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277
Am. J. Trop. Med. Hyg., 83(2), 2010, pp. 277–284
doi:10.4269/ajtmh.2010.09-0623
Copyright © 2010 by The American Society of Tropical Medicine and Hygiene
INTRODUCTION
Aedes aegypti is a vector of several human pathogens
including the viruses responsible for dengue, yellow fever, and
chikungunya. This mosquito species has a cosmotropical dis-
tribution and is established in the majority, if not all, of the
countries in the Americas.
1 The Cayman Islands are located
in the western Caribbean, south of Cuba. The country con-
sists of three islands, Grand Cayman, Cayman Brac, and Little
Cayman with the majority of the population living in Grand
Cayman. Although Ae. aegypti is not considered endemic to
the Cayman Islands, this species has been continually present
in Grand Cayman since 2002, and occasional specimens have
been collected from Cayman Brac. There have been several
cases of imported dengue, but local transmission is very rare
with the only recorded case occurring in 2005. However, with
the vector established, the climatic conditions favorable, and
with frequent travel between the Cayman Islands and den-
gue endemic areas, there is an ever present risk of a dengue
outbreak. Therefore, like past introductions of this species, the
discovery of Ae. aegypti in 2002 stimulated an aggressive erad-
ication campaign by the Mosquito Research and Control Unit
(MRCU), an agency of the Cayman Islands Government. This
campaign has not achieved the level of success expected and
the reasons for this need to be explored.
The Dengue Prevention Campaign in Grand Cayman
focuses on monitoring the urban centers of George Town and
West Bay. Data are collected from a network of 670 ovipots,
which are supplemented by yard-to-yard surveys carried out
by crews who collect larval samples for identification. Crews
eliminate breeding sites by emptying any unnecessary sources
of standing water and treat those that remain with larvicide.
The organophosphate temephos, and insect growth regulator
methoprene, were used in rotation until late 2006 when teme-
phos was replaced with Bacillus thuringiensis israelensis ( Bti ).
In addition, yards with the greatest number of larval finds over
the course of the previous year are targeted for external resid-
ual wall treatment with lambda-cyhalothrin or bifenthrin used
in rotation. In cases of imported dengue fever, areas surround-
ing the homes of the patient are thermally fogged using per-
methrin to reduce adult numbers within the risk area.
In addition to the Dengue Prevention Campaign the MRCU
use an array of insecticides and control methods to reduce nui-
sance biting mosquitoes notably Ochlerotatus taeniorhynchus ,
the Salt Marsh Mosquito, that plagues the swamps that cover
over 50% of the islands. This currently involves three pre-
hatch campaigns annually in which temephos or methoprene
are applied aerially in rotation to large swamp areas to reduce
numbers of larvae when swamp levels rise caused by rain or
high tide. This is supplemented by aerial adulticiding with per-
methrin if unexpectedly high numbers of adult mosquitoes are
observed. There is also extensive private sector use of insec-
ticides with many homes employing pest control services or
using aerosols to control cockroaches, ants, termites, centi-
pedes, and scorpions.
Resistance to insecticides is common in Ae. aegypti . In the
Caribbean, resistance to DDT developed as early as 1955.
2
Organophosphate resistance is also widespread in the region
3– 7
and pyrethroid resistance has been reported in Puerto Rico,
8
Dominican Republic,
3 British Virgin Islands,
6 Cuba, 7 and
Martinique.
9 Two major mechanisms are thought to be largely
responsible for insecticide resistance: changes in the target site
or increases in the rates of insecticide detoxification. Both of
these mechanisms have been implicated in conferring resis-
tance to insecticides in Ae. aegypti. For example, elevated lev-
els of esterases have been associated with temephos resistance
in Trinidad,
10 British Virgin Islands,
6 and Cuba
7 and several
cytochrome P450 genes have been found over-expressed in
pyrethroid-resistant populations of Ae. aegypti . 11, 12 Multiple
substitutions in the target site of DDT and the pyrethroid
insecticides, the voltage-gated sodium channel on the insects’
neurones, have also been described,
9, 13, 14 often referred to as
kdr mutations (describing the knockdown resistance pheno-
type). However, only one of these, a valine to isoleucine sub-
stitution at codon 1016, has been clearly linked to insecticide
resistance.
13
Rising levels of insecticide resistance in the region combined
with strong Caribbean transport links, increased urbanization,
and heavy pesticide usage on the island make it imperative
that the MRCU take a proactive approach to insecticide resis-
tance monitoring and management. A pilot study in November
2006 found low levels of resistance to the organophosphate
temephos in Ae. aegypti in Grand Cayman and prompted a
change in larviciding policy to introduce Bti . Here, we report
the results of a larger survey of the insecticide resistance status
of the local Ae. aegypti population and describe the underlying
mechanisms responsible for this resistance.
Pyrethroid Resistance in Aedes aegypti from Grand Cayman
Angela F. Harris , Shavanthi Rajatileka , and Hilary Ranson *
The Mosquito Research and Control Unit, Grand Cayman, Cayman Islands, British West Indies; Vector Group,
Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, United Kingdom
Abstract. The Grand Cayman population of Aedes aegypti is highly resistant to DDT and pyrethroid insecticides.
Glutathione transferase, cytochrome P450, and esterase levels were increased in the Grand Cayman population relative to
a susceptible laboratory strain, but synergist studies did not implicate elevated insecticide detoxification as a major cause
of resistance. The role of target site resistance was therefore investigated. Two substitutions in the voltage-gated sodium
channel were identified, V1016I in domain II, segment 6 (IIS6) (allele frequency = 0.79) and F1534C in IIIS6 (allele fre-
quency = 0.68). The role of the F1534C mutation in conferring resistance to insecticides has not been previously estab-
lished and so a tetraplex polymerase chain reaction assay was designed and used to genotype mosquitoes that had been
exposed to insecticides. The F1534C mutation was strongly correlated with resistance to DDT and permethrin.
* Address correspondence to Hilary Ranson, Vector Group, Liverpool
School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA,
United Kingdom. E-mail: Hranson@liverpool.ac.uk
278 HARRIS AND OTHERS
MATERIALS AND METHODS
Mosquito strains. Aedes aegypti larvae were collected
from field surveillance sites in George Town and West Bay,
Grand Cayman in January 2008. The collections were pooled
and reared to adults in the insectary at the MCRU. The F
1
generation was used for insecticide bioassays and the F
2
generation for the biochemical assays. Two insecticide sus-
ceptible strains were used in the study: the Rockefeller strain,
an insecticide susceptible strain of Caribbean origin that has
been in colony since the early 1930s,
15 and the New Orleans
strain, originally colonized by the Centers for Disease Control
and Prevention (CDC).
Further larval field collections were made from West Bay,
George Town, and East End in February and March 2008.
These were reared to adults and then frozen for later molecu-
lar analysis.
Insecticide bioassays. Larval bioassays were performed
according to World Health Organization (WHO) guidelines
16 ;
briefly, 1 mL of temephos (Chemservice, West Chester, PA)
dissolved in ethanol was added to 249 mL distilled water
containing 25 third- to fourth-instar larvae. Five different
concentrations between 0.0015 and 0.06 mg/L temephos and
an ethanol-only control were tested in triplicate on different
days. Mortality was scored in each group over a 24-hour test
period. Mosquitoes with abnormal appearance or that were
unable to swim to the surface were counted as dead. Any
larvae that had pupated during the course of the experiment
were disregarded from the totals. The lethal concentration that
kills 50% (LC
50 ) values was calculated using Log dose Probit
(LdP) Line software (Ehabsoft, Cairo, Egypt ).
Adult bioassays were carried out on 1–3-day-old mosqui-
toes using WHO insecticide susceptibility test kits using papers
supplied by WHO: 4% DDT, 0.75% permethrin, 0.05% delta-
methrin, and 0.05% lambda-cyhalothrin. The exposure time
was varied to determine the Lethal Time that kills 50% of the
population (LT
50 ). Control assays, in which mosquitoes were
exposed to papers impregnated with carrier oil only, were
conducted in parallel. After exposure mosquitoes were trans-
ferred to a holding tube and supplied 10% sugar solution on
a cotton pad. Mortality was scored over a 24-hour test period;
LT
50 values were from log time versus probit mortality lines
generated using Log dose Probit (Ldp) line software.
The effect of pre-exposure to the synergist, piperonyl butox-
ide (PBO) on permethrin-induced mortality was also assessed.
Adult 1–3-day-old females were exposed to papers impreg-
nated with 4% PBO or to control papers and then immedi-
ately exposed to 0.75% permethrin for a further 2 hours using
WHO susceptibility test kits. Mortality was scored after 24
hours. Over 100 mosquitoes were used in each assay.
Biochemical assays. Esterase activities were measured
using the model substrates α- and β-naphthyl acetate and
para-nitrophenyl acetate (PNPA). Glutathione transferase
(GST) activity was measured using chlorodinitrobenzine
(CDNB). Cytochrome P450 levels were determined using
heme peroxidase and acetylcholinesterase activities were
determined, according to the methods described by Penilla.
17
Fifty individual, 3-day-old females from both the Cayman
strain and the New Orleans strain were used in each assay.
Protein levels were quantified using the QuantiPro BCA Assay
Kit (Sigma-Aldrich, St. Louis, MO) and the enzyme activities/
mg protein were calculated as in Penilla.
17 One-tailed Mann-
Whitney tests were used to compare the enzyme activities in
the Cayman and New Orleans strains.
Partial sequencing of the Ae. aegypti sodium channel. D N A
was extracted from individual mosquitoes using the method
of Livak.
18 The polymerase chain reaction (PCR) primer pairs
shown in Table 1 were designed to amplify four exons of the
voltage-gated sodium channel, exons 20, 21, 22, and 31, which
encode domain II subunit 4, 5, and 6, and domain III, subunit 6.
14
The PCR reactions were carried out in a volume of 25 μL
with final concentrations of 2.5 mM MgCl
2 , 0.2–0.4 mM each
dNTPs, 0.5 μM forward and reverse primers, 2.5 U Ta q poly-
merase , and 1% of the total genomic DNA extracted from a
single mosquito as template. Cycling conditions were as fol-
lows: for primer sets AaNa20 and AaNa21 initial denaturation
of 95°C for 5 min followed by 35 cycles of 94°C for 30 sec,
62°C for 30 sec, and 72°C for 1 min, then a final elongation
at 72°C for 10 min. For primer set AaNa31 conditions were
the same except the annealing temperature was 59°C. Cycling
conditions for the Ae2021a primers were 95°C for 5 min fol-
lowed by 35 cycles of 94°C for 30 sec, 60°C for 45 sec, and 72°C
for 2 min followed by a final elongation stage of 72°C for
7 min. The PCR products were visualized by gel electrophore-
sis and then sequenced directly by Macrogen, (Seoul, Korea).
The sequences were assembled and aligned using Lasergene
(DNAstar, Madison, WI ).
Kdr genotyping. The hot oligonucleotide ligation assay
(HOLA) method described in Rajatileka
19 was used to
genotype the Cayman Islands populations for the V1016I
mutation. A second amino acid substitution, F1534C, was
detected in the sequenced regions of the sodium channel of
Ae. aegypti from Grand Cayman and a tetra primer PCR assay
was designed to genotype mosquitoes at this locus ( Figure 1 ).
Table 1
Sequences of primers used for partial amplification of the Aedes aegypti sodium channel gene in the current study
Region amplified Primer name Sequence (5′–3′) Product size (bp)
Exon 20 AaNa20F CCCATTGCTGCCTAAACACT 321
AaNa20R CTTTTCGCAGTCGTTGATGA
Exon 21 AaNa21F AGACAATGTGGATCGCTTCC 175
AaNa21R CACTACGGTGGCCAAAAAGA
Exon 21 22 (including Intron) Ae2021aF
19 ATTGTATGCTTGTGGGTG 457
Ae2021aR
19 GCGTTGGCGATGTTC
Exon 31 AaNa31F GACTCGCGGGAGGTAAGTT 500
AaNa31R CCGTCTGCTTGTAGTGATCG
AaEx31P TCGCGGGAGGTAAGTTATTG 350
AaEx31Q GTTGATGTGCGATGGAAATG
AaEx31wt CCTCTACTTTGTGTTCTTCATCATCTT 231
AaEx31mut GCGTGAAGAACGACCCGC 163
279
PYRETHROID RESISTANCE IN AEDES AEGYPTI FROM GRAND CAYMAN
Figure 1. Diagnostic polymerase chain reaction (PCR) for F534C sodium channel mutation. Panel A: shows the partial sequence of the Aedes
aegypti sodium channel with the position of the primers used in the assay marked. Exonic regions are shown in grey with the amino acid translation
above the sequence data, boxed text indicates the position of the primers and the mutation detected in the Cayman population is indicated in black.
Panel B: shows a schematic of the tetraplex PCR assay indicating the expected product sizes. Panel C: provides an example of the results obtained.
Lane 1: contains a 100-bp ladder and lanes 2–7: contain PCR products obtained using template from a single mosquito. The amino acid sequence at
position 1534, as deduced by the results of this tetraplex assay and confirmed by sequencing, is indicated above each lane.
280 HARRIS AND OTHERS
Table 2
Larval bioassays with temephos *
Sample size
LC
50 mg/L
(95% upper and lower limits)
LC
90 mg/L
(95% upper and lower limits)
RR at the LC
50 vs.
Rockefeller strain
RR at the LC
50 vs.
NO strain
Rockefeller 2006 341 0.0059 (0.0054–0.0065) 0.011 (0.0099–0.013) – –
F
1 Cayman strain 2006 262 0.017 (0.015–0.02) 0.037 (0.031–0.045) 2.88 1.21
New Orleans 2008 315 0.014 (0.012–0.017) 0.045 (0.035–0.064)
F
1 Cayman strain 2008 427 0.023 (0.021–0.025) 0.043 (0.039–0.049) 3.89 1.64
* The Rockefeller and New Orleans strains are two long established laboratory insecticide susceptible strains that were used as controls in 2006 and 2008, respectively.
RR = resistance ratio.
Table 3
Bioassay results for New Orleans and Cayman population of Aedes
aegypti exposed to pyrethroid insecticides
Sample size LT
90 RR
Permethrin
(0.75%)
New Orleans 265 7 min
Cayman 331 3077 min 434
Deltamethrin
(0.05%)
New Orleans 88 6 min
Cayman 106 177 min 29
Lambda-cyhalothrin
(0.05%)
New Orleans 100 < 5 min * > 41.2
Cayman 143 206 min
* The New Orleans strain was killed very rapidly by lambda-cyhalothrin making it difficult
to calculate an accurate resistance ratio for this insecticide.
RR = resistance ratio.
In this assay, the flanking primers amplify a control band of 350
bp. Two internal allele-specific primers were designed to give
PCR products of either 231 bp (“wild-type” phenylalanine
allele) or 167 bp (“mutant” cysteine allele) by forming PCR
primer pairs with the flanking primers. Each PCR reaction
(25 μL) contained 2.5 mM MgCl
2 , 0.4 mM each dNTPs, 0.5
μM each primer, 2.5 U Ta q polymerase , and 1% of the total
genomic DNA extracted from a single mosquito as template
and the cycling conditions were 95°C for 5 min followed by 35
cycles of 94°C for 30 sec, 63°C for 30 sec, and 72°C for 30 sec,
and a final elongation at 72°C for 10 min. The PCR products
were resolved on a 2% agarose gel and a 100-bp ladder
(Hyperladder IV, Bioline, MA ) was used for sizing.
After validating this allele-specific PCR on templates of
known sequence, the assay was used to genotype 150 mosqui-
toes collected from Grand Cayman. An additional 200 mosqui-
toes that had been exposed to the LT
50 for permethrin or DDT
were also genotyped to test for genotype:phenotype associa-
tion (Fisher’s exact test). Tests for Hardy Weinberg equilib-
rium were performed using Genepop version 4.0.
20
RESULTS
Bioassays. A low level of resistance to temephos was detected
in field populations of Ae. aegypti from Grand Cayman in
2006 and this was the stimulus for the current study. In 2008,
the resistance level based on the LC
50 of the local population
had increased slightly from 0.017 to 0.023 mg/L, a 1.3-fold
increase ( P < 0.01), despite the withdrawal of temephos for
larviciding in Grand Cayman in 2006 ( Table 2 ). Calculations
of the resistance ratios for temephos are complicated by the
significant variations in the LC
50 of the two susceptible strains
( P < 0.01) (see Discussion).
Very high levels of resistance to DDT and pyrethroid insec-
ticides are present in Cayman Ae. aegypti . All of the Cayman
Ae. aegypti population survived 1 hour exposure to the WHO
pyrethroid impregnated papers. When comparing LT
90 times
for the Cayman versus the New Orleans strain the resistance
ratios (RR), are 434, 29, and > 41.2 for permethrin, deltame-
thrin, and lambda-cyhalothrin, respectively ( Table 3 ). The New
Orleans strain showed 86% mortality after 1 hour exposure to
DDT (100% after 75 min), whereas the Cayman strain was
able to withstand exposure in excess of 8 hours at which point
only 11% mortality was observed (data not shown). The very
low levels of mortality induced by DDT exposure precluded
an accurate determination of the RR for this insecticide.
Pre-exposure to the synergist piperonyl butoxide had no
significant effect on permethrin mortality ( P = 0.16) (data not
shown). The effect of PBO on DDT mortality was not assessed.
Biochemical assays. Elevated levels of esterases (with all
three substrates), cytochrome P450s, and GSTs were found
in the Cayman population compared with the susceptible
New Orleans strain ( Figure 2 ). The greatest increase was
observed in the esterase assays with median activity in the
Cayman strain 4.74, 3.57, and 3.97 times than the New Orleans
strain with PNPA, α-naphthol, and β-naphthol, respectively.
The corresponding fold changes for GST and, P450, are 1.98
and 2.63. A one-tailed Mann-Whitney test to determine the
significance of the increase in activity in each of these enzymes
results in P values of < 0.0001.
For the insensitive acetylcholine assay remaining AchE
activity was less than 30% for all individuals, suggesting that
this is not a major resistance mechanism in the Cayman Islands
population ( Figure 3 ). There was no significant difference in
the percentage of remaining AchE activity in the Cayman or
New Orleans strains ( P = 0.2453).
Kdr alleles. Partial DNA sequencing of the voltage-gated
sodium channel identified two amino acid substitutions in
the Cayman population compared with the susceptible New
Orleans strain. The first, a valine to isoleucine substitution
found at codon 1016, domain II, subunit 5, has been reported
elsewhere in Latin America
13 and shown to be associated with
resistance to pyrethroids. The second substitution was at codon
1534 where a single base pair substitution changes the codon
from TTC to TGC resulting in a phenylalanine to cysteine sub-
stitution in domain III, subunit 6 (note numbering of residues
is based on the reference sequence from Musca domestica , 21
exon assignment is based on the annotation of the Ae. aegypti
sodium channel gene in Chang
14 ). Given the importance
of this subunit in the binding of pyrethroid insecticides (see
below) we predicted that this amino acid substitution may be
associated with insecticide resistance. Hence, we developed
a new, simple, allele-specific PCR assay to screen for this
mutation in Ae. aegypti . The assay works on the same principles
as the assay developed by Martinez-Torres
22 for detecting the
L1014F kdr mutations in Anopheles gambiae and can readily
distinguish all three genotypes (SS, RS, and RR) ( Figure 1B ).
The new tetraplex PCR to detect F1534C and the HOLA
assay to detect V1016I were used to determine the frequency
281
PYRETHROID RESISTANCE IN AEDES AEGYPTI FROM GRAND CAYMAN
of these two substitutions in Grand Cayman. Fifty mosqui-
toes from three areas of the Island (East End, George Town,
and West Bay) were genotyped at both loci. The two loci were
in genotypic equilibrium. The overall frequency of the 1016I
allele was 0.79 ( Table 4 ). The East End and West Bay popu-
lation were in Hardy Weinberg equilibrium but the George
Town population had an excess of heterozygotes. The overall
frequency of the 1534C allele was 0.68. Significant deviations
from Hardy Weinberg equilibrium were observed in West Bay
only, which also had an excess of heterozygotes at this locus
( Table 4 ).
To determine the correlation between the genotypes at
codons 1016 and 1534 and resistance to insecticides, the off-
spring of adults reared from wild caught Ae. aegypti larvae were
exposed to either 4% DDT for 24 hours or 0.75% permethrin
for 2 hours and 50 surviving and 50 dead mosquitoes (for
codon 1016) or 100 surviving and dead (for codon 1534) were
genotyped ( Table 5 ). The 1016I mutation was positively associ-
ated with permethrin survival ( P = 0) but not survival to DDT
( P = 0.145). The 1534C mutation was strongly associated with
survival to both insecticides ( P = 0). Individuals homozygous
for both resistance alleles (1015I and 1534C) survived per-
methrin exposure, but this double homozygous genotype was
not associated with DDT survival.
DISCUSSION
The Ae. aegypti population in the Cayman Islands is highly
resistant to DDT and pyrethroid insecticides. The DDT resis-
tance was first reported in the Caribbean in the 1950s and
contributed to the failure of the Ae. aegypti eradication cam-
paign.
2, 23 Resistance to DDT persists in the region despite
the fact that the use of this insecticide for Aedes control was
largely phased out in the 1960s when organophosphate insec-
ticides became available. As discussed below, it is possible that
DDT resistance is being maintained in the population by selec-
tion with pyrethroid insecticides as both shares the same tar-
get site. The level of resistance to pyrethroids in the Cayman
Islands population is particularly high. The discriminating
doses for adult Ae. aegypti set by the WHO ( http://www.who
.int/whopes/resistance/en/ ) are a 1 hour exposure to 0.25% per-
methrin or 0.03% lambda-cyhalothrin (no discriminating dose
is set for deltamethrin for Ae. aegypti ). In this study. less than
80% mortality was observed after a 1 hour exposure to higher
concentrations of insecticide (0.75% permethrin and 0.05%
lambda-cyhalothrin) and hence the Cayman Islands population
Figure 2. Boxplots of results from biochemical assays. The median activity is shown by a horizontal bar; the box denotes the upper and lower
quartiles. The vertical lines show the full range of the data set. Panel A = GST assay using CDNB; B = esterase assay using PNPA; C = esterase assay
using α and β naphthol and D = P450 assay using heme peroxidase. Results are expressed as μmole/min/mg protein with the exception of the P450
assay, which is expressed as mg of cytochrome C equivalents/mg protein.
Figure 3. Histogram showing acetylcholinesterase activity in the
presence of propoxur. In both the New Orleans and Grand Cayman
populations, remaining AchE activity was less than 30% for all indi-
viduals, suggesting that insensitive acetylcholinesterase is not a major
resistance mechanism in the Cayman Islands population.
282 HARRIS AND OTHERS
would clearly be defined as pyrethroid resistant by WHO
standards. When compared with the susceptible New Orleans
strain, the resistance ratios of the Cayman Islands population
are 29- to 434-fold and these resistance levels are higher than
reported in neighboring islands in the Caribbean . For exam-
ple, resistance ratios of 4.7-fold to deltamethrin were reported
in Ae. aegypti from Cuba in 2001
7 and 35-fold resistance to
permethrin was recorded in a population from Martinique in
2003.
9 However, care should be taken when comparing resis-
tance ratios between different studies as the value obtained
will be dependent on the susceptible strain used. This can be
clearly seen in the results for the larval temephos bioassays in
the current study. If the resistance ratios obtained in 2006 and
2008 are compared, it appears that temephos resistance has
decreased after the cessation of use of this insecticide in the
Dengue Prevention Campaign. However, the actual LC
50 for
temephos increased in the Cayman Islands population between
2006 and 2008. Nevertheless, the Cayman Islands population
of Ae. aegypti is considerably more susceptible to temephos
(LC
50 0.023 mg/L) than populations from Cuba (LC
50 0.0713
mg/L
7 ), and British Virgin Islands (LC
50 0.0603 mg/L
6 ).
The biochemical assays indicate elevated levels of all three
of the major detoxification enzyme families in the Cayman
Islands population relative to the New Orleans strain. However,
pre-exposure to the synergist PBO, which acts as a general
inhibitor of cytochrome P450s and esterases,
24, 25 did not sig-
nificantly increase the level of permethrin-induced mortality.
This synergist data suggest that enhanced metabolism is not a
major cause of permethrin resistance in this population and it
is possible that the elevated levels of P450 observed may be
caused by differences between the Cayman and New Orleans
strains that are unrelated to their resistance status. Several
recent studies using the Ae. aegypti Detox chip have identi-
fied elevated expression of CYP9 P450s and Epsilon GSTs in
multiple pyrethroid-resistant strains
11 (Rajatileka and others,
unpublished data). The DDT resistance in Ae. aegypti is asso-
ciated with elevated activity of the Epsilon GST, GSTE2, and
this enzyme is very efficient at detoxifying this insecticide.
26
Further transcriptomic and metabolism studies are needed to
determine whether metabolic resistance is contributing to the
resistance phenotype in the Cayman Islands population.
This study provides evidence for the role of two sodium
channel mutations in conferring resistance to both DDT and/
or permethrin in Ae. aegypti . The first of these, a V1016I substi-
tution, in domain II, segment 6 (IIS6), has been reported previ-
ously in the Caribbean and was found at a high frequency (0.79)
in Grand Cayman. An alternative glycine substitution at this
position has been found in populations from South East Asia
but this was not present in the Cayman Islands population.
9, 19
The Cayman Islands population was fixed for the ATA codon
encoding isoleucine, at position 1011 and neither the valine or
methionine substitutions that have been detected in Ae. aegypti
from Latin American and Thai populations
9, 13, 19 were found.
The presence of the 1016I allele was significantly correlated
with survival to permethrin but not with DDT. The frequency
of this allele increases dramatically in response to selection
with pyrethroids in the laboratory
13 and a recent field study in
Mexico identified a rapid increase in frequency of this allele
in the last decade.
27 Models of the interaction of pyrethroid
and DDT insecticides with the sodium channel predict that
residues in the helices IIS5 and IIIS6 play a key role in bind-
ing of insecticides.
28 These regions of the sodium channel were
therefore amplified and sequenced from bioassay survivors
to search for any additional mutations that may be associated
with resistance to these insecticide classes. A substitution in
codon 1534 within IIIS6 from TTC to TGC, resulting in the
replacement of phenylalanine with cysteine, was detected and
a tetraplex PCR reaction was developed and used to assess
the correlation of this mutation with the resistance pheno-
type. All of the permethrin survivors and 46/49 DDT survivors
were homozygous for the cysteine allele. This allele is present
at a high frequency in the Cayman Islands population (allele
frequency = 0.68) and so the numbers of “wild-type” pheny-
lalanine homozygotes in the bioassayed individuals were low
( N = 7), but all of these were killed by insecticide exposure.
The 1534C allele is largely recessive with heterozygotes
being overwhelmingly found within the dead subset of the bio-
assayed mosquitoes. Not all cysteine homozygote individuals
survived insecticide exposure but it should be noted that for
DDT, mosquitoes were exposed to insecticide for 24 hours and
then held for a further 24 hours and hence some of this mortal-
ity may not be induced by insecticide exposure alone.
Table 5
Kdr genotypes and allele frequencies for Grand Cayman Aedes aegypti that survived or died after a 24-hour exposure to 4% DDT or a 2-hour
exposure to 0.75% permethrin *
1016 1534 Double homozygotes
V/V V/I I/I Freq I F/F F/C C/Cs Freq C V/V & F/F I/I & C/C
DDT Alive 0 9 10 0.76 P = 0.145 0 3 46 0.97 P = 0 0 9
Dead 0 16 7 0.65 3 20 27 0.74 0 6
Permethrin Alive 0 12 14 0.77 P = 0 0 0 50 1.0 P = 0 0 14
Dead 2 22 0 0.46 4 35 11 0.57 2 0
* Fisher’s exact test was used to test for correlation between genotype and phenotype.
Table 4
Genotypes and resistance allele frequencies of three Grand Cayman populations of Aedes aegypti for the V1016I and F1534C mutations *
Population
1016 1534
V/V V/I I/I Freq I P value F/F F/C C/C Freq C P value
East End 3 18 28 0.76 1.00 6 21 22 0.66 0.758
George Town 0 25 24 0.74 0.021 1 19 30 0.79 0.667
West Bay 0 9 32 0.89 1.0 1 36 9 0.59 0.000
Grand Cayman 3 52 84 0.79 8 76 61 0.68
* Tests for Hardy Weinberg Equilibrium were applied to the data and the P values are shown. The final row shows the combined analysis for all three populations.
283
PYRETHROID RESISTANCE IN AEDES AEGYPTI FROM GRAND CAYMAN
Several additional amino acid substitutions have been iden-
tified in the voltage-gated sodium channel of Ae. aegypti but for
the majority of these (G923V, L982W, I1011M, V1016G,
9 and
D1763Y
14 ), there is little evidence associating these mutations
with resistance. Hence, to date the only two sodium channel
mutations with a clear association with resistance to insecti-
cides are the 1016I and 1534C substitutions described in this
study. Preliminary screening of Ae. aegypti populations from
South East Asia indicate that the 1534C mutation has a wide-
spread geographical distribution (Rajatileka S, unpublished
data). Substitutions in an alternative phenylalanine residue in
IIIS6, F1538, have been associated with pyrethroid resistance
in the southern cattle tick, Boophilus microplus 29 and the
two-spotted spider mite, Tetranychus urticae . 30 Recently, site
directed mutagenesis has been used in an attempt to delin-
eate the role of residues in this helix in pyrethroid binding.
31
This study found that replacement of the F1538 residue
(referred to as F1518 in the Du study
31 ) with alanine almost
completely abolished pyrethroid binding. However, an alanine
replacement of F1534 had no effect. The substitution observed
at residue 1534 in the Cayman Islands Ae. aegypti population
replaces phenylalanine with a polar, hydrophilic cysteine, and
this may potentially have a more profound effect on the prop-
erties of the channel than an alanine substitution. In any case
the results from this study strongly suggest that this F1534C
substitution is very important in conferring resistance to pyre-
throid and DDT insecticides.
The high level of resistance in Ae. aegypti poses a signifi-
cant threat to the MRCUs Dengue Prevention Campaign.
It is not yet known whether the Ae. aegypti population that
arrived on the island in 2002 already contained the resis-
tance alleles detected in the current study or whether
resistance has arisen as a result of the intensive use of pyre-
throid insecticides by both the control program and house-
holders on the island. However, the high frequency of the
kdr alleles suggests that alternatives to pyrethroid insec-
ticides should be considered to control Ae. aegypti in the
Cayman Islands.
Received October 15, 2009. Accepted for publication April 19, 2010.
Financial support: This work was partially funded by Adapco, Bayer
Environmental Science and Central Life Sciences. We thank William
Petrie (Director, MRCU), Alan Wheeler and the staff at the MRCU,
Grand Cayman. Thanks also to Evangelia Morou and Patricia Penilla
for advice on the biochemical assays.
Authors’ addresses: Angela F. Harris, Mosquito Research and Control
Unit 99, Grand Cayman, Cayman Islands, E-mail: angela.harris@gov
.ky . Shavanthi Rajatileka and Hilary Ranson, Vector Group, Liverpool
School of Tropical Medicine, Pembroke Place, Liverpool, UK, E-mails:
Msc1sr@liv.ac.uk and Hranson@liverpool.ac.uk .
REFERENCES
1. Gubler DJ , 2002 . The global emergence/resurgence of arboviral
diseases as public health problems . Arch Med Res 33: 330 – 342 .
2. Brown AW , 1986 . Insecticide resistance in mosquitoes: a pragmatic
review . J Am Mosq Control Assoc 2: 123 – 140 .
3. Mekuria Y , Gwinn TA , Williams DC , Tidwell MA , 1991 . Insecticide
susceptibility of Aedes aegypti from Santo-Domingo,
Dominican-Republic. J Am Mosq Control Assoc 7: 69 – 72 .
4. Rawlins SC , Wan JO , 1995 . Resistance in some Caribbean popula-
tions of Aedes aegypti to several insecticides . J Am Mosq
Control Assoc 11: 59 – 65 .
5. Rawlins SC , 1998 . Spatial distribution of insecticide resistance in
Caribbean populations of Aedes aegypti and its significance .
Rev Panam Salud Publica 4: 243 – 251 .
6. Wirth MC , Georghiou GP , 1999 . Selection and characterization of
temephos resistance in a population of Aedes aegypti from
Tortola, British Virgin Islands. J Am Mosq Control Assoc 15:
315 – 320 .
7. Rodriguez MM , Bisset J , De Fernandez DM , Lauzan L , Soca A ,
2001 . Detection of insecticide resistance in Aedes aegypti
(Diptera: Culicidae) from Cuba and Venezuela . J Med Entomol
38: 623 – 628 .
8. Hemingway J , Boddington RG , Harris J , Dunbar SJ , 1989 .
Mechanisms of insecticide resistance in Aedes aegypti ( L. )
(Diptera, Culicidae) from Puerto-Rico. Bull Entomol Res 79:
123 – 130 .
9. Brengues C , Hawkes NJ , Chandre F , McCarroll L , Duchon S ,
Guillet P , Manguin S , Morgan JC , Hemingway J , 2003 . Pyrethroid
and DDT cross-resistance in Aedes aegypti is correlated with
novel mutations in the voltage-gated sodium channel gene .
Med Vet Entomol 17: 87 – 94 .
10. Vaughan A , Chadee DD , Ffrench-Constant R , 1998 . Biochemical
monitoring of organophosphorus and carbamate insecticide
resistance in Aedes aegypti mosquitoes from Trinidad . Med Vet
Entomol 12: 318 – 321 .
11. Strode C , Wondji CS , David JP , Hawkes NJ , Lumjuan N , Nelson
DR , Drane DR , Karunaratne S , Hemingway J , Black WC ,
Ranson H , 2008 . Genomic analysis of detoxification genes in
the mosquito Aedes aegypti . Insect Biochem Mol Biol 38:
113 – 123 .
12. Marcombe S , Poupardin R , Darriet F , Reynaud S , Bonnet J , Strode C ,
Brengues C , Yebakima A , Ranson H , Corbel V , David JP , 2009 .
Exploring the molecular basis of insecticide resistance in the
dengue vector Aedes aegypti : a case study in Martinique Island
(French West Indies) . BMC Genomics 10: 494 .
13. Saavedra-Rodriguez K , Urdaneta-Marquez L , Rajatileka S ,
Moulton M , Flores AE , Fernandez-Salas I , Bisset J , Rodriguez M ,
McCall PJ , Donnelly MJ , Ranson H , Hemingway J , Black WC ,
2007 . A mutation in the voltage-gated sodium channel gene
associated with pyrethroid resistance in Latin American Aedes
aegypti . Insect Mol Biol 16: 785 – 798 .
14. Chang C , Shen WK , Wang TT , Lin YH , Hsu EL , Dai SM , 2009 .
A novel amino acid substitution in a voltage-gated sodium
channel is associated with knockdown resistance to permethrin
in Aedes aegypti . Insect Biochem Mol Biol 39: 272 – 278 .
15. Coto MM , Lazcano JA , De Fernandez DM , Soca A , 2000 .
Malathion resistance in Aedes aegypti and Culex quinquefascia-
tus after its use in Aedes aegypti control programs . J Am Mosq
Control Assoc 16: 324 – 330 .
16. WHO , 2005 . Guidelines for Laboratory and Field Testing of
Mosquito Larvicides . WHO/CDS/WHOPES/GCDPP/2005.13.
17. Penilla RP , Rodriguez AD , Hemingway J , Torres JL , Arredondo-
Jimenez JI , Rodriguez MH , 1998 . Resistance management
strategies in malaria vector mosquito control. Baseline data for
a large-scale field trial against Anopheles albimanus in Mexico.
Med Vet Entomol 12: 217 – 233 .
18. Livak KJ , 1984 . Organization and mapping of a sequence on the
drosophila-melanogaster X-chromosome and Y-chromosome
that is transcribed during spermatogenesis . Genetics 107:
611 – 634 .
19. Rajatileka S , Black WC IV , Saavedra-Rodriguez K , Trongtokit Y ,
Apiwathnasorn C , McCall PJ , Ranson H , 2008 . Development
and application of a simple colorimetric assay reveals wide-
spread distribution of sodium channel mutations in Thai popu-
lations of Aedes aegypti . Acta Trop 108: 54 – 57 .
20. Rousset F , 2008 . GENEPOP’007: a complete re-implementation
of the GENEPOP software for Windows and Linux . Molecular
Ecology Resources 8: 103 – 106 .
21. Williamson MS , Martinez-Torres D , Hick CA , Devonshire AL ,
1996 . Identification of mutations in the housefly para-type
sodium channel gene associated with knockdown resistance
( kdr ) to pyrethroid insecticides . Mol Gen Genet 252: 51 – 60 .
22. Martinez-Torres D , Chandre F , Williamson MS , Darriet F , Berge
JB , Devonshire AL , Guillet P , Pasteur N , Pauron D , 1998 .
Molecular characterization of pyrethroid knockdown resis-
tance ( kdr ) in the major malaria vector Anopheles gambiae s.s .
Insect Mol Biol 7: 179 – 184 .
284 HARRIS AND OTHERS
23. Brown AW , Pal R , 1971 . Insecticide resistance in arthropods .
Public Health Pap 38: 1 – 491 .
24. Khot AC , Bingham G , Field LM , Moores GD , 2008 . A novel assay
reveals the blockade of esterases by piperonyl butoxide . Pest
Manag Sci 64: 1139 – 1142 .
25. Sun YP , Johnson ER , 1960 . Synergistic and antagonistic actions of
insecticide-synergist combinations and their mode of action .
J Agric Food Chem 8: 261 – 266 .
26. Lumjuan N , McCarroll L , Prapanthadara LA , Hemingway J ,
Ranson H , 2005 . Elevated activity of an Epsilon class glutathi-
one transferase confers DDT resistance in the dengue vector,
Aedes aegypti . Insect Biochem Mol Biol 35: 861 – 871 .
27. Ponce García G , Flores AE , Fernandez-Salas I , Saavedra-
Rodriguez K , Reyes-Solis G , Lozano-Fuentes S , Bond JG ,
Casas-Martínez M , Ramsay JM , García-Rejón J , Domínguez-
Galera M , Ranson H , Hemingway J , Eisen L , Black WC IV ,
2009 . Recent rapid rise of a permethrin knock down resistance
allele in Aedes aegypti in Mexico . PLoS Negl Trop Dis 3: e531 .
28. O’Reilly AO , Khambay BP , Williamson MS , Field LM , Wallace
BA , Davies TG , 2006 . Modelling insecticide-binding sites
in the voltage-gated sodium channel . Biochem J 396:
255 – 263 .
29. He HQ , Chen AC , Davey RB , Ivie GW , George JE , 1999 .
Identification of a point mutation in the para-type sodium
channel gene from a pyrethroid-resistant cattle tick . Biochem
Biophys Res Commun 261: 558 – 561 .
30. Tsagkarakou A , Van Leeuwen T , Khajehali J , Ilias A , Grispou M ,
Williamson MS , Tirry L , Vontas J , 2009 . Identification of pyre-
throid resistance associated mutations in the para sodium chan-
nel of the two-spotted spider mite Tetranychus urticae (Acari:
Tetranychidae) . Insect Mol Biol 18: 583 – 593 .
31. Du Y , Lee JE , Nomura Y , Zhang TX , Zhorov BS , Dong K , 2009 .
Identification of a cluster of residues in transmembrane seg-
ment 6 of domain III of the cockroach sodium channel essen-
tial for the action of pyrethroid insecticides . Biochem J 419:
377 – 385 .