Efficient production of a highly active lysozyme from European flat oyster Ostrea edulis.

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZdm) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZdm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 103 U mL-1 to 2.11 × 104 U mL-1 in shake flask culture, and eventually reaching 2.05 × 105 U mL-1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme.

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Effect of carboxymethyl chitosan on the detoxification and biotransformation of paralytic shellfish toxins in oyster Ostrea rivularis.

Economic bivalve ingested toxic algae causes frequent human poisoning events. To explore new compounds that can accelerate the depuration of toxins in shellfish, we investigated the detoxification of the paralytic shellfish toxins (PSTs) and the biotransformation pathway of PSTs during detoxification by the application of three treatments to a toxic bloom, Alexandrium minutum (A. minutum). The detoxification effect of Platymonas subcordiformis (PS) mixed with carboxymethyl chitosan (CMC) group is significantly better than the starving group in each oyster tissues. The toxicity of viscera which occupied 78.95% of total toxicity reduced to 155 MU/100g after 13 days' depuration experiment. And adding CMC could significantly achieve rapid detoxification and effectively reduce the STX to 0.07 µmol/100 g in viscera. Meanwhile, PSTs underwent biotransformation during the depuration process, which mainly manifested as GTX1/4→GTX2/3→STX, GTX2→dcGTX2. This study explored a new strategy for toxin depuration in shellfish.

Accumulation and biotransformation dynamics of the neurotoxic complex, saxitoxin, in different life stages of Ostrea chilensis.

The neurotoxic complex saxitoxin, is a group of marine toxins that historically has significantly impacted human health and the ability to utilize marine resources. A steady increase in the distribution and intensity of Alexandrium catenella blooms in Chile, and around the world, has caused major ecological and socioeconomic impacts, putting this type of dinoflagellate, and its toxicity, in the spotlight. Ostrea chilensis is a commercially and ecologically important resource harvested from wild populations and farmed in centers of southern Chile, where it is exposed to large harmful algal blooms of the type that can cause paralysis in humans. This study contributes to our understanding about the transfer of toxins from A. catenella cells to juvenile and adult Ostrea chilensis by tracking transformations of the neurotoxic complex until it reaches its most stable molecular form in the intracellular environment of O. chilensis tissues. These biotransformations are different in O. chilensis juveniles and adults, indicating a differentiated response for these two life stages of this bivalve species. These studies can be used for similar analyses in other ecologically and commercially important species of filter feeding organisms, providing greater understanding of the specific interactions of bivalves in scenarios of toxic dinoflagellate proliferations (e.g. A. catenella blooms).

Changes in the biochemical and nutrient composition of seafood due to ocean acidification and warming.

Ocean acidification and warming may threaten future seafood production, safety and quality by negatively impacting the fitness of marine species. Identifying changes in nutritional quality, as well as species most at risk, is crucial if societies are to secure food production. Here, changes in the biochemical composition and nutritional properties of the commercially valuable oysters, Magallana gigas and Ostrea edulis, were evaluated following a 12-week exposure to six ocean acidification and warming scenarios that were designed to reflect the temperature (+3 °C above ambient) and atmospheric pCO2 conditions (increase of 350-600 ppm) predicted for the mid-to end-of-century. Results suggest that O. edulis, and especially M. gigas, are likely to become less nutritious (i.e. containing lower levels of protein, lipid, and carbohydrate), and have reduced caloric content under ocean acidification and warming. Important changes to essential mineral composition under ocean acidification and warming were evident in both species; enhanced accumulation of copper in M. gigas may be of concern regarding consumption safety. In light of these findings, the aquaculture industry may wish to consider a shift in focus toward species that are most robust to climate change and less prone to deterioration in quality, in order to secure future food provision and socio-economic benefits of aquaculture.

Anti-fatigue and anti-oxidant activities of oyster (Ostrea rivularis) hydrolysate prepared by compound protease.

Oyster, which is rich in protein and widely used as a marine traditional Chinese medicine, was believed to have good curative effects in health care and on chronic diseases. This study was designed to evaluate the anti-fatigue and anti-oxidant effects of oyster hydrolysate. Oyster meat (OM) was hydrolyzed with a complex protease, and oyster hydrolysate (OH) was separated by a 6 kDa ultrafiltration membrane into two fractions, OH-I (<6 kDa) and OH-II (≥6 kDa). The anti-fatigue effects of OM, OH, OH-I and OH-II groups were first investigated, and then the antioxidant activities of OH-I and OH-II were further analyzed. Anti-fatigue experimental results showed that OH-I displayed the strongest activity among the four groups. Compared to the control group, OH-I significantly prolonged swimming time (67.79%), increased the content of muscle glycogen (45.65%) and liver glycogen (49.01%), and reduced the content of blood urea nitrogen (BUN) (18.44%) (P < 0.05). Meanwhile, OH-I showed excellent chemical and cellular antioxidant activities, especially when the concentration increased; its antioxidant activity was significantly better than that of OH-II (P < 0.05). Results of an amino acid analysis showed that OH-I was rich in branched-chain amino acids (10.84 g per 100 g), Glu (8.63 g per 100 g), Tau (1.68 g per 100 g), Asp (5.02 g per 100 g) and Arg (3.61 g per 100 g), which were expected to contribute to its antioxidant and anti-fatigue activities.

Proteomic profile of Ostrea edulis haemolymph in response to bonamiosis and identification of candidate proteins as resistance markers.

European flat oyster Ostrea edulis populations have suffered extensive mortalities caused by bonamiosis. The protozoan parasite Bonamia ostreae is largely responsible for this disease in Europe, while its congener B. exitiosa has been detected more recently in various European countries. Both of these intracellular parasites are able to survive and proliferate within haemocytes, the main cellular effectors of the immune system in molluscs. Two-dimensional electrophoresis was used to compare the haemolymph protein profile between Bonamia spp.-infected and non-infected oysters within 3 different stocks, a Galician stock of oysters selected for resistance against bonamiosis, a non-selected Galician stock and a selected Irish stock. Thirty-four proteins with a presumably relevant role in the oyster-Bonamia spp. interaction were identified; they were involved in major metabolic pathways, such as energy production, respiratory chain, oxidative stress, signal transduction, transcription, translation, protein degradation and cell defence. Furthermore, the haemolymph proteomic profiles of the non-infected oysters of the 2 Galician stocks were compared. As a result, 7 proteins representative of the non-infected Galician oysters selected for resistance against bonamiosis were identified; these 7 proteins could be considered as candidate markers of resistance to bonamiosis, which should be further assessed.

Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae.

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30 d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.

Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: morphology and functional aspects.

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses.

Kinetic study of the bioaccumulation of heavy metals (Cu, Pb, and Cd) in Chinese domestic oyster Ostrea plicatula.

Much attention has been given to heavy metal contamination in coastal waters and on seafood safety induced by bioaccumulation of aquatic organisms in China. To obtain the kinetic characteristics of heavy metal bioaccumulation in Chinese native organisms, the accumulation and elimination of three heavy metals (Cu, Pb, and Cd) in domestic oyster Ostrea plicatula were investigated in both laboratory and field conditions. The kinetic parameters of bioaccumulation, including the uptake rate constant (K(1)), elimination rate constant (K(2)), bioconcentration factors (BCFs), and biological half-life (t(1/2)), were estimated through nonlinear curve fitting using a two-compartment model. The results showed that the Pb and Cd metal concentration data perfectly fitted the two-compartment model, and good agreement was obtained between the predicted data and those obtained by the goodness-of-fit test. However, the Cu data did not fit the model. The modeling results indicated that the Pb and Cd concentrations in O. plicatula could reflect the contamination status of seawater and that O. plicatula is a potentially dependable indicator for monitoring heavy metal (Pb and Cd) contamination and seafood safety in the coastal areas of China.

Profiles and levels of fatty acid esters of okadaic acid group toxins and pectenotoxins during toxin depuration. Part II: blue mussels (Mytilus edulis) and flat oyster (Ostrea edulis).

Bivalve molluscs accumulate toxins of the okadaic acid (OA) and pectenotoxin (PTX) groups, which are frequently found in Dinophysis spp. Transformation of the OA-group toxins into fatty acid ester derivatives (often designated "DTX3") is common in many bivalve species but the degree to which these toxins are transformed vary between species, and is also depending on the parent toxin involved. In this paper, detailed profiles and levels of fatty acid esters of OA, DTX1, DTX2 and PTX2 SA were studied in blue mussels (Mytilus edulis) and European flat oysters (Ostrea edulis), collected during a bloom of Dinophysis spp. and after 3 and 6 weeks of depuration. Analysis of samples by HPLC-MS/MS and HPLC-MS(2) revealed some differences in identity and abundance of fatty acid moieties of the OA-group esters between species, but the 16:0 fatty acid esters dominated in both oysters and mussels, which is in accordance with the free fatty acid profiles in these species. A wider range of PTX2 SA-esters were detected compared to esters of the OA-group toxins in both mussels and oysters, and in oysters, both 14:0, 18:4 and 20:5 fatty acid side chains were more common than 16:0. OA-group toxins were esterified to a larger degree in oysters (83-93%) compared to mussels (21-41%), and in mussels a higher proportion of OA was esterified compared to DTX1 and DTX2. Contrary to what was observed for OA-group toxins, PTX2 SA was esterified to a larger degree in mussels (81%) compared to oysters (64%). Calculations of depuration rates for all individual esters of each parent compound showed that the esters of DTX1 depurated significantly slower from both mussels and oysters compared to esters of OA, DTX2 and PTX2 SA, but overall the depuration rates of esters of both toxin group were highly similar for both species. This indicated that differences in depuration rates are not causing the large species-specific differences in levels and profiles of these toxins. Instead, the results for the OA-group toxins suggested that a higher rate of esterification in oysters is the main factor causing the observed differences in the proportion of esters to free toxin. For PTX2 SA, large differences in ester profiles and a higher proportion of esters of PTX2 SA in mussels compared to oysters suggested differential assimilation and metabolic rate processes for the PTXs compared to OA-group toxins between these species. Hence, although produced by the same Dinophysis species, conclusions about the dynamics of one toxin group based on results from the other group should be avoided in future studies.

Protamines and other basic proteins from spermatozoa of molluscs.

The basic proteins obtained from spermatozoa of different species of the phylum Mollusca have been extracted and fractionated. The amino acid analysis and electrophoretic mobility of these proteins show a considerable variation in the types and relative amounts of the components present in different species. In some case-(Gibbula, Haliotis, Loligo, Octopus) the main components are similar to the protamines found in the salmonid fishes, although they appear to be larger in size (40-80 amino acids) and show significant differences in amino acid composition. In other cases (Mytilus, Chiton) a complex mixture of proteins is present, which including somatic-like histones and proteins intermediate in size and composition betweeln protamines and histones. Other molluscs (Ostrea, Spisula, Patella) also contain proteins intermediate in composition between protamines and histones, but their molecular weight appears to be larger than in histones. In Eledone a complex mixture of proteins containing cystine is obtained, with some components rich in arginine. In most species, somatic-like histones are also present. Their type and relative amount are different in each species. The significance of these results towards an understanding of the evolutionary history of these proteins is discussed. It is suggested that these proteins evolved from histone precursors.