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Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis. [ABSTRACT FROM AUTHOR]

The chloride channel 7 gene (CLC7) of the Hong Kong oyster Crassostrea hongkongensis was cloned and named ChCLC7. The cDNA was 2 572 bp in length, with a 5′ non-coding region containing 25 bp, a 3′ non-coding region containing 327 bp, and an open reading frame of 2 298 bp. ChCLC7 has 96.8% and 92.1% homology with CLC7 of Crassostrea gigas and Crassostrea virginica, respectively, and it was clustered with CLC7 of C. gigas and C. virginica. QRT-PCR showed that ChCLC7 was expressed in all eight tissues, with the highest in adductor muscle and second in gill. The ChCLC7 expression pattern in gill was altered significantly under high salinity stress with an overall upward and then downward trend. After RNA interference, the expression of ChCLC7 and survival rate of oyster under high salinity stress was reduced significantly, and so did the concentration of hemolymph chloride ion in 48–96 h after RNA interference. We believed that ChCLC7 could play an important role in osmoregulation of C. hongkongensis by regulating Cl− transport. This study provided data for the analysis of molecular mechanism against oyster salinity stress. [ABSTRACT FROM AUTHOR]

The use of an appropriate amount of anesthetic can prevent the sacrifice of oysters during experimental sampling or seedling rearing. In this study, we assessed the effects of air exposure and three concentrations of magnesium chloride (10, 40, and 70 g L–1) on the anesthetic efficiency for Crassostrea hongkongensis. We examined the recuperative capacity of the oysters post-tissue sampling. The results indicated that 36 h of air exposure could significantly enhance the anesthetic efficiency of C. hongkongensis while ensuring the oysters’ survival. A 40 g L–1 solution of magnesium chloride had the optimal anesthetic effect on C. hongkongensis across all oyster sizes. In addition, smaller oysters (119.10 g) had a higher relaxation rate than larger oysters (191.00 g). The oysters anesthetized with 40 g L–1 magnesium chloride also demonstrated the best recovery. No deaths were observed among the non-anesthetized oysters, anesthetized-recovered oysters, or non-recovered oysters during a seven-day post-farming period. In terms of live sampling, the control group (100 %), gonad group (100 %), mantle group (98.67 %), gill group (89.33 %), and adductor muscle group (97.33 %) all showed high survival rates after anesthesia. Observation of tissue growth post-sampling revealed the presence of focal green areas in tissues between days 7 and 14. By day 30, the adductor muscle and mantle tissues had fully healed, and the regrown tissue in the gills had approached its original length. A 40 g L–1 magnesium chloride solution had no significant effects on gametes in terms of fertilization rate, growth rate, and attachment rate. The present findings indicated that C. hongkongensis could be effectively anesthetized using a 40 g L–1 magnesium chloride solution following 36 h of aerial exposure. Post-anesthesia tissue and gamete sampling did not result in significant mortality, and the tissues had fully recovered within 30 days post-sampling. The anesthesia did not have any adverse effects on the progeny.

Copyright of China Condiment is the property of China Condiment and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Caspase-8, a member of the caspase family, is an initiating caspase and plays a crucial role in apoptosis. In this study, the full-length cDNA of caspase8-like (CASP8-like) was isolated from Crassostrea hongkongensis (C. hongkongensis) by RACE-PCR. ChCASP8-like contained a 1599-bp open reading frame (ORF) encoding 533 amino acids with two conserved death effector domains (DEDs) and a cysteine aspartase cysteine structural domain (CASc). Amino acid sequence comparison showed that ChCASP8-like shared the highest identity (85.4%) with CASP8-like of C. angulata. The tissue expression profile showed that ChCASP8-like was constitutively expressed in gills, hepatopancreas, mantle, adductor muscle, hemocytes and gonads, and was significantly upregulated in hemocytes, hepatopancreas and gills under hyper-salinity stress. The apoptosis-related genes, including ATR, CHK1, BCL-XL, CASP8-like, CASP9 and CASP3, were significantly activated by hyper-salinity stress, but were remarkably inhibited by ChCASP8-like silencing. The caspase 8 activity was increased by 1.7-fold after hyper-salinity stress, and was inhibited by 9.4% by ChCASP8-like silencing. Moreover, ChCASP8-like silencing clearly alleviated the apoptosis resulting from hyper-salinity stress. These results collectively demonstrated that ChCASP8-like played a crucial role in inducing apoptosis against hyper-salinity stress. [ABSTRACT FROM AUTHOR]

Simple Summary: A new interspecific backcross ([(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀]) variety was bred by the South China Sea Institute of Oceanology and named "South China No. 1". This study aims to explore the effects of stocking density on the growth performance of "South China No. 1", and compare the growth performance and genetic diversity to that of C. hongkongensis in Beibu Gulf. The results showed that oysters bred under a stocking density of 20 oysters/substrate had better growth manifestation. It was found that the shell height and total weight of "South China No. 1" cultured in Fangchenggang were significantly higher than that of those in Beihai and Qinzhou from September 2018 to November 2018. In addition, the shell height and shell width of "South China No. 1" were significantly higher than that of C. hongkongensis. In the Hardy–Weinberg equilibrium test, for the seven populations and ten microsatellites, in 10 of the 70 groups, the segregation distortion was significant. These results suggest that a stocking density of 20 oysters/substrate can promote the shell height, shell width and total weight of "South China No. 1". Fangchenggang is a suitable place to cultivate the "South China No. 1" breed according to the total weight and sum of all algal genus abundances. Crassostrea hongkongensis is an economically important bivalve found in various parts of the South China Sea. A new interspecific backcross ([(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀]) variety was bred by the South China Sea Institute of Oceanology which named "South China No. 1". This study aims to explore the effects of stocking density on the growth performance of "South China No. 1", compared their growth performance and genetic diversity to C. hongkongensis, and found the best place breeding site for "South China No. 1" in Beibu Gulf. The results showed that stocking a density of 20 oysters/substrate can significantly increase the shell height, shell width, total weight, survival rate, daily shell height gain and daily body mass gain. It was found that the shell height and total weight of "South China No. 1" cultured in Fangchenggang were significantly higher than that of those in Beihai and Qinzhou from September 2018 to November 2018. Similarly, the shell width of oysters in Fangchenggang and Qinzhou was also significantly higher in September 2018, and the interaction between site and stocking density had significant effects on the shell width in March 2018 and November 2018. In addition, the shell height and shell width of "South China No. 1" were significantly higher than that of C. hongkongensis in all three sites. At all three sites, the phytoplankton community structure was mostly dominated by Bacillariophyta. In the Hardy–Weinberg equilibrium test, for the seven populations and ten microsatellites, in 10 of the 70 groups, the segregation distortion was significant. These results suggest that a stocking density of 20 oysters/substrate can promote the shell height, shell width and total weight of "South China No. 1" in Beibu Gulf, China. "South China No. 1" has better growth performance compared with C. hongkongensis. Fangchenggang is a suitable place to cultivate the "South China No. 1" breed according to the total weight and sum of all algal genus abundances. The results of this study can be used as a reference to further understand the stocking density and genetic diversity of the "South China No. 1" breed in Beibu Gulf, China. [ABSTRACT FROM AUTHOR]

The estuarine Crassostrea hongkongensis is the major cultured oyster species in the South China, and mass mortality induced by hyper-salinity has heavily damaged the oyster farming, especially during dry season. To benefit the genetic improvement of hyper-salinity tolerance, three genes related to the intracellular concentration of glycine (Gly) that functions as important osmolyte were identified and investigated in C. hongkongensis. Serine hydroxymethyltransferase 1 in C. hongkongensis (CHSHMT1) was found to be upregulated in gill since 48 h transferred from 18‰ to 30‰ filtered seawater (FSW). While glycine dehydrogenase (CHGLDC) and ALA synthase gene (CHALAS) were significantly upregulated in oyster from 8 to 48 h exposed to 6‰ FSW when compared with that in 18‰ and 30‰ FSW. And the expression of the three genes agreed with that Gly content decreased under hypo-osmotic stress and increased under hyper-osmotic stress. RNA in situ hybridization (ISH) revealed that CHSHMT1 and CHGLDC were mainly expressed in the ciliated columnar cell of gill filament and epithelial cell of water tube wall, where oyster gill directly contacts with seawater when the animal is filtering. But positive signal of CHALAS mRNA showed no significant difference in intensity between various gill cells. Based on these findings, CHSHMT1, CHGLDC and CHALAS were supposed to contribute to the variation of Gly content under osmotic stress. Subsequent RNA interference (RNAi) confirmed the function of CHSHMT1 in the transformation from serine (Ser) to Gly under hyper-osmotic stress. Moreover, CHGLDC and CHALAS were found to be significantly upregulated when CHSHMT1 was silenced under hyper-osmotic stress. This was hypothesized that the expression of CHGLDC and CHALAS might be suppressed by CHSHMT1, but more evidence should be provided. This work enriched the understanding of oyster osmolyte system that prevent cell shrinking or swelling under osmotic stress and provided potential genes for the breeding of Hong Kong oyster strain with hyper-salinity tolerance.

Crassostrea hongkongensis (C. hongkongensis) is one of the three most commonly cultivated oyster species in China. Seasonal hypoxia is one of the most serious threats to its metabolism, reproductive behavior, and survival. To investigate the effects of hypoxia stress on the antioxidant capacity and energy metabolism of C. hongkongensis, the total antioxidant capacity (T-AOC), glycogen content, and enzyme activities (phosphofructokinase, PFK; pyruvate kinase, PK; phosphoenolpyruvate carboxykinase, PEPCK) of oysters were determined under normoxic (DO 6 ± 0.2 mg/L) and hypoxic (DO 1.5 mg/L) conditions at 0 h, 6 h, 48 h, and 72 h. We also determined the T-AOC, glycogen content, and enzyme activities of oysters under reoxygenation (recovered to normoxia for 24 h). To further examine the potential molecular regulatory mechanism of hypoxic adaptation, a transcriptome analysis was conducted on the gill of C. hongkongensis under normoxia (N, 72 h), hypoxia (H, 72 h), and reoxygenation (R). After being exposed to hypoxia for 6 h, the T-AOC, glycogen content, and enzyme activities of PK, PFK, and PEPCK in C. hongkongensis were significantly decreased. However, after prolonging the duration of hypoxia exposure for 72 h, the T-AOC, glycogen content, and enzyme activities increased compared to that of 48 h. After 24 h reoxygenation, the T-AOC, glycogen content, and enzyme activity of PK and PFK returned to close to initial levels. In addition, a transcriptome analysis discovered 6097 novel genes by mapping the C. hongkongensis genome with the clean reads. In total, 352 differentially expressed genes (DEGs) were identified in the H vs. N comparison group (235 upregulated and 117 downregulated genes). After recovery to normoxia, 292 DEGs (134 upregulated and 158 downregulated genes) were identified in the R vs. N comparison group, and 632 DEGs were identified (253 upregulated and 379 downregulated genes) in the R vs. H comparison group. The DEGs included some hypoxia-tolerant genes, such as phosphoenolpyruvate carboxykinase (PEPCK), mitochondrial (AOX), tyramine beta-hydroxylase (TBH), superoxide dismutase (SOD), glutathione S-transferase (GST), and egl nine homolog 1 isoform X2 (EGLN1). Additionally, DEGs were significantly enriched in the KEGG pathways that are involved in hypoxia tolerance, including the metabolism of xenobiotics by cytochrome P450 pathways and the HIF-1 signaling pathway. Then, we selected the five hypoxic-tolerant candidate DEGs for real-time quantitative polymerase chain reaction (RT-qPCR) validation, and the results were consistent with the transcriptome sequencing data. These discoveries have increased our understanding of hypoxia tolerance, recovery ability after reoxygenation, and molecular mechanisms governing the responses to hypoxia in C. hongkongensis.

Copyright of Journal of Hydrobiology / Shuisheng Shengwu Xuebao is the property of Editorial Department of Journal of Hydrobiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear. Methods: The full length of ChFasL was identified and cloned based on the genome of Crassostrea hongkongensis. Quantitative PCR was used to detect the relative expression of ChFasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of ChFasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dualfluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after ChFasL silencing was identified by RNA interference technology. Results: We identified a novel FasL gene from C. hongkongensis and named it ChFasL. We found that ChFasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. ChFasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by V. alginolyticus or S. haemolyticus, its relative expression in hemocytes increased significantly, suggesting that ChFasL was deeply engaged in the immune response process of C. hongkongensis to external microbial stimulation. The results of subcellular localization showed that ChFasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- ChFasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of ChFasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group. Conclusion: These results comprehensively confirmed the important role of ChFasL in the apoptosis process of C. hongkongensis, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves. [ABSTRACT FROM AUTHOR]

Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Caspase-8, a member of the caspase family, is an initiating caspase and plays a crucial role in apoptosis. In this study, the full-length cDNA of caspase8-like (CASP8-like) was isolated from Crassostrea hongkongensis (C. hongkongensis) by RACE-PCR. ChCASP8-like contained a 1599-bp open reading frame (ORF) encoding 533 amino acids with two conserved death effector domains (DEDs) and a cysteine aspartase cysteine structural domain (CASc). Amino acid sequence comparison showed that ChCASP8-like shared the highest identity (85.4%) with CASP8-like of C. angulata. The tissue expression profile showed that ChCASP8-like was constitutively expressed in gills, hepatopancreas, mantle, adductor muscle, hemocytes and gonads, and was significantly upregulated in hemocytes, hepatopancreas and gills under hyper-salinity stress. The apoptosis-related genes, including ATR, CHK1, BCL-XL, CASP8-like, CASP9 and CASP3, were significantly activated by hyper-salinity stress, but were remarkably inhibited by ChCASP8-like silencing. The caspase 8 activity was increased by 1.7-fold after hyper-salinity stress, and was inhibited by 9.4% by ChCASP8-like silencing. Moreover, ChCASP8-like silencing clearly alleviated the apoptosis resulting from hyper-salinity stress. These results collectively demonstrated that ChCASP8-like played a crucial role in inducing apoptosis against hyper-salinity stress.

Crassostrea hongkongensis is an economically important bivalve found in various parts of the South China Sea. A new interspecific backcross ([(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀]) variety was bred by the South China Sea Institute of Oceanology which named “South China No. 1”. This study aims to explore the effects of stocking density on the growth performance of “South China No. 1”, compared their growth performance and genetic diversity to C. hongkongensis, and found the best place breeding site for “South China No. 1” in Beibu Gulf. The results showed that stocking a density of 20 oysters/substrate can significantly increase the shell height, shell width, total weight, survival rate, daily shell height gain and daily body mass gain. It was found that the shell height and total weight of “South China No. 1” cultured in Fangchenggang were significantly higher than that of those in Beihai and Qinzhou from September 2018 to November 2018. Similarly, the shell width of oysters in Fangchenggang and Qinzhou was also significantly higher in September 2018, and the interaction between site and stocking density had significant effects on the shell width in March 2018 and November 2018. In addition, the shell height and shell width of “South China No. 1” were significantly higher than that of C. hongkongensis in all three sites. At all three sites, the phytoplankton community structure was mostly dominated by Bacillariophyta. In the Hardy–Weinberg equilibrium test, for the seven populations and ten microsatellites, in 10 of the 70 groups, the segregation distortion was significant. These results suggest that a stocking density of 20 oysters/substrate can promote the shell height, shell width and total weight of “South China No. 1” in Beibu Gulf, China. “South China No. 1” has better growth performance compared with C. hongkongensis. Fangchenggang is a suitable place to cultivate the “South China No. 1” breed according to the total weight and sum of all algal genus abundances. The results of this study can be used as a reference to further understand the stocking density and genetic diversity of the “South China No. 1” breed in Beibu Gulf, China.

The Hong Kong oyster, Crassostrea hongkongensis, is an estuarine bivalve with remarkable commercial value in South China, and the increase of salinity in estuaries during the dry season has posed a major threat to the oyster farming. To explore the global transcriptional response to salinity stress, a whole-transcriptome analysis was performed with the gills of oysters in 6‰, 18‰, and 30‰ filtered seawater. Overall, 2243, 194, 371, and 167 differentially expressed mRNAs (DEmRNAs), differentially expressed long non-coding RNAs (DElncRNAs), differentially expressed circular RNAs (DEcircRNAs), and differentially expressed microRNAs (DEmiRNAs) were identified, respectively. Based on GO enrichment and KEGG pathway analysis, these important DEmRNAs, DElncRNAs, DEcircRNAs, and DEmiRNAs were predicted to be mainly involved in amino acids metabolism, microtubule movement, and immune defense. This demonstrated the complexity of dynamic transcriptomic profiles of C. hongkongensis in response to salinity fluctuation. The regulatory relationships of DEmiRNAs-DEmRNAs, DElncRNAs-DEmiRNAs, and DEcircRNAs-DEmiRNAs were also predicted, and finally, a circRNA-associated competing endogenous RNA (ceRNA) network was constructed, consisting of six DEcircRNAs, eight DEmiRNAs, and five DEmRNAs. The key roles of taurine and hypotaurine metabolism and phenylalanine metabolism were highlighted in this ceRNA network, which was consistent with the major contribution of free amino acids to intracellular osmolality and cell volume regulation. Collectively, this study provides comprehensive data, contributing to the exploration of coding and non-coding RNAs in C. hongkongensis salinity response. The results would benefit the understanding of the response mechanism of bivalves against salinity fluctuation, and provide clues for genetic improvement of C. hongkongensis with hyper-salinity tolerance. [ABSTRACT FROM AUTHOR]

Abstract Background Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation. Results Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels. Conclusions In this study, we generated a first “knowledge landscape” of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. Video Abstract

Abstract Background Infectious keratitis, a medical emergency with acute and rapid disease progression may lead to severe visual impairment and even blindness. Herein, an antimicrobial polypeptide from Crassostrea hongkongensis, named URP20, was evaluated for its therapeutic efficacy against keratitis caused by Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infection in rats, respectively. Methods A needle was used to scratch the surface of the eyeballs of rats and infect them with S. aureus and E.coli to construct a keratitis model. The two models were treated by giving 100 μL 100 μM URP20 drops. Positive drugs for S. aureus and E. coli infection were cefazolin eye drops and tobramycin eye drops, respectively. For the curative effect, the formation of blood vessels in the fundus was observed by a slit lamp (the third day). At the end of the experiment, the condition of the injured eye was photographed by cobalt blue light using 5 μL of 1% sodium fluorescein. The pathological damage to corneal tissues was assessed using hematoxylin–eosin staining, and the expression level of vascular endothelial growth factor (VEGF) was detected by immunohistochemistry. Results URP20 alleviated the symptoms of corneal neovascularization as observed by slit lamp and cobalt blue lamp. The activity of S. aureus and E.coli is inhibited by URP20 to protect corneal epithelial cells and reduce corneal stromal bacterial invasion. It also prevented corneal thickening and inhibited neovascularization by reducing VEGF expression at the cornea. Conclusion URP20 can effectively inhibit keratitis caused by E.coli as well as S. aureus in rats, as reflected by the inhibition of corneal neovascularization and the reduction in bacterial damage to the cornea.

Oysters are nutritious and tasty but difficult to store. Drying can extend the storage period of oysters and give them a unique flavor. In this study, the effects of four drying procedures, namely, vacuum freeze drying (VFD), vacuum drying (VD), natural sun-drying (NSD), and hot air drying (HAD), on the flavor characteristics of oysters (Crassostrea hongkongensis) were investigated using blanched oysters as a control (CK). Results showed that HAD produced more free amino acids than the other methods, but VFD retained the most flavor nucleotides. Compared with cold drying (VFD), hot drying (VD, NSD, and HAD) increased the abundance of organic acids, betaine, and aroma substances. Glutamic acid, alanine, AMP, hexanal, octanal, heptanal, (E, E)-2,4-heptadienal, (E)-2-decenal, nonanal, etc., are defined as the characteristic flavor compounds of dried oysters, with umami, sweet, green, fatty, and fruity aromas being the main organoleptic attributes of dried oysters. Glutamic acid, glycine, betaine, IMP, pentanal, ethyl heptanoate, (E, Z)-2,4-nonadienal, 1-octen-3-one, 2-hexenal, 2-octenal, hexanal, decanal were defined as markers to distinguish different drying methods. Overall, HAD showed improved flavor qualities and characteristics and was better suited for the highly commercialized production of dried oysters. [ABSTRACT FROM AUTHOR]

In the present study, the nutrient composition of Crassostrea hongkongensis from two oyster fattening areas in Qinzhou Bay, Guangxi was evaluated. And then the metabolism-related gene expression in the mantle, adductor muscle and gill tissues of C. hongkongensis was analyzed, and the correlation between nutrient composition and gene expression was discussed. The results showed that the contents of cholesterol, vitamin A, vitamin E, zinc and some amino acids including phenylalanine, histidine, glutamic acid and alanine of C. hongkongensis in Qinzhou Port fattening area were significantly higher than those in Dafeng River fattening area (P0.05). The heat map of Spearman correlation showed that the gene expression of P5CS, GP and SREBP were positively correlated with the contents of multiple nutrient composition and amino acids, in which the gene expression of GP was significantly positively correlated with energy, fat, cholesterol and vitamins (P

Background: Viruses play critical roles in the marine environment because of their interactions with an extremely broad range of potential hosts. Many studies of viruses in seawater have been published, but viruses that inhabit marine animals have been largely neglected. Oysters are keystone species in coastal ecosystems, yet as filter-feeding bivalves with very large roosting numbers and species co-habitation, it is not clear what role they play in marine virus transmission and coastal microbiome regulation. Results: Here, we report a Dataset of Oyster Virome (DOV) that contains 728,784 nonredundant viral operational taxonomic unit contigs (≥ 800 bp) and 3473 high-quality viral genomes, enabling the first comprehensive overview of both DNA and RNA viral communities in the oyster Crassostrea hongkongensis. We discovered tremendous diversity among novel viruses that inhabit this oyster using multiple approaches, including reads recruitment, viral operational taxonomic units, and high-quality virus genomes. Our results show that these viruses are very different from viruses in the oceans or other habitats. In particular, the high diversity of novel circoviruses that we found in the oysters indicates that oysters may be potential hotspots for circoviruses. Notably, the viruses that were enriched in oysters are not random but are well-organized communities that can respond to changes in the health state of the host and the external environment at both compositional and functional levels. Conclusions: In this study, we generated a first "knowledge landscape" of the oyster virome, which has increased the number of known oyster-related viruses by tens of thousands. Our results suggest that oysters provide a unique habitat that is different from that of seawater, and highlight the importance of filter-feeding bivalves for marine virus exploration as well as their essential but still invisible roles in regulating marine ecosystems. APUP9ceFw5rgMggB2pWS5V Video Abstract [ABSTRACT FROM AUTHOR]

[Display omitted] • Oyster quality best preserved within 3 days of air exposure at 4 °C. • Metabolomic analysis reveals 38 differentially abundant metabolites during early exposure. • Enzyme activities and metabolites show adaptive responses within 3-day window. • Subtle changes in flavor compounds observed within optimal consumption period. Post-harvest air exposure is unavoidable during oyster transportation and storage, yet the physiological tolerance limits and underlying metabolic responses of commercially important oyster species remain poorly understood. While previous studies have focused on immediate post-harvest quality changes, there is limited knowledge about the time-dependent metabolic adaptations that determine product quality during extended air exposure. This study investigated the physiological and metabolic responses of Crassostrea hongkongensis during air exposure at 4 °C, focusing on identifying the optimal period for quality preservation. Using a combination of survival analysis, enzyme activity assays, and metabolomic profiling, we examined oysters exposed to air for up to 18 days, with particular emphasis on the critical first three days. Survival analysis showed 100 % survival rate at 4 °C through day 7, with mortality beginning thereafter, compared to significant mortality observed at 25 °C (complete mortality by day 7) and 37 °C (complete mortality by day 2). Analysis of antioxidant enzyme activities revealed complex, time-dependent changes, with robust responses observed within the first three days, indicating effective stress management. Metabolomic analysis identified 38 differentially abundant metabolites throughout the exposure period. Notably, the metabolic profile at day 3 showed a tendency to revert towards the control state, suggesting a temporary adaptive response. Key findings included stability in total antioxidant capacity (T-AOC) levels during the initial three days and subtle changes in flavor-related compounds, such as slight decreases in glutamate and aspartate levels. Correlation analyses revealed intricate interactions between enzyme activities and metabolites, highlighting complex stress response mechanisms. The relationship between T-AOC and key osmolytes underscored their critical role in maintaining cellular redox balance during the initial exposure period. Our findings suggest that the optimal window for maintaining C. hongkongensis quality during air exposure at 4 °C is within the first three days. During this period, oysters demonstrate effective adaptive responses, maintaining key quality attributes and nutritional value. Beyond this timeframe, the risk of quality degradation increases significantly. These results have important implications for the oyster industry, providing evidence-based guidelines for post-harvest handling, transportation, and storage practices. We recommend limiting air exposure during cold storage to no more than 3 days to ensure optimal product quality and consumer satisfaction. [ABSTRACT FROM AUTHOR]

Oysters have evolved an effective and powerful innate immune system to recognize foreign substances and eliminate invaders through orchestrated immune responses such as programmed cell death (PCD), including apoptosis, autophagy, necroptosis, and pyroptosis. Both apoptosis and autophagy have been considered to be crucial mechanisms for oyster defence. However, the role of pyroptosis, a gasdermin (GSDM)-dependent programmed cell death pathway that is critical for defence against infection, in oysters remains unclear. In this study, we investigated the activity of Crassostrea hongkongensis GSDME (Ch GSDME), which is cleaved by inflammatory C.hongkongensis caspase-1 (Ch CASP1). qRT–PCR analysis showed that Ch CASP1 and Ch GSDME were widely expressed in oyster developmental stages and tissues, and responded to Vibrio coralliilyticus challenge. Meanwhile, hemocyte death was reduced after knockdown of Ch CASP1 or Ch GSDME by RNA interference (RNAi). Following injection of V. coralliilyticus , the cell death rate of the knockdown Ch CASP1 or Ch GSDME group was significantly lower than that of the control group. In vitro , coexpressing Ch CASP1 and Ch GSDME in HEK293T cells resulted in pyroptotic morphology, a bubble-like membrane, and a truncated protein (Ch GSDME N-terminal). Subsequently, immunofluorescence revealed that Ch GSDME-NT oligomerized at the cell membrane, inducing bubble-like membrane formation, thereby causing low cytotoxicity. Luciferase reporter gene analysis revealed that Ch CASP1 significantly upregulated Nuclear factor kappa-B (NF-κB) and modestly upregulated the p53 signalling pathway. Taken together, our study demonstrated that the pyroptosis-related genes Ch CASP1 and Ch GSDME mediate pyroptosis in HEK293T cells and play important roles in cell death during V. coralliilyticus infection in C. hongkongensis. • ChCASP1 and ChGSDME were first isolated and cloned from C. hongkongensis. • RNAi of ChCASP1 or ChGSDME resulted in decrease of hemocytes death rate in C. hongkongensis. • Overexpression of ChCh CASP1 and Ch GSDME produced Ch GSDME-NT and induced pyroptosis in HEK293T cells. • Ch GSDME-NT mediates pyroptosis in HEK293T cells. • Ch CASP1 significantly up-regulates NF-κB-Luc reporter and modestly up-regulated p53-Luc reporter in HEK293T cells. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) and polychlorinated biphenyls (PCBs) are known with high persistence and toxicity, posing urgent threats to food safety and human health. However, little is known about the synergistic effect of MPs on PCBs bioaccumulation on Crassostrea hongkongensis. In the present study, diverse types of MPs were analyzed on sea water and C. hongkongensis sampled from three distinct estuary sites, and film-shaped MPs were discovered to be preferentially ingested by the oysters. Interestingly, the content of MPs and PCBs showed negative correlation (R2 = 0.452, p < 0.001) in the oysters sampled from site 2. Upon MPs and PCBs co-treatment, the in vivo accumulation of PCBs in C. hongkongensis was inhibited by 25.90 % when compared to the group treated with PCBs solely. PCBs stresses significantly induced the expression of genes of CYP2C31 , GST , SOD and HSP70 in C. hongkongensis , while, the elevated state was compromised when co-treated with PCBs. The present research alleviates concerns about the potential effects of MPs on promoting PCBs bioaccumulation and provide a better understanding of the combined impact of MPs and PCBs on C. hongkongensis. [Display omitted] • Monitoring MPs and PCBs content in C. hongkongensis of aquacultural areas of South China. • C. hongkongensis preferentially ingest film-type MP in all three sampling aquacultural areas. • MPs' and PCBs' accumulation in C. hongkongensis onsite was negatively correlated. • PE/PCBs co-treatment aroused mutually inhibited accumulation of the two pollutants in oysters. • PE/PCBs co-presence caused deteriorated oxidative stresses in C. hongkongensis. [ABSTRACT FROM AUTHOR]

• A total of 51 known miRNAs and 95 novel miRNAs were identified from the nine small RNA libraries. • 6 down-regulated DE miRNAs responded to the hypo-salinity stress. • 1 up-regulated and 5 down-regulated DE miRNAs responded to the hyper-salinity stress. • 931 and 768 potential target genes were respectively predicted from the hypo- and hyper-salinity stress. • Genes involved in the vesicle-mediated transport and metal ion binding responded to the hypo-salinity stress. • Genes related to the signal transduction and metabolic process responded to the hyper-salinity stress. The Hong Kong oyster, Crassostrea hongkongensis , is a significant bivalve species with economic importance. It primarily inhabits the estuarine intertidal zones in southern China, making it susceptible to salinity fluctuations. Consequently, investigating the molecular mechanisms governing salinity regulation in C. hongkongensis is essential. In this study, we conducted miRNA-seq on C. hongkongensis to compare miRNA expression differences under varying salinities (5‰, 25‰, and 35‰). The miRNA sequencing revealed 51 known miRNAs and 95 novel miRNAs across nine small RNA libraries (S5, S25, and S35). Among these miRNAs, we identified 6 down-regulated differentially expressed (DE) miRNAs in response to hypo-salinity stress (5‰), while 1 up-regulated DE miRNA and 5 down-regulated DE miRNAs were associated with hyper-salinity stress (35‰). Additionally, we predicted 931 and 768 potential target genes for hypo- and hyper-salinity stress, respectively. Functional gene annotation indicated that the target genes under hypo-salinity stress were linked to vesicle-mediated transport and metal ion binding. Conversely, those under hyper-salinity stress were primarily involved in signal transduction and metabolic processes. These findings have provided insights into the regulatory role of miRNAs, their potential target genes and associated pathways in oyster hypo- and hyper-salinity stress, which establish a foundation for future studies on the roles of miRNAs in salinity acclimation mechanisms in C. hongkongensis. [ABSTRACT FROM AUTHOR]

The lipids of the oyster (Crassostrea hongkongensis) have a special physiological activity function, which is essential to maintain human health. However, comprehensive research on their lipids species and metabolism is not so common. In our study, based on the high-performance liquid chromatography/quadrupole time-of-flight mass spectrometer (HPLC/Q-TOF-MS), the non-targeted lipidomics research of Crassostrea hongkongensis fresh and dried products was determined. Meanwhile, we analyzed its lipid outline, screened the differences between the lipid molecules of Crassostrea hongkongensis fresh and dried products, and determined the lipid metabolic pathway. Results showed that 1,523 lipid molecules were detected, in which polyunsaturated fatty acids mostly existed in such lipids as phosphoglyceride. Through the multivariate statistical analysis, according to the conditions of P < 0.05, FC > 2 or FC < 0.05, and VIP > 1.2, 239 different lipid molecules were selected, including 37 fatty acids (FA), 60 glycerol phospholipids (GP), 20 glycerin (GL), 38 sheath lipids (SP), 31 steroid lipids (ST), 36 polyethylene (PK), and 17 progesterone lipids (PR). Combined with the Kyoto Encyclopedia of Genes and Genomes (KEGG), the differential lipid molecules were analyzed to mainly determine the role of the glycerin phospholipid metabolic pathway. As a whole, the results of this study provide the theoretical basis for the high-value utilization of oysters and are helpful to the development of oysters' physiological activity functions and deep utilization.

Crassostrea hongkongensis (Hong Kong oyster), a commercially valuable bivalve thriving in the estuaries along the northern coast of the South China Sea, is threatened by the increase of salinity during the dry season. It is essential to improve its hyper-salinity tolerance, which requires a clear understanding of the mechanism to regulate its osmotic balance. For this purpose, 25 free amino acids (FAA), the most critical intracellular osmolyte in bivalves, were quantified in gill, adductor muscle, and hemolymph of C. hongkongensis during abrupt salinity changes (18–6, 12, 24 and 30‰) by high-performance liquid chromatography-tandem mass spectrometry. Taurine, alanine and glycine were dominant FAA, contributing to total FAA contents that varied with salinities, no matter in gill, muscle or hemolymph. Under hypo-osmotic stress, total FAA contents decreased in gill and adductor muscle but increased in hemolymph, probably resulting from the extrusion of some FAA from tissues to prevent swelling of cells. And taurine contributed most significantly to the hypo-salinity response, which was mainly reflected by the remarkable increase in hemolymph at 8 h after the exposure to 6‰. By contrast, total FAA contents increased in gill, muscle, and hemolymph when transferred to hyper-salinity, suggesting the assumption that it increases the osmolality and subsequently prevents the cell shrinking. Alanine and glycine contributed most significantly to hyper-salinity response. Of the two, the former was more sensitive to hyper-osmotic stress for its rapid increase in gill within 8 h after the exposure to 24 and 30‰. An inverse pattern with most FAA was presented by ornithine, the intermediate of the urea cycle, which was probably associated with the FAA metabolism. This study identified the major FAA as intracellular osmolytes in C. hongkongensis and would benefit the further understanding of the regulatory mechanisms of oyster salinity acclimation.

Background: Infectious keratitis, a medical emergency with acute and rapid disease progression may lead to severe visual impairment and even blindness. Herein, an antimicrobial polypeptide from Crassostrea hongkongensis, named URP20, was evaluated for its therapeutic efficacy against keratitis caused by Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infection in rats, respectively. Methods: A needle was used to scratch the surface of the eyeballs of rats and infect them with S. aureus and E.coli to construct a keratitis model. The two models were treated by giving 100 μL 100 μM URP20 drops. Positive drugs for S. aureus and E. coli infection were cefazolin eye drops and tobramycin eye drops, respectively. For the curative effect, the formation of blood vessels in the fundus was observed by a slit lamp (the third day). At the end of the experiment, the condition of the injured eye was photographed by cobalt blue light using 5 μL of 1% sodium fluorescein. The pathological damage to corneal tissues was assessed using hematoxylin–eosin staining, and the expression level of vascular endothelial growth factor (VEGF) was detected by immunohistochemistry. Results: URP20 alleviated the symptoms of corneal neovascularization as observed by slit lamp and cobalt blue lamp. The activity of S. aureus and E.coli is inhibited by URP20 to protect corneal epithelial cells and reduce corneal stromal bacterial invasion. It also prevented corneal thickening and inhibited neovascularization by reducing VEGF expression at the cornea. Conclusion: URP20 can effectively inhibit keratitis caused by E.coli as well as S. aureus in rats, as reflected by the inhibition of corneal neovascularization and the reduction in bacterial damage to the cornea. [ABSTRACT FROM AUTHOR]

Summary: A novel natural zinc‐enriched polysaccharide: OZPs‐1 was isolated from oyster (Crassostrea hongkongensis), with 922.18 μg g−1 of zinc. The physicochemical properties and structural features of OZPs‐1 were systematically studied. The results showed OZPs‐1 was predominantly composed of glucose, with a molecular weight ranging from 4 to 2044 kDa. OZPs‐1 showed favourable thermal stability. NMR analysis showed that there was no sulphate substitution and C‐6 substitution in OZPs‐1, while C‐4 might be the binding site of zinc and polysaccharide. UV, FTIR and XRD spectra suggested ‐OH and ‐COOH group might be involved in the binding of zinc to polysaccharides in OZPs‐1 and there were no ZnO and sulphate substitutions in OZPs‐1. These conclusions indicated that zinc might bind with ‐OH and ‐COOH in C‐4 of polysaccharides and impart the stable structure and favourable physicochemical properties of OZPs‐1, which could facilitate the application of OZPs‐1 as natural organic zinc supplements. [ABSTRACT FROM AUTHOR]

Protein content, a vital component determining the nutritional quality of oysters, is unevenly distributed in different parts of oyster. In this study, the spatial distribution (visceral mass, mantle, gill, and adductor) patterns and structural characteristics of proteins, including water–soluble proteins (WSP), salt–soluble proteins (SSP), acid–soluble proteins (ASP) and alkali–soluble proteins (ALSP) of oysters (Crassostrea hongkongensis) were investigated with the amino acid analyzer, circular dichroism spectroscopy (CD), fourier transform infrared spectroscopy (FTIR), and fluorescence spectroscopy. The results showed that oyster proteins were mainly distributed in the visceral mass and mantle. The protein composition was WSP, SSP, ALSP, and ASP in descending order, which conformed to the ideal amino acid pattern. Variations in secondary structure, molecular weight distribution, and thermal denaturation temperatures of the oyster proteins were observed. SSP had wider bands (16–270 kDa) than those of ASP (30–37 kDa) and ALSP (66–270 kDa). Among the four proteins, the SSP of the mantle showed the highest thermal stability (87.4 °C), while ALSP of the adductor muscle had the lowest the lowest the peak denaturation temperature (Tm) (53.8 °C). The proportions of secondary structures in oyster proteins were different, with a higher proportion of solid protein β–folds, and the exposure of aromatic amino acid residues and disulfide bonds and the microenvironment in which they were located were also different. [ABSTRACT FROM AUTHOR]

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Interspecific hybridization of oysters is a highly efficient breeding strategy that can produce commercially viable phenotypes. Crassostrea hongkongensis , the primary cultivar in southern China, faces increasing summer mortality risks and slow growth rates, which elevate commercial risks for farmers. C. iredalei , a commercially cultivated species in Southeast Asia, has been expanding its ecological niche northward in recent years, providing opportunities for interspecific hybridization with C. hongkongensis. This study conducted a 2 × 2 factorial cross between C. hongkongensis and C. iredalei (II: C. iredalei ♀ × C. iredalei ♂, HH: C. hongkongensis ♀ × C. hongkongensis ♂; IH: C. iredalei ♀ × C. hongkongensis ♂ , HI: C. hongkongensis ♀ × C. iredalei ♂) and set up 6 salinity treatments (14, 17, 20, 23, 26 and 29 ppt) to explore the optimal salinity for embryo hatching. The results showed that the hybridization was successful, despite bidirectional fertilization and pronounced asymmetry in fertilization success. The optimal hatching salinity for C. iredalei and C. hongkongensis was 23 ppt and 17–20 ppt, respectively, while the optimal salinity for reciprocal hybrids was 20 ppt. Notably, the hatching rate of IH was lower than that of the control group at the optimal salinity. During the pelagic larval stage, hybrid larvae exhibited significant hybrid inferiority, with slower growth rates and higher mortality rates compared to intraspecific hybridization. It is worth noting that IH failed to overcome genetic incompatibility, resulting in complete mortality during the pelagic larval stage and failure to enter the growth stage. At the 360th day of the growth period, HI hybrids grown in Sanbaimen Sea exhibited significant hybrid vigor in shell height (15.65 %), and in Daya Bay Marine Biology Research Station, Chinese Academy of Sciences (MBRS), they showed significant single-parent hybrid advantages in shell height (28.44 %) and cumulative survival rate (58.11 %) compared to C. hongkongensis. In terms of gonadal development, HI hybrids were able to produce functional and viable gametes, although with some delay compared to their parents. The hybrid offspring inherited the phenotypic traits of their parents, with a grayish-yellow flesh color intermediate between the two oyster species. HI is a valuable genetic resource for oyster breeding, and its growth potential should be further explored to meet commercialization demands. • Artificial hybridization of C. hongkongensis and C. iredalei was firstly reported; • Successfully identified hybrid offspring using capillary electrophoresis; • While fertilization in hybrid experiments is bidirectional, IH (C. iredalei ♀ × C. hongkongensis ♂) is incapable of surpassing the umbo larval stage; • The growth performance of HI (C. hongkongensis ♀ × C. iredalei ♂) has a single parent heterosis compared to C. hongkongensis. • HI (C. hongkongensis ♀ × C. iredalei ♂) has delayed gonadal development, but gamete morphology and function are normal. [ABSTRACT FROM AUTHOR]

Crassostrea hongkongensis (C. hongkongensis) is an important economic shellfish in marine aquaculture, but suffers from severe infection of Vibrio parahaemolyticus (V. Parahemolyticus). The study on immune strategy against Vibrio infection will help to enhance the disease resistance of C. hongkongensis. In this study, after V. parahaemolyticus challenge, the immune enzyme activities and transcriptome were analyzed to explain the immune strategy of C. hongkongensis. The immune enzyme activities analysis showed that all of superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM) activities and malondialdehyde (MDA) content were significantly increased among 3 and 12 h post-infection, indicating that V. parahaemolyticus had stimulated the immune response of C. hongkongensis. Transcriptome analysis showed that a total of 636,295,318 clean readings were generated from the libraries of control and infection groups. Comparing with the control groups (C0h), 147 differentially expressed genes (DEGs) were identified in T3h group, 392 DEGs in T6h group, 47 DEGs in T12h group, and 423 DEGs in T24h group. The time-series expression profiles of DEGs showed that the rising and failing trends were significantly enriched in 2 clusters, including 158 DEGs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that immune-related DEGs were annotated into 20 categories, including Toll and Imd signaling pathway, IL-17 signaling pathway, Toll like receptor (TLR) pathway and c-type lectin receptor signaling pathway. Since multiple immune-related DEGs were enriched in TLR signal pathway, qRT-PCR was employed to validate the expression of genes in TLR pathway. The qRT-PCR results showed that all of six detected genes were significantly upregulated, including TLR4, MyD88, IRAK4, TRAF6, IL17 and COX2. The data indicated that TLR4-MyD88 pathway took part in the immune response of C. hongkongensis against Vibrio infection.

Oysters are nutritious and tasty but difficult to store. Drying can extend the storage period of oysters and give them a unique flavor. In this study, the effects of four drying procedures, namely, vacuum freeze drying (VFD), vacuum drying (VD), natural sun-drying (NSD), and hot air drying (HAD), on the flavor characteristics of oysters (Crassostrea hongkongensis) were investigated using blanched oysters as a control (CK). Results showed that HAD produced more free amino acids than the other methods, but VFD retained the most flavor nucleotides. Compared with cold drying (VFD), hot drying (VD, NSD, and HAD) increased the abundance of organic acids, betaine, and aroma substances. Glutamic acid, alanine, AMP, hexanal, octanal, heptanal, (E, E)-2,4-heptadienal, (E)-2-decenal, nonanal, etc., are defined as the characteristic flavor compounds of dried oysters, with umami, sweet, green, fatty, and fruity aromas being the main organoleptic attributes of dried oysters. Glutamic acid, glycine, betaine, IMP, pentanal, ethyl heptanoate, (E, Z)-2,4-nonadienal, 1-octen-3-one, 2-hexenal, 2-octenal, hexanal, decanal were defined as markers to distinguish different drying methods. Overall, HAD showed improved flavor qualities and characteristics and was better suited for the highly commercialized production of dried oysters.

Interspecific hybridization plays a significant role in the genetic improvement of oysters in aquaculture, with the main objective being to obtain progeny that inherit the desirable traits of both parents and establish a stable inheritance pattern. Crassostrea hongkongensis is well-known for its large size and excellent meat quality. However, it exhibits poor resistance to external environmental changes and a high mortality rate. To address these shortcomings, this study conducted a 2 × 2 interspecific hybridization between Crassostrea dianbaiensis and C. hongkongensis. D larvae from C. hongkongensis ♀ × C. dianbaiensis ♂ (HD) and C. dianbaiensis ♀ × C. hongkongensis ♂ (DH) crosses were successfully hatched. However, the hatching index of both groups was lower than that of C. hongkongensis ♀ × C. hongkongensis ♂ (HH) and C. dianbaiensis ♀ × C. dianbaiensis ♂ (DD) groups. The average shell height of DH on the 15th day was only 111.50 ± 6.31 μm, and all individuals were dead by the 18th day. Despite experiments being repeated many times, no attached DH larvae were obtained. Compared with intraspecific hybridization, HD grew slower in the larval stage and had a lower survival rate, but the shell height of HD was slightly higher than that of HH on the 450th day (P > 0.05). The cumulative survival rate of HD throughout the growth period was higher than that of HH and DD, demonstrating a clear hybrid advantage in terms of relative yield and survival rate, especially higher than that of C. hongkongensis. Moreover, the gonads of HD can develop normally and produce gametes, and the color of the inner shell and body is between that of C. hongkongensis and C. dianbaiensis. These results indicate that HD effectively improved the survival rate of C. hongkongensis , demonstrating its great application potential and promising prospects in aquaculture. • Artificial hybridization of C. hongkongensis and C. dianbaiensis was firstly reported. • Hybrid offspring were successfully confirmed by ITS2 molecular markers. • Fertilization is bidirectional and produce normal D larvae, but DH larvae will be dead within 18 days. • HD hybrids exhibited notable heterosis in term of both survival and yield traits. [ABSTRACT FROM AUTHOR]

Gender differences in the hemocyte immune response of Hong Kong oyster Crassostrea hongkongensis to Vibrio harveyi and lipopolysaccharide (LPS) infection exist. To determine if a gender difference also exists, we use a 1H NMR-based metabolomics method to investigate responses in C. hongkongensis hepatopancreas tissues to V. harveyi and LPS infection. Both infections induced pronounced gender- and immune-specific metabolic responses in hepatopancreas tissues. Responses are mainly presented in changes in substances involved in energy metabolism (decreased glucose, ATP, and AMP in males and increased ATP and AMP in LPS-infected females), oxidative stress (decreased glutathione in males and decreased tryptophan and phenylalanine and increased choline and proline in LPS-infected females), tricarboxylic acid (TCA) cycle (decreased α-ketoglutarate acid and increased fumarate in LPS-infected males, and decreased fumarate in LPS-infected females), and osmotic regulation (decreased trigonelline and increased taurine in V. harveyi-infected males and decreased betaine in V. harveyi-infected females). Results suggest that post-spawning-phase male oysters have a more significant energy metabolic response and greater ability to cope with oxidative stress than female oysters. We propose that the impact of oyster gender should be taken into consideration in the aftermath of oyster farming or oyster disease in natural seas. [ABSTRACT FROM AUTHOR]

Ammonia nitrogen, a major oxygen-consuming pollutant in the environment, can adversely affect aquatic organisms such as fish, bivalves, and crustaceans. We investigated the toxic effects of ammonia nitrogen on the Hong Kong oyster, Crassostrea hongkongensis, using flow cytometry and 1H nuclear magnetic resonance metabolomics. Exposure to ammonia nitrogen caused time- and concentration-dependent alterations in various immune parameters in hemocytes and impaired the metabolic profiles of the gills. We observed changes in the rate of apoptosis, esterase activity, lysosomal mass, hemocyte counts, phagocytic activity, and mitochondrial mass. Exposure affected metabolic pathways involved in energy metabolism, osmotic balance, and oxidative stress. We concluded that ammonia nitrogen induces metabolic and hematological dysfunction in C. hongkongensis, and our findings provide insights into the biochemical defense strategies of bivalves exposed to acute high-concentration ammonia nitrogen.

The Hong Kong oyster (Crassostrea hongkongensis) lives in the brackish waters of estuaries in the intertidal zone and has a high resistance to low levels of salinity, but low resistance to higher levels. Juveniles are more sensitive to high levels of salinity than adults. To better understand the mechanisms underlying these differences, we performed a high-salinity stress analysis of juvenile and adult Hong Kong oysters. Trait analysis between groups showed that Na+-K+-ATPase activity, T-AOC, betaine, taurine and glycogen content of the high-salinity group were almost all higher than that of the control group at 8 h under high-salt stress, but lower than that of the control group at 96 h. The differences are more significant in Adult population. The results indicate that these indexes are related to the salinity adaptability of oysters. Enrichment analysis and protein-protein interaction (PPI) network analysis of the differentially expressed genes (DEGs) screened from transcriptomic data showed that their functions were mainly related to the oxidation-reduction process, immune response, protein ubiquitination and ATP synthesis. Furthermore, a weighted gene co-expression network analysis (WGCNA) of all samples showed that the hub gene functions were mainly associated with the oxidation-reduction process and immune response. These results indicate that immune response-related and antioxidant genes play key roles in the adaptability of Hong Kong oysters to high salinity. This study provides new insights and enriches our current understanding of the salinity adaptation mechanisms in oysters.

Hydrolase genes play an important role in the immune defense system in mollusks. We found a new hydrolase gene in Crassostrea hongkongensis. To explore its function, we cloned and characterized the hydrolase gene (Chhd) from C. hongkongensis. The full-length cDNA of Chhd is 936 bp, which is composed of a 531 bp open reading frame (ORF), a 92 bp 5′ -untranslated region (UTR) and a 313 bp 3′ UTR, the ORF encodes a polypeptide of 176 amino acids which contains a hydrolase_2 domain. qRT-PCR results showed that Chhd was ubiquitously expressed in tissues of C. hongkongensis, and the expression was highest in the mantle. After bacterial challenge, the expression of Chhd in hemocytes was significantly up-regulated. Recombinant pEGFP-N1-Chhd was distributed in the cytoplasm of HEK293T cells. The results of prokaryotic expression showed that pET28a-Chhd protein could express in large quantities. The expression of Chhd in total protein of C. hongkongensis was detected by western blot assay. In addition, Chhd inhibited the activities of NF-κB/p21-Luc in HEK293T cells, and RNA interference of Chhd significantly reduced the apoptosis rate of C. hongkongensis hemocytes. Taken together, our finding indicated that Chhd was involved in immunity and apoptosis regulation of C. hongkongensis, and would provide basic data for understanding the immune mechanism of shellfish.

Integrins, a family of cell adhesion transmembrane receptors, mediate cell adhesion, migration, proliferation, apoptosis, and phagocytosis. In the present study, an integrin ChIntα 4 from Crassostrea hongkongensis was characterized to investigate its role in defensing against pathogenic bacterium Vibrio alginolyticus. The full-length cDNA sequence of ChIntα 4 was 3572 bp with an open reading frame (ORF) of 3168 bp, which encoded a polypeptide with 1055 amino acids. The mRNA expression of ChIntα 4 in the hemocytes was significantly up-regulated at 6 h and 24 h post V. alginolyticus stimulation (p < 0.01). The recombinant Ch Intα 4 protein could agglutinate the rabbit red blood cells and Gram-negative bacteria V. alginolyticus and Escherichia coli. Moreover, the phagocytic activity of the hemocytes was significantly down-regulated from 46.9% to 32.7% when blocked with anti- Ch Intα 4 antibody, and it was significantly up-regulated from 42.7% to 59.5% post transfection with pCI-neo- ChIntα 4 plasmid (p < 0.05). In conclusion, these findings demonstrated that Ch Intα 4 might be involved in resisting V. alginolyticus infection and regulating phagocytosis as a cell adhesion receptor in C. hongkongensis. • The sequence of ChIntα 4 was firstly cloned from Crassostrea hongkongensis. • The mRNA level of ChIntα 4 showed positive response post Vibrio stimulation. • Ch Intα 4 protein agglutinated the erythrocyte and Gram-negative bacteria. • Ch Intα 4 protein was demonstrated to regulate phagocytosis of the hemocytes. [ABSTRACT FROM AUTHOR]

Hong Kong oyster (Crassostrea hongkongensis) is one of the main species of economic shellfish cultivated in the coastal areas of southern China. The cultivation of this shellfish may be adversely impacted by Vibrio parahaemolyticus , a harmful pathogenic bacterium for many mariculture species, as it usually exists on the surface of Hong Kong oysters. Although previous studies have discovered that oysters rely on non-specific immune system to fight pathogen invasion, the genes corresponding to the complex immune system against Vibrio is still not fully elucidated. Therefore, we conducted a transcriptome analysis on the gill from Hong Kong oysters at two time points (i.e., 12 h and 24 h after V. parahaemolyticus or PBS challenge) to identify potential immune genes against V. parahaemolyticus infection. A total of 61779 unigenes with the average length of 1221 bp were obtained, and the annotation information of 39917 unigenes were obtained from Nr, SwissProt, KEGG and COG/KOG. After a pairwise comparison between V. parahaemolyticus or PBS challenge at the two time points, three groups of differentially expressed genes induced by V. parahaemolyticus were captured and analyzed. GO and KEGG analyses showed that multiple immune-related genes played an important role in pathogen infection, including HSP70, PCDP3 and TLR4. Furthermore, genes annotation indicated that LITAF, TNFSF10, Duox2 and big defensin family are also involved in immune regulation. Our study provides a reference for further exploration the molecular mechanism that defenses the pathogen infection regarding the identified immune-related genes in Hong Kong oysters. • RNA-seq was used to investigate the immune response of Hong Kong oyster against V. parahaemolyticus infection in our study. • We mainly focused on the analysis of three groups of differentially expressed genes induced by V. parahaemolyticus. •Our results reflected potential role of multiple immune-related genes in the immune response of Hong Kong oyster. [ABSTRACT FROM AUTHOR]

Background: Crassostrea hongkongensis is an important mariculture shellfish with a relatively narrow distribution range. Recently, larger wild oysters were identified as C. hongkongensis from Sanmen bay in East China Sea. No natural distribution had been reported for this species here, and its origin remains unknown. Methods and results: We assembled the complete 18,617 bp circular mitochondrial genome of C. hongkongensis from Sanmen bay by next generation sequencing. It included 12 protein-coding genes, 23 tRNAs, and two rRNAs. The A/T content of the mitogenome was higher than its G/C content. Similar values and features were previously found for five other specimens of C. hongkongensis, and were comparable to those of other congeneric species. A phylogenetic analysis based on the 12 protein-coding genes and complete mitochondrial sequence indicated that the six specimens of C. hongkongensis formed a monophyletic group and shared a sister group relationship with C. ariakensis, C. nippona, C. sikamea, C. angulata, C. gigas, and C. iredalei, whereas specimens from the Sanmen bay area clustered later with the five other C. hongkongensis individuals, sharing a sub-clade. The newly sequenced mitogenome had more singleton sites than previously published C. hongkongensis mitogenomes. Conclusions: Crassostrea hongkongensis may be a native species, and the species' range extends further to the north than previously known. Our data may therefore contribute to a better understanding of the species diversity and conservation of Crassostrea oysters. [ABSTRACT FROM AUTHOR]

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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

Copyright of South China Fisheries Science is the property of South China Fisheries Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background Perkinsosis, a disease caused by the protist Perkinsus, is responsible for mass mortalities of many molluscan species worldwide. The rapid, early and accurate detection of Perkinsus infection is necessary to react to outbreaks, and manage disease transmission. Current methods for diagnosis of Perkinsus spp. are time-consuming or require professional equipment and experienced personnel, rendering them unsuitable for field application. Recombinase polymerase amplification (RPA) assay is a highly sensitive and selective isothermal amplification technique that operates at temperatures of 37–42 °C, requires minimal sample preparation, and is capable of amplifying as low as 1–10 target DNA copies in less than 20 minutes. Methods We report a novel RPA assay that amplifies the internal transcriber spacer (ITS) region of P. beihaiensis, which, followed by rapid detection of amplicons using a lateral flow (LF) strip, enables easy visualization of results by the naked eye. Results The LF-RPA assay successfully amplified P. beihaiensis DNA using a set of primers of 20–25 bp in length. After incubation at 37 °C for 25 min, results were read within 5 min by the naked eye on a lateral flow strip. Our LF-RPA assay was comparably sensitive to qPCR assay, and capable of detecting as few as 26 copies of P. beihaiensis DNA. Cross-amplification occurred with other two Perkinsus species, P. olseni and P. chesapeaki, but not with other potential pathogen taxa in culture environments. We compared the performance of LF-RPA, conventional PCR and qPCR assays on 60 oyster samples. While LF-RPA assay results were 86.2% as sensitive, 77.4% as specific, and generally in agreement with those of conventional PCR results, they were more (93.3%) sensitive, (86.7%) specific, and agreed better with qPCR assay results. Future research should focus on developing simple DNA extraction methods that do not require professional laboratories and complicated extraction procedures, to facilitate application of this LF-RPA assay in the field. Conclusions Our LF-RPA assay provides a rapid and efficient method for detecting species of Perkinsus. This novel assay has potential to be used in field applications.

Protein content, a vital component determining the nutritional quality of oysters, is unevenly distributed in different parts of oyster. In this study, the spatial distribution (visceral mass, mantle, gill, and adductor) patterns and structural characteristics of proteins, including water–soluble proteins (WSP), salt–soluble proteins (SSP), acid–soluble proteins (ASP) and alkali–soluble proteins (ALSP) of oysters (Crassostrea hongkongensis) were investigated with the amino acid analyzer, circular dichroism spectroscopy (CD), fourier transform infrared spectroscopy (FTIR), and fluorescence spectroscopy. The results showed that oyster proteins were mainly distributed in the visceral mass and mantle. The protein composition was WSP, SSP, ALSP, and ASP in descending order, which conformed to the ideal amino acid pattern. Variations in secondary structure, molecular weight distribution, and thermal denaturation temperatures of the oyster proteins were observed. SSP had wider bands (16–270 kDa) than those of ASP (30–37 kDa) and ALSP (66–270 kDa). Among the four proteins, the SSP of the mantle showed the highest thermal stability (87.4 °C), while ALSP of the adductor muscle had the lowest the lowest the peak denaturation temperature (Tm) (53.8 °C). The proportions of secondary structures in oyster proteins were different, with a higher proportion of solid protein β–folds, and the exposure of aromatic amino acid residues and disulfide bonds and the microenvironment in which they were located were also different.

An enrichment protocol was used to isolate and characterize tri-nucleotide microsatellite markers in Crassostrea hongkongensis, an economically important and extensively cultured oyster species along the southern coastal areas of China. Thirty tri-nucleotide microsatellite loci were assessed in 32 individuals from a wild population. The number of alleles ranged from 2 to 12, averaging 7.63 alleles per locus. The observed and expected heterozygosity values ranged from 0.031 to 0.844 (averaging 0.579) and 0.062 to 0.881 (averaging 0.665), respectively. Six loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction. BLAST analysis showed that the tri-nucleotide microsatellite markers randomly developed from the genome of C.hongkongensis have a higher correlation with functional genes than di-nucleotide markers. Cross-amplification in C. ariakensis and C. gigas was performed and 21 loci showed reasonable polymorphism between C. hongkongensis and C. ariakensis, while 15 loci showed reasonable polymorphism between C. hongkongensis and C. gigas. The tri-nucleotide microsatellite markers identified in this study have greater potential for future use in gene mapping, molecular-marker assisted selection and comparative linkage mapping than previously identified di-nucleotide markers. Additionally, we found two microsatellite loci that can effectively identify the purebred and hybrid offspring of these three oyster species.

The Hong Kong oyster (Crassostrea hongkongensis) is endemic and one of the most important economic and extensively cultured oyster species along the southern coastal areas of China. However, limited information is available about its genetic variation and genetic structure. In this study, we analyzed genetic variation at twelve microsatellite DNA loci in eleven locations throughout the distribution of C. hongkongensis in the Sourthern China. Similarly to other cupped oyster species, a high level of genetic diversity was observed in all sampled populations. Populations exhibited significant genetic differentiation, and could be divided into four genetically distinct and geographically related groups: Guangxi group, the eastern Guangdong group, the western Guangdong group and Fujian group. The highest local differentiation was observed between the samples from Guangxi versus Guangdong and Fujian, which are separated by the Leizhou Peninsula. In addition, significant genetic differentiation was observed between the samples from the western Guangdong versus the eastern Guangdong, which are separated by the Pearl River estuary. Geographic isolation is one of the most important factors causing genetic differentiation among populations of C. hongkongensis. Additionally, the differentiation between some populations may be caused by salinity of seawater and runoff from the Pearl River. The significant genetic differentiation among populations implies that translocation of individuals for aquaculture should be properly managed for the preservation of genetic diversity and eventual local adaptation in natural populations.