Population-specific responses in eastern oysters exposed to low salinity in the northern Gulf of Mexico.

Eastern oysters, Crassostrea virginica, are facing rapid environmental changes in the northern Gulf of Mexico and can respond to these changes via plasticity or evolution. Plastic responses can immediately buffer against environmental changes, although this buffering may impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigated the roles of plastic and evolved responses of C. virginica acclimated to low salinity using a common garden experiment with four populations exposed to two salinities. We used three transcriptomic analyses (edgeR, PERMANOVA and WGCNA) combined with physiology data to identify the effect of genotype (population), environment (salinity) and the genotype-environment interaction on both whole-organism and molecular phenotypes. We demonstrate that variation in gene expression is mainly driven by population, with relatively small changes in response to salinity. In contrast, the morphology and physiology data reveal that salinity has a larger influence on oyster performance than the population of origin. All analyses lacked signatures of the genotype×environment interaction and, in contrast to previous studies, we found no evidence for population-specific responses to low salinity. However, individuals from the highest salinity estuary displayed highly divergent gene expression from that of other populations, which could potentially drive population-specific responses to other stressors. Our findings suggest that C. virginica largely rely on plasticity in physiology to buffer the effects of low salinity, but that these changes in physiology do not rely on large persistent changes in gene expression.

Survival and growth of triploid eastern oysters, Crassostrea virginica, produced from wild diploids collected from low-salinity areas.

Triploid Eastern oysters have been reported to suffer greater mortalities than diploids when exposed to low-salinity (<5) conditions in the U.S. Gulf of Mexico and Atlantic estuaries. As such, the effect of broodstock parentage was investigated on the low-salinity tolerance of triploid progeny produced by mating diploid females (collected from three Louisiana estuaries differing in salinity regimes) with male tetraploids at two hatcheries. Diploid crosses were also produced using the wild broodstocks to verify expected differences in low-salinity tolerance among diploid progeny and between ploidy levels. All progeny were deployed at low and moderate-salinity (averages of 9.3 and 19.4) field sites to monitor monthly growth and mortality. Sex ratio, gametogenic stage, gonad-to-body ratio, condition index, and Perkinsus marinus infection were also measured periodically at both field sites Although high triploid mortality at the low-salinity site prevented complete analysis, results indicated that diploid parentage had little effect on triploid survival at low salinity. Broodstock parentage affected diploid mortality and growth, although results did not match with predictions made based on historical salinity at broodstock collection sites. Ploidy level had the largest effect on triploid survival and growth followed by the hatchery site where the oysters were produced.

Tolerance of northern Gulf of Mexico eastern oysters to chronic warming at extreme salinities.

The eastern oyster, Crassostrea virginica, provides critical ecosystem services and supports valuable fishery and aquaculture industries in northern Gulf of Mexico (nGoM) subtropical estuaries where it is grown subtidally. Its upper critical thermal limit is not well defined, especially when combined with extreme salinities. The cumulative mortalities of the progenies of wild C. virginica from four nGoM estuaries differing in mean annual salinity, acclimated to low (4.0), moderate (20.0), and high (36.0) salinities at 28.9 °C (84 °F) and exposed to increasing target temperatures of 33.3 °C (92 °F), 35.6 °C (96 °F) or 37.8 °C (100 °F), were measured over a three-week period. Oysters of all stocks were the most sensitive to increasing temperatures at low salinity, dying quicker (i.e., lower median lethal time, LT50) than at the moderate and high salinities and resulting in high cumulative mortalities at all target temperatures. Oysters of all stocks at moderate salinity died the slowest with high cumulative mortalities only at the two highest temperatures. The F1 oysters from the more southern and hypersaline Upper Laguna Madre estuary were generally more tolerant to prolonged higher temperatures (higher LT50) than stocks originating from lower salinity estuaries, most notably at the highest salinity. Using the measured temperatures oysters were exposed to, 3-day median lethal Celsius degrees (LD50) were estimated for each stock at each salinity. The lowest 3-day LD50 (35.1-36.0 °C) for all stocks was calculated at a salinity of 4.0, while the highest 3-day LD50 (40.1-44.0 °C) was calculated at a salinity of 20.0.

Lack of genotype-by-environment interaction suggests limited potential for evolutionary changes in plasticity in the eastern oyster, Crassostrea virginica.

Eastern oysters in the northern Gulf of Mexico are facing rapid environmental changes and can respond to this change via plasticity or evolution. Plasticity can act as an immediate buffer against environmental change, but this buffering could impact the organism's ability to evolve in subsequent generations. While plasticity and evolution are not mutually exclusive, the relative contribution and interaction between them remains unclear. In this study, we investigate the roles of plastic and evolved responses to environmental variation and Perkinsus marinus infection in Crassostrea virginica by using a common garden experiment with 80 oysters from six families outplanted at two field sites naturally differing in salinity. We use growth data, P. marinus infection intensities, 3' RNA sequencing (TagSeq) and low-coverage whole-genome sequencing to identify the effect of genotype, environment and genotype-by-environment interaction on the oyster's response to site. As one of first studies to characterize the joint effects of genotype and environment on transcriptomic and morphological profiles in a natural setting, we demonstrate that C. virginica has a highly plastic response to environment and that this response is parallel among genotypes. We also find that genes responding to genotype have distinct and opposing profiles compared to genes responding to environment with regard to expression levels, Ka/Ks ratios and nucleotide diversity. Our findings suggest that C. virginica may be able to buffer the immediate impacts of future environmental changes by altering gene expression and physiology, but the lack of genetic variation in plasticity suggests limited capacity for evolved responses.

Divergence in salinity tolerance of northern Gulf of Mexico eastern oysters under field and laboratory exposure.

The eastern oyster, Crassostrea virginica, is a foundation species within US Gulf of Mexico (GoM) estuaries that has experienced substantial population declines. As changes from management and climate are expected to continue to impact estuarine salinity, understanding how local oyster populations might respond and identifying populations with adaptations to more extreme changes in salinity could inform resource management, including restoration and aquaculture programs. Wild oysters were collected from four estuarine sites from Texas [Packery Channel (PC): 35.5, annual mean salinity, Aransas Bay (AB): 23.0] and Louisiana [Calcasieu Lake (CL): 16.2, Vermilion Bay (VB): 7.4] and spawned. The progeny were compared in field and laboratory studies under different salinity regimes. For the field study, F1 oysters were deployed at low (6.4) and intermediate (16.5) salinity sites in Alabama. Growth and mortality were measured monthly. Condition index and Perkinsus marinus infection intensity were measured quarterly. For the laboratory studies, mortality was recorded in F1 oysters that were exposed to salinities of 2.0, 4.0, 20.0/22.0, 38.0 and 44.0 with and without acclimation. The results of the field study and laboratory study with acclimation indicated that PC oysters are adapted to high-salinity conditions and do not tolerate very low salinities. The AB stock had the highest plasticity as it performed as well as the PC stock at high salinities and as well as Louisiana stocks at the lowest salinity. Louisiana stocks did not perform as well as the Texas stocks at high salinities. Results from the laboratory studies without salinity acclimation showed that all F1 stocks experiencing rapid mortality at low salinities when 3-month oysters collected at a salinity of 24 were used and at both low and high salinities when 7-month oysters collected at a salinity of 14.5 were used.

Transgenerational plasticity and the capacity to adapt to low salinity in the eastern oyster, Crassostrea virginica.

Salinity conditions in oyster breeding grounds in the Gulf of Mexico are expected to drastically change due to increased precipitation from climate change and anthropogenic changes to local hydrology. We determined the capacity of the eastern oyster, Crassostrea virginica, to adapt via standing genetic variation or acclimate through transgenerational plasticity (TGP). We outplanted oysters to either a low- or medium-salinity site in Louisiana for 2 years. We then crossed adult parents using a North Carolina II breeding design, and measured body size and survival of larvae 5 dpf raised under low or ambient salinity. We found that TGP is unlikely to significantly contribute to low-salinity tolerance since we did not observe increased growth or survival in offspring reared in low salinity when their parents were also acclimated at a low-salinity site. However, we detected genetic variation for body size, with an estimated heritability of 0.68 ± 0.25 (95% CI). This suggests there is ample genetic variation for this trait to evolve, and that evolutionary adaptation is a possible mechanism through which oysters will persist with future declines in salinity. The results of this experiment provide valuable insights into successfully breeding low-salinity tolerance in this commercially important species.

Transcriptomic signatures of temperature adaptation in the eastern oyster Crassostrea virginica.

The large geographic distribution of the eastern oyster, Crassostrea virginica, makes it an ideal species to test how populations have adapted to latitudinal gradients in temperature. Despite inhabiting distinct thermal regimes, populations of C. virginica near the species' southern and northern geographic range show no population differences in their physiological response to temperature. In this study, we used comparative transcriptomics to understand how oysters from either end of the species' range maintain enantiostasis across three acclimation temperatures (10, 20, and 30°C). With this approach, we identified genes that were differentially expressed in response to temperature between individuals of C. virginica collected from New Brunswick, Canada and Louisiana, USA. We observed a core set of genes whose expression responded to temperature in both populations, but also an even larger set of genes with expression patterns that were unique to each population. Intriguingly, the genes with population-specific responses to temperature had elevated FST and Ka/Ks ratios compared to the genome-wide average. In contrast, genes showing only a response to temperature were found to only have elevated FST values suggesting that divergent FST may be due to selection on linked regulatory regions rather than positive selection on protein coding regions. Taken together, our results suggest that, despite coarse-scale physiological similarities, natural selection has shaped divergent gene expression responses to temperature in geographically separated populations of this broadly eurythermal marine invertebrate.

Heat shock protein 70 levels and post-harvest survival of eastern oysters following sublethal heat shock in the laboratory or conditioning in the field.

A major problem of storing and shipping eastern oysters (Crassostrea virginica) from the Northern Gulf of Mexico in summer and early fall is their elevated mortality. A study was therefore conducted to determine whether heat shocking the oysters or conditioning them to aerial exposure prior to harvest could reduce their mortality during cold storage. Increasing the levels of stress proteins in bivalves has been shown to reduce their mortality when exposed to additional stressors. In this study, the levels of heat shock protein 70 (HSP70) proteins and cumulative mortality during cold storage, out of water, of market-sized oysters were measured, in summer, following (1) sublethal heat shocks (41 °C, 1 h) in the laboratory or (2) 3 weeks to 6 weeks of daily exposures to air (0 h, ~ 10 h, or ~ 18 h) in the field. In total, four heat shock and two aerial exposure studies were done. Consistently, heat shocks or 6 weeks of daily aerial exposures increased HSP70 levels in oysters but did not reduce their mortality during cold storage. Three weeks of daily aerial exposure did not increase HSP70 levels and only marginally reduced mortality; a significant reduction in cumulative mortality occurred in one of the aerial exposure studies after 7 days of cold storage (0 h [26%], ~ 18 h [8%]). In conclusion, upregulation of HSP70 proteins or aerial exposure during grow-out was not an effective tool in reducing the mortality of oysters harvested in summer and held in cold storage.

Testing plasma subtilisin inhibitory activity as a selective marker for dermo resistance in eastern oysters.

Recent findings have suggested that eastern oyster plasma possesses inhibitors of the protease subtilisin, which play a role in the host defense against Perkinsus marinus, a protist parasite causing dermo. A study was conducted to determine whether plasma subtilisin inhibitory activity (PSIA) could be used as a selective marker in breeding programs for dermo resistance. Eastern oysters Crassostrea virginica from 2 wild Louisiana populations shown to differ in dermo resistance were collected and their PSIA was measured. Three groups of oysters were established to spawn from each population. One group was composed of randomly sampled oysters (i.e. unselected) and the other 2 groups were composed of oysters with the highest or lowest PSIA. After spawning, progenies were deployed in October 2014 in a dermo endemic area and sampled quarterly for 2 yr to measure their mortality, growth, P. marinus infection intensity, condition index, PSIA, and the gene expression of 3 subtilisin inhibitors (cvSI-1, cvSI-2, and cvSI-3). Oyster cumulative mortalities of the progenies of all groups increased both years from April to October, concomitant with increasing P. marinus infection intensities. Mortalities and P. marinus infection intensities differed markedly between the 2 populations, but differences between the unselected and selected groups of each population were limited. Measurements of PSIA and cvSI-1, cvSI-2, and cvSI-3 gene expressions between the progenies of all groups showed few differences. CvSI-1 gene expression in surviving oysters of the most susceptible population was increased at the end of the study, adding additional support to the potential role of cvSI-1 defense against P. marinus.

Identifying factors inducing trophozoite differentiation into hypnospores in Perkinsus species.

Trophozoites of species of Perkinsus in host tissues readily differentiate into hypnospores when incubated in Ray's fluid thioglycollate medium (RFTM). In contrast, hypnospores have rarely been observed in vivo, and when reported they have been associated with dying hosts. The objective of this study was to determine what altered environmental conditions trigger the differentiation of Perkinsus trophozoites into hypnospores. In the first part of the study, cultured P. chesapeaki trophozoites were exposed to lowered oxygen, acidic pH, increased nutrient levels, heat shock, or osmotic shock conditions, and hypnospore density was measured. Acidic pH, lowered oxygen, or increased nutrient levels significantly increased P. chesapeaki hypnospore formation. In the second part of the study, P. olseni and P. marinus trophozoites were exposed to acidic pH, lowered oxygen, or increased nutrient levels resulting in hypnospore formation in P. olseni but not P. marinus. This study demonstrated that changes in environmental conditions consistent with changes expected in decaying tissues or with RFTM incubation induce trophozoite differentiation. The response of the cultured trophozoites varied between species and between isolates of the same species.

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae).

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.

Increasing the in vitro proliferation rate of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis.

Perkinsus mediterraneus is an alveolate parasite first described in Ostrea edulis from the Balearic Islands (Mediterranean Sea, Spain), and little is known about its biology or the disease it causes. Continuous in vitro cultures of P. mediterraneus have recently been established in the protein-deficient culture medium JL-ODRP-2F to facilitate its study. Parasite proliferation rate in vitro however was low, with densities increasing 2- to 6-fold between subcultures at 6-week intervals. To increase the proliferation rate of P. mediterraneus cultures to rates similar to other Perkinsus species, various culture conditions (temperature, osmolality, pH, O(2), and CO(2) concentrations), culture procedures (seeding density and frequency of medium changes), concentrations of medium components, and addition of medium supplements (oyster tissue lysate, oyster plasma, animal sera, growth factors, and hormones) were tested. All treatments were evaluated by measuring parasite densities after 2 weeks of culture. The greatest increase in parasite densities, a 35-fold increase over the cell seeding density and 18 times that of the control (cells without supplementation), occurred in medium supplemented with 1,000 µg/mL of O. edulis tissue lysate. P. mediterraneus proliferation was also significantly increased by oyster tissue lysate concentration as low as 125 µg/mL.

Characterization of the major plasma protein of the eastern oyster, Crassostrea virginica, and a proposed role in host defense.

The major plasma protein of the eastern oyster, Crassostrea virginica, was purified, characterized and named dominin. SDS-PAGE analyses revealed that dominin consistently made up more than 40% of eastern oyster plasma and extrapallial fluid proteins. Three different forms of dominin were observed under non-reducing conditions. PCR and RACE primers designed from partial amino acid sequences obtained by tandem mass spectrometry of purified dominin identified 720bp of complete cDNA encoding 192 amino acid residues. Based on the deduced amino acid sequence of mature dominin, its molecular mass was calculated to be 19,389Da and was lower than the molecular mass of purified dominin measured by MALDI. This difference is likely due to post-translational modifications of dominin as the purified protein was found to be glycolysated, phosphorylated and likely sulfated. The amino acid sequence showed high similarity to the major plasma protein of the Pacific oyster (Crassostrea gigas), cavortin, and of the green-lipped mussel (Perna canaliculus), pernin, and to a recently described protein labeled as an extracellular superoxide dismutase from the Sydney rock oyster Saccostrea glomerata. While dominin was found to possess a Cu/Zn superoxide dismutase (SOD) domain, the domain was not completely conserved which explained why purified dominin lacked SOD activity. Dominin mRNA was detected in hemocytes by in situ hybridization and its expression measured by quantitative real time RT-PCR was significantly higher in winter than summer. Although the function(s) of dominin and homologous proteins is uncertain, the reported ability of cavortin to sequester iron and possibly limit the availability of this essential metal to pathogens suggests a potential role in host defense for this group of dominant plasma proteins. Other possible functions of dominin in antioxidation, wound repair, metal transport and shell mineralization are discussed leading us to conclude that dominin is likely a multifunctional protein.

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica.

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 degrees C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 degrees C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 degrees C: 0% viability), 60 days (3.5 ppt, 10 degrees C: 0% viability) and 90 days (7 ppt, 2 degrees C: 0.6+/-0.7%; 7 ppt, 10 degrees C: 0.2+/-0.2%).

A new lysozyme from the eastern oyster, Crassostrea virginica, and a possible evolutionary pathway for i-type lysozymes in bivalves from host defense to digestion.

BACKGROUND: Lysozymes are enzymes that lyse bacterial cell walls, an activity widely used for host defense but also modified in some instances for digestion. The biochemical and evolutionary changes between these different functional forms has been well-studied in the c-type lysozymes of vertebrates, but less so in the i-type lysozymes prevalent in most invertebrate animals. Some bivalve molluscs possess both defensive and digestive lysozymes. RESULTS: We report a third lysozyme from the oyster Crassostrea virginica, cv-lysozyme 3. The chemical properties of cv-lysozyme 3 (including molecular weight, isoelectric point, basic amino acid residue number, and predicted protease cutting sites) suggest it represents a transitional form between lysozymes used for digestion and immunity. The cv-lysozyme 3 protein inhibited the growth of bacteria (consistent with a defensive function), but semi-quantitative RT-PCR suggested the gene was expressed mainly in digestive glands. Purified cv-lysozyme 3 expressed maximum muramidase activity within a range of pH (7.0 and 8.0) and ionic strength (I = 0.005-0.01) unfavorable for either cv-lysozyme 1 or cv-lysozyme 2 activities. The topology of a phylogenetic analysis of cv-lysozyme 3 cDNA (full length 663 bp, encoding an open reading frame of 187 amino acids) is also consistent with a transitional condition, as cv-lysozyme 3 falls at the base of a monophyletic clade of bivalve lysozymes identified from digestive glands. Rates of nonsynonymous substitution are significantly high at the base of this clade, consistent with an episode of positive selection associated with the functional transition from defense to digestion. CONCLUSION: The pattern of molecular evolution accompanying the shift from defensive to digestive function in the i-type lysozymes of bivalves parallels those seen for c-type lysozymes in mammals and suggests that the lysozyme paralogs that enhance the range of physiological conditions for lysozyme activity may provide stepping stones between defensive and digestive forms.

Serine protease inhibitor cvSI-1 potential role in the eastern oyster host defense against the protozoan parasite Perkinsus marinus.

The serine protease inhibitor cvSI-1, purified from plasma of eastern oysters, inhibited the proliferation of the protozoan parasite Perkinsus marinus in vitro. In situ hybridization located cvSI-1 gene expression in basophil cells of the digestive tubules and cvSI-1 expression measured by real-time quantitative reverse transcriptase polymerase chain reaction was several hundred folds greater in digestive glands than in other organs examined or circulating hemocytes. cvSI-1 gene expression was also significantly greater in winter than in summer. Finally, cvSI-1 gene expression and plasma protease inhibitory activity in oysters selected for increased resistance to P. marinus were significantly greater than in unselected oysters. These findings support the hypothesis that cvSI-1 plays a role in eastern oyster host defense against P. marinus possibly through inhibition of parasite proliferation.

Evidence indicating the existence of a novel family of serine protease inhibitors that may be involved in marine invertebrate immunity.

A new serine protease inhibitor, designated cvSI-2, was purified and characterized from the plasma of the eastern oyster, Crassostrea virginica. CvSI-2 inhibited the serine protease subtilisin A in a slow-tight binding manner, with an overall dissociation constant Ki* of 0.18 nM. It also inhibited perkinsin, the major extracellular protease of the oyster protozoan parasite Perkinsus marinus. Sequencing of cvSI-2 cloned cDNA revealed an open reading frame of 258 bp encoding a polypeptide of 85 amino acids, with the 18 N-terminal amino acids forming a signal peptide. The mature cvSI-2 molecule predicted consisted of 67 amino acids with 12 cysteine residues and a calculated molecular mass of 7202.96 Da. Overall 91% of the cvSI-2 amino acid sequence predicted from cDNA was confirmed by tandem mass spectrometry sequencing of purified cvSI-2. In addition, serine 43 and a threonine substitution at this position were observed. CvSI-2 amino acid sequence showed a 38% identity and 54% similarity with that of cvSI-1, the first protease inhibitor purified and characterized from a bivalve mollusc. Like cvSI-1, cvSI-2 gene was expressed in the basophil cells of digestive tubules. BLAST search found multiple ESTs from the eastern oyster, Pacific oyster, Mediterranean mussel, and sea vase, a tunicate, which could encode proteins with sequences similar to cvSI-1 and cvSI-2. Our findings indicate that cvSI-1 and cvSI-2 are members of a novel family of serine protease inhibitors in bivalve molluscs and perhaps other marine invertebrates, which share the characteristic cysteine array C-X(4-9)-C-X(4-6)-C-X(7)-C-X(4)-C-T-C-X(6-9)-C-X(5)-C-X(3-7)-C-X(6-10)-C-X(4)-C-X-C.

In vitro effects of growth factors and hormones on three Perkinsus species and increased proliferation of P. marinus during cloning.

Clonal cultures are essential for the genotypic and phenotypic characterization of Perkinsus species but their cloning, especially of P. marinus, can be tedious. The use of a growth factor and hormone supplement to facilitate cloning was, therefore, investigated. Many of the 16 supplements tested significantly increased P. marinus and P. olseni proliferation but only two significantly increased P. chesapeaki proliferation. The concentration of the most effective supplement for all three Perkinsus species (i.e., endothelial cell growth supplement, ECGS) and medium dilution were then optimized for P. marinus cultured at low densities. Finally, the advantage of using conditioned culture medium, a feeder layer, and ECGS alone and in different combinations to improve cloning of P. marinus were compared. Using conditioned culture medium, a feeder layer and ECGS in combination, each cell (N=7) seeded singly yielded clonal cultures with 253+/-167 cells after 21 days. In contrast, only 4 out of 7 cells seeded singly in culture medium yielded clonal cultures with 5+/-4 cells after 21 days.

Continuous culture of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis, and characterization of its morphology, propagation, and extracellular proteins in vitro.

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.

cDNA cloning and tissue expression of plasma lysozyme in the eastern oyster, Crassostrea virginica.

The cDNA sequence of a 17,861 Da lysozyme first purified from plasma of eastern oysters (Crassostrea virginica) was identified and its complete amino acid sequence deduced. The amino acid sequence of the plasma lysozyme, designated cv-lysozyme 1, contained both a unique and a conserved region when compared to the amino acid sequences of other bivalve lysozymes. In situ hybridisation located cv-lysozyme 1 gene expression in mantle and gill cells in standard histological sections. Quantitative real-time RT-PCR detected cv-lysozyme 1 expression in all organs examined and circulating haemocytes. The number of cv-lysozyme 1 mRNA transcripts was particularly high in mantles and labial palps suggesting those organs are the main sites of cv-lysozyme 1 synthesis. Cv-lysozyme 1 enzyme activity measured by lysing Micrococcus lysodeikticus bacteria and expressed in units per gram tissue was highest in mantles, labial palps and gills. Most cv-lysozyme 1 enzyme activity in oysters was found in plasma. Cv-lysozyme 1 main organs of synthesis, its abundance in plasma and its strong antimicrobial properties suggest its main role is in oyster host defences.