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.

Molecular cloning and characterization of a cyclin B gene on the ovarian maturation stage of black tiger shrimp (Penaeus monodon).

The techniques of homology cloning and anchored PCR were used to clone the cyclin B gene from black tiger shrimp. The full length cDNA of black tiger shrimp cyclin B (btscyclin B) contained a 5' untranslated region (UTR) of 102 bp, an ORF of 1,206 bp encoding a polypeptide of 401 amino acids with an estimated molecular mass of 45 kDa and a 3' UTR of 396 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btscyclin B was homological to the cyclin B of other species and even the mammalians. Two conserved signature sequences of cyclin B gene family were found in the btscyclin B deduced amino acid sequence. The temporal expressions of cyclin B gene in the different tissues, including liver, ovary, muscle, brain stomach, heart and intestine, were measured by RT-PCR. mRNA expression of cyclin B could be detected in liver, ovary, muscle, brain, stomach, heart and strongest in the ovary, but almost not be detected in the intestine. In ovarian maturation stages, the expression of btscyclin B was different. The result indicated that btscyclin B was constitutive expressed and played an important role in the cell division stage.

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.

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 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.

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.

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.

Characterization and functional study of nuclear factor erythroid 2-related factor 2 (Nrf2) in black tiger shrimp (Penaeus monodon).

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a member of the Cap'n'collar basic region leucine zipper (CNC-bZIP) transcription factor family, and is activated by diverse oxidants, pro-oxidants, antioxidants and chemopreventive agents. The full-length cDNA of Nrf2 from Penaeus monodon (PmNrf2; 2024 bp long with 729 bp coding region, GenBank accession no. MW390830) was cloned. The 242-amino-acid polypeptide encoded by this gene had a predicted molecular mass of 27.80 kDa. Sequence homology and phylogenetic analysis showed that PmNrf2 was similar to the insect Cap'n'Collar (CNC) transcription factor and mammalian Nrf2. Tissue expression profile analyzed by quantitative real-time RT-PCR (qRT-PCR) demonstrated that PmNrf2 was constitutively expressed in all examined tissues, with the highest expression observed in the intestines and the weakest expression observed in the hemocyte. PmNrf2 expression profiles were detected in the hepatopancreas of shrimp after bacterial challenge. The results suggested that PmNrf2 was involved in the responses to bacterial challenge, but the temporal expression pattern trend of PmNrf2 differed between the gram-negative and gram-positive bacterial challenges in the shrimp hepatopancreas. The recombinant PmNrf2 protein was expressed and purified through affinity chromatography. Furthermore, an anti-PmNrf2 polyclonal antibody was obtained, which was able to clearly detect PmNrf2 protein expression in the hepatopancreas of shrimp. Knockdown of PmNrf2 by RNA interference (RNAi) resulted in a reduction in the expression of PmGPx gene. Taken together, the results of our study indicated that PmNrf2 played a role in regulation the transcription of PmGPx antioxidant enzyme genes.

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.

Comprehensive expression analysis of the beta integrin from Penaeus monodon indicating its participation in innate immunity and ammonia nitrogen stress response.

The aim of the present study was to investigate the function of the beta integrin (PmItgb) in Penaeus monodon. The 3011 bp cDNA sequence of PmItgb was cloned from P. monodon using rapid amplification of cDNA ends (RACE) PCR. Phylogenetic tree analyses indicated that the amino acid sequence of PmItgb should be merged into Fenneropenaeus chinensis (93%). Quantitative real-time PCR (q RT-PCR) revealed that PmItgb mRNA was highly expressed in the hemocytes. In addition, with regard to developmental stages, PmItgb showed significantly higher expression in oosperm, nauplius IV, zoea I and III, and post larval stages than that in other development stages. PmItgb expression in the shrimp epidermis was higher in the postmolt (B) stage, and lower in other molting stages. We also found that Vibrio harveyi and V. anguillarum challenge enhanced PmItgb expression in the hepatopancreas and gills. When PmItgb was inhibited, innate immunity-related genes such as ALF, crustin 1, crustin 7, penaeidin 3, and penaeidin 5 were significantly down-regulated. Furthermore, we demonstrated that PmItgb knock-down by specific dsRNA reduced bacterial clearance. In high ammonia nitrogen concentrations, PmItgb was significantly up-regulated in the hepatopancreas and gills. After PmItgb was silenced, the rate of mortality owing to high ammonia nitrogen concentrations decreased; the expression of related anti-apoptotic genes was up-regulated, and that of the apoptotic genes was slightly down-regulated. These results suggested that PmItgb may be involved in shrimp innate immunity and mediate apoptosis of hepatopancreatic cells induced by high ammonia nitrogen environments.

Interferon regulatory factor 2 plays a positive role in interferon gamma expression in golden pompano, Trachinotus ovatus (Linnaeus 1758).

In fish, interferon (IFN) regulatory factor 2 (IRF2) is a regulator of the type I IFN-dependent immune response, thereby playing a crucial role in innate immunity. However, the specific mechanism by which IRF2 regulates type II IFN in fish remains unclear. In the present study, first, to analyse the potential role of golden pompano (Trachinotus ovatus) IRF2 (ToIRF2) in the immune response, the mRNA level of ToIRF2 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) after parasite infection. ToIRF2 was upregulated at early time points in both local infection sites (skin and gill) and system immune tissues (liver, spleen, and head-kidney) after stimulation with Cryptocaryon irritans. Second, to investigate the modulation effect of ToIRF2 on type II IFN (interferon gamma, IFNγ) expression, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The expression level of IFNγ-5 was highest among the five truncated mutants in response to ToIRF2, indicating that the core promoter region was located from -189 bp to +120 bp, which included the IRF2 binding sites. Mutation analyses showed that the activity of the ToIFNγ promoter dramatically decreased after the targeted mutation of the M1, M2 or M3 binding sites. Additionally, electrophoretic mobile shift assay (EMSA) confirmed that IRF2 interacted with the M1 binding site in the ToIFNγ promoter region to dominate ToIFNγ expression. Finally, overexpressing ToIRF2 in vitro notably increased ToIFNγ and the transcription of several type II IFN/IRF-based signalling pathway genes. These results suggested that ToIRF2 might be involved in the host defence against C. irritans infection and contribute to a better understanding of the transcriptional mechanisms by which ToIRF2 regulates type II IFN in fish.

Structural and expression analysis of golden pompano Trachinotus ovatus IRF5 and its role in regulation of type I IFN.

The interferon regulatory factor 5 (IRF5) is a mediator of the type I IFN signalling pathways, thereby playing a key role in innate immunity. However, the detailed mechanism through which IRF5 regulates type I IFN in fish remains unclearly. In the present study, we first describe the identification of IRF5 (ToIRF5) from golden pompano (Trachinotus ovatus) and its features at the genomic sequence and expression level. The genomic DNA sequence consists of eight exons and seven introns. The full-length ToIRF5 cDNA is composed of 2, 059 bp and encodes for 499 amino acid polypeptides. The putative protein sequence shares 66.3%-82.9% identity to fish IRF5 and possesses three representative conserved domains (a DNA-binding domain (DBD) at the N-terminus, an IRF-associated domain (IAD), and a virus-activated domain (VAD) at the C-terminus) and one highly variable domain (middle region (MR)). Furthermore, the ToIRF5 transcript is constitutively expressed in all examined tissues, with higher levels observed in the immune relevant tissues. The mRNA levels of ToIRF5 are increased by polyinosinic: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin stimulation in the immune- and nonimmune-related tissues. The subcellular localization indicates that ToIRF5 is mainly localized in the cytoplasm with or without poly (I: C) induction. In addition, to explore whether ToIRF5 is a modulator of ToIFNa3, promoter analysis is performed. The region from -200 bp to +1 bp is identified as the core promoter by different truncated mutants of ToIFNa3. Mutation analyse declares that the activity of the ToIFNa3-5 promoter significantly decreases after targeted mutation of M2 binding sites. Moreover, overexpression of ToIRF5 in vitro memorably aggrandizes the expression of some IFN/IRF-based signalling pathway genes. These results provide new insights into the roles of teleost IRF5 in transcriptional mechanisms of type I IFN in the immunity process.

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.

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.

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.

Genome-wide comparative analysis ofbone morphogenetic proteins: genomic structure, phylogeny, and expression patterns in the golden pompano,Trachinotus ovatus(Linnaeus, 1758).

Bone morphogenetic proteins (BMPs) play important roles in various physiological processes, especially during the formation and maintenance of various organs. In this study, we first obtained and characterized twenty BMP genes from the Trachinotus ovatus genome (designated as ToBMPs). Sequence alignment and phylogenetic analysis both indicated that the predicted amino acid sequences of ToBMP were highly conserved with corresponding homologs of other species. Moreover, a comparative analysis was performed with seven representative vertebrate genomes and found difference in number of BMP3 genes in different species, which three members, BMP3a, BMP3b-1, and BMP3b-2, existed in diploid T. ovatus, but there were four and two members in tetraploidized Cyprinus carpio (BMP3a-1, BMP3a-2, BMP3b-1, and BMP3b-2) and diploid Danio rerio (BMP3a and BMP3b), respectively. The amino acid alignment and genomic structure analysis of ToBMP3 also suggested that the BMP3 gene had expanded in T. ovatus. Furthermore, tissue expression patterns were assessed for the small intestine, liver, white muscle, brain, spleen, fin, gill, head kidney, stomach, blood, and gonads. It was discovered that BMP1, BMP2, BMP3a, BMP4, BMP6, BMP7b, BMP11, and BMP16 were ubiquitously expressed in all the tissues tested. To study the regulatory function of BMP in response to the intake of different types of food, the expression changes in BMP mRNAs were detected by qRT-PCR, and the results showed that the majority of the BMP genes had the highest mRNA levels in the small intestine and liver after ingesting pelleted feed. Our data provide a useful resource for further studies on how paralogous genes may have different expression profiles in T. ovatus.

Ecdysone inducible gene E75 from black tiger shrimp Penaeus monodon: Characterization and elucidation of its role in molting.

Molting is controlled by ecdysteroids, which are synthesized and secreted by the Y-organ in crustaceans. Ecdysone inducible gene, E75, is an early-response gene in the 20-hydroxyecdysone (20E) signaling pathway, with crucial roles in arthropod development. Complementary DNA (cDNA) encoding Penaeus monodon E75 (PmE75) was cloned using RT-PCR and RACE. PmE75 cDNA was 3526 bp long and encoded a 799-amino acid protein. Tissue distribution analysis showed that PmE75 was expressed ubiquitously in selected tissues, and was relatively abundant in the epidermis, muscle, and hepatopancreas. Developmental expression revealed that PmE75 was expressed throughout its life cycle. Silencing PmE75 significantly decreased PmE75 expression. Shrimps injected with PBS and dsGFP started molting on Day 7 and had almost completed molting on Day 9, whereas dsPmE75-injected shrimp presented no signs of molting. These results suggested that PmE75 might be involved in molting. In situ hybridization results support this hypothesis. To explore the role of 20E and eyestalks in the regulation of molting in P. monodon, exogenous 20E injection and eyestalk ablation (ESA) were performed, and showed that 20E can induce the transcription and expression of PmE75 in the hepatopancreas, epidermis, and muscle, which were significantly elevated after ESA. These results provide further insights into our understanding of molting.

C-type lectin response to bacterial infection and ammonia nitrogen stress in tiger shrimp (Penaeus monodon).

C-type lectins (CTLs) 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 shrimp, C-type lectins subfamily contain a large number of members with different functions that need to research in deep. In this present study, a new type of CTL, PmCL1 with 861 bp long full-length cDNA, that encodes a protein with 164-amino acid from a 495-bp open reading frame, was isolated and characterized from tiger shrimp (Penaeus monodon). The mRNA transcript of PmCL1 showed the highest expression in the hepatopancreas, whereas it was barely detected in the ovary. After the shrimp were stimulated by Vibrio harveyi and Vibrio anguillarum, PmCL1 expression in the hepatopancreas and gill was significantly upregulated. A carbohydrate-binding assay revealed the specificity of PmCL1 for pathogen-associated molecular patterns (PAMPs) that included peptidoglycan (PGN) and lipopolysaccharide (LPS), and saccharides that included d-glucose, galactosamine, α-lactose, treholose, and d-mannose. Recombinant PmCL1 agglutinated gram-positive (Staphylococcus aureus) and gram-negative bacteria (V. harveyi, V. anguillarum, Vibrio alginolyticus, Vibrio parahemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) in the presence of calcium ions and enhanced the efficiency of clearing the invading bacteria. Collectively, our results suggested that PmCL1 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, as well as the response towards ammonia nitrogen stress.

Functional characterization of interferon regulatory factor 2 and its role in the transcription of interferon a3 in golden pompano Trachinotus ovatus (Linnaeus 1758).

Similar to mammals, fish possess interferon (IFN) regulatory factor 2 (IRF2)-dependent type I IFN responses. Nevertheless, the detailed mechanism through which IRF2 regulates type I IFNa3 remains largely unknown. In the present study, we first identified two genes from golden pompano (Trachinotus ovatus), IRF2 (ToIRF2) and IFNa3 (ToIFNa3), in the IFN/IRF-based signalling pathway. The open reading frame (ORF) sequence of ToIRF2 encoded 335 amino acids possessing four typical characteristic domains, including a conserved DNA-binding domain (DBD), an interferon association domain 2 (IAD2), a transcriptional activation domain (TAD), and a transcriptional repression domain (TRD). Furthermore, transcripts of ToIRF2 were significantly upregulated after stimulation by polyinosinic: polycytidylic acid [poly (I:C)], lipopolysaccharide (LPS) and flagellin in immune-related tissues (blood, liver, and head-kidney). Moreover, to investigate whether ToIRF2 was a regulator of ToIFNa3, promoter analysis was performed. The results showed that the region from -896 bp to -200 bp is defined as the core promoter using progressive deletion mutations of IFNa3. Additionally, ToIRF2 overexpression led to a clear time-dependent enhancement of ToIFNa3 promoter expression in HEK293T cells. Mutation analyses indicated that the activity of the ToIFNa3 promoter significantly decreased after targeted mutation of M4/5 binding sites. Electrophoretic mobile shift assays (EMSAs) verified that IRF2 interacted with the binding site of the ToIFNa3 promoter region to regulate ToIFNa3 transcription. Last, the promoter activity of ToIFNa3-2 was more responsive to treatment with poly (I:C) than LPS and flagellin. Furthermore, overexpression of ToIRF2 in vitro obviously increased the expression of several IFN/IRF-based signalling pathway genes after poly (I:C) abduction. In conclusion, the present study provides the first evidence of the positive regulation of ToIFNa3 transcription by ToIRF2 and contributes to a better understanding of the transcriptional mechanisms of ToIRF2 in fish.

Functional characterization of IRF8 regulation of type II IFN in golden pompano (Trachinotus ovatus).

Interferon regulatory factor 8 (IRF8) increases type I IFN transcription levels by binding to IFN promoters, thereby playing a role in innate immunity. Nevertheless, the detailed mechanism through which IRF8 regulates type II IFN in fish remains ambiguous. In the present study, two genes from the golden pompano (Trachinotus ovatus), IRF8 (ToIRF8) and IFN gamma (ToIFNγ), were identified in the IFN/IRF-based signalling pathway. The full-length ToIRF8 cDNA was composed of 2,141 bp and encoded a 421 amino acid polypeptide; the genomic DNA was 2,917 bp in length and consisted of 8 exons and 7 introns. The putative protein showed the highest sequence identity (90-92%) with fish IRF8 and possessed a DNA-binding domain (DBD), an IRF-association domain (IAD) and a nuclear localization signal (NLS) motif consistent with those of IRF8 in other vertebrates. Furthermore, the ToIRF8 transcripts were expressed in all examined tissues of healthy fish, with higher levels observed in the central nervous and immune relevant tissues. They were upregulated by polyinosinic acid: polycytidylic acid [poly (I: C)], lipopolysaccharide (LPS) and flagellin treatments in the blood, liver, intestine and kidney. The results from assays of subcellular localization showed that ToIRF8 was localized to the cytoplasm. Moreover, to investigate whether ToIRF8 was a regulator of ToIFNγ, a promoter analysis was performed using progressive deletion mutations of ToIFNγ. The results indicated that the region from -601 bp to -468 bp includes the core promoter. Mutation analyses indicated that the activity of the ToIFNγ promoter significantly decreased after the targeted mutation of the M1-M3 binding sites. Additionally, overexpressed ToIRF8 in vitro notably increased the expression of several IFN/IRF-based signalling pathway genes. These results suggest that IRF8 is vital in the defence of T. ovatus against bacterial infection and contributes to a better understanding of the transcriptional mechanisms of ToIRF8 on type II IFN in fish.