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

ABSTRACT: 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.

SUBMITTER: Balabanidou V

PROVIDER: S-EPMC4995928 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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

Balabanidou Vasileia V Kampouraki Anastasia A MacLean Marina M Blomquist Gary J GJ Tittiger Claus C Juárez M Patricia MP Mijailovsky Sergio J SJ Chalepakis George G Anthousi Amalia A Lynd Amy A Antoine Sanou S Hemingway Janet J Ranson Hilary H Lycett Gareth J GJ Vontas John J

Proceedings of the National Academy of Sciences of the United States of America 20160720 33

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 ...[more]

PMID: 27439866

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Project description:BackgroundThe emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported.MethodsThe protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR.ResultsStrong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (<or= 5%).ConclusionThis study showed a widespread resistance to permethrin in An. gambiae populations from southern Benin, with a significant increase of kdr frequency compared to what was observed previously in Benin. The low frequency of Ace-1 recorded in all populations is encouraging for the use of bendiocarb as an alternative insecticide to pyrethroids for IRS in Benin.

Project description:Published data revealed that two of the 243 structural cuticular proteins of Anopheles gambiae, CPLCG3 and CPLCG4, are implicated in insecticide resistance and a third, CPF3, has far higher transcript levels in M than in S incipient species. We studied the distribution of transcripts for these three genes in the tissues of An. gambiae and the location of the proteins in the cuticle itself to gain information about how these cuticular proteins contribute to their important roles. Our data are consistent with CPLCG3/4 contributing to a thicker cuticle thus slowing penetration of insecticides and CPF3 possibly having a role in the greater desiccation tolerance of the M form.Using RT-qPCR, we established the temporal expression of the genes and by in situ hybridization we revealed the main tissues where their mRNAs are found. Electron microscopy immunolocalization, using secondary antibodies labeled with colloidal gold, allowed us to localize these proteins within different regions of the cuticle.The temporal expression of these genes overlaps, albeit with higher levels of transcripts from CPF3 in pharate adults and both CPLCG3 and CPLCG4 are higher in animals immediately after adult eclosion. The main location of mRNAs for all three genes is in appendages and genitalia. In contrast, the location of their proteins within the cuticle is completely different. CPF3 is found exclusively in exocuticle and CPLCG3/4 is restricted to the endocuticle. The other CPF gene expressed at the same times, CPF4, in addition to appendages, has message in pharate adult sclerites.The temporal and spatial differences in transcript abundance and protein localization help to account for An. gambiae devoting about 2% of its protein coding genes to structural cuticular proteins. The location of CPLCG3/4 in the endocuticle may contribute to the thickness of the cuticle, one of the recently appreciated components of insecticide resistance, while the location of CPF3 might be related to the greater desiccation resistance of the M form.

Project description:Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

Project description:BackgroundVector control activities, namely long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS), have contributed significantly to the decreasing malaria burden observed in The Gambia since 2008. Nevertheless, insecticide resistance may threaten such success; it is important to regularly assess the susceptibility of local malaria vectors to available insecticides.MethodsIn the transmission seasons of 2016 and 2017, Anopheles gambiae (s.l.) larvae were sampled in or around the nine vector surveillance sentinel sites of the Gambia National Malaria Control Programme (GNMCP) and in a few additional sampling points. Using WHO susceptibility bioassays, female adult mosquitoes were exposed to insecticide-impregnated papers. Molecular identification of sibling species and insecticide resistance molecular markers was done on a subset of 2000 female mosquitoes.ResultsA total of 4666 wild-caught female adult mosquitoes were exposed to either permethrin (n = 665), deltamethrin (n = 744), DDT (n = 1021), bendiocarb (n = 990) or pirimiphos-methyl (n = 630) insecticide-impregnated papers and control papers (n = 616). Among the 2000 anophelines, 1511 (80.7%) were Anopheles arabiensis, 204 (10.9%) Anopheles coluzzii, 75 (4%) Anopheles gambiae (s.s.), and 83 (4.4%) An. gambiae (s.s.) and An. coluzzii hybrids. There was a significant variation in the composition and species distribution by regions and year, P = 0.009. Deltamethrin, permethrin and DDT resistance was found in An. arabiensis, especially in the coastal region, and was mediated by Vgsc-1014F/S mutations (odds ratio = 34, P = 0.014). There was suspected resistance to pirimiphos-methyl (actellic 300CS) in the North Bank Region although only one survivor had the Ace-1-119S mutation.ConclusionsAs no confirmed resistance to bendiocarb and actellic 300CS was detected, the national malaria control programme can continue using these insecticides for IRS. Nevertheless, the detection of Ace-1 119S mutation warrants extensive monitoring. The source of insecticide pressure driving insecticide resistance to pyrethroids and DDT detected at the coastal region should be further investigated in order to properly manage the spread of resistance in The Gambia.

Project description:BackgroundThe emergence of insecticide resistance is a major threat to malaria control programmes in Africa, with many different factors contributing to insecticide resistance in its vectors, Anopheles mosquitoes. CYP6M2 has previously been recognized as an important candidate in cytochrome P450-mediated detoxification in Anopheles. As it has been implicated in resistance against pyrethroids, organochlorines and carbamates, its broad metabolic activity makes it a potential agent in insecticide cross-resistance. Currently, allelic variation within the Cyp6m2 gene remains unknown.MethodsHere, Illumina whole-genome sequence data from Phase 2 of the Anopheles gambiae 1000 Genomes Project (Ag1000G) was used to examine genetic variation in the Cyp6m2 gene across 16 populations in 13 countries comprising Anopheles gambiae and Anopheles coluzzii mosquitoes. To identify whether these alleles show evidence of selection either through potentially modified enzymatic function or by being linked to variants that change the transcriptional profile of the gene, hierarchical clustering of haplotypes, linkage disequilibrium, median joining networks and extended haplotype homozygosity analyses were performed.ResultsFifteen missense biallelic substitutions at high frequency (defined as > 5% frequency in one or more populations) are found, which fall into five distinct haplotype groups that carry the main high frequency variants: A13T, D65A, E328Q, Y347F, I359V and A468S. Despite consistent reports of Cyp6m2 upregulation and metabolic activity in insecticide resistant Anophelines, no evidence of directional selection is found occurring on these variants or on the haplotype clusters in which they are found.ConclusionThese results imply that emerging resistance associated with Cyp6m2 is potentially driven by distant regulatory loci such as transcriptional factors rather than by its missense variants, or that other genes are playing a more significant role in conferring metabolic resistance.

Project description:BackgroundSelection pressure from continued exposure to insecticides drives development of insecticide resistance and changes in resting behaviour of malaria vectors. There is need to understand how resistance drives changes in resting behaviour within vector species. The association between insecticide resistance and resting behaviour of Anopheles gambiae sensu lato (s.l.) in Northern Ghana was examined.MethodsF1 progenies from adult mosquitoes collected indoors and outdoors were exposed to DDT, deltamethrin, malathion and bendiocarb using WHO insecticide susceptibility tests. Insecticide resistance markers including voltage-gated sodium channel (Vgsc)-1014F, Vgsc-1014S, Vgsc-1575Y, glutathione-S-transferase epsilon 2 (GSTe2)-114T and acetylcholinesterase (Ace1)-119S, as well as blood meal sources were investigated using PCR methods. Activities of metabolic enzymes, acetylcholine esterase (AChE), non-specific β-esterases, glutathione-S-transferase (GST) and monooxygenases were measured from unexposed F1 progenies using microplate assays.ResultsSusceptibility of Anopheles coluzzii to deltamethrin 24 h post-exposure was significantly higher in indoor (mortality = 5%) than outdoor (mortality = 2.5%) populations (P = 0.02). Mosquitoes were fully susceptible to malathion (mortality: indoor = 98%, outdoor = 100%). Susceptibility to DDT was significantly higher in outdoor (mortality = 9%) than indoor (mortality = 0%) mosquitoes (P = 0.006). Mosquitoes were also found with suspected resistance to bendiocarb but mortality was not statistically different (mortality: indoor = 90%, outdoor = 95%. P = 0.30). Frequencies of all resistance alleles were higher in F1 outdoor (0.11-0.85) than indoor (0.04-0.65) mosquito populations, while Vgsc-1014F in F0 An. gambiae sensu stricto (s.s) was significantly associated with outdoor-resting behaviour (P = 0.01). Activities of non-specific β-esterase enzymes were significantly higher in outdoor than indoor mosquitoes (Mean enzyme activity: Outdoor = : 1.70/mg protein; Indoor = 1.35/mg protein. P < 0.0001). AChE activity was also more elevated in outdoor (0.62/mg protein) than indoor (0.57/mg protein) mosquitoes but this was not significant (P = 0.08). Human blood index (HBI) was predominantly detected in indoor (18%) than outdoor mosquito populations (3%).ConclusionsThe overall results did not establish that there was a significant preference of resistant malaria vectors to solely rest indoors or outdoors, but varied depending on the resistant alleles present. Phenotypic resistance was higher in indoor than outdoor-resting mosquitoes, but genotypic and metabolic resistance levels were higher in outdoor than the indoor populations. Continued monitoring of changes in resting behaviour within An. gambiae s.l. populations is recommended.

Dataset Information

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

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

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