Occurrence and Seasonal Monitoring of Domoic Acid in Three Shellfish Species from the Northern Adriatic Sea.

As filter feeders, bivalves and ascidians can accumulate contaminants present in the environment and pass them on to higher food chain levels as vectors. The consumption of bivalves contaminated with the potent neurotoxin domoic acid (DA) can cause amnesic shellfish poisoning in humans. The aim of this study was to determine seasonal differences in occurrence and accumulation of this phycotoxin in European oysters (Ostrea edulis Linnaeus, 1758) (n = 46), Queen scallops (Aequipecten opercularis Linnaeus, 1758) (n = 53), and edible ascidians of the Microcosmus spp. (n = 107), originating from the same harvesting area in the Northern Adriatic Sea. The quantification was performed using ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) preceded by derivatization with dansyl chloride. DA was found in very low concentrations throughout the year, with a maximum value of 810 µg/kg in Queen scallops. This study reveals differences in the occurrence and accumulation of DA between Queen scallops and the other two investigated species (oysters and ascidians) and the highest concentrations during the colder part of the year. Even though DA was detected in all of them, Queen scallops showed higher DA accumulation compared to the other two (p < 0.001), hence representing a sentinel species suitable for the monitoring of DA level in seafood.

Quantitative and comparative study of plasmalogen molecular species in six edible shellfishes by hydrophilic interaction chromatography mass spectrometry.

Shellfishes contain plasmalogens correlating to the functions of brain, heart, etc. Herein, a mild acid hydrolysis and hydrophilic interaction chromatography (HILIC) tandem mass spectrometry method was developed for analyzing plasmalogens in six shellfish species. A total of 19 plasmalogen molecular species were successfully identified, including nine phosphatidylcholine plasmalogen (plasPC), seven phosphatidylethanolamine plasmalogen (plasPE), and three phosphatidylserine plasmalogen (plasPS). The quantitative results indicated that mussel (32 µg·mg-1) possessed the highest content of plasmalogens, followed by oyster (21 µg·mg-1) and razor clam (15 µg·mg-1). The statistic models showed that the plasPE P-18:0/20:5 (m/z 748), plasPE P-16:0/22:2 & P-18:0/20:2 (m/z 754) and plasPS were the most contributing difference between shellfishes. The results indicated that this method was sensitive and precise to determine plasmalogens in shellfish, and mussel was demonstrated to be a good choice for the large-scale preparation of plasmalogens.

Is pallial mucus involved in Ostrea edulis defenses against the parasite Bonamia ostreae?

Bonamia ostreae is an intrahemocytic parasite that has been responsible for severe mortalities in the flat oyster Ostrea edulis since the 1970́s. The Pacific oyster Crassostrea gigas is considered to be resistant to the disease and appears to have mechanisms to avoid infection. Most studies carried out on the invertebrate immune system focus on the role of hemolymph, although mucus, which covers the body surface of molluscs, could also act as a barrier against pathogens. In this study, the in vitro effect of mucus from the oyster species Ostrea edulis and C. gigas on B. ostreae was investigated using flow cytometry. Results showed an increase in esterase activities and mortality rate of parasites exposed to mucus from both oyster species. In order to better understand the potential role of mucus in the defense of the oyster against parasites such as B. ostreae, liquid chromatography and tandem mass spectrometry were used to describe and compare mucus protein composition from both species. In all oyster species, pallial mucus contains a high level of proteins; however, O. edulis mucus produced a variety of proteins that could be involved in the immune response against the parasite, including Cu/Zn extracellular superoxide dismutase, thioxiredoxin, peroxiredon VI, heat shock protein 90 as well as several hydrolases. Conversely, a different set of antioxidant proteins, hydrolases and stress related proteins were identified in mucus from C. gigas. Our results suggest an innate immunity adaptation of oysters to develop a specific response against their respective pathogens. The mucosal protein composition also provides new insights for further investigations into the immune response in oysters.

Calcium carbonate unit realignment under acidification: A potential compensatory mechanism in an edible estuarine oyster.

Ocean acidification (OA) is well-known for impairing marine calcification; however, the end response of several essential species to this perturbation remains unknown. Decreased pH and saturation levels (Ω) of minerals under OA is projected to alter shell crystallography and thus to reduce shell mechanical properties. This study examined this hypothesis using a commercially important estuarine oyster Magallana hongkongensis. Although shell damage occurred on the outmost prismatic layer and the undying myostracum at decreased pH 7.6 and 7.3, the major foliated layer was relatively unharmed. Oysters maintained their shell hardness and stiffness through altered crystal unit orientation under pH 7.6 conditions. However, under the undersaturated conditions (ΩCal ~ 0.8) at pH 7.3, the realigned crystal units in foliated layer ultimately resulted in less stiff shells which indicated although estuarine oysters are mechanically resistant to unfavorable calcification conditions, extremely low pH condition is still a threat to this essential species.

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.

Polysaccharide from Ostrea rivularis attenuates reproductive oxidative stress damage via activating Keap1-Nrf2/ARE pathway.

The purpose of this study was to investigate the effects of Ostrea rivularis polysaccharide (ORP) against testicular oxidative stress injury via kelch-like ECH-associated protein 1-nuclear erythroid 2-related factor 2/antioxidant response element (Keap1-Nrf2/ARE) pathway. In pharmacological experiments in vivo, ORP administration could dose-dependently inhibit body and testicular weight loss, ameliorate epididymal sperm quality and protect reproductive impairment in cyclophosphamide-induced male Balb/c mice. Moreover, the mechanism in vivo might be elucidated that ORP could increase expression level of Nrf2 and its downstream ARE gene battery in the testis, promote production of corresponding antioxidative enzymes and protein, and enhance Keap1-Nrf2/ARE signaling pathway to avoid male reproductive dysfunction. In addition, ORP treatment could improve survival capacity of H2O2-induced TM4 cells and its antioxidant mechanism in vitro also had been verified to activate Keap1-Nrf2/ARE signaling pathway. Overall, these results showed that ORP as a potent antioxidant could reduce reproductive oxidative stress damage related to Keap1-Nrf2/ARE pathway.

Oyster (Ostrea plicatula Gmelin) polysaccharides intervention ameliorates cyclophosphamide-Induced genotoxicity and hepatotoxicity in mice via the Nrf2-ARE pathway.

In this study, we explored the protective effects of oyster (Ostrea plicatula Gmelin) polysaccharides (OPS) against genotoxicity and liver injury induced by cyclophosphamide (CP) in BALB/c mice. OPS was administered to mice at doses of 100 and 200mg/kg for 7 consecutive days, then 50mg/kg CP was injected via abdomen. Then mice were sacrificed and samples were collected. Bone marrow micronuclei (MN) and polychromatic erythrocytes (PCE): normochromatic erythrocytes (NCE) ratio were calculated to evaluate CP induced genetic toxicity. Activites of transaminase and antioxidants in serum as well as liver histopathology were examined to assess the severity of liver damage. We further investigate the molecular mechanism by Western blot analysis. When CP induced group pretreated with OPS, the generation of MN was obviously reduced accompanying by the restoration of PCE: NCE ratio. We also found that pretreatment of mice with OPS markedly reduced the release of serum alanine transaminase (ALT) and aspartate transferase (AST). Histological examination and grading evaluation also showed that OPS could significantly attenuated CP-induced liver damage. At the same time, OPS supplementation attenuated CP-induced oxidative stress as evident by the alternation of malondialdehyde (MDA) and superoxide dismutase (SOD) activity. Furthermore, CP induced mice showed the downregulation of Nrf2 (nuclear factor E2 - related factor 2) - ARE (antioxidant response element) and the downstream genes i.e. NAD(P)H: quinine oxidoreductase-1 (NQO - 1) and Hemoxygenase-1 (HO - 1), which were obviously reversed by OPS pretreatment. In conclusion, OPS protects against the genotoxicity and hepatotoxicity induced by CP in vivo. The beneficial effect may depend on activation of Nrf2 - ARE pathway and subsequent suppression of oxidative stress and genetic toxicity.

Liquid Chromatography with a Fluorimetric Detection Method for Analysis of Paralytic Shellfish Toxins and Tetrodotoxin Based on a Porous Graphitic Carbon Column.

Paralytic shellfish toxins (PST) traditionally have been analyzed by liquid chromatography with either pre- or post-column derivatization and always with a silica-based stationary phase. This technique resulted in different methods that need more than one run to analyze the toxins. Furthermore, tetrodotoxin (TTX) was recently found in bivalves of northward locations in Europe due to climate change, so it is important to analyze it along with PST because their signs of toxicity are similar in the bioassay. The methods described here detail a new approach to eliminate different runs, by using a new porous graphitic carbon stationary phase. Firstly we describe the separation of 13 PST that belong to different groups, taking into account the side-chains of substituents, in one single run of less than 30 min with good reproducibility. The method was assayed in four shellfish matrices: mussel (Mytillus galloprovincialis), clam (Pecten maximus), scallop (Ruditapes decussatus) and oyster (Ostrea edulis). The results for all of the parameters studied are provided, and the detection limits for the majority of toxins were improved with regard to previous liquid chromatography methods: the lowest values were those for decarbamoyl-gonyautoxin 2 (dcGTX2) and gonyautoxin 2 (GTX2) in mussel (0.0001 mg saxitoxin (STX)·diHCl kg(-1) for each toxin), decarbamoyl-saxitoxin (dcSTX) in clam (0.0003 mg STX·diHCl kg(-1)), N-sulfocarbamoyl-gonyautoxins 2 and 3 (C1 and C2) in scallop (0.0001 mg STX·diHCl kg(-1) for each toxin) and dcSTX (0.0003 mg STX·diHCl kg(-1) ) in oyster; gonyautoxin 2 (GTX2) showed the highest limit of detection in oyster (0.0366 mg STX·diHCl kg(-1)). Secondly, we propose a modification of the method for the simultaneous analysis of PST and TTX, with some minor changes in the solvent gradient, although the detection limit for TTX does not allow its use nowadays for regulatory purposes.

Purification, preliminary characterization and bioactivities of polysaccharides from Ostrea rivularis Gould.

In this study, purification, preliminary characterization and biological activities of water-soluble polysaccharides from Ostrea rivularis Gould (ORP) were investigated. Firstly, crude ORP was extracted by enzyme-assisted extraction and then sequentially purified by chromatography of DEAE-52 and Sephadex G-100, producing one main purified fractions of ORPp. Furthermore, the preliminary characterization of ORPp was studied, and its antioxidant and spermatogenesis activities were evaluated. Experimental results showed that ORPp was mainly composed of glucose (76.3%) and galactose (23.7%). The average molecular weight of ORPp was 118 kDa. Besides, ORPp showed strong antioxidant activities in vitro. For the experiments of antioxidant activities in vivo, ORPp can significantly inhibited the formation of MDA in rats' serums, and raised the activities of antioxidant enzymes and the level of total antioxidant capacity (TAOC). Furthermore, ORPp could significantly increase the weights of male rats' sexual organs, promote sperm motility and raise epididymal sperm counts. These results suggest that ORPp could be a new source of natural antioxidants and spermatogenic agent with its potential usage in developing novel supplements and medicines.

[Study on medication regularity of grand master of traditional Chinese medicine YAN Zheng-hua's Ostreae Concha-containing prescriptions based on data mining].

In this study, prescriptions were collected to establish a database based on the traditional Chinese medicine inheritance support system (TCMISS). Such data-mining methods as software's statistical statement module, data analysis module and apriori algorithm were used to analyze the frequency of single drug, the frequency of drug combination, the association rules and the core drug combinations of ostreae concha-containing prescriptions. The results showed that Ostreae Concha-containing prescriptions were frequently used to treat insomnia, vertigo, stomach-ache and other syndromes. The frequently used drugs included Ossis Mastodi Fossilia, Parched Ziziphi Spinosae Semen, Polygoni Multiflori Caulis, Salviae Miltiorrhizae Radix and Paeoniae Rubra Radix. The frequently used drug combinations included "Ostreae Concha and Ossis Mastodi Fossilia", "Ostreae Concha and Parched Ziziphi Spinosae Semen", and "Ostreae Concha and Polygoni Multiflori Caulis". The drug association rules with the confidence coefficient of more than 0. 95 included "Ossis Mastodi Fossilia-->Ostreae Concha", "Cocos Poria-Ossis Mastodi Fossilia-Ostreae Concha", "Ossis Mastodi Fossilia-Parched Ziziphi Spinosae Semen-->Ostreae Concha", and "Salviae Miltiorrhizae Radix-Ossis Mastodi Fossilia--Ostreae Concha".

Crystallographic reorganization of the calcitic prismatic layer of oysters.

The calcitic columnar prisms of pteriomorphian bivalves have the crystallographic c-axis oriented perpendicular to the shell surface and the a-axes rotated without any preferential orientation. In oysters, SEM, XRD and EBSD analyses show that individual prisms initially have their a-axes randomly oriented but are able to progressively orient them parallel to those of their neighbors. This ability is apparently confined to groups, such as oysters and scallops, in which prisms are internally constituted by smaller lath-like crystal units. We have developed a competition model - not between prisms, but between the lath-like secondary units of prisms - which is based on differences in the inclination of laths relative to the shell growth surface. Units having a growth component which coincides with the growth direction protrude faster from the growth surface and out-compete those which are not favorably oriented, which reduces the overall dispersion of the a-axes of the prismatic lamella. The extent of re-alignment increases with the relative inclination of the growth surface and the length attained by the prisms. Oysters are the only group in which these two characters are pronounced enough to provide a measurable re-alignment. The proposed competition model is unprecedented in biomaterials and reveals how important crystal growth processes are in microstructure organization.

Design, synthesis and structure-activity study of shorter hexa peptide analogues as HIV-1 protease inhibitors.

Inhibition of HIV-1 protease enzyme can render the Human Immunodeficiency Virus (HIV-1) non-infectious in vitro. Previous studies have shown that several shorter peptides were discovered as HIV-1 protease inhibitors. In this context, a series of shorter synthetic hexapeptides, Leu-Leu-Glu-Tyr-Val-Xaa (Xaa=Phe, Met, Tyr and Trp), were designed. The synthesized hexa peptides were screened for their HIV-1 protease inhibition. These peptides showed moderately good HIV-1 protease inhibition when compared to acetyl pepstatin.

Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules.

We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10(-3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.

A new adenosyl-alkaloid from Ostrea rivularis.

A new adenosyl-alkaloid, ostrerine A, has been isolated along with an amino acid, tryptophan and a ribonucleoside, 2'-deoxythymidine from the Quanzhou marine mollusk, Ostrea rivularis, and the structures were elucidated by 1D and 2D NMR experiments, including (1)H-(1)H COSY, TOCSY, NOESY, HMQC, and HMBC methods.

The red tide event in El Salvador, August 2001-January 2002

A red tide event occurred in El Salvador from August 2001 to January 2002. National health authorities usually measured toxin levels in Ostrea iridescens, however other species were analyzed during this microalgae bloom: Anadara similis, Anadara tuberculosa and Modiolus sp. El Salvador authorities consider 400 mouse units/100 g the highest value that is safe for human health. During this period toxin levels in O. iridescens and Modiolus sp. increased from values under 400 to 3 977 and 15 468 mouse units/100 g, respectively. Persistent and higher levels were recorded in oyster and mussel banks on the west part of the country. The Ministry of Health and Social Assistance treated 41 slight to moderate intoxications associated to bivalve mollusks consumption

Hubo una marea roja en El Salvador desde agosto de 2001 hasta enero de 2002. Las autoridades de salud usualmente medían niveles de toxinas en Ostrea iridescens, pero durante esta proliferación de microalgas se estudió también Anadara similis, Anadara tuberculosa y Modiolus sp. En El Salvador se consideran 400 unidades ratón/ 100 g como el límite sobre el cual el consumo de moluscos representa riesgo para la salud humana. Durante este período se detectó que los niveles de toxinas en O. iridescens y Modiolus sp. variaron desde niveles menores a 400 hasta 3 977 y 15 468 unidades ratón/100 g, respectivamente. Los niveles más altos y persistentes se registraron en los bancos de ostras y mejillones de la zona oeste del país. El Ministerio de Salud y Asistencia Social atendio 41 intoxicaciones de leves a moderadas atribuidas al consumo de moluscos bivalvos

The red tide event in El Salvador, August 2001-January 2002.

A red tide event occurred in El Salvador from August 2001 to January 2002. National health authorities usually measured toxin levels in Ostrea iridescens, however other species were analyzed during this microalgae bloom: Anadara similis, Anadara tuberculosa and Modiolus sp. El Salvador authorities consider 400 mouse units/100 g the highest value that is safe for human health. During this period toxin levels in 0. iridescens and Modiolus sp. increased from values under 400 to 3977 and 15,468 mouse units/100 g, respectively. Persistent and higher levels were recorded in oyster and mussel banks on the west part of the country. The Ministry of Health and Social Assistance treated 41 slight to moderate intoxications associated to bivalve mollusks consumption.