ABSTRACT
Objective: To evaluate 11 Cuban native Bacillus (B.) thuringiensis isolates in order to select one with the best larvicidal activity against Aedes (Ae.) aegypti and low cytotoxicity. Methods: The cry and cyt genes of the isolates (A21, A51, L95, L910, M29, R84, R85, R87, R89, U81 and X48) were amplified by PCR. The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays. Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice. Results: The cyt1 (Aa, Ab, Ba), cyt2, cry4aA, cry4Ba, cry11 (Aa, Ba, Bb) and cry10 genes were identified in all native Cuban isolates. The larvicidal activity (LC90) of seven isolates was affected by the presence of organic matter in the water, while A21, A51, L910, R84, U81 and X48 had better LC50, LC90, LC95 than the 266/2 9-VI-98 control strain. The LC50 of two isolates was affected by the presence of NaCl and A21, A51, R85 isolate had better larvicidal activity than the 266/2 9-VI-98 control strain. In terms of toxicity against macrophages, the extracts of nine isolates were less cytotoxic than the control strains. Conclusions: Native isolate A21 had the main virulence factors against Ae. aegypti larvae, displayed a good larvicidal activity in presence of different factors related with Ae. aegypti breeding sites, and had low citotoxicity against macrophages. These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.
ABSTRACT
Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.
Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.
Subject(s)
Humans , Animals , Bacillus thuringiensis/genetics , Aedes , Insecticides , Culicidae , Pest Control, Biological , Ecosystem , Mosquito Vectors , LarvaABSTRACT
Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.
Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.