Aquaculture Performance and Genetic Diversity of a New [(Crassostrea hongkongensis ♀ × C. gigas ♂) ♂ × C. hongkongensis ♀] Variety of the Oyster "South China No. 1" in Beibu Gulf, China.

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.

Resistance of an intertidal oyster(Saccostrea mordax)to marine heatwaves and the implication for reef building.

Marine heatwaves (MHWs) have a significant impact on intertidal bivalves and the ecosystems they sustain, causing the destruction of organisms' original habitats. Saccostrea mordax mainly inhabits the intertidal zone around the equator, exhibiting potential tolerance to high temperatures and maybe a species suitable for habitat restoration. However, an understanding about the tolerance mechanism of S. mordax to high temperatures is unclear. It is also unknown the extent to which S. mordax can tolerate repeated heatwaves of increasing intensity and frequency. Here, we simulated the effects of two scenarios of MHWs and measured the physiological and biochemical responses and gene expression spectrum of S. mordax. The predicted responses varied greatly across heatwaves, and no heatwave had a significant impact on the survival of S. mordax. Specifically, there were no statistically significant changes apparent in the standard metabolic rate and the activities of enzymes of the oyster during repeated heatwaves. S. mordax exposed to high-intensity heatwaves enhanced their standard metabolic rate to fuel essential physiological maintenance and increasing activity of SOD and expression of HSP70/90. These strategies are presumably at the expense of functions related to immunity and growth, as best exemplified by significant depressions in activities of enzymes (NaK, CaMg, T-ATP, and AKP) and expression levels of genes (Rab, eEF-2, HMGR, Rac1, SGK, Rab8, etc.). The performance status of S. mordax tends to improve by implementing a suite of less energy-costly compensatory mechanisms at various levels of biological organization when re-exposed to heatwaves. The adaptive abilities shown by S. mordax indicate that they can play a crucial role in the restoration of oyster reefs in tropical seas.

Identification and involvement of DAX1 gene in spermatogenesis of boring giant clam Tridacna crocea.

DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenital critical region on X chromosome gene 1), a key sex determinant in various species, plays a vital role in gonad differentiation and development and controls spermatogenesis. However, the identity and function of DAX1 are still unclear in bivalves. In the present study, we identified a DAX1 (designed as Tc-DAX1) gene from the boring giant clam Tridacna crocea, a tropical marine bivalve. The full length of Tc-DAX1 was 1877 bp, encoding 462 amino acids, with a Molecular weight of 51.81 kDa and a theoretical Isoelectric point of 5.87 (pI). Multiple sequence alignments and phylogenetic analysis indicated a putative ligand binding domain (LBD) conserved regions clustered with molluscans DAX1 homologs. The tissue distributions in different reproductive stages revealed a dimorphic pattern, with the highest expression trend in the male reproductive stage, indicating its role in spermatogenesis. The DAX1 expression data from embryonic stages shows its highest expression profile (P < 0.05) in the zygote stage, followed by decreasing trends in the larvae stages (P > 0.05). The localization of DAX1 transcripts has also been confirmed by whole mount in situ hybridization, showing high positive signals in the fertilized egg, 2, and 4-cell stage, and gastrula. Moreover, RNAi knockdown of the Tc-DAX1 transcripts shows a significantly lower expression profile in the ds-DAX1 group compared to the ds-EGFP group. Subsequent histological analysis of gonads revealed that spermatogenesis was affected in a ds-DAX1 group compared to the ds-EGFP group. All these results indicate that Tc-DAX1 is involved in the spermatogenesis and early embryonic development of T. crocea, providing valuable information for the breeding and aquaculture of giant clams.

Chromosome-level genome assembly and annotation of rare and endangered tropical bivalve, Tridacna crocea.

Tridacna crocea is an ecologically important marine bivalve inhabiting tropical coral reef waters. High quality and available genomic resources will help us understand the population structure and genetic diversity of giant clams. This study reports a high-quality chromosome-scale T. crocea genome sequence of 1.30 Gb, with a scaffold N50 and contig N50 of 56.38 Mb and 1.29 Mb, respectively, which was assembled by combining PacBio long reads and Hi-C sequencing data. Repetitive sequences cover 71.60% of the total length, and a total of 25,440 protein-coding genes were annotated. A total of 1,963 non-coding RNA (ncRNA) were determined in the T. crocea genome, including 62 micro RNA (miRNA), 58 small nuclear RNA (snRNA), 83 ribosomal RNA (rRNA), and 1,760 transfer RNA (tRNA). Phylogenetic analysis revealed that giant clams diverged from oyster about 505.7 Mya during the evolution of bivalves. The genome assembly presented here provides valuable genomic resources to enhance our understanding of the genetic diversity and population structure of giant clams.

A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL.

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 dual-fluorescence 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.

Comparison of biochemical composition, nutritional quality, and metals concentrations between males and females of three different Crassostrea sp.

Gametogenesis can significantly affect the biochemical composition of oysters, but little research on the difference between sexes. Therefore, we conducted the first in-depth study on the composition differences between males and females of three different Crassostrea sp.. The results showed that females had higher glycogen, lipid, Cu and Zn contents than males, while males had higher protein and taurine contents than females at maturity, which might be related to special meiosis pattern of eggs and less energy was required for female gametogenesis. In addition, both males and females had well-balanced essential amino acid compositions. The omega-3: omega-6 (n-3: n-6) ratio of males was significantly higher than that of females, indicating that the nutritional quality of males was higher. These results provide a reliable and refined theoretical and research basis for revealing the nutritional quality, extracting beneficial ingredients, and developing functional food of Crassostrea sp., and provide data support for the sex-regulated breeding of oysters.

Adaptive Bird-like Genome Miniaturization During the Evolution of Scallop Swimming Lifestyle.

Genome miniaturization drives key evolutionary innovations of adaptive traits in vertebrates, such as the flight evolution of birds. However, whether similar evolutionary processes exist in invertebrates remains poorly understood. Derived from the second-largest animal phylum, scallops are a special group of bivalve molluscs and acquire the evolutionary novelty of the swimming lifestyle, providing excellent models for investigating the coordinated genome and lifestyle evolution. Here, we show for the first time that genome sizes of scallops exhibit a generally negative correlation with locomotion activity. To elucidate the co-evolution of genome size and swimming lifestyle, we focus on the Asian moon scallop (Amusium pleuronectes) that possesses the smallest known scallop genome while being among scallops with the highest swimming activity. Whole-genome sequencing of A. pleuronectes reveals highly conserved chromosomal macrosynteny and microsynteny, suggestive of a highly contracted but not degenerated genome. Genome reduction of A. pleuronectes is facilitated by significant inactivation of transposable elements, leading to reduced gene length, elevated expression of genes involved in energy-producing pathways, and decreased copy numbers and expression levels of biomineralization-related genes. Similar evolutionary changes of relevant pathways are also observed for bird genome reduction with flight evolution. The striking mimicry of genome miniaturization underlying the evolution of bird flight and scallop swimming unveils the potentially common, pivotal role of genome size fluctuation in the evolution of novel lifestyles in the animal kingdom.

Serum amyloid protein (SAA) as a healthy marker for immune function in Tridacna crocea.

Serum amyloid protein (SAA) is known as an acute reactive protein of innate immunity in mammals. However, in invertebrates, the role of SAA in innate immunity is still unclear. In this study, a full-length cDNA of the SAA gene (named TcSAA) was cloned from Tridacna crocea, mollusca. The gene includes a 193 bp 5' untranslated region (UTR) and a 129 bp 3' UTR sequence, and the open reading frame (ORF) with 393 bp nucleotides encodes a polypeptide of 130 amino acids. TcSAA contains a typical signal peptide and an SAA functional domain. The mRNA expression of TcSAA was detected in all 12 selected tissues and 7 different developmental stages. Furthermore, the expression of TcSAA was increased quickly in hemocytes after challenge with V. coralliilyticus or LPS. Furthermore, rTcSAA could bind V. coralliilyticus and V. alginolyticus, and the protein could reduce the lethality rate of the clams from 80% to 55% which caused by V. coralliilyticus about 48 h after injection. In summary, these results indicate that TcSAA may act as a marker for monitoring health and protecting T. crocea.

Comparative Genomics Reveals Evolutionary Drivers of Sessile Life and Left-right Shell Asymmetry in Bivalves.

Bivalves are species-rich mollusks with prominent protective roles in coastal ecosystems. Across these ancient lineages, colony-founding larvae anchor themselves either by byssus production or by cemented attachment. The latter mode of sessile life is strongly molded by left-right shell asymmetry during larval development of Ostreoida oysters such as Crassostrea hongkongensis. Here, we sequenced the genome of C. hongkongensis in high resolution and compared it to reference bivalve genomes to unveil genomic determinants driving cemented attachment and shell asymmetry. Importantly, loss of the homeobox gene Antennapedia (Antp) and broad expansion of lineage-specific extracellular gene families are implicated in a shift from byssal to cemented attachment in bivalves. Comparative transcriptomic analysis shows a conspicuous divergence between left-right asymmetrical C. hongkongensis and symmetrical Pinctada fucata in their expression profiles. Especially, a couple of orthologous transcription factor genes and lineage-specific shell-related gene families including that encoding tyrosinases are elevated, and may cooperatively govern asymmetrical shell formation in Ostreoida oysters.

MDM2 is involved in the regulation of p53 expression in the immune response of oyster Crassostrea hongkongensis.

MDM2 (mouse double-minute) and p53 form a negative feedback loop and play a prominent role in preventing the induction of uncontrolled apoptosis. To better understand their potential roles in oyster Crassostrea hongkongensis, MDM2 and p53 homologs were first isolated and cloned in C. hongkongensis (named ChMDM2 and Chp53), and their mRNA expression patterns in tissues and developmental stages were analyzed. Multiple sequence alignment analysis and phylogenetic analysis of ChMDM2 and Chp53 displayed a high degree of homology and conservation. In addition, exposure to Vibrio coralliilyticus resulted in DNA damage and apoptosis in the hemocytes of C. hongkongensis, and found that the mRNA expression level of ChMDM2 was decreased, while the relative expression of Chp53 was significantly increased in the hemocytes and gills. Furthermore, fluorescence from ChMDM2-EGFP and Chp53-Red were found to be distributed in the nucleus of HEK293T cells. Besides, dual-luciferase reporter assays showed that ChMDM2 antagonized with Chp53 and participates in p53 signaling pathway. In addition, the interaction between ChMDM2 and Chp53 was confirmed strongly by Co-immunoprecipitation assays. Furthermore, the results of RNAi showed that ChMDM2 and Chp53 participated in apoptosis which induced infection of V. coralliilyticus. Taken together, our results characterized the features of ChMDM2 and Chp53, which played a critical role in apoptosis of C. hongkongensis.

Assessment of the juvenile vulnerability of symbiont-bearing giant clams to ocean acidification.

Ocean acidification (OA) severely affects marine bivalves, especially their calcification processes. However, very little is known about the fate of symbiont-bearing giant clams in the acidified oceans, which hinders our ability to develop strategies to protect this ecologically and economically important group in coral reef ecosystems. Here, we explored the integrated juvenile responses of fluted giant clam Tridacna squamosa (Lamarck, 1819) to acidified seawater at different levels of biological organization. Our results revealed that OA did not cause a significant reduction in survival and shell growth performance, indicating that T. squamosa juveniles are tolerated to moderate acidification. Yet, significantly reduced net calcification rate demonstrated the calcifying physiology sensitivity to OA, in line with significant declines in symbiont photosynthetic yield and zooxanthellae density which in turn lowered the amount of energy supply for energetically expensive calcification processes. Subsequent transcriptome sequencing and comparative analysis of differentially expressed genes revealed that the regulation of calcification processes, such as transport of calcification substrates, acid-base regulation, synthesis of organic matrix in the calcifying fluid, as well as metabolic depression were the major response to OA. Taken together, the integration of physiological and molecular responses can provide a comprehensive understanding of how the early life history stages of giant clams respond to OA and make an important leap forward in assessing their fate under future ocean conditions.

Transcriptome analysis reveals acclimation responses of pearl oysters to marine heatwaves.

Marine heatwaves (MHWs) are weather-timescale extreme events in the oceans and can have devastating effects on marine bivalves and ecosystems they support, with considerable socio-economic consequences. Yet, the extent to which marine bivalves have the capacity to acclimate and adapt to MHWs remains unknown. Understanding molecular responses to MHWs is imperative to develop strategies for conservation of ecologically and economically important marine organisms. Here, using RNA-Seq, we investigate how various MHWs scenarios elicit molecular changes in threatened and vulnerable pearl oysters, Pinctada maxima (Jameson). Acute exposure of MHWs - mimicked by rapid increases of seawater temperature from 24 °C to 28 °C and 32 °C, respectively - significantly affected the expression levels of metabolic and immune-related genes, with thermal stress-responsive genes especially like HSP20, HSP70 and HSP90 being remarkably up-regulated. Following repeat exposure to MHWs, encouragingly, pearl oysters exhibited evident acclimation responses, as best exemplified by significantly lowered expression levels of key stress-responsive genes involved in metabolism and immunity in comparison to those observed during acute exposure. Findings of the present study provide a better understanding of molecular processes underpinning the acclimation and adaptation of marine bivalves to MHWs in the context of climate change.

Impacts of marine heatwaves on pearl oysters are alleviated following repeated exposure.

Marine heatwaves (MHWs) have occurred with increasing duration, frequency and intensity in the past decade in the South China Sea, posing serious threats to marine ecosystems and fisheries. However, the impact of MHWs on marine bivalves - one of the most ecologically and economically important fauna in coastal ecosystems - remains largely unknown. Here, we investigated physiological responses of the pearl oyster, Pinctada maxima inhabiting a newly identified climate change hotspot (Beibu Gulf, South China Sea) to short-lasting and repeatedly-occurring MHWs scenarios. Following 3-day exposure to short-lasting MHWs scenarios with water temperature rapidly arising from 24 °C to 28 °C, 32 °C and 36 °C, respectively, mortality rates of pearl oysters increased, and especially they suffered 100% mortality at 36 °C. Activities of enzymes including acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione (GSH) and level of malondialdehyde (MDA) increased significantly with increasing intensity and duration of MHWs, indicating thermal stress responses. When exposed to repeatedly-occurring MHWs scenarios, mortality rates of pearl oysters increased slightly, and thermal stress responses were alleviated, as exemplified by significant decreases in ACP, AKP, GSH and MDA activities compared with those during short-lasting MHWs scenarios, demonstrating the potential of P. maxima to acclimate rapidly to MHWs. These findings advance our understanding of how marine bivalves respond to MHWs scenarios varying in duration, frequency, and intensity.

Repeated exposure to simulated marine heatwaves enhances the thermal tolerance in pearl oysters.

In an era of unprecedented climate change, marine heatwaves (MHWs) are projected to increase in frequency, intensity, and duration, severely affecting marine organisms and fisheries and causing profound ecological and socioeconomic impacts. However, very little is known about effects of MHWs on ecologically and economically important bivalve species. Here, we investigate how pearl oysters, Pinctada maxima (Jameson), respond to MHWs under various simulated scenarios. Acute 3-day exposure to MHWs, mimicked by increasing the ambient seawater temperature from 24°C to 28°C, 32°C, and 36°C, respectively, induced significant changes (initially sustained increase and late decrease) in activities of antioxidant enzymes (GSH-Px, SOD, CAT, MDA, and T-AOC) and biomineralizaiton-related enzymes (AKP and ACP). Likewise, energy-metabolizing enzymes (NKA, CMA, and T-ATP) showed remarkable acute responses, with significant increases occurring at the beginning and end of thermal exposure. Following repeated exposure to MHWs, without exception, acute responses of assayed enzymes to MHWs were significantly alleviated, implying that pearl oysters have the ability to implement more efficient and less costly energy-utilizing strategies to compensate for thermal stress induced physiological interferences. These findings indicate that marine bivalves can respond plastically and acclimate rapidly to MHWs and pave the way for predicting the fate of this important taxonomic groups in rapidly changing oceans.

Sequencing and analysis of the complete mitochondrial genome of Hippopus porcellanus.

In this study, the complete mitochondrial genome of Hippopus porcellanus was reported. The whole mitochondrial genome was 21,565bp in length with a typical mitochondrial genomic structure including 13 protein-coding genes, 23 transfer RNA genes, 2 ribosomal RNA genes and 1 control region (D-loop). Mitogenome base composition was biased toward A + T content, at 60.3%. A phylogenetic tree based on complete mitogenome sequences revealed that, H. porcellanus is closely related to H. hippopus, both of which belong to the genus Hippopus.

The genome of Nautilus pompilius illuminates eye evolution and biomineralization.

Nautilus is the sole surviving externally shelled cephalopod from the Palaeozoic. It is unique within cephalopod genealogy and critical to understanding the evolutionary novelties of cephalopods. Here, we present a complete Nautilus pompilius genome as a fundamental genomic reference on cephalopod innovations, such as the pinhole eye and biomineralization. Nautilus shows a compact, minimalist genome with few encoding genes and slow evolutionary rates in both non-coding and coding regions among known cephalopods. Importantly, multiple genomic innovations including gene losses, independent contraction and expansion of specific gene families and their associated regulatory networks likely moulded the evolution of the nautilus pinhole eye. The conserved molluscan biomineralization toolkit and lineage-specific repetitive low-complexity domains are essential to the construction of the nautilus shell. The nautilus genome constitutes a valuable resource for reconstructing the evolutionary scenarios and genomic innovations that shape the extant cephalopods.

Seasonal variations in biochemical composition and nutritional quality of Crassostrea hongkongensis, in relation to the gametogenic cycle.

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).

Tetraploid induction of Crassostrea hongkongensis and C. sikamea by inhibiting the polar body 1 release in diploid fertilized eggs.

The production of an all-triploid population by mating tetraploid males with diploid females is the best and most fundamental method for the large-scale production of triploid oysters. Obtaining a stable tetraploid population is essential for guaranteed production in industrialized triploid cultivation. C. hongkongensis and C. sikamea are important oyster breeding species in southern China, and have great economic value. However, there are not any published data on inducing tetraploid C. hongkongensis or C. sikamea. Therefore, we investigated tetraploid induction in these two oyster species by inhibiting the PB1 release in diploid fertilized eggs using Cytochalasin B (CB) under 31 °C, 15 ‰ salinity. The results confirmed that the optimal tetraploid induction conditions for C. hongkongensis were a CB concentration of 0.50 mg/L with induction starting at 9.0 min after fertilization, and stopping at 21.0 min after fertilization; the induction efficiency index reached 0.123 under these conditions. The optimal tetraploid induction conditions for C. sikamea were a CB concentration of 0.50 mg/L, with induction starting at 7.5 min after fertilization and stopping at 18 min after fertilization; the induction efficiency index could be as high as 0.281 under these conditions. However, we confirmed that the tetraploid rate decreased with larval growth, and no tetraploids were detected in the juvenile period of either C. hongkongensis or C. sikamea. This may be attributed to the very low survival of the tetraploid larvae induced by this method, especially as most tetraploid larvae died during the first three days. In summary, it is simple to directly induce tetraploid C. hongkongensis and C. sikamea larvae by inhibiting the PB1 release of diploid zygotes, but the low survival rate makes it challenging to obtain viable juvenile tetraploids.

First report of the mitochondrial DNA sequences of the giant clam, Tridacna gigas (Tridacnidae Tridacna).

In this study, we present the first complete mitochondrial genome sequence of the giant clam Tridacna gigas. The total length of the mitogenome is 19,558 bp. It contains the typical mitochondrial genomic structure, including 13 protein-coding genes, 23 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). Mitogenome base composition is biased toward A + T content, at 57.6%. A phylogenetic tree based on complete mitogenome sequences revealed that, within the genus Tridacna, T. gigas is closely related to T. derasa.