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.

Review of the Hispaniolan Parachondria (Chondropomorus) complex (Gastropoda: Littorinoidea: Annulariidae).

The Parachondria (Chondropomorus) complex in Hispaniola is reviewed. Nineteen species are recognized including eight new species: Parachondria anatolensis n. sp., Parachondria arcisensis n. sp., Parachondria daedalus n. sp., Parachondria heatheraikenae n. sp., Parachondria isabellinus n. sp., Parachondria muchai n. sp., Parachondria silvaticus n. sp., and Parachondria stigmosus n. sp. Distributional and habitat notes are given for additional taxa. Chondropoma marinum "Weinland" Reeve, 1863, is regarded as a nomen dubium. Chondropoma (Chondropomorus) moroni Bartsch, 1946, is reidentified as Crossepoma emilianum (Weinland, 1862). Chondropoma simplex Pfeiffer, 1852, regarded by Bartsch (1946) as a Chondropomorus, is considered a Chondropoma.

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.

A revision of the Annulariidae of Central America (Gastropoda: Littorinoidea).

Twenty annulariid taxa are reviewed from Central America, including three new species and one new genus. One species is regarded as an incertae sedis and two as mislabeled lots of Cuban origin. Many species are highly endemic. Although not speciose, Central America has a high diversity of conchological forms and may represent the ancestral source of annulariids in general.

New taxa and distributional notes on Abbottella and related taxa (Gastropoda: Littorinoidea: Annulariidae).

Seven new species of Abbottella and related taxa are described from Hispaniola: Abbottella calliotropis n. sp., Abbottella diadema n. sp., Abbottella dichroa n. sp., Abbottella nitens n. sp., Abbottella paradoxa n. sp., Abbottella tenebrosa n. sp., and Leiabbottella thompsoni n. sp. Distributional and habitat notes are given for additional taxa.

Analysis and cytologic characterization of hemocytes from freshwater mussels (Quadrula sp.).

BACKGROUND: Freshwater mussels are among the most endangered taxa in North America and minimally invasive techniques to evaluate their health are needed. OBJECTIVE: The objective of this study was to develop a standardized approach for identifying and enumerating the cellular components of freshwater mussel hemolymph. METHODS: Hemocyte clumping, total hemocyte count, and hemocyte morphology were compared in untreated hemolymph or hemolymph treated with formalin, sodium citrate, sodium heparin, EDTA, water, or l-cysteine. Morphology was then used to categorize hemocytes and perform a 100-cell differential. RESULTS: Treatment with formalin or >25 mg/mL l-cysteine reduced hemocyte clumping, although only formalin significantly increased the total hemocyte count. However, formalin also induced crenation that impaired hemocyte identification. Both EDTA and sodium citrate-induced hemocyte degranulation while sodium citrate and >40 mg/mL l-cysteine-induced cell lysis. Hemocytes could be categorized into 2 groups of granulocytes (eosinophilic or basophilic) and 2 groups of agranulocytes (large or small) for performing a cytologic differential. The differential was not significantly altered by anticoagulant treatments providing cell morphology was adequate for obtaining a differential. Eosinophilic granulocytes predominated (59%) with fewer large agranulocytes (27%) and basophilic granulocytes (13%). Small agranulocytes comprised 2% of the total population. CONCLUSIONS: No single treatment provided an optimal method to evaluate freshwater mussel hemolymph. Maximal hemocyte counts were obtained following formalin treatment. l-cysteine reduced clumping and maintained hemocyte morphology for performing a cytologic differential. These techniques provide a standardized approach for the hematologic evaluation of freshwater mussels.