Freshwater mussels (Unionida) are suffering mass mortality events worldwide, but the causes remain enigmatic. Here, we describe an analysis of bacterial loads, community structure, and inferred metabolic pathways in the hemolymph of pheasantshells (Actinonaias pectorosa) from the Clinch River, USA, during a multi-year mass mortality event. Bacterial loads were approximately 2 logs higher in moribund mussels (cases) than in apparently healthy mussels (controls). Bacterial communities also differed between cases and controls, with fewer sequence variants (SVs) and higher relative abundances of the proteobacteria Yokenella regensburgei and Aeromonas salmonicida in cases than in controls. Inferred bacterial metabolic pathways demonstrated a predominance of degradation, utilization, and assimilation pathways in cases and a predominance of biosynthesis pathways in controls. Only two SVs correlated with Clinch densovirus 1, a virus previously shown to be strongly associated with mortality in this system: Deinococcota and Actinobacteriota, which were associated with densovirus-positive and densovirus-negative mussels, respectively. Overall, our results suggest that bacterial invasion and shifts in the bacterial microbiome during unionid mass mortality events may result from primary insults such as viral infection or environmental stressors. If so, bacterial communities in mussel hemolymph may be sensitive, if generalized, indicators of declining mussel health.

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 Wisconsin portions of the upper Mississippi River basin and compared the results to those from mussel populations experiencing a mortality event in the Clinch River in Virginia and Tennessee. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy bacterial species identified from the Clinch River was Yokenella regensburgei, which occurred in relatively high prevalence during the mortality event but was absent from samples acquired afterward. Its role in the mortality event, if any, is unknown but deserves further investigation. We suggest that future studies of freshwater mussel health incorporate hemolymph as a sample type due to its relative separation from the aquatic environment, its role in the circulatory system, and the fact that it can be collected nonlethally.

Freshwater mussels (Unionida) are globally imperiled, in part due to largely unexplained mass mortality events (MMEs). While recent studies have begun to investigate the possibility that mussel MMEs in the Eastern USA may be caused by infectious diseases, mussels in the Western USA have received relatively little attention in this regard. We conducted a two-year epidemiologic investigation of the role of viruses in ongoing MMEs of the Western pearlshell (Margaritifera falcata) and the Western ridged mussel (Gonidea angulata) in the Chehalis River and Columbia River watersheds in the Western USA. We characterized viromes of mussel hemolymph from 5 locations in 2018 and 2020 using metagenomic methods and identified 557 viruses based on assembled contiguous sequences, most of which are novel. We also characterized the distribution and diversity of a previously identified mussel Gammarhabdovirus related to pathogenic finfish viruses. Overall, we found few consistent associations between viruses and mussel health status. Variation in mussel viromes was most strongly driven by location, with little influence from date, species, or health status, though these variables together only explained ~1/3 of variation in virome composition. Our results demonstrate that Western freshwater mussels host remarkably diverse viromes, but no single virus or combination of viruses appears to be associated with morbidity or mortality during MMEs. Our findings have implications for the conservation of imperiled freshwater mussels, including efforts to enhance natural populations through captive propagation.

The pirate perch Aphredoderus sayanus is a relatively small fish species found in rivers throughout much of the eastern United States. Due to their cryptic nature, relatively little is known regarding their parasite fauna. A survey of pirate perch from the upper Mississippi River revealed 2 novel myxozoans. Hennegoides flockae n. sp. was observed in heavily infected gills where the lamellae featured irregular expansion by bulbous myxozoan polysporic plasmodia, typically affecting the middle to distal half of the filaments. When severe, infection of sequential filaments was such that the filaments were fused, forming what appeared as multicystic/lobular parasitic aggregates subdivided by fine epithelial cords. The total myxospore length of Hennegoides flockae was 35.4–46.4 (41.3 ± 3.3) and the spore body, asymmetrically ovoid in valvular view, was 15.4–18.7 (17.0 ± 0.7) × 7.1–8.7 (7.9 ± 0.4). Henneguya marcquenskiae n. sp. was observed in the liver with plasmodia present randomly and infrequently in the hepatocellular parenchyma. The total myxospore length for Henneguya marcquenskiae was 39.5–55.9 (48.4 ± 4.2), with the spore body being lanceolate, 13.9–16.5 (15.4 ± 0.7) × 7.1–9.0 (8.3 ± 0.5). Phylogenetic analysis of the SSU rRNA gene placed both Hennegoides flockae and Henneguya marcquenskiae as sisters to each other in a clade containing other Myxozoans known to infect the gills of esocids, percids, and centrarchids. These parasites represent the first reports of Henneguya and Hennegoides from pirate perch, with the latter being the first report of this genus outside of the Asian continent.

Freshwater mussels of the order Unionida are among the most endangered animal groups globally, but the causes of their population decline 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, Virginia and Tennessee, 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 the 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 muckets (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, 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 of M. clinchi n. sp. and evaluation of broodstock and their progeny for microsporidia would therefore be prudent.

Freshwater mussels (Unionida) are among the world’s most imperiled taxa, but the relationship between freshwater mussel mortality events and infectious disease is largely unstudied. We surveyed viromes of a widespread and abundant species (mucket, Actinonaias ligamentina; syn: Ortmanniana ligamentina) experiencing a mortality event of unknown etiology in the Huron River, Michigan, in 2019–2020 and compared them to viromes from mucket in a healthy population in the St. Croix River, Wisconsin and a population from the Clinch River, Virginia and Tennessee, where a mortality event was affecting the congeneric pheasantshell (Actinonaias pectorosa; syn: Ortmanniana pectorosa) population. We identified 38 viruses, most of which were associated with mussels collected during the Huron River mortality event. Viral richness and cumulative viral read depths were significantly higher in moribund mussels from the Huron River than in healthy controls from each of the three populations. Our results demonstrate significant increases in the number and intensity of viral infections for freshwater mussels experiencing mortality events, whereas individuals from healthy populations have a substantially reduced virome comprising a limited number of species at low viral read depths.