During an epidemiologic survey following a mortality event of freshwater mussels in 2018 in the Embarrass River, Wisconsin, USA, we identified a novel microsporidian parasite in the ovary of mucket (Actinonaias ligamentina), plain pocketbook (Lampsilis cardium), and fatmucket (Lampsilis siliquoidea) (Unioinidae). Histopathology showed round-to-oval microsporidial spores in the cytoplasm of oocytes in 60% (3/5) of mucket, 100% (4/4) of plain pocketbook and 50% (1/2) of fatmucket. On transmission electron microscopy, mature spores were round to oval, measured 4.13 +/- 0.64 µm (3.14–5.31) long by 2.88 +/-0.37 µm (2.36–3.68) wide. Spores had a thin electron dense exospore with a spiky coat, a thick electron lucent endospore, diplokaryotic nuclei, a polar vacuole, and 27–28 polar filaments arranged in 1 to 3 rows. Sequencing of the small subunit rRNA produced a 1356 bp sequence most similar to Pseudonosema cristatellae, and phylogenetic analysis grouped it with freshwater Neopereziida. The morphologic and ultrastructural characteristics did not match those of Pseudonosema sp. and a new genus and species, Hirsutonosema embarrassi n. gen., n. sp., were designated. Additional studies could evaluate host susceptibility, distribution, seasonality, transmission, and lethal or sub-lethal effects of this parasite to freshwater mussels.

Freshwater mussels of the order Unionida are among the most endangered animal groups globally, but the causes of population declines are often enigmatic with little known about the role of disease. In 2018, we collected wild adult pheasantshell (Actinonaias pectorosa) and mucket (Actinonaias ligamentina) during an epidemiologic survey investigating an ongoing mussel mass mortality event in the Clinch River, USA. Histopathology and transmission electron microscopy showed a novel microsporidian parasite primarily infecting the ovary of pheasantshell. Sequencing of the small subunit rRNA gene produced a 1333 bp sequence with greatest similarity to Pseudonosema cristatellae (AF484694.1; 86.36%; e-value = 0), a microsporidium infecting the freshwater bryozoan (Cristatella mucedo). Microsporidia were observed in 65% (17/26) of the examined female pheasantshell (A. pectorosa) and in no (0/2) female mucket (A. ligamentina), and occurred at mortality and non-mortality sites. Our findings indicate that a novel parasite, Microsporidium clinchi n. sp., is present in pheasantshell in the Clinch River, USA, and while likely not a cause of mass mortality, could reduce fecundity and recruitment in this declining population and threaten the success of reintroductions. Surveillance for M. clinchi n. sp. and evaluation of brood stock and their progeny for microsporidia would therefore be prudent.

There is a fundamental knowledge gap on the distribution, prevalence, intensity, and ecology of salmonid myxozoan parasites in the Lake Sammamish watershed, Washington. To address this knowledge gap, we tested water samples for Ceratonova shasta, Parvicapsula minibicornis and Tetracapsuloides bryosalmonae DNA from 84 sites distributed throughout the Lake Sammamish watershed in fall 2019 and 74 sites in spring 2020. Our surveillance identified zones with high waterborne parasite loads and provides a proof of concept for this approach that could be expanded throughout the larger Lake Washington watershed.

Spectaclecase (Margaritifera monodonta) is a federally endangered freshwater mussel species that has experienced a 55% reduction in range (USFWS 2014) and is currently concentrated in three rivers in the Midwest of the United States (Gasconade, Meramec Rivers, MO, and St. Croix River, WI). Its preference for living under large rocks and boulders has limited detection of new populations by traditional survey methods. Environmental DNA technology has been used to detect invasive and rare species, but its use for detection of rare, benthic-dwelling species in large flowing systems has been limited. Here, we propose using environmental DNA to identify presumable sites for discovery of M. monodonta. We designed a M. monodonta-specific qPCR assay and tested it using M. monodonta-housed tank water and water samples from two known mussel beds on the St. Croix River and three known mussel beds on the Mississippi River. We observed higher overall detection rate on the St. Croix River (30.2%) compared to the Upper Mississippi River (0.60%). We also observed higher eDNA detection rates (73.3-93.1%) in 2018 for samples collected during the larval release period in May compared to samples collected in August after reproduction stopped (55.6-70.8%) on the St. Croix River. We tested samples collected at three distances downstream of the two mussel beds found in the St. Croix River, but we did not observe a significant effect of distance on our detection rates. However, we did observe greater detection rates for samples collected near the bottom compared to at the surface. Our results indicate that this novel qPCR assay can successfully detect M. monodonta eDNA and could be utilized to rapidly screen locations to guide intensive physical searches for populations in riverine systems.

Spectaclecase (Margaritifera monodonta) is a federally endangered freshwater mussel species that has experienced a 55% reduction in range (USFWS 2014) and is currently concentrated in three rivers in the Midwest of the United States (Gasconade, Meramec Rivers, MO, and St. Croix River, WI). Its preference for living under large rocks and boulders has limited detection of new populations by traditional survey methods. Environmental DNA technology has been used to detect invasive and rare species, but its use for detection of rare, benthic-dwelling species in large flowing systems has been limited. Here, we propose using environmental DNA to identify presumable sites for discovery of M. monodonta. We designed a M. monodonta-specific qPCR assay and tested it using M. monodonta-housed tank water and water samples from two known mussel beds on the St. Croix River and three known mussel beds on the Mississippi River. We observed higher overall detection rate on the St. Croix River (30.2%) compared to the Upper Mississippi River (0.60%). We also observed higher eDNA detection rates (73.3-93.1%) in 2018 for samples collected during the larval release period in May compared to samples collected in August after reproduction stopped (55.6-70.8%) on the St. Croix River. We tested samples collected at three distances downstream of the two mussel beds found in the St. Croix River, but we did not observe a significant effect of distance on our detection rates. However, we did observe greater detection rates for samples collected near the bottom compared to at the surface. Our results indicate that this novel qPCR assay can successfully detect M. monodonta eDNA and could be utilized to rapidly screen locations to guide intensive physical searches for populations in riverine systems.

Data from metabarcoding assays to detect a suite of mussel species using mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes sequences.

Data from metabarcoding assays to detect a suite of mussel species using mitochondrial DNA regions of the cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit (ND1) genes sequences.

This dataset includes microsomal ERDO data from an assay done with liver samples from several fish species that are found in Arizona at sites that are being assessed for PBDE contamination. The data was created in September and October 2016.

This dataset includes microsomal ERDO data from an assay done with liver samples from several fish species that are found in Arizona at sites that are being assessed for PBDE contamination. The data was created in September and October 2016.

The diagnosis of bacterial disease in freshwater unionid mussels has been hindered by a lack of baseline information regarding the microbial communities associated with these animals. In this study, we cultured and identified bacteria from the hemolymph of stable mussel populations from the upper Mississippi River basin and compared results to mussel populations associated with a mortality event in the Clinch River, VA and TN. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy isolate was identified from the Clinch River. Yokenella regensbergei was found with relatively high prevalence during the mortality event but was absent from post-mortality samples. Its role, if any, in the mortality event is unknown and deserves further investigation. We suggest that future studies of freshwater mussel health utilize hemolymph samples due to its relative disconnect with the aquatic environment, role in the circulatory system and nonlethal nature of collection.