Avalia??o dos perfis prot?ico e lip?dico na resposta de Rhipicephalus microplus ? infec??o com fungos

• Autor(a) principal: Angelo, Isabele da Costa
• Data de Publicação: 2011
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRRJ
• Texto Completo: https://tede.ufrrj.br/jspui/handle/jspui/3422
• topic: Pept?deos antimicrobianos; Controle biol?gico; metabolismo lip?dico; Antimicrobial peptides; biological control; lipid metabolism; Medicina Veterin?ria

The study evaluated the protein and lipid profiles of Rhipicephalus microplus engorged females after infection by Metarhizium anisopliae, Beauveria bassiana and Fusarium oxysporum. The treatments were immersion or inoculation of conidial suspension in R. microplus. The hemolymph was collected 24 and 48 hours after treatment. The cell-free hemolymph was separated of hemocytes by centrifugation and hemocytes resuspended in phosphate buffer pH 7.2. The amount of total protein was determined in both fractions ofhemolymph and hemocytes were quantified. The cell-free hemolymph was filtered through a 100 kDa and 10 kDa membranes, and analyzed by electrophoresis and liquid chromatography (HPLC). The proteome of cell-free hemolymph (treatment by injection) was evaluated by 2DPAGE. Changes were observed in amount total protein and the amount of hemocytes, but no difference was observed in the electrophoretic profile (1D-PAGE) of the cell-free hemolymph. In haemocytes, the entomopathogens reduced the amount of serpins, while F. oxysporum caused increased. In 2D-PAGE variations were observed in both expression and presence/absence of protein between the groups. The cell-free hemolymph antimicrobial activity was tested against Escherichia coli and Staphylococcus aureus and against the fungus used in the treatment of engorged ticks. The hemolymph collected 48 hours after the immersion treatment with B. bassiana apresented activity anti-B. bassiana with 48 hours of evaluation. This hemolymph was subjected to Superose column to HPLC and peak was collected and analyzed on the analytical column C18. The fractions were collected from the C18 and its apresented activity anti-B. bassiana, but showed no activity against Candida albicans. These fractions were analyzed by Maldi-Tof and most of them had in common an ion with m/z 1,119.5; however, other ions may be involved with this activity antimicrobial. The lipids present in cell-free hemolymph, in the hemocyte and fat body were extracted and analyzed by thin layer chromatography (TLC) or HPTLC for neutral lipids and phospholipids. The classes of neutral lipids in the cell-free hemolymph were cholesterol ester, cholesterol (CHO) and fatty acids (FA), which have varied depending on the fungus used, type of treatment and observation time. Phospholipids found were phosphatidylcholine and phosphatidylethanolamine, its were not significantly altered after the fungal infection. In hemocytes, the same classes of lipids were found and B. bassiana modified phospholipids, while M. anisopliae s.l. altered FA and CHO. The fat body showed, in addition to these classes of neutral lipids, the triglycerides, which increased significantly 48 hours after inoculation with M. anisopliae s.l. The lipase activity in fat body was measured and it was demonstrated that increased activity 48 hours after inoculation, mainly in the group inoculated with Metarhizium. Therefore, the results showed alterations related to the proteins expression in the hemocytes and the cell-free hemolymph after inoculation with fungi, immunosuppression of hemocytes and antimicrobial peptides induction after infection with B. bassiana, besides changes in the lipid profile of R. microplus after infection. However, further studies are necessary to understand these changes.