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The normalised expression ratio of each contig against the RSP7 gene is represented on the primary vertical axis while the secondary vertical axis represents FUMOZ-R/Kela fold change of each contig.
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Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field str...
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Speciation with gene flow may be aided by reduced recombination helping to build linkage between genes involved in the early stages of reproductive isolation. Reduced recombination on chromosome X has been implicated in speciation within the Anopheles gambiae complex, species of which represent the major Afrotropical malaria vectors. The most recen...
Citations
... A detailed description of the sequence capture array is provided by Hearn et al. 2022 [35]. Briefly, the array was designed using a mix of de novo assembled An. funestus transcripts [37,38] and up-regulated genes in pyrethroid resistant populations [21,39]. The entire genomic regions of the major QTLs associated with pyrethroid resistance which are the 120kb BAC clone of the rp1 containing the major CYP6 P450 cluster on the 2R chromosome arm as well as the 113kb BAC clone sequence for the rp2 on the 2L chromosome arm were also included. ...
Cross-resistance to insecticides in multiple resistant malaria vectors is hampering resistance management. Understanding its underlying molecular basis is critical to implementation of suitable insecticide-based interventions. Here, we established that the tandemly duplicated cytochrome P450s, CYP6P9a/b are driving carbamate and pyrethroid cross-resistance in Southern African populations of the major malaria vector Anopheles funestus. Transcriptome sequencing revealed that cytochrome P450s are the most over-expressed genes in bendiocarb and permethrin-resistant An. funestus. The CYP6P9a and CYP6P9b genes are overexpressed in resistant An. funestus from Southern Africa (Malawi) versus susceptible An. funestus (Fold change (FC) is 53.4 and 17 respectively), while the CYP6P4a and CYP6P4b genes are overexpressed in resistant An. funestus in Ghana, West Africa, (FC is 41.1 and 17.2 respectively). Other up-regulated genes in resistant An. funestus include several additional cytochrome P450s (e.g. CYP9J5, CYP6P2, CYP6P5), glutathione-S transferases, ATP-binding cassette transporters, digestive enzymes, microRNA and transcription factors (FC
... The sequence capture array was designed prior to the release of the An. funestus genome assembly, using a mix of de novo assembled An. funestus transcripts (Crawford et al., 2010;Gregory et al., 2011) selected from previous pyrethroid resistance microarray experiments Riveron et al., 2013). Among these were heat shock proteins (HSPs), Odorant Binding Proteins and immune response genes such as serine peptidases, Anopheles gambiae detoxification genes sequences (282 genes) and all target-site resistance genes sequences from An. funestus. ...
Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi‐omics approach, followed‐up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field‐applicable markers to better track resistance Africa‐wide.
... Because AC microsatellite variants have much lower coverage than their AT counterpart, the 454-platform may experience difficulty in sequencing AC regions of homopolymers in variants and therefore yield low accuracy and variant detection Plasmodium genomes [80,81]. Owing to its long read capacity, 454-platform is used to generate de novo sequences in Anopheles funectus [82], and P. cynomolgi [83] in transcriptome profiling of pyrethroid resistant mosquitoes from China [75], transcriptome analysis of female An. albimanus [84] and analysis of species and genotype diversity in Gabon [85]. ...
Malaria is one of the most severe life-threatening human vector-borne diseases
worldwide today, leading to high mortality. Children under the age of five and pregnant women
in many developing countries are the most vulnerable groups. However, significant reduction has
been achieved globally. This is owed to the advancement in technology. Whole genome sequencing
(WGS) is such a high throughput technology, which provides unprecedented relevant information
concerning malaria parasite genomes used to study malaria pathology. Here, we present the roles
of WGS in malaria elimination. This review also found insufficient availability of WGS within sub Saharan Africa which bears the highest malaria burden and proposed that if malaria elimination is to
be achieved in this region, laboratories should be strategically equipped with WGS machines, where
clinical isolates could be received and processed affordably within the region.
... The thickness and composition of the cuticle have been identified as a critical determinant of insecticide resistance due to its role in reducing insecticide penetration (Balabanidou et al. 2018). Over expression of genes associated with formation and maintenance of the cuticle have been reported in insecticide resistant populations of medically relevant species including An. gambiae (Awolola et al. 2009, Balabanidou et al. 2016, Yahouédo et al. 2017, An. funestus (Giles, Diptera: Culicidae) (Gregory et al. 2011) and Culex pipiens pallens (Linnaeus, Diptera: Culicidae) (Pan et al. 2009, Fang et al. 2015. The cuticle has also been associated with resistance in Ae. aegypti including in larvae (David et al. 2010, Riaz et al. 2013. ...
Arboviruses including dengue, Zika, and chikungunya are amongst the most significant public health concerns worldwide. Arbovirus control relies on the use of insecticides to control the vector mosquito Aedes aegypti (Linnaeus), the success of which is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of Ae. aegypti larvae from field populations of Ae. aegypti with differential susceptibility to temephos originating from two Colombian urban locations, Bello and Cúcuta, previously reported to have distinctive disease incidence, socioeconomics, and climate. We demonstrated that an exclusive field-to-lab (Ae. aegypti strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes including detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long noncoding RNAs, and chromatin modelling among the differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to temephos. The results from the sampling triangulation approach here contribute a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity, increasing the number of potential targets for the control of insecticide resistant mosquitoes and widening our knowledge base on the complex phenotypic network of the Ae. aegypti response to insecticides.
... Upregulation of the GSTE2 gene is associated with resistance to both permethrin and deltamethrin, as well as DDT, in An. gambiae and An. coluzzii [70,72,76], An. funestus [29,71,74] and Ae. aegypti [77][78][79], and allele frequencies for targetsite mutations in the voltage-gated sodium channel gene, Vgsc, have been shown to be useful partial predictors of resistance in An. gambiae (s.l.) [35]. ...
Background
It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach.
Methods
Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations.
Results
We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations.
Conclusion
Importantly, the prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin and permethrin was correlated across malaria vector populations, and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.
... Upregulation of the GSTE2 gene is associated with resistance to both permethrin and deltamethrin, as well as DDT, in An. gambiae and An. coluzzii [71,73,77], An. funestus [29,72,75] and Ae. aegypti [78][79][80], and allele frequencies for target site mutations in the voltage-gated sodium channel gene, Vgsc, have been shown to be useful partial predictors of resistance in An. gambiae s.l. ...
The primary malaria control intervention in high burden countries is the deployment of long-lasting insecticide-treated nets (LLINs) treated with pyrethroids, alone or in combination with a second active ingredient or synergist. It is essential to understand whether the impact of pyrethroid resistance can be mitigated by switching between different pyrethroids or whether cross-resistance precludes this. Structural diversity within the pyrethroids could mean some compounds are better able to counteract the resistance mechanisms that have evolved in malaria vectors. Here we consider variation in vulnerability to the P450 enzymes that confer metabolic pyrethroid resistance in Anopheles gambiae s.l. and Anopheles funestus. We assess the relationships among pyrethroids in terms of their binding affinity to key P450s and the percent depletion by these P450s, in order to identify which pyrethroids diverge from the others. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. We found that etofenprox, which lacks the common structural moiety of other pyrethroids, potentially diverges from the commonly deployed pyrethroids in terms of P450 binding affinity and resistance in malaria vector populations, but not depletion by the P450s tested. These results are supplemented by an analysis of resistance to the same pyrethroids in Aedes aegypti populations, which also found etofenprox diverges from the other pyrethroids in terms of resistance in wild populations. In addition, we found that bifenthrin, which also lacks the common structural moiety of most pyrethroids, diverges from the commonly deployed pyrethroids in terms of P450 binding affinity and depletion by P450s. However, resistance to bifenthrin in vector populations is largely untested. The prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin, and permethrin was correlated across malaria vector populations and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance.
... This chip contains the 4 × 44 array (A-MEXP-2374) [10] plus an additional 15,527 expressed sequence tags (ESTs) generated from a transcriptome sequence analysis of An. funestus [19]. Overall, each array is incorporated with 60-mer probes designed from 8540 ESTs (2 probes for each EST) generated from An. funestus transcriptome 454 sequencing [20], a set of 2850 An. funestus cDNAs from GenBank (2 probes for each EST), a set of P450 genes (3 probes for each gene) from the rp1 and rp2 QTL BAC sequence [21,22], and the 13,000 transcripts of the complete An. gambiae genome. Also, all of the An. ...
... The expression pattern of known resistant-associated genes was evaluated in resistant samples compared to the unexposed control using the RT-PCR. This analysis was performed for some selected metabolic genes that were either identified in the microarray analysis or previously associated with permethrin and DDT resistance in An. funestus mosquitoes [8,11,20,21]. Metabolic genes analysed are glutathione S-transferase family (GSTe2, GSTd3, GSTd1-5); cytochrome P450 genes (CYP6P9a, CYP6P9b, CYP6P4a, CYP6P4b, CYP6M7, CYP6AA1, CYP4C27, CYP9K1); aldehyde oxidase (Ald oxi) and trypsin. This experiment was conducted using three batches of a pool of 10 resistant R perm and R DDT , unexposed control and FANG mosquitoes [10]. ...
Background:
Understanding the mechanisms used by Anopheles mosquitoes to survive insecticide exposure is key to manage existing insecticide resistance and develop more suitable insecticide-based malaria vector control interventions as well as other alternative integrated tools. To this regard, the molecular basis of permethrin, DDT and dieldrin resistance in Anopheles funestus (sensu stricto) at Akaka-Remo was investigated.
Methods:
Bioassays were conducted on 3-5-day-old adult An. funestus (s.s.) mosquitoes for permethrin, DDT and dieldrin susceptibility test. The molecular mechanisms of mosquito resistance to these insecticides were investigated using microarray and reverse transcriptase PCR techniques. The voltage-gated sodium channel region of mosquitoes was also screened for the presence of knockdown resistance mutations (kdr west and east) by sequencing method.
Results:
Anopheles funestus (s.s.) population was resistant to permethrin (mortality rate of 68%), DDT (mortality rate of 10%) and dieldrin (mortality rate of 8%) insecticides. Microarray and RT-PCR analyses revealed the overexpression of glutathione S-transferase genes, cytochrome P450s, esterase, trypsin and cuticle proteins in resistant mosquitoes compared to control. The GSTe2 was the most upregulated detoxification gene in permethrin-resistant (FC = 44.89), DDT-resistant (FC = 57.39) and dieldrin-resistant (FC = 41.10) mosquitoes compared to control population (FC = 22.34). The cytochrome P450 gene, CYP6P9b was also upregulated in both permethrin- and DDT-resistant mosquitoes. The digestive enzyme, trypsin (hydrolytic processes) and the cuticle proteins (inducing cuticle thickening leading to reduced insecticides penetration) also showed high involvement in insecticide resistance, through their overexpression in resistant mosquitoes compared to control. The kdr east and west were absent in all mosquitoes analysed, suggesting their non-involvement in the observed mosquito resistance.
Conclusions:
The upregulation of metabolic genes, especially the GSTe2 and trypsin, as well as the cuticle proteins is driving insecticide resistance of An. funestus (s.s.) population. However, additional molecular analyses, including functional metabolic assays of these genes as well as screening for a possible higher cuticular hydrocarbon and lipid contents, and increased procuticle thickness in resistant mosquitoes are needed to further describe their distinct roles in mosquito resistance.
... Previous studies on the molecular mechanisms of insecticide resistance have been performed at the transcriptional level, cDNA microarray [12,13], RNA-seq [14,15], leading to exciting progress with regard to the transcriptional basis of insecticide resistance. Dramatic progress has been made in identifying targetsite insensitivity in mosquitoess, such as Anopheles gambiae [16][17][18], An. arabiensis [19], Aedes albopictus [20], Culex pipiens [18,21]. ...
... In previous studies, some cuticle genes have been identi ed overexpressed in pyrethroid resistant strains of several mosquito species in An. stephensi [41], An. funestus [14] and Cx. pipiens pallens [34] and bed bug, Cimex lectularius [39,42,43]. ...
... In this study, P450 proteins whose expression levels changed signi cantly belonged primarily to the different families. In addition, data from many other studies are highlighting a key role for P450 in insecticide detoxi cation in Anopheles funestus [14], Culex quinquefasciatus [57,58]. Elevated levels of P450 activity are frequently observed in pyrethroid-resistant malaria vectors in Africa [59][60][61][62][63][64][65][66][67], in line with other insect pests such as the white y [68] and cockroach [69], over-expression of cytochrome P450s contribute to resistance to neonicotinoid insecticides in the hemipterans Bemisia tabaci and Myzus persicae [70,71]. ...
Background: Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of insecticide resistance of important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. As opposed to target site resistance, other mechanisms are far from being fully understood.
Results: Susceptible strain of Cx. pipiens pallen showed elevated resistance levels to after 25 generations insecticide-selected, through bioinformatics analysis allowed detecting 2,502 proteins, of which 1513 were differentially expression in insecticide-selected strains as compared to the susceptible strain. Finally, midgut differential expression protein profiles and 62 proteins were selected for verification of differential expression using parallel reaction monitoring strategy.
Conclusions Significant molecular resources were developed for Cx. pipiens pallen potential candidates involved in metabolic resistance as well as those participating in lower penetration or sequestration of insecticide. Global protein profiles of change to three insecticide strains combined with midgut profiles revealed multiple insecticide resistance mechanisms operate simultaneously in resistant insects of Cx. pipiens pallens. Future research that is targeted towards RNA interference on the identified metabolic targets such as cuticular, cytochrome P450s and glutathione S-transferase proteins could lay the foundation for a better understanding of the genetic basis of insecticide resistance in Cx. pipiens pallen.
... For example, 12 full-length CYP enzymes were discovered in the transcriptome of Zygaena filipendulae, many of which became candidates for the biosynthesis of cyanogenic glucosides (Zagrobelny et al., 2009), defense chemicals that are widespread in plants that are sequestered by several insect specialists or, in the case of Z. filipendulae, also synthesized de novo. A lifestage comprehensive Anopheles funestus transcriptome was sequenced with 454 technologies (Gregory et al., 2011), and this work helped in the identification of a homolog of the An. gambiae CYP4H18 as the most differentially expressed contig in a pyrethroid-resistant strain. ...
This book introduces transcriptomics and presents an array of its uses in entomology, past and present. It starts with a thorough introduction to how transcriptomics works, its history, and some of its broad-stroke uses in insect science (Chapter 1). This chapter is followed by an exhaustively comprehensive look at how transcriptomics analysis is performed, and the software packages available to help one go from next-generation sequencing data to an interpretable dataset (Chapter 2). It continues with reviews of how transcriptomics has been used within larger subfields of entomology, showing different applications of the basic techniques covered. Transcriptomics and other next-generation sequencing technologies are ushering in radical new ways to approach pest management by finding new targets for control, genes responsible for pesticide resistance, and even novel pathogens (Chapter 3). This is exemplified by the many studies done on the aphids, often with non-model but economically significant species for whom genomic data does not exist, which have succeeded in finding critical genes involved in aphid-plant interactions and host specificity and finding targets for biocontrol by blocking transcription of key genes (Chapter 4). Identifying new pathogens has been particularly important for honey bees ( Apis mellifera ), where transcriptomics revealed several new pathogens with potential links to Colony Collapse Disorder (Chapter 5). Discoveries within insects can have implications throughout biology: researchers use transcriptomics to accelerate the discovery of certain large yet conserved gene families, such as the hyper-diverse and multifunctional cytochrome P450s of interest to toxicologists, physiologists, agriculturalists, pharmacologists, and more (Chapter 6). The book ends with case studies going into more depth on how transcriptomics has been used to reveal more specific facets of a particular system. The cases presented include untangling insect-microbe interactions in cryptic parasitoid wasps ( Megastigmus spermotrophus ) (Chapter 7), identifying conserved insect digestive enzymes from the silverfish ( Ctenolepisma longicaudata ) transcriptome (Chapter 8), discovering the function of mysterious organs in the stick insects (Phasmatodea) (Chapter 9), and using functional transcriptomics to describe the chemical defenses of the bombardier beetle ( Brachinus elongatulus ) (Chapter 10).
... 66 Cuticular resistance has been mentioned often recently in Anopheles mosquitoes. [67][68][69][70][71] In Ae. aegypti, differential expression of genes encoding cuticle proteins has been reported in mosquito larvae. 60,72 The overexpression of multiple genes encoding cuticle proteins has been found in mosquito larvae that were selected with imidacloprid for several generations in the laboratory. ...
The mosquito Aedes aegypti is a transmission vector for dangerous epidemic diseases in humans. Insecticides have been used as the most general vector control method in the world. However, Ae. aegypti have developed many resistant mechanisms such as reduced neuronal sensitivity to insecticides (target-site resistance), enhanced insecticide metabolism (metabolic resistance), altered transport, sequestration, and other mechanisms. It has become a major problem for vector control programs. Transcriptome sequencing and bioinformatic analysis were used to compare transcription levels between a susceptible strain (Bora7) and a resistant strain (KhanhHoa7) collected from the field. A total of 161 million Illumina reads, including 66,076,678 reads from the Bora7 strain and 69,606,654 reads from the KhanhHoa7 strain, were generated and assembled into 11,174 genes. A comparison of the KhanhHoa7 transcriptome to that of Bora7 showed 672 upregulated genes and 488 downregulated genes. We identified the highly upregulated genes: cytochrome P450 4C1, 4C3, 4C21, 4D1, 4D1 isoform X2, 4D2, 4D2 isoform X2, 4G15, 6A2, 6A8, 6D3, and 9E2; Glutathione S transferase (GST1), UGT1-3, 1-7, 2B15, and 2B37; ABC transporter F family member 4 and ABC transporter G family member 20. Interestingly, there was a significant increase in the expression of the genes such as CYP9E2 (8.3-fold), CYP6A8 (5.9-fold), CYP6D3 (5.4-fold), CYP4C21 (5.4-fold), CYP4G15 (5.2-fold), GST1 (3.5-fold), and ABC transporter 4 (2.1-fold). Our results suggested a potential relationship between the expression of the genes in metabolic processes and insecticide resistance in the studied strain. These results may contribute to the understanding of the mechanisms of insecticide resistance in Ae. aegypti.
