Epinephelus Moara Research Articles

Environmentally relevant concentrations of Mn2+ disrupts the endocrine regulation of growth in juvenile Yunlong groupers (Epinephelus moara♀×Epinephelus lanceolatus♂)

Even though manganese is a bioelement essential for metabolism, excessive manganese levels in water can be detrimental to fish development and growth. Therefore, the aim of this study was to evaluate the effects of Mn2+ (0, 0.5,1, 2, and 4 mg·L−1) exposure for 30 d on the growth performance, growth hormone/insulin-like growth factor (GH/IGF) axis, hypothalamic-pituitary-thyroid (HPT) axis, and monoaminergic neurotransmitters of Epinephelus moara♀×Epinephelus lanceolatus♂(Yunlong grouper). Compared with the control and low Mn2+concentration groups of (0.5 and 1 mg·L−1), the high concentration of Mn2+ (4 mg·L−1) significantly reduced body weight (BW), body length (BL), weight gain rate (WGR), and specific growth rate (SGR), increased the feed coefficient rate (FCR) and mortality of Yunlong groupers (P < 0.05). Further, the levels of GH and IGF, along with the expression of ghra and ghrb were significantly reduced after exposure to 2 and 4 mg·L−1 Mn2+for 30 d, whereas the expression of sst5 was significantly up-regulated after exposure to 2 and 4 mg·L−1 Mn2+for 20 and 30 days. Moreover, Mn2+exposure increased thyroid hormone (T3) and thyroid stimulating hormone (TSH) contents, accompanied by increased mRNA levels of dio1 and dio2, however, the T4 level was decreased. Finally, dopamine (DA) and serotonin (5-HT) levels significantly decreased after long-term exposure to higher concentrations of Mn2+, and the levels their metabolites changed as well, suggesting that the synthesis and metabolism of DA and 5-HT were affected. Accordingly, changes in the GH/IGF and HPT axes-related parameters may be the cause of growth inhibition in juvenile groupers under Mn2+ exposure, indicating that the relationship between endocrine disorder and growth inhibition should not be ignored.

Polybrominated diphenyl ethers as hitchhikers on microplastics: Sorption behaviors and combined toxicities to Epinephelus moara

Emerging pollutants, such as microplastics (MPs) and polybrominated diphenyl ethers (PBDEs) may pose a serious threat to human health and ecological safety. However, little is known about the MP-mediated PBDEs exposures and their combined toxicities towards farmed fishes. This study investigated the sorption behaviors of two typical PBDEs (BDE-47 and BDE-209) to MPs of different polymer types (PE, PS, PHA and PHB), and examined their combined toxic effects on grouper (Epinephelus moara) by determining the change of oxidative stress markers and comparing gene expression difference through high-throughput sequencing. Our results demonstrated that the sorption of PBDEs on MPs were polymer type-dependent and the sorption capacities were in the order of PHA>PHB>PS>PE. The combined exposures of MPs and PBDEs led to more severe disturbance on the oxidative system compared with individual exposure. The activity of superoxide dismutase (SOD) and the content of glutathione were decreased, while the activity of catalase (CAT) and the content of malondialdehyde were increased. The disorder of oxidative system can influence the growth of groupers. High-throughput sequencing confirmed that pathways of ferroptosis, IL-17 and PPAR expressed differently under combined exposure of MPs and BDE-47. IL-17 pathway related genes were inhibited, while genes in PPAR pathway were upregulated. The combined exposure brought more severe effect on grouper's gene expression compared with individual exposure. GPX-related genes and CAT gene in the liver were up-regulated, while SOD-related genes were down-regulated. Our results demonstrated that the combined toxicity of MPs and PBDEs can pose a non-neglectable threat to aquaculture development and food safety, and gained a primary insight into the potential risk of MPs to farmed fishes.

Effect of long-term manganese exposure on oxidative stress, liver damage and apoptosis in grouper Epinephelus moara ♀ × Epinephelus lanceolatus ♂

Manganese is an indispensable trace element, however, it may be present at high concentrations in water and sediments of aquatic ecosystems due to natural and anthropogenic activities, and can interfere with physiological and biochemical mechanisms in fish. This study was conducted to determine the toxic effects associated with exposure to Mn2+ (0, 0.5, 1, 2, and 4 mg/L) for 30 d, regarding liver damage and apoptosis in Yunlong grouper (Epinephelus moara♀×E. lanceolatus♂). Expression of superoxide dismutase (sod) and catalase (cat) genes in the liver was significantly increased on days 10 and 20 following Mn2+ exposure (4 mg/L), but was reduced on day 30. Similarly, expression of glutathione peroxidase (gpx) and glutathione reductase (gr) genes was elevated after 10 d of exposure to 2 and 4 mg/L Mn2+, but decreased after 20 and 30 d. After 30 d of exposure to high concentrations (2 and 4 mg/L) of Mn2+, liver tissue showed hepatic sinusoidal gap congestion, dilatation, cell vacuolation, and necrosis. In addition, the activities of alanine aminotransferase (ALT) and aspartate transaminase (AST) as well as 8-hydroxydeoxyguanosine (8-OHdG) levels were significantly increased after Mn2+ exposure. Moreover, Mn2+ exposure altered the expression pattern of some pivotal genes associated to apoptosis (p53, bax, bcl-2, apaf-1, caspase-9, and caspase-3), which suggested that Mn2+ exposure induces apoptosis through the mitochondrial pathway. The above results showed that excessive Mn2+ induced apoptosis and liver damage in grouper through elicitation of oxidative stress. These insights help elucidate the mechanism by which Mn2+ induces toxicity in marine fish, and provide a new perspective regarding the detrimental effects of heavy metals in fish.

Bioaccumulation of manganese and its effects on oxidative stress and immune response in juvenile groupers (Epinephelus moara ♀ × E. lanceolatus ♂)

We evaluated the effects of Mn in juvenile Yunlong groupers (Epinephelus moara ♀ × E. lanceolatus ♂). The groupers were exposed to Mn2+ (0, 0.5, 1, 2, and 4 mg/L) for 30 days after which they were assessed. The results indicate the accumulation of Mn in fish depended on dose and time. Mn2+ accumulation in tissues occurred in the following order: liver > gills > intestine > muscle. The concentrations of SOD and CAT in the fish significantly increased after 10 and 20 days of treatment with 4 mg/L Mn2+ but decreased after 30 days. Similarly, GSH and GPx levels increased after 10 days of exposure to 2 and 4 mg/L Mn2+ but decreased after 20 and 30 days of exposure. Additionally, malondialdehyde levels significantly increased after exposing the fish to 2 and 4 mg/L Mn2+ for 10, 20, and 30 days. In addition, liver HSP70 and HSP90 levels significantly increased at days 20 and 30 in all fish exposed to Mn2+. In addition, when Mn2+ concentration was 1, 2, and 4 mg/L, liver C3 and C4 levels were significantly increased after 10, 20, and 30 days. Conversely, the levels of LZM and IgM significantly decreased. Mn2+ also significantly upregulated the expression of genes associated with immunity (tlr3, tnf-α, il-1β, and il-6) in the fish, which suggests that it induces immunotoxicity by altering the immune response. Overall, the findings showed that Mn2+ can disrupt grouper health by bioaccumulating in the fish and subsequently inducing oxidative stress and immune responses. These results can help elucidate the mechanism by which manganese induces toxicity in marine fish. Additionally, they provide a new perspective regarding the detrimental effects of heavy metals in fish.

Construction of high-density linkage maps and QTL mapping for growth-related traits in F1 hybrid Yunlong grouper (Epinephelus moara♀ × E. lanceolatus♂)

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.

Comparison of the gut microbiota composition between asymptomatic and diseased Epinephelus moara ♀ × Epinephelus lanceolatus ♂ with nervous necrosis virus infection

Yunlong grouper (Epinephelus moara ♀ × Epinephelus lanceolatus ♂) is a new hybrid grouper with high economic value because it has shown some superiority in terms of growth, temperature tolerance, deformity resistance and some nutritional indicators compared with its parents. Currently, the aquaculture industry of grouper is plagued by viral diseases, and there is still no effective control method. Gut microbiomes have a significant impact on the digestion, immunity, metabolism, health and behaviours. In this study, 16S rRNA gene sequencing was performed to characterize the gut microbiota of yunlong grouper, and the differences in community composition between the asymptomatic and diseased fish naturally infected with nervous necrosis virus (NNV) were compared. A total of 3262 OTUs with ≥97% sequence similarity of 31 bacterial phyla were identified. In all grouper samples, the most abundant phylum was Proteobacteria and the most abundant genus was Blastopirellula, the predicted functions in gut bacteria were mainly involved with metabolism. There were no significant differences between two groups of yunlong grouper in α-diversity analysis. Specific bacterial taxa difference for each group from the phyla to the genus level was identified using LEfSe analysis. This is the first report of comparison of the gut microbiota between asymptomatic and diseased yunlong grouper under NNV infection. These findings provide an understanding about the interaction between intestinal microbiota and host under NNV infection, and provide targets for future probiotics screening.

Comparative transcriptome analyses reveal changes of gene expression in larvae hatched from fresh and cryopreserved kelp grouper (Epinephelus moara) embryos

For the first time in fish, this study investigated the changes of gene expression in larvae hatched from fresh and cryopreserved embryos using RNA-seq. The transcriptome of kelp grouper from 1-day larvae hatched from fresh and cryopreserved embryos were obtained by high-throughput sequencing and analyzed by KEGG and GO database. The analysis results showed that 1815 unigenes were significantly differentially expressed (1165 up-regulated vs 650 down-regulated). Significantly down-regulated genes were enriched for eye development, cranial nerve development, sensory light stimulation and neurotransmitter transport terms, predicting that dehydration/rehydration or freezing/thawing during cryopreservation may affect development of the central nervous system, including the primordia of the eye. Moreover, many down-regulated genes were involved in calcium ion regulation, which may affect cell structure, as well as cell proliferation and differentiation. Meanwhile, gene expression of some proteins that protect neurons from the toxic effects of high concentrations of extracellular neurotransmitters significantly decreased, further suggesting that cryopreservation may have an effect on the central nervous system of larvae. In addition, huge amounts of primary data provided in present paper contribute to the genome and proteome databases of the grouper. These results will help other researchers better understand the effect of cryopreservation on fish embryos and improve exploration of the molecular mechanism of fish embryo cryoinjury.

Complete mitochondrial genome of the hybrid grouper Hyporthodus septemfasciatus (♀)×Epinephelus moara (♂) (Perciformes, Serranidae) and results of a phylogenetic analysis

The complete mitochondrial genome of the hybrid grouper Hyporthodus septemfasciatus (♀)×Epinephelus moara (♂) was obtained by next-generation sequencing. The mitochondrial genome was 16,499 bp long, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region (D-loop). The overall base composition is 28.62% A, 28.27% C, 16.27% G, and 26.84% T with 55.46% A + T. In the maximum-likelihood (ML) phylogenetic analysis, the hybrid grouper belonged to the same clade as H. septemfasciatus (maternal inheritance).

The complete mitochondrial genome of the hybrid grouper Epinephelus moara (♀)×Epinephelus tukula (♂), and phylogenetic analysis in subfamily Epinephelinae

The mitochondrial genome (mitogenome) of hybrid grouper Epinephelus moara (♀)×Epinephelus tukula (♂), a new hybrid progeny, can provide valuable information for analyzing phylogeny and molecular evolution. In this study, the mitogenome was analyzed using PCR amplification and sequenced, then the phylogenetic relationship of E. moara (♀)×E. tukula (♂) and 35 other species were constructed using Maximum Likelihood and Neighbor-Joining methods with the nucleotide sequences of 13 conserved protein-coding genes (PCGs). The complete mitogenome of E. moara (♀)×E. tukula (♂) was 16 695 bp in length, which contained 13 PCGs, 2 rRNA genes, 22 tRNA genes, a replication origin and a control region. The composition and order of these genes were consistent with most other vertebrates. Of the 13 PCGs, 12 PCGs were encoded on the heavy strand, and ND6 was encoded on the light strand. The mitogenome of the E. moara (♀)×E. tukula (♂) had a higher AT nucleotide content, a positive AT-skew and a negative GC-skew. All protein initiation codons were ATG, except for COX and ND4 (GTG), ATP6 (CTG), and ND3 (ATA). ND2, COXII, ND3, ND4 and Cytb had T as the terminating codon, COXIII’s termination codon was TA, and the remaining PCGs of that were TAA. All tRNA genes, except for the lacking DHU-arm of tRNASer(AGN), were predicted to form a typical cloverleaf secondary structure. In addition, sequence similarity analysis (99% identity) and phylogenetic analysis (100% bootstrap value) indicated that the mitochondrial genome was maternally inherited. This study provides mitogenome data for studying genetic, phylogenetic relationships and breeding of grouper.

A Chromosomal-scale Reference Genome of the Kelp Grouper Epinephelus moara.

The kelp grouper, Epinephelus moara, is a carnivorous coral reef fish widely distributing in the Indo-Pacific Oceans. Compared to other grouper species, E. moara is featured for its wide tolerance to environmental stresses, such as temperature and salinity. In addition, it is popularly used as a breeding species for hybrid grouper production. Here, we report the de novo sequencing and assembly of E. moara genome using a combination of Illumina pair-ended, PacBio and Hi-C technologies. We generated a 1.08Gb genome assembly with a scaffold N50 of 44.93Mb and contig N50 of 2.22Mb. The scaffolds and contigs were clustered and oriented into 24 chromosomes, and 24,135 protein-coding genes were predicted, among which 96.8% were annotated. More than 95.6% of the conserved complete genes were successfully retrieved by BUSCO analysis. Comparative genomic analyses showed that some expanded gene families in the E. moara genome were significantly enriched in innate immune pathways. The E. moara genome provides a valuable resource for genetic improvement and genomic breeding of groupers, as well as evolutionary and comparative study with other grouper species.

Comparison of Morphological Characteristics and Its Parent Species Hybrid Grouper between a Epinephelus moara ♀ and E. lanceolatus ♂

Comparison of Morphological Characteristics and Its Parent Species Hybrid Grouper between a Epinephelus moara ♀ and E. lanceolatus ♂

Gastric evacuation and changes in postprandial blood biochemistry, digestive enzymes, and appetite-related genes in juvenile hybrid grouper (Epinephelus moara ♀ × E. lanceolatus ♂)

The proportion of remaining stomach meal (PSR), gastric evacuation model, serum cortisol and glucose levels, intestinal lipase, amylase, and chymotrypsin activities and the expression levels of neuropeptide Y (npy), orexin (ore) and leptins (lepa, lepb) of the hybrid grouper (Epinephelus moara ♀ × E. lanceolatus ♂) during gastric evacuation were evaluated to determine the optimal feeding interval. Results showed that PSR significantly decreased by more than 60%, 70%, and 90% at postprandial 6 h, 9 h and 12 h, respectively. The cubic root curve was the most supported model and fitted all PSR data. The cortisol and glucose levels were significantly varied, and the highest values were obtained at postprandial 6 h. Cortisol and the glucose levels then gradually decreased from postprandial 9 h to postprandial 12 h. Intestinal lipase and chymotrypsin activities showed similar results to cortisol, whereas the amylase activity was not significantly different during gastric evacuation. The hypothalamic npy and ore mRNA levels were gradually up-regulated within postprandial 6 h, and then significantly down-regulated from postprandial 9 to postprandial 12 h. By contrast, hepatic lepa and lepb mRNA levels remained high levels within postprandial 6 h and significantly decreased from postprandial 9 to postprandial 12 h. These results indicated that food digestion in the intestine peaked at postprandial 6 h. Afterward, appetite returned and fully recovered at about postprandial 12 h. Therefore, on the basis of the aforementioned results and considering the fish management, postprandial 12 h is the optimal feeding interval of hybrid grouper (Epinephelus moara ♀ × E. lanceolatus ♂). This feeding interval offers potential savings in time and labor spent in cultivating hybrid grouper in captivity.

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.

Photodegradation Elevated the Toxicity of Polystyrene Microplastics to Grouper (Epinephelus moara) through Disrupting Hepatic Lipid Homeostasis

Microplastics (MPs) have caused increasing global concerns due to their detrimental effects on marine ecosystems. However, the role of photodegradation in altering toxicity of MPs to marine organisms is poorly understood. We therefore investigated the photolytic transformation of pristine polystyrene fragments (P-PS) by 60-day ultraviolet (UV) irradiation, and compared the toxicity of P-PS, photodegraded PS (PD-PS), and commercially available polystyrene microbeads (C-PS) to juvenile grouper (Epinephelus moara). Photodegradation reduced the size from ∼55.9 μm of P-PS to ∼38.6 μm of PD-PS, even produced nanoparticles (∼75 nm) with a yield of 7.03 ± 0.37% (w/w), and induced surface oxidation and formation of persistent free radicals (e.g., CO•, COO•). Also, endogenous pollutants (chemical additives and polymer fragments) were leached out. Thus, PD-PS had the highest growth inhibition and lipidosis-driven hepatic lesions of grouper, followed by P-PS and C-PS, which was mainly explained by increased hepatic bioaccumulation of MPs/NPs and released endogenous toxicants. Furthermore, oxidative stress-triggered mitochondrial depolarization, suppression of fatty acid oxidation and transport, and promotion of inflammation were identified as the key mechanisms for the enhanced hepatotoxicity after photodegradation. This work provides new insight into the potential hazard and harm of MPs in marine environments after photodegradation.

Comparison of the intestinal microbiota composition and function in healthy and diseased Yunlong Grouper

Maintaining stabilization of the intestinal microbiota is important in preventing bacterial diseases in cultured fish. At present, there have been no reports on the composition and functional analysis of intestinal microbiota in Yunlong Grouper (Epinephelus moara♀ × Epinephelus lanceolatus♂). In this study we analyzed and compared the intestinal microbiota composition of healthy and diseased pond-reared fish to discern the functional profile of a healthy status. The richness and diversity of the intestinal microbiota did not differ significantly between diseased and healthy fish, yet the abundance of predominant phyla like the Proteobacteria were upregulated in the diseased Yunlong Grouper. At the genus level, a significant reduction of Cetobacterium was observed in the intestinal tracts of diseased fish, as Pseudomonas became the most dominant bacterium. To compare the intestinal microorganism abundances between the two health groups of fish, we first screened the gut bacteria and discerned 4 phyla and 12 genera to designate a healthy status in Yunlong Grouper. The environmental bacterial community influenced composition of the intestinal microbiota in Yunlong Grouper, and the intestinal microbiota of diseased fish was more susceptible to the influence of the culture water. In addition, the prediction of functional genes by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) indicated that the intestinal microbiota of Yunlong Grouper is related mainly to the terms “metabolism, environmental information processing, genetic information processing, human diseases, and cellular processing; moreover, the functions of the intestinal microbiota differed between the different health states of this fish. The overall results indicate that the occurrence of disease can affect the composition and function of the intestinal microbiota in a cultured fish.

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.

Dietary protein requirement of juvenile kelp grouper ( Epinephelus moara )

Dietary protein requirement of juvenile kelp grouper Epinephelus moara was investigated through a feeding trial. Experimental diets with graded crude protein (CP) levels (33.01%, 38.54%, 45.21%, 50.71%, 56.10% and 63.09% of dry matter respectively) were formulated. Six triplicate groups of fish (20 individuals per replicate with initial mean weight 6.00 g) were fed with each diet for 8 weeks. Best growth performance of fish was detected in 56.10% CP diet. The specific growth rate (SGR) significantly elevated with increasing dietary CP level to 50.71%, but there was no significant difference thereafter (p < .05). The feed conversion ratio (FCR) decreased significantly with dietary CP levels from 33.01% to 56.10% (p < .05). Glucose (GLU) and total protein (TP) concentrations in plasma had an increasing trend with dietary protein increasing. In the 33.01% CP group, plasma triglyceride (TG) content was significantly higher (1.67 mmol/L) than that in other dietary treatments (0.65–1.14 mmol/L). The lowest alanine transaminase (ALT) activity was observed in the 56.10% CP group (163.16 U/L). Crude lipid content in the muscle and liver was significantly elevated with increasing dietary protein levels (p < .05). The glycogen content in the liver decreased significantly as CP levels increased (p < .05). The fish fed diet with higher CP level (56.10% and 63.09%) had significantly higher energy retention (ER) and lipid retention (LR) than other treatments. Based on the broken-line regression analysis of SGR and FCR, the optimal dietary protein requirement for juvenile kelp grouper is 54.61%–56.22%.

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.

Time-series transcriptomic analysis of the kelp grouper Epinephelus moara in response to low salinity stress

ABSTRACTThe Kelp grouper Epinephelus moara is one of the most widely consumed and economically important marine fish in China. The species can tolerate a wide range of salinity, but genomic resources are not available, and the molecular mechanisms underlying adaptation to salinity at the transcriptomic level remain largely unclear. In this study, the transcriptomic responses of the liver of E. moara under low salinity were investigated using the Illumina digital gene expression system. After de novo assembly, 499,356 transcripts were generated and contributed 445,068 unigenes. A total of 14, 19, 33 and 3101 genes were differentially expressed following exposure to low salinity stress for 2, 6, 24 and 48 h, respectively. Only two genes were differentially expressed in all groups. Four genes related to metabolism and ambient salinity adaption were randomly selected to validate the differentially expressed genes (DEGs) by real-time PCR. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyse the functional significance of DEGs, including those responding to salinity through diverse biological processes, cellular components, molecular functions, and pathways associated with metabolic and osmotic responses. This work provides new insight into the response to salinity challenges in E. moara, and the findings expand our knowledge of the molecular basis of metabolic regulation mechanisms in this species. Additionally, the transcriptional data provide a valuable resource for future molecular and genetic studies on E. moara.

Effect of vitrification solutions on survival rate of cryopreserved Epinephelus moara embryos

Embryo cryopreservation is important for long-term preservation of germplasm and assisted reproduction. However, it is still very difficult to obtain viable embryos from cryopreserved fish embryos. In this study, embryos of Epinephelus moara were used to investigate the effects of various cryopreservation methods. Embryos in stages 10 pairs somite (10S), 18 pairs somite (18S), 22 pairs somite (22S), tail-bud (TB), embryo twitching (ET) and pre-hatch (PH) were treated with five-step equilibrium penetration in 40% PMG3T vitrification solution, which contained 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Trehalose. We found that 18S, 22S, TB and ET stage embryos had higher survival rates and were more tolerant to the vitrification solution. Five-step equilibrium treatments on the embryos at the tail-bud stage were performed using two vitrification solutions: 40% PMG3T and 40% PMG3S, which consisted of 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Sucrose. The embryonic survival rate under PMG3S treatment (63.36%) was significantly higher than PMG3T treatment (43.93%) (P < 0.05). PMG3S and PMG3T with concentrations of 35%, 40% and 45% were tested on tail-bud stage embryos. Higher concentration of the vitrification solution led to significantly lower embryonic survival rate (P < 0.05). The survival rate was 36.79–72.05% in PMG3S, and 37.11–55.18% in PMG3T, and there were non-significant differences in embryonic development and malformation rates among the groups treated with different concentrations. The embryonic normal development rates in PMG3S and PMG3T were 21.27% and 11.04%, and the malformation rates were 36.13% and 31.04%, respectively. The optimum treatment condition was 40 min using 40% PMG3S on embryos at the tail-bud stage. Both PMG3S and PMG3T were used for cryopreserving embryos at 16 pairs somite, tail-bud and ET stage in liquid nitrogen, where we obtained 190 surviving embryos, and 44 fishes underwent normal development and hatched. The survival rate of cryopreserved embryos was 5.15%, the normal development rate was 1.31%, and the malformation rate was 3.66%. We found that PMG3S and PMG3T were effective for cryopreservation of Epinephelus moara embryos. The results provide a foundation for further explorations of fish embryo cryopreservation techniques.