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Chitinase is a key enzyme in many organisms that functions as a defense against chitin-coated pathogens or as a digestive enzyme to hydrolyze dietary chitin. Chitin is also essential for the formation of organic frameworks, involves in various biological processes, including shell formation. However, the function of chitinase of Crassostrea hongkongensis has not yet been determined. In this study, we obtained a full Ch-chit sequence from C. hongkongensis. The full length of Ch-chit cDNA encoded a predicted 470-amino acid protein, contained a signal peptide, a Glyco 18 domain, a low complexity domain, and a transmembrane domain. Phylogenetic analysis showed that Ch-chit clustered with Chit3-lp of other bivalves. In different stages of larvae development, the expression of Ch-chit in the trochophore larvae was the highest and was significantly higher than that in other stages (P

Gender differences in individual immune responses to external stimuli have been elucidated in many invertebrates. However, it is unclear if gender differences do exist in the Hong Kong oyster Crassostrea hongkongensis, one of the most valuable marine species cultivated along the coast of South China. To clarify this, we stimulated post-spawning adult C. hongkongensis with Vibrio harveyi and lipopolysaccharide (LPS). Gender-based differences in some essential functional parameters of hemocytes were studied via flow cytometry. Obvious gender-, subpopulation-, and immune-specific alterations were found in the hemocyte immune parameters of C. hongkongensis. Three hemocyte subpopulations were identified: granulocytes, semi-granulocytes, and agranulocytes. Granulocytes, the chief phagocytes and major producers of esterase, reactive oxygen species, and nitric oxide, were the main immunocompetent hemocytes. Immune parameter alterations were notable in the accumulation of granulocyte esterase activities, lysosomal masses, nitric oxide levels, and granulocyte numbers in male oysters. These results suggest that post-spawning-phase male oysters possess a more powerful immune response than females. Gender and subpopulation differences in bivalve immune parameters should be considered in the future analysis of immune parameters when studying the impact of pathogenic or environmental factors.

Variations in the biochemical composition and nutritional quality with annual changes in gonad development were investigated to identify the optimal harvesting time of C. hongkongensis. The glycogen levels in the mantle, muscle, and gonad-visceral mass were significantly lower in June than in December, associated with changes in the expressions of ChGS and ChGP. Protein content consistently exceeded 52% of dry weight. The only significant change in protein levels was an increase between April and June in the gonad-visceral mass, which was associated with the gonadal transition from proliferation to maturation. Moreover, C. hongkongensis consistently had a well-balanced essential amino acid profile, meeting the essential amino acid requirements of preschool children. The lipid content and fatty acid composition of C. hongkongensis varied with the reproductive cycle, but the omega-3:omega-6 ratio was consistently higher than those of C. gigas and C. virginica. In summary, the optimal harvest time of C. hongkongensis was during the inactive stage of most gonads (from August to February at Beihai).

The changes in myofibrillar protein, muscle microstructure, and lipid oxidation of oysters (Crassostrea hongkongensis) treated with chitosan-based coatings (CP-0°C) or aseptic water (CG-0°C) for 20 days storage at 0°C were investigated. Compared to the CG-0°C at day 20, the higher total sulfhydryl group content (83.24%), unfrozen water content (39.24%), and Ca2+-ATPase activity (96.79%) along with the lower myofibrillar protein solubility (80.15%) indicated that CP-0°C protected oysters from protein denaturation. Similarly, the CP-0°C better maintained muscle microstructure shown by the relatively higher quality of muscle and less ice crystals. Meanwhile, CP-0°C prevented lipid hydrolysis and oxidation, as evidenced by the lower contents of fatty acid (FA) content (total content of 12.16 ± 1.02 mg/g wet weight at day 20), peroxide value (POV) (15.13 meq oxygen/Kg lipid at day 20), and thiobarbituric acid-reactive substances (TBARS) (1.79 mg MDA eq/Kg sample at day 20) in CP-0°C. These results imply that CP-0°C could protect the quality of chilled oysters stored at 0°C.

Nanoparticulate and ionic Zn have potential impacts on the detoxification systems of organisms, and Gst genes play key roles in the detoxification of xenobiotics. In this study, we cloned the ChGstα and ChGstκ genes of C. hongkongensis, and studied their expression in gills under nanoparticulate and ionic Zn stress. The results showed that the coding sequences of the ChGstα and ChGstκ genes were 684 and 675 bp, respectively, and had no signal peptide; ChGstα was cytoplasmic, while ChGstκ was mitochondrial. The two genes were expressed in all 8 tested samples, with the most abundant expression observed in hemocytes for ChGstα and digestive glands for ChGstκ. After ZnCl2 or ZnoNP challenge, the expression of ChGstα decreased significantly in the ZnCl2 groups, and its expression was higher in the ZnoNP groups than in the ZnCl2 groups. The expression of ChGstκ was significantly decreased in the ZnCl2 and ZnoNP groups, and its expression was higher in the ZnoNP groups than in the ZnCl2 groups except at 3 h post metal Zn stress, which suggested that ChGstα and ChGstκ were more sensitive to ZnoNP than ZnCl2.

In this study, the effects of high hydrostatic pressure (HHP) and steam on biochemical composition and non-volatile taste active compounds of oysters Crassostrea hongkongensis were investigated. The moisture content in steamed oysters significantly decreased when compared to raw samples, subsequently their crude protein, crude lipid, glycogen and ash contents (% wet weight) were all increased (P 0.05). In addition, though the moisture content in HHP oysters decreased, no significant differences were observed in proximate compositions compared to raw oysters, except crude protein. There were no significant differences in saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) profiles between raw and HHP oysters, however, C20:3n6 content in HHP oysters was significantly higher than that in raw samples (P 0.05). The PUFA profile of steamed oysters, mostly contributed by n-3 PUFA, was significantly higher than that of both raw and HHP samples (P 0.05). Major free amino acids (FAA) (taste activity value, TAV 1) in oysters with three treatments were alanine, glycine, glutamic acid and histidine, and their contents were significantly higher in raw and HHP groups than that in steamed group. The 5'-inosine monophosphate (IMP) and 5'-guanosine monophosphate (GMP) in HHP and steamed oysters decreased compared to raw samples, while AMP content in steam oysters were significantly increased (P 0.05). The equivalent umami concentration (EUC) of oysters of raw, HHP and steamed groups were 8.80, 3.66 and 1.44 g MSG/100 g, respectively, with significant differences observed among different treatments (P 0.05). Succinic acid was the major organic acid in raw and HHP oysters, while lactic acid was the major organic acid in steamed groups. Further, Na

The oyster Crassostrea hongkongensis is an ideal biomonitor due to its widespread distribution along the coast of Southern China and the ability to hyperaccumulate metals including Cu and Zn. In this study, we conducted the first investigation of the molecular responses to metal hyperaccumulation based on quantitative shotgun proteomics technique and genome information. Gill tissue of oysters collected from the uncontaminated environment (Site 1, 59.6 μg/g and 670 μg/g dry weight for Cu and Zn) displayed significant protein profile differentiation compared to those from a moderately contaminated (Site 2, 1,465 μg/g and 10,170 μg/g for Cu and Zn) and a severely contaminated environment (Site 3, 3,899 μg/g and 39,170 μg/g for Cu and Zn). There were 626 proteins identified to be differentially expressed at Site 3 but only 247 proteins at Site 2. Oysters from a moderately contaminated estuary (Site 2) displayed fewer effects as compared to oysters under severe contamination, with fluctuated small molecule metabolism and enhanced translation process. At Site 3, the induction of reactive oxygen species (ROS) was the main toxicity under the extremely high level of metal stress, which resulted in protein damage. Additionally, the impaired structure of cytoskeleton and modified membrane tracking process at Site 3 oysters led to the blockage or less efficient protein or macromolecule distribution within cells. Nonetheless, proteomic analysis in this study revealed that oysters could partly alleviate the adverse metal effects by boosting the translation process, enhancing the ability to recycle the misfolded proteins, and enhancing the potential to eliminate the excess ROS. Our study demonstrated an adaptive potential of oysters at the protein level to survive under conditions of metal hyper-accumulation.

This study is the first systematic comparison of the biochemical composition and nutritional quality between diploid and triploid Hong Kong oysters, Crassostrea hongkongensis. Results showed that in the reproductive season, the glycogen content in five tissues (gill, mantle, adductor muscle, labial palps and gonad) was significantly higher (P < 0.05) in triploids than in diploids, with odds ratios (ORs) of 96.26, 60.17, 72.59, 53.56, and 128.52%, respectively. In the non-reproductive phase, significant differences in glycogen content (P < 0.05) between diploid and triploid oysters existed only in gill and gonad. In both diploid and triploid Hong Kong oysters, quantitative real-time PCR analysis of the glycogen synthesis gene (ChGS) and glycogen phosphorylase gene (ChGP) showed that the gene expression patterns matched the pattern of variation in glycogen content. Moreover, in both the reproductive and the non-reproductive phases, triploid Hong Kong oysters had a well balance of essential amino acids and were thus a well source of high-quality protein. Surprisingly, in both phases, significantly higher (P < 0.05) percentages of four essential fatty acids (α-linolenic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid) were observed in triploids than in diploids. Additionally, the ratio of n-3/n-6 polyunsaturated fatty acids (PUFAs) was much higher in triploids than that in diploids. Variations in Biochemical composition were consistent with the relative expression of the citrate synthase gene (ChCS) and the α-ketoglutarate dehydrogenase gene (ChKD), which are key enzyme genes of the tricarboxylic acid cycle. Overall, the triploid Hong Kong oyster has a better nutritional value and taste than the diploid in terms of glycogen content, protein quality and fatty acid content.

A Y-box binding protein ChYB-1 was discovered as a ChHsc70 promoter-associated protein in Crassostrea hongkongensis by DNA-affinity purification and mass spectrometry analysis. The overexpression of ChYB-1 in heterologous HEK293T cells led to clear enhancement of ChHsc70 promoter expression, while ChYB-1 depletion correlated with significant reduction of ChHsc70 transcription in the hemocytes of C. hongkongensis. Quantitative Real-time PCR analysis revealed that both ChHsc70 and ChYB-1 were transcriptionally responsive to external chemical or physical stressors. This indicates a plausible correlation between ChHsc70 and ChYB-1 in the genetic regulatory pathway triggered by external stresses. This study presents the first evidence of positive regulator for Hsc70 transcription and contributes to a better understanding of the regulatory mechanisms governing Hsc70 expression.

Pearl River Estuary (PRE) is the largest estuary in Southern China and there has been an increasing concern of metal pollution due to regional industrialization. In this study, we investigated the spatial variation of metal pollution (Ag, As, Cd, Cu, Pb, and Zn) as well as their subcellular handling in the oyster Crassostrea hongkongensis. Hot spots of metal contamination in the oysters were found in different sites, suggesting that there were different sources of metals in the estuary associated with industrial activity. Metals differed in their subcellular bindings in the oysters from different locations. Metal distribution in the biologically detoxified fraction decreased for Cu but increased for Zn with increasing contamination in the oysters. For Zn, there was a significant difference in its two detoxification pools (metal-rich granules and metallothionein-like proteins) in response to Zn contamination. The high Cd concentrations in oysters may carry a high Cd hazard to the consumers.

Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.

Despite salinity has been well documented for its significant effects on the bioaccumulation of many trace elements in biomonitors, no calibration method has been proposed to reduce such influences. For the first time, the present study established a novel method to calibrate biomonitoring data against salinity. Relationships between trace element concentration in oyster Crassostrea hongkongensis and the biological proxy for salinity were quantified based on laboratory exposure experiments. The method was then verified by the biomonitoring data of Pearl River Estuary (PRE). Tissue concentrations of trace elements (Cu, Zn, Ag, Cd, Pb, Cr, As, Se, and Ni) and major cations (Na, Mg, K, and Ca) in oysters exposed at 4 salinities (5, 12, 20, and 28psu) and low concentrations for 6weeks were measured to establish such quantitative relationships. Tissue Na, Mg, and K could be the proxy for salinity, while Na was the best one. Negative correlations between tissue concentrations of trace elements and Na after exposure were observed for metal cations such as Cu, Zn, Ag, Cd, and Pb, while tissue As, Se, and Ni were positively correlated with Na. In PRE, salinity significantly influenced the bioaccumulation of trace elements even under the multifactor-affected field conditions. The calibration method applied to the biomonitoring of PRE was verified to be feasible, and effectively reduced the influences of salinity. Therefore, calibration against salinity could facilitate the interpretation, comparability, and analysis of biomonitoring data.