Effects of Different Salinity Stress on the Transcriptomic Responses of Freshwater Crayfish (Procambarus clarkii, Girard, 1852).

Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.

First transcriptome profiling in gill and hepatopancrease tissues of Metapenaeus ensis in response to acute ammonia-N stress.

The greasyback shrimp, Metapenaeus ensis, suffers from ammonia-N stress during intensive factory aquaculture. Optimizing ammonia-N stress tolerance has become an important issue in M. ensis breeding. The metabolic and adaptive mechanisms of ammonia-N toxicity in M. ensis have not been comprehensively understood yet. In this study, a large number of potential simple sequence repeats (SSRs) in the transcriptome of M. ensis were identified. Differentially expressed genes (DEGs) in the gill and hepatopancreas at 24 h post-challenges under high concentrations of ammonia-N treatment were detected. We obtained 20,108,851-27,681,918 clean reads from the control and high groups, assembled and clustered a total of 103,174 unigenes with an average of 876 bp and an N50 of 1189 bp. Comparative transcriptome analyses identified 2000 different expressed genes in the gill and 2010 different expressed genes in the hepatopancreas, a large number of which were related to immune function, oxidative stress, metabolic regulation, and apoptosis. The results suggest that M. ensis may counteract ammonia-N toxicity at the transcriptome level by increasing the expression of genes related to immune stress and detoxification metabolism, and that selected genes may serve as molecular indicators of ammonia-N. By exploring the genetic basis of M. ensis' ammonia-N stress adaptation, we constructed the genetic networks for ammonia-N adaptation. These findings will accelerate the understanding of M. ensis' ammonia-N adaptation, contribute to the research of future breeding, and promote the level of factory aquaculture of M. ensis.

Transcriptome and molecular regulatory mechanisms analysis of gills in the black tiger shrimp Penaeus monodon under chronic low-salinity stress.

Background: Salinity is one of the main influencing factors in the culture environment and is extremely important for the survival, growth, development and reproduction of aquatic animals. Methods: In this study, a comparative transcriptome analysis (maintained for 45 days in three different salinities, 30 psu (HC group), 18 psu (MC group) and 3 psu (LC group)) was performed by high-throughput sequencing of economically cultured Penaeus monodon. P. monodon gill tissues from each treatment were collected for RNA-seq analysis to identify potential genes and pathways in response to low salinity stress. Results: A total of 64,475 unigenes were annotated in this study. There were 1,140 upregulated genes and 1,531 downregulated genes observed in the LC vs. HC group and 1,000 upregulated genes and 1,062 downregulated genes observed in the MC vs. HC group. In the LC vs. HC group, 583 DEGs significantly mapped to 37 signaling pathways, such as the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway; in the MC vs. HC group, 444 DEGs significantly mapped to 28 signaling pathways, such as the MAPK signaling pathway, Hippo signaling pathway and calcium signaling pathway. These pathways were significantly associated mainly with signal transduction, immunity and metabolism. Conclusions: These results suggest that low salinity stress may affect regulatory mechanisms such as metabolism, immunity, and signal transduction in addition to osmolarity in P. monodon. The greater the difference in salinity, the more significant the difference in genes. This study provides some guidance for understanding the low-salt domestication culture of P. monodon.

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.

Transcriptome analysis of hepatopancreas in penaeus monodon under acute low pH stress.

The decrease of seawater pH can affect the metabolism, acid-base balance, immune response and immunoprotease activity of aquatic animals, leading to aquatic animal stress, impairing the immune system of aquatic animals and weakening disease resistance, etc. In this study, we performed high-throughput sequencing analysis of the hepatopancreas transcriptome library of low pH stress penaeus monodon, and after sequencing quality control, a total of 43488612-56271828 Clean Reads were obtained, and GO annotation and KEGG pathway enrichment analysis were performed on the obtained Clean Reads, and a total of 395 DEGs were identified. we mined 10 differentially expressed and found that they were significantly enriched in the Metabolic pathways (ko01100), Biosynthesis of secondary metabolites (ko01110), Nitrogen metabolism (ko00910) pathways, such as PIGA, DGAT1, DGAT2, UBE2E on Metabolic pathways; UGT, GLT1, TIM genes on Biosynthesis of secondary metabolites; CA, CA2, CA4 genes on Nitrogen metabolism, are involved in lipid metabolism, induction of oxidative stress and inflammation in the muscular body of spot prawns. These genes play an important role in lipid metabolism, induction of oxidative stress and inflammatory response in the muscle of the shrimp. In summary, these genes provide valuable reference information for future breeding of low pH-tolerant shrimp.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Identification of multifunctionality of the PmE74 gene and development of SNPs associated with low salt tolerance in Penaeus monodon.

Members of the E74-like factor (ELF) subfamily are involved in the immune stress process of organisms by regulating immune responses and the development of immune-related cells. PmE74 of Penaeus monodon was characterized and functionally analyzed in this study. The full length of PmE74 was 3106 bp, with a 5'-UTR of 297 bp, and a 3'-UTR of 460 bp. The ORF (Open reading frame) was 2349 bp and encoded 782 amino acids. Domain analysis showed that PmE74 contains a typical Ets domain. Multiple sequence alignment and phylogenetic tree analysis showed that PmE74 clustered with Litopenaeus vannamei E74 and displayed significant similarity (98.98%). PmE74 was expressed in all tissues tested in P. monodon, with the highest levels of expression observed in the testis, intestine, and epidermis. Different pathogen stimulation studies have revealed that PmE74 expression varies in response to different pathogen stimuli. A 96-h acute low salt stress study revealed that PmE74 in the hepatopancreas was upregulated and downregulated in the salinity 17 group and considerably downregulated in the salinity 3 group, whereas PmE74 in gill tissue was considerably downregulated in both groups. Further, by knocking down PmE74 and learning the trends of its linkage genes PmAQP1, PmNKA, PmE75, PmFtz-f1, PmEcR, and PmRXR in response to low salt stress, it was further indicated that PmE74 could have a vital role in the regulation of low salt stress. The SNP test revealed that PmE74-In1-53 was significantly associated with low salt tolerance traits in P. monodon (P < 0.05). The findings of this study can aid in the advancement of molecular marker-assisted breeding in P. monodon, as well as provide fundamental data and methodologies for further investigation of its low salt tolerance strains in P. monodon.

Comparative transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

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.

Characterization, expression and function analysis of the TLR3 gene in golden pompano (Trachinotus ovatus).

Toll-like receptors (TLRs)are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many fishes, Toll-like receptors subfamily contain a large number of members with different functions that need to research in deep. In the present study, the full-length cDNA of TLR3 from the golden pompano, Trachinotus ovatus, was cloned and characterized. The full length of ToTLR3 cDNA was 3710 bp including an open reading frame of 2760 bp encoding a peptide of 919 amino acids. The derived amino acids sequence comprised of 14 leucine-rich repeats (LRR), capped with LRRCT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). Multiple sequence alignment and phylogenetic analysis revealed that ToTLR3 shared the highest similarity to the teleost fish and suggested ToTLR3 is fairly conservative in evolution process. Tissues distribution analysis indicated that ToTLR3 showed a tissue-specific variation with high expression in blood and liver. After the fish were stimulated by poly(I:C), flagellin and LPS, ToTLR3 expression in the liver, intestine, blood, kidney, skin and muscle was significantly upregulated in a time-depended manner, especially in immune related tissues such as liver, blood and kidney. Binding assay revealed the specificity of rToTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included Vibrio harveyi, V. vulnificus, V. anguillarum, Photobacterium damselae, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and PolyI:C, LPS, Flagellin, and PGN. In addition, a luciferase reporter assay showed that overexpression ToTLR3 significantly increased NF-κB activity. Collectively, our results suggested that ToTLR3 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, and transmiss the danger signal to downstream signaling pathways.

Transcriptome reveals the important role of metabolic imbalances, immune disorders and apoptosis in the treatment of Procambarus clarkii at super high temperature.

Temperature is an important environmental factor in the living environment of crustaceans. Changes in temperature can affect their normal growth and metabolism and even cause bacterial disease. Currently, the potential anti-reverse molecular reaction mechanism of crustaceans during high-temperature conditions has not yet been fully understood. Therefore, in this study, we characterised the transcriptome of Procambarus clarkii using RNA sequencing and performed a comparison between super-high-temperature treated samples and controls. After assembly and annotation, 81,097 unigenes with an average length of 069 bp and 358 differentially expressed genes (DEGs) were identified. Among these DEGs, 264 were differentially upregulated and 94 were differentially downregulated. To obtain comprehensive gene function information, we queried seven databases, namely, Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO to annotate gene functions. Transcriptome analysis revealed that the identified DEGs have significant effects on immune-related pathways, including lysosomal and phagosomal pathways, and that super-high-temperature conditions can cause disease in P. clarkii. Some significantly downregulated genes are involved in oxidative phosphorylation and the PPAR signalling pathway; this suggests a metabolic imbalance in P. clarkia during extreme temperature conditions. In addition, elevated temperature changed the expression patterns of key apoptosis genes XIAP, CASP2, CASP2, CASP8, and CYTC, thereby confirming that high-temperature conditions caused immune disorders, metabolic imbalance, and, finally, triggered apoptosis. Our results provide a useful foundation for understanding the molecular mechanisms underlying the responses of P. clarkii during high-temperature conditions.

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.

Transcription factor pparαb activates fads2s to promote LC-PUFA biosynthesis in the golden pompano Trachinotus ovatus (Linnaeus 1758).

Fatty acyl desaturase 2 (fads2) is a rate-limiting enzyme in long chain polyunsaturated fatty acids (LC-PUFAs) biosynthesis. In mammals, the lipid metabolism is modulated by a transcription factor, peroxisome proliferator-activated receptor alpha ß (pparαß); however, the detailed mechanism via pparαß regulates fads2 remains unclear in fish. In the present study, we identified the sequence features of Trachinotus ovatus fatty acyl desaturase 2a (Tofads2a) and fatty acyl desaturase 2b (Tofads2b), which both encoded 442 amino acid polypeptides containing cytochrome-b5-like domains and three representative histidine-rich domains. The Phylogenetic and genome organization analyses revealed characteristic phylogeny: the majority of fads2s exhibited a highly conserved exon/intron architecture. Tissue expression patterns by quantitative real-time PCR (qRT-PCR) showed that the two Tofads2s were prominently expressed in the brain. A nutritional study indicated that the transcription of the two Tofads2s was significantly implicated by treatment with a 1: 1 ratio of fish oil: soybean oil (FO:SO) in the liver and brain. Furthermore, functional characterization in yeast demonstrated that both Tofads2a and Tofads2b possessed Δ4/Δ5/Δ8 desaturation activity. Furthermore, promoter activity assays showed that the expressions of the two Tofads2s were actively regulated by pparαß. Moreover, mutation analyses showed that the M1 and M4/M5 binding sites of pparαß were functionally vital for binding to Tofads2a and Tofads2b promoters, respectively. Transcriptional activities of the two Tofads2s promoters were significantly reduced after targeted mutation of M1 or M4/M5. Electrophoretic mobile shift assays (EMSAs) verified that pparαß interacted with the M1 binding site in Tofads2a promoter to accommodate Tofads2a transcription. Briefly, pparαß plays an important role in Tofads2 expression and may promote the LC-PUFAs biosynthesis by regulating the expression of two Tofads2s.

Functional Analysis of IRF1 Reveals its Role in the Activation of the Type I IFN Pathway in Golden Pompano, Trachinotus ovatus (Linnaeus 1758).

Interferon (IFN) regulatory factor 1 (IRF1), a transcription factor with a novel helix-turn-helix DNA-binding domain, plays a crucial role in innate immunity by regulating the type I IFN signaling pathway. However, the regulatory mechanism through which IRF1 regulates type I IFN in fish is not yet elucidated. In the present study, IRF1 was characterized from golden pompano, Trachinotus ovatus (designated ToIRF1), and its immune function was identified to elucidate the transcriptional regulatory mechanism of ToIFNa3. The full-length complementary DNA (cDNA) of IRF1 is 1763 bp, including a 900-bp open reading frame (ORF) encoding a 299-amino-acid polypeptide. The putative protein sequence has 42.7-71.7% identity to fish IRF1 and possesses a representative conserved domain (a DNA-binding domain (DBD) at the N-terminus). The genomic DNA sequence of ToIRF1 consists of eight exons and seven introns. Moreover, ToIRF1 is constitutively expressed in all examined tissues, with higher levels being observed in immune-relevant tissues (whole blood, gill, and skin). Additionally, Cryptocaryon irritans challenge in vivo increases ToIRF1 expression in the skin as determined by Western blotting (WB); however, protein levels of ToIRF1 in the gill did not change significantly. The subcellular localization indicates that ToIRF1 is localized in the nucleus and cytoplasm with or without polyinosinic/polycytidylic acid (poly (I:C)) induction. Furthermore, overexpression of ToIRF1 or ToIFNa3 shows that ToIRF1 can notably activate ToIFNa3 and interferon signaling molecule expression. Promoter sequence analysis finds that several interferon stimulating response element (ISRE) binding sites are present in the promoter of ToIFNa3. Additionally, truncation, point mutation, and electrophoretic mobile shift (EMSA) assays confirmed that ToIRF1 M5 ISRE binding sites are functionally important for ToIFNa3 transcription. These results may help to illuminate the roles of teleost IRF1 in the transcriptional mechanisms of type I IFN in the immune process.

MultiplexSSR: A pipeline for developing multiplex SSR-PCR assays from resequencing data.

Next-generation sequencing has greatly promoted the investigation of single nucleotide polymorphisms, while studies of simple sequence repeats are sharply decreasing. However, simple sequence repeats still present some advantages in conservation genetics. In this study, an end-to-end pipeline referred to as MultiplexSSR was established to develop multiplex PCR assays in batches with highly polymorphic simple sequence repeats for capillary platforms from resequencing data. The distribution of single sequence repeats in the genome, the error profiles of genotypes and allelotypes, and the increase in the allele length range depending on the number of individuals were investigated. A total of 98% of single sequence repeats presented lengths of less than 100 bp. The error rate of the genotyping and allelotyping of dimeric patterns was ten times higher than those for other patterns. The error rate of allelotyping was less than that of genotyping. The allele length range reached approximate saturation with 10 individuals. This pipeline uses allele numbers to select highly polymorphic loci, masks loci with variation, and applies in silico PCR to improve primer specificity. The application of the developed multiplex SSR-PCR assays validated the pipeline's robustness, showing higher polymorphism and stability for the developed simple sequence repeats and a lower cost for genotyping and providing low-depth resequencing data from less than a dozen individuals for the development of markers. This pipeline fills the gap between next-generation sequencing and multiplex SSR-PCR.

Functional characterization of NK-lysin in golden pompano Trachinotus ovatus (Linnaeus 1758).

NK-lysin is an important part of the innate immune defence system and plays an important role in resisting the invasion of pathogenic microorganisms. In this study, NK-lysin from golden pompano (Trachinotus ovatus) was characterized and its expression in response to Photobacterium damselae was investigated. The full-length NK-lysin cDNA was 731 bp, which comprised a 5'-UTR of 63 bp, an ORF of 444 bp, and a 3'-UTR of 224 bp, and encoded 147 amino acids; NK-lysin consisted of a conserved saposin B domain and six conserved cysteines that formed three pairs of disulfide bonds. The genomic organization of NK-lysin was also determined and the gene consisted of four introns and five exons. The predicted promoter region of ToNK-lysin contained several putative transcription factor binding sites. Quantitative real-time (qRT-PCR) analysis indicated that ToNK-lysin was ubiquitously expressed in all examined tissues; the highest mRNA levels were observed in the skin, kidney and intestine, while the lowest expression level was detected in the stomach. After P. damselae stimulation, the expression level of NK-lysin mRNA was significantly upregulated in various tissues of golden pompano. In addition, SDS-PAGE showed that the molecular mass of recombinant NK-lysin expressed in pGEX-6P-1 was approximately 37 kDa. The purified recombinant protein showed antibacterial activity against gram-positive and gram-negative bacteria. The results indicate that golden pompano NK-lysin has potential antimicrobial roles in fish innate immunity.