Linking optimization and ecological models in a decision support tool for oyster restoration and management.

Restoration of ecologically important marine species and habitats is restricted by funding constraints and hindered by lack of information about trade-offs among restoration goals and the effectiveness of alternative restoration strategies. Because ecosystems provide diverse human and ecological benefits, achieving one restoration benefit may take place at the expense of other benefits. This poses challenges when attempting to allocate limited resources to optimally achieve multiple benefits, and when defining measures of restoration success. We present a restoration decision-support tool that links ecosystem prediction and human use in a flexible "optimization" framework that clarifies important restoration trade-offs, makes location-specific recommendations, predicts benefits, and quantifies the associated costs (in the form of lost opportunities). The tool is illustrated by examining restoration options related to the eastern oyster, Crassostrea virginica, which supported an historically important fishery in Chesapeake Bay and provides a range of ecosystem services such as removing seston, enhancing water clarity, and creating benthic habitat. We use an optimization approach to identify the locations where oyster restoration efforts are most likely to maximize one or more benefits such as reduction in seston, increase in light penetration, spawning stock enhancement, and harvest, subject to funding constraints and other limitations. This proof-of-concept Oyster Restoration Optimization model (ORO) incorporates predictions from three-dimensional water quality (nutrients-phytoplankton zooplankton-detritus [NPZD] with oyster filtration) and larval transport models; calculates size- and salinity-dependent growth, mortality, and fecundity of oysters; and includes economic costs of restoration efforts. Model results indicate that restoration of oysters in different regions of the Chesapeake Bay would maximize different suites of benefits due to interactions between the physical characteristics of a system and nonlinear biological processes. For example, restoration locations that maximize harvest are not the same as those that would maximize spawning stock enhancement. Although preliminary, the ORO model demonstrates that our understanding of circulation patterns, single-species population dynamics and their interactions with the ecosystem can be integrated into one quantitative framework that optimizes spending allocations and provides explicit advice along with testable predictions. The ORO model has strengths and constraints as a tool to support restoration efforts and ecosystem approaches to fisheries management.

Real-time PCR investigation of parasite ecology: in situ determination of oyster parasite Perkinsus marinus transmission dynamics in lower Chesapeake Bay.

Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica on the East Coast of the United States. Transmission dynamics of this parasite were investigated in situ for 2 consecutive years (May through October) at 2 lower Chesapeake Bay sites. Compared to previous studies where seasonal infection patterns in oysters were measured, this study also provided parasite water column abundance data measured using real-time PCR. As previously observed, salinity and temperature modulated parasite transmission dynamics. Using regression analysis, parasite prevalence, oyster mortalities and parasite water column abundance were significantly positively related to salinity. Perkinsus marinus weighted prevalence in wild oysters and parasite water column abundance both were significantly related to temperature, but the responses lagged 1 month behind temperature. Parasite water column abundance was the highest during August (up to 1,200 cells/l) and was significantly related to P. marinus weighted prevalence in wild oysters, and to wild oyster mortality suggesting that parasites are released in the environment via both moribund and live hosts (i.e. through feces). Incidence was not significantly related to parasite water column abundance, which seems to indicate the absence of a linear relationship or that infection acquisition is controlled by a more complex set of parameters.

Matrix metalloproteinase-like activity from hemocytes of the eastern oyster, Crassostrea virginica.

Investigation of oyster blood cell lysate revealed one prominent band of proteolytic activity when analyzed using gelatin and collagen impregnated polyacrylamide gel electrophoresis. The proteolytic activity was inhibited by 1,10 phenanthroline and EDTA, but not by other proteinase inhibitors. Maximal activity was shown at pH 8.2 and the molecular weight of the protein responsible for the activity was estimated to be 68 kDa. Proteolytic activity was also measured by fluorescence assays containing hemocyte lysate and fluorescein-labeled gelatin, type I or type IV collagen. Characteristics of this proteolytic activity suggest that an invertebrate matrix metalloproteinase is responsible.

Acidification of the phagosome in Crassostrea virginica hemocytes following engulfment of zymosan.

Phagocytic hemocytes are responsible for engulfing and internally degrading foreign organisms within the hemolymph and tissue of the eastern oyster, Crassostrea virginica. Since rapid acidification of the phagosome lumen is typically essential for activation of hydrolytic and reactive oxygen intermediate (ROI) producing enzymes in vertebrate cells, we measured phagosomal pH in oyster hemocytes by using the emission fluorescence of two fluorescent probes, rhodamine and Oregon Green 488 (OG 488), conjugated to zymosan to determine whether oyster hemocyte phagosomes become acidified after phagocytosis of zymosan. The average pH of 1079 phagosomes within 277 hemocytes 1 h after phagocytosis of zymosan was 3.9 +/- 0.03. Observations of 141 hemocytes with internalized zymosan by light microscopy revealed that, over a 60-min time period, 51% of highly granular hemocytes became partially granular, and 29% became agranular. In addition, 83% of partially granular hemocytes containing zymosan at time = 0 became agranular within 60 min. A comparison revealed that the phagosomes of agranular hemocytes were much more acidic (pH 3.1 +/- 0.02) than those of highly granular hemocytes (4.9 +/- 0.02; P < 0.05). These values are significantly lower than most reported in the literature for blood cells from metazoan organisms.

Acute osmotic tolerance of cultured cells of the oyster pathogen Perkinsus marinus (Apicomplexa:Perkinsida).

Cultured Perkinsus marinus cells were exposed for 24 hr to salinities of 0, 3, 6, 9, 12 and 22 ppt at temperatures of 1, 5, 10, 15 and 28 degrees C in artificial seawater (ASW) and to the same salinities at 28 degrees C in ASW with the osmotic concentration adjusted with sucrose to the equivalent of 22 ppt. At 28 degrees C mortality increased as salinity decreased below 22 ppt. Mortality was greater than 99% at 0 ppt and greater than 90% at 3 ppt. Mortality was 70% at 6 ppt, 43% at 9 ppt and 20% at 12 ppt. Mortality was low (< 5%) and equal to that at 22 ppt in all treatments where osmotic concentration was maintained with sucrose. Mortality occurred rapidly, within 5 min of exposure to experimental conditions. In the region where mortality was most sensitive to salinity changes (6-12 ppt), lower temperature caused an increase in mortality, but the temperature effect was significant only at 9 ppt.

Prebedtime activity and sleep satisfaction of short and long sleepers.

Significant correlations between prebedtime activities and sleep satisfaction varied with sleep length in 115 college students: 15 short sleepers who listened to music were less satisfied with sleep, while 18 long sleepers who snacked on milk and sweets were more satisfied with sleep. Longer sleepers may substitute alcohol for food as they grow older.

Increased Reactive Oxygen Intermediate Production by Hemocytes Withdrawn from Crassostrea virginica Infected with Perkinsus marinus.

Perkinsus marinus is a protozoan parasite responsible for a major infectious disease of the Eastern oyster, Crassostrea virginica. Nonspecific immunity was assayed in oysters with known intensities of infection so that the physiological responses of the host elicited by the parasite could be better understood. This report describes the capacity of hemocytes to generate reactive oxygen intermediates during the progression of the disease. The hemocytes constitute the major internal defense effector system of oysters, and cytotoxic oxygen species are thought to play central roles in antimicrobial activities of hemocytes and other phagocytic cells. Production of oxyradicals by both resting and phagocytically stimulated hemocytes was quantified by luminol-augmented chemiluminescence. Hemocytes from oysters with heavy Perkinsus infections produced significantly higher levels of chemiluminescence than their counterparts withdrawn from lightly or moderately infected individuals. Furthermore, in addition to a higher chemiluminescent activity per cell, the total circulating hemocyte count was elevated in the heavily infected animals. Therefore, advanced cases of this disease seem to be characterized by hemocyte activation and recruitment, with concomitant exuberant production of hemocyte-derived reactive oxygen intermediates. The resultant oxidant load may participate in the pathogenesis of the disease.

Evidence of lactate dehydrogenase-B allozyme effects in the teleost, Fundulus heteroclitus.

The evolutionary significance of protein polymorphisms has long been debated. Exponents of the balanced theory advocate that selection operates to maintain polymorphisms, whereas the neoclassical school argues that most genetic variation is neutral. Some studies have suggested that protein polymorphisms are not neutral, but their significance has been questioned because one cannot eliminate the possibility that linked loci were responsible for the observed differences. Evidence is presented that an enzymatic phenotype can affect carbon flow through a metabolic pathway. Glucose flux differences between lactate dehydrogenase-B phenotypes of Fundulus heteroclitus were reversed by substituting the Ldh-B gene product of one homozygous genotype with that of another.

Peptidergic transmission in the brain. VI. Behavioral consequences of central activation.

Having described a peptidergic transmitter system in the rat brain, we now begin to evaluate its behavioral function. We stimulated cell bodies in the medial amygdaloid nucleus (AME) with indwelling bilateral electrodes. These cell bodies contain a vasopressin-like peptide and send fibers to the hippocampus where the peptide is released upon stimulation. There the peptide inhibits hippocampal output in the awake rat just as it does in the anesthetized rat and in the rat brain slice. The stimulation reorganizes behavior with the same latency and duration as the hippocampal effect. For about 15-20 minutes after the brief stimulus, rats remain motionless with eyes wide open. This "freezing" state is punctuated by episodes of exploratory behavior. The stimulus appears to have a positive affective quality. Review of the literature in light of the present results indicates a probable role for this peptidergic system in the generation of sexual behavior in male rats.

Peptidergic transmission in the brain. IV. Sex hormone dependence in the vasopressin/oxytocin system.

The medial amygdaloid nucleus (AME) synthesizes a peptide similar to arginine vasopressin and projects peptidergic fibers to the ipsilateral hippocampus in the male rat. In previous studies, we have shown that the peptide acts as a transmitter and, using in vitro and in vivo electrophysiology, we have characterized its mechanism of action. Previous anatomical work has shown that the peptidergic fibers are more dense in male rats than females and are obliterated entirely by castration. Here we report the results of our attempt to find an electrophysiological correlate of these anatomical findings. First, we show specific mediation by a vasopressin- or oxytocin-like peptide by use of a structural vasotocin antagonist. Then we show that castration obliterates the peptidergic signal in males. However, we were unable to find any sex difference that corresponded to the male/female disparity noted in the density of the peptidergic fibers. The strength, nature and stimulus-response characteristics were the same between males and females. Apart from a very subtle difference in the duration of the signal, no physiological correlate of the sexual dimorphism could be found with our techniques. We conclude that the neurophysiology partly complements the anatomy and biochemistry of this system.

Biological Activity of Biosynthetic Rainbow Trout Growth Hormone in the Eastern Oyster, Crassostrea virginica.

Juvenile oysters were exposed to seawater containing 10-9, 10-8, and 10-7 M biosynthetic rainbow trout growth hormone (rtGH); the treatment was applied for one five-hour period per week for five weeks. At the end of the five weeks, the animals treated with the two highest concentrations of hormone were significantly longer and had dry tissue weights 50% greater than did the lowest treatment group or the control group. Continuing in vitro experiments on isolated oyster tissue showed that the hormone treatment significantly increased oxygen consumption. Boiled hormone had no effect. In both sets of experiments (whole animals and isolated tissues), the % dry wt (dry wt/wet wt) was significantly higher in all animals and tissues that responded to rtGH. The results demonstrate that rtGH has biological activity in oyster tissues, and this activity may be directly associated with growth regulation in the whole animal. The results further show that bivalve growth is not directly limited by environmental parameters.

Serum hormone levels associated with spawning activity in the mummichog, Fundulus heteroclitus.

Daily collections of the mummichog, Fundulus heteroclitus, from field populations during the first 45 days of the breeding season revealed a semilunar cycle in the sperm index. Peaks in the sperm index were preceded by 6 days with peaks in the serum testosterone concentration. Bihourly sampling of field populations during a 72-hr period at the new moon showed both diel cycling and an upward trend in serum progesterone levels in female mummichogs. Male mummichogs had 12-hr cycles in serum 17 beta-hydroxy-4-androstene-3,11-dione levels, the peaks of which preceded high tide by 4 hr. The physiological significance of these cycles are discussed.

Pyruvate dehydrogenase complex from ribbed mussel gill mitochondria.

The pyruvate dehydrogenase complex has been demonstrated in high speed pellet preparations from sonicated ribbed mussel gill mitochondria. The activity of the complex is inhibited by low chloride (less than 100 mM) concentrations, EDTA (1 mM), succinate, ATP, and NAD/NADH ratios below 4. Inhibition by EDTA is relieved by addition of 10 mM MgCl2-1 mM CaCl2. ATP inhibition was enhanced by NaF and reversed by high Mg++ concentrations in the absence of NaF. Pyruvate and thiamine pyrophosphate inhibited the inactivation by ATP. The nonhydrolyzable ATP analog AMP-PNP caused inhibition of the overall catalytic activity that was identical to ATP. Factors involved in the ATP inhibition and Mg++ reversal are lost with freezing or cold storage. Preliminary results using gamma-32P-ATP indicate that a protein kinase that phosphorylates the alpha subunit of E1 (pyruvate dehydrogenase) from the mammalian PDC is associated with the gill PDC. The activity of the complex may be regulated by a phosphorylation/dephosphorylation mechanism and by the relative levels of substrates, products, and other metabolites in the mitochondria.

Amino acid metabolism in euryhaline bivalves: regulation of glycine accumulation in ribbed mussel gills.

Glycine levels in isolated ribbed mussel (Modiolus demissus) gill tissue increased slightly and decreased markedly when incubated at high and low salinities, respectively. Low levels of the enzymes involved in the biosynthesis of serine from triose phosphate intermediates, the serine hydroxymethyltransferase, and serine dehydrase were detected in gill tissue homogenates. Experiments using gill tissue incubated with (U-14C)-glycine and (U-14C)-serine indicated interconversion between serine and glycine and transfer of label to alanine, asparate, glutamate, CO2, organic acids, and protein. Glyoxylate was metabolized more slowly than glycine and was probably converted to glycine for catabolism. Studies using (1-14C)-glycine and (2-14C)-glycine with isolated gill tissue and mitochondria indicated that the mitochondrial glycine cleavage enzyme was the major route of glycine catabolism. Metabolic controls activating or inhibiting the glycine cleavage enzyme regulate tissue glycine accumulation and catabolism during hypersalinity or hyposalinity stress.

Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves.

Aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), malic enzyme (ME), malate dehydrogenase (MDH), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (PEPCK) activities in cytosolic and mitochondrial fractions of gill tissue from Modiolus demissus (ribbed mussel), Mytilus edulis (sea mussel), Crassostrea virginica (oyster) and Mercenaria mercenaria (quahog) were determined using enzyme assay and starch gel electrophoresis combined with subcellular fractionation. AAT showed distinct mitochondrial and cytosolic isozymes in gills of all these animals. Although ALAT showed distinct mitochondrial and cytosolic isozymes in the gills of oysters, sea mussels and quahogs, only the mitochondrial ALAT was evident in ribbed mussel gill tissue. PK and PEPCK were cytosolic in all these preparations. ME was found only in the mitochondrial fraction of ribbed mussel and quahog gill tissue whereas sea mussel gills showed distinct cytosolic and mitochondrial ME isozymes. With oyster gills, the "cytosolic ME" was electrophoretically identical to the mitochondrial ME indicating that in vivo, the ME is probably mitochondrial. MDH showed distinct cytosolic and mitochondrial isozymes in all bivalve gills tested.