Rapid Evaporative Ionization Mass Spectrometry (REIMS): a Potential and Rapid Tool for the Identification of Insecticide Resistance in Mosquito Larvae.

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain, and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionization mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cúcuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analyzed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin, and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% (±0.01) accuracy, between mosquitoes of field and lab origin with 89% (±0.01) accuracy and between susceptible and resistant larvae with 85% (±0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools.

The impact of pyrethroid resistance on the efficacy of insecticide-treated bed nets against African anopheline mosquitoes: systematic review and meta-analysis.

BACKGROUND: Pyrethroid insecticide-treated bed nets (ITNs) help contribute to reducing malaria deaths in Africa, but their efficacy is threatened by insecticide resistance in some malaria mosquito vectors. We therefore assessed the evidence that resistance is attenuating the effect of ITNs on entomological outcomes. METHODS AND FINDINGS: We included laboratory and field studies of African malaria vectors that measured resistance at the time of the study and used World Health Organization-recommended impregnation regimens. We reported mosquito mortality, blood feeding, induced exophily (premature exit of mosquitoes from the hut), deterrence, time to 50% or 95% knock-down, and percentage knock-down at 60 min. Publications were searched from 1 January 1980 to 31 December 2013 using MEDLINE, Cochrane Central Register of Controlled Trials, Science Citation Index Expanded, Social Sciences Citation Index, African Index Medicus, and CAB Abstracts. We stratified studies into three levels of insecticide resistance, and ITNs were compared with untreated bed nets (UTNs) using the risk difference (RD). Heterogeneity was explored visually and statistically. Included were 36 laboratory and 24 field studies, reported in 25 records. Studies tested and reported resistance inconsistently. Based on the meta-analytic results, the difference in mosquito mortality risk for ITNs compared to UTNs was lower in higher resistance categories. However, mortality risk was significantly higher for ITNs compared to UTNs regardless of resistance. For cone tests: low resistance, risk difference (RD) 0.86 (95% CI 0.72 to 1.01); moderate resistance, RD 0.71 (95% CI 0.53 to 0.88); high resistance, RD 0.56 (95% CI 0.17 to 0.95). For tunnel tests: low resistance, RD 0.74 (95% CI 0.61 to 0.87); moderate resistance, RD 0.50 (95% CI 0.40 to 0.60); high resistance, RD 0.39 (95% CI 0.24 to 0.54). For hut studies: low resistance, RD 0.56 (95% CI 0.43 to 0.68); moderate resistance, RD 0.39 (95% CI 0.16 to 0.61); high resistance, RD 0.35 (95% CI 0.27 to 0.43). However, with the exception of the moderate resistance category for tunnel tests, there was extremely high heterogeneity across studies in each resistance category (chi-squared test, p<0.00001, I² varied from 95% to 100%). CONCLUSIONS: This meta-analysis found that ITNs are more effective than UTNs regardless of resistance. There appears to be a relationship between resistance and the RD for mosquito mortality in laboratory and field studies. However, the substantive heterogeneity in the studies' results and design may mask the true relationship between resistance and the RD, and the results need to be interpreted with caution. Our analysis suggests the potential for cumulative meta-analysis in entomological trials, but further field research in this area will require specialists in the field to work together to improve the quality of trials, and to standardise designs, assessment, and reporting of both resistance and entomological outcomes.

Identifying permethrin resistance loci in malaria vectors by genetic mapping.

Identification of the major loci responsible for insecticide resistance in malaria vectors would aid the development and implementation of effective resistance management strategies, which are urgently needed to tackle the growing threat posed by resistance to the limited insecticides available for malaria control. Genome-wide association studies in the major malaria vector, Anopheles gambiae, have been hindered by the high degree of within-population structuring and very low levels of linkage disequilibrium hence we revisited the use of quantitative trait loci (QTL) mapping to study resistance phenotypes in this vector species. Earlier work, identified two major QTL associated with pyrethroid resistance in A. gambiae s.s. from East Africa using genetic crossing of laboratory-colonized resistant and susceptible strains. In this study, we report the results from genetic mapping of pyrethroid resistance in three isofemale pedigrees established from wild-caught female A. gambiae s.s. mosquitoes from Benin. We identified two QTL on chromosomes 2L and 3R in these field populations, in similar genomic locations to the QTL identified in laboratory strains. The relative merits of two alternative study designs are discussed and suggestions made for future genetic mapping studies of insecticide resistance in mosquitoes.

Expansive and Diverse Phenotypic Landscape of Field Aedes aegypti (Diptera: Culicidae) Larvae with Differential Susceptibility to Temephos: Beyond Metabolic Detoxification.

Arboviruses including dengue, Zika, and chikungunya are amongst the most significant public health concerns worldwide. Arbovirus control relies on the use of insecticides to control the vector mosquito Aedes aegypti (Linnaeus), the success of which is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of Ae. aegypti larvae from field populations of Ae. aegypti with differential susceptibility to temephos originating from two Colombian urban locations, Bello and Cúcuta, previously reported to have distinctive disease incidence, socioeconomics, and climate. We demonstrated that an exclusive field-to-lab (Ae. aegypti strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes including detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long noncoding RNAs, and chromatin modelling among the differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to temephos. The results from the sampling triangulation approach here contribute a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity, increasing the number of potential targets for the control of insecticide resistant mosquitoes and widening our knowledge base on the complex phenotypic network of the Ae. aegypti response to insecticides.

Can piperonyl butoxide enhance the efficacy of pyrethroids against pyrethroid-resistant Aedes aegypti?

BACKGROUND: Pyrethroid resistance can be considered the main threat to the continued control of many mosquito vectors of disease. Piperonyl butoxide (PBO) has been used as a synergist to help increase the efficacy of certain insecticides. This enhancement stems from its ability to inhibit two major metabolic enzyme systems, P450s and non-specific esterases, and to enhance cuticular penetration of the insecticide. OBJECTIVE: To compare the mortality of a characterized resistant Aedes aegypti strain, Nha Trang, from Vietnam and the susceptible laboratory strain Bora Bora on netting with the pyrethroid deltamethrin (DM) alone and in combination with PBO. METHODS: Resistance mechanisms were characterized using molecular and bioassay techniques; standard PCR was used to test for the kdr target site mutation. Potential genes conferring metabolic resistance to DM were identified with microarray analysis using the Ae. aegypti 'detox chip'. These data were analysed alongside results from WHO susceptibility tests. P450, CYP9J32, was significantly overexpressed in the DM-resistant strain compared with the susceptible Bora Bora strain. Another five genes involved with oxidative stress responses in mosquitoes were also significantly overexpressed. The Nha Trang strain was homozygous for two kdr mutations. WHO cone bioassays were used to investigate mortality with incorporated DM-treated nets with and without PBO. PBO used in combination with DM resulted in higher mortality than DM alone. CONCLUSION: Synergists may have an important role to play in the future design of vector control products in an era when alternatives to pyrethroids are scarce.

Climatic and socio-economic factors supporting the co-circulation of dengue, Zika and chikungunya in three different ecosystems in Colombia.

Dengue, Zika and chikungunya are diseases of global health significance caused by arboviruses and transmitted by the mosquito Aedes aegypti, which is of worldwide circulation. The arrival of the Zika and chikungunya viruses to South America increased the complexity of transmission and morbidity caused by these viruses co-circulating in the same vector mosquito species. Here we present an integrated analysis of the reported arbovirus cases between 2007 and 2017 and local climate and socio-economic profiles of three distinct Colombian municipalities (Bello, Cúcuta and Moniquirá). These locations were confirmed as three different ecosystems given their contrasted geographic, climatic and socio-economic profiles. Correlational analyses were conducted with both generalised linear models and generalised additive models for the geographical data. Average temperature, minimum temperature and wind speed were strongly correlated with disease incidence. The transmission of Zika during the 2016 epidemic appeared to decrease circulation of dengue in Cúcuta, an area of sustained high incidence of dengue. Socio-economic factors such as barriers to health and childhood services, inadequate sanitation and poor water supply suggested an unfavourable impact on the transmission of dengue, Zika and chikungunya in all three ecosystems. Socio-demographic influencers were also discussed including the influx of people to Cúcuta, fleeing political and economic instability from neighbouring Venezuela. Aedes aegypti is expanding its range and increasing the global threat of these diseases. It is therefore vital that we learn from the epidemiology of these arboviruses and translate it into an actionable local knowledge base. This is even more acute given the recent historical high of dengue cases in the Americas in 2019, preceding the COVID-19 pandemic, which is itself hampering mosquito control efforts.

A potential global surveillance tool for effective, low-cost sampling of invasive Aedes mosquito eggs from tyres using adhesive tape.

BACKGROUND: The international movement of used tyres is a major factor responsible for global introductions of Aedes invasive mosquitoes (AIMs) (Diptera: Culicidae) that are major disease vectors (e.g. dengue, Zika, chikungunya and yellow fever). Surveillance methods are restricted by expense, availability and efficiency to detect all life stages. Currently, no tested method exists to screen imported used tyres for eggs in diapause, the life stage most at risk from accidental introduction. Here we test the efficiency of adhesive tape as an affordable and readily available material to screen tyres for eggs, testing its effect on hatch rate, larval development, DNA amplification and structural damage on the egg surface. RESULTS: We demonstrated that the properties of adhesive tape can influence pick up of dormant eggs attached to dry surfaces. Tapes with high levels of adhesion, such as duct tape, removed eggs with high levels of efficiency (97% ± 3.14). Egg numbers collected from cleaned used tyres were found to explain larval hatch rate success well, particularly in subsequent larval to adult emergence experiments. The strength of this relationship decreased when we tested dirty tyres. Damage to the exochorion was observed following scanning electron microscopy (SEM), possibly resulting in the high variance in the observed model. We found that five days was the optimal time for eggs to remain on all tested tapes for maximum return on hatch rate success. Tape type did not inhibit amplification of DNA of eggs from three, five or ten days of exposure. Using this DNA, genotyping of AIMs was possible using species-specific markers. CONCLUSIONS: We demonstrated for the first time that adhesive tapes are effective at removing AIM eggs from tyres. We propose that this method could be a standardised tool for surveillance to provide public health authorities and researchers with an additional method to screen tyre cargo. We provide a screening protocol for this purpose. This method has a global applicability and in turn can lead to increased predictability of introductions and improve screening methods at high risk entry points.

Working towards a Co-Ordinated Approach to Invasive Mosquito Detection, Response and Control in the UK.

The United Kingdom (UK) has reported a single detection of the eggs of the invasive mosquito vector Aedes albopictus in each of the three years from 2016 to 2018, all in southeast England. Here, we report the detection of mosquito eggs on three occasions at two sites in London and southeast England in September 2019. Mosquito traps were deployed at 56 sites, in England, Scotland, Wales, and Northern Ireland, as part of a coordinated surveillance programme with local authorities, Edge Hill University, and government departments. Response to each detection was coordinated by Public Health England's (PHE) local health protection teams, with technical support from PHE's Medical Entomology group, and control conducted by the respective local authority. Control, including source reduction and larviciding, was conducted within a 300 metre radius of the positive site. The response followed a National Contingency Plan for Invasive Mosquitoes: Detection of Incursions. Although the response to these incidents was rapid and well co-ordinated, recommendations are made to further develop mosquito surveillance and control capability for the UK.

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies).

BACKGROUND: The yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood. RESULTS: The present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR. CONCLUSION: These results suggest that the high level of insecticide resistance found in Ae. aegypti mosquitoes from Martinique island is the consequence of both target-site and metabolic based resistance mechanisms. Insecticide resistance levels and associated mechanisms are discussed in relation with the environmental context of Martinique Island. These finding have important implications for dengue vector control in Martinique and emphasizes the need to develop new tools and strategies for maintaining an effective control of Aedes mosquito populations worldwide.

Quantitative trait loci mapping of genome regions controlling permethrin resistance in the mosquito Aedes aegypti.

The mosquito Aedes aegypti is the principal vector of dengue and yellow fever flaviviruses. Permethrin is an insecticide used to suppress Ae. aegypti adult populations but metabolic and target site resistance to pyrethroids has evolved in many locations worldwide. Quantitative trait loci (QTL) controlling permethrin survival in Ae. aegypti were mapped in an F(3) advanced intercross line. Parents came from a collection of mosquitoes from Isla Mujeres, México, that had been selected for permethrin resistance for two generations and a reference permethrin-susceptible strain originally from New Orleans. Following a 1-hr permethrin exposure, 439 F(3) adult mosquitoes were phenotyped as knockdown resistant, knocked down/recovered, or dead. For QTL mapping, single nucleotide polymorphisms (SNPs) were identified at 22 loci with potential antixenobiotic activity including genes encoding cytochrome P450s (CYP), esterases (EST), or glutathione transferases (GST) and at 12 previously mapped loci. Seven antixenobiotic genes mapped to chromosome I, six to chromosome II, and nine to chromosome III. Two QTL of major effect were detected on chromosome III. One corresponds with a SNP previously associated with permethrin resistance in the para sodium channel gene and the second with the CCEunk7o esterase marker. Additional QTL but of relatively minor effect were also found. These included two sex-linked QTL on chromosome I affecting knockdown and recovery and a QTL affecting survival and recovery. On chromosome II, one QTL affecting survival and a second affecting recovery were detected. The patterns confirm that mutations in the para gene cause target-site insensitivity and are the major source of permethrin resistance but that other genes dispersed throughout the genome contribute to recovery and survival of mosquitoes following permethrin exposure.

Expression of the cytochrome P450s, CYP6P3 and CYP6M2 are significantly elevated in multiple pyrethroid resistant populations of Anopheles gambiae s.s. from Southern Benin and Nigeria.

BACKGROUND: Insecticide resistance in Anopheles mosquitoes is threatening the success of malaria control programmes. This is particularly true in Benin where pyrethroid resistance has been linked to the failure of insecticide treated bed nets. The role of mutations in the insecticide target sites in conferring resistance has been clearly established. In this study, the contribution of other potential resistance mechanisms was investigated in Anopheles gambiae s.s. from a number of localities in Southern Benin and Nigeria. The mosquitoes were sampled from a variety of breeding sites in a preliminary attempt to investigate the role of contamination of mosquito breeding sites in selecting for resistance in adult mosquitoes. RESULTS: All mosquitoes sampled belonged to the M form of An. gambiae s.s. There were high levels of permethrin resistance in an agricultural area (Akron) and an urban area (Gbedjromede), low levels of resistance in mosquito samples from an oil contaminated site (Ojoo) and complete susceptibility in the rural Orogun location. The target site mutation kdrW was detected at high levels in two of the populations (Akron f = 0.86 and Gbedjromede f = 0.84) but was not detected in Ojoo or Orogun. Microarray analysis using the Anopheles gambiae detox chip identified two P450s, CYP6P3 and CYP6M2 up regulated in all three populations, the former was expressed at particularly high levels in the Akron (12.4-fold) and Ojoo (7.4-fold) populations compared to the susceptible population. Additional detoxification and redox genes were also over expressed in one or more populations including two cuticular pre-cursor genes which were elevated in two of the three resistant populations. CONCLUSION: Multiple resistance mechanisms incurred in the different breeding sites contribute to resistance to permethrin in Benin. The cytochrome P450 genes, CYP6P3 and CYP6M2 are upregulated in all three resistant populations analysed. Several additional potential resistance mechanisms were also identified that warrant further investigation. Metabolic genes were over expressed irrespective of the presence of kdr, the latter resistance mechanism being absent in one resistant population. The discovery that mosquitoes collected from different types of breeding sites display differing profiles of metabolic genes at the adult stage may reflect the influence of a range of xenobiotics on selecting for resistance in mosquitoes.

Cross-induction of detoxification genes by environmental xenobiotics and insecticides in the mosquito Aedes aegypti: impact on larval tolerance to chemical insecticides.

The effect of exposure of Aedes aegypti larvae to sub-lethal doses of the pyrethroid insecticide permethrin, the organophosphate temephos, the herbicide atrazine, the polycyclic aromatic hydrocarbon fluoranthene and the heavy metal copper on their subsequent tolerance to insecticides, detoxification enzyme activities and expression of detoxification genes was investigated. Bioassays revealed a moderate increase in larval tolerance to permethrin following exposure to fluoranthene and copper while larval tolerance to temephos increased moderately after exposure to atrazine, copper and permethrin. Cytochrome P450 monooxygenases activities were induced in larvae exposed to permethrin, fluoranthene and copper while glutathione S-transferase activities were induced after exposure to fluoranthene and repressed after exposure to copper. Microarray screening of the expression patterns of all detoxification genes following exposure to each xenobiotic with the Aedes Detox Chip identified multiple genes induced by xenobiotics and insecticides. Further expression studies using real-time quantitative PCR confirmed the induction of multiple CYP genes and one carboxylesterase gene by insecticides and xenobiotics. Overall, this study reveals the potential of xenobiotics found in polluted mosquito breeding sites to affect their tolerance to insecticides, possibly through the cross-induction of particular detoxification genes. Molecular mechanisms involved and impact on mosquito control strategies are discussed.

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.

A Point Mutation V419L in the Sodium Channel Gene from Natural Populations of Aedes aegypti Is Involved in Resistance to λ-Cyhalothrin in Colombia.

Resistance to pyrethroids in mosquitoes is mainly caused by target site insensitivity known as knockdown resistance (kdr). In this work, we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene in Aedes aegypti mosquitoes from three Colombian municipalities. A partial region coding for the sodium channel gene from resistant mosquitoes was sequenced, and a simple allele-specific PCR-based assay (AS-PCR) was used to analyze mutations at the population level. The previously reported mutations, V1016I and F1534C, were found with frequencies ranging from 0.04 to 0.41, and 0.56 to 0.71, respectively, in the three cities. Moreover, a novel mutation, at 419 codon (V419L), was found in Ae. aegypti populations from Bello, Riohacha and Villavicencio cities with allelic frequencies of 0.06, 0.36, and 0.46, respectively. Interestingly, the insecticide susceptibility assays showed that mosquitoes from Bello were susceptible to λ-cyhalothrin pyrethroid whilst those from Riohacha and Villavicencio were resistant. A positive association between V419L and V1016I mutations with λ-cyhalothrin resistance was established in Riohacha and Villavicencio. The frequency of the F1534C was high in the three populations, suggesting that this mutation could be conferring resistance to insecticides other than λ-cyhalothrin, particularly type I pyrethroids. Further studies are required to confirm this hypothesis.

Discovery of a single male Aedes aegypti (L.) in Merseyside, England.

BACKGROUND: The mosquito Aedes aegypti (L.) is found in tropical and sub-tropical regions where it is the major vector of dengue fever, yellow fever, chikungunya and more recently Zika virus. Given its importance as a vector of arboviruses and its propensity to be transported to new regions, the European Centre for Disease Prevention and Control (ECDC) has placed Ae. aegypti on a list of potentially invasive mosquito species. It was previously reported in the United Kingdom (UK) in 1865 and 1919 but did not establish on either occasion. It is now beginning to reappear in European countries and has been recorded in the Netherlands (not established) and Madeira (Portugal), as well as southern Russia, Georgia and Turkey. RESULTS: During summer 2014, a single male Ae. aegypti was captured during mosquito collections in north-western England using a sweep net. Morphological identification complimented by sequencing of the ITS2 rDNA, and cox1 mtDNA regions, confirmed the species. Following confirmation, a programme of targeted surveillance was implemented around the collection site by first identifying potential larval habitats in greenhouses, a cemetery, a farm and industrial units. Despite intensive surveillance around the location, no other Ae. aegypti specimens were collected using a combination of sweep netting, larval dipping, mosquito magnets, BG sentinel traps and ovitraps. All species collected were native to the UK. CONCLUSION: The finding of the single male Ae. aegypti, while significant, presents no apparent disease risk to public health, and the follow-up survey suggests that there was no established population. However, this report does highlight the need for vigilance and robust surveillance, and the requirement for procedures to be in place to investigate such findings.

Deltamethrin Resistance Mechanisms in Aedes aegypti Populations from Three French Overseas Territories Worldwide.

BACKGROUND: Aedes aegypti is a cosmopolite mosquito, vector of arboviruses. The worldwide studies of its insecticide resistance have demonstrated a strong loss of susceptibility to pyrethroids, the major class of insecticide used for vector control. French overseas territories such as French Guiana (South America), Guadeloupe islands (Lesser Antilles) as well as New Caledonia (Pacific Ocean), have encountered such resistance. METHODOLOGY/PRINCIPAL FINDINGS: We initiated a research program on the pyrethroid resistance in French Guiana, Guadeloupe and New Caledonia. Aedes aegypti populations were tested for their deltamethrin resistance level then screened by an improved microarray developed to specifically study metabolic resistance mechanisms. Cytochrome P450 genes were implicated in conferring resistance. CYP6BB2, CYP6M11, CYP6N12, CYP9J9, CYP9J10 and CCE3 genes were upregulated in the resistant populations and were common to other populations at a regional scale. The implication of these genes in resistance phenomenon is therefore strongly suggested. Other genes from detoxification pathways were also differentially regulated. Screening for target site mutations on the voltage-gated sodium channel gene demonstrated the presence of I1016 and C1534. CONCLUSION /SIGNIFICANCE: This study highlighted the presence of a common set of differentially up-regulated detoxifying genes, mainly cytochrome P450 genes in all three populations. GUA and GUY populations shared a higher number of those genes compared to CAL. Two kdr mutations well known to be associated to pyrethroid resistance were also detected in those two populations but not in CAL. Different selective pressures and genetic backgrounds can explain such differences. These results are also compared with those obtained from other parts of the world and are discussed in the context of integrative research on vector competence.

Underpinning sustainable vector control through informed insecticide resistance management.

BACKGROUND: There has been rapid scale-up of malaria vector control in the last ten years. Both of the primary control strategies, long-lasting pyrethroid treated nets and indoor residual spraying, rely on the use of a limited number of insecticides. Insecticide resistance, as measured by bioassay, has rapidly increased in prevalence and has come to the forefront as an issue that needs to be addressed to maintain the sustainability of malaria control and the drive to elimination. Zambia's programme reported high levels of resistance to the insecticides it used in 2010, and, as a result, increased its investment in resistance monitoring to support informed resistance management decisions. METHODOLOGY/PRINCIPAL FINDINGS: A country-wide survey on insecticide resistance in Zambian malaria vectors was performed using WHO bioassays to detect resistant phenotypes. Molecular techniques were used to detect target-site mutations and microarray to detect metabolic resistance mechanisms. Anopheles gambiae s.s. was resistant to pyrethroids, DDT and carbamates, with potential organophosphate resistance in one population. The resistant phenotypes were conferred by both target-site and metabolic mechanisms. Anopheles funestus s.s. was largely resistant to pyrethroids and carbamates, with potential resistance to DDT in two locations. The resistant phenotypes were conferred by elevated levels of cytochrome p450s. CONCLUSIONS/SIGNIFICANCE: Currently, the Zambia National Malaria Control Centre is using these results to inform their vector control strategy. The methods employed here can serve as a template to all malaria-endemic countries striving to create a sustainable insecticide resistance management plan.

Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti.

Mosquitoes are vectors of several major human diseases and their control is mainly based on the use of chemical insecticides. Resistance of mosquitoes to organochlorines, organophosphates, carbamates and pyrethroids led to a regain of interest for the use of neonicotinoid insecticides in vector control. The present study investigated the molecular basis of neonicotinoid resistance in the mosquito Aedes aegypti. A strain susceptible to insecticides was selected at the larval stage with imidacloprid. After eight generations of selection, larvae of the selected strain (Imida-R) showed a 5.4-fold increased tolerance to imidacloprid while adult tolerance level remained low. Imida-R larvae showed significant cross-tolerance to other neonicotinoids but not to pyrethroids, organophosphates and carbamates. Transcriptome profiling identified 344 and 108 genes differentially transcribed in larvae and adults of the Imida-R strain compared to the parental strain. Most of these genes encode detoxification enzymes, cuticle proteins, hexamerins as well as other proteins involved in cell metabolism. Among detoxification enzymes, cytochrome P450 monooxygenases (CYPs) and glucosyl/glucuronosyl transferases (UDPGTs) were over-represented. Bioassays with enzyme inhibitors and biochemical assays confirmed the contribution of P450s with an increased capacity of the Imida-R microsomes to metabolize imidacloprid in presence of NADPH. Comparison of substrate recognition sites and imidacloprid docking models of six CYP6s over-transcribed in the Imida-R strain together with Bemisia tabaci CYP6CM1vQ and Drosophila melanogaster CYP6G1, both able to metabolize imidacloprid, suggested that CYP6BB2 and CYP6N12 are good candidates for imidacloprid metabolism in Ae. aegypti. The present study revealed that imidacloprid tolerance in mosquitoes can arise after few generations of selection at the larval stage but does not lead to a significant tolerance of adults. As in other insects, P450-mediated insecticide metabolism appears to play a major role in imidacloprid tolerance in mosquitoes.

Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti.

BACKGROUND: The mosquito Aedes aegypti is one of the most important disease vectors because it transmits two major arboviruses, dengue and yellow fever, which cause significant global morbidity and mortality. Chemical insecticides form the cornerstone of vector control. The organophosphate temephos a larvicide recommended by WHO for controlling Ae. aegypti, however, resistance to this compound has been reported in many countries, including Brazil. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify genes implicated in metabolic resistance in an Ae. aegypti temephos resistant strain, named RecR, through microarray analysis. We utilized a custom 'Ae. aegypti detox chip' and validated microarray data through RT-PCR comparing susceptible and resistant individuals. In addition, we analyzed gene expression in 4(th) instar larvae from a reversed susceptible strain (RecRev), exposed and unexposed to temephos. The results obtained revealed a set of 13 and 6 genes significantly over expressed in resistant adult mosquitoes and larvae, respectively. One of these genes, the cytochrome P450 CYP6N12, was up-regulated in both stages. RT-PCR confirmed the microarray results and, additionally, showed no difference in gene expression between temephos exposed and unexposed RecRev mosquitoes. This suggested that the differences in the transcript profiles among the strains are heritable due to a selection process and are not caused by immediate insecticide exposure. Reversal of temephos resistance was demonstrated and, importantly, there was a positive correlation between a decrease in the resistance ratio and an accompanying decrease in the expression levels of previously over expressed genes. Some of the genes identified here have also been implicated in metabolic resistance in other mosquito species and insecticide resistant populations of Ae. aegypti. CONCLUSIONS/SIGNIFICANCE: The identification of gene expression signatures associated to insecticide resistance and their suppression could greatly aid the development of improved strategies of vector control.

Impact of glyphosate and benzo[a]pyrene on the tolerance of mosquito larvae to chemical insecticides. Role of detoxification genes in response to xenobiotics.

The effect of exposure of Aedes aegypti larvae for 72h to sub-lethal concentrations of the herbicide glyphosate and the polycyclic aromatic hydrocarbon benzo[a]pyrene on their subsequent tolerance to the chemical insecticides imidacloprid, permethrin and propoxur, detoxification enzyme activities and transcription of detoxification genes was investigated. Bioassays revealed a significant increase in larval tolerance to imidacloprid and permethrin following exposure to benzo[a]pyrene and glyphosate. Larval tolerance to propoxur increased moderately after exposure to benzo[a]pyrene while a minor increased tolerance was observed after exposure to glyphosate. Cytochrome P450 monooxygenases activities were strongly induced in larvae exposed to benzo[a]pyrene and moderately induced in larvae exposed to imidacloprid and glyphosate. Larval glutathione S-transferases activities were strongly induced after exposure to propoxur and moderately induced after exposure to benzo[a]pyrene and glyphosate. Larval esterase activities were considerably induced after exposure to propoxur but only slightly induced by other xenobiotics. Microarray screening of 290 detoxification genes following exposure to each xenobiotic with the DNA microarray Aedes Detox Chip identified multiple detoxification and red/ox genes induced by xenobiotics and insecticides. Further transcription studies using real-time quantitative RT-PCR confirmed the induction of multiple P450 genes, 1 carboxy/cholinelesterase gene and 2 red/ox genes by insecticides and xenobiotics. Overall, this study reveals the potential of benzo[a]pyrene and glyphosate to affect the tolerance of mosquito larvae to chemical insecticides, possibly through the cross-induction of particular genes encoding detoxification enzymes.