Our current understanding of the early developmental characteristics and growth traits of the potato grouper (Epinephelus tukula) is limited but necessary for the development of large-scale aquaculture farming of this species. To investigate this, this study used artificially propagated E. tukula and systematically observed embryonic and morphological development processes of both larvae and juvenile fish and analyzed the growth characteristics during the young developmental stage. Under water temperature and salinity conditions of 23.9 ± 0.4°C and 28-30 ppt, respectively, the fertilized eggs completed embryonic development in 25 h 34 min, sequentially passing through seven typical stages: fertilization, cleavage, blastula, gastrula, neurula, organogenesis, and hatching. The newly hatched larvae had an average total length of 1.60 ± 0.17 mm. The yolk sac and oil globule were visible at 1 dph (days post-hatching); at 3 dph they began exogenous feeding; the prototype of the “trident” appeared at 9 dph; by 30 dph, the fins were almost fully developed and the fish entered the juvenile stage; by 35 dph, the second dorsal fin spine and pelvic fin spine began to shrink; by 55 dph, spots and scales covered the entire body, and the fish entered the young developmental stage. After 105 dph, average body weight increased from 34.134 ± 5.85 g to 135.28 ± 20.11 g, and average total length increased from 13.41 ± 0.81 cm to 21.29 ± 1.15 cm. The best-fit model between average total length (TL) and age was the quadratic function TL = -0.001t² + 0.283t - 11.018 (R² = 0.982). The results of this study showed that the E. tukula has a relatively fast embryonic development rate, typical morphological changes in larval and juvenile stages, and rapid early growth in the young developmental stage, while the specific growth rate decreased with prolonged culture time. This study provides relevant biological information for the artificial propagation, fry rearing, and selective breeding of this species.

To systematically evaluate the growth heterosis and genetic characteristics of the hybrid offspring E. moara ♀ × E. tukula ♂, this study assessed the phenotypic growth pattern through a comparative breeding experiment and cytogenic and molecular genetic characteristics through integrated chromosomal karyotype and mitogenomic analyses. The results showed that at 9 months of age, the body weight of the hybrid offspring reached 137.08 ± 26.41 g, which was 1.50 times that of the maternal E. moara (91.60 ± 18.63 g), but significantly lower than that of the paternal E. tukula (178.19 ± 32.74 g), exhibiting a mid-parent growth heterosis (1.62%). The chromosome number of the hybrid offspring was 2n=48, with a karyotype formula of 2sm+1st+45t, NF=51, and an intermediate chromosomal composition between the parents. The mitochondrial genome was 16,405 bp, and its gene composition, arrangement order, base composition and codon usage preference were highly consistent with those of E. moara. A phylogenetic analysis further supported that its mitochondrial genome strictly followed maternal inheritance. This study provides a theoretical foundation for evaluating the breeding potential and germplasm characteristics of E. moara ♀ × E. tukula ♂ hybrids.

The blacktip grouper E. fasciatus is a kind of precious economic fish, and is a valuable marine fish for aquaculture. At present, there are no reports on the genome sequence of this species, which has restricted the mechanism analysis of its important economic traits and molecular breeding. In this study, we present the first high-quality telomere-to-telomere genome assembly and annotation of E. fasciatus with no gaps, generated using a combination of the Oxford Nanopore Technologies (ONT) reads, Pacific Biosciences (PacBio) high-fidelity (HiFi) long reads, high-throughput chromosome conformation capture (Hi-C) reads, and Illumina short-read sequencing reads. The final assembly spans 898.71 Mb, with a scaffold N50 of 38.50 Mb and 99.90% of the assembled genome anchored onto 24 pseudo-chromosomes. Genome annotation predicted approximately 39.49% of repetitive sequences and 26,191 protein-coding genes. This reference genome serves as a valuable resource for molecular marker development and future evolutionary, ecological, and functional genomic studies in E. fasciatus.

Cryopreservation is an important method for protecting germplasm resources. Dehydration caused by cryoprotectants or frozen during cryopreservation affects the development of embryos and results in tail deformity. To elucidate the molecular mechanisms underlying dehydration in oviparous embryos, whole-transcriptome sequencing was performed for Oplegnathus punctatus embryos exposed to sucrose-induced dehydration for 0 (OpC), 60 (Op60), or 120 min (Op120). Differentially expressed mRNAs (DE mRNAs), lncRNAs (DE lncRNAs), and miRNAs (DE miRNAs) for OpC-vs-Op60, OpC-vs-Op120, and Op60-vs-Op120 were obtained. The functional analysis of DE mRNAs, DE lncRNAs, and DE miRNAs revealed that pathways related to energy metabolism, free amino acids, the cell cycle, and the cytoskeleton are involved in the response of dehydration. Abnormal osteoclast differentiation may be associated with tail deformities resulting from dehydration. Key genes were identified, including PFKFB3, ALDO, glnA, CDKN1B, CLDN4, ITGB1, HB-EGF, FOS, JUN, NCOA2 and Hsp70. A competitive endogenous RNA (ceRNA) regulatory network was constructed, ceRNA-associated interaction pairs of DE mRNAs and noncoding RNAs were predicted. These results provide an important reference for the dehydration (osmotic stress) response of oviparous embryos and a solid foundation for further analyses of cryopreservation mechanisms in fish embryos.

Epinephelus awoara is an important germplasm resource characterized by its high economic value and ecological significance. In this study, the low-temperature tolerance of E. awoara was characterized. Compared to E. fuscoguttatus, E. awoara exhibited superior cold tolerance, ceasing feeding at 12 degrees C, which is 7 degrees C lower than E. fuscoguttatus. Subsequently, genomic and transcriptomic analyses were employed to elucidate the underlying mechanisms of its adaptation to low temperatures. A high-quality genome assembly of E. awoara was obtained, and gene family changes and positively selected genes were identified through comparative genomic analysis. A significant number of low-temperature response genes were identified in the liver tissue of E. awoara. The results indicated an increased expression of genes associated with fatty acid metabolism, REDOX regulation, and immune-related pathways under cold stress. This study represents the first systematic analysis of the molecular mechanisms of low-temperature adaptation in E. awoara at the genomic and transcriptomic levels, providing an important theoretical basis for its cold tolerance breeding.

To investigate the early developmental characteristics and mitochondrial inheritance patterns of the hybrid offspring of a paternal Plectropomus leopardus and maternal Epinephelus fasciatus, the study systematically mapped the embryonic developmental trajectory and mitochondrial genome of the new germplasm. Results revealed that the fertilized hybrid eggs completed embryonic development within 28h55min, with the newly hatched larvae measuring 2.05 ± 0.37 mm in total length. The mitochondrial genome length of the hybrid was 16,570 bp, preserving 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. The hybrid's mitochondrial gene composition and arrangement showed high consistency with that of the maternal E. fasciatus. Concurrently, the co-linearity, Ka/Ks, and phylogenetic tree analyses collectively indicate that the hybrid progeny has a closer genetic relationship with the maternal parent, supporting the mitochondrial maternal inheritance of this species. This study details the embryonic development and mitochondrial inheritance of an intergeneric hybrid grouper germplasm, providing significant molecular biological evidence for grouper hybrid breeding, germplasm resource identification, and genetic diversity conservation.

Epinephelus fasciatus,a small grouper,is valued for its delectable flesh and substantial market value.This study utilized wild red grouper parents to examine artificial breeding,embryonic and post-embryonic development,and juvenile fish cultivation under industrialized conditions in northern China.At present,no documented studies exist on the artificial breeding,embryonic and post-embryonic development,and feed conversion processes of E.fasciatus under local factory conditions in China.This study focused on fertilized eggs and juvenile fish of E.fasciatus artificially bred in factories and recorded their embryonic development and instances of rapid growth.The study also explored the relationship between juvenile fish development and feed conversion,providing a scientific reference for the industrial breeding and aquaculture of E.fasciatus and its high-value utilization. The parent experiments,including fish cultivation,fertilization,and hatching,were conducted at Laizhou Mingbo Aquatic Products Co.,Ltd.For egg collection,female red groupers with stage Ⅳgonad development were selected for artificial induction.The oxytocin used was a mixture of HCG and LHRH-A3,which was administered at dosages of 350 IU/kg and 20 μg/kg of fish weight,respectively.Two days before sperm collection,a halved mixture was injected into mature male fish to promote sperm production.To obtain fertilized eggs,mature eggs were first collected in a dry plastic basin by compressing the abdomen,while sperm with high microscopic vitality were simultaneously collected for artificial insemination.Dry insemination was performed at a sperm-to-egg volume ratio of 1∶500.After stirring,the eggs were allowed to stand for 5 min.The eggs were then washed with an equal volume of seawater and allowed to stand for 5 min.The floating eggs were collected and incubated in an incubation bucket with seawater maintained at a water temperature of(23.4±0.8)℃,dissolved oxygen levels greater than 6 mg/L,and a salinity range of 28-30.Upon completion of the embryonic hole closure period,the eggs were collected with gauze and immediately transferred to a breeding pool with an egg-laying volume of 2-3 mL/m3. Starting with fertilized eggs,10 floating eggs were regularly removed from the hatching bucket,and an optical microscope(Nikon E200)was used to observe the embryonic development process.Photos were captured,and the time and developmental characteristics of each developmental stage were recorded.The time point of each developmental stage was defined when two-thirds of the embryos reached this stage.The fertilized eggs completed embryonic development within 31 h and 12 min under water temperature and salinity conditions of(23.4±0.8)℃ and 28-30,respectively. Starting with the initial hatching of fry,fish fry exhibiting good growth from the breeding pool were regularly selected.A microscope(Nikon E200),dissecting microscope(Olympus),and Image View software were used to capture photos,measure total length,and record the developmental stages and characteristics of the fry and juveniles.During this period,samples were collected daily from 0 to 10 days post-hatching(dph),every 2 days from 10 dph to 20 dph,and every 5 days from 20 dph to 40 dph.Fish fry(3-5)was randomly selected at each time point to measure their total length and record their growth status.One-way ANOVA was performed on growth data using SPSS 27.0,with significant differences between groups compared using the least significant difference(LSD)and Duncan tests.Origin Pro 2022 software was employed for figure creation. The post-embryonic developmental sequences were divided into the early larval(0-3 dph),late larval(4-29 dph),juvenile(30-54 dph),and young(＞55 dph)stages.At 3 dph,the larvae were fed S-grade rotifers.At 9-20 dph,L-grade rotifers were provided.At 20-30 dph,brine shrimp larvae were provided.Starting at 31 dph,the fish larvae transitioned from animal-based feed to compound feed.Compared to the early developmental stage,the growth rate,phenotype,scales,and body color of the larvae rapidly increased after transitioning to compound feed.This indicated a high acceptance of compound feed during the transitioning stage.The feeding frequency was twice daily,occurring at 08:00 and 16:00.The dimensions of the cultivation tank were 6 m × 8 m× 2.5 m,with a water flow exchange rate of 8 m3/h.Dissolved oxygen levels exceeded 6 mg/L,salinity ranged from 28 to 30,and water temperature varied between 25 ℃ and 28 ℃. At present,although red groupers can be artificially reproduced,cultivation technology remains imperfect,and large-scale breeding has not yet been realized.This study systematically investigated embryonic developmental processes and early morphological changes in red grouper larvae produced using artificial insemination under industrial conditions.The early developmental patterns of metamorphosis,growth,and early body color changes were systematically documented,providing a reference for the artificial cultivation and scientific feeding of early fry and juveniles to mitigate mortality risk.

The allotriploid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂, is an economically valuable aquaculture species owing to its fast growth rate and desirable meat quality. Despite its many favorable traits, there is still a lack of records on allotriploid grouper mitochondrial genome. In this study, we determined and analyzed the complete mitochondrial genome of the triploid hybrid grouper Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂. The mitogenome was 16,610 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a control region. The order and composition of these genes were similar to other vertebrates. The overall base composition was 29.11 % for A, 28.40 % for C, 15.65 % for G, and 26.84 % for T, with a higher A + T content (55.95 %). Phylogenetic analysis showed that the allotriploid grouper, Epinephelus fuscoguttatus ♀ × Epinephelus tukula ♂, was most closely related to Epinephelus fuscoguttatus. This study provides the first mitochondrial genome record of allotriploid grouper, which will facilitate future research on evolution and phylogeny of Epinephelidae.

To develop superior grouper aquaculture varieties, in this study, a intergeneric hybrid breed was constructed by crossing a male Plectropomus leopardus, with a female parent Epinephelus fasciatus. Here, we researched embryonic development and mitochondrial composition of the new hybrid germplasm. Results revealed that the fertilization and hatching rate were 85.87 ± 5.22% and 70.37 ± 0.33%, respectively. Hatching occurred 28 h 55 min after fertilization at 24.8 ± 0.5 °C, and the newly hatched larvae were 2.05 ± 0.37 mm in total length. The mitochondrial genome length of the hybrid was 16,570 bp, preserving 13 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, and 22 transfer RNA (tRNA) genes. The mitochondrial gene composition and arrangement of the hybrid were very similar to those of the maternal E. fasciatus. Additionally, analyses including co-linearity, Ka/Ks ratio, and phylogenetic tree all demonstrated that the hybrid’s mitochondria are inherited from the mother. Furthermore, the genetic distance between the hybrid and species from the maternal genus is shorter than that between the hybrid and the paternal P. leopardus. This study provides detailed insights into the embryonic development and mitochondrial inheritance of an intergeneric hybrid grouper, offering valuable molecular biological evidence to support grouper hybrid breeding, germplasm identification, and the conservation of genetic diversity.

Vibrio anguillarum acts as an infectious agent in the aquaculture industry that causes a fatal hemolytic septicaemic disease in fish and shellfish. Viral nervous necrosis (VNN) disease seriously impacts the healthy development of the aquaculture industry. While the detrimental effects of V. anguillarum and NNV have been widely researched on freshwater and marine fish, whether they affect DNA replication in fish is unclear. In this study, we used Jinhu grouper as a model to investigate the influence of V. anguillarum and NNV on their DNA replication. By RNA-seq analysis in conjunction with gene set enrichment analysis, we found a significant upregulation of genes related to DNA replication in the liver and spleen of the Jinhu grouper after V. anguillarum infection while a prominent downregulation of genes related to DNA replication in the brain of the Jinhu grouper after NNV infection. We identified 27, 29 and 18 key genes involved in DNA replication that may respond to V. anguillarum and NNV infection and selected and identified six DNA replication genes (pole, pole2, pold1, pola1, pcna and mcm2). In addition, our results indicated that these genes exhibit tissue-specific expression patterns, with an increasing pattern of expression when V. anguillarum infected and a decreasing pattern when NNV infected. These results may provide valuable understanding on the underlying mechanisms of V. anguillarum and NNV infection in fish.

The tomato hind grouper (Cephalopholis sonnerati) is an emerging aquaculture species, with significant commercial value and promising farming potential. To advance the theoretical framework for artificial breeding, this study systematically investigated the embryogenesis, early larval morphology, growth patterns, and heritable traits of the species. The results indicated fertilization and hatching rates of 88.67 ± 3.93% and 79.67 ± 7.55%, respectively, with an average egg diameter of 0.87 ± 0.02 mm. Hatching occurred 22:55 h after fertilization at 24.80 ± 0.70 °C, corresponding to 568.42 degree-hours, and the newly hatched larvae measured 2.09 ± 0.12 mm in total length. Interestingly, as the fish grows, the color of its head patches shifts from yellow to red, providing a visible trait that can be used for early growth screening. After 15 months of graded rearing, marked growth disparities were observed among individuals originating from the same clutch, with the fast-growing group weighing 457.12 ± 58.68 g, which was 2.9 times greater than that of the slow-growing group. These findings underscore the potential of C. sonnerati as a valuable aquaculture species. Future efforts should prioritize enhanced broodstock selection and the development of fast-growing germplasm to increase its cultivation potential.

Backcrossing serves as an effective tool for interspecific gene introgression, facilitating germplasm improvement. To develop superior grouper aquaculture varieties, in this study, a backcrossed breed was constructed by crossing a male hybrid grouper, KGGG (Epinephelus moara ♀ × E. lanceolatus ♂), with a female parent kelp grouper (KG; E. moara). Here, we observed embryonic, larval development, and morphological changes in KG × KGGG. Additionally, the total length and body weight of the backcrossed breed and maternal parent were compared at 160 d post hatching (dph). The results showed that fertilization and the hatching rate were 73.23 ± 4.23% and 51.74 ± 3.67%, respectively, and the egg size was 0.89 ± 0.03 mm. Hatching occurred 23:19 h after fertilization at 25 ± 1 °C, and the newly hatched larvae were 1.94 ± 0.13 mm in length. Furthermore, at 160 dph, the total length and body weight of the backcrossed breed were 1.2-fold and 1.9-fold greater, respectively, when compared with those of the maternal parent. Importantly, it is possible that backcrossed KG × KGGG could become a dominant strain in grouper aquaculture practices.

Vibrio anguillarum serves as a pathogenic organism in aquaculture, leading to a lethal hemolytic septicemia in aquatic species. Whereas little study has evaluated the molecular mechanism of the infection caused by V. anguillarum in Jinhu grouper. In this study, analysis of the transcriptome was conducted on the liver and spleen tissues from Jinhu groupers infected with V. anguillarum infection. We identified 2978 DEGs in the liver group and 2506 DEGs in spleen group, including 1689 and 1502 up-regulated genes and 1289 and 1004 down-regulated genes, respectively. Gene set enrichment analysis revealed a significant reduction in genes associated with metabolism such as carbon metabolism and glycolysis/gluconeogenesis in the liver, while upregulation of genes linked to the above pathways as well as in the citrate cycle in the spleen. In addition, the upregulated genes in the liver and spleen are both enriched in the cell cycle. Subsequent investigation into the principal DEGs implicated in the TLR pathways showed that V. anguillarum infection may activate the TLR pathway by overexpression of the tlr5 and promote the synthesis of proinflammatory cytokines il1β and il-8. Among these, 11 genes related to metabolism, cell cycle and immunity were selected and characterized. Overall, our research indicates that V. anguillarum can affect the metabolism and cell cycle while also triggering immune defense reactions in Jinhu grouper.

Deoxynivalenol (DON) is a type of mycotoxin that widely contaminates aquatic feed. Although the detrimental effects of DON on fish have been investigated, the specific impacts of DON on the proliferation of zebrafish PAC2 fibroblasts remain unclear. In this study, it was found that DON significantly suppressed the proliferation of zebrafish PAC2 fibroblasts in a dose-dependent manner. Through RNA-seq, GSEA, molecular docking and immunofluorescence analysis, we discovered that genes related to the ribosome, ECM and DNA replication were significantly down-regulated. Interestingly, we verified that DON binds to ribosomes and DON exposure could interfere with the mitosis of zebrafish embryonic fibroblasts and arrest the cell cycle. Additionally, we found that DON treatment activated autophagy-animals and mitophagy-animals signaling pathway. This study might offer valuable perspectives on the mechanisms beneath DON toxicity in zebrafish PAC2 fibroblasts.

Leopard coral grouper (Plectropomus leopardus), possessing a distinct red body color, is an important species in commercial markets; however, the high ratio of black individuals under intensive cultivation has limited the commercial value of the species. To dissect the regulatory mechanisms underlying the red skin trait in P. leopardus, gene expression and DNA methylation modifications were compared between red and black skin tissues after astaxanthin treatment. Astaxanthin effectively increased the redness value a* and body weight. Multi-omics analyses revealed the crucial roles of pathways related to antioxidants and lipid metabolism, particularly “Tyrosine metabolism”, “Melanogenesis”, “Fatty acid metabolism”, “Fatty acid elongation”, and “Biosynthesis of unsaturated acids”, in red skin coloration. A molecular network for the regulation of red skin coloration in P. leopardus was constructed, and pmel, tyr, tyrp1a, tyrp1b, dct, slc24a5, wnt1, acsl4, elovl1, elovl6l.1, elovl6l.2, and elovl7 were identified as key genes. Notably, pmel, acsl4, and elovl7 were negatively regulated by differential DNA methylation. Our results provide new insight into the molecular and epigenetic mechanisms of body color variation, representing a significant step towards breeding for the red skin trait in P. leopardus.

Grouper (Epinephelus sp.) is one of the most economically important aquaculture species in China and Southeast Asian countries. Nervous necrosis virus (NNV) infection causes significant mortality in grouper aquaculture. Detection of anti-NNV antibodies using the ELISA method has been reported as effective in preventing vertical transmission of NNV in sea bass and flounder. In this study, IgM antibodies were purified from the serum of groupers and subsequently used to immunize rabbits to obtain a secondary antibody. Additionally, the NNV capsid protein (CP) was cloned, over-expressed, and purified in Escherichia coli. Consequently, an ELISA method was developed to detect NNV-specific antibodies in groupers by utilizing purified recombinant NNV capsid protein (CP) and horseradish peroxidase (HRP)-labeled rabbit anti-grouper antibody. This ELISA system was applied in three asymptomatic grouper hatcheries in China, revealing significant variations in antibody levels. Our ELISA method can effectively detect NNV-specific antibodies and is simpler than other methods, making it suitable for subsequent large-scale applications in grouper.

Jinhu groupers, the hybrid offspring of tiger groupers (Epinephelus fuscoguttatus) and potato groupers (Epinephelus tukula), have excellent heterosis in fast growth and strong stress resistance. However, compared with the maternal tiger grouper, Jinhu groupers show delayed gonadal development. To explore the interspecific difference in gonadal development, we compared the transcriptomes of brain, pituitary, and gonadal tissues between Jinhu groupers and tiger groupers at 24-months old. In total, 3034 differentially expressed genes (DEGs) were obtained. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that the osteoclast differentiation, oocyte meiosis, and ovarian steroidogenesis may be involved in the difference in gonadal development. Trend analysis showed that the DEGs were mainly related to signal transduction and cell growth and death. Additionally, differences in expression levels of nr4a1, pgr, dmrta2, tbx19, and cyp19a1 may be related to gonadal retardation in Jinhu groupers. A weighted gene co-expression network analysis revealed three modules (i.e., saddlebrown, paleturquoise, and greenyellow) that were significantly related to gonadal development in the brain, pituitary, and gonadal tissues, respectively, of Jinhu groupers and tiger groupers. Network diagrams of the target modules were constructed and the respective hub genes were determined (i.e., cdh6, col18a1, and hat1). This study provides additional insight into the molecular mechanism underlying ovarian stunting in grouper hybrids.

Heterosis of growth traits in economic fish has benefited the production of aquaculture for many years, yet its genetic and molecular basis has remained obscure. Nowadays, a new germplasm of hybrid Jinhu grouper (Epinephelus fuscoguttatus ♀ × E. tukula ♂), abbreviated as EFT, exhibiting paternal-biased growth heterosis, has provided an excellent model for investigating the potential regulatory mechanisms of heterosis. We integrated transcriptome and methylome to unravel the changes of gene expression, epigenetic modification, and subgenome dominance in EFT compared with maternal E. fuscoguttatus. Integration analyses showed that the heterotic hybrids showed lower genomic DNA methylation levels than the purebred parent, and the up-regulated genes were mostly DNA hypomethylation. Furthermore, allele-specific expression (ASE) detected paternal subgenome dominance-regulated paternal-biased heterosis, and paternal bias differentially expressed genes (DEGs) were wholly up-regulated in the muscle. Multi-omics results highlighted the role of lipid metabolism, particularly “Fatty acid synthesis”, “EPA biosynthesis”, and “Signaling lipids”, in EFT heterosis formation. Coherently, our studies have proved that the eicosapentaenoic acid (EPA) of EFT was greater than that of maternal E. fuscoguttatus (8.46% vs. 7.46%). Finally, we constructed a potential regulatory network for control of the heterosis formation in EFT. Among them, fasn, pparg, dgat1, igf1, pomca, fgf8a, and fgfr4 were identified as key genes. Our results provide new and valuable clues for understanding paternal-biased growth heterosis in EFT, taking a significant step towards the molecular basis of heterosis.

The Jinhu grouper, a hybrid of tiger grouper (Epinephelus fuscoguttatus female) and potato grouper (E. tukula male), is a new germplasm with excellent characters. To understand the molecular mechanism underlying the responses of Jinhu grouper and tiger grouper to temperature stress, we performed comparative transcriptome analyses of liver, muscle, and brain tissues at three temperatures (18 C-degrees, 24 C-degrees, and 30 C-degrees). In total, 28,331 genes and 4380 differentially expressed genes among temperature treatments were obtained using the Illumina NovaSeq 6000 platform. GO and KEGG enrichment analyses indicated that binding, catalytic activity, metabolic process, carbon metabolism, complement and coagulation cascade, AMPK signaling pathway, FoxO signaling pathway, and ECM-receptor interaction may be involved in temperature adaptation in tiger grouper and Jinhu grouper. We also found differences in the expression of several PPAR signaling pathway-related genes (such as acsl1, slc27a2, and ppard) in tiger grouper and Jinhu grouper, and HSP genes (hsp30, hsp70, hsp90aa1, hsp90b1, hspa5, hspa4, hspa8, hspa9, and hspa13) were highly expressed in the brain tissue of Jinhu grouper. It is speculated that Jinhu grouper has better temperature tolerance than tiger grouper. In addition, a weighted gene co-expression network analysis was used to identify target modules (i.e., EF.greenyellow, EF.sienna3, EF.brown, EFET.salmon, EFET.red, and EFET.pink) significantly associated with temperature stress in the liver, muscle, and brain tissues of tiger grouper and Jinhu grouper, and a series of hub genes were detected in the network diagrams of these six target modules. These results deepen our understanding of the complex molecular mechanisms underlying the response to temperature stress in tiger grouper and Jinhu grouper.

The giant grouper fish (Epinephelus lanceolatus), one of the largest and rarest groupers, is a fast-growing economic fish. Grouper sperm is often used for cross-breeding with other fish and therefore sperm cryopreservation is important. However, freezing damage cannot be avoided. Herein, we performed a transcriptome analysis to compare fresh and frozen sperm of the giant grouper with frozen storage times of 0, 23, 49, and 61 months. In total, 1911 differentially expressed genes (DEGs), including 91 in El-0-vs-El-23 (40 upregulated and 51 downregulated), 251 in El-0-vs-El-49 (152 upregulated and 69 downregulated), and 1569 in El-0-vs-El-61 (984 upregulated and 585 downregulated), were obtained in the giant grouper sperm. DEGs were significantly increased at 61 months of cryopreservation (p < 0.05). GO and KEGG enrichment analyses of the DEGs revealed significant enrichment in the pilus assembly, metabolic process, MAPK signaling pathway, apoptosis, and P53 signaling pathway. Time-series expression profiling of the DEGs showed that consistently upregulated modules were also significantly enriched in signaling pathways associated with apoptosis. Four genes, scarb1, odf3, exoc8, and atp5f1d, were associated with mitochondria and flagella in a weighted correlation network analysis. These genes may play an important role in the response to sperm freezing. The experimental results show that long-term cryopreservation results in freezing damage to the giant grouper sperm. This study provides rich data for studies of the mechanism underlying frozen fish sperm damage as well as a technical reference and evaluation index for the long-term cryopreservation of fish sperm.