Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.     

Characterisation of DDT and pyrethroid resistance in Trinidad and Tobago populations of Aedes aegypti

Insecticide resistance is an important factor in the effectiveness of Aedes aegypti control and the related spread of dengue. The objectives of this study were to investigate the status of the organochlorine dichlorodiphenyltrichloroethane (DDT) and pyrethroid (permethrin and deltamethrin) resistance in Trinidad and Tobago populations of Ae. aegypti and the underlying biochemical mechanisms. Nine populations of Ae. aegypti larvae from Trinidad and Tobago were assayed to DDT and PYs using the Centers for Disease Control and Prevention (CDC) time-mortality-based bioassay method. A diagnostic dosage (DD) was established for each insecticide using the CAREC reference susceptible Ae. aegypti strain and a resistance threshold (RT), time in which 98-100% mortality was observed in the CAREC strain, was calculated for each insecticide. Mosquitoes which survived the DD and RT were considered as resistant, and the resistance status of each population was categorised based on the WHO criteria with mortality <80% indicative of resistance. Biochemical assays were conducted to determine the activities of α and β esterases, mixed function oxidases (MFO) and glutathione-S-transferases (GST) enzymes which are involved in resistance of mosquitoes to DDT and PYs. Enzymatic activity levels in each population were compared with those obtained for the CAREC susceptible strain, and significant differences were determined by Kruskal-Wallis and Tukey's non-parametric tests (P<0.05). The established DDs were 0.01 mg l(-1), 0.2 mg l(-1) and 1.0 mg l(-1) for deltamethrin, permethrin and DDT, respectively; and the RTs for deltamethrin, permethrin and DDT were 30, 75 and 120 min, respectively. All Ae. aegypti populations were resistant to DDT (<80% mortality); two strains were incipiently resistant to deltamethrin and three to permethrin (80-98% mortality). Biochemical assays revealed elevated levels of α-esterase and MFO enzymes in all Ae. aegypti populations. All, except three populations, showed increased levels of β-esterases; and all populations, except Curepe, demonstrated elevated GST levels.Metabolic detoxification of enzymes is correlated with the manifestation of DDT and PY resistance in Trinidad and Tobago populations of Ae. aegypti. The presence of this resistance also suggests that knock down (kdr)-type resistance may be involved, hence the need for further investigations. This information can contribute to the development of an insecticide resistance surveillance programme and improvement of resistance management strategies aimed at combatting the spread of dengue in Trinidad and Tobago.     

Comparison of the insecticide susceptibilities of laboratory strains of Aedes aegypti and Aedes albopictus

A susceptible strain of Aedes albopictus derived from the Gainesville strain (Florida, USA) was established in our laboratory. The larvicidal efficacies of the neurotoxic insecticides temephos, permethrin and the pure cis and trans-permethrin isomers and the microbial insecticide Bacillus thuringiensis israelensis (Bti) against Ae. albopictus were estimated and compared to a susceptible strain of Aedes aegypti. The larvicidal effect of insect growth regulator pyriproxyfen was also evaluated in both mosquito strains. The median lethal concentration/median emergency inhibition values for Ae. aegypti and Ae. albopictus, respectively, were: temephos, 3.058 and 6.632 ppb, permethrin, 3.143 and 4.933 ppb, cis-permethrin, 4.457 and 10.068 ppb, trans-permethrin, 1.510 and 3.883 ppb, Bti, 0.655 and 0.880 ppb and pyriproxyfen, 0.00774 and 0.01642 ppb. Ae. albopictus was more tolerant than Ae. aegypti to all six larvicides evaluated. The order of susceptibility for Ae. aegypti was pyriproxyfen > Bti > trans-permethrin > temephos > permethrin > cis-permethrin and for Ae. albopictus was pyriproxyfen > Bti > trans-permethrin > permethrin > temephos > cis-permethrin. Because both species can be found together in common urban, suburban and rural breeding sites, the results of this work provide baseline data on the susceptibility of Ae. albopictus to insecticides commonly used for controlling Ae. aegypti in the field.     

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.     

Determination of the resistance to organophosphate, carbamate, and pyrethroid insecticides in Panamanian Anopheles albimanus (Diptera: Culicidae) mosquitoes

Introduction. The susceptibility of Anopheles albimanus to organophosphates, carbamates and pyrethroid insecticides was unknown in the Panama communities of Aguas Claras, Pintupo and Puente Bayano, located in the Amerindian Reservation of Madungandi. This region is considered a malaria transmission area, where An. albimanus is the main vector. Objective. The resistance to organophosphate insecticides, carbamates and pyrethroids was evaluated in field populations of the Anopheles albimanus in Panama. Materials and methods. Progeny of An. albimanus collected in three localities in the indigenous Madugandi region were exposed to bioassays of susceptibility to organophosphate insecticides (fenitrothion, malathion and chlorpyrifos), the carbamate (propoxur) and pyrethroids (deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin). The protocols were in accordance with those established for adult mosquitoes by World Health Organization. Results. The three strains of the An. albimanus were resistant to the pyrethroid insecticides deltamethrin, lambdacyhalothrin, cyfluthrin and cypermethrin. Susceptibility remained for the organophosphate insecticides fenitrothion, malathion, chlorpyrifos, and the carbamate insecticide propoxur. Conclusion. The results provided important information to the vector control program, contributing to the application of new strategies on the use of insecticides, and thereby lengthening the life of the insecticide in use.     

Host feeding responses of Aedes aegypti (L.) exposed to deltamethrin

Escape responses of mated and unmated nulliparous Aedes aegypti mosquitoes were compared using three different concentrations of deltamethrin in the presence or absence of a live animal host using an excito-repellency (ER) test system. Both insecticide contact (excitation) and non-contact (repellency) test configurations were compared. For contact trials, mated mosquitoes showed similar escape movements among the three concentrations when host stimuli were absent. Significant differences in responses were seen between the lower concentrations of (LC(50) and LC(75) ) deltamethrin with and without hosts present (P<0.05). Presence or absence of host stimuli produced no significant differences in escape response for unmated females when exposed to the highest concentration (LC(90) ) of deltamethrin. Our findings indicate that as deltamethrin concentrations decrease to sublethal levels, mating status and host cues play a more significant role in escape behavior. Therefore, insemination can influence the outcome of feeding success and flight movement of nulliparous female Ae. aegypti in contact with deltamethrin and in the presence of live host stimuli. The ER assay system serves as a useful tool for observing excitation and repellency responses of Ae. aegypti to insecticides in the presence or absence of other environmental and biological cues that can affect mosquito behavior.     

Susceptibility and fitness cost of Aedes albopictus on their survivability after the exposure to the insecticide

The present study was undertaken to investigate the lethal and sub-lethal effects of insecticides on the life history of Aedes albopictus mosquitoes from urban and sub-urban areas in Penang, Malaysia. The female mosquitoes aged two to five days old were exposed to; (1) diagnostic dose (lethal dose) at 5% malathion and 0.75% permethrin; and (2) sub-lethal concentration of 1.5% malathion, and 0.2% permethrin, respectively and adult female survivors from sub-lethal concentrations were evaluated for fitness parameters. Fecundity, fertility, adult longevity, development time and survival of mosquitoes were the crucial point in their life history had studied. Aedes albopictus species from urban Sg. Dua strain has developed high resistance towards 0.75% permethrin and was confirmed on resistance after 24 h of reading. While sub-urban Batu Maung strain is still susceptible to both 5% malathion and 0.75% permethrin. At the sub-lethal dose, we discovered 0.2% permethrin insecticide have significantly more effects on the fitness cost of Ae. albopictus as compared to 1.5% malathion; with decreasing on fecundity, lesser time was needed to reach each development stages, and more male adult was emerged compared to female mosquitoes for both urban and sub-urban strains. Whereas, malathion insecticide only affected the number of eggs laid by the parent mosquitoes and the development time to reach adult stages. Even though, Ae. albopictus mosquitoes had developed resistance towards permethrin resulted in decreasing mortality, but subsequent effects on their fitness cost still continued on the first generation. Thus, it will benefit in reducing the transmission of mosquito-borne diseases.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti)

Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar‐insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose‐permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose‐permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar‐insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field‐collected strains was characterized by probit analysis of dosage‐response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage‐response patterns were similar, indicating that the sugar‐insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.     

Multiple insecticide resistance in Aedes aegypti (Diptera: Culicidae) populations compromises the effectiveness of dengue vector control in French Guiana

In French Guiana, pyrethroids and organophosphates have been used for many years against Aedes aegypti. We aimed to establish both the resistance level of Ae. aegypti and the ultra low volume spray efficacy to provide mosquito control services with practical information to implement vector control and resistance management. Resistance to deltamethrin and fenitrothion was observed. In addition, the profound loss of efficacy of AquaK'othrine? and the moderate loss of efficacy of Paluthion? 500 were recorded. Fenitrothion remained the most effective candidate for spatial application in French Guiana until its removal in December 2010. Further investigation of the mechanism of resistance to deltamethrin demonstrated the involvement of mixed-function oxidases and, to a lesser extent, of carboxylesterases. However, these observations alone cannot explain the level of insecticide resistance we observed during tube and cage tests.     

Resistance of Aedes aegypti from the state of São Paulo,Brazil, to organophosphates insecticides

Since the reintroduction of Aedes aegypti in the state of S o Paulo, in the middle of the 1980-decade, organophosphate insecticides are being used to control the dengue vector. In 1996, an annual program for monitoring the susceptibility of Ae. aegypti to the insecticides was implemented. Some of the results of this monitoring program are presented. Ae. aegypti populations from ten localities have been submitted to bioassays with the diagnostic dose of temephos and fenitrothion. Only two (Mar lia and Presidente Prudente) remain susceptible to both insecticides and one (Santos) exhibits true resistance. Ae. aegypti from the remaining localities showed an incipient altered susceptibility. Resistance ratios varied from 1.2 to 2.9 for temephos and from 1.5 to 3.2 to fenitrothion, indicating moderate levels of resistance. Biochemical assays did not detect alterations in the enzyme acetilcholinesterase, but indicated that resistance is associated with esterases.     

Impact of deltamethrin-resistance in Aedes albopictus on its fitness cost and vector competence

BackgroundAedes albopictus is one of the most invasive species in the world as well as the important vector for mosquito-borne diseases such as dengue fever, chikungunya fever and zika virus disease. Chemical control of mosquitoes is an effective method to control mosquito-borne diseases, however, the wide and improper application of insecticides for vector control has led to serious resistance problems. At present, there have been many reports on the resistance to pyrethroid insecticides in vector mosquitoes including deltamethrin to Aedes albopictus. However, the fitness cost and vector competence of deltamethrin resistant Aedes albopictus remain unknown. To understand the impact of insecticide resistant mosquito is of great significance for the prevention and control mosquitoes and mosquito-borne diseases.Methodology/Principal findingsA laboratory resistant strain (Lab-R) of Aedes albopictus was established by deltamethrin insecticide selecting from the laboratory susceptible strain (Lab-S). The life table between the two strains were comparatively analyzed. The average development time of Lab-R and Lab-S in larvae was 9.7 days and 8.2 days (P < 0.005), and in pupae was 2.0 days and 1.8 days respectively (P > 0.05), indicating that deltamethrin resistance prolongs the larval development time of resistant mosquitoes. The average survival time of resistant adults was significantly shorter than that of susceptible adults, while the body weight of resistant female adults was significantly higher than that of the susceptible females. We also compared the vector competence for dengue virus type-2 (DENV-2) between the two strains via RT-qPCR. Considering the results of infection rate (IR) and virus load, there was no difference between the two strains during the early period of infection (4, 7, 10 day post infection (dpi)). However, in the later period of infection (14 dpi), IR and virus load in heads, salivary glands and ovaries of the resistant mosquitoes were significantly lower than those of the susceptible strain (IR of heads, salivary glands and ovaries: P < 0.05; virus load in heads and salivary glands: P < 0.05; virus load in ovaries: P < 0.001). And then, fourteen days after the DENV-2-infectious blood meal, females of the susceptible and resistant strains were allow to bite 5-day-old suckling mice. Both stains of mosquito can transmit DENV-2 to mice, but the onset of viremia was later in the mice biting by resistant group as well as lower virus copies in serum and brains, suggesting that the horizontal transmission of the resistant strain is lower than the susceptible strain. Meanwhile, we also detected IR of egg pools of the two strains on 14 dpi and found that the resistant strain were less capable of vertical transmission than susceptible mosquitoes. In addition, the average survival time of the resistant females infected with DENV-2 was 16 days, which was the shortest among the four groups of female mosquitoes, suggesting that deltamethrin resistance would shorten the life span of female Aedes albopictus infected with DENV-2.Conclusions/SignificanceAs Aedes albopictus developing high resistance to deltamethrin, the resistance prolonged the growth and development of larvae, shorten the life span of adults, as well as reduced the vector competence of resistant Aedes albopictus for DENV-2. It can be concluded that the resistance to deltamethrin in Aedes albopictus is a double-edged sword, which not only endow the mosquito survive under the pressure of insecticide, but also increase the fitness cost and decrease its vector competence. However, Aedes albopictus resistant to deltamethrin can still complete the external incubation period and transmit dengue virus, which remains a potential vector for dengue virus transmission and becomes a threat to public health. Therefore, we should pay high attention for the problem of insecticide resistance so that to better prevent and control mosquito-borne diseases.     

Evaluation of boric acid as toxic sugar bait against resistant Aedes aegypti mosquitoes

Current methods of broad area application of contact insecticides used in mosquito control are becoming less effective, primarily due to resistance within mosquito populations. New methods that can deliver ingestible insecticides are being investigated as a means to mitigate resistance. This study evaluated insecticide delivery through toxic sugar baits (TSB) and resulting mortality of susceptible and resistant strains of Aedes aegypti. Two Ae. aegypti strains were evaluated using a 1% boric acid TSB: the susceptible Orlando 1952 (ORL) strain and the resistant Puerto Rican (PR) strain. The TSB resulted in high mortality for both ORL and PR strain of Ae. aegypti. Average mortality of female mosquitoes given TSB was 90.8% for PR and 99.3% for ORL. Our study suggests that targeting resistant mosquitoes with ingestible insecticides through TSBs could be a viable alternative to current mosquito control strategies and should be considered when developing an integrated vector management program.     

The role of olfaction in host preference by Aedes (Stegomyia) simpsoni and Ae. aegypti

Host preference in females of the mosquitoes Aedes simpsoni (Theo.) and Aedes aegypti (L) appears to be based largely on responses to specific odours at short range, with the site of olfaction being the antennae. In blank Y-olfactometer tests, 48 % of an anthropophilic (llobi) strain of Ae. aegypti responded to one arm and 52% to the other. However, presented with a man's hand in one arm of the olfacto-meter and a whole rat, Arvicanthis niloticus , in the other, 65% responded to man and 35% to the rat. With the same strain in a blank Gouck's type of olfactometer, the response was very poor, only 1 % of the mosquitoes in the olfactometer responded at all, again with no preference for either side. On the introduction of the same hosts into the Gouck's olfactometer, the total response was c. 31 %, of which 72 % responded to man and 28 % to the rat. Elimination of some visual cues affected neither the total response nor the preference. Removing one antenna, reduced the preference for man from about 70 % in the normal mosquitoes to about 58%. The total response remained approximately the same as in the normal mosquitoes. Removing both antennae, in both Ae. aegypti and Ae. simpsoni , caused a sharp depression in the total response and in host discrimination.     

The first report of Aedes (Stegomyia) albopictus in Haiti

Aedes albopictus was found in six of the 10 departments of Haiti and in 14 of the 35 communes surveyed. The survey found the larvae of Ae. albopictus in 13 different types of containers. Used tires and tins were by far the most common breeding sites used by this mosquito species. At the breeding sites, Ae. albopictus was associated with other mosquito species, such as Aedes aegypti, Culex nigripalpus and Aedes mediovittatus. The highest proportion of association was with Ae. aegypti. This study represents the first report of Ae. albopictus in Haiti.     

Laboratory testing of a lethal ovitrap for Aedes aegypti

Laboratory tests were conducted to determine the feasibility of making the mosquito ovitrap lethal to Aedes aegypti (L.) when they attempt to oviposit in the trap. Heavy-weight velour paper strips (2.54 x 11 cm) were used as an alternative to the wooden paddle normally provided as a substrate for mosquito oviposition. The paper strips were pretreated with insecticide solutions and allowed to dry before being used in oviposition cups of 473 ml capacity, filled with water initially to within 2.5 cm of the brim. Insecticides chosen for their quick knock-down efficacy were bendiocarb 76% WP (1.06 mg a.i./strip) and four pyrethroids: permethrin 25% WP (0.16 mg a.i./strip), deltamethin 4.75% SC (0.87 mg a.i./strip), cypermethrin 40% WP (2.81 mg a.i./strip), and cyfluthrin 20% WP (0.57 mg a.i./ strip). For experimental evaluation, two oviposition cups (one with an insecticide-treated strip and one with an untreated strip) were placed in cages (cubic 30 cm) with gravid female Ae. aegypti mosquitoes (aged 6-8 days) from a susceptible laboratory strain. Mortality-rates of female mosquitoes were 45% for bendiocarb, 47% for permethrin, 98% for deltamethrin, 100% for cypermethrin, and 100% for cyfluthrin. Young instar larvae added to the treated cups died within 2h. After water evaporation from the cups for 38 days, fresh mosquito females had access to previously submerged portions of the velour paper paddle, and mortality rates of 59% or more occurred. Cups that had water (360 ml) dripped into them, to simulate rain, produced female mosquito mortality rates of > 50% and all larvae died within 3 h of being added. These tests demonstrate that the ovitrap can be made lethal to both adults and larvae by insecticidal treatment of the ovistrip. Field efficacy trials are underway in Brazil to access the impact of this simple, low-cost, environmentally benign approach on populations of the dengue vector Ae. aegypti.     

Identification of Carboxylesterase Genes Implicated in Temephos Resistance in the Dengue Vector Aedes aegypti

Background

Thailand is currently experiencing one of its worst dengue outbreaks in decades. As in most countries where this disease is endemic, dengue control in Thailand is largely reliant on the use of insecticides targeting both immature and adult stages of the Aedes mosquito, with the organophosphate insecticide, temephos, being the insecticide of choice for attacking the mosquito larvae. Resistance to temephos was first detected in Aedes aegypti larvae in Thailand approximately 25 years ago but the mechanism responsible for this resistance has not been determined.

Principal Findings

Bioassays on Ae. aegypti larvae from Thailand detected temephos resistance ratios ranging from 3.5 fold in Chiang Mai to nearly 10 fold in Nakhon Sawan (NS) province. Synergist and biochemical assays suggested a role for increased carboxylesterase (CCE) activities in conferring temephos resistance in the NS population and microarray analysis revealed that the CCE gene, CCEae3a, was upregulated more than 60 fold in the NS population compared to the susceptible population. Upregulation of CCEae3a was shown to be partially due to gene duplication. Another CCE gene, CCEae6a, was also highly regulated in both comparisons. Sequencing and in silico structure prediction of CCEae3a showed that several amino acid polymorphisms in the NS population may also play a role in the increased resistance phenotype.

Significance

Carboxylesterases have previously been implicated in conferring temephos resistance in Ae aegypti but the specific member(s) of this family responsible for this phenotype have not been identified. The identification of a strong candidate is an important step in the development of new molecular diagnostic tools for management of temephos resistant populations and thus improved control of dengue.     

Genomic analysis of detoxification genes in the mosquito Aedes aegypti

Annotation of the recently determined genome sequence of the major dengue vector, Aedes aegypti, reveals an abundance of detoxification genes. Here, we report the presence of 235 members of the cytochrome P450, glutathione transferase and carboxy/cholinesterase families in Ae. aegypti. This gene count represents an increase of 58% and 36% compared with the fruitfly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, respectively. The expansion is not uniform within the gene families. Secure orthologs can be found across the insect species for enzymes that have presumed or proven biosynthetic or housekeeping roles. In contrast, subsets of these gene families that are associated with general xenobiotic detoxification, in particular the CYP6, CYP9 and alpha esterase families, have expanded in Ae. aegypti. In order to identify detoxification genes associated with resistance to insecticides we constructed an array containing unique oligonucleotide probes for these genes and compared their expression level in insecticide resistant and susceptible strains. Several candidate genes were identified with the majority belonging to two gene families, the CYP9 P450s and the Epsilon GSTs. This 'Ae. aegypti Detox Chip' will facilitate the implementation of insecticide resistance management strategies for arboviral control programmes.     

Changes in enzyme titres with age in four geographical strains of Aedes aegypti and their association with insecticide resistance

Abstract. The enzymes acetylcholinesterase, glutathione S-transferase (GST), glucose 6-phosphate dehydrogenase (G6PD), and general esterases were assayed in four strains of Aedes aegypti mosquitoes aged between 1 and 30 days. Microtitre plate methods were used to assay activity in the homogenates of individual mosquitoes. The levels of GST and G6PD declined with the age of the mosquitoes, while the activity for the other enzymes remained constant. Soluble protein content was also found to decline with mosquito age in all the strains. Insecticide bioassays showed that two strains (Trinidad and Virtudes) of Ae. aegypti were resistant to DDT, deltamethrin and malathion, whereas two other strains (Bangkok and Indian) were susceptible to all four classes of insecticides tested. Higher esterase activity levels in the resistant compared to the susceptible strains were assumed to be the cause of organophosphate resistance. The combination of DDT and deltamethrin resistance in two strains with normal GST and G6PD characteristics suggests that a kdr-type nerve insensitivity mechanism may be involved.