Abstract
Hemocytes, cells present in the hemocoel, are involved in the immune response of arthropods challenged with entomopathogens. The present study established the best methodology for harvesting hemocytes from Rhipicephalus microplus and evaluated the number of hemocytes in addition to histological analysis from ovaries of fungus-infected females and tested the virulence of GFP-fungi transformants. Different centrifugation protocols were tested, and the one in which presented fewer disrupted cells and higher cell recovery was applied for evaluating the effect of Metarhizium spp. on hemocytes against R. microplus. After processing, protocol number 1 (i.e., hemolymph samples were centrifuged at 500×g for 3 min at 4 °C) was considered more efficient, with two isolates used (Metarhizium robertsii ARSEF 2575 and Metarhizium anisopliae ARSEF 549), both wild types and GFP, to assess their virulence. In the biological assays, the GFP-fungi were as virulent as wild types, showing no significant differences. Subsequently, hemocyte quantifications were performed after inoculation, which exhibited notable changes in the number of hemocytes, reducing by approximately 80% in females previously treated with Metarhizium isolates in comparison to non-treated females. Complementarily, 48 h after inoculation, in which hemolymph could not be obtained, histological analysis showed the high competence of these fungi to colonize ovary from ticks. Here, for the first time, the best protocol (i.e., very low cell disruption and high cell recovery) for R. microplus hemocyte obtaining was established aiming to guide directions to other studies that involves cellular responses from ticks to fungi infection.
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Acknowledgments
We thank Coordenacão de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) from Brazil. We thank Dr. Diva Denelle Spadacci Morena (Butantan Institute, Sao Paulo, Brazil) for helping with the histological studies and Dr. Donald Roberts (Utah State University, Utah, USA) for providing the fungal isolates previously supplied by Dr. Raymond St. Leger (University of Maryland). Vania R.E.P. Bittencourt is a CNPq researcher.
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de Paulo, J.F., Camargo, M.G., Coutinho-Rodrigues, C.J.B. et al. Rhipicephalus microplus infected by Metarhizium: unveiling hemocyte quantification, GFP-fungi virulence, and ovary infection. Parasitol Res 117, 1847–1856 (2018). https://doi.org/10.1007/s00436-018-5874-y
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DOI: https://doi.org/10.1007/s00436-018-5874-y