A sensory appendage protein protects malaria vectors from pyrethroids.

Pyrethroid-impregnated bed nets have driven considerable reductions in malaria-associated morbidity and mortality in Africa since the beginning of the century1. The intense selection pressure exerted by bed nets has precipitated widespread and escalating resistance to pyrethroids in African Anopheles populations, threatening to reverse the gains that been made by malaria control2. Here we show that expression of a sensory appendage protein (SAP2), which is enriched in the legs, confers pyrethroid resistance to Anopheles gambiae. Expression of SAP2 is increased in insecticide-resistant populations and is further induced after the mosquito comes into contact with pyrethroids. SAP2 silencing fully restores mortality of the mosquitoes, whereas SAP2 overexpression results in increased resistance, probably owing to high-affinity binding of SAP2 to pyrethroid insecticides. Mining of genome sequence data reveals a selective sweep near the SAP2 locus in the mosquito populations of three West African countries (Cameroon, Guinea and Burkina Faso) with the observed increase in haplotype-associated single-nucleotide polymorphisms mirroring the increasing resistance of mosquitoes to pyrethroids reported in Burkina Faso. Our study identifies a previously undescribed mechanism of insecticide resistance that is likely to be highly relevant to malaria control efforts.

ABCH2 transporter mediates deltamethrin uptake and toxicity in the malaria vector Anopheles coluzzii.

Contact insecticides are primarily used for the control of Anopheles malaria vectors. These chemicals penetrate mosquito legs and other appendages; the first barriers to reaching their neuronal targets. An ATP-Binding Cassette transporter from the H family (ABCH2) is highly expressed in Anopheles coluzzii legs, and further induced upon insecticide exposure. RNAi-mediated silencing of the ABCH2 caused a significant increase in deltamethrin mortality compared to control mosquitoes, coincident with a corresponding increase in 14C-deltamethrin penetration. RT-qPCR analysis and immunolocalization revealed ABCH2 to be mainly localized in the legs and head appendages, and more specifically, the apical part of the epidermis, underneath the cuticle. To unravel the molecular mechanism underlying the role of ABCH2 in modulating pyrethroid toxicity, two hypotheses were investigated: An indirect role, based on the orthology with other insect ABCH transporters involved with lipid transport and deposition of CHC lipids in Anopheles legs which may increase cuticle thickness, slowing down the penetration rate of deltamethrin; or the direct pumping of deltamethrin out of the organism. Evaluation of the leg cuticular hydrocarbon (CHC) content showed no affect by ABCH2 silencing, indicating this protein is not associated with the transport of leg CHCs. Homology-based modeling suggested that the ABCH2 half-transporter adopts a physiological homodimeric state, in line with its ability to hydrolyze ATP in vitro when expressed on its own in insect cells. Docking analysis revealed a deltamethrin pocket in the homodimeric transporter. Furthermore, deltamethrin-induced ATP hydrolysis in ABCH2-expressing cell membranes, further supports that deltamethrin is indeed an ABCH2 substrate. Overall, our findings pinpoint ABCH2 participating in deltamethrin toxicity regulation.

Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

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.

Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae.

Resistance in Anopheles gambiae to members of all 4 major classes (pyrethroids, carbamates, organochlorines, and organophosphates) of public health insecticides limits effective control of malaria transmission in Africa. Increase in expression of detoxifying enzymes has been associated with insecticide resistance, but their direct functional validation in An. gambiae is still lacking. Here, we perform transgenic analysis using the GAL4/UAS system to examine insecticide resistance phenotypes conferred by increased expression of the 3 genes-Cyp6m2, Cyp6p3, and Gste2-most often found up-regulated in resistant An. gambiae We report evidence in An. gambiae that organophosphate and organochlorine resistance is conferred by overexpression of GSTE2 in a broad tissue profile. Pyrethroid and carbamate resistance is bestowed by similar Cyp6p3 overexpression, and Cyp6m2 confers only pyrethroid resistance when overexpressed in the same tissues. Conversely, such Cyp6m2 overexpression increases susceptibility to the organophosphate malathion, presumably due to conversion to the more toxic metabolite, malaoxon. No resistant phenotypes are conferred when either Cyp6 gene overexpression is restricted to the midgut or oenocytes, indicating that neither tissue is involved in insecticide resistance mediated by the candidate P450s examined. Validation of genes conferring resistance provides markers to guide control strategies, and the observed negative cross-resistance due to Cyp6m2 gives credence to proposed dual-insecticide strategies to overcome pyrethroid resistance. These transgenic An. gambiae-resistant lines are being used to test the "resistance-breaking" efficacy of active compounds early in their development.

Actions of Camptothecin Derivatives on Larvae and Adults of the Arboviral Vector Aedes aegypti.

Mosquito-borne viruses including dengue, Zika, and Chikungunya viruses, and parasites such as malaria and Onchocerca volvulus endanger health and economic security around the globe, and emerging mosquito-borne pathogens have pandemic potential. However, the rapid spread of insecticide resistance threatens our ability to control mosquito vectors. Larvae of Aedes aegypti were screened with the Medicines for Malaria Venture Pandemic Response Box, an open-source compound library, using INVAPP, an invertebrate automated phenotyping platform suited to high-throughput chemical screening of larval motility. We identified rubitecan (a synthetic derivative of camptothecin) as a hit compound that reduced A. aegypti larval motility. Both rubitecan and camptothecin displayed concentration dependent reduction in larval motility with estimated EC50 of 25.5 ± 5.0 µM and 22.3 ± 5.4 µM, respectively. We extended our investigation to adult mosquitoes and found that camptothecin increased lethality when delivered in a blood meal to A. aegypti adults at 100 µM and 10 µM, and completely blocked egg laying when fed at 100 µM. Camptothecin and its derivatives are inhibitors of topoisomerase I, have known activity against several agricultural pests, and are also approved for the treatment of several cancers. Crucially, they can inhibit Zika virus replication in human cells, so there is potential for dual targeting of both the vector and an important arbovirus that it carries.

Potential for Zika virus transmission by mosquitoes in temperate climates.

Mosquito-borne Zika virus (ZIKV) transmission has almost exclusively been detected in the tropics despite the distributions of its primary vectors extending farther into temperate regions. Therefore, it is unknown whether ZIKV's range has reached a temperature-dependent limit, or if it can spread into temperate climates. Using field-collected mosquitoes for biological relevance, we found that two common temperate mosquito species, Aedes albopictus and Ochlerotatus detritus, were competent for ZIKV. We orally exposed mosquitoes to ZIKV and held them at between 17 and 31°C, estimated the time required for mosquitoes to become infectious, and applied these data to a ZIKV spatial risk model. We identified a minimum temperature threshold for the transmission of ZIKV by mosquitoes between 17 and 19°C. Using these data, we generated standardized basic reproduction number R0-based risk maps and we derived estimates for the length of the transmission season for recent and future climate conditions. Our standardized R0-based risk maps show potential risk of ZIKV transmission beyond the current observed range in southern USA, southern China and southern European countries. Transmission risk is simulated to increase over southern and Eastern Europe, northern USA and temperate regions of Asia (northern China, southern Japan) in future climate scenarios.

Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.

Aedes aegypti CCEae3A carboxylase expression confers carbamate, organophosphate and limited pyrethroid resistance in a model transgenic mosquito.

Insecticide resistance is a serious threat to our ability to control mosquito vectors which transmit pathogens including malaria parasites and arboviruses. Understanding the underlying mechanisms is an essential first step in tackling the challenges presented by resistance. This study aimed to functionally characterise the carboxylesterase, CCEae3A, the elevated expression of which has been implicated in temephos resistance in Aedes aegypti and Aedes albopictus larvae. Using our GAL4/UAS expression system, already established in insecticide-sensitive Anopheles gambiae mosquitoes, we produced transgenic An. gambiae mosquitoes that express an Ae. aegypti CCEae3A ubiquitously. This new transgenic line permits examination of CCEae3A expression in a background in which there is not a clear orthologue in Vectorbase and allows comparison with existing An. gambiae GAL4-UAS lines. Insecticide resistance profiling of these transgenic An. gambiae larvae indicated significant increases in resistance ratio for three organophosphate insecticides, temephos (6), chloropyriphos (6.6) and fenthion (3.2) when compared to the parental strain. Cross resistance to adulticides from three major insecticide classes: organophosphates (malathion, fenitrothion and pirimiphos methyl), carbamates (bendiocarb and propoxur) and pyrethroid (alpha-cypermethrin) was also detected. Resistance to certain organophosphates and carbamates validates conclusions drawn from previous expression and phenotypic data. However, detection of resistance to pirimiphos methyl and alphacypermethrin has not previously been formally associated with CCEae3A, despite occurring in Ae. aegypti strains where this gene was upregulated. Our findings highlight the importance of characterising individual resistance mechanisms, thereby ensuring accurate information is used to guide future vector control strategies.

Demasculinization of the Anopheles gambiae X chromosome.

BACKGROUND: In a number of organisms sex-biased genes are non-randomly distributed between autosomes and the shared sex chromosome X (or Z). Studies on Anopheles gambiae have produced conflicting results regarding the underrepresentation of male-biased genes on the X chromosome and it is unclear to what extent sexual antagonism, dosage compensation or X-inactivation in the male germline, the evolutionary forces that have been suggested to affect the chromosomal distribution of sex-biased genes, are operational in Anopheles. RESULTS: We performed a meta-analysis of sex-biased gene expression in Anopheles gambiae which provides evidence for a general underrepresentation of male-biased genes on the X-chromosome that increased in significance with the observed degree of sex-bias. A phylogenomic comparison between Drosophila melanogaster, Aedes aegypti and Culex quinquefasciatus also indicates that the Anopheles X chromosome strongly disfavours the evolutionary conservation of male-biased expression and that novel male-biased genes are more likely to arise on autosomes. Finally, we demonstrate experimentally that transgenes situated on the Anopheles gambiae X chromosome are transcriptionally silenced in the male germline. CONCLUSION: The data presented here support the hypothesis that the observed demasculinization of the Anopheles X chromosome is driven by X-chromosome inactivation in the male germline and by sexual antagonism. The demasculinization appears to be the consequence of a loss of male-biased expression, rather than a failure in the establishment or the extinction of male-biased genes.

The Anopheles gambiae alpha-tubulin-1b promoter directs neuronal, testes and developing imaginal tissue specific expression and is a sensitive enhancer detector.

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.

Optimization of the Gal4-UAS system in an Anopheles gambiae cell line.

The development of the bipartite Gal4-UAS system in Anopheles gambiae would improve the functional characterization of genes in this important malaria vector. Towards this aim, we used Gal4 driver plasmids to successfully activate expression of the reporter gene, luciferase, from UAS responder plasmids when cotransfected into an An. gambiae cell line. To optimize Gal4-regulated gene expression in mosquitoes, we compared the efficiency of a series of alternative Gal4 transactivators to drive reporter gene expression from responder plasmids incorporating different numbers of tandemly arrayed Gal4 binding sites or upstream activation sequences (UAS). The results indicated that the native Gal4 is only weakly active in these cells. Modified forms of Gal4, including those carrying minimal VP16 activation domains, as well as a deleted form of Gal4, give up to 20-fold greater activity than the native protein, when used in conjunction with a responder plasmid having 14 UAS repeats. The identification of Gal4-UAS vectors that are efficiently expressed in a mosquito cell line should facilitate the transfer of this versatile expression system to An. gambiae, and potentially to other insects of medical importance.

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria.

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

Automated phenotyping of mosquito larvae enables high-throughput screening for novel larvicides and offers potential for smartphone-based detection of larval insecticide resistance.

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

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.

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.

Laboratory transmission potential of British mosquitoes for equine arboviruses.

BACKGROUND: There has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses important for both equine and human health, as measured by the presence of viral RNA in saliva at different time points after taking an infective blood meal. RESULTS: The following wild-caught British mosquitoes were evaluated for their potential as vectors of zoonotic equine arboviruses: Ochlerotatus detritus for Venezuelan equine encephalitis virus (VEEV) and Ross River virus (RRV), and Culiseta annulata and Culex pipiens for Japanese encephalitis virus (JEV). Production of RNA in saliva was demonstrated at varying efficiencies for all mosquito-virus pairs. Ochlerotatus detritus was more permissive for production of RRV RNA in saliva than VEEV RNA. For RRV, 27.3% of mosquitoes expectorated viral RNA at 7 days post-infection when incubated at 21 °C and 50% at 24 °C. Strikingly, 72% of Cx. pipiens produced JEV RNA in saliva after 21 days at 18 °C. For some mosquito-virus pairs, infection and salivary RNA titres reduced over time, suggesting unstable infection dynamics. CONCLUSIONS: This study adds to the number of Palaearctic mosquito species that demonstrate expectoration of viral RNA, for arboviruses of importance to human and equine health. This work adds to evidence that native mosquito species should be investigated further for their potential to vector zoonotic mosquito-borne arboviral disease of equines in northern Europe. The evidence that Cx. pipiens is potentially an efficient laboratory vector of JEV at temperatures as low as 18 °C warrants further investigation, as this mosquito is abundant in cooler regions of Europe and is considered an important vector for West Nile Virus, which has a comparable transmission ecology.

New insecticide screening platforms indicate that Mitochondrial Complex I inhibitors are susceptible to cross-resistance by mosquito P450s that metabolise pyrethroids.

Fenazaquin, pyridaben, tolfenpyrad and fenpyroximate are Complex I inhibitors offering a new mode of action for insecticidal malaria vector control. However, extended exposure to pyrethroid based products such as long-lasting insecticidal nets (LLINs) has created mosquito populations that are largely pyrethroid-resistant, often with elevated levels of P450s that can metabolise and neutralise diverse substrates. To assess cross-resistance liabilities of the Complex I inhibitors, we profiled their susceptibility to metabolism by P450s associated with pyrethroid resistance in Anopheles gambiae (CYPs 6M2, 6P3, 6P4, 6P5, 9J5, 9K1, 6Z2) and An. funestus (CYP6P9a). All compounds were highly susceptible. Transgenic An. gambiae overexpressing CYP6M2 or CYP6P3 showed reduced mortality when exposed to fenpyroximate and tolfenpyrad. Mortality from fenpyroximate was also reduced in pyrethroid-resistant strains of An. gambiae (VK7 2014 and Tiassalé 13) and An. funestus (FUMOZ-R). P450 inhibitor piperonyl butoxide (PBO) significantly enhanced the efficacy of fenpyroximate and tolfenpyrad, fully restoring mortality in fenpyroximate-exposed FUMOZ-R. Overall, results suggest that in vivo and in vitro assays are a useful guide in the development of new vector control products, and that the Complex I inhibitors tested are susceptible to metabolic cross-resistance and may lack efficacy in controlling pyrethroid resistant mosquitoes.

Two functionally distinct CYP4G genes of Anopheles gambiae contribute to cuticular hydrocarbon biosynthesis.

Cuticular hydrocarbon (CHC) biosynthesis is a major pathway of insect physiology. In Drosophila melanogaster the cytochrome P450 CYP4G1 catalyses the insect-specific oxidative decarbonylation step, while in the malaria vector Anopheles gambiae, two CYP4G paralogues, CYP4G16 and CYP4G17 are present. Analysis of the subcellular localization of CYP4G17 and CYP4G16 in larval and pupal stages revealed that CYP4G16 preserves its PM localization across developmental stages analyzed; however CYPG17 is differentially localized in two distinct types of pupal oenocytes, presumably oenocytes of larval and adult developmental specificity. Western blot analysis showed the presence of two CYP4G17 forms, potentially associated with each oenocyte type. Both An. gambiae CYP4Gs were expressed in D. melanogaster flies in a Cyp4g1 silenced background in order to functionally characterize them in vivo. CYP4G16, CYP4G17 or their combination rescued the lethal phenotype of Cyp4g1-knock down flies, demonstrating that CYP4G17 is also a functional decarbonylase, albeit of somewhat lower efficiency than CYP4G16 in Drosophila. Flies expressing mosquito CYP4G16 and/or CYP4G17 produced similar CHC profiles to 'wild-type' flies expressing the endogenous CYP4G1, but they also produce very long-chain dimethyl-branched CHCs not detectable in wild type flies, suggesting that the specificity of the CYP4G enzymes contributes to determine the complexity of the CHC blend. In conclusion, both An. gambiae CYP4G enzymes contribute to the unique Anopheles CHC profile, which has been associated to defense, adult desiccation tolerance, insecticide penetration rate and chemical communication.

Opening the toolkit for genetic analysis and control of Anopheles mosquito vectors.

Anopheles is the only genus of mosquitoes that transmit human malaria and consequently the focus of large scale genome and transcriptome-wide association studies. Genetic tools to define the function of the candidate genes arising from these analyses are vital. Moreover, genome editing offers the potential to modify Anopheles population structure at local and global scale to provide complementary tools towards the ultimate goal of malaria elimination. Major breakthroughs in Anopheles genetic analysis came with the development of germline transformation and RNA interference technology. Yet, the field has been revolutionised again by precise genome editing now possible through site-specific nucleases. Here we review the components of the current genetic toolkit available to study Anopheles, focusing particularly on how these technical advances are used to gain insight into malaria transmission and the design of genetic methods to control Anopheles vectors.