Search

The complete mitochondrial genomes of the Southeast Asian freshwater pufferfishes, Pao abei and Pao suvattii, were reconstructed using the MGISEQ platform. The genomes were 16,448 bp and 16,449 bp in length, each made up of 37 mitochondrial genes (13 CDSs, 22 tRNAs, and two rRNAs) and putative control region. It is suggested that an accumulation of complete mitochondrial genome sequences can contribute to resolve the taxonomic status of Pao species.

A complete mitogenome of T. lineatus from Lake Turkana in the Kenyan part was determined. It had a length of 16 470 bp, including 37 genes as found in teleosts with the typical gene order in these fishes. Mitogenomic comparison and phylogenetic analysis supported not only the morphology-based recognition of the pufferfish specimen from Lake Turkana as the Nile pufferfish, but also the identification of the Chinese specimen recently recorded as T. lineatus and its African source. The mitochondrial genome here amplified for the sample of T. lineatus from Lake Turkana also provides conclusive molecular evidence for the monophyletic nature of Tetraodon s.s., as it serves as a reference genome of this species used to clarify ambiguity in its identification in previous molecular studies. [ABSTRACT FROM AUTHOR]

The complete mitochondrial genomes of the Southeast Asian freshwater pufferfishes, Pao abei and Pao suvattii, were reconstructed using the MGISEQ platform. The genomes were 16,448 bp and 16,449 bp in length, each made up of 37 mitochondrial genes (13 CDSs, 22 tRNAs, and two rRNAs) and putative control region. It is suggested that an accumulation of complete mitochondrial genome sequences can contribute to resolve the taxonomic status of Pao species. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties. [ABSTRACT FROM AUTHOR]

Vibriosis is the most common bacterial diseases and brings great economic loss on aquaculture. Vibrio parahaemolyticus ( V. parahaemolyticus) , a gram-negative bacterium, has been identified as one main pathogens of Vibriosis. The pathogenic mechanism of V. parahaemolyticus is not entirely clear now. In our study, a model of V. parahaemolyticus infection of green-spotted puffer fish ( Tetraodon nigroviridis ) was established . T. nigroviridis were injected intraperitoneally (i.p.) with 200 μL of V. parahaemolyticus (8 × 10 10 CFU/mL). V. parahaemolyticus infection caused 64% mortality and infected some organs of T. nigroviridis . Histopathology studies revealed V. parahaemolyticus infection induced tissue structural changes, including adipose hollow space in the liver. Immunohistochemistry showed V. parahaemolyticus were present in infected tissue such as liver, head kidney and spleen. In livers of T. nigroviridis infected by V. parahaemolyticus , the alkaline phosphatases (ALP) activity first gradually increased and then backed to normal level, a trend that was on the contrary to the expression profile of the miR-29b. Quantitative real-time PCR analysis showed that the expression level of TLR1, TLR2, TLR5, TLR9, TLR21, NOD1, NOD2 and IL-6 in response to V. parahaemolyticus infection decreased compared to that of non-infected fish. The establishment of the T. nigroviridis model of V. parahaemolyticus infection further confirmed V. parahaemolyticus spreads through the blood circulation system primary as an extracellular pathogen. Meanwhile, liver is an important target organ when infected by V. parahaemolyticus . miR-29b in liver was involved in the progress of liver steatosis during V. parahaemolyticus infection. Moreover, V. parahaemolyticus infection in vivo may have an effect of immunosuppression on host. [ABSTRACT FROM AUTHOR]

Interferon gamma (IFNγ) is a Th1 cytokine that plays a very important role in almost all phases of immune and inflammatory responses. In this study, we explored the functions of IFNγ1 and IFNγ2 of Tetraodon nigroviridis . Treating T. nigroviridis spleen and head kidney cells in vitro with recombinant T. nigroviridis IFNγ1 protein (r Tn IFNγ1) or recombinant T. nigroviridis IFNγ2 protein (r Tn IFNγ2) enhanced their nitric oxide responses. Both r Tn IFNγ1 and r Tn IFNγ2 also induced the expression of interferon-stimulated gene 15 (ISG15), a common anti-viral gene, although the expression of the interferon-inducible Mx gene was markedly inhibited by r Tn IFNγ1 and was induced by r Tn IFNγ2. The in vivo effects of r Tn IFNγ1 and r Tn IFNγ2 on Vibrio parahaemolyticus ( V. parahaemolyticus ) infection were assessed by intraperitoneally injecting r Tn IFNγ1 or r Tn IFNγ2 (100 ng) and V. parahaemolyticus (8 × 10 10 CFU/mL) into T. nigroviridis. A comparison of the group treated only with V. parahaemolyticus and those also treated with r Tn IFNγ1 or r Tn IFNγ2 showed that neither of these IFNγs protected T. nigroviridis from V. parahaemolyticus infection. However, r Tn IFNγ1 more rapidly and robustly promoted inflammatory responses compared with r Tn IFNγ2, whereas r Tn IFNγ2 was involved in inducing the host to develop a more effective response earlier during the later stage of a V. parahaemolyticus infection. Moreover, microRNA-29b (miR-29b) expression is inversely correlated with IFNγ2 expression in T. nigroviridis . [ABSTRACT FROM AUTHOR]

Background: TAARs (trace amine-associated receptors) are among the principal receptors expressed by the olfactory epithelium. We used the recent BROAD Institute release of the genome sequences of five representative fishes of the cichlid family to establish the complete TAAR repertoires of these species and to compare them with five other fish TAAR repertoires. Results: The genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were analyzed by exhaustive TBLASTN searches with a set of published TAAR gene sequences used as positive bait. A second TBLASTN analysis was then performed on the candidate genes, with a set of non-TAAR class A GPCR (G protein-coupled receptors) used as negative bait. The resulting cichlid repertoire contained 44 complete TAAR genes from O. niloticus, 18 from P. nyererei, 23 from H. burtoni, 12 from N. brichardi and 20 from M. zebra, plus a number of pseudogenes, edge genes and fragments. A large proportion of these sequences (80%) consisted of two coding exons, separated in all but two cases by an intron in the interloop 1 coding sequence. We constructed phylogenetic trees. These trees indicated that TAARs constitute a distinct clade, well separated from ORs (olfactory receptors) and other class A GPCRs. Also these repertoires consist of several families and subfamilies, a number of which are common to fugu, tetraodon, stickleback and medaka. Like all other TAARs identified to date, cichlid TAARs have a characteristic two-dimensional structure and contain a number of amino-acid motifs or amino acids, such cysteine, in particular conserved positions. Conclusions: Little is known about the functions of TAARs: in most cases their ligands have yet to be identified, partly because appropriate methods for such investigations have not been developed. Sequences analyses and comparisons of TAARs in several animal species, here fishes living in the same environment, should help reveal their roles and whether they are complementary to that of ORs. [ABSTRACT FROM AUTHOR]

Background: To help understand the molecular mechanisms underlying the remarkable phenotypic diversity displayed by cichlids, the genome sequences of O. niloticus, P. nyererei, H. burtoni, N. brichardi and M. zebra were recently determined. Here, we present the contents of the olfactory receptor (OR) repertoires in the genomes of these five fishes. Results: We performed an exhaustive TBLASTN search of the five cichlid genomes to identify their OR repertoires as completely as possible. We used as bait a set of ORs described in the literature. The cichlid repertoires thereby extracted contained large numbers of complete genes (O. niloticus 158; H. burtoni 90; M. zebra 102; N. brichardi 69; P. nyererei 88), a small numbers of pseudogenes and many “edge genes” corresponding to incomplete genes located at the ends of contigs. A phylogenetic tree was constructed and showed these repertoires include a large number of families and subfamilies. It also allowed the identification of a large number of OR analogues between cichlids with very high amino-acid identity (≥99%). Nearly 9% of the full-length cichlid OR genes are composed of several coding exons. This is very unusual for vertebrate OR genes. Nevertheless, the evidence is strong, and includes the donor and acceptor splice junction sequences; also, the positions of these genes in the phylogenetic tree indicate that they constitute subfamilies well apart from non-OR G protein-coupled receptor families. Conclusions: Cichlid OR repertoires are made up of a larger number of genes and fewer pseudogenes than those in other teleosts except zebrafish. These ORs share all identified properties common to all fish ORs; however, the large number of families and subfamilies, each containing few ORs implies that they have evolved more rapidly. This high level of OR diversity is consistent with the substantial phenotypic diversity that characterizes cichlids. [ABSTRACT FROM AUTHOR]

Highlights: [•] Two IFNGR1s were identified in first fish other than Cypriniformes. [•] First mapped two IFNGR1 loci to different H. sapiens and G. gallus chromosomes. [•] Suggested the presence of two IFNGR1s is a common phenomenon in teleosts. [•] Provided in vitro evidence to support the ligand/receptor model from morpholino D. rerio. [Copyright &y& Elsevier]

Expression profiles of claudin-6, -10d and -10e in the euryhaline teleost fish Tetraodon nigroviridis revealed claudin-6 in brain, eye, gill and skin tissue, while claudin-10d and -10e were found in brain, gill and skin only. In fishes, the gill and skin are important tissue barriers that interface directly with surrounding water, but these organs generally function differently in osmoregulation. Therefore, roles for gill and skin claudin-6, -10d and -10e in the osmoregulatory strategies of T. nigroviridis were investigated. In the gill epithelium, claudin-6, -10d and -10e co-localized with Na+-K+-ATPase immunoreactive (NKA-ir) ionocytes, and differences in sub-cellular localization could be observed in hypoosmotic (freshwater, FW) versus hyperosmotic (seawater, SW) environments. Claudin-10d and -10e abundance increased in the gills of fish acclimated to SW versus FW, while claudin-6 abundance decreased in the gills of fish acclimated to SW. Taken together with our knowledge of claudin-6 and -10 function in other vertebrates, data support the idea that in SW-acclimated T. nigroviridis, these claudins are abundant in gill ionocytes, where they contribute to the formation of a Na+ shunt and 'leaky' epithelium, both of which are characteristic of salt-secreting SW fish gills. Skin claudin- 10d and -10e abundance also increased in fish acclimated to SW versus those in FW, but so did claudin-6. In skin, claudin-6 was found to co-localize with NKA-ir cells, but claudin-10d and -10e did not. This study provides direct evidence that the gill epithelium contains salinity-responsive tight junction proteins that are abundant primarily in ionocytes. These same proteins also appear to play a role in the osmoregulatory physiology of the epidermis. [ABSTRACT FROM AUTHOR]

Abstract: Induction of interferons (IFNs) produces an innate immune response through activation of the JAK-STAT signaling pathway. Type I IFN signaling activates downstream gene expression through the IFN-stimulated gene factor 3 (ISGF3) complex, while type II IFN (IFN-γ) signaling is mediated through active STAT1 protein. The IFN target gene Mx is involved in the defense against viral infection. However, the mechanism by which Tetraodon (pufferfish) Mx is regulated by IFN signaling has not been identified. In this study, we describe the cloning and expression of Tetraodon STAT1, STAT2, and IFN regulatory factor 9 (IRF9). By combining constitutively-active STAT1 (STAT1-JH1) and STAT2 (STA2-JH1) fusion proteins with IRF9, we demonstrate that a constitutively-active ISGF3 complex increases the transcriptional activity of the Tetraodon Mx promoter via direct binding to two IFN-stimulated response element (ISRE) sites. In addition, a constitutively-active TnIRF9-S2C containing a fusion of the C-terminal region of STAT2 and IRF9 also activated the Mx promoter through binding to the ISRE sites. Furthermore, constitutively-active STAT1-JH1 elevates Mx promoter activity through two IFN gamma-activated sequence (GAS) elements. The Mx promoter is also activated by constitutively-active TnIRF9-S2C and STAT1-JH1 protein, as determined using an in vivo luciferase assay. We conclude that the Tetraodon Mx gene is activated via Type I (IFN-1) and Type II (IFN-γ) signaling. These results provide mechanistic insights into the role of IFN signaling in teleosts, and the in vivo luciferase assay may be suitable as a tool for studying induction and regulation by IFNs in teleost fish. [Copyright &y& Elsevier]

The fatty acid-binding protein ( fabp) genes belong to the multigene family of intracellular lipid-binding proteins. To date, 12 different FABPs have been identified in vertebrate genomes. Owing to the teleost-specific genome duplication event, many fishes have duplicated copies of the fabp genes. Here, we identified and characterized the fabp genes of spotted green pufferfish ( Tetraodon nigroviridis). Seven fabp genes were identified, out of which, two were retained in the pufferfish genome as duplicated copies. Each putative pufferfish Fabp protein shares greatest sequence identity and similarity with their teleost and tetrapod orthologs, and clustered together as a distinct clade in phylogenetic analysis. Conserved gene synteny was evident between the pufferfish fabp genes and the orthologs of human, zebrafish, three-spined stickleback, and medaka FABP/fabp genes, providing evidence that the duplicated copies of pufferfish fabp genes most likely arose as a result of the teleost-specific genome duplication event. The differential tissue-specific distribution of pufferfish fabp transcripts suggests divergent spatial regulation of duplicated pairs of fabp genes. [ABSTRACT FROM AUTHOR]

Abstract: The nuclear envelopes surrounding the oocyte germinal vesicles of lower vertebrates (fish and frog) are supported by the lamina, which consists of the protein lamin B3 encoded by a gene found also in birds but lost in the lineage leading to mammals. Like other members of the lamin family, goldfish lamin B3 (gfLB3) contains two putative consensus phosphoacceptor p34cdc2 sites (Ser-28 and Ser-398) for the M-phase kinase to regulate lamin polymerization on the N- and C-terminal regions flanking a central rod domain. Partial phosphorylation of gfLB3 occurs on Ser-28 in the N-terminal head domain in immature oocytes prior to germinal vesicle breakdown, which suggests continual rearrangement of lamins by a novel lamin kinase in fish oocytes. We applied the expression-screening method to isolate lamin kinases by using phosphorylation site Ser-28-specific monoclonal antibody and a vector encoding substrate peptides from a goldfish ovarian cDNA library. As a result, SRPK1 was screened as a prominent lamin kinase candidate. The gfLB3 has a short stretch of the RS repeats (9-SRASTVRSSRRS-20) upstream of the Ser-28, within the N-terminal head. This stretch of repeats is conserved among fish lamin B3 but is not found in other lamins. In vitro phosphorylation studies and GST-pull down assay revealed that SRPK1 bound to the region of sequential RS repeats (9–20) with affinity and recruited serine into the active site by a grab-and-pull manner. These results indicate SRPK1 may phosphorylate the p34cdc2 site in the N-terminal head of GV-lamin B3 at the RS motifs, which have the general property of aggregation. [Copyright &y& Elsevier]

Lysine carbamylation, a post-translational modification, facilitates metal coordination for specific enzymatic activities. We have determined structures of the vertebrate dihydropyrimidinase from Tetraodon nigroviridis (TnDhp) in various states: the apoenzyme as well as two forms of the holoenzyme with one and two metals at the catalytic site. The essential active-site structural requirements have been identified for the possible existence of four metal-mediated stages of lysine carbamylation. Only one metal is sufficient for stabilizing lysine carbamylation; however, the post-translational lysine carbamylation facilitates additional metal coordination for the regulation of specific enzymatic activities through controlling the conformations of two dynamic loops, Ala69-Arg74 and Met158-Met165, located in the tunnel for the substrate entrance. The substrate/product tunnel is in the "open form" in the apo-TnDhp, in the "intermediate state" in the monometal TnDhp, and in the "closed form" in the dimetal TnDhp structure, respectively. Structural comparison also suggests that the C-terminal tail plays a role in the enzymatic function through interactions with the Ala69- Arg74 dynamic loop. In addition, the structures of the dimetal TnDhp in complexes with hydantoin, N-carbamyl-β-alanine, and N-carbamyl-β-amino isobutyrate as well as apo-TnDhp in complex with a product analog, N-(2-acetamido)-iminodi-acetic acid, have been determined. These structural results illustrate how a protein exploits unique lysines and the metal distribution to accomplish lysine carbamylation as well as subsequent enzymatic functions. [ABSTRACT FROM AUTHOR]

Inflammatory bowel disease (IBD) has long been a worldwide health care problem with a persistently increasing incidence. Although its clinical features have been well described, its etiology and pathogenesis remain unclear. IL-16 is a chemoattractant cytokine with various effects on cellular activities and diseases. However, the involvement of IL-16 in IBD remains poorly understood. In this study, to our knowledge we report for the first time the mechanism by which IL-16 induces intestinal inflammation by upregulating the expression of oligopeptide transporter member 1 (PepT1) in a Tetraodon nigroviridis fish model. The dextran sodium sulfate–induced colitis model in this species revealed that IL-16 levels significantly increase accompanied by elevations in PepT1 in the colon. Moreover, the signs of colitis were dramatically attenuated by IL-16 depletion using anti–IL-16 Abs. In vivo IL-16 administration induced remarkable intestinal inflammation with typical ulcerative colitis–like features, including histologic damage, inflammatory cell infiltration, increased myeloperoxidase activity, and proinflammatory cytokines expression, which corresponded with significant PepT1 upregulation in the colon. The IL-16–induced PepT1 expression and its upregulated fMLF transport were also demonstrated in vitro. To our knowledge, our study provides the first evidence of the connection between IL-16 and PepT1, which provides new insights into the molecular mechanism underlying IBD development. Additionally, this study suggests that fish species are an attractive model for studying IBD. By providing a better understanding of IL-16 biology from fish to mammals, this study should aid the development of IL-16–based therapies for IBD. [ABSTRACT FROM AUTHOR]

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation. [ABSTRACT FROM AUTHOR]

The COL5A1 gene, a member of the clade B fibrillar collagen gene family, was recently shown to contain two alternatively spliced exons (64A and 64B) that encode 23 amino acids in the carboxyl-terminal propeptide. The two are identical in length, very similar in sequence, and used in a mutually exclusive fashion because of the small intron that separates them. Each COL5A1 allele uses both exons, but a given transcript will contain only one of the two exons. The sequences in other species are highly conserved at the amino acid level. The expression profile of the two isoforms was determined from analysis of RNA levels in a panel of murine tissues. While both isoforms were found in all tissues studied, actively proliferating tissues (liver, lung) used isoform B more often, while a less mitotically active tissue, brain, had a higher proportion of exon 64A. The high degree of conservation between the two exons is consistent with a regional genomic duplication. The presence of the two isoforms as far back as pufferfish (tetraodon) implies an important functional significance. The exact role determined by the two sequences is not known, but involvement in the determination of chain composition of mature type V collagen or regulation of cell activity is possible, given the differences in tissue distribution. [ABSTRACT FROM AUTHOR]

Abstract: This study proposes a new fishery resource recovery strategy and empirically examines its feasibility. The proposed strategy is unique because it may help fishermen recover fishery resources by closing for the first several months of the fishing season without decreasing their average annual landings. If demand is elastic, it is feasible that they could recover biomass without reducing average annual revenue. To demonstrate the validity of this strategy, this study empirically examines the ocellate puffer in the vicinity of Japan, as it has experienced a drastic depletion of biomass. The results indicate that if fishing were to be prohibited, for example, between October and February (current fishing season is between October and March), ocellate puffer recovery in the first year would be 30,453kg, which would have the same effect as an 18.4% reduction in the current average landings. The price elasticity for ocellate puffer is elastic, which implies not only that fishery revenue would not decrease, but that the strategy could be a promising means of recovering ocellate puffer stocks. [Copyright &y& Elsevier]

Members of the Fox gene family of transcriptional regulators are essential for animal development and have been extensively studied in vertebrates. The mouse and human genomes contain at least 40 FOX genes which are divided into 19 subclasses based on the sequence similarity of the highly conserved forkhead domain. Using the genome sequence of the Takifugu rubripes and Tetraodon nigroviridis, we examined the genomic complement of fox genes in these organisms to gain insight into the evolutionary relationship of this gene family. We identified 53 fox genes in Tetraodon nigroviridis and Takifugu rubripes genome by searching the forkhead domain. These genes are divided into 18 subclasses as follows: 8 fox genes in subclass O; 6 in subclass P ; 4 in subclasses D, J, and N; 3 in subclasses A, B, C, E, F, and I; 2 in subclasses K, L, and Q; and 1 in subclasses G, H, M, and R. Together with the forkhead domain sequences of human, chicken, frog, zebrafish, medaka, and Caenorhabditis elegans, the phylogenetic relationship of the fox genes in Takifugu rubripes and Tetraodon nigroviridis were analyzed and compared. The genes structure, general features, and the three-dimensional model of these genes were also discussed. [ABSTRACT FROM AUTHOR]

Parasitological examination of the freshwater fish Tetraodon palembangensis Bleeker (Tetraodontidae, Tetraodontiformes) (n = 19), imported along with other ornamental fishes from Thailand into the Czech Republic in October 1998, revealed the presence of male specimens of the nematode Dichelyne ( Dichelyne) hartwichi Moravec, Wolter et Körting, 1999 (Cucullanidae) (prevalence 11%, intensity 1). Their detailed study using light and scanning electron microscopy (the latter used for the first time in this species) enabled the redescription of this hitherto little-known species and the recognition of considerable morphological and biometrical intraspecific variability. The finding of this species in T. palembangensis represents a new host record. [ABSTRACT FROM AUTHOR]

Abstract: A growing family of cellular proteins encoding for caspase activation and the recruitment domain (CARD) plays a crucial role in immunity by sensing viral infections and signaling antiviral immune defenses. We obtained a MAVS-like protein (named TnMAVS) from Tetradon nigroviridis, which contains a CARD domain, a pro-rich domain, and a TM domain similar to human MAVS. A fluorescence assay showed that TnMAVS was located in the cytoplasm and near by the membrane, and not the mitochondria in FHM cells. As such, it was considered as a new member of MAVS. The TnMAVS was highly expressed in the liver and muscle of T. nigroviridis. In the spleen, TnMAVS was down-regulated when the fish was treated with polyinosinic:polycytidylic acid or challenged with ISKNV, but was not affected by PGN or LPS. The dual luciferase reporter assay revealed that TnMAVS overexpression resulted in the activation of the interferon-sensitive response element and NF-κB signal pathways. In addition, a characteristic TRAF3-associated peptide PVQD was found in the TnMAVS sequence. Co-immunoprecipitation assays indicated that TnMAVS could interact with zfTRAF3 in eukaryotic cells. [Copyright &y& Elsevier]

Recent genome-wide analyses have revealed patterns of positive selection acting on protein-coding genes in humans and mammals. To assess whether the conclusions drawn from these analyses are valid for other vertebrates and to identify mammalian specificities, I have investigated the selective pressure acting on protein-coding genes of the puffer fishes Tetraodon and Takifugu. My results indicate that the strength of purifying selection in puffer fishes is similar to previous reports for murids but stronger in hominids, which have a smaller population size. Gene ontology analyses show that more than half of the biological processes targeted by positive selection in mammals are also targeted in puffer fishes, highlighting general patterns for vertebrates. Biological processes enriched with positively selected genes that are shared between mammals and fishes include immune and defense responses, signal transduction, regulation of transcription and several of their descendent terms. Mammalian-specific processes displaying an excess of positively selected genes are related to sensory perception and neurological processes. The comparative analyses also revealed that, for both mammals and fishes, genes encoding extracellular proteins are preferentially targeted by positive selection, indicating that adaptive evolution occurs more often in the extra-cellular environment rather than inside the cell. Moreover, I present here the first genome-wide characterization of neutrally-evolving regions of protein-coding genes. This analysis revealed an unexpectedly high proportion of genes containing both positively selected motifs and neutrally-evolving regions, uncovering a strong link between neutral evolution and positive selection. I speculate that neutrally-evolving regions are a major source of novelties screened by natural selection. [ABSTRACT FROM AUTHOR]

CD4CD25Foxp3 regulatory T cells (Treg cells) are critical for the maintenance of peripheral tolerance, and the suppression of autoimmune diseases and even tumors. Although Treg cells are well characterized in humans, little is known regarding their existence or occurrence in ancient vertebrates. In the present study, we report on the molecular and functional characterization of a Treg-like subset with the phenotype CD4-2CD25-likeFoxp3-like from a pufferfish ( Tetraodon nigroviridis) model. Functional studies showed that depletion of this subset produced an enhanced mixed lymphocyte reaction (MLR) and nonspecific cytotoxic cell (NCC) activity in vitro, as well as inflammation of the intestine in vivo. The data presented here will not only enrich the knowledge of fish immunology but will also be beneficial for a better cross-species understanding of the evolutionary history of the Treg family and Treg-mediated regulatory networks in cellular immunity. [ABSTRACT FROM AUTHOR]

Abstract: This study assessed the relationship between the occurrence and function of intronless or single exon genes (SEG) in the genome of five teleost species and their phylogenetic distance. The results revealed that Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculeatus and Danio rerio genomes are respectively comprised of 2.83%, 3.42%, 4.49%, 4.35% and 4.02% SEGs. These SEGs encode for a variety of family proteins including claudins, olfactory receptors and histones that are essential for various biological functions. Subsequently, we predicted and annotated SEGs in three European sea bass, Dicentrarchus labrax chromosomes that we have sequenced, and compared results with those of stickleback (G. aculeatus) homologous chromosomes. While the annotation features of three D. labrax chromosomes revealed 78 (5.30%) intronless genes, comparisons with G. aculeatus showed that SEG composition and their order varied significantly among corresponding chromosomes, even for those with nearly complete synteny. More than half of SEGs identified in most of the species have at least one ortholog multiple exon gene in the same genome, which provides insight to their possible origin by retrotransposition. In spite of the fact that they belong to the same lineage, the fraction of predicted SEGs varied significantly between the genomes analyzed, and only a low fraction of proteins (4.1%) is conserved between all five species. Furthermore, the inter-specific distribution of SEGs as well as the functional categories shared by species did not reflect their phylogenetic relationships. These results indicate that new SEGs are continuously and independently generated after species divergence over evolutionary time as evidenced by the phylogenetic results of single exon claudins genes. Although the origin of SEGs cannot be inferred directly from the phylogeny, our results provide strong support for the idea that retrotransposition followed by tandem duplications is the most probable event that can explain the expansion of SEGs in eukaryotic organisms. [Copyright &y& Elsevier]

In water of varying ion content, the gills and kidney of fishes contribute significantly to the maintenance of salt and water balance. However, little is known about the molecular architecture of the tight junction (TJ) complex and the regulation of paracellular permeability characteristics in these tissues. In the current studies, puffer fish ( Tetraodon biocellatus) were acclimated to freshwater (FW), seawater (SW) or ion-poor freshwater (IPW) conditions. Following acclimation, alterations in systemic endpoints of hydromineral status were examined in conjunction with changes in gill and kidney epithelia morphology/morphometrics, as well as claudin TJ protein mRNA abundance. T. biocellatus were able to maintain endpoints of hydromineral status within relatively tight limits across the broad range of water ion content examined. Both gill and kidney tissue exhibited substantial alterations in morphology as well as claudin TJ protein mRNA abundance. These responses were particularly pronounced when comparing fish acclimated to SW versus those acclimated to IPW. TEM observations of IPW-acclimated fish gills revealed the presence of cells that exhibited the typical characteristics of gill mitochondria-rich cells (e.g. voluminous, Na-K-ATPase-immunoreactive, exposed to the external environment at the apical surface), but were not mitochondria-rich. To our knowledge, this type of cell has not previously been described in hyperosmoregulating fish gills. Furthermore, modifications in the morphometrics and claudin mRNA abundance of kidney tissue support the notion that spatial alterations in claudin TJ proteins along the nephron of fishes will likely play an important role in the regulation of salt and water balance in these organisms. [ABSTRACT FROM AUTHOR]

The article presents a study which clarifies Mitochondrion-Rich (MR) cells in Tetraodon nigroviridis. It states that MR cells were observed from Tetraodon nigroviridis gill filaments and was subject to whole-mount immunofluorescent staining. It concludes that changes in the phenotypes of the gill MR cells are crucial for responses to salinity challenges in pufferfish.

Abstract: Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. We have previously established a high mortality ISKNV infection model of zebrafish (Danio rerio). In this study, a nonlethal Tetraodon nigroviridis model of ISKNV infection was established. ISKNV infection did not cause lethal disease in Tetraodon but could infect almost all the organs of this species. Electron microscopy showed ISKNV particles were present in infected tissues. Immunofluorescence and quantitative real-time PCR analysis showed that nearly all the virions and infected cells were cleared at 14d postinfection. The expression profiles of interferon-γ and tumor necrosis factor-α gene in response to ISKNV infection were significantly different in Tetraodon and zebrafish. The establishment of the nonlethal Tetraodon model of ISKNV infection can offer a valuable tool complementary to the zebrafish infection model for studying megalocytivirus disease, fish immune systems, and viral tropism. [ABSTRACT FROM AUTHOR]

Abstract: In this paper, we report the cloning and characterization of the STAT6 gene from the pufferfish, Tetraodon nigroviridis. The TnSTAT6 gene is composed of 20 exons and 19 introns. The exon–intron organization of this gene is similar to that of HsSTAT6 except for the exons encoding the C-terminal transactivation domain. The full-length complementary (c)DNA of TnSTAT6 encodes a 794-amino acid protein that is 31% identical to human STAT6. We generated a constitutively active TnSTAT6-JH1 by fusing the kinase domain of carp JAK1 to the C-terminal end of TnSTAT6 and demonstrated that the fusion protein has specific DNA-binding ability and can activate a reporter construct carrying multiple copies of mammalian IL-4-response elements. Interestingly, TnSTAT6-JH1 associated with and phosphorylated TnSTAT6 on Tyr661. Mutation of this residue, Y661W, in TnSTAT6 abolished its association with TnSTAT6-JH1. This is consistent with the importance of the corresponding Tyr641 of HsSTAT6 in tyrosine phosphorylation and dimer formation. On the other hand, treatment of mammalian IL-4 did not induce tyrosine phosphorylation of wild-type TnSTAT6, suggesting that both the divergent N-terminal domain and coiled-coiled domain of TnSTAT6 may affect the interaction of TnSTAT6 with mammalian IL-4 receptor complexes. [Copyright &y& Elsevier]

Abstract: A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na+–K+–ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability. [Copyright &y& Elsevier]

Abstract: In fishes, variation in paracellular permeability is important for regulating salt and water balance. Paracellular permeability is maintained by TJs in vertebrate epithelia. This study examined the spatial distribution and effects of salinity on claudin-3 isoform mRNA expression and abundance along the gastrointestinal (GI) tract of the euryhaline puffer fish (Tetraodon nigroviridis) and related these to morphological heterogeneity of the TJ complex. The puffer fish GI tract was divided into three regions (anterior, middle and posterior) and four isoforms of claudin-3 (Tncldn3a, Tncldn3b, Tncldn3c and Tncldn3d) were found to be expressed in each section. The effect of freshwater (FW) or seawater (SW) acclimation on regional 1) Tncldn3 isoform mRNA abundance, 2) TJ complex morphology and 3) Na+–K+-ATPase (NKA) activity was examined. In situ hybridization indicated that all Tncldn3 isoforms localized to the mucosal epithelium in the intestine. The mRNA abundance of Tncldn3 isoforms varied spatially along the GI tract. Furthermore, region as well as isoform specific alterations in mRNA abundance could be observed along the GI tract in response to salinity change. Qualitative TEM observations suggested that the depth of TJ complexes increased from anterior to posterior along the GI tract and that TJ complexes in the GI tract of FW fish were deeper than those in SW. NKA activity increased from anterior to posterior in fish acclimated to FW, whereas activity in fish acclimated to SW was uniformly high along the length of the intestine. Taken together data; (1) suggest a progressive decrease in epithelial permeability from anterior to posterior along the longitudinal axis of the puffer fish GI tract, (2) indicate that claudin-3 protein isoforms may play a role in regulating paracellular movement of solutes across this epithelium, and (3) provide further evidence that claudin-3 proteins are involved in the homeostatic control of salt and water balance in fishes. [Copyright &y& Elsevier]

Genes encoding for claudin-8 and -27 tight junction proteins in the euryhaline puffer fish ( Tetraodon nigroviridis) were identified using its recently sequenced genome. Phylogenetic analysis indicated that multiple genes encoding for claudin-8 proteins (designated Tncldn8a, Tncldn8b, Tncldn8c and Tncldn8d) arose by tandem gene duplication. In contrast, both tandem and whole genome duplication events appear to have generated genes encoding for claudin-27 proteins (designated Tncldn27a, Tncldn27b, Tncldn27c and Tncldn27d). Tncldn8 and Tncldn27 mRNA were widely distributed in Tetraodon, suggesting involvement in various physiological processes. All Tncldn8 and Tncldn27 genes were expressed in gill and skin tissue (i.e., epithelia exposed directly to the external environment). A potential role for claudin-8 and -27 proteins in the regulation of hydromineral balance in Tetraodon was investigated by examining alterations in mRNA abundance in select ionoregulatory tissue of fish acclimated to freshwater (FW) and seawater (SW). In FW or SW, Tetraodon exhibited alterations in Na+-K+-ATPase activity (a correlate of transcellular transport) typical of a euryhaline teleost fish. Simultaneously, tissue and gene specific alterations in Tncldn8 and Tncldn27 transcript abundance occurred. These data provide some insight into the duplication history of cldn8 and cldn27 genes in fishes and suggest a possible role for claudin-8 and -27 proteins in the osmoregulatory strategies of euryhaline teleosts. [ABSTRACT FROM AUTHOR]

Abstract: The spotted green pufferfish (Tetraodon nigroviridis) is an important genetics model animal due to its small, well-mapped genome. However, only wild-caught juveniles and adults are available to researchers. A lack of gametes, fertilized eggs, developing embryos, and other early life stages hampers development of the full potential of T. nigroviridis as a model research species. We report on successful spawning trials using a novel induced spawning technique, ovarian lavage. Chorulon® (human chorionic gonadotropin, hCG) was injected into a catheter inserted into the oviduct at a rate of 3 μl/g body weight. In one trial, a female paired with a male spawned in an aquarium at about 72 h post-treatment. In other trials, females were hand-stripped of eggs at 36 h post-treatment. There were 3680 eggs/g of eggs and females produced up to 24% of their body weight in eggs. Hatch resulted from all trials on the 4th day post-fertilization. Ovarian lavage is a simple method for administering spawning hormones, uses a catheter technique similar to that frequently performed to determine egg maturity in broodstock, and eliminates the need for injection. [Copyright &y& Elsevier]

Using zebrafish intestinal fatty acid-binding protein 2 ( FABP2) mRNA sequence as the initial query probe, four highly homologous Paralichthys olivaceus EST sequences were retrieved from Genbank database. The assembled full-length cDNA contains the open reading frame of P. olivaceus FABP2 gene, which was validated by subsequent RT-PCR cloning. In the coding region, the average GC content is 56%, but it would reach 76.8% if only counting for the third base of the codons. The deduced P. olivaceus FABP2 polypeptide contains 132 amino acids (aa), with a predicted molecular size of 15.3 kD and pI at 6.74. This protein multiple-alignment has shown that this peptide is 75.7% identical to the corresponding homologous protein in Danio rerio. Among the 7 aa that are essential for FABP2 function, 3 were found to be conserved among P. olivaceus, Danio rerio, Tetraodon nigroviridi, Rattus norvegicus, and Homo sapiens. The study provides essential information on molecular evolution and function of FABP family. [ABSTRACT FROM AUTHOR]

Na+/K+-ATPase (NKA) is a ubiquitous membrane-bound protein crucial for teleost osmoregulation. The enzyme is composed of two essential subunits, a catalytic a subunit and a glycosylated 3 subunit which is responsible for membrane targeting of the enzyme. In mammals, seven FXYD members have been found. FXYD proteins have been identified as the regulatory protein of NKA in mammals and elasmobranchs, it is thus interesting to examine the expression and functions of FXYD protein in the euryhaline teleosts with salinity-dependent changes of gill NKA activity. The present study investigated the expression and distribution of the FXYD protein in gills of seawater (SW)- or freshwater (FW)-acclimated euryhaline pufferfish (Tetraodon nigroviridis). The full-length pufferfish FXYD gene (pFXYD) was confirmed by RT-PCR. pFXYD was found to be expressed in many organs including gills of both SW and FW pufferfish. pFXYD mRNA abundance in gills, determined by real-time PCR, was significantly higher in FW fish than in SW fish. An antiserum raised against a partial amino acid sequence of pFXYD was used for the immunoblots of gill homogenates and a major band at 13kDa was detected. The relative amounts of pFXYD protein and mRNA in gills of SW and FW pufferfish were identical, but opposite to the expression levels of NKA. Immunofluorescent staining of frozen sections demonstrated that pFXYD was colocalized to NKA-immunoreactive cells in the gill filaments. In addition, interaction between pFXYD and NKA was demonstrated by co-immunoprecipitation. Taken together, salinity-dependent expression of pFXYD protein and NKA, as well as the evidence for their colocalization and interaction in pufferfish gills suggested that pFXYD regulates NKA activity in gills of euryhaline teleosts upon salinity challenge. [ABSTRACT FROM AUTHOR]

Large numbers of sequence elements have been identified to be highly conserved among vertebrate genomes. These highly conserved elements (HCEs) are often located in or around genes that are involved in transcription regulation and early development. They have been shown to be involved in cis-regulatory activities through both in vivo and additional computational studies. We have investigated the structural relationships between such elements and genes in six vertebrate genomes human, mouse, rat, chicken, zebrafish and tetraodon and detected several thousand cases of conserved HCE-gene associations, and also cases of HCEs with no common target genes. A few examples underscore the potential significance of our findings about several individual genes. We found that the conserved association between HCE/HCEs and gene/genes are not restricted to elements by their absolute distance on the genome. Notably, long-range associations were identified and the molecular functions of the associated genes do not show any particular overrepresentation of the functional categories previously reported. HCEs in close proximity are found to be linked with different set of gene/genes. The results reflect the highly complex correlation between HCEs and their putative target genes. [ABSTRACT FROM AUTHOR]

Abstract: Clock (Circadian locomotor output cycle kaput) was the first vertebrate circadian clock gene identified in a mouse forward genetics mutagenesis screen. It encodes a bHLH-PAS protein that is highly conserved throughout evolution. Tetrapods also have the second Clock gene, Clock2 or Npas2 (Neuronal PAS domain protein 2). Conversely, the fruit fly, an invertebrate, has only one clock gene. Interrogation of the five teleost fish genome databases revealed that the zebrafish and the Japanese pufferfish (fugu) each have three clock genes, whereas the green spotted pufferfish (tetraodon), the Japanese medaka fish and the three-spine stickleback each have two clock genes. Phylogenetic and splice site analyses indicated that zebrafish and fugu each have two clock1 genes, clock1a and clock1b and one clock2; tetraodon also have clock1a and clock1b but do not have clock2; and medaka and stickleback each have clock1b and one clock2. Genome neighborhood analysis further showed that clock1a/clock1b in zebrafish, fugu and tetraodon is an ancient duplicate. While the dN/dS ratios of these three fish clock duplicates are all <1, indicating that purifying selection has acted upon them; the Tajima relative rate test showed that all three fish clock duplicates have asymmetric evolutionary rates, implicating that one of these duplicates have been under positive selection or relaxed functional constraint. These results support the view that teleost fish clock genes were generated from an ancient genome-wide duplication, and differential gene loss after the duplication resulted in retention of different ancient duplicates in different teleost fishes, which could have contributed to the evolution of the distinct fish circadian clock mechanisms. [Copyright &y& Elsevier]

Abstract: The eye lens of the Antarctic toothfish living in the −2 °C Southern Ocean is cold-stable. To investigate the molecular basis of this cold stability, we isolated, cloned and sequenced 22 full length crystallin cDNAs. We found two α crystallins (αA, αB), six β crystallins (βA1, βA2, βA4, βB1, βB2, βB3) and 14 γ crystallins (γN, γS1, γS2, γM1, γM3, γM4, γM5, γM7, γM8a, γM8b, γM8c, γM8d, γM8e, and γM9). Alignments of α, β and γ with other known crystallin sequences indicate that toothfish α and β crystallins are relatively conserved orthologues of their vertebrate counterparts, but the toothfish and other fish γM crystallins form a distinct group that are not orthologous to mammalian γ crystallins. A preliminary Fingerprinted Contig analysis of clones containing crystallin genes screened from a toothfish BAC library indicated α crystallin genes occurred in a single genomic region of ~266 kbp, β crystallin genes in ~273 kbp, while the γ crystallin gene family occurred in two separate regions of ~180 and ~296 kbp. In phylogenetic analysis, the γM isoforms of the ectothermic toothfish displayed a diversity not seen with endothermic mammalian γ crystallins. Similar to other fishes, several toothfish γ crystallins are methionine-rich (γM isoforms) which may have predisposed the toothfish lens to biochemically attenuate γ crystallin hydrophobicity allowing for cold adaptation. In addition to high methionine content, conservation of αβ crystallins both in sequence and abundance suggests greater functional constraints relative to γ crystallins. Conversely, reduced constraints upon γ crystallins could have allowed for greater evolutionary plasticity resulting in increased polydispersity of γ crystallins contributing to the cold-stability of the Antarctic toothfish lens. [Copyright &y& Elsevier]

Claudins are a large family of integral transmembrane tight junction (TJ) proteins involved in regulating the permeability of the paracellular pathway. In these studies, we clone and describe the tissue distribution of four claudin-3 genes (designated Tnc/dn3a, Tncldn3b, Tncldn3c, and Tncldn3d) from the euryhaline spotted green puffer fish Tetraodon nigroviridis and examine the response of Tetraodon and Tncldn3 mRNAs to salinity variation (freshwater, FW; seawater, SW; and hypersaline seawater, HSW). In Tetraodon, genes encoding for claudin-3 Ti proteins are widely expressed, suggesting that claudin-3 proteins participate in regulating paracellular permeability across various epithelia within fishes. Of particular note is the widespread distribution of Tncldn3 genes in tissues that regulate hydromineral balance (gills, skin, kidney, and intestine). Renal and intestinal tissues express all four Tncldn3 genes, while the gills and skin specifically express Tncldn3a and Tncldn3c. In response to salinity variation, Tetraodon exhibits characteristics typical of a euryhaline fish species: moderate changes in blood osmolality and muscle moisture content; alterations in gill, kidney, and intestinal Na+-K+-ATPase activity; and unaltered Na+-K+-ATPase activity in the integument. In conjunction with these changes, Tncldn3 mRNA expression exhibits marked and significant salinity-dependent alterations that are both tissue and gene specific. Overall, our data suggest that a decreased abundance of claudin-3 in Tetraodon occurs in "leakier" epithelia and that claudin-3 TJ proteins will likely play an important role in the maintenance of hydromineral balance across osmoregulatory epithelia of euryhaline fishes. [ABSTRACT FROM AUTHOR]

Abstract: The DHPC (DNA Hilbert–Peano curve) is a new tool for visualizing large-scale genome sequences by mapping sequences into a two-dimensional square. It utilizes the space-filling function of Hilbert–Peano mapping. By applying a Gauss smoothing technique and a user-defined color function, a large-scale genome sequence can be mapped into a two-dimensional color image. In the calculated DHPCs, many genome characteristics are revealed. In this article we introduce the method and show how DHPCs may be used to identify regions of different base composition. The power of the method is demonstrated by presenting multiple examples such as repeating sequences, degree of base bias, regions of homogeneity and their boundaries, and mark of annotated segments. We also present several genome curves generated by DHPC to demonstrate how DHPC can be used to find previously unidentified sequence features in these genomes. [Copyright &y& Elsevier]