The intertidal zone is a dynamic environment that fluctuates with the 12.4-h tidal and 24-h light/dark cycle to predictably alter food availability, temperature, air exposure, wave action, oxygen partial pressure, and osmotic conditions. Intertidal sessile bivalves exhibit behavioral or physiological changes to minimize the persistent challenges of fluctuating environmental conditions, such as adjusting gaping behavior and heart rate. At the cellular level, transcriptomic studies on mussels’ baseline circadian and circatidal rhythms have determined that the circadian rhythm is the dominant transcriptional rhythm. However, as proteins reflect the basic molecular phenotype of an organism and their abundance may differ greatly from that of mRNA, these methods could fail to detect important cyclical changes in the proteome that cope with the regular stress of tidal rhythms. For this study, we acclimated intertidal Mytilus californianus to circadian (12:12 h light/dark cycle) and circatidal (6:6 h tidal cycle) conditions in a tidal simulator and sampled gill tissue from mussels every 2 h for 48 h for proteomic analysis. Approximately 86% of the proteins that were detected exhibited rhythmicity over the time course. The circadian cycle primarily determined the cyclic abundance of energy metabolism proteins, pivoting around the transition to the nighttime high tide. The tidal cycle contributed to alterations in cytoskeletal components, ER protein processing and vesicular trafficking, extracellular matrix and immune proteins, and oxidative stress and chaperoning proteins. We also found evidence that post-translational modifications may be important for driving these rhythms, as acetylation and phosphorylation motifs were enriched in the rhythmic proteins and we identified rhythms in elements of methylation, mitochondrial peptide processing, and acylation. These dynamic changes in proteins across numerous functional categories indicate that the combination of circadian and tidal cycles drive complex cellular changes to coordinate processes in a changing environment. This variation clearly shows that differential expression studies and biomonitoring efforts cannot assume a static baseline of cellular conditions in intertidal mussels.

Since their recent introductions into Florida waters, three nonnative species [Perna viridis Linnaeus, 1758 (Asian green mussel), Mytella charruana d'Orbigny, 1846 (charru mussel) and Megabalanus coccopoma Darwin, 1854 (pink titan acorn barnacle)] have expanded both north and south along the Atlantic coast. Very little research has been done to understand how these nonnative species interact with the native eastern oyster (Crassostrea virginica Gmelin, 1791), which is a keystone species that provides important ecological services and economic benefits. To test the potential effects of P. viridis, M. charruana and M. coccopoma on C. virginica, I addressed the following questions: 1a) Does the presence of nonnative species decrease oyster larval settlement? 1b) Do oyster larvae avoid settling on oyster shells to which nonnative species are attached? 2a) Do nonnative species decrease survival of juvenile oysters (spat)? and 2b) Do nonnative species hinder spat growth? My manipulative experiments showed that the tested nonnative species influenced settlement, growth and survival of C. virginica in unique ways. Megabalanus coccopoma decreased the total number of settled oyster larvae, but did not influence larval preference or survival and growth of spat. Perna viridis negatively influenced larval settlement and oyster larvae avoided settling on shells of P. viridis. Mytella charruana had no influence on the total number of settled larvae but oyster larvae avoided settling on oyster shell with M. charruana or on the mussel shells themselves. Furthermore, both nonnative mussels negatively affected the survival of juvenile oysters, but only M. charruana reduced spat growth. These three nonnative species should be classified as invasive species because all had negative effects on the native oyster C. virginica.

The present thesis work is focused on the low-temperature transformation of food industry wastes like mussel shells into nanocrystalline ions-substituted hydroxyapatite powder, having similarities with natural bone apatite, on the consolidation of such powder by cold sintering, and on the physicochemical characterization of the raw materials, synthesised powders and sintered pellets. Nonetheless the evaluation of the mechanical and biological properties was carried out to address cold sintered bodies to possible scaffolds for bone tissue engineering applications. Mussel shells, like other biogenic source of calcium carbonate/phosphate, have the attractive of being a “zero”-cost raw material because they are a waste, but also of having trace elements (Mg, Na, Sr, etc.) which, if found in a bioceramic, have a positive effect on the biological properties. Therefore, mussel shell-derived hydroxyapatite could resemble the mineralized bone tissue, being natural apatite nanometric, ion substituted and with low crystalline tenor. In the first part of the manuscript, two production methods were explored: mechanochemistry and dissolution-precipitation synthesis. Mechanochemistry was carried out at room temperature by directly mixing crushed mussel shells with phosphoric acid in a ball mill. Nanocrystalline multi-ions substituted hydroxyapatite was produced after 4 h of milling and drying at 150°C. Conversely, dissolution-precipitation synthesis was carried out in two steps: the dissolution of crushed mussel shells by adding phosphoric and chloric acid occurred at room temperature, whereas the precipitation of calcium phosphates induced by soda solution, occurred at 45°C. Dissolution-precipitation was further implemented to produce a homogeneous composite material in a single-step by introducing chitosan (in a 2/5/10 wt%) during the dissolution step. The idea was to produce a composite material able to mimic the natural bone tissue composition. In the second part of the manuscript, cold sintering was investigated for the consolidation of the synthesised hydroxyapatite and hydroxyapatite-based composites at a maximum temperature of 200 °C to avoid phase transformation, limit grain growth and preserve the osteoconduction of the bioceramic materials. The effect of the main process parameters such as solvent amount, pressure, temperature and holding time was discussed. Pressure-solution creep and plastic deformation were pointed out as the fundamental consolidation mechanisms in cold sintering, the pressure playing the major role. With a synergistic combination of pressure (600 MPa), temperature (200°C) and liquid phase (20 wt%) it was possible to consolidate hydroxyapatite above 80% relative density in only 15 min. Furthermore, pressure and temperature act a complementary agent during cold sintering. In fact, it was possible to consolidate nanometric HAp and HAp/chitosan composites above 90% relative density by increasing the applied pressure up to 1.5 GPa at room temperature. The mechanical properties of cold sintered pellets were investigated, and resulted in a flexural bending strength and Vickers microhardness, respectively, of 45 MPa and 1.1 GPa for pure hydroxyapatite and of 55 MPa and 0.8 GPa for HAp/chitosan composite. In the frame of bone tissue engineering applications, cold sintered bodies were also preliminarily tested in vitro to establish their bioactivity, their cellular viability through cytotoxicity assessment, and the ability to sustain cells adhesion, osteogenic differentiation. And extracellular matrix mineralization.

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Feeding selectivity by the rainbow mussel (Villosa iris) was examined for three age groups; 2-3 days old, 50-53 days old, and 3-6 years old. The mussels were fed an algal diet consisting of Scenedesmus quadricauda (22.3 - 44.5 μm), Nannochloropsis oculata (2.8 – 8.1 μm), and Selenastrum capricornutum (3.6 – 8.5 μm) in equal cell densities. The change in relative abundance of each algal species within feeding chambers over a 5 hr feeding trial was used to discern selectivity. At the conclusion of the feeding trials, the gut contents of mussels were analyzed for preferential ingestion. The mussels selected for N. oculata and S. capricornutum over S. quadricauda (p < 0.05). This may be an indication of particle size-dependent selection. Feeding trials also suggest that selectivity by the rainbow mussel does not change with age. Gut content analyses showed a preferential ingestion of algae, in the sequence N. oculata, S. capricornutum, then S. quadricauda. The suitability of two algal diets, S. quadricauda and N. oculata, for rearing captive juveniles of V. iris in 145-L recirculating culture systems was compared. Juveniles were fed their assigned diet at a density rate of approximately 30,000 cells/ml for 42 days, and sampled weekly for percent survival and shell length. Regardless of diet, juvenile survival decreased rapidly after 21 days, and growth did not exceed approximately 450 μm. High mortality rates and slow growth of juveniles was likely due to inadequate diets. Juveniles that were fed S. quadricauda lacked chlorophyll coloration in their guts, indicating that the juveniles did not ingest this species of algae. Colonies of S. quadricauda were likely too large for the juveniles to ingest. The gut content of juveniles fed N. oculata showed chlorophyll coloration, indicating that the juveniles ingested this species, but N. oculata may have been difficult for the juveniles to assimilate. Under the culture conditions provided, survival and growth did not compare favorably to those of other studies with V. iris. Newly metamorphosed juveniles of V. iris were reared in 145-L recirculating culture systems containing sediment (< 600 μm) of two depths, 5 mm and 15 mm. Mussels were fed a bi-algal diet of Nannochloropsis oculata and Neochloris oleoabundans. Survival differed significantly between treatments (p=0.04), and was higher for juveniles reared in 5 mm of sediment over a 40-day period. Growth was not significantly different between treatments. After 40 days, juveniles achieved a mean length of approximately 578 μm in both treatments. Survival and growth of juveniles compared favorably to those of other culture studies using juveniles of V. iris. A shallow layer of sediment is recommended for the culture of juvenile mussels. / Master of Science

River-floodplain connectivity is the degree of water-driven transport of matter, energy, and organisms between rivers and their floodplains. Recent advancement of high-resolution lidar data and numerical modeling is helpful to explore river-floodplain connectivity precisely to improve our predictions of sediment transport and deposition on floodplains. In the present work, we studied floodplain sediment transport and deposition, and juvenile mussel settling in three river systems in the US. A two-dimensional hydrodynamic model was developed and simulated model results were coupled with field measurements to study river-floodplain systems of the East Fork White River in Indiana, South River in Virginia, and Dan River in North Carolina. Results show that the East Fork White River in Indiana is capable of supplying sand to the channels on the floodplain and these floodplain channels can transport sand in suspension and gravel as bedload. These floodplain channels are supply limited under the current hydrologic regime and identified as net erosional. On the South River floodplain in Virginia, incorporating hydrologic flowpaths as an explicit measure of river-floodplain connectivity can improve predictions of floodplain sediment deposition. Three regression models were developed incorporating flow pathways and the best model was applied to hydrodynamic model results to create a spatial map of floodplain sedimentation rate. The deposition map highlights how floodplain topography and river-floodplain connectivity affect sedimentation rates and can help inform the development of floodplain sediment budgets. Lastly, streamflow conditions were investigated in the Dan River, North Carolina as they affect juvenile freshwater mussel settling. Two uplooking velocity sensors on the river bed were deployed and hydraulic parameters were measured for a 7-mo period in May-November 2019 to estimate the juvenile mussel settling. Results show that juvenile freshwater mussels as large as 280-508 µm could always be suspended during our study period and not be able to settle onto the river bed at the location of our velocity sensors. Therefore, the flow and shear velocity during our study period was high enough to prohibit the recruitment of juvenile freshwater mussels from settling out of suspension at the sensor locations. Modest flow obstructions such as large boulders, downed trees, or large wood that create downstream wakes may be important features that provide suitable conditions for the settling of juvenile freshwater mussels onto the river bed. Furthermore, low flows have been increasing since the year 2000 which may be exacerbating the decline in freshwater mussel populations. / Doctor of Philosophy / Human civilization has developed near rivers due to the wide range of benefits provided by rivers. Rivers provide food, water, and energy to more than 2.7 billion people around the world. However, the health of the rivers is degrading rapidly to meet the increasing demand of the growing population. We studied water, sediment, and mussel transport in the three rivers in the US: East Fork White River in Indiana, South River in Virginia, and Dan River in North Carolina. These rivers play an important role in agriculture, water supply, sediment, and nutrient transport of the surrounding environment. Our research work on East Fork White River in Indiana, USA shows that the area directly adjacent to the river is eroding, which is important information for river managers and policymakers. As part of that work, we identified the potential of various sizes of sediment to move over this area at different flows and developed a method to predict the largest sediment size that could be moved in water and hopping along the ground. This method is also applicable to other areas along rivers and the coast. We estimated the sediment deposition rate, deposition volume, and prepared a spatial map of the sediment deposition pattern for the South River floodplain in Virginia. From this map, deposition hot spots could be identified. We estimated that 66% of the sediment deposited adjacent to the South River was located in 32% of the area. This information will be helpful for understanding how sediment and sediment-associated pollutants deposit around rivers. Our work on the Dan River in North Carolina was focused on freshwater mussels. Our results showed that juvenile freshwater mussels could not have settled onto the river bed at the location of our measurements. Historical data reveal that freshwater mussels are declining at an alarming rate in that river, posing a threat to the river environment. We identified that streamflow has been increasing over the last two decades, which could be a potential cause of declining freshwater mussels.

Host fishes for the state-endangered Tennessee heelsplitter (Lasmigona holstonia) and state-threatened black sandshell (Ligumia recta) were identified through induced infestations of glochidia on potential hosts. Largemouth bass (Micropterus salmoides), yellow perch (Perca flavescens), convict cichlid (Cichlasoma nigrofasciatum), platy (Xiphophorus maculatus), green sunfish (Lepomis cyanellus), rock bass (Ambloplites rupestris), redbreast sunfish (Lepomis auritus), and white perch (Morone americana) were identified as suitable hosts for L. recta. The banded sculpin (Cottus carolinae) and rock bass were identified as hosts for L. holstonia; striped shiner (Luxilus chrysocephalus), central stoneroller (Campostoma anomalum), and warpaint shiner (Luxilus coccogenis) were identified as potential hosts. Additionally, rock bass and bluegill (Lepomis macrochirus) were identified as potential hosts for the state-endangered spectaclecase (Cumberlandia monodonta), with numerous encysted glochidia present at 11 days postinfestation when the fish died. Recirculating culture systems of different design were tested for suitability in juvenile mussel culture. In one system (high maintenance), juveniles of the wavy-rayed lampmussel, Lampsilis fasciola, were kept in culture dishes, and in the other system (low maintenance), the juveniles were kept in culture beds. At the end of the 16-wk culture period, the 31.3% (± 15.4) survival exhibited in the dish culture system was significantly greater than the 3.1% (± 2.8) survival in the bed culture system (P< 0.01, Tukey-Kramer). However, mussels grown in the bed system exhibited significantly greater growth (1.4 ± 0.50 mm height, 1.8 ± 0.76 mm length) than those grown in the dish system (0.86 ± 0.19 mm height, 1.1 ± 0.27 mm length) (P<0.01, Tukey-Kramer). Using the high maintenance dish culture system and juveniles of L. fasciola, the influence of high (4.1%) and low (2.5 %) organic content in substrate, and high (250 mg/L CaCO₃) and low (50 mg/L CaCO₃) water hardness levels were assessed on growth and survival. After 15 wk, juveniles in the high water hardness treatment exhibited significantly greater survival and growth (44.2 ± 9.3% survival, 1.5 ± 0.28 mm height, 2.1 ± 0.41 mm length) than those in the low water hardness treatment (9.0 ± 7.9% survival, 1.3 ± 0.25 mm height, 1.8 ± 0.37 mm length) (P<0.01, Tukey- Kramer). Juveniles in the high organic substrate exhibited similar growth (1.41 ± 0.24 mm height, 1.96 +- 0.37 mm length) to those in the low organic substrate (1.39 ± 0.28 mm height, 1.94 ± 0.42 mm length). Juveniles grown in high hardness and high organic substrate had similar survival (27.4 +- 9.2%) to those in low organic substrate (25.8 ± 8.1 %). For the culture of L. fasciola juveniles, I recommend using a culture system that is cleaned regularly (weekly), receives a consistent (daily) supply of algal food, has relatively even flow, and from which juveniles are easily sampled. I recommend culturing them in relatively hard water (~ 250 mg/L CaCO₃), in a substratum with some organic content. These recommendations may warrant modification for the culture of other freshwater mussel species. / Master of Science

There is no single methodology toward freshwater conservation planning, and few analytical tools exist for summarizing ecological risks at a landscape scale. I constructed a relative risk model, the Ecological Risk Index (ERI), to combine the frequency and severity of human-induced stressors with mappable land and water use data to evaluate impacts to five major biotic drivers: energy sources, physical habitat, flow regime, water quality, and biotic interactions. It assigns 3 final risk rankings based on a user-specified spatial grain. In a case study of the 5 major drainages within the upper Tennessee River basin (UTRB), U.S.A, differences in risk patterns among drainages reflected dominant land uses, such as mining and agriculture. A principal components analysis showed that localized, moderately severe threats accounted for most of the threat composition differences among watersheds. Also, the relative importance of threats is sensitive to the spatial grain of the analysis. An evaluation of the ERI procedures showed that the protocol is sensitive to how extent and severity of risk are defined, and threat frequency-class criteria strongly influenced final risk rankings. Multivariate analysis tested for model robustness and assessed the influence of expert judgment by comparing my original approach to a quantile-based approach. Results suggest that experts were less likely to assign catchments to high-risk categories than was the quantile approach, and that 3 final risk rankings were appropriate. I evaluated the influence of land use on freshwater ecosystems by studying the relationship between land cover changes and the persistence of freshwater mussels. First, historical species data were collected and the Upper Tennessee River Mussel Database (UTRMD) was constructed. The UTRMD contains >47,400 species records from 1963-2008 distributed across nearly 2,100 sampling sites. My study suggests that 30 years of land cover change does not explain observed freshwater mussel declines. Quantitative surveys are recommended basin-wide to provide more accurate information about mussel distribution and abundance. Lastly, results suggest that streams with repeated mussel surveys have increasing populations, including active recruitment in several beds. Additional quantitative surveys since 2004 have probably provided more accurate species and population counts, although actual population sizes are still uncertain. / Ph. D.