Metarhizium anisopliae s.l. modulation of lipid metabolism during tick infection is independent of AMPK and ERK pathways.

Despite the importance of fat body in metabolism of arthropods, studies in ticks are scarce. This study evaluated the lipid composition and activation of extracellular signal-regulated protein kinase (ERK) and AMP-activated protein kinase (AMPK) enzymes in Rhipicephalus microplus fat body after infection with different isolates of the fungus Metarhizium anisopliae sensu lato (Metschnikoff, 1879) Sorokin, 1883. The isolates CG 32, GC 112, GC 148, GC 347, and GC 629 were inoculated as viable or non-viable conidia in the ticks. The engorged females were dissected, and their fat bodies were collected 24 and 48 h after infection. The lipid composition was assessed by thin layer chromatography, and enzyme activation was detected by Western blotting with antibodies against p-AMPK and p-ERK. The study showed increased levels of triacylglycerol 24 and 48 h and fatty acid after 48 h after inoculation with different isolates of viable fungi in the tick's hemocoel. Detection of the active form of ERK was demonstrated only after inoculation with non-viable conidia of all isolates tested. The active form of AMPK, only isolate CG 112 was able to activate with viable or non-viable conidia, whereas isolates CG 32 and CG 629 were able to activate with non-viable conidia. This study provides the first report about changes in important metabolic pathways in ticks infected with entomopathogenic fungi and suggests that the lipid content is modulated by non-usual pathways. However, further studies may be necessary for a better elucidation of this interaction.

The influence of conidial Pr1 protease on pathogenicity potential of Metarhizium anisopliae senso latu to ticks.

Pr1 is a subtilisin-like protease produced by Metarhizium spp. entomopathogenic fungi, and it is recognized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, correlation was sought between mortality of tick larvae and conidial Pr1 levels of one Metarhizium anisopliae senso latu (s.l.) isolate (CG 148). Conidia with different levels of pr1 gene expression and enzymatic activity were obtained by producing them on either artificial medium (to yield low Pr1 activity) or on Rhipicephalus microplus cadavers (to yield high Pr1 activity). Conidial proteolytic activity was assessed using N-suc-ala-ala-pro-phe-ρNA as the chromogenic substrate, and pr1 expression was profiled by qPCR using three genes (gpd, try, and tef) as reference genes. Pr1 enzymatic (proteolytic) activity on conidia obtained from tick cadavers was 36 U mg(-1) in comparison to 4 U mg(-1) on conidia from PDA medium. Also, pr1 gene expression level was ten times higher in conidia from tick cadavers compared to PDA medium. Bioassays of M. anisopliae s.l. CG 148 spores with elevated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant change percent mortality of tick larvae). The minimal levels of Pr1 on conidia produced on artificial medium was adequate to afford high levels of virulence, and the elevated amounts of the enzyme on tick-cadaver-produced conidia did not induce elevated larval mortality. As long as some Pr1 activity was present, fungal virulence of isolate CG 148 against tick larvae was not elevated by increased levels of conidial Pr1.

Neutral lipid composition changes in the fat bodies of engorged females Rhipicephalus microplus ticks in response to fungal infections.

The tick's fat body plays an essential role in energy storage and utilization. This study aimed to analyze the fat body neutral lipid composition in Rhipicephalus microplus engorged females. In the first study (physiological profile of untreated ticks), the lipid analysis took place over the course of 4 days; the engorged females were incubated at optimal conditions and their fat bodies were dissected daily. Fat body lipid analysis after fungal infection with Metarhizium anisopliae sensu lato (s.l.) or Beauveria bassiana s.l. was performed with four groups: one without any treatment, one that was inoculated with a solution of 0.1 % Tween 80 in water, and two groups that were inoculated with M. anisopliae or B. bassiana conidial suspensions. The fat bodies were dissected 24 and 48 h after infection. Lipid analysis was conducted by thin-layer chromatography on a silica plate. The results of the physiological profile showed that the amounts of triacylglycerol (TAG) and free cholesterol (CHO) decreased with time, whereas cholesterol ester (CHOE) increased on the second and fourth days. Following M. anisopliae or B. bassiana infection, there was an increase in the amount of CHO after 24 h, whereas the other lipid classes were not altered. M. anisopliae caused an increase in CHOE and TAG and a reduction in CHO at 48 h after infection; however, B. bassiana infection did not cause significant alterations in the concentrations of these lipids. M. anisopliae and B. bassiana infection changed the fat body metabolism of engorged female R. microplus ticks. This study provides the first report of changes in the neutral lipid composition of the R. microplus fat body.

Commercial formulation of Metarhizium anisopliae for the control of Rhipicephalus microplus in a pen study.

The present study evaluated, for the first time, the effect of the commercial formulation Metarril(®) SP Organic of Metarhizium anisopliae plus 10% mineral oil to control Rhipicephalus microplus in a pen study. Three groups were formed with six animals each: the first group was exposed to Metarril(®) plus 10% mineral oil and 1% Tween 80; the second group was exposed to sterile distilled water, mineral oil and Tween 80 (oil control group); and the third group received no treatment (control group). The fungal formulation contained 1 × 10(8)conidiaml(-1). Each animal was sprayed with 3L of formulation. Fallen ticks were counted daily and a sample of 20 engorged females per day was incubated for assessment of biological parameters. Throughout the study period, Metarril(®) oil-based formulation showed an efficacy ranging from 19.20% to 67.39% in comparison with the control group; and from 8.18% to 61.38% in comparison with the oil control group. The average efficacy of Metarril(®) oil-based formulation was 47.74% and 40.89% in comparison with control and oil control groups, respectively. Changes in the biological parameters of engorged R. microplus females were observed in the first three days after treatment, with a significant reduction in hatching percentage and egg production index. We concluded that Metarril(®) SP Organic plus 10% mineral oil was efficient against R. microplus in pen studies. However, further in vivo studies are required to increase the efficacy and to establish a protocol for the use of this product in the field against the cattle tick.

Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus.

The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

Virulence potential of Metarhizium anisopliae s.l. isolates on Rhipicephalus (Boophilus) microplus larvae.

The use of entomopathogenic fungi to control arthropods has been reported worldwide for decades. The aim of the present study was to evaluate the virulence of 30 Metarhizium anisopliae s.l. Brazilian isolates from different geographical regions, hosts or substrates on the larvae of Rhipicephalus (Boophilus) microplus ticks under in vitro conditions to the selection of virulent isolates in order to be further used in biological control programs. The current study confirmed the lethal action of M. anisopliae s.l. isolates on R. (B.) microplus larvae with different mortality levels, usually directly proportional to the conidia concentration. No relationship was found between the origin of the isolate and its virulence potential or between the virulence potential and conidia production. Three isolates (CG 37, CG 384 and IBCB 481) caused a high percentage of larval mortality, reaching LC(50) at 10(6) conidia ml(-1), thus requiring a lower conidia concentration to cause an approximately 100% larval mortality. The results of this study suggest that these three isolates are the most promising for use in programs aimed at microbial control in the field.

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions.

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.