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Rhipicephalus microplus is an important tick in tropical regions due to the high economic losses caused by its parasitism. Metarhizium anisopliae and Beauveria bassiana are well-known entomopathogenic fungi that can afflict R. microplus ticks. The development of new targets and strategies to control this parasite can be driven by studies of this tick's physiology. Recently, it was reported that when exposed to adverse physiological conditions, ticks can activate fermentative pathways, indicating transition from aerobic to anaerobic metabolism. Nevertheless, the precise mechanism by which entomopathogenic fungi influence R. microplus metabolism has not been clarified, limiting understanding of the tick-fungus association. Thus, the present study aimed to evaluate the effect of infection of ticks by M. anisopliae and B. bassiana on the amount of selected carboxylic acids present in the hemolymph, enabling increased understanding of changes previously reported. The results showed preservation in the concentrations of oxalic, lactic, and pyruvic acids in the hemolymph 24 and 48 h after dropping from cattle; while there were variations in the concentration of these carboxylic acids after infection of female ticks to M. anisopliae and B. bassiana. Significant increases were observed in the concentration of oxalic and lactic acids and significant reduction of pyruvic acid for both observation times (24 and 48 h) after infection by entomopathogenic fungi. These results indicate that B. bassiana and M. anisopliae infection alters the basal metabolism of R. microplus females, resulting in the activation of fermentative pathways.

In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

Aiming to characterize the potential off-target effects of fluazuron on ticks, biochemical analyses were conducted to evaluate changes in the carbohydrate metabolism of Rhipicephalus (Boophilus) microplus ticks after exposure to fluazuron. Hemolymph and fat body were collected from female ticks before and after (4, 8 and 15 days) exposure to fluazuron. Spectrophotometric analyses were done to quantify glucose concentration and lactate dehydrogenase (LDH) activity in the hemolymph and the concentration of glycogen in the tick's fat body. High Performance Liquid Chromatography (HPLC) was employed to determine the concentration of carboxylic acids in the hemolymph and to evaluate changes in intermediary metabolic processes requiring oxygen consumption. Increases in the levels of LDH activity and lactic acid concentration indicated that fluazuron enhanced fermentative metabolism in ticks. Exposure to fluazuron was also found to increase glucose concentrations in the hemolymph over time, although no significant differences were noted daily. In addition to expanding the body of knowledge about the mode of action of fluazuron, investigations into these mechanisms may also be useful in discovering new and as yet unexplored secondary effects.

This study evaluated the effects of destruxin A on Rhipicephalus (Boophilus) microplus females, since this toxin is one of the likely causes of high mortality induced by the entomopathogenic fungus Metarhizium anisopliae in arthropods. Ticks were immersed or inoculated with different concentrations of destruxin A. Despite the doses applied, there were no deaths or significant alterations in oviposition between the groups treated with destruxin A and the control groups. No other external effect caused by destruxin, such as tetanic paralysis, was observed in these engorged female ticks after the treatment.

The current study investigated the biology of nymphs of the first and second instars of Argas (Persicargas) miniatus. Nymphs were deprived of food for 15, 30 or 60 days and held at 27 ± 1 °C and 80 ± 10% relative humidity (controlled conditions) or at room conditions of temperature and relative humidity. Nymphs of first instar deprived of food for 15 or 30 days molted to second and third instars in both controlled and room conditions. Nymphs of the first instar deprived of food for 60 days had 28 and 37% mortality in controlled and room conditions, respectively; and survivors did not attach to the host. Nymphs of the second instar, deprived of food for 60 days, molted either to the third instar or to males after feeding on Gallus gallus, and the nymphs of the third instar developed to adults (42.42% males and 36.36% females when nymphs were held in controlled temperature and humidity conditions, and 40.54% males and 48.65% females when nymphs were held in room conditions). The remainder of the nymphs molted to the fourth instar and then molted to females. In conclusion, the nymphal starvation period of 60 days determined the number of nymph instars in the life cycle of A. miniatus under the experimental conditions studied.

Stomoxys calcitrans control is primarily based on the use of chemical products; however, new methods for control have been investigated. Biological control has been proposed as a promising alternative to traditional chemicals. The current study evaluated three methods in vitro to treat S. calcitrans eggs with the fungus Metarhizium anisopliae and assessed the virulence of this fungus to immature stages of this fly. In the first method of egg exposure, eggs were immersed in conidial suspensions and transferred to Petri plates with high humidity. A high mortality was observed in both treated and control groups. In the second method, the eggs were transferred to rearing medium right after immersion in conidial suspensions; the suspensions with highest conidial concentrations, 10(7) and 10(8) conidia mL(-1), caused 96.25% and 100% mortality, respectively. In the third method, the eggs were sprayed with conidial suspensions on the rearing medium. A high mortality was observed at 10(7) and 10(8) conidia mL(-1), 78.3% and 100%, respectively. The second method had the lowest mortality in the control group and was considered the most useful method for evaluating the effect of entomopathogenic fungi on S. calcitrans eggs. While eggs were very susceptible to M. anisopliae infection, larvae and pupae were not.

The use of the entomopathogenic fungus, Metarhizium anisopliae, to control ticks is a promising alternative to chemical acaricides, but both chemical and biological products could also work together in an integrated management. The current study evaluated the performance of M. anisopliae and its combination with deltamethrin to control a Boophilus microplus strain resistant to pyrethroid. Calves were held in individual pens and artificially infested with B. microplus. They were treated with an aqueous conidial suspension of M. anisopliae, a deltamethrin solution, or both fungus and deltamethrin combined. After treatment, engorged females that naturally dropped off from calves were counted daily and evaluated in the laboratory. The mean mortality rate was 32.57% in the groups treated with M. anisopliae, 38.58% in those groups treated with deltamethrin, and 30.92% in those treated with fungus and chemical combined. Additionally, ticks that dropped off from calves after 2 days had a lower nutrient index (NI) and egg production index (EPI) than those that dropped off at a later day after treatment. This indicates that M. anisopliae may reduce B. microplus population only for a few days after treatment when a simple aqueous conidial suspension was used.

The aim of the present study was to evaluate the capacity of the fungus Metarhizium anisopliae to control Boophilus microplus tick in pen trials. Infested calves were held in individual pen and treated with fungus suspension through aspersion bath. The results were evaluated based on ticks' mortality rate for 28 days after treatment, and on the analysis of biology of tick's samples which were transferred to an incubation chamber. It was reported 33% of mortality during the total period analyzed, and the production of eggs and nutritional rates were decreased only for a short period after treatment.