Avaliação do perfil protéico da postura de fêmeas de Rhipicephalus microplus infectadas por Metarhizium anisopliae.

Abstract

Rhipicephalus microplus ticks are an important research target because it causes severe economic losses. Parasite control, in general, is based on the use of chemical acaricides. Entomopathogenic fungi are potential candidates as biocontrol agents due to its dispersibility, its ability to penetrate through the arthropods’s cuticle and the relatively specificity to arthropods, contributing to the preservation of non-target organisms. Therefore, studies of fungal infection mechanism and of the effects of the infection on oocytes maturation and on nutrients availability to the embryo are aspects demanded to be explored for further comprehension of the fungi infection, as well as, the ticks’ defense mechanisms. These investigations would significantly contribute to advances in tick control. The present study evaluates possible changes in the protein profile of eggs layed by R. microplus females infected with the entomopathogenic fungus Metarhizium anisopliae s.l. Ma 959 isolate. Bioassay was composed by five groups: four groups treated with M. anisopliae s.l. and one control group. Each group lead 50 females. Ticks were immersed in 1 ml of 0.1% Tween 80 aqueous conidial suspension at 1×108, 1 × 107, 1 × 106 or 1 × 105 conidia ml−1 for three minutes. The control group was immersed in 1 ml of 0.1% Tween 80 aqueous solution with no conidia. Eggs were separated daily, for the first ten days of oviposition and stored at – 20°C. Egg masses were separately homogenized, ground with lyses buffer and centrifuged. The protein content of supernatant was determined by the method of Lowry with modifications. Samples were analyzed through a 12%-polyacrylamide electrophoresis gel under denaturing conditions (SDS-PAGE). There was no significant (p ≤ 0.05) difference in the total protein content between the eggs from infected and non-infected groups. Nineteen protein bands were observed on the SDS-PAGE. No difference was observed in the bands profile between the control and the infected groups. In adition, no differences was observed in the profiles of groups treated with different concentrations of conidia. Eggs from females infected with the highest fungal concentration or from non-infected females were used for HPLC analysis. Samples were pre-purified by solid phase extraction. Elution steps were performed with 5, 40, and 80% acetonitrile (ACN). Samples were then submitted to a HPLC using an analytic C18 reverse-phase column. The antimicrobial activity of samples collected by HPLC was tested against Candida albicans and M. anisopliae s.l. Nine samples reduced C. albicans growth over than 35%. The fraction with the highest antimicrobial activity to C. albicans was collected from eggs from day 8 of oviposition of infected engorged females. This fraction was pre-purified in 5% of ACN, caused 80.94% reduction of growth of C. albicans and was not observed in any other chromatogram. There was no antimicrobial activity against M. anisopliae s.l. The present study reportes, for the first time, induced response in eggs from R. microplus females infected by the entomopathogenic fungus M. anisopliae s.l.