How Dopamine Influences Survival and Cellular Immune Response of Rhipicephalus microplus Inoculated with Metarhizium anisopliae.

Dopamine (DA) is a biogenic monoamine reported to modulate insect hemocytes. Although the immune functions of DA are known in insects, there is a lack of knowledge of DA's role in the immune system of ticks. The use of Metarhizium anisopliae has been considered for tick control, driving studies on the immune response of these arthropods challenged with fungi. The present study evaluated the effect of DA on the cellular immune response and survival of Rhipicephalus microplus inoculated with M. anisopliae blastospores. Exogenous DA increased both ticks' survival 72 h after M. anisopliae inoculation and the number of circulating hemocytes compared to the control group, 24 h after the treatment. The phagocytic index of tick hemocytes challenged with M. anisopliae did not change upon injection of exogenous DA. Phenoloxidase activity in the hemolymph of ticks injected with DA and the fungus or exclusively with DA was higher than in untreated ticks or ticks inoculated with the fungus alone, 72 h after treatment. DA was detected in the hemocytes of fungus-treated and untreated ticks. Unveiling the cellular immune response in ticks challenged with entomopathogenic fungi is important to improve strategies for the biological control of these ectoparasites.

Innovative granular formulation of Metarhizium robertsii microsclerotia and blastospores for cattle tick control.

The tick Rhipicephalus microplus poses a serious threat to the cattle industry, resulting in economic losses aggravated by tick resistance to chemical acaricides. Strains of Metarhizium spp., a well-known group of entomopathogenic fungi, can contribute to managing this ectoparasite. We explored two novel granular, microsclerotia- or blastospores-based formulations of Metarhizium robertsii for R. microplus control under semi-field conditions. Fungal persistence in soil was also observed for 336 days. The experiment used pots of Urochloa decumbens cv. Basilisk grass, treated with 0.25 or 0.5 mg of granular formulation/cm2 (25 or 50 kg/ha) applied to the soil surface prior to transferring engorged tick females onto the treated soil. The fungal granules yielded more conidia with subsequent sporulation under controlled indoor conditions than in the outdoor environment, where the levels of fungus rapidly declined over time. Metarhizium-root colonization ranged from 25 to 66.7% depending on the propagule and rate. Fungal formulations significantly reduced the number of tick larvae during the humid season, reaching at least 64.8% relative efficacy. Microsclerotia or blastospores-granular formulations of M. robertsii can reduce the impact of R. microplus, and thus prove to be a promising tool in the control of ticks.

In vitro efficacy of two commercial products of Metarhizium anisopliae s.l. for controlling the cattle tick Rhipicephalus microplus.

The effects of two different products - Metarril® SP Organic (dry conidia) and Metarril® SC Organic (emulsifiable concentrated conidia in vegetable oil) - on eggs, larvae and Rhipicephalus microplus engorged females were here explored. Three concentrations (108, 107, and 106 conidia mL-1) for both products were prepared in water + 0.1% Tween® 80 (v/v); afterward, bioassays were carried out for all R. microplus stages by immersion in suspensions (Metarril® SP) or formulations (Metarril® SC). Metarril® SP suspensions showed low efficacy and did not affect biological parameters of treated engorged females; for eggs and larvae, only slight decreases in hatchability and larvae population were observed. Despite a delay in germination, Metarril® SC presented better results; for females, reductions in Egg Mass Weight (EMW) and Egg Production Index (EPI) were reported. On eggs, 108 conidia mL-1 increased Incubation Period (IP), shortened Hatching Period (HP) and decreased hatchability by up to 61%; for larvae, 107 and 108 conidia mL-1 reached 99.6 and 100% larval mortality respectively, 10 days after fungal exposure. Thus, further studies involving the use of oil-based formulations for ticks such as Metarril® SC need to be performed, especially to control the most susceptible stages (eggs and larvae).

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies.

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals' leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick's immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female's body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gené's organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes' disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Rhipicephalus microplus infected by Metarhizium: unveiling hemocyte quantification, GFP-fungi virulence, and ovary infection.

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.