Search

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]

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]

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]

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]

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]