Introduction

Increasing concerns about degraded environmental conditions and public health worries are stimulating research on biological control of arthropod pests. A group of insect-pathogenic fungi, the Metarhizium anisopliae species complex (Bischoff et al. 2009), has been widely studied both as biocontrol agents of arthropods and as models for elucidating host-pathogen infection processes (Chandler et al. 2000; Roberts and St Leger 2004; Fernandes et al. 2012). Arthropod-pathogenic (AP) fungi have worldwide distribution, ranging from the subarctic to the tropics (Zimmermann 2007) and hosts that include insects, ticks, and plant roots (Costa et al. 2002; Roberts and St Leger 2004; Fernandes et al. 2010; Sasan and Bidochka 2012). Laboratory tests using M. anisopliae senso latu (s.l.) species against Rhipicephalus microplus (Canestrini 1888) ticks, commonly known as the cattle tick, indicate that these fungi may be effective alternatives to chemical acaricides (Bittencourt et al. 1992, 1994; Castro et al. 1997; Camargo et al. 2012; Quinelato et al. 2012).

To infect arthropods, AP fungal conidia need to adhere, germinate, and penetrate through host cuticle, and these fungi produce enzymes (primarily proteases, lipases, and chitinases) to degrade the most abundant constituents of cuticle (Schrank and Vainstein 2010) to reach the host’s hemocoel. Fungal virulence to a targeted arthropod pest is suspected to be determined by several factors, including fungal production of enzymes and toxins, as well as the microorganism’s ability to evade or inhibit the host’s immune system. Studies have been conducted to understand, at the molecular level, infection of insects by M. anisopliae s.l. (Fang and Bidochka 2006; Wang et al. 2009); with ticks, however, there is virtually no published information on the molecular aspects of infection.

Pr1 is a protease produced by AP fungi to hydrolyze cuticle proteins of host. Pr1 is upregulated two times in the Metarhizium spp. infection cycle: firstly, during the initial stages of infection, including in the appressorium, the structure formed by the fungus in preparation for host colonization to hydrolyze host cuticular proteins (St Leger et al. 1989; St Leger et al. 1991a), and secondly, during the final stages of pathogenesis (conidiation) to facilitate hyphal emergence through insect cuticle (Small and Bidochka 2005); this emergence is necessary for completion of the fungal pathogenic cycle, viz., allowing the mass production of conidia on the surface of the arthropod cadaver, thereby facilitating wide-scale conidial dispersion—which may increase levels of biocontrol.

Multiple (serial) passages of AP fungi on artificial media is widely known to eventually lead to degradation of spore production levels and reduced virulence to its arthropod host. Interestingly, passage through arthropod hosts almost immediately restores these “lost” traits (Kawakami 1960; Fargues and Robert 1983). Accordingly, differences in conidia produced on ticks (= in vivo conidia) in contrast to those produced on artificial media (= in vitro conidia) was examined in the hope of providing critical tool(s) for improving fungal biological control of ticks. The current study evaluated pr1 expression and Pr1 proteolytic activity of M. anisopliae s.l. conidia produced on artificial medium or on R. microplus female cadavers to identify the levels of the cuticle-invasion protease Pr1 from conidia of each substrate.

Material and methods

Fungal isolate

The M. anisopliae s.l. used in the present study was CG 148 isolated from Deois flavopicta (Homoptera: Cercopidae) in Goiânia, Goiás, Brazil. It was obtained from the Brazilian Agriculture and Livestock Research Enterprise (EMBRAPA), Genetic Resources and Biotechnology (CENARGEN) station in Brasilia, DF, Brazil. Stock culture plates of potato dextrose agar medium (PDA) were incubated at 25 ± 1 °C and ≥80 % relative humidity (RH) for 14 days, and then stored at 4 °C. The isolate CG 148 was chosen based on previous information on the virulence of this fungus for tick larvae, reported by Quinelato et al. (2012). CG 148-fungus isolate yielded moderate to high virulence against tick larvae (Quinelato et al. 2012).

Fungal cultivation on artificial medium and ticks

Conidia of M. anisopliae s.l. CG 148 were produced on either PDA or R. microplus ticks. For artificial culture, conidia were inoculated on PDA medium and incubated at 25 ± 1 °C and ≥80 % RH for 15 days; for cultivation on ticks, CG 148 conidial suspension (1 × 108 conidia mL−1) was inoculated into engorged R. microplus females using a microinsulin needle (30 gauge).

Conidia were harvested from culture plates by scraping the medium surface with a scalpel blade and suspended in 100 mL of polyoxyethylene sorbitan monooleate (Tween 80®, Sigma Chemical Co., St. Louis, MO, USA) solution (0.01 % v/v). The conidial suspension was homogenized for 1 min using a vortex mixer, quantified in a hemocytometer and adjusted to 1.0 × 108 conidia mL−1. Engorged R. microplus females were from naturally infested bovines that had not been treated with chemical acaricides for more than 2 months. The engorged females were collected from stall floors and washed in a 1 % sodium hypochlorite solution for cuticular asepsis. Then, they were rinsed in sterile distilled water and dried on sterile paper towels. Inoculation was by injecting into each females tick 5 μL of fungal suspension via the foramen between the capitulum and the anterior end of scutum (Johns et al. 1998). Eight hundred females were inoculated and incubated at 25 ± 1 °C and ≥80 % RH for 15 days, which resulted to 100 % infection.

Conidial viability was determined by plating an aliquot (~50 μL) of the conidia suspension on PDA medium plus 0.05 % chloramphenicol, followed by incubation at 25 ± 1 °C and ≥80 % RH. Conidial germination was observed by microscope (×200) after 24 h (Alves 1998).

Harvest of conidia

Conidia produced on PDA medium were harvested from plates after 15 days of growth and placed in plastic microtubes. Conidia produced on ticks were removed by placing the infected ticks on sieves (final pore size = 1.18 mm; Bertel®) on a sieve shaker (Laboratory Test Sieve; Bertel®) at maximum vibration for 15 min. Conidia from PDA or from ticks were separated into seven aliquots: three aliquots of 60 mg for pr1 expression assay, two aliquots of 37 mg for proteolytic activity assay, and two aliquots of 80 mg for larval bioassay. Samples for pr1 gene expression assay were stored in 500-μL RNAlater solution (Ambion®). Conidial production experiments were performed two times.

Specific subtilisin-like protease (Pr1) activity

Conidia obtained from medium or from female ticks were suspended in an extraction buffer (Tris–HCl 50 mM, pH 8.0, containing 0.25 % Triton X-100, 1:2.5 w/v) (Santi et al. 2010). The suspensions were shaken vigorously for 5 min, and the resulting supernatant filtered through a 25-mm diameter filter, 0.2-mm pore size (Millipore® USA), to remove conidia. The obtained filtrates were used to measure conidial proteolytic activity using N-suc-ala-ala-pro-phe-ρNA as the chromogenic substrate, as follows: 5 μL of filtrate, 5 μL of 10 mM N-suc-ala-ala-pro-phe-ρNA (Sigma®) and 90 μL of 0.1 mol L−1 Tris–HCl buffer pH 8.0 (Santi et al. 2010). Kinetic assays were incubated at 37 °C for 30 min, after which absorbance was measured at 405 nm in a plate spectrophotometer (Multiskan GO® Thermo Scientific). The enzyme activity was calculated from a standard curve of ρ-nitroaniline (ρNA), with each sample expressed as micromolars per minute of reaction per milligrams of protein; the assay was repeated two times with each sample in triplicate. Total protein was quantified according to Lowry et al. (1951), using bovine serum albumin as standard. Data were analyzed via analysis of variance (ANOVA) followed by Tukey test using BioEstat® software, version 4.0. P values less than 0.05 were considered significant (Sampaio 2002).

Pr1 gene expression

The expression of the subtilisin-like protease (pr1) gene was profiled using three genes (gpd, try, and tef) as reference genes (Table 1) (Fang and Bidochka 2006). RNA was isolated using RNeasy Mini Kit (QIAGEN®) according to manufacturer’s instructions and concentrations measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific® USA). Complementary DNA (cDNA) was synthesized using High Capacity RNA-to-cDNA kit (Life Technologies®) following manufacturer’s protocol. The cDNA was subsequently diluted with DEPC-treated water (Life Technologies® USA) to 25 ng μL−1. Real-time PCR (qPCR) amplification mixtures (12 μL) contained 25 ng cDNA template, 2× Power SYBR GreenPCR Master Mix (6 μL; Life Technologies®) and 150 nM each of the forward and reverse primers (Life Technologies®) (Table 1). The reaction was performed with a StepOne Plus (Life Technologies® USA) real-time PCR system. The PCR protocol was as follows: 10-min activation/denaturation step at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. Fluorescence was measured at each polymerization step. The PCR amplifications were conducted two times with each cDNA analyzed in triplicate in each of two tests.

Table 1 Name, primer sequences, and PCR efficiency of the genes used in the present study
Full size table

The expression stability of the reference genes, as well as expression of the pr1 gene, was evaluated by geNorm qbasePLUS (available from http://www.biogazelle.com/). Detailed information on this application can be found in Vandesompele et al. (2002).

Larval bioassay

Engorged R. microplus females were collected and incubated for oviposition following the same methodology reported in “Fungal cultivation on artificial medium and ticks” section. Aliquots of 50 mg eggs (approximately 1000 eggs) were collected from day 1 to day 10 of oviposition, placed in test tubes sealed with cotton plugs and observed daily for 20–25 days to estimate percent hatch. Tubes with less than 98 % hatch were discarded. The larval treatment with conidia from PDA medium or from ticks was performed on the 15th day after larval hatching (approximately 40 days after oviposition). Suspensions were prepared as described in “Fungal cultivation on artificial medium and ticks” section. Conidia were quantified and adjusted to 1.0 × 108 conidia mL−1 according to Alves (1998). Conidial viability was determined as reported in “Fungal cultivation on artificial medium and ticks” section.

Each bioassay consisted of three groups (control group; group treated with conidia from PDA, and group treated with conidia from ticks). A Tween 80® solution (0.01 % v/v) was used to treat the control groups. Each group had eight test tubes, each containing approximately 1000 R. microplus larvae. Experiments were conducted by injecting 1 mL of conidial suspension into each test tube. The larvae were held immersed in the injected fluid for 3 min, and the test tube was then inverted until all of the conidial suspension had been absorbed by the cotton plug. The tubes were maintained at 27 ± 1 °C and ≥80 % RH in the dark. Larval mortality was recorded at 5-day intervals for 30 days. The percent larval mortality for each tube was visually estimated by microscopic observation (×20), with the estimates expressed as percentages varying from 0 to 100 % in 1 % intervals. Lack of larval ability to move was recorded as “dead.” Bioassays were repeated two times.

The mean mortalities of the larvae were compared using the nonparametric Kruskal-Wallis test for statistical differences, followed by Student-Newman-Keuls (SNK) test for comparison between the means. Data analyses were conducted using BioEstat® software, version 4.0. P values less than or equal to 0.05 were considered significant (Sampaio 2002).

Reisolation of Metarhizium anisopliae s.l

Dead larvae from all treatment groups were placed on PDA in Petri plates for 14 days at 25 ± 1 °C and ≥80 % RH to allow fungal growth and conidiogenesis. The macromorphology and micromorphology of the fungal colonies on fungus treated larvae were examined for the fungal characteristics (Tulloch 1976) that, if present, confirm the identity as M. anisopliae s.l.

Results

Conidial viability

Conidia of M. anisopliae isolate CG 148 used for treatment of adult females and tick larvae had approximately 100 % germination after incubation for 24 h at 25 ± 1 °C and ≥80 % RH.

Specific subtilisin-like protease (Pr1) activity

Specific Pr1 enzyme activity of surface extracts of conidia produced on artificial medium (PDA) or on infected ticks were statistically (P ≤ 0.05) different, viz., proteolytic activity of 4 U mg−1 for conidia produced on artificial medium and 36.2 U mg−1 (nine times higher) for conidia from tick cadavers.

Pr1 gene expression

The expression of three reference genes were used to clearly show the upregulation of pr1 in conidia produced on adult ticks (in contrast to artificial medium). All three reference genes demonstrated high target stability (average geNorm M ≤0. 5); i.e., geNorm M values of 0.218 for gpd, 0.237 for try, and 0.291 for tef. On the other hand, pr1 was ten times more expressed in conidia produced on tick cadavers than on artificial medium (Fig. 1). PCR efficiencies are given in Table 1.

Fig. 1
figure 1

Relative expression levels (with standard deviations) of the gene pr1 from conidia cultivated on artificial medium (PDA) or on cadavers of Rhipicephalus microplus females (ticks). The genes gpd, tef, and try were used as reference genes. Relative quantities were based on expression levels in conidia produced on PDA (geNorm qbasePLUS)

Full size image

Fungal virulence against R. microplus larvae

Larvae in the control group remained at least 99 % alive. Ticks exposed to conidia produced on either tick cadavers or PDA medium had significantly (P ≤ 0.05) higher mortalities than those in the control (untreated) group. Although conidia from tick cadavers induced the highest mortality levels, there was not a statistically significant difference (P ≥ 0.05) between mortalities from the two sources of conidia (Table 2).

Table 2 Mean percent (%) mortality and standard deviation of Rhipicephalus microplus larvae treated with conidia from artificial medium (PDA) or female tick cadavers (ticks)
Full size table

Culture of M. anisopliae s.l. from bioassay cadavers

R. microplus larvae from the fungus-treated groups were incubated at high humidity to encourage the development of fungal colonies. The fungus morphology was characterized according to Tulloch (1976). The isolated fungus colonies universally presented the key morphological features consistent with M. anisopliae s.l. R. microplus larvae from the control group neither died nor exhibited fungal growth.

Discussion

The biological control of ticks using entomopathogenic fungi has been studied repeatedly in recent decades (e.g., Bittencourt et al. 1992, 1994, 1995, 1999; Samish and Rehacek 1999; Samish et al. 2004; Fernandes and Bittencourt 2008; Angelo et al. 2010; Fernandes et al. 2011; Perinotto et al. 2012), and this approach is frequently presented as a promising alternative to the use of chemical products. Nevertheless, further research on improving the effectiveness of these microorganisms against acari is needed, including a detailed knowledge on the key factors for the fungal virulence.

Pr1 is a subtilisin-like protease that has been proposed as an important factor in initiating fungal infection via penetration through the arthropod host’s cuticle (St Leger et al. 1986, 1988, 1989; Campos et al. 2005; Small and Bidochka 2005; Fang and Bidochka 2006; Dhar and Kaur 2010). It is well recognized that Metarhizium spp. can produce a large number of enzymes, and one of these, Pr1, probably is required to digest the arthropod-cuticle complex during the initial stages of infection, i.e., during appressorium and infection-peg formation. Thus, the question why would the presence of this enzyme be important to nongerminated conidia? According to St Leger et al. (1991b), the ease with which these enzymes can be extracted from spores using buffers, in addition to its activity on appropriate substrates, indicates that there is enzyme activity at the spore surface. This might provide ways for the germinating spore to obtain nutrients and also achieve some preliminary modifications of host cuticle surface before or at germination.

Low, but detectable, levels of subtilisin Pr1 activity were present when M. anisopliae mycelia were cultivated in liquid minimal medium (inorganic nitrogen only), the mycelium discarded, and the liquid assayed (Dhar and Kaur 2010). This suggests that Pr1 synthesis is constitutive. Growing the fungus in liquid medium containing colloidal chitin (2 %) as the sole carbon and nitrogen source afforded increased levels of Pr1 activity in the medium (Dhar and Kaur 2010). In contrast to this study on the effects of culture substrate on Pr1 presence in liquid media, the current study compared the effects of culture substrates (PDA medium or tick cadavers) on Pr1 enzyme activity detectable on the outside of conidia, which revealed considerably higher enzyme activity on conidia produced on tick-cadaver substrate. Accordingly, there was a direct relationship between culture substrate and Pr1 enzyme production levels. PDA, the artificial medium, has very low nitrogen levels in comparison to adult ticks. Confirmation of the findings on Pr1 enzyme activity in relation to culture substrates was sought by genetic analysis. Three genes were used as reference genes to analyze changes in conidial pr1 expression from PDA medium or tick cadavers. The results were as follows: pr1 gene expression was ten times higher in conidia obtained from ticks, which was in agreement with the enzymatic results presented above and, importantly, change in substrate did not alter levels of any of the reference genes (Fig. 1).

We show here, for the first time, that spores of M. anisopliae s.l. have pr1 gene expression, as well as Pr1 specific activity, increased when the fungus is cultivated in ticks. A wide arsenal of enzyme activities has been detected on M. anisopliae s.l. spore surfaces. This enzymatic diversity contributes to fungal survival and ecological fit and may be correlated to the adaptation of the fungus to a broad array of habitats (Santi et al. 2010). Nevertheless, increased Pr1activity on conidia and elevated pr1 expression levels were not associated with significant (P ≥ 0.05) changes (up or down) in larval mortality. It is well known that virulence of AP fungi may be markedly reduced after successive passages on artificial medium (e.g., Kawakami 1960; Fargues and Robert 1983). Currently, it is not known if attenuated virulence following multiple artificial medium passages involves reduction in Pr1 levels on conidia. This may be a possibility, but our findings of little correlation between conidial Pr1 levels and virulence to ticks would speak against this concept.

Experiments to identify additional differences between tick-passaged and nonpassaged fungal strains, looking for modulation of other virulence factors (such as lipases and chitinases), are needed. It is also important to note that, unlike ticks, insects are much more susceptible to entomopathogenic fungi, once conidial concentrations around 106 conidia mL−1 are already enough to successfully control insect larvae (Alves 1998). If tested against insects, tick-cadaver-produced conidia would probably yield better results in comparison with artificial-media-produced conidia. Nevertheless, conidia concentrations used for insects has no, or very little, effect against ticks (Quinelato et al. 2012).

Although Pr1 activity levels on conidia were expected to be related to fungal virulence for ticks, our findings did not concur. Similarly, recent literature shows that the presence of Pr1 genes and the activity of these proteases from some M. anisopliae isolates could not be associated with the virulence against the two insect pests (Rosas-García et al. 2014). The levels of Pr1 associated with conidia produced on artificial (PDA) medium was 9–10-fold lower than on cadaver-produced conidia; nevertheless, the minimal amount on the former was adequate to induce the same high levels of tick mortality as the latter. It is suggested that the elevated levels of Pr1 on tick-cadaver-produced conidia might have their effects merged in the virulence stability of this fungal isolate due to the enormous amount of spores (108 conidia mL−1) commonly used to control ticks. We conclude that, under the conditions of our tests, the virulence level of M. anisopliae s.l. against ticks is not directly related to conidial Pr1 levels.