Pest Management Science
Pest Manag Sci 63:882–889 (2007)
Acaricide resistance and synergism
between permethrin and amitraz against
susceptible and resistant strains of Boophilus
microplus (Acari: Ixodidae)†
Andrew Y Li,1∗ Andrew C Chen,1 Robert J Miller,2 Ronald B Davey2 and
John E George1
1 USDA,
2 USDA,
ARS, Knipling-Bushland US Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX 78028, USA
ARS, Cattle Fever Tick Research Laboratory, 22675 N. Moorefield Road, Edinburg, TX 78541, USA
Abstract: The control of the southern cattle tick, Boophilus microplus (Canestrini), in Mexico and many other
countries relies on chemical acaricides. Boophilus microplus has developed resistance to all major classes of
acaricides in recent years. To gain a better understanding of the resistance and to develop resistance management
strategies that benefit both Mexican ranchers and USDA’s cattle fever tick eradication program (CFTEP), the
authors used larval bioassay techniques to determine levels of resistance to permethrin and amitraz and then
evaluated synergism between these two acaricides in one susceptible laboratory tick strain and four resistant
strains originating from Mexico and Brazil. To examine mechanisms of resistance to permethrin in these strains,
the frequency of a mutated sodium channel gene was determined using a PCR assay. The tick strains from
Mexico and Brazil demonstrated 49.4- to over 672.2-fold resistance to permethrin, and up to 94.5-fold resistance
to amitraz. While the San Roman strain from Mexico was the most permethrin-resistant strain, the Santa
Luiza strain from Brazil was the most amitraz-resistant strain. A significant correlation was found between the
permethrin resistance ratio and the allelic frequency of the sodium channel mutation. Significant synergism
between permethrin and amitraz was found when one acaricide was tested in the presence of another. Synergism
ratios ranged from 1.5 to 54.9 when amitraz was tested as a synergist for permethrin. Similar synergism ratios were
obtained when permethrin was tested as a synergist for amitraz. Permethrin caused virtually no mortality in the
San Roman strain, even at the highest concentration (3294 µg cm−2 ). Adding amitraz (11.0 µg cm−2 ) to permethrin
led to a dramatic increase in larval mortality, even at very low concentrations of permethrin.
2007 Society of Chemical Industry
Keywords: acaricide; synergism; permethrin; amitraz; cattle tick; sodium channel mutation
1 INTRODUCTION
The southern cattle tick, Boophilus microplus
(Canestrini), is a damaging ectoparasite of cattle and
the key vector of bovine babesioses (Texas fever) which
once devastated the US cattle industry.1,2 This pest
was eradicated from the southern United States in
the 1940s after an intensive eradication campaign that
lasted over three decades.1,3,4 The US Department of
Agriculture (USDA) has since maintained an active
cattle fever tick eradication program (CFTEP) along
the US–Mexican border to prevent the reintroduction of B. microplus via cattle exported to the USA
from Mexico, where B. microplus remains endemic
and continues to cause serious economic damage.
One critical component of the CFTEP is the systematic treatment of all cattle imported from Mexico
in total immersion vats charged with coumaphos, an
organophosphate (OP) acaricide, to eliminate ticks
that the cattle may carry. In Mexico, B. microplus
has developed resistance to coumaphos and other
acaricides in past decades owing to intensive use of
chemical acaricides.3,5 – 9 Resistance to OP acaricides
first developed in the 1980s in Mexico, and resistance
to pyrethroids emerged in the 1990s.5,10,11 Amitraz,
a formamidine acaricide, was introduced along with
pyrethroids to control OP-resistant ticks in Mexico in
1986.10,12 Initially, amitraz was not widely used owing
to its higher cost, but its use became more prevalent
and intensive after pyrethroid resistance was discovered in 1993.13 The first case of amitraz resistance
in B. microplus from Mexico was confirmed in 2001
at a ranch in the state of Tabasco.12 Presently, many
tick populations are resistant to multiple classes of
acaricides in Mexico.14
Different pesticide resistance management strategies, including rotation of pesticides, and mixtures of
∗
Correspondence to: Andrew Y Li, USDA, ARS, Knipling-Bushland US Livestock Insects Research Laboratory, 2700 Fredericksburg Road, Kerrville, TX
78028, USA
E-mail: Andrew.Li@ars.usda.gov
†
This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation by the USDA for
its use.
(Received 27 September 2006; revised version received 31 January 2007; accepted 15 March 2007)
Published online 30 July 2007; DOI: 10.1002/ps.1417
This article is a US Government work and is in the public domain in the USA. Pest Manag Sci 1526–498X/2007/$30.00
Resistance and synergism between permethrin and amitraz in B. microplus
pesticides or synergists, have been reported to be effective in controlling resistant insect pests.15 – 20 However,
acaricide rotation may no longer be a good option for
tick control in some areas of Mexico, as many tick populations have developed resistance to multiple classes
of acaricides, including OP, pyrethroid and amitraz.
Alternatively, there has been success in the application
of mixtures of insecticides to suppress insect pests that
are resistant to the application of a single insecticide.
For example, the whitefly, Bemisia tabaci Gennadius,
a serious pest of cotton, melons and vegetables in
Arizona, developed high levels of resistance to both
pyrethroid and OP insecticides as a result of heavy
use of such compounds.21 To overcome the resistance problem, mixtures of various insecticides with
different modes of action were evaluated to control
resistant white flies.19,22 An increase of over 1000-fold
in the toxicity of fenpropathrin, a pyrethroid, to B.
tabaci was observed when acephate, an OP insecticide,
was co-applied with the pyrethroid.19 The mixture
formulation of fenpropathrin and acephate provided
an effective control to highly resistant B. tabaci populations when it was adopted by cotton growers in
Arizona.22 Similarly, mixtures of OP and pyrethroid
insecticides have been shown to be effective in the
control of the cotton bollworm, Helicoverpa armigera
(Hübner), and the southern house mosquito, Culex
quinquefasciatus Say.20,23
Although OPs, pyrethroids and amitraz have now
been used widely to control B. microplus in Mexico,
the potential of the acaricide mixture strategy for
tick control has not yet been explored. Synergism
between pyrethroid and formamidine insecticides has
been previously reported in several insect species.24,25
It is unknown whether such synergism exists between
these two classes of acaricides in ticks. The authors
have maintained in the laboratory a susceptible
strain and several resistant strains of B. microplus
that originated from Mexico and Brazil. These tick
strains provided an opportunity for detailed studies
on resistance mechanisms and possible interaction
between different classes of acaricides in B. microplus.
The objectives of this study were to determine levels of
resistance to permethrin and amitraz in B. microplus,
correlate allelic frequencies of a sodium channel
mutation that confers pyrethroid resistance with levels
of resistance determined by bioassays and evaluate
synergism between permethrin and amitraz to assess
its usefulness in tick resistance management.
2 MATERIALS AND METHODS
2.1 Ticks
Five strains of B. microplus were used in this study.
The Muñoz strain was established at the USDA Cattle
Fever Tick Research Laboratory (CFTRL) in 1999
from an outbreak of B. microplus ticks in Zapata
County, Texas. The Muñoz strain was susceptible
to all major classes of acaricides tested, and therefore
was used as a susceptible reference strain to determine
Pest Manag Sci 63:882–889 (2007)
DOI: 10.1002/ps
the level of resistance in other tick strains.7,26,27 The
OP-resistant Pesqueria strain was collected in 2000 at
the US port of entry in Reynosa, Tamaulipas, Mexico,
by USDA Veterinary Service inspectors from cattle
originating in Pesqueria, Nuevo Leon, Mexico. The
Santa Luiza strain was an amitraz-resistant tick strain
collected from a ranch in Brazil, and was maintained
at the Mexican National Parasitology Laboratory,
Jiutepec, Morelos, Mexico, before being established at
CFTRL in Mission, Texas, in 2000. The OP-resistant
San Roman strain was collected from a ranch in
Champoton, Campeche, Mexico, and was established
at the CFTRL in 1998. The pyrethroid-resistant
San Felipe strain was collected from a ranch in the
state of Tamaulipas, Mexico, and was established
at the CFTRL in 1996. The San Felipe strain was
challenged with permethrin, the San Roman strain
with coumaphos, the Santa Luiza strain with amitraz
and the Pesqueria strain with diazinon to increase or
maintain resistance to the respective acaricides during
their laboratory colonization and maintenance.6 – 8 The
procedures for rearing ticks on cattle, maintaining
non-parasitic stages in the laboratory and challenging
larvae with acaricides were similar to those previously
described.28,29
2.2 Chemicals
Amitraz 125 g L−1 EC (Taktic ; NOR-AM Chemical
Company, Wilmington, DE) was used in this study.
Technical-grade permethrin [92.2% active ingredient
(AI); cis:trans ratio = 1:3] was obtained from FMC
(Philadelphia, PA). Three synergists used in this study,
triphenylphosphate (TPP) (an inhibitor of esterases),
piperonyl butoxide (PBO) (an inhibitor of cytochrome
P450 monooxygenases) and diethyl maleate (DEM)
(an inhibitor of glutathione-S-transferases), were
purchased from Aldrich (Milwaukee, WI).
2.3 Larval bioassays
A slightly modified version of the larval packet test
(LPT) recommended by FAO30 was used to determine
permethrin toxicity to tick larvae, levels of permethrin
resistance and the effect of synergists on permethrin
toxicity.6 Larvae that were 12–16 days old were used
for all bioassays. A stock solution of permethrin
was made by dissolving technical-grade permethrin
in trichloroethylene (Sigma, St Louis, MO). The top
concentration was prepared by adding a volume of
the stock solution to a mixture of trichloroethylene
and olive oil (Sigma) with a final 2:1 ratio. Serial
dilutions from the top concentration were made using
a diluent of two parts trichloroethylene and one
part oil. A volume of 0.7 mL of each dilution was
applied to a Whatman No. 1 filter paper (7.5 × 8.5
cm; Whatman, Maidstone, Kent, UK). Three filter
papers were prepared for each dilution. Treated filter
papers were placed in a fume hood for 2 h, to allow
trichloroethylene to evaporate, before being folded
in half and sealed with bulldog clips on both sides.
Approximately 100 larvae were placed into each
883
AY Li et al.
packet, and the top was sealed immediately with
another bulldog clip. Packets were then held in an
environmental chamber at 27 ± 2 ◦ C, 90% RH for
24 h. Packets were removed from the environmental
chamber, and mortality was determined by counting
live and dead larvae.
A modified FAO larval packet test (LPT)8,26,31 was
used for all amitraz bioassays in this study. Pieces
(7.5 × 8.5 cm) of nylon fabric (type 2320; Cerex
Advanced Fabrics, Pensacola, FL) were used as the
substrate instead of the Whatman filter papers.
2.4 Synergism study
When amitraz was evaluated as a synergist for
permethrin, the modified FAO bioassay technique
for permethrin was used. A concentration of amitraz
that would cause <25% mortality was first determined
with the filter paper bioassay technique for each tick
strain. The appropriate concentration of amitraz was
then used as the diluent to prepare serial dilutions
of permethrin for a particular tick strain. When
permethrin was evaluated as a synergist for amitraz, the
modified FAO bioassay technique with nylon fabric
as substrate for amitraz was used. A concentration
of permethrin that would cause little or low larval
mortality was first determined with the nylon fabric
bioassay technique for each tick strain. The permethrin
solution was then used as the diluent to prepare serial
dilutions of amitraz for a particular tick strain.
Effects of three synergists, TPP, PBO and DEM,
on permethrin toxicity to B. microplus larvae were
evaluated only in the San Roman strain by adding
one of the synergists to the diluent to achieve a
constant concentration of 109.8 µg cm−2 on substrate,
the highest concentration at which no larval mortality
in B. microplus was observed when applied alone
(unpublished data). Diluent with that constant
concentration of the synergist was used to make serial
dilutions of permethrin.
2.5 Detection of the sodium channel mutation
Larvae used to detect the sodium channel mutation
were either from the same generation when bioassays
were conducted or from generations immediately
before or after. Tick larvae were individually ground in
20 µL of 0.1× TE (Tris-EDTA, 0.1–1 mM, pH 8.0)
buffer in microcentrifuge tubes with polypropylene
pestles for approximately 30 s. A quantity of 100 µL
of Chelex (BioRad Laboratories, Hercules, CA; 5%
suspension in 0.1× TE buffer) was immediately
added, incubated for 15 min at 55 ◦ C, then at
room temperature for 30 min and finally centrifuged
for 5 min at 12 000 × g at room temperature. The
supernatant was removed as the DNA template
and stored at −20 ◦ C until use. For detection of a
T → A mutation conferring pyrethroid resistance in
B. microplus, a gel-based PCR assay was used.32,33
2.6 Data analysis
Probit analysis of dose–mortality data was performed
using POLO-PC.34 The resistance ratio (RR) was
calculated by dividing the LC50 (µg cm−2 ) of a
particular tick strain by the LC50 of the reference
Muñoz strain. The synergism ratio (SR) of one
acaricide caused by another acaricide was calculated by
dividing the LC50 of the bioassay using one acaricide
alone by the LC50 using the mixture of that acaricide
and the other acaricide (synergist). The difference
between LC50 estimates was designated as significant
if the 95% confidence intervals (CIs) did not overlap.
Mean mortalities at the same acaricide concentrations
with and without the presence of the other acaricide
or a synergist were compared with the t-test using the
JMP software.35
3 RESULTS
3.1 Resistance to permethrin
Results of probit analysis of concentration–mortality
data of permethrin with and without amitraz in the
five strains of B. microplus are summarized in Table 1.
Compared with the susceptible Muñoz strain, all
Mexican strains and the Brazilian strain (Santa Luiza)
had varying levels of resistance to permethrin, with
RRs ranging from 49.4 to >672.2. The San Roman
Table 1. Resistance to permethrin and synergism of permethrin toxicity to tick larvae by amitraz in various strains of Boophilus microplus
Tick strain
Muñoz
Pesqueria
Santa Luiza
San Felipe
San Roman
Acaricides
n
Slope (± SE)
χ 2 (df)
LC50 (µg cm−2 ) (CI)a
Permethrin
+ amitraz (0.4 µg cm−2 )
Permethrin
+ amitraz (2.8 µg cm−2 )
Permethrin
+ amitraz (11.0 µg cm−2 )
Permethrin
+ amitraz (1.1 µg cm−2 )
Permethrin
+ amitraz (11.0 µg cm−2 )
1639
1550
2467
2379
2164
1937
1410
1181
2435
2793
3.2(±0.2)
4.7(±0.3)
0.9(±0.1)
1.0(±0.1)
4.6(±0.2)
8.6(±0.4)
0.7(±0.1)
0.9(±0.1)
–
–
32.8 (19)
55.4 (16)
61.5 (19)
41.8 (19)
162.5 (6)
42.0 (15)
45.9 (19)
53.8 (19)
–
–
4.9 (4.4–5.5)
2.4 (2.1–2.7)∗
241.8 (190.3–338.2)
129.9 (89.2–179.7)∗
444.5 (382.8–601.1)
178.4 (168.8–188.1)∗
3125.8 (1639.5–8354.9)
2127.3 (1076.3–5150.7)
>3294.0
∼60.0∗
RRb
SRc
1.0
2.0
49.4
1.9
90.7
2.5
637.9
1.5
>672.2
∼54.9
a
CI = confidence interval.
RR = resistance ratio.
c
SR = synergism ratio.
∗
indicates significant difference from permethrin alone.
b
884
Pest Manag Sci 63:882–889 (2007)
DOI: 10.1002/ps
Resistance and synergism between permethrin and amitraz in B. microplus
strain demonstrated the highest permethrin resistance.
There was virtually no mortality when larvae were
exposed to the highest concentration (3294 µg cm−2 )
of permethrin used in the tick bioassay.
3.2 Frequency of the sodium channel mutation
Results of PCR analysis of tick larvae in all five strains
of B. microplus utilized are summarized in Table 2.
The susceptible Muñoz strain had few larvae carrying
a copy of the mutated gene. The allelic frequency of
the sodium channel mutation was the lowest (7.3%)
among all strains, and the mutated copy only appeared
in a heterozygous form. The San Roman strain had
the highest allelic frequency of the mutated gene
(100%), and all larvae tested were homozygous for
the mutation. The Pesqueria strain had a mutated
allelic frequency of 42.7%, and most appeared in the
heterozygous form. No copies of the mutated gene
were detected in the Santa Luiza strain, although
this tick strain demonstrated a 90.7-fold resistance to
permethrin. Overall, there was a significant correlation
between the LC50 estimates and the sodium channel
mutation frequencies (r 2 = 0.827, P < 0.05).
3.3 Effects of amitraz and other synergistic
compounds on permethrin toxicity
Amitraz alone, at a given concentration (see Table 1),
caused no mortality in the Muñoz strain, very low
mortality (2.4%) in the Santa Luiza strain and
higher mortalities in other strains (12.6%, 15.3%
and 22.1% in Pesqueria, San Felipe and San Roman
strains respectively). The addition of amitraz to
permethrin produced increased toxicity of permethrin
to tick larvae in all strains, with SRs ranging from
1.5 to >54.9. The most significant synergism of
permethrin toxicity by amitraz was observed in the
San Roman strain (Table 1). Permethrin alone, even
at the highest concentration used (3294 µg cm−2 ),
caused virtually no mortality in this strain. None
of the traditional synergists tested (TPP, PBO and
DEM) had any effect on permethrin toxicity in the
San Roman strain (data not shown). The inclusion
of amitraz (11 µg cm−2 ) in the permethrin bioassay
dramatically increased permethrin toxicity in the
San Roman strain. When PBO (109.8 µg cm−2 ) was
added to diluent containing amitraz (11 µg cm−2 )
for permethrin bioassay, further increase in tick
Table 2. Allelic frequency of the sodium channel mutation (scm)
detected with the PCR assay in various strains of Boophilus microplus
Genotype
Tick
strain
Muñoz
Pesqueria
Santa Luiza
San Felipe
San Roman
Allelic scm
Generation
n
SS
SR
RR
frequency (%)
f-19
f-14
f-13
f-41
f-30
48
48
48
48
48
41
10
48
4
0
7
35
0
11
0
0
3
0
33
48
7.3
42.7
0.0
80.2
100.0
Pest Manag Sci 63:882–889 (2007)
DOI: 10.1002/ps
Table 3. Effects of amitraz and PBO on permethrin toxicity in larvae of
the San Roman strain of Boophilus microplus
Mortality (%) (± SD)
Permethrin
concentration
(µg cm−2 )
Permethrin
Permethrin +
amitraza
Permethrin +
amitraza + PBOb
3294.0
1647.0
823.5
411.8
205.9
102.9
51.5
25.7
12.9
0
1.2(±1.4)
1.5(±2.6)
0
1.0(±1.7)
0
1.2(±2.0)
0
0
0
0
87.7(±8.6)
68.0(±17.7)
73.6(±6.0)
72.4(±23.0)
68.5(±11.8)
78.7(±5.1)
44.3(±13.1)
6.2(±2.6)
4.6(±2.7)
22.1(±7.3)
75.4(±9.4)
83.2(±15.1)
78.1(±1.4)
72.3(±13.3)
62.7(±15.5)
77.1(±13.0)
72.9(±7.2)∗
62.1(±4.7)∗
65.5(±7.1)∗
42.0(±31.5)∗
a
Amitraz concentration = 11.0 µg cm−2 .
PBO concentration = 109.8 µg cm−2 .
∗ indicates significant difference from other treatments in row (t-test,
P < 0.001).
b
mortality was observed, particularly at low permethrin
concentrations tested (12.9–51.5 µg cm−2 ) (Table 3).
3.4 Resistance to amitraz
The results of probit analysis of concentration–mortality data of amitraz with and without
permethrin in five strains of B. microplus included
in this study are summarized in Table 4. Compared
with the susceptible reference strain (Muñoz strain),
the San Roman and the San Felipe strains were slightly
less susceptible to amitraz (RR = 2.0 and 1.4 respectively), the Pesqueria strain was moderately resistant
(RR = 18.0) and the Santa Luiza strain was the most
resistant (RR = 94.5).
3.5 Effects of permethrin on amitraz toxicity
When permethrin was tested as a synergist of amitraz,
permethrin alone (2.2 µg cm−2 ) caused very high
(63%) mortality in the Muñoz strain, and therefore
the data were excluded. No bioassay was conducted
to test permethrin as a synergist for amitraz in the
San Roman strain. In other tick strains evaluated,
permethrin alone, at a given concentration (see
Table 4), caused very low mortality (2.5–3.1%) in
the Pesqueria and Santa Luiza strains, and a slightly
higher mortality (21.9%) in the San Felipe strain.
The addition of permethrin to amitraz resulted in
increased toxicity of amitraz to tick larvae in all
strains, with SRs ranging from 1.5 to 54.0. The
most dramatic synergism of amitraz toxicity by
permethrin was observed in the Santa Luiza strain
(Tables 4 and 5). Amitraz alone caused virtually
no mortality at concentrations of 6.9 µg cm−2 and
lower. When permethrin (109.8 µg cm−2 ) was added
to amitraz bioassay, the same amitraz concentration
(6.9 µg cm−2 ) produced 85.3% mortality.
885
AY Li et al.
Table 4. Resistance to amitraz and synergism of amitraz toxicity to tick larvae by permethrin in various strains of Boophilus microplus
Acaricides
n
Slope (± SE)
χ 2 (df)
LC50 (µg cm−2 ) (CI)a
Amitraz
Amitraz
Amitraz
+ permethrin (549 µg cm−2 )
Amitraz
+ permethrin (2.8 µg cm−2 )
Amitraz
+ permethrin (109.8 µg cm−2 )
2265
1969
1626
1465
1811
2110
2019
2660
1.6(±0.1)
1.9(±0.1)
2.9(±0.2)
1.6(±0.2)
1.9(±0.1)
2.1(±0.1)
2.2(±0.1)
1.1(±0.1)
311.0 (22)
54.2 (22)
33.6 (13)
53.0 (13)
43.3 (22)
68.0 (22)
181.6 (19)
162.9 (25)
0.4 (0.1–0.7)
0.8 (0.7–1.0)∗
0.6 (0.5–0.7)
0.1 (0.0–0.2)∗
7.2 (6.0–8.4)
4.9 (4.0–5.9)∗
37.8 (27.4–50.2)
0.7 (0.3–1.3)∗
Tick strain
Muñoz
San Roman
San Felipe
Pesquria
Santa Luiza
RRb
SRc
1.0
2.0
1.5
6.0
18.0
1.5
94.5
54.0
a
CI = confidence interval.
RR = resistance ratio.
c SR = synergism ratio.
∗ indicates significant difference from amitraz alone.
b
Table 5. Effects of permethrin on amitraz toxicity in larvae of the
Santa Luiza strain of Boophilus microplus
Mortality (%) (± SD)
Amitraz concentration
(µg cm−2 )
219.6
109.8
54.9
27.5
13.7
6.9
3.4
1.7
0.9
Permethrin only
0
Amitraz
Amitraz +
permethrina
100.0(±0.0)
88.8(±0.5)
57.7(±23.7)
39.6(±12.3)
28.6(±26.3)
2.6(±1.1)
5.7(±7.5)
3.4(±2.6)
–
–
0.3(±0.5)
100.0(±0.0)
98.5(±1.8)∗
98.3(±2.9)∗
96.3(±4.2)∗
94.0(±3.5)∗
85.3(±4.5)∗
60.4(±12.6)∗
61.8(±27.1)∗
70.5(±12.7)∗
3.1(±1.6)
1.5(±2.6)
a
Permethrin concentration = 109.8 µg cm−2 .
indicates significant difference from other treatment in row (t-test,
P < 0.001).
∗
4 DISCUSSION
Results from this and previous studies have shown
that resistance to pyrethroids and amitraz coexists in
the tick strains from Mexico and Brazil. Double or
possibly triple resistance to all three major classes
of acaricides (OPs, pyrethroids and amitraz) has
become increasingly prevalent in Mexico,7,8,14 and
mechanisms of resistance to these commonly used
acaricides have been extensively studied in recent
years.36,37 Both the sodium channel mutation and
metabolic detoxification mechanisms are known to be
responsible for pyrethroid resistance.32,36,37 Although
a significant correlation was found between the
permethrin resistance ratio and the allelic frequency of
the sodium channel mutation in this study, metabolic
mechanisms of resistance, instead of insensitive target
sites, were presumed to play a major role in Santa Luiza
strain owing to the lack of a sodium channel mutation.
Results of further synergist bioassays with TPP, PBO
and DEM suggest the existence of enhanced metabolic
detoxification mechanisms involving esterases and
mixed-function oxidases (Li et al., unpublished data).
The San Felipe strain was described previously by
886
Miller et al.6 as having >1000-fold resistance to
permethrin. He et al.32 identified a sodium channel
mutation in this and another highly pyrethroidresistant strain (Corrales). This led to the development
of a PCR assay for detecting this resistance gene.33
Since its laboratory colonization, this tick strain has
maintained a high level of pyrethroid resistance and a
high allelic frequency of the mutated sodium channel
gene (80.2%), similar to that previously reported.33
Challenges of larvae with permethrin in the laboratory
over the years failed to eliminate wild-type susceptible
allele from this tick strain. The failure to eliminate the
susceptible allele from this tick strain was likely caused
by the relatively low concentration (109.8 µg cm−2 )
of permethrin used, which was apparently sufficient
to maintain resistance level but not high enough to
eliminate all heterozygotes possessing the susceptible
allele.
The San Roman strain has been the most OPresistant tick strain and has been repeatedly challenged
to maintain its high level of resistance to coumaphos
since its laboratory colonization. Although this tick
strain has not been exposed to pyrethroids since its
collection from Mexico in 1998, it has maintained a
very high level of resistance to permethrin. The highest
level of permethrin resistance in the San Roman
strain is well supported by PCR data indicating 100%
frequency of homozygous mutant alleles (Table 2).
The total lack of synergism of permethrin toxicity
by the three traditional synergists (TPP, PBO and
DEM) suggests that metabolic detoxification is not
involved in permethrin resistance in this particular
tick strain, and a sodium channel mutation is the
sole mechanism conferring permethrin resistance. It is
likely that individual ticks collected from Mexico for
the establishment of the San Roman strain were all
homozygous resistant genotype (RR) for the sodium
channel mutation. The mutated gene was fixed and
remained stable in the colony even without pressure
of selection with permethrin during the entire period
of its laboratory colonization (>30 generations).
Compared with permethrin or amitraz alone,
enhanced toxicity by one acaricide was observed when
the other acaricide was added in both susceptible
and resistant tick strains. To test possible synergistic
Pest Manag Sci 63:882–889 (2007)
DOI: 10.1002/ps
Resistance and synergism between permethrin and amitraz in B. microplus
effects of mixtures of these two acaricides, only
concentrations of permethrin or amitraz that caused
relatively low mortalities (<25%) were used when it
was tested as a synergist.
It should be pointed out that probit analysis may
be problematic when the ‘control mortality’ was
higher than 10% in bioassays where one acaricide
was used as a ‘synergist’ for the other acaricide.
Also, because different concentrations of the ‘synergist
acaricide’ were used for different tick strains, it may
not be appropriate directly to compare the SRs of the
same acaricide among the tick strains. Nevertheless,
adding amitraz to permethrin or permethrin to amitraz
significantly increased the toxicity of permethrin or
amitraz alone to tick larvae in both susceptible and
resistant strains. Results from this study provide
positive confirmation of the synergism between
permethrin and amitraz in larvae of B. microplus.
Although the mode of action of pyrethroids is general knowledge, the mode of action of amitraz is not
well defined. It has been proposed that amitraz and
other formamidine pesticides exert their toxic effect on
pest species by binding to the octopamine receptor of
the central nervous system, and possibly also by inhibition of monoamine oxidases.38,39 Synergistic effects
of formamidines on pyrethroids have been previously
reported in the tobacco budworm, Heliothis virescens
F., and the housefly, Musca domestica L.40 – 42 Three
different mechanisms by which synergism between
formamidine and pyrethroid insecticides occurs have
been proposed for these insect species.42 – 45 Synergism of pyrethroids by chlordimeform, a formamidine,
in the tobacco budworm was thought to be caused
by enhanced uptake of pyrethroids as a result of
exposure to chlordimeform.43,44 The same mechanism was also suggested for the housefly, where
formamidine exposure of houseflies caused a 2.5-fold
increase in uptake of a radiolabeled pyrethroid.42 Liu
and Plapp45 conducted a further study on houseflies
with and without the kdr mutation, by measurements of radiolabeled Saxitoxin (STX) binding to
the nerve membrane sodium channel from untreated
and chlordimeform-treated flies. They found that
formamidines reduced the number of STX binding components and decreased the concentration at
which saturation of STX binding occurred. They concluded that formamidines act as target-site synergists
of pyrethroids by modifying binding cooperativity in
target tissues. The third mechanism was proposed
by Usmani et al.24 who reported that amitraz pretreatment of the bollworm, Helicoverpa zea (Boddie),
before treatment of third-instar larvae with permethrin
resulted in a decreased rate of pyrethroid metabolism
when compared with larvae treated with permethrin
alone. In vitro metabolism experiments also confirmed
that amitraz and some of its metabolites inhibited
degradation of permethrin.25
In the present study, the authors were unable to
determine the exact mechanisms by which synergism
between permethrin and amitraz occurred. However,
Pest Manag Sci 63:882–889 (2007)
DOI: 10.1002/ps
it was demonstrated that amitraz (11.0 µg cm−2 )
dramatically synergized permethrin toxicity in the
San Roman strain, the most permethrin-resistant
strain with 100% homozygous resistant genotype.
Larval mortality quickly reached a plateau (around
70–80%), but never reached 100% even at the
highest permethrin concentration tested (Table 3). A
similar pattern was also observed when permethrin
was tested as a synergist for amitraz (Table 5). When
PBO was added to the permethrin and amitraz
mixture, even higher tick mortalities were observed at
low permethrin concentrations (12.9–51.5 µg cm−2 )
(Table 3). PBO has been shown to synergize amitraz
toxicity to tick larvae,8 possibly by inhibiting oxidasebased detoxification of amitraz. Amitraz itself has
been shown to inhibit monoamine oxidase in B.
microplus,39 which was regarded as one possible mode
of action of amitraz. It is reasonable to suggest that
the synergism of permethrin and amitraz mixture was
further increased by PBO through PBO inhibition
of oxidase activity in tick larvae, leading to reduced
metabolism of amitraz and/or permethrin.
Synergism between permethrin and amitraz reported
in this study may be caused by one or more of the
three mechanisms proposed by previous researchers.
Regardless, the finding of synergism between permethrin and amitraz in tick larvae has significant
implications for the management of acaricide resistance in B. microplus. New formulations of permethrin
and amitraz mixture could be used to control tick
populations that are highly resistant to one or both
acaricides. However, the ratio of each component in
the mixture has to be further tested and determined on
the basis of field efficacy trials. The acaricide mixture
strategy should be approached with caution as it has
been demonstrated in some insects that targeted pests
can also develop resistance to insecticide mixtures.22
ACKNOWLEDGEMENTS
The authors thank Dave Krska and Michael Moses
for their excellent technical assistance in bioassays,
and other members of the USDA-ARS-CFTRL who
maintained tick strains and handled animals. Thanks
also to two anonymous reviewers for their critical
review of the manuscript, and to Drs Pia Untalan,
Simon Yu and Hunter White for reviewing an early
draft of this manuscript.
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