Metarhizium is an entomopathogenic fungus widely employed in the biological control of arthropods. Hemocytes present in the hemolymph of invertebrates are the cells involved in the immune response of arthropods. Despite this, knowledge about Rhipicephalus microplus hemocytes morphological aspects as well as their role in response to the fungal infection is scarce. The present study aimed to analyze the hemocytes of R. microplus females after Metarhizium robertsii infection, using light and electron microscopy approaches associated with the cytotoxicity evaluation. Five types of hemocytes (prohemocytes, spherulocytes, plasmatocytes, granulocytes, and oenocytoids) were described in the hemolymph of uninfected ticks, while only prohemocytes, granulocytes, and plasmatocytes were observed in fungus-infected tick females. Twenty-four hours after the fungal infection, only granulocytes and plasmatocytes were detected in the transmission electron microscopy analysis. Hemocytes from fungus-infected tick females showed several cytoplasmic vacuoles with different electron densities, and lipid droplets in close contact to low electron density vacuoles, as well as the formation of autophagosomes and subcellular material in different stages of degradation could also be observed. M. robertsii propagules were more toxic to tick hemocytes in the highest concentration tested (1.0 × 10 8 conidia mL −1 ). Interestingly, the lowest fungus concentration did not affect significantly the cell viability. Microanalysis showed that cells granules from fungus-infected and uninfected ticks had similar composition. This study addressed the first report of fungal cytotoxicity analyzing ultrastructural effects on hemocytes of R. microplus infected with entomopathogenic fungi. These results open new perspectives for the comprehension of ticks physiology and pathology, allowing the identification of new targets for the biological control.

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Metarril SP Organic is a product based on the fungus Metarhizium anisopliae, which was developed for controlling agricultural pests. The present study evaluated the effect of Metarril SP Organic plus 10% mineral oil, for controlling Rhipicephalus microplus under field conditions. Three groups were formed: Control group, which received no treatment; Oil control group, which was bathed only with water, Tween 80 and mineral oil; and Metarril group, bathed in the oil-based formulation Metarril SP Organic. Two treatments per group were performed and to verify the effect of the treatments, all R. microplus ticks between 4.5 and 8.0mm in length on the left side of the cattle were counted on days +7, +14 and +21 after each treatment, and a sample of engorged females was collected for evaluation of biological parameters. The Metarril SP Organic oil formulation showed efficacy ranging from 8.53 to 90.53%. The average efficacy of the oil-based formulation of Metarril SP Organic was 75.09 and 46.59% compared with the groups Control and Oil control, respectively. There were no significant changes in biological parameters of engorged R. microplus females collected from animals. Although there was no significant difference in the amount of ticks between the Oil control and Metarril groups, it is believed that the association of mineral oil with Metarril SP Organic product is effective in R. microplus tick control in field. Thus, this association has potential to be used in strategic control programs of cattle tick.

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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