Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae.

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete, with resistance levels reaching up to 132-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

Rapid identification of mosquito species and age by mass spectrometric analysis.

BACKGROUND: A rapid, accurate method to identify and to age-grade mosquito populations would be a major advance in predicting the risk of pathogen transmission and evaluating the public health impact of vector control interventions. Whilst other spectrometric or transcriptomic methods show promise, current approaches rely on challenging morphological techniques or simple binary classifications that cannot identify the subset of the population old enough to be infectious. In this study, the ability of rapid evaporative ionisation mass spectrometry (REIMS) to identify the species and age of mosquitoes reared in the laboratory and derived from the wild was investigated. RESULTS: The accuracy of REIMS in identifying morphologically identical species of the Anopheles gambiae complex exceeded 97% using principal component/linear discriminant analysis (PC-LDA) and 84% based on random forest analysis. Age separation into 3 different age categories (1 day, 5-6 days, 14-15 days) was achieved with 99% (PC-LDA) and 91% (random forest) accuracy. When tested on wild mosquitoes from the UK, REIMS data could determine the species and age of the specimens with accuracies of 91 and 90% respectively. CONCLUSIONS: The accuracy of REIMS to resolve the species and age of Anopheles mosquitoes is comparable to that achieved by infrared spectroscopy approaches. The processing time and ease of use represent significant advantages over current, dissection-based methods. Importantly, the accuracy was maintained when using wild mosquitoes reared under differing environmental conditions, and when mosquitoes were stored frozen or desiccated. This high throughput approach thus has potential to conduct rapid, real-time monitoring of vector populations, providing entomological evidence of the impact of alternative interventions.

In vivo functional validation of the V402L voltage gated sodium channel mutation in the malaria vector An. gambiae.

BACKGROUND: Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations. RESULTS: We identify the presence of the V402L-I1527T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso, at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast to L995F however, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions. CONCLUSION: The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening.

CRISPR/Cas9 modified An. gambiae carrying kdr mutation L1014F functionally validate its contribution in insecticide resistance and combined effect with metabolic enzymes.

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Bioassay and molecular monitoring of insecticide resistance status in Aedes albopictus populations from Greece, to support evidence-based vector control.

BACKGROUND: Aedes albopictus has a well-established presence in southern European countries, associated with recent disease outbreaks (e.g. chikungunya). Development of insecticide resistance in the vector is a major concern as its control mainly relies on the use of biocides. Data on the species' resistance status are essential for efficient and sustainable control. To date the insecticide resistance status of Ae. albopictus populations from Greece against major insecticides used in vector control remains largely unknown. METHODS: We investigated the insecticide resistance status of 19 Ae. albopictus populations from 11 regions of Greece. Bioassays were performed against diflubenzuron (DFB), Bacillus thuringiensis var. israelensis (Bti), deltamethrin and malathion. Known insecticide resistance loci were molecularly analysed, i.e. voltage-gated sodium channel (VGSC) mutations associated with pyrethroid resistance; presence and frequency of carboxylesterases 3 (CCEae3a) and 6 (CCEae6a) gene amplification associated with organophosphate (OP) resistance and; chitin synthase-1 (CHS-1) for the possible presence of DFB resistance mutations. RESULTS: Bioassays showed full susceptibility to DFB, Bti and deltamethrin, but resistance against the OP malathion (range of mortality: 55.30-91.40%). VGSC analysis revealed a widespread distribution of the mutations F1534C (in all populations, with allelic frequencies between 6.6-68.3%), and I1532T (in 6 populations; allelic frequencies below 22.70%), but absence of V1016G. CCE gene amplifications were recorded in 8 out of 11 populations (overall frequency: 33%). Co-presence of the F1534C mutation and CCEae3a amplification was reported in 39 of the 156 samples analysed by both assays. No mutations at the CHS-1 I1043 locus were detected. CONCLUSIONS: The results indicate: (i) the suitability of larvicides DFB and Bti for Ae. albopictus control in Greece; (ii) possible incipient pyrethroid resistance due to the presence of kdr mutations; and (iii) possible reduced efficacy of OPs, in a scenario of re-introducing them for vector control. The study highlights the need for systematic resistance monitoring for developing and implementing appropriate evidence-based control programmes.

Isolation and transcriptomic analysis of Anopheles gambiae oenocytes enables the delineation of hydrocarbon biosynthesis.

The surface of insects is coated in cuticular hydrocarbons (CHCs); variations in the composition of this layer affect a range of traits including adaptation to arid environments and defence against pathogens and toxins. In the African malaria vector, Anopheles gambiae quantitative and qualitative variance in CHC composition have been associated with speciation, ecological habitat and insecticide resistance. Understanding how these modifications arise will inform us of how mosquitoes are responding to climate change and vector control interventions. CHCs are synthesised in sub-epidermal cells called oenocytes that are very difficult to isolate from surrounding tissues. Here we utilise a transgenic line with fluorescent oenocytes to purify these cells for the first time. Comparative transcriptomics revealed the enrichment of biological processes related to long chain fatty acyl-CoA biosynthesis and elongation of mono-, poly-unsaturated and saturated fatty acids and enabled us to delineate, and partially validate, the hydrocarbon biosynthetic pathway in An. gambiae.

The bodies of insects are encased in an exoskeleton or cuticle that is key for their survival. The cuticle helps protect insects against damage, prevents water loss and can defend against pesticides. A better understanding of the role of the cuticle for survival in mosquitoes and other insects could lead to new ways to prevent the spread of diseases such as malaria. The cuticle is coated with various molecules from a group of chemicals called hydrocarbons. This coating is made by specialized cells called oenocytes and helps to protect insects. Hydrocarbons can also influence communications between certain insects by acting as recognition signals. In mosquitoes, oenocytes make several hydrocarbons using a set of processes that are not well understood, and the types of hydrocarbons they make can vary between individuals of the same species. It is unclear how this mixture of hydrocarbons is generated and how differences in the mixture can determine how mosquitoes adapt to their surroundings. Grigoraki et al. studied the genes that were active in isolated oenocytes from the mosquito Anopheles gambiae, which carries the parasite that causes malaria. The study revealed a set of genes which are highly active in oenocytes and control the production of fatty acids, a group of molecules used to make hydrocarbons. Other genes involved in creating hydrocarbons were also found. Grigoraki et al. further investigated a specific gene called FAS1899 and showed that loss of this gene reduces overall hydrocarbon production by 25%. Additionally, genes for transporting and recycling molecules and for producing fats were also shown to be active, which may indicate that oenocytes have a variety of unexplored roles besides making hydrocarbons. Grigoraki et al. identify the genes involved in producing the hydrocarbon coating of mosquitoes and demonstrate their significance. Further work is needed to understand the precise roles of each of these genes and how they are regulated to adapt the hydrocarbon coating to different situations. This can help explain how the hydrocarbon coating changes in mosquitoes, for example in response to the use of insecticides or climate change. This information is important to adapt and develop new tools to improve mosquito control.

Identification and detection of a novel point mutation in the Chitin Synthase gene of Culex pipiens associated with diflubenzuron resistance.

BACKGROUND: Diflubenzuron (DFB) is one of the most used insecticides in mosquito larval control including that of Culex pipiens, the proven vector of the recent West Nile Virus epidemics in Europe. Two mutations (I1043L and I1043M) in the chitin synthase (CHS) putative binding site of DFB have been previously reported in Cx. pipiens from Italy and associated with high levels of resistance against this larvicide. METHODOLOGY/PRINCIPAL FINDINGS: Here we report the identification of a third mutation at the same I1043 position of the CHS gene resulting in the substitution of Isoleucine to Phenylalanine (I1043F). This mutation has also been found in agricultural pests and has been functionally validated with genome editing in Drosophila, showing to confer striking levels (>15,000 fold) of DFB resistance. The frequency of the I1043F mutation was found to be substantially higher in Cx. pipiens mosquitoes surviving DFB doses largely exceeding the recommended field dose, raising concerns about the future efficient use of this insecticide. We monitored the presence and frequency of DFB mutations in Cx. pipiens mosquitoes from several Mediterranean countries, including Italy, France, Greece, Portugal and Israel. Among the Cx. pipiens populations collected in Northern Italy all but one had at least one of the three DFB mutations at allele frequencies reaching 93.3% for the I1043M, 64.8% for the I1043L and 10% for the I1043F. The newly reported I1043F mutation was also identified in two heterozygote individuals from France (4.2% allelic frequency). In contrast to Italy and France, no DFB resistant mutations were identified in the Cx. pipiens mosquitoes sampled from Greece, Portugal and Israel. CONCLUSIONS/SIGNIFICANCE: The findings of our study are of major concern for mosquito control programs in Europe, that rely on the use of a limited number of available larvicides, and highlight the necessity for the development of appropriate Insecticide Resistance Management (IRM) programs, to ensure the sustainable use of DFB.

Overexpression of an alternative allele of carboxyl/choline esterase 4 (CCE04) of Tetranychus urticae is associated with high levels of resistance to the keto-enol acaricide spirodiclofen.

BACKGROUND: Spirodiclofen is an acaricide that targets lipid biosynthesis by inhibiting acetyl-coenzyme A carboxylase. Spirodiclofen resistance in spider mites has been previously documented and was associated with overexpression of CYP392E10, a cytochrome P450 mono-oxygenase that metabolizes spirodiclofen. However, additional mechanisms have been suggested in several studies and a carboxyl/choline esterase gene, CCE04, was shown to be overexpressed in two genetically different strains, SR-VP and SR-TK, both exhibiting high spirodiclofen resistance levels. RESULTS: We identified two different CCE04 alleles in both resistant strains, CCE04SR-VP and CCE04London , with CCE04SR-VP being highly overexpressed. Isoelectric focusing analysis confirmed the overexpression of a single esterase isozyme, while copy number and random fragment length polymorphism analysis revealed that CCE04SR-VP overexpression was more likely due to selection for the CCE04SR-VP allele rather than gene amplification. Both CCE04 alleles were functionally expressed using the Pichia expression system. Functional enzyme assays revealed only limited kinetic differences between CCE04 isoforms for model substrates. In addition, inhibition/competition experiments with spirodiclofen suggested a similar interaction with both enzymes, whereas its active metabolite, spirodiclofen enol, did not inhibit enzyme activity. CONCLUSION: Our study suggests that selection with spirodiclofen results in enrichment of a specific allele of CCE04 (CCE04SR-VP ) in two genetically independent strains, which is highly overexpressed. Based on kinetic enzyme data, however, quantitative rather than qualitative differences between CCE04SR-VP and CCE04London seem more likely to be involved in resistance. Our findings are discussed in the light of a possible spirodiclofen resistance mechanism, with sequestration of spirodiclofen by CCE04SR-VP being a likely hypothesis. © 2019 Society of Chemical Industry.

Insect cuticle: a critical determinant of insecticide resistance.

Intense use of insecticides has resulted in the selection of extreme levels of resistance in insect populations. Therefore understanding the molecular basis of insecticide resistance mechanisms becomes critical. Penetration resistance refers to modifications in the cuticle that will eventually slow down the penetration of insecticide molecules within insects' body. So far, two mechanisms of penetration resistance have been described, the cuticle thickening and the altering of cuticle composition. Cuticular modifications are attributed to the over-expression of diversified genes or proteins, which belong to structural components (cuticular proteins mainly), enzymes that catalyze enzymatic reactions (CYP4G16 and laccase 2) or ABC transporters that promote cuticular translocation. In the present review we summarize recent studies and discuss future perspectives.

Author Correction: Striking diflubenzuron resistance in Culex pipiens, the prime vector of West Nile Virus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities.

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; kdr) pyrethroid resistance was detected in 2004 in Anopheles coluzzii (formerly known as the M form of the Anopheles gambiae complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that kdr alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in kdr frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan.

Striking diflubenzuron resistance in Culex pipiens, the prime vector of West Nile Virus.

Culex pipiens mosquitoes cause severe nuisance and transmit human diseases including West Nile. Vector control by insecticides is the main tool to prevent these diseases and diflubenzuron is one of the most effective mosquito larvicides used in many places. Here, high levels of resistance were identified in Cx. pipiens from Italy, with a Resistance Ratio of 128 fold. The phenotype was associated with mutations at amino acid I1043 (I1043M and I1043L) of the Chitin synthase gene, which showed significantly higher frequency in bioassay survivors. Both mutations have been introduced in the Drosophila melanogaster chitin synthase gene using the genome editing method CRISPR/Cas9 and validated to confer significant levels of resistance, although at different levels. The I→M mutation results in a Resistance Ratio >2,900 fold and the I→L mutation >20 fold. Two PCR based diagnostics were developed for monitoring of the resistant mutations in field populations. The findings are of major concern for public health given the importance of diflubenzuron in mosquito control in many places, the intensity of the resistance phenotype and the limited availability of alternative larvicides.

Insecticide resistance is mediated by multiple mechanisms in recently introduced Aedes aegypti from Madeira Island (Portugal).

BACKGROUND: Aedes aegypti is a major mosquito vector of arboviruses, including dengue, chikungunya and Zika. In 2005, Ae. aegypti was identified for the first time in Madeira Island. Despite an initial insecticide-based vector control program, the species expanded throughout the Southern coast of the island, suggesting the presence of insecticide resistance. Here, we characterized the insecticide resistance status and the underlying mechanisms of two populations of Ae. aegypti from Madeira Island, Funchal and Paúl do Mar. METHODOLOGY/PRINCIPAL FINDINGS: WHO susceptibility bioassays indicated resistance to cyfluthrin, permethrin, fenitrothion and bendiocarb. Use of synergists significantly increased mortality rates, and biochemical assays indicated elevated activities of detoxification enzymes, suggesting the importance of metabolic resistance. Microarray-based transcriptome analysis detected significant upregulation in both populations of nine cytochrome P450 oxidase genes (including four known pyrethroid metabolizing enzymes), the organophosphate metabolizer CCEae3a, Glutathione-S-transferases, and multiple putative cuticle proteins. Genotyping of knockdown resistance loci linked to pyrethroid resistance revealed fixation of the 1534C mutation, and presence with moderate frequencies of the V1016I mutation in each population. CONCLUSIONS/SIGNIFICANCE: Significant resistance to three major insecticide classes (pyrethroid, carbamate and organophosphate) is present in Ae. aegypti from Madeira Island, and appears to be mediated by multiple mechanisms. Implementation of appropriate resistance management strategies including rotation of insecticides with alternative modes of action, and methods other than chemical-based vector control are strongly advised to delay or reverse the spread of resistance and achieve efficient control.

Analysis of population structure and insecticide resistance in mosquitoes of the genus Culex, Anopheles and Aedes from different environments of Greece with a history of mosquito borne disease transmission.

Greece has been recently affected by several mosquito borne diseases with the West Nile Virus (WNV) outbreak in 2010 being one of the largest reported in Europe. Currently at the epicenter of an economic and refugee crisis and visited by over 16 million tourists a year the integrated management of diseases transmitted by mosquitoes is a public health and economic priority. Vector control programs rely mainly on insecticides, however data on insecticide resistance and the mosquito fauna is essential for successful applications. We determined the mosquito species composition and population dynamics in areas of increased vulnerability to vector borne disease transmission, as well as investigated the resistance status of major nuisance and disease vectors to insecticides. High mosquito densities were recorded in Thessaloniki and Evros, with Aedes caspius, a nuisance species, Culex pipiens, a known vector of WNV and Anopheles hyrcanus a potential vector of malaria being among the most prevalent species. Both vector species populations reached their peak in late summer. Aedes albopictus was recorded at high densities in Thessaloniki, but not in Evros. Notably, Cx. pipiens hybrids, which show an opportunistic biting behavior and are suspected to be involved in the transmission of the WNV, were recorded in considerable numbers in Thessaloniki and Attica. Culex pipiens and An. hyrcanus, but not Ae. caspius mosquitoes, showed moderate levels of resistance to deltamethrin. The presence of resistance in areas not exposed to vector control indicates that other factors could be selecting for resistance, i.e. pesticide applications for agriculture. Both L1014F and L101C kdr mutations were detected in Cx. pipiens populations. Anopheles hyrcanus resistance was not associated with mutations at the L1014 site. The Ace-1 mutations conferring insensitivity to organophosphates and carbamates were detected at low frequencies in all Cx. pipiens populations. Increased activity of P450s and esterases was found in Cx. pipiens individuals from Thessaloniki. Our study contributes evidence for sustainable and efficient vector control strategies and the prevention of disease outbreaks.

Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin.

BACKGROUND: Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. PRINCIPAL FINDINGS: Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. SIGNIFICANCE: The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies.

Functional and immunohistochemical characterization of CCEae3a, a carboxylesterase associated with temephos resistance in the major arbovirus vectors Aedes aegypti and Ae. albopictus.

Temephos is a major organophosphate (OP) larvicide that has been used extensively for the control of Aedes albopictus and Aedes aegypti, the major vectors for viral diseases, such as dengue fever, zika and chikungunya. Resistance to temephos has been recently detected and associated with the upregulation of carboxylesterases (CCEs) through gene amplification, in both species. Here, we expressed the CCEae3a genes which showed the most striking up-regulation in resistant Aedes strains, using the baculovirus system. All CCEae3a variants encoded functional enzymes, with high activity and preference for p-nitrophenyl butyrate, a substrate that was shown capable to differentiate temephos resistant from susceptible Aedes larvae. Enzyme kinetic studies showed that CCEae3as from both Ae. aegypti and Ae. albopictus (CCEae3a_aeg and CCEae3a_alb, respectively) strongly interact with temephos oxon and slowly released the OP molecule, indicating a sequestration resistance mechanism. No difference was detected between resistant and susceptible CCEae3a_aeg variants (CCEae3a_aegR and CCEae3a_aegS, respectively), indicating that previously reported polymorphism is unlikely to play a role in temephos resistance. HPLC/MS showed that CCEae3as were able to metabolize temephos oxon to the temephos monoester [(4-hydroxyphenyl) sulfanyl] phenyl O,O-dimethylphosphorothioate. Western blot and immunolocalization studies, based on a specific antibody raised against the CCEae3a_alb showed that the enzyme is expressed at higher levels in resistant insects, primarily in malpighian tubules (MT) and nerve tissues.

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus.

BACKGROUND: The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. PRINCIPAL FINDINGS: Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. SIGNIFICANCE: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.