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The Powell River is located in southwestern Virginia and northeastern Tennessee, USA and supports a diverse freshwater mussel assemblage of 29 extant species. Throughout the river major ion and trace element concentrations have increased over the last several decades due to extensive surface coal-mining in the headwaters in Virginia. As watershed area affected by mining has increased, mussel populations have declined, especially in Virginia where populations have been severely reduced or extirpated. The upper watershed now has been extensively mined for coal, causing widespread effects on water and sediment quality. To investigate how mining may be affecting mussel populations, I first conducted a laboratory bio-assay to assess the effects of elevated major ions and the trace element nickel (Ni) on growth and survival of juvenile mussels, including one common species (Villosa iris) and one endangered species (Epioblasma capsaeformis). No significant differences in overall survival between treatments and control were observed for either species over a 70 day test period. Total growth was not significantly different between treatments and control for either species. However, overall growth varied significantly (p=0.009) between species, with V. iris (2.49 mm) exhibiting greater growth compared to E. capsaeformis (1.97 mm). Results suggest that major ion chronic toxicity alone or in combination with Ni at or below my test concentration is not a likely source of toxicity to juvenile mussels in the Powell River. Secondly, I conducted a field study in the Powell River using two cohorts of juveniles of Villosa iris to assess the effects of trace elements and PAH contamination related to mining on mussel survival and growth. Specific conductance was elevated throughout the Powell River, where site means ranged from 450 to 900 µS/cm. While mortality was high at all eight sites it was not significantly different among these sites (p>0.28); however, growth of juvenile mussels was significantly higher (p<0.001) in the lower river in Tennessee. Regression analysis showed significant relationships (p<0.001) of river kilometer with temperature, specific conductance, and aqueous major ion concentrations. A principal component analysis (PC) was conducted on all trace element data. Growth of Cohort 1 on Day 106 was best explained by the PC dominated by aqueous major ion concentrations (p<0.0001, R2= 0.65) and growth of Cohort 2 on Day 106 was best explained by specific conductance (p<0.0001, R2= 0.68). Growth of Cohort 2 at Day 423 was best explained by tissue trace element concentration PC1 and PC2 (p<0.0001, R2= 0.73). This study suggests major ions and select trace elements (Ba, Ni, Fe, Se, and Sr) in the Powell River are negatively affecting the growth of freshwater mussels and that the source of these contaminants is primarily from mining in the headwaters. / Master of Science / The Powell River is located in southwestern Virginia and northeastern Tennessee, USA and supports a diverse freshwater mussel assemblage of 29 extant species. Throughout the river major ion and trace element concentrations have increased over the last several decades. As watershed area affected by coal mining has increased mussel populations have declined, especially in Virginia where populations have been severely reduced or extirpated. The upper Powell River watershed has been extensively mined for coal, causing widespread decline in the river’s water and sediment quality. My study consisted of a laboratory and field exposure to assess the toxicity of the mining related contaminants, such as major ions, trace elements, and polycyclic aromatic hydrocarbons (PAHs) to freshwater mussels. Further, the study investigated the concentrations of these contaminants in the river and their effects on the survival and growth of exposed juvenile mussels. In my laboratory study, mussels of a common species (Villosa iris) and an endangered species (Epioblasma capsaeformis) showed no effect when exposed to a suite of major ions and the trace element Ni similar to levels measured in the Powell River. When juvenile Villosa iris were exposed in the Powell River at eight sites in Virginia and Tennessee, high rates of mortality were observed at all eight sites and growth of juveniles showed a significant spatial trend, with higher growth observed downstream in Tennessee. Water quality analysis revealed increased concentrations of major ions at all sites but concentrations of trace elements were generally below EPA water quality criteria. Further, many of the major ions and trace elements trended spatially with higher concentrations measured in the headwaters in Virginia and lower concentrations observed downstream in Tennessee. Statistical analysis revealed that major ions and trace elements (Ba, Ni, Fe, Se, and Sr) may have negatively affected growth of exposed mussels. This study revealed that laboratory conditions may not adequately be representing river conditions and that in the river major ions and trace elements likely are negatively effecting growth and survival of freshwater mussels. This study revealed that conditions in the Powell River likely are not suitable for mussel reintroduction and that mining is the main source of the contaminants in the river.

Understanding the respiratory physiology and energy metabolism is important for establishing the dissolved oxygen (DO) requirements of freshwater mussels, determining the metabolite(s) indicative of environmental stress, and interpreting environmental conditions based on physiological indicators of mussels. Three studies were undertaken to address these questions. The first study was conducted with seven mussel species collected from reservoir and riverine habitats. The two objectives were to determine the diurnal patterns of valve gaping of freshwater mussels from different habitats, and to monitor heart rate changes of a mussel species that exhibited the diurnal gaping. The results showed that night gaping is evident for the mussels collected from lentic areas, but not for those collected from lotic areas. The heart rate of Pyganodon grandis increased when they gaped. The second study was conducted with nine species of freshwater mussels from different habitats. The three objectives were to determine the patterns (i.e., regulator and conformer) of oxygen consumption (OC) rate under declining DO, evaluate the effects of temperature on ability to regulate OC under declining DO, and finally to use this information to predict DO criteria for maintaining freshwater mussels in captivity. The results showed that mussels living in the habitats subjected to low DO have a better ability to regulate the OC and were more tolerant to hypoxia. The third study assessed three mussel species from different habitats with different abilities to regulate OC under low DO. The two objectives in this study were to identify the energetic metabolite changes under different DO levels and air exposure for the three species, and to determine the appropriate tissue(s) and metabolite(s) to use for estimating the stress in mussels. The results showed that different biochemical strategies were used by Villosa iris, Elliptio complanata, and Pyganodon grandis. Villosa iris had the lowest anaerobic capacity. Elliptio complanata had a more efficient anaerobic metabolism, and P. grandis reduced energy metabolism under low DO and air exposure. posterior adductor muscle, gill and mantle were good tissues for evaluating hypoxic stress. The mantle tissue had the highest glycogen store and was the best tissue for non-lethal study of physiological condition. / Ph. D.

Three spined, mussel species occur in the United States along the Atlantic slope; James spinymussel (Pleurobema collina), Tar spinymussel (Elliptio steinstansana), and Altamaha spinymussel (E. spinosa). The James spinymussel was listed as endangered in 1988, and was until recently considered to be endemic to the James River basin (Clarke and Neves 1984; USFWS 1990). Biologists with the North Carolina Department of Transportation (NCDOT) discovered spinymussel populations in the Dan and Mayo rivers in NC in 2000 and 2001, respectively. The U.S. Fish & Wildlife Service (USFWS) tentatively identified this species as Pleurobema collina. My project proposed by the Virginia Cooperative Fish and Wildlife Research Unit to the USFWS and the Virginia Transportation Research Council, determined where P. collina resides in VA and what the extent of its range is within the state. An informal preliminary survey design for P. collina was used during the summer of 2002 and simple random sampling was deployed in 2003-2004 surveys to provide a good basis for comparison to gauge the efficiency of the informal sampling design. In 2002, a total of 116 person-hours were spent surveying 39 localities on the Mayo, Dan, and Smith rivers. A total of 96 P. collina was observed in the South Fork of the Mayo River, Patrick and Henry counties, VA. A documented range of 24 rkm was established in the South Fork Mayo River. During the summers of 2003 and 2004, a total of 228 person-hours were spent surveying 38 equal-area river reaches (10, 000 m2) on the mainstems of the Dan, Smith, South Mayo, and Banister rivers. No specimens of P. collina (live or relic shells) were detected. A simple random sampling approach was designed to be easy, relatively quick and cost effective, applicable to most rivers, and to provide actual numbers for comparison. Negative results were only reported after 6 person-hours of searching within each randomly selected, equal-area river reach had been expended. P. collina was declared absent from the VA random sites surveyed in 2003-2004 with a confidence of ~90%. A genetic characterization of four extant populations of P. collina was conducted to assess its taxonomic affinity and to resolve conservation issues related to recovery planning and management actions. The populations were examined for phenotypic variation, and were characterized phylogenetically using DNA sequences. A comprehensive analysis was performed for both separate and combined mitochondrial (357 bp of cytochrome-b, 916 bp of ND-1) and nuclear (502 bp of ITS-1) DNA sequences. Based on comprehensive molecular, morphological, and life history data, populations of P. collina sampled from the Dan River sub-drainage do not warrant separate species designation from P. collina sampled from the James River drainage. / Master of Science

Due to the increasing need to provide refugia for freshwater mussels impacted by anthropogenic activities and exotic species, facilities should be identified and protocols developed for holding mussels in captivity. White Sulphur Springs National Fish Hatchery (WSSNFH), White Sulphur Springs, WV, has held freshwater mussels for nearly eight years, and has the potential to become an important refugium and propagation facility for conservation of mussels in the Ohio River Basin and elsewhere. The goal of this study was to determine the feasibility of holding adult freshwater mussels in long-term captivity at WSSNFH by evaluating survival, energy reserves, and gametogenesis of captive mussels in a recirculating pond system. I relocated three mussel species in the summer of 2001 and 10 mussel species in the summer of 2002 to a recirculating pond system (reservoir and raceway) at the hatchery. Water quality parameters of pH, alkalinity, hardness, temperature, and dissolved oxygen; and algal concentrations were measured periodically from summer 2001 to summer 2003. Annual survival rates of 10 species were estimated (August 2002 to August 2003) using the program MARK. Glycogen, protein, and lipid concentrations in mantle tissue of three captive species (Actinonaias ligamentina, Cyclonaias tuberculata, and Tritogonia verrucosa) were compared to those of wild mussels in the New River. Gametogenic activity and synchrony in A. ligamentina and C. tuberculata were compared between captive and wild mussels. Water quality parameters, with the exception of temperature, were within desirable ranges for most of the study. Temperatures of > 28° C were observed for several days during summers 2002 and 2003. Algal concentrations averaged 1903 cells ml-1 in the raceway (range: 300 to 4658 cells ml-1), which is comparable to algal concentrations reported for nearby rivers. The overall survival rate for 10 freshwater mussel species held in the raceway for one year was 77%. Villosa vanuxemensis had the highest survival rate (96%), and Lampsilis cardium had the lowest survival rate (31%). Although there were fluctuations in glycogen, protein, and lipid levels over 2 yr, there were no overall differences in energy substrates between captive and wild mussels at the end of the study. Captivity did not appear to have a negative affect on gametogenesis. Captive C. tuberculata spawned within the expected time frame between January and June, but slightly earlier than their wild counterparts in the New River. Due to the infestation of the gonads of both captive and wild A. ligamentina by digenean trematodes, little gametogenesis was observed. However, captive holding did not appear to have an effect on trematode infestation rates. From these results, I conclude that captive holding conditions in the recirculating pond system at WSSNFH were adequate for long-term holding of a wide range of mussel taxa. I recommend that WSSNFH continue to be used as an adult holding facility. Further research should be conducted to determine food and habitat preferences of freshwater mussel species in captivity so that optimal holding conditions can be provided for each species. / Master of Science

The use of glass racks and suspended pocket nets for holding freshwater mussels collected from the Ohio River and relocated to lined-ponds was studied over 3 years. Survival of mussels in ponds was 73 % after 1 y, 44 % after 2 y, and 5 % after 3 y. The glycogen levels of mussels in ponds for ly were significantly greater than that of mussels in ponds after 2.5 y and 3 y, indicating a chronic decline in body condition in mussels. Despite the presence of a diverse and dense assemblage of algae and organic detritus in the ponds, the stomachs examined at 3 y were empty and the bodies were emaciated. In the laboratory, I determined the amount of algae cleared by the rainbow mussel, <i>Villosa iris</i> (Lea, 1829) fed different algal rations, and estimated the algae concentration needed to maintain mussels in captivity. Filtration rate in the first feeding hour was highest in ration B (1.0 mg dry wt L⁻¹) and lowest in ration C (3.4 mg dry wt L⁻¹). After 1 h, filtration rates declined in ration B but increased in rations C and A. <i>V.iris</i> likely achieved gut satiation in the first hour using maximum filtration (712.5 mL h⁻¹g⁻¹) and then decreased filtration (259 mL h⁻¹g⁻¹) thereby regulating ingestion rate during the following 2 h. I estimate, therefore, that K <i>V. iris</i> daily maintenance requirement for carbon is 8.2 mg C (1.2 x 10⁹ cells of N. oleoabundans) or ca. 2.4% of dry body weight. Assimilation efficiencies (AE) and carbon budgets also were established for the rainbow mussel, <i>Villosa iris</i> (Lea 1829), using radio-labeled cultures of <i>Neochloris oleoabundans</i> (Chantanachat and Bold 1962) at three cell concentrations. Approximately 70% of the ingested carbon was assimilated (assimilation efficiency) by V. iris fed 5 x 10⁵ cells mL⁻¹) (3.4 mg dry weight L⁻¹). At 5 x 10⁴ cells' mL⁻¹) (0.34 mg dry weight L⁻¹), AE was 47.5 %. At 5 X 10³ cells mL⁻¹) (0.034 mg dry weight L⁻¹), AE was 40%. V. iris had the greatest amount of energy available for growth, reproduction, and body condition in captivity at 3.4 mg dry weight L⁻¹). The gross composition (protein, carbohydrate (CHO), and lipid) of four algae (Bracteacoccus grandis, Neochloris oleoabundans, and Scenedesmus quadricauda, and Phaeodactylum tricomutum) was examined at four different phases of growth. The CHO content (% algal dry wt) increased with growth phase (age of the algal culture) with the exception of B. grandis. N. oleoabundans and P. tricomutum contained the greatest CHO content (33.07 ± 6.89% and 39.37 ± 3.07%, respectively) at late stationary phase. The total lipid content increased with growth phase for N. oleoabundans and P. tricomutum. Lipid content of B. grandis decreased with age, and S. quadricauda showed no difference in lipid content (% algal dry wt) between growth phase. N. oleoabundans' lipid content (31.85 ± 9.4%) was greater than all other species. Generally, there was no effect of phase on the sterol content, with the exception of the sterol content of <i>S. quadricauda</i> increased with growth phase. The mean sterol content of the four algae ranged 1.0 % ± 0.4 to 1.8 ± 1.8 of the total lipid dry wt; maximum sterol % of lipid was 5 % for <i>Scenedesmus</i> and 4.4% for <i>B. grandis</i>. There was no effect of growth phase or species on the protein content (% of algal dry wt). The protein content ranged 60.6 ± 17.1 to 70.3 ± 9.5 % of algal dry wt. / Ph. D.

The Clinch River, Virginia is known for high aquatic biodiversity, particularly Unionidae which are declining at remarkable rates. Studies conducted on the mainstem have only addressed effects of point-source stressors (various toxic spills and effluents from the Clinch River Plant (CRP), Carbo, Virginia) that have been introduced into the Clinch River. It is hypothesized that the tributaries of the Clinch River deliver a variety of stressors to the mainstem, which may affect the diverse fauna. The aquatic health of 19 upper Clinch River tributaries, Virginia, was assessed via ecotoxicological ratings that indicated the least healthy catchments were associated with mining activity (Dumps, Russell and Coal Creeks). Tributaries were categorized by land use and mining streams were significantly different from agricultural and forested streams (F = 9.63, p<0.0001). Tributaries with ecotoxicological ratings (ETR) <80 from 100 were deemed suboptimal and thus studied further. Using identical response variables and upstream and downstream sites, resulting ETRs for nine streams indicated no model significance regarding land use, year, or site. Variability within treatments and low sampling sizes contributed to lack of significance, and results indicate that future studies need to be designed incorporating sites with analogous land use stressors. This first assessment of upper Clinch River tributaries indicates the catchments requiring remediation are Dumps, Russell and Coal Creeks, while tributaries requiring extensive evaluations are Big, Lick, Swords, Big Spring, Guest River, Cavitts and Middle Creeks. Tributaries that were deemed healthy (ETRs >80) were Big Stony, Copper, Indian, Stock, Little River and Cove Creeks. / Master of Science

Distribution of the James spinymussel (Pleurobema collina) and aspects of its life history were studied from June 1987 to October 1989. Field and laboratory observations were used to determine gravidity and glochidial release periods, fish host requirements, age class structure, growth characteristics, and juvenile survival. Stream surveys conducted at 243 sites in 20 counties revealed new populations of this species in three subdrainages (Catawba Creek, Pedlar River, and Mechums River), of the James River, greatly expanding its known range. Female P. collina were gravid from late May through early August 1988 and 1989 in Craig Creek, Virginia, and the South Fork Potts Creek, West Virginia. Glochidia were released from late June through early July in the South Fork Potts Creek, at mean water temperatures between 21 and 25 °C, and discharges between 0.013 and 0.050 m l/sec. Field observations and laboratory tests confirmed that cyprinids serve as fish hosts for P. collina glochidia. Glochidia of the James spinymussel were identified on the common shiner (Notropis cornutus), rosyside dace (Clinostomus funduloides), bluehead chub (Nocomis leptocephalus), pumpkinseed (Lepomis gibbosus), and fantail darter (Etheostoma flabellare) in the South Fork Potts Creek. Eleven fish families were tested for suitability as fish hosts in the laboratory, but only the following 7 species served as hosts: bluehead chub (N. leptocephalus), rosyside dace (C. funduloides), satinfin shiner (N. analostanus), rosefin shiner (N. ardens), central stoneroller (Campostoma anomalum), blacknose dace (Rhinichthys atratulus), and mountain redbelly dace (Phoxinus oreas). Juvenile P. collina, fed a mixture of three green algae species in chambers with and without silt, lived 50 days. Juveniles in silt grew significantly faster than those not in silt after 18 days. Age and growth characteristics of adults were obtained by thin-sectioning valves collected in muskrat middens in Dicks and Johns creeks, Virginia. Ages ranged between 3 and 19 yr, and a strong year class was identified at age 11. Mean annual mortality rate was calculated to be 9.8%. As predicted by the von Bertalanffy growth equation, P. collina reaches a maximum length of 74.4 mm, and annual growth increments decrease from 7.04 mm/yr (age 1) to 0.88 mm/yr (age 19). As judged by qualitative surveys, current populations of P. collina in Craig Creek, Johns Creek, and the South Fork Potts Creek appear to be self-sustaining. Populations in Catawba and Patterson creeks appear to be small and are probably in jeopardy of being extirpated. The status of the James spiny mussel in Dicks Creek, Rocky Run, and Pedlar and Mechums rivers is unknown since these streams were not extensively surveyed. / Master of Science

Four life stages of freshwater mussels were tested for their sensitivity to copper and a metal-containing effluent. This permitted an assessment of the variability in sensitivity among the life stages in order to identify those stages most threatened in the wild from copper exposure. Glochidia, while held within the marsupia of the adult, the released or isolated glochidia, the encysted glochidia, and the gravid mussel were tested. Little effect on glochidia was detected following 30- day artificial stream exposures of gravid adults to 19.1 ug Cu/L and an effluent containing an average of 23.9 ug Cu/L. Isolated glochidia were killed by copper concentrations ranging from 20-80 ug/L in 24-hour exposures, with sensitivity increasing with hardness and temperature. Encysted glochidia were resistant to exposures up to 400 ug Cu/L. No significant effect on metamorphosis to the stage was detected. This is likely due to the encapsulation of the glochidium by the host fish. Juvenile mussels reduced their activity during 24-hour exposures to copper concentrations as low as 17 to 24 ug/L and concentrations of 30 to 42 ug/L caused mortality. Juvenile mussels and glochidia within the marsupia are probably the two most sensitive stages in the life cycle of the freshwater mussel. Copper pollution will have its greatest impact in the summer, during periods of high water temperature and low flow. / Master of Science

Freshwater mussels have played an important role in the history of human culture and also in ecosystem functioning. But during the past several decades, the abundance and diversity of mussel species has declined all over the world. To address the urgent need to maintain and restore populations of endangered freshwater mussels, quantitative population dynamics modeling is needed to evaluate population status and guide the management of endangered freshwater mussels. One endangered mussel species, the oyster mussel (Epioblasma capsaeformis), was selected to study its population dynamics for my research. The analysis was based on two datasets, length frequency data from annual surveys conducted at three sites in Clinch River: Wallen Bend (Clinch River Mile 192) from 2004-2010, Frost Ford (CRM 182) from 2005 to 2010 and Swan Island (CRM 172) from 2005 to 2010, and age-length data based on shell thin-sections. Three hypothetical scenarios were assumed in model estimations: (1) constant natural mortality; (2) one constant natural mortality rate for young mussels and another one for adult mussels; (3) age-specific natural mortality. A Bayesian approach was used to analyze the age-structured models and a Bayesian model averaging approach was applied to average the results by weighting each model using the deviance information criterion (DIC). A risk assessment was conducted to evaluate alternative restoration strategies for E. capsaeformis. The results indicated that releasing adult mussels was the quickest way to increase mussel population size and increasing survival and fertility of young mussels was a suitable way to restore mussel populations in the long term. The population of E. capsaeformis at Frost Ford had a lower risk of decline compared with the populations at Wallen Bend and Swan Island. Passive integrated transponder (PIT) tags were applied in my fieldwork to monitor the translocation efficiency of E. capsaeformis and Actinonaias pectorosa at Cleveland Islands (CRM 270.8). Hierarchical Bayesian models were developed to address the individual variability and sex-related differences in growth. In model selection, the model considering individual variability and sex-related differences (if a species has sexual dimorphism) yielded the lowest DIC value. The results from the best model showed that the mean asymptotic length and mean growth rate of female E. capsaeformis were 45.34 mm and 0.279, which were higher than values estimated for males (42.09 mm and 0.216). The mean asymptotic length and mean growth rate for A. pectorosa were 104.2 mm and 0.063, respectively. To test for the existence of individual and sex-related variability in survival and recapture rates, Bayesian models were developed to address the variability in the analysis of the mark-recapture data of E. capsaeformis and A. pectorosa. DIC was used to compare different models. The median survival rates of male E. capsaeformis, female E. capsaeformis and A. pectorosa were high (>87%, >74% and >91%), indicating that the habitat at Cleveland Islands was suitable for these two mussel species within this survey duration. In addition, the median recapture rates for E. capsaeformis and A. pectorosa were >93% and >96%, indicating that the PIT tag technique provided an efficient monitoring approach. According to model comparison results, the non-hierarchical model or the model with sex--related differences (if a species is sexually dimorphic) in survival rate was suggested for analyzing mark-recapture data when sample sizes are small. / Master of Science