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Abstract: The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection. [Copyright &y& Elsevier]

Abstract: The characterization of chitinase genes and enzymes is an important step toward global understanding of the chitinolytic system in entomopathogenic fungi. Chitinase CHIT30 from Metarhizium anisopliae var. anisopliae (strain E6) has both endo- and exochitinase activities and is a potential determinant of pathogenicity. Serum anti-CHIT30 specifically detected this chitinase amongst five isoenzymes shown in glycol-chitin activity gels. Chitinase CHIT30 secretion is upregulated by chitin, tick cuticle and low concentrations of N-acetylglucosamine (0.25%) and is downregulated by both high N-acetylglucosamine (1%) and glucose (1%) concentrations. Chitinase CHIT30 was produced at tick cuticle during fungal infection. The chi3 gene was assigned to code chitinase CHIT30 in M. anisopliae var. anisopliae. [Copyright &y& Elsevier]

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• M. anisopliae s.l. isolate was low tolerant to menadione (MND). • Half of MND LC 50 was used as a sublethal dose in the experiments. • Changes in the colony and conidial enzymatic activities were noticed. • Better efficacy against R. microplus with fungal LC 50 and LT 50 reductions. • Mycelial secretome reveals a specific increase in some Pr1 isoforms. Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC 50 by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC 50) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC 50 and LT 50 , particularly at 108 conidia mL−1. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC 50), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species. [ABSTRACT FROM AUTHOR]