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 recog- nized as heavily involved in the initial steps of the fungal invasion of arthropod-host cuticles. In the current study, cor- relation was sought between mortality of tick larvae and co- nidial 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 pro- ducing 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 sub- strate, 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 medi- um. Bioassays of M. anisopliae s.l. CG 148 spores with ele- vated Pr1 proteolytic activity and gene expression levels did not demonstrate increased virulence (= significant changeper- cent 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 en- zyme on tick-cadaver-produced conidia did not induce elevat- ed 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.