Identificação molecular de Metarhizium sp. em adultos de Aedes aegypti infectados
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Data
2023-07-28
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Universidade Federal de Goiás
Resumo
The use of entomopathogenic fungi has been an alternative of biorational control to control Aedes aegypti. A device containing mycoinsecticide based on Metarhizium humberi IP 46 for the focal control of A. aegypti adults is in developing. The evaluation of the effectiveness of this device under field conditions can be done with the aid of sensitive methods for detecting IP 46 in diseased mosquitoes captured in the field. To standardizing a methodology to detect IP 46 DNA present in A. aegypti adults after fungal exposure, adults were exposed to IP 46 formulations under laboratory or semi-field conditions for up to 8 or 14 days, respectively. In laboratory testing, the DNA extraction buffers by Saghai-Maroof et al. (1984) (I), Raeder and Broda (1985) (II) and Doyle and Doyle (1987) (III) were evaluated. For the semi-field test, the most efficient buffer in DNA extraction was used in the treated samples. The extractions and amplifications were confirmed by electrophoresis. The TEF-intron region was amplified by PCR using primers EF1T and EF2T. For positive control, use IP 46 mycelium, and for negative control, use unexposed mosquitoes. PCR products with positive amplification were sequenced. The results of the sequencing appreciated that, with buffer I, it was possible to extract DNA from IP 46 present in A. aegypti with incubation of 0, 6 or 8 days at 75% relative humidity (RH) and on 2 or 8 days more of 98% RH. With buffer II, fungal DNA transmission occurred in mosquitoes incubated for 4, 6 or 8 days at 75% RH and for 2, 4, 6 or 8 days at more than 98% RH. IP 46 DNA was obtained from mosquitoes incubated for 2, 6 or 8 days at 75% RH and 1, 2, 4 or 8 days at more than 98% RH, using buffer III. Buffer II showed the most consistent results, so it was used in field trials. With this methodology, it was possible to detect IP 46 in live or dead mosquitoes at different chances of fungal infection. The primers used in the PCR were specific for M. humberi, with no amplification of DNA from othermicroorganisms present in the tested mosquitoes. The buffer described by Raeder and Broda (1985) (II) was the most efficient in the transmission of M. humberi IP 46 DNA in A. aegypti adults, showing potential to be used in a technique for monitoring the effectiveness of the device under conditions field. The evaluation of the protocol in semi-field samples still needs to be done in a more standardized way.
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Fungo entomopatogênico, Mosquito, Extração, DNA, PCR, Detecção, Entomopathogenic fungus, Mosquito, Extraction, DNA, PCR, Detection
Citação
SILVA, Fernanda Cristina Soares da. Identificação molecular de Metarhizium sp. em adultos de Aedes aegypti infectados. 2023. 22 f. Trabalho de Conclusão de Curso (Bacharelado em Farmácia) - Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, 2023.