Aswani K. Volety

THE ROLE OF OYSTERS IN HABITAT USE OF OYSTER REEFS BY RESIDENT FISHES AND DECAPOD CRUSTACEANS

Abstract To assess the role of live oysters in providing habitat, community metrics of resident fishes and decapod crustaceans were compared among 3 habitat treatments: live oyster clusters; cleaned, articulated shell and sand bottom. Sampling was conducted during three seasonally wet and three seasonally dry months using 1-m2 lift nets deployed on an intertidal oyster reef in the Caloosahatchee estuary, Florida. Metrics used to assess relative habitat value included organism density, biomass and species richness. Species-specific comparisons were also made. Results indicate that organism density, biomass and richness were all greater for treatments with shell (live oyster clusters or cleaned, articulated shell) compared with the sand-bottom (no-shell) treatment. Two patterns emerged from species-specific comparisons: (1) species found in live and articulated shell (e.g., flatback mud crab, green porcelain crab) might require shelter; and (2) species found in association with articulated, cleaned shell (i.e., frillfin goby) might use empty oyster boxes for spawning substrate. There was little evidence to suggest that any of the decapods or fishes present were specifically selecting habitat with living oysters present.

Suppression of chemiluminescence of eastern oyster (Crassostrea virginica) hemocytes by the protozoan parasite Perkinsus marinus

Experiments were conducted to determine the ability of the protistan parasite, Perkinsus marinus, to inhibit chemiluminescence of hemocytes from the eastern oyster, Crassostrea virginica. Luminol-enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen intermediates (ROI) generated by oyster hemocytes using zymosan as a stimulant. To determine whether P. marinus suppresses ROI evoked from zymosan-stimulated hemocytes, live or heat killed P. marinus in filtered estuarine water (YRW) (salinity = 20 ppt) were added to (1) zymosan-stimulated hemocytes after CL reached its peak, or (2) hemocytes at the same time as zymosan, and reduction of CL responses were recorded. In both tests, controls received only estuarine water. Live P. marinus meronts significantly suppressed ROI production by zymosan-stimulated hemocytes. The suppression of ROI production was dose dependent. Suppression of ROI production from zymosan-stimulated hemocytes by heat killed P. marinus was significantly less than by live P. marinus. Similarly, CL of hemocytes was reduced, though not significantly when hemocytes were exposed to YRW preincubated with P. marinus. When P. marinus meronts were used as a stimulant, no CL response was elicited. Results of this study suggest that P. marinus cells are able to suppress ROI release from oyster hemocytes, thus evading this component of the hosts defense.

Host-parasite interactions: Marine bivalve molluscs and protozoan parasites, Perkinsus species.

This review assesses and examines the work conducted to date concerning host and parasite interactions between marine bivalve molluscs and protozoan parasites, belonging to Perkinsus species. The review focuses on two well-studied host-parasite interaction models: the two clam species, Ruditapes philippinarum and R. decussatus, and the parasite Perkinsus olseni, and the eastern oyster, Crassostrea virginica, and the parasite Perkinsus marinus. Cellular and humoral defense responses of the host in combating parasitic infection, the mechanisms (e.g., antioxidant enzymes, extracellular products) employed by the parasite in evading host defenses as well as the role of environmental factors in modulating the host-parasite interactions are described.

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field — contaminated sediments

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.

Reactive Oxygen Species in Unstimulated Hemocytes of the Pacific Oyster Crassostrea gigas: A Mitochondrial Involvement

The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster’s hemocytes have been reported to produce reactive oxygen species (ROS), even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψm) of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψm in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor) had no effect on ROS production. Incubation with antimycin A (complex III inhibitor) resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψm and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

A HABITAT SUITABILITY INDEX MODEL FOR THE EASTERN OYSTER (CRASSOSTREA VIRGINICA), A TOOL FOR RESTORATION OF THE CALOOSAHATCHEE ESTUARY, FLORIDA

Abstract A tool in the form of a habitat suitability index model (HSI) for the eastern oyster, Crassostrea virginica, was adapted to evaluate and compare the effects of alternative restoration plans in southwest Florida. A component of a large forecasting model, this tool simulates system response by examining the impact of freshwater inputs into the system. The eastern oyster is a good indicator species for modeling because of its sedentary nature and its susceptibility to natural and artificial changes. In addition, oysters form a complex three-dimensional reef structure, which provides habitat and food for numerous species of fish and invertebrates. The model focuses on salinity, temperature, depth, substrate, and high flow frequency as the particular requirements to determine habitat suitability for the eastern oyster. A geographic information system (GIS) incorporates the oyster HSI model, which includes larval and adult components, to determine responses spatially and temporally to facilitate the decision making process. This paper evaluates four hydrologic and land use scenarios for the C-43 West Basin Reservoir Project. Model results indicate that the Preferred Flow scenario and the future conditions with the Comprehensive Everglades Restoration Plan have higher HSI values then either the existing conditions or the future without the Comprehensive Everglades Plan.

Effects of salinity, heavy metals and pesticides on health and physiology of oysters in the Caloosahatchee Estuary, Florida

The Caloosahatchee Estuary has been exhibiting signs of impaired ecological health due to the extensive hydrological alteration, agricultural land use, and increasing watershed development. This project investigated the responses of the American oyster, Crassostrea virginica at five locations in the Caloosahatchee River in relation to salinity changes, levels of heavy metals, pesticides, and PCBs in the water as well as in the oyster tissue. Individual heavy metal and organochlorine pesticide concentrations in oysters varied significantly between sampling locations and sampling months. PCB concentrations in oyster tissues were below detection limits as were the metal, pesticide and PCB concentrations in water at all the sampling locations. Both heavy metal and pesticide concentrations decreased with increasing distance downstream indicating upstream source of contaminants. The highly pathogenic oyster parasite Perkinsus marinus infection intensity (level) and prevalence (% infected oysters), condition index, spat recruitment, and gonadal index showed a seasonal trend varying with spawning activity and increased downstream. However, juvenile oyster growth was higher at upstream estuarine locations. Oyster responses varied more with seasonal programming (salinity), rather than due to contaminant levels. While significant correlations were noted between some oyster responses and metal concentrations in oyster tissues, overall metal concentrations were low compared to national averages. It appears that oyster health in the Caloosahatchee River is influenced more by freshwater inflow and resulting salinity fluctuations, rather than due to the measured contaminants.

Functional and metabolic characterization of hemocytes of the green mussel, Perna viridis: in vitro impacts of temperature

The green mussel, Perna viridis, is a bivalve mollusk native to Asia and was recently introduced to Florida, USA. Since its first observation in 1999 in Tampa Bay, Florida, green mussel population has expanded considerably, to reach the Atlantic coast of Florida, Georgia and South Carolina. Most of currently available studies about the ecology and biology of green mussels were performed in the Indian and Pacific oceans. Very recently, it has been suggested that due to a weak low temperature resistance, green mussels might have already reached the Northern edge of their distribution in the USA. However, there is currently an obvious lack of data about the adaptation capacities of Perna viridis to environmental conditions in Florida, especially at the physiological and cellular levels. In the present work, we determined and characterized the populations of circulating hemocytes, and the cellular components of hemolymph involved in various physiological functions, including immunity. Two main populations were characterized, hyalinocytes and granulocytes. Granulocytes accounted for 60% of circulating cells, and displayed higher phagocytic capacities, lysosomal content and basal oxidative metabolism than hyalinocytes. Hemocyte parameters were not influenced by the size of green mussels. In addition, hemocytes were subjected to acute temperature challenges (10, 20 and 30 °C) and their immune-related functions and metabolism analyzed. Our results showed that 10 °C represent a stressful condition for the Floridian green mussels, as depicted by a low phagocytosis capacity and an increase of oxidative metabolism.

INFLUENCE OF SALINITY ON THE HABITAT USE OF OYSTER REEFS IN THREE SOUTHWEST FLORIDA ESTUARIES

Abstract To gauge the influence of salinity on the habitat value of oyster reefs, spatial and seasonal patterns of the presence of reef-resident fishes and decapods were assessed in the Caloosahatchee, Estero, and Faka-Union estuaries of Southwest Florida. Lift nets (1 m2) containing 5 L of oyster clusters were deployed on intertidal reefs at three sites along the salinity gradient of each estuary. Nets were deployed during three seasonally dry and three seasonally wet months for a period of 30 d. Oyster densities were estimated at each site and a number of community metrics were calculated as a measure of habitat use (e.g., organism density, biomass, diversity, dominance, richness). Several metrics increased downstream in one or more systems (e.g., organism density, biomass, diversity) and in general appeared to be more related to salinity than to the density of living oysters present. Although organism density was higher during the wet season for all three systems, biomass was higher during the dry season in the Caloosahatchee. In the Caloosahatchee and the Estero, measures of biodiversity tended to be higher during the dry season. These results suggest that the salinity requirements of the organisms that inhabit oyster reefs should be considered in the planning of oyster-reef restoration or enhancement projects or in the management or alteration of freshwater inflow into estuaries.

Greater hemocyte bactericidal activity in oysters (Crassostrea virginica) from a relatively contaminated site in Pensacola Bay, Florida

Bivalve mollusks such as Crassostrea virginica inhabiting polluted estuaries and coastal areas may bioaccumulate high concentrations of contaminants without apparent ill effects. However, changes in putative internal defense activities have been associated with contaminant accumulation in both experimental and long-term field exposures. In an effort to elucidate these relationships, 40 oysters were collected from Bayou Chico (BC) and East Bay (EB) in Pensacola Bay, FL, two estuaries known to differ in the type and magnitude of chemical contaminants present. Oyster tissue concentrations of metals, tri- and dibutyltin (TBT, DBT), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) were measured in individual oysters, as were hemocyte counts (HCs), hemocyte bacterial killing indices (KI), serum lysozyme (LYS) and serum protein (PRO) levels. Average HC, KI, LYS and PRO were significantly higher in BC oysters, which also had significantly higher tissue concentrations of total trace metals, butyltins (BTs), PAHs, PCBs, pesticides, and Mn, Cu, Zn and Sn. EB oysters had low organic contaminant levels and no detectable BTs, but significantly higher concentrations of Al, Cr, Fe, Ag, Cd, and Hg. Simple correlation analysis between specific defense measurements and specific chemical analytes showed specific positive relationships that corroborated previous findings in other FL estuaries. Canonical correlation analysis was used to examine relationships between defense measurements and tissue metals using linearly combined sets of variables. Results were also consistent with previous findings-the highest possible canonical correlation was positive: r=0.864, P<0.0019 among canonical variables composed of HC, KI and LYS for defense, and Fe, Cu, Ag, Cd, Sb, Sn, Ni, Pb and Hg for metals.