**1. Introduction**

#### **1.1. Response of aquatic organisms to exposure to toxic substances**

Exposure to toxic compounds has not only lethal but also important sublethal effects upon affected individuals. Successful identification of molecular mechanisms underlying response to toxic exposure depends upon development and use of a suitable set of assays, and several approaches are potentially available. While various biomarkers [1, 2] or gene expression [3, 4] assays have been demonstrated for marine mollusks in recent years, there has been no parallel work for freshwater mussels, many of which are of conservation concern. More fundamentally, adaptation of existing assays to freshwater mussels is not entirely attractive because a focus on candidate genes of known function certainly will miss genes or biochemical pathways that are not known. A preferable approach would be to screen global gene expression and thereby identify genes of interest not only in known but also in unknown pathways. Three approaches are available. Microarrays are available for several marine mollusks, and have in some cases been used for purposes of characterizing responses to environmental stressors including toxins or other bioactive compounds (e.g., marine mussels [5–8], Manila clam *Venerupis philippinarum* [9]). There are no existing microarrays for freshwater mussels, however, and construction of such screening platforms would require a large body of work developing and characterizing expressed sequence tags for hundreds to thousands of genes. Subtractive hybridization has been applied to other mollusks, such as the invasive zebra mussel *Dreissena polymorpha* [10], Pacific oyster *Crassostrea gigas* [11], and peppery furrow shell *Scrobicularia plana* [12], and has been shown a viable but technically challenging approach to identifying differentially expressed genes. Next-generation DNA-sequencing technology has made it possible to cost-effectively screen expression of all genes transcribed in tissues of interest, even in species for which prior knowledge of the genome is lacking. The approach is tantamount to sequencing all the RNAs produced in that tissue; hence, the approach is referred to as RNAseq. In our context, RNAseq makes quantitative comparison of gene expression in selected tissues among toxin-challenged and control individuals possible. Against this background, the goal of this study was to relate gene expression end points in a representative freshwater mussel, pheasantshell *A. pectorosa*, to exposure to arsenate and sulfate, two pollutants resulting from coal combustion and mining, respectively.

#### **1.2. Freshwater mussels**

Freshwater mussels (Class Bivalvia: Family Unionidae) have their center of biodiversity in the southeastern United States. However, many regionally important species face a variety of threats, including exposure to toxic compounds. In particular, mussels of the Clinch and Powell River systems of southwest Virginia have been heavily impacted by runoff, leachates, or spills of materials related to coal extraction, processing, and use for electric power generation. Given the continued operation of coal extraction and processing facilities, the shift from deepto surface-mining practices, and the increase in coal-bed gas extraction wells in the Clinch and Powell River system, defensible biological assays for assessing the impacts on key components of this aquatic ecosystem must be developed. These assays could provide critical information for assessing the impacts of future toxic events and thereby allow characterization of potential long-term effects of resource extraction on freshwater mussel populations. This project was conducted to show the response of freshwater mussels to selected physiological stressors. Within this context, the objective of the study was to screen gene-transcriptional markers in a laboratory study under controlled conditions, focusing upon arsenate and sulfate, two contaminants related to coal mining and processing. Identification of such genetic markers could prove essential for use in future nonlethal determinations of contaminant impacts to federally listed mussels.

#### **1.3. Contaminant selection**

**1. Introduction**

100 Organismal and Molecular Malacology

**1.2. Freshwater mussels**

**1.1. Response of aquatic organisms to exposure to toxic substances**

Exposure to toxic compounds has not only lethal but also important sublethal effects upon affected individuals. Successful identification of molecular mechanisms underlying response to toxic exposure depends upon development and use of a suitable set of assays, and several approaches are potentially available. While various biomarkers [1, 2] or gene expression [3, 4] assays have been demonstrated for marine mollusks in recent years, there has been no parallel work for freshwater mussels, many of which are of conservation concern. More fundamentally, adaptation of existing assays to freshwater mussels is not entirely attractive because a focus on candidate genes of known function certainly will miss genes or biochemical pathways that are not known. A preferable approach would be to screen global gene expression and thereby identify genes of interest not only in known but also in unknown pathways. Three approaches are available. Microarrays are available for several marine mollusks, and have in some cases been used for purposes of characterizing responses to environmental stressors including toxins or other bioactive compounds (e.g., marine mussels [5–8], Manila clam *Venerupis philippinarum* [9]). There are no existing microarrays for freshwater mussels, however, and construction of such screening platforms would require a large body of work developing and characterizing expressed sequence tags for hundreds to thousands of genes. Subtractive hybridization has been applied to other mollusks, such as the invasive zebra mussel *Dreissena polymorpha* [10], Pacific oyster *Crassostrea gigas* [11], and peppery furrow shell *Scrobicularia plana* [12], and has been shown a viable but technically challenging approach to identifying differentially expressed genes. Next-generation DNA-sequencing technology has made it possible to cost-effectively screen expression of all genes transcribed in tissues of interest, even in species for which prior knowledge of the genome is lacking. The approach is tantamount to sequencing all the RNAs produced in that tissue; hence, the approach is referred to as RNAseq. In our context, RNAseq makes quantitative comparison of gene expression in selected tissues among toxin-challenged and control individuals possible. Against this background, the goal of this study was to relate gene expression end points in a representative freshwater mussel, pheasantshell *A. pectorosa*, to exposure to arsenate and

sulfate, two pollutants resulting from coal combustion and mining, respectively.

Freshwater mussels (Class Bivalvia: Family Unionidae) have their center of biodiversity in the southeastern United States. However, many regionally important species face a variety of threats, including exposure to toxic compounds. In particular, mussels of the Clinch and Powell River systems of southwest Virginia have been heavily impacted by runoff, leachates, or spills of materials related to coal extraction, processing, and use for electric power generation. Given the continued operation of coal extraction and processing facilities, the shift from deepto surface-mining practices, and the increase in coal-bed gas extraction wells in the Clinch and Powell River system, defensible biological assays for assessing the impacts on key components of this aquatic ecosystem must be developed. These assays could provide critical information The coal industry is a potential source of numerous contaminants to aquatic environments. In several Virginia watersheds, habitat for imperiled freshwater mussel populations exists in close proximity to coal industry-associated activities. These activities include mining and coal-fired electric power plants with on-site storage of coal combustion residue (CCR). Two contaminants were selected for this study based on their association with surface mining (sulfate, SO<sup>4</sup> 2-) or CCR storage (arsenate, As(V)). The contaminants As and SO<sup>4</sup> 2- were selected due to their environmental relevance and the potential for concentrations to become elevated due to activities related to coal mining and power generation.

Freshwater bivalves appear to be relatively tolerant to acute effects of As; the estimated 96-h LC50 for Asian clam *Corbicula fluminea* is 20,740 µg/L As, with no mortality observed at concentrations up to 5000 µg/L for 21 days [13]. In surface waters, the majority of total As will be As(V) under oxygenated conditions. However, investigations of sublethal effects of As on bivalves have generally focused on As(III). Exposures of bivalves to As(III) have demonstrated histological effects including increased damage to digestive gland tissue [14] and biochemical effects including alteration of levels of adenosine triphosphate [15], inhibition of the detoxification enzyme catalase [14], and changes in the activity of glutathione-S-transferase (stimulation and inhibition) [14, 16]. Detoxification of As(III) and As(V) involves reduced glutathione and glutathione-dependent enzymes, and effects on this system in bivalves have been shown to be variable and dependent on both concentration and chemical speciation [14, 16]. The sublethal effects of environmentally relevant concentrations of As(V) on freshwater mussels are currently unknown.

Freshwater bivalves also appear to be relatively tolerant to acute effects of SO<sup>4</sup> 2-. Soucek and Kennedy [17] determined a sulfate 96-h LC50 for grooved fingernail clam *Sphaerium simile* of 2078 mg/L. However, exposure of *C. fluminea* to 1500 mg/L SO<sup>4</sup> 2- reduced feeding rates over a 4-week period [18].

For each contaminant, a relatively 'high' concentration was selected based on worst-case conditions previously measured in heavily impacted environments. The concentration of SO<sup>4</sup> 2- (1250 mg/L) was based on low-flow measurements up to 1200 mg/L in a notoriously polluted river in West Virginia, USA [19]. The high concentration of As (1000 µg/L) was based on measurements in pore-water downstream of a massive CCR spill (up to 1200 µg/L; [20]). For both chemicals, the selected concentrations were not expected to cause mortality based on results of acute and chronic toxicity studies conducted with other bivalves (*S. simile* and *C. fluminea;* [13, 17, 18, 21]). The majority of toxicity tests with As have been conducted with arsenite (As(III)), the more toxic form of As. However, due to the oxygenated environment in our exposure system, mussels were exposed to As as arsenate (As(V)). Hence, the purpose of this study was to determine whether As (as As(V)) and SO<sup>4</sup> 2- caused sublethal changes in freshwater mussels, the nature of these changes, and the relationships between biochemical, histological, and genetic markers.
