PLOS NEGLECTED TROPICAL DISEASES
RESEARCH ARTICLE
Expansions of chemosensory gene orthologs
among selected tsetse fly species and their
expressions in Glossina morsitans morsitans
tsetse fly
Joy M. Kabaka1,2*, Benson M. Wachira1,3, Clarence M. Mang’era ID4, Martin K. Rono5,
Ahmed Hassanali3, Sylvance O. Okoth1, Vincent O. Oduol6, Rosaline W. Macharia7, Grace
A. Murilla1, Paul O. Mireji ID1,5*
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OPEN ACCESS
Citation: Kabaka JM, Wachira BM, Mang’era CM,
Rono MK, Hassanali A, Okoth SO, et al. (2020)
Expansions of chemosensory gene orthologs
among selected tsetse fly species and their
expressions in Glossina morsitans morsitans
tsetse fly. PLoS Negl Trop Dis 14(6): e0008341.
https://doi.org/10.1371/journal.pntd.0008341
Editor: Peter John Myler, Seattle Biomedical
Research Institute, UNITED STATES
Received: September 17, 2019
Accepted: May 1, 2020
Published: June 26, 2020
Copyright: © 2020 Kabaka et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All RNA-Seq data
files are available from the NCBI database
(accession number(s) PRJNA343267 and
PRJNA343269).
Funding: None of the authors received direct
funding for the research but were supported by
Fogarty International Center of the National
Institutes of Health (FIC-NIH) grants
R03TW009444 and, D43TW007391, and NIH/
NIAID supported grant U01AI115648 to Professor
1 Biotechnology Research Institute—Kenya Agricultural and Livestock Research Organization, Kikuyu,
Kenya, 2 Department of Biochemistry, Microbiology and Biotechnology, School of Pure and Applied
Sciences, Kenyatta University, Ruiru Campus, Nairobi, Kenya, 3 Department of Chemistry, School of Pure
and Applied Sciences, Kenyatta University, Ruiru Campus, Nairobi, Kenya, 4 Department of Biochemistry
and Molecular Biology, Egerton University, Njoro Campus, Egerton, Kenya, 5 Centre for Geographic
Medicine Research—Coast, Kenya Medical Research Institute, Kilifi, Kenya, 6 Department of Biochemistry,
University of Nairobi, Nairobi, Kenya, 7 Center for Bioinformatics and Biotechnology, University of Nairobi,
Nairobi, Kenya
* jmanyasa@gmail.com (JMK); Mireji.paul@gmail.com (POM)
Abstract
Tsetse fly exhibit species-specific olfactory uniqueness potentially underpinned by differences in their chemosensory protein repertoire. We assessed 1) expansions of chemosensory protein orthologs in Glossina morsitans morsitans, Glossina pallidipes, Glossina
austeni, Glossina palpalis gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis
tsetse fly species using Café analysis (to identify species-specific expansions) and 2) differential expressions of the orthologs and associated proteins in male G. m. morsitans antennae and head tissues using RNA-Seq approaches (to establish associated functional
molecular pathways). We established accelerated and significant (P<0.05, λ = 2.60452e-7)
expansions of gene families in G. m. morsitans Odorant receptor (Or)71a, Or46a, Ir75a,d,
Ionotropic receptor (Ir) 31a, Ir84a, Ir64a and Odorant binding protein (Obp) 83a-b), G. pallidipes Or67a,c, Or49a, Or92a, Or85b-c,f and Obp73a, G. f. fuscipes Ir21a, Gustatory receptor (Gr) 21a and Gr63a), G. p. gambiensis clumsy, Ir25a and Ir8a, and G. brevipalpis Ir68a
and missing orthologs in each tsetse fly species. Most abundantly expressed transcripts in
male G. m. morsitans included specific Or (Orco, Or56a, 65a-c, Or47b, Or67b,
GMOY012254, GMOY009475, and GMOY006265), Gr (Gr21a, Gr63a, GMOY013297 and
GMOY013298), Ir (Ir8a, Ir25a and Ir41a) and Obp (Obp19a, lush, Obp28a, Obp83a-b
Obp44a, GMOY012275 and GMOY013254) orthologs. Most enriched biological processes
in the head were associated with vision, muscle activity and neuropeptide regulations,
amino acid/nucleotide metabolism and circulatory system processes. Antennal enrichments
(>90% of chemosensory transcripts) included cilium-associated mechanoreceptors,
chemo-sensation, neuronal controlled growth/differentiation and regeneration/responses to
stress. The expanded and tsetse fly species specific orthologs includes those associated
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Serap Aksoy, Yale School of Public Health, Yale
University, NH,CT, USA. The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Expanded chemosensory gene orthologs among selected tsetse species
with known tsetse fly responsive ligands (4-methyl phenol, 4-propyl phenol, acetic acid,
butanol and carbon dioxide) and potential tsetse fly species-specific responsive ligands (2oxopentanoic acid, phenylacetaldehyde, hydroxycinnamic acid, 2-heptanone, caffeine,
geosmin, DEET and (cVA) pheromone). Some of the orthologs can potentially modulate
several tsetse fly species-specific behavioral (male-male courtship, hunger/host seeking,
cool avoidance, hygrosensory and feeding) phenotypes. The putative tsetse fly specific chemosensory gene orthologs and their respective ligands provide candidate gene targets and
kairomones for respective downstream functional genomic and field evaluations that can
effectively expand toolbox of species-specific tsetse fly attractants, repellents and other
tsetse fly behavioral modulators.
Author summary
Tsetse flies are insect vectors of sleeping sickness in humans and nagana in livestock in
sub-Sahara Africa. Tsetse flies identify their hosts (preferred and non-preferred) by detecting and processing odor cues emitted by the hosts in their environment. Tsetse flies use
chemosensory proteins and associated pathways in their antennae to identify these cues.
In this study, we identified expansions of these chemosensory protein in six tsetse fly species (Glossina morsitans morsitans, Glossina pallidipes, Glossina austeni, Glossina palpalis
gambiensis, Glossina fuscipes fuscipes and Glossina brevipalpis) with different known hosts.
We also identified potential ligands to these proteins based on fruit fly (Drosophila melanogaster) orthologs. With G. m. morsitans as an example, we identified the proteins and
associated molecular pathways preferentially expressed in tsetse fly antennae. These proteins may be responsible for the tsetse fly species-specific host discrimination, with the
ligands eliciting species-specific behavioral responses in the flies. The expressed orthologs
may be functionally important in odor detection in tsetse fly and lay down useful groundwork for downstream functional genomics R&D for more effective tsetse fly species-specific odor attractants and repellents for routine tsetse fly control operations.
Introduction
Human African Trypanosomiasis (HAT) constitutes one of the most neglected tropical diseases (NTDs) with devastating health and economic consequences in sub-Sahara Africa [1,2].
On the other hand, African Animal Trypanosomiasis (AAT) is rampant in livestock inhabiting
tsetse-infested areas throughout the continent. The AAT cause death of about three million
cattle each year [3], and in terms of agricultural Gross Domestic Product (GDP), loss of about
US$ 4.75 billion per year [3]. The HAT and AAT causative trypanosomes are transmitted by
different groups of tsetse species. Tsetse control is considered an effective approach and constitutes the corner stone in trypanosomiasis suppression [4,5]. Tsetse fly species belong to Glossina genus and are generally restricted to sub-Saharan Africa. Twenty-three species and eight
sub-species of tsetse flies are recognized [6,7]. These species are divided into Morsitans, Palpalis and Fusca clade sub-genera, described by respective savanna, riverine/lacustrine and forest
ecological niches they occupy. The Morsitans group consists of five species that include Glossina morsitans morsitans and Glossina pallidipes restricted to savannah grassland and Glossina
austeni occupying coastal woodlands [8]. This group is adapted to drier habitats than Palpalis
and Fusca [9] and preferentially feeds on livestock and wildlife. They are thus important
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Expanded chemosensory gene orthologs among selected tsetse species
vectors of African Animal Trypanosomiasis (AAT) also known as nagana. On the other hand,
Palpalis group consists of five species, including Glossina palpalis gambiensis and Glossina fuscipes fuscipes in West, Central and East Africa. These species are predominant vectors of
Human African Trypanosomosis (HAT), also known as sleeping sickness, despite their preferential predilection to feeding on reptiles and ungulates. Fusca group consist of 13 species
largely inhabiting damp evergreen forests of West Africa (except Glossina brevipalpis) and are
mainly associated with livestock. Glossina brevipalpis is of limited medical and agricultural significance and occurs discontinuously in other parts of sub-Saharan Africa [6].
These tsetse fly species exhibit different olfactory uniqueness, which partly accounts for their
gradation of preferences for their particular hosts. This olfactory uniqueness (and visual
responses) has been exploited in designing effective tsetse fly bait technologies that consist of synthetic blends of attractants and repellents that mimic those of their natural hosts and non-hosts
respectively [10–13]. These technologies are especially applicable for G. m. morsitans and G. pallidipes but not G. austeni (among savanna species) [14] and palpalis group. For example, G. pallldipes, G. m. morsitans and to some extent G. brevipalpis are attracted to traps baited with POCA
(3-n-propylphenol, 1-octen-3-ol, 4-cresol and acetone) and to which G. austeni poorly responds
[15–17]. Molecular bases of these natural differential responses are poorly understood but may
be underpinned by differences in their chemosensory apparatus. The chemosensory apparatus
facilitate reception of odorants and tastants, and consist of Odorant-binding proteins (Obps),
Odorant-degrading enzymes (Odes), Odorant receptors (Ors), Ionotropic receptors (Irs), Gustatory receptors (Grs), Chemosensory proteins (Csps), Sensory neuron membrane proteins
(Snmps) and CD36-like pheromone sensors [18–24]. These chemosensory proteins mediate
decoding of ecological odors and odorant specific behavioral responses in insect hosts. These
responses include seeking for hosts, location of oviposition sites, searching for mates, and detecting and escaping from potential predators. The Obp transport pheromone molecules and general
odorants to Ors [25]. The Ors are odorant-gated ion channels composed of an odorant-binding
subunit and olfactory co-receptor Orco [26,27]. The Irs have higher specificity to volatiles than
Ors, detecting specific variety of odors, such as acids, aldehydes, amines and humidity [20,28].
The Ir25a and Ir8a are putative conserved Ir co-receptors [23]. The Grs discern odor tastes and
contact pheromones [29]. Only two Snmp subfamilies (Snmp 1 and Snmp 2) have been identified
in insects, where Snmp1 is expressed in pheromone-sensitive Olfactory Receptor Neurons
(ORNs) while Snmp 2 is expressed in supporting cells [30–32]. Some of these chemosensory proteins are present in non-canonical chemosensory organs, such as legs [33,34], wings [35,36] and
pheromone glands [37], where only a subset of Irs are specifically expressed in D. melanogaster
antennae [20]. Among tsetse flies, genomes of G. pallidipes, G. m. morsitans, G. austeni, G. p. gambiensis, G. f. fuscipes and G. brevipalpis (representative of the different clades/sub-general) have
been sequenced [38], and their respective chemosensory proteins annotated [39–41].
Here we report on 1) expansions of chemosensory protein orthologs in six tsetse fly species/
subspecies (G. pallidipes, G. m. morsitans, G. austeni, G. p. gambiensis, G. f. fuscipes and G. brevipalpis) to identify species-specific expansions and 2) differential expressions of these and
associated proteins in antennae and head tissues G. m. morsitans to establish probable functional pathways influencing host seeking behaviors in this specie.
Materials and methods
Differential expansions of D. melanogaster chemosensory gene orthologs
among tsetse flies
We obtained complete D. melanogaster gene set release 79 (Drosophila_melanogaster. BDGP6.
pep.all.fa) from Ensembl project [42] in fasta format. We then isolated D. melanogaster
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chemosensory genes from the gene set by searching and retrieving flybase [43] chemosensory
gene IDs in the gene set using “Odorant receptor”, “Gustatory receptor”, “Ionotropic receptor”, “Odorant-binding protein”, “Sensory neuron membrane protein” and “Glutamate receptor” Linux bash regular expressions. For Csp orthologs, we extracted D. melanogaster IDs from
Macharia et al., (2016) [40]. We separately obtained VectorBase Release VB-2019-02 homologs
(gene trees) of disease vectors from VectorBase database [44] in OrthoXML formats. The gene
trees were pre-computed by Gene Orthology/Paralogy prediction pipeline in VectorBase [44]
that identified gene duplications within species and speciation events. We probed the VectorBase homologs for ortholog groups (gene families) with the D. melanogaster chemonsensory
genes (flybase IDs) to identify their respective tsetse flies (G. austeni, G. f. fuscipes, G. p. gambiensis, G. brevipalpis, G. pallidipes and G. m. morsitans) orthologs. We identified presence of
the individual genes in each gene family (ortholog group) and species. Gene families with
accelerated gene expansions were pre-computed through Computational Analysis of gene
Family Evolution (CAFE) [45] in VectorBase [44]. We considered the VectorBase [44] precomputed gene expansions/contractions reliable since they are 1) community reviewed and
adopted and with stable ortholog IDs and 2) regularly updated (with new gene-sets and
genomes). We also conducted Principal Component Analysis (PCA) in R using FactoMineR
and Factoextra packages with species-specific gene counts as input data to establish relationship between the expanded/contracted chemosensory genes (Ors, Irs, Grs and Obps) and
tsetse species.
Transcriptional expression of D. melanogaster chemosensory gene
orthologs in male G. m. morsitans
We employed high throughput Illumina based RNA-Seq approach to establish expression profiles of the D. melanogaster chemosensory gene orthologs in male G. m. morsitans. We established expression levels of the orthologs in the antennae and in relation to the head libraries.
We isolated and sequenced RNA from antennae or head tissues from colony reared G. m. morsitans as described previously [46]. Briefly, we fed teneral male G. m. morsitans (1–3 days old)
on defibrinated bovine blood meal (their initial blood meal post-eclosion) (commercially supplied by Hemostat Laboratories, Dixon, CA, USA) to putatively prime their chemosensory system. We then extracted their antennae in two independent biological replicates (from 50 flies
each) using liquid nitrogen-based method of Menuz et al. (2014) [47] 72 hrs post-feeding. We
envisaged that the 72 hrs deprivation of blood meal (food) would biologically prime potential
host seeking chemosensory apparatus in the flies and enhance RNA-seq detection of chemosensory gene expressions, specifically those associated with hunger/host seeking.
The G. m. morsitans show marked die1changes in their biting activity in the field, with their
peak activity in the morning and afternoon [48,49]. We thus snap froze individual tsetse flies
in liquid nitrogen in the morning (09:30 hrs) and carefully hand-dissected their antennae from
the head into 1.5 ml microfuge tubes kept cold in liquid nitrogen. We then isolated RNA by
mechanically crushing the antennae with disposable RNAseq-free plastic pestles in TRIzol
reagent (Invitrogen, Carlsbad, USA) following the manufacturer’s protocol. We removed
traces of potential carry over DNA (that could potentially confound our RNA-Seq analysis) by
digesting possible contaminating genomic DNAs (gDNA) in the total RNA using TURBO
DNase (Ambion life technologies, TX, USA) following manufacturer’s instructions. We confirmed removal of the gDNA from total RNA by qualitative assessment of PCR amplicons
from final RNA samples using tsetse fly specific beta-tubulin gene primers as documented in
Bateta et al. (2017) [46]. We verified quality and integrity of RNA samples using Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) following manufacturer’s instructions. cDNA was
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then generated from the RNA using Illumina TruSeq RNA Sample Preparation Kit (Illumina,
Hayward, CA, USA) and the cDNA (75 bp single-end read) and sequenced on Illumina HiSeq
2500 at Yale University Center of Genome Analysis (YCGA), New Haven, CT, USA. We similarly prepared head transcriptomes from two independent biological replicates (50 flies each)
from 72 hrs starved 40 days old males. We deposited all transcriptome sequences at the
Sequence Read Archive (SRA) under study accession numbers PRJNA343267 and
PRJNA343269 for the antennae and head libraries respectively.
Expression profiles of D. melanogaster chemosensory gene orthologs in
male G. m. morsitans antennae and head libraries
We established quality of the reads in each individual transcriptome library using FastQC
(Babraham Bioinformatics) software package (http://www.bioinformatics.babraham.ac.uk/
projects/fastqc/)). We then used the FastQC results to clean (trimm) the reads using CLC
genomic workbench version 10 software (CLC Bio, Aarhus, Denmark) through settings that
permitted 1) removal of low quality sequences (limit = 0.05), 2) removal of ambiguous nucleotides (maximum 2 nucleotides allowed), 3) removal of terminal nucleotides (10 nucleotides
from the 5’ end and 1 nucleotide from the 3’ end) and 4) removal of sequences on length (minimum length 15 nucleotides, maximum length 1000 nucleotides). We then mapped the cleaned
reads on to G. m. morsitans transcripts gene-set version 1.9 from Vectorbase [44] using CLC
genomic workbench version 10 software (CLC Bio, Aarhus, Denmark) thorough settings that
permitted 1) mismatch cost of 2, 2) insertion/deletion cost of 3, 3)length fraction of 0.8, 4) similarity fraction of 0.8, 5) maximum number of reads per hit of 10, and 6) strand specificity set
as both strands.
From the mappings, we established reads mapping per transcript and reads per kilobase of
transcripts per Million mapped reads (RPKM), a normalized index of relative gene expression
associated with each transcript (including chemosensory genes) in the gene-set for individual
transcriptomes [50]. We then established differentially expressed transcripts between the
antennae and the head transcriptomes by comparing the reads mapped in the genes sets from
respective transcriptomes using edgeR software [51,52]. We considered transcripts validly differentially expressed if they had at least two-fold changes, p-value corrected False Detection
Rate (FDR) < 0.05 and one Counts Per Million (CPM) coverage to mitigate against type I statistical errors. We then determined antennae or head enriched molecular processes using
canonical Gene Set Enrichment Analysis (GSEA) using WEB-based GEne SeT AnaLysis
Toolkit (WebGestalt) [53]. Since WebGestalt database did not include tsetse flies, but D. melanogaster gene set, we obtained homologs of the entire G. m. morsitans gene-set in D. melanogaster through Basic Alignment Search Tool (BLAST) analysis of protein sequences (Blastp) [54]
of the G. m. morsitans gene-set against those of D. melanogaster and accepted hits with evalue < 0.001 as significantly homologous. We then used these D. melanogaster homologs as
proxy in WebGestalt to assess enrichment of their associated G. m. morsitans homologs. We
used the FDR corrected p-value ranked D. melanogaster homolog gene-sets of differentially
expressed G. m. morsitans transcripts as input for the analysis [55]. We considered selection of
5–2000 Entrez Gene IDs, FDR < 0.05, 1000 permutations and 20 categories with the outputted
leading-edge genes default parameters for the analysis. Through GSEA, we separated and identified significantly enriched non-redundant biological processes, cellular components and
molecular function Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes,
KEGG, PANTHER, Reactome, pathways and Database of Protein, Chemical and Genetic
Interactions (BioGRID) network [56–61]. Next, we identified antennae or head (tissue) specific chemosensory genes by mapping the global most differentially (based on fold change)
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and abundantly (based on CPM) or significantly expressed (based on p-value) transcripts in
MA or volcano plots respectively using edgeR software package [52,62] in R software [63]. We
considered chemosensory genes with fold changes (FC) � 1.25 as of chemosensory biological
significance as previously documented [64].
Results
Expansions of chemosensory gene orthologs among tsetse fly species
We identified 60 each of Ors, Irs or Grs, 51 Obps, seven GluR and two Snmps (excluding isoforms) in D. melanogaster [43] and four Csps [40], with 58, 34, 13, 22, 2 and 3 orthologs (VectorBase gene trees, Release VB-2019-02) [44] respectively among the tsetse fly species (S1
Table). Café gene expansion analysis [45] revealed significant (P<0.05, λ = 2.60452e-7) accelerated expansions of several gene families/clusters including VBGT00190000010263 (Or71a and
Or46a), VBGT00190000009736 (Ir75a,d, Ir31a, Ir84a and Ir64a) and VBGT00190000009994
(Obp83a-b) in G. m. morsitans, VBGT00840000047907 (Or67a,c, Or49a, Or92a, Or85b-c,f) and
VBGT00190000013627 (Obp73a) in G. pallidipes, VBGT00190000012412 (Ir21a) and
VBGT00190000010879 (Gr21a and Gr63a) carbon dioxide receptors orthologs [65] in G. f. fuscipes,
VBGT00820000046003 (clumsy, Ir25a and Ir8a) in G. p. gambiensis and VBGT00190000013104
(Ir68a) in G. brevipalpis (S1 Table). No gene families were significantly expanded in G. austeni.
We also identified several orthologs that were missing/absent in specific tsetse fly species (S1
Table). The Ir76b ortholog was absent in four tsetse fly species (G. p. gambiensis, G. m. morsitans, G. pallidipes and G. brevipalpis) while Gr33a was missing in G. brevipalpis. Both Gr32a and
Gr68a were missing in G. brevipalpis and G. m. morsitans. The Gr64a-f, Gr5a, Gr43a, Obp56a/
d/e and Or71a orthologs were absent in all tsetse fly species. The Snmp1, Or67d and Obp19a
and Orco ortholog appeared to be conserved across all tsetse fly species. Our PCA analysis
revealed a general positive correlation between tsetse species across four chemosensory groups
(Ors, Irs, Grs or Obps). Additionally, Gr and Ir orthologs appeared to be positively correlated
(S1 Fig panels B2 and B3) in relation to a unique G. m. morsitans cluster (S1 Fig panels A2 and
A3).
Expression profiles of chemosensory ortholog transcripts in male G. m.
morsitans antennae
The RNA-Seq of the antennae and head libraries yielded 23.3 to 17.9 million reads from
respective libraries. We successfully mapped 51.0 to 69.6% of these reads onto G. m. morsitans
transcripts where we established about 88.4% unique mappings of the reads to specific transcripts (Fig 1). We have summarized expressions profiles of the chemosensory orthologs in Fig
2. Orco, Or56a, 65a-c, Or47b and Or67b, and three G. m. morsitans specific orthologs
(GMOY012254, GMOY009475, and GMOY006265) were among most abundantly expressed
transcripts with Or33a-c orthologs exhibiting the least expression. Expressions of the members
of the significantly expanded Ors gene families were marginal. Only six Gr orthologs were
expressed among which Gr21a and Gr63a orthologs (carbon dioxide receptors) [65] and
related two G. m. morsitans specific (GMOY013297 and GMOY013298) orthologs were abundantly expressed. The putative conserved core-receptors (Ir8a and Ir25a) and Ir41a were
among the most abundantly expressed Irs orthologs. All but Ir75a-c expanded Ir orthologs
were expressed. Most abundantly expressed Obp orthologs include Obp19a, lush, Obp28a,
Obp83a-b Obp44a and two G. m. morsitans specific (GMOY012275 and GMOY013254)
orthologs. Among these, Obp83a-b were among the significantly expanded Obp families. Both
Snmps (Snmp 1 and Snmp 2) and Csp2 were also abundantly expressed.
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Fig 1. Summary of processing and mapping statistics of RNA-Seq reads from male G. m. morsitans antennae and head transcriptomes.
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Enriched pathways between male G. m. morsitans antennae and head
libraries
Our Gene Set Enrichment Analysis (GSEA) of transcripts between the antennae and head
libraries revealed several enriched pathways and processes between these tissues (Table 1, S2
Table). Our GoSlim GO analysis component of the GSEA assigned 85.4% of our transcripts to
biological process, cellular components and molecular function ontologies (S2 Table). The
most predominantly enriched biological processes between the antennae and head include
metabolic processes, biological regulations, multicellular organismal processes, developmental
processes and responses to stimuli. Most of these biological processes appeared to be localized
in the membrane, macromolecular complex and nucleus cellular components, and were predominantly involved in protein binding, nucleic acid binding, ion binding and hydrolase
activity molecular functions (S2 Table). More specifically, most enriched biological processes
in the head were associated with vision, muscle activity and associated structural proteins and
neuropeptide regulations, amino acid/nucleotide metabolism and circulatory system processes. The enriched cellular components were predominantly associated with vision and muscular functions. On the other hand, most enriched antennal biological processes were ciliumassociated mechanoreceptors, chemo-sensation, neuronal controlled growth and
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Fig 2. Expression profiles of D. melanogaster chemosensory gene orthologs in male G. m. morsitans antennae 72 hrs post feeding.
https://doi.org/10.1371/journal.pntd.0008341.g002
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Table 1. Summary of Canonical Gene-set Enrichment Analysis (GSEA) of differentially expressed transcripts between male G. m. morsitans tsetse fly antennae and
head transcriptomes.
Functional Database
Name
Class
Gene
Biological
Ontology
Process
Tissue
Tissue
Head
Annotation
Process ID
Head
Component
General Function
Size
L
ES
NES
P
Value
FDR
GO:0050953
Sensory perception of light stimulus
Vision
59
22 0.898
1.883 0.000
0.000
GO:0007186
G-protein coupled receptor
signaling pathway
Vision
162
56 0.796
1.869 0.000
0.000
GO:0032101
Regulation of response to external
stimulus
Vision
101
8
0.801
1.833 0.000
0.000
GO:0010927
Cellular component assembly
involved in morphogenesis
Muscle activity
108
18 0.773
1.765 0.000
0.001
GO:0042440
Pigment metabolic process
Vision
115
18 0.735
1.692 0.000
0.004
GO:0009628
Response to abiotic stimulus
Vision
360
36 0.682
1.689 0.000
0.003
GO:0003012
Muscle system process
Muscle activity
27
12 0.879
1.676 0.000
0.005
GO:0044057
Regulation of system process
Neuropeptide muscle
regulations
48
12 0.795
1.661 0.000
0.007
GO:0043473
Pigmentation
Vision
103
18 0.706
1.610 0.003
0.025
GO:0006730
One-carbon metabolic process
Vision
15
5
0.910
1.607 0.000
0.024
GO:0003013
Circulatory system process
Neuropeptide
regulations
40
12 0.784
1.590 0.005
0.032
Antennae GO:0044782
Cellular
Description
Statistics
Cilium organization
Mechanoreception
62
22 -0.839 2.170 0.000
0.000
GO:0031503
Protein complex localization
Mechanoreception
30
12 -0.849 1.903 0.000
0.001
GO:0007606
Sensory perception of chemical
stimulus
Chemo-sensation
124
57 -0.628 1.806 0.000
0.006
GO:0035218
Leg disc development
Growth/differentiation
87
15 -0.665 1.781 0.000
0.007
GO:0030705
Cytoskeleton-dependent
intracellular transport
Mechanoreception
66
11 -0.676 1.751 0.000
0.012
GO:0030031
Cell projection assembly
Mechanoreception
112
35 -0.624 1.742 0.005
0.011
GO:0031099
Regeneration
Repair/response to
stress
18
4
0.015
-0.828 1.711 0.000
GO:0019898
Extrinsic component of membrane
Vision
72
11 0.870
1.891 0.000
0.000
GO:0016028
rhabdomere
Vision
34
17 0.955
1.886 0.000
0.000
GO:0043292
Contractile fiber
Muscle activity
50
20 0.871
1.822 0.000
0.000
GO:0015629
Actin cytoskeleton
Vision/Muscle activity
99
18 0.794
1.807 0.000
0.000
GO:0098796
Membrane protein complex
Vision
233
10 0.690
1.689 0.000
0.001
GO:0098858
Actin-based cell projection
Vision
22
4
0.861
1.600 0.002
0.012
GO:0031984
Organelle sub-compartment
Vision
86
9
0.684
1.515 0.007
0.046
Cilium
Chemo-sensation/
Mechanoreception
80
30 -0.846 2.256 0.000
0.000
GO:0031252
Cell leading edge
Chemo-sensation
52
28 -0.811 2.005 0.000
0.000
GO:0005815
Microtubule organizing center
Mechanoreception/
Muscle activity
111
20 -0.666 1.849 0.000
0.001
Antennae GO:0005929
Molecular
Head
Calmodulin binding
Vision/Muscle activity
43
6
1.706 0.000
0.009
Function
Antennae GO:0005549
GO:0005516
Odorant binding
Chemo-sensation
49
35 -0.843 2.170 0.000
0.834
0.000
Pathway
KEGG
Head
dme04745
Phototransduction—fly—
Drosophila melanogaster (fruit fly)
Vision
25
14 0.954
0.000
Analysis
Panther
Head
P00057
Wnt signaling pathway
Vision
62
8
0.827
1.749 0.000
0.000
P00031
Inflammation mediated by
chemokine and cytokine signaling
pathway
Vision/Muscle activity
26
5
0.895
1.716 0.000
0.002
P00044
Nicotinic acetylcholine receptor
signaling pathway
Vision/Muscle activity
38
9
0.845
1.705 0.000
0.002
1.772 0.000
(Continued )
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Table 1. (Continued)
Functional Database
Name
Class
Reactome
Network
Analysis
�
Tissue
Tissue
Head
PPI_BIOGRID Head
Annotation
Process ID
Description
Statistics
General Function
Size
L
ES
NES
P
Value
FDR
P00016
Cytoskeletal regulation by Rho
GTPase
Vision/Muscle activity
21
4
0.911
1.680 0.000
0.004
P00004
Alzheimer disease-presenilin
pathway
Vision/Muscle activity
25
5
0.848
1.593 0.005
0.026
P00012
Cadherin signaling pathway
Muscle activity
25
3
0.839
1.585 0.003
0.025
P00042
Muscarinic acetylcholine receptor 1
and 3 signaling pathway
Vision/Neuropeptide
regulations
20
7
0.836
1.568 0.011
0.033
P04374
5HT2 type receptor mediated
signaling pathway
Vision
18
7
0.841
1.567 0.014
0.030
P00028
Heterotrimeric G-protein signaling
pathway-rod outer segment
phototransduction
Vision
5 3
0.991
1.563 0.000
0.031
4
0.904
1.778 0.000
0.000
R-DME-1852241 Organelle biogenesis and
maintenance
Vision
38
R-DME-2514856 The phototransduction cascade
Vision
12
6
0.961
1.687 0.000
0.025
R-DME-5620920 Cargo trafficking to the periciliary
membrane
Vision
15
4
0.956
1.683 0.000
0.018
R-DME-5617833 Cilium Assembly
Vision
15
4
0.956
1.674 0.000
0.018
R-DME-5620916 VxPx cargo-targeting to cilium
Vision
12
4
0.965
1.655 0.000
0.026
R-DME-2514859 Inactivation, recovery and
regulation of the phototransduction
cascade
Vision
12
6
0.961
1.644 0.000
0.029
R-DME-2187338 Visual phototransduction
Vision
14
6
0.957
1.644 0.000
0.025
R-DME-76002
Platelet activation, signaling and
aggregation
Vision/Muscle activity
47
9
0.784
1.640 0.000
0.024
R-DME-71291
Metabolism of amino acids and
derivatives
Metabolism
57
19 0.761
1.634 0.000
0.027
R-DME-2672351 Stimuli-sensing channels
Vision
9 3
0.954
1.622 0.000
0.034
R-DME-500792
Vision
14
4
0.920
1.618 0.002
0.034
PPI_BIOGRID
M119
Muscle activity
33
16 0.872
1.711 0.000
0.004
PPI_BIOGRID
M37
Muscle activity
71
20 0.767
1.683 0.000
0.004
PPI_BIOGRID
M80
Vision
12
8
1.643 0.000
0.017
GPCR ligand binding
0.957
Non-Redundant
https://doi.org/10.1371/journal.pntd.0008341.t001
differentiation, and regeneration/responses to stress, while enriched cellular components were
associated with chemo-sensation, mechano-reception and muscular activities. Most enriched
molecular functions in the head and antennae were associated with vision/muscular activities
and chemo-sensation, respectively. The KEGG pathway analysis revealed enrichment of
vision-associated pathways. Similarly, PANTHER pathway analysis also identified vision, in
addition to neuropeptide signaling and muscular associated activities among the most
enriched pathways in the head. We identified similar outcomes from our protein-protein
interactions BIOGRID analysis in the head library. The Reactome pathway analysis identified
vision and amino acids and derivative metabolism pathways predominating in the head transcriptome. We did not identify pathways or networks significantly enriched in the antennae
library.
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Differentially expressed transcripts between male G. m. morsitans antennae
and head libraries
Our search for both differentially (FC > 2) and abundantly expressed (CPM > 1) transcripts
between the head and antennae libraries identified 2179 and 2158 transcripts respectively differentially expressed (FDR corrected p value < 0.05) between each library as summarized in
our MA plot (Fig 3). Among these transcripts, at least 52 transcripts were most differentially
and abundantly expressed (log FC > 2 and Average log CPM > 10) in both libraries. These
transcripts were predominantly associated with vision, iron transport, metabolism and signal
transduction in the head. In the antennae, the transcripts were involved in odor sensing and
clearing, fatty acid synthesis and regulation of feeding behavior and locomotor activity (S3
Table). Analysis of both differentially (FC) and significantly expressed (p-value) transcripts
between the head and antennae libraries identified 49 and 61 transcripts as most significantly
expressed (FC >10 or <-5, and–log10 p-value > 25) in the head and antennae libraries respectively as summarized in our volcano plot (Fig 4). Overall, about 40 and 52 percent of the transcripts were associated with vision (head) and chemo-sensation (antennae) respectively. Most
significantly expressed transcripts in the head library were functionally associated with energy
mobilization, feeding, immunity, cytoskeleton integrity, amino acid metabolism, endocrine
signaling and neuronal development and support. In the antennae, most significantly
expressed transcripts were functionally associated chemo-sensation, metabolism, and cell proliferation, regulation of gene expression, signal transduction, anatomical integrity, neuron
integrity/development and mechanoreception (S3 Table).
Differential expression of chemosensory gene transcripts between male G.
m. morsitans antennae and head libraries
When we considered fold change greater than 1.25 as of biological chemosensory significance
[64], most (> 90%) chemosensory transcripts showed significantly higher expressions in the
antennae than in the head (Fig 5). Among these, significantly expressed chemosensory transcripts (p-value < 1e-20) in the antennae include several Obp (Lush, Obp19a, Obp28a,
Obp59a, Obp83a/b and Obp84a), Ir (Ir25a, Ir31a, Ir40a, Ir41a, Ir64a, Ir75a, Ir76b, Ir84a, Ir8a
and Ir92a), Or (Orco, Or7a, Or13a, Or43a, Or45a, Or47b, Or63a/c/d and Or85d), Gr (Gr21a),
Csp [Csp2 (a10) and Csp4 (Phk-3)] and Snmp1 orthologs. Specifically, most significantly
expressed transcripts were predominantly Obp orthologs. On the other hand, we identified a
subset of obp (Obp8a, Clumsy, Obp99c Obp83cd), Or (Or85e, Or71a), Grs (Gr2a, Gr28b) and
Csp4 (Phk-3) orthologs with significantly higher expression in the head than in the antennae
libraries.
Discussion
In this study, we profiled expansions of chemosensory gene orthologs among six tsetse fly species/subspecies (G. pallidipes, G. m. morsitans, G. austeni, G. p. gambiensis, G. f. fuscipes and G.
brevipalpis) and employed RNA-seq to discern differential expressions of the orthologs and
associated proteins in antennae and head tissues male G. m. morsitans. Our café analysis for
gene expansion revealed significant accelerated expansion of 4-methyl phenol and 4-propyl
phenol responsive Or71a [66] in G. m. morsitans. The 4-methyl phenol and 4-propyl phenol
are known G. m. morsitans and G. pallidipes attractants present in natural ox odor [17,67].
These findings probably account for the observed differential responses of these species to synthetic blends of these odors [68]. On the other hand, expansions of Ir75a,d, Ir31a, Ir84a and
Ir64a orthologs in G. m. morsitans suggest differential odor-tuning and responses to acetic
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Fig 3. MA plot showing abundantly and differentially expressed transcripts between the male G. m. morsitans head and antennae transcriptomes. Dots
indicate points-of-interest that display individual transcript abundance (x axis) and fold-change (y axis). Red dots indicate transcripts with fold-changes of
two or more (log2 � 1) and False Detection Rate (FDR) corrected p values of less than 0.05 (significant) between the head and antennae transcriptomes. Black
dots indicate transcripts with non-significant changes between the transcriptomes.
https://doi.org/10.1371/journal.pntd.0008341.g003
acid and 2-oxopentanoic acid in this species [69–73]. Acetic acid component of the vertebrate
breath is an attractant of most hematophagous vectors while 2-oxopentanoic acid elicit a landing response from Anopheles gambiae [74]. Whether there is enhanced attraction and landing
behavior in G. m. morsitans in the presence of these kairomones remains to be determined.
Expansion of Ir84a in G. m. morsitans may also indicate enhanced response to phenylacetaldehyde and male-male courtship [75] in this tsetse fly specie relative to the other species. Expansion of hunger responsive Obp83a ortholog [76] in G. m. morsitans suggest enhanced host
seeking persistence in this specie relative to the other species. The G. pallidipes appears to be
characterized by potentially muted responses to feeding stimulating hydroxycinnamic acids
linked to missing Or71a [77], but enhanced responses to butanol, 2-heptanone and ketones
lactones and phenolic compounds associated with the expanded Or49a [78,79], Or67a [80],
Or85f [81] and Or85c [82] orthologs. The responses to butanol, lactones, ketones and phenolic
compounds have been evaluated in development of baits used routinely in field control of G.
pallidipes. Carbon dioxide receptors Gr21a and Gr63a orthologs [65] were expanded in G. f.
fuscipes and most abundantly expressed in male G. m. morsitans antennae. These findings are
indicative of the heavier investment by G. f. fuscipes than other tsetse flies in carbon dioxide
detection and consequently host location [83]. The potential impact of the expansion (in G. f.
fuscipes) of the Ir21a required for cool avoidance behavior [84] is not clear, but may be tied to
the humid and warm habitat preference in the G. f. fuscipes lacustrine habitats. The Gr64a-f,
Gr5a and Gr43a sugar receptor orthologs [85,86] were conspicuously absent in tsetse flies, consistent with our previous finding [40], a phenomenon attributable to exclusive sugar deficient
blood diet in tsetse flies. The G. brevipaplis specific expansions of hygrosensory behavior mediating Ir68a ortholog [87] suggest potential behavioral responses to these and related odor cues
specific to this tsetse fly. We did not identify expansion of Or67d in tsetse flies, contrary to previous reports [39,40].
We identified several missing/absent or conserved tsetse fly species specific orthologs with
potential implications on respective tsetse species phenotypes. Absent Gr33a ortholog responsive to nonvolatile repulsive chemicals, including N,N-diethyl-meta-toluamide (DEET)
[88,89] in G. brevipalpis and marginal expression of Gr66a ortholog in male G. m. morsitans
antennae, suggest diminished responses in these species to some repellents. This phenomenon
is further supported by absence of another caffeine and DEET responsive Gr32a ortholog
[88,89] and courtship pheromone associated Gr68a ortholog [90] in G. brevipalpis and G. m.
morsitans. The missing Ir76b ortholog in four tsetse fly species (G. p. gambiensis, G. m. morsitans, G. pallidipes and G. brevipalpis) suggests that these tsetse species may have reduced
responses to Ir76b ortholog mediated feeding preferences for amino acids [73] relative to
remaining tsetse fly species. The conspicuous absence of Obp56a,d,e orthologs in tsetse flies,
point to possible reduction in their responses to the associated pheromones [91]. Geosmin
responsive Or56a ortholog [92] was most abundantly expressed Or after Orco in the G. m.
morsitans antennae. Since Geosmin is a microbial odorant that alerts flies of presence of harmful microbes and induces avoidance behavior [92], the findings suggest potential repellence of
tsetse flies by Geosmin and associated compounds, which can form a basis for a search for
tsetse fly specific repellents. Conserved Gr2a, Gr28b and Gr66a orthologs across most species
supports a notion of general aversion of salts [93], caffeine, DEET and some amino acids (theophylline, threonine and valine) [88,94–97] among the vectors. The Snmp1 ortholog
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Fig 4. Volcano plot showing abundantly and significantly expressed transcripts between the male G. m. morsitans
head and antennae transcriptomes. Dots indicate points-of-interest that display fold-changes (x axis) and statistical
significance (-log10 of p value, y axis) in transcripts between the head and antennae transcriptomes. Red dots indicate
transcripts with fold-changes of two or more (log2 � 1) and False Detection Rate (FDR) corrected p values of less than
0.05 and are indicate transcripts with significant changes between the transcriptomes. Black dots represent transcripts
with non-significant changes between the transcriptomes.
https://doi.org/10.1371/journal.pntd.0008341.g004
associated with detection of pheromones appears to be conserved across all the tsetse fly species, which in concert with similarly conserved Or67d and Orco orthologs, are functionally
associated with detection of lipid-derived pheromones [98,99]. Other conserved pheromone
responsive orthologs, include male-specific pheromone 11-cis-vaccenyl acetate (cVA)
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Fig 5. Volcano plot showing abundantly and significantly expressed chemosensory gene orthologs between the male G. m.
morsitans head and antennae transcriptomes. Dots indicate points-of-interest that display fold-changes (x axis) and statistical
significance (-log10 of p value, y axis) in transcripts between the head and antennae transcriptomes. Red dots indicate transcripts with
fold-changes of two or more (log2 � 1) and False Detection Rate (FDR) corrected p values of less than 0.05 and are indicate transcripts
with significant changes between the transcriptomes. Black dots represent transcripts with non-significant changes between the
transcriptomes.
https://doi.org/10.1371/journal.pntd.0008341.g005
responsive lush and Obp19a [100] (absent in G. austeni) and l-carvone, 2-heptanone and acetophenone responsive Obp83a [101]. Lush, Or67d, Or83c and Obp83a were predominantly
expressed in male G. m. morsitans antennae. We identified Ir93a ortholog in G. austeni contrary to previous findings [40]. Overall, we identified potential tsetse fly specific receptors and
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semiochemicals/ligands for downstream functional validations that can be employed to
expand the toolbox of tsetse fly attractants, repellents and regulators.
Our gene and pathway enrichment analyses suggest that male G. m. morsitans head and
antennae are predominately involved with vision and olfaction (odor sensing and clearing)
respectively. In addition to the classical and canonical olfaction pathways, we also established
fatty acid synthesis and associated xenobiotic responsive cytochrome P450 (Cyp6g1/2,
Cyp304a1) and Glutathione S transferase pathways preferentially enriched in the antennae. Similar observations have been made in cutworm moth (Agrotis ipsilon) antennae [102] and may
indicate significant investment in odor/pheromone clearing [103], probably as a strategy for
faster desensitization of antennae responses in the absence or disengagement with relevant cues.
Other enriched pathways and transcripts included lush, lush-like Obp19a, Obp28a and Obp83a/
b, Obp84a, Or7a and Snmp1 that are associated with responses to pheromones [91,104]. The
antennae transcriptome appears to be dominated with abundant, differentially expressed Ir75ac, Ir31a, Ir84a, Ir41a, Ir92a and Gr21a orthologs, functionally associated with responses to various odor cues including acetic acid, 2-oxopentanoic acid [70–72], pyridine, 1,4-diaminobutane,
cadaverine, spermidine, pyrrolidine [72], phenylacetyaldehyde [26], ammonia [20] and carbon
dioxide [65]. Some of the cues, such as butanol, carbon dioxide and acetic acid are documented
odor cues in the breath of the tsetse fly vertebrate hosts and are actively employed by tsetse fly in
host location [10,15], suggesting that the rest might perform similar functions in nature.
The antennae were also enriched with transcripts associated with cilium mechanoreceptors/locomotor activity, indicating possible significant role of antennae in the detection of
kinetic energy (energy of movement, e.g. touch, sound, vibration, changing pressure) or
potential energy (e.g. gravity) and hence guiding physical orientation of the fly. Stress induced
neuronal controlled growth and differentiation and regeneration pathways were also enriched
in the antennae, suggesting important role of the antennae in modulating responses of the fly
to fluctuations in oxygen levels, temperature and redox state [105]. In addition to vision gene,
the head was enriched with muscle and associated structural proteins, and energy mobilization
potentially associated with feeding, as well as neuropeptide regulations associated with modification of nervous and endocrine systems. Most differential and abundantly expressed head
specific chemosensory transcripts were also functionally associated with feeding. These
included Obp8a involved in food perception [106] and host location [107], and Gr28a/b and
Gr2a linked to regulation of aversion to high-salt associated diet [93]. Phenotypic roles of
other head-specific chemosensory transcripts, such as Csp2 (a10) and Csp4 (Phk-3), Clumsy,
Obp99c Obp83cd, Or85e, Or71a and Csp4 (Phk-3), remain to be elucidated. Other than vision,
olfaction and associated molecular processes, other processes appear to dominate physiological
and molecular functions in the head and antennae libraries, respectively, indicating other
functional roles of these tissues. Since these tissues (antennae and head) where extracted in the
morning, the transcriptional responses coincided with the peak activity of the tsetse flies and
hence reflect chemosensory and visual processes associated with host finding behavior predominant in that duration. Since our gene analyses were focused on antennae from male G. m.
morsitans, our gene expression results were potentially biased toward male tsetse flies and G.
m. morsitans subspecies. It would therefore be prudent to further assess for similar response in
the remaining five tsetse fly species/subspecies, both gender and at different physiological
states that influence their olfactory responses.
Conclusions
We identified tsetse fly specific chemosensory gene orthologs and their putative ligands, as
potential candidates for downstream functional genomic and field validations. The validations
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Expanded chemosensory gene orthologs among selected tsetse species
could yield new tsetse fly attractants, repellents and pheromones with potential in incremental
improvements of current tsetse fly control strategies. We also identified major sensory pathways and processes potentially active in the tsetse fly antennae and head that can be exploited
in modulating tsetse fly behavior.
Supporting information
S1 Fig. Principal Component Analysis (PCA)-based clustering of gene orthologs showing
differences in number of expanded/contracted orthologs between the six tsetse fly species.
(A) Clustering of chemosensory orthologs between tsetse species (B) Clustering of individual
orthologs within chemosensory gene families.
(TIF)
S1 Table. Counts of chemosensory gene orthologs among fruit fly (D. melanogaster) and
selected tsetse fly species.
(XLSX)
S2 Table. Canonical Gene-set Enrichment Analysis (GSEA) Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Panther and Reactome pathways, and ProteinProtein Interactions BIOGRID network statistics for the differentially expressed transcripts between male G. m. morsitans antennae and head transcriptomes.
(XLSX)
S3 Table. Annotations of most abundantly or significantly differentially expressed transcripts between male G. m. morsitans antennae and head transcriptomes.
(XLSX)
Acknowledgments
We are thankful to Ms. Yineng Wu for technical assistance during this study.
Author Contributions
Conceptualization: Paul O. Mireji.
Data curation: Paul O. Mireji.
Formal analysis: Clarence M. Mang’era, Paul O. Mireji.
Funding acquisition: Grace A. Murilla, Paul O. Mireji.
Investigation: Joy M. Kabaka, Paul O. Mireji.
Methodology: Paul O. Mireji.
Project administration: Sylvance O. Okoth, Grace A. Murilla, Paul O. Mireji.
Resources: Sylvance O. Okoth, Grace A. Murilla, Paul O. Mireji.
Software: Clarence M. Mang’era, Paul O. Mireji.
Supervision: Ahmed Hassanali, Paul O. Mireji.
Visualization: Clarence M. Mang’era, Paul O. Mireji.
Writing – original draft: Joy M. Kabaka, Paul O. Mireji.
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Expanded chemosensory gene orthologs among selected tsetse species
Writing – review & editing: Joy M. Kabaka, Benson M. Wachira, Clarence M. Mang’era, Martin K. Rono, Ahmed Hassanali, Sylvance O. Okoth, Vincent O. Oduol, Rosaline W.
Macharia, Grace A. Murilla, Paul O. Mireji.
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