The eastern oyster (Crassostrea virginica Gmelin 1791) has great ecological and economic importance but populations have declined, especially in Chesapeake Bay, to historically low numbers. Hatcheries strive to produce oysters with beneficial characteristics for supplementation and commercial purposes, both natural and stimulated mass spawning. Unequal contribution of parents in mass spawnings potentially can lead to high levels of inbreeding and a loss of beneficial characteristics in offspring. In this study, we determined microsatellite genotypes for parents (n^parents =24, 49, and 77 parents) and progeny (n=96 each) of three hatchery-produced families and used the data for parental assignment. We observed the presence of more than two alleles per locus for some offspring, yet because genetic analysis software only allows for a maximum of two alleles per locus, we chose the two alleles with the strongest signals. For the three parent “populations,” 71% of alleles had frequencies of <0.05 and observed heterozygosities were lower than expected by an average factor of 0.27. Inbreeding within the various parent populations was similar across the three families ranging from F^IS 0.26–0.43. In all three families, the offspring exhibited greater levels of genetic diversity and lower inbreeding levels than the parents (F^IS 0.14–0.21), and in some cases offspring exhibited alleles that were not present in the parents. Variance in the number of offspring produced per parent was observed for all families and in general, <10% of potential parents (generally 2-5 females and 1-3 males) produced >10% of the offspring. Reproductive success for spawning parents, N^b, determined by three methods, ranged from 0.07 to 0.27. As the number of parents per family increased, a higher proportion of parents failed to produce offspring. Across all three families, the average effective number of breeders was N^b = 7.1 and the level of reproductive success was inversely proportional to the number of potential parents. This finding implies that to maintain high levels of diversity and beneficial characteristics in the offspring (and to avoid the chance of unintentional inbreeding), hatcheries should perform more spawnings with fewer parents.

There have long been concerns about the negative impacts of recreational boating activity in the Indian River Lagoon system (IRL), especially in Mosquito Lagoon (ML), the northernmost part of the IRL. My research is focused on the impacts of boat wakes on intertidal reefs formed by the eastern oyster, Crassostrea virginica. There has been a 24% loss of oyster habitat in ML since 1943, where natural oyster reefs have been replaced by dead oyster reefs which do not serve the same ecological function. While there is anecdotal and correlative evidence that this loss is a result of boat wakes, no studies to date have confirmed dead reefs can be a direct result of boat wakes. Therefore, I addressed the following questions: (1) What wake heights are generated by a range of boat types, and (2) What amount of oyster movement and erosion occurs as a result of these boat wakes? A series of boat pass experiments addressed the first question; these results were utilized in experiments at Florida Institute of Technology's wave tank to observe sediment erosion and oyster movement as a result of specific wake heights. Model selection was used for both the field and wave tank experiments to determine which variables contributed most to explaining the wake heights, erosion, and oyster movement that occurred. Wake heights ranging from 0.05 cm to 20.80 cm were documented contacting the oyster reefs from the boat passes, with a mean of 2.95 cm. Boat type was less important than speed or distance when determining wake height. My wave tank results document that wake heights as small as 2 cm contacting oysters are capable of moving individual and clusters of oysters. Minimum distances for boats to travel in order to maintain wakes smaller than 2 cm at reefs are suggested for management purposes based on regression equations. This could minimize the amount of movement that occurs when oysters are subjected to boat wakes. The results of this study can help resource managers implement boating policies in Mosquito Lagoon, and contribute greatly to conserving this important ecosystem engineer.

Aquatic animals, such as fish and shellfish, provide important economic and nutritional benefits for human society. Due to overexploitation of natural fish sources through traditional wild-caught fisheries, aquaculture (generally described as fish farming or culturing) has grown into an economically important industry. A major focus area for the aquaculture field is related to sustainability by ensuring the health and welfare of the aquatic animals. Communities of microorganisms inhabiting the various niches of a given host comprise its microbiome and provide several key health benefits. The microbiome impacts nutrient acquisition, gut homeostasis, protection against pathogens, and immune system modulation. Therefore, much attention has been placed on studying how various culturing conditions and host factors impact the microbiomes of aquatic animals. Here, multiple studies were conducted to elucidate the impacts of various parameters on the microbiomes of rainbow trout, steelhead trout, and Nile tilapia, including dietary supplementation, administration of probiotics and animal age. Though there is a significant correlation between the diet fed to fish and their microbiome communities, small dietary changes such as the inclusion of a dried and lysed yeast product, acting as a protein source alternative to unsustainable fishmeal did not significantly alter the intestinal adherent microbiome of rainbow trout. Moreover, an optimal percentage of yeast replacement that did not negatively impact weight gain for the aquaculture-raised fish was identified, suggesting its efficacy for the industry. Similarly, the intestinal adherent microbiomes of steelhead trout were not significantly altered by diet supplementation with a Bacillus subtilis probiotic. The total microbiome of steelhead trout (mucosa combined with digesta) was instead significantly changed when they were only fed the probiotic additive at an early stage of intestinal development. This change in the microbiome of steelhead trout correlated with a significant increase in weight gain compared to fish only fed the probiotic during later stages of intestinal development. These findings also corroborate previous observations wherein the intestinal microbiome of fish varies during their developmental stages but then stabilizes over time. Determining the core set of bacteria present in fish microbiomes, independent of treatment variables, is another important factor when considering attempts to manipulate the microbiome. To that end, a literature review was conducted in which the phyla Firmicutes, Proteobacteria and, to a lesser extent, Actinobacteria, Bacteroides, and Tenericutes were identified as likely members of the rainbow trout core microbiome. Bacterial families identified as part of the core phyla included Lactobacilliaceae that are commonly used as probiotics and Mycoplasmataceae that lack cell walls. Preventing dysbiosis of the rainbow trout microbiomes will be crucial to ensuring the health of the fish hosts and increasing longevity and profitability of the aquaculture industry. Another important aquaculture-raised species is the Eastern oyster. This animal is critical for the ecological health of the Chesapeake Bay, and it is also an important source of revenue. A significant portion of the revenue flow is the harvest and sale of live oysters for consumption. Unfortunately, consumption of raw or undercooked oysters is the most common route of infection by the human pathogen Vibrio parahaemolyticus (VP) as oysters are a natural reservoir for VP. This bacterium is responsible for a debilitating acute gastroenteritis with potential to cause fatal septicemia. Despite efforts to mitigate infection by this CDC-reportable pathogen, cases continue to increase. The understudied host-microbe relationship between the Eastern oyster and VP has been implicated as a path to research for potential future therapeutics. A novel culturing system for oysters was created using fermentation jars within a BSL-2 ready biosafety cabinet. Using this system, the effect of harvest season was tested against the inoculation efficiency of VP. It was found that higher native Vibrio levels within the oysters were present during the summer compared to the winter. Moreover, addition of the bacteriostatic antibiotic chloramphenicol (Cm) enabled a higher inoculation efficiency by VP during both the summer and winter compared to oysters not exposed to the antibiotic. During the winter, exposure to Cm led to the highest inoculation efficiency (~100%). These findings confirm the importance of the existing microbial communities against exogenous inoculation. Therefore, a year-long study was conducted to investigate the microbiome of oysters during each season. This pan-microbiome study identified a significant impact of harvest season on the microbiome structure. An increased diversity, including higher levels of Cyanobacteriaceae, was observed during the summer. Whereas an increase in Arcobacteriaceae was observed during the winter. Bacteria that persisted throughout the year included Mycoplamataceae and Spirochaeteacae; these families may represent potential members of the Eastern oyster core microbiome. Further work is needed to study the localization patterns of VP within oysters. Such work includes further optimization of immunohistochemistry (IHC) and intracellular colonization assay methods under development here. Collectively, studies of the oyster-microbe interactions will help improve aquaculture methods and identify mitigation targets to reduce VP-related clinical infections. / Doctor of Philosophy / Fish and shellfish provide important economic and nutritional benefits for human society across the globe. Unfortunately, over-fishing of traditional sources of fish and shellfish has led to a reduced supply for world markets, even as the human population increases. Aquaculture, or fish farming, has been around for centuries, but its role in society has significantly increased in the past 50 years. It currently provides about half of fish and other aquatic products on the market today. To better maintain and increase the sustainability and profitability of this industry, more focus is being placed on the health of the fish. The microbiome is the collection of communities of microorganisms, including bacteria, fungi, and archaea, that inhabit various environments including animal hosts. The majority of this dissertation focuses on the impact of factors like diet and age on the microbiomes of aquaculture-raised animals, especially fish. Dietary changes such as the addition of dried yeast-products had a significant impact on fish health but not on the microbiome communities. However, a common probiotic, Bacillus subtilis, did significantly increase not only the growth rate of trout but it also significantly altered the total intestinal microbiome found in the feces and the intestinal mucosal layer. Moreover, it was found that early exposure of the animals to the probiotic had enhanced benefits even though the microbiome appeared to stabilize over time as the fish developed. Maintaining or improving the microbiomes of fish, paying close attention to the microbes that exist as part of a core group of bacteria always present, is vital to ensuring fish health and understanding vertebrate host-microbe relationships. Thus, an analysis of the core microbiome of trout was performed. The final set of projects within this dissertation focused on the relationship between the Eastern oyster, a mollusk native to the Chesapeake Bay, and the bacterial human pathogen Vibrio parahaemolyticus (VP). VP is the leading cause of seafood-borne acute gastroenteritis worldwide, and efforts are needed to mitigate the increasing rate of human infections. Therefore, a simple system using fermentation jars within the laboratory biosafety cabinet was designed to enable safe culture of oysters that were exposed to VP under experimentally controlled conditions. Oysters harvested during the summer naturally harbored higher amounts of native Vibrio organisms in contrast to the winter oysters that harbored much lower levels. A separate microbiome analysis revealed large shifts in the oyster microbiome between summer and winter, although some microbes were continually present. The lower levels of existing Vibrio species detected in winter oysters may have allowed for the higher efficiency of inoculation of winter animals by VP. In fact, these winter animals had Vibrio microbiomes that were completely dominated by the inoculated strain which will enable future work to observe the pattern by which VP localizes, or colonizes, the oysters. Ultimately, these efforts may lead to the development of future disease mitigation strategies against VP.

Native oysters have been promoted as a means to improve water quality in Chesapeake Bay. This project added important insights into the potential of oyster aquaculture to process and remove nutrients from Bay waters. Results clarified that nutrient removal of nitrogen (N), phosphorous (P), and carbon (C) through harvest of cultivated oyster biomass can be quantified and modeled with high levels of statistical confidence. A simple, yet accurate, method is now available for estimating the amount of nutrients removed via harvesting aquacultured oysters. Based on model estimates, 106 harvest sized oysters (76 mm TL) remove 132 kg TN, 19 kg TP, and 3,823 kg TC. Previous work suggested that potentially substantial quantities of N may be removed through enhancement of the coupled nitrification-denitrification pathway in sediments as a result of oyster biodeposition. Using 15N and N2/Ar methods to measure N2 production in sediments, encompassing direct denitrification (DNF), coupled nitrification- denitrification, and anaerobic ammonium oxidation (anammox) pathways, at two oyster aquaculture sites and two reference sites (no aquaculture), we found that oyster biodeposition did not accelerate sediment N removal. We estimate sediment N removal rates via N2 production at an oyster cultivation site producing 5 x 105 oysters (1750 m2) to range from 0.49-12.60 kg N yr-1, compared to 2.27-16.72 kg N yr-1 at a reference site of the same area; making the contribution of oyster cultivation to N removal via sediment N2 production inconsequential as a policy initiative for Chesapeake Bay eutrophication mitigation. Molecular approaches and direct abundance measures have improved our understanding of the sediment microbial community response to oyster biodeposition. Overall, sediments impacted by oyster biodeposition had a significantly different denitrifying community composition than sediments a few meters away or at the non-aquaculture reference sites. Bacterial abundance in sediments was determined by site rather than by oyster biodeposition. No apparent effects of oyster biodeposition were evident in nitrifying bacterial abundance patterns at either site, indicating that oyster biodeposition does not enhance coupled nitrification-denitrification by increasing the abundance of nitrifiers in sediments.

Widely regarded as a keystone species and ecosystem engineer, the eastern oyster Crassostrea virginica plays a vital role in estuarine environments. Complex, three-dimensional oyster reefs act as havens for biodiversity and contribute to ecological processes. Recently, concern for this resource has arisen in Mosquito Lagoon, Florida, the southernmost limit along the Atlantic coast for undisturbed, intertidal reefs of C. virginica. Since the 1990s, intense recreational boating activity has caused atypical dead margins (mounds of disarticulated shells) to emerge on the seaward edges of oyster reefs located along major navigational channels. Once dead margins are formed, little is known about their influence on biotic composition and interactions on oyster reefs. This study focused on the affect of dead margins on: (1) mobile species biodiversity and distribution, (2) reef architecture, and (3) the affect of structural variables on predation of juvenile oysters. To determine if dead margins influenced the biodiversity of mobile species on oyster reefs, lift nets (1 m2) were deployed within Mosquito Lagoon for one year (June 2004 - June 2005). These nets (5/site) were deployed on the back-reef areas of six reefs (3 reference reefs and 3 reefs containing dead margins). To simulate reef habitat, one and a half liters of live oysters were placed within each net. Lift nets were checked monthly and surveyed for all mobile species. The resulting data were assimilated into a species inventory containing 65 species of fishes, mollusks, crustaceans, worms, and echinoderms. The two most abundant species present on reefs in Mosquito Lagoon were the big-claw snapping shrimp Alpheus heterochaelis, a filter-feeder, and the flat-back mud crab Eurypanopeus herbstii, a predator of oyster spat. Contrary to expections, analyses of community metrics showed that dead margins did not significantly affect the biodiversity of back-reef areas on oyster reefs. Modified lift nets (0.25 m2) were placed on six different oyster reefs (3 reference reefs and 3 containing dead margins) to test if dead margins affected the distribution of mobile species inhabiting oyster reefs. Nine nets were arranged to cover three separate areas of each reef: the fore-reef (3 nets), mid-reef (3 nets), and back-reef (3 nets). Half a liter of oyster shells were placed inside each net. These nets were checked weekly, for five weeks and species richness, density, and biomass were recorded. Analyses revealed that all community metrics were significantly higher on reference reefs than reefs affected with dead margins. Further, a significant drop in all three metrics was seen on the mid-reef area of affected reefs. The absence of species on this area is hypothesized to be due to a lack of water, shade, and habitat complexity. To document architectural differences, two types of transects were run along five reference reefs and five reefs with dead margins. First, quadrat transects determined the percent of live oysters, the percent of shell clusters, topographic complexity (using chain links), and the angle of shells on each reef type. Transect lines were stretched parallel to the water line and covered all three reef areas (fore-reef, mid-reef, and back-reef). The results showed reference reefs to have approximately four-fold more live oysters, approximately twice as many shell clusters, and significantly greater topographic complexity. Numbers of live oysters and shell clusters were greater on the fore-reef and back-reef areas of both reef types. Second, laser transects were used to record reef profiles and the slope of fore-reef areas. Transect lines were stretched perpendicular to the water line and every 20 cm the distance between the lagoon bottom and reef top was measured. Vertical reef profiles and fore-reef slopes were significantly different between reference reefs and reefs with dead margins. Dead margins compressed reef widths, increased center peaks, and increased slopes on the fore-reef area by two-fold. Lastly, field experiments were conducted to determine the affect of dead margins on the vulnerability of oyster spat to predation. Structural variables (e.g. shell orientation, single versus shell clusters, reef slope) were manipulated and effects on oyster mortality were observed. Three predators were tested: the blue crab Callinectes sapidus, the common mud crab Panopeus herbstii, and the Atlantic oyster drill Urosalpinx cinerea. Structural variables did not have a significant influence on oyster mortality; however, a significant difference was established between predators. Panopeus herbstii consumed the most juvenile oysters, followed by U. cinerea and then C. sapidus. Together, these findings document ecological implications of dead margins on C. virginica reefs and reinforce the urgent need for enhanced regulations and restoration. If the intensity of recreational boating remains unregulated, dead margins will continue to increase. Thus, in order to maintain the diversity and productivity of Mosquito Lagoon, it is crucial to fully understand how dead margins alter the biogenic habitat and biotic communities of oyster reefs.

A demographic population simulation model was developed to examine alternative fishery management strategies and their likely effects on the probability of extirpation of local eastern oyster (Crassostrea virginica) populations in the Chesapeake Bay. Management strategies include varying the minimum shell length-at-harvest, harvest rate, and rate and frequency of stocking of oyster seed with respect to varying salinities and oyster population densities. We also examined the rate of disease-mediated mortality that can be tolerated by a viable population. High density populations at low salinity sites remained viable under a 100% harvest rate and 76.6 minimum shell length-at-harvest due to increased fertilization efficiency in high densities, which increased reproduction. Low density populations at low salinity sites remained viable when harvest rate was set at 0.5 and minimum shell length-at-harvest was set at 85 mm. Neither reducing harvest rate nor minimum shell length-at-harvest produced a viable population at high salinity sites. The effects of disease-mediated mortality were too great for these management options to decrease the probability of extirpation to zero. Supplemental stocking conducted regularly reduced extirpation probabilities to zero and pulse stocking (every five to ten years) did as well, although it required a much larger number of oysters to be stocked. Decreasing disease-mediated mortality rates by 20% in high density populations and by 80% in low density populations reduced the probability of extirpation to zero, suggesting the degree of genetic improvement needed to rebuild eastern oyster populations in the Chesapeake Bay. Culture of a non-native species, such as the Suminoe oyster (Crassostrea ariakensis), could supplement harvest of the declining eastern oyster fishery in Chesapeake Bay. Because of possible ecological impacts from introducing a fertile non-native species, introduction of sterile triploid oysters has been proposed. However, recent data show that a small percentage of triploid individuals progressively revert toward diploidy, introducing the possibility Suminoe oyster might establish self-sustaining populations. To assess the risk of Suminoe oyster populations becoming established in Chesapeake Bay, a demographic population model was developed. Inputs modeled included: salinity, stocking density, reversion rate, reproductive potential, natural and harvest mortality, growth rates, and effects of various management strategies, including harvest criteria. Results showed decreased probability of a Suminoe oyster population becoming self-sustaining when oysters are grown at low salinity sites, certainty of harvest is high, minimum shell length-at-harvest is small, and stocking density is low. Results of the model suggest management strategies that will decrease the probability of a Suminoe oyster population becoming self-sustaining. Policy makers and fishery managers can use the model to predict potential outcomes of policy decisions, supporting the ability to make science-based policy decisions about the proposed introduction of triploid Suminoe oysters into the Chesapeake Bay. / Master of Science

<p>Curiosity is a powerful tool, and combined with the ability to observe the natural world, grants humankind an unique opportunity, the opportunity to wonder why. Why do things exist?, why do they do the things they do?, why is this even possible?</p> <p>Research in our lab is focused on the basic understanding and potential application of biological materials, in particular, biological adhesives produced by marine organisms such as oysters. Oysters produce a cement-like material that is able to withstand the dynamic conditions found in coastal environments. The focus of this dissertation is to lay the basis of the characterization of new biological materials by observing and analyzing its physical properties, to measure the performance of the material in natural conditions and finally to identify the basic components that give the material the properties that we observe. The end goal of this project is to understand the properties of this material so we are able to develop a synthetic system that is able to imitate, as close as possible, what we find in nature. These results, and more importantly, the new questions that emerge from this research, provide a first look at the adhesive system of oysters leading the way to new discoveries in the future.</p>

With the majority of Earth’s population living in coastal areas, estuarine ecosystems have been particularly affected by anthropogenic disturbances. My dissertation research focused on three interrelated types of human disturbance that affect estuaries: Anthropogenic alteration of freshwater inflow, the introduction of invasive species, and habitat alteration. Using the LoxahatcheeRiver(Jupiter, FL) as a model system, my goal was to understand how these disturbances affect estuarine organisms, particularly fishes. One of the most ecologically harmful disturbances affecting estuaries is anthropogenic alteration of freshwater inflow (and resulting changes in salinity patterns). To identify effects of freshwater inflow on the behavior of an ecologically and economically important fish (common snook Centropomus undecimalis), I conducted a 19-month acoustic telemetry study. Common snook were more abundant and made more frequent upstream migrations during the wet season, but freshwater inflow did not appear to be the proximate cause for these behaviors. Increased estuarine salinity resulting from anthropogenic flow alteration may have facilitated the second type of disturbance that I address in this dissertation; the invasion of non-native Indo-Pacific lionfish into estuarine habitats. During the course of my dissertation research, I documented the first ever estuarine invasion by non-native lionfish. Using mark-recapture, I identified high site fidelity in lionfish, a trait that may aid future control efforts. The extremely low minimum salinity tolerance that I identified in lionfish appears to have allowed the species to colonize far upriver in estuaries with anthropogenically modified salinity patterns. Anthropogenic salinity alteration has also led to a severe degradation of oyster reef habitats in theLoxahatcheeRiver. As a foundation species, oysters provide food, shelter, and nursery habitat for a wide variety of estuarine organisms, including many ecologically and economically important fishes. Increasingly, degraded oyster reef habitats have been the focus of restoration efforts. I identified a relatively rapid (< 2 years) convergence between restored and natural oyster reef communities, and documented the importance of vertical relief in restoration success. My dissertation research is critical for the management and conservation of coastal rivers inFlorida, while more broadly informing restoration and management decisions in many other estuarine and coastal ecosystems.

Crassostrea virginica, the eastern oyster, is a native keystone species that inhabits many coastal and estuarine ecosystems along the Atlantic seaboard. Introduction of the eastern oyster into estuarine areas with limited current populations is gaining popularity as a pro-active approach to improve estuarine water quality. In November 2014 and April 2015, a total of five pilot oyster reef treatments were deployed in Brevard County: bagged adult oysters (grown by community members under their docks through oyster gardening) collected in fall 2014 and spring 2015, bagged clean shell, oyster restoration mats, and empty plots (control). Locations of deployment included a Merritt Island impoundment (Marsh Harbor), Nicol Park (Port St. John), and Scout Island (Melbourne Beach). Prior to deployment, we collected morphometric data (shell length, weight) on all gardened oysters. Abiotic factors including salinity, air and water temperature, and wind speed were collected monthly. During quarterly sampling at each site, morphometric data were collected for all live oysters, surviving and newly recruited. Results show survival of gardened oysters and natural recruitment differed by and depended greatly on the within-site location of each reef. In areas with no recruitment and limited gardened oyster survival, regular deployment of gardened oysters is needed for long term success. In areas with natural recruitment, bagged, clean shell or oyster restoration mats are most successful. Future restoration sites should be tested prior to any large-scale oyster deployments.