Prevalence of the microsporidian parasite Thelohania contejeani in crayfish across five Ohio watersheds

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Emily E. Gremling, Steven C. Churchin, Tyler A. Koncsol, Leslie A. Riley, Robert G. Verb, Katherine L. Krynak and Kenneth J. Oswald, School of Science, Technology, and Math, Ohio Northern University, Ada, OH, Emily M. Imhoff, Zoology, Cincinnati Museum Center, Cincinnati, OH

Background/Question/Methods
Thelohania contejeani is a parasitic microsporidian that causes porcelain disease (PD) in crayfish. The spores of T. contejeani infect the host's muscle tissues, resulting in loss of movement and ultimately, death. The first report of this newly emergent pathogen from Ohio was in 2019. This study utilized molecular methods to survey for the presence of T. contejeani across five Ohio watersheds, the Muskingum, Scioto, Little Miami, Great Miami, and Maumee Rivers. In 2019, crayfish (N=31) were collected via seine and dip nets only from the Muskingum and Maumee Rivers, but in 2020 (N=97), crayfish from all five watersheds were surveyed for the presence of PD. Following collection, the fourth right walking leg was dissected from individuals and stored in 95% ethanol for subsequent molecular genetic analyses. Total genomic DNA (gDNA) was extracted from legs using a Qiagen DNeasy Blood and Tissue Kit®. The ITS1 gene of microsporidia was then amplified via the polymerase chain reaction (PCR) using gDNA as template. Successful microsporidia amplifications then served as template in a subsequent PCR reamplification using primers specific for T. contejeani. Rates of infection were then estimated by calculating the proportion of individuals positive for T. contejeani.
Results/Conclusions
In 2019, 3% of crayfish sampled were positive for T. contejeani (N=12), a result attributable to a single individual sampled from the Muskingum River. No infected crayfish were found in the Maumee River in 2019 (N=19). In 2020, the Maumee River (16.7%; N=24) and the Great Miami River (17.6%; N=17) had crayfish positive for T. contejeani, but PD-infected crayfish were not found in the Muskingum (N=26), Scioto (N=10), nor Little Miami Rivers (N=20) in 2020. DNA sequencing will be conducted to confirm all T. contejeani infections. Additionally, crayfish will be DNA barcoded to identify the host species since crayfish might be differentially susceptible to PD, information that could be utilized for future research and conservation efforts.