Contribuição da microbiota bacteriana para o processo digestivo e desenvolvimento da lagarta da soja (Anticarsia gemmatalis) e caracterização de bactérias proteolíticas associadas ao seu trato intestinal

• Autor(a) principal: Visôtto, Liliane Evangelista
• Data de Publicação: 2007
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/343
• topic: Anticarsia gemmatalis; Soja; Bactérias proteolíticas; Anticarsia gemmatalis; Soybean; Proteolytic bacteria; CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA

Interactions between intestinal bacteria and insects have been extensively studied in systems such as termites and aphids. However, little is known about the association between bacteria and the order Lepidoptera, which comprises major phytophagous insects that cause heavy agricultural losses. A number of authors believe that the lack of specialized structures and the rapid food transit in the intestinal tract of Lepidopteras may cause the little contribution of microorganisms to nutrition and digestion of these insects, whereas other authors suggest that the microbiota provides nutrients and contributes to insect metabolism. This difference raises particular interest in studies on functionality of microbiota associated with Lepidopteras. The velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), becomes therefore a suitable model for studies on physiological processes involved in the association. This work sought to examine whether the microbiota may affect hydrolytic enzyme activity in A. gemmatalis intestine, as well as developmental biological parameters. Tetracycline, chloramphenicol and a combination of these two antibiotics along with ampicillin were added to the artificial diet at five specific dose levels (0; 30; 40; 50; 60 and 70 µg/mL). Larvae were fed until reach pupal stage. Tetracycline was found to be the most efficient antibiotic for suppression of bacterial growth, with substantial inhibition at the tested doses. Microscopic analyses showed that the intestine of A. gemmatalis fed on control diet harbors abundant but not diverse microbiota. Few bacteria were found in the lumen of larvae fed on 70 µg / mL tetracycline, confirming the result found by the conventional plating method. Enzymatic activities of the intestinal extract from larvae fed on tetracycline, on fourteen substrates, showed that the reduction in microbiota significantly affected the hydrolysis of L-BApNA and dithiopropanol tributyrate. Tetracycline at the dose of 70 µg / mL produced the largest inhibitory effect. Analysis of enzymatic profile was also carried out over the post-embryonic development of larvae fed on control diet and diet with 70 µg / mL tetracycline. Maximum proteolytic, amidolytic and lipase activities were found between the 4th and 6th instars in larvae presenting intact microbiota, whereas a constant profile was found in larvae fed on tetracycline. These results can be correlated with premature pupation, low emergence and increased mortality in larvae with reduced microbiota. The results suggest that the presence of microbiota exerts influence on the digestive process and development of A. gemmatalis, possibly contributing to protease and lipase production. Because protein digestion was affected, culturable bacteria showing proteolytic capacity were isolated from the intestinal tract, identified and characterized. All the isolates were Gram-positive and identified as Bacillus subtilis, Bacillus sp., Enterococcus sp. and Staphylococcus sp.. Analyses of origin proteases from the isolates were carried out with different substrates. All bacteria were efficiently capable of degrading the tested substrates, except Staphylococcus sp.. These results, besides contributing to the scientific knowledge on the insect-microorganism interaction, serve as a complement to future research on new methods of pest control.