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Sterility caused by triploidization holds promising commercial implications in aquaculture. In this study, we conducted a thorough investigation into the spermatogenesis of triploid male oysters (Crassostrea gigas) to evaluate their fertility. Histological analysis and immunostaining revealed the presence of numerous immature spermatocytes and a markedly low abundance of mature spermatozoa in triploid males, indicating a robust blockade in spermatogenesis. Ultrastructural analysis and apoptosis analysis demonstrated that triploid males underwent normal spermatogonial mitosis during early spermatogenesis, like diploids. Analysis of phosphorylated histone H2AX (γ-H2AX) displayed heightened γ-H2AX staining in developed triploid male gonads, with a significantly higher number of apoptotic cells confirmed by the TUNEL assay compared to diploid counterparts. These findings suggest a potential arrest at the zygotene stage, preventing the completion of the zygotene-pachytene transition and ultimately leading to cell apoptosis. Flow cytometry results confirmed aneuploidy in spermatozoa from triploid oysters, containing approximately 1.5 times the DNA content of spermatozoa from diploid oysters. Notably, the sperm head size and flagella length in triploid oysters surpassed those in their diploid counterparts. Occasional observations of two-tailed spermatozoa in triploid males were noted. This study provides novel insights into the blocked spermatogenesis in triploid oysters, emphasizing the sterility of triploid male oysters and its relevance in aquaculture practices. • Spermatogenesis in triploid oysters undergoes meiotic prophase I arrest, failing to progress beyond the zygotene stage. • The meiotic arrest induces cell apoptosis and restricts the growth of gonadal tubules. • Triploid males generate a small number of abnormal aneuploid spermatozoa, which may lead to failed fertilization outcomes. [ABSTRACT FROM AUTHOR]

Genome editing using CRISPR/Cas9 methodology holds much potential to accelerate the genetic improvement in aquaculture, and has been extensively tested and applied to various aquaculture species. However, screening and identifying successfully edited individuals based on sensitive, low-cost, and rapid methods remains challenging. Here, we developed and optimized a high-resolution melting analysis (HRM) assay to screen CRISPR/Cas9-edited larval oysters. The target amplification size range was optimized to 100–200 bp. Compared with fluorescent dyes SYBR Green and SYTO-9, saturated dye EvaGreen showed high stability and repeatability when used in PCR master mixes. The HRM assay was sensitive enough to detect a temperature shift when a mixture of 10% mutant colonies and 90% WT colonies were present. Sanger sequencing analysis confirmed that oysters with indel mutations could be successfully identified by the HRM method. Together, these data indicate that the HRM assay developed in this study is a reproducible, sensitive, and efficient method for rapidly screening and identifying gene-edited oysters. • The HRM is sensitive enough to detect a mixture of 10% mutant colonies and 90% WT colonies were present. • Amplification size is between 100 and 200 bp, and the saturated dye EvaGreen Mix demonstrated repeatability and stability. • The use of HRM to screen CRISPR/Cas9 oysters has not been reported previously. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas), as the most productive cultured shellfish in the world, has considerable economic importance and value. Yet research on its sex determination remains lacking. Here, a novel HMG-box-containing homolog of the sox gene was uncovered in C. gigas (named CgSox-like). Alignment with the HMG-box of sox genes from other animals revealed that CgSox-like had a high similarity to the Sox gene of scallops. Among different adult tissues, CgSox-like was mainly expressed in the gonads. qPCR analysis revealed that CgSox-like and CgSox8 , a sox gene family gene mainly expressed in male gonads, shared similar expression patterns, with elevated expression as the male gonads developed. RNA interference and dual luciferase reporter assays demonstrated that CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. In addition, analysis of CgSox-like and CgSox8 methylation patterns indicated that both genes were in hypomethylation state during the gonadal development. Finally, demethylase inhibitors resulted in elevated expression levels of CgSox-like and CgSox8 genes, suggesting that epigenetics was engaged in modulating the expression of sex-related genes in the Pacific oyster. Taken together, the novel gene CgSox-like was implicated in the sex determination or gonadal development process in male oysters. These results will provide valuable information for the investigation of sex determination mechanism and sex control breeding. • CgSox-like is involved in sex determination or gonadal development in oysters. • CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. • DNA methylation participates in the regulation of sex determination in Crassostrea gigas. [ABSTRACT FROM AUTHOR]

Transportation stress is considered the major cause of production and economic losses in aquaculture. Here, we examined the effects of dried ginger extract (GE) and 1,8-cineole (CIN) on juvenile largemouth bass (Micropterus salmoides) subjected to simulated transport by quantifying serum metabolites, antioxidant enzyme activity, and haematological features in conjunction with whole transcriptome analysis. Under transportation stress, the addition of dried ginger extract (20 μg/L) or 1,8-cineole (30 μg/L) significantly reduced serum cortisol levels in juvenile largemouth bass. Additionally, CIN administration was associated with a significant increase in total protein and glucose levels in serum, and reduced alkaline phosphatase (AKP) and alanine aminotransferase (ALT) enzyme activity. GE and CIN significantly increased serum lysozyme activity, glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC), thereby counteracting lipid peroxidation. Transportation stress-induced malondialdehyde (MDA) production was reduced under exposure to GE and CIN, while white blood cell (WBC) counts were increased in the treatment group compared with controls. Whole transcriptome analysis of M. salmoides liver tissue identified 533 significantly differentially expressed genes (DEGs) between the CIN and transport-only control treatment groups. DEGs were mainly enriched in endocrine system, immune system and metabolic pathways. This study demonstrates that dried ginger extract or 1,8-cineole can potentially regulate M. salmoides response to transportation stress, thereby providing novel insights into the mechanisms underlying immune response and antioxidant pathways during sedation, supporting 1,8-cineole as an effective potential antioxidant agent in aquaculture. • Impact of dried ginger extract (GE) and 1,8-cineole (CIN) in largemouth bass was assessed. • GE (20 μg/L) and CIN (30 μg/L) reduced cortisol and increased glucose in serum of juvenile largemouth bass during transport. • CIN (30 μg/L) increased antioxidant activity and WBC counts in largemouth bass. • CIN alleviated stress responses via regulating endocrine, immune systems and carbohydrate metabolic pathway. [ABSTRACT FROM AUTHOR]

Triploid oysters generate fewer gametes than diploid oysters and exhibit varying degrees of gonad development. The expression of related genes, such as those involved in gametogenesis and energy metabolism, may influence reproductive potential. In addition, microRNAs (miRNAs) are important regulators of gene expression. However, the contribution of miRNAs has not yet been assessed in sterile triploid Pacific oysters (Crassostrea gigas). We therefore obtained extensive miRNA and gene expression data to identify potentially critical miRNA-mRNA interactions that are linked to sterility in triploid C. gigas. Here, we found miR-263-x to be significantly downregulated in the gonads of sterile females. The upregulated target genes were associated with lipid droplet formation, which revealed the likely involvement of nutrient accumulation during gonadal development as regulated by miR-263-x in female sterile triploids. In males, the upregulated miRNAs (miR-1992-y and miR-2001-x) were shown to regulate spermatogenesis-related gene expression, including flagellar formation, and cell proliferation and migration. Our results suggest that energy redistribution regulated by miRNAs may be critical for gonad development in females, indicating that sterile triploids undergo a significant reduction in gamete number and show an increased accumulation of nutrients in the gonads. Our findings further indicate that decreased expression of genes related to spermatogenesis regulated by miRNAs is important in gonadal dysgenesis in sterile triploids. These results shed light on the epigenetic regulatory mechanisms governing sterility in triploid bivalves. • miRNAs played a vital role in the impaired gametogenesis of triploid Pacific oysters. • miR-263-x was associated with the regulation of lipogenesis in female triploid gonads. • Spermatogenesis-related genes were negatively regulated by miR-1992-y and miR-2001-x. [ABSTRACT FROM AUTHOR]

Triploid oysters with their large size and consistent meat quality have become a major component of the oyster aquaculture industry worldwide. It has been observed that triploid Pacific oysters (Crassostrea gigas) showed high levels of variation and sex-specific differences in sterility. Here, we analyzed mitosis and meiosis during gametogenesis in triploid Pacific oysters and the expression of key cell cycle regulatory proteins, namely members of the cyclin family (A, B, D, E). We found that in female C. gigas , expression of cyclin D and E in gonad of triploids were higher than those in diploids, while expression of cyclin A and B were significantly suppressed in the gonads of triploids. To test whether mitosis of gonia might be arrested at G1/S or S/G2 phase in triploids, we compared EdU labeling in gonadal cells of triploid and diploid oysters. The results showed a similar number of EdU-labelled cells in gonads of triploid and diploid females, indicating that gonia in triploids successfully entered the S-phase as diploids. In male triploids, gonadal expression of the four cyclin genes at the mature stage were similar to that in male diploids at the growth stage with numerous spermatogenic cells. The number of EdU-labelled cells in triploid male gonads at the mature stage was significantly more than that in diploids, suggesting more proliferating cells in triploid gonads in males. Collectively, these data inidate that the sterility of triploid females is likely caused by the failure of germ cells to enter G2-phase during mitosis. In addition, we speculate that gonadal development of triploid males was arrested at the growth stage and defective spermatogenesis might be associated with disruption of meiosis. • The sterility of triploid females was related to the germ cells disrupted at mitosis. • The β gonia in triploid females could enter the S-phase but might fail to move into the G2-phase. • The gonad development of triploid males was arrested at the growth stage. • The sterility of male triploids might be associated with disruption of meiosis. [ABSTRACT FROM AUTHOR]

The enrichment of the fish feeds with the essential amino acids like methionine is a common strategy in Aquaculture. Leaching of the amino acids in the water environment and rapid absorption from the Gastro-Intestinal tract are some issues with the free amino acids supplementation strategy. The study aimed to investigate the use of Solid Lipid Microparticles as microencapsulation system for an essential amino acid, methionine. Solid Lipid Microparticles (SLMs) of two lipids, Trimyristin (TM) and Tristearin (TS) were fabricated using a melt emulsification method to encapsulate methionine and compared for their leaching protection and release characteristics in vitro and in vivo. Leaching study performed in the simulated water environment showed that the microparticles were leaching free. In simulated Gastro-Intestinal medias, release was minimal in the gastric media for both systems, while Trimyristin based microparticles showed better release in the simulated intestinal media than Tristearin based microparticles. Oral delivery study in Tilapia by mixing SLMs with the feed showed that the encapsulated methionine can provide slow release compared to the free methionine. Among the two lipids, Trimyristin based SLMs were found to be better digested by the fish. The encapsulation of methionine in lipids can be a suitable alternative for traditional free supplementation strategy for preventing leaching losses and better absorption characteristics for enhanced utilization by the fish. • Solid Lipid Microparticles (SLMs) are suitable for encapsulation of Methionine. • The microparticles prevented leaching of Methionine in water. • The SLMs showed slow release of Methionine. • Shorter carbon chain lipid, Trimyristin showed better release properties in gastrointestinal tract. • In vivo absorption study in Tilapia demonstrated the slow-release ability of the SLMs. [Display omitted] [ABSTRACT FROM AUTHOR]

Breakthroughs in polyploidy technologies have enabled the scaling-up of the commercial utilization of phenotypically-sterile triploid Crassostrea gigas. Compared with diploid oysters, gonad development in triploids is retarded, but not absent. Although histological methods of characterizing gonadal development in triploid C. gigas has been reported, the selection of sampling sites and densities might lead to bias when taking seasonal variations into consideration. Moreover, previously-employed classification systems were found to be incompatible with our long-term observations. In this study, by sampling Pacific oysters at different seasons, over several years and from different locations, we were able to establish a new classification system for gonadal development in triploid Pacific oyster based on histological analysis. Based on the types of germ cells, triploids were grouped into female (divided into female α and female β), male and hermaphrodites during the reproductive season. Surprisingly, female α was partially sterile, showing active gametogenesis and few abnormal germ cells, which were defined as β gonia. Unlike female α, triploid female β had the most severely retarded gonad along with abnormal morphological features in the gonia. We inferred that β gonia were correlated with female triploid sterility. Despite the presence of numerous gametes in triploid male gonads, the fecundity of triploid males was still severely reduced compared with that of diploids. In addition, maturation arrest of spermatogenesis in triploid males was detected histologically. Based on the types of germ cells and the presence of β gonia, hermaphrodites were further divided into hermaphrodite I and hermaphrodite II. An almost male-like triploid was redefined as a hermaphrodite triploid. The gonadal development of triploids from different sites was mostly similar. As shown via annual histological analysis, the increase in the percentage of female α and the decrease in the percentage of female β matched the seasonal development of the oyster gonad. Hence, we speculated that gonad development in female triploid oysters was delayed. • A new classification system for triploid C. gigas was established. • The sex of triploid C. gigas could be identified at early gametogenesis. • The fecundity of triploid males was overestimated. • Some male-like triploids were hermaphrodite. • We hypothesized β gonia lead to sterility of female triploids, whose gonad development delayed. [ABSTRACT FROM AUTHOR]

FOXL2 and DMRT1 are transcription factors that play critical roles in sex differentiation and gonadal development in model organisms for both vertebrates and invertebrates. However, studies on Foxl2 and Dmrt1 have been restricted to levels of gene expression and localization in adult molluscs because of limitations to experimental conditions. Here, non-invasive RNA interference was conducted in order to investigate the roles of Foxl2 and Dmrt1 in sex differentiation and gonadal development in the Pacific oyster Crassostrea gigas. The results showed that feeding oysters with dsRNA of Foxl2 could knockdown gene expression corresponding to Foxl2 by 62% - 82% and led to a decrease in gonadal coverage area, hindering development in female gonads. Similar knockdown results (58% - 70%) were obtained in male oysters subjected to Dmrt1 RNAi, causing 54% of the gonads to failing to differentiate. These findings for Foxl2 and Dmrt1 in C. gigas together with the regulation of Esr expression indicated that both genes are involved in gonadal development through an estrogen signaling pathway. The inhibitory effects of DMRT1 on Foxl2 supported a hypothesis of antagonism between Foxl2 and Dmrt1 in the sex-determining pathway in oysters. The results of this study have deepened the understanding of the Foxl2 and Dmrt1 functioning underlying bivalve gonadal development and sex differentiation, thus providing useful data for the control of sex in aquaculture. • The non-invasive RNA interference was conducted to investigate the roles of Foxl2 and Dmrt1. • The gonadal differentiation failed in partial oysters after the RNAi of Foxl2 and Dmrt1. • Foxl2 and Dmrt1 were involved in the gonadal development through estrogen signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract: Rapid, sensitive, and specific detection of pathogens is important for effective disease control in aquaculture. However, the use of conventional microbiological methods for pathogen detection is often limited by the time required to complete the assays. The aim of this study was to develop a DNA microarray assay to detect 8 species of pathogenic bacteria, i.e., Vibrio harveyi, V. alginolyticus, V. parahaemolyticus, V. anguillarum, V. cholerae, Nocardia seriolae, Aeromonas hydrophila, and Streptococcus iniae, in cultured aquatic animals. Probes were designed according to a variable region of the bacterial 16S rRNA gene and gryB sequences, and 23 probes were finally selected by hybridization with 25 bacterial type cultures. The microarray sensitivity for V. anguillarum detection in healthy fish blood was ≥100CFU/mL. Pathogenic bacteria in blood samples from 21 diseased aquacultural samples were analyzed in a blinded fashion by using the microarray, conventional culture, and 16S rRNA gene sequencing methods; the results of the microarray assay agreed well with those of the conventional culture and 16S rRNA gene sequencing methods. The results of this study show that the microarray method described is suitable for preliminary diagnoses or for confirmation of the main potential pathogens in aquatic animals. [Copyright &y& Elsevier]

Abstract: Five cultured populations of Pacific oyster, Crassostrea gigas, were examined at seven polymorphic microsatellite loci to assess the levels of allelic diversity and heterozygosity within and differentiation among the populations. The genetic variability of the seven loci was high for all the populations. The average numbers of alleles per locus ranged from 19.1 to 26.0, while the average observed and expected heterozygosity ranged from 0.474 to 0.616, and from 0.916 to 0.949, respectively. F st and R st values showed significant genetic differentiation among the five cultured populations. Genetic differences between the populations were also detected by pairwise comparison based on allelic distribution. The Neighbor-joining tree topology constructed on the basis of genetic distances between populations showed a clear separation of the northern three populations and southern two populations, suggesting that geographically separated cultured populations of C. gigas could be genetically differentiated unless seeds are transplanted among them. The useful information on the genetic variation and differentiation in cultured populations obtained in this study can be applied for future genetic improvement by selective breeding, and to design suitable management guidelines for the Pacific oyster. [Copyright &y& Elsevier]

Gain and loss-of-function analyses are powerful genetic approaches to uncover gene functions in basic and applied research. However, the genetic analysis in bivalve molluscs has been challenged by the lack of effective gene promoters for driving gene overexpression. The cosmopolitan Pacific oyster (Crassostrea gigas) is an economically important marine bivalve and a representative model species for ecological, evolutionary, and developmental studies. Here, we isolated the elongation factor1-α (EF-1α) gene promoter from the Pacific oyster and compared the promoter activity with the commonly used cytomegalovirus (CMV) and Xenopus EF-1α promoters in oyster embryos. We found that the 3465 bp DNA sequence prior to the ATG start codon in oyster EF-1α was able to drive enhanced green fluorescent protein (EGFP) expression in oyster embryos. In contrast, CMV and Xenopus EF-1α promoters failed to direct EGFP expression in oyster embryos. It appeared that the first intron sequence in oyster EF-1α gene was required for the promoter activity. DNA construct without the first intron located upstream of the ATG start codon failed to drive EGFP expression in oyster embryos. Collectively, these studies indicate that the endogenous EF-1α promoter is preferred promoter for driving gene expression in bivalves. • The first endogenous ubiquitous promoter that induces gene expression in bivalves. • CMV and Xenopus EF-1α promoters failed to direct EGFP expression in oyster embryos. • The first intron sequence in oyster EF -1α gene was required for the promoter activity. [ABSTRACT FROM AUTHOR]

Amphibians' gut and skin microbiota are vital for host health. Ulcers may affect the skin microbiota, with unknown impacts on the gut microbiota. Skin diseases could influence both, but research on their interrelationships is limited. The cutaneous and gut microbiota of healthy and ulcerated Rana dybowskii were analyzed with Illumina sequencing. The result showed healthy frogs showed greater alpha diversity in skin microbiota than ulcerated ones, with no gut microbiota difference. Bray-Curtis and unweighted UniFrac distances revealed distinct skin microbiotas in healthy versus ulcerated groups and significant gut microbiota differences only in Bray-Curtis. Linear discriminant analysis of effect size (LEfSe) showed that ulcerated skin groups were enriched in Proteobacteria and healthy in Actinobacteria. A variety of analyses revealed that diseased frogs, unlike healthy ones, exhibit similar skin and gut microbiota, suggesting disease impacts microbiota relationships and composition. Using the indicator method, identify key bacterial species indicating healthy or diseased status in the frog's skin and gut microbiota. There were significant differences in skin and gut microbiota functions between diseased and healthy frogs, with disease impacting bacterial community structure and physiological functions. Research on R. dybowskii indicated disease aligns the skin and gut microbiota, affecting composition and function, with distinct bacterial indicators for health and disease status. This research underscores the complex interplay between skin and gut microbiota in amphibian health, highlighting implications for disease understanding and conservation strategies. • Pioneering impact analysis of skin ulcers on gut microbiota similarity • Discovery of distinct microbiotas in healthy and ulcerated amphibians • Uncovering high skin microbiota derivation from gut in ulcerated frogs • Unique indicator method to identify genera linked to health conditions. • Revealing correlation between skin microbiota composition and physiological functions [ABSTRACT FROM AUTHOR]

Crassostrea nippona has recently been recognized as a preferred alternative to C. gigas in summer due to its firm flesh and high glycogen content. To promote industrial development, the mass selective breeding program was initiated to improve growth performance of C. nippona in 2014. Although a steady genetic gain of approximately 10% for growth traits was achieved in each of the first three generations of the selected strains, little is known about the cumulative genetic progress in desired traits contributed by the selective breeding in the genetic improvement program. In this study, the aquaculture performance of the selected population was comprehensively evaluated by comparing the production performance and reproductive traits of the progeny (WF1 and SF4) from the wild population and the third-generation selected line in commercial farming environments. The results showed that the diameters of eggs from both populations were at similar levels, but the fertilization and hatching rates of the progeny from the selected lines were significantly higher than those of the wild population (P < 0.05). At larval stages, the shell height and cumulative survival rate (CSR) of individuals from SF4 were consistently superior to that of WF1, especially on day 6 post-fertilization, in which CSR of the improved C. nippona was increased by 25.08% compared to that of wild oysters. Meanwhile, a significant 12.10% enhancement in the metamorphosis rate was observed in SF4 when compared with WF1. During the grow-out stage, SF4 displayed distinct advantages in growth performance and survival compared to the wild strain, with 23.19, 33.48, 34.18 and 79.11% improvement in shell height, body weight, CSR and final yield at harvest on day 800, respectively. Meanwhile, relative brood amount (RBA) associated with initial development of offspring was found to be significantly higher in SF4 compared with WF1 (P < 0.05). Remarkably, the gonadal development, sex ratios and biochemical composition of oysters from both populations were at similar levels at final harvest, implying that high-intensity artificial selection had no significant effect on reproductive characteristics and nutritional quality of C. nippona. The encouraging results in the present study suggest that the overall genetic progress contributed by genetic breeding is considerable in the fast-growing strain of C. nippona , and such gains are expected to continue to increase with further selective breeding. • Genetic progress obtained in C. nippona after three-generation mass selection is considerable. • Production performance of improved C. nippona was superior to that of wild individuals. • Survival of improved C. nippona was significantly enhanced, especially at the early larval stage. • Selection for growth had no clear effect on gonad development and nutritional quality of C. nippona. [ABSTRACT FROM AUTHOR]

Shell coloration in many molluscs is highly variable. A shell color segregation study with progenies from a full factorial cross generated among Pacific oysters exhibiting distinct shell colors (golden, white and black) was conducted to investigate the inheritance of the golden shell color and its correlation with dark pigmentation. Random samples from twenty-three full-sib families were obtained and the shell coloration of offspring within each family was recorded. Results revealed that golden coloration was inherited in a different pattern from dark pigmentation, indicating its different genetic basis. Dark pigmentation was identified as a foreground color while golden or white color were background ones. The locus controlling background colors has two alleles with the allele for golden background being dominant to the allele for white background. In addition, the overlying foreground pigmentation of shells with a golden background was significantly lighter than that of shells with a white background, which suggested an epistatic effect of background color on shell foreground pigmentation. All these findings will facilitate the selection of elite oyster lines with desired shell coloration for aquaculture. [ABSTRACT FROM AUTHOR]

The Pacific oyster ( Crassostrea gigas ) is one of the most important farmed oysters worldwide. To improve breeding progress, research was done to identify molecular markers linked to shell pigmentation, which has been viewed as a key factor for product value. An F 1 -segregating population exhibiting a bimodal distribution of shell pigmentation intensity was obtained by crossing two wild oysters with opposite shell pigmentation. Genomic DNA from nine individuals with lightest shell pigmentation and nine individuals with darkest shell pigmentation were equally pooled for amplified fragment length polymorphism (AFLP) screening. In bulked segregant analysis, six out of 225 selective primer pair combinations produced seven polymorphic fragments tightly associated with shell pigmentation across the segregating population. The seven AFLP markers were mapped onto a single linkage group, and 80% of phenotypic variance could be explained by this locus. In conversion of the seven fragments into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a co-dominant SCAR marker, named SP-170. The SCAR marker was integrated into a previously constructed linkage map. The SCAR marker obtained in this study will be useful for marker-assisted selection of the Pacific oyster. [ABSTRACT FROM AUTHOR]

One of the most important physiological process in the life cycle of most bivalves is the sexual reproduction required for species propagation. Understanding the molecular regulatory mechanisms of gender determination and gonad development have potential application in bivalve aquaculture, but there is still limited knowledge about this issue. The Pacific oyster Crassostrea gigas is a commercially important bivalve in aquaculture worldwide, as well as a representative specie to study the sexual development in bivalves for its complicated sexuality, including dioecy, sex change and rare hermaphroditism. In this study, an integrated analysis of miRNA and mRNA expression profiles of oyster gonads at different gametogenesis stages was performed to explore their possibly regulatory patterns in sexual development of C. gigas. Based on small RNA sequencing, a total of 122 miRNAs were identified, including 53 conserved and 69 novel miRNAs. Six miRNAs exhibited sex-specific pattern that expressed exclusively from either male or female gonads with increasing abundance throughout the gonadal development. Moreover, 287 putative targets of the six sex-biased miRNAs were predicted by combining previous transcriptome data. GO and KEGG enrichment analysis of target genes revealed that these sex-biased miRNAs might participate in the regulation of gametogenesis. Further, potential miRNA-mRNA interactions based on six sex-biased miRNAs were collected. The homolog of a key participator in testis development, meiotic recombination protein SPO11 in C. gigas (CgSPO11), was predicted as a target of female-biased miRNA lgi-miR-96a. This negative miRNA-mRNA expression relationship was validated in additional samples by quantitative real-time PCR (qRT-PCR) and the regulation of CgSPO11 by lgi-miR-96a was preliminarily confirmed by a dual-luciferase reporter assay. This study provides information regarding the gonad miRNAs of C. gigas and leads to the discovery of miRNA-mRNA interactions in gonad development, which will facilitate further investigations in molecular mechanisms of bivalve sex differentiation. • A total of 122 miRNAs were identified during the Pacific oyster gonad development. • Six sex-biased miRNAs might participate in the regulation of oyster gametogenesis. • The CgSPO11 gene is a target of lgi-miR-96a. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas) is a widely cultured shellfish species with great economic value; however, estimation of heritabilities for meat composition traits meat composition have not been studied with molecular pedigree. In this study, the heritabilities of six meat composition traits were studied using near-infrared reflectance spectroscopy (NIRS) in the golden shell strain of Pacific oyster. All 30 families used in this study were produced by nested mating. In total, progeny from crosses among 10 sires and 30 dams were communally reared and pedigree was reconstructed with six microsatellite markers. A total of 608 offspring were harvested at 16 months of age and unambiguously assigned to their respective parents. We estimated the heritability at 0.41 ± 0.19 for glycogen content, 0.51 ± 0.09 for total protein content, 0.89 ± 0.18 for total fat content, 0.40 ± 0.11 for zinc content, 0.87 ± 0.06 for selenium content and 0.65 ± 0.13 for ash content. Remarkably, there were moderate negative genetic correlations between protein and both glycogen (−0.65 ± 0.11) and fat content (−0.40 ± 0.09) as well as between shell height and glycogen content (−0.68 ± 0.11). Overall, this study demonstrated that in meat composition traits could be improved through selective breeding under commercial conditions. The correlations among composition traits and growth traits should be taken into consideration. • Genetic parameters for meat composition traits in the golden shell color strain of C. gigas was estimated. • The meat composition traits were determined with near-infrared reflectance spectroscopy (NIRS) technology. • The families of oysters were fostered communally and reconstructed with six microsatellite makers. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas) is one of the most important species which has the largest production among all cultured shellfish. Recently, several shell color strains of C. gigas have been developed to improve the commercial values, however the inheritance of shell pigmentation is still unclear. A total of 29 families cross-mated between different pigmented oysters were produced to investigate the inheritance of the shell pigmentation and purple pigmentation. Random samples from each family were obtained and their shell pigmentation were recorded. Results revealed that shell pigmentation was controlled by two genetic locus, among which one is responsible for secretion of pigmentation and the other is responsible for distribution mode of pigmentation. The locus controlling the secretion of pigmentation has two alleles with the allele for the presence of pigmentation being dominant to the allele for the absence of pigmentation. Similarly, another locus controlling the distribution mode of pigmentation has two alleles with the allele for striped distribution being dominant to the allele for solid distribution. In addition, one independent locus with two alleles was suggested to control the purple-striped pigmentation, which one allele for shell devoid of purple pigmentation is dominant to another allele for shell with purple pigmentation. The findings will provide valuable information for the efficient selective breeding of shell color strains in the Pacific oyster. • A two-locus model for inheritance of shell pigmentation were demonstrated. • The allele for secretion of pigmentation is dominant to the other allele for absence of pigmentation. • The allele for striped distribution of pigmentation is dominant to the allele for solid distribution of pigmentation. • An indenpendent loci was found to control the inheritance of purple striped pigmentation. [ABSTRACT FROM AUTHOR]

Rare breeds represent a valuable resource for current or future market demands, but their low census compromises the genetic variability and future utilization of these breeds. Whether genetic variability of rare breeds with low initial genetic variation can be maintained during an intense mass selection becomes the key to applying mass selection to the genetic improvement of these breeds. The genetic variability among three generations of successive mass selection of the orange-shell line of Crassostrea gigas (MS1–MS3) were evaluated by both 20 nuclear microsatellite loci and mitochondrial cytochrome oxidase I sequences (mtCOI) compared to four wild populations. In this study, the orange-shell line exhibited very low genetic variability. Only one mtCOI haplotype was detected in all individuals of MS1–MS3. Significant reductions in average number of alleles (N a : 69.55–76.92%), allelic richness (A r : 68.17–74.91%) and expected heterozygosity (H e : 34.21–39.24%) as well as increased mean pairwise genetic relatedness (R : 6.87–25.79 times) were observed in MS1–MS3 when compared to wild populations. However, the genetic variability of orange-shell line with very low initial variation successfully maintained during three generations of mass selection. No significant difference in N a (3.60–4.40), A r (3.51–4.08) and H e (0.48–0.50) occurred among MS1–MS3. Pedigree reconstructions (no full-sib group from MS1–MS3 was larger than 16% of the whole group) revealed artificial spawning used in this study has a better control over contribution of gametes than traditional mass spawning. In addition, effective population size of MS1–MS3 calculated by linkage disequilibrium methods increased from 29.3 to 67.0 indicating the linkage disequilibrium decays over time. This study provides important insights in the genetic consequences of a rare variant line of C. gigas with very low genetic variation over generations of mass selection. This will provide a reference for carrying out genetic improvement programs on rare breeds where small populations are inevitable. • This study is the first to report genetic change of rare variant of oysters over several generations of mass selection. • The initial variation of orange-shell line was lower than that of most other strains of mollusk in previous studies. • The genetic variability of orange-shell line successfully maintained during three generations of intense mass selection. • This will provide a reference for carrying out genetic improvement programs on rare breeds. [ABSTRACT FROM AUTHOR]

Crassostrea nippnoa is a potential aquaculture species because of its unique flavor, delicious taste and edibility during the summer, while C. gigas is a major global aquaculture species due to the fast growth rate, high disease resistance and strong environmental adaptability. This study firstly described the reciprocal hybridization between the two oyster species, and four crosses were produced: C. gigas ♀ × C. gigas ♂ (GG), C. gigas ♀ × C. nippona ♂ (GN), C. nippona ♀ × C. gigas ♂ (NG) and C. nippona ♀ × C. nippona ♂ (NN). The embryonic development of reciprocal crosses was similar to that of the pure crosses. However, fertilization rates were obviously different between the reciprocal hybrids and parental species, where the fertilization rates of reciprocal hybrids were significantly lower than those of parental species. At larval stages, the survival rates of the hybrid cohorts were significantly lower than those of inbred cohorts, and the critical stage of reciprocal hybrids where highest mortality occurred was the umbo period. The growth rate of GN progeny was not different from the inbred cohorts, while the growth rate of the NG progeny was higher than that of inbred cohorts. Furthermore, both hybrid cohorts had normal metamorphosis and produced viable spats. During the spat stage, the survival rates of reciprocal hybrids were not different from that of GG spat but were much higher than that of the NN spat. The growth rates of reciprocal hybrids were comparable to that of GG spat but significantly higher than that of NN spat. The shell height of the reciprocal crosses GN and NG were increased by 46.66% and 60.75% compared with the NN cross at day 360, respectively. The reciprocal hybrids had significant survival advantage and growth heterosis compared to NN cohort. The results demonstrated that the cross NG was deemed to be the better hybridization combination for genetic improvement, which could have a potential utilization for oyster aquaculture. • C. nippona was successfully hybridized with Pacific oyster. • Reciprocal hybrids exhibited larger growth heterosis and survival advantage at spat stage. • The cross NG was deemed to be the better hybridization combination in oyster genetic improvement. [ABSTRACT FROM AUTHOR]

Abstract The Pacific oyster (Crassostrea gigas) is one of the highly fecund marine aquaculture species and has a large variance in reproductive success, these characteristics can result in loss of genetic diversity and inbreeding especially when conducting mass selection. In the current study, we investigated the level of genetic diversity and effective population size over three successive mass-selected generations in the black shell strain of C. gigas assessed by 11 microsatellites as well as mitochondrial COI region. The results showed that the genetic variation over three generations were maintained since no detectable depression of expected heterozygosity (H e = 0.647–0.681), number of alleles (N = 5.6–6.0) and haplotypes (N h = 2). The difference in alleles and haplotypes number between the base population (BP) and three mass selective generations (M5-M7) could be mainly due to the loss during preceding family selection process. Pair-wise F ST values along with AMOVA analysis from both markers indicated unremarkable differentiation within each generation including BP. There was no deleterious effect on genetic diversity and population structure for mass selected generations imposed by our artificial breeding practice. The N eb estimation for M5, M6 and M7 was 29.8, 40.7 and 52.7, respectively. The different number of broodstocks used for each generation implied that using a balanced sex ratio and large size of broodstock as well as low selection pressure would help to increase the effective population size and avoid high level of inbreeding. The estimated linkage disequilibrium-based effective breeders size (N eb) was significantly lower than sex-ratio correction effective population size (N e), which indicated high variance in family contribution. It is suggested during mass spawning to simply undertake several mini-spawn groups and then pool the embryos to produce next generation, which can be a hedge to the high variance in reproductive success, thus, the loss of effective populations size and diversity. Other practical strategies against the loss of genetic diversity were also discussed, however, it remains to be investigated how diverse selection and follow-up procedures will benefit retaining genetic variability. This study will provide an insight into the level of genetic diversity and effective population size within mass-selected black shell line and enable a better understanding of how efficient current breeding practices are at maintaining genetic variation. This information can be applied for future selective breeding program and in the design of suitable management guidelines for sustainable breeding of C. gigas. Highlights • Genetic diversity and effective population size over successive mass-selected generations in the black shell strain were assessed. • There was no detectable loss of genetic diversity revealed by 11 microsatellites and mtDNA COI region. • A large size and balanced sex-ration of broodstock and lower selection pressure would alleviate effective population size loss. • It is suggested to simply undertake mini-spawning during mass spawning to against high variance in reproductive success. [ABSTRACT FROM AUTHOR]

Shell color polymorphism is common in nature for the marine mollusks. The Pacific oyster Crassostrea gigas , is widely cultured and one of the most economically important bivalve species with various shell color variants. Here, a total of 1061 single nucleotide polymorphism markers, developed from expressed sequence tags (EST-SNPs), were used to construct two genetic linkage maps based on reciprocal-cross between black shell and white shell oysters (MF-A and MF-B) respectively. The two separate linkage maps were combined into an integrated map spanning 947.31 cM in total length. The integrated map was composed of 351 EST-SNP markers distributing on 10 linkage groups, with an average interval of 2.78 cM between adjacent markers. For the MF-A, 11 quantitative trait loci (QTLs) for shell color traits and five QTLs for growth traits were detected, explaining 8.1%–13.8% (mean = 11.76%) and 10.4%–11.0% (mean = 10.66%) phenotypic variance respectively. For the MF-B, nine QTLs for shell color traits and eight QTLs for growth traits were detected, explaining 8.8%–21.7% (mean = 12.84%) and 8.4%–9.4% (mean = 8.88%) phenotypic variance respectively. QTLs clustering was found on the linkage group 2 of the MF-A and the linkage group 8 of the MF-B, and the QTLs showed that pleiotropism could affect at most three traits. Three shared-QTLs associated with shell color traits were identified on the integrated map, and one of them (qA- a * 4) was significantly homologous to C. gigas calmodulin-like protein. The QTLs identified in the present study could be useful in finding candidate genes for the shell color and growth-related traits in future, and potentially applied to marker-assisted selection breeding programs for C. gigas . [ABSTRACT FROM AUTHOR]

The pacific oyster ( Crassostrea gigas ) is one of the most widely farmed aquatic animal species and of great economic importance. Mantle edge color in C . gigas is highly variable and considered as a new potential trait for a better commercial value. In this study, heritability for mantle edge pigmentation and its correlation with shell pigmentation were explored in the white-shell strain of C . gigas with mixed-family method. A total of 460 offspring raised under communal conditions were successfully assigned to their parents using four multiplex PCR protocols based on 11 microsatellite loci. Mantle edge pigmentation was measured through assay of melanin content using spectrophotometric analysis. Animal model heritability estimate were 0.215 ± 0.092 for mantle edge pigmentation. The genetic and phenotypic correlations between two pigment traits were 0.980 ± 0.094 and 0.423 ± 0.042, respectively. Pearson analyses for average mantle edge and shell pigmentation of family also showed strong and significant correlation ( r = 0.729, P = 0.000, n = 29). Chi-square analyses showed that mantle edge pigmentation of family 7 segregated into “lighter” and “darker” groups in a 3:1 ratio ( P = 0.833), suggesting that a major locus might control mantle edge pigment trait. The results obtained in this study will benefit selective breeding of C . gigas with desired mantle and shell pigmentation. [ABSTRACT FROM AUTHOR]

The Pacific oyster ( Crassostrea gigas ) is an important aquatic animal species with enormous production and economic benefits, but heritability estimation for shell color applying mixed-family approach and computer vision system has not been performed. In this study, heritability for shell color-related traits was estimated in the golden shell strain of Pacific oyster by fostering a single cohort of 30 families in a nested mating design consisting of 10 sires and 30 dams. After parentage assignment inference based on six microsatellite markers, 690 offspring were included in the subsequent analyses. Using an animal model, at 16 months of age, heritability estimates were 0.10 ± 0.12 for L ⁎ , 0.41 ± 0.16 for a ⁎ , 0.23 ± 0.15 for b ⁎ and 0.33 ± 0.19 for Δ E . Genetic correlation between shell color-related traits and growth-related traits were generally inconspicuous ranging from − 0.02 to 0.11. Conclusions can be obtained from our study that favourable improvement in aquatic selective breeding would be predictable for the genetic potential of the golden shell color in this strain of Pacific oyster, whereas independent selection conducted at the same time rather than correlative selection would be feasible in the improvement of both types of traits. [ABSTRACT FROM AUTHOR]

Crassostrea nippona has recently been identified as a potential aquaculture species for its high glycogen content and delicate flavor in summer when Crassostrea gigas suffer from low meat quality. In 2014, we initiated the selective breeding program to improve growth traits of C. nippona through successive generations of mass selection, yet the genetic impact of an intense artificial selection on the genetic variability of C. nippona was not fully understood. In this study, the genetic diversity and genetic structure of three generations of mass-selected lines (G1-G3) and three wild populations were investigated using both 15 microsatellite loci and mitochondrial COI sequences (mtCOI). The selected lines exhibited no significant decrease in the average number of alleles (N a : 7.53–9.87), observed heterozygosity (H o : 0.58–0.64), expected heterozygosity (H e : 0.67–0.71) and alleles richness (A r : 6.35–7.75), compared with those of the wild populations. The abundant genetic diversity of selected lines was successfully maintained during three generations of mass selection due to no detectable loss. The effective population sizes (N e − lin) estimated by linkage disequilibrium methods for G1, G2 and G3 were 64.1, 25.3 and 47.4, respectively. Moreover, little genetic differentiation within the selected lines was observed in AMOVA analysis (global F ST : 0.005, P > 0.05) and significant genetic differentiation among the wild populations was revealed by global F ST (0.15, P < 0.01), pairwise F ST (0.095–0.211), Nei's D (0.260–0.730) and clustering results, which might suggest genetically isolated populations occurring in these sampling locations. To maximize future selective breeding efforts, a larger scale of broodstock and multi-line breeding strategy along with alleviating selection intensity is recommended against the reduction in the genetic diversity and effective population size in subsequent breeding practices. This study contributes to an increasing understanding of the efficiency of current breeding procedures in maintaining genetic variation and provides insight into future genetic improvement programs of C. nippona. • This study is the first to investigate the genetic diversity of C. nippona using both microsatellites and mtCOI. • Microsatellite analysis revealed no significant difference in genetic diversity between selected and wild populations. • The genetic diversity of the selected lines was successfully maintained over successive generations of mass selection. • Little genetic differentiation among the selected lines was observed based on the microsatellite result. [ABSTRACT FROM AUTHOR]

The Iwagaki oyster (Crassostrea nippona) is a potential aquaculture species with delicious taste and edibility in summer when the Pacific oyster (Crassostrea gigas) suffer from low meat quality. Although there is an increasing interest in hatchery mass production and field grow-out among farmers, large-scale expansion of C. nippona aquaculture has been compromised by unimproved growth performance. To improve the growth rate of C. nippona , we initiated the mass selective breeding program for fast growth in 2014 and estimated the genetic parameters of growth traits in the first three successive mass-selected generations. Each selected line exhibited better growth performance than the corresponding control group at all sampling dates. At grow-out stage, the average selection response (SR), realized heritability (h R 2) and genetic gain (GG) for shell height was 0.45–0.56, 0.27–0.42 and 9.20–11.12% in the three mass selected lines, respectively. A significant increase in GG for shell height was observed in the third-generation selected line compared with that in the first-generation (P < 0.05). Meanwhile, the average SR (0.44–0.49) and GG (11.07–12.46%) for body weight of C. nippona were observed in the selective breeding program after three successive generations of mass selection. The encouraging results obtained in this study suggest that genetic variances still remain at a relatively high level in the breeding populations of C. nippona , and demonstrate the feasibility of improving the growth traits by selective breeding for C. nippona. • Mass selection for fast growth of Crassostrea nippona obtained considerable selection responses. • Continuous and stable genetic gains for growth were observed during an intense mass selection. • Realized heritability of growth traits proved to possess great potential for genetic improvements. [ABSTRACT FROM AUTHOR]

Recently, mass mortality of cultured Pacific oyster (Crassostrea gigas) has occurred frequently, which has aroused our attention to the stress tolerance of oyster. Hybridization is regarded as an effective way to produce genetic improvement in aquaculture, which can effectively introduce improved characteristics to the hybrids. In the present study, we evaluated the physiological and immune responses at different temperatures (16–36 °C) and salinities (15–35 psu) in two strains of C. gigas , a fast-growing strain "Haida No. 1" (HH) and an inbreeding line with orange shell color (OO), and their reciprocal hybrids HO (HH♀ × OO♂), OH (OO♀ × HH♂). Oxygen consumption rate (OCR), ammonia-N excretion rate (AER), superoxide dismutase activity (SOD), catalase activity (CAT) and contents of malondialdehyde (MDA) were determined on day 10 of the temperature and salinity exposure. Results showed higher stress resistance ability of the hybrids than their parents against environment challenge. For physiological parameters, the OCR of OH strain increased in the experimental temperature range, while the maximum values of HO, HH and OO strains were 2.013, 2.193 and 1.994 mg g−1 h−1 respectively at 31 °C; OCR and AER of OH strain was significantly higher than that of the other strains at lower salinity (P < 0.05). For immune parameters, the overall SOD and CAT activity of hybrids were lower than that of the other strains under temperature treatments. Besides, the overall MDA content of OH strain was lowest at experimental temperature and salinity, and the other three strains' MDA level ranked as: OO > HH > HO. Overall, the results suggest that environment changes could significantly affect the physiological and immune status of oyster, and hybrids may be more resistant to environment stresses than their parents. This study provides physiological and immune evidences for interpreting the stress resistant heterosis in this oyster hybrid system, which could help us in a better understanding and utilization of heterosis in oyster aquaculture. • Hybridization improved the stress resistance of oyster under temperature and salinity challenge. • Oyster hybrids are more active in physiological responses. • Hybrids gained advantage in some immune parameters compared to their parents. [ABSTRACT FROM AUTHOR]

Comparative genomics has become an important strategy for transferring genetic information from a well-characterized model species to one that is less described and can facilitate in functional gene mining and genomic evolution. Crassostrea angulata and Crassostrea gigas are closed related species that they can hybridize to produce fertile offspring, and C. gigas has accumulated a considerable number of genetic and genomic resources while C. angulata lags far behind. In the current study, 684 single nucleotide polymorphism (SNP) markers developed from C. gigas expressed sequence tags (ESTs) were used for transferability test in C. angulata , and 635 loci were successfully amplified. Then a genetic linkage map of C. angulata was for the first time constructed using these transferable SNP markers. A total of 988 transferable SNP markers were used for polymorphism screening in a mapping family, and 273 loci were distributed on the genetic map which was composed of 10 linkage groups and estimated 1040.29 cM in total length. The average spacing between markers was 3.96 cM. By comparing the shared EST-SNP marker locations on homologous linkage groups, comparative mapping analysis between the C. angulata genetic map and an integrated map of C. gigas revealed putative macro-collinearity in the two oyster genomes with little difference in markers order. These transferable SNP markers could be applied in genetic diversity and population structure studies, and the genetic map would be useful for quantitative trait loci mapping and genetic improvement through marker-assisted selection for C. angulata. • A total of 635 transferable SNP markers were developed for C. angulata from EST sequences of C. gigas. • The first genetic linkage map of C. angulata was constructed composing of 273 transferable EST-SNPs. • High macro-collinearity was revealed by the shared markers on C. gigas and C. angulata genetic maps. [ABSTRACT FROM AUTHOR]

The Pacific oyster is one of the most economically important species in global aquaculture. However, the production of C. gigas has been compromised by widespread and severe summer mortality events in recent years. To explore the possibility of obtaining a new hybrid line with rapid growth and high summer survival rate traits through hybridization, we performed a systematic comparative study on heterosis for growth and survival of C. gigas (GG) , C. angulata (AA) and their reciprocal hybrids of C. gigas × C. angulata (GA) and C. angulata × C. gigas (AG) for 450 days in northern China. The fertilization and D-stage rates of reciprocal hybrids were lower than those of parental species, but obvious heterosis for growth, survival and metamorphosis were observed at larval stages. Meanwhile, the hybrid cohorts showed high heterosis for growth and survival at both juvenile and adult stages. Moreover, compared with growth, heterosis was higher in survival with a mid-parent heterosis ranged from 12.75% to 110.20% throughout the whole period. At day 450, the cumulative survival rate of hybrid GA increased by nearly 34% compared with GG, with a single patent heterosis I (GA/GG) of 191.14%. Notably, the final yield of hybrid crosses was significantly higher than that of parental lines, with a mid-parent heterosis of 182.72% and I (GA/GG) of 239.46%. To investigate the relationship between summer mortality and high temperature tolerance, the survivability of reciprocal hybrids and parental species through exposure to acute increasing temperature stresses for 96 h were compared. The reciprocal hybrids exhibited greater thermotolerance than the parental species, followed the order of GA > AG > AA > GG. Overall, our study confirmed that GA showed the greatest heterosis in terms of growth, survival and thermotolerance, which could be used as the preferred alternative variety in oyster farming in northern China. • The cumulative survivals of GA increased by nearly 34% compared with GG at day 450, with a single patent heterosis I (GA/GG) of 191.14%. • The mid-parent heterosis H and single parent heterosis I (GA / GG) for final yield of the hybrids were 182.72% and 239.46% respectively. • The reciprocal hybrids exhibited higher thermal tolerance than the parental species, followed the order of GA > AG > AA> GG. • The hybrid of GA has a large potential to become the preferred alternative variety in oyster farming in northern China. [ABSTRACT FROM AUTHOR]

Molluscan shellfish have a diverse form of shell coloration and sex determination, understanding the genetic mechanism under these two traits is crucial to their germplasm exploitation and conservation. In our breeding program, a novel orange-shell variant of C. gigas was obtained, and an F2 family was constructed by crossing an orange-shell male with a black-shell female. This F2 family provided an ideal material for QTL mapping of orange shell color and sex traits of C. gigas simultaneously. Here, a high-density genetic linkage map containing 1799 SNP markers was constructed using this F2 family by genotyping-by-sequencing. These markers were distributed on 10 linkage groups and the total map size was 828.31 cM, with an average marker interval of 0.58 cM. Based on this map, six chromosome-level significant QTLs associated with orange shell color were detected, with the phenotypic variance explained ranging from 9.10% to 12.30%. Within the QTL regions, four candidate genes, 3-ketoacyl-CoA thiolase A, fibroblast growth factor receptor 4, poly [ADP-ribose] polymerase tankyrase-1 and tripartite motif-containing protein 45-like, related to the organic matrix of mollusk shells or collagen were identified. Additionally, only one genome-wide significant sex-related QTL was detected, which explained 19.30% of phenotypic variance. Two sex-related genes, proteasome subunit beta type-3 and G-protein coupled estrogen receptor 1 were identified within the QTL region. The QTLs and candidate genes identified in this study provide valuable resources for further investigations on the molecular mechanisms of shell color and sex in C. gigas. • A high-density genetic linkage map containing 1799 SNP markers was constructed for C. gigas using an F2 family. • Six chromosome-level significant QTLs associate with orange shell color were detected in C. gigas. • Only one genome-wide significant sex-related QTL was detected in C. gigas. • Different description methods of phenotype have a great impact on the result of QTL mapping. [ABSTRACT FROM AUTHOR]