Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227bp, 1194bp and 1155bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9kDa, 44.4kDa and 43.6kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.

Mammalian POU5F1 (also known as OCT4) is an essential transcription factor that induces and controls stemness in the inner cell mass and embryonic stem (ES) cells. Its expression results from intricate regulatory networks involving its 5' upstream DNA elements and numerous transcription factors. Pou5f3, the ortholog of POU5F1, has been identified in non-mammalians including fish. However, little is known about the molecular mechanisms controlling its expression up to date. Here we report the promoter activity and regulation of Nile tilapia (Oreochromis niloticus) pou5f3 (Onpou5f3) in fish early-stage embryos and ES cells. A 3.1-kb Onpou5f3 promoter region was cloned, analyzed and constructed into pT2AL-GFP vector. Multiple potential regulatory elements including potential octamer sequence for Pou domain and retinoic acid-responsive elements were found in the 5' upstream region. In vivo and in vitro transfection assays reveal that the 3.1-kb DNA sequence was sufficient to drive strong GFP expression in blastula-stage embryos and ES cells, but low or undetectable expression in either late developmental stage embryos or differentiated cells, suggesting the feasibility as a tool to monitor the pluripotency state in fish stem cells. Deletion luciferase assays reveal that the region from -726 to -219 contains positive regulatory elements, whereas both the regions from -3056 to -1306 and -1306 to -729 contain negative regulatory elements. Notably, just like mammalian POU5F1, OnPou5f3 significantly enhanced its own expression in a dose-dependent manner, whereas RA treatment dramatically reduced its expression. Taken together, our study not only provides a tool for monitoring the pluripotency state of fish stem cells in vitro, but also experimentally demonstrates the molecular mechanisms underlying the Pou5f1 homolog expression might be conserved to some content between mammals and fish.

The Nile tilapia Sox9b gene is characterized as a homolog of the mammalian Sox9 gene, which exhibits testis-biased expression and is involved in testis development. However, the transcriptional regulation of the Sox9b gene is poorly understood. In this study, we demonstrated that the male sex differentiation gene doublesex and mab-3 related transcription factor 1 (Dmrt1) was predominantly expressed in the Nile tilapia testis and that Dmrt1 knockdown in the Nile tilapia decreased the expression of the Sox9b gene in the testis. An in silico analysis predicted that the proximal promoter of the Nile tilapia Sox9b gene had two potential cis-regulatory elements (CREs) for the Dmrt1 transcription factor. Together, a luciferase reporter analysis and site-directed mutagenesis revealed that Dmrt1 increased the transcriptional activity of the Nile tilapia Sox9b promoter via a specific CRE near the translation start site. A chromatin immunoprecipitation (ChIP) analysis and an electrophoretic mobility shift assay (EMSA) confirmed that Dmrt1 could directly bind to this specific CRE for Dmrt1 in the Nile tilapia Sox9b promoter. Taken together, our results demonstrate that the male sex-differentiation factor Dmrt1 positively regulates the transcription of the Nile tilapia Sox9b gene by directly binding to a specific CRE within the Sox9b promoter.

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

The nuclear receptor (NR) superfamily, which is divided into 7 subfamilies, constitutes one of the largest classes of transcription factors. In this study, through comprehensive database search, we identified all NRs (including 4 novel members) from the tilapia (75), common carp (137), zebrafish (73), fugu (73), tetraodon (72), stickleback (70), medaka (69), coelacanth (55), spotted gar (51) and elephant shark (50). For 21 NRs, two duplicates were found in teleosts, while only one in tetrapods. These duplicates, except those of DAX1, SHP and GCNF found in the elephant shark, were derived from 3R (third round of genome duplication). The linkage duplication of 5 syntenic blocks (comprising 14 duplicated NR couples) in teleosts further supported their 3R origin. Based on transcriptome data from adult tilapia, 53 NRs were found to be expressed in more than one tissue (brain, head kidney, heart, liver, kidney, muscle, ovary and testis), and 4 were tissue-specific, indicating their essential roles in the corresponding tissue. Based on the XX and XY gonadal transcriptome data from four developmental stages, 65 NRs were detected in gonads, with 21, 31, 11 and 29 expressed sexual dimorphically at 5, 30, 90 and 180days after hatching, respectively. The expression of four selected genes was examined by in situ hybridization (ISH) and quantitative PCR (qPCR) to validate the spatial and temporal expression profiles of NRs. Comparative analyses of the expression profiles of duplicated NRs revealed divergence in gene expression as well as gene function. Our results demonstrated that NRs may play important roles in sex determination and gonadal development in teleosts.

Hypoxia is one of the critical environmental stressors for fish in aquatic environments. Although accumulating evidences indicate that gene expression is regulated by hypoxia stress in fish, how genes undergoing differential gene expression and/or alternative splicing (AS) in response to hypoxia stress in heart are not well understood. Using RNA-seq, we surveyed and detected 289 differential expressed genes (DEG) and 103 genes that undergo differential usage of exons and splice junctions events (DUES) in heart of a hypoxia tolerant fish, Nile tilapia, Oreochromis niloticus following 12h hypoxic treatment. The spatio-temporal expression analysis validated the significant association of differential exon usages in two randomly selected DUES genes (fam162a and ndrg2) in 5 tissues (heart, liver, brain, gill and spleen) sampled at three time points (6h, 12h, and 24h) under acute hypoxia treatment. Functional analysis significantly associated the differential expressed genes with the categories related to energy conservation, protein synthesis and immune response. Different enrichment categories were found between the DEG and DUES dataset. The Isomerase activity, Oxidoreductase activity, Glycolysis and Oxidative stress process were significantly enriched for the DEG gene dataset, but the Structural constituent of ribosome and Structural molecule activity, Ribosomal protein and RNA binding protein were significantly enriched only for the DUES genes. Our comparative transcriptomic analysis reveals abundant stress responsive genes and their differential regulation function in the heart tissues of Nile tilapia under acute hypoxia stress. Our findings will facilitate future investigation on transcriptome complexity and AS regulation during hypoxia stress in fish.

Prkaca consists of the catalytic subunit alpha protein kinase A (PKA), which is involved in many cellular processes. In this study, the cDNA and genomic sequences of prkaca in tilapia hybrids (Oreochromis mossambicus × Oreochromis hornorum) were cloned and analysed. The results showed the prkaca gene consists of 11 exons and 10 introns, and its protein contains 351 amino acid residues and is clustered with Oreochromis niloticus, Maylandia zebra and Haplochromis burtoni first in a phylogenetic tree. Amino acid alignment indicates that prkaca shares the highest identity (100%) to Oreochromis niloticus, Maylandia zebra and Haplochromis burtoni. Two CpG islands of prkaca were found by MethPrimer software, and 32 CG sites were found in the proximal promoter. The methylation level of prkaca in the hybrids (0.31%) was significantly lower than that of their parents (0.94% and 3.43%) in kidney tissue (P < 0.05). The gene expression levels and DNA methylation levels of prkaca in muscle and kidney tissues of the tilapia hybrids were detected by quantitative real-time PCR and bisulfite sequencing PCR and showed a negative correlation under saline-alkali stress. The results of this research demonstrated that DNA methylation levels and prkaca mRNA expression levels were inversely correlated under saline-alkali stress, implying that heterosis is likely accompanied by DNA methylation alterations. This research provides new clues for further investigations of DNA methylation and heterosis in hybrid fish.

Beta-defensins (β-defensins) are small cationic amphiphilic peptides that are widely distributed in plants, insects, and vertebrates, and are important for their antimicrobial properties. In this study, the β-defensin (Onβ-defensin) gene of the Nile tilapia (Oreochromis niloticus) was cloned from spleen tissue. Onβ-defensin has a genomic DNA sequence of 674 bp and produces a cDNA of 454 bp. Sequence alignments showed that Onβ-defensin contains three exons and two introns. Sequence analysis of the cDNA identified an open reading frame of 201 bp, encoding 66 amino acids. Bioinformatic analysis showed that Onβ-defensin encodes a cytoplasmic protein molecule containing a signal peptide. The deduced amino acid sequence of this peptide contains six conserved cysteine residues and two conserved glycine residues, and shows 81.82% and 78.33% sequence similarities with β-defensin-1 of fugu (Takifugu rubripes) and rainbow trout (Oncorhynchus mykiss), respectively. Real-time quantitative PCR showed that the level of Onβ-defensin expression was highest in the skin (307.1-fold), followed by the spleen (77.3-fold), kidney (17.8-fold), and muscle (16.5-fold) compared to controls. By contrast, low levels of expression were found in the liver, heart, intestine, stomach, and gill (<3.0-fold). Artificial infection of tilapia with Streptococcus agalactiae (group B streptococcus [GBS] strain) resulted in a significantly upregulated expression of Onβ-defensin in the skin, muscle, kidney, and gill. In vitro antimicrobial experiments showed that a synthetic Onβ-defensin polypeptide had a certain degree of inhibitory effect on the growth of Escherichia coli DH5α and S. agalactiae. The results indicate that Onβ-defensin plays a role in immune responses that suppress or kill pathogens.

CD2BP2 (CD2 cytoplasmic tail binding protein 2), one of several proteins interacting with the cytoplasmic tail of CD2, plays a crucial role in CD2-triggered T cell activation and nuclear splicing. The studies on CD2BP2 have tended to be confined to a few mammals, and little information is available to date regarding fish CD2BP2. In this paper, a CD2BP2 gene (On-CD2BP2) was cloned from Nile tilapia, Oreochromis niloticus. Sequence analysis showed that the full length of On-CD2BP2 cDNA was 1429 bp, containing a 5'untranslated region (UTR) of 111 bp, a 3'-UTR of 193 bp and an open reading frame of 1125 bp which is encoding 374 amino acids. Two important structural features, a GYF domain and a consensus motif GPFXXXXMXXWXXXGYF were detected in the deduced amino acid sequence of On-CD2BP2, and the deduced genomic structure of On-CD2BP2 was similar to the known CD2BP2. The mRNA expression of On-CD2BP2 in various tissues of Nile tilapia was analyzed by fluorescent quantitative real-time PCR. In healthy Nile tilapia, the On-CD2BP2 transcripts were mainly detected in the head kidney and spleen. While vaccinated with inactivated Streptococcus agalactiae, the On-CD2BP2 mRNA expression was significantly up-regulated in the head kidney, spleen and brain 48 h post immunization. Moreover, there was a clear time-dependent expression pattern of On-CD2BP2 after immunization and the expression reached the highest level at 24h in the brain and 48 h in the head kidney and spleen. This is the first report of proving the presence of a CD2BP2 ortholog in fish, and investigating its tissue distribution and expression profile in response to bacterial stimulus. These findings indicated that On-CD2BP2 may play an important role in the immune response to bacteria in Nile tilapia.

Primordial germ cells (PGCs) are specified by maternally provided determinants in fish. PGCs migrate then into prospective gonadal sites during early development and give rise to germ cell lineage. PGC disrupted animals do not sexually mature which has a range of commercial as well as environmental benefits. To find potential target genes for sterilisation of Nile tilapia, relative mRNA abundance patterns and tissue distribution of four nanos, two piwil, dnd1, vasa and three pum genes were investigated during ontogenic development from unfertilised eggs to newly hatched larvae and in adult tissues, respectively. The ontogenic pattern of RNA abundance revealed that all the investigated gene transcripts are maternally deposited to varying degrees, except for nanos2 which is not expressed in eggs. The ontogenic patterns of relative RNA abundance could be grouped into three categories. The first one, including nanos3, piwil1, piwil2, dnd1 and vasa, showed abundant transcript levels during early developmental stages which are then degraded during the period of maternal to zygotic transition between blastula and gastrula stages with a reduction in expression of four to five orders of magnitude by hatching stage. Another, including pum2 and pum3, showed similar patterns to the first group, but the transcript levels are reduced by only two orders of magnitude. The third group, including nanos1a, nanos1b and pum1, was characterised by a zygotic increase. nanos2 had no detectable transcripts until hatching stage. The tissue screening of nanos1a, nanos1b, pum1, pum2 and pum3 showed that they are expressed in various tissues, implying their potential pleiotropic effects in these tissues apart from gonads. In contrast, nanos3, piwil1, piwil2, dnd1 and vasa appeared to be exclusively expressed in gonads (both ovary and testis), and nanos2 showed testis-specific expression. Based on these results nanos3, piwil1, piwil2, dnd1 and vasa were prioritised among the 11 selected genes as potential target genes for sterilisation in Nile tilapia as they have no significant zygotic expression during embryogenesis, they are expressed exclusively in gonads and maternally deposited. These features suggest a potential role of these genes in the specification and maintenance of PGCs during the ontogenic development of Nile tilapia.

Herein, Nile tilapia thioredoxin-interacting protein (On-TXNIP) and selenoprotein P (On-SEPP) cDNAs were cloned and characterized. The full-length On-TXNIP cDNA contained 2 arrestin domains, 2 conserved cysteine residues that bind to thioredoxin to inhibit thioredoxin function, and 2 PPXY motifs, which negatively regulate the protein by stimulating binding to E3 ubiquitin ligase. The On-SEPP cDNA contained 17 selenocysteines (Sec) encoded by the TGA codon, which can be recognized as either a stop codon or a Sec codon. The On-SEPP cDNA also carried 2 typical SECIS elements located in the 3'UTR that are important for selenocysteine translation. Evolutionary analyses of both the On-TXNIP and On-SEPP genes revealed that these genes are closely related to the TXNIP and SEPP genes in zebrafish (Danio rerio), with amino acid similarities of 91.8% and 61.9%, respectively. A normal tissue distribution analysis indicated that the On-TXNIP and On-SEPP genes were ubiquitously expressed in all tissues examined, and the highest expression levels of these genes were observed in peripheral blood leukocytes (PBLs) and the trunk kidney, respectively. The expression levels of On-TXNIP and On-SEPP transcripts were acutely and chronically analyzed following the injection of fish with 1, 10 or 100mg/kg silver nanoparticles (Ag NPs). Significant up-regulation of On-TXNIP and On-SEPP transcripts was observed in the liver, spleen, and head kidney at the early phase of Ag NP exposure (hours 6 through 48). Down-regulation of On-SEPP transcripts was clearly observed in the liver at weeks 1 to 4. Histopathology analysis demonstrated that the fish livers exhibited a dramatic infiltration of Kupffer cells, elevated bi-nucleated cells, expanded sinusoidal blood congestion and severe necrosis in a dose-dependent manner. Based on these findings, coupling of the expression analysis of these two cellular stress response genes and histopathological observation of fish exposed to Ag NPs should be reliable for the assessment of Ag NP contamination in teleost fish.

Steroidogenic factor 1 (sf1) (officially designated as nuclear receptor subfamily 5 group A member 1 [NR5A1]) is an important regulator of gonad development. Previous studies on sf1 in fish have been limited to cloning and in vitro expression experiments. In this study, we used antisense RNA to down-regulate sf1 transcription and sf1 protein expression. Down-regulation of sf1 resulted in an increase in body weight and inhibition of gonadal development in both males and females with the consequent lower gonadosomatic index compared to fish in the control group. Hematoxylin-eosin staining of the gonads of fish with down-regulated sf1 revealed fewer seminiferous tubules and sperm in the testis of males. In addition, the oocytes were mainly stage II and many of them were atretic follicle. We conducted comparative transcriptome and proteome analyses between the sf1-down-regulated group and the control group. These analyses revealed multiple gene-protein pairs and pathways involved in regulating the observed changes, including 44 and 74 differently expressed genes and proteins in males and females, respectively. The results indicated that dysfunctional retinal metabolism and fatty acid metabolism could be causes of the observed weight gain and gonad abnormalities in sf1-down-regulated fish. These findings demonstrate the feasibility of using antisense RNA for gene editing in fish. This methodology allows the study gene function in species less amenable to gene editing as for example aquaculture species with long life cycles.

Taste receptors (TRs) are seven trans-membrane G protein-coupled receptors as well as the interface between internal and external milieus, which playing pivotal roles in nutrient identification and acquisition. To better understand the scope and function of tr gene family in common carp, one of the most economic and important breeding fish species, which has undergone an additional round of whole genome duplication (WGD), we characterized 13 tr gene homologues including eight type I and five type II taste receptor genes from common carp genome, which were more than any other teleosts. Phylogenetic and syntenic analysis revealed the evolution dynamics of tr gene family, which was highly conserved, though gene duplication and gene loss do exist recently. Furthermore, the motif and dN/dS analyses indicated that these receptors were under different negative selection pressure. Additionally, the gene expression divergences were observed in 12 health tissues of common carp, with a relatively high level in barbel and head kidney, demonstrating tissue-specific expression of tr genes in the tetraploidized genome. The overarching goals of this study were to identify the abundance of tr genes in common carp, compare the gene divergence among species with varied feeding habits and provide genomic resources for future studies on teleost taste sensation.

Growth hormone is an essential polypeptide required for normal growth and development of vertebrates. In this report, striped catfish (Pangasianodon hypophthalmus) growth hormone gene and cDNA were isolated by reverse transcriptase-polymerase chain reaction. The striped catfish growth hormone (scGH) encoding gene contains 5 exons and 4 introns. The cDNA sequence of the scGH gene contains a 603bp open reading frame and encodes for a 200-aa protein consisting of a putative 22-aa signal peptide and the mature 178-aa protein. The recombinant histidine-tagged scGH protein which expressed in Escherichia coli as inclusion bodies was unfolded, refolded and purified to near-homogeneity by Ni(2+)-NTA chromatography. Analysis of the secondary structure content by CD spectroscopy showed that the α-helical content of the refolded scGH is 55%. Elucidation of the folding pathway of scGH by fluorescence spectroscopy showed that denaturation transition of scGH is coincident and cooperative, consistent with the two-state denaturation mechanism. The purified scGH was biologically active and exhibited growth-promoting activity in striped catfish, but not tilapia.

The evolution of apolipoprotein B (Apob) has been intensely researched due to its importance during lipid transport. Mammalian full-length apob100 can be post-transcriptionally edited by the enzyme apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like complex-one (Apobec1) resulting in a truncated Apob, known as Apob48. Whilst both full-length and truncated forms of Apob are important for normal lipid homeostasis in mammals, there is no evidence for the presence of apob mRNA editing prior to the divergence of the mammals, yet, non-mammalian vertebrates appear to function normally with only Apob100. To date, the majority of the research carried out in non-mammalian vertebrates has focused on chickens with only a very limited number examining apob mRNA editing in fish. This study focused on the molecular evolution of Apobec1 and Apob in order to ascertain if apob mRNA editing occurs in eels, a basal teleost which represents an evolutionarily important animal group. No evidence for the presence of Apobec1 or the ability for eel apob to be edited was found. However, an important link between mutant mice and the evident hypertriglyceridemia in the plasma of non-mammalian vertebrates was made. This study has provided imperative evidence to help bridge the evolutionary gap between fish and mammals and provides further support for the lack of apob mRNA editing in non-mammalian vertebrates.

Hypoxia is one of the major challenges in aquaculture industry. Breeding of fish tolerant to hypoxia is an important task in genetic improvement of aquaculture species. The Asian seabass, Lates calcarifer, is an important foodfish species. We identified and characterized the hypoxia-inducible factor inhibitor (HIF1αn) gene in the Asian seabass. The full-length cDNA sequence of the HIF1αn was 3425 bp, with an ORF of 1065 bp, encoding 354 amino acids. The genomic sequence of the gene was 8667 bp in length, and contained eight exons and seven introns. Phylogenetic analysis of the gene in fish and tetrapods revealed that the HIF1αn in the Asian seabass was closely related to that of tilapia (Oreochromis niloticus). The HIF1αn was highly up-regulated in the gill, spleen and heart after 3.5-hours hypoxia treatment. We identified three SNPs in the third and fourth introns of the HIF1αn gene. The SNP (i.e. SNP 9332241 (C/T)) in the fourth intron was significantly (P < 0.01) associated with hypoxia tolerance. This SNP might be useful in selecting Asian seabass for improved tolerance to hypoxia. Our data also provide useful information for further detailed analysis of the function of the HIF1αn gene in hypoxia tolerance.

Tumor necrosis factor alpha-induced protein 8-like 2 (TNFAIP8L2) is a newly described negative immune regulator, whose enigmatic biological functions are not clearly understood. In the present study, the TNFAIP8L2 homolog of rock bream (Oplegnathus fasciatus) was identified and characterized. The genomic composition of rock bream TNFAIP8L2 (~6.7 kb) represented a tripartite arrangement in which three exons are interrupted by two introns. The rock bream TNFAIP8L2 transcript (1974 bp) possessed a coding sequence of 561 bp encoding a peptide of 186 amino acids. The predicted rock bream TNFAIP8L2 protein was 21.1kDa and revealed the typical features of known TNFAIP8L2 members including the DED-like domain. Rock bream TNFAIP8L2 was composed of six α-helices and demonstrated a distinct folding pattern of the TNFAIP8L2 family. It showed a certain degree of homology and phylogenetic relationship with the corresponding tilapia counterpart. Based on an interspecies genomic organizational comparison of TNFAIP8L2 orthologs, they could be classified into two classes, with teleost and non-teleost origin respectively. While teleost TNFAIP8L2s manifest a tripartite arrangement, non-teleost counterparts demonstrate a dipartite structure suggesting the loss of an intron during the post-piscine speciation. Promoter proximal region of rock bream TNFAIP8L2 consisted of multiple immune responsive cis-regulatory elements. Analysis of basal transcription in eleven tissues revealed its constitutive expression in examined tissues with highest magnitude in the head kidney. The modulated temporal mRNA expression of rock bream TNFAIP8L2 in head kidney post-challenges with stimulants (LPS and poly I:C) and pathogens (Streptococcus iniae and irido virus) was stimulant-specific. Additionally, a drastic down-regulation of rock bream TNFAIP8L2 mRNA level occurred in blood cells collected from experimentally injured animals, and it was accompanied by a contemporaneous down-regulation of cytokines, TNF-α and TGFβ3. All these findings imply that rock bream TNFAIP8L2 is potentially responsible for immune and inflammatory modulation in rock bream.

The sea bass Dicentrarchus labrax is the center of interest of an increasing number of basic or applied research investigations, even though few genomic or transcriptomic data is available. Current public data only represent a very partial view of its transcriptome. To fill this need, we characterized brain and liver transcriptomes in a generalist manner that would benefit the entire scientific community. We also tackled some bioinformatics questions, related to the effect of RNA fragment size on the assembly quality. Using Illumina RNA-seq, we sequenced organ pools from both wild and farmed Atlantic and Mediterranean fishes. We built two distinct cDNA libraries per organ that only differed by the length of the selected mRNA fragments. Efficiency of assemblies performed on either or both fragments size differed depending on the organ, but remained very close reflecting the quality of the technical replication. We generated more than 19,538Mbp of data. Over 193million reads were assembled into 35,073 contigs (average length=2374bp; N50=3257). 59% contigs were annotated with SwissProt, which corresponded to 12,517 unique genes. We compared the Gene Ontology (GO) contig distribution between the sea bass and the tilapia. We also looked for brain and liver GO specific signatures as well as KEGG pathway coverage. 23,050 putative micro-satellites and 134,890 putative SNPs were identified. Our sampling strategy and assembly pipeline provided a reliable and broad reference transcriptome for the sea bass. It constitutes an indisputable quantitative and qualitative improvement of the public data, as it provides 5 times more base pairs with fewer and longer contigs. Both organs present unique signatures consistent with their specific physiological functions. The discrepancy in fragment size effect on assembly quality between organs lies in their difference in complexity and thus does not allow prescribing any general strategy. This information on two key organs will facilitate further functional approaches.

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.