Dissecting multitrophic interactions: The relationships among Entomophthora, their dipteran hosts, and associated bacteria

Interactions with microorganisms across the parasite-mutualist continuum shape the biology of insects at all levels - from individual traits to populations to communities. However, the understanding of pathogens infecting non-model insect species in natural ecosystems, or their interactions with other insect-associated microorganisms, is fragmentary. Here, we tested a conceptually novel approach - the simultaneous sequencing of insect, fungal, and bacterial marker gene amplicons - as a means of dissecting interactions among entomopathogenic fungi in the genus Entomophthora and their dipteran hosts in South Greenland. We aimed to describe the taxonomic diversity of Entomophthora, their dipteran hosts, and the bacterial diversity within a set of field-collected dead insects exhibiting signs of Entomophthora infection. Across nine collected dipteran species, we identified multiple Entomophthora genotypes, with strong but not perfect patterns of host-specificity across the five targeted marker regions. Additionally, we found consistent differences in bacterial community composition among fungus-killed fly species and sampling sites. Our results substantially expand the knowledge of Entomopthora diversity and host associations while providing the very first insights into associated bacteria and their potential roles. We also conclude that multi-target amplicon sequencing can be a powerful tool for addressing broad questions about biological interactions in diverse natural communities.