Functional characterization of a Glutathione-S-Transferase Epsilon class (GSTE2) from Aedes aegypti

Made available in DSpace on 2016-05-19T13:08:20Z (GMT). No. of bitstreams: 2 2014helvecio-e.pdf: 1469299 bytes, checksum: daf2db6f3397fb4c6483926fef50bc16 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife. PE, Brasil.As Glutationa-S-Transferases (GSTs) em insetos desempenham um papel fundamental na metabolização de inseticidas químicos, e provavelmente estão envolvidas na proteção contra o estresse oxidativo decorrente da exposição a xenobióticos. O objetivo do trabalho foi a caracterização funcional do gene GSTE2 em linhagens de Aedes aegypti com diferentes perfis de susceptibilidade ao temephos. Foram usadas uma colônia susceptível (RecLab) e outra resistente, (RecR). Larvas de ambas as linhagens foram divididas em dois grupos: exposto ao temephos com concentrações subletais e não exposto. Os indivíduos sobreviventes foram usados em ensaios enzimáticos para medir a atividade das GSTs totais contra os substratos CDNB (padrão) e o 4-HNE, um produto endógeno resultante da peroxidação de lipídeos. Adicionalmente, foi feito o sequenciamento do cDNA deste gene em amostras das duas linhagens e a sua expressão foi investigada. A GSTE2 das duas linhagens foi expressa em sistema heterólogo e purificada para avaliação da atividade metabólica contra o 4-HNE, através de testes de biocatálise. Os resultados revelaram que a atividade enzimática da GST usando o CDNB foi normal para RecLab, em ambas as condições estudadas, porém, para RecR houve alteração na atividade de GST, para os dois grugo estudados . Usando o 4-HNE como substrato, as duas linhagens apresentaram um perfil enzimático alterado para GST em relação à Rock, com uma resposta aumentada após a exposição ao temephos. Foram identificados polimorfismos que diferenciam as duas linhagens. Os resultados de expressão gênica indicaram que as larvas resistentes apresentam níveis de expressão significativamente maiores do que as susceptíveis, e em RecR a expressão caiu após a exposição, sugerindo o envolvimento dessa enzima nos processos de resistência metabólica na linhagem RecR. Esses dados abrem novas perspectivas de monitoramento da resistência metabólica em Ae. aegypti.2016-09-3

Exposure to Zika and chikungunya viruses impacts aspects of the vectorial capacity of Aedes aegypti and Culex quinquefasciatus.

Zika (ZIKV) and chikungunya (CHIKV) are arboviruses that cause infections in humans and can cause clinical complications, representing a worldwide public health problem. Aedes aegypti is the primary vector of these pathogens and Culex quinquefasciatus may be a potential ZIKV vector. This study aimed to evaluate fecundity, fertility, survival, longevity, and blood feeding activity in Ae. aegypti after exposure to ZIKV and CHIKV and, in Cx. quinquefasciatus exposed to ZIKV. Three colonies were evaluated: AeCamp (Ae. aegypti-field), RecL (Ae. aegypti-laboratory) and CqSLab (Cx. quinquefasciatus-laboratory). Seven to 10 days-old females from these colonies were exposed to artificial blood feeding with CHIKV or ZIKV. CHIKV caused reduction in fecundity and fertility in AeCamp and reduction in survival and fertility in RecL. ZIKV impacted survival in RecL, fertility in AeCamp and, fecundity and fertility in CqSLab. Both viruses had no effect on blood feeding activity. These results show that CHIKV produces a higher biological cost in Ae. aegypti, compared to ZIKV, and ZIKV differently alters the biological performance in colonies of Ae. aegypti and Cx. quinquefasciatus. These results provide a better understanding over the processes of virus-vector interaction and can shed light on the complexity of arbovirus transmission

The susceptibility of Aedes aegypti populations displaying temephos resistance to Bacillus thuringiensis israelensis: a basis for management

Submitted by Kamylla Nascimento ([email protected]) on 2017-12-06T14:14:58Z No. of bitstreams: 1 art. The susceptibility of Aedes - araujo.pdf: 242752 bytes, checksum: e29f1758d49dd108fde1fb351e80080a (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2017-12-06T14:28:15Z (GMT) No. of bitstreams: 1 art. The susceptibility of Aedes - araujo.pdf: 242752 bytes, checksum: e29f1758d49dd108fde1fb351e80080a (MD5)Made available in DSpace on 2017-12-06T14:28:15Z (GMT). No. of bitstreams: 1 art. The susceptibility of Aedes - araujo.pdf: 242752 bytes, checksum: e29f1758d49dd108fde1fb351e80080a (MD5) Previous issue date: 2013Este trabalho foi apoiado pelo Conselho Nacional de Pesquisa (CNPq PRONEX-Rede Dengue 550116 / 2010-9, CNPq 590154 / 2011-7, CNPq 472491 / 2012-1), Fundação de Apoio à Pesquisa do Estado de Pernambuco (subvenção FACEPE-PPSUS APQ 1361-4.00 / 08) e Fundação Oswaldo Cruz / Vice-Presidência de Pesquisa e Laboratórios de Referência / Serviço de Referência para Controle de Culicídeos Vetores / Centro de Pesquisas Aggeu Magalhães-PE (FIOCRUZ / VPPLR / SRCCV / CPqAM-PE) .Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundaçao Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Aedes aegypti is the vector of dengue virus, and its control is essential to prevent disease transmission. Among the agents available to control this species, biolarvicides based on Bacillus thuringiensis serovar israelensis (Bti) are an effective alternative to replace the organophosphate temephos for controlling populations that display resistance to this insecticide. The major goal of this study was to determine the baseline susceptibility of Brazilian Ae. aegypti populations to Bti, taking into account their background in terms of larvicide exposure, status of temephos resistance and the level of activity of detoxifying enzymes involved in metabolic resistance to insecticides

Fitness cost in field and laboratory Aedes aegypti populations associated with resistance to the insecticide temephos

Abstract Background The continued use of chemical insecticides in the context of the National Program of Dengue Control in Brazil has generated a high selective pressure on the natural populations of Aedes aegypti, leading to their resistance to these compounds in the field. Fitness costs have been described as adaptive consequences of resistance. This study evaluated the biological and reproductive performance of A. aegypti strains and a field population resistant to temephos, the main larvicide used for controlling mosquitoes. Methods Comparative tests were performed with a resistant field population from the municipality of Arcoverde, Pernambuco State, Brazil, with a high rate of temephos resistance (RR = 226.6) and three isogenetic laboratory strains from the same origin (Araripina municipality, Pernambuco): RecR (RR = 283.6); RecRNEx (RR = 250.5), a strain under a process of resistance reversion; and RecRev (RR = 2.32), a reversed susceptible strain used as an experimental control. Results Our study revealed that the absence of selective pressure imposed by exposure to temephos, for five consecutive generations, led to a discrete reduction of the resistance ratio and the response of the detoxifying enzymes. Most of the 19 biological parameters were impaired in the resistant strains and field population. The analysis of the fertility life table confirmed the presence of reproductive disadvantages for the resistant individuals. Similarly, the longevity, body size, and total energetic resources were also lower for the resistant females, except for the last two parameters in the field females (Arcoverde). In contrast, the sex ratio and embryonic viability suffered no interference in all strains or population evaluated, regardless of their status of resistance to temephos. Conclusions The reproductive potential and survival of the resistant individuals were compromised. The parameters most affected were the larval development time, fecundity, net reproduction rate, and the generational doubling time. These fitness costs in the natural population and laboratory strains investigated are likely associated with maintaining the metabolic mechanism of resistance to temephos. Our results show that despite these costs, the highly temephos resistant populations can compensate for these losses and successfully overcome the control actions that are based on the use of chemical insecticides

Polymorphisms in GSTE2 is associated with temephos resistance in Aedes aegypti

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism

Screening Aedes aegypti (Diptera: Culicidae) Populations From Pernambuco, Brazil for Resistance to Temephos, Diflubenzuron, and Cypermethrin and Characterization of Potential Resistance Mechanisms

Submitted by Paulo Silva ([email protected]) on 2019-09-24T13:43:48Z No. of bitstreams: 1 Screening Aedes aegypti.pdf: 320276 bytes, checksum: 95794516356ff200a7bad3d9123f0aa3 (MD5)Approved for entry into archive by Paulo Silva ([email protected]) on 2019-09-24T14:23:47Z (GMT) No. of bitstreams: 1 Screening Aedes aegypti.pdf: 320276 bytes, checksum: 95794516356ff200a7bad3d9123f0aa3 (MD5)Made available in DSpace on 2019-09-24T14:23:47Z (GMT). No. of bitstreams: 1 Screening Aedes aegypti.pdf: 320276 bytes, checksum: 95794516356ff200a7bad3d9123f0aa3 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil / Universidade Federal de Pernambuco. Centro Acadêmico do Agreste. Núcleo de Ciências da Vida. Caruaru, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Universidade Federal de Santa Catarina. Departamento de Biologia Celular, Embriologia e Genética – BEG. Florianópolis, SC, Brasil.Departamento de Saúde de Pernambuco. Recife, PE, Brasil.Departamento de Saúde de Pernambuco. Recife, PE, Brasil.Secretaria Municipal de Saúde do Recife, Recife, PE, Brasil.Secretaria de Saúde do Distrito de Fernando de Noronha. Fernando de Noronha, PE, Brasil.Secretaria de Saúde do Distrito de Fernando de Noronha. Fernando de Noronha, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Departamento de Entomologia. Recife, PE, Brasil.Resistance to chemical insecticides detected in Aedes aegypti (L.) mosquitoes has been a problem for the National Dengue Control Program (PNCD) over the last years. In order to provide deeper knowledge of resistance to xenobiotics, our study evaluated the susceptibility profile of temephos, diflubenzuron, and cypermethrin insecticides in natural mosquito populations from the Pernambuco State, associating these results with the local historical use of such compounds. Furthermore, mechanisms that may be associated with this particular type of resistance were characterized. Bioassays with multiple temephos and diflubenzuron concentrations were performed to detect and quantify resistance. For cypermethrin, diagnostic dose assays were performed. Biochemical tests were carried out to quantify the activity of detoxification enzymes. In addition, a screening of mutations present in the voltage-gated sodium channel gene (NaV) was performed in samples previously submitted to bioassays with cypermethrin. The populations under study were resistant to temephos and showed a positive correlation between insecticide consumption and the resistance ratio (RR) to the compound. For diflubenzuron, the biological activity ratio (BAR) ranged from 1.3 to 4.7 times, when compared to the susceptible strain. All populations showed resistance to cypermethrin. Altered enzymatic profiles of alpha, p-nitrophenyl acetate (PNPA) esterases and glutathione-S-transferases were recorded in most of these samples. Molecular analysis demonstrated that Arcoverde was the only population that presented the mutated form 1016Ile/Ile. These findings show that the situation is critical vis-à-vis the effectiveness of mosquito control using chemical insecticides, since resistance to temephos and cypermethrin is widespread in Ae. aegypti from Pernambuco