrian texts date back to about 5000 years
(Kamboj 2000). Natural product extracts of therapeutic uses are of immense
importance as reservoirs of structural and chemical diversity. A review on national
pharmacopoeias from several countries reveals that at least 120 distinct chemical
substances from different plants have utility as life-saving drugs (Chauhan and
Tiwari 2003). Natural products are obtained from plants, microbes and animals.
Among these, plants are more reliable and useful because of easy availability and
higher concentration of constituents. Some of the plant products nowadays are
used either in their natural form or as derivatives were used originally for other
purposes, such as arrow poisons, as part of the religious or other rituals and even
as cosmetics (Gupta 1993). Terpenes are among the most widespread diversified
and chemically interesting group of natural products that are typically found in
higher plants, mosses, liverworts, algae and lichens, although some are of insect or
microbial origin (Langenheim 1994). The plants of family Rutaceae consist of 162
genera and 1650 species and are generally shrubs, trees or sometimes herbs, with
aromatic volatile oils contained in glands visible at the surface of leaves (Throne
2007). The family Rutaceae is of great economic importance as the source of citrus
fruits for commerce such as the citrons, lemons, limes, oranges, pomelos (Genus:
Citrus), the kumquat (Fortunella) and the trifoliate orange (Poncirus). Rutaceae
also includes many ornamental plants such as Choisya ternata, Murraya panicu-
lata and Severinia buxifolia which are cultivated for their glossy green foliage,
sweet-scented flowers or bright attractive fruits (Nair and Nayar 1997). In India,
the family Rutaceae is represented by 29 genera and 114 species (Karthikeyan
2000). In Uttarakhand, the family Rutaceae is represented by 15 genera and 29
species (Uniyal et al. 2007).
The genus Skimmia, belonging to the family Rutaceae, contain about seven or
eight species distributed in the Himalayas, East Asia and the Philippines (Gaur
1999). They are generally evergreen shrubs and small trees. The plant is strongly
scented and locally used to manufacture incense sticks (Nair and Nayar 1977).
Skimmia species are used as ornamentals, condiments, food-flavouring agent and
238 M. Gondwal et al.
also in the manufacturing of scenting soaps. The leaves are used as insecticides,
pesticides and also in the treatment of smallpox, cold, fever and headache (Ahmed
et al. 2004; Qureshi et al. 2009). Skimmia species show antibacterial, antifungal and
antifeedant activity (Sampurna and Nigam 1979; Ahmad and Sultana 2003). The
genus Skimmia is a rich source of essential oils, coumarins and alkaloids (Wu 1987;
Rahman et al. 1998; Razdan et al. 1988). The principal constituent of essential oil,
‘linalyl acetate’, is very important in the manufacture of soaps, cosmetics, perfum-
ery and flavouring (Skaria et al. 2007). The essential oil is antiseptic and found to be
effective against Staphylococcus and Streptococcus bacteria. Some feed-deterrent
and antitumour constituents have also been reported from Skimmia species
(Escoubas et al. 1992; Hashi 1991). Leaves are used as incense in various religious
Hindu rites, eaten in curries by hill tribes and also used for flavouring food in
Kashmir (Skaria et al. 2007). In China, it is used for darkening hair and also for hair
washing (Skaria et al. 2007). The soot obtained from the burning of leaves is inhaled
for the treatment of body pain, fever and flu by the local population of Hazara,
Pakistan (Sultana et al. 2002).
Skimmia anquetilia N.P. Taylor & Airy Shaw (Rutaceae) is an aromatic ever-
green erect or creeping shrub (up to 1.5 m high) found in the subalpine region of the
Garhwal Himalayas (Gaur 1999). There are about 7–8 species of genus Skimmia
distributed in the Himalayas, East Asia and the Philippines, out of which five spe-
cies are found in India. It is locally known as Nair patti, Nayalpatti or Nihar in
Kumaoun; Kasturchara or Gurlpatta in Jaunsar; Nair in Garhwal, Patar and Nar,
near Kashmir; Nar, Barru, Shalangli or Patrang in Punjab; and Kedar patti in various
hill regions (Goel et al. 1989). It has the following characteristics: evergreen, usu-
ally dioecious or monoclinous, unarmed, leaves alternate, simple, terminal inflores-
cences, thyrsiform, sepals (3 or) 4 or 5(–7), distinct or basally connate, petals (3 or)
4 or 5(–7), imbricate in bud, stamens (3 or) 4 or 5(–7), distinct, rudimentary in
female flowers, with a fleshy drupaceous berry fruit, with 1–5 one-seeded leathery
pyrenes and with seeds that are ovoid to ellipsoid. Five to six species are found in
the East, South and Southeast Asia (Zhang et al. 2008).
Different concentrations of flower oil and leaf oil of Skimmia anquetilia were pre-
pared in water containing Tween 20 as surfactant as given below:
The studies on egg-laying behaviour of flower and leaf oil of Skimmia anquetilia
were carried out with Caryedon serratus (Olivier). Nucleus culture of Caryedon
serratus (Olivier) was obtained from IICT Hyderabad and maintained on ground-
nuts in the incubator at 35 ± 2 °C and 70% relative humidity at the Department of
Chemistry, G. B. Pant University of Agriculture and Technology, Pantnagar.
11.6 C
hoice Experiment and Observation
for Adult Emergence
To investigate the egg-laying behaviour of flower and leaf oil of Skimmia anquetilia
oil against Caryedon serratus (Olivier), open choice experiment was conducted at
room temperature (35 ± 2 °C) (Sundria and Kumar 2004). For these experiments,
glass petri dishes (diameter 180 mm) were used. On each glass petri dish, the five
equidistant circles were marked and labelled as C1, C2, C3, C and U where C1 stands
for concentration (0.5%), C2 stands for concentration (1.0%), C3 stands for concentra-
tion (1.5%) of oils, C stands for solvent control (water + tween 20) and U stands for
untreated groundnuts (Figs. 11.3 and 11.4). Ten unshelled groundnuts were used for
each treatment. The unshelled groundnuts were dipped in different concentrations of
oils and control and dried at room temperature to evaporate the solvent. Then, the
treated and untreated groundnuts were placed in their respective labelled circles. Each
treatment was replicated thrice. Freshly immersed five pairs of insects, i.e. five males
and five females of Caryedon serratus (Olivier), were released in the centre of the
petri dish per replication. The petri dish was finally covered and left for mating and
egg laying. The number of eggs and number of dead insects were counted after each
24 h till all insects were dead. The groundnuts which contained eggs were removed
and replaced with freshly treated groundnuts. The removed groundnuts were collected
in separate boxes, respectively, and development of eggs to adults was observed.
Fig. 11.3 Choice
experiment of flower oil of
Skimmia anquetilia
240 M. Gondwal et al.
Fig. 11.4 Choice
experiment of leaf oil of
Skimmia anquetilia
The present studies on egg-laying behaviour of essential oils of flowers and leaves
of Skimmia anquetilia against Caryedon serratus (Olivier) by choice experiment
were carried out on three concentrations of oils (0.5, 1.0 and 1.5%), solvent control
(water + tween 20) and control (untreated) groundnuts. It was observed that the
number of eggs decreased as the concentration of oil increased. The maximum
numbers of eggs was observed on solvent control (Tables 11.1 and 11.2).
In flower and leaf oil, almost similar trend for egg-laying response was observed.
These results have also been shown in Figs. 11.5 and 11.6.
In flower oil maximum numbers of eggs were observed on solvent control fol-
lowed by C1, untreated, C2 and C3 groundnuts.
The observations on the number of eggs laid per day in each replication of flower
oil have clearly shown that egg laying decreased day by day and the maximum
number of eggs was laid in first 6 days and then was almost arrested. Similar trend
was also observed in solvent control (water + tween 20) and control (untreated)
groundnuts.
In the case of leaf oil, surprisingly the number of eggs laid on the first day was
less as compared to the second, third and fourth day. The egg laying was arrested
completely on the eighth day. Similar trend was observed in solvent control
(water + tween 20) and control (untreated) groundnuts. The results have been
showed in Figs. 11.7 and 11.8.
In both the cases, the maximum number of eggs was observed on solvent control
and minimum number of eggs on maximum-tested concentration (1.5%) of oil.
Generally, female insects prefer egg laying where more food security is available
for development of future generation. In this study, it has been clearly shown that
higher concentration of oil reduces egg laying. Similar results on neem oil have
been reported by Atta and Ahmed.
Table 11.1 No. of eggs laid by Caryedon serratus on unshelled groundnuts treated with 0.5 %, 1.0 % & 1.5 % concentrations (C1, C2 & C3) of essential oil from flower treated with control and untreated of
Skimmia anquetilia.
Table 11.2 No. of eggs laid by Caryedon serratus on unshelled groundnuts treated with 0.5 %, 1.0 % & 1.5 % concentrations (C1, C2 & C3) of essential oil from flower treated with control and untreated of
Skimmia anquetilia.
Untreated C1
18% 20%
Solvent Control C2
24% 21%
C3
17%
Fig. 11.5 No. of eggs (%) on unshelled groundnuts treated with flower essential oil of Skimmia
anquetilia
Untreated C1
18% 20%
Solvent Control C2
24% 21%
C3
17%
Fig. 11.6 No. of eggs (%) on unshelled groundnuts treated with leaf essential oil of Skimmia
anquetilia
40
35
30
No of Eggs
25
C1
20 C2
C3
15
Control
10 Untreated
0
1 2 3 4 5 6 7 8 9 10 11 12
No of Days
Fig. 11.7 No. of eggs on unshelled groundnuts per day treated with flower essential oil of Skimmia
anquetilia
244 M. Gondwal et al.
45
40
35
30
No of eggs
C1
25
C2
20
C3
15
10 Control
5 Untreated
0
1 2 3 4 5 6 7 8
No of Days
Fig. 11.8 No. of eggs on unshelled groundnuts per day treated with leaf essential oil of Skimmia
anquetilia
DNA makers have made a significant contribution to rapid rise of molecular studies
of genetic relatedness, phylogeny, population dynamics or gene and genome map-
ping in insects over the last 15 years (Loxdale and Lushai 1998; Avise 2000;
Severson et al. 2001; Heckel 2003). Many improvements have been made to enhance
power of resolution (ability to reveal more informative polymorphisms from less
number of loci), reproducibility, cost and time consumption in developing and scor-
ing the marker loci. Since then, application of DNA markers in entomology has
gone through and is still undergoing a perceptible change in continuously accom-
modating new technologies for robust and less expensive genotyping methods. The
unparalleled advancements in modern molecular biology, particularly in those of
DNA marker technology, have already created a wealth of technical know-how that
finds useful applications of these markers especially in molecular ecology research
in insects (Hoy 2003). Usually, mitochondrial DNA (mtDNA) has been a choice of
marker for studying genetic variations in insect species. In addition, mtDNA
sequences are often transferred to the nucleus, called nuclear mtDNA (Numt).
Variations in copy number and size of Numts are also used to assess the interspecific
diversity of these loci in insects (Richly and Leister 2004). Microsatellites are also
used as popular markers in insect studies because of high profusion and highly vari-
able nature of their loci in genome. However, the introduction of random amplified
polymorphic DNA (RAPD) technique (Williams et al. 1990). The use of PCR (poly-
merase chain reaction)-based fingerprinting assays gained popularity generally
because of the easy-to-perform and easy-to-score procedures for these marker loci.
But, because RAPD markers suffer from poor reproducibility, the use of these
11 Egg-Laying Behaviour of Caryedon serratus (Olivier) on the Essential Oils 245
markers in insect ecological studies was limited (Black 1993). The advancement of
amplified fragment length polymorphism (AFLP) (Vos et al. 1995) technology was
adopted as a better substitute to generate more numbers of multiloci reproducible
markers, more reliable than RAPD markers. Today, molecular marker technology
has reached a new height with the power and the precision of modern genomic tools.
High-throughput genotyping methods are now available that can be used for
genome-wide mutation screening in hundreds or even thousands of individuals (as
much as 300,000 genotypes) as quickly as in a day.
DNA markers such as mtDNA, RAPD, AFLP, microsatellites and ESTs have been
used as popular marker systems in insect genetics research. Although there are natu-
ral advantages and disadvantages linked with each marker systems, the choice of
applying them depends upon the objectives of a study. An additional class of mark-
ers generated by arbitrarily primed PCR-based DNA fingerprinting methods, such
as RAPD, DNA amplification polymorphisms (DAF) and arbitrarily primed PCR
(AP-PCR), are easy to achieve and comparatively easy to score (Black 1993).
However, these markers, because of poor reliability and reproducibility, are not
appropriate for population studies (Black 1993).
Sembène et al. (2010) and Vos et al. (1995) extracted, amplified and sequenced DNA
with standard protocols described elsewhere. According to them, each sequence was
obtained from the DNA of a single seed—beetle. The abdomen, elytra and antennae
were kept apart to avoid contamination by fungi and nematodes and to permit conse-
quent morphological observations. A partial cytochrome B (Cyt. B) end region was
PCR-amplified using the primers 5′-TATGTACTACCATGAGGACAAATATC-3′ and
5′-ATTACACCTCCTAATTTATTAGGAAT-3′. The ribosomal DNA was targeted for
PCR, amplified and sequenced with primer CIL (5′ GCGTTCGAARTGCGATGATCAA
3′) and CIU (5′GTAGGTGAACCTGCAGAAGG3′). For both markers, PCR amplifi-
cation was performed in 25 μL reaction volume, 2.5 μL enzyme buffer supplied by the
manufacturer, 2.5 mM MgCl2, 0.6 unit of Taq polymerase (Promega), 17.5 pM of each
primer, 25 nM of each DNTP and 1 μL of DNA extract. They obtained 518 bp of the
partial Cyt. B gene in 30 C. serratus populations. The arrangement was basic and
involved no insertions. The sequences could be clearly aligned and showed 22 different
haplotypes due to 51 polymorphic sites. Of these sites, 94% were stinginess informative.
The number of nucleotide differences in pairwise comparisons of C. serratus popula-
tions ranged from 0% to 16.1% due to a large part of C. serratus sampled on C. sieberi-
ana and the others. Within the same host species, the number ranged from 0% to 0.02%.
246 M. Gondwal et al.
Conclusions
In both the essential oils of S. anquetilia, the maximum number of eggs was
observed in solvent control and minimum number of eggs in maximum-tested
concentration (1.5%) of oil. These studies have clearly shown that higher con-
centration of oil reduced egg laying. Similar results on neem oil have been
reported. The essential oils of flowers and leaves of Skimmia anquetilia sup-
pressed egg laying by females of Caryedon serratus; this effect increases with
increase in concentration of oil, but there was no effect on further development
of eggs to adults. Essential oils are complex mixture of terpenoids with different
quantitative makeup, and due to synergetic effects of constituents, they can be
used as safe, eco-friendly and alternative source of synthetic antifeedant and
insecticides. Essential oils extracted from C. schoenanthus, L. multiflora and O.
americanum have proved lethal concentration (CL50) on adult beetle C. serratus
thus testifying their efficacy. The egg-laying ability of female C. serratus in the
presence of essential oils was disturbed and even inhibited. The reduction of
females’ lifespan equally affected reduction of the number of laid eggs. This
inhibition of the egg laying may be attributed to the physiological disturbance
caused by essential oils on the females, and in addition, it was reported that
monoterpenes inhibit the oviposition of females. Also the egg laying by females
of Bruchidae (C. maculatus and C. subinnotatus PIC) is inhibited by the pres-
ence of the essential oil vapours. Essential oil vapours extracted from aromatic
plants may cause physiological dysfunction which disturbs the normal function-
ing of insect ovarioles. This situation may block the sphincters that are likely to
push eggs towards the genital opening for their emission. The evaluation of the
persistence of essential oils indicated that the effectiveness of oils decreases with
the duration of treatment. Similar results were reported on the cowpea beetle C.
maculatus.
References
Abbas HK (2005) Aflatoxin and food safety. CRC, Boca Raton, FL, p 587
Adeyemi SA (1968) Storage entomology. In: Proceedings of Agricultural Society of Nigeria,
vol. 34
Ahmad KF, Sultana N (2003) Studies on bioassay directed antifungal activity of medicinal plants
Calotropis procera, Skimmia laureola, Peltophorum pterocarpum and two pure natural com-
pounds ulopterol and 4-methoxy-1-methyl-3-(2’S-hydroxy-3’-ene butyl)-2-quinolone. J Chem
Soc Pak 25:328–330
Ahmed E, Arshad M, Ahmad M, Saeed M, Ishaque M (2004) Ethnopharmacological survey of
some medicinally important plants of Galliyat areas of NWEP, Pakistan. Asian J Plant Sci
3:410–415
Aribisala OA (1993) Raw material revolution and impact on industrialization in Nigeria. Mednet,
Lagos, p 150
Ashle J (1993) Drought and crop adaptation. In: Rowland RJ (ed) Dry land farming in Africa.
Macmillan Education, London, p 10
Atta EHA, Ahmed A (2002) Comparative effects of some botanicals for the control of the seed
weevil Caryedon serratus Olivier (Col., Bruchidae). J Appl Entomol 126:577–582
11 Egg-Laying Behaviour of Caryedon serratus (Olivier) on the Essential Oils 247
Avise JC (2000) Phylogeography: the history and formation of species. Harvard University Press,
Cambridge, MA, p 684
Beghmin J, Diop N, Sewadah M (2003) The impact of groundnut trade liberalization. Tim Hill
Publishing Company Limited India, Mumbai, pp 241–242
Black WC (1993) PCR with arbitrary primers: approach with care. Insect Mol Biol 2:1–6
Chauhan SK, Tiwari G (2003) Effect of sources and levels of nitrogen on partitioning of androgra-
pholide in kalmegh (Andrographis paniculata (Burm. F.) Wall. ex Nees.) Indian J Plant Physiol
8:60–62
Chaytor AC, See MT, Hansen JA, de Souza ALP, Middleton TF, Kim SW (2011) Effects of chronic
exposure of diets with reduced concentrations of aflatoxin and deoxynivalenol on growth and
immune status of pigs. J Anim Sci 89:124–135
Cragg GM, Newman DJ (2001) Medicinals for the milennia. Ann NY Acad Sci 953:3–25
Davey PM (1958) The effect of insect infestation on the quality of decorticated groundnuts with
special reference to storage at high and low humidities. Trop Sci 1:296–307
Devi DR, Rao NV (2005) Note on the performance of different groundnut pod protectants against
groundnut bruchid, Caryedon serratus (Olivier). Legum Res 28:229–230
Diaollo A, Huignard J (1993) Oviposition of four strains of Caryedon serratus (Olivier).
(Coloeptera: Bruchidae) in the presence of pods or seeds of their wild and cultivated host
plants. J Afr Zool 107:113–120
Diaz GJ, Murcia HW, Cepeda SM (2010) Bioactivation of aflatoxin B1 by turkey liver micro-
somes: responsible cytochrome P450 enzymes. Br Poult Sci 51:828–837
Escoubas P, Fukushi Y, Lajide L, Mizutani J (1992) A new method for fast isolation of antifeedant
compounds from complex mixture. J Chem Ecol 18:1819–1832
Essono G, Ayodele M, Akoa A, Foko J, Filtenborg O, Olembo S (2009) Aflatoxin-producing
Aspergillus spp. and aflatoxin levels in stored cassava chips as effected by processing practices.
Food Control 20:648–654
Gaur RD (1999) Flora of the District Garhwal Northwest Himalaya (with ethnobotanical notes).
Trans Media, Srinagar Garhwal
Goel CL, Shiva MP, Mehra SN, Rai YC, Badola KC (1989) Production of essential oil from S.
laureola (S. orborescens) in Uttar Pradesh. Indian Perfum 33:161–164
Green AA (1960) The control of insects infesting groundnuts after harvest in Gambia. Trop Sci
2:130–133
Gupta R (1993) Medicinal and Aromatic plants in India. RAPA Publication, Bangkok, pp 117–180
Hall DW (1954) The quality of the groundnuts from Gambia with special reference to insect infes-
tation. Colon PI Anim Prod 4:227–235
Harish G, Holajjer P, Savaliya SD, Gedia MV (2012) Population density on damage of groundnut
by Caryedon serratus. Annu Plant Prot Sci 20:217–219
Hashi M (1991) Antitumour effects and anticomplementary effects of tree polysaccharides.
Bulletin of the Forestry and Forest Products Research Institute, Ibaraki, pp 121–148
Heckel DG (2003) Genomics in pure and applied entomology. Annu Rev Entomol 48:235–260
Hifnawy MS, Mangoud AM, Eissa MH, Edin EN, Mostafa Y, Abouel-Magd Y, Sabee EI, Amin I,
Ismail A, Morsy TA, Mahrous S, Afefy AF, El-Shorbagy E, El-Sadawy M, Ragab H, Hassan
MI, El-Hady G, Saber M (2004) The role of aflatoxin-contaminated food materials and HCV
in developing hepatocellular carcinoma in Al-Sharkia Governorate. J Egypt Soc Parasitol
34:479–488
Hoy MA (2003) Insect molecular genetics, 2nd edn. Academic/Elsevier, San Diego, CA
Iheshiulor OOM, Esonu BO, Chuwuka OK, Omede AA, Okoli IC, Ogbuewu IP (2011) Effects of
mycotoxins in animal nutrition: a review. Asian Australas J Anim Sci 5:19–33
Issoufou O, Roger NC, Dona D, Wendgoundi G (2016) Insecticide activity of essential oils on the
development of eggs and adult of Caryedon serratus olivier (Coleoptera: Chrysomelidae), pest
of stored groundnut. J Agric Ecol Res Int 9:1–10
Kamboj VP (2000) Herbal medicine. Curr Sci 78:35–39
Karthikeyan S (2000) A statistical analysis of flowering plants of India. In: Singh NP et al (eds)
Flora of India, Introductory Vol, Part II. Botanical Survey of India, Calcutta, pp 201–217
248 M. Gondwal et al.
Kumari DA, Kumar ST, Reddy VS (1998) Management of groundnut bruchid, Caryedon serratus
(Olivier) with botanicals in stored groundnut. Pest Manag Econ Zool 6:127–131
Kurtzman CP, Horn BW, Hesseltine CW (1987) Aspergillus nomius, a new aflatoxin-producing
species related to Aspergillus flavus and Aspergillus tamarii. Antonie Van Leeuwenhoek
53:147–158
Langenheim JH (1994) Higher plant terpenoids: A phytocentric overview of their ecological roles.
J Chem Ecol. 20:1223–1280
Loxdale HD, Lushai G (1998) Molecular markers in entomology. Bull Entomol Res 88:577–600
Murkerji G, Chatterjee S (1957) Morphology of the genital structures of some of the Bruchidae
(Lariidae) of Indian and Ceylon and their economic importance. Indian J Entomol 13:1–28
Nair KN, Nayar MP (1977) Rutaceae. In: Hajra PK, Nair VJ, Daniel P (eds) Flora of India.
Botanical Survey of India, Calcutta, pp 259–408
Nair KN, Nayar MP (1997) Rutaceae. In: Hajra PK, Nair UJ, Daniel P (eds) Flora of India, vol 4.
Botanical Survey of India, Calcutta, pp 259–261
Negi DS, Sharma R, Dashmana PP, Negi P, Mundrawal R, Kalia S (2006) Antifeedant activity of
some Rutaceae family plants against forest pests/insects. Acta Cienc Indica 32:209
Nyilra NZ (1988) Pest of grain legumes and their control in Uganda. Academic, London, pp 22–24
Oaya CS, Malgwi AM, Samaila AE (2012) Damage potential and loss caused by the groundnut
bruchid caryedon serratus olivier (Coleoptera: Bruchidae) on stored groundnut and tamarind in
Yola. IOSR J Agric Vet Sci 1:58–62
Ouedraogo I, Hema SA, Guenda W, Dakouo D (2016) Influence of host plants on the development
of Caryedon serratus olivier (Coleoptera: Chrysomelidae, Bruchinae), insect pest of groundnut
stocks in Burkina Faso. Adv Entomol 4:279–292
Prates HT, Santos JP, Waquil JM, Fabris JD, Oliveria AB, Foster JE (1998) Insecticidal activity of
monoterpenes against Rhyzopertha dominica (F.) and Tribolium castneum (Herbst). J Stored
Prod Res 34:243–249
Qureshi RA, Ghufran MA, Gilani SA, Yousaf Z, Abbas G, Batool A (2009) Indigenous medicinal
plants used by local women in Southern Himalayan regions of Pakistan. Pak J Bot 41:19–25
Rahman AU, Sultana N, Jahan S, Choudhary MI (1998) Phytochemical Studies on S. laureola. Nat
Prod Res 12:223–229. http://www.informaworld.com/smpp/title~content=t713398545~db=all
~tab=issueslist~branches=12-v1212
Razdan TK, Harkar S, Qadri B, Qurishi MA, Khuroo MA (1988) Lupene derivatives from S. lau-
reola. Phytochemistry 27:1890–1892
Reddy TY, Reddy VR, Anbumozhi V (2003) Physiological responses of groundnut (Arachis hypo-
gaea L.) to drought stress and its amelioration: a critical view. Plant Growth Regul 41:75–88
Richly E, Leister D (2004) NUMTs in sequenced eukaryotic genomes. Mol Biol Evol 21:1081–1084
Sampurna T, Nigam SS (1979) Antibacterial study of some Indian essential oils. Indian Perfum
23:205–207
Sembène M, Kébé K, Delobel A, Rasplus JY (2010) Phylogenetic information reveals the peculiar-
ity of Caryedon serratus (Coleoptera, Chrysomelidae, Bruchinae) feeding on Cassia sieberi-
ana DC (Caesalpinioideae). Afr J Biotechnol 9:1470–1480
Severson D, Brown W, Knudson SE, D. L. (2001) Genetic and physical mapping in mosquitoes:
molecular approaches. Annu Rev Entomol 46:183–219
Singh T (1977) A key to the northwest Indian bruchids. Ent Mon Mag 113:219–231
Skaria BP (2007) Aromatic Plants, New India Publishing Agency, Pitam Pura, New Delhi, P 210
Southgate BJ, Pope RP (1957) The ground seed beetle. A Study of its identity and taxonomic posi-
tion. J Nat Hist 10:669–672
Sultana N, Khan MR, Choudhary MI (2002) Triterpene and Coumarins from Skimmia laureola.
Natural Product Letters, 16, 305-313
Sundria MM, Kumar A (2004) Biology of groundnuts bruchid, Caryedon serratus (OL) on differ-
ent test hosts. Annu Plant Prot Sci 12:9–12
Taranu IE, Marin DP, Burlacu R, Pinton P, Damian V, Oswald I (2010) Comparative aspects of
in vitro proliferation of human and porcine lymphocytes exposed to mycotoxins. Arch Anim
Nutr 64:383–393
11 Egg-Laying Behaviour of Caryedon serratus (Olivier) on the Essential Oils 249
Abstract
Chinese caterpillar fungus, Cordyceps sinensis, is a traditional Chinese medicine
that parasitize Hepialidae larvae and grows on mountain at an altitude of 3000 m
high. After infection, the larvae become rigid, latent feel the humidity in the
Cordyceps topsoil depth of 10 cm, which is formed by a stiff end pumping out
insects long rod-like stroma (i.e., Cordyceps sinensis fruiting and sclerotium of
dead insects (larvae corpse) to form a composite) when the spring comes and
snow melt. It is mainly produced in Qinghai, Tibet, Sichuan, Yunnan, Gansu,
Guizhou, and other provinces and autonomous regions of the alpine zone and
snow-capped mountains and plains.
Z. Pan
School of Biology and Basic Medical Science, Soochow University, Suzhou 215123, China
e-mail: pzh1971@163.com
that isolated from Yunnan by Chinese bent neck Diqing (Tolypocladium sinensis
C.L. Li) on Cordyceps sinensis, its chemical composition and natural Cordyceps is
very similar; Liu Xijin (1989) from Sichuan Kangding production of Cordyceps
stroma and sclerotium, isolated strains by multiple batches, large quantity, in many
ways, China of colony and sporulation characteristics, and reported by Shen Nanying
et al. are basically the same, they will be the strain named Chinese Hirsutella
(Hirsutella sinensis, Liu, Guo, Yu & Zeng; Dai Ruqin (1989) from Yunnan, Qinghai,
Diqing) Longhua strains named Paecilomyces hepiali (Paecilomyces hepiali, Chen
& Dai Liang Zongqi (1991)) from Lixian of Sichuan Cordyceps in sclerotium iso-
lated from another Hyphomycetes, named China Chrysosporium (Chrysosporium
sinensis Liang); Wang Wei (1997) isolated from Yunnan Lijiang Cordyceps to a new
species, named China Verticil (Verticillium sinensis Wang sp. nov).
12.1.2 Functions
reaction, and the presence of GMF and IL-6 was confirmed in the supernatant. The
above results showed that the expression of mice orally fed with HW can activate
macrophages to regulate the IL-6, at the same time improve the hematopoietic growth
factors (such as Peyer’s patch cells secreted GM-CSF and IL-6) expression, the latter
effect on the immune system, which play the regulating role of the immune system.
Chen et al. reported that the use of PKA and PKC inhibitors in MA-10 mice
Leydig tumor cells to do the prognosis, the amount of steroids to reduce the
amount of 61%. Moreover, the production of acute steroid production-induced
protein (StAR) was induced by the dose and concentration of Cordyceps sinensis.
The expression was inhibited by PKA and PKC inhibitors. It was showed that the
cells stimulated by the activation of PKA and PKC signal transduction pathway
stimulated the production of steroids, which mediated immune responses. Kon
based on the mycelium of HW is on the antifatigue and anti-stress effect were
found in mice fed with 150 and 300 mg-kg-1-d-1 HW after the swimming endur-
ance was significantly prolonged by 75–90 min and with relieve fatigue. When the
mice were fed with the 150 mg-kg-1-d-1 8 h, the stress of the adrenal gland,
spleen, thymus, and thyroid gland were inhibited when the 48 h was in a state of
stress. HW as the brake parameters were also significantly inhibited the increase
of total cholesterol and decreased the level of alkaline phosphatase. These effects
were related to the improvement of immune function. Studies have reported that
Cordyceps aqueous extract could significantly inhibit mice spleen cells to con-
canavalin A (ConA), LPS proliferation, reduce the delayed hypersensitivity
induced by allogenic antigen.
12.1.2.2 Hepatoprotective
Protective effects of CC14 and (TAA) on the prevention and treatment of CS in mice
with liver injury. Results show that CC14- or TAA-induced liver injury in mice after
oral administration of CS liposome, all of which can cause liver damage of ALT
have different degrees of decline and the decline range and dose related. Pathological
sections of liver tissue in mice showed that there was no significant improvement in
the liver tissue of CC14 damage induced by CS liposome, which may be due to the
irreversible liver injury induced by TAA in mice.
12.1.2.4 Antitumor
12.1.3.1 Cordycepin
Cordycepin is 3′-deoxy adenosine or deoxyadenosine (3-deoxyadenosine), nucleic
acid derivative containing nitrogen glycosides, is a purine alkaloid, and is a kind of
nucleoside antibiotics. It is an important active component of Cordyceps sinensis,
which has significant pharmacological effects. As early as 1970s in the twentieth
century, it was found that the effect of the inhibition of tumor, anti-plasmodium, and
mRNA translation was found to have a role in the inhibition of tumor. A 1990s study
found that plays an important role in adding the expression of adenosine deaminase
inhibitor on the antitumor activity of cordycepin, to achieve a breakthrough, the
United States will start in 1997 for a period of cordycepin in clinical trials, treatment
of acute anterior B and T lymphocytic leukemia patients, at the same time, cordyce-
pin also exhibit antifungal strong, anti HIV-virus type, and selective inhibition of
clostridium bacteria activity. The synthesis of cordycepin can interfere with RNA
and DNA cells, inhibit abnormal cells (cancer cells) of the division and different as
the difference in cell RNA polymerase tool, and has the special effect to protect life
and repair gene, genetic code, now cordycepin in the United States as anticancer,
antiviral drugs has entered the three phases of beds.
At present, the domestic and foreign reports that the artificial synthesis of
Chinese caterpillar fungus can also be extracted from the Chinese caterpillar fun-
gus, but the yield is very low, only a small amount of the product supply of Chinese
caterpillar fungus, the market price is 2 070 000 dollars/kg (more than 98%). It can
be expected that with the application of Cordyceps in the field of medicine and the
development of related products, market demand will be greatly increased, so the
market potential is huge, its extraction and purification methods of research are
worth the attention.
the molecular weight is about 15,000. Shen Min reported by the gel filtration method
to measure Cordyceps polysaccharide molecular weight of 43,000, was composed of
mannose, galactose, and glucose =10.3:3.6:1. Su Pu in Tibetan Medicine Research
reported in the separation of two kinds of polysaccharides from Cordyceps sinensis,
a molecular weight of approximately 23,000. The monosaccharide composition of
D-mannitol and D-galactose, the molar ratio of 3:5; another and Shen Min reported
the same. Yuan Jianguo and other kinds of polysaccharides were separated into seven
groups, the molecular weight of these seven kinds of polysaccharides and the com-
position and molar ratio of the sugars were different. Sasaki et al. confirmed that the
antitumor activity of the fungal polysaccharide was related to the molecular weight,
only when the molecular weight is more than 16,000; it has the antitumor activity.
Cordyceps sinensis grows in the mountains at 3000–5000 m above sea level. From
a worldwide perspective, Cordyceps is only distributed in four countries such as
China, Nepal, Bhutan, and India. (Fig. 12.2)
In China, the main Cordyceps bat moth larvae parasitized in Qinghai Tibet
Plateau alpine environment, mainly distributed in Qinghai, Yunnan, Sichuan, Tibet,
and Gansu provinces, including Qinghai Cordyceps best quality, maximum yield.
According to records, in the 1980s the annual production of Cordyceps sinensis in
Qinghai province is 30~35t, accounting for about 70~80% of the national
Fig. 12.2 The
environment of Cordyceps
sinensis growth. The
Cordyceps sinensis is the
worm being infected by
Cordyceps sinensis and the
fruiting body being grow
out of the ground when the
snow to melt causes by
more and more warmth
weather on mountain
260 Z. Pan
production, the main producing areas of Yushu and Golog, two states, especially in
the highest yield of Yushu Cordyceps sinensis.
Cordyceps resources are increasingly scarce, therefore, most of the current use of
Cordyceps sinensis fungus fermentation of hyphae.
12.1.6.5 Cylinders
Cordyceps produced from Qinghai, isolated by the Navy Medical Research Institute
and the Shanghai Cooperation with the Third Factory made of “Cordyceps” cap-
sule, attending primary thrombocytopenia and chronic obstructive pulmonary dis-
ease. In addition, Baoding pharmaceutical plant made of Cordyceps “liver Ganbao”
capsule, the treatment of chronic hepatitis; Kunming Kangfu pharmaceutical fac-
tory trial production of “Cordyceps sinensis” capsules, have a role in arrhythmia. In
addition, there are treatment of liver disease, “off Austria Ling” in Cordyceps and so
on. In recent years, with the rapid development of nutrition and health products,
Cordyceps food series of development has attracted wide attention, the development
of varieties is increasing. Its medicinal value is more and more people are happy.
We study the ultimate goal of Cordyceps sinensis is to use them for the benefit of
mankind. One of the foster entity is one of the goals that people pursue. Cordyceps
cultivation of artificial research has been nearly 40 years, and now it is difficult to
cultivate our laboratory fruiting body (whether artificial or insects on the basis of the
body), Cordyceps sinensis appears to be large-scale artificial cultivation to achieve
There is a long way to go. However, with the completion of the genome sequencing,
joint research in different areas of scientific research and attention from all sectors
of society, we look forward to large-scale cultivation of Cordyceps sinensis bacteria
can be achieved as soon as possible.
262 Z. Pan
Cordyceps militaris (L.) Link, also known as Cordyceps militaris (Cordyceps mili-
taris (L.) Link), is also known as Cordyceps militaris (North Cordyceps),
Ascensycota belonging to the fungus community, Hypocreales, clavicipitaceae,
Widely distributed species, all provinces and autonomous regions are distributed,
the child seat in the spring to fall in semi-buried in the forest or under the foliage of
Lepidoptera insect pupae grow.
Cordyceps as a traditional precious traditional Chinese medicine, our people
have long been aware of its medicinal value. Li Shizhen (1518) in the “Compendium
of Materia Medica,” pointed out that the cicada can attending “pediatric Tianjian
epilepsy, cry at night, palpitations.” According to King of soldiers (1986) research,
when the “cicadas” includes the cicada Cordyceps (C.sobolifera) and Cordyceps
militaris and other parasitic on the body or the pupa body of Cordyceps. Cordyceps
militaris cultivation since the success of pharmacological and toxicological aspects
of research has been widely carried out. Studies have shown that Cordyceps milita-
ris and Cordyceps sinensis has a very similar role, non-toxic side effects, Cordyceps
sinensis can be used as a substitute (Cordyceps sinensis sace). In recent years, with
the people of Cordyceps militaris health care efficacy and a variety of medicinal
value awareness, its development and utilization of research attention, and in
genomics, pharmacology, active ingredients and product development and other
aspects of a great deal progress.
Cordyceps militaris is Cordyceps militaris infected silkworm pupae, then pupa was
hardening, the appropriate temperature and humidity in the fruiting bodies formed
under the growth of insect compound (Fig. 12.2).
12 Research Advancement of Insect Origin Fungus Cordyceps 263
The results showed that the genome of Cordyceps militaris was 32.2 Mb smaller
than that of Metarhizium anisopliae and found that more than 5000 expressed
sequence tags were predicted to encode 9684 Protein gene, gene number:
AEVU00000000; interproScan analysis identified 2736 conserved proteins.
Successful determination of the genome of Cordyceps militaris has brought great
value to the study of Cordyceps militaris (Fig. 12.3).
In the study of functional genes, Zheng Z carried out the transgenic research of
Cordyceps sinensis in 2011, and concluded that Agrobacterium can be used as a tool
to transgene into Cordyceps militaris. In 2012, Zheng and Z cloned and analyzed
the cDNA of cytochrome C oxidase gene of Cordyceps militaris (ORF) encodes a
530-amino-acid protein. In 2012, Zhou XW et al. Cloned and expressed the SOD
gene of Cordyceps sinensis. Xiong, C. In 2013, It was found that the activity of
glutathione peroxidase (GPX) directly affected the activity of Cordyceps militaris.
In the classification of bacteria, no systematic research on Cordyceps militaris
has been found. The main difficulty is that the strains with different morphologies
and traits are found, and the resource collection and preservation are difficult and
the coefficient of variation is too large.
12.2.3 Effect
have a direct effect, indicating the role of cordycepin anti-tumor immunity mecha-
nism. Cordycepin enrichment of Cordyceps militaris is a promising candidate in
cancer immune adjuvant;
12.2.3.2 Anti-Microbial
Cordyceps militaris on a variety of pathogenic microorganisms were inhibited.
Cordyceps has been found active ingredient in the antibacterial activity of the main
component of cordycepin. Ahn, YJ reported in 2000, ten micrograms of cordycepin
dose can inhibit Clostridium paraputrificum and Clostridium perfringens,
Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis,
Lactobacillus acidophilus and Lactobacillus casei and other common pathogens.
research reports, typical of Zhu, S. and J. with different doses of FCM and DCM in
2013 after oral administration in 15 days, in the cyclophosphamide (CY) induced
immunosuppressive mice; in vitro, spleen cell extraction and overlapping with CY
from healthy mice, then cultured with different doses the FCM or DCM; Cordyceps
polysaccharide (CMP) content of cordycepin, adenosine, total phenolic (TP) and
total flavonoids (TF) on FCM and DCM were determined. FCM was significantly
higher than DCM in the spleen and thymus index, spleen lymphocyte activity, mac-
rophage function, promoting IL-2, IFN and in vivo and in vitro. The contents of
CMP and TF in FCM were significantly higher than those in DCM. Conclusion:
These results suggest that FCM is superior to DCM in enhancing immunity. Cui
Xinying et al. (2004) reported that Cordyceps can significantly improve the weight
of mice spleen and thymus, and the amount and phagocytic activity of peritoneal
macrophages.
12.3.1 Historical
12.3.2.2 Sclerotia
Sclerotia, the cicadas cicadas larvae infected with Cordyceps bacteria after the para-
site, sclerotia formed by the three-tier structure, the outermost of the milky white
called “bacteria was” structure, thickness 0.5 mm, top grade gold cicada Layer
Fig. 12.4 Medicinal
Mushroom Cordyceps
Cicadae. Being
Composition by fruiting
body like flower with
orange color and Cicada
12 Research Advancement of Insect Origin Fungus Cordyceps 267
completely wrapped worm; the middle layer is the shell of the cicada larvae, the
name of medicine in the “cicada”; the innermost layer of “mycelium” that is trans-
formed from the cicada’s nutrients. Therefore, nature has this three-tier structure
and carry full of cicada flower spore powder Need for the cicadas is very rare.
Sclerotia long kidney shape, slightly curved, about 2.5–3.5 cm, diameter 1–1.4 cm,
the shape of cicada larvae. Rod body with 1–2 rod-shaped sub-blocks, also known
as spore stems, long or curled, branched or unbranched, long 3–7 cm, diameter
3–4 mm, the original eco-egg white, dried milky white, Some are dark brown, the
top slightly swollen, the surface of powdery cicada spore powder, the shape of
flowers.
Containing liver sugar, Cordyceps acid, a variety of alkaloids and ergosterol and so
on. Japan isolated from the parasite part of the alkali-soluble polysaccharides of
cicada, cicadae contains a large number of chitin and nitrogen, and its function to
reduce rhabdomyolysis, and blocking the ganglion.
The taste: Gan; cold; non-toxic.
Herb source: for the ergot fungus Corynebacterium parvum spores stems stem
beam, the great seat of the grass and a total parasitic worm body.
The chemical composition of the Chinese herbal medicine: the great cicadae
fruit contains galactomannan (galactomannan), from D-mannose (D-mannose) and
268 Z. Pan
12.3.5 Characterstics
1. Corynebacterium parachyposis: This product from the parasite and its head out
of the composition of the spore stalk. Body length oval, slightly curved, about
3 cm, diameter 1–4 cm, the surface of brown, mostly gray mycelium coating, the
head of the cluster bundle spores. Sporophyll-branched or unbranched, 1.6–6 cm
long, subequal to solid and sessile; robust minister elliptic, elliptic or spindle-
shaped, 5–8 mm long, 2–3 mm in diam. Diameter 1–2 mm, brown to dark brown.
Crisp, easy to break, the body is full of white or white soft material like. Gas
slightly fragrant, tasteless.
2. Large cicada: This product from the worm and its front-end sub-base composi-
tion. 1–2 cm in length, branched or unbranched, 3–7 cm long, brown; head swol-
len, tapering, 4–6 mm long, 6.5–7 mm in diameter, with small dots on the surface
Of the orifice), the stem diameter of 4–5 mm. Worm body white, the body cov-
ered with white hyphae. Crisp, easy to break. Gas micro, tasteless.
3. Corynebacterium coryneform bacterium sporophyte bottle-shaped, central
enlargement, terminal tapering or suddenly narrow, long 5–8 μm, diameter
2–3 μm often clustered in the bundle of silk, shaped like a petal. Conidia oblong,
spindle-shaped or narrow kidney-shaped, long 5–14 μm, diameter 1.8–3.5 μm,
with 1–3 fat droplets.
4. Large cicada grass seat head cross-section: subcapsular shell embedded in the
sub-seat, bottle-shaped, long 350–540 μm, diameter 125–300 μm; subcapsular
cylindrical, flat spherical cap length 262.5–378 μm, diameter 6.2–9.1 μm; asco-
spores slender filamentous, multiple septum, ruptured rectangular small segment
length 3.5–5.2 μm, diameter 1.7–2.6 μm.
12.3.6 Functionality
Studies at home and abroad have shown that cicada has many functions, such as
improving immunity, resisting fatigue, protecting kidney, improving sleep, resisting
tumor, protecting liver, resisting radiation and eyesight, and is a magic ancient
Chinese medicine. The contents of arsenic, mercury, lead and other toxic heavy
metals were similar to those of natural Cordyceps sinensis, but the contents of arse-
nic, mercury, lead and other heavy metals were not detected in artificially cultured
270 Z. Pan
12.3.6.1 Tonic
Chen Wanqun et al. reported that the results show that cicada and a variety of
Cordyceps in the main components of amino acids similar to the content of more
consistent. Scholars have recognized a variety of amino acids is one of the material
basis for tonic strong, pharmacological experiments show that a variety of Cordyceps
and Cordyceps amino acids have different levels of beneficial effects.
12.3.6.2 Anti-Fatigue
Cicada spent decoction can significantly extend the swimming time of mice, signifi-
cantly increased atmospheric hypoxia and high temperature conditions in the sur-
vival time, prove that cicada has anti-fatigue and anti-stress.
12.3.6.3 Hypnosis
Cicada spent mice 1 h after the determination of the number of autonomous activi-
ties within 10 min significantly less than the control group; cicada can significantly
prolong the sleep time of mice, shorten the disappearance of pentobarbital sodium
righting reflex time; cicadas can also increase Mice in the unit time to sleep. This
shows that cicada has better sedative and hypnotic effects. At the same time studies
have shown that artificial culture and the role of natural cicada close.
12.3.6.4 Analgesia
The results showed that cicadae had significant effects on the chemical and thermal
pain of mice, and the effects of cicadella on chemical and thermal pain in mice were
significant Inhibition. It is proved that the cicada has good antipyretic and analgesic
effects. This experiment and Chen Zhu and others experiments have proved that
cicadae artificial culture products also have the same effect.
12.3.6.5 Immunization
The cicadae strains were artificially fermented to produce cicadae hyphae, and the
polysaccharides from the cicadae were extracted. The Cordyceps sinensis polysac-
charide was used as the positive control and the polysaccharides of Grifola frondosa
as the reference. The lymphatic transformation test, Ea and E roses test, Specific
immune rosette test (macrophage phagocytosis test, anti-sheep red blood cell
(SRBC) antibody titer test, the results show that the polysaccharide has a significant
role in improving the immune function.
Shanghai University of Traditional Chinese Medicine Longhua Hospital
Professor Chen Yiping, director of clinical Rudy and other clinical application con-
firmed that cicadas has reduced blood, urine creatinine, increased endogenous cre-
atinine clearance rate, improve serum protein content and reduce urinary protein
excretion and other functions. Therefore, early and mid-term efficacy of patients
12 Research Advancement of Insect Origin Fungus Cordyceps 271
with chronic renal insufficiency. After further study confirmed: cicadae renal tubu-
lointerstitial lesions have a good effect, can protect the renal tubular cells
Na + -K + -ATPase, reduce cell lysosomes and lipid peroxidation damage, improve
renal hemodynamics, Reduce endothelial cell damage and blood clotting. So that
can improve kidney function cicada.
Cicadas are mainly found in China’s Sichuan, Jiangsu, Zhejiang, Fujian, but Anhui,
eastern Yunnan Province.
The land is distributed. In Zhejiang, the growth of bamboo hills, 80–500 m
above sea level, gentle terrain, canopy density is higher, loose soil, humidity,
ground covered with litter, and often bamboo forest activities of certain woodland,
Generally can be taken to the cicada. On the contrary, the steep slope, Dicranopteris
dichotoma, thatched, soil compaction, there is little occurrence of cicada. On the
source of the three rivers in Yunnan Province, the results of the study, more than
2500 m above sea level, cicadas disappeared. The broad-leaved forest below
2500 m in altitude or the Cyclobalanopsis glauca, Castanea henryi, Pinus yunna-
nensis, Abies and other needle and broadleaf mixed forest, canopy density, soil
loose, litter layer thickness of some forest land can be collected Cicada specimens.
The host is mostly small cicadas and mantis. Pure coniferous forest found no
cicada.
Fig. 12.5 The
environment of Medicinal
Mushroom Cordyceps
Cicadae growth. The
Medicinal Mushroom
Cordyceps Cicadae is the
worm of Cicada being
infected by Cordyceps
militaris and the fruiting
body being grow out of the
ground
Liangshan Cordyceps look like the child is more than branches or single branches,
slender and hard, upright and tortuous, born out by the host mouth.
Fig. 12.7 The
environment of Cordyceps
liangshanensis Zang, Liu et
Hu. Growth. The
Cordyceps liangshanensis
Zang, Liu et Hu is the
worm being infected by
Cordyceps militaris and
the fruiting body being
grow out of the ground
Parasitic on Lepidoptera larvae, more common in the area below 1500 m above sea
level, especially in the bamboo plexus in the worm body (Fig. 12.7).
Edible fungus. In the goods. Sichuan area market farmers to sell this on behalf of the
authentic Cordyceps. Because of fiber quality, so the quality inferior times. The highest
274 Z. Pan
value of the domestic medicinal Cordyceps produced in Tibet, Nagqu, Qamdo and
Qinghai Yushu, Guoluo these origin, which originated at an altitude of 3000–4000 m
line, anti-cancer natural cordycepin and the highest content of natural amino acids.
Sichuan Liangshan Yi Autonomous Prefecture drug testing had to separate the hypha in
the potato agar culture medium, hyphae community white, stunted, not by the seat.
12.5.1 Introduction
Gurney Cordyceps is a summer born in the broadleaf forest buried in the larvae of
Lepidoptera insects in 1983, Gurney Cordyceps first reported in China, Liang Zongqi
and other strains of its separation, an asexual to determine the nutritional composition of
pharmacological Experiments and liquid fermentation and other aspects made a lot of
research work, indicating that Gurney Cordyceps in improving the body immunity, pro-
mote sleep and enhance memory, analgesia and so has a very important role. With the
continuous improvement of experimental technology, Gurney Cordyceps still has great
potential for applied research. In this paper, the biological characteristics, chemical con-
stituents and pharmacological effects of Cordyceps sinensis were summarized, with a
view to further understanding of the biological characteristics of Cordyceps sinensis and
related products to provide the basis for in-depth research and development.
Gurney Cordyceps general length 10 ~ 90 mm, coarse 5 ~ 6 mm. Gurney worm
grass seat from the head of the host to give birth, solitary, bifurcation or clusters of
12 Research Advancement of Insect Origin Fungus Cordyceps 275
Fig. 12.8 Cordyceps
gunnii (Berk.) Berk. Being
Composition by fruiting
body with orange color and
worm
students. Generally longer than the worm body 4 ~ 14 cm, base white, coarse, up
gradually fine. The head is generally gray to gray-black, long oval to cylindrical,
forming a sub-head (Fig. 12.8). Immature Gurney Cordyceps pale yellow surface,
sub-seat inside the full, sub-seat head white. Mature Gurney caterpillar fungus sur-
face brown, sub-seat filling gradually into a hollow, the entire sub-head rough,
brown, length 1.5 ~ 2.5 cm. There are many sub-capsule shell formation of tiny
particles, ripe Gurney grass seat and handle the clear boundaries, ascus shell oval-
shaped or ampoule-shaped, 700 ~ 9l0 μm × 200 ~ 300 μm, buried, mature exposed
orifice.
spontaneous activity time of mice, Can prolong the sleep time of pentobarbital
sodium model mice, and show obvious analgesic effect.
functioning of the brain; also contains excitatory amino acids such as glutamic acid,
aspartic acid, glycine, etc. are Selectivity and transmitter receptors, can activate the
NMI) _R channel to open channel Ca Ca, Ca intracellular increase in regulation of
excitatory synaptic transmission and development may be involved in learning and
memory synapses.
Character identification. This product is the parasite and its head out of the sub-
blocks. Worms like silkworm, 3–4 cm long, 4–5 mm in diameter, head reddish yel-
low or purple-black, white surface, there are 20–30 links, near the head has three
pairs of feet, tail one, four pairs, the valve punctate, black, scrape off the outer white
membrane, showing brown or chestnut brown body worm skin; crisp, easy to break,
section flat, yellow-white. Apex rounded, 1–1.2 cm in length, 3–6 mm in diam., Tan
brown, with more than 2 mm in diameter, Gray or gray-black, with vertical lines;
crisp, easy to break, loose section or empty deflated. Gas smell, taste slightly salty
(sclerotia) or light (sub-seat). Sub-seat cross-section: subcapsular shell embedded in
the sub-seat, flask-shaped or sole shape, length 325–585 μm, diameter 65–156 μm;
subcapsule length 304–398 μm, 3–5 μm in diameter, ascospores linear, 182–325 μm,
diameter 1.5–2 μm, transverse septum was not obvious; wall mycelium arranged
closely; mycelial mycelium arranged loose.
At this stage of the study of Cordyceps sinensis has made gratifying achievements.
Gurney Cordyceps with enhance immunity, promote sleep and enhance memory,
analgesic and other effects, so in medicine and health care products development
has a high value. Among them, Guizhou on the development of ancient Chinese
caterpillar fungus has been walking in the forefront of China, Guizhou Chitianhua
Group Co., Ltd. and Guizhou University to develop ancient Nigeria Cordyceps
resources, and now has achieved great success, has developed the A Fu Le
Cordyceps wine and tea And other ancient Nigeria Cordyceps products. Guizhou
Renhuai Maotai Hanfang Liquor Industry Co., Ltd., has developed a variety of
alcoholic liquor such as Emperor Maojiu and Fu Guo Liquor. It uses aging liquor
and joins valuable raw materials such as Cordyceps sinensis. The main use of
ancient Nigeria Cordyceps is rich in nutrients, Gurney Cordyceps and wine with
wine brewing, and its products have a certain therapeutic value. However, until
now, the study of bioactive compounds of Cordyceps militaris has not been deep
enough. In this case, the development of new Cordyceps sinensis and other Chinese
herbal medicine has important significance, such as compound Chinese caterpillar
fungus capsule, Compound Chinese caterpillar fungus and so on, take the tradi-
tional Chinese medicine as the medicine lead, directs the Cordyceps active sub-
stance to give full play to its medicinal effect, is develops the ancient Chinese
Cordyceps a new way. However, because Goni Cordyceps has a lot of biological
activity, it should increase its independent research and development, so as to give
full play to its unique effect.
In short, the study of Cordyceps sinensis need to increase its efforts to clear its
pharmacological properties and the correlation between the active substances to
accelerate the transformation of research results, and shorten its cycle. Now the
understanding of Cordyceps sinensis and Cordyceps research is not so in-depth and
thorough, but the study found that some active ingredients of Cordyceps Cordyceps
sinensis is much higher than the Cordyceps sinensis, how to develop and use this
valuable resource to play its pharmacological effects, which Gurney Cordyceps for
12 Research Advancement of Insect Origin Fungus Cordyceps 279
Bacteria: strains are mostly from the nature of the ancient Chinese caterpillar fun-
gus, according to conventional isolation and culture. According to the research,
Cordyceps fungus sexual occurrence process, the need for a certain degree of
humidity to meet the complex physiological changes in the required water, and the
temperature and humidity there is a correlation between, in a certain humidity, the
temperature changes slowly or more constant, Is not conducive to extract the fruit-
ing body. Sexual occurrence must be through the low temperature and variable tem-
perature treatment Pathways: Gurney Cordyceps is Cordyceps spores contact larvae
infection death, in order to grow fruiting bodies. It was observed that the host 4–5
instar larvae of the highest infection rate. Mature larvae rarely infected, three instar
larvae are not infected. How to grasp the timing, the average person is also very dif-
ficult to grasp. In the dip problem, the artificial breeding of insects better conditions,
the worm is too strong, strong antibacterial, difficult to infection. Conditions are
poor, then the invasion of insects cause death, both of which will fail.
Mimic the origin of the ecological environment: that is, at an altitude of 3500–
5000 m on the mountains with the temperature, humidity, light, soil, vegetation and
other conditions, which is generally difficult to do. It is also the above reasons, most
people cannot plant Gurney absolute Cordyceps.
Large capsule Cordyceps, traditional Chinese medicine name. For the ergot fungi
large group of Cordyceps ophioglossoides (Ehrenb.) Link of the seat. Distributed in
Jiangsu, Guangxi, Sichuan, Yunnan and other places. With blood, bleeding, men-
struation effect. Commonly used in the blood bank, irregular menstruation.
Large capsule Cordyceps: also known as Cordyceps trees, large group capsule
grass. Sub-base by the root, multi-branched hyphae fixed in the soil on the host, the
ground part of the high 2–8 cm. Shank coarse 1–2.5 mm, less branched, dark green
to purple-brown, with vertical lines. Head oval, obovate to rod-shaped, 5–13 mm
long, 3–5 mm thick, dark brown, dry nearly black (Fig. 12.9). Ascus shell oval,
(600–650) μm × 300 μm, orifice protruding (300–400) μm × (7–8) μm. Spore linear,
transparent colorless, with most of the diaphragm, mature to break into
(3–4) μm × (2–2.5) μm small segment.
280 Z. Pan
Fig. 12.9 Cordyceps
ophioglossoides. Being
Composition by fruiting
body with orange color and
worm
Parasitic in bamboo or oak forest loose soil under the large group of bacteria
(Elaphomyces granulatus Fr.) on the fruiting bodies. Distributed in Jiangsu,
Guangxi, Sichuan, Yunnan and other places.
Seat length of 2–6 cm, the roots of mycorrhizal residual root. Head oval, obovate or
rod-shaped, 5–13 mm long, 3–5 mm in diameter, dark brown or dark brown; handle
diameter 1–2.5 mm, less branched, dark green, with vertical lines. Crisp, easy to
break, section dark brown. Gas slightly fishy, tasteless.
Further reading
Ahn YJ, Park SJ, Lee SG et al (2000) Cordycepin: selective growth inhibitor derived from liquid
culture of Cordyceps militaris against Clostridium spp. J Agric Food Chem 48(7):2744–2748
Cannon PF, Kirk PM (2007) Fungal families of the world. CabinetMaker:456
Chang Y, Hsu WH, Lu WJ et al (2015) Inhibitory mechanisms of CME-1, a novel polysaccha-
ride from the mycelia of Cordyceps sinensis, in platelet activation. Curr Pharm Biotechnol
16(5):451–461
Chen Y, Wang W, Yang Y et al (1997) Genetic divergence of Cordyceps sinensis as estimated by
random amplified polymorphic DNA analysis. Acta Genet Sin 24(5):410–416
Chen Y-C, Huang Y-L, Huang B-M (2005) Cordyceps sinensis mycelium activates PKA and
PKC signal pathways to stimulate steroidogenesis in MA-10 mouse Leydig tumor cells. Int J
Biochem Cell Biol 37(1):214–223
Cho HJ, Cho JY, Rhee MH et al (2007) Inhibitory effects of cordycepin (3´-deoxyadenosine), a
component of Cordyceps militaris, on human platelet aggregation induced by thapsigargin. J
Microbiol Biotechnol 17(7):1134–1138
Choi HN, Jang YH, Kim MJ et al (2014) Cordyceps militaris alleviates non-alcoholic fatty liver
disease in mice. Nutr Res Pract 8(2):172–176
12 Research Advancement of Insect Origin Fungus Cordyceps 281
Das SK, Masuda M, Hatashita M et al (2008) A new approach for improving cordycepin produc-
tivity in surface liquid culture of Cordyceps militaris using high-energy ion beam irradiation.
Lett Appl Microbiol 47(6):534–538
Hong T, Cui LK, Wen J et al (2015) Cordycepin protects podocytes from injury mediated by
complements complex C5b-9. Sichuan Da Xue Xue Bao Yi Xue Ban 46(2):173–178. 227
Hur H (2008) Chemical Ingredients of Cordyceps militaris. Mycobiology 36(4):233–235
Jagger DV, Kredich NM, Guarino AJ (1961) Inhibition of Ehrlich mouse ascites tumor growth by
cordycepin. Cancer Res 21(2):216–220
Jeong MH, Lee CM, Lee SW et al (2013) Cordycepin-enriched Cordyceps militaris induces
immunomodulation and tumor growth delay in mouse-derived breast cancer. Oncol Rep
30(4):1996–2002
Jing Y, Cui X, Chen Z et al (2014) Elucidation and biological activities of a new polysaccharide
from cultured Cordyceps militaris. Carbohydr Polym 102:288–296
Jing Y, Zhu J, Liu T et al (2015) Structural characterization and biological activities of a novel
polysaccharide from cultured Cordyceps militaris and its sulfated derivative. J Agric Food
Chem 63(13):3464–3471
Jong-Ho KOH, Kwang-Won YU, Hyung-Joo SUH, Yang-Moon CHOI, Tae-Seok AHN (2002)
Activationofmacrophages and the intestinal immune system by an orally administered decoc-
tion fromcultured mycelia of Cordyceps sinensis. Biosci Biotechnol Biochem 66:407–411
Koh J-H, Kim K-M, Kim J-M et al (2003) Antifatigue and antistress effect of the hot-water fraction
from mycelia of Cordyceps sinensis. Biol Pharm Bull 26(5):691–694
Kuo HC, Su YL, Yang HL et al (2005) Identification of Chinese medicinal fungus Cordyceps
sinensis by PCR-single-stranded conformation polymorphism and phylogenetic relationship. J
Agric Food Chem 53(10):3963–3968
Lee DH, Kim HH, Lim DH et al (2015a) Effect of Cordycepin-Enriched WIB801C from Cordyceps
militaris Suppressing Fibrinogen Binding to Glycoprotein IIb/IIIa. Biomol Ther 23(1):60–70
Lee H, Kim YJ, Kim HW et al (2006) Induction of apoptosis by Cordyceps militaris through acti-
vation of caspase-3 in leukemia HL-60 cells. Biol Pharm Bull 29(4):670–674
Lee JS, Kwon DS, Lee KR et al (2015b) Mechanism of macrophage activation induced by polysac-
charide from Cordyceps militaris culture broth. Carbohydr Polym 120:29–37
Liang HH, Cheng Z, Yang XL et al (2008) Genetic diversity and structure of Cordyceps sinensis
populations from extensive geographical regions in China as revealed by inter-simple sequence
repeat markers. J Microbiol 46(5):549–556
Liao Y, Ling J, Zhang G et al (2015) Cordycepin induces cell cycle arrest and apoptosis by induc-
ing DNA damage and up-regulation of p53 in Leukemia cells. Cell Cycle 14(5):761–771
Lu Q, Mei W, Luo S et al (2015) Apoptosis of Bel-7402 human hepatoma cells induced by a
ruthenium(II) complex coordinated by cordycepin through the p53 pathway. Mol Med Rep
11(6):4424–4430
Mao XB, Zhong JJ (2004) Hyperproduction of cordycepin by two-stage dissolved oxygen con-
trol in submerged cultivation of medicinal mushroom Cordyceps militaris in bioreactors.
Biotechnol Prog 20(5):1408–1413
Pan BS, Wang YK, Lai MS et al (2015) Cordycepin induced MA-10 mouse Leydig tumor cell
apoptosis by regulating p38 MAPKs and PI3K/AKT signaling pathways. Sci Rep 5:13372
Stensrud Q, Schumacher T, Shalchian-Tabrizi K et al (2007) Accelerated nrDNA evolution and
profound AT bias in the medicinal fungus Cordyceps sinensis. Mycol Res 111(4):409–415
Wang L, Xu N, Zhang J et al (2015a) Antihyperlipidemic and hepatoprotective activities of residue
polysaccharide from Cordyceps militaris SU-12. Carbohydr Polym 131:355–362
Wang Y, Liu D, Wang W et al (2015b) Cordyceps sinensis polysaccharide inhibits PDGF-BB-
induced inflammation and ROS production in human mesangial cells. Carbohydr Polym
125:135–145
Wei HP, Ye XL, Zhang HY et al (2008) Investigations on cordycepin production by solid culture of
Cordyceps militaris. Zhongguo Zhong Yao Za Zhi 33(19):2159–2162
Wen L, Tang YL, Yin QF et al (2005) Assays on nutrient and effective ingredients in different parts
of Cordyceps militaris. Zhongguo Zhong Yao Za Zhi 30(9):659–661
282 Z. Pan
Xiang F, Lin L, Hu M et al (2016) Therapeutic efficacy of a polysaccharide isolated from Cordyceps
sinensis on hypertensive rats. Int J Biol Macromol 82:308–314
Xie CY, Gu ZX, Fan GJ et al (2009) Production of cordycepin and mycelia by submerged fer-
mentation of Cordyceps militaris in mixture natural culture. Appl Biochem Biotechnol
158(2):483–492
Xiong C, Xia Y, Zheng P et al (2013) Increasing oxidative stress tolerance and subculturing stabil-
ity of Cordyceps militaris by overexpression of a glutathione peroxidase gene. Appl Microbiol
Biotechnol 97(5):2009–2015
Yang J, Zhang W, Shi P et al (2005) Effects of exopolysaccharide fraction (EPSF) from a cultivated
Cordyceps sinensis fungus on c-Myc, c-Fos, and VEGF expression in B16 melanoma-bearing
mice. Pathol Res Pract 201(11):745–750
Yang JL, Xiao W, He HX et al (2008) Molecular phylogenetic analysis of Paecilomyces hepiali
and Cordyceps sinensis. Acta Pharm Sin 43(4):421–426
Yang X, Li Y, He Y et al (2015) Cordycepin alleviates airway hyperreactivity in a murine model of
asthma by attenuating the inflammatory process. Int Immunopharmacol 26(2):401–408
Zhang Y, Xu L, Zhang S et al (2009) Genetic diversity of Ophiocordyceps sinensis, a medicinal
fungus endemic to the Tibetan Plateau: implications for its evolution and conservation. BMC
Evol Biol 9(1):290
Zheng P, Xia Y, Xiao G et al (2011a) Genome sequence of the insect pathogenic fungus Cordyceps
militaris, a valued traditional Chinese medicine. Genome Biol 12(11):R116
Zheng Z, Huang C, Cao L et al (2011b) Agrobacterium tumefaciens-mediated transformation
as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris. Fungal Biol
115(3):265–274
Zheng Z, Jiang K, Huang C et al (2012) Cordyceps militaris (Hypocreales: Cordycipitaceae):
transcriptional analysis and molecular characterization of cox1 and group I intron with putative
LAGLIDADG endonuclease. World J Microbiol Biotechnol 28(1):371–380
Zhou XW, Wang XF, Li QZ (2012) Expression and characteristic of the Cu/Zn superox-
ide dismutase gene from the insect parasitizing fungus Cordyceps militaris. Mol Biol Rep
39(12):10303–10311
Zhu SJ, Pan J, Zhao B et al (2013) Comparisons on enhancing the immunity of fresh and dry
Cordyceps militaris in vivo and in vitro. J Ethnopharmacol 149(3):713–719
Zhu ZY, Liu XC, Dong FY et al (2016) Influence of fermentation conditions on polysaccharide
production and the activities of enzymes involved in the polysaccharide synthesis of Cordyceps
militaris. Appl Microbiol Biotechnol 100(9):3909–3921
Application of Recombinant Insect
Products in Modern Research: 13
An Overview
Mohd Yusuf
Abstract
Biotechnology enables the genetic engineering through gene modification,
broadening the range of natural products, as far production and application of
transgenic products, such as implant coatings, scaffolds for tissue engineering,
wound dressing devices, as well as drug delivery systems. In the present sce-
nario, recombinant technology including the expression of DNA and gene modi-
fication or simple genetic manipulation to several host organisms, involving
bacteria, yeast, plants, insect cells, mammalian cells, and transgenic animals
seems to have tremendous and promising future research opportunities. In this
chapter, an attempt is made onto modern research initiatives using recombinantly
produced insect products and applications.
13.1 Introduction
Recombinant DNA technology is one of the most emerging and innovative tech-
nologies applied commonly for medicine, agriculture, and industry to produce new
genetically engineered products that are of high specific value. Interestingly, several
expression systems have been developed for the production of rDNA products with
strong positive ethnopharmacological correlation with the traditional knowledge as
well as therapeutic potential, including bacteria, yeast, bird cells, insect cells, mam-
malian cells, etc. which possess specific platforms (Dossey 2010). Insects, since
thousands of years, provided many valuable natural substances, including honey,
silk, and other products (i.e. jelly, wax, etc.). Also, insect secretions and ground-up
M. Yusuf
Department of Chemistry, YMD College, M. D. University, Nuh, Haryana 122107, India
e-mail: yusuf1020@gmail.com
bodies have commonly been used as an important folklore medicine around the
globe, for example, India, China, and European, African, and American countries.
Literature published have described variable alluring accounts on insect-based folk
medicine in India, Zaire, and Bolivian Amazon and other regions toward the use of
insects and their secreted products as folk medicine for a huge range of treatments
including allergy, anemia, arthritis, and bronchitis and some antivenoms (Dyck
et al. 2003; Ratcliffe et al. 2011; Cherniack 2010; Ratcliffe et al. 2014).
Some studies have been published onto insect-derived natural products and their
latent abilities in targeted drug development and treatment for various human dis-
eases (Ratcliffe et al. 2011; Cherniack 2010). Among them, transgenic insect cells
are known to be realized in cost-effective recombinant material production as a new
expression system (Grzelak 1995; Kuwana et al. 2014). In this chapter transgenic
insect-derived natural products and their potent applications are discussed through
current researches.
Human civilization has utilized insect silks since thousands of years because of their
advantages and comfortness. Silks are protein materials produced by a wide range
of insects and spider species for applications requiring high-performance fibers.
The most common example is the use of reeled silkworm, Bombyx mori, to produce
textiles which are used as a suture biomaterial for centuries, and also farmed silk-
worm silk in recent years, reprocessed into many forms such as films, gels, and
sponges for biomedical applications. Despite the more promising and inherent abili-
ties of insect-derived silk, spiders have not been domesticated for large scale or even
industrial applications, since farming the spiders is not commercially viable due to
their highly territorial and cannibalistic nature (Vepari and Kaplan 2007). There are
chiefly two kinds of silk proteins which are derived, such as spiders and
honeybees.
production of mottled silks with divergent mechanical properties, and most of them
make rather elaborate nests, traps, and cocoons using typically more than one type
of silk-producing glands, spigots, and ducts (Scheibel 2004). However, the natural
spiders cannot cover the current global needs, and therefore, the recombinant DNA
technology exists to handle the situation for silk production on a super large scale,
and one may reasonably assume the possibility of achieving the mass production of
recombinant proteins in an approach similar to the desirable silk (Kuwana et al.
2014; Tomita 2011; Scheibel 2004).
Structurally, spider silks are natural polymers consist of three clarified domains
(Fig. 13.1), for example, repetitive middle-core domain and non-repetitive
N-terminal and C-terminal domains. Generally, silks differ in primary sequence,
physical properties, and functions, for example, dragline silks used to build frames,
radii, and lifelines are known for dazzling mechanical properties including elastic-
ity, strength, and toughness (Gatesy et al. 2001; Tokareva et al. 2013). The most
studied silk is dragline silk that shows remarked elasticity as well as strength. In a
common way, N. Clavipes, the golden orb-spider produces dragline silk in the major
ampullate gland. Also, it has been found that dragline silk is a protein complex,
Sons 2013
286 M. Yusuf
Table 13.1 Potential use of silk biopolymers in medicine (Ratcliffe et al. 2011; Tomita 2011;
Teulé et al. 2012a; An et al. 2012; Ding et al. 2014; Hu et al. 2012; Zhang et al. 2013; Demain and
Vaishnav 2009)
Type of silk proteins Potent use
Nanoparticles Delivery of drugs to cancer cells
B. mori porous materials For repair of cartilage, bone, ligaments, tendons, vascular
tissue, nerves, and corneas and as wound dressings
Silk-heparin support Vascular tissue growth application
Copolymer blocks Transfection of target cancer cells
Small, globular units with Improved tumor cell-specific transfection
tumor-homing peptides (THP)
Ionic complexes of nano-scaled Further improved tumor cell-specific transfection
silk with THP
Silk hydrogels Treatment of breast cancer
Antibiotic-loaded silk hydrogels Prevention and treatment of infection
Electrically stimulated silk films Enhancement of neural growth
Vitamin E-loaded silk Skin tissue regeneration
nanofibrous mats
Silk protein matrices Thermostabilization of vaccines
proportions silk proteins are massively generated and gathered high repute applica-
tions in genetic engineering to meet the copious demands for industrial espionage
and also in rapid progress are being made in the development of silk for use in medi-
cine (Table 13.1) (Ratcliffe et al. 2011; Zhang et al. 2013). Hence, spider silks are
also favorable as commence biomaterials (Demain and Vaishnav 2009; Omenetto
and Kaplan 2010). Furthermore, with a broader screening of other naturally fibrous
proteins, insect silks can be grouped into more than 23 independently evolved lin-
eages with different secondary structures such as cross-beta sheets, coiled coils, or
polyglycine II structures that may be utilized to the next generation through various
novel applied sectors.
Even so, in a consequent study, silkworms transformed with chimeric silkworm/
spider silk genes spin composite silk fibers that were reported with improved
mechanical properties. In this study, engineered transgenic silkworms were sub-
jected to express the synthetic A2S814 spider silk gene in an effort to produce com-
posite fibers consisting, at least in part, of the synthetic spider silk protein, and
results showed that transgenic silkworms encoding synthetic spider silk proteins
can, indeed, spin composite silk fibers with improved mechanical properties, rela-
tive to the fibers produced by the parental animals tested (Teulé et al. 2012b).
Similar to spider silks (i.e., Bombyx mori), honeybees also secrete four different
types of small coiled-coil proteins with a molecular weight of about 30 kDa and
were found to be stable in water. These proteins are non-repetitive and rich in ala-
nine residues chiefly. The ingenuity of science continues to amaze with the
288 M. Yusuf
transient honeybee silk recently produced as biomaterials for the transport and
drug delivery and also in tissue engineering. Nevertheless, the particular interest is
being under consideration about the silk from bees, ants, and hornets of
Hymenoptera type (Sutherland et al. 2010). The hymenopteran silk has the molec-
ular structure of type α-helical proteins assembled into a tetrameric coiled confor-
mation and also fundamental design to the β-sheet plesiomorphic crystallites that
dominate the silkworm cocoon and spider dragline silks reported (Atkins 1967;
Sutherland et al. 2011). The spatial and sequential arrangement of repetitive units/
amino acids arises within the proteins, and these features promote self-assembly
and formation of structural hierarchy in consequence to material-related functional
roles (Sutherland et al. 2011; Poole et al. 2013). For example, recombinantly
accessed Asiatic honeybee silk was obtained from Apis cerana (Shi et al. 2008).
A flexible and solvent-stable fiber production was described by Poole et al. (2013).
In this method after concentrated recombinant honeybee protein solutions were
extruded into a methanol bath, dried, drawn in aqueous methanol, then covalently
cross-linked using dry heat and solvent-stable and flexible fibers were fabricated.
In another study, biomimetic spinning system was employed for recombinant pro-
duction and purification of the four full-length unmodified honeybee silk proteins
in E. coli at substantial yields of 0.2–2.5 g/L under the correct conditions the
recombinant proteins self-assembled to reproduce the native coiled-coil structure
(Weisman et al. 2010).
In the present scenario, naturally originated biomimetic biomaterials are much
attentive and noteworthy targets for tissue engineering and other biomedical
applications. In a study, honeybee silk membranes were described to have bright
exploration toward their applicability for tissue engineering and found capabili-
ties such as biodegradation and considerable mechanical and biological properties
(Kumar et al. 2016). In addition, honeybee recombinant silk proteins have recently
gained interest as materials for bioengineering and nanomedicine as they possess
several features with significant functionality, biocompatibility, and degradability
(Corchero et al. 2014).
Collagen is one of the most abundant proteins found in tissues of animals, including
tendons, ligaments, and skin, and is ubiquitous throughout the animal kingdom,
where it comprises some 28 diverse molecules that form the extracellular matrix
within organisms, and many of those recently discovered are present in tissues in
small. In general, collagen consists of different chain compositions depending on
the specific types and is composed of three left-handed helices disheveled to form a
right-handed triple helix with (GPX)n, as commonest amino acids, in which X is
any amino acid other than proline, glycine, or hydroxyproline (Sutherland et al.
2013; Browning et al. 2012). Although recombinant collagen proteins could be pro-
duced by bacterial and insect cells as standard expression systems, they facilitate in
13 Application of Recombinant Insect Products in Modern Research: An Overview 289
opening the new opportunistic door to manufacture. For example, triple-helical pro-
collagen from recombinant type-III procollagen was produced in a study by co-
expressing together with mammalian P4H in the yeast cell, Pichia pastoris (Vuorela
et al. 1997). Another study was carried out on insect cells as expression carrier, and
as a consequence it was observed that insect cells possess a very low level of endog-
enous P4H activity; co-transfection of such cells with a combination of baculovirus
vectors encoding human type-III procollagen and both α- and β-subunits of P4H
yielded up to 60 μg/mL cellular procollagen (Lamberg et al. 1996). However, triple-
helical collagen molecules showed to have an ability to assemble into favorable
supramolecular structures that form the basis of commercial uses of collagen in real
industrial sectors, such as food, tissue engineering, and medical applications
(Sutherland et al. 2013; Lamberg et al. 1996).
13.4 Cantharidin
O
CH3
O O
CH3
Fig. 13.2 Chemical structure of cantharidin O
290 M. Yusuf
Undoubtedly, insects are one of the best repertoire sources for antimicrobial pep-
tides (AMPs), and their potent antimicrobial activity to address the threat of
multidrug-resistant pathogens, in vitro and in vivo, has encouraged their develop-
ment as alternatives to conventional antibiotics (Dossey 2010; Ratcliffe et al. 2011).
Chemically insect-derived AMPs are mainly cationic (although anionic forms do
exist to a significant extent), facilitate their binding through electrostatic force to
negatively charged bacterial and tumor cell walls, and are also amphipathic in their
folded state with hydrophilic and hydrophobic regions mediating their solubility in
phospholipid cell membranes. These interactions of the AMPs resulted disruptive
capabilities (Ratcliffe et al. 2014; Ntwasa 2012).
Insect AMPs can be classified on the basis of their structure or function and can
be categorized into three groups, as under:
Linear 𝛼-helical AMPs are present in a wide range of insect orders, including cole-
opterans, dipterans, and lepidopterans, for example, cecropins, moricin, sarcotoxin,
and melittin, out of which cecropin is the firstly discovered AMP produced by lar-
vae of the giant silk moth Hyalophora cecropia, prototype 𝛼-helical linear AMP,
active against Gram-negative bacteria such as E. coli. In general, cecropins contain
a tryptophan residue at or near the N-terminus, a long N-terminal amphiphilic
α-helix, a shorter and more hydrophobic α-helix at the C-terminus, and an amidated
C-terminal residue. Furthermore, cecropins are found to be active against Gram-
positive and Gram-negative bacteria, viruses, protozoans, fungi, nematodes, and
tumor cells (Ratcliffe et al. 2014; Gaspar et al. 2013). Consequently, some new
additional cecropin-like peptides such as enbocin, sarcotoxins, and hyphancin with
selective antimicrobial potential against both Gram-positive and Gram-negative
bacteria (Ratcliffe et al. 2011, 2014; Gaspar et al. 2013; Mylonakis et al. 2016).
From ancient times around the globe, honey has been obtained from bees and used
as important folklore medication for several ailments and diseases. Surprisingly,
despite the success of insects in terms of numbers and diversity, recently, the potent
activity of honey has been extensively studied. Interestingly, honey was found to
have brilliant antimicrobial performance against several antibiotic-resistant human
pathogens (Ratcliffe et al. 2011; Cherniack 2010). The presence of large number of
bioactive phenolic compounds (Table 13.2) in honey has gaining popularity to the
use of honey in clinical practices and trials and is evidently found to be a great
potential, for example, wound care anticancer, antimicrobial, antispasmodic, and
other beneficial abilities (Ratcliffe et al. 2014; Mylonakis et al. 2016).
Bee venoms include a large range of practically unexplored compounds awaiting
discovery and development into the future medicines, for example, some ant and
parasitoid wasp venoms may contain even more than 75 more different components.
Nevertheless, bee venom therapy also has been used in folk medicine for many
thousands of years to treat several ailments from arthritis, skin diseases, multiple
sclerosis, rheumatism, cancer, infections, and pain (Ratcliffe et al. 2014; Mylonakis
et al. 2016; Al-Waili et al. 2011).
Table 13.2 Bioactive phenols present in honey (Ratcliffe et al. 2014; Mylonakis et al. 2016)
Class of phenolic
compounds Typical examples
Phenolic acids Caffeic acid
Coumarins Coumarin
Tannins Ellagic acid
Flavonols Quercetin, kaempferol, galangin, fisetin, myricetin, etc.
Flavanones Hesperidin
Flavones Apigenin, acacetin, chrysin, luteolin, genkwanin, wogonin, and tricetin
292 M. Yusuf
In the present work, insect-derived natural products based on rDNA technology are
discussed which are of great interest in recent era, such as proteins/peptides (silks,
collagen, cantharidin, AMPs) and others (honey, venom, etc.). Spider dragline and
honeybee silks having significant functionality, biocompatibility, and degradability
possess high tensile strength, flexible performance, and excellent mechanical prop-
erties suitable for significant biomedical applications. Collagen and cantharidin pro-
teins are also found applicable toward food, tissue, and medical engineering.
Furthermore, antimicrobial peptides (AMPs) with potent antimicrobial activity
toward multidrug-resistant pathogens, in vitro and in vivo, have encouraged their
development as alternatives to conventional antibiotics. The significant step toward
the progression in transient organisms surely suggests new directions to emulate in
the pursuit of new high-performance, multifunctional materials generated with
green platforms that integrate with living systems that could be used for various
applications, including cosmetic, diagnostic, animal therapeutic, and human thera-
peutic uses. Thus, the spectrum of tunable functional materials is expected to grow
exponentially in the next years and fulfill the demands of new targeted, sustained
drug delivery platforms and functional scaffolds using recombinant DNA
technology.
References
Al-Waili NS, Salom K, Al-Ghamdi AA (2011) Honey for wound healing, ulcers, and burns; data
supporting its use in clinical practice. Sci World J 11:766–787
An B, Jenkins JE, Sampath S et al (2012) Reproducing natural spider silks’ copolymer behavior in
synthetic silk mimics. Biomacromolecules 13:3938–3948
Atkins EDT (1967) A four-strand coiled-coil model for some insect fibrous proteins. J Mol Biol
24:139–141
Brooks AE, Steinkraus HB, Nelson SR et al (2005) An investigation of the divergence of major
ampullate silk fibers from Nephila clavipes and Argiope aurantia. Biomacromolecules
6:3095–3099
Browning MB, Dempsey D, Guiza V et al (2012) Multilayer vascular grafts based on collagen-
mimetic proteins. Acta Biomater 8:1010–1021
Cherniack EP (2010) Bugs as drugs, part 1: insects. The “new” alternative medicine for the 21st
century? Altern Med Rev 15(2):124–135
Choudary PV, Kamita SG, Maeda S (1995) Expression of foreign genes in Bombyx mori larvae
using baculovirus vectors. Methods Mol Biol 39:243–264
Corchero JL, Vázquez E, García-Fruitós E et al (2014) Recombinant protein materials for bioengi-
neering and nanomedicine. Nanomedicine 9(18):2817–2828
Dang YJ, Zhu CY (2013) Oral bioavailability of cantharidin-loaded solid lipid nanoparticles.
BMC Chin Med 8:1. doi:10.1186/1749-8546-8-1
Demain AL, Vaishnav P (2009) Production of recombinant proteins by microbes and higher organ-
isms. Biotechnol Adv 27(3):297–306
Ding D, Guerette PA, Hoon S et al (2014) Biomimetic production of silk-like recombinant squid
sucker ring teeth proteins. Biomacromolecules 15(9):3278–3289
Dossey AT (2010) Insects and their chemical weaponry: new potential for drug discovery. Nat Prod
Rep 27(12):1737–1757
13 Application of Recombinant Insect Products in Modern Research: An Overview 293
Dyck MK, Lacroix D, Pothier F et al (2003) Making recombinant proteins in animals different
systems, different applications. Trends Biotechnol 21:394–399
Fahnestock SR, Irwin SL (1997) Synthetic spider dragline silk proteins and their production in
Escherichia coli. Appl Microbiol Biotechnol 47:23–32
Gaspar D, Veiga AS, Castanho MARB (2013) From antimicrobial to anticancer peptides: a review.
Front Microbiol 4:294. doi:10.3389/fmicb.2013.00294
Gatesy J, Hayashi C, Motriuk D et al (2001) Extreme diversity, conservation, and convergence of
spider silk fibroin sequences. Science 291(5513):2603–2605
Ghaffarifar F (2010) Leishmania major: In vitro and in vivo anti-leishmanial effect of cantharidin.
Exp Parasitol 126(2):126–129
Gosline JM, Denny MW, DeMont ME (1984) Spider silk as rubber. Nature 309:551–552
Grzelak K (1995) Control of expression of silk protein genes. Comp Biochem Physiol B Biochem
Mol Biol 110:671–681
Higashiya S, Topilina NI, Ngo SC et al (2007) Design and preparation of b-sheet forming repeti-
tive and block-copolymerized polypeptides. Biomacromolecules 8:1487–1497
Hu X, Vasanthavada K, Kohler K et al (2006) Molecular mechanisms of spider silk. Cell Mol Life
Sci 63:1986–1999
Hu X, Cebe P, Weiss AS et al (2012) Protein-based composite materials. Mater Today 15(5):208–215
Huemmerich D, Helsen CW, Quedzuweit S et al (2004) Primary structure elements of spider drag-
line silks and their contribution to protein solubility. Biochemist 43:13604–13612
Humenik M, Scheibel T, Smith A (2011) Spider silk understanding the structure-function relation-
ship of a natural fiber. Prog Mol Biol Transl Sci 103:131–185
Kumar M, Jain D, Bhardwaj N et al (2016) Native honeybee silk membrane: a potential matrix for
tissue engineering and regenerative medicine. RSC Adv 6(59):54394–54403
Kuwana Y, Sezutsu H, Nakajima KI et al (2014) High-toughness silk produced by a trans-
genic silkworm expressing spider (Araneus ventricosus) dragline silk protein. PloS One
9(8):e105325, 1-11
Lamberg A, Helaakoski T, Myllyharju J et al (1996) Characterization of human type III collagen
expressed in a baculovirus system production of a protein with a stable triple helix requires
coexpression with the two types of recombinant prolyl 4-hydroxylase subunit. J Biol Chem
271(20):11988–11995
Lewis RV (2006) Spider silk: ancient ideas for new biomaterials. Chem Rev 106:3762–3774
Lissina E, Young B, Urbanus ML et al (2011) A systems biology approach reveals the role of a
novel methyltransferase in response to chemical stress and lipid homeostasis. PLoS Genet
7(10):e1002332
Maeda S, Kawai T, Obinata M et al (1985) Production of human alpha-interferon in silkworm
using a baculovirus vector. Nature 315:592–594
Meyer DE, Chilkoti A (2002) Genetically encoded synthesis of protein-based polymers with pre-
cisely specified molecular weight and sequence by recursive directional ligation: examples
from the elastin-like polypeptide system. Biomacromolecules 3:357–367
Moed L, Shwayder TA, Chang MW (2001) Cantharidin revisited: a blistering defense of an ancient
medicine. Arch Dermatol 137(10):1357–1360
Mylonakis E, Podsiadlowski L, Muhammed M, Vilcinskas A (2016) Diversity, evolution and
medical applications of insect antimicrobial peptides. Philos Trans R Soc B 371:20150290.
doi:10.1098/rstb.2015.0290
Ntwasa M (2012) Cationic peptide interactions with biological macromolecules. In: Abdelmohsen
K (ed) Binding Protein. InTech, Croatia, pp 139–164
Omenetto FG, Kaplan DL (2010) New opportunities for an ancient material. Science
329(5991):528–531
Poole J, Church JS, Woodhead AL et al (2013) Continuous production of flexible fibers from trans-
genically produced honeybee silk proteins. Macromol Biosci 13(10):1321–1326
Poppel AK, Vogel H, Wiesner J, Vilcinskas A (2015) Antimicrobial peptides expressed in medic-
inal maggots of the blow fly Lucilia sericata show combinatorial activity against bacteria.
Antimicrob Agents Chemother 59:2508–2514
294 M. Yusuf
Rabotyagova OS, Cebe P, Kaplan DL (2010) Role of polyalanine domains in β-sheet formation in
spider silk block copolymers. Macromol Biosci 10:9–59
Rahnamaeian M, Vilcinskas A (2012) Defense gene expression is potentiated in transgenic barley
expressing antifungal peptide Metchnikowin throughout powdery mildew challenge. J Plant
Res 125:115–124
Ratcliffe NA, Mello CB, Garcia ES et al (2011) Insect natural products and processes: new treat-
ments for human disease. Insect Biochem Mol Biol 41(10):747–769
Ratcliffe N, Azambuja P, Mello CB (2014) Recent advances in developing insect natural products
as potential modern day medicines. Evid Based Complement Alternat Med 2014:1–21
Scheibel T (2004) Spider silks: recombinant synthesis, assembly, spinning, and engineering of
synthetic proteins. Microb Cell Fact 3:1–14
Shi J, Lua S, Du N, Liu X, Song J (2008) Identification, recombinant production and structural
characterization of four silk proteins from the Asiatic honeybee Apis cerana. Biomaterials
29:2820–2828
Sutherland TD, Young J, Weisman S et al (2010) Insect silk: one name, many materials. Annu Rev
Entomol 55:171–188
Sutherland TD, Church JS, Hu X et al (2011) Single honeybee silk protein mimics properties of
multi-protein silk. PLoS One 6(2):e16489
Sutherland TD, Peng YY, Trueman HE et al (2013) A new class of animal collagen masquerading
as an insect silk. Sci Rep 3:2864
Teulé F, Addison B, Cooper AR et al (2012a) Combining flagelliform and dragline spider silk
motifs to produce tunable synthetic biopolymer fibers. Biopolymers 97:418–431
Teulé F, Miao YG, Sohn BH et al (2012b) Silkworms transformed with chimeric silkworm/spider
silk genes spin composite silk fibers with improved mechanical properties. Proc Natl Acad Sci
109(3):923–928
Tokareva O, Michalczechen-Lacerda VA, Rech EL et al (2013) Recombinant DNA production of
spider silk proteins. Microb Biotechnol 6(6):651–663
Tomita M (2011) Transgenic silkworms that weave recombinant proteins into silk cocoons.
Biotechnol Lett 33(4):645–654
Vepari C, Kaplan DL (2007) Silk as a biomaterial. Prog Polym Sci 32:991–1007
Vuorela A, Myllyharju J, Nissi R et al (1997) Assembly of human prolyl 4-hydroxylase and type
III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coex-
pression with collagen and assembly of a stable collagen requires coexpression with prolyl
4-hydroxylase. EMBO J 16(22):6702–6712
Wang GS (1989) Medical uses of mylabris in ancient China and recent studies. J Ethnopharmacol
26(2):147–162
Wang C, Patwardhan SV, Belton DJ et al (2006) Novel nanocomposites from spider silk-silica
fusion (chimeric) proteins. Proc Natl Acad Sci U S A 103:9428–9433
Weisman S, Haritos VS, Church JS et al (2010) Honeybee silk: recombinant protein production,
assembly and fiber spinning. Biomaterials 31(9):2695–2700
Zhang J, Pritchard E, Hu X et al (2013) Stabilization of vaccines and antibiotics in silk and elimi-
nating the cold chain. Proc Natl Acad Sci U S A 109(30):11981–11986
Structure, Regulation, and Potential
Applications of Insect Chitin- 14
Metabolizing Enzymes
Abstract
Chitin is a vital component of insect exoskeleton and peritrophic matrix and
because of this reason a potential target for insecticidal agents. Chitin-
metabolizing enzymes, viz., chitin synthases and chitinases, belong to the glyco-
side hydrolase superfamily (GH18). Chitin synthases are involved in deposition
of new cuticle during molting and also ideal for development of insecticidal
agents. Chitinases are considered as an essential enzyme for insect growth and
development being involved in molting and various other physiological pro-
cesses, i.e., cuticle turnover, regulation of abdominal contraction and wing
expansion, digestion, immunity, and natural defense. Chitinases possess multi-
domain architecture, i.e., chitin-binding domain, Ser-/Thr-rich linker domains,
catalytic domains, fibronectin, and mucin-like domains. Knockdown of both the
enzymes resulted into irregularities in metamorphosis. Diverse group of
chitinase-like proteins have also been detected in insect species that possess
chitin-binding domains but do not exhibit catalytic activity. Development of chi-
tinases as defensive agents against chitin-bearing insect pests and pathogens will
generate new knowledge and innovative processes for biocontrol
advancements.
14.1 Introduction
Chitin is the second most abundant insoluble structural polymer in nature following
cellulose and is composed of linear chains of β-1,4-linked N-acetyl-D-glucosamine
(GlcNAc) residues. It is the essential component of fungal cell wall, house dust
mites, exoskeletons of crabs, shrimp and insects, parasitic nematodes, and digestive
tracts of many insects (Pillai et al. 2009). In insects (Arthropoda), chitin is mainly
found in the exoskeleton and peritrophic matrix (PM). The PM is a structure in the
insect’s gut, which consists mainly of proteins and glycosaminoglycans embedded
in a chitinous matrix that protect insects against the mechanical damage and patho-
gens (Chapman 1998). The development of the cuticle which is the complex exo-
skeleton with diverse functions is the key factor responsible for the evolutionary
success of insects (Wittkopp and Beldade 2009). Apart from these two structures,
chitin has also been identified as a component of the salivary glands, trachea, tra-
cheiolas, ovaries, dermal glands, eggs, and eggshell in insects. The growth and
development in insects are regulated by the metabolism of chitin in the cuticle and
the PM. In insects, the rigidity of the cuticle although provides physical support and
protection, it also results in the restriction of growth and development (Tetreau et al.
2015). This restriction is compensated by molting, i.e., a complex and multistep
cascade including a series of enzymes and cofactors. Therefore, these chitin-related
enzymes and proteins play a key role in the insect’s metabolism and help in their
growth, development, and survival. On the basis of the function, the enzymes
involved in chitin metabolism can be divided into three major categories, i.e., syn-
thetic (chitin synthases), modifying (chitin deacetylases, to enzymatically alter chi-
tin by deacetylation), and the degradative enzymes (chitinases and
N-acetylglucosaminidases, to degrade chitin by hydrolytic process) (Chen 1987;
Merzendorfer and Zimoch 2003). Among these, chitin synthases and chitinases are
considered as the key enzymes and widely studied. Chitinases can be further divided
into endochitinases (EC 3.2.1.14) and exochitinases (EC 3.2.1.52) on the basis of
their mode of action. The former cleave within the chitin polymer to release long-
chain chitin oligosaccharides, and the latter releases short-chain oligomers.
Modular structure of CHS has been predicted and studied by various researchers.
According to the modular structure given by Arakane et al. (2004), CHS are large
membrane-integrated enzymes with various domains that are important for subcel-
lular localization and activation. Like cellulose synthases, CHS has multiple trans-
membrane (TM) domains work as the transport channel for chitin deposition in the
outer membrane (Morgan et al. 2013). CHS has three unique domains, N-terminal
moderately conserved sequence domain, highly conserved catalytic domain, and the
C-terminal module with multiple transmembrane segments. The catalytic domain
contains several stretches of highly conserved amino acid sequences. There are dif-
ferent patterns of transmembrane segments of the N-terminal domain in different
species of insects. In spite of domains, CHS also possesses several conserved motifs
like EDR, QRRRW, and WGTRE (Morgan et al. 2013; Muthukrishnan et al. 2016;
Moussian et al. 2005) (Fig. 14.1) in which EDR and QRRRW are closed to the
active site (Merzendorfer 2006). The insect CHS has molecular mass in the range of
160–180 kDa and exhibit a slightly acidic isoelectric point between 6.1 and 6.7
(Merzendorfer and Zimoch 2003). On the basis of available crystal structure of a
bacterial cellulose synthase, Dorfmueller et al. (2014) reported the chitooligosac-
charide synthase model encoded by the bacterial NodC gene. In this study they
proposed about the membrane spanning region of NodC protein and traversing of
the lipid bilayer in three different orders, i.e., outside to inside, inside to outside, and
outside to inside. On the basis of crystal structure of eight GT2 enzymes,
Chitin
Transmembrane N-terminal C-terminal
helices domain domain
Extracellular
Intracellular
UDP-GlcNAc
Binding Extrusion motif
UDP + Pi sites site
Catalytic
domain
Fig. 14.1 Hypothetical model of the tripartite domain organization of the insect chitin synthase
298 M. Kumar et al.
Muthukrishnan et al. (2016) modeled an insect synthase and reported the presence
of a long tunnel at the active site that is a remarkable difference between the NodC
structure and that of the cellulose synthases and CHS. This long tunnel accommo-
dates sugars of elongation chains of cellulose and chitin, whereas the NodC enzyme
has a closed pocket which can accommodate only a pentasaccharide chain. The
study also indicated that six transmembrane helices (TMH) are involved in the for-
mation of narrow channel. The helix of the TMS region controls the entry of the
chitin-conducting channel as it is attached to the cytosolic side of the membrane
(Van Leeuwen et al. 2012; Demaeght et al. 2014). Muthukrishnan et al. (2012) have
predicted the structural model of the tripartite domain organization of Drosophila
DmCHS1. According to the model (Fig. 14.1), N-terminal domain contains eight
TMH which vary from 7 to 10 among different insect species. The domain facing
toward the cytoplasm is the middle catalytic domain which contains the catalytic
sites like nucleotide-binding sites, donor saccharide-binding site, acceptor
saccharide-binding site, and the product-binding site, and the domain C contains
(5 + 2) conserved TMH. This model suggested that polymer synthesis occurs in the
cytosol and further the chitin chain is translocated across the membrane by the help
of five TMS clusters and the extrusion motif SWGTR. In the model, one helix
appeared not to span the membrane but is attached to the cytosolic side of the plasma
membrane assist in predicting the intracellular orientation of the C-terminus.
CHS plays a leading role in the biosynthesis of chitin. CHS requires UDP-N-
acetylglucosamine (UDP-GlcNAc) as the activated sugar donor in the formation
of the chitin in insects (Merzendorfer 2011; Moussian 2008). The biosynthetic
pathway of chitin synthesis in insects starts with trehalose and ends with the chitin
(Cohen 1987; Tharanathan and Kittur 2003). The major site for the chitin biosyn-
thesis in insects is the epidermis and the midgut (Tellam et al. 2000). The epider-
mal cells cause the deposition of new cuticle during molting, and the midgut cells
are related to the PM formation during feeding (Reynolds 1987; Zimoch et al.
2005). This biosynthesis can be grouped into three major steps. The first step deals
with the formation of polymer in which enzymes’ catalytic domain facing toward
the cytoplasmic sites plays a crucial role. In the second step of biosynthesis, the
nascent polymer translocated across the membrane followed by the third step
where the process completes by the formation of crystalline microfibrils
(Merzendorfer and Zimoch 2003; Muthukrishnan et al. 2012). These microfibrils
further combine with other sugars and proteins to develop the insect’s cuticle and
PM. There are three key enzymes that act as the rate-limiting factors in the chitin
biosynthesis pathway, i.e., glutamine-fructose-6-P aminotransferase (EC 2.6.1.16),
UDP-GlcNAc pyrophosphorylase (EC 2.7.7.23), and chitin synthase (EC
2.4.1.16). Chitin biosynthesis pathway is published and well discussed in the book
chapter by Kramer and Muthukrishnan (2009). In the biosynthesis pathway
(Fig. 14.2), trehalose is the extracellular source of sugar and acted upon by
14 Structure, Regulation, and Potential Applications of Insect Chitin-Metabolizing 299
Glucose
Hexokinase
Glucose--6-phosphate
Glucose-6-P isomerase
Fructose-6-phosphate
Glutamine-fructose-6-P
aminotransferase
Gtucosamine-6-phosphate
Glucosamine-6-P N-
acetyltransferase
N-acetylglucosamine 6-P
Phospho-N-acetyl-
glucosamine mutase
N-acetylglucosamine 1-P
UDP-N-acetylglucosamine
UDP-N-acetylglucosamine
Chitin synthase
Chitin
O O
HO H
NAc OH
O O
OH O OH
O NAc
UDP
O O
HO
NAc H NAc
OH
O O OH
OH O O O
O NAc OH
UDP
O O
HO
NAc H
OH OH NAc
O O O OH
O
OH O O O
O NAc
NAc OH
UDP
O O
HO
NAc H NAc OH NAc
OH OH
O O O
O
O O
OH O O
O
O NAc OH
NAc
OH
UDP
Donor A B C D
14.2.3 Regulation
14.2.4 Applications
CHS is the enzyme responsible for the synthesis of chitin in insect exoskeleton.
CHS inhibitors are gaining huge importance in therapeutic medicine. CHS has also
role in plant infection process. The utilization of pesticides is crucial for crop, pub-
lic hygiene, and pest control. The present commercial pesticides are facing the prob-
lems of resistance. To properly check and control the problems arising from the
302 M. Kumar et al.
insufficient pest control, there is a need to search for novel compounds capable of
acting at new target sites. CHS is essential for insect growth and development, and
thus it also serves as the ideal target for insecticides.
Arakane et al. (2008) have reported the role of CHS in red flour beetle, T. casta-
neum, and conferred that CHS is required for embryonic and adult development as
well as for other types of molting. RNA interference (RNAi)-mediated knockdown
of CHS genes can be an efficient pest control approach (Harris and Fuhrman 2002;
Mansur et al. 2014; Veronico et al. 2001). Apart from CHS, enzymes like acetylcho-
linesterase, cytochrome P450, amino peptidase N, allatostatin, allatotropin, trypto-
phan oxygenase, arginine kinase, etc. can be regulated by RNAi approach (Kola
et al. 2015). Zhang et al. (2010), on the basis of the observation that chitin synthesis
can be blocked by dsRNA in mosquitoes, established a process to create a systemic
knockdown of CHS gene expression in Anopheles gambiae larvae by nourishing
with nanoparticles containing chitosan and dsRNA specific for the target gene. Yang
et al. (2016) showed the potentiality of developing effective insecticides by repress-
ing CHS1 and chitinase10 genes by miR-71 and miR-263 in locusts, which resulted
into blocking of molting and alterations in the chitin content. Zhang and Yan Zhu
(2013) reported slight in vitro inhibition of A. gambiae CHS activity by the employ-
ment of diflubenzuron and nikkomycin Z at the concentration of 2.5 μmol/L. But
they did not find any in vivo inhibition at any concentration by polyoxin D. In a
study conducted by Chen et al. (2013), the full-length cDNA-encoding chitin syn-
thase 2 (BdCHS2) was cloned and characterized Bactrocera dorsalis. The study
proposed that BdCHS2 could play a major role in the regulation of midgut chitin
content and, thus, affects the growth and development of B. dorsalis. Peptidyl
nucleosides and acylureas are known CHS inhibitors (Abo-Elghar et al. 2004;
Wilson and Cryan 1997; Ruiz-Herrera and San-Blas 2003; Tellam and Eisemann
2000). The former contain polyoxins and nikkomycins, whereas the latter exhibit
high CHS activity with compounds like diflubenzuron and novaluron (Merzendorfer
2006; De Cock and Degheele 1998; Soltani et al. 1993). Fontoura et al. (2012) have
reported the efficiency of chitin synthesis inhibitor compound, novaluron, against
organophosphate-resistant Aedes aegypti.
14.3 Chitinases
Chitinase genes from various insect species have been cloned and studied to get
more information about structure, gene variants, and related functions. Kim et al.
(1998) cloned the cDNAs encoding chitinases from B. mori and Hyphantria cunea
and studied their gene expression during metamorphosis. Southern blot analysis
further divulged that B. mori genome contains only one chitinase gene, while
H. cunea genome has one or two chitinase gene copies. The chitinase cDNA of
B. mori and H. cunea encoded 63.4 and 62 kDa proteins that contain 565 and 553
amino acids, respectively. Both the chitinases exhibit 75% and 77–80% homology
with chitinase from M. sexta. The enzymes were N-glycosylated, and the B. mori
enzyme has three potential N-glycosylation sites at the amino acid residues 86–89,
NFTS; 304–307, NATG; and 398–401, NYTV, whereas two potential sites were
present at the amino acid residues 86–89, NFTA and 304–307, NATG in H. cunea
chitinase. The presence of amino acid residues at conserved positions is necessarily
required for activation of chitinases that secreted as inactive zymogens in insects.
Lysine or arginine residues are present at conserved positions in the Tenebrio moli-
tor chitinase 5 (Royer et al. 2002). Similarly, lysine residues are also important for
activation of A. gambiae chitinases (Shen and Jacobs-Lorena 1997).
306 M. Kumar et al.
of proteolytic enzymes in the molting fluid that coincided with the beginning of
degradation suggested the possible role of proteolysis in activation of chitinases
(Samuels and Reynolds 1993). Similar results were detected in (Shen and Jacobs-
Lorena 1997) A. gambiae, where whole gut extracts show chitinase activity after
treatment with trypsin that cleaves at Lys-31 or Lys-32. Lysine or arginine residues
are also present at conserved positions in the T. molitor chitinase 5.
Multiple chitinases are observed to be produced by entomopathogenic fungi, and
some of them act synergistically with proteases to degrade insect cuticle. However,
involvement of chitinase in insect fungus pathogenesis has not been fully decoded.
A 33 kDa endochitinase, Bbchit1, was produced by Beauveria bassiana; Bbchit1
exhibited significant similarity to Trichoderma harzianum endochitinase Chit36Y
and putative endochitinase from Streptomyces avermitilis and Streptomyces coeli-
color (Fang et al. 2005). However, Bbchit1 had low identity levels to other chitinase
genes from entomopathogenic fungi. The presence of putative CreA/Crel carbon
catabolic repressor-binding domains in the regulatory sequence was consistent with
glucose suppression of Bbchit1. Insect bioassays indicated that the enhanced
expression of Bbchit1 is related with the increased virulence of B. bassiana for
aphids.
Chitinase family genes in insects are functionally specialized, primarily during
the molting process. Among the large family of chitinase-like proteins present in
the red flour beetle, T. castaneum (Tc), TcCHT5 was found to be required only for
pupal–adult molting (Zhu et al. 2008a). Downregulation of TcCHT5 by injection
of enzyme-specific dsRNA into larvae resulted into a lethal phenotype. The larvae
had metamorphosed into pupae and then pharate adults with incomplete adult
eclosion. Deterrence of embryo hatch, larval molting, pupation, and adult meta-
morphosis by specific knockdown of TcCHT10 indicated its vital role these pro-
cesses. Another chitinase-like protein, TcCHT7, was essentially required for
abdominal contraction and wing/elytra extension immediately after pupation,
while its role was dispensable for larval–larval molting, pupation, and adult eclo-
sion. TcIDGF4 is also a chitinase-like protein that contributed to adult eclosion.
Knockdown of other chitinase-like proteins like imaginal disk growth factor 2
(IDGF2) has not resulted into phenotypic effects. The studies on T. castaneum
chitinases provided a biological rationale for the presence of a large assortment of
chitinase-like proteins.
Locusta migratoria secreted duplicated chitinase 5 genes among which LmCht5-1
was observed to be expressed in hindgut while LmCht5-2 in integument, foregut,
hindgut, and fat bodies (Li et al. 2015a). Furthermore, LmCht5-2 is not vital for
development and survivorship of the locust. Similar expression patterns of LmCht5-1
and LmCht5-2 from the fourth-instar nymphs to the adults suggested their similar
regulation and response to the active molting hormone, 20-hydroxyecdysone.
Chitinase variants from E. sinensis exhibit differential expression patterns
implied to their distinct biological functions during growth, development, and
reproductive stages (Li et al. 2015b). EsCht1 from group I might play a role in the
digestion of chitin-containing food, while EsCht2 from group III has a role in the
degradation of chitinous cuticle during molting for growth and during the
308 M. Kumar et al.
postembryonic development. EsCht3 from group III potentially had dual roles in the
digestion of chitin-containing food and defense against chitin-bearing pathogens.
EsCht3, EsCht4, and EsCht6 are potentially required reproductive molting as evi-
dent by their overexpression in the reproductive system. Enhanced EsCht2 mRNA
expression in the cuticle and EsCht4 and EsCht6 mRNA expression in the hepato-
pancreas were 108-fold, 19-fold, and 12-fold which was observed in the premolt as
compared to the intermolt stage, respectively, during molting (Li et al. 2015b).
Sex-related variations in properties of chitin present in male and female grass-
hopper species revealed that the α-chitin present in all is similar in terms of thermal
properties and crystalline index (Kaya et al. 2015). Two major differences observed
in chitin with respect to gender were the presence of more amount of chitin in males
than females with dry weight ranging from 4.71% to 11.84% and occurrence of
nanofibers (25–90 nm) and nanopores (90–250 nm) in the male chitin surface.
Nanofibers were observed only in Melanogryllus desertus females.
The cuticle forms an apical extracellular matrix (ECM) that covers exposed
organs, such as the epidermis, trachea, and gut, for organizing morphogenesis and
protection of insects. Cuticle proteins and chitin are involved in the formation of
extracellular matrix. Chitinases (Chts) and imaginal disc growth factors (Idgfs)
were observed to be essential for larval and adult molting in Drosophila (Pesch
et al. 2016). Depletion of Cht and Idgf resulted into deformed cuticle, larval, and
adult molting defects and insufficient protection against wounding and bacterial
infection that led to early lethality. Cht2/Cht5/Cht7/Cht9/Cht12 and idgf1/idgf3/
idgf4/idgf5/idgf6 are needed for organizing proteins and chitin matrix at the apical
cell surface (Pesch et al. 2016). Chts are required for extracellular matrix formation,
while idgfs act as structural proteins to maintain the extracellular matrix scaffold
against chitinolytic degradation. Chts and Idgfs play analogous roles in ECM
dynamics across the insect taxa put forwarded them as new targets for species-
specific pest control.
Chitin has also been identified in the compound eyes of arthropods, where it is
considered as a part of the visual system. Corneal lens of dragonfly (Sympetrum
fonscolombii) contains 20.3% chitin and presents the α-form for increased
mechanical strength (Kaya et al. 2016a). Scanning electron micrographs revealed
that the outer part of corneal lenses consisted of long chitin fibrils with regular
arrays of papillary structures, while the smoother inner part had concentric lamel-
lated chitin formation with shorter chitin nanofibrils. The presence of chitin in the
compound eyes paves the way to design chitin-based optical materials.
Physicochemical characteristics and chitin content were observed to variate with
developmental stages in Vespa crabro (Kaya et al. 2016b). With the growth, chitin
content of V. crabro was gradually increased from 2.1% to 10.3% with altered
surface properties. From larval stage to pupal stage, threefold increase in chitin
deposition was observed. The house dust mite (HDM) allergen Der p 18 belongs
to the glycoside hydrolase family 18 chitinases (Resch et al. 2016). The allergen
exhibits weak chitin-binding activity and is mainly present in the PM of HDM gut
and to a lower extent in fecal pellets. The allergen can be utilized for developing
diagnostic test panels for HDM allergy.
14 Structure, Regulation, and Potential Applications of Insect Chitin-Metabolizing 309
A range of chitinase-like proteins are also present in various insect species that lack
catalytic activity. Insect chitinase-like proteins were observed to be encoded by a
large and diverse group of genes as suggested by bioinformatics-based investigation
of three insect species, i.e., D. melanogaster, A. gambiae, and T. castaneum (Zhu
et al. 2008c). Sixteen, 16, and 13 putative chitinase-like genes have been identified
in the genomic databases of T. castaneum, D. melanogaster, and A. gambiae,
respectively. Based on the phylogenetic analyses, the chitinase-like proteins have
been classified into five groups. Among the groups, groups I–III are each repre-
sented by only a single gene in each species, while multiple genes encode group IV
and group V chitinase-like proteins. Group I chitinases are secretory proteins, are
abundant in molting fluid and/or integument, and contain single copy of catalytic
and chitin-binding domain (ChBD) connected by an S-/T-rich linker polypeptide.
Group II chitinases possess multiple catalytic and ChBDs and are unusually larger-
sized secreted proteins. Group III chitinases are predicted to be membrane-anchored
proteins that contain two catalytic domains. The most divergent is group IV chitin-
ases that usually lack a ChBD and/or an S-/T-rich linker domain. These are present
as secreted proteins in gut or fat body. Putative chitinase-like imaginal disc growth
factors (IDGFs) included in the group V.
Glycoside hydrolase family 18 involves chitinase-like proteins (CLPs) that pos-
sess structural similarity to chitinases but lack enzymatic activity (Kucerova et al.
2016). CLPs are observed to be upregulated in several human disorders that affect
regenerative and inflammatory processes, but very little is known about their normal
physiological function. Kucerova et al. (2016) showed imaginal disc growth factor
3 (IDGF3), a CLP from D. melanogaster, which plays an immune-protective role
during entomopathogenic nematode infections. Whole-genome transcriptional
310 M. Kumar et al.
analysis of nematode-infected wild type and Idgf3 mutant larvae revealed that
IDGF3 also has roles in repression of Jak/STAT and wingless signaling. IDGF3 has
multiple roles in innate immunity. It is an essential component required for the for-
mation of hemolymph clots to seal wounds. Vertebrate and invertebrate CLP pro-
teins supposed to have analogous function and a broad impact on inflammatory
reactions and infections. Further, genetic analysis of CLP will help to elucidate
molecular basis of CLP functions.
The importance of chitinolytic enzymes for insect, nematode, and fungal growth
has raised the concerns regarding their employment not only as biopesticides or
chemical defense proteins in transgenic plants but also as microbial biocontrol
agents. Insect pests and pathogenic fungi can be biologically controlled by target-
ing chitin present in their extracellular matrices and cell wall. Chitinases can be
exploited as insect control agents due to their ability to degrade chitin associated
with the PM or exoskeleton. cDNA of chitinase from cotton leaf worm (Spodoptera
littoralis) has been synthesized, and its tolerance against insects was increased by
transgenic maize plant system (Osman et al. 2015). Insect chitinase transcripts
and proteins were expressed in transgenic maize plants, and their functional integ-
rity was confirmed using insect bioassays. The bioassays employing transgenic
maize plants against Sesamia cretica (corn borer) revealed that ~50% of the
insects present on transgenic plants were died owing to their enhanced resistance
against S. cretica.
In planta expression of chitinase RNAi effectors using a recombinant plant virus
can be potentially employed to control Mythimna separata, a prevalent corn pest in
China (Bao et al. 2016). Fragments of the M. separata chitinase sequences were
cloned into a virus vector in order to produce RNA interference (RNAi) effectors
during virus infection and replication in plants. Expression of target MseChi1 and
MseChi2 genes were downregulated by 76 and 45 %, respectively, when the infected
plants were fed to M. separata.
The filamentous fungus, I. fumosorosea, is a chitinase producing promising
insect biocontrol agent (Huang et al. 2016). The chitinase (chit1) gene from
I. fumosorosea encoded for a 423 amino acid (46kDa) long protein present as a
single copy in the genome and an important virulence factor. Chit1 gene knockout
strains of I. fumosorosea (ΔIfchit1) displayed minor alterations in mycelial growth,
increased temperature sensitivity, delayed sporulation, and increased conidial pro-
duction. Also, ΔIfchit1 exhibits decreased infectivity, i.e., increased LC50 (three-
fold to fourfold) and a significantly delayed time to death (LT50 from 3 to 6 days)
as revealed by insect bioassays using Plutella xylostella (diamond back moth).
Chitinase (HaCHI) gene was selected as a potential target to develop sustainable
and environmental-friendly methods for crop improvement and protection against
devastating agricultural insect pest, H. armigera (Reddy and Rajam 2016). The
enzyme is critically required for molting and metamorphosis.
14 Structure, Regulation, and Potential Applications of Insect Chitin-Metabolizing 311
Chitozymes have been thoroughly explored during the past decades because of their
wide spectrum of applications and growing interests in insect biotechnology. Chitin
is an important part of insect exoskeleton and PM, whereas chitinase appeared as a
critical enzyme for growth and development. Apart from chitin, many other compo-
nents are also present in insect cuticle. Interaction of chitin with other components
of extracellular matrix will redefine the integrity of chitin as an essential exoskele-
ton component. Expression of chitin synthesizing and metabolizing enzymes is
associated with various stages of insect growth and development advocating their
diverse physiological functions. Research on the multiplicity of chitin synthases and
chitinases will provide an idea about their relation with insect growth. Cloning of
the genes would provide recombinant enzymes and sub-domains, which can be uti-
lized to study regulation and specific functions of multiple enzyme forms. Chitin
synthases are comparatively less studied enzymes, and because of that the research
on chitin biosynthesis is still in its infancy. Initiation and elongation processes
whether involved along with the essential precursor molecules need to be explored
to understand the detailed mechanism of chitin synthesis. This would also support
development of novel insecticidal agents. Catalytic mechanism of multiple enzyme
forms will give further insight to the chitin synthesis and degradation in relation to
the molting. Furthermore, exploration and characterization of chitinase-like pro-
teins may provide some novel targets to be exploited as biocides. The significance
of chitinases as biocontrol agents can further be explored by developing transgenic
plants expressing chitinase genes against insect and plant pathogens. Enhancing the
gene expression levels along with combination of other insecticidal agents may fur-
ther improve the potentiality of chitinase in biocontrol of pests and pathogens.
Moreover, improved understanding of structure, catalysis, and biochemistry of
these largely unexploited resources will accelerate their utilization in various bio-
technological processes.
References
Abdel-Banat BMA, Kameyama Y, Yoshioka T, Koga D (1999) Purification and characterization of
a 54 kDa chitinase from Bombyx mori. Insect Biochem Mol Biol 30:107–117
Abo-Elghar GE, Fujiyoshi P, Matsumura F (2004) Significance of the sulfonylurea receptor (SUR)
as the target of diflubenzuron in chitin synthesis inhibition in Drosophila melanogaster and
Blattella germanica. Insect Biochem Mol Biol 34:743–752
Ahmad T, Rajagopal R, Bhatnagar RK (2003) Molecular characterization of chitinase from
polyphagous pest Helicoverpa armigera. Biochem Biophys Res Commun 310:188–195
Arakane Y, Muthukrishnan S (2010) Insect chitinase and chitinase-like proteins. Cell Mol Life Sci
67:201–216
Arakane Y, Zhu Q, Matsumiya M, Muthukrishnan S, Kramer KJ (2003) Properties of catalytic,
linker and chitin-binding domains of insect chitinase. Insect Biochem Mol Biol 33:631–648
Arakane Y, Hogenkamp DG, Zhu YC et al (2004) Characterization of two chitin synthase genes of
the red flour beetle, Tribolium castaneum, and alternate exon usage in one of the genes during
development. Insect Biochem Mol Biol 34:291–304
312 M. Kumar et al.
Arakane Y, Muthukrishnan S, Kramer K et al (2005) The Tribolium chitin synthase genes TcCHS1
and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix.
Insect Mol Biol 14:453–463
Arakane Y, Specht CA, Kramer KJ et al (2008) Chitin synthases are required for survival, fecun-
dity and egg hatch in the red flour beetle, Tribolium castaneum. Insect Biochem Mol Biol
38:959–962
Aronson NN, Halloran BA, Alexyev ME, Amable L, Madura JD (2003) Family 18 chitinase oli-
gosaccharide substrate interaction: subsite preference and anomer selectivity of S. marcescens
chitinase A. J Biochem 376:87–95
Bade ML (1974) Localisation of molting chitinase in insect cuticle. Biochim Biophys Acta
372:474–477
Bao W, Cao B, Zhang Y, Wuriyanghan H (2016) Silencing of Mythimna separata chitinase
genes via oral delivery of in planta-expressed RNAi effectors from a recombinant plant virus.
Biotechnol Lett 38:1961–1966. doi:10.1007/s10529-016-2186-0
Bortone K, Monzingo AE, Ernst S, Robertus JD (2002) The structure of an allosamidin complex
with the Coccidioides immitis chitinase defines a role for a second acid residue in substrate-
assisted mechanism. J Mol Biol 320:293–302
Brameld KA, Shrader WD, Imperiali B, Goddard WA (2002) Substrate assistance in the mecha-
nism of family 18 chitinases: theoretical studies of potential intermediates and inhibitors. J Mol
Biol 280:913–923
Chapman RF (1998) The insects: structure and function. Cambridge University Press, New York
Chen AC (1987) Chitin metabolism. Arch Insect Biochem Physiol 6:267–277
Chen L, Yang W-J, Cong L et al (2013) Molecular cloning, characterization and mRNA expression
of a chitin synthase 2 gene from the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae).
Int J Mol Sci 14:17055–17072
Chen L, Liu T, Zhou Y, Chen Q, Shen X, Yang Q (2014a) Structural characteristics of an insect
group I chitinase, an enzyme indispensable to moulting. Acta Crystallogr D Biol Crystallogr
70:932–942
Chen L, Zhou Y, Qu M, Zhao Y, Yang Q (2014b) Fully deacetylated chitooligosaccharides act as
efficient glycoside hydrolase family 18 chitinase inhibitors. J Biol Chem 289:17932–17940
Cohen E (1987) Chitin biochemistry: synthesis and inhibition. Annu Rev Entomol 32:71–93
Coutinho PM, Deleury E, Davies GJ et al (2003) An evolving hierarchical family classification for
glycosyltransferases. J Mol Biol 328:307–317
De Cock A, Degheele D (1998) Buprofezin: a novel chitin synthesis inhibitor affecting specifi-
cally planthoppers, whiteflies and scale insects. In: Insecticides with novel modes of action.
Springer, Berlin Heidelberg, pp 74–91
Demaeght P, Osborne EJ, Odman-Naresh J et al (2014) High resolution genetic mapping uncovers
chitin synthase-1 as the target-site of the structurally diverse mite growth inhibitors clofente-
zine, hexythiazox and etoxazole in Tetranychus urticae. Insect Biochem Mol Biol 51:52–61
Dorfmueller HC, Ferenbach AT, Borodkin VS et al (2014) A structural and biochemical model of
processive chitin synthesis. J Biol Chem 289:23020–23028
Fang W, Leng B, Xiao Y, Jin K, Ma J, Fan Y, Feng J, Yang X, Zhang Y, Pei Y (2005) Cloning of
Beauveria bassiana chitinase gene bbchit1 and its application to improve fungal strain viru-
lence. Appl Environ Microbiol 71:363–370
Filho BPD, Lemos EA, Secundino NEC, Pascoa V, Pereira ST (2002) Presence of chitinase and β
-N-acetylglucosaminidase in the Aedes aegypti chitinolytic system involving peritrophic matrix
formation and degradation. Insect Biochem Mol Biol 32:1723–1729
Fitches E, Wilkinson H, Bell H, Bown DP, Gatehouse JA, Edwards JP (2004) Cloning, expression
and functional characterisation of chitinase from larvae of tomato moth (Lacanobia olera-
cea): a demonstration of the insecticidal activity of insect chitinase. Insect Biochem Mol Biol
34:1037–1050
Fontoura NG, Bellinato DF, Valle D et al (2012) The efficacy of a chitin synthesis inhibitor against
field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem Inst Oswaldo
Cruz 107:387–395
14 Structure, Regulation, and Potential Applications of Insect Chitin-Metabolizing 313
Fukamizo T (2000) Chitinolytic enzymes: catalysis, substrate binding, and their application. Curr
Protein Pept Sci 1:105–124
Harris MT, Fuhrman JA (2002) Structure and expression of chitin synthase in the parasitic nema-
tode Dirofilaria immitis. Mol Biochem Parasitol 122:231–234
Hogenkamp DG, Arakane Y, Zimoch L et al (2005) Chitin synthase genes in Manduca sexta: char-
acterization of a gut-specific transcript and differential tissue expression of alternately spliced
mRNAs during development. Insect Biochem Mol Biol 35:529–540
Honda Y, Kitaoka M, Tokuyasu K, Sasaki C, Fukamizo T et al (2003) Kinetic studies on the
hydrolysis of N-acetylated and N-deacetylated derivatives of 4-methylumbelliferyl chitobio-
side by the family 18 chitinases ChiA and ChiB from Serratia marcescens. J Biochem (Tokyo)
133:253–258
Huang X, Zhang H, Zen KC, Muthukrishnan S, Kramer KJ (2000) Homology modeling of the
insect chitinase catalytic domain–oligosaccharide complex and the role of a putative active site
tryptophan in catalysis. Insect Biochem Mol Biol 30:107–117
Huang Z, Hao Y, Gao T, Huang Y, Ren S, Keyhani NO (2016) The Ifchit1 chitinase gene acts as
a critical virulence factor in the insect pathogenic fungus Isaria fumosorosea. Appl Microbiol
Biotechnol 100:5491–5503
Ibrahim GH, Smartt CT, Kiley LM et al (2000) Cloning and characterization of a chitin synthase
cDNA from the mosquito Aedes aegypti. Insect Biochem Mol Biol 30:1213–1222
Kaya M, Tozak KÖ, Baran T et al (2013) Natural porous and nano fiber chitin structure from
Gammarus argaeus (Gammaridae Crustacea). Excli J (In eCollection) 12:503–510
Kaya M, Lelesius E, Nagrockaite R, Sargin I, Arslan G, Mol A, Baran T, Can E, Bitim B (2015)
Differentiations of chitin content and surface morphologies of chitins extracted from male and
female Grasshopper species. PLoS One 10:1–14
Kaya M, Sargin I, Al-jafa I, Erdogan S, Arslan G (2016a) Characteristics of corneal lens chitin in
dragonfly compound eyes. Int J Biol Macromol 89:54–61
Kaya M, Sofi K, Sargin I, Mujtabaa M (2016b) Changes in physicochemical properties of chitin
at developmental stages (larvae, pupa and adult) of Vespa crabro (wasp). Carbohydr Polym
145:64–70
Kim MG, Shin SW, Bae KS, Kim SC, Park HY (1998) Molecular cloning of chitinase cDNAs
from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea. Insect Biochem
Mol Biol 28:163–171
Koga D, Funakoshi T, Mizuki K, Ide A, Kramer KJ, Zen KC, Choi H, Muthukrishnan S (1992)
Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm
Bombyx mori and the tobacco hornworm Manduca sexta. Insect Biochem Mol Biol 22:305–311
Kola VSR, Renuka P, Madhav MS et al (2015) Key enzymes and proteins of crop insects as candi-
date for RNAi based gene silencing. Front Physiol 6:119–134
Kramer KJ, Koga D (1986) Insect chitin: physical state, synthesis, degradation and metabolic
regulation. Insect Biochem 16:851–877
Kramer KJ, Muthukrishnan S (1997) Insect chitinases: molecular biology and potential use as
biopesticides. Insect Biochem Mol Biol 27:897–900
Kramer KJ, Muthukrishnan S (2005) Chitin metabolism in insects. In: Gilbert LI, Iatrou K, Gill S
(eds) Comprehensive molecular insect science, vol 4. Elsevier, Oxford, pp 111–144
Kramer K, Muthukrishnan S (2009) Chitin metabolism in insects. In: Gillbert LI (ed) Insect devel-
opment: morphogenesis, molting and metamorphosis. Academic Press, London, pp 497–530
Kramer KJ, Corpuz LM, Choi H, Muthukrishnan S (1993) Sequence of a eDNA and expression of
the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochem Mol Biol
23:691–701
Kucerova L, Broz V, Arefin B, Maaroufi HO, Hurychova J, Strnad H, Zurovec M, Theopold U
(2016) The Drosophila chitinase-like protein IDGF3 is involved in protection against nema-
todes and in wound healing. J Innate Immun 8:199–210
Li D, Zhang J, Wang Y, Liu X, Ma E, Sun Y, Li S, Zhu KY, Zhang J (2015a) Two chitinase 5
genes from Locusta migratoria: molecular characteristics and functional differentiation. Insect
Biochem Mol Biol 58:46–54
314 M. Kumar et al.
Royer V, Fraichard S, Bouhin H (2002) A novel putative insect chitinase with multiple catalytic
domains: hormonal regulation during metamorphosis. Biochem J 366:921–928
Ruiz-Herrera J, San-Blas G (2003) Chitin synthesis as a target for antifungal drugs. Curr Drug
Targets Infect Disord 3:77–91
Samuels RI, Reynolds SE (1993) Moulting fluid enzymes of the tobacco hornworm, Manduca
sexta: timing of proteolytic and chitinolytic activity in relation to pre-ecdysial development.
Arch Insect Biochem Physiol 24:33–44
Saxena IM, Brown RM Jr, Fevre M et al (1995) Multidomain architecture of beta-glycosyl trans-
ferases: implications for mechanism of action. J Bacteriol 177:1419
Shen Z, Jacobs-Lorena M (1997) Characterization of a novel gut-specific chitinase gene from the
human malaria vector Anopheles gambiae. J Biol Chem 272:28895–28900
Soltani N, Chebira S, Delbecque J, Delachambre J (1993) Biological activity of flucycloxuron, a
novel benzoylphenylurea derivative, on Tenebrio molitor: comparison with diflubenzuron and
triflumuron. Experientia 49:1088–1091
Stern R, Jedrzejas MJ (2008) Carbohydrate polymers at the center of life’s origins: the importance
of molecular processivity. Chem Rev 108:5061–5085
Suzuki K, Taiyoji M, Sugawara N, Nikaidou N, Hernissat B, Watanabe T (1999) The third chitin-
ase gene (ChiC) of Serratia marcescens 2170 and the relationship of its product to other bacte-
rial chitinases. Biochem J 343:587–596
Tellam RL (1996) Protein motifs in filarial chitinases: an alternative view. Parasitol Today
12:291–292
Tellam RL, Eisemann C (2000) Chitin is only a minor component of the peritrophic matrix from
larvae of Lucilia cuprina. Insect Biochem Mol Biol 30:1189–1201
Tellam RL, Vuocolo T, Johnson SE, Jarmey J, Pearson RD (2000) Insect chitin synthase. Eur J
Biochem 267:6025–6043
Tetreau G, Cao X, Chen Y-R, Muthukrishnan S, Jiang H et al (2015) Overview of chitin metabo-
lism enzymes in Manduca sexta: identification, domain organization, phylogenetic analysis
and gene expression. Insect Biochem Mol Biol 62:114–126
Tharanathan RN, Kittur FS (2003) Chitin – the undisputed biomolecule of great potential. Crit Rev
Food Sci Nutr 43:61–87
Van Leeuwen T, Demaeght P, Osborne EJ et al (2012) Population bulk segregant mapping uncov-
ers resistance mutations and the mode of action of a chitin synthesis inhibitor in arthropods.
Proc Natl Acad Sci 109:4407–4412
Veronico P, Gray L, Jones J et al (2001) Nematode chitin synthases: gene structure, expression and
function in Caenorhabditis elegans and the plant parasitic nematode Meloidogyne artiellia.
Mol Gen Genomics 266:28–34
Wilson TG, Cryan JR (1997) Lufenuron, a chitin synthesis inhibitor, interrupts development of
Drosophila melanogaster. J Exp Zool 278:37–44
Wittkopp PJ, Beldade P (2009) Development and evolution of insect pigmentation: genetic mecha-
nisms and the potential consequences of pleiotropy. Semin Cell Dev Biol (A Special Edition
on Biosensors and Development of Pigment Cells and Pigment Patterns). Elsevier 20:65–71
Wu Q, Liu T, Yang Q (2013) Cloning, expression and biocharacterization of OfCht5, the chitinase
from the insect Ostrinia furnacalis. Insect Sci 20:147–157
Yang B, Zhang M, Li L, Pu F, You W, Ke C (2015) Molecular analysis of atypical family 18 chitin-
ase from fujian Oyster crassostrea angulata and its physiological role in the digestive system.
PLoS One 10(6):1–13
Yang M, Wang Y, Jiang F et al (2016) miR-71 and miR-263 jointly regulate target genes chitin
synthase and chitinase to control Locust molting. PLoS Genet 12:e1006257
Yeager AR, Finney NS (2004) The first direct evaluation of the two-active site mechanism for
chitin synthase. J Organomet Chem 69:613–618
Zechel DL, Withers SG (2000) Glycosidase mechanisms: anatomy of a finely tuned catalyst. Acc
Chem Res 33:11–18
Zhang X, Yan Zhu K (2013) Biochemical characterization of chitin synthase activity and inhibition
in the African malaria mosquito, Anopheles gambiae. Insect Sci 20:158–166
316 M. Kumar et al.
Zhang X, Zhang J, Zhu K (2010) Chitosan/double stranded RNA nanoparticle mediated RNA
interference to silence chitin synthase genes through larval feeding in the African malaria mos-
quito (Anopheles gambiae). Insect Mol Biol 19:683–693
Zhang J, Zhang X, Arakane Y, Muthukrishnan S, Kramer KJ, Ma E, Zhu KY (2011) Comparative
genomic analysis of chitinase and chitinase-like genes in the African malaria mosquito
(Anopheles gambiae). PLoS One 6:e19899
Zhang D, Chen J, Yao Q, Pan Z, Chen J, Zhang W (2012) Functional analysis of two chitinase
genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by
RNA interference. Arch Insect Biochem Physiol 79:220–234
Zhu Q, Deng Y, Vanka P, Brown SJ, Muthukrishnan S et al (2004) Computational identifica-
tion of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics
20:161–169
Zhu Q, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S (2008a) Characterization of recom-
binant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect
Biochem Mol Biol 38:467–477
Zhu Q, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S (2008b) Functional specializa-
tion among insect chitinase family genes revealed by RNA interference. Proc Natl Acad Sci
105:6650–6655
Zhu Q, Arakane Y, Banerjee D, Beeman RW, Kramer KJ, Muthukrishnan S (2008c) Domain orga-
nization and phylogenetic analysis of the chitinase-like family of proteins in three species of
insects. Insect Biochem Mol Biol 38:452–466
Zhu KY, Merzendorfer H, Zhang W et al (2016) Biosynthesis, turnover, and functions of chitin in
insects. Annu Rev Entomol 61:177–196
Zhuo W, Fang Y, Kong L et al (2014) Chitin synthase A: a novel epidermal development regulation
gene in the larvae of Bombyx mori. Mol Biol Rep 41:4177–4186
Zimoch L, Hogenkamp D, Kramer K, Muthukrishnan S, Merzendorfer H (2005) Regulation of
chitin synthesis in the larval midgut of Manduca sexta. Insect Biochem Mol Biol 35:515–527
Correlation of Insects with Forensic
Sciences 15
Mian Sahib Zar and Moli Huang
Abstract
This chapter portrays the importance of insects in forensic sciences and high-
lights that how the insects are used as evidence in the court and how they can
assist in solving crimes. Forensic science plays a key role in the investigation of
crimes and terrorism. Forensic entomology is one of the emerging fields of
forensic sciences which aids in legal investigation. Postmortem interval or time
since death is the time elapsed from death to the discovery of the corpse. Various
methods are used to estimate postmortem intervals. They include algor mortis,
livor mortis, rigor mortis, and chemical and enzymatic changes. Insects also play
a significant role in estimation of time since death which is a prime concern in
the field of forensic medicine. Insects are considered to be the first visitors on any
decomposing dead matter. Forensic entomology is a field that is highly neglected
around the world so far. Much research work is required to flourish this field for
the purpose of forensic investigation. The basic challenge for forensic entomolo-
gists is the identification of insects at larval stages as they all look similar. The
taxonomic keys for identification at this level are still unavailable. There is a
need to cover the drawbacks of morphological identification of these species
especially if the adult form of insect is not available.
15.1 Introduction
Forensic (Latin word; meaning “in open court” or “public”) is a field of science that
solves legal issues in both criminal and in civil cases (Jobling and Gill 2004). Insects
are the most abundant living creatures that exist on the dry lands of earth. Though
few have some aquatic adaptations as well, mainly they tend to like land masses.
Insects are invertebrates of class Insecta belonging to phylum Arthropoda and king-
dom Animalia in the domain of Eukaryota. Usually not classified as animals, insects
are most diverse group having more than six to ten million extant species, making
them more than half of all the known living organisms, though the described species
are more than a million. The word “insect” comes from Latin word “insectum”
meaning divided into segments. Their study is called entomology, having been
derived from the Greek word “entomon” meaning “cut into two or cut in segments.”
General characteristics are an exoskeleton composed of chitin; three parts of the
body, i.e., the head, thorax, and abdomen; three pairs of legs; compound eyes and a
pair of antennae; and in some two wings as well (Triplehorn and Johnson 2004).
Insects are one tough group of living organisms which are highly adaptable and
exist in nearly any habitat. In their role as part of the ecosystem, they are both ben-
eficial and a nuisance for other organisms including humans. The beneficial roles
are production of honey, wax, silk, shellac, or parts of cosmetics. Some are used as
good source of protein-rich food. The nuisance factors are insects being vectors for
harmful agents of many diseases of man and animals. Also the insect bites, sting, or
infestation can cause serious illnesses or even death. Common examples of insects
are ants, flies, mosquitoes, termites, beetles, wasps, cockroaches, lice, fleas, butter-
flies, etc. (Lord and Rodrigues 1989).
The development is variable, but mainly the insects develop through a process of
holometabolism, i.e., going through a complete process of metamorphosis that
includes four stages of oviposition (or in some viviparous species by direct laying
of larvae), larval stage, pupa, and then imago or adult insect. Insects play an impor-
tant role in the scavenging of the decomposed dead organic matter other than bacte-
ria and fungi. As processors of dead animals, they consume most of the carcasses if
they are uncovered, otherwise if covered the carcasses mummify. The two special-
ized features of smell for detection and locomotion for flight help insects to approach
dead matter and consume it. The gases and body fluids including blood produce
specific odors that attract many scavengers including insects. They are capable of
consuming all carcasses except bones. The pattern of succession varies with sea-
sons, habitats, and countries, but the basic pattern of succession is constant around
the world (Lord and Rodrigues 1989).
Insects are first visitors from external environment for any dead matter, including
human corpses. Whether humans face natural death or die under some foul play, the
succession of insects is as same as in other dead matter. This leads to yet another
special type of study known as “forensic entomology” which can be defined as the
study of insects succeeding dead remains to aid legal investigations. The field
includes medicolegal, urban, and pests. The sequential colonization of a corpse as
part of decomposition saga and the rates of development of their offsprings are
15 Correlation of Insects with Forensic Sciences 319
analyzed and reported to find out the cause and manner of death as well to narrow
down the search of suspect (Amendt et al. 2004).
The predictable metamorphosis of insects acts as a perfect timeline to find out
how long it has been for a corpse being dead. This predictability is well utilized by
forensic entomologists to measure the time since death or postmortem interval in
cases of suspicious deaths where bodies are found unattended and gone through a
certain stage of decomposition (Villet and Amendt 2011).
These insects also make a part of circumstantial evidence at the scene of crime
whether found alive or dead, facilitating forensic scientists to rule out many aspects
of crime including foul play, addiction, neglect, infestation, suicides, offense, and
murders. A trained forensic scientist or a crime scene investigator should be well
aware of stages of decomposition and the sequence of entomological signs on the
dead body as well as at the scene of crime. So, forensic scientists, entomologists,
and investigators should be well equipped to collect proper evidence, its proper
handling, transport, and methods to deal with such evidence alive or dead (Lord and
Rodrigues 1989).
The role of insects in assisting decomposition and helping natural process of organic
matter consumption has been studied in the past centuries. Many paintings, figu-
rines, and drawings in previous literature or artwork suggest the observation of
decomposition process by the artists and scientists in the past. The first forensic
entomological evidence-related medicolegal case has been reported in medicolegal
textbook Hsi Yuan Lu (The Washing Away of Wrongs) by Chinese lawyer and death
investigator Sung Tz’u in the thirteenth century. The case described is of a stabbing
of a man near the rice field. The investigator asked all the farmers to gather at a place
and asked them to put down their sickles on the floor. All sickles seemed clean, but
blowflies gathered on one of the sickle that still had invisible traces of blood. When
investigated from the tool owner, he confessed to the murder.
Carl Von Linne in 1767 made observation that three flies destroyed a horse faster
than a lion by producing large numbers of maggots. Then in the eighteenth and
nineteenth century, French and German observed that buried bodies are consumed
by insects. French physician Bergeret in 1855 published the first case report that
included estimation of postmortem interval by making use of modern forensic ento-
mology. Colonization interval estimation started in the nineteenth century as men-
tioned in the famous book of La faune de cadavres by Pierre Megnin. This method
spreads to Canada and the United States and Europe giving rise to species lists and
monograph publications in 1920 (Benecke 2004). Again in 1935 forensic entomol-
ogy was successfully utilized in the most famous Buck Ruxton case. In 1958 a valu-
able study on insects and their relationships with decay rates was reported by Reed.
In the twentieth century, much work has been done on the taxonomy, and many
new species have been discovered, and their life cycles have been studied in detail.
Regarding the medicolegal importance of insects’ presence at scene of crime and
320 M.S. Zar and M. Huang
the time of colonization, much work has been done in Europe specially. The new
trend is to describe the life cycles of forensically important insects under different
biogeographical environments to rule out foul play, neglect, assaults, and murders,
and much success has been achieved to this end lately. Still the science of forensic
entomology needs more encouragement for further researches in this area to improve
our judicial and forensic system.
The use of insects in medicolegal investigations hides in the fact that they have been
studied extensively with already reported life cycles that are constant for each spe-
cies, so with the help of this available data, a fair and accurate prediction can be
given for the sequence and time of events that lead to a crime, assault, or murder.
The group of interest for forensic investigators and forensic entomologists is arthro-
pods and so the insects. Once they infest the scene or body, they naturally start a
biological clock that keeps ticking until they are found, collected, and reared to give
an accurate time since colonization.
Many factors affect the study of insects especially regarding a medicolegal scene
of crime. Most important factor is the environmental temperature. The colder the
weather, the longer is the life cycle and vice versa. Other facts affect the life cycles
too, including humidity, seasons, rainfalls, sunlight, shade, and off course not to
mention the food source. The limitations are the arthropods present at the scene but
advantageous as well, as the collection is easier and limited to those species.
To recognize and learn the manifestations of decomposition and to associate
these with insects present at a particular stage of decomposition need knowledge of
entomology and its training. The first presence of insects is in the form of eggs on
the decomposing dead matter or body. It is technically the first visible stage and
looks like rice-like particles on the body orifices initially. The other initial stage can
be threadlike worms at same locations if some insect species has directly laid worms
as in flesh flies. Rather than ignoring the collection of live specimen, it should be
started at the very moment (Shin et al. 2015). Though the following facts shouldn’t
be ignored:
• The stage of decomposition of the dead remains as algor mortis, livor mortis, and
rigor mortis has to be noted and properly photographed.
• Egg deposition on the body orifices or flexures has to be observed before han-
dling of corpse.
• See if larvae are present, and if they are, they have not dispersed far from the
body.
• Eggs when seen should be collected; same is true for larvae, pupae, or dead
young flies at the scene of crime.
• Make sure no entomological evidence is lost, and proper supplies and equipment
should be part of crime scene investigator’s equipment list.
Table 15.2 Information largely helps investigators to collect insects from crime scene
Insect group Forensic importance
Necrophagous flies Most primary and important isolatable
Diptera: species for forensic investigation for
Calliphoridae (blowflies; blue and green postmortem interval estimations
bottle → oviparous)
Sarcophagidae (flesh flies → viviparous)
Parasites and predators of necrophagous species Second most important group
Coleoptera (beetles)
Parasites of dipteran larva (necrophages)
Omnivorous species Adversely affect investigations by eating
Wasps, ants, beetles Necrophagous species
Feed on corpse and arthropods
Adventives species Less valuable insects
Use corpse as extension of normal habitat
Collembolan, spiders, centipedes
Acari
Fungi feeders
322 M.S. Zar and M. Huang
important insects after the death of a person, while the time since colonization is
actually the estimated age of insects collected from the body. Blowflies are the first
individual to arrive in the crime scene as they accumulate within minutes to few
hours (Greenberg 1991).
The larvae of different species of insects develop at different times or rates even
under similar temperature regimes. After hatching of eggs, the larvae go through
three growth phases called “instars” separated by molting of larval skin. After the
third instar stage, the larva goes into post-feeding stage or prepupal stage. It is also
the wandering stage of post-feeding larva where the larva leaves the wet environment
of flesh and body and seeks a drier place to pupate and rest. Then it goes through
metamorphosis and converted into imago or adult stage of insect (Fig. 15.1). For
complete developmental analysis, available temperature data nearest the crime scene
along with the life cycle of that species from former research should be considered.
Various methods are used worldwide to calculate time since death by entomo-
logical evidence. Some are stages of succession, age-dependent changes in the
intestinal contents, onstage invasion, developmental patterns, weights of larvae,
isomegalen/isomorphen diagrams, fly eggs, from insect’s gut contents, from
cuticular hydrocarbons, width, from accumulated degree days/hours (ADD/
ADH), aging blowfly eggs through gene expression, ontogenetic study, effect of
body length and crawling speed, larval dispersal, length of larvae, pupae, inter-
nal morphological analysis of pupae, new simulation model, differential gene
expression during metamorphosis, estimation of age with 3D micro-computed
tomography, and volatile organic compounds released by larvae and pupae
(Sharma et al. 2015).
ADULT
SEVERAL
WEEKS
PUPA EGGS
9 – 15 DAYS 20 – 28 HRS
LARVA LARVA
THIRD INSTAR FIRST INSTAR
100 – 360 HRS 18 – 34 HRS
LARVA
SECOND INSTAR
16 – 28 HRS
Fig. 15.1 Life cycle of insects (adapted from Krikken and Huijbregts (2001))
15 Correlation of Insects with Forensic Sciences 323
The insects revealed the original location of crime when the body is removed from
the actual place due to the absence of species of a particular place. This could help
the police investigator that crime is committed somewhere else. The place where the
body is found is of utmost importance for circumstantial findings as well as for col-
lection and detection of entomological fauna. A detailed and precise description of
the locality where the body is found gives an idea of the habitat to the forensic
entomologists. Written notes along with photographs are necessary to assess the
habitat type, terrain, vegetation, soil, and exposure of the body to sunlight or rain.
The climatic conditions profoundly affect the faunal succession on the corpse.
Temperature is the most important factor followed by climatic data and weather
conditions like rainfall, cloud cover, wind speed, and direction. When properly col-
lected forensically important insects prove to be a powerful tool for detection of
homicide, sudden deaths, assault, and violent crimes (Varatharajan and Sen 2000).
The insects are useful source for linking the origin of drugs in drug trafficking cases
(Varatharajan and Sen 2000).
Insects when feed on cadaver tissues (Fig. 15.2) will also ingest and store toxicologi-
cal substances consumed by the person just before death or stored in tissues for a
N
Sarcophaga sp Sarcophaga Maggot Chrysoma Sp
Fig. 15.2 Morphology of few insects found on corpses (adapted from Divya and Sathe (2015))
324 M.S. Zar and M. Huang
longer time. Successful extraction of these substances from the insects can solve cases
of substance abuse, suicide, or poisoning especially when the corpse is decomposed
beyond performing toxicological analysis on tissues. Though the amount of sub-
stances can be difficult to calculate to accuracy, however, qualitative results can still
lead to useful information where crime scenes are concerned. The forensic insect is
the source of detecting cases of narcotics when corpse is putrefied to identify. GC-MS
and HPLC are techniques utilize to find toxins in insects present on corpse. So the
detection of drugs in chitinized insect coins the term forensic entomotoxicology. The
presence of toxicological material that causes alteration of growth pattern of arthro-
pods also demonstrated to play key role in determining PMI (Joseph et al. 2011).
The fact that entomology experts are dramatically decreasing since 1990s, the iden-
tification of arthropods on morphological level is getting difficult nowadays. DNA
typing since 1985 is becoming a powerful identification tool in the field of forensic
sciences and medicine due to the following reasons (Jeffreys et al. 1985):
• Huge diagnostic information are obtained for crime investigation through molec-
ular techniques as compared to older methods like blood group typing.
• DNA is present in all biological tissues except RBCs, which can be easily used
for forensic investigation.
• DNA is much resistant to outside degradation than other biological molecules
like proteins.
• Molecular biology allows the use of DNA for forensic investigation even if DNA
is strongly degraded and broken into short pieces.
It can be useful if DNA settings of medicolegal centers are able to support foren-
sic entomologists with DNA typing to get genetic fingerprints of insect specimen.
The usual specimen is human DNA, so finding services for insect DNA typing can
be unusual. The main goal nowadays is to find suitable PCR primers or sequencing
sites for insect identification on species level (not on individual level). So the possi-
ble targets are all types of repetitive DNA like random amplified polymorphic DNA
(RAPDs), STRs, and all mini-satellite DNA, as well as non-repetitive DNA and
unique sites on mtDNA (mitochondrial DNA). Insects have noncoding mtDNA
region that contains high proportions of adenine and thymine bases that are useful
for DNA typing in forensic entomology. Fly mtDNA has considerable biological
information that makes it easy to design PCR primers and to interpret the results of
any study on new fly species. The base sequence of protein-coding genes like cyto-
chrome oxidase subunits 1 and 2 (CO1+2) may help in identification of species (Rein
2001). The activity of flies at a crime scene can lead to artifacts (small spots) which
are valuable sources of DNA typing. Sometimes those traces might be the only
source of information for DNA typing (Kulstein et al. 2015). Necrophagous and
hematophagous arthropods and their excretion products might also be used as a
source of victim DNA to assist in identification of the crime (Campobasso et al. 2005).
15 Correlation of Insects with Forensic Sciences 325
Both human and nonhuman DNA profiles can be obtained from insect, as fly artifacts
may contain DNA from corpses which might give valuable information in cases
where a corpse had been removed (Mehus and Vaughan 2013).
At the scene of crime, forensic scientists, medicolegal investigators, and police offi-
cers have to cooperate to rule out foul play at crime scene as well as to reach the
motive of crime and cause of death. By properly collecting forensically important
evidences including the entomological evidence, not only postmortem interval can
be estimated but other causes like child abuse or neglect and elderly neglect can be
evaluated as well. This can lead to conviction of not only parents of neglected chil-
dren or adult kids of neglected old people but also of welfare workers involved.
In a particular case of a 41-year-old physician’s death, maggots were found in
only one eye socket, which was unusual. A bed light (40 W light bulb) had been
switched on in the bedroom. All other lights were switched off and no direct sun-
light could enter the room. Maggots flee light so obviously they invaded the eye that
was farther from the light source. As the mummification of the body progressed,
food source restricted, so maggots moved to the eye on which light was shining
(Rein 2001).
15.3.7.1 Lesions
It started during the nineteenth century when bite patterns of cockroaches and ants
became a topic of interest. The abrasions of the skin caused by these insects were
mistaken for signs of poisoning, e.g., the insect tracks are mistaken for trickling of
acid down the chin and neck or bite marks of beetles sometimes resemble a gunshot
wound even. In the 1930s it was reported that maggots can enter spongiosa of the
long bones to reach the bone marrow by creeping through foramina nutricia.
Dermestid beetles feed on dried-up corpses, while bacon and corpse beetles will
cause lesions that may resemble close-range gunshot wounds.
Ideally a forensic entomologist should be part of crime scene unit. The challenges
of the crime scene can be unpredictable and never ideal. So, the crime scene mobile
unit should be well equipped to collect all critical data including entomological
evidence. Samples should be collected from on, in, and beneath the corpse. Collect
adult flies with hand nets. After collection samples must be subdivided into pre-
served and alive samples to be reared to adult insects. Immature insects or larvae are
reared on beef liver or small musculature pieces from the corpse. The following
table adapted from Byrd (2010) shows supplies and equipment required for collect-
ing entomological data:
Table 15.3 (continued)
Item Reason
Plastic “yogurt” or “bait” containers: 16–64 oz. For collecting and shipping larvae
in size
Aluminum foil o hold live larvae and food source
T
during shipment (precut potato
wrappers work well)
Vermiculite (or dirt from scene) For filling the bottom of the larval
containers to allow for migration and
to absorb excess fluids during
shipment
Plastic specimen containers: 4–8 oz. size Additional collection containers
Paper labels (adhesive and nonadhesive, heavy bond Nonadhesive: used to label inside of
paper) preserved and live specimen
containers
Adhesive: for labeling outside of
containers
Graphite pencil For making labels (preservation fluids
will cause ink to smear)
Small hand trowel or garden spade For sampling soil and digging for
migrating larvae or pupae in outdoor
death scenes
Thermometer: digital Used for taking temperature of area
around the body and maggot mass
Photographic equipment (including scales) Need to capture forensically relevant
photographs: establishing mid-range
and close-ups (of insects)
Chemicals: ethyl acetate, ethanol, KAA Used to kill insects
Paper towel For cleaning jars, cleaning utensils,
and drying hands after disinfecting
Disposable gloves For personal protection
Sifting screens Used to process soil samples for
collecting insects and insect artifacts
Form for death scene investigation For record keeping
Shipping containers: styrofoam containers with lids Used to ship collected specimens to
are best because they are insulated. Corrugated appropriate experts
cardboard boxes are cheap and commonly used
1. Data collected
2. Time collected
3. Location of remains
4. Area of body infested
5. Name, address, and telephone number of collector
328 M.S. Zar and M. Huang
1. Take clear and good photographs of areas of collection of insects. There can be
a rapid change in the decomposed area infested. The bite marks of mites should
be marked on living persons.
2. Always take photographs without using flash because the maggots will be
“flashed out” or appear “just white nothing” especially on digital photos.
3. A metric and inch scale should be used for every single picture.
4. One spoon full of insects from at least three different locations on dead corpse
and crime scene must be collected in three different and properly labeled jars.
5. Put half of insects in 98% ethanol. Do not use hand cleaning alcohol or forma-
lin. Killed insects can be stored frozen with or without alcohol.
6. Kill the insects in boiling water (tea water) before storing in alcohol.
7. Take living insects and refrigerate (do not freeze). They should be covered with
a fabric so they can breathe. Transport insects to a biologist within 1–2 days.
Separate white from brown larvae and brown from adults or post-feeding adults.
8. Properly label insect evidence with location, time, date, and initials.
9. If any query arises at the crime scene regarding entomological evidence, call an
entomologist.
10. Identification of infesting species should be done by an experienced entomolo-
gist by using proper keys. Still much identification is carried out by determina-
tion of third instar larvae of unknown species by characteristics of their mouth
parts (Benecke 2004).
Forensic entomology is an emerging field in which the insects are used as evidence
to determine the death time of the corpse. It has become the most important tool in
crime investigation. Unfortunately this felid is still poorly understood due to the
lack of expertise, resources, knowledge, and awareness. There is need to incorporate
this area in the curriculum of universities and forensic research institutes as an
important tool to have insight in forensic investigation of cadavers found in the
crime scene. Much research work on forensic entomology is required to flourish this
field for the purpose of forensic investigation.
15 Correlation of Insects with Forensic Sciences 329
Conclusions
This chapter describes the correlation of insects with forensic science and
emphasizes that how the insects were used as evidence in the court and how they
can assist in solving crimes. In addition, it describes the history, habitat, and key
roles of insects in forensic sciences. Further it highlights the need and impor-
tance of forensic entomology in crime investigation.
References
Amendt J, Krettek R, Zehner R (2004) Forensic entomology. Nature Science 91:51–65
Benecke M (2004) Forensic entomology: arthropods and corpses. In: Tsokos M (ed) Forensic
pathology reviews, vol ΙΙ. Humana, Totowa, NJ, pp 207–240
Byrd JH, Castner JL (2010) Forensic Entomology: The Utility of Arthropods in Legal Investigations.
2nd Edition. CRC Press, Boca Raton, 681
Campobasso CP et al (2005) Forensic genetic analysis of insect gut contents. Am J Forensic Med
Pathol 26(2):161–165
Divya JK, Sathe TV (2015) Diversity occurrence and development of forensic insects in Dog canis
domesticus L Carcass from Kolhapur India. Int J Pharm Bio Sci 6:498–506
Gomes L, Godoy WAC, Von Zuben CJ (2006) A review of postfeeding larval dispersal in blowflies:
implications for forensic entomology. Naturwissenschaften 93(5):207–215
Greenberg B (1991) Flies as forensic indicators. J Med Entomol 28(5):565–577
Jeffreys AJ, Wilson V, Thein SL (1985) Individual-specific ‘fingerprints’ of human DNA. Nature
316(6023):76–79
Jobling MA, Gill P (2004) Encoded evidence: DNA in forensic analysis. Nat Rev Genet
5(10):739–751
Joseph I et al (2011) The use of insects in forensic investigations: an overview on the scope of
forensic entomology. J Forensic Dent Sci 3(2):89–91
Krikken J, Huijbregts J (2001) Insects as forensic informants: the Dutch experience and procedure.
Proc Exp Appl Entomol 12:159–164
Kulstein G, Amendt J, Zehner R (2015) Blow fly artifacts from blood and putrefaction fluid on
various surfaces: a source for forensic STR typing. Entomol Exp Appl 157(3):255–262
Lord WD, Rodrigues WC (1989) Forensic entomology: the use of insects in the investigation of
homicide and untimely death
Mehus JO, Vaughan JA (2013) Molecular identification of vertebrate and hemoparasite DNA within
mosquito blood meals from eastern North Dakota. Vector Borne Zoonotic Dis 13(11):818–824
Rein BM (2001) Purely unilateral occurrence of blowfly maggots in the face of a decomposing
body. Arch Kriminol 208:182–185
Sharma R, Kumar Garg R, Gaur JR (2015) Various methods for the estimation of the post mortem
interval from Calliphoridae: a review. Egypt J Forensic Sci 5(1):1–12
Shin SE et al (2015) The first survey of forensically important entomofauna collected from medi-
colegal autopsies in South Korea. Biomed Res Int 2015:6
Triplehorn CA, Johnson NF (2004) Borror and DeLong’s introduction to the study of insects.
Thomson Brooks/Cole, Belmont, p 888
Varatharajan R, Sen A (2000) Role of entomology in forensic sciences. Curr Sci 78(5):544–545
Villet MH, Amendt J (2011) Advances in entomological methods for death time estimation. In:
Turk EE (ed) Forensic pathology reviews. Humana, New York, 213–217s
Nanoparticles as Precious Stones
in the Crown of Modern 16
Molecular Biology
M. Rajesh Kumar and P. Joice Sophia
Abstract
The interdisciplinary field of research on biosystems at the nanoscale involving
physical sciences, molecular engineering, biology, biotechnology, and medicine
supplements the knowledge of synthesizing new drugs, targeted delivery, regen-
erative medicine, and neuromorphic engineering forms the booming research in
the present society. The present chapter deals with the role of nanoparticles in
modern molecular biology. This is an interesting area of research that creates
great impact on the healthcare of the society. The prime focus is to give the
reader a historic background of nanomaterial application in biology and medi-
cine. We have also provided the overview of most recent developments in this
field leading to discussion of hard road to commercialization.
16.1 Introduction
M. Rajesh Kumar (*)
College of Chemistry, Chemical Engineering and Material Science, Soochow University,
Suzhou 215123, China
e-mail: rajeshkumar_vgm@yahoo.com
P. Joice Sophia
Department of Nanoscience and Technology, Bharathiar University,
Coimbatore 641046, India
e-mail: joicesophia@gmail.com
organisms (Elsaesser and Howard 2012; Prabhu and Poulose 2012). This ability
results primarily from their small size thereby allowing them to penetrate physio-
logical barriers and travel within the circulatory systems of a host (Panessa-Warren
et al. 2006; Li et al. 2010; Chen et al. 2016). The vast majority causes little ill effect
and goes unnoticed, but occasionally an intruder will cause appreciable harm to the
organism. The most advanced of the toxic intruders are viruses, composed of nucleic
acid-based structures that allow them not only to interfere with biological systems
but also in parasitically exploit cellular processes to replicate themselves (Ravindran
et al. 2016; Franzen and Lommel 2009). A growing number of recent studies show,
however, that nano- and microorganisms might play a vital role in many chronic
diseases where infection pathogens have not been suspected, diseases that were
previously attributed only to genetic factors and lifestyle (He and Shi 2009; Zheng
et al. 2016). These diseases include leukemia (caused by viruses from the retrovirus
and herpesvirus families) (Nisole et al. 2005; Walther and Stein 2000; Jarrett 2006),
cervical cancer (Papillomavirus) (DiMaio and Liao 2006; Muñoz et al. 2003; Bosch
et al. 1995), liver cancer (hepatitis virus), gastric ulcer (Helicobacter pylori)
(Kusters et al. 2006; Blaser et al. 1995; Tomb et al. 1997), nasopharyngeal cancer
(Epstein-Barr virus) (Zur Hausen et al. 1970; Lo et al. 1999; Burgos 2005), kidney
stones (nanobacteria) (Çiftçioglu et al. 1999; Kramer et al. 2000; Kajander and
Ciftcioglu 1998), severe acquired respiratory syndrome SARS (coronavirus) (Peiris
et al. 2003; Rota et al. 2003; Marra et al. 2003; Kahn 2006), heart disease (Chlamydia
pneumonia) (Patel et al. 1995; Danesh et al. 1997; Bachmaier et al. 1999; Kol and
Santini 2004), juvenile diabetes (Coxsackievirus) (Atkinson et al. 1994; Horwitz
et al. 1998; Fohlman and Friman 1993), Alzheimer’s disease (Chlamydia pneu-
moniae) (Balin et al. 1998; Hammond et al. 2010; Itzhaki et al. 2004), pediatric
obsessive-compulsive disorder (Streptococcal bacteria) (Swedo et al. 1998; Mell
et al. 2005; Lynch et al. 2006), psychotic disorders (bornavirus) (Nunes et al. 2008;
Miranda et al. 2006), and prion diseases such as mad cow disease (proteins-prions)
(Chesebro 1998; Legname et al. 2004; Janka and Maldarelli 2004).
Nanotechnology is the manipulation of matter at the scale of 1–100 nanometers.
Using nanotechnology, we can control molecules at an atomic level and create
materials with unique properties. A nanometer is 10−9 (a billionth) of a meter. The
prefix nano has the Greek meaning of dwarf. As a reference point, a hair is approxi-
mately 100,000 nanometers. A red blood cell is approximately 10,000 nanometers.
Fundamentally the properties of materials can be changed by nanotechnology. We
can arrange molecules in a way that they do not normally occur in nature. The mate-
rial strength along with electronic and optical properties of materials could be
altered with the aid of nanotechnology.
Molecular biology explores cells and their characteristics, parts, and chemical pro-
cesses which pay special attention to how molecules control cell’s activities and
growth (Crick 1970). Looking at the molecular machinery of life that began in the
16 Nanoparticles as Precious Stones in the Crown of Modern Molecular Biology 333
early 1930s, but truly modern molecular biology emerged with the uncovering of
the structure of DNA in the 1960s (Meyer 2003; Morange and Cobb 2000). As a
science that studies interactions between the molecular components that carry out
various biological processes in living cells, an important idea in molecular biology
states that information flow in organisms follows a one-way street: genes are tran-
scribed into RNA, and RNA is translated into proteins. The molecular components
make up biochemical pathways that provide the cells with energy, facilitate process-
ing “messages” from outside the cell itself, generate new proteins, and replicate the
cellular DNA genome. For example, molecular biologists study how proteins inter-
act with RNA during “translation” (the biosynthesis of new proteins), the molecular
mechanism behind DNA replication, and how genes are turned on and off, a process
called “transcription” (Lodish et al. 1995).
The birth and development of molecular biology were driven by the collaborative
efforts of physicists, chemists, and biologists. As mentioned, modern molecular
biology emerged with the discovery of the double helix structure of DNA. The 1962
Nobel Prize in Physiology or Medicine was awarded jointly to Francis H. Crick,
James D. Watson, and Maurice H. F. Wilkins “for their discoveries concerning the
molecular structure of nucleic acids and its significance for information transfer in
living material” (Palenik et al. 2003; Scott and Thompson 2011). Advances and
discoveries in molecular biology continue to make major contributions to medical
research and drug development (Lipinski and Hopkins 2004).
DNA sequencing and synthesis are two sides of the same coin, the “read” and
“write” functions of genetic material (Shendure and Ji 2008; Chan 2005). This field
and its requisite technology took off in the 1990s with the Human Genome Project’s
effort to sequence billions of bases to unlock a new era of genetically informed
medicine (Sawicki et al. 1993; Venter et al. 2001). The resulting science is still a
work in progress – it turns out the genetic code a more complicated one than antici-
pated – but the technologies and companies helped it spawn are an impressive leg-
acy. The Integrated DNA Technologies (IDT) got its start during the Human Genome
Project, as it produced single nucleotides (the As, Ts, Cs, and Gs that comprise the
genetic code) and short oligonucleotide chains (or “oligos”) to facilitate a massive
sequencing effort around the world (Burns et al. 1996). Of course, sequencing tech-
nology has advanced dramatically in the intervening decades, but “you still need
oligos to do the sequencing,” explains Jerry Steele, IDT’s Director of Marketing,
“especially in the next generation sequencing space, sequencing and DNA synthesis
go hand in hand.” The current sequencing method of choice is Illumina, a process
that frequently returns millions of bases of DNA sequence by reading distinct step-
wise fluorescent signals associated with each base in a massively parallel array
(Schadt et al. 2010; Nakamura et al. 2011). To distinguish genetic material from
different samples (a few hundred are often run on the same plate), scientists label
each sample’s DNA extract with a distinct barcode (Hebert et al. 2003). With each
barcode comprised of about ten nucleotides, the demand for synthetic DNA chains
in the sequencing process is substantial (deWaard et al. 2008).
Unlike other biotech companies prioritizing longer constructs or gene variants,
IDT specializes in relatively short oligos. These chains are used not only in Illumina
334 M. Rajesh Kumar and P. Joice Sophia
barcoding but also as primers, consistent patches of sequence that may border
unknown regions and facilitate polymerase chain reaction (PCR)-based amplifica-
tion (Bhargava et al. 2013). Both techniques – “next-generation” Illumina sequenc-
ing and primer-based amplification – are staples of any self-respecting applied or
research-based microbiology laboratory, as they allow researchers to identify con-
stituent organisms or confirm a gene’s presence. With such short sequences, a single
nucleotide discrepancy could mean the difference between two Illumina samples
from opposite ends of the world or between a gene native to the Firmicutes or the
Proteobacteria. It’s a small margin for error, “so every base better be right,” explains
Steele. “As we’ve grown, it’s just a matter of maintaining that consistency on a
larger scale.” In the spirit of not fixing something that needs no repairs, IDT shipped
an entire fabrication room from its headquarters in Coralville, Iowa, to Belgium
when that facility was being built.
Fundamental as they are to modern biology, oligos are used every day in thou-
sands of laboratories around the world, often in innovative ways that the company
itself may not have predicted. “The things that people are doing with DNA are really
inspiring,” noted Steele. One of his favorite cases involves low-impact prenatal
tests: rather than a painful and invasive amniocentesis, “we’ve discovered that now
because of sequencing, we can see the baby’s DNA in a blood draw from the
mother.” Improved sequencing fidelity and throughput are expanding the resolution
of the technique, and Steele envisions scientists using next-generation sequencing
to detect cancer cells from the bloodstream as an early diagnostic tool. “Biology is
really leaving the lab and coming into the real world,” Steele explains, “and it’s
going to improve a lot of lives.”
and biocompatible electronic systems for detection and control, implants of wire-
less systems, neuroprostheses, and parts of the neural system form the part of
molecular biology applications (PROKOP 2001; Gillies et al. 2002; Mercanzini
et al. 2010; Tokárová et al. 2013). There is an interesting classification of nano-
technology in two classes, “wet” nanotechnology (living biosystems) and “dry”
nanotechnology. The systematic approaches are important in engineering man-
made objects at the nanoscale for integrating them into large-scale structures
which are similar to that of nature (Sarikaya et al. 2003; Alivisatos 2004).
Interestingly, this booming field is also known as biomimicry or bionics where
researchers get the idea from nature by mimicking them to be explored in various
applications (Chakrabarti and Shu 2010; Quinn and Gaughran 2010). Few exam-
ples of such concepts can be lotus effect, pearl effect, Gecko effect, butterfly
wings, Namib desert beetle, spider web, and so on (Bhushan 2009; Pereira et al.
2015). These biomimetic ideas were adopted for the successful fabrication of
Velcro, self-cleaning tiles, desert fog harvesting, water filters, adhesives, nano-
paints, etc. (Bhushan and Jung 2011; Vierra 2011; Ivanić et al. 2015; Diamanti and
Pedeferri 2015; Weiler and Goel 2015). Some of the companies that are involved
Table 16.1 Examples of companies commercializing nanomaterials for bio- and medical
applications
Company Major area of activity Technology
Advectus Life Drug delivery Polymeric nanoparticles engineered to
Sciences Inc. carry antitumor drug across the blood-
brain barrier
Alnis Biosciences, Biopharmaceutical Biodegradable polymeric nanoparticles for
Inc. drug delivery
Argonide Membrane filtration Nanoporous ceramic materials for
endotoxin filtration, orthopedic and dental
implants, and DNA and protein separation
BASF Toothpaste Hydroxyapatite nanoparticles seem to
improve dental surface
Biophan MRI shielding Nanomagnetic/carbon composite materials
Technologies, Inc. to shield medical devices from RF fields
Capsulution Pharmaceutical coatings Layer-by-layer polyelectrolyte coatings,
NanoScience AG to improve solubility of 8–50 nm
drugs
Dynal Biotech Magnetic beads
Eiffel Technologies Drug delivery Reducing size of the drug particles to
50–100 nm
EnviroSystems, Inc. Surface disinfectant Nanoemulsions
Evident Technologies Luminescent biomarkers Semiconductor quantum dots with amine
or carboxyl groups on the surface,
emission from 350 to 2500 nm
Immunicon Tracking and separation Magnetic core surrounded by a polymeric
of different cell types layer coated with antibodies for capturing
cells
(continued)
336 M. Rajesh Kumar and P. Joice Sophia
Table 16.1 (continued)
Company Major area of activity Technology
KES Science and Airocide filters Nano-TiO2 to destroy airborne pathogens
Technology, Inc.
NanoBio Corporation Pharmaceutical Antimicrobial nanoemulsions
NanoCarrier Co., Ltd Drug delivery Micellar nanoparticles for encapsulation of
drugs, proteins, DNA
NanoPharm AG Drug delivery Polybutylcyanoacrylate nanoparticles are
coated with drugs and then with surfactant
and can go across the blood-brain barrier
Nanoplex Nanobarcodes for
Technologies, Inc. bioanalysis
Nanoprobes, Inc. Gold nanoparticles for Gold nanoparticles bioconjugate for TEM
biological markers and/or fluorescent microscopy
Nanoshpere, Inc. Gold biomarkers DNA barcode attached to each nanoprobe
for identification purposes; PCR is used to
amplify the signal and also catalytic silver
deposition to amplify the signal using
surface plasmon resonance
NanoMed Drug delivery Nanoparticles for drug delivery
Pharmaceutical, Inc.
Oxonica Ltd Sunscreens Doped transparent nanoparticles to
effectively absorb harmful UV and convert
it into heat
pSivida Ltd Tissue engineering, Exploiting material properties of
implants, drug and gene nanostructured porous silicone
delivery, biofiltration
Smith & Nephew Acticoat bandages Nanocrystal silver is highly toxic to
pathogens
Quantum Dot Luminescent biomarkers Bioconjugated semiconductor quantum
Corporation dots
16.1.3.1 Nanomedicine
Nanomedicine is a field of medical science employing nanotechnology whose appli-
cations are increasing more and more, thanks to nanorobots and biological machines
which become very useful tools to develop this area of knowledge (Andrew 2000;
Wagner et al. 2006; Riehemann et al. 2009). Nanomedicine is the application of nan-
otechnology in different areas of medicine and biology (Andrew 2000). Nanomedicine
ranges from the biomedical applications of nanomaterials and biological devices to
nanoelectronic biosensors and even possible future applications of molecular nano-
technology such as biological machines (Nie 2010; Vinogradov and Wei 2012; Jain
and Stylianopoulos 2010). Current problems for nanomedicine involve understand-
ing the issues related to toxicity and environmental impact of nanoscale materials
(Nel et al. 2006; Hardman 2006; Dreher 2004; Colvin 2003; Jia et al. 2005). The size
of nanomaterials is similar to that of most biological molecules and structures; there-
fore, nanomaterials can be useful for both in vivo and in vitro biomedical research
and applications (Salata 2004). More than just an extension of “molecular medicine,”
nanomedicine will employ molecular machine systems to address medical problems
and will use molecular knowledge to maintain and improve human health at the
molecular scale (Freitas 2002; Freitas 2005; Kostarelos 2006; Bogunia-Kubik and
Sugisaka 2002). Nanomedicine will have extraordinary and far-reaching implications
for the medical profession, in the definition of disease and in the diagnosis and treat-
ment of medical conditions including aging, and ultimately for the improvement and
extension of natural human biological structure and function.
16.1.3.2 Nanoinsecticide
An insecticide is a substance used to kill insects. They include ovicides and larvi-
cides used against insect eggs and larvae, respectively. Insecticides are used in agri-
culture, medicine, industry, and by consumers. Insecticides are claimed to be a
major factor behind the increase in agricultural twentieth century’s productivity
(Perry et al. 2013). Nearly all insecticides have the potential to significantly alter
ecosystems; many are toxic to humans; some concentrate along the food chain.
Nanostructures are classified based on the number of dimensions, which are not
confined to the nanoscale range (<100 nm) (Kelsall et al. 2005):
Materials wherein all the dimensions are measured within the nanoscale (no dimen-
sions, or 0-D, are larger than 100 nm). The most common representation of zero-
dimensional nanomaterials is nanoparticles or quantum dots. Nanoparticles or
quantum dots can be amorphous or crystalline, single crystalline or polycrystalline,
metallic, ceramic, or polymeric, and composed of single or multi-chemical ele-
ments; exhibit various shapes and forms; and exist individually or are incorporated
in a matrix.
In 2-D nanostructures, two of the dimensions are not confined in the nanoscale.
Two-dimensional nanomaterials exhibit platelike shapes. Two-dimensional nano-
materials include nanofilms, nanolayers, and nanocoatings. Two-dimensional nano-
materials can be amorphous or crystalline, metallic, ceramic, or polymeric, deposited
on a substrate, integrated in a surrounding matrix material, made up of various
chemical compositions, and used as a single layer or as multilayer structures.
Bulk nanomaterials are materials that are not confined to the nanoscale in any
dimension and are known as 3-D nanostructures. These materials are thus character-
ized by having three arbitrarily dimensions above 100 nm. Materials possess a
nanocrystalline structure or involve the presence of features at the nanoscale. In
terms of nanocrystalline structure, bulk nanomaterials can be composed of a multi-
ple arrangement of nanosize crystals, most typically in different orientations. With
respect to the presence of features at the nanoscale, 3-D nanomaterials can contain
dispersions of nanoparticles, bundles of nanowires, and nanotubes as well as
multinanolayers.
The following table shows a list of nanomedical technologies (Freitasjr 2005)
(Table 16.2).
16 Nanoparticles as Precious Stones in the Crown of Modern Molecular Biology 339
Solid-state synthesis generally involves a heat treatment step in order to achieve the
desired crystal structure, which is followed by media milling (Cao 2004). While it
is generally believed that it is difficult for the lower limit of the average particle size
to be much below 100 nm, recent innovations by established companies in the
industry may prove otherwise. In particular, the Netzsch LMZ-25 ZETA II System
and the Dyno-Mill ECM may push the envelope on what mechanical attrition can
do to reduce the particle size.
Judging by the contents of publications, the scientific community has not shown
much enthusiasm for mechanical attrition processes for nanoparticle synthesis, perhaps
due to issues pertaining to impurity pickup, lack of control on the particle size distribu-
tion, and inability to tailor precisely the shape and size of particles in the 10–30 nm
range as well as the surface characteristics. Nonetheless, in several instances a modi-
fied version of mechanical attrition has been used to synthesize oxide nanoparticles.
Dry milling was used to induce chemical reactions through ball-powder collisions that
resulted in forming nanoparticles within a salt matrix. Particle agglomeration was min-
imized by the salt matrix, which then was removed by a simple washing procedure.
Most soft chemical methods are carried out in liquid media, based on the reac-
tants dissolving, diffusion, and crystallization process. Therefore, soft chemical
methods have other characteristics including:
Every method has its limitation. It is very difficult to synthesize certain chemi-
cals, for example, solid nitride, by soft chemical methods. However, as environmen-
tal protection has become a larger issue, soft chemistry, which is also called “green
chemistry,” has quietly evolved into a major phenomenon. The soft chemical method
is further classified as follows:
16.4 Applications
• Fluorescent biological labels (Bruchez et al. 1998; Chan and Nie 1998; Wang
et al. 2002)
• Drug and gene delivery (Mah et al. 2000; Pantarotto et al. 2003)
342 M. Rajesh Kumar and P. Joice Sophia
Fluorescent labeling is known for its nondestructive nature and high sensitivity.
This has made it one of the most widely used methods for labeling and tracking
biomolecules (Sahoo 2012). Fluorescent labels can be hybridized to mRNA to
help visualize interaction and activity, such as mRNA localization. An antisense
strand labeled with the fluorescent probe is attached to a single mRNA strand and
can then be viewed during cell development to see the movement of mRNA
within the cell (Weil et al. 2010). Advantages of these labels include a smaller
size with more variety in color. They can be used to tag proteins of interest more
selectively by various methods including chemical recognition-based labeling,
such as utilizing metal-chelating peptide tags, and biological recognition-based
labeling utilizing enzymatic reactions (Jung et al. 2013). However, despite their
wide array of excitation and emission wavelengths as well as better stability,
synthetic probes tend to be toxic to the cell and so are not generally used in cell
imaging studies. Silica nanoparticles and other different nanoparticles were
widely employed in near-infrared-to-visible upconversion fluorescent applica-
tions for imaging and targeted delivery in cancer treatment (Jiang et al. 2009;
Taton et al. 2000; Ow et al. 2005).
Drug delivery systems are engineered technologies for the targeted delivery and/or
controlled release of therapeutic agents (Allen and Cullis 2004). Drugs have long
been used to improve health and extend lives. The practice of drug delivery has
changed dramatically in the past few decades, and even greater changes are antici-
pated in the near future. Biomedical engineers have contributed substantially to our
understanding of the physiological barriers to efficient drug delivery, such as trans-
port in the circulatory system and drug movement through cells and tissues; they
have also contributed to the development of several new modes of drug delivery that
have entered clinical practice. Yet, with all of this progress, many drugs, even those
discovered using the most advanced molecular biology strategies, have unaccept-
able side effects due to the drug interacting with healthy tissues that are not the
target of the drug. Side effects limit our ability to design optimal medications for
16 Nanoparticles as Precious Stones in the Crown of Modern Molecular Biology 343
Advances in DNA sequencing technology have made it possible for scientists all
over the world to sequence complete microbial genomes rapidly and efficiently.
Access to the DNA sequences of entire microbial genomes offers new opportunities
to analyze and understand microorganism at the molecular level. Scientists are able
to detect pathogens in biological tissues and study variations in gene expression in
response to the pathogenic invasion. These responses help in designing novel
approaches for microbial pathogen detection and drug development. Identification
of certain microbial pathogens as etiologic agents responsible for chronic diseases
is leading to new treatments and prevention strategies for these diseases.
Each species of pathogens carries with it unique DNA or RNA signatures that
differentiate it from other organisms. One of the challenges is to develop this
DNA signature for each microorganism of interest for rapid and specific detec-
tion. Pathogen detection has become an important part of research in many fields
like biodefense, animal healthcare, food safety, diagnostics, pathology, clinical
research, forensics, and drug discovery. For biodefense, accurate analytical tech-
niques for discovering pathogenic agents are needed. Animal healthcare com-
munity uses pathogen detection to develop various diagnostic tests that are rapid,
reliable, and highly sensitive for effective control and treatment of diseases of
animals. In diagnostics, the technique is employed to detect or identify infec-
tious agents, toxins, parasites, metabolic disorders, and genetic susceptibility/
resistance.
344 M. Rajesh Kumar and P. Joice Sophia
The word protein is derived from the Greek proteios, meaning “of the first rank.” The
term was coined in 1838 by the Swedish scientist Jöns Berzelius, to reflect the impor-
tance of this group of molecules. SDS polyacrylamide gel electrophoresis (SDS-
PAGE) involves the separation of proteins based on their size. By heating the sample
under denaturing and reducing conditions, proteins become unfolded and coated with
SDS detergent molecules, acquiring a high net negative charge that is proportional to
the length of the polypeptide chain. When loaded onto a gel matrix and placed in an
electric field, the negatively charged protein molecules migrate toward the positively
charged electrode and are separated by a molecular sieving effect. After visualization
by a protein-specific staining technique, the size of a protein can be estimated by com-
parison of its migration distance with that of a standard of known molecular weight.
After protein transfer from an SDS-PAGE gel to a membrane, the remaining pro-
tein-free sites on the membrane must be blocked. This prevents the primary or second-
ary antibody from binding directly to the membrane and giving rise to a high
background signal. Several blocking reagents are in common use, including nonfat
dried milk, BSA, and casein. After blocking, the primary antibody is added and allowed
to bind to the protein. After washing (which removes nonspecifically bound antibody),
the secondary antibody is added, to detect where the primary antibody has bound.
After another wash step, the location of the secondary antibody (and therefore the
primary antibody and the protein of interest) is determined by adding a substrate for the
enzyme conjugated to the secondary antibody. Substrates are available that give rise to
a colored compound (chromogenic detection), or to the emission of light (chemilumi-
nescent detection), at the reaction site. The use of an antibody that reacts specifically
with an epitope commonly introduced into a recombinant protein eliminates the need
for a protein-specific antibody and allows the use of one antibody for the detection of
all proteins containing this feature. Coupling a reporter enzyme directly to such anti-
bodies eliminates the need for a secondary antibody and delivers significant time
savings.
cells more sensitive to the effects of radiation and certain anticancer drugs.
Techniques that may bring local tissues to quite high temperatures, such as radio-
frequency ablation, are not usually what is meant by “hyperthermia.” When com-
bined with radiation therapy, it is called thermoradiotherapy. Whole-body
hyperthermia has also been found to be helpful for depression (Hanusch et al. 2013).
It is also promoted for use in the treatment of chronic Lyme disease.
Cell biologists research the intricate relationship between structure and function at
the molecular, subcellular, and cellular levels. However, a complex biological sys-
tem such as a biochemical pathway can only be understood after each one of its
components has been analyzed separately. Only if a biomolecule or cellular compo-
nent is pure and biologically still active can it be characterized and its biological
functions elucidated.
Fractionation procedures purify proteins and other cell constituents. In a series of
independent steps, the various properties of the protein of interest solubility, charge,
size, polarity, and specific binding affinity are utilized to fractionate it or separate it
progressively from other substances. Three key analytical and purification methods
are chromatography, electrophoresis, and ultracentrifugation. Each one relies on
certain physicochemical properties of biomolecules.
MRI contrast agents are a group of contrast media used to improve the visibility
of internal body structures in magnetic resonance imaging (MRI). The most
commonly used compounds for contrast enhancement are gadolinium based.
Such MRI contrast agents shorten the relaxation times of atoms within body tis-
sues following oral or intravenous administration. In MRI scanners, sections of
the body are exposed to a very strong magnetic field causing primarily the hydro-
gen nuclei (“spins”) of water in tissues to be polarized in the direction of the
magnetic field. An intense radio-frequency pulse is applied that tips the magne-
tization generated by the hydrogen nuclei in the direction of the receiver coil
where the spin polarization can be detected. Random molecular rotational oscil-
lations matching the resonance frequency of the nuclear spins provide the “relax-
ation” mechanisms that bring the net magnetization back to its equilibrium
position in alignment with the applied magnetic field. The magnitude of the spin
polarization detected by the receiver is used to form the MR image but decays
with a characteristic time constant known as the T1 relaxation time. Water pro-
tons in different tissues have different T1 values, which is one of the main sources
of contrast in MR images. A contrast agent usually shortens, but in some instances
increases, the value of T1 of nearby water protons, thereby altering the contrast
in the image.
346 M. Rajesh Kumar and P. Joice Sophia
Tumor cell migration is a key step underlying cancer cell dissemination and metas-
tasis and is controlled by extracellular signaling-mediated dynamic cytoskeletal and
cell matrix adhesion remodeling. Using a phagokinetic track (PKT) assay in combi-
nation with multi-parametric image analysis and highly motile H1299 adenocarci-
noma cells, they have screened 1429 upstream kinase signaling components and
downstream adhesion and cytoskeletal regulators that determine tumor cell migra-
tory behavior: speed, directionality, and persistence. Thirty significant genes were
validated by live cell imaging random tumor cell migration, which was associated
with modulation of focal adhesion dynamics. For eight genes, a significant associa-
tion with metastasis-free survival in breast cancer patients was observed, SHC1,
SRPK1, NEK2, ITGB3BP, and MAP 3K8 being most significant. Also, high SRFS
protein kinase 1 (SRPK1) protein expression on breast cancer tissue microarrays
was associated with poor disease outcome. SRPK1 expression was highest in basal-
like breast cancer cell lines and depletion of SRPK1 inhibited breast cancer cell
motility and focal adhesion dynamics. Finally, in an orthotopic mammary tumor
metastasis model, stable knockdown of SRPK1 in lung metastatic variant
MDA-MB-231 basal-like breast cancer cells reduced lung metastasis formation
(Wies van Roosmalen et al 2015). This study provides a comprehensive information
resource on the molecular determinants of tumor cell migration in close association
with a clinical significant role in breast cancer progression.
As mentioned above, the fact that nanoparticles exist in the same size domain as
proteins makes nanomaterials suitable for biotagging or biolabelling. However, size
is just one of the many characteristics of nanoparticles that itself is rarely sufficient
if one is to use nanoparticles as biological tags. In order to interact with biological
target, a biological or molecular coating or layer acting as a bioinorganic interface
should be attached to the nanoparticle.
16.5 Summary
References
Alivisatos P (2004) The use of nanocrystals in biological detection. Nat Biotechnol 22(1):47–52
Allen TM, Cullis PR (2004) Drug delivery systems: entering the mainstream. Science
303(5665):1818–1822
Andrew AM (2000) Nanomedicine, volume 1: basic capabilities, by Robert A. Freitas Jr., Landes
Bioscience, Austin, Texas, 1999, xxi+ 509 pp., ISBN 1-57059-645-X Index (Hardback,
$89.000), Cambridge University Press. doi: 10.1017/S0263574700232827
Atkinson MA et al (1994) Cellular immunity to a determinant common to glutamate decarboxyl-
ase and coxsackie virus in insulin-dependent diabetes. J Clin Investig 94(5):2125
Bachmaier K et al (1999) Chlamydia infections and heart disease linked through antigenic mim-
icry. Science 283(5406):1335–1339
Balin BJ et al (1998) Identification and localization of Chlamydia pneumoniae in the Alzheimer's
brain. Med Microbiol Immunol 187(1):23–42
Bennett-Woods D (2006) Nanotechnology in medicine: implications of converging technologies
on humanity. Development 49(4):54–59
Bernardi M et al (2009) Development of metal oxide nanoparticles by soft chemical method.
Ceram Int 35(1):463–466
Bhargava V et al (2013) Quantitative transcriptomics using designed primer-based amplification.
Sci Rep 3:1740
Bhushan B (2009) Biomimetics: lessons from nature–an overview. Philos Trans R Soc Lond A
Math Phys Eng Sci 367(1893):1445–1486
Bhushan B, Jung YC (2011) Natural and biomimetic artificial surfaces for superhydrophobicity,
self-cleaning, low adhesion, and drag reduction. Prog Mater Sci 56(1):1–108
Blaser MJ, Chyou P, Nomura A (1995) Age at establishment of Helicobacter pylori infection and
gastric carcinoma, gastric ulcer, and duodenal ulcer risk. Cancer Res 55(3):562–565
Bogunia-Kubik K, Sugisaka M (2002) From molecular biology to nanotechnology and nanomedi-
cine. Biosystems 65(2):123–138
Bosch FX et al (1995) Prevalence of human papillomavirus in cervical cancer: a worldwide per-
spective. J Natl Cancer Inst 87(11):796–802
Bruchez M et al (1998) Semiconductor nanocrystals as fluorescent biological labels. Science
281(5385):2013–2016
Burgos JS (2005) Involvement of the Epstein-Barr virus in the nasopharyngeal carcinoma patho-
genesis. Med Oncol 22(2):113–121
Burns MA et al (1996) Microfabricated structures for integrated DNA analysis. Proc Natl Acad
Sci 93(11):5556–5561
Cao G (2004) Synthesis, properties and applications. World Scientific, Singapore
Chakrabarti A, Shu L (2010) Biologically inspired design. Artif Intel Eng Des Anal Manuf
24(04):453–454
Chan EY (2005) Advances in sequencing technology. Mutat Res Fundam Mol Mech Mutagen
573(1):13–40
Chan WC, Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection.
Science 281(5385):2016–2018
Chen G et al (2016) Nanochemistry and nanomedicine for nanoparticle-based diagnostics and
therapy. Chem Rev 116(5):2826–2885
Chesebro B (1998) BSE and prions: uncertainties about the agent. Science 279(5347):42–43
Çiftçioglu N et al (1999) Nanobacteria: an infectious cause for kidney stone formation. Kidney Int
56(5):1893–1898
Colvin VL (2003) The potential environmental impact of engineered nanomaterials. Nat Biotechnol
21(10):1166–1170
Crick F (1970) Central dogma of molecular biology. Nature 227(5258):561–563
Danesh J, Collins R, Peto R (1997) Chronic infections and coronary heart disease: is there a link?
Lancet 350(9075):430–436
348 M. Rajesh Kumar and P. Joice Sophia
De La Isla A et al (2003) Nanohybrid scratch resistant coatings for teeth and bone viscoelasticity
manifested in tribology. Mater Res Innov 7(2):110–114
deWaard JR et al (2008) Assembling DNA barcodes. In: Environmental genomics. Humana,
New York, pp 275–294
Diamanti MV, Pedeferri M (2015) Bioinspired self-cleaning materials. In: Biotechnologies and
biomimetics for civil engineering. Springer, Cham, pp 211–234
DiMaio D, Liao JB (2006) Human papillomaviruses and cervical cancer. Adv Virus Res 66:125–159
Disci-Zayed D (2016) Green synthesis of nanoparticles. Christian-Albrechts Universität Kiel, Kiel
Dreher KL (2004) Health and environmental impact of nanotechnology: toxicological assessment
of manufactured nanoparticles. Toxicol Sci 77(1):3–5
Edelstein R et al (2000) The BARC biosensor applied to the detection of biological warfare agents.
Biosens Bioelectron 14(10):805–813
Elsaesser A, Howard CV (2012) Toxicology of nanoparticles. Adv Drug Deliv Rev 64(2):129–137
Ferrari M (2005) Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer
5(3):161–171
Fohlman J, Friman G (1993) Is juvenile diabetes a viral disease? Ann Med 25(6):569–574
Franzen S, Lommel SA (2009) Targeting cancer with ‘smart bombs’: equipping plant virus
nanoparticles for a ‘seek and destroy’ mission. Nanomedicine 4(5):575–588
Freitas RA (2002) The future of nanofabrication and molecular scale devices in nanomedicine. In:
Studies in health technology and informatics. IOS, Amsterdam, pp 45–60
Freitas RA (2005) Current status of nanomedicine and medical nanorobotics. J Comput Theor
Nanosci 2(1):1–25
Freitasjr R (2005) What is nanomedicine? Nanomed nanotechnol Biol Med 1:2
Gillies G et al (2002) A spinal cord surrogate with nanoscale porosity for in vitro simulations of
restorative neurosurgical techniques. Nanotechnology 13(5):587
Hammond CJ et al (2010) Immunohistological detection of Chlamydia pneumoniae in the
Alzheimer's disease brain. BMC Neurosci 11(1):1
Hanusch K-U et al (2013) Whole-body hyperthermia for the treatment of major depression: asso-
ciations with thermoregulatory cooling. Am J Psychiatr 170(7):802–804
Hardman R (2006) A toxicologic review of quantum dots: toxicity depends on physicochemical
and environmental factors. Environ Health Perspect 114:165–172
He X-S, Shi W-Y (2009) Oral microbiology: past, present and future. Int J Oral Sci 1(2):47
Hebert PD, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proc R
Soc Lond B Biol Sci 270(1512):313–321
Horwitz MS et al (1998) Diabetes induced by Coxsackie virus: initiation by bystander damage and
not molecular mimicry. Nat Med 4(7):781–785
Huang X et al (2007) Gold nanoparticles: interesting optical properties and recent applications in
cancer diagnostics and therapy. Nanomedicine 2(5):681–693
Iravani S (2011) Green synthesis of metal nanoparticles using plants. Green Chem 13(10):2638–2650
Itzhaki R et al (2004) Infiltration of the brain by pathogens causes Alzheimer’s disease. Neurobiol
Aging 25(5):619–627
Ivanić K-Z, Tadić Z, Omazić MA (2015) Biomimicry – an overview. Holistic Approach Environ
5(1):19–36
Jain RK, Stylianopoulos T (2010) Delivering nanomedicine to solid tumors. Nat Rev Clin Oncol
7(11):653–664
Janka J, Maldarelli F (2004) Prion diseases: Update on mad cow disease, variant creutzfeldt-jakob
disease, and the transmissible spongiform encephalopathies. Curr Infect Dis Rep 6(4):305–315
Jarrett RF (2006) Viruses and lymphoma/leukaemia. J Pathol 208(2):176–186
Jia G et al (2005) Cytotoxicity of carbon nanomaterials: single-wall nanotube, multi-wall nano-
tube, and fullerene. Environ Sci Technol 39(5):1378–1383
Jiang S et al (2009) NIR-to-visible upconversion nanoparticles for fluorescent labeling and tar-
geted delivery of siRNA. Nanotechnology 20(15):155101
Jung D et al (2013) Chemical biology-based approaches on fluorescent labeling of proteins in live
cells. Mol BioSyst 9(5):862–872
Kah JCY et al (2007) Early diagnosis of oral cancer based on the surface plasmon resonance of
gold nanoparticles. Int J Nanomedicine 2(4):785
16 Nanoparticles as Precious Stones in the Crown of Modern Molecular Biology 349
Kahn JS (2006) The widening scope of coronaviruses. Curr Opin Pediatr 18(1):42–47
Kajander EO, Ciftcioglu N (1998) Nanobacteria: an alternative mechanism for pathogenic intra-
and extracellular calcification and stone formation. Proc Natl Acad Sci 95(14):8274–8279
Kelsall RW, Hamley IW, Geoghegan M (2005) Nanoscale science and technology. Wiley Online
Library, New York
Kneuer C et al (2000) A nonviral DNA delivery system based on surface modified silica-
nanoparticles can efficiently transfect cells in vitro. Bioconjug Chem 11(6):926–932
Kol A, Santini M (2004) Infectious agents and atherosclerosis: current perspectives and unsolved
issues. Ital Heart J 5(5):350–357
Kostarelos K (2006) The emergence of nanomedicine: a field in the making. Nanomedicine
1(1):1–3
Kramer G, Klingler HC, Steiner GE (2000) Role of bacteria in the development of kidney stones.
Curr Opin Urol 10(1):35–38
Kusters JG, van Vliet AH, Kuipers EJ (2006) Pathogenesis of Helicobacter pylori infection. Clin
Microbiol Rev 19(3):449–490
Legname G et al (2004) Synthetic mammalian prions. Science 305(5684):673–676
Li M et al (2010) Physiologically based pharmacokinetic modeling of nanoparticles. ACS Nano
4(11):6303–6317
Lipinski C, Hopkins A (2004) Navigating chemical space for biology and medicine. Nature
432(7019):855–861
Lo YD et al (1999) Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients
with nasopharyngeal carcinoma. Cancer Res 59(6):1188–1191
Lodish H et al (1995) Molecular cell biology, vol 3. Scientific American, New York
Lynch N et al (2006) PANDAS (paediatric autoimmune neuropsychiatric disorder associated with
streptococcal infection). Ir Med J 99(5):155–155
Ma J et al (2003) Biomimetic processing of nanocrystallite bioactive apatite coating on titanium.
Nanotechnology 14(6):619
Mah C et al (2000) Microsphere-mediated delivery of recombinant AAV vectors in vitro and
in vivo. Mol Ther 1:S239
Majoros IJ et al (2008) Current dendrimer applications in cancer diagnosis and therapy. Curr Top
Med Chem 8(14):1165–1179
Marra MA et al (2003) The genome sequence of the SARS-associated coronavirus. Science
300(5624):1399–1404
McIntire LV (2002) World technology panel report on tissue engineering. Ann Biomed Eng
30(10):1216–1220
Mell LK, Davis RL, Owens D (2005) Association between streptococcal infection and obsessive-
compulsive disorder, Tourette's syndrome, and tic disorder. Pediatrics 116(1):56–60
Mendonça G et al (2008) Advancing dental implant surface technology–from micron-to nanotop-
ography. Biomaterials 29(28):3822–3835
Mercanzini A et al (2010) Controlled release nanoparticle-embedded coatings reduce the tissue
reaction to neuroprostheses. J Control Release 145(3):196–202
Meyer SC (2003) DNA and the origin of life: information, specification, and explanation. Design
and Public Education, Darwinism, pp 223–285
Miranda HC et al (2006) Detection of Borna disease virus p24 RNA in peripheral blood cells from
Brazilian mood and psychotic disorder patients. J Affect Disord 90(1):43–47
Molday RS, Mackenzie D (1982) Immunospecific ferromagnetic iron-dextran reagents for the
labeling and magnetic separation of cells. J Immunol Methods 52(3):353–367
Morange M, Cobb M (2000) A history of molecular biology. Harvard University Press, Cambridge,
MA
Muñoz N et al (2003) Epidemiologic classification of human papillomavirus types associated with
cervical cancer. N Engl J Med 348(6):518–527
Nakamura K et al (2011) Sequence-specific error profile of Illumina sequencers. Nucleic Acids
Res 39(13):gkr344
Nam J-M, Thaxton CS, Mirkin CA (2003) Nanoparticle-based bio-bar codes for the ultrasensitive
detection of proteins. Science 301(5641):1884–1886
Nel A et al (2006) Toxic potential of materials at the nanolevel. Science 311(5761):622–627
350 M. Rajesh Kumar and P. Joice Sophia
Nie S (2010) Understanding and overcoming major barriers in cancer nanomedicine. Nanomedicine
5(4):523–528
Nisole S, Stoye JP, Saïb A (2005) TRIM family proteins: retroviral restriction and antiviral defence.
Nat Rev Microbiol 3(10):799–808
Nunes SOV et al (2008) RNA from Borna disease virus in patients with schizophrenia, schizoaf-
fective patients, and in their biological relatives. J Clin Lab Anal 22(4):314–320
Ow H et al (2005) Bright and stable core-shell fluorescent silica nanoparticles. Nano Lett
5(1):113–117
Palenik GJ, Jensen WP, Suh I-H (2003) The history of molecular structure determination viewed
through the Nobel prizes. J Chem Educ 80(7):753
Pandey A et al (2008) Nanoscience and their biological importance: human health and disease. Dig
J Nanomater Biostruct 3(3):141–146
Panessa-Warren B et al (2006) Biological cellular response to carbon nanoparticle toxicity. J Phys
Condens Matter 18(33):S2185
Pantarotto D et al (2003) Immunization with peptide-functionalized carbon nanotubes enhances
virus-specific neutralizing antibody responses. Chem Biol 10(10):961–966
Parak WJ et al (2002) Cell motility and metastatic potential studies based on quantum dot imaging
of phagokinetic tracks. Adv Mater 14(12):882–885
Patel P et al (1995) Association of Helicobacter pylori and Chlamydia pneumoniae infections with
coronary heart disease and cardiovascular risk factors. BMJ 311(7007):711–714
Peer D et al (2007) Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol
2(12):751–760
Peiris J et al (2003) Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet
361(9366):1319–1325
Pereira P, Monteiro G, Prazeres D (2015) General aspects of biomimetic materials. In:
Biotechnologies and biomimetics for civil engineering. Springer, Cham, pp 57–79
Perry AS et al (2013) Insecticides in agriculture and environment: retrospects and prospects.
Springer Science & Business Media, Berlin
Prabhu S, Poulose EK (2012) Silver nanoparticles: mechanism of antimicrobial action, synthesis,
medical applications, and toxicity effects. Int Nano Lett 2(1):1–10
PROKOP A (2001) Bioartificial organs in the twenty-first century. Ann N Y Acad Sci 944(1):472–490
Quinn S, Gaughran W (2010) Bionics – an inspiration for intelligent manufacturing and engineer-
ing. Robot Comput Integr Manuf 26(6):616–621
Ravindran MS et al (2016) Opportunistic intruders: how viruses orchestrate ER functions to infect
cells. Nat Rev Microbiol 14(7):407
Riehemann K et al (2009) Nanomedicine – challenge and perspectives. Angew Chem Int Ed
48(5):872–897
Rizzello L et al (2011) Impact of nanoscale topography on genomics and proteomics of adherent
bacteria. ACS Nano 5(3):1865–1876
Roco MC (2003) Nanotechnology: convergence with modern biology and medicine. Curr Opin
Biotechnol 14(3):337–346
Rota PA et al (2003) Characterization of a novel coronavirus associated with severe acute respira-
tory syndrome. Science 300(5624):1394–1399
Roy I et al (2005) Optical tracking of organically modified silica nanoparticles as DNA carriers: a
nonviral, nanomedicine approach for gene delivery. Proc Natl Acad Sci U S A 102(2):279–284
Sahoo H (2012) Fluorescent labeling techniques in biomolecules: a flashback. RSC Adv
2(18):7017–7029
Salata OV (2004) Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2(1):1
Sarikaya M et al (2003) Molecular biomimetics: nanotechnology through biology. Nat Mater
2(9):577–585
Sawicki MP et al (1993) Human genome project. Am J Surg 165(2):258–264
Schadt EE, Turner S, Kasarskis A (2010) A window into third-generation sequencing. Hum Mol
Genet 19(R2):R227–R240
Scott J, Thompson G (2011) The discovery of the structure of DNA. In: Nobel prizes that changed
medicine. Imperical College Press, London, p 89
16 Nanoparticles as Precious Stones in the Crown of Modern Molecular Biology 351
Graphical Abstract
17.1 Introduction
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Fig. 17.1 Comparison of various objects in terms of size
system for exploring the world of colloid solutions. Gold NPs have a huge range of
applications, like charge storage, sensor and electronic device fabrication. The
methodologies for the synthesis of monodisperse metal NPs of particular size and
shape are still limited and challenging the scientific community. There is a limited
knowledge of mechanisms of NPs formation. The reason for this is the difficulty
faced in real-time observation of the formation of NPs. Nucleation and growth pro-
cesses are often very fast in the synthesis of metal NPs. Therefore, making direct
observations is very difficult. Further, literature reported that the melting point of
metal nanoparticles depends upon the size. Smaller the size of metal nanoparticles,
the more will be the surface area (Kamlesh et al. 2012a, b; Prashant 2013; Prashant
et al. 2011) (Fig. 17.1).
There are various routes for the synthesis of metal nanoparticles, and they are
physical, chemical, biological and hybrid. Generally in physical methods, two
approaches, up-down and down-up, have been studied. Herein, solid-phase synthesis
of nanoparticles is taken into consideration. In chemical method, synthesis of NPs
has been carried out in the solution phase (Fig. 17.2a). Sometimes, there is a need to
use stabiliser to stabilise metal nanoparticle in the solution phase. In biological
method, there is no need of stabiliser because the biological extract itself works as
stabiliser. Hybrid method uses the combination of two or more than two techniques
for the synthesis of the nanoparticle, and this methodology is the most effective one.
Various methodologies for synthesising stable metal nanoparticles have been
reported. Many research groups specially focus on synthesis methods for creating
nanoparticles like physical vapour deposition, chemical vapour deposition, sol-gel
method, Radio frequency (RF) plasma method, pulsed laser method, thermolysis and
solution combustion method. Various metal nanoparticles have wide range of use in
the living and nonliving systems (Kamlesh et al. 2012a, b; 2014, 2017; Pradeep et al.
2011; Prashant 2013; Prashant et al. 2008a, b; 2009a, b, c, 2011, 2017). A general
schematic representation of metal nanoparticle can be given in Fig. 17.2b. Herein
stabiliser has been used for the synthesis of metal nanoparticle.
356 P. Singh et al.
a
Amino Acid
Bacteria
Fungi
Algae
Plants
Viruses
DNA Solvothermal
RNA Chemical
Pollens Methods of Galvanic
Proteins Synthesis Electrochemical
S-layer (Nanomaterial) Sol-gel
Peptide Replacement
Oligonueleotide Mioelle based
Vapor deposition
Thermal
Decomposition
Ultrasonication
Radiolysis
Spray Pyrolysis
Precursor Nanoparticle
Solution Reducing in solution
Stabilizer phase
agent
Fig. 17.2 (a) Methodologies used for the synthesis of nanoparticles. (b) Schematic representation
of metal nanoparticle in solution
There are several applications of metal NPs as mentioned in Fig. 17.3, but in this
section, only few of them are explained (Herrero et al. 2010; Larguinho and Baptista
2011; Niikura et al. 2013; Yao et al. 2012; Zhang et al. 2012).
Defence
Environment Textiles
Aerospace Agriculture
Applications of Information
Energy Technology
metal NPs
Automotive Materials
Chemicals Telecom
Medicines &
Pharmaceut
icals
Fig. 17.3 Application of metal NPs in various disciplines (Prashant et al. 2017)
Biomedical Applications of Metal NPs From, first use of metal NPs in ancient
times in various medical treatments, paintings, etc and the claims for the use of
gold NPs in medical sciences. Literature reported the interesting developments
for the use of gold NPs in the delivery of various vaccines into the human body.
Au NPs have been used in the finding of deadly poisons, in the testing of vaccines,
etc. Au NPs are also used in biomedical analyses. Gold NPs are attractive and
efficient material for various testing. This technique gives visual proof of the
availability of an analyte in a liquid sample. Au NPs have superiority due to their
high stability, sensitivity and reproducibility. Gold NPs have the ability to be lay-
ered with particular organic ligands, DNA. It makes easier to engineer nanostruc-
tures and to modify properties for different applications. Au NPs have been used
to locate the tumours, and when exposed to X-rays, kill the tumour (Prashant et al.
2012a, b, c; 2014; Pradeep et al. 2011; Prashant 2013; Prashant et al. 2009a, b,
2011, 2017).
cytotoxicity effects. Au NPs have been explored for various applications in medical
science. Researchers have also studied the uptake of gold nanoparticles by mam-
malian cells by pinocytosis and its compartmentalisation in lysosomal bodies. It
was reported that the gold NPs do not show any visible cytotoxicity to the human
cell lines until 100 μM concentration. These reports clearly suggest that the gold
NPs are biocompatible. Thus, Au NPs can be used in various applications in
nanomedicine.
17.3 S
ynthesis and Characterisation of Some Metal
Nanoparticles (Ajeet et al. 2008; Chaichi and Alijanpour
2014; Kamlesh et al. 2012a, b, 2014, 2017; Pradeep et al. 2011;
Prashant 2013; Prashant et al. 2008a, b, c, 2009a, b, c, 2011,
2017; Scheeren et al. 2006; Xu et al. 2012, 2013;
Yang et al. 2014; Zapp et al. 2014)
In a typical methodology, in a 10 mL round bottom flask, 5 mL of the above syn-
thesised ionic liquid and 10 mg of tetrachloroauric acid (HAuCl4) were taken and
stirred for 10 min (yellow colour), and the mixture was treated with more metha-
nolic solution of sodium borohydride (20 mg in 10 mL of methanol). A ruby red-
coloured solution was obtained from yellow-coloured solution of HAuCl4
indicating the formation of gold in zero oxidation state. Stirring was continued for
another 6 hours for the reduction of Au (III) to Au (0). Then the solution obtained
was centrifuged for 10 min at 10,000 rpm, and the supernatant was discarded. The
centrifuged pellet was washed with ethanol, and the nanoparticles were analysed
for characterisation using powder X-ray diffraction (XRD), transmission electron
microscopy (TEM), quasi-elastic light scattering (QELS) and UV-visible tech-
niques to determine the shape, size and oxidation state of gold nanoparticles.
Similarly, the synthesis of Cu and Ag NPs in ionic liquid (1-butyl-5-carboxy-
methyl-4-(2-cyano-ethyl)-4H-tetrazolium bromide) (Scheme 17.1b and c, respec-
tively) has been carried out.
17 Nanotechnology and Its Impact on Insects in Agriculture 359
a OH b OH c OH
O Br O Br O Br
NC NC NC
N N N
N N N
NN N N N N
Au NPs
200
150
100
50
0
20 30 40 50 60 70 80
2 Theta value (degree)
300
Intensity peak
200
c
100
0
5 10 15 20 25 30 35 40 45 50 55 60
2 Theta value (degree)
a b c
Fig. 17.5 TEM pictures of (a) Au, (b) Cu and (c) Ag NPs
determines the shape and size of the Au nanoparticles. On the analysis of TEM
data, it was observed that the range of size of gold NPs is 20–30 nm and the par-
ticles are spherical in nature as in Fig. 17.5a. On the analysis of TEM picture of
Cu NPs, shape of the NPs appeared spherical and the bar present in the picture
corresponds to 100 nm and corresponds to approximately two nanoparticles;
therefore, the average particle size of the particles is 50–60 nm (Fig. 17.5b).
Transmission electron micrographs of the sample clearly indicate that the size of
Ag NPs is approximately 50 nm with spherical shape (Fig. 17.5c).
monodispersive in nature and the average size of the NPs was found out to be
55.47 nm, which is in correlation with TEM results as explained above. QELS pic-
tures (Fig. 17.6c) gave clear indication that Ag NPs are monodispersive and the aver-
age size of the particles is 57.31 nm, which is again in correlation with TEM results.
a b c
Fig. 17.6 QELS pictures of (a) Au, (b) Cu and (c) Ag NPs
a 0.6
b
0.9
Absorbance
0.5 0.8
Absorbance
0.7
0.6
0.4 0.5
0.4
0.3
0.3
0.2
0.1
0.2 0
450 475 500 525 550 575 600 450 500 550 600 650 700
Wavelength (nm) Wavelength (nm)
c 0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0
300 350 400 450 500 550 600 650 700 750 800
Wavelength (nm)
Fig. 17.7 UV-visible spectra of (a) Au, (b) Cu and (c) Ag NPs
362 P. Singh et al.
Ag NPs, is obtained and gave a characteristic peak of 460 nm and indicates the size
of NPs is in the range of 50–60 nm (Fig. 17.7c). As the size of NPs decreases, λmax
also increases or shifts to higher wavelength.
Insects are counted as one of the major animal populations with a long successful
evaluative history. They can even be found in all possible environments throughout
the world. Their success can be attributed to several important evolutionary aspects
like wings, malleable exoskeleton, high reproductive potential, habit diversification,
desiccation-resistant eggs and metamorphosis. On the other hand, many insects are
known as vectors of many diseases. Insects damages crop plantations or wood struc-
tures, and serious health and economic issues were caused by insects. In order to
combat the various losses that are caused by insects on agriculture, several chemi-
cals have been used to kill them or inhibit their reproduction and feeding habits
(Buonasera et al. 2009; Ebrahimi et al. 2011; Ghormade et al. 2011; Ginet et al.
2011; Ginjupalli and Baldwin 2013; Grillo et al. 2016; Hake et al. 2007; Jokar et al.
2016; Kah and Hofmann 2014). Nanopesticides encompass a great variety of prod-
ucts and cannot be considered as a single category. Nanopesticides can consist of
organic ingredients and inorganic ingredients. The aims of nanoformulations con-
sist of (1) increasing the apparent solubility of poorly soluble active ingredient and
(2) releasing the active ingredient in a slow/targeted manner as well protecting the
active ingredient against premature degradation. Literature also defines nanopesti-
cides as any formulation that intentionally includes elements in the nanoscale size
range and has novel properties. The advances in science and technology in the last
two decades were made in several areas of insecticide usage. It includes either
development of more effective and non-persistent pesticides and new ways of appli-
cation, like controlled release formulation. One of the most promising uses of nano-
technology is to promote a more efficient assembly of the active compound in a
matrix in order to protect core materials from adverse reactions due to factors like
air or light. An outcry is exhibited against the use of pesticides due to their hazard-
ous effects on human as well as environment. Insecticides are proven to be a cheap
approach to managing disease-carrying, crop-destroying, and residential pest
insects. Chemical insecticides have a long evolutionary history spanning sulphur as
a fumigant against vermin to current neonicotinoids. Neonicotinoids are introduced
as a new class of insecticides in 1991. They control the agonist effects of the nico-
tinic ACh receptor in insects. These chemicals also act reversibly on the α4β2 sub-
type of mammalian nicotinic ACh receptors and display some chronic toxicity in
mammals. Hence, their use to protect a variety of agricultural crops has increased
during the last 20 years (Hubert et al. 2007; Hummel et al. 2013; Kah et al. 2014,
2016; Khandelwal et al. 2016; Khodadoust et al. 2013; Kookana et al. 2016; Kumar
et al. 2016). The development of novel plant-protection products has received
greater attention than other applications, such as those related to nanosensors or
fertilisers (Hubert et al. 2007; Hummel et al. 2013; Kah et al. 2014, 2016; Khandelwal
17 Nanotechnology and Its Impact on Insects in Agriculture 363
et al. 2016; Khodadoust et al. 2013; Kookana et al. 2016; Kumar et al. 2016;
Mirabelli et al. 2016; Mishra et al. 2016) (Table 17.1).
formulation sector as well as definitions and illustrations for the different nanopes-
ticide types can be found (Mirabelli et al. 2016; Mishra et al. 2016; Narayanan et al.
2017; Nuruzzaman et al. 2016; Periasamy et al. 2009; Petosa et al. 2016; Wang et al.
2015, 2016; Yu et al. 2007; Zheng et al. 2016) (Table 17.2).
Table 17.2 Efficacy and toxicity of nanoformulation reported in literature published between
October 2011 and October 2013
Efficacy of the nanoformulation compared to that
Type of AI of a commercial formulation or the pure AI References
Neem oil IC50 decreased with droplet size Anjali et al. (2012)
Permethrin Greater efficacy than pure AI against larvae Kumar et al. (2013)
(LC50) (24 h), 0.006 and 0.020 mg/L, respectively
Glyphosate Similar or slightly greater efficacy than Jiang et al. (2012)
commercial (roundup)
Beta-cyfluthrin Greater efficacy than commercial formulation Loha et al. (2012)
when evaluated over a long period
Acephate Greater efficacy than commercial formulation Choudhury et al.
(biochemical assays, in vitro and field trials) (2012)
Emamectin Similar and slightly higher insecticidal activity Qian et al. (2011)
than commercial formulation
Lansiumamide B Higher nematicidal activity than pure AI Yin et al. (2012)
Neem oil Cytotoxicity (human lymphocyte): alginate Jerobin et al. (2012)
< starch < polyethylene glycol formulation
Ametryn Lower toxicity than pure AI Grillo et al. (2012)
Atrazine and Efficacy in open orchard demonstrated during Abreu et al. (2012)
simazine nanogel adverse season
Essential oil Greater efficacy than free oil Silva et al. (2011)
Copper Synergistic effect between chitosan nanogels Brunel et al. (2013)
and copper
Thiamethoxam Efficient at 50% of the recommended dosage Xiang et al. (2013)
Pyrifluquinazon Delayed efficacy compared to pure AI Kang et al. (2012)
Etofenprox Prolonged effect compared to commercial Hwang et al. (2011)
formulation
Chlorfenapyr Insecticidal activity of silica nanoparticles Song et al. (2012)
formulation was twice as high as that of
microparticles
Naphthylacetic Better growth protection than pure AI after 7 days Mingming et al. (2013)
acid at the highest concentration tested
Validamycin Most Prolonged activity compared to pure AI Qing et al. (2013)
Inorganic silica Efficacy at similar rates than commercial Debnath et al. (2011)
as AI silica diatomaceous earth for stored gains
TiO2 Better or on per efficacy compared to standard Debnath et al. (2012)
treatment and Paret et al. (2013a)
Silver Fungicidal activity against 18 plant pathogens Paret et al. (2013b)
Copper Greater efficacy than Cu oxychloride Kim et al. (2012)
Aluminium Similar or greater insecticidal activity than most Mondal and Mani
effective commercial available diatomaceous earth (2012) and Stadler
formulation et al. (2010)
LC 50, EC 50: Concentration required to observed 50% mortality or effect, respectively
17 Nanotechnology and Its Impact on Insects in Agriculture 365
17.6 A
pplication of NPs As Insecticides
(Tables 17.1 and 17.2)
17.6.1 Silica
Silicon has long been known to increase the plant tolerance of various abiotic and
biotic stresses. Silica nanoparticles have therefore naturally been suggested as
potential candidates for increasing the control over a range of agricultural pests.
It was observed that there is higher insect mortality from treatment with silica
nanoparticles (15–30 nm) than with bulk silica (100–400 nm). The similar effi-
cacy of silica nanoparticles with different coatings indicated a mechanical mode
of action that could be enhanced for smaller particles. A second study, however,
indicated that silica nanoparticles coated with 3-mercaptopropyltriethoxysilane
were more efficient than those coated with hexamethyldisilazane. In one case, the
effect was not related to size since the former nanoparticles (29–37 nm) were
larger than the latter (15–20 nm). The application rates were generally compara-
ble with those recommended for commercially available diatomaceous formula-
tions (0.5–2 g/kg), and hence the additional costs involved in engineering
nanoparticles may not be justified by the slight increase in efficacy (Belz et al.
2017; Ellison et al. 2017; Laranjeira et al. 2017; Xiang et al. 2017; Yao et al. 2017;
Zheng et al. 2017).
The antimicrobial activity of titanium dioxide is well recognised, and several stud-
ies have suggested that applying titanium dioxide to crops can suppress bacterial
and fungal pathogens. The antibacterial potential of photocatalytic nanoscale tita-
nium dioxide has recently been tested. Nanoscale titanium dioxide has been used
either alone or doped with silver or zinc, against the causal agent for bacterial spot
disease in tomatoes and roses. Greenhouse and field trials showed that using tita-
nium dioxide/zinc could result in significantly reduced bacterial spot severity com-
pared to using untreated controls. The overall efficacy was better than the standard
treatments for management of the diseases. The main advantage of the titanium
dioxide/zinc formulation presented is its potential to lower ecological and toxico-
logical risks, compared to currently use copper-based treatments (Abreu et al. 2012;
Anjali et al. 2012; Bhagat et al. 2013; Brunel et al. 2013; Choudhury et al. 2012;
Grillo et al. 2012; Hwang et al. 2011; Jerobin et al. 2012; Jiang et al. 2012; Kang
et al. 2012; Kumar et al. 2013; Loha et al. 2012; Qian et al. 2011; Silva et al. 2011;
Xiang et al. 2013; Yin et al. 2012).
17.6.3 Silver
Silver is known for a long time for its antimicrobial properties, and several in vitro
studies have demonstrated that nanosilver can significantly inhibit the growth of
plant pathogens in a dose-dependent manner. Recently people demonstrated the
in vitro activity of nanosilver against 18 plant pathogens. Where possible uses are
17 Nanotechnology and Its Impact on Insects in Agriculture 367
coatings for fruit bags or as treatments for cut flowers are conceivable, the applica-
tion of nanosilver to crops that are likely to enter the food chain is more question-
able. Possible benefits of nanosilver over synthetic fungicides have been suggested
in view of the cost of nanosilver, the uncertainties associated with its toxicity.
According to the latest regulatory developments and the public perceptions, it is
unlikely that formulations of nanosilver for open field application will be devel-
oped. This conclusion is corroborated by the lack of recent publications on the
subject (Debnath et al. 2011, 2012; Hwang et al. 2011; Kim et al. 2012; Mingming
et al. 2013; Mondal and Mani 2012; Paret et al. 2013a; Song et al. 2012; Stadler
et al. 2010, 2012).
17.6.4 Copper
Zinc oxide nanoparticles are very much important due to their utilisation in gas sen-
sors, biosensors, cosmetics, drug delivery systems and so forth. ZnO NPs also have
remarkable optical, physical and antimicrobial properties and therefore have great
potential to enhance agriculture. The zinc oxide nanoparticles are integrated in pest
management programmes as alternative to chemical insecticides where they are
considered safe for humans compared with synthetic insecticide. ZnO NPs can be
synthesised by several chemical methods such as precipitation method, vapour
transport method and hydrothermal process. The biogenic synthesis of ZnO NPs by
using different plant extracts is also common nowadays.
This green synthesis is quite safe and eco-friendly compared to chemical synthe-
sis. Several novel inventions of different nanoparticles and nanomaterials are capa-
ble to diminish the environmental problems. Nanopesticides develop and explore
the possibility of nanotechnology, which accentuate the concept of particle size
reduction and its properties. Nanoencapsulation is another part of nanotechnology
in which the pesticide is coated by a matrix and the size of the pesticide reduces up
to the nano size. Therefore, the nanoencapsulation helps to minimise the doses to
get maximum effect on the target organisms. In recent years, it is emphasised on the
application of nanotechnology in insect pest management. Technologies like encap-
sulation and controlled release system (CRS) have, therefore, modernised the appli-
cation of biocides. Nanotechnology-based insecticides are devised by a number of
companies. These formulations embrace nanoparticles of size 120–250 nm size
range being more efficiently water soluble as compared to existing pesticides. In
India, the relevance of nanotechnology in pest management has just started in the
last two decades.
Mosquito-borne diseases are one of the world’s most health perilous problems.
Numerous mosquito species belonging to genera Aedes, Anopheles, and Culex are
the common vectors which cause various diseases like dengue, yellow fever,
malaria, filariasis, Japanese encephalitis, etc.; lot of efforts have been made to con-
trol the mosquitoes. Initially chemical insecticides like dichlorodiphenyltrichloro-
ethane (DDT), benzene hexachloride (BHC), malathion, etc., have been used to
control the mosquito inhabitants, but these chemicals cause ill effects on the envi-
ronment and non-target organisms and are also non-biodegradable in nature.
Therefore, environment friendly NPs were used to control the mosquito population
instead of using chemical NPs. Since antiquity plant products have been exposed to
display not only their pharmacological benefits but also for other biological proper-
ties including fungicidal, microbial, insecticidal and pesticidal activities. Therefore,
the botanical pesticides were initiated for further research as they are effective, eco-
friendly, easily biodegradable and non-toxic to non-target organisms. The encapsu-
lated plant-based nanopesticides have the following advantages: they can be easily
taken by target organisms and are more active in action as compared to synthetic
nanopesticides. Plant-based nanopesticides are eco-friendly since they are biode-
gradable. Pests and vectors were unsuccessful to develop resistance against
17 Nanotechnology and Its Impact on Insects in Agriculture 369
17.7 O
bjection of Regulators on the Usage
of Nanoagrochemicals
Literature revealed that the majority of publications originated from mainly Asia,
from China and India, followed by the United States. About 55% of the nanopesti-
cides investigated were insecticides, then fungicides (30%) and followed by herbi-
cides (15%). In general, the hypothesis is as follows: the smaller the size, the higher
is the reactivity. Therefore, more reactive materials cannot be agrochemicalised.
Most of the nanopesticides come under “nano”, but literature reported that 100 nm
size is the boundary that has been recommended for regulatory purposes. There are
several considerable issues relating to the definition of nanoparticles and how the
criteria proposed could apply to nanopesticides. Most importantly, a definition
based on size alone would exclude many recent so-called nanoformulations and, on
the other hand, include products that have been on the market for decades without
posing particular. The European Union (EU) initiative of a repository for nanoma-
terials (EC 2014), therefore, comes with the risk to further confuse consumers by
including ingredients that have been used for decades without previously being
classified as “nano”. In this context, it may be more useful to speak about nano-
enabled or formulation technology, rather than focusing only on the nanoparticles
and how they should be defined (Balaji et al. 2015, 2017; Buonasera et al. 2009;
Diaz-Blancas et al. 2016; Ghormade et al. 2011; Grillo et al. 2016; Hu et al. 2016;
Hubert et al. 2007; Hummel et al. 2013; Kah 2015; and Hofmann 2014; Kah et al.
2016; Kalayou et al. 2016; Khandelwal et al. 2016; Khodadoust et al. 2013; Kookana
et al. 2016; Kumar et al. 2016; Mirabelli et al. 2016; Mishra et al. 2016; Nuruzzaman
et al. 2016; Periasamy et al. 2009) (Fig. 17.8).
Literature has reported many nanoagrochemicals, and they do not fit due to certain
limitations. Most of the nanoagrochemicals have low agronomic relevance, and the
rest has association with unacceptable risks without any advantages. Engineered
nanoparticles have attained lots and lots of interest in many areas which have very
low potential for large-scale agricultural applications. Nanoagrochemicals having
organic-based delivery systems have been used for food or pharmaceutical applica-
tions. However, they are economically incompetitive in comparison to agrochemi-
cals. Many studies have been reported about the potency of products whether they
370 P. Singh et al.
35
30
25
20
15
10
n d n
a pi
d
io sio
de
n
e
lic
+A
Li
se
rs
io
on
Si ul
xi
ba
s
e
al
id
al
ro
ul
sp ol em
et
w
em
yd
er
ed
llo no
M
od S
lym
H
ho
us
ro
an Na
le
ic
us
al
Po
ub
N
M
et
ro
do
M
Po
d
re
ye
La
Fig. 17.8 Chart diagram for the different agrochemicals in different span of time
are capable to compete with the present formulations (costs and performance). As a
whole, nanoagrochemicals will soon emerge consisting of “nano” formulations of
ingredients (Balaji et al. 2015, 2017; Buonasera et al. 2009; Diaz-Blancas et al.
2016; Ghormade et al. 2011; Grillo et al. 2016; Hu et al. 2016; Hubert et al. 2007;
Hummel et al. 2013; Kah and Hofmann 2014; Kookana et al. 2016; Kumar et al.
2016; Mirabelli et al. 2016; Mishra et al. 2016; Nuruzzaman et al. 2016; Periasamy
et al. 2009).
Development of new formulations has remain an interesting research area.
Literature reported that there is a need to formulate agrochemicals of specific
applications. With increasing pressure of regulatory agencies, the researchers
have focused on the optimisation application and delivery to authorised active
ingredients. Researchers are working on the formulation to find out new solutions
for targeting agrochemical activity. Keeping the view to maintain colloidal stabil-
ity as well to avoid the phase separation during storage, many formulations con-
tain structures having size less than 100 nm. Therefore, researchers have access to
the advanced instruments or technology that permits clear picture. This facilitates
the synthesis and modifications for a separate purpose. Nano-based products have
the capability to support a better management of agricultural inputs. Hence, the
marketing of formulation of the active molecules on the market should not be
based on its size, but also depends on evaluation of new risks and benefits involved
therein (Balaji et al. 2015; Buonasera et al. 2009; Diaz-Blancas et al. 2016;
Ghormade et al. 2011; Grillo et al. 2016; Hu et al. 2016; Kookana et al. 2016;
Kumar et al. 2016; Mirabelli et al. 2016; Mishra et al. 2016).
17 Nanotechnology and Its Impact on Insects in Agriculture 371
Literature reported that the researchers are actively engaged in developing and test-
ing a series of insecticide formulations based on amphiphilic copolymers. They
found that the active ingredient (AIs) in water were less active than from commer-
cial formulations including imidacloprid, thiamethoxam, carbofuran, thiram and
β-cyfluthrin. It has been observed that the release rates increases on increasing
molecular weight of Polyethylene glycol (PEG). It was found that the release of
β-cyfluthrin from the nanoformulation has occurred in 1–20 days while its release
from the available commercial formulation happened in 4–5 days. The high efficacy
may be explained by a slow release of AIs as well as protection of the active ingre-
dients, but in case of nanoemulsions, the reasons for showing the less toxicity to
non-target organisms are still not clear (Buonasera et al. 2009; Diaz-Blancas et al.
2016; Efremenko et al. 2017; Grillo et al. 2016; Kah and Hofmann 2014; Kookana
et al. 2016; Momeni and Nabipour 2015).
To date the formulations reported in this category involve the following: (1) using
mesoporous silica as a carrier for decreasing the rate of slow release or (2) incorpo-
rating TiO2 onto a polymer matrix to accelerate the photodegradation of the organic
AIs. Few new formulations have been reported and have used nanoparticle of silica
or calcium carbonate as carriers. A slow release of an organic AI has been shown.
These nanoformulations have reported to increase the activity of the AIs, but no
generalisations are yet possible due to the very different natures of the AIs investi-
gated (Abreu et al. 2012; Anjali et al. 2012; Bhagat et al. 2013; Brunel et al. 2013;
Choudhury et al. 2012; Debnath et al. 2011, Debnath et al. 2012; Grillo et al. 2012;
Hwang et al. 2011; Jerobin et al. 2012; Jiang et al. 2012; Kang et al. 2012; Kim et al.
2012; Kumar et al. 2013; Loha et al. 2012; Mingming et al. 2013; Mondal and Mani
2012; Paret et al. 2013a; Qian et al. 2011; Qing et al. 2013; Sarkar et al. 2012;
Table 17.3 Micro-, nano- emulsion, entrapment
372
Silva et al. 2011; Song et al. 2012; Stadler et al. 2010, 2012; Xiang et al. 2013; Yin
et al. 2012).
Conclusions
Nanotechnology and nanoscience has attracted the scientists and researchers
working on different areas. Nanoformulations are expected to have strong impacts
on the fate of active ingredient and to find new ingredients whose environmental
fate is not well studied. The present status cannot permit a fair evaluation of the
merits and demerits that come from exploration of nanopesticides. There is a need
to use the advance instrumentation to detect, characterise and quantify the active
ingredient and adjuvants emanating from nanoformulations. There is urgency of
thorough risk assessments channel for the nanopesticides. Further there is a need
to carry out the research on environmental fate as well to study it under different
conditions so risk evaluation of nanoparticles can be done.
References
Abreu FOMS, Oliveira EF, Paula HCB, de Paula RCM (2012) Chitosan/cashew gum nanogels for
essential oil encapsulation. Carbohydr Polym 89(4):1277–1282
Adak T, Kumar J, Shakil NA, Walia S (2012) Development of controlled release formulations
of imidacloprid employing novel nano-ranged amphiphilic polymers. J Environ Sci Health B
47(3):217–225
Ajeet K, Prashant S, Amit S, Arnab D, Ramesh C, Subho M (2008) Nano-sized copper as an
efficient catalyst for one pot three component synthesis of thiazolidine-2, 4-dione derivatives.
Catal Commun 10:17–22
Anjali CH, Sharma Y, Mukherjee A, Chandrasekaran N (2012) Neem oil (Azadirachta indica)
nanoemulsion—a potent larvicidal agent against Culex quinquefasciatus. Pest Manag Sci
68(2):158–163
Balaji AP, Mishra P, Suresh Kumar RS, Ashu A, Margulis K, Magdassi S, Mukherjee A,
Chandrasekaran N (2015) The environmentally benign form of pesticide in hydrodispersive
nanometric form with improvedefficacyagainstadultmosquitoesatlowexposureconcentrations.
Bull Environ Contam Toxicol 95:734–739
Balaji AP, Sastry TP, Manigandan S, Mukherjee A, Chandrasekaran N (2017) Environmental benig-
nity of a pesticide in soft colloidal hydrodispersive nanometric form with improved toxic preci-
sion towards the target organisms than non-target organisms. Sci Total Environ 579:190–201
Belz J, Castilla-Ojo N, Sridhar S, Kumar R (2017) Radiosensitizing silica nanoparticles encapsu-
lating docetaxel for treatment of prostate cancer. Methods Mol Biol 1530:403–409
Bhagat D, Samanta SK, Bhattacharya S (2013) Efficient management of fruit pests by pheromone
nanogels. Sci Rep 3:1294
Brunel FE, Gueddari NE, Moerschbacher BM (2013) Complexation of copper(II) with chitosan
nanogels: toward control of microbial growth. Carbohydr Polym 92(2):1348–1356
Buonasera K, D’Orazio G, Fanali S, Dugo P, Mondello L (2009) Separation of organophosphorus
pesticides by using nano-liquid chromatography. J Chromatogr A 1216:3970–3976
Chaichi MJ, Alijanpour SO (2014) Chitosan-induced Au/Ag nanoalloy dispersed in IL and appli-
cation in fabricating an ultrasensitive glucose biosensor based on luminol-H2O2-Cu2+/IL che-
miluminescence system. J Photochem Photobiol B 140:41–48
Choudhary SR, Pradhan S, Goswami A (2012) Preparation and characterisation of acephate nano-
encapsulated complex. Nanosci Methods 1(1):9–15
374 P. Singh et al.
Debnath N, Das S, Seth D, Chandra R, Bhattacharya SC, Goswami A (2011) Entomotoxic effect
of silica nanoparticles against Sitophilus oryzae (L.) J Pest Sci 84(1):99–105
Debnath N, Mitra S, Das S, Goswami A (2012) Synthesis of surface functionalized silica nanopar-
ticles and their use as entomotoxic nanocides. Powder Technol 221:252–256
Diaz-Blancas V, Medina DI, Padilla-Ortega E, Bortolini-Zavala R, Olvera-Romero M,
LunaBarcenas G (2016) Nanoemulsion formulations of fungicide tebuconazole for agricultural
applications. Molecules 21:1271
Ebrahimi M, Es’haghi Z, Samadi F, Hosseini MS (2011) Ionic liquid mediated solgel sorbents
for hollow fiber solidphase microextraction of pesticide residues in water and hair samples. J
Chromatogr A 1218:8313–8321
Efremenko EN, Lyagin IV, Klyachko NL, Bronich T, Zavyalova NV, Jiang Y, Kabanov AV (2017)
A simple and highly effective catalytic nanozyme scavenger for organophosphorus neurotox-
ins. J Control Release 247:175–181
Ellison PA, Chen F, Goel S, Barnhart TE, Nickles RJ, DeJesus OT, Cai W (2017) Intrinsic and
stable conjugation of thiolated mesoporous silica nanoparticles with radioarsenic. ACS Appl
Mater Interfaces 9(8):6772–6781. https://doi.org/10.1021/acsami.6b14049
Ghormade V, Deshpande MV, Paknikar KM (2011) Perspectives for nano-biotechnology enabled
protection and nutrition of plants. Biotechnol Adv 29:792–803
Ginet N, Pardoux R, Adryanczyk G, Garcia D, Brutesco C, Pignol D (2011) Single-step production
of a recyclable nanobiocatalyst for organophosphate pesticides biodegradation using function-
alized bacterial magnetosomes. PLoS One 6:e21442
Ginjupalli GK, Baldwin WS (2013) The time- and age-dependent effects of the juvenile
hormone analog pesticide, pyriproxyfen on Daphnia magna reproduction. Chemosphere
92:1260–1266
Grillo R, dos Santos NZ, Maruyama CR, Rosa AH, de Lima R, Fraceto LF (2012) Poly(epsilon-
caprolactone) nanocapsules as carrier systems for herbicides: physico-chemical characteriza-
tion and genotoxicity evaluation. J Hazard Mater 231:1–9
Grillo R, Abhilash PC, Fraceto LF (2016) Nanotechnology applied to bio-encapsulation of pesti-
cides. J Nanosci Nanotechnol 16:1231–1234
Hake H, Ben-Zur R, Schechter I, Anders A (2007) Fast optical assessment of pesticide coverage
on plants. Anal Chim Acta 596:1–8
Herrero MA, Guerra J, Myers VS, Gomez MV, Crooks RM, Prato M (2010) Gold dendrimer
encapsulated nanoparticles as labeling agents for multiwalled carbon nanotubes. ACS Nano
4:905–912
Hu X, Zheng P, Meng G, Huang Q, Zhu C, Han F, Huang Z, Li Z, Wang Z, Wu N (2016) An
ordered array of hierarchical spheres for surface-enhanced Raman scattering detection of traces
of pesticide. Nanotechnology 27:384001
Hubert J, Stejskal V, Munzbergova Z, Hajslova J, Arthur FH (2007) Toxicity and efficacy of
selected pesticides and new acaricides to stored product mites (Acari: Acaridida). Exp Appl
Acarol 42:283–290
Hummel HE, Langner SS, Eisinger MT (2013) Pheromone dispensers, including organic polymer
fibers, described in the crop protection literature: comparison of their innovation potential.
Commun Agric Appl Biol Sci 78:233–252
Hwang IC, Kim TH, Bang SH, Kim KS, Kwon HR, Seo MJ et al (2011) Insecticidal effect of con-
trolled release formulations of etofenprox based on nano-bio technique. J Fac Agric Kyushu
Univ 56(1):33–40
Itoh H, Naka K, Chujo Y (2004) Synthesis of gold nanoparticles modified with ionic liquid based
on the imidazolium cation. J Am Chem Soc 126:3026–3027
Jerobin J, Sureshkumar RS, Anjali CH, Mukherjee A, Chandrasekaran N (2012) Biodegradable
polymer based encapsulation of neem oil nanoemulsion for controlled release of Aza-A.
Carbohydr Polym 90(4):1750–1756
Jiang LC, Basri M, Omar D, Rahman MBA, Salleh AB, Rahman RNZRA et al (2012) Green
nano-emulsion intervention for water-soluble glyphosate isopropylamine (IPA) formulations in
controlling Eleusine indica (E. indica). Pestic Biochem Physiol 102(1):19–29
17 Nanotechnology and Its Impact on Insects in Agriculture 375
Jokar M, Safaralizadeh MH, Hadizadeh F, Rahmani F, Kalani MR (2016) Design and evalua-
tion of an apta-nano-sensor to detect Acetamiprid in vitro and in silico. J Biomol Struct Dyn
34:2505–2517
Kah M (2015) Nanopesticides and nanofertilizers: emerging contaminants or opportunities for risk
mitigation? Front Chem 3:64
Kah M, Hofmann T (2014) Nanopesticide research: current trends and future priorities. Environ
Int 63:224–235
Kah M, Machinski P, Koerner P, Tiede K, Grillo R, Fraceto LF, Hofmann T (2014) Analysing the
fate of nanopesticides in soil and the applicability of regulatory protocols using a polymer-
based nanoformulation of atrazine. Environ Sci Pollut Res Int 21:11699–11707
Kah M, Weniger AK, Hofmann T (2016) Impacts of (nano) formulations on the fate of an insec-
ticide in soil and consequences for environmental exposure assessment. Environ Sci Technol
50(20):10960–10967
Kalayou S, Granum C, Berntsen HF, Groseth PK, Verhaegen S, Connolly L, Brandt I, de Souza GA,
Ropstad E (2016) Label-free based quantitative proteomics analysis of primary neonatal porcine
Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE. J Proteome 137:68–82
Kamlesh K, Prashant S, Gopal M (2012a) Synthesis and characterization of rosiglitazone loaded
magnetic nanopolymer. Int J Green Nanotechnol 4:339–344
Kamlesh K, Prashant S, Gopal M (2012b) A facile one pot synthesis of collagen protected gold
nanoparticles using Na–malanodialdehyde. Mater Lett 79:199–201
Kamlesh K, Prashant S, Gopal KM (2014) Ionic liquid stabilized Metal NPs & their role a potent
catalyst. In: Tiwari A (ed) Advanced Materials. WILEY-Scrivener Publisher, Chapter 14,
549–564
Kamlesh K, Prashant S, Chandra M (2017) Metal (Au, Ag & Cu) NPs in ionic liquid: Potential
Catalytic system for organic reactions. J Nanomed Nanotechnol 8(6):1–11
Kang MA, Seo MJ, Hwang IC, Jang C, Park HJ, YM Y et al (2012) Insecticidal activity and feed-
ing behavior of the green peach aphid, Myzus persicae, after treatment with nano types of
pyrifluquinazon. J Asia Pac Entomol 15(4):533–541
Kaushik P, Shakil NA, Kumar J, Singh MK, Yadav SK (2013) Development of controlled release
formulations of thiram employing amphiphilic polymers and their bioefficacy evaluation in
seed quality enhancement studies. J Environ Sci Health B 48(8):677–685
Khandelwal N, Barbole RS, Banerjee SS, Chate GP, Biradar AV, Khandare JJ, Giri AP (2016)
Budding trends in integrated pest management using advanced micro- and nano-materials:
challenges and perspectives. J Environ Manag 184:157–169
Khodadoust S, Ghaedi M, Hadjmohammadi MR (2013) Dispersive nano solid material-ultrasound
assisted microextraction as a novel method for extraction and determination of bendiocarb and
promecarb: response surface methodology. Talanta 116:637–646
Kim SW, Jung JH, Lamasal K, Kim YS, Min JS, Lee YS (2012) Antifungal effects of silver
nanoparticles (AgNPs) against various plant pathogenic fungi. Mycobiology 40(1):53–58
Kookana RS, Boxall AB, Reeves PT, Ashauer R, Beulke S, Chaudhry Q, Cornelis G, Fernandes
TF, Gan J, Kah M, Lynch I, Ranville J, Sinclair C, Spurgeon D, Tiede K, Van den Brink PJ
(2016) Nanopesticides: guiding principles for regulatory evaluation of environmental risks. J
Agric Food Chem 62:4227–4240
Kumar RSS, Shiny PJ, Anjali CH, Jerobin J, Goshen KM, Magdassi S et al (2013) Distinctive
effects of nano-sized permethrin in the environment. Environ Sci Pollut Res 20(4):2593–2602
Kumar PM, Murugan K, Madhiyazhagan P, Kovendan K, Amerasan D, Chandramohan B, Dinesh D,
Suresh U, Nicoletti M, Alsalhi MS, Devanesan S, Wei H, Kalimuthu K, Hwang JS, Lo Iacono A,
Benelli G (2016) Biosynthesis, characterization, and acute toxicity of Berberis tinctoria-fabricated
silver nanoparticles against the Asian tiger mosquito, aedes albopictus, and the mosquito preda-
tors toxorhynchites splendens and mesocyclops thermocyclopoides. Parasitol Res 115:751–759
Laranjeira M, Shirosaki Y, Yoshimatsu Yasutomi S, Miyazaki T, Monteiro FJ (2017) Enhanced
biosafety of silica coated gadolinium based nanoparticles. J Mater Sci Mater Med 28:46
Larguinho M, Baptista PV (2011) Gold and silver nanoparticles for clinical diagnostics from
genomics to proteomics. J Proteome 75:2811–2823
376 P. Singh et al.
Loha KM, Shakil NA, Kumar J, Singh MK, Adak T, Jain S (2011) Release kinetics of β-cyfluthrin
from its encapsulated formulations in water. J Environ Sci Health B 46(3):201–206
Loha KM, Shakil NA, Kumar J, Singh M, Srivastava C (2012) Bio-efficacy evaluation of nano-
formulations of β-cyfluthrin against Callosobruchus maculatus (Coleoptera: Bruchidae).
J Environ Sci Health B 47(7):687–691
Mingming A, Yuncong Z, Shun H, Deguang L, Pingliang L, Jianqiang L et al (2013) Preparation
and characterization of 1-naphthylacetic acid-silica conjugated nanospheres for enhancement
of controlled-released performance. Nanotechnology 24(3):035601–035608
Mirabelli MF, Wolf JC, Zenobi R (2016) Pesticide analysis at ppt concentration levels: coupling
nano-liquid chromatography with dielectric barrier discharge ionization-mass spectrometry.
Anal Bioanal Chem 408:3425–3434
Mishra P, Balaji AP, Swathy JS, Paari AL, Kezhiah M, Tyagi BK, Mukherjee A, Chandrasekaran
N (2016) Stability assessment of hydro dispersive nanometric permethrin and its biosafety
study towards the beneficial bacterial isolate from paddy rhizome. Environ Sci Pollut Res Int
23:24970–24982
Momeni S, Nabipour I (2015) A simple green synthesis of palladium nanoparticles with sargassum
alga and their electrocatalytic activities towards hydrogen peroxide. Appl Biochem Biotechnol
176:1937–1949
Mondal KK, Mani C (2012) Investigation of the antibacterial properties of nanocopper against
Xanthomonas axonopodis pv. punicae, the incitant of pomegranate bacterial blight. Ann
Microbiol 62(2):889–893
Narayanan N, Gupta S, Gajbhiye VT, Manjaiah KM (2017) Chemosphere optimization of iso-
therm models for pesticide sorption on biopolymer-nanoclay composite by error analysis.
Parasitol Res 173:502–511
Niikura K, Matsunaga T, Suzuki T, Kobayashi S, Yamaguchi H, Orba Y, Kawaguchi A, Hasegawa
H, Kajino K, Ninomiya T, Ijiro K, Sawa H (2013) Gold nanoparticles as a vaccine platform:
influence of size and shape on immunological responses in vitro and in vivo. ACS Nano
7:3926–3938
Nuruzzaman M, Rahman MM, Liu Y, Naidu R (2016) Nanoencapsulation, nano-guard for pesti-
cides: a new window for safe application. J Agric Food Chem 64:1447–1483
Paret ML, Palmateer AJ, Knox GW (2013a) Evaluation of a light-activated nanoparticle formu-
lation of titanium dioxide with zinc for management of bacterial leaf spot on rosa ‘Noare.
Hortscience 48(2):189–192
Paret ML, Vallad GE, Averett DR, Jones JB, Olson SM (2013b) Photocatalysis: effect of light-
activated nanoscale formulations of TiO2 on Xanthomonas perforans and control of bacterial
spot of tomato. Phytopathology 103(3):228–236
Periasamy AP, Umasankar Y, Chen SM (2009) Nanomaterials - acetylcholinesterase enzyme
matrices for organophosphorus pesticides electrochemical sensors: a review. Sensors (Basel)
9:4034–4055
Petosa AR, Rajput F, Selvam O, Ohl C, Tufenkji N (2016) Assessing the transport potential of
polymeric nanocapsules developed for crop protection. Water Res 111:10–17
Pradeep K, Prashant S, Kamlesh K, Subho M, Ramesh C (2011) A green approach for the syn-
thesis of gold nanotriangles using aqueous leaf extract of Callistemon viminalis. Mater Lett
65:595–597
Prashant S (2013) A simple, rapid, and green synthesis of capped gold nanospheres and nanorods
using aqueous extract of azolla. Int J Green Nano 1:1–5
Prashant S, Sunil K, Anju K, Rashmi K, Ramesh C (2008a) A novel route for the synthesis of
indium nanoparticles in ionic liquid. Mater Lett 62:4164–4166
Prashant S, Anju K, Rashmi K, Ramesh C (2008b) Copper nanoparticles in ionic liquid: an easy
and efficient catalyst for the coupling of thiazolidine-2,4-dione, aromatic aldehyde and ammo-
nium acetate. Catal Commun 9:1618–1623
Prashant S, Anju K, Rashmi K, Ramesh C (2008c) Copper nanoparticles in an ionic liquid: an effi-
cient catalyst for the synthesis of bis-(4-hydroxy-2-oxothiazolyl)methanes. Tetrahedron Lett
49:727–730
17 Nanotechnology and Its Impact on Insects in Agriculture 377