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In this study, Cromileptes altivelis embryos were successfully preserved using non-permeable cryoprotectants for the first time. We compared the toxicity of single non-permeable cryoprotectants and combination of non-permeable cryoprotectants on C. altivelis embryos. Five non-permeable cryoprotectants at 1 M (i.e., trehalose, sucrose, lactose, fructose, and glucose) were used to treat embryos in the somite, tail-bud, or heart-beating stages for 1 h. Survival rates and rates of normal embryonic development after treatment with trehalose, sucrose, or lactose were all greater than 86.97% and 86.01%, respectively. Levels of toxicity of trehalose, sucrose, and lactose were lower than those of fructose and glucose in C. altivelis embryos. Sucrose and trehalose were combined at different ratios (total concentration 1 M) and used to treat embryos for 1 h. Rates of survival and normal development for the somite, tail-bud, or heart-beating stages did not significantly differ among groups treated with sucrose + trehalose at ratios of 1:1 (S1T1), 1:3 (S1T3), and 3:1 (S3T1). Embryos treated with S1T1 had a little higher survival rate than those of the other treatment ratios(P > 0.05). Survival rates of embryos treated with T1S1, sucrose, and trehalose were 93.86 ± 4.17%, 91.58 ± 3.15%, and 92.79 ± 2.45%, respectively. S1T1 was less toxic than sucrose or trehalose alone under the same conditions. Various cryopreservation-related factors (e.g., the cryoprotectant solution, embryonic stage, equilibrium time, thawing temperature, and time) were screened. The optimal protocol was the treatment of embryos at the tail-bud and heart-beating stages with 1 M S1T1 for 2–10 min and thawing at 26 °C for 10 s after freezing. In total, 21 viable embryos were recovered after freezing and thawing, with a survival rate of 0.21–7.55% and maximum survival time of 5 days. Our results demonstrate that it is feasible to cryopreserve C. altivelis embryos using non-permeable cryoprotectants and provide a reference for the use of combinations of cryoprotectants for the species. • 21 viable C. altivelis embryos were obtained after frozen-thawing for the first time. • The maximum survival rates and time of viable embryos were 7.55% and 5 days. • Non-permeable antifreeze mixtures(S1T1) were used for embryos cryopreservation. • The toxicity of trehalose or sucrose at 1 M was lower than fructose and glucose. [ABSTRACT FROM AUTHOR]

In this study, Cromileptes altivelis embryos were successfully preserved using non-permeable cryoprotectants for the first time. We compared the toxicity of single non-permeable cryoprotectants and combination of non-permeable cryoprotectants on C. altivelis embryos. Five non-permeable cryoprotectants at 1 M (i.e., trehalose, sucrose, lactose, fructose, and glucose) were used to treat embryos in the somite, tail-bud, or heart-beating stages for 1 h. Survival rates and rates of normal embryonic development after treatment with trehalose, sucrose, or lactose were all greater than 86.97% and 86.01%, respectively. Levels of toxicity of trehalose, sucrose, and lactose were lower than those of fructose and glucose in C. altivelis embryos. Sucrose and trehalose were combined at different ratios (total concentration 1 M) and used to treat embryos for 1 h. Rates of survival and normal development for the somite, tail-bud, or heart-beating stages did not significantly differ among groups treated with sucrose + trehalose at ratios of 1:1 (S1T1), 1:3 (S1T3), and 3:1 (S3T1). Embryos treated with S1T1 had a little higher survival rate than those of the other treatment ratios(P > 0.05). Survival rates of embryos treated with T1S1, sucrose, and trehalose were 93.86 ± 4.17%, 91.58 ± 3.15%, and 92.79 ± 2.45%, respectively. S1T1 was less toxic than sucrose or trehalose alone under the same conditions. Various cryopreservation-related factors (e.g., the cryoprotectant solution, embryonic stage, equilibrium time, thawing temperature, and time) were screened. The optimal protocol was the treatment of embryos at the tail-bud and heart-beating stages with 1 M S1T1 for 2–10 min and thawing at 26 °C for 10 s after freezing. In total, 21 viable embryos were recovered after freezing and thawing, with a survival rate of 0.21–7.55% and maximum survival time of 5 days. Our results demonstrate that it is feasible to cryopreserve C. altivelis embryos using non-permeable cryoprotectants and provide a reference for the use of combinations of cryoprotectants for the species. • 21 viable C. altivelis embryos were obtained after frozen-thawing for the first time. • The maximum survival rates and time of viable embryos were 7.55% and 5 days. • Non-permeable antifreeze mixtures(S1T1) were used for embryos cryopreservation. • The toxicity of trehalose or sucrose at 1 M was lower than fructose and glucose. [ABSTRACT FROM AUTHOR]

The Jinhu grouper, a hybrid of tiger grouper (Epinephelus fuscoguttatus ♀) and potato grouper (E. tukula ♂), 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, 24 °C, and 30 °C). 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. • RNA-seq was performed on three tissues of two groupers under temperature stress. • The expression of PPAR signaling pathway was different in two groupers. • Several hsp genes were highly expressed in the brain tissue of Jinhu grouper. • Modules and hub genes associated with temperature stress were identified by WGCNA. [ABSTRACT FROM AUTHOR]

Hybridization is a widely used and effective method in grouper breeding. In this study, we constructed full-sib families of brown-marbled grouper (Epinephelus fuscoguttatus), Jinhu grouper (E. fuscoguttatus ♀ × E. tukula ♂), and Hulong grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂) and compared the growth characteristics under industrial conditions. The body weight of Jinhu grouper was 1.49 times that of Hulong grouper (P < 0.05) and the body weight of Jinhu grouper was 2.03 times that of E. fuscoguttatus (P < 0.05). Moreover, the survival rate of Jinhu grouper was significantly higher than those of Hulong grouper and E. fuscoguttatus (P < 0.05). The relationships between body length and body weight (W = aLb) in Jinhu grouper, Hulong grouper, and E. fuscoguttatus were similar, and both Jinhu grouper and Hulong grouper may inherit the growth pattern of E. fuscoguttatus. The b values were all <3, indicating allometric relationships at this stage. In a comparison of the low temperature tolerance, E. fuscoguttatus and Jinhu grouper stopped feeding at 19 °C and 16 °C, respectively, and 11 °C and 9 °C were the half-lethal temperatures, respectively. A histopathological analysis showed that the degree of damage in the gill, muscle, and liver tissues increased as the temperature decreased. The activities of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), and glutathione peroxidase (GSH-Px) in the liver decreased initially and then increased as the temperature decreased. The malondialdehyde (MDA) contents in the liver increased initially and decreased thereafter. Lysozyme (LZM) activity in the serum increased initially and then decreased, and immunoglobulin M (IgM) levels did not vary significantly with respect to temperature (P > 0.05). In conclusion, Jinhu grouper was characterized by a rapid growth rate, high survival rate, and low temperature resistance. This hybrid grouper is suitable for industrial culture in China, with broad potential applications • Growth and survival were better in Jinhu grouper than in E. fuscoguttatus and Hulong grouper. • Behavioral, histological, and enzyme responses to low temperature stress were studied. • The half-lethal temperature was lower in Jinhu grouper than in E. fuscoguttatus. [ABSTRACT FROM AUTHOR]

QTL mapping is an effective method for marker assisted selection (MAS) in animal breeding, and high-density linkage genetic maps are necessary for fine mapping of QTLs. In this study, high-density genetic maps of hybrid Epinephelus fuscoguttatus (♀) and Epinephelus tukula (♂) progeny (which demonstrated improved growth rates compared with the female parent) were constructed using RAD technology. Sex-average, female-specific, and male-specific linkage maps were composed of 24 linkage groups (LGs). The genetic map developed in this study had the highest number of SNPs among reported genetic maps for groupers. The sex-average linkage map contained 2759 bin makers (150,468 SNPs) and spanned 1627.28 cM, with an average marker interval of 0.59 cM. The female-specific (E. fuscoguttatus) linkage map contained 648 bin makers (38,269 SNPs) and spanned 1414.68 cM, with an average marker interval of 2.22 cM. The male-specific (E. tukula) linkage map contained 2126 bin makers (112,015 SNPs) and spanned 1839.88 cM, with an average marker interval of 0.86 cM. In total, 45 QTLs comprising 318 SNPs for growth-related traits were identified, including 10 QTLs for body weight, 6 QTLs for full length, 7 QTLs for body length, 14 QTLs for head length, 5 QTLs for body height, and 3 QTLs for body thickness. In addition, 56 candidate genes related to growth, metabolism, bone formation, muscle development, and neural regulation were identified, including eight genes associated with growth in other fish. Among these, gpr126 , fam20a , fam20c , sox9b , foxm1 , foxl1 , myf6 , zbtb38 , and sl should be a focus of further research. These results provide a foundation for fine QTL mapping in grouper, and theoretical guidance for exploring the causes of heterosis, which is of great significance for MAS in the breeding of hybrids and male and female parents. • The genetic map of Epinephelus fuscoguttatus , Epinephelus tukula and their hybrid progeny were constructed. • The genetic map in this study had the highest number of SNPs among reported genetic maps for groupers. • Forty-five QTLs comprising 318 SNPs for growth-related traits were identified. • Fifty-six candidate genes related to growth, metabolism, bone formation, and neural regulation were obtained. [ABSTRACT FROM AUTHOR]

Grouper is an economically important fish in China. However, it exhibits a high frequency of skeletal abnormalities, particularly vertebral deformities. The molecular mechanisms underlying fish vertebral deformities are still poorly understood. In this study, a HiSeq™ 4000 platform (Illumina) was used to analyze the transcriptomic profiles of the brain, pituitary, and vertebrae from normal fish (NF) and fish with lordosis (LF) of Yunlong grouper. A total of 87,888 unigenes were assembled with lengths that varied from 201 to 28,922 bp and a N50 length of 2670 bp. A total of 36,268 unigenes were functionally annotated by BLAST alignments. A total of 2875 significantly differentially expressed genes (DEGs) were identified between the NF group and the LF group, including 706 upregulated unigenes and 2169 downregulated unigenes in LF. GO and KEGG pathway enrichment analyses showed that DNA binding, transmembrane receptor activity, cytokine receptor interaction, neuroactive ligand-receptor interaction, calcium signaling pathway and ECM-receptor interaction HIF-1 signaling pathway, and mineral absorption may be involved in the formation of vertebral deformities. Furthermore, weighted gene co-expression network analyses, including three modules (turquoise, yellow, and blue), significantly positively corrected with vertebral deformities. A network map that included these three modules enabled the identification of a series of hub genes, including claudin-22-like (cldn22), fibronectin type III domain-containing protein 1 isoform X2 (fndc1l2), E3 ubiquitin-protein ligase NRDP1-like (rnf41), and Catenin alpha-2 (ctnna1). We found that the levels of most genes in the blue module were closely related to the expression of parvalbumin, thymic CPV3-like isoform X2 (ocm), platelet glycoprotein Ib alpha chain (gp1ba), and matrix metalloproteinase-9 (mmp9), suggesting that this module is associated with skeletal development. Some uncharacterized genes associated with known bone-related genes, including Unigene0067643, Unigene0056862, and Unigene0059867, were detected by a weighted gene co-expression network analysis. A detailed functional investigation of these networks and genes will further improve our understanding of the molecular mechanisms that underlie the formation of lordosis in fish. • Skletal abnormality is one of the most challenging problems for grouper aquaculture industry. • Transcriptome sequencing was performed on the brain, pituitary and vertebrae to study the lordosis of Yunlong grouper. • WGCNA enabled us to uncover networks and new genes involved in vertebrae development. • Changes in the expression of some genes might disrupt the dynamic balance of vertebrae processes, resulting in lordosis. [ABSTRACT FROM AUTHOR]

Owing to the increasing market demand and emerging grouper aquaculture industry, close attention has been paid to genetic breeding for growth traits in grouper. The construction of linkage maps for quantitative trait loci (QTL) analyses is an essential step for marker-assisted selection (MAS) programs. In this study, we report the first high-density linkage map of Yunlong grouper (Epinephelus moara ♀ × E. lanceolatus ♂) constructed by sequencing 118 F1 progenies in a full-sib family using restriction site associated DNA (RAD) technology. For the female and male maps, 12,307 and 12,340 single nucleotide polymorphism (SNP) markers were anchored onto 24 linkage groups, spanning 1909.37 and 1933.61 cM, respectively, with an average marker interval of 0.16 cM. QTL mapping revealed 39 significant QTLs for three growth traits. Candidate genes participating in ubiquitin modification, cell adhesion, G-protein coupled receptors, cell proliferation, and calcium transport were identified from these QTLs, including itgb1 , col1a1 , pdgfc , bmpr1a , pcdhgc5, and cacna1b. The results of this study provide a foundation for genomic and genetic studies and further analyses of the heterosis of some growth traits, thereby facilitating MAS in Yunlong grouper. • High-density linkage maps with 12,307 and 12,340 SNP markers were constructed in F1 hybrid Yunlong grouper. • Thirty-nine QTLs were found to associated with three growth-related traits. • Forty-four candidate genes related to multiple growth-regulatory pathways were identified in significant QTL regions. • This study provides a valuable resource for heterosis study and marker-assisted selection breeding in Yunlong grouper. [ABSTRACT FROM AUTHOR]