Contaminants of emerging concern in the Maumee River and their effects on freshwater mussel physiology.

Contaminants of emerging concern pose a serious hazard to aquatic wildlife, especially freshwater mussels. The growing number of contaminants in aquatic systems requires scientists and managers to prioritize contaminants that are most likely to elicit a biological response for further monitoring and toxicological testing. The objectives of this study were to identify a sub-category of contaminants most likely to affect Pyganodon grandis and to describe alterations in metabolites and gene expression between various sites. Mussels were deployed in cages for two weeks at four sites along the Maumee River Basin, Ohio, USA. Water samples were analyzed for the presence of 220 contaminants. Hemolymph samples were collected for metabolomics and analyzed using mass spectrometry. Contaminants that significantly covaried with metabolites were identified using partial least-squares (PLS) regression. Tissue samples were collected for transcriptomics, RNA was sequenced using an Illumina HiSeq 2500, and differential expression analysis was performed on assembled transcripts. Of the 220 targeted contaminants, 69 were detected in at least one water sample. Of the 186 metabolites detected in mussel hemolymph, 43 showed significant differences between the four sites. The PLS model identified 44 contaminants that significantly covaried with changes in metabolites. A total of 296 transcripts were differentially expressed between two or more sites, 107 received BLAST hits, and 52 were annotated and assigned to one or more Gene Ontology domains. Our analyses reveal the contaminants that significantly covaried with changes in metabolites and are most likely to negatively impact freshwater mussel health and contribute to ongoing population declines in this group of highly endangered animals. Our integration of "omics" technologies provides a broad and in-depth assessment of the short-term effects of contaminants on organismal physiology. Our findings highlight which contaminants are most likely to be causing these changes and should be prioritized for more extensive toxicological testing.

Freshwater mussels (Unionidae) brought into captivity exhibit up-regulation of genes involved in stress and energy metabolism.

Approximately two thirds of freshwater mussel species in the United States and Canada are imperiled, and populations are declining rapidly. Translocation and captive management are commonly used to mitigate losses of freshwater mussel biodiversity, but these conservation tools may result in decreased growth and increased mortality. This study uses RNA-Seq to determine how translocation into captivity affects gene expression in Amblema plicata. Mussels were collected from the Muskingum River in Ohio, USA and brought into a captive holding facility. RNA was extracted from gill tissue 11 months post translocation from mussels in captivity and the Muskingum River on the same day. RNA was sequenced on an Illumina HiSeq 2500, and differential expression analysis was performed on de novo assembled transcripts. More than 1200 transcripts were up-regulated in captive mussels, and 246 were assigned functional annotations. Many up-regulated transcripts were involved in energy metabolism and the stress response, such as heat shock proteins and antioxidants. More than 500 transcripts were down-regulated in captive mussels, and 41 were assigned functional annotations. We observed an over-representation of down-regulated transcripts associated with immune response. Our work suggests that A. plicata experienced moderate levels of stress and altered energy metabolism and immune response for at least 11 months post translocation into captivity.

Nontargeted metabolomics reveals biochemical pathways altered in response to captivity and food limitation in the freshwater mussel Amblema plicata.

Effective conservation of freshwater mussels (Mollusca: Bivalvia: Unionidae), one of the most endangered groups of animals in North America, is compromised by limited knowledge of their health. We address this gap in knowledge by characterizing the metabolic profile of Amblema plicata in the wild and in response to captivity and food limitation. Eight mussels brought into captivity from the wild were isolated for 18 days without a food source. Hemolymph samples were taken prior to, and 9 and 18 days after the start of the experiment; these samples were analyzed by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. We detected and identified 71 biochemicals in the hemolymph of freshwater mussels; of these, 49 showed significant changes during captivity and/or food limitation (p<0.05). Fasting resulted in severe metabolite depletion. Captive (but fed) mussels experienced changes similar to (albeit less severe than) fasting mussels, suggesting that mussels may experience nutritional deficiency under common captive conditions. A. plicata responded to food limitation stress by preferentially using energy reserves for maintenance rather than growth. Carbohydrate and energy metabolism exhibited down-regulation in captive, food-limited, and wild mussels. Lipid metabolism was up-regulated in captive/food-limited mussels and unchanged in wild mussels. Amino acid metabolism was up-regulated in wild mussels and down-regulated in captive/food-limited mussels. Nucleotide metabolism was up-regulated in the wild mussels, down-regulated in food-limited mussels, and unchanged in captive mussels. The different responses between treatment groups suggest potential for nucleotide metabolism as a biomarker of health status for freshwater mussels.