NGS (Illumina Miseq) of ITS rRNA gene of signal crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas, intestine) and samples of its environment (water, sediment)

Increasing number of studies indicate the important role of microbiome during biological invasions, since it affects various interactions between host and environment. However, the majority of studies focus on bacteriome, insufficiently addressing other microbiome components such as mycobiome. Microbial fungi are among the most damaging pathogens in freshwater crayfish populations, with both native and invasive crayfish species being susceptible to fungal colonization and possible infection. Invading crayfish may transmit novel fungal species to native populations, but also, dispersal process and characteristics of the novel environment may affect the composition of invaders' mycobiome, directly and indirectly affecting their fitness and invasion success. This study analyzes the mycobiome of a very successful invader in Europe, the signal crayfish, using ITS rRNA amplicon sequencing approach. We explored the mycobiome of four types of crayfish samples (exoskeletal biofilm, hemolymph, hepatopancreas, intestine), compared them to mycobiomes of environmental samples (water, sediment), and searched for differences in fungal biodiversity and abundance between upstream and downstream segments of the signal crayfish' invasion range in Korana River, Croatia. Small number of ASVs was obtained in hemolymph and hepatopancreas samples, pointing to small fungal abundance in these tissues. Thus, only exoskeleton, intestine, sediment and water samples were analysed further and significant differences were recorded between their mycobiomes, confirming their uniqueness. Generally, environmental samples showed higher biodiversity than crayfish-associated samples. Intestinal mycobiome showed significantly lower richness compared to other mycobiomes. Comparison between different river segments revealed significant differences in diversity of sediment and exoskeletal mycobiome (but not in water and intestinal mycobiome), indicating that sediment mycobiome (i.e. environment) at least partly shapes the mycobiome of crayfish exoskeleton. This study presents the first metagenomic data on crayfish-associated fungal communities accross different tissues, valuable in a light of general absence of studies on crayfish mycobiome. We demonstrate significant differences in the signal crayfish exoskeletal mycobiome along the invasion range, suggesting that different local environmental conditions may shape the exoskeletal mycobiome during range expansion, while mycobiome of internal organ (intestine) remained more stable. Our findings represent the first insight into mycobiome of a successful invasive crayfish species, and offer a baseline for assessing how mycobiome contributes to invader's overall health and its further invasion success.