Metabolomics and lipidomics reveal the effects of the toxic dinoflagellate Alexandrium catenella on immune cells of the blue mussel, Mytilus edulis.

The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes - the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella.

Development and Evaluation of High-Density SNP Arrays for the Eastern Oyster Crassostrea virginica.

The eastern oyster Crassostrea virginica is a major aquaculture species for the USA. The sustainable development of eastern oyster aquaculture depends upon the continued improvement of cultured stocks through advanced breeding technologies. The Eastern Oyster Breeding Consortium (EOBC) was formed to advance the genetics and breeding of the eastern oyster. To facilitate efficient genotyping needed for genomic studies and selection, the consortium developed two single-nucleotide polymorphism (SNP) arrays for the eastern oyster: one screening array with 566K SNPs and one breeders' array with 66K SNPs. The 566K screening array was developed based on whole-genome resequencing data from 292 oysters from Atlantic and Gulf of Mexico populations; it contains 566,262 SNPs including 47K from protein-coding genes with a marker conversion rate of 48.34%. The 66K array was developed using best-performing SNPs from the screening array, which contained 65,893 oyster SNPs including 22,984 genic markers with a calling rate of 99.34%, a concordance rate of 99.81%, and a much-improved marker conversion rate of 92.04%. Null alleles attributable to large indels were found in 13.1% of the SNPs, suggesting that copy number variation is pervasive. Both arrays provided easy identification and separation of selected stocks from wild progenitor populations. The arrays contain 31 mitochondrial SNPs that allowed unambiguous identification of Gulf mitochondrial genotypes in some Atlantic populations. The arrays also contain 756 probes from 13 oyster and human pathogens for possible detection. Our results show that marker conversion rate is low in high polymorphism species and that the two-step process of array development can greatly improve array performance. The two arrays will advance genomic research and accelerate genetic improvement of the eastern oyster by delineating genetic architecture of production traits and enabling genomic selection. The arrays also may be used to monitor pedigree and inbreeding, identify selected stocks and their introgression into wild populations, and assess the success of oyster restoration.

Toward a Holistic Understanding and Models of Nonphotochemical Quenching Effects on In Vivo Fluorometry of Chlorophyll a in Coastal Waters.

Nonphotochemical quenching (NPQ) is known to depress in vivo fluorescence (IVF) of chlorophyll a (Chla) in aquatic environments, which makes it difficult to interpret the hour-to-hour variations in Chla measured by in situ fluorometers. We hypothesized that ratios between quenched and unquenched IVF are a function of both NPQ and photochemical quenching. In this study, two diatom model species Thalassiosira pseudonana (CCMP1335) and Thalassiosira weissflogii (CCMP1047) incubated under a sinusoidal light:dark cycle were studied; IVF was recorded continuously, and Chla and photo-physiological variables were measured seven times a day. The maximal decline in Chla-specific IVF (IVFB ) attributable to quenching was 50% under the experimental settings. An NPQ and photochemical quenching-based modeling equation exhibited a better match to the measured IVFB than equations representing the sole NPQ effect. Photochemical quenching induced by measuring light beam varied substantially during the day, and the part of the model for this process is excitation intensity-dependent (which is differed between models of in situ fluorometers, implying no straightforward method to correct Chla for all instrument models, instrument-specific parameterization is required). The forms of the IVFB -light relationship are discussed as well. The findings foster a holistic understanding of NPQ effects on in vivo Chla fluorometry.

Dynamic energy budget modeling of Atlantic surfclam, Spisula solidissima, under future ocean acidification and warming.

A dynamic energy budget (DEB) model integrating pCO2 was used to describe ocean acidification (OA) effects on Atlantic surfclam, Spisula solidissima, bioenergetics. Effects of elevated pCO2 on ingestion and somatic maintenance costs were simulated, validated, and adapted in the DEB model based upon growth and biological rates acquired during a 12-week laboratory experiment. Temperature and pCO2 were projected for the next 100 years following the intergovernmental panel on climate change representative concentration pathways scenarios (2.6, 6.0, and 8.5) and used as forcing variables to project surfclam growth and reproduction. End-of-century water warming and acidification conditions resulted in simulated faster growth for young surfclams and more energy allocated to reproduction until the beginning of the 22nd century when a reduction in maximum shell length and energy allocated to reproduction was observed for the RCP 8.5 scenario.

Perkinsus marinus suppresses in vitro eastern oyster apoptosis via IAP-dependent and caspase-independent pathways involving TNFR, NF-kB, and oxidative pathway crosstalk.

The protozoan parasite Perkinsus marinus causes Dermo disease in eastern oysters, Crassostrea virginica, and can suppress apoptosis of infected hemocytes using incompletely understood mechanisms. This study challenged hemocytes in vitro with P. marinus for 1 h in the presence or absence of caspase inhibitor Z-VAD-FMK or Inhibitor of Apoptosis protein (IAP) inhibitor GDC-0152. Hemocytes exposure to P. marinus significantly reduced granulocyte apoptosis, and pre-incubation with Z-VAD-FMK did not affect P. marinus-induced apoptosis suppression. Hemocyte pre-incubation with GDC-0152 prior to P. marinus challenge further reduced apoptosis of granulocytes with engulfed parasite, but not mitochondrial permeabilization. This suggests P. marinus-induced apoptosis suppression may be caspase-independent, affect an IAP-involved pathway, and occur downstream of mitochondrial permeabilization. P. marinus challenge stimulated hemocyte differential expression of oxidation-reduction, TNFR, and NF-kB pathways. WGCNA analysis of P. marinus expression in response to hemocyte exposure revealed correlated protease, kinase, and hydrolase expression that could contribute to P. marinus-induced apoptosis suppression.

Differences in Specific Mass Density Between Dinoflagellate Life Stages and Relevance to Accumulation by Hydrodynamic Processes.

One previously unstudied aspect of differences between sexual and asexual life stages in large-scale transport and accumulation is density (mass per unit volume) of cells in each life stage. The specific density was determined for Scrippsiella lachrymosa cells in medium with and without nitrogen (N) enrichment through density-gradient centrifugation. Growth medium without N addition is often called "encystment medium" when used for the purpose of resting cyst formation in cyst-forming dinoflagellates; mating gametes are usually seen after 2-3 days. Significant differences in specific density were found after 2 days in encystment medium simultaneously with the observation of typical gamete swimming behavior and mating. The specific density of cells in encystment medium was 1.06 g · cm-3 ; whereas, the specific density of cells in growth medium was 1.11 g · cm-3 . Cells in encystment medium were found to have significantly increased lipid content, reduced chlorophyll content, and reduced internal complexity. The findings may explain differential transport of less dense and chemotactically aggregating gametes into surface blooms in contrast to denser vegetative cells that perform daily vertical migration and do not aggregate. Passive accumulation of non-migrating gametes into layers in stagnant water also can be explained, as well as sinking of zygotes when the storage of highly dense starch increases. Resting cysts had a density of over 1.14 g · cm-3 and would sink to become part of the silt fraction of the sediment. We suggest that differences in behavior and buoyancy between sexual and asexual life stages cause differences in cell accumulation, and therefore large-scale, environmental transport could be directly dependent upon life-cycle transitions.

Effect of pluronic block polymers and N-acetylcysteine culture media additives on growth rate and fatty acid composition of six marine microalgae species.

The efficiency of microalgal biomass production is a determining factor for the economic competitiveness of microalgae-based industries. N-acetylcysteine (NAC) and pluronic block polymers are two compounds of interest as novel culture media constituents because of their respective protective properties against oxidative stress and shear-stress-induced cell damage. Here we quantify the effect of NAC and two pluronic (F127 and F68) culture media additives upon the culture productivity of six marine microalgal species of relevance to the aquaculture industry (four diatoms-Chaetoceros calcitrans, Chaetoceros muelleri, Skeletonema costatum, and Thalassiosira pseudonana; two haptophytes-Tisochrysis lutea and Pavlova salina). Algal culture performance in response to the addition of NAC and pluronic, singly or combined, is dosage- and species-dependent. Combined NAC and pluronic F127 algal culture media additives resulted in specific growth rate increases of 38%, 16%, and 24% for C. calcitrans, C. muelleri, and P. salina, respectively. Enhanced culture productivity for strains belonging to the genus Chaetoceros was paired with an ~27% increase in stationary-phase cell density. For some of the species examined, culture media enrichments with NAC and pluronic resulted in increased omega-3-fatty acid content of the algal biomass. Larval development (i.e., growth and survival) of the Pacific oyster (Crassostrea gigas) was not changed when fed a mixture of microalgae grown in NAC- and F127-supplemented culture medium. Based upon these results, we propose that culture media enrichment with NAC and pluronic F127 is an effective and easily adopted approach to increase algal productivity and enhance the nutritional quality of marine microalgal strains commonly cultured for live-feed applications in aquaculture. KEY POINTS: • Single and combined NAC and pluronic F127 culture media supplementation significantly enhanced the productivity of Chaetoceros calcitrans and Chaetoceros muelleri cultures. • Culture media enrichments with NAC and F127 can increase omega-3-fatty acid content of algal biomass. • Microalgae grown in NAC- and pluronic F127-supplemented culture media are suitable for live-feed applications.

Quantification and Valuation of Nitrogen Removal Services Provided by Commercial Shellfish Aquaculture at the Subwatershed Scale.

Eutrophication is a global environmental challenge, and diverse watershed nitrogen sources require multifaceted management approaches. Shellfish aquaculture removes nitrogen, but the extent and value of this ecosystem service have not been well-characterized at the local scale. A novel approach was employed to quantify and value nitrogen reduction services provided by the shellfish aquaculture industry to a municipality. Cultivated hard clam and eastern oyster nitrogen removal in Greenwich Bay, Connecticut, was valued using the replacement cost methodology and allocated by municipal nitrogen source. Using the preferred analysis allocating replacement costs by nitrogen source, aquaculture-based removal of 14 006 kg nitrogen was valued at $2.3-5.8 (2.3-6.4€) million year-1. This nitrogen removal represents 9% of the total annual Greenwich-specific nitrogen load, 16% of the combined nonpoint sources, 38% of the fertilizer sources, 51% of the septic sources, 98% of the atmospheric deposition to the watershed, or 184% of the atmospheric deposition to the embayments that discharge to Greenwich Bay. Our approach is transferable to other coastal watersheds pursuing nitrogen reduction goals, both with and without established shellfish aquaculture. It provides context for decisions related to watershed nitrogen management expenditures and suggests a strategy to comprehensively evaluate mechanisms to achieve nitrogen reduction targets.

Energetic response of Atlantic surfclam Spisula solidissima to ocean acidification.

In this study, we assessed the Atlantic surfclam (Spisula solidissima) energy budget under different ocean acidification conditions (OA). During 12 weeks, 126 individuals were maintained at three different ρCO2 concentrations. Every two weeks, individuals were sampled for physiological measurements and scope for growth (SFG). In the high ρCO2 treatment, clearance rate decreased and excretion rate increased relative to the low ρCO2 treatment, resulting in reduced SFG. Moreover, oxygen:nitrogen (O:N) excretion ratio dropped, suggesting that a switch in metabolic strategy occurred. The medium ρCO2 treatment had no significant effects upon SFG; however, metabolic loss increased, suggesting a rise in energy expenditure. In addition, a significant increase in food selection efficiency was observed in the medium treatment, which could be a compensatory reaction to the metabolic over-costs. Results showed that surfclams are particularly sensitive to OA; however, the different compensatory mechanisms observed indicate that they are capable of some temporary resilience.

Effects of marine harmful algal blooms on bivalve cellular immunity and infectious diseases: A review.

Bivalves were long thought to be "symptomless carriers" of marine microalgal toxins to human seafood consumers. In the past three decades, science has come to recognize that harmful algae and their toxins can be harmful to grazers, including bivalves. Indeed, studies have shown conclusively that some microalgal toxins function as active grazing deterrents. When responding to marine Harmful Algal Bloom (HAB) events, bivalves can reject toxic cells to minimize toxin and bioactive extracellular compound (BEC) exposure, or ingest and digest cells, incorporating nutritional components and toxins. Several studies have reported modulation of bivalve hemocyte variables in response to HAB exposure. Hemocytes are specialized cells involved in many functions in bivalves, particularly in immunological defense mechanisms. Hemocytes protect tissues by engulfing or encapsulating living pathogens and repair tissue damage caused by injury, poisoning, and infections through inflammatory processes. The effects of HAB exposure observed on bivalve cellular immune variables have raised the question of possible effects on susceptibility to infectious disease. As science has described a previously unrecognized diversity in microalgal bioactive substances, and also found a growing list of infectious diseases in bivalves, episodic reports of interactions between harmful algae and disease in bivalves have been published. Only recently, studies directed to understand the physiological and metabolic bases of these interactions have been undertaken. This review compiles evidence from studies of harmful algal effects upon bivalve shellfish that establishes a framework for recent efforts to understand how harmful algae can alter infectious disease, and particularly the fundamental role of cellular immunity, in modulating these interactions. Experimental studies reviewed here indicate that HABs can modulate bivalve-pathogen interactions in various ways, either by increasing bivalve susceptibility to disease or conversely by lessening infection proliferation or transmission. Alteration of immune defense and global physiological distress caused by HAB exposure have been the most frequent reasons identified for these effects on disease. Only few studies, however, have addressed these effects so far and a general pattern cannot be established. Other mechanisms are likely involved but are under-studied thus far and will need more attention in the future. In particular, the inhibition of bivalve filtration by HABs and direct interaction between HABs and infectious agents in the seawater likely interfere with pathogen transmission. The study of these interactions in the field and at the population level also are needed to establish the ecological and economical significance of the effects of HABs upon bivalve diseases. A more thorough understanding of these interactions will assist in development of more effective management of bivalve shellfisheries and aquaculture in oceans subjected to increasing HAB and disease pressures.

Bioextractive Removal of Nitrogen by Oysters in Great Bay Piscataqua River Estuary, New Hampshire, USA.

Eutrophication is a challenge to coastal waters around the globe. In many places, nutrient reductions from land-based sources have not been sufficient to achieve desired water quality improvements. Bivalve shellfish have shown promise as an in-water strategy to complement land-based nutrient management. A local-scale production model was used to estimate oyster (Crassostrea virginica) harvest and bioextraction of nitrogen (N) in Great Bay Piscataqua River Estuary (GBP), New Hampshire, USA, because a system-scale ecological model was not available. Farm-scale N removal results (0.072 metric tons acre-1 year-1) were up-scaled to provide a system-wide removal estimate for current (0.61 metric tons year-1), and potential removal (2.35 metric tons year-1) at maximum possible expansion of licensed aquaculture areas. Restored reef N removal was included to provide a more complete picture. Nitrogen removal through reef sequestration was ~ 3 times that of aquaculture. Estimated reef-associated denitrification, based on previously reported rates, removed 0.19 metric tons N year-1. When all oyster processes (aquaculture and reefs) were included, N removal was 0.33% and 0.54% of incoming N for current and expanded acres, respectively. An avoided cost approach, with wastewater treatment as the alternative management measure, was used to estimate the value of the N removed. The maximum economic value for aquaculture-based removal was $105,000 and $405,000 for current and expanded oyster areas, respectively. Combined aquaculture and reef restoration is suggested to maximize N reduction capacity while limiting use conflicts. Comparison of removal based on per oyster N content suggests much lower removal rates than model results, but model harvest estimates are similar to reported harvest. Though results are specific to GBP, the approach is transferable to estuaries that support bivalve aquaculture but do not have complex system-scale hydrodynamic or ecological models.

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

Oyster transcriptome response to Alexandrium exposure is related to saxitoxin load and characterized by disrupted digestion, energy balance, and calcium and sodium signaling.

Harmful Algal Blooms are worldwide occurrences that can cause poisoning in human seafood consumers as well as mortality and sublethal effets in wildlife, propagating economic losses. One of the most widespread toxigenic microalgal taxa is the dinoflagellate Genus Alexandrium, that includes species producing neurotoxins referred to as PST (Paralytic Shellfish Toxins). Blooms cause shellfish harvest restrictions to protect human consumers from accumulated toxins. Large inter-individual variability in toxin load within an exposed bivalve population complicates monitoring of shellfish toxicity for ecology and human health regulation. To decipher the physiological pathways involved in the bivalve response to PST, we explored the whole transcriptome of the digestive gland of the Pacific oyster Crassostrea gigas fed experimentally with a toxic Alexandrium minutum culture. The largest differences in transcript abundance were between oysters with contrasting toxin loads (1098 transcripts), rather than between exposed and non-exposed oysters (16 transcripts), emphasizing the importance of toxin load in oyster response to toxic dinoflagellates. Additionally, penalized regressions, innovative in this field, modeled accurately toxin load based upon only 70 transcripts. Transcriptomic differences between oysters with contrasting PST burdens revealed a limited suite of metabolic pathways affected, including ion channels, neuromuscular communication, and digestion, all of which are interconnected and linked to sodium and calcium exchanges. Carbohydrate metabolism, unconsidered previously in studies of harmful algal effects on shellfish, was also highlighted, suggesting energy challenge in oysters with high toxin loads. Associations between toxin load, genotype, and mRNA levels were revealed that open new doors for genetic studies identifying genetically-based low toxin accumulation.

Acute exposure to TiO2 nanoparticles produces minimal apparent effects on oyster, Crassostrea virginica (Gmelin), hemocytes.

The response of oyster (Crassostrea virginica) hemocytes was studied following exposure to anatase nanoparticles (ca. 7.4nm), surface-coated rutile nanocomposites (UV-Titan M212, ca. 86nm) and bulk titanium dioxide (TiO2) particles (anatase and rutile crystalline forms; 0.4-0.5µm). Hemocytes were collected from oysters and exposed to one of the four particle types at concentrations of 0.1, 0.5, and 1.0mg/L under dark and environmentally-relevant light conditions for periods of two and four hours. Hemocyte mortality, phagocytosis, and reactive oxygen species (ROS) production were then evaluated using flow-cytometric assays. Bulk and nanoparticulate TiO2 had little effect on viability of oyster hemocytes or on production of ROS. Significant changes in phagocytosis occurred after exposure to anatase nanoparticles for 4h under dark conditions, and UV-Titan for 2h under light conditions. Results demonstrate that TiO2 particles (bulk or nanoscale) produce minimal effects on hemocyte biomarkers examined following acute, in vitro exposures.

Role of Shellfish Aquaculture in the Reduction of Eutrophication in an Urban Estuary.

Land-based management has reduced nutrient discharges; however, many coastal waterbodies remain impaired. Oyster "bioextraction" of nutrients and how oyster aquaculture might complement existing management measures in urban estuaries was examined in Long Island Sound, Connecticut. Eutrophication status, nutrient removal, and ecosystem service values were estimated using eutrophication, circulation, local- and ecosystem-scale models, and an avoided-costs valuation. System-scale modeling estimated that 1.31% and 2.68% of incoming nutrients could be removed by current and expanded production, respectively. Up-scaled local-scale results were similar to system-scale results, suggesting that this up-scaling method could be useful in bodies of water without circulation models. The value of removed nitrogen was estimated using alternative management costs (e.g., wastewater treatment) as representative, showing ecosystem service values of $8.5 and $470 million per year for current and maximum expanded production, respectively. These estimates are conservative; removal by clams in Connecticut, oysters and clams in New York, and denitrification are not included. Optimistically, the calculation of oyster-associated removal from all leases in both states (5% of bottom area) plus denitrification losses showed increases to 10%-30% of annual inputs, which would be higher if clams were included. Results are specific to Long Island Sound, but the approach is transferable to other urban estuaries.

Cultivation of the Ribbed Mussel (Geukensia demissa) for Nutrient Bioextraction in an Urban Estuary.

Shellfish aquaculture is gaining acceptance as a tool to reduce nutrient over enrichment in coastal and estuarine ecosystems through the feeding activity of the animals and assimilation of filtered particles in shellfish tissues. This ecosystem service, provided by the ribbed mussel (Geukensia demissa), was studied in animals suspended from a commercial mussel raft in the urban Bronx River Estuary, NY, in waters closed to shellfish harvest due to bacterial contamination. Naturally occurring populations of ribbed mussels were observed to be healthy and resilient in this highly urbanized environment. Furthermore, mussels grown suspended in the water column contained substantially lower concentrations of heavy metals and organic contaminants in their tissues than blue mussels (Mytilus edulis) collected at a nearby benthic site. Spat collection efforts from shore and within the water column were unsuccessful; this was identified as a key bottleneck to future large-scale implementation. Filtration experiments indicated that a fully stocked G. demissa raft would clear an average 1.2 × 107 L of Bronx River Estuary water daily, removing 160 kg of particulate matter from the water column, of which 12 kg would be absorbed into mussel digestive systems. At harvest, 62.6 kg of nitrogen would be sequestered in mussel tissue and shell. These values compare favorably to other resource management recovery methods targeting agricultural and stormwater nitrogen sources.

Characterizing seston in the Penobscot River Estuary.

The Penobscot River Estuary is an important system for diadromous fish in the Northeast United States of American (USA), in part because it is home to the largest remnant population of Atlantic salmon, Salmo salar, in the country. Little is known about the chemical and biological characteristics of seston in the Penobscot River Estuary. This study used estuarine transects to characterize the seston during the spring when river discharge is high and diadromous fish migration peaks in the Penobscot River Estuary. To characterize the seston, samples were taken in spring 2015 for phytoplankton identification, total suspended matter (TSM), percent organic TSM, chlorophyll a, particle size (2 µm-180 µm), particulate carbon and nitrogen concentrations, and stable carbon and nitrogen isotopes. The estuarine profiles indicate that TSM behaved non-conservatively with a net gain in the estuary. As phytoplankton constituted only 1/1000 of the particles, the non-conservative behavior of TSM observed in the estuary was most likely not attributable to phytoplankton. Particulate carbon and nitrogen ratios and stable isotope signals indicate a strong terrestrial, allochthonous signal. The seston in the Penobscot River Estuary was dominated by non-detrital particles. During a short, two-week time period, Heterosigma akashiwo, a phytoplankton species toxic to finfish, also was detected in the estuary. A limited number of fish samples, taken after the 2015 Penobscot River Estuary bloom of H. akashiwo, indicated frequent pathological gill damage. The composition of seston, along with ichthyotoxic algae, suggest the need for further research into possible effects upon resident and migratory fish in the Penobscot River Estuary.

Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi).

We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction.

Bioconversion of (13)C-labeled microalgal phytosterols to cholesterol by the Northern Bay scallop, Argopecten irradians irradians.

Bivalve mollusks lack de novo cholesterol biosynthesis capabilities and therefore rely upon dietary sources of sterols for rapid growth. Microalgae that constitute the main source of nutrition for suspension-feeding bivalves contain a diverse array of phytosterols, in most cases lacking cholesterol. Rapid growth of bivalves on microalgal diets with no cholesterol implies that some phytosterols can satisfy the dietary requirement for cholesterol through metabolic conversion to cholesterol, but such metabolic pathways have not been rigorously demonstrated. In the present study, stable isotope-labeled phytosterols were used to supplement a unialgal diet of Rhodomonas sp. and their biological transformation to cholesterol within scallop tissues was determined using (13)C-NMR spectroscopy. Scallops efficiently dealkylated ∆(5) C29 (24-ethyl) sterols to cholesterol, and the only C28 sterol that was dealkylated efficiently possessed the 24(28)-double bond. Non-metabolized dietary phytosterols accumulated in the soft tissues. Observed formation of ∆(5,7) sterols (provitamin D) from ∆(5) sterols may represent initiation of steroid hormone (possibly ecdysone) biosynthesis. These findings provide a key component necessary for formulation of nutritionally complete microalgal diets for hatchery production of seed for molluscan aquaculture.

Essential Fatty Acid Assimilation and Synthesis in Larvae of the Bivalve Crassostrea gigas.

Essential fatty acids (EFA) are important for bivalve larval survival and growth. The purpose of this study was to quantitatively assess for the first time through a mass-balance approach dietary EFA incorporation and synthesis within Crassostrea gigas larvae. A first experiment was carried out using two microalgae, Tisochrysis lutea (T) and Chaetoceros neogracile (Cg), as mono- and bi-specific diets. A second experiment using a similar design was performed to confirm and extend the results obtained in the first. Flow-through larval rearing was used for accurate control of food supply and measurement of ingestion. Non-methylene-interrupted fatty acids were synthetized from precursors supplied in the diet: 16:1n-7 and 18:1n-9, mediated by Δ5 desaturase. Moreover, this Δ5 desaturase presumably allowed larvae to convert 20:3n-6 and 20:4n-3 to 20:4n-6 and 20:5n-3, respectively, when the product EFA were poorly or not supplied in the diet, as when larvae were fed T exclusively. Under our experimental conditions, none of the diets induced 22:6n-3 synthesis; however, 22:6n-3 incorporation into larval tissues occurred selectively under non-limiting dietary supply to maintain optimal levels in the larvae. This combination of flow-through larval rearing and biochemical analysis of FA levels could be applied to additional dietary experiments to precisely define optimal levels of EFA supply.