Genomic and Organismal Studies to Elucidate the Mechanisms of Infectivity of Entomopathogenic Fungi to Ticks

The overall goal of this research was to elucidate the factors affecting early development of Metarhizium spp. (previously named M. anisopliae) on ticks or tick cuticle extracts and the molecular basis of these early infection processes. The original objectives were: 1. Characterize the pre-penetration events (adhesion, germination and appressorium formation) of spores of M. anisopliae strains with high or low virulence during tick infection. 2. Create GFP-expressing strains of M. anisopliae tick pathogens having high and low virulence to compare their progress of infection by microscopy. 3. Use microarray analyses, primarily with existing M. anisopliae EST sequences in GenBank, to identify and characterize fungal genes whose expression is regulated in response to host cuticle extracts. Objective 3 was later modified (as approved by BARD) to use RNAseq to characterize the early stages of fungal gene expression during infection of intact host cuticles. This new method provides a massively larger and more informative dataset and allows us to take advantage of a) recently published genomes of Metarhizium robertsii and M. acridum for RNAseq data analysis, and b) newly developed and highly efficient cDNA sequencing technologies that are relatively low cost and, therefore, allow deep sequencing of multiple transcriptome samples. We examined pre-penetration and penetration events that differentiate high and low virulence strains of Metarhizium spp., focusing on spore adhesion, germination, appressorium formation, and penetration of tick integuments. Initiation of fungal infection was compared on susceptible and resistant tick species at different tick developmental stages. In vitro studies comparing the effects of protein and fatty acid profiles from tick cuticle extracts demonstrated that resistant tick cuticles contain higher concentrations of specific lipids that inhibit fungal development than do susceptible tick cuticles, suggesting one mechanism of Ixodidae resistance to fungal entomopathogens (Objective 1). We used molecular markers to determine that the three M. anisopliae strains from Israel that we studied actually were three distinct species. M. brunneum is highly virulent against the tick Rhipicephalus annulatus, M. pingshaense and M. robertsii are intermediate in virulence, and M. majus is of low virulence. We transformed all four Metarhizium species to express GFP and used them in pathogenicity assays against diverse tick species. Key findings were that a) resistant ticks inhibit Metarhizium infection prior to hemocoel invasion by reducing fungal viability on the cuticle surface (Objective 2), as was supported by the in vitro studies of Objective 1, and b) Metarhizium kills susceptible ticks after cuticle penetration but prior to hemocoel colonization. Transcriptome studies of the most virulent species, M. brunneum, are in progress and include analyses of ungerminated conidia and conidia germination and development on a low nutrient medium or on susceptible R. annulatus exoskeleton (Objective 3). We anticipate these studies will contribute to identifying fungal genetic factors that increase virulence and speed of kill and may help reveal tick chemistries that could be included in biocontrol formulations to increase efficacy. Methodologies developed to screen tick cuticle extracts for ability to support conidia germination and development may help in the selection of wild fungi with increased virulence against resistant ticks. The overall knowledge gained should contribute not only to the improvement of tick control but also to the control of other blood-sucking arthropods and related plant pests. Use of bio-based agents for controlling arthropods will contribute to a healthier, more sustainable environment and serve a growing number of organic food farmers.