Influência da microbiota e dos patógenos de carrapatos na ação de fungos entomopatogênicos

Abstract

Reports on the use of entomopathogenic fungi in the biological control of ticks have shown good efficacy, however the importance of tick’s microbiota, including tick-borne pathogens, during fungal infection remains largely unexplored. Therefore, this thesis aimed to evaluate the influence of the microbiota of Rhipicephalus microplus on its survival under the action of Metarhizium anisopliae; evaluate composition and diversity of bacteria in the gut of R. microplus after treatment with M. anisopliae; in addition, to analyze whether the administration of antibiotics is compatible with the use of entomopathogenic fungi. Moreover, using the model of Ixodes scapularis and Borrelia burgdorferi, the objective was to compare the survival and engorgement of I. scapularis infected or not with B. burgdorferi after treatment with M. anisopliae; to analyze the immune response of the groups mentioned above by the expression of the Myd88 daptor molecule in “Toll-like” receptors; evaluate the colonization of B. burgdorferi in ticks treated or not with M. anisopliae through expression of the flaB gene; to investigate the influence of co-infection of B. burgdorferi and M. anisopliae in the microbiota of I. scapularis by the expression of the 16S gene. For tests with R. microplus, partially engorged females were removed from the body of a calf and artificially fed with blood from the same animal. Tetracycline administration was performed by adding the antibiotic into the blood by the time of feeding, thus forming four groups: females fed only with pure bovine blood (C) and blood+tetracycline (T); and two other groups that received the same diet, only blood or blood + tetracycline, but each of these groups was topically treated with M. anisopliae LCM S04 (F and T+F, respectively). For the studies with I. scapularis, nymphs infected or not with B. burgdorferi were inoculated with M. anisopliae ARSEF 549. The first assays were carried out to evaluate the survival of nymphs after exposure to the fungus; in the following tests, the nymphs were in contact with mice to feed ad libitum. The fed nymphs were weighed on the and dissected and the RNA from the guts was extracted and transformed into cDNA for gene expression analysis. Administration of tetracycline to R. microplus females did not alter tick survival. Furthermore, the disruption of tick microbiota and the use of antibiotics, did not impair fungal action, and both groups treated with M. anisopliae showed similar survival curves. The group T+F had the bacterial composition with the highest diversity. Inoculation of M anisopliae in I. scapularis infected or not with B. burgdorferi was not different; engorgement of I. scapularis nymphs was not affected by fungal treatment in ticks infected or not with B. burgdorferi. The relative expression of the flaB gene associated with the colonization of B. burgdorferi in nymphs remained similar with or without fungal treatment. Still among the same groups, the number of bacteria through the relative expression of the 16S gene was not altered after treatment with M. anisopliae; however, nymphs infected with B. burgdorferi, and fungus treated showed a higher relative expression of the Myd88 gene. Thus, the results shown here reiterate the need for further studies related to the multiple interactions between ticks, tick pathogens and entomopathogenic fungi, with the aim of understanding the complexity and to design efficient biological control programs.