A Novel Gonadotropic Microsporidian Parasite (Microsporidium clinchi n. sp.) Infecting a Declining Population of Pheasantshell Mussels (Actinonaias pectorosa) (Unioinidae) from the Clinch River, USA

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.

Mussel Mass Mortality and the Microbiome: Evidence for Shifts in the Bacterial Microbiome of a Declining Freshwater Bivalve

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 morality 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.

Guiding Species Recovery through Assessment of Spatial and Temporal Population Genetic Structure of Two Critically Endangered Freshwater Mussel Species (Bivalvia: Unionidae)

The Cumberlandian Combshell (Epioblasma brevidens) and Oyster Mussel (E. capsaeformis) are critically endangered freshwater mussel species native to the Tennessee and Cumberland River drainages, major tributaries of the Ohio River in the eastern United States. The Clinch River in northeastern Tennessee (TN) and southwestern Virginia (VA) harbors the only remaining stronghold population for either species, containing tens of thousands of individuals per species; however, a few smaller populations are still extant in other rivers. We collected and analyzed genetic data to assist with population restoration and recovery planning for both species. We used an 888 base-pair sequence of the mitochondrial NADH dehydrogenase 1 (ND1) gene and ten nuclear DNA microsatellite loci to assess patterns of genetic differentiation and diversity in populations at small and large spatial scales, and at a 9-year (2004 to 2013) temporal scale, which showed how quickly these populations can diverge from each other in a short time period. Intraspecific mitochondrial DNA (mtDNA) and microsatellite DNA variation was higher in E. capsaeformis than in E. brevidens. These two species have maintained quite different levels of genetic diversity within their Clinch River stronghold and in their smaller peripheral populations in the Big South Fork Cumberland and Nolichucky rivers, TN. For instance, with only three mtDNA haplotypes detected overall across populations, E. brevidens’ capacity for maintaining genetic diversity appears to be less than that of E. capsaeformis, which had 18 haplotypes. At the relatively small spatial scales (15-30 kilometers) investigated in the Clinch River, demes of both species exhibited minimal genetic differentiation in either the 2004 or 2013 sampling periods, typically <0.02 based on FST and <0.1 based on Jost’s D. Our genetic data suggest that mussels at the numerous shoals in a 32-kilometer section of the Clinch River comprise a single, large population of each respective species with very high gene-flow among individual demes. However, we also observed a high level of genetic differentiation among demes at the 9-year temporal scale, with differentiation metrics for E. brevidens (D = 0.47 and FST = 0.12) and E. capsaeformis (D = 0.31 and FST = 0.05) proving higher than the within-year values. This result strongly suggests that genetic drift is playing an important role in allele frequency change over time in these populations. At the spatial and temporal scales investigated in this study, various demographic, life history, and environmental factors are influencing maintenance of genetic variation and need to be considered during conservation planning for each species.

A Comparison of Systematic Quadrat and Capture-Mark-Recapture Sampling Designs for Assessing Freshwater Mussel Populations

Our study objective was to compare the relative effectiveness and efficiency of quadrat and capture-mark-recapture (CMR) sampling designs for monitoring mussels. We collected data on a recently reintroduced population of federally endangered Epioblasma capsaeformis and two nonlisted, naturally occurring species—Actinonaias pectorosa and Medionidus conradicus—in the Upper Clinch River, Virginia, over two years using systematic quadrat and CMR sampling. Both sampling approaches produced similar estimates of abundance; however, precision of estimates varied between approaches, years, and among species, and further, quadrat sampling efficiency of mussels detectable on the substrate surface varied among species. CMR modeling revealed that capture probabilities for all three study species varied by time and were positively associated with shell length, that E. capsaeformis detection was influenced by sex, and that year-to-year apparent survival was high (>96%) for reintroduced E. capsaeformis. We recommend that monitoring projects use systematic quadrat sampling when the objective is to estimate and detect trends in abundance for species of moderate to high densities (>0.2/m2), whereas a CMR component should be incorporated when objectives include assessing reintroduced populations, obtaining reliable estimates of survival and recruitment, or producing unbiased population estimates for species of low to moderate densities (≤0.2/m2).