Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention.

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4+T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.

Expression and functional analysis of Fushi Tarazu transcription factor 1 (FTZ-F1) in the regulation of steroid hormones during the gonad development of Fujian Oyster, Crassostrea angulata.

Crassostrea angulata, a major shellfish cultivated in Southern China, has experienced a notable surge in commercial value in recent years. Understanding the molecular mechanisms governing their reproductive processes holds significant implications for advancing aquaculture practices. In this study, we cloned the orphan nuclear receptor gene, Fushi Tarazu transcription factor 1 (FTZ-F1), of C. angulata and investigated its functional role in the gonadal development. The full-length cDNA of FTZ-F1 spans 2357 bp and encodes a protein sequence of 530 amino acids. Notably, the amino acid sequence of FTZ-F1 in C. angulata shares remarkable similarity with its homologues in other species, particularly in the DNA-binding region (>90%) and ligand-binding region (>44%). In C. angulata, the highest expression level of FTZ-F1 was observed in the ovary, exhibiting more than a 200-fold increase during the maturation stage compared to the initiation stage (P < 0.001). Specifically, FTZ-F1 was mainly expressed in the follicular cells surrounding the oocytes of C. angulata. Upon inhibiting FTZ-F1 gene expression in C. angulata through RNA interference (RNAi), a substantial reduction in the expression of genes involved in the synthesis of sex steroids in the gonads, including 3ß-HSD, Cyp17, and follistatin, was observed. In addition, estradiol (E2) and testosterone (T) levels also showed a decrease upon FTZ-F1 silencing, resulting in a delayed gonadal development. These results indicate that FTZ-F1 acts as a steroidogenic factor, participating in the synthesis and regulation of steroid hormones and thus playing an important role in the reproductive and endocrine systems within oysters.

Microplastics in coastal farmed oyster (Crassostrea angulata) shells: Abundance, characteristics, and diversity.

One of the most concerning emerging pollutants is microplastics (MPs), which can infiltrate soft tissues of organisms by ingestion, adhesion, and fusing and may even become embedded in biominerals. However, very little evidence is available about MPs in biominerals found in the wild. This study detected the abundance and characteristics of MPs in the shells of farmed oysters (Crassostrea angulata) off the coast of Taiwan and discussed the distribution, accumulation, and diversity in the oyster shells. The results showed that MPs were ubiquitous in oyster shells, with an average abundance of 0.70 ± 0.40 MPs/g. MPs abundance was significantly (p < 0.01) higher in small oyster shells (shell length < 6.5 cm, weight 5-10 g) and inorganic (CaCO3) fraction (HCl digestion) than in large oyster shells (>6.5 cm, 10-25 g) and an organic fraction (H2O2 digestion), respectively. However, there was no significant difference in MPs abundance between the top and bottom shells (p > 0.05). MPs with a size <2 mm accounted for 78.5 %, fibrous MPs for 93.7 %, and rayon for 89.5 %. The MPs diversity integrated index (MPDII) in oyster shells was low (0.27), and the small and fibrous MPs seemed more easily embedded in biominerals. The findings confirm the presence of MPs in oyster shells in coastal environments. In addition, oyster shells may contain higher amounts of MPs than soft tissues 4-5 times, which needs to be confirmed. Further revealing the distribution and accumulation of MPs in water/terrestrial biominerals will help to understand the fate of MPs in the environment.

Comparative proteomic and phosphoproteomic analysis reveals differential heat response mechanism in two congeneric oyster species.

High-temperature stress caused by global climate change poses a significant threat to marine ectotherms. This study investigated the role of protein phosphorylation modifications in the molecular regulation network under heat stress in oysters, which are representative intertidal organisms that experience considerable temperature changes. Firstly, the study compared the extent of thermal damage between two congeneric oyster species, the relative heat-tolerant Crassostrea angulata (C. angulata) and heat-sensitive Crassostrea gigas (C. gigas), under sublethal temperature (37 °C) for 12 h, using various physiological and biochemical methods. Subsequently, the comparative proteomic and phosphoproteomic analyses revealed that high-temperature considerably regulated signal transduction, energy metabolism, protein synthesis, cell survival and apoptosis, and cytoskeleton remodeling through phosphorylation modifications of related receptors and kinases. Furthermore, the protein kinase A, mitogen-activated protein kinase 1, tyrosine-protein kinase Src, and serine/threonine kinase AKT, exhibiting differential phosphorylation modification patterns, were identified as hub regulators that may enhance glycolysis and TCA cycle to increase the energy supply, distribute protein synthesis, inhibit Caspase-dependent apoptosis activated by endogenous mitochondrial cytochrome release and maintain cytoskeletal stability, ultimately shaping the higher thermal resistance of C. angulata. This study represents the first investigation of protein phosphorylation dynamics in marine invertebrates under heat stress, reveals the molecular mechanisms underlying the differential thermal responses between two Crassostrea oysters at the phosphorylation level, and provides new insights into understanding phosphorylation-mediated molecular responses in marine organisms during environmental changes and predicting the adaptive potential in the context of global warming.

Ocean acidification drives gut microbiome changes linked to species-specific immune defence.

Ocean acidification (OA) has important effects on the intrinsic phenotypic characteristics of many marine organisms. Concomitantly, OA can alter the extended phenotypes of these organisms by perturbing the structure and function of their associated microbiomes. It is unclear, however, the extent to which interactions between these levels of phenotypic change can modulate the capacity for resilience to OA. Here, we explored this theoretical framework assessing the influence of OA on intrinsic (immunological responses and energy reserve) and extrinsic (gut microbiome) phenotypic characteristics and the survival of important calcifiers, the edible oysters Crassostrea angulata and C. hongkongensis. After one-month exposure to experimental OA (pH 7.4) and control (pH 8.0) conditions, we found species-specific responses characterised by elevated stress (hemocyte apoptosis) and decreased survival in the coastal species (C. angulata) compared with the estuarine species (C. hongkongensis). Phagocytosis of hemocytes was not affected by OA but in vitro bacterial clearance capability decreased in both species. Gut microbial diversity decreased in C. angulata but not in C. hongkongensis. Overall, C. hongkongensis was capable of maintaining the homeostasis of the immune system and energy supply under OA. In contrast, C. angulata's immune function was suppressed, and the energy reserve was imbalanced, which might be attributed to the declined microbial diversity and the functional loss of essential bacteria in the guts. This study highlights a species-specific response to OA determined by genetic background and local adaptation, shedding light on the understanding of host-microbiota-environment interactions in future coastal acidification.

Genome-Wide Association Analysis of Heat Tolerance in F2 Progeny from the Hybridization between Two Congeneric Oyster Species.

As the world's largest farmed marine animal, oysters have enormous economic and ecological value. However, mass summer mortality caused by high temperature poses a significant threat to the oyster industry. To investigate the molecular mechanisms underlying heat adaptation and improve the heat tolerance ability in the oyster, we conducted genome-wide association analysis (GWAS) analysis on the F2 generation derived from the hybridization of relatively heat-tolerant Crassostrea angulata ♀ and heat-sensitive Crassostrea gigas ♂, which are the dominant cultured species in southern and northern China, respectively. Acute heat stress experiment (semi-lethal temperature 42 °C) demonstrated that the F2 population showed differentiation in heat tolerance, leading to extremely differentiated individuals (approximately 20% of individuals die within the first four days with 10% survival after 14 days). Genome resequencing and GWAS of the two divergent groups had identified 18 significant SNPs associated with heat tolerance, with 26 candidate genes located near these SNPs. Eleven candidate genes that may associate with the thermal resistance were identified, which were classified into five categories: temperature sensor (Trpm2), transcriptional factor (Gata3), protein ubiquitination (Ube2h, Usp50, Uchl3), heat shock subfamily (Dnajc17, Dnaja1), and transporters (Slc16a9, Slc16a14, Slc16a9, Slc16a2). The expressional differentiation of the above genes between C. gigas and C. angulata under sublethal temperature (37 °C) further supports their crucial role in coping with high temperature. Our results will contribute to understanding the molecular mechanisms underlying heat tolerance, and provide genetic markers for heat-resistance breeding in the oyster industry.

Identification of the Immunoglobulin E Epitope of Arginine Kinase, an Important Allergen from Crassostrea angulata.

Arginine kinase (AK) was identified as an allergen in Crassostrea angulata. However, little information is available about its epitopes. In this study, AK from C. angulata was registered to the World Health Organization/International Union of Immunological Societies allergen nomenclature committee to be named as Cra a 2. The immunoglobulin G/immunoglobulin E-binding capacity of Cra a 2 was significantly reduced after chemical denaturation treatment. Further, eight linear mimotopes and five conformational mimotopes of Cra a 2 were obtained using phage panning. In addition to six linear epitopes that have been identified, two linear epitopes were verified by a synthetic peptide, of which L-Cra a 2-2 was conserved in shellfish. Four conformational epitopes were verified by site-directed mutation, among which mutation of C-Cra a 2-1 affected the structure and reduced the immunoreactivity of Cra a 2 most significantly. Overall, the identified epitopes may lay a foundation for the development of hypoallergenic oyster products through food processing.

Characterization, Epitope Identification, and Cross-reactivity Analysis of Tropomyosin: An Important Allergen of Crassostrea angulata.

Oyster is a common shellfish product in China, which is associated with food allergy. However, there is still lack of research on allergens in oysters. In this study, tropomyosin (TM), an important allergen of Crassostrea angulata, was purified and identified by mass spectrometry. Subsequently, TM was cloned and expressed, with a sequence of size 852 bp, encoding 284 amino acid residues. The results of circular dichroism, digestion assay, inhibition enzyme-linked immunosorbent assay, and basophil activation test showed that recombinant TM had similar physicochemical properties and immunological properties to native TM. Furthermore, two conformational mimotopes were obtained and 10 IgE linear epitopes were verified. Meanwhile, different degrees of cross-reactivity were observed between C. angulata TM and the other 8 shellfish TMs using antibodies and serological analysis, which may relate to the 3 conserved epitope regions. These findings are expected to provide a theoretical basis for the molecular diagnosis of oyster allergy and cross-reactivity among shellfish.

Influence of seasonality on the quality of oysters from the Sado and Mira rivers.

There is a lack of knowledge about the influence of seasonality on the microbial and physicochemical quality of oysters in Sado and Mira rivers. Water, sediment, and oysters (Crassostrea angulata and Crassostrea gigas) were collected for microbiological, nutritional, and sensory analyses. The microbiological water quality and the oyster shell contamination were better during the warmer months. No seasonal effect was observed on sediments and on oyster meat. A good physicochemical and nutritional quality was also observed, with high content of polyunsaturated fatty acids, including omega-3 fatty acids, resulting in good lipid quality indices. From the sensory evaluation, both oysters' species were well scored and presented the highest scores (4) in parameters such as cream-ivory colour, sea smell, firmness and juiciness. These attributes denote the freshness degree at the time of the tasting, reflecting the quality of the bivalve.

Construction and analysis of the chromosome-level haplotype-resolved genomes of two Crassostrea oyster congeners: Crassostrea angulata and Crassostrea gigas.

BACKGROUND: The Portuguese oyster Crassostrea angulata and the Pacific oyster C. gigas are two major Crassostrea species that are naturally distributed along the Northwest Pacific coast and possess great ecological and economic value. Here, we report the construction and comparative analysis of the chromosome-level haplotype-resolved genomes of the two oyster congeners. FINDINGS: Based on a trio-binning strategy, the PacBio high-fidelity and Illumina Hi-C reads of the offspring of the hybrid cross C. angulata (♂) × C. gigas (♀) were partitioned and independently assembled to construct two chromosome-level fully phased genomes. The assembly size (contig N50 size, BUSCO completeness) of the two genomes were 582.4 M (12.8 M, 99.1%) and 606.4 M (5.46 M, 98.9%) for C. angulata and C. gigas, respectively, ranking at the top of mollusk genomes with high contiguity and integrity. The general features of the two genomes were highly similar, and 15,475 highly conserved ortholog gene pairs shared identical gene structures and similar genomic locations. Highly similar sequences can be primarily identified in the coding regions, whereas most noncoding regions and introns of genes in the same ortholog group contain substantial small genomic and/or structural variations. Based on population resequencing analysis, a total of 2,756 species-specific single-nucleotide polymorphisms and 1,088 genes possibly under selection were identified. CONCLUSIONS: This is the first report of trio-binned fully phased chromosome-level genomes in marine invertebrates. The study provides fundamental resources for the research on mollusk genetics, comparative genomics, and molecular evolution.