Details: Freshwater mussels are among North America’s most imperiled species. Die-offs are increasingly recognized as population threats, with etiologies frequently undetermined. Minimal health and disease data exists for freshwater mussels. Detailed plans and descriptions of techniques for thorough and rapid diagnostics to guide a targeted die-off response are lacking. This project’s objectives were to develop die-off response protocols in coordination with partners nationwide and establish and compare baseline health parameters for freshwater mollusks in Indiana waterways. Study species included native Fatmucket (Lampsilis siliquoidea) and Plain Pocketbook (Lampsilis cardium) and non-native Asian clam (Corbicula fluminea), all common in Indiana. Methods involved: 1) collection of mollusks (20 per species per site) from three Wildcat Creek drainage sites under assessment for mussel translocation suitability, 2) determination of microbial populations (viral, bacterial, parasitic, and fungal) and antibiotic resistance of bacteria cultured, and 3) assay of hemolymph and tissue samples to determine analyte levels (including metabolomics, glycogen, stable isotopes, contaminants) and histologic tissue evaluation. Using common species, this study allowed for optimization of techniques and protocols for use in diagnostic response to die-offs of potentially endangered species. Preliminary results begin to establish baseline health parameters of multiple species at varied sites which is critical for interpretation of results in the event of a die-off. Analysis of results, compared between species and sites and to water quality parameters, will add to assessment of the suitability of the three sites for translocations, evaluate potential interspecies competition based on dietary composition comparison, identify potential pathogens associated with Asian clam that might threaten native species, and increase understanding of antimicrobial resistance in aquatic environments. Expansion of this pilot study, replicating the protocols and incorporating additional locations, species, and seasons over time, will establish a comprehensive program for understanding challenges to mussel populations while informing management and conservation approaches, including for population restoration. It may generate data to develop risk mitigation strategies for microbes and contaminants that are determined to contribute to freshwater mussel morbidity and mortality.