Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali.
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ABSTRACT:
SUBMITTER: Main BJ
PROVIDER: S-EPMC4615556 | biostudies-literature | 2015 Oct
REPOSITORIES: biostudies-literature
Publications
Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali.
Main Bradley J BJ Lee Yoosook Y Collier Travis C TC Norris Laura C LC Brisco Katherine K Fofana Abdrahamane A Cornel Anthony J AJ Lanzaro Gregory C GC
Molecular ecology 20151006 20
In certain cases, a species may have access to important genetic variation present in a related species via adaptive introgression. These novel alleles may interact with their new genetic background, resulting in unexpected phenotypes. In this study, we describe a selective sweep on standing variation on the X chromosome in the mosquito Anopheles coluzzii, a principal malaria vector in West Africa. This event may have been influenced by the recent adaptive introgression of the insecticide resist ...[more]
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Project description:BackgroundMalaria mortality rates in sub-Saharan Africa have declined significantly in recent years as a result of increased insecticide-treated bed net (ITN) usage. A major challenge to further progress is the emergence and spread of insecticide resistance alleles in the Anopheles mosquito vectors, like An. coluzzii. A non-synonymous mutation in the para voltage-gated sodium channel gene reduces pyrethroid-binding affinity, resulting in knockdown resistance (kdr). Metabolic mechanisms of insecticide resistance involving detoxification genes like cytochrome P450 genes, carboxylesterases, and glutathione S-transferases are also important. As some gene activity is tissue-specific and/or environmentally induced, gene regulatory variation may be overlooked when comparing expression from whole mosquito bodies under standard rearing conditions.ResultsWe detected complex insecticide resistance in a 2014 An. coluzzii colony from southern Mali using bottle bioassays. Additional bioassays involving recombinant genotypes from a cross with a relatively susceptible 1995 An. coluzzii colony from Mali confirmed the importance of kdr and associated increased permethrin resistance to the CYP9K1 locus on the X chromosome. Significant differential expression of CYP9K1 was not observed among these colonies in Malpighian tubules. However, the P450 gene CYP6Z1 was overexpressed in resistant individuals following sublethal permethrin exposure and the carboxylesterase gene COEAE5G was constitutively overexpressed.ConclusionsThe significant P450-related insecticide resistance observed in the 2014 An. coluzzii colony indicates that ITNs treated with the P450 inhibitor piperonyl butoxide (PBO) would be more effective in this region. The known insecticide resistance gene CYP6Z1 was differentially expressed exclusively in the context of sublethal permethrin exposure, highlighting the importance of tissue-specificity and environmental conditions in gene expression studies. The increased activity of the carboxylesterase COEAE5G in the resistant An. coluzzii colony suggests resistance to other insecticides like organophosphates. Additional gene expression studies involving other tissues (e.g. fat body) would provide a more comprehensive view of genes underlying metabolic insecticide resistance in An. coluzzii from Mali. Identifying genetic markers linked to these regulatory alleles is an important next step that would substantially improve insecticide resistance surveillance and population genetic studies in this important vector species.
| S-EPMC5885317 | biostudies-literature
Project description:The poorly understood mechanisms of dry season persistence of Anopheles spp. mosquitoes through the dry season in Africa remain a critical gap in our knowledge of Plasmodium disease transmission. While it is thought that adult mosquitoes remain in a dormant state throughout this seven-month dry season, the nature of this state remains unknown and has largely not been recapitulated in laboratory settings. To elucidate possible connections of this state with microbial composition, the whole body microbiomes of adult mosquitoes in the dry and wet seasons in two locations of Mali with varying water availability were compared by sequencing the 16S ribosomal RNA gene. These locations were a village near the Niger River with year-round water sources (N'Gabakoro, "riparian"), and a typical Sahelian area with highly seasonal breeding sites (Thierola Area, "Sahelian"). The 16S bacterial data consisted of 2057 sequence variants in 426 genera across 184 families. From these data, we found several compositional differences that were seasonally and spatially linked. Counter to our initial hypothesis, there were more pronounced seasonal differences in the bacterial microbiome of riparian, rather than Sahelian areas. These seasonal shifts were primarily in Ralstonia, Sphingorhabdus, and Duganella spp. bacteria that are usually soil and water-associated, indicating these changes may be from bacteria acquired in the larval environment, rather than adulthood. In Sahelian dry season mosquitoes, there was a unique intracellular bacteria, Anaplasma, which likely was acquired through non-human blood feeding. Cytochrome B analysis of blood meals showed greater heterogeneity in host choice of An. coluzzii independent of season in the Thierola area compared to N'Gabakoro (77.5% vs. 94.6% human-origin blood meal, respectively), indicating a relaxation of anthropophily. Overall, this exploratory study provides valuable indications of spatial and seasonal differences in bacterial composition which help refine this difficult to study state.
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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.
| S-EPMC8146665 | biostudies-literature
Project description:Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.
| S-EPMC8741062 | biostudies-literature
Project description:BackgroundThe Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations of Anopheles coluzzii from Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.MethodsBloodfed female Anopheles gambiae s.l. resting indoor, were collected at N'djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) and ace-1 markers were investigated by TaqMan genotyping.ResultsAnopheles coluzzii was the major vector found in N'djamena (100%) and Massakory (~ 94%). No Plasmodium was found in 147 bloodfed F0 An. coluzzii (82 from N'djamena and 65 from Massakory). High intensity pyrethroid resistance was observed with mortalities of < 2% for permethrin, deltamethrin and etofenprox, and with < 50% and < 60% dead following exposure to 10× diagnostic doses of deltamethrin and permethrin, respectively. For both sites, < 10% mortalities were observed with DDT. Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations, implicating CYP450s (mortality = 13.6% for permethrin, χ2 = 22.8, df = 1, P = 0.0006; mortality = 13.0% for deltamethrin, χ2 = 8.8, df = 1, P < 0.00031). Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs; and a 100% recovery of susceptibility following exposure to the roof of PermaNet®3.0, containing piperonylbutoxide. Both populations were susceptible to malathion, but high bendiocarb resistance was observed in Massakory population. The absence of ace-1 mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and < 20% respectively. Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism.ConclusionsMultiple resistance in An. coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.
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Project description:BackgroundThe wetlands used for some agricultural activities constitute productive breeding sites for many mosquito species. Thus, the agricultural use of insecticide targeting other pests may select for insecticide resistance in malaria mosquitoes. The purpose of this study is to clarify some knowledge gaps on the role of agrochemicals in the development of insecticide resistance in malaria vectors is of utmost importance for vector control.MethodsUsing the CDC bottle test and the log-probit analysis, we investigated for the first time the resistance levels of Anopheles coluzzii mosquitoes to neonicotinoids, insecticides used exclusively for crop protection in Côte d'Ivoire. The study was conducted in two agricultural regions (Tiassale and Gagnoa) and one non-agricultural region (Vitre) between June and August 2017 using clothianidin, acetamiprid and imidacloprid.ResultsMosquito populations from Tiassale and Gagnoa (agricultural settings) were determined to be resistant to acetamiprid with mortality rates being < 85% at 24 h post-exposure. In Vitre (non-agricultural area) however, the mosquito population was susceptible to acetamiprid. In all three localities, mosquito populations were resistant to imidacloprid (mortality rates were 60% in Vitre, 37% in Tiassale, and 13% in Gagnoa) and completely susceptible to clothianidin (100% mortality). An. coluzzii represented 100% of mosquito collected in Gagnoa, 86% in Tiassale and 96% in Vitre.ConclusionsThis study provides strong evidence that agricultural use of insecticides can cause insecticide resistance in malaria vector populations. Insecticide resistance driven by agrochemical usage should be considered when vector control strategies are developed.
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Project description:BackgroundAnopheles coluzzii and Anopheles arabiensis belong to the Anopheles gambiae complex and are among the major malaria vectors in sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species.FindingsIn this study, we produced de novo chromosome-level genome assemblies for A. coluzzii and A. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 and 256.8 Mb of the total assemblies for A. coluzzii and A. arabiensis, respectively. Each assembly consists of 3 chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomic coordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in A. coluzzii and A. arabiensis.ConclusionThe new chromosome-level assemblies will facilitate functional and population genomic studies in A. coluzzii and A. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.
| S-EPMC7957348 | biostudies-literature
Project description:Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multi-allelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and may form the basis for improved surveillance methods.
| S-EPMC10432508 | biostudies-literature
Project description:The emergence and spread of insecticide resistance among the main malaria vectors is threatening the effectiveness of vector control interventions in Senegal. The main drivers of this resistance in the Anopheles gambiae complex (e.g., An. gambiae and Anopheles coluzzii) remains poorly characterized in Senegal. Here we characterized the main target site and metabolic resistances mechanisms among the An. gambiae and An. coluzzii populations from their sympatric and allopatric or predominance area in Senegal. Larvae and pupae of An. gambiae s.l. were collected, reared to adulthood, and then used for insecticides susceptibility and synergist assays using the WHO (World Health Organisation) test kits for adult mosquitoes. The TaqMan method was used for the molecular characterization of the main target site insecticide resistance mechanisms (Vgsc-1014F, Vgsc-1014S, N1575Y and G119S). A RT-qPCR (Reverse Transcriptase-quantitative Polymerase Chaine Reaction) was performed to estimate the level of genes expression belonging to the CYP450 (Cytochrome P450) family. Plasmodium infection rate was investigated using TaqMan method. High levels of resistance to pyrethroids and DDT and full susceptibility to organophosphates and carbamates where observed in all three sites, excepted a probable resistance to bendiocarb in Kedougou. The L1014F, L1014S, and N1575Y mutations were found in both species. Pre-exposure to the PBO (Piperonyl butoxide) synergist induced a partial recovery of susceptibility to permethrin and full recovery to deltamethrin. Subsequent analysis of the level of genes expression, revealed that the CYP6Z1 and CYP6Z2 genes were over-expressed in wild-resistant mosquitoes compared to the reference susceptible strain (Kisumu), suggesting that both the metabolic resistance and target site mutation involving kdr mutations are likely implicated in this pyrethroid resistance. The presence of both target-site and metabolic resistance mechanisms in highly pyrethroid-resistant populations of An. gambiae s.l. from Senegal threatens the effectiveness and the sustainability of the pyrethroid-based tools and interventions currently deployed in the country. The Kdr-west mutation is widely widespread in An. coluzzii sympatric population. PBO or Duo nets and IRS (Indoor Residual Spraying) with organophosphates could be used as an alternative measure to sustain malaria control in the study area.
| S-EPMC7760107 | biostudies-literature
Project description:BackgroundInformation on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon.ResultsGenome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450.ConclusionsThese findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.
| S-EPMC10210336 | biostudies-literature