RESUMO
BACKGROUND: Malaria control is heavily reliant on the use of insecticides that target and kill the adult female Anopheline vector. The intensive use of insecticides of the pyrethroid class has led to widespread resistance in mosquito populations. The intensity of pyrethroid resistance in some settings in Africa means mosquitoes can contact bednets treated with this insecticide class multiple times with minimal mortality effects. Furthermore, both ageing and diel cycle have been shown to have large impacts on the resistance phenotype. Together, these traits may affect other aspects of vector biology controlling the vectorial capacity or fitness of the mosquito. RESULTS: Here we show that sublethal exposure of a highly resistant Anopheles coluzzii population originally from Burkina Faso to the pyrethroid deltamethrin results in large and sustained changes to transcript expression. We identify five clear patterns in the data showing changes to transcripts relating to: DNA repair, respiration, translation, behaviour and oxioreductase processes. Further, we highlight differential regulation of transcripts from detoxification families previously linked with insecticide resistance, in addition to clear down-regulation of the oxidative phosphorylation pathway both indicative of changes in metabolism post-exposure. Finally, we show that both ageing and diel cycle have major effects on known insecticide resistance related transcripts. CONCLUSION: Sub-lethal pyrethroid exposure, ageing and the diel cycle results in large-scale changes in the transcriptome of the major malaria vector Anopheles coluzzii. Our data strongly supports further phenotypic studies on how transcriptional changes such as reduced expression of the oxidative phosphorylation pathway or pyrethroid induced changes to redox state might impact key mosquito traits, such as vectorial capacity and life history traits.
Assuntos
Anopheles , Inseticidas , Malária , Piretrinas , Envelhecimento/genética , Animais , Anopheles/genética , Burkina Faso , Feminino , Resistência a Inseticidas/genética , Inseticidas/toxicidade , Controle de Mosquitos , Mosquitos Vetores/genética , Piretrinas/toxicidade , TranscriptomaRESUMO
BACKGROUND: Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes' legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. RESULTS: Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. CONCLUSIONS: The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.
Assuntos
Anopheles , Inseticidas , Piretrinas , Animais , Anopheles/genética , Humanos , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Perna (Membro) , Mosquitos Vetores/genética , Proteômica , Piretrinas/toxicidade , TranscriptomaRESUMO
The role of ATP-binding cassette (ABC) transporters in conferring insecticide resistance has received much attention recently. Here we identify ABC transporters differentially expressed in insecticide-resistant populations of the malaria vector, Anopheles gambiae. Although we found little evidence that the orthologues of the multidrug resistance proteins described in other species are associated with resistance in An. gambiae we did identify a subset of ABC proteins consistently differentially expressed in pyrethroid-resistant populations from across Africa. We present information on the phylogenetic relationship, primary sites of expression and potential role of ABC transporters in mediating the mosquito's response to insecticides. Furthermore we demonstrate that a paralogous group of eight ABCG transporters, clustered on chromosome 3R, are highly enriched in the legs of An. gambiae mosquitoes, consistent with a proposed role for this ABC subfamily in transport of lipids to the outer surface of the cuticle. Finally, antibodies raised against one of the most highly expressed ABC transporters in adult females, ABCG7 (AGAP009850), localized this transporter to the pericardial cells. These data will help prioritize members of this gene family for further localization and functional validation studies to identify the in vivo function of these transporters in the mosquito and determine whether elevated expression of members of this family contribute to insecticide resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Anopheles/fisiologia , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Piretrinas/farmacologia , Regulação para Cima , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Anopheles/genética , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Família Multigênica/genética , FilogeniaRESUMO
Malaria control is dependent on the use of longlasting insecticidal nets (LLINs) containing pyrethroids. A new generation of LLINs containing both pyrethroids and the synergist piperonyl butoxide (PBO) has been developed in response to increasing pyrethroid resistance in African malaria vectors, but questions remain about the performance of these nets in areas where levels of pyrethroid resistance are very high. This study was conducted in two settings in southwest Burkina Faso, Vallée du Kou 5 and Tengrela, where Anopheles gambiae s.l. (Diptera: Culicidae) mortality rates in World Health Organization (WHO) discriminating dose assays were < 14% for permethrin and < 33% for deltamethrin. When mosquitoes were pre-exposed to PBO in WHO tube assays, mortality rates increased substantially but full susceptibility was not restored. Molecular characterization revealed high levels of kdr alleles and elevated levels of P450s previously implicated in pyrethroid resistance. In cone bioassays and experimental huts, PBO LLINs outperformed the pyrethroid-only equivalents from the same manufacturers. Blood feeding rates were 1.6-2.2-fold lower and mortality rates were 1.69-1.78-fold greater in huts with PBO LLINs vs. non-PBO LLINs. This study indicates that PBO LLINs provide greater personal and community-level protection than standard LLINs against highly pyrethroid-resistant mosquito populations.
Assuntos
Anopheles , Resistência a Inseticidas , Mosquiteiros Tratados com Inseticida , Inseticidas , Butóxido de Piperonila , Piretrinas , Animais , Anopheles/genética , Bioensaio , Burkina Faso , DNA/genética , DNA/isolamento & purificação , Feminino , Habitação , Humanos , Malária/prevenção & controle , Masculino , Mutação , RNA/análise , RNA/isolamento & purificação , TranscriptomaRESUMO
The U.K. has not yet experienced a confirmed outbreak of mosquito-borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito-borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito-borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field-collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7-21 days post-infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/transmissão , Insetos Vetores/virologia , Ochlerotatus/virologia , Aedes/fisiologia , Aedes/virologia , Animais , Encefalite Japonesa/virologia , Inglaterra , Feminino , Temperatura Alta , Humanos , Insetos Vetores/fisiologia , Ochlerotatus/fisiologiaRESUMO
The mosquito Aedes aegypti is the main vector of Dengue and Yellow Fever flaviviruses. The organophosphate insecticide temephos is a larvicide that is used globally to control Ae. aegypti populations; many of which have in turn evolved resistance. Target site alteration in the acetylcholine esterase of this species has not being identified. Instead, we tracked changes in transcription of metabolic detoxification genes using the Ae. aegypti 'Detox Chip' microarray during five generations of temephos selection. We selected for temephos resistance in three replicates in each of six collections, five from Mexico, and one from Peru. The response to selection was tracked in terms of lethal concentrations. Uniform upregulation was seen in the epsilon class glutathione-S-transferase (eGST) genes in strains from Mexico prior to laboratory selection, while eGSTs in the Iquitos Peru strain became upregulated after five generations of temephos selection. While expression of many carboxyl/cholinesterase esterase (CCE) genes increased with selection, no single esterase was consistently upregulated and this same pattern was noted in the cytochrome P450 monooxygenase (CYP) genes and in other genes involved in reduction or oxidation of xenobiotics. Bioassays using glutathione-S-transferase (GST), CCE and CYP inhibitors suggest that various CCEs instead of GSTs are the main metabolic mechanism conferring resistance to temephos. We show that temephos-selected strains show no cross resistance to permethrin and that genes associated with temephos selection are largely independent of those selected with permethrin in a previous study.
Assuntos
Aedes/genética , Resistência a Inseticidas , Inseticidas/farmacologia , Seleção Genética , Temefós/farmacologia , Aedes/efeitos dos fármacos , Aedes/crescimento & desenvolvimento , Aedes/metabolismo , Animais , Perfilação da Expressão Gênica , Larva/efeitos dos fármacos , Larva/genética , Larva/metabolismo , México , Análise de Sequência com Séries de Oligonucleotídeos , Peru , Reação em Cadeia da Polimerase em Tempo Real , Transcrição GênicaRESUMO
Prevention of malaria transmission throughout much of Africa is dependent on bednets that are impregnated with pyrethroid insecticides. Anopheles arabiensis is the major malaria vector in Chad and efforts to control this vector are threatened by the emergence of pyrethroid resistance. WHO bioassays revealed that An. arabiensis from Ndjamena is resistant to pyrethroids and dichlorodiphenyltrichloroethane (DDT) but fully susceptible to carbamates and organophosphates. No 1014F or 1014S kdr alleles were detected in this population. To determine the mechanisms that are responsible for resistance, genetic crosses were established between the Ndja strain and an insecticide susceptible population from Mozambique. Resistance was inherited as an autosomal trait and quantitative trait locus (QTL) mapping identified a single major locus on chromosome 2R, which explained 24.4% of the variance in resistance. This QTL is enriched in P450 genes including 25 cytochrome P450s in total. One of these, Cyp6p4 is 22-fold upregulated in the Ndja strain compared with the susceptible. Piperonyl butoxide (PBO) synergist and biochemical assays further support a role for P450s in conferring pyrethroid resistance in this population.
Assuntos
Anopheles/genética , Sistema Enzimático do Citocromo P-450/genética , Resistência a Inseticidas/genética , Malária/genética , Animais , Anopheles/efeitos dos fármacos , Chade , Mapeamento Cromossômico , Diclorodifenildicloroetano/toxicidade , Expressão Gênica/efeitos dos fármacos , Humanos , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/genética , Inseticidas/farmacologia , Malária/transmissão , Piretrinas/toxicidade , Locos de Características Quantitativas/genéticaRESUMO
Changes in gene expression before, during and after five generations of permethrin laboratory selection were monitored in six strains of Aedes aegypti: five F(2)-F(3) collections from the Yucatán Peninsula of Mexico and one F(2) from Iquitos, Peru. Three biological replicate lines were generated for each strain. The response to selection was measured as changes in the lethal and knockdown permethrin concentrations (LC(50), KC(50)) and in the frequency of the Ile1,016 substitution in the voltage-gated sodium channel (para) gene. Changes in expression of 290 metabolic detoxification genes were measured using the 'Aedes Detox' microarray. Selection simultaneously increased the LC(50), KC(50) and Ile1,016 frequency. There was an inverse relationship between Ile1,016 frequency and the numbers of differentially transcribed genes. The Iquitos strain lacked the Ile1,016 allele and 51 genes were differentially transcribed after selection as compared with 10-18 genes in the Mexican strains. Very few of the same genes were differentially transcribed among field strains but 10 cytochrome P(450) genes were upregulated in more than one strain. Laboratory adaptation to permethrin in Ae. aegypti is genetically complex and largely conditioned by geographic origin and pre-existing target site insensitivity in the para gene. The lack of uniformity in the genes that responded to artificial selection as well as differences in the direction of their responses challenges the assumption that one or a few genes control permethrin metabolic resistance. Attempts to identify one or a few metabolic genes that are predictably associated with permethrin adaptation may be futile.
Assuntos
Aedes/metabolismo , Inseticidas , Permetrina , Seleção Genética , Aedes/genética , Animais , Feminino , Perfilação da Expressão Gênica , Inativação Metabólica/genética , Resistência a Inseticidas/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição GênicaRESUMO
Aedes aegypti (L.) (Diptera: Culicidae) control programmes in Cuba rely on the application of the organophosphate temephos for larval control. Hence, the monitoring of resistance to this insecticide is an essential component of such programmes. Here, 15 field populations from different municipalities of Havana City were assayed for resistance to temephos. High levels of resistance were detected in all strains and resistance ratios were highly correlated with esterase activity (P = 0.00001). Populations from three municipalities were tested in both 2006 and 2008; resistance and esterase activities both significantly increased during this 2-year period. Synergist studies demonstrated that neither glutathione transferases nor monooxygenases were associated with the increase in resistance to temephos in this period. The duration of the efficacy of commercial formulations of temephos in controlling Ae. aegypti populations in Havana City was reduced by the high level of temephos resistance observed; hence these data are of clear operational significance for the dengue control programme in Cuba. New integrated strategies to avoid further increases in temephos resistance in Cuba are necessary.
Assuntos
Aedes/efeitos dos fármacos , Esterases/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Temefós/farmacologia , Aedes/enzimologia , Animais , Cuba , Demografia , Larva/efeitos dos fármacos , Fatores de TempoRESUMO
The numbers of glutathione S-transferase, cytochrome P450 and esterase genes in the genome of the hymenopteran parasitoid Nasonia vitripennis are about twice those found in the genome of another hymenopteran, the honeybee Apis mellifera. Some of the difference is associated with clades of these families implicated in xenobiotic resistance in other insects and some is in clades implicated in hormone and pheromone metabolism. The data support the hypothesis that the eusocial behaviour of the honeybee and the concomitant homeostasis of the nest environment may obviate the need for as many gene/enzyme systems associated with xenobiotic metabolism as are found in other species, including N. vitripennis, that are thought to encounter a wider range of potentially toxic xenobiotics in their diet and habitat.
Assuntos
Hidrolases de Éster Carboxílico/genética , Sistema Enzimático do Citocromo P-450/genética , Variação Genética , Glutationa Transferase/genética , Filogenia , Vespas/enzimologia , Animais , Hidrolases de Éster Carboxílico/metabolismo , Mapeamento Cromossômico , Análise por Conglomerados , Biologia Computacional , Sistema Enzimático do Citocromo P-450/metabolismo , Genômica , Glutationa Transferase/metabolismo , Modelos Genéticos , Receptores Odorantes/metabolismo , Especificidade da Espécie , Xenobióticos/metabolismoRESUMO
Anopheles funestus Giles is one of the major African malaria vectors. It has previously been implicated in a major outbreak of malaria in KwaZulu/Natal, South Africa, during the period 1996 to 2000. The re-emergence of this vector was associated with monooxygenase-based resistance to pyrethroid insecticides. We have identified a gene from the monooxygenase CYP6 family, CYP6P9, which is over expressed in a pyrethroid resistant strain originating from Mozambique. Quantitative Real-Time PCR shows that this gene is highly over expressed in the egg and adult stages of the resistant strain relative to the susceptible strain but the larval stages showed almost no difference in expression between strains. This gene is genetically linked to a major locus associated with pyrethroid resistance in this A. funestus population.
Assuntos
Anopheles/enzimologia , Sistema Enzimático do Citocromo P-450/biossíntese , Insetos Vetores/enzimologia , Resistência a Inseticidas , Piretrinas , África Subsaariana , Animais , Anopheles/genética , Anopheles/crescimento & desenvolvimento , Sequência de Bases , Northern Blotting , Sistema Enzimático do Citocromo P-450/genética , Feminino , Insetos Vetores/genética , Insetos Vetores/crescimento & desenvolvimento , Inseticidas , Isoenzimas , Masculino , Dados de Sequência Molecular , Permetrina , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-expressing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K(m) = 0.13 microM; K(cat) = 1.5 min(-1). The IC(50) values of a wide range of compounds were measured. Pyrethroids cypermethrin and permethrin produced low IC(50) values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the herbivorous phase of the life-cycle.
Assuntos
Anopheles/enzimologia , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Insetos Vetores/enzimologia , Inseticidas/farmacologia , Piretrinas/farmacologia , Laranja de Acridina , Sequência de Aminoácidos , Animais , Anopheles/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , DNA/química , DNA/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Concentração Inibidora 50 , Insetos Vetores/genética , Resistência a Inseticidas , Inseticidas/farmacocinética , Isoenzimas , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Piretrinas/farmacocinética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de SequênciaRESUMO
Increasing insecticide resistance in malaria-transmitting vectors represents a public health threat, but underlying mechanisms are poorly understood. Here, a data integration approach is used to analyse transcriptomic data from comparisons of insecticide resistant and susceptible Anopheles populations from disparate geographical regions across the African continent. An unbiased, integrated analysis of this data confirms previously described resistance candidates but also identifies multiple novel genes involving alternative resistance mechanisms, including sequestration, and transcription factors regulating multiple downstream effector genes, which are validated by gene silencing. The integrated datasets can be interrogated with a bespoke Shiny R script, deployed as an interactive web-based application, that maps the expression of resistance candidates and identifies co-regulated transcripts that may give clues to the function of novel resistance-associated genes.
Assuntos
Anopheles/efeitos dos fármacos , Anopheles/genética , Proteínas de Insetos/genética , Resistência a Inseticidas , Transcriptoma , Animais , Anopheles/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Inseticidas/farmacologiaRESUMO
The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed.
Assuntos
Aedes/enzimologia , Sistema Enzimático do Citocromo P-450/fisiologia , Proteínas de Insetos/fisiologia , Xenobióticos/farmacologia , Adaptação Biológica , Aedes/efeitos dos fármacos , Aedes/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Meio Ambiente , Expressão Gênica , Genoma de Inseto , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/enzimologia , Dados de Sequência Molecular , Família Multigênica/fisiologia , Folhas de Planta/metabolismo , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
Insect class I glutathione S-transferases (GSTs) were expressed from cDNA obtained from larvae of the Thai malaria vector. Anopheles dirus in a PCR RACE (rapid amplification of cDNA ends) reaction using a primer to the conserved N-terminal region of An. gambiae class I GSTs and a synthetic oligo d(T)-adaptor primer. Seven different plasmids, resulting from sub-cloning of an original single 0.7 Kb PCR band, were picked at random and sequenced. Four of these were clearly GSTs on the basis of putative amino acid sequence conservation. All the sequences had a conserved N-terminal region, but were highly divergent at the C-terminus. The variability in the PCR products suggests that there is a high level of GST class I isoenzyme variability in larval An. dirus. One of the subclones from the PCR reaction contained a full coding region of the cDNA for GST. This had a putative amino acid sequence which was 76 and 91% identity to the An. gambiae GST class I, agGST 1-5 and agGST 1-6 respectively, but only 48% identity to agGST 1-2. The catalytically active enzyme, expressed in Escherichia coli, was strongly immuno-cross reactive with antisera raised against the two An. gambiae class I GSTs. The expressed enzyme was purified to homogeneity from an E. coli cell lysate by S-hexylglutathione agarose affinity chromatography. The enzyme had a high specific activity with CDNB, and also used DCNB and ethacrynic acid as substrates. In addition, it had peroxidase and DDTase activity and its activity with CDNB, was strongly inhibited by a range of organophosphorus and pyrethroid insecticides. This is consistent with the predicted role of this GST class in insecticide resistance.
Assuntos
Anopheles/enzimologia , Anopheles/genética , Glutationa Transferase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli/genética , Expressão Gênica , Glutationa Transferase/classificação , Glutationa Transferase/metabolismo , Humanos , Insetos Vetores/enzimologia , Insetos Vetores/genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Malária/transmissão , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Pyrethroid insecticide resistance in Anopheles gambiae sensu stricto is a major concern to malaria vector control programmes. Resistance is mainly due to target-site insensitivity arising from a single point mutation, often referred to as knockdown resistance (kdr). Metabolic-based resistance mechanisms have also been implicated in pyrethroid resistance in East Africa and are currently being investigated in West Africa. Here we report the co-occurrence of both resistance mechanisms in a population of An. gambiae s.s. from Nigeria. Bioassay, synergist and biochemical analysis carried out on resistant and susceptible strains of An. gambiae s.s. from the same geographical area revealed >50% of the West African kdr mutation in the resistant mosquitoes but <3% in the susceptible mosquitoes. Resistant mosquitoes synergized using pyperonyl butoxide before permethrin exposure showed a significant increase in mortality compared with the non-synergized. Biochemical assays showed an increased level of monooxygenase but not glutathione-S-transferase or esterase activities in the resistant mosquitoes. Microarray analysis using the An. gambiae detox-chip for expression of detoxifying genes showed five over-expressed genes in the resistant strain when compared with the susceptible one. Two of these, CPLC8 and CPLC#, are cuticular genes not implicated in pyrethroid metabolism in An. gambiae s.s, and could constitute a novel set of candidate genes that warrant further investigation.
Assuntos
Anopheles/genética , Resistência a Inseticidas/genética , Larva/genética , Mutação Puntual/genética , Animais , Anopheles/efeitos dos fármacos , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Malária/genética , Malária/transmissão , Controle de Mosquitos , Nigéria , Análise de Sequência com Séries de Oligonucleotídeos , Piretrinas/farmacologiaRESUMO
A large scale microarray (20k MMC1) from the African malaria vector Anopheles gambiae was used to monitor gene expression in insecticide resistant and susceptible strains of the Asian mosquito Anopheles stephensi. Heterologous hybridization at slightly reduced stringency yielded approximately 7000 significant signals. Thirty-six putative genes were differentially transcribed between the pyrethroid-resistant (DUB-R) and the susceptible (BEECH) strains. The expression profiles of selected transcripts were verified by real-time PCR. A gene putatively involved in the thickening of the adult cuticle showed the most striking up-regulation in DUB-R. A more specialized microarray containing 231 An. gambiae genes putatively involved in insecticide detoxification was used to further analyse classical insecticide resistance genes. Three glutathione S-transferase (GST) transcripts, one esterase and a cytochrome P450 were up-regulated in the resistant strain, while two peroxidases were down-regulated.
Assuntos
Anopheles/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Proteínas de Insetos/metabolismo , Resistência a Inseticidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Pyrethroids are commonly used as mosquito adulticides and evolution of resistance to these compounds is a major threat to public health. 'Knockdown resistance' to pyrethroids (kdr) is frequently caused by nonsynonymous mutations in the voltage-gated sodium channel transmembrane protein (para) that reduce pyrethroid binding. Early detection of kdr is critical to the development of resistance management strategies in mosquitoes including Aedes aegypti, the most prevalent vector of dengue and yellow fever viruses. Brengues et al. described seven novel mutations in hydrophobic segment 6 of domain II of para in Ae. aegypti. Assays on larvae from strains bearing these mutations indicated reduced nerve sensitivity to permethrin inhibition. Two of these occurred in codons Iso1011 and Val1016 in exons 20 and 21 respectively. A transition in the third position of Iso1011 encoded a Met1011 replacement and a transversion in the second position of Val1016 encoded a Gly1016 replacement. We have screened this same region in 1318 mosquitoes in 32 additional strains; 30 from throughout Latin America. While the Gly1016 allele was never detected in Latin America, we found two new mutations in these same codons. A transition in the first position of codon 1011 encodes a Val replacement while a transition in the first position of codon 1016 encodes an Iso replacement. We developed PCR assays for these four mutations that can be read either on an agarose gel or as a melting curve. Selection experiments, one with deltamethrin on a field strain from Santiago de Cuba and another with permethrin on a strain from Isla Mujeres, Mexico rapidly increased the frequency of the Iso1016 allele. Bioassays of F(3) offspring arising from permethrin susceptible Val1016 homozygous parents and permethrin resistant Iso1016 homozygous parents show that Iso1016 segregates as a recessive allele in conferring kdr. Analysis of segregation between alleles at the 1011 and 1016 codons in the F(3) showed a high rate of recombination even though the two codons are only separated by a ~250 bp intron. The tools and information presented provide a means for early detection and characterization of kdr that is critical to the development of strategies for resistance management.
Assuntos
Aedes/efeitos dos fármacos , Aedes/genética , Proteínas de Insetos/genética , Mutação Puntual , Canais de Sódio/genética , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Cruzamentos Genéticos , Primers do DNA/genética , Feminino , Frequência do Gene , Genes de Insetos , Genótipo , Resistência a Inseticidas/genética , Inseticidas/farmacologia , América Latina , Masculino , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Piretrinas/farmacologia , Homologia de Sequência do Ácido NucleicoRESUMO
The diverse habitats and diets encountered during the life cycle of an Anopheles mosquito have necessitated the development of extensive families of detoxification enzymes. Expansion of the three detoxification enzyme families (cytochrome P450s, carboxylesterases and glutathione transfereases), has occurred in mosquitoes compared with Drosophila, however, very little is known regarding the developmental expression of theses genes. Using a custom made microarray we determined the expression profile of the detoxification genes in adults, larvae and pupae of the malaria vector A. gambiae. The expression of approximately one quarter of these genes was developmentally regulated. The expression profile of each of these genes and the information this data provides on putative functions of the mosquito detoxification enzymes is discussed.