Genomic structures of insulin-like growth factor from golden pompano (Trachinotus ovatus) and their expression responses to the feed types.

Golden pompano is an important aquaculture product in the coastal regions of southern China, which is highly dependent on insulin-like growth factor (IGF) for various biological processes. The cDNAs of ToIGF1, ToIGF2, and ToIGF3 are 1718 bp, 1658 bp, and 2272 bp in length, respectively, with corresponding amino acid sequences of 185 aa, 215 aa, and 194 aa. These sequences consist of 5 parts, including the signal peptide, the B domain, the C domain, the A domain, the D domain, and the E domain, which are also found in other species. While ToIGF1 has no SSR polymorphism, ToIGF2 and ToIGF3 have 3 and 1 SSR polymorphism sites, respectively. In terms of tissue expression, ToIGF1 is predominantly expressed in the liver, ToIGF2 shows its highest expression in the gills, and ToIGF3 also shows its highest expression in the gills, but no expression in the liver and spleen. These tissue distribution results suggest that ToIGFs are not only present in growth-related tissues such as the brain, muscle, and liver, but also in reproductive tissues, tissues that regulate osmotic pressure, and tissues related to food intake. This observation is consistent with other bony fish species and highlights the extensive biological functions of ToIGFs that need to be further explored and exploited. In addition, the expression levels of ToIGFs were found to be different in the different dietary groups, including the pelleted food group, the frozen squid group, and the frozen fish group. In the pelleted diet group, ToIGF1 and ToIGF2 were highly expressed in the liver and intestinal tissues, followed by the frozen fish group. These results suggest that the type of diet can affect the body's energy metabolism by influencing tissue expression of growth-related genes, which in turn affects individual growth.

Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of -415 to +192 bp and -311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.

Genome-wide identification of heat shock protein gene family and their responses to pathogen challenge in Trachinotus ovatus.

Heat Shock Proteins (HSPs) are a widely distributed family of proteins produced in response to heat and other stresses. To develop a deeper understanding of the mechanisms governing expression of HSPs in the bony fish Trachinotus ovatus, we carried out a whole genome analysis and identified 43 HSP genes. Based on their phylogenetic relationships with Danio rerio, Seriola dumerili, and Seriola lalandi, they were divided into four subfamilies: HSP20, HSP60, HSP70, and HSP90. We performed an analysis of the predicted physicochemical properties and subcellular localization of proteins encoded by these genes. The chromosomal localization results showed that the HSP genes are distributed across 20 chromosomes of T. ovatus.These genes were found to be expressed in different tissues, and they showed differential expression in the immune response against Streptococcus agalactiae. However, there was no significant differential expression in the different skin tissue locations of T. ovatus after infection by Cryptocaryon irritans Brown. This study provides basic information for further research on the evolution and structure and function of HSPs in teleosts.

Identification of the NOD-like receptor family of golden pompano and expression in response to bacterial and parasitic exposure reveal its key role in innate immunity.

This study presents a genome-wide identification of NOD-like receptors (NLRs) in the golden pompano, key to its innate immunity. We identified 30 ToNLRs, analyzing their chromosomal positions, characteristics, evolutionary relationships, evidence of positive selection, and synteny with the yellowtail kingfish. Our findings categorize these NLRs into three main subgroups: NLRA, NLRC, and the distinct ToNLRX1. Post-exposure to Streptococcus agalactiae, most ToNLRs increased expression in the spleen, whereas NLRC3like13, NLRC3like16, and NLRC3like19 so in the kidneys. Upon Cryptocaryon irritans exposure, we categorized our groups based on the site of infection into the control group (BFS), the trophont-attached skin (TAS), and the nearby region skin (NRS). ToAPAF1 and ToNOD1 expressions rose in the NRS, in contrast to decreased expressions of ToNLRC5, ToNWD1 and ToCIITA. Other ToNLRs showed variable expressions in the TAS. Overall, this research lays the groundwork for further exploration of innate immunity in the golden pompano.

Genome-Wide Identification of Trachinotus ovatus Antimicrobial Peptides and Their Immune Response against Two Pathogen Challenges.

Golden pompano, Trachinotus ovatus, as a highly nutritious commercially valuable marine fish, has become one of the preferred species for many fish farmers due to its rapid growth, wide adaptability, and ease of feeding and management. However, with the expansion of aquaculture scale, bacterial and parasitic diseases have also become major threats to the golden pompano industry. This study, based on comparative genomics, shows the possibility of preferential evolution of freshwater fish over marine fish by analyzing the phylogenetic relationships and divergence times of 14 marine fish and freshwater fish. Furthermore, we identified antimicrobial peptide genes from 14 species at the genomic level and found that the number of putative antimicrobial peptides may be related to species evolution. Subsequently, we classified the 341 identified AMPs from golden pompano into 38 categories based on the classification provided by the APD3. Among them, TCP represented the highest proportion, accounting for 23.2% of the total, followed by scolopendin, lectin, chemokine, BPTI, and histone-derived peptides. At the same time, the distribution of AMPs in chromosomes varied with type, and covariance analysis showed the frequency of its repeat events. Enrichment analysis and PPI indicated that AMP was mainly concentrated in pathways associated with disease immunity. In addition, our transcriptomic data measured the expression of putative AMPs of golden pompano in 12 normal tissues, as well as in the liver, spleen, and kidney infected with Streptococcus agalactiae and skin infected with Cryptocaryon irritans. As the infection with S. agalactiae and C. irritans progressed, we observed tissue specificity in the number and types of responsive AMPs. Positive selection of AMP genes may participate in the immune response through the MAPK signaling pathway. The genome-wide identification of antimicrobial peptides in the golden pompano provided a complete database of potential AMPs that can contribute to further understanding the immune mechanisms in pathogens. AMPs were expected to replace traditional antibiotics and be developed into targeted drugs against specific bacterial and parasitic pathogens for more precise and effective treatment to improve aquaculture production.

Transcriptomics Reveal the Effects of Breeding Temperature on Growth and Metabolism in the Early Developmental Stage of Platax teira.

The growth, development, and survival of fish, especially in the early stages of development, is influenced by a complex of environmental factors, among which temperature is one of the most important. Although the physiological effects of environmental stress in fish have been extensively studied, the molecular mechanisms are poorly understood. However, recent advances in transcriptomic techniques have facilitated the study of the molecular mechanisms of environmental stress responses in aquatic species. Here, we aimed to elucidate the effects of breeding temperatures (21, 24, 27, and 30 °C) on the growth and nutrient metabolism in the early developmental stage of Platax teira, using transcriptomic techniques. Transcriptomic analysis identified 5492, 6937, and 4246 differentially expressed genes (DEGs) in the 21 vs. 24 °C, 27 vs. 24 °C, and 30 vs. 24 °C comparisons, respectively, most of which were involved in cell processes, single organism, metabolism, catalytic activity, and cell part, based on gene ontology (GO) functional annotations. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were mainly enriched in pathways related to metabolism of matter and energy, protein digestion and absorption, and glucose and lipid metabolism. Additionally, the expression of genes related to energy, lipid, and glucose metabolism in the fish liver was upregulated under a low-temperature condition (21 °C), although increasing the temperature within the acceptable threshold improved nutrient metabolism and growth in the fish. Meanwhile, nutrient metabolism and growth were suppressed by an extremely high temperature (30 °C) owing to oxidative stress. Overall, it was shown that nutrient metabolism pathways were involved in thermal stress responses in P. teira, and the optimal breeding temperature range was 24-27 °C. Through transcriptomics, the regulatory mechanism of larval development in P. teira under different growth temperatures was elucidated, with the goal of establishing a theoretical basis for industrial breeding.

The regulatory mechanisms of IRF7 mediated by the type I IFN signalling pathway against Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782).

Interferon regulatory factor 7 (IRF7) regulates type I interferon (IFN) genes via combining to the ISRE region in the immune response against bacteria. Streptococcus iniae is one of the dominant pathogenic bacteria of yellowfin seabream, Acanthopagrus latus. However, the regulatory mechanisms of A. latus IRF7 (AlIRF7) mediated by the type I IFN signalling pathway against S. iniae was ambiguously. In the present study, IRF7, and two IFNa3s (IFNa3 and IFNa3-like) were authenticated from A. latus. The total length of AlIRF7 cDNA is 2142 bp, containing a 1314 bp open reading frame (ORF) encoding an inferred 437 amino acids (aa). Three typical regions, a serine-rich domain (SRD), a DNA-binding domain (DBD), and an IRF association domain (IAD), are conserved in AlIRF7. Furthermore, AlIRF7 is fundamentally expressed in various kinds of organs, with high levels in the spleen and liver. Additionally, S. iniae challenge promoted AlIRF7 expression in the spleen, liver, kidney, and brain. AlIRF7 is confirmed to be located at the nucleus and cytoplasm by overexpression of AlIRF7. Moreover, truncation mutation analyses shows that the regions, -821 bp to +192 bp and -928 bp to +196 bp, were known as core promoters from AlIFNa3 and AlIFNa3-like, respectively. The point mutation analyses and electrophoretic mobile shift assay (EMSA) verified that AlIFNa3 and AlIFNa3-like transcriptions are depended on the M2/5 and M2/3/4 binding sites with AlIRF7 regulation, respectively. Additionally, an overexpression experiment showed that AlIRF7 can dramatically decrease the mRNA levels of two AlIFNa3s and interferon signalling molecules. These results suggest that two IFNa3s may mediate the regulation of AlIRF7 in the immune responses of A. latus against S. iniae infection.

Cryopreservation of black seabream (Acanthopagrus schlegelii) sperm.

In recent years, biotechnology has had a significant impact on the aquaculture industry, particularly in the field of breeding. Molecular selection breeding has emerged as a novel approach to breeding. Reducing the cost of genetic information for individuals with desirable traits after breeding has become an important research direction. Cryopreservation technology allows bypassing time and space constraints in genetic breeding, simplifying broodstock management. This study presents a detailed cryopreservation method for black seabream sperm, evaluating extender type, glucose concentration, cryoprotectant type and concentration, sperm-dilution ratio, and cooling protocols. Sperm motility parameters were analyzed using computer-assisted sperm analysis (CASA) before and after two days of freezing. This involved using an RS solution with a glucose concentration of 15 g/L and adding a 5% final concentration of EG as the sperm cryoprotectant. After mixing the sperm and solution at a ratio of 1:2, we subjected it to 5 min fumigation at 5 cm above the liquid nitrogen surface before plunging it into the nitrogen. Sperm motility reached 85.46 ± 7.32% after two days. Various enzymatic activities showed changes over 20 days post-cryopreservation. This improved cryopreservation protocol for black seabream sperm is beneficial for genetic breeding and reproduction and provides reference for studying the cryodamage mechanisms of black seabream sperm.

Molecular characterization of TNF-ß and IFN-γ in yellowfin seabream (Acanthopagrus latus, Hottuyn, 1782) and their immune responses to density stress during transport.

The inflammatory cytokines TNF-ß and IFN-γ are important mediators of the vertebrate inflammatory response and coordinators of the immune system in regard to NF-κB signalling pathways. In this study, the TNF-ß and IFN-γ genes of yellowfin seabream, Acanthopagrus latus were identified, and the multiple sequence alignments, evolutionary relationships and gene expressions of the two genes were also determined. AlTNF-ß contained a 762 bp open reading frame (ORF) encoding 253 amino acids, while AlIFN-γ contained a 582 bp ORF encoding 193 amino acids. An amino-acid sequence alignment analysis showed that these proteins have highly conserved transmembrane structural domains among teleosts. Moreover, AlTNF-ß has a close affinity with TNF-ß of yellowfin seabream while AlIFN-γ has a high evolutionary correlation with A. regius and Sparus aurata. In addition, the mRNAs of AlTNF-ß and AlIFN-γ are widely expressed in various tissues. AlTNF-ß is highly expressed in gill and intestinal tissues, and the mRNA levels of AlIFN-γ are higher in spleen, skin, and gill tissues than in other tissues. Under transportation density stress, the mRNA level of AlTNF-ß was significantly elevated in the intestine of the high-density group, while AlTNF-ß transcription in the gills did not vary significantly among the density groups. Furthermore, AlIFN-γ expression was increased in liver, intestinal, and gill tissues under high transportation density. The results of this study show that TNF-ß and IFN-γ expression in yellowfin seabream is greatly affected by density stress. The density of 125 per bag for 4-5 cm fry or 1200 per bag for 1-2 cm fry is most suitable for the transportation of live fish. These results might provide a reference for further studies on the immunomodulatory response process and auxiliary function of immune stress of TNF and IFN genes in fish under density stress.

Characterization of the MMP9 Gene and Its Association with Cryptocaryon irritans Resistance Traits in Trachinotus ovatus (Linnaeus, 1758).

The MMPs are endogenous proteolytic enzymes that require zinc and calcium as cofactors. MMP9 is one of the most complex matrix metalloproteinases in the gelatinase family and has many biological functions. In mammals, mmp9 is thought to be closely associated with cancer. However, studies in fish have rarely been reported. In this study, to understand the expression pattern of the ToMMP9 gene and its association with the resistance of Trachinotus ovatus to Cryptocaryon irritans, the sequence of the MMP9 gene was obtained from the genome database. The expression profiles were measured by qRT-PCR, the SNPs were screened by direct sequencing, and genotyping was performed. The ToMMP9 gene contained a 2058 bp ORF encoding a putative amino acid sequence of 685 residues. The homology of the ToMMP9 in teleosts was more than 85%, and the genome structure of ToMMP9 was conserved in chordates. The ToMMP9 gene was expressed in different tissues of healthy individuals and was highly expressed in the fin, the gill, the liver and the skin tissues. The ToMMP9 expression in the skin of the infected site and its adjacent sites increased significantly after C. irritans infection. Two SNPs were identified in the ToMMP9 gene, and the SNP (+400A/G) located in the first intron was found to be significantly associated with the susceptibility/resistance to C. irritans. These findings suggest that ToMMP9 may play an important role in the immune response of T. ovatus against C. irritans.

Characteristics of microplastic pollution in golden pompano (Trachinotus ovatus) aquaculture areas and the relationship between colonized-microbiota on microplastics and intestinal microflora.

Microplastic (MPs) pollution is a global marine environmental problem. The effects of MPs on the gut microbiota of aquatic organisms have received considerable attention. For example, microbes colonizing MPs in pond cultures alter the structure and function of the intestinal microbes of shrimp and fish. It was hypothesized that bacteria on MPs in natural mariculture areas also interact with the intestinal flora of golden pompano (Trachinotus ovatus) because biofilms can form on the surface of MPs during long-term floating in seawater. To our knowledge, this study is the first to investigate MPs pollution in T. ovatus aquaculture. DNA sequencing and bioinformatics analysis confirmed the effect of microbial colonization of MPs on the intestinal flora of T. ovatus. The MPs detected in the gut wet weight (w.w.) of golden pompano (546 ± 52 items/g) were mainly pellets and fragments of blue or green, whereas the sediment MPs dry weight (d.w.) (4765 ± 116 items/kg) were mainly black fibers. The MPs richness in the sediment gradually increased from the open-sea aquaculture area to the estuarine aquaculture area and was positively correlated with the MPs richness in the intestinal tract of golden pompano. MPs 20-200 µm were the most common in the gut and sediment. The intake of MPs increased the abundance of Proteobacteria and decreased that of Firmicutes in the intestinal flora. The functional compositions of MP-colonizing microbes and gut microbiota were similar, suggesting that the two communities influence each other. Network analysis further confirmed this and revealed that Vibrio plays a key role in the intestinal flora and surface microorganisms of MPs. Overall, the intake of MPs by aquatic animals not only affects the intestinal flora and intestinal microbial function, but also poses potential risks to aquaculture.

Effects of cysteine addition to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus).

As the precursor of taurine, cysteine serves physiological functions, such as anti-oxidative stress and immune improvement. Investigation of cysteine and its derivatives has made positive progress in avian and mammalian species, yet the study and application of cysteine in aquatic animals are relatively rare. Therefore, we evaluated the effects of supplementing a low-fishmeal diet with various levels of cysteine on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae of the juvenile golden pompano (Trachinotus ovatus). According to our study, exogenous supplementation with 0.6-1.2% cysteine greatly increased the final body weight (FBW) and specific growth rate (SGR) of golden pompano compared to the control group. Under the present conditions, the optimum dietary cysteine supplementation level for golden pompano was 0.91% based on the polynomial regression analysis of SGR. Meanwhile, we found that the Nrf2/Keap1/HO-1 signaling pathway was notably upregulated with the increase of exogenous cysteine, which increased antioxidant enzyme activity in serum and gene expression in the intestine and reduced the level of reactive oxygen species (ROS) in the serum of golden pompano. In addition, morphological analysis of the midgut demonstrated that exogenous cysteine improved muscle thickness and villi length, which suggested that the physical barrier of the intestine was greatly strengthened by cysteine. Moreover, cysteine increased the diversity and relative abundance of the intestinal flora of golden pompano. Cysteine suppressed intestinal NF-κB/IKK/IκB signaling and pro-inflammatory cytokine mRNA levels. Conversely, intestinal anti-inflammatory cytokine gene expression and serum immune parameters were upregulated with the supplementary volume of cysteine and improved intestine immunity. Further, exogenous cysteine supplementation greatly reduced the mortality rate of golden pompano challenged with S. agalactiae. In general, our findings provide more valuable information and new insights into the rational use of cysteine in the culture of healthy aquatic animals.

Effects of exogenous taurine supplementation on the growth, antioxidant capacity, intestine immunity, and resistance against Streptococcus agalactiae in juvenile golden pompano (Trachinotus ovatus) fed with a low-fishmeal diet.

Taurine has various biological functions in fish, playing an essential role in growth, resistance to oxidative stress, and intestine immunity. Here, we evaluated the effects of exogenous taurine added to low-fishmeal diets on the growth, anti-oxidative stress, intestine immunity, and Streptococcus agalactiae resistance in juvenile golden pompano (Trachinotus ovatus). Our study showed that exogenous taurine supplementation of 1.2% (T3 group) greatly enhanced the weight gain rate and specific growth rate (SGR) of juvenile golden pompano, significantly upregulating growth-related factor expression in the brain and liver, as well as the levels of growth-related parameters in the serum. Polynomial regression analysis using SGR estimated the optimal dietary taurine level for golden pompano at 1.18%. Moderate exogenous taurine also increased the muscular thickness and villus length within the intestine, maintained intestinal physical barrier stability, activated the Nrf2/Keap-1/HO-1 signaling pathway, increased intestinal antioxidant enzyme gene expression and antioxidant enzyme activity in the serum, and upregulated immunoglobulin and complement levels in parallel with declining reactive oxygen species (ROS) levels in the serum. Antioxidant factor expression was also upregulated in the intestine. Furthermore, supplementation suppressed NF-κB signaling and intestinal pro-inflammatory cytokine gene expression, increased anti-inflammatory cytokine gene expression, and improved intestine immunity. Finally, taurine supplementation improved the survival rate of golden pompano challenged with S. agalactiae. Overall, our findings provide additional information and support for the rational use of taurine in healthy aquatic animal farming.

Detection and identification of marine fish mislabeling in Guangzhou's supermarkets and sushi restaurants using DNA barcoding.

In this study, DNA barcoding was applied to identify the distinct species of fish products in Guangzhou supermarkets and sushi restaurants in order to confirm whether products were correctly labeled. Samples were analyzed using mitochondrial cytochrome C oxidase subunit I (CO I) gene as the target. Our results showed that the CO I gene of all 139 samples examined was successfully amplified by PCR. When sequenced, 30 samples (21.58%) were mislabeled as the wrong species, 11 samples had insufficient information provided on the label to determine if the labeling was correct (7.91%), and four samples failed sequencing (2.88%). We also found that the use of proper labels for fish products in sushi restaurants was higher than that in supermarkets. As a simple, rapid, and efficient technology, DNA barcoding can be widely used for species identification of fish products. Our work shows that regulation of the labeling of fish products, as we evaluated in Guangzhou and other markets in China, is needed on a global scale.

Effects of acute ammonia exposure and recovery on the antioxidant response and expression of genes in the Nrf2-Keap1 signaling pathway in the juvenile golden pompano (Trachinotus ovatus).

Continuous exposure to high levels of ammonia can cause oxidative damage to fish tissues and organs. To date, the mechanism by which juvenile golden pompano (Trachinotus ovatus) are poisoned by ammonia exposure has not been thoroughly elucidated. although the mechanisms of ammonia toxicity are not well described for the pompano, many other studies presented these effects to other fish species. So an overview would be given. First, an acute ammonia nitrogen toxicity experiment on juvenile golden pompano obtained a 96-h half-lethal concentration (96 h LC50) of 26.9 mg/L. In the ammonia exposure experiment, fish were sampled at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h after exposure to ammonia water (26.93 mg/L). The results showed that with the prolonged ammonia nitrogen exposure, plasma cortisol (COR), total cholesterol (TC), glutamic-pyruvic transaminase (ALT), glutamic oxalacetic transaminase (AST) and malonaldehyde (MDA) levels continued to rise, while glucose (GLU) levels first increased and later gradually decreased after 12 h. The activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in the liver and the mRNA expression levels of antioxidant genes (SOD, CAT, and GPX) first increased and subsequently decreased with increasing exposure time. Through microscopic observation, it was found that the degree of liver damage increased with increasing stress time and was most serious at 96 h. In the post-poison recovery experiment, the fish exposed to ammonia were transferred to clean water, and samples were taken at 24 h, 48 h, 72 h and 96 h after recovery. The results showed that with the increasing recovery time, each index recovered to the initial level to varying degrees, but the recovery time of 96 h was not enough for the fish to return to the normal level. We also examined the regulation of the Nrf2-Keap1 signaling pathway by the molecular mechanism of the antioxidant defense system. The results of this analysis showed that there was a positive correlation between Nrf2 and liver antioxidant gene expression levels, while there was a negative correlation between Keap1 and liver antioxidant gene expression levels, which may be observed because Nrf2 plays a key role in inducing antioxidant genes, and Keap1 may hinder the response to Nrf2. These results may provide a deeper and more comprehensive understanding of the impact of ammonia exposure on fish and help to provide a foundation for managing the healthy reproduction of juvenile fish.

Gill oxidative damage caused by acute ammonia stress was reduced through the HIF-1α/NF-κb signaling pathway in golden pompano (Trachinotus ovatus).

This study aimed to investigate the intoxication mechanism of golden pompano (Trachinotus ovatus) exposed to high ammonia levels and the effects on the immune and antioxidant mechanisms of gills. Juvenile golden pompano was exposed to ammonia (total ammonia: 26.9 mg/L) to induce 96 h of ammonia stress, and a 96 h recovery experiment was performed after poisoning. Then, we evaluated hematological parameters, the histological structure and the expression of related genes. In this experiment, continuous exposure to high levels of ammonia led to a significant increase in plasma alkaline phosphatase (ALP), acid phosphatase (ACP) and lactate dehydrogenase (LDH) levels (P < 0.05), and the levels of triiodothyronine (T3) and tetraiodothyronine (T4) were significantly reduced (P < 0.05). Moreover, the expression of antioxidant genes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) increased (P < 0.05). These results indicate that ammonia activates the active osmotic regulatory mechanism of fish gills and participates in defense and immune responses. However, with prolonged exposure to ammonia, the balance of the defense system is disrupted, leading to oxidative damage and inflammation of the gill tissue. This research not only helps elucidate the intoxication mechanism of golden pompano by ammonia at the molecular level but also provides a theoretical basis for further research on detoxification mechanisms.

A chromosome-level genome assembly of the yellowfin seabream (Acanthopagrus latus; Hottuyn, 1782) provides insights into its osmoregulation and sex reversal.

The yellowfin seabream Acanthopagrus latus is the economically most important Sparidae fish in the northern South China Sea. As euryhaline fish, they are perfect model for investigating osmoregulatory mechanisms in teleosts. Moreover, the reproductive biology of hermaphrodites has long been intriguing; however, little information is known about the molecular pathways underlying their sex change. Here, we report a chromosome level reference genome of A. latus generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The draft genome of yellowfin seabream was 806 Mb, with 732 Mb scaffolds anchored on 24 chromosomes. The contig N50 and scaffold N50 were 2.6 Mb and 30.17 Mb, respectively. The assembly is of high integrity and includes 92.23% universal single-copy orthologues based on benchmarking universal single-copy orthologs (BUSCO) analysis. A total of 19,631 protein-coding genes were functionally annotated in the reference genome. Moreover, ARRDC3 and GSTA gene families which related to osmoregulation underwent an extensive expansion in two euryhaline sparids fish genomes compared to other teleost genomes. Moreover, integrating sex-specific transcriptome analyses, several genes related to the transforming growth factor beta (TGF-ß) signalling pathway involved in sex differentiation and development. This genomic resource will not only be valuable for studying the osmoregulatory mechanisms in estuarine fish and sex determination in hermaphrodite vertebrate species, but also provide useful genomic tools for facilitating breeding of the yellowfin seabream.

Functional characterization, tissue distribution and nutritional regulation of the Elovl4 gene in golden pompano, Trachinotus ovatus (Linnaeus, 1758).

The elongases of very long-chain fatty acids (Elovls) are involved in the rate-limiting of the carbon chain elongation reaction in fatty acid (FA) biosynthesis in vertebrates. One member of the Elovls family, Elovl4, has been regarded as a critical enzyme involved in the biosynthesis pathway of polyunsaturated fatty acids (PUFAs). To explore the role of Elovl4 in PUFA synthesis in Trachinotus ovatus, the cDNA of the Elovl4b gene is cloned from T. ovatus (ToElovl4b). The ORF of ToElovl4b was 918 bp and encoded 305 amino acid (aa) protein sequences. Sequence alignment showed that the deduced amino acids contained significant structural features of the Elovl4 family, such as a histidine box motif (HXXHH), multiple transmembrane domains and an endoplasmic reticulum (ER) retention signal. Moreover, phylogenetic analysis revealed that ToElovl4b was highly conserved with that of Rachycentron canadum Elovl4b. Moreover, heterologous expression in yeast demonstrated that ToElovl4b could efficiently elongate 18:2n-6, 18:3n-6 and 20:5n-3 FAs up to 20:2n-6, 20:3n-6 and 22:5n-3, respectively. Furthermore, the tissue expression profile indicated that mRNA expression of ToElovl4b was higher in the gonads and brain than in other tissues. Additionally, nutritional regulation suggested the highest mRNA levels of ToElovl4b in liver and brain were under feeding with 1:1 FO-SO (fish oil, FO; soybean oil, SO) and 1:1 FO-CO (corn oil, CO)), respectively. These new insights were useful for understanding the molecular basis and regulation of LC-PUFA biosynthesis in fish.

Toll-Like Receptor 5 of Golden Pompano Trachinotus ovatus (Linnaeus 1758): Characterization, Promoter Activity and Functional Analysis.

Toll-like receptors (TLRs), as important pattern recognition receptors, represent a significant component of fish immune systems and play an important role in resisting the invasion of pathogenic microorganisms. The TLR5 subfamily contains two types of TLR5, the membrane form of TLR5 (TLR5M) and the soluble form of TLR5 (TLR5S), whose detailed functions have not been completely elucidated. In the present study, we first identified two genes, TLR5M (ToTLR5M) and TLR5S (ToTLR5S), from golden pompano (Trachinotus ovatus). The full-length ToTLR5M and ToTLR5S cDNA are 3644 bp and 2329 bp, respectively, comprising an open reading frame (ORF) of 2673 bp, encoding 890 amino acids, and an ORF of 1935 bp, encoding 644 amino acids. Both the ToTLR5s possess representative TLR domains; however, only ToTLR5M has transmembrane and intracellular TIR domains. Moreover, the transcription of two ToTLR5s was significantly upregulated after stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and flagellin in both immune-related tissues (liver, intestine, blood, kidney, and skin) and nonimmune-related tissue (muscle). Furthermore, the results of bioinformatic and promoter analysis show that the transcription factors GATA-1 (GATA Binding Protein 1), C/EBPalpha (CCAAT Enhancer Binding Protein Alpha), and ICSBP (Interferon (IFN) consensus sequence binding protein) may play a positive role in moderating the expression of two ToTLR5s. Overexpression of ToTLR5M and ToTLR5S notably increases NF-κB (nuclear factor kappa-B) activity. Additionally, the binding assay revealed that two rToTLR5s can bind specifically to bacteria and pathogen-associated molecular patterns (PAMPs) containing Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, Escherichia coli, Photobacterium damselae, Staphylococcus aureus, Aeromonas hydrophila, LPS, poly(I:C), flagellin, and peptidoglycan (PGN). In conclusion, the present study may help to elucidate the function of ToTLR5M/S and clarify their possible roles in the fish immune response to bacterial infection.

Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) from golden pompano Trachinotus ovatus (Linnaeus 1758).

The liver-expressed antimicrobial peptide-2 (LEAP-2) is an important component of the innate immune defense system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, LEAP-2 from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length LEAP-2 cDNA was 1758 bp, which comprised a 5'-UTR of 250 bp, an ORF of 321 bp, and a 3'-UTR of 1187 bp, encoding 106 amino acids. LEAP-2 consisted of a conserved saposin B domain and four conserved cysteines that formed two pairs of disulphide bonds. The genomic organization of LEAP-2 was also determined and shown to consisted of three introns and two exons. The predicted promoter region of ToLEAP-2 contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that LEAP-2 was ubiquitously expressed in all examined tissues, with higher mRNA levels observed in the muscle, liver, spleen, and kidney. After P. damselae stimulation, the expression level of LEAP-2 mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant LEAP-2 expressed in pET-32a was approximately 23 kDa. The purified recombinant protein showed antibacterial activity against Gram-positive and Gram-negative bacteria. Luciferase reporters were constructed for five deletion fragments of different lengths from the promoter region (-1575 bp to +251 bp), and the results showed that L3 (-659 bp to +251 bp) presented the highest activity, and it was therefore defined as the core region of the LEAP-2 promoter. The seven predicted transcription factor binding sites were deleted by using PCR technology, and the results showed that the mutation of the USF transcription factor binding site caused the activity to significantly decrease. The results indicate that golden pompano LEAP-2 potentially exhibits antimicrobial effects in fish innate immunity.