Galactosylation of glycoconjugates using Pacific oyster ß-1,3-galactosyltransferases.

The Pacific oyster (Magallana gigas) exhibits an extensive diversity of N- and O-linked glycoconjugates, offering significant potential for biotechnological applications. Through genomic data mining, we have identified and characterized a suite of ß-1,3-galactosyltransferase enzymes, pivotal for the synthesis of glycan structures. Out of ten cloned gene candidates, six enzymes were successfully expressed recombinantly in Escherichia coli. Four of these enzymes exhibited measurable catalytic activity in the transfer of galactose to various acceptor substrates. Notably, MgB3GalT1 demonstrated the highest efficiency, achieving a 91.2 % conversion rate. This enzyme was proficient in glycosylating diverse glycan structures, including Core 2 O-glycans and several di-, tri-, and tetra-antennary complex N-glycan standards. Mass spectrometric analysis confirmed the successful modification of N-glycans. These findings open new approaches for utilizing oyster-derived enzymes in glycan-based therapeutics and molecular glycoengineering, highlighting their utility in synthetic applications and biotechnological advancements.

Molecular cloning, expression pattern of ß-carotene 15,15-dioxygenase gene and association analysis with total carotenoid content in pearl oyster Pinctada fucata martensii.

ß-carotene-15,15-dioxygenase is an enzyme involved in carotenoid metabolism to catalyze oxidative cleavage of ß-carotene at its central double bond to two molecules of retinal in intestinal cells of vertebrate. In this study, we cloned and characterized ß-carotene-15,15-dioxygenase in pearl oyster Pinctada fucata martensii (PmßCDOX). The full length of PmßCDOX gene was 1802 bp, including 1554 bp of the open reading frame (ORF) that encoded 517 amino acids, a 5'UTR of 134 bp and a 3' UTR of 114 bp. PmßCDOX was expressed at various tissues with highest level in hepatopancreas. Eighteen and fifteen single nucleotide polymorphisms (SNPs) were separately obtained in the exon and promoter of PmßCDOX. Eight SNPs (six SNPs in the exon and two SNPs in the promoter region) were significantly associated to total carotenoid content (TCC) (P < .05). The eight SNPs of significantly associated TCC were divided three haploblocks. Haplotypes CCTT had larger TCC than other haplotypes. The present results suggest that PmßCDOX is involved in carotenoid metabolism in pearl oyster. Our study will be helpful for development gene marker in selective breeding programs for TCC trait of the species.

UDP-Glucose 4-Epimerase and ß-1,4-Galactosyltransferase from the Oyster Magallana gigas as Valuable Biocatalysts for the Production of Galactosylated Products.

Uridine diphosphate galactose (UDP-galactose) is a valuable building block in the enzymatic synthesis of galactose-containing glycoconjugates. UDP-glucose 4-epimerase (UGE) is an enzyme which catalyzes the reversible conversion of abundantly available UDP-glucose to UDP-galactose. Herein, we described the cloning, expression, purification, and biochemical characterization of an unstudied UGE from the oyster Magallana gigas (MgUGE). Activity tests of recombinantly expressed MgUGE, using HPLC (high-performance liquid chromatography), mass spectrometry, and photometric assays, showed an optimal temperature of 16 °C, and reasonable thermal stability up to 37 °C. No metal ions were required for enzymatic activity. The simple nickel-affinity-purification procedure makes MgUGE a valuable biocatalyst for the synthesis of UDP-galactose from UDP-glucose. The biosynthetic potential of MgUGE was further exemplified in a coupled enzymatic reaction with an oyster-derived ß-1,4-galactosyltransferase (MgGalT7), allowing the galactosylation of the model substrate para-nitrophenol xylose (pNP-xylose) using UDP-glucose as the starting material.

Cholinesterase activity in the cup oyster Saccostrea sp. exposed to chlorpyrifos, imidacloprid, cadmium and copper.

In the present study, the sensitivity and concentration dependence of three functionally-defined components of cholinesterase activity (total: T-ChE; eserine-sensitive: Es-ChE; and eserine-resistant: Er-ChE) were quantified in the gill, digestive gland and adductor muscle of the tropical cup oyster Saccostrea sp., following acute (96h) aqueous exposure to commercial formulations of the organophosphate (OP) insecticide chlorpyrifos and the neonicotinoid (NN) imidacloprid (concentration range: 0.1-100mg/L), as well as to dissolved cadmium and copper (concentration range: 1-1000µg/L). Oysters (1.5-5.0cm shell length), field-collected from a boating marina in Santa Marta, Colombia (Caribbean Sea) were exposed in the laboratory to each substance at five concentrations. T-ChE, Es-ChE, and Er-ChE activity were quantified in the three tissues in pools of 5 individuals (3 replicates per concentration), before and after inhibition with the total cholinesterase inhibitor eserine (physostigmine, 100µM). Oysters exposed to chlorpyrifos, imidacloprid and Cd showed reduced T-ChE and Es-ChE activity in gills at highest exposure concentrations, with Es-ChE activity being inhibited proportionally more so than T-ChE, whereas Er-ChE activity showed no significant concentration-response. Digestive gland also showed diminished T-ChE, Es-ChE and Er-ChE activity for highest chlorpyrifos and Cd concentrations relative to controls, but an increase of T-ChE and Er-ChE activity at the highest imidacloprid concentration (100mg/L). For Cu, T-ChE, Es-ChE and Er-ChE activities in gills and digestive gland were elevated relative to controls in oysters exposed to Cu concentrations > 100µg/L. In adductor muscle, T-ChE, Es-ChE and Er-ChE activity showed no apparent pattern for any of the four xenobiotics and concentration levels tested. Although this study confirms acute (96h) concentration-dependent reduction of tissue T-ChE and Es-ChE activity in gills and digestive glands of Saccostrea sp. exposed to high concentrations of chlorpyrifos (100mg/L), significant changes in T-ChE, Es-ChE and Er-ChE were also caused by exposure to Cd and Cu at concentrations > 100µg/L and by exposure to imidacloprid (100mg/L), indicating that cholinesterase activity is not a specific biomarker of organophosphate exposure in this species, but, rather, a biomarker of diverse xenobiotic exposure.

Integrated assessment of biomarker responses and microbiological analysis of oysters from São Luís Island, Brazil.

This study was conducted to evaluate the use of biochemical biomarkers and microbiological analysis to identify levels of oyster contamination at different ports in São Luís Island (Maranhão), Brazil. Oysters were analyzed for total coliforms, thermotolerant coliforms, Escherichia coli and Aeromonas spp. In addition, tissue was removed from the digestive gland to determine the glutathione-S-transferase (GST) and catalase (CAT) activity. The highest percentage of microbiological contamination of oyster samples occurred during the rainy season. The activity of GST and catalase in oysters was also higher in the rainy season, coinciding with the greatest abundance of total and thermotolerant coliforms. Among the prospective biomarkers, GST showed the best results for identification of areas with higher levels of contamination.

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas†.

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

Angiotensin I-converting enzyme inhibitor derived from cross-linked oyster protein.

Following cross-linking by microbial transglutaminase, modified oyster proteins were hydrolyzed to improve inhibitory activity against angiotensin-converting enzyme (ACE) inhibitory activity with the use of a single protease, or a combination of six proteases. The oyster hydrolysate with the lowest 50% ACE inhibitory concentration (IC50) of 0.40 mg/mL was obtained by two-step hydrolysis of the cross-linked oyster protein using Protamex and Neutrase. Five ACE inhibitory peptides were purified from the oyster hydrolysate using a multistep chromatographic procedure comprised of ion-exchange, size exclusion, and reversed-phase liquid chromatography. Their sequences were identified as TAY, VK, KY, FYN, and YA, using automated Edman degradation and mass spectrometry. These peptides were synthesized, and their IC50 values were measured to be 16.7, 29.0, 51.5, 68.2, and 93.9 µM, respectively. Toxicity of the peptides on the HepG2 cell line was not detected. The oyster hydrolysate also significantly decreased the systolic blood pressure of spontaneously hypertensive rats (SHR). The antihypertensive effect of the oyster hydrolysate on SHR was rapid and long-lasting, compared to commercially obtained sardine hydrolysate. These results suggest that the oyster hydrolysate could be a source of effective nutraceuticals against hypertension.

A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE.

Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells.

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 degrees C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 +/- 0.65 microM and 129.27 +/- 0.38 microM with the substrates 4-methylumbelliferyl-N-N'-diacetylchitobiose and alpha-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.

Inhibitor of REL/NF-KappaB is regulated in Sydney rock oysters in response to specific double-stranded RNA and Vibrio alginolyticus, but the major immune anti-oxidants EcSOD and Prx6 are non-inducible.

Invertebrates, such as oysters have mechanisms to recognize different microbial associated molecular patterns (MAMPs) and recognition of these antigens triggers signaling pathways for the transcription of immune-related proteins. Components of the Rel/NF-(capital KA, Cyrillic)B pathway have been identified in mollusks but the role of different MAMPs in the activation of this pathway is poorly understood. In the current study, the Sydney rock oyster (Saccostrea glomerata) was injected intramuscularly with a range of different MAMPs. Oysters were sampled over a 24 h period and the difference in expression of genes associated with immunity was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA extracted from hemocytes. The results showed that oysters can discriminate between different nucleic acids and double-stranded RNA sequences for activation of the Rel/NF-(capital KA, Cyrillic)B pathway in S. glomerata. Polyinosinic-polycytidylic acid (poly I:C) up-regulated expression of inhibitor of Rel/NF-(capital KA, Cyrillic)B (I(capital KA, Cyrillic)B) by 26-fold and interferon-inhibiting cytokine (IK) by 2.2-fold, whereas polycytidylic acid-polyguanylic acid (poly G:C) and double stranded Vibrio alginolyticus genomic DNA (dsDNA) did not. I(capital KA, Cyrillic)B was also up-regulated 3.8-fold higher for oysters stimulated with V. alginolyticus bacterial cells suggesting that lipopolysaccharide (LPS) also activates the Rel/NF-(capital KA, Cyrillic)B pathway. The expressions of extracellular superoxide dismutase (EcSOD) and peroxiredoxin 6 (Prx6) were not significantly affected by dsRNA, dsDNA or V. alginolyticus bacterial cells suggesting that these two genes, previously demonstrated to be important in the resistance of S. glomerata to disease are not directly inducible by these MAMPs. This is the first study investigating differences in expression kinetics of immune genes in response to challenge from different MAMPs in this economically important bivalve species.

Differential expression of genes encoding anti-oxidant enzymes in Sydney rock oysters, Saccostrea glomerata (Gould) selected for disease resistance.

Sydney rock oysters (Saccostrea glomerata) selectively bred for disease resistance (R) and wild-caught control oysters (W) were exposed to a field infection of disseminating neoplasia. Cumulative mortality of W oysters (31.7%) was significantly greater than R oysters (0.0%) over the 118 days of the experiment. In an attempt to understand the biochemical and molecular pathways involved in disease resistance, differentially expressed sequence tags (ESTs) between R and W S. glomerata hemocytes were identified using the PCR technique, suppression subtractive hybridisation (SSH). Sequencing of 300 clones from two SSH libraries revealed 183 distinct sequences of which 113 shared high similarity to sequences in the public databases. Putative function could be assigned to 64 of the sequences. Expression of nine ESTs homologous to genes previously shown to be involved in bivalve immunity was further studied using quantitative reverse-transcriptase PCR (qRT-PCR). The base-line expression of an extracellular superoxide dismutase (ecSOD) and a small heat shock protein (sHsP) were significantly increased, whilst peroxiredoxin 6 (Prx6) and interferon inhibiting cytokine factor (IK) were significantly decreased in R oysters. From these results it was hypothesised that R oysters would be able to generate the anti-parasitic compound, hydrogen peroxide (H(2)O(2)) faster and to higher concentrations during respiratory burst due to the differential expression of genes for the two anti-oxidant enzymes of ecSOD and Prx6. To investigate this hypothesis, protein extracts from hemolymph were analysed for oxidative burst enzyme activity. Analysis of the cell free hemolymph proteins separated by native-polyacrylamide gel electrophoresis (PAGE) failed to detect true superoxide dismutase (SOD) activity by assaying dismutation of superoxide anion in zymograms. However, the ecSOD enzyme appears to generate hydrogen peroxide, presumably via another process, which is yet to be elucidated. This corroborates our hypothesis, whilst phylogenetic analysis of the complete coding sequence (CDS) of the S. glomerata ecSOD gene is supportive of the atypical nature of the ecSOD enzyme. Results obtained from this work further the current understanding of the molecular mechanisms involved in resistance to disease in this economically important bivalve, and shed further light on the anomalous oxidative processes involved.

Association among growth, food consumption-related traits and amylase gene polymorphism in the Pacific oyster Crassostrea gigas.

To examine further a previously reported association between amylase gene polymorphism and growth in the Pacific oyster Crassostrea gigas, ecophysiological parameters and biochemical and molecular expression levels of alpha-amylase were studied in Pacific oysters of different amylase genotypes. Genotypes that previously displayed significantly different growth were found to be significantly different for ingestion and absorption efficiency. These estimated parameters, used in a dynamic energy budget model, showed that observed ingestion rates (unlike absorption efficiencies) allowed an accurate prediction of growth potential in these genotypes. The observed association between growth and amylase gene polymorphism is therefore more likely to be related to ingestion than to absorption efficiency. Additionally, relative mRNA levels of the two amylase cDNAs were also strongly associated with amylase gene polymorphism, possibly reflecting variation in an undefined regulatory region, although no corresponding variation was observed in specific amylase activity. Amylase gene sequences were determined for each genotype, showing the existence of only synonymous or functionally equivalent non-synonymous polymorphisms. The observed associations among growth, food consumption-related traits and amylase gene polymorphism are therefore more likely to be related to variation in the level of amylase gene expression than to functional enzymatic variants.

Control of Listeria monocytogenes and Salmonella anatum on the surface of smoked salmon coated with calcium alginate coating containing oyster lysozyme and nisin.

This study investigated the antimicrobial effect of oyster lysozyme with or without nisin added to calcium alginate (CaAlg) coated on the surface of smoked salmon against Listeria monocytogenes and Salmonella anatum. L. monocytogenes or S. anatum inoculated smoked salmon samples (1 g) were dipped into CaAlg with either oyster lysozyme (OysL) or hen egg white lysozyme (HEWL), with or without added nisin (N), then stored at 4 degrees C for 35 d. Our results indicated that the effectiveness of oyster lysozyme or hen egg white lysozyme was enhanced when added to calcium alginate coatings. After 35 d at 4 degrees C the growth of L. monocytogenes and S. anatum was suppressed in the range of 2.2 to 2.8 log CFU/g with CaAlgNOysL or CaAlgNHEWL coatings compared to the control nontreated samples. There was no significant difference between oyster lysozyme and hen egg white lysozyme treatments against L. monocytogenes or S. anatum inoculated on the surface of salmon. Calcium alginate coatings containing lysozyme with nisin or without could be used to reduce the growth of L. monocytogenes and S. anatum on the surface of ready-to-eat smoked salmon at refrigerated temperatures.

Phenoloxidase-associated cellular defence in the Sydney rock oyster, Saccostrea glomerata, provides resistance against QX disease infections.

The enzyme phenoloxidase is a critical component of the immunological defence of invertebrates. Previously, we have shown that the activity of phenoloxidase in Sydney rock oysters (Saccostrea glomerata) correlates with the severity of QX disease outbreaks. The aetiological agent of QX disease is the opportunistic protozoan parasite, Marteilia sydneyi. In this study, we examined the response of oyster haemocytes to challenge with M. sydneyi. Granular haemocytes were able to rapidly phagocytose parasite sporonts. Phagocytosis stimulated intracellular associated phenoloxidase activity that led to the complete melanisation of phagosomes. Significant differences in phagocytic indexes and phenoloxidase activities were observed between oysters selected for resistance to QX disease (QXR) and non-selected wild-type oysters. The data suggest that phagocytosis and cellular melanisation are critical defensive responses of Sydney rock oysters infected with M. sydneyi.

Effects of noradrenaline on immunological activity in Sydney rock oysters.

This study investigates the effects of noradrenaline injection on a range of immunological activities in Sydney rock oysters (Saccostrea glomerata). Noradrenaline caused a decrease in most of the immunological parameters tested. Phenoloxidase activities in both whole hemolymph and serum decreased significantly within the first 60 min of noradrenaline injection, as did the total frequency of hemocytes in hemolymph, differential hemocyte frequencies, the frequency of phenoloxidase positive cells and phagocytic activity. All of these parameters started to return to normal levels within 120 min. In contrast, the total protein content of hemolymph, which also decreased after noradrenaline injection continued to decline throughout the experimental period. In vitro studies found that superoxide and peroxide production by hemocytes increased in the presence of noradrenaline, but acid phosphatase activity decreased significantly. Additional experiments showed that noradrenaline secretion was stimulated by altered salinity, altered temperature and physical agitation. This suggests that stressors commonly associated with oyster farming may result in noradrenaline-based stress responses that suppress immunological activity.

Induction of phenoloxidase and other immunological activities in Sydney rock oysters challenged with microbial pathogen-associate molecular patterns.

This study investigates the effects of two pathogen-associated molecular patterns (PAMPs), LPS and zymosan, on the Sydney rock oyster (Saccostrea glomerata) immune system. Phenoloxidase and phagocytic activities, total and differential haemocyte frequencies, as well as peroxide and superoxide concentrations were measured after the injection of lipopolysaccharide and zymosan. All of the immunological parameters were induced by both PAMPs. Phenoloxidase (monophenolase and diphenolase) and phagocytic activities, as well as the frequencies of phenoloxidase-positive haemocytes, hyalinocytes and granulocytes in the haemolymph, increased within 24 h of PAMP injection. Values for all of these parameters peaked within 48 h of challenge and began to decrease to levels that were indistinguishable from those of controls within 96h. The only exception to this pattern was diphenolase activity, which remained elevated for at least 96 h. Control saline injections that lacked PAMPs also induced responses in most of the parameters measured. However, reactions to saline injections were of far lower magnitude compared to those induced by PAMPs. All of the data suggest that the phenoloxidase and phagocytic systems of oysters are inducible components of the Sydney rock oyster immune system, and that induction is primarily due to increased frequencies of specialised haemocytes in the haemolymph.

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata).

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca2+ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (Km=4.45+/-1.46 mM) than for the diphenol, l-DOPA (Km=10.27+/-1.33 mM). Maximum enzyme activity was evident at 37 degrees C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.

A tolloid homologue from the Pacific oyster Crassostrea gigas.

The genes governing mesoderm specification have been extensively studied in vertebrates, arthropods and nematodes. The latter two phyla belong to the Ecdysozoan clade but little is understood of the role that these genes might play in the development of the other major protostomal clade, the Lophotrochozoa. As part of a wider project to analyze the functions associated with transforming growth factor beta superfamily members in Lophotrochozoa, we have cloned a gene encoding a tolloid homologue from the bivalve mollusc Crassostrea gigas. Tolloid is a key developmental protein that regulates the activity of bone morphogenetic proteins (BMPs). We have determined the intron-exon structure of the gene encoding C. gigas tolloid and have compared it with those of homologous genes from both protostomes and deuterostomes. In order to analyze the functionality of oyster tolloid the zebrafish embryo has been employed as a reporter organism and we show that over-expression of this protein results in the ventralization of zebrafish embryos at 24h post fertilization. The expression of the C. gigas tolloid gene during embryonic and larval development as well as in adult tissues is also explored.

Estimation of the critical effect level for pollution prevention based on oyster embryonic development toxicity test: the search for reliability.

In spite of the consideration that toxicity testing is a reduced approach to measure the effects of pollutants on ecosystems, the early-life-stage (ELS) tests have evident ecological relevance because they reflect the possible reproductive impairment of the natural populations. The procedure and validation of Crassostrea rhizophorae embryonic development test have shown that it meets the same precision as other U.S. EPA tests, where EC(50) is generally used as a toxicological endpoint. However, the recognition that EC(50) is not the best endpoint to assess contaminant effects led U.S. EPA to recently suggest EC(25) as an alternative to estimate xenobiotic effects for pollution prevention. To provide reliability to the toxicological test results on C. rhizophorae embryos, the present work aimed to establish the critical effect level for this test organism, based on its reaction to reference toxicants, by using the statistical method proposed by Norberg-King (Inhibition Concentration, version 2.0). Oyster embryos were exposed to graded series of reference toxicants (ZnSO(4) x 7H(2)O; AgNO(3); KCl; CdCl(2)H(2)O; phenol, 4-chlorophenol and dodecyl sodium sulphate). Based on the obtained results, the critical value for C. rhizophorae embryonic development test was estimated as EC(15). The present research enhances the emerging consensus that ELS tests data would be adequate for estimating the chronic safe concentrations of pollutants in the receiving waters. Based on recommended criteria and on the results of the present research, zinc sulphate and 4-chlorophenol have been pointed out, among the inorganic and organic compounds tested, as the best reference toxicants for C. rhizophorae ELS-test.

Breeding for QX disease resistance negatively selects one form of the defensive enzyme, phenoloxidase, in Sydney rock oysters.

QX disease in Sydney rock oysters (Saccostrea glomerata) is caused by the paramyxean protozoan, Marteilia sydneyi. Disease outbreaks occur during summer (January to May) and can result in up to 95% mortality. The New South Wales Department of Primary Industries has been selectively breeding S. glomerata for resistance to QX disease since 1996. Previous work suggests that this breeding program has specifically affected the defensive phenoloxidase enzyme system of oysters. The current study more thoroughly characterises the effect of selection on the different forms of phenoloxidase found in oyster populations. Native polyacrylamide gel electrophoresis (native-PAGE) identified five discrete types of phenoloxidase in non-selected (wild type) and fourth generation QX disease resistant (QXR4) oysters. One electrophoretically distinct form of phenoloxidase, POb, is significantly less frequent in resistant oysters when compared to the wild type population. The frequency of POb also decreased in both the wild type and QXR4 populations over the course of a QX disease outbreak. This suggests that possession of POb makes oysters susceptible to QX disease and that breeding for resistance has resulted in negative selection against this form of phenoloxidase.