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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.

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.

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

This project evaluated the feasibility of salvaging zebra mussel-infested freshwater mussels from their native habitat by determining 1) how zebra mussel infestation affects unionid feeding and body condition, 2) how starvation in quarantine affects body condition of unionids, and 3) what feeding regime maintain unionid condition in quarantine. The effects of zebra mussel infestation on two mussel species were evaluated through glycogen analyses of mantle tissue and gut content analysis. Specimens of Amblema p. plicata (Say, 1817) and Quadrula p. pustulosa (I. Lea, 1831), collected from a heavily infested reach of the Ohio River in 1996, had significantly lower (p<0.05) glycogen levels than specimens collected from a low-infestation reach upstream. In 1996 and 1997, heavily infested Amblema p. plicata and Quadrula p. pustulosa had significantly less (p<0.01) organic matter and fewer algal cells in their guts than lightly infested specimens. In addition, gut contents of individual A. p. plicata contained significantly less (p<0.05) organic matter and fewer algal cells than the combined gut contents of all zebra mussels (18-33 mm in length) attached to their shells. Gut analyses also revealed significant diet overlap between native unionids and infesting zebra mussels. Thus, competitive interactions or interference by zebra mussels likely reduced unionid ingestion and consequently reduced glycogen stores. During quarantine, unionids salvaged from a lightly infested area and starved for 30 days had glycogen levels that declined dramatically. After 30 days without supplemental feeding, mean glycogen levels of A. p. plicata declined 85%, and mean glycogen levels of Q. p. pustulosa declined 70%. Thus, feeding of unionids is necessary to maintain their condition during lengthy quarantine. To determine the best feeding regime for unionids in quarantine, assimilation efficiencies and carbon budgets were established for the rainbow mussel, Villosa iris (Lea, 1829), using radio-labelled cultures of Neochloris oleoabundans (Chantanachat and Bold 1962) at three cell concentrations. Assimilation efficiencies for Villosa iris at 100,000cells/ml, 10,000 cells/ml, and 1,000 cells/ml were similar (45-56%); however, regardless of these similarities, assimilation efficiencies from this study indicate that Neochloris oleoabundans is readily assimilated (~50% AE) by Villosa iris . In addition, total assimilation was maximized at 100,000cells/ml, which indicates that Villosa iris has the greatest amount of energy available for growth, reproduction, and maintenance of condition in captivity at this cell concentration. During a second quarantine experiment, specimens were provided with 100,000cells/ml of N. oleoabundans twice per day. Initial mean glycogen levels for Amblema p. plicata (9.4 + 2.4 mg/g) and Quadrula p. pustulosa collected from ORM 175.5 in July 1997 were not significantly different (p>0.3) than the mean glycogen levels of A. p. plicata and Q. p. pustulosa collected from the same site in July 1996. Glycogen stores of unionids entering quarantine, therefore, were similar in both the starvation and controlled feeding experiments. After 7, 14, and 30 days of controlled feeding in quarantine, mean glycogen levels of A. p. plicata and Q. p. pustulosa did not change significantly (p>0.1). Thus, quarantine protocol for salvaged native mussels should include the feeding of algae to captive specimens to sustain glycogen levels prior to relocation. / Master of Science

Freshwater mussel culture has become an attractive enterprise because of its application for restoring depleted freshwater mussel populations, for rearing adults to support shell and cultured pearl industries, and for environmental monitoring and assessment. This study focused on testing indoor and outdoor culture techniques to rear newly metamorphosed juveniles of the rainbow mussel (Villosa iris). Tanks and floating containers were used outdoors, and sediment beds and floating containers were used in indoor culture experiments. Culture experiments with indoor sediment beds produced modest growth rates and variable survival rates for up to 4 months. Shell lengths of juveniles increased from 0.38 - 0.41 mm to 0.93 - 3.22 mm, 1.45 - 7.05 times original size; survival rates ranged from 1.27 % to 51.0 %. Experiments with indoor floating containers resulted in an increase of juvenile shell lengths from 0.38 - 0.40 mm to 0.86 - 2.07 mm, an 86 - 207 % increase; survival rates were 23.3 % to 27.0 % after 3 months. All indoor culture experiments combined water flow, inoculation of algae, fertilization, lighting and a pre-operation phase. Experimental results suggested that indoor floating container culture was a useful method to rear newly metamorphosed juvenile mussels, and the indoor sediment bed culture technique may also be suitable with modification, especially if filamentous algae can be controlled. Both outdoor tank and floating container culture experiments yielded poor results, which were probably caused by unsuitable culture conditions. Differences in juvenile growth rates (P < 0.001) and survival (P < 0.001) between floating container experiments I and II, which used the same culture techniques, were attributed to differences in culture conditions; water temperature (P < 0.001), dissolved oxygen (P < 0.01) and hardness (P < 0.01). Water temperature also contributed to a higher growth (P < 0.001) and survival rate (P < 0.001) in sediment bed culture II. The extensive occurrence of filamentous algae on sediments affected results and caused lower growth (P < 0.001) and survival rates (P <0.001) of juveniles in the sediment bed culture experiment III, IV and V. Finally, absence of water flow may have influenced growth and survival of juvenile V. iris, a naturally riverine species. Lack of currents near the bottom of tank and floating containers where juveniles resided likely contributed to complete mortality of juveniles in the outdoor culture experiments. / 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