Estudo morfo-fisiológico de hemócitos de fêmeas ingurgitadas de Rhipicephalus microplus infectadas por Metarhizium spp. marcado com GFP

Abstract

Metarhizium spp. is a fungus widely used in the biological control of arthropods. Hemocytes, cells present in the hemolymph, are involved in the immune response of these ectoparasites, especially when challenged by pathogens. Tick methods for cells obtainment face difficulties and the knowledge about morphology of Rhipicephalus microplus hemocytes are very scarce, as well as the cell’s responses to infections. The present study had as objectives the establishment of a better protocol for obtaining hemocytes from Rhipicephalus microplus engorged females, morphological analysis of hemocytes and their conditions when infected by arthropodopathogenic fungi GFP. Different spin speeds were tested, and the method with higher cell viability and high cell concentration was applied after fungi physical inoculation into R. microplus engorged females. In the biological assay, the parameters of egg production, the incubation period of the larvae, the nutrition index and the percentage of control were evaluated followed by immersion of tick females using fungal suspension. After inoculation, hemolymph collection was performed and the hemocytes separated from plasma, resuspended in PBS and quantified in a Neubauer chamber. Hemocytes were submitted to hematoxylin and eosin and PAS staining and analyses of transmission electron microscopy were performed. For standardization, a centrifugation at 500 × g for three minutes at 4 ° C was considered more efficient and applied after inoculation of fungi. Bioassay results of the two fungal isolates yielded very similar data, demonstrating that GFP molecular marking did not influence on fungi virulence against R. microplus. Hemocyte’s quantification after fungal inoculation showed that all isolates were able to decrease the concentration by approximately 80% in comparison to hemocytes from uninfected females. Under physiological conditions, five types of hemocytes (prohemocytes, granulocytes, spherulocytes, plasmatocytes and oenocytes) were identified and when females were infected by Metarhizium, these cells were presented in a cell death process and some cells presented fungus internalized in cytoplasm, suggesting a presence phagocytosis process. It was suggested that infection by entomopathogenic fungi can generate changes in the R. microplus hemocytes and directly influence on the concentration of these cells. The present study was the first to perform transmission electron microscopy on hemocytes of this species of tick and was the first to describe the cellular alterations due to fungal infection.