Entomopathogenic fungus treatment changes the gut bacterial diversity of Rhipicephalus microplus ticks.

BACKGROUND: Ticks are obligate bloodsucking parasites responsible for significant economic losses and concerns with human and animal health, mainly due to the transmission of pathogens. Entomopathogenic fungi have been intensively studied as an alternative strategy for tick control that can be used in combination with synthetic acaricides in the integrated management of ticks. Here, we investigated how the gut bacterial community of Rhipicephalus microplus is shaped after Metarhizium anisopliae treatment and how the tick susceptibility to the fungus is affected after disrupting gut bacterial microbiota. METHODS: Partially engorged tick females were artificially fed with pure bovine blood or blood plus tetracycline. Two other groups received the same diet and were topically treated with M. anisopliae. The guts were dissected, and the genomic DNA was extracted 3 days after the treatment; the V3-V4 variable region of the bacterial 16S rRNA gene was amplified. RESULTS: The gut of ticks that received no antibiotic but were treated with M. anisopliae exhibited lower bacterial diversity and a higher occurrence of Coxiella species. The Simpson diversity index and Pielou equability coefficient were higher in the gut bacterial community when R. microplus were fed with tetracycline and fungus-treated. Ticks from fungus-treated groups (with or without tetracycline) exhibited lower survival than untreated females. Previous feeding of ticks with the antibiotic did not change their susceptibility to the fungus. Ehrlichia spp. were not detected in the gueated groups. CONCLUSIONS: These findings suggest that myco-acaricidal action would not be impacted if the calf hosting these ticks is under antibiotic therapy. Moreover, the hypothesis that entomopathogenic fungi can affect the bacterial community in the gut of R. microplus engorged females is endorsed by the fact that ticks exposed to M. anisopliae exhibited a dramatic reduction in bacterial diversity. This is the first report of an entomopathogenic fungus affecting the tick gut microbiota.

Encapsulation of entomopathogenic fungal conidia: evaluation of stability and control potential of Rhipicephalus microplus.

The use of chemical acaricides is the primary strategy to control tick infestations. Nonetheless, chemical resistance in ticks has been reported. Thus, complementary methods such as biological control using entomopathogenic fungi (EPF) have been investigated. EPF, although efficient, have their viability compromised when applied under natural conditions, which indicates that formulation development is essential. Some researchers have demonstrated the efficacy of ionic gelation in protecting EPF against deleterious abiotic factors. In the present study, we conducted the ionic gelation technique to encapsulate Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) conidia in 2% (EC 2%) and 3% (EC 3%) sodium alginate. Next, the quantity and viability of encapsulated conidia (EC) were determined. The morphology of particles was characterized by using Scanning Electron Microscopy (SEM). EC and non-encapsulated conidia (NEC) were stored at room temperature (26.8 °C) and in the freezer (-11.9 °C) to shelf-life testing. For UV-B irradiance tolerance and thermotolerance tests, EC and NEC were exposed to UV-B (6.0 or 8.0 kJ m - 2) and heat (42 ºC). In addition, biological parameters of Rhipicephalus microplus Canestrini (Acari: Ixodidae) engorged females exposed to EC were evaluated. The particles presented a spherical shape, more homogeneous (EC 2%) or heterogeneous (EC 3%). Encapsulation decreased (4.8×) the conidial concentration and did not affect their viability. On the other hand, encapsulation increased the shelf life of conidia at room temperature as well as their UV-B tolerance and thermotolerance (6 h). The fungal particles decreased the biological parameters of females more significantly than the NEC. As far as we know, we reported for the first time the use of the ionic gelation to encapsulate entomopathogenic fungi toward controlling R. microplus.

Beauveria bassiana interacts with gut and hemocytes to manipulate Aedes aegypti immunity.

BACKGROUND: Mosquito-borne diseases affect millions of people. Chemical insecticides are currently employed against mosquitoes. However, many cases of insecticide resistance have been reported. Entomopathogenic fungi (EPF) have demonstrated potential as a bioinsecticide. Here, we assessed the invasion of the EPF Beauveria bassiana into Aedes aegypti larvae and changes in the activity of phenoloxidase (PO) as a proxy for the general activation of the insect innate immune system. In addition, other cellular and humoral responses were evaluated. METHODS: Larvae were exposed to blastospores or conidia of B. bassiana CG 206. After 24 and 48 h, scanning electron microscopy (SEM) was conducted on the larvae. The hemolymph was collected to determine changes in total hemocyte concentration (THC), the dynamics of hemocytes, and to observe hemocyte-fungus interactions. In addition, the larvae were macerated to assess the activity of PO using L-DOPA conversion, and the expression of antimicrobial peptides (AMPs) was measured using quantitative Real-Time PCR. RESULTS: Propagules invaded mosquitoes through the midgut, and blastopores were detected inside the hemocoel. Both propagules decreased the THC regardless of the time. By 24 h after exposure to conidia the percentage of granulocytes and oenocytoids increased while the prohemocytes decreased. By 48 h, the oenocytoid percentage increased significantly (P < 0.05) in larvae exposed to blastospores; however, the other hemocyte types did not change significantly. Regardless of the time, SEM revealed hemocytes adhering to, and nodulating, blastospores. For the larvae exposed to conidia, these interactions were observed only at 48 h. Irrespective of the propagule, the PO activity increased only at 48 h. At 24 h, cathepsin B was upregulated by infection with conidia, whereas both propagules resulted in a downregulation of cecropin and defensin A. At 48 h, blastospores and conidia increased the expression of defensin A suggesting this may be an essential AMP against EPF. CONCLUSION: By 24 h, B. bassiana CG 206 occluded the midgut, reduced THC, did not stimulate PO activity, and downregulated AMP expression in larvae, all of which allowed the fungus to impair the larvae to facilitate infection. Our data reports a complex interplay between Ae. aegypti larvae and B. bassiana CG 206 demonstrating how this fungus can infect, affect, and kill Ae. aegypti larvae.

Inhibition of Dopamine Activity and Response of Rhipicephalus microplus Challenged with Metarhizium anisopliae.

Dopamine modulates ticks and insect hemocytes and links these arthropods' nervous and immune systems. For the first time, the present study analyzed the effect of a dopamine receptor antagonist on the survival, biological parameters, phagocytic index, and dopamine detection in the hemocytes of ticks challenged by Metarhizium anisopliae. The survival and egg production index of Rhipicephalus microplus were negatively impacted when ticks were inoculated with the antagonist and fungus. Five days after the treatment, the survival of ticks treated only with fungus was 2.2 times higher than ticks treated with the antagonist (highest concentration) and fungus. A reduction in the phagocytic index of hemocytes of 68.4% was observed in the group inoculated with the highest concentration of the antagonist and fungus compared to ticks treated only with fungus. No changes were detected in the R. microplus levels of intrahemocytic dopamine or hemocytic quantification. Our results support the hypothesis that dopamine is crucial for tick immune defense, changing the phagocytic capacity of hemocytes and the susceptibility of ticks to entomopathogenic fungi.

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.