RNA-seq analyses of changes in the Anopheles gambiae transcriptome associated with resistance to pyrethroids in Kenya: identification of candidate-resistance genes and candidate-resistance SNPs.

BACKGROUND: The extensive use of pyrethroids for control of malaria vectors, driven by their cost, efficacy and safety, has led to widespread resistance. To favor their sustainable use, the World Health Organization (WHO) formulated an insecticide resistance management plan, which includes the identification of the mechanisms of resistance and resistance surveillance. Recognized physiological mechanisms of resistance include target site mutations in the para voltage-gated sodium channel, metabolic detoxification and penetration resistance. Such understanding of resistance mechanisms has allowed the development of resistance monitoring tools, including genotyping of the kdr mutation L1014F/S in the para gene. METHODS: The sequence-based technique RNA-seq was applied to study changes in the transcriptome of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from the Western Province of Kenya. The resulting gene expression profiles were compared to data in the most recent literature to derive a list of candidate resistance genes. RNA-seq data were analyzed also to identify sequence polymorphisms linked to resistance. RESULTS: A total of five candidate-resistance genes (AGAP04177, AGAP004572, AGAP008840, AGAP007530 and AGAP013036) were identified with altered expression between resistant and susceptible mosquitoes from West and East Africa. A change from G to C at position 36043997 of chromosome 3R resulting in A101G of the sulfotransferase gene AGAP009551 was significantly associated with the resistance phenotype (odds ratio: 5.10). The kdr L1014S mutation was detected at similar frequencies in both phenotypically resistant and susceptible mosquitoes, suggesting it is no longer fully predictive of the resistant phenotype. CONCLUSIONS: Overall, these results support the conclusion that resistance to pyrethroids is a complex and evolving phenotype, dependent on multiple gene functions including, but not limited to, metabolic detoxification. Functional convergence among metabolic detoxification genes may exist, with the role of each gene being modulated by the life history and selection pressure on mosquito populations. As a consequence, biochemical assays that quantify overall enzyme activity may be a more suitable method for predicting metabolic resistance than gene-based assays.

Probing functional polymorphisms in the dengue vector, Aedes aegypti.

BACKGROUND: Dengue is the most prevalent arboviral disease world-wide and its primary vector is the mosquito Aedes aegypti. The current lack of commercially-available vaccines makes control of vector populations the only effective strategy to prevent dengue transmission. Aedes aegypti geographic populations exhibit great variability in insecticide resistance and susceptibility to dengue infection. The characterization of single nucleotide polymorphisms (SNPs) as molecular markers to study quantitatively this variation is needed greatly because this species has a low abundance of microsatellite markers and limited known restriction fragments length polymorphisms (RFLPs) and single-strand conformation polymorphism (SSCP) markers. RESULTS: We used RNA-seq to characterize SNPs in three Ae. aegypti strains, including the Liverpool (LVP) strain, from which the current genome annotation is derived. We identified 131,764 unique genome locations with at least one alternative nucleotide to what is reported in the reference annotation. These comprised changes in both open-reading frames (ORFs) and untranslated regions (UTRs) of transcripts. An in depth-look at sequence variation in immunity genes revealed that those associated with autophagy, MD2-like receptors and Peptidoglycan Recognition Proteins had more sequence variation in their 3'UTRs than mutations associated with non-synonymous changes. This supports the conclusion that these genes had maintained their functional specificity while being adapted to different regulatory domains. In contrast, a number of peroxidases, serpins and Clip-domain serine proteases exhibited conservation of putative UTR regulatory sequences while displaying diversification of the ORFs. Transcriptome evidence also was found for ~2500 novel transcriptional units (NTUs) not annotated in the reference genome. CONCLUSIONS: The transcriptome-wide assessment of within and inter-strain polymorphisms in Ae. aegypti adds considerably to the number of molecular markers available for genetic studies in this mosquito. Additionally, data supporting NTU discovery emphasizes the need for continuous amendments of the reference genome annotation.

Comparative transcriptome analyses of deltamethrin-resistant and -susceptible Anopheles gambiae mosquitoes from Kenya by RNA-Seq.

Malaria causes more than 300 million clinical cases and 665,000 deaths each year, and the majority of the mortality and morbidity occurs in sub-Saharan Africa. Due to the lack of effective vaccines and wide-spread resistance to antimalarial drugs, mosquito control is the primary method of malaria prevention and control. Currently, malaria vector control relies on the use of insecticides, primarily pyrethroids. The extensive use of insecticides has imposed strong selection pressures for resistance in the mosquito populations. Consequently, resistance to pyrethroids in Anopheles gambiae, the main malaria vector in sub-Saharan Africa, has become a major obstacle for malaria control. A key element of resistance management is the identification of resistance mechanisms and subsequent development of reliable resistance monitoring tools. Field-derived An. gambiae from Western Kenya were phenotyped as deltamethrin-resistant or -susceptible by the standard WHO tube test, and their expression profile compared by RNA-seq. Based on the current annotation of the An. gambiae genome, a total of 1,093 transcripts were detected as significantly differentially accumulated between deltamethrin-resistant and -susceptible mosquitoes. These transcripts are distributed over the entire genome, with a large number mapping in QTLs previously linked to pyrethorid resistance, and correspond to heat-shock proteins, metabolic and transport functions, signal transduction activities, cytoskeleton and others. The detected differences in transcript accumulation levels between resistant and susceptible mosquitoes reflect transcripts directly or indirectly correlated with pyrethroid resistance. RNA-seq data also were used to perform a de-novo Cufflinks assembly of the An. gambiae genome.