Microsporidian diversity and host associations in aquatic invertebrate communities of South Bohemian fish ponds revealed by metabarcoding

Microsporidians are highly specialised intracellular parasites adapted to an obligate parasitic lifestyle. Although more than 1,500 species have been described to date, their true diversity is likely much higher, particularly in poorly-studied aquatic environments. Here, we applied an optimised tissue-based metabarcoding approach to investigate the diversity and distribution of microsporidians in aquatic invertebrates from three nearby fish ponds in South Bohemia in the Czech Republic. We analysed more than two thousand individuals of chironomids and oligochaetes, which represent important aquatic invertebrate hosts of microsporidians, and compared microsporidian communities across ponds and host morphogroups. In total, 66 microsporidian OTUs were detected across both host groups. Metabarcoding and phylogenetic analyses revealed broad sequence diversity within the studied localities, with parasite sequences clustering into four main clades. The high proportion of OTUs without close matches to described taxa (more than 95%) highlights substantial undocumented diversity of microsporidians in freshwater ecosystems. The occurrence of shared OTUs amongst different invertebrate taxa is consistent with multi-host strategies and/or complex life cycles. Overall, our findings emphasise the ecological complexity of microsporidians in aquatic habitats. Further experimental work, including parasite localisation and transmission studies, is needed to determine whether the observed host associations represent active infections or incidental detection.