Avaliação da atividade larvicida da microbiota intestinal cultivável de mosquitos Aedes spp.
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Data
2021-03-30
Autores
Porto, Mariana Cristina de Freitas
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Universidade Estadual Paulista (Unesp)
Resumo
Mosquitos são grandes responsáveis por sérios problemas de saúde pública no mundo e transmissores de diversas arboviroses. Até o momento, não existem vacinas ou medicamentos eficazes disponíveis contra essas arboviroses, dessa maneira, o controle vetorial ainda é a principal estratégia de intervenção para reduzir a disseminação dos principais arbovírus urbanos transmitidos pelos mosquitos do gênero Aedes. No entanto, a eliminação dos mosquitos tornou-se muito desafiadora no ambiente urbano moderno, onde a crescente ocorrência de resistência a inseticidas tornou o uso de tais compostos químicos cada vez menos eficazes. Neste contexto, o desenvolvimento de novos produtos é extremamente necessário. Recentemente, a microbiota intestinal do mosquito provou ser um ótimo recurso utilizando bactérias com atividades antivirais e entomopatogênicas. O objetivo deste trabalho foi identificar e analisar a atividade larvicida de bactérias cultiváveis isoladas da microbiota intestinal de mosquitos Aedes aegypti e Aedes albopictus coletados em Avaré e Botucatu/SP. As bactérias isoladas do intestino do mosquito foram purificadas em meios seletivos e foram realizadas extrações de DNA e a identificação foi realizada a partir da amplificação e sequenciamento do fragmento do rDNA 16S. Posteriormente, avaliou-se a ação larvicida das bactérias que foram identificadas e isoladas sobre larvas do segundo estágio de Ae. aegypti (Rockefeller), em condições de laboratório. Os bioensaios foram preparados com dez larvas, em triplicata, colocadas em placas de seis poços, contendo os pellets de cultura bacteriana resuspendida em 5ml de PBS 1X e o controle negativo contendo 5ml de PBS 1X. As suspensões bacterianas foram ajustadas para DO600= 1.0. Os bioensaios foram mantidos no insetário à temperatura de 28°C, umidade relativa de 80 ± 5% e fotoperíodo de 12 horas. Os resultados do bioensaio larvicida foram registrados diariamente após a inoculação do meio bacteriano nos poços contendo as larvas por quatro dias. Foram identificadas 26 diferentes espécies de bactérias de 10 gêneros. Os gêneros Bacillus (30,76%) e Chryseobacterium (15,38%) apresentaram a maior porcentagem dos isolados obtidos. Nossas análises revelaram que três isolados bacterianos, Bacillus sp. (1C1-1), Chryseobacterium cucumeris (27D12) e Chryseobacterium indologenes (POOL 4D), apresentaram elevada atividade na mortalidade nas larvas de Ae. aegypti (Rockfeller), sendo 70%, 80% e 90% , respectivamente após 96 horas. Essas bactérias por apresentarem mais de 50% de mortalidade em 96 horas foram utilizadas em bioensaios para estimar as doses letais (DL50) contra larvas de segundo estágio de Ae. aegypti. Considerando as avaliações nos intervados de 24, 48, 72 e 96 horas, verificou-se que o isolado Chryseobacterium indologenes (POOL 4D) obteve maior toxicidade, para os quais foram obtidos os menores valores de DL50 nos quatro intervalos de leitura. Nossos resultados demonstram que o uso dessas bactérias podem ser uma alternativa viável no controle dos vetores futuramente.
Mosquitoes are largely responsible for serious public health problems in the world and transmitters of several arboviruses. To date, there are no effective vaccines or drugs available against these arboviruses, thus, the vector control is still the main intervention strategy to reduce the spread of the main urban arboviruses transmitted by mosquitoes of the genus Aedes. However, mosquito elimination has become very challenging in the modern urban environment, where the increasing occurrence of resistance to insecticides has made the use of such chemical compounds less and less effective. In this context, the development of new products is extremely necessary. Recently, the mosquito's intestinal microbiota proved to be a great resource using bacteria with antiviral and entomopathogenic activities. The objective of this work was to identify and analyze the larvicidal activity of cultivable bacteria isolated from the intestinal microbiota of Aedes aegypti and Aedes albopictus mosquitoes collected in Avaré and Botucatu/SP. The bacteria isolated from the mosquito's intestine were purified in selective media and DNA extractions were performed. and identification was carried out by amplifying and sequencing the 16S rDNA fragment. Subsequently, the larvicidal action of the bacteria that were identified and isolated on larvae of the second stage of Ae. aegypti (Rockefeller), under laboratory conditions, placed in six-well plates, containing bacterial culture pellets resuspended in 5ml of 1X PBS and the negative control containing 5ml of 1X PBS. The bacterial suspensions were adjusted to OD600= 1.0. The bioassays were maintained in the insectarium at a temperature of 28°C, a relative humidity of 80 ± 5% and a photoperiod of 12 hours. The results of the larvicidal bioassay were recorded daily after inoculation of the bacterial medium in the wells containing the larvae for four days. 26 different species of bacteria from 10 genera were identified. 26 different species of bacteria from 10 genera were identified. The genera Bacillus (30.76%) and Chryseobacterium (15.38%) presented the highest percentage of the isolates obtained. Our analyzes revealed that three bacterial isolates, Bacillus sp. (1C1-1), Chryseobacterium cucumeris (27D12) e Chryseobacterium indologenes (POOL 4D), showed high activity in mortality in larvae of Ae. aegypti (Rockfeller), 70%, 80% and 90%, respectively. These bacteria, for having more than 50% mortality in 96 hours, were used in bioassays to estimate lethal concentrations (LD50) against second stage larvae of Ae. aegypti. Considering the evaluations in the intervals of 24, 48, 72 and 96 hours, it was found that the isolate Chryseobacterium indologenes (POOL 4D) obtained greater toxicity, for which the lowest LD50 values were obtained in the four reading intervals. Our results demonstrate that the use of these bacteria may be a viable alternative to control vectors in the future.
Mosquitoes are largely responsible for serious public health problems in the world and transmitters of several arboviruses. To date, there are no effective vaccines or drugs available against these arboviruses, thus, the vector control is still the main intervention strategy to reduce the spread of the main urban arboviruses transmitted by mosquitoes of the genus Aedes. However, mosquito elimination has become very challenging in the modern urban environment, where the increasing occurrence of resistance to insecticides has made the use of such chemical compounds less and less effective. In this context, the development of new products is extremely necessary. Recently, the mosquito's intestinal microbiota proved to be a great resource using bacteria with antiviral and entomopathogenic activities. The objective of this work was to identify and analyze the larvicidal activity of cultivable bacteria isolated from the intestinal microbiota of Aedes aegypti and Aedes albopictus mosquitoes collected in Avaré and Botucatu/SP. The bacteria isolated from the mosquito's intestine were purified in selective media and DNA extractions were performed. and identification was carried out by amplifying and sequencing the 16S rDNA fragment. Subsequently, the larvicidal action of the bacteria that were identified and isolated on larvae of the second stage of Ae. aegypti (Rockefeller), under laboratory conditions, placed in six-well plates, containing bacterial culture pellets resuspended in 5ml of 1X PBS and the negative control containing 5ml of 1X PBS. The bacterial suspensions were adjusted to OD600= 1.0. The bioassays were maintained in the insectarium at a temperature of 28°C, a relative humidity of 80 ± 5% and a photoperiod of 12 hours. The results of the larvicidal bioassay were recorded daily after inoculation of the bacterial medium in the wells containing the larvae for four days. 26 different species of bacteria from 10 genera were identified. 26 different species of bacteria from 10 genera were identified. The genera Bacillus (30.76%) and Chryseobacterium (15.38%) presented the highest percentage of the isolates obtained. Our analyzes revealed that three bacterial isolates, Bacillus sp. (1C1-1), Chryseobacterium cucumeris (27D12) e Chryseobacterium indologenes (POOL 4D), showed high activity in mortality in larvae of Ae. aegypti (Rockfeller), 70%, 80% and 90%, respectively. These bacteria, for having more than 50% mortality in 96 hours, were used in bioassays to estimate lethal concentrations (LD50) against second stage larvae of Ae. aegypti. Considering the evaluations in the intervals of 24, 48, 72 and 96 hours, it was found that the isolate Chryseobacterium indologenes (POOL 4D) obtained greater toxicity, for which the lowest LD50 values were obtained in the four reading intervals. Our results demonstrate that the use of these bacteria may be a viable alternative to control vectors in the future.
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Palavras-chave
Controle vetorial, Aedes, Microbiota, Bactérias, Bioensaio larvicida, Vector control, Bacteria, Larvicidal bioassay