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The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis.

ABSTRACT: BACKGROUND: The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays. METHODS: WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays. RESULTS: Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands. CONCLUSION: The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands.

SUBMITTER: Jones CM

PROVIDER: S-EPMC3895773 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis.

Jones Christopher M CM Haji Khamis A KA Khatib Bakari O BO Bagi Judit J Mcha Juma J Devine Gregor J GJ Daley Matthew M Kabula Bilali B Ali Abdullah S AS Majambere Silas S Ranson Hilary H

Parasites & vectors 20131206

<h4>Background</h4>The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays.<h4 ...[more]

PMID: 24314005

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Project description:Pyrethroid resistance has been slower to emerge in Anopheles arabiensis than in An. gambiae s.s and An. funestus and, consequently, studies are only just beginning to unravel the genes involved. Permethrin resistance in An. arabiensis in Lower Moshi, Tanzania has been linked to elevated levels of both P450 monooxygenases and ?-esterases. We have conducted a gene expression study to identify specific genes linked with metabolic resistance in the Lower Moshi An. arabiensis population.Microarray experiments employing an An. gambiae whole genome expression chip were performed on An. arabiensis, using interwoven loop designs. Permethrin-exposed survivors were compared to three separate unexposed mosquitoes from the same or a nearby population. A subsection of detoxification genes were chosen for subsequent quantitative real-time PCR (qRT-PCR).Microarray analysis revealed significant over expression of 87 probes and under expression of 85 probes (in pairwise comparisons between permethrin survivors and unexposed sympatric and allopatric samples from Dar es Salaam (controls). For qRT-PCR we targeted over expressed ABC transporter genes (ABC '2060'), a glutathione-S-transferase, P450s and esterases. Design of efficient, specific primers was successful for ABC '2060'and two P450s (CYP6P3, CYP6M2). For the CYP4G16 gene, we used the primers that were previously used in a microarray study of An. arabiensis from Zanzibar islands. Over expression of CYP4G16 and ABC '2060' was detected though with contrasting patterns in pairwise comparisons between survivors and controls. CYP4G16 was only up regulated in survivors, whereas ABC '2060' was similar in survivors and controls but over expressed in Lower Moshi samples compared to the Dar es Salaam samples. Increased transcription of CYP4G16 and ABC '2060' are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis.Increased transcription of a P450 (CYP4G16) and an ABC transporter (ABC 2060) are linked directly and indirectly respectively, with permethrin resistance in Lower Moshi An. arabiensis. Our study provides replication of CYP4G16 as a candidate gene for pyrethroid resistance in An. arabiensis, although its role may not be in detoxification, and requires further investigation.

Project description:BackgroundIndoor residual spraying and insecticide-treated nets are among the key malaria control intervention tools. However, their efficacy is declining due to the development and spread of insecticide resistant vectors. In Ethiopia, several studies reported resistance of An. arabiensis to multiple insecticide classes. However, such data is scarce in irrigated areas of the country where insecticides, pesticides and herbicides are intensively used. Susceptibility of An. gambiae s.l. to existing and new insecticides and resistance mechanisms were assessed in Arjo-Didessa sugarcane plantation area, southwestern Ethiopia.MethodsAdult An. gambiae s.l. reared from larval/pupal collections of Arjo-Didessa sugarcane irrigation area and its surrounding were tested for their susceptibility to selected insecticides. Randomly selected An. gambiae s.l. (dead and survived) samples were identified to species using species-specific polymerase chain reaction (PCR) and were further analyzed for the presence of knockdown resistance (kdr) alleles using allele-specific PCR.ResultsAmong the 214 An. gambiae s.l. samples analyzed by PCR, 89% (n = 190) were An. amharicus and 9% (n = 20) were An. arabiensis. Mortality rates of the An. gambiae s.l. exposed to deltamethrin and alphacypermethrin were 85% and 86.8%, respectively. On the other hand, mortalities against pirmiphos-methyl, bendiocarb, propoxur and clothianidin were 100%, 99%, 100% and 100%, respectively. Of those sub-samples (An. amharicus and An. arabiensis) examined for presence of kdr gene, none of them were found to carry the L1014F (West African) allelic mutation.ConclusionAnopheles amharicus and An. arabiensis from Arjo-Didessa sugarcane irrigation area were resistant to pyrethroids which might be synergized by extensive use of agricultural chemicals. Occurrence of pyrethroid resistant malaria vectors could challenge the ongoing malaria control and elimination program in the area unless resistance management strategies are implemented. Given the resistance of An. amharicus to pyrethroids, its behavior and vectorial capacity should be further investigated.

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Project description:Malaria transmission is dependent on the propensity of Anopheles mosquitoes to bite humans (anthropophily) instead of other dead end hosts. Recent increases in the usage of Long Lasting Insecticide Treated Nets (LLINs) in Africa have been associated with reductions in highly anthropophilic and endophilic vectors such as Anopheles gambiae s.s., leaving species with a broader host range, such as Anopheles arabiensis, as the most prominent remaining source of transmission in many settings. An. arabiensis appears to be more of a generalist in terms of its host choice and resting behavior, which may be due to phenotypic plasticity and/or segregating allelic variation. To investigate the genetic basis of host choice and resting behavior in An. arabiensis we sequenced the genomes of 23 human-fed and 25 cattle-fed mosquitoes collected both in-doors and out-doors in the Kilombero Valley, Tanzania. We identified a total of 4,820,851 SNPs, which were used to conduct the first genome-wide estimates of "SNP heritability" for host choice and resting behavior in this species. A genetic component was detected for host choice (human vs cow fed; permuted P = 0.002), but there was no evidence of a genetic component for resting behavior (indoors versus outside; permuted P = 0.465). A principal component analysis (PCA) segregated individuals based on genomic variation into three groups which were characterized by differences at the 2Rb and/or 3Ra paracentromeric chromosome inversions. There was a non-random distribution of cattle-fed mosquitoes between the PCA clusters, suggesting that alleles linked to the 2Rb and/or 3Ra inversions may influence host choice. Using a novel inversion genotyping assay, we detected a significant enrichment of the standard arrangement (non-inverted) of 3Ra among cattle-fed mosquitoes (N = 129) versus all non-cattle-fed individuals (N = 234; χ2, p = 0.007). Thus, tracking the frequency of the 3Ra in An. arabiensis populations may be of use to infer selection on host choice behavior within these vector populations; possibly in response to vector control. Controlled host-choice assays are needed to discern whether the observed genetic component has a direct relationship with innate host preference. A better understanding of the genetic basis for host feeding behavior in An. arabiensis may also open avenues for novel vector control strategies based on driving genes for zoophily into wild mosquito populations.

Dataset Information

The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis.

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The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis.

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