Genome-wide transcriptional analyses in Anopheles mosquitoes reveal an unexpected association between salivary gland gene expression and insecticide resistance.

BACKGROUND: To combat malaria transmission, the Ugandan government has embarked upon an ambitious programme of indoor residual spraying (IRS) with a carbamate class insecticide, bendiocarb. In preparation for this campaign, we characterized bendiocarb resistance and associated transcriptional variation among Anopheles gambiae s.s. mosquitoes from two sites in Uganda. RESULTS: Gene expression in two mosquito populations displaying some resistance to bendiocarb (95% and 79% An. gambiae s.l. WHO tube bioassay mortality in Nagongera and Kihihi, respectively) was investigated using whole-genome microarrays. Significant overexpression of several genes encoding salivary gland proteins, including D7r2 and D7r4, was detected in mosquitoes from Nagongera. In Kihihi, D7r4, two detoxification-associated genes (Cyp6m2 and Gstd3) and an epithelial serine protease were among the genes most highly overexpressed in resistant mosquitoes. Following the first round of IRS in Nagongera, bendiocarb-resistant mosquitoes were collected, and real-time quantitative PCR analyses detected significant overexpression of D7r2 and D7r4 in resistant mosquitoes. A single nucleotide polymorphism located in a non-coding transcript downstream of the D7 genes was found at a significantly higher frequency in resistant individuals. In silico modelling of the interaction between D7r4 and bendiocarb demonstrated similarity between the insecticide and serotonin, a known ligand of D7 proteins. A meta-analysis of published microarray studies revealed a recurring association between D7 expression and insecticide resistance across Anopheles species and locations. CONCLUSIONS: A whole-genome microarray approach identified an association between novel insecticide resistance candidates and bendiocarb resistance in Uganda. In addition, a single nucleotide polymorphism associated with this resistance mechanism was discovered. The use of such impartial screening methods allows for discovery of resistance candidates that have no previously-ascribed function in insecticide binding or detoxification. Characterizing these novel candidates will broaden our understanding of resistance mechanisms and yield new strategies for combatting widespread insecticide resistance among malaria vectors.

Association mapping by pooled sequencing identifies TOLL 11 as a protective factor against Plasmodium falciparum in Anopheles gambiae.

BACKGROUND: The genome-wide association study (GWAS) techniques that have been used for genetic mapping in other organisms have not been successfully applied to mosquitoes, which have genetic characteristics of high nucleotide diversity, low linkage disequilibrium, and complex population stratification that render population-based GWAS essentially unfeasible at realistic sample size and marker density. METHODS: We designed a novel mapping strategy for the mosquito system that combines the power of linkage mapping with the resolution afforded by genetic association. We established founder colonies from West Africa, controlled for diversity, linkage disequilibrium and population stratification. Colonies were challenged by feeding on the infectious stage of the human malaria parasite, Plasmodium falciparum, mosquitoes were phenotyped for parasite load, and DNA pools for phenotypically similar mosquitoes were Illumina sequenced. Phenotype-genotype mapping was carried out in two stages, coarse and fine. RESULTS: In the first mapping stage, pooled sequences were analysed genome-wide for intervals displaying relativereduction in diversity between phenotype pools, and candidate genomic loci were identified for influence upon parasite infection levels. In the second mapping stage, focused genotyping of SNPs from the first mapping stage was carried out in unpooled individual mosquitoes and replicates. The second stage confirmed significant SNPs in a locus encoding two Toll-family proteins. RNAi-mediated gene silencing and infection challenge revealed that TOLL 11 protects mosquitoes against P. falciparum infection. CONCLUSIONS: We present an efficient and cost-effective method for genetic mapping using natural variation segregating in defined recent Anopheles founder colonies, and demonstrate its applicability for mapping in a complex non-model genome. This approach is a practical and preferred alternative to population-based GWAS for first-pass mapping of phenotypes in Anopheles. This design should facilitate mapping of other traits involved in physiology, epidemiology, and behaviour.

Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi.

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.

Candidate-gene based GWAS identifies reproducible DNA markers for metabolic pyrethroid resistance from standing genetic variation in East African Anopheles gambiae.

Metabolic resistance to pyrethroid insecticides is widespread in Anopheles mosquitoes and is a major threat to malaria control. DNA markers would aid predictive monitoring of resistance, but few mutations have been discovered outside of insecticide-targeted genes. Isofemale family pools from a wild Ugandan Anopheles gambiae population, from an area where operational pyrethroid failure is suspected, were genotyped using a candidate-gene enriched SNP array. Resistance-associated SNPs were detected in three genes from detoxification superfamilies, in addition to the insecticide target site (the Voltage Gated Sodium Channel gene, Vgsc). The putative associations were confirmed for two of the marker SNPs, in the P450 Cyp4j5 and the esterase Coeae1d by reproducible association with pyrethroid resistance in multiple field collections from Uganda and Kenya, and together with the Vgsc-1014S (kdr) mutation these SNPs explained around 20% of variation in resistance. Moreover, the >20 Mb 2La inversion also showed evidence of association with resistance as did environmental humidity. Sequencing of Cyp4j5 and Coeae1d detected no resistance-linked loss of diversity, suggesting selection from standing variation. Our study provides novel, regionally-validated DNA assays for resistance to the most important insecticide class, and establishes both 2La karyotype variation and humidity as common factors impacting the resistance phenotype.

Insecticide-induced leg loss does not eliminate biting and reproduction in Anopheles gambiae mosquitoes.

Recent successes in malaria control have been largely attributable to the deployment of insecticide-based vector control tools such as bed nets and indoor residual spraying. Pyrethroid-treated bed nets are acutely neurotoxic to mosquitoes, inducing symptoms such as loss of coordination, paralysis, and violent spasms. One result of pyrethroid exposure often seen in laboratory tests is mosquito leg loss, a condition that has thus far been assumed to equate to mortality, as females are not expected to blood feed. However, whilst limb loss is unlikely to be adaptive, females with missing limbs may play a role in the propagation of both their species and pathogens. To test the hypothesis that leg loss inhibits mosquitoes from biting and reproducing, mosquitoes with one, two, or six legs were evaluated for their success in feeding upon a human. These experiments demonstrated that insecticide-induced leg loss had no significant effect upon blood feeding or egg laying success. We conclude that studies of pyrethroid efficacy should not discount mosquitoes that survive insecticide exposure with fewer than six legs, as they may still be capable of biting humans, reproducing, and contributing to malaria transmission.

Identification, Validation, and Application of Molecular Diagnostics for Insecticide Resistance in Malaria Vectors.

Insecticide resistance is a major obstacle to control of Anopheles malaria mosquitoes in sub-Saharan Africa and requires an improved understanding of the underlying mechanisms. Efforts to discover resistance genes and DNA markers have been dominated by candidate gene and quantitative trait locus studies of laboratory strains, but with greater availability of genome sequences a shift toward field-based agnostic discovery is anticipated. Mechanisms evolve continually to produce elevated resistance yielding multiplicative diagnostic markers, co-screening of which can give high predictive value. With a shift toward prospective analyses, identification and screening of resistance marker panels will boost monitoring and programmatic decision making.

Patterns of hybrid loss of imprinting reveal tissue- and cluster-specific regulation.

BACKGROUND: Crosses between natural populations of two species of deer mice, Peromyscus maniculatus (BW), and P. polionotus (PO), produce parent-of-origin effects on growth and development. BW females mated to PO males (bwxpo) produce growth-retarded but otherwise healthy offspring. In contrast, PO females mated to BW males (POxBW) produce overgrown and severely defective offspring. The hybrid phenotypes are pronounced in the placenta and include POxBW conceptuses which lack embryonic structures. Evidence to date links variation in control of genomic imprinting with the hybrid defects, particularly in the POxBW offspring. Establishment of genomic imprinting is typically mediated by gametic DNA methylation at sites known as gDMRs. However, imprinted gene clusters vary in their regulation by gDMR sequences. METHODOLOGY/PRINCIPAL FINDINGS: Here we further assess imprinted gene expression and DNA methylation at different cluster types in order to discern patterns. These data reveal POxBW misexpression at the Kcnq1ot1 and Peg3 clusters, both of which lose ICR methylation in placental tissues. In contrast, some embryonic transcripts (Peg10, Kcnq1ot1) reactivated the silenced allele with little or no loss of DNA methylation. Hybrid brains also display different patterns of imprinting perturbations. Several cluster pairs thought to use analogous regulatory mechanisms are differentially affected in the hybrids. CONCLUSIONS/SIGNIFICANCE: These data reinforce the hypothesis that placental and somatic gene regulation differs significantly, as does that between imprinted gene clusters and between species. That such epigenetic regulatory variation exists in recently diverged species suggests a role in reproductive isolation, and that this variation is likely to be adaptive.