**Author details**

Abundances of lesions in digestive cells were negatively correlated with genes BRAFL-DRAFT\_118372 (histamine N-methyltransferase activity) and CGI\_10002926 (uncharacterized protein with unknown function) [47–49]. A negative correlation between gene regulation of histamine methylation and lesions in digestive cells seems reasonable, because with increasing abundance of lesions, destruction of cytoplasmic organelles probably occurs. A positive correlation between the expression of Sp-Hypp\_8991 and digestive lesions may be related to

Although there has been some limited application in marine mollusks, and one study has identified candidate stress-response genes in the freshwater mussel *Elliptio complanata* [50], RNAseq has not been evaluated for its utility for monitoring of the responses of freshwater mussels to coal-related environmental contaminants. Successful execution of this project provides proof of principle for using RNAseq technology to approach issues of toxicogenomics in freshwater mussels. Among key findings, our results collectively support the view that substantial changes of gene expression occur before dramatic changes in biochemical and histological effects. Our understanding of how changes in gene expression result in physiological and histological effects on the organism is hampered by the current situation in which many

RNAseq-based assessment of global gene expression identified candidate markers for quantitative polymerase chain reaction (qPCR) assays, which could be developed and validated for rapidly assessing single-gene responses to exposure to toxic compounds. As noted above, Hamadeh et al. [40] demonstrated that chemicals from the same class of compounds give rise to discernible gene expression profiles that bear more similarity to each other than to patterns corresponding to exposure to compounds from a different class [40]. It would be useful for a comprehensive database for gene expression responses to environmental pollutants to be developed toward recognizing compound-specific expression profiles and molecular signatures of stress responses. Additional, well-controlled laboratory studies would be appropriate for the purpose of defining defensible biomarkers for toxicant-induced harm in freshwater mussels. Ultimately, practical work would apply (modifications of) the respective approaches to a range of sites in the Clinch, Powell, and other aquatic ecosystems.

This work was supported by a grant from the US Fish and Wildlife Service. The participation of EMH in this project was supported in part by the Virginia Agricultural Experiment Station under the US Department of Agriculture Hatch Program. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. The findings and conclusions in this article are those of the authors and do not

necessarily represent the views of the US Fish and Wildlife Service.

activities to repair nuclear damage incurred during treatment and captive holding.

**4.2. Implications for future research**

112 Organismal and Molecular Malacology

freshwater mollusk genes remain unannotated.

**Acknowledgements**

Pawel Michalak1 , Lin Kang1 , Serena Ciparis2,3, William Henley<sup>2</sup> , Jess Jones2,3, Andrew Phipps<sup>2</sup> and Eric Hallerman2 \*


2 Department of Fish and Wildlife Conservation, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA

3 U.S. Fish and Wildlife Service, Blacksburg, Virginia, USA
