Secretomic analysis of Beauveria bassiana related to cattle tick, Rhipicephalus microplus, infection.

Beauveria bassiana is widely studied as an alternative to chemical acaricides in controlling the cattle tick Rhipicephalus microplus. Although its biocontrol efficiency has been proved in laboratory and field scales, there is a need to a better understanding of host interaction process at molecular level related to biocontrol activity. In this work, applying a proteomic technique multidimensional protein identification technology (MudPIT), the differential secretome of B. bassiana induced by the host R. microplus cuticle was evaluated. The use of the host cuticle in a culture medium, mimicking an infection condition, is an established experimental model that triggers the secretion of inducible enzymes. From a total of 236 proteins, 50 proteins were identified exclusively in infection condition, assigned to different aspects of infection like host adhesion, cuticle penetration and fungal defense, and stress. Other 32 proteins were considered up- or down-regulated. In order to get a meaningful global view of the secretome, several bioinformatic analyses were performed. Regarding molecular function classification, the highest number of proteins in the differential secretome was assigned in to hydrolase activity, enzyme class of all cuticle-degrading enzymes like lipases and proteases. These activities were also further validated through enzymatic assays. The results presented here reveal dozens of specific proteins and different processes potentially implicated in cattle tick infection improving the understanding of molecular basis of biocontrol of B. bassiana against R. microplus.

Metarhizium anisopliae for controlling Rhipicephalus microplus ticks under field conditions.

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.

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.

Physiological changes in Rhipicephalus microplus (Acari: Ixodidae) experimentally infected with entomopathogenic fungi.

Carbohydrate metabolism plays an important role in the physiology and maintenance of energy stores within living organisms. However, when organisms are exposed to adverse physiological conditions, such as during pathogenic infection, these organisms begin to use alternative substrates (proteins and lipids) for energy production. This paper studied the carbohydrate metabolism of Rhipicephalus microplus after infection with Beauveria bassiana and Metarhizium anisopliae. The parameters evaluated were glucose concentration, enzymatic activities of lactate dehydrogenase (LDH), alanine aminostransferase (ALT) and aspartate aminostransferase (AST), amounts of uric acid and urea in the hemolymph, and amount of glycogen in the fat body. The results showed changes in nitrogenous products, including an increase in the amount of urea detected 48 h after infection with both fungi. The enzymatic activities of LDH, ALT, and AST were increased after infection. The amount of glucose was increased 24 h after infection with B. bassiana and was reduced 48 h after infection with both fungi. The amount of glycogen in the fat body was reduced at different times of infection with both fungi. These results demonstrate, for the first time, the changes in carbohydrate metabolism of R. microplus after infection with M. anisopliae and B. bassiana and contribute to a better understanding of this host-parasite relationship. Together with knowledge of diseases that affect these ticks and their susceptibility to entomopathogens, an understanding of tick physiology will be necessary for the effective implementation of current biological control methods and will assist in the discovery of new methods to control this ectoparasite.

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.

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.

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.

Virulence of Isaria sp. and Purpureocillium lilacinum to Rhipicephalus microplus tick under laboratory conditions.

Rhipicephalus microplus (Canestrini) is an ectoparasite accountable for great economic losses. The use of entomopathogenic fungi to control arthropods has shown promising responses. The present study evaluated the virulence of Isaria farinosa (Holmsk.) Fr., Isaria fumosorosea (Wize) Brown and Smith, and Purpureocillium lilacinum (=Paecilomyces lilacinus) (Thom.) Samson to engorged females, eggs, and larvae of R. microplus. There were four treatment groups (10(5), 10(6), 10(7), and 10(8) conidia ml(-1)) and the control group (water and Tween 80, 0.1% v/v). The treatment was based on immersion of the specimen in 1 ml of the suspension or control solution. The study observed changes in egg viability and larval mortality after treatment. The results showed that I. farinosa, P. lilacinum, and I. fumosorosea caused alterations in the biological parameters of R. microplus ticks. I. fumosorosea presented the greatest potential to control R. microplus engorged females in vitro, causing a 49% decrease in nutritional index. All fungal isolates presented significant reduction in the egg production index. I. farinosa reduced the hatching percentage if the eggs were treated with the two highest conidial concentrations. All conidial concentrations of I. fumosorosea were able to reduce the hatching percentage significantly. All tested isolates showed pathogenicity toward unfed R. microplus larvae. As far as we know, this is the first study reporting the effect in vitro of I. farinosa, I. fumosorosea, and P. lilacinum to different developmental stages of R. microplus ticks.

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.

Efficiency of Lecanicillium lecanii to control the tick Rhipicephalus microplus.

Rhipicephalus microplus, known as the cattle tick, causes serious economic losses in the Brazilian cattle industry each year. Traditional parasite control is primarily based on the use of chemical acaricides, which unfortunately have many negative side effects. Biological control is seen as a promising alternative to chemical acaricide use. This study evaluates the entomopathogenic fungus Lecanicillium lecanii for effectiveness in controlling engorged females, eggs, and larvae of R. microplus. Conidial formulations of L. lecanii, isolate CG 420, were prepared in both oil (15% mineral oil) and aqueous suspensions. Ticks were immersed in a 1ml oil-based conidial suspension at 1x10(8)conidiaml(-1) or one of several aqueous conidial suspensions at 1x10(5), 10(6), 10(7) or 10(8)conidiaml(-1). The control groups were immersed in water or oil solutions with no conidia. Treatments with aqueous conidial suspensions were conducted with 10 ticks per group (1x10(5), 10(6), 10(7), 10(8)conidiaml(-1) or control) whereas the oil treatments used 30 ticks per group (1x10(8)conidiaml(-1) or control). Bioassays were repeated twice on different days with different batches of conidia. After treatment, the biological parameters of engorged females were evaluated, while eggs and larvae were evaluated taking into consideration hatchability and mortality, respectively. The results indicate that L. lecanii has the potential to control engorged females, eggs and larvae of R. microplus. Better results were observed when conidial oil-based suspension was used. In general, engorged females treated with 1x10(8)conidiaml(-1) oil suspensions died before laying eggs, resulting in 97.6% of tick control. As far as we know, this is the first report of the effects of L. lecanii on R. microplus tick.

Performance of Metarhizium anisopliae and Its combination with deltamethrin against a pyrethroid-resistant strain of Boophilus microplus in a stall test.

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.