Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

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.(AU)

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.(AU)

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

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.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

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.

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.(AU)

Effects of BMP-4 and FSH on growth, morphology and mRNA expression of oocyte-secreted factors in cultured bovine secondary follicles

This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.

Efeito da temperatura de descongelação na integridade de espermatozoides criopreservados de cães / The effect of the thawing temperature on the integrity of canine cryopreserved sperm

Avaliou-se a influência da temperatura de descongelação na integridade de espermatozoides criopreservados de cães. Foram utilizados reprodutores das raças Basset Hound (n=3) e Rottweiler (n=3), submetidos a colheitas de sêmen por manipulação peniana. As amostras de sêmen foram descongeladas a 37ºC/1min (G1) ou 70ºC/6s (G2) e avaliadas quanto à motilidade progressiva, vigor e integridade do acrossoma após 0, 30 e 60 minutos de incubação (37ºC), e ultraestrutura espermática imediatamente após a descongelação. Em todos os tempos de incubação, a motilidade progressiva dos espermatozoides descongelados a 70ºC por 6s (74,6%) foi mais alta (P<0,05) que a dos descongelados a 37ºC por 1min (64,6%). O vigor espermático não diferiu (P>0,05) entre os grupos, e o porcentual de gametas com acrossomas íntegros foi maior (P<0,05) nos espermatozoides do G1 do que no G2. Lesões ultraestruturais foram identificadas nos espermatozoides descongelados de ambos os grupos, em maior quantidade nos gametas do G2. Conclui-se que amostras congeladas de sêmen de cães devam ser descongeladas a 37ºC por 1min.(AU)

Aiming to evaluate the influence of the thawing temperature on the viability of canine cryopreserved sperm, Basset Hound (n=3) and Rottweiler (n=3) dogs were used, submitted to semen collected through manual manipulation. Semen samples were thawed at 37ºC during 1min (G1) or at 70ºC during 6s (G2), and evaluated for progressive motility, vigor and acrosome integrity, after 0, 30 e 60 minutes of incubation (37ºC), and sperm ultrastructure immediately after thawing. In all incubation times, the average of progressive motility was higher (P<0.05) in samples from G2 Group (74.6%) than from G1 (64.6%). Sperm vigor had no difference (P>0.05) between groups, and the percentage of gametes with intact acrosome was higher (P<0.05) on sperm cells from G1 than from G2. Ultrastructural changes were identified on dog sperm from both groups, and were observed in higher quantity in gametes from G2 Group. It can be concluded that samples of frozen dog sperm must be thawed at 37°C for 1min.(AU)