Mussel mass mortality in the Clinch River, USA: metabolomics detects affected pathways and biomarkers of stress.

Biologists monitoring freshwater mussel (order Unionida) populations rely on behavioral, often subjective, signs to identify moribund ("sick") or stressed mussels, such as gaping valves and slow response to probing, and they lack clinical indicators to support a diagnosis. As part of a multi-year study to investigate causes of reoccurring mortality of pheasantshell (Ortmanniana pectorosa; synonym Actinonaias pectorosa) in the Clinch River, Virginia and Tennessee, USA, we analyzed the hemolymph metabolome of a subset of mussels from the 2018 sampling period. Mussels at the mortality sites were diagnosed in the field as affected (case) or unaffected (control) based on behavioral and physical signs. Hemolymph was collected in the field by non-lethal methods from the anterior adductor muscle for analysis. We used ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectroscopy to detect targeted and untargeted metabolites in hemolymph and compared metabolomic profiles by field assessment of clinical status. Targeted biomarker analysis found 13 metabolites associated with field assessments of clinical status. Of these, increased gamma-linolenic acid and N-methyl-l-alanine were most indicative of case mussels, while adenine and inosine were the best indicators of control mussels. Five pathways in the targeted analysis differed by clinical status; two of these, purine metabolism and glycerophospholipid metabolism, were also indicated in the untargeted analysis. In the untargeted nalysis, 22 metabolic pathways were associated with clinical status. Many of the impacted pathways in the case group were catabolic processes, such as degradation of amino acids and fatty acids. Hierarchical clustering analysis matched clinical status in 72% (18 of 25) of mussels, with control mussels more frequently (5 of 16) not matching clinical status. Our study demonstrated that metabolomic analysis of hemolymph is suitable for assessing mussel condition and complements field-based indicators of health.

Freshwater Mussels Show Elevated Viral Richness and Intensity during a Mortality Event.

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.

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 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.

Using silver and bighead carp cell lines for the identification of a unique metabolite fingerprint from thiram-specific chemical exposure.

Conservation biology often requires the control of invasive species. One method is the development and use of biocides. Identifying new chemicals as part of the biocide registration approval process can require screening millions of compounds. Traditionally, screening new chemicals has been done in vivo using test organisms. Using in vitro (e.g., cell lines) and in silico (e.g., computer models) methods decrease test organism requirements and increase screening speed and efficiency. These methods, however, would be greatly improved by better understanding how individual fish species metabolize selected compounds. We combined cell assays and metabolomics to create a powerful tool to facilitate the identification of new control chemicals. Specifically, we exposed cell lines established from bighead carp and silver carp larvae to thiram (7 concentrations) then completed metabolite profiling to assess the dose-response of the bighead carp and silver carp metabolome to thiram. Forty one of the 700 metabolomic markers identified in bighead carp exhibited a dose-response to thiram exposure compared to silver carp in which 205 of 1590 metabolomic markers exhibited a dose-response. Additionally, we identified 11 statistically significant metabolomic markers based upon volcano plot analysis common between both species. This smaller subset of metabolites formed a thiram-specific metabolomic fingerprint which allowed for the creation of a toxicant specific, rather than a species-specific, metabolomic fingerprint. Metabolomic fingerprints may be used in biocide development and improve our understanding of ecologically significant events, such as mass fish kills.