RESUMO
BACKGROUND: Anopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations. METHODS: Adult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR-RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species. RESULTS: A total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR-RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. SPECIMENS: None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection. CONCLUSION: All An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.
Assuntos
Anopheles , Inseticidas , Malária , Piretrinas , Animais , Feminino , Anopheles/genética , Acetilcolinesterase , Alelos , Etiópia , Mosquitos Vetores/genética , Inseticidas/farmacologia , Resistência a Inseticidas/genéticaRESUMO
Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [14C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4 RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.
Assuntos
Abelhas/genética , Sistema Enzimático do Citocromo P-450/genética , Varroidae/efeitos dos fármacos , Animais , Abelhas/efeitos dos fármacos , Abelhas/parasitologia , Cumafos/efeitos adversos , Cumafos/farmacologia , Inativação Metabólica/efeitos dos fármacos , Inseticidas/efeitos adversos , Inseticidas/farmacologia , Taxa de Depuração Metabólica/genética , Varroidae/patogenicidadeRESUMO
BACKGROUND: The emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts. METHODS: Insecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain. RESULTS: Populations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed. CONCLUSIONS: Anopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids. Moreover, some mosquito populations exhibited resistance to propoxur and possible resistance to bendiocarb. Target site mutation kdr L1014F was detected in all mosquito populations while elevated levels of glutathione-S-transferases (GSTs) was detected in some mosquito populations. The reduced susceptibility of An. arabiensis to propoxur and bendiocarb, which are currently used for indoor residual spraying (IRS) in Ethiopia, calls for continuous resistance monitoring, in order to plan and implement evidence based insecticide resistance management.
Assuntos
Anopheles/genética , Resistência a Inseticidas/genética , Inseticidas , Animais , Anopheles/enzimologia , Colinesterases/genética , Colinesterases/metabolismo , DDT , Etiópia , Controle de Mosquitos , Nitrilas , Reação em Cadeia da Polimerase , Propoxur , Piretrinas , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Insect ryanodine receptors (RyR) are the molecular target-site for the recently introduced diamide insecticides. Diamides are particularly active on Lepidoptera pests, including tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae). High levels of diamide resistance were recently described in some European populations of T. absoluta, however, the mechanisms of resistance remained unknown. In this study the molecular basis of diamide resistance was investigated in a diamide resistant strain from Italy (IT-GELA-SD4), and additional resistant field populations collected in Greece, Spain and Brazil. The genetics of resistance was investigated by reciprocally crossing strain IT-GELA-SD4 with a susceptible strain and revealed an autosomal incompletely recessive mode of inheritance. To investigate the possible role of target-site mutations as known from diamondback moth (Plutella xylostella), we sequenced respective domains of the RyR gene of T. absoluta. Genotyping of individuals of IT-GELA-SD4 and field-collected strains showing different levels of diamide resistance revealed the presence of G4903E and I4746M RyR target-site mutations. These amino acid substitutions correspond to those recently described for diamide resistant diamondback moth, i.e. G4946E and I4790M. We also detected two novel mutations, G4903V and I4746T, in some of the resistant T. absoluta strains. Radioligand binding studies with thoracic membrane preparations of the IT-GELA-SD4 strain provided functional evidence that these mutations alter the affinity of the RyR to diamides. In combination with previous work on P. xylostella our study highlights the importance of position G4903 (G4946 in P. xylostella) of the insect RyR in defining sensitivity to diamides. The discovery of diamide resistance mutations in T. absoluta populations of diverse geographic origin has serious implications for the efficacy of diamides under applied conditions. The implementation of appropriate resistance management strategies is strongly advised to delay the further spread of resistance.
Assuntos
Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas , Mariposas/fisiologia , Mutação Puntual , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Sequência de Aminoácidos , Animais , Benzamidas/farmacologia , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Mariposas/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sulfonas/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
BACKGROUND: Indoxacarb is an important active ingredient extensively used for the control of Tuta absoluta, a major tomato pest, playing a particular role in insecticide resistance management schemes. RESULTS: Reduced susceptibility to indoxacarb was identified (1794-fold resistance) through toxicological bioassays in a field population from Greece and evolved rapidly to resistance after short laboratory selection. Combined bioassays with synergists and biochemical analysis suggested only a partial involvement of detoxification enzymes in the resistant phenotype. To investigate the role of target-site resistance, segment 6 of domain IV of the sodium channel in T. absoluta was cloned and the sequences compared between susceptible and indoxacarb-resistant T. absoluta insects. The presence of the F1845Y and the V1848I indoxacarb resistance mutations was detected and was strongly associated with the phenotype. These amino acid substitutions correspond to recently characterised indoxacarb resistance mutations in diamondback moth (Plutella xylostella). Robust and accurate PCR-RFLP assays were subsequently developed and successfully validated for detecting both indoxacarb resistance mutations in field T. absoluta populations. CONCLUSION: The identification of indoxacarb resistance mutations and the development of diagnostic tools will allow early detection of indoxacarb resistance, facilitating implementation of appropriate resistance management strategies, thus delaying the spread of resistance. © 2016 Society of Chemical Industry.
Assuntos
Análise Mutacional de DNA , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Oxazinas/farmacologia , Canais de Sódio/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Bioensaio , Técnicas de Genotipagem , Inseticidas/farmacologia , Canais de Sódio/químicaRESUMO
BACKGROUND: The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. PRINCIPAL FINDINGS: Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. SIGNIFICANCE: The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.
Assuntos
Aedes/enzimologia , Carboxilesterase/genética , Insetos Vetores/enzimologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Temefós/farmacologia , Aedes/efeitos dos fármacos , Aedes/genética , Animais , Sequência de Bases , Carboxilesterase/metabolismo , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Insetos Vetores/efeitos dos fármacos , Insetos Vetores/genética , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNA , Regulação para CimaRESUMO
BACKGROUND: Bendiocarb was introduced for the first time for Indoor Residual Spraying (IRS) in Tanzania in 2012 as part of the interim national insecticide resistance management plan. This move followed reports of increasingly alarming levels of pyrethroid resistance across the country. This study used the insecticide quantification kit (IQK) to investigate the intra-operational IRS coverage and quality of spraying, and decay rate of bendiocarb on different wall surfaces in Kagera region. METHODS: To assess intra-operational IRS coverage and quality of spraying, 104 houses were randomly selected out of 161,414 sprayed houses. A total of 509 samples (218 in Muleba and 291 in Karagwe) were obtained by scraping the insecticide samples from wall surfaces. To investigate decay rate, 66 houses (36 in Muleba and 30 in Karagwe) were selected and samples were collected monthly for a period of five months. Laboratory testing of insecticide concentration was done using IQK(TM) [Innovative Vector Control Consortium]. RESULTS: Of the 509 samples, 89.5% met the World Health Organization (WHO) recommended concentration (between 100-400 mg/m(2)) for IRS target dosage. The proportion of samples meeting WHO standards varied between Karagwe (84.3%) and Muleba (96.3%) (p < 0.001). Assessment of quality of spraying at house level revealed that Muleba (84.8%) had a significantly higher proportion of households that met the expected target dosage (100-400 mg/m(2)) compared to Karagwe (68.9%) (p < 0.001). The quality of spraying varied across different wall substrates in both districts. Evaluation of bendiocarb decay showed that the proportion of houses with recommended concentration declined from 96.9%, 93.5% and 76.2% at months one, two, and three post IRS, respectively (p-trend = 0.03). The rate of decay increased in the fourth and fifth month post spraying with only 55.9% and 26.3% houses meeting the WHO recommendations, respectively. CONCLUSION: IQK is an important tool for assessing IRS coverage and quality of spraying. The study found adequate coverage of IRS; however, residual life of bendiocarb was observed to be three months. Results suggest that in order to maintain the recommended concentrations with bendiocarb, a second spray cycle should be carried out after three months.
Assuntos
Técnicas de Química Analítica/métodos , Inseticidas/química , Inseticidas/farmacologia , Fenilcarbamatos/química , Fenilcarbamatos/farmacologia , Animais , Habitação , Controle de Mosquitos/métodos , Propriedades de Superfície , TanzâniaRESUMO
BACKGROUND: One of the largest West Nile virus outbreaks in Europe occurred in Greece in 2010. Use of insecticides against Culex pipiens was substantially scaled up, as an emergency tool. Although mosquito control has been based on insecticides for several decades in Greece, insecticide resistance data are not available. RESULTS: An examination was made of the resistance status of 13 Cx. pipiens populations from five regional units in Greece against four insecticides used for its control over a 3 year period. Bioassays demonstrated susceptibility of most populations to all insecticides, except for temephos in some regions, and deltamethrin and diflubenzuron on one occasion each. The authors also monitored the frequency of the pyrethroid target-site resistance mutations L1014F (kdr), as well as G119S and F290V in the Ace1 gene. Ace1 insensitivity mutations were found at low frequencies and always in heterozygocity. However, the frequency of kdr pyrethroid resistance mutations was higher (up to 63.0% in Thessaloniki). CONCLUSIONS: The high frequency of kdr mutations indicates a risk that needs to be addressed, should the use of pyrethroids be further extended. There was no strong evidence of significant resistance levels against Bacillus thuringiensis var. israelensis and diflubenzuron. Continued monitoring of insecticide resistance is recommended for the application of appropriate management tactics.
Assuntos
Culex/genética , Insetos Vetores/genética , Controle de Mosquitos , Animais , Bacillus thuringiensis , Diflubenzuron , Grécia , Resistência a Inseticidas/genética , Larva , Nitrilas , Piretrinas , Temefós , Vírus do Nilo OcidentalRESUMO
Helicoverpa armigera has been controlled effectively with chemical insecticides in the major cotton crop production areas of northern Greece for many years. However, a resurgence of the pest was observed in 2010, which significantly affected crop production. During a 4-year survey (2007-2010), we examined the insecticide resistance status of H. armigera populations from two major and representative cotton production areas in northern Greece against seven insecticides (chlorpyrifos, diazinon, methomyl, alpha-cypermethrin, cypermethrin, gamma-cyhalothrin and endosulfan). Full dose-response bioassays on third instar larvae were performed by topical application. Lethal doses at 50% were estimated by probit analysis and resistance factors (RF) were calculated, compared to a susceptible laboratory reference strain. Resistance levels were relatively moderate until 2009, with resistance ratios below 10-fold for organophosphates and carbamates and up to 16-fold for the pyrethroid alpha-cypermethrin. However, resistance rose to 46- and 81-fold for chlorpyrifos and alpha-cypermethrin, respectively in 2010, when the resurgence of the pest was observed. None of the known pyrethroid resistance mutations were found in the pyrethroid-resistant insects. The possible association between resistance and H. armigera resurgence in Greece is discussed.
Assuntos
Gossypium/parasitologia , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Doenças das Plantas/parasitologia , Animais , Clorpirifos/farmacologia , Endossulfano/farmacologia , Grécia , Mariposas/fisiologia , Nitrilas/farmacologia , Piretrinas/farmacologiaRESUMO
BACKGROUND: Dibenzoylhydrazine (DBH) compounds have been applied successfully as environmentally safe insecticides against lepidopteran larvae and ground-dwelling coleopterans, but their potential to combat mosquito larvae is largely unknown. Here, toxicity tests of three commercial DBHs (tebufenozide, methoxyfenozide and halofenozide) and one experimental DBH (KU-106) against larvae of Anopheles gambiae, the major vector for human malaria, are reported. RESULTS: Based on calculated median larvicidal concentration (LC50 ) values at 5 days of treatment, KU-106 (760 nM) showed an activity against Anopheles larvae similar to that of commercial halofenozide. Induction of the early-late gene hr3 and docking studies of DBHs in the ligand-binding pocket of the modelled Anopheles ecdysone receptor indicated that toxicity is caused by the activation of the ecdysone regulatory cascade causing a premature lethal moult. CONCLUSIONS: As a result of the similar toxicity exhibited by the experimental compound KU-106 to that shown by commercial products, the present study demonstrated that the use of DBH compounds to combat harmful dipteran insects, such as mosquitoes, remains unexplored and invites further systematic toxicity tests using other derivatives of the DBH class of compounds.
Assuntos
Anopheles/efeitos dos fármacos , Hidrazinas/toxicidade , Inseticidas/toxicidade , Animais , Anopheles/crescimento & desenvolvimento , Hidrazinas/química , Inseticidas/química , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Dose Letal MedianaRESUMO
BACKGROUND: Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. METHODOLOGY/PRINCIPAL FINDINGS: We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. CONCLUSIONS/SIGNIFICANCE: The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.
Assuntos
Aedes/enzimologia , Colorimetria/métodos , DDT , Glutationa Transferase/metabolismo , Resistência a Inseticidas , Parasitologia/métodos , Animais , Compostos Cromogênicos/metabolismo , Hidrocarbonetos Iodados/metabolismo , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR-RLFP assay developed in this study.
Assuntos
Acaricidas/farmacologia , Acetilcolinesterase/genética , Resistência a Medicamentos , Organofosfatos/farmacologia , Mutação Puntual , Tetranychidae/efeitos dos fármacos , Tetranychidae/enzimologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Sequência de Aminoácidos , Animais , Europa (Continente) , Dados de Sequência Molecular , Alinhamento de Sequência , Tetranychidae/genéticaRESUMO
The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K(cat) = 3.2 pmol/min/pmol P450, K(m) = 36 microM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.
Assuntos
Sistema Enzimático do Citocromo P-450/química , Hemípteros/enzimologia , Imidazóis/farmacologia , Proteínas de Insetos/química , Resistência a Inseticidas , Inseticidas/farmacologia , Nitrocompostos/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hemípteros/química , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Imidazóis/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/química , Modelos Moleculares , Dados de Sequência Molecular , Neonicotinoides , Nitrocompostos/química , Alinhamento de SequênciaRESUMO
BACKGROUND: A major problem of crop protection in Crete, Greece, is the control of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) with chemical insecticides owing to the rapid development of resistance. The aim of this study was to investigate the establishment of resistance and the underlying mechanisms to major insecticide classes with classical bioassays and known biochemical resistance markers. RESULTS: During a 2005-2007 survey, 53 Q biotype populations were collected. Application history records showed extensive use of neonicotinoids, organophosphates, carbamates and pyrethroids. High resistance levels were identified in the majority of populations (>80%) for imidacloprid (RF: 38-1958x) and alpha-cypermethrin (RF: 30-600x). Low resistance levels (RF < 12) were observed for pirimiphos-methyl. A strong correlation between resistance to imidacloprid and the number of applications with neonicotinoids was observed. Significant correlations were observed between COE and P450-dependent monoxygenase activity with resistance to alpha-cypermethrin and imidacloprid respectively. A propoxur-based AChE diagnostic test indicated that iAChE was widespread in most populations. Resistance levels for alpha-cypermethrin were increased when compared with a previous survey (2002-2003). Differentiation of LC(50) values between localities was observed for imidacloprid only. CONCLUSION: Bemisia tabaci resistance evolved differently in each of the three insecticides studied. Imidacloprid resistance seems less established and less persistent than alpha-cypermethrin resistance. The low resistance levels for pirimiphos-methyl suggest absence of cross-resistance with other organophosphates or carbamates used.
Assuntos
Hemípteros/efeitos dos fármacos , Resistência a Inseticidas , Inseticidas/farmacologia , Animais , Grécia , Hemípteros/classificação , Hemípteros/enzimologia , Imidazóis/farmacologia , Proteínas de Insetos/metabolismo , Neonicotinoides , Nitrocompostos/farmacologia , Oxigenases/metabolismo , Piretrinas/farmacologiaRESUMO
BACKGROUND: The most important pest of olive orchards worldwide is the olive fruit fly Bactrocera oleae (Gmelin). Its control in Greece has been based on organophosphates (OPs), but their intense use has led to the development of resistance. A test previously developed to monitor the trait may not be as robust as originally thought. The pyrethroid alpha-cypermethrin has recently been registered for bait sprays, as an alternative to OPs. RESULTS: The susceptibility of 20 B. oleae populations to alpha-cypermethrin was examined. Variation was observed in their response, with LD(50) ranging from 0.14 to 3.28 ng insect(-1) and resistance factors from 2.3 to 54.7. Resistance mechanisms were investigated. Cytochrome P450 monoxygenase activities showed an association with resistance. Sequences in the domain IIS4-IIS6 of the B. oleae para-type sodium channel were also analysed, but no resistance-associated mutations were identified. Finally, a novel diagnostic assay able to reliably monitor the frequency of the iAChE G488S resistance mutation was developed. CONCLUSION: This is the first attempt to evaluate the efficacy of alpha-cypermethrin against B. oleae from Greece. Data showed that it can be used effectively, but also highlighted the importance of continuous monitoring. The IIS4-IIS6 sodium channel region is the default area in which to look for resistance mutations if target-site resistance to pyrethroids arises. The application of the novel iAChE molecular diagnostic may facilitate the introduction of pyrethroids alongside OPs currently in use.
Assuntos
Proteínas de Insetos/genética , Inseticidas/farmacologia , Mutação , Peptidil Dipeptidase A/genética , Reação em Cadeia da Polimerase/métodos , Piretrinas/farmacologia , Tephritidae/efeitos dos fármacos , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Sequência de Bases , Clonagem Molecular , Análise Mutacional de DNA/métodos , Grécia , Controle de Insetos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Dados de Sequência Molecular , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/metabolismo , Polimorfismo de Fragmento de Restrição , Estrutura Terciária de Proteína , Canais de Sódio/química , Canais de Sódio/genética , Canais de Sódio/metabolismo , Tephritidae/química , Tephritidae/enzimologia , Tephritidae/genéticaRESUMO
A glutathione S-transferase (GST) from the mosquito Aedes aegypti (aagste2), selected in the field as a major metabolic resistance enzyme for this parasite vector, was employed to produce a highly specific assay for the determination of DDT [1,1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene]. Detection is based on the pH change occurring in an appropriate buffer system by the concomitant release of H(+) during the aagste2-catalyzed dehydrochlorination reaction and is monitored potentiometrically or colorimetrically in the presence of a pH marker. The theoretical limit of detection (LOD) of the assay is 3.8 microg/ml, and the linear range of quantification is 12 to 250 microg/ml. The method does not recognize biologically inactive DDT analogues or major DDT photodegradants and breakdown molecules, and it is highly specific for the insecticidal p.p'DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane]. The biosensor was validated with a number of insecticide swabs from DDT-sprayed surfaces and found to be reproducible and reliable as compared with high-performance liquid chromatography (HPLC) (correlation coefficient R(2)=0.98). Given the current expansion of DDT residual sprayings in many regions of Africa as a key strategic intervention for malaria vector control, this simple assay to monitor DDT levels for vector control spraying programs could have an important impact on malaria control.
Assuntos
DDT/análise , DDT/metabolismo , Liases/metabolismo , Concentração de Íons de Hidrogênio , Potenciometria , Sensibilidade e Especificidade , Especificidade por Substrato , Propriedades de SuperfícieRESUMO
To explore the feasibility of developing inhibitors of signaling by opioid receptors and other G protein-coupled receptors (GPCRs) that use the same G protein pool, we investigated the capacity of a minigene encoding the third intracellular loop of the delta-opioid receptor (delta-i3L) to act as competitive antagonist of the receptor-G protein interface interaction. In delta-i3L-expressing cells, the peptide blocked high-affinity agonist binding to both the delta- and the mu-opioid (delta-OR and mu-OR) and attenuated opioid and alpha2-adrenergic receptor (alpha2AR)-dependent [35S]guanosine-5'-O-(3-thio)triphosphate binding. Furthermore, delta-i3L expression resulted in inhibition of delta-, mu-OR-, and alpha2AR-receptor-mediated cAMP accumulation, whereas the cAMP response produced by activation of the beta2-adrenergic receptor was unaffected, suggesting that the inhibitory effects of delta-i3L expression were selective for Gi/Go proteins. Moreover, although delta-i3L expression also attenuated drastically phospholipase C accumulation and Ca2+ release following mu- and delta-OR stimulation, it failed to inhibit carbachol-mediated stimulation of inositol phosphate accumulation in M1-muscarinic receptor-expressing human embryonic kidney 293 cells. Finally, we also examined the effects of delta-i3L expression on the regulation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway. Our results demonstrate that, although ERK activation by mu- and delta-ORs is attenuated by the presence of delta-i3L, ERK activation mediated by alpha2AR remained unaffected. Collectively, our data demonstrate that the delta-i3L can be used as potent inhibitor of G protein signaling for various GPCRs that use a common pool of G proteins.