Desenvolvimento de um meio quimicamente definido e avaliação do efeito de um peptídeo tóxico em drosophila melanogaster

Abstract

Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbon dioxide and are involved in the bioavailability of nitrogen and in defense mechanisms against predators and pathogens in plants. They are present in plant, fungi and bacteria, but are not synthesized by animals. Canavalia ensiformis, a leguminous plant, have three urease isoforms: JBURE-I, JBURE-II and CNTX, being JBURE-I or just JBU the most abundant. The insecticidal activity of JBU depends on the release of a peptide – Jaburetox (JBTX) – that is cleaved by the insects cathepsins enzymes. JBTX have fungitoxic, entomotoxic, neurotoxic and antimicrobial effects. The toxicity of JBTX was described for insects of the order Lepidoptera, Blattodea e Hemiptera wich the neurotoxic effect is due to the binding of the peptide to neuronal cells, increasing UDP-N-acetylglucosamine activity and inhibiting nitric oxide synthase. It was also related that JBTX acts on the Malpighian tubules of Rhodnius prolixus, inhibiting diuresis by altering the cGMP levels and acting in the transmembrane potential. However, there are no investigations about the toxicity in Drosophila melanogaster in the literature. D. melanogaster has been a model organism for almost a century, showing a range of advantages such as short life cycle, low mantaining costs, easy to handle, besides having its genome sequenced e its proteins annotated, which makes proteomics approaches viable. In this work, we aimed to stablish a chemically defined medium, as well as elect the best additive for a better rate of oviposition. Also, we wanted to investigate how JBTX acts on the central nervous system of D. melanogaster larvae. The chemically defined medium that allowed the full development of the flies was the medium composed only of agar, sucrose, yeast and methyl paraben, which fed the larvae of D. melanogaster until they reached the adult stage. The additive with the best oviposition rate was ethanol (5%), presenting approximately 700 eggs, unlike the control that presented approximately 500 eggs and propionic acid (1%) with approximately 200 eggs, thus demonstrating that an additive more similar to natural habitat of oviposition is more attractive. One-dimensional electrophoresis was performed to visualize significant changes in the electrophoretic profile of the central nervous system of D. melanogaster larvae on the action of JBTX. However, due to an aberrant electrophoresis, no quantitative or qualitative analysis of the proteins was possible. to visualize the presence of proteins in the gel.