Chronic low salinity stress rescued masculinization effect in farmed Cynoglossus semilaevis population.

Salinity, being an indispensable abiotic factor crucial for the survival of marine organisms, has demonstrated diverse alterations globally in response to the current trend of global warming. In this study, the effect of chronic low salinity stress on teleosts' sex differentiation was investigated using Cynoglossus semilaevis, an economically important fish with both genetic and environmental sex determination system. The cultivation experiment was conducted employing artificially simulated seawater of 20 ppt and ambient sea water of 30 ppt to rear juveniles C. semilaevis. Throughout the experiment, the growth performance was assessed and the histology of gonadal development was examined, a significantly lower masculinization rate was observed in LS group. To gain further insights, transcriptome analysis was conducted using raw reads obtained from 53 libraries derived from gonads of 55 days post fertilization (dpf) and 100 dpf juveniles in both LS and CT groups. GO/KEGG enrichment were further proceeded, Terms and pathways involved in reproduction ability, germ cell proliferation, immune function, steroid metabolism etc., were illuminated and a possible crosstalk between HPI and HPG axis was proposed. WGCNA was conducted and two hub genes, hspb8-like and Histone H2A.V were exhibited to be of great significance in the changes of masculinization rate. Our findings provided solid reference for sex differentiation study of GSD + ESD species in a constantly changing ocean environment, as well as practice guiding significance for the environmental management for the culture of C. semilaevis.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos.

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4-6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

A chromosome-level genome assembly of the potato grouper (Epinephelus tukula).

The potato grouper, Epinephelus tukula, is one of the largest coral reef teleost, and it is an important germplasm resource for selection and cross breeding. Here we report a potato grouper genome assembly generated using PacBio long-read sequencing, Illumina sequencing and high-throughput chromatin conformation capture (Hi-C) technology. The genome size was 1.13 Gb, with a total of 508 contigs anchored into 24 chromosomes. The scaffold N50 was 42.65 Mb. For the genome models, our assembled genome contained 98.11% complete BUSCO with the vertebrata_odb9 database. One more copies of Gh and Hsp90b1 were identified in the E. tukula genome, which might contribute to its fast growth and high resistance to stress. In addition, 435 putative antimicrobial peptide (AMP) genes were identified in the potato grouper. This study provides a good reference for whole genome selective breeding of the potato grouper and for future development of novel marine drugs.

Intensive masculinization caused by chronic heat stress in juvenile Cynoglossus semilaevis: Growth performance, gonadal histology and gene responses.

The sea temperature has been observed to chronically increase during the past decades, leaving unpredictable influences to the marine biological resources. Thus, it is of vital significance to study the biological responses of ocean inhabited organisms with the artificially stimulated heat stress environment. Cynoglossus semilaevis provides us with an ideal model to study the influence of chronic heat stress on the sexual differentiation in marine teleosts for its genetic sex determination (GSD) + environmental effected (EE) sex determination system. In this study, the comparative experiment was conducted employing heated seawater (HT group) and ambient seawater (CT group) to cultivate juvenile C. semilaevis respectively. Significant differences were exhibited in growth performance and a delayed germ cell development effect was found in pseudomales formed under chronic heat stress. Using transcriptome analysis, the transcription profile of 55 days post fertilization (dpf) and 100 dpf juveniles' gonads were studied. A total of 47 libraries were constructed with an average mapping rate of 94.63% after assembling. GO and KEGG enrichment were proceeded using DEGs screened out between (1) pseudomale gonads at 55 dpf and 100 dpf in HT and CT group (2) pseudomale and female gonads at 55 dpf and 100 dpf in HT and CT group. Terms and pathways involved in steroid stimulation, reproduction ability, germ cell proliferation et al. were shed light on. The expression pattern of 29 DEGs including amh, hsp90b1, pgr et al. were also provided to supplement the results of functional enrichment. Weighted gene co-expression networks analysis (WGCNA) was constructed and hspb8-like, histone H2A.V were exhibited to play vital roles in the heat-induced masculinization. Our findings facilitate the understanding for transcriptional variations in intensive masculinization cause by chronic heat stress of C. semilaevis and provide referable study of the influences on the teleosts in elevated sea temperature.

Characterization of the complete mitochondrial genome of the hybrid grouper (Cromileptes altivelis♀ × Epinephelus tukula♂) with phylogenetic consideration.

Here we isolated and characterized the complete mitochondrial genome of the hybrid grouper (Cromileptes altivelis♀ × Epinephelus tukula♂). It is 16,503 bp long and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The nucleotide composition is 29.08% of A, 29.03% of C, 15.66% of G and 26.23% of T, with 55.31% A + T. The phylogenetic analysis by neighbor-joining (NJ) method reveals that the hybrid offspring has a closer relationship to C. altivelis.

The complete mitochondrial genome of the hybrid offspring Epinephelus awoara♀ × Epinephelus tukula♂.

The complete mitochondrial genome of hybrid grouper from Epinephelus awoara (♀) ×E. tukula (♂) was obtained by PCR amplification. The circular genome was 16,801 bp in length, consisting of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region (D-loop region). The overall base composition was as follows: A: 28.46%, T: 27.27%, C: 27.27%, G: 16.49%. The new results may provide valuable data for the genetic and taxonomic research on artificial hybrid grouper.

Gene expression changes in response to low temperatures in embryos of the kelp grouper, Epinephelus moara.

The kelp grouper Epinephelus moara has high economic value and is popular in fisheries and aquaculture in China. In the previous study, we treated the embryos at 16-22 somite stage at 4 °C, -25.7 °C, -140 °C and -196 °C, and successfully obtained surviving embryos in each group. To better understand the molecular changes affected by the low temperatures, we conducted a comparative transcriptome analysis among embryos exposed at 4 °C for 30 min, embryos exposed at -25.7 °C for 30 min and the control group. qPCR assays were conducted for the validation. Signal transduction pathways were highly enriched for the differentially expressed genes. c-Fos, c-Jun, JunD, GADD45, involved in MAPK signaling pathway, were upregulated when embryos were treated at low temperatures. As immediate early genes, Egr-1a and b, and IER2, that respond quickly to the environment stress, their expression increased as well. Hsp70 showed similar expression pattern as immediate early genes. Meanwhile, transcription factors Sox, HES, TFIID, muscle movement and protein synthesis-related genes were downregulated. Taken together, our findings suggest that cooling disrupts gene expression patterns in E. moara embryos. The differentially expressed genes may be involved in cellular resistance against low temperatures, possibly through neural activation, apoptosis, proliferation, differentiation, cellular recovery and heat shock regulation. This study also provides transcriptome dataset of E. moara embryos exposed to cold temperatures for future studies focusing on the molecular effects of cryopreservation.

Optimization of vitrification factors for embryo cryopreservation of kelp grouper (Epinephelus moara).

Cryopreservation of marine fish embryos causes to severe cryogenic damage, and to date, adults have not been reared from embryos that were cryopreserved. Here, we optimized vitrification factors to improve the survival and hatching rate of kelp grouper (Epinephelus moara) embryos after cryopreservation. We screened the effects of 11 vitrification solution concentrations (25-50%) on the survival rate of embryos at four developmental stages (16S, 18S, 22S, TB). We investigated the effects of different equilibration time (25-45min) on the survival rate and the influence of vitrification solutions on embryonic volume. In addition, we tested the effects of treating embryos at five different developmental stages (4-6S, 16S, 22S, TB, HB) with different vitrification solutions (35% PMG3S and 35% PMG3T), prechilling temperature (-5 °C and 4 °C) and prechilling time. In total, 9855 embryos were cryopreserved at 10 developmental stages, from optic capsule stage to pre-hatch stage. We found that kelp grouper embryos performed best at equilibration time of 30 min. Embryos at the tail-bud stage exhibited greater tolerance to vitrification than other stages. Vitrification solutions that contained sucrose showed better survival rates compared to embryos treated with vitrification solutions containing trehalose. Pre-chilling treatment improved viability before freezing, but did not improve viability after freezing. In the most optimal condition we identified in this study, the average survival, normal development and malformation rates of cryopreserved embryos were 6.32%, 2.36% and 3.49%, and 39.85% of the surviving embryos that were cryopreserved hatched. The hatched larvae gradually died at day 12 of cultivation, where the longest surviving individuals lived for 16 days. This study provides valuable data for improving survival and hatching rate of cryopreserved grouper embryos, and provides references for further exploring techniques in fish embryo cryopreservation.

Alternations in oxidative stress, apoptosis, and innate-immune gene expression at mRNA levels in subadult tiger puffer (Takifugu rubripes) under two different rearing systems.

Tiger puffer (Takifugu rubripes) is one of the major aquaculture fish species in China due to its high economic value. In this study, the transcriptions of hepatic antioxidant enzyme, stress, apoptosis, and immune-related genes of sub-adult tiger puffers (Takifugu rubripes) were evaluated under two different rearing systems [offshore sea cage aquaculture system (OSCS) and recirculating aquaculture system (RAS)]. Results showed that the mRNA expression levels of the antioxidant enzyme (mn-sod, cu/zn-sod, gpx, and gr) and stress-related (hsp70 and hsp90) genes of male tiger puffers reared in the OSCS were significantly higher than female fish reared in the OSCS and fish reared in the RAS. The anti-apoptotic gene bcl2 exhibited the similar results. By contrast, the mRNAs of the pro-apoptotic genes (p53, caspase8, caspase9, and caspase3) of male tiger puffers reared in the OSCS were significantly lower than female fish reared in the OSCS and fish reared in the RAS. Male tiger puffers reared in the OSCS displayed significantly higher complement components (c3) and inflammatory cytokine (il-6) mRNAs, whereas B-cell activating factor (baf) and tumor necrosis factor α (tnf-α) mRNAs remained unchanged. Meanwhile, the mRNA levels of pro-apoptotic (bax, caspase8) and immunity-related (c3, il-6 and il-7) genes of female tiger puffers reared in the OSCS were significantly lower and higher than female fish reared in the RAS, respectively. In conclusion, the hepatic antioxidant, anti-apoptosis, and innate immunity of tiger puffers reared in the OSCS were better than fish in the RAS, male tiger puffer obtained the best values. These results expand the knowledge on the combined RAS and OSCS alternative aquaculture model for tiger puffers and aid in their management in captive.

Transcriptome comparative analysis of immune tissues from asymptomatic and diseased Epinephelus moara naturally infected with nervous necrosis virus.

Epinephelus moara is an economically important fish in Southeast Asian countries but is suffering from nervous necrosis virus (NNV) infection. A deeper understanding of the host-NNV interaction mechanisms makes sense for disease control, however, at present, the pathogenesis of natural NNV infection and the resistance mechanism in host remains poorly understood. In this study, asymptomatic and diseased E. moara with clinical symptoms of viral nervous necrosis (VNN) from a grouper farm were both detected with a positive RT-PCR signal of NNV, then transcriptome sequencing of their immune tissues (liver, spleen and kidney) were performed for comparation analysis. The de novo assemblies yielded 53,789 unigenes which had a length varied from 201 to 19,675 bp and a N50 length of 2115 bp, and 29,451 unigenes were functionally annotated, with 83, 250 and 5632 unigenes being differentially expressed in liver, spleen and kidney respectively. KEGG pathway enrichment analysis of the DEGs showed many DEGs were enriched in immune related pathways. Although the expression of class I major histocompatibility complex (MHC) was significantly higher in three immune tissues of the diseased grouper, many immune related genes, including humoral immune molecules (such as antibodies), the cellular mediated cytotoxic molecules (such as perforin) and some adhesion related genes were down regulated in the diseased grouper. Our results provided many unigenes that might play important roles in NNV resistance for further research. Furthermore, a total of 8666 unigenes containing 11,623 simple sequence repeats (SSRs) were identified, which provided useful information for screening molecular markers associated with NNV resistance in E. moara.

A chromosome-level genome assembly of the giant grouper (Epinephelus lanceolatus) provides insights into its innate immunity and rapid growth.

The giant grouper (Epinephelus lanceolatus) is the largest coral reef teleost, with a native range that spans temperate and tropical waters in the Pacific and the Indian Oceans. It is cultured artificially and used as a breeding species in aquaculture due to its rapid growth rate. Here we report a giant grouper genome assembled at the chromosome scale from sequences generated using Illumina and high-throughput chromatin conformation capture (Hi-C) technology. The assembly comprised 1.086 Gb, with 98.4% of the scaffold sequences anchored into 24 chromosomes. The contig and scaffold N50 values were 119.9 kb and 46.2 Mb, respectively. The assembly is of high integrity, including 96.4% universal single-copy orthologues based on BUSCO analysis. Through chromosome-scale evolution analysis, we identified alignments of six giant grouper chromosomes to three stickleback chromosomes and some of the genes located within the breakpoints of reshuffling events may related to development and growth. From the 24,718 protein-coding genes, we found that several gene families related to innate immunity and glycan biosynthesis were significantly expanded in the giant grouper genome compared to other teleost genomes. In addition, we identified several genes related to the hormone signalling pathway and innate immunity that have experienced positive selection or accelerated evolution, implicating their roles in immune defence and fast growth of the species. The high-quality genome assembly will provide a valuable genomic resource for further biological and evolutionary studies, and useful genomic tools for breeding of the giant grouper.

A novel C-type lectin from spotted knifejaw, Oplegnathus punctatus possesses antibacterial and anti-inflammatory activity.

C-type lectin is a type of carbohydrate-binding protein and plays significant roles in innate immune response against pathogen infection. To date, thousands of C-type lectin had been identified in teleost. In the present study, we isolated a novel isoform of C-type lectin (OppCTL) from spotted knifejaw (Oplegnathus punctatus). The OppCTL encoded a typical Ca2+-dependent carbohydrate-binding protein, and was mainly expressed in liver in a tissue specific fashion. The expression of OppCTL was significantly up-regulated following Vibrio anguillarum infection in vivo, suggesting involvement in immune response. Hemagglutination analysis showed that the recombinant OppCTL (rOppCTL) could agglutinate erythrocyte from Mus musculus, Oplegnathus punctatus, Sebastes schlegelii and Paralichthys olivaceus. The rOppCTL could bind and agglutinate all tested bacteria. The rOppCTL possessed capacities of calcium-dependent agglutination to all tested bacteria. Sugar binding assay revealed that rOppCTL could also bind to the glycoconjugates of the bacterial surface, including lipopolysaccharide and peptidoglycan. Interestingly, Dual-luciferase analysis revealed that OppCTL could inhibit the activity of NF-κB in HEK-293T cells after OppCTL overexpression. Taken together, these results indicate that OppCTL has immune activity capable of defending invading pathogens and possesses potential immunoregulatory activity, enriching our understanding of the function of C-type lectin.

Spotted knifejaw (Oplegnathus punctatus) MyD88: Intracellular localization, signal transduction function and immune responses to bacterial infection.

Myeloid differentiation factor 88 (MyD88) links members of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of NF-κB as a "bridge" molecular in response to exogenous pathogen, but the function in spotted knifejaw (Oplegnathus. punctatus), a commercial fish in China, is still unknown. We present a functional analysis of spotted knifejaw MyD88 (OppMyD88) with a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus and suggest that MyD88 is important for the activation of TLR-mediated NF-κB with the synergy between domains. Subcellular localization showed that OppMyD88 was distributed in the cytoplasm in a condensed form. Tissues expression profiling analysis showed that OppMyD88 ubiquitously expressed in all tested tissues with the highest expression in the liver, as determined by real-time PCR. The expression of OppMyD88 significantly upregulated in the liver, spleen, kidney and gills within 120 h post Vibrio anguillarum infection. Moreover, we further confirmed that over-expressed OppMyD88 could also induce apoptosis. These results indicate that OppMyD88 might possess important roles in defense against microbial infection and other biological processes in spotted knifejaw similar to those in mammals, which will deepen our understandings in innate immunity of spotted knifejaw.

The complete mitochondrial genome of the hybrid offspring Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂.

The hybrid offspring Epinephelus fuscoguttatus ♀ × E. tukula ♂ showed heterosis in terms of growth and disease resistance. The mitochondrial genome is 16,629 bp in length and consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region. The difference of total length is mainly caused by the difference of length in non-coding region. The nucleotide base composition is A = 29.15%, G = 15.62%, T = 26.91%, C = 28.32%, A + T = 56.06%, and C + G = 43.94%. The phylogenetic analysis using neighbour-joining (NJ) method showed that the hybrid offspring has a closer relationship to E. fuscoguttatus × E. lanceolatus.

Characterization of the complete mitochondrial genome of the hybrid grouper Epinephelus akaara ♀ × Epinephelus tukula ♂, with its phylogenetic analysis.

Epinephelus akaara ♀ × Epinephelus tukula ♂ is an economically important fish. The mitochondrial genome of the hybrid grouper had a double-stranded DNA molecule with the length of 16,928 bp and consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. The gene composition of the hybrid grouper mitochondrial genome was similar to that of most other vertebrates. Furthermore, phylogenetic analysis by maximum-likelihood (ML) method, based on the nucleotide sequences of 13 protein-coding genes, showed that the hybrid grouper has the closer relationship to Epinephelus akaara and confirmed that the mitochondrial genome is maternally inherited.

Functional characterization of Cynoglossus semilaevis R-spondin2 and its role in muscle development during embryogenesis.

R-spondin2 (Rspo2) is a member of the R-spondin family, which plays important roles in cell proliferation, cell fate determination and organogenesis. Rspo2 exhibits important functions during embryonic development and muscle maintenance in adult human, mouse and Xenopus. In the present study, the tongue sole Cynoglossus semilaevis Rspo2 (CsRspo2) gene was isolated and characterized, and its role in muscle development during embryogenesis was studied. Our results showed that CsRspo2 expression was abundant during gastrulation and significantly high during somite formation, but then decreased markedly after hatching. CsRspo2 expression was high in brain and gill, moderate in heart, ovary and testis, and almost undetectable in muscle and other tissues. Moreover, the potential involvement of Rspo2 in muscle development was investigated. We found that overexpression of CsRspo2 mRNA in zebrafish embryos resulted in slow development and abnormal muscle formation at the embryonic stage. Our work provides a fundamental understanding of the structure and potential functions of CsRspo2 during muscle development.

Cynoglossus semilaevis Rspo3 Regulates Embryo Development by Inhibiting the Wnt/ß-Catenin Signaling Pathway.

Cynoglossus semilaevis is an important economic fish species and has long been cultivated in China. Since the completion of its genome and transcriptome sequencing, genes relating to C. semilaevis development have been extensively studied. R-spondin 3 (Rspo3) is a member of the R-spondin family. It plays an important role in biological processes such as vascular development and oncogenesis. In this study, we cloned and characterized the expression patterns and functions of C. semilaevisRspo3. Initial structural and phylogenetic analyses revealed a unique FU3 domain that exists only in ray-finned fish RSPO3. Subsequent embryonic expression profile analysis showed elevating expression of Rspo3 from gastrulation to the formation of the eye lens, while, in tail bud embryos, Rspo3 expression was significantly high in the diencephalon and mesencephalon. The overexpression of C. semilaevis Rspo3 in Danio rerio embryos resulted in a shortened rostral⁻caudal axis, edema of the pericardial cavity, stubby yolk extension, and ecchymosis. Vascular anomalies were also observed, which is consistent with Rspo3 role in vascular development. Drug treatment and a dual-luciferase reporter assay confirmed the inhibitory role of C. semilaevis Rspo3 in D. rerio Wnt/ß-catenin signaling pathway. We further concluded that the FU2, FU3, and TSP1 domains regulate the maternal Wnt/ß-catenin signaling pathway, while the FU1 domain regulates the zygotic Wnt/β-catenin signaling pathway. This study enriches Rspo3 research in non-model animals and serves as the basis for further research into the interactions between Rspo and the Wnt/ß-catenin signaling pathway.

Ammonia-oxidizing bacteria and archaea within biofilters of a commercial recirculating marine aquaculture system.

While biofilters are widely used to metabolize ammonia and other wastes in marine recirculating aquaculture systems, the ammonia-oxidizing bacterial and archaeal communities have not been characterized across a diversity of production systems. Using a metagenomics approach, we characterized the ammonia-oxidizing microbiological community of biofilters in a commercial recirculating marine aquaculture system producing hybrid grouper (Epinephelus lanceolatus × E. fuscoguttatus). Cloning and sequencing of the amoA gene showed that nitrifying bacteria included Nitrosomonas europea, N. stercoris, N. cryotolerans, N. eutropha, N. estuarii, eight strains of N. marina, and 15 strains not associated with described species. Nitrifying archaea included eight strains of Nitrosopumilus maritimus, N. koreensis, N. piranensis, N. adriaticus, undescribed congeners, and other undescribed archaea. The species composition of the bacterial and especially the archaeal communities was beyond that yet reported for aquaculture biofilters. While ammonia flux through the respective communities has yet to be estimated, the diverse environmental adaptations of the bacterial and archaeal communities suggest resilience of function under a range of environmental conditions.

Expression pattern and functional analysis of R-spondin1 in tongue sole Cynoglossus semilaevis.

R-spondin 1 (Rspo1) is a potential female-determining gene in mammals that could regulate the Wnt/ß-catenin signaling pathway. The deletion of Rspo1 causes sex reversal in females. To investigate sexual determination and differentiation, we cloned and analyzed the Rspo1 gene in Cynoglossus semilaevis. Phylogenetic and gene structure analyses revealed that Rspo1 gene exhibited high sequence conservation and contained an N-terminal signal peptide, two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat, and a C-terminal region enriched with basic charged amino acids. qRT-PCR revealed that Rspo1 expressed sexual dimorphism in gonad, with higher expression levels in the ovary than in the testis, thus, suggesting the involvement of Rspo1 in gonad differentiation. In situ hybridization results demonstrated that Rspo1 was expressed in premature germ cells, including spermatogonia and spermatocytes in the testis and stage II and stage III oocytes in the ovary. The methylation levels in two CpG sites of Rspo1 promoter significantly differed among females, males, and pseudomales. After 30days of exposure to high temperature, the expression of Rspo1 significantly decreased in female individuals, some of which were prone to males. However, no difference of Rspo1 gene expression was observed between the control group and high-temperature group in males. These preliminary findings suggested that Rspo1 played a crucial role in sex determination and development. This study laid the groundwork for further sex control breeding techniques in C. semilaevis.

Effects of cryopreservation at various temperatures on the survival of kelp grouper (Epinephelus moara) embryos from fertilization with cryopreserved sperm.

Fish embryo cryopreservation is highly important for the long-term preservation of genomic and genetic information; however, few successful cases of fish embryo cryopreservation have been reported over the past 60 years. This is the first study to use Epinephelus moara embryos from fertilization with cryopreserved sperm as experimental material. Embryos that developed to the 16-22 somite stage and tail-bud stage were treated with the vitrification solution PMG3T according to a five-step equilibration method and cryopreserved at various temperatures and storage duration. Only 19.9 ± 9.2% of 16-22 somite stage embryos and 1.3 ± 1.1% of tail-bud stage embryos survived when cooled at 4 °C for 60 min. In total, 8.0 ± 3.0% of 16-22 somite stage embryos survived when cooled at -25.7 °C for 30 min, 22.4 ± 4.7% of tail-bud stage embryos survived after 45 min of cooling at -25.7 °C, and none survived after 60 min. Only 2.0 ± 2.7% of embryos survived when cryopreserved at -140 °C for 20 min. However, 9.7% of tail-bud stage embryos survived after cryopreservation in liquid nitrogen (-196 °C) for 2 h. Most surviving embryos developed normally. Embryonic volume decreased and spherical segments appeared when embryos were treated with higher concentrations of vitrification solution. Additionally, the volume recovered gradually after rinsing with sucrose and seawater. This is the first estimate of the survival of E. moara embryos and larvae after cryopreservation. These findings provide a foundation for further explorations of fish embryo cryopreservation techniques.