TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.

Integrating Genomic and Chromosomal Data: A Cytogenetic Study of Transancistrus santarosensis (Loricariidae: Hypostominae) with Characterization of a ZZ/ZW Sex Chromosome System.

The plecos (Loricariidae) fish represent a great model for cytogenetic investigations due to their variety of karyotypes, including diploid and polyploid genomes, and different types of sex chromosomes. In this study we investigate Transancistrus santarosensis a rare loricariid endemic to Ecuador, integrating cytogenetic methods with specimens' molecular identification by mtDNA, to describe the the species karyotype. We aim to verify whether sex chromosomes are cytologically identifiable and if they are associated with the accumulation of repetitive sequences present in other species of the family. The analysis of the karyotype (2n = 54 chromosomes) excludes recent centric fusion and pericentromeric inversion and suggests the presence of a ZZ/ZW sex chromosome system at an early stage of differentiation: the W chromosome is degenerated but is not characterized by the presence of differential sex-specific repetitive DNAs. Data indicate that although T. santarosensis has retained the ancestral diploid number of Loricariidae, it accumulated heterochromatin and shows non-syntenic ribosomal genes localization, chromosomal traits considered apomorphic in the family.

Species tree analyses and speciation-based species delimitation support new species in the relict catfish family Diplomystidae and provide insights on recent glacial history in Patagonia.

Diplomystidae is an early-diverged family of freshwater catfish endemic to southern South America. We have recently collected five juvenile specimens belonging to this family from the Bueno River Basin, a basin which the only previous record was a single juvenile specimen collected in 1996. This finding confirms the distribution of the family further South in northern Patagonia, but poses new questions about the origin of this population in an area with a strong glacial history. We used phylogenetic analyses to evaluate three different hypotheses that could explain the origin of this population in the basin. First, the population could have originated in Atlantic basins (East of the Andes) and dispersed to the Bueno Basin after the Last Glacial Maximum (LGM) via river reversals, as it has been proposed for other population of Diplomystes as well as for other freshwater species from Patagonia. Second, the population could have originated in the geographically close Valdivia Basin (West of the Andes) and dispersed south to its current location in the Bueno Basin. Third, regardless of its geographic origin (West or East of the Andes), the Bueno Basin population could have a longer history in the basin, surviving in situ through the LGM. In addition, we conducted species delimitation analyses using a recently developed method that uses a protracted model of speciation. Our goal was to test the species status of the Bueno Basin population along with another controversial population in Central Chile (Biobío Basin), which appeared highly divergent in previous studies with mtDNA. The phylogenetic analyses showed that the population from the Bueno Basin is more related to Atlantic than to Pacific lineages, although with a deep divergence that predated the LGM, supporting in situ survival rather than postglacial dispersal. In addition, these analyses also showed that the species D. nahuelbutaensis is polyphyletic, supporting the need for a taxonomic reevaluation. The species delimitation analyses supported two new species which are described using molecular diagnostic characters: Diplomystes arratiae sp. nov. from the Biobío, Carampangue, and Laraquete basins, maintaining D. nahuelbutaensis valid only for the Imperial Basin, and Diplomystes habitae sp. nov. from the Bueno Basin. This study greatly increases the number of species within both the family Diplomystidae and Patagonia, and contributes substantially to the knowledge of the evolution of southern South American freshwater biodiversity during its glacial history. Given the important contribution to the phylogenetic diversity of the family, we recommend a high conservation priority for both new species. Finally, this study highlights an exemplary scenario where species descriptions based only on DNA data are particularly valuable, bringing additional elements to the ongoing debate on DNA-based taxonomy.

Characterization of fifteen key genes involved in iron metabolism and their responses to dietary iron sources in yellow catfish Pelteobagrus fulvidraco.

BACKGROUND: Iron is an essential metal element for organisms, whose metabolism is regulated by many genes and also dietary iron sources. However, the characterization, distribution and the responses of iron metabolism-related genes to different iron sources were not clear in fish. METHODS: The full-length cDNA sequences of fifteen iron metabolism-relevant genes (tf, tfr1, hp, fpn1, ho1, ho2, tfr2, hjv, hepcidin, fth, ftl, ftm, irp1, irp2 and hif2α.) were obtained via 3' and 5' RACE PCR from yellow catfish, a widely distributed freshwater teleost in China and other Asian countries. Their molecular characterizations were analyzed via the bioinformatic methods. Real-time quantitative PCR was used to explore their mRNA distribution in nine tissues. Their mRNA expression responses in four tissues (heart, brain, kidney and gill) were explored in yellow catfish fed diets with five iron sources, including ferrous sulfate (FeSO4), ferrous bisglycinate (Fe-Gly), ferrous chloride (FeCl2), ferric citrate (Fe-CA) and ferric oxide nanoparticles (Fe2O3NPs). RESULTS: Compared with mammals and other teleost, these members shared similar domains. Their mRNAs were expressed in nine tested tissues, but mRNA levels varied. Yellow catfish fed the diets containing Fe-Gly and Fe2O3NPs had higher iron contents in heart, brain, kidney and gill. Meantime, different dietary iron sources addition affected their mRNA expression differentially in brain, heart, kidney and gill. It should be pointed out that only three biological replicate tanks were used in the present feeding treatment, and more biological replicate tanks (more than five) should be emphasized in further researches. CONCLUSION: Taken together, our study identified fifteen iron metabolism-relevant genes, explored their mRNA expression in nine tissues, and their mRNA expression in the responses to different dietary iron sources in four tissues, indicating their important regulatory function in iron metabolism and homeostasis.

Characterization and tissue expression of twelve selenoproteins in yellow catfish Pelteobagrus fulvidraco fed diets varying in oxidized fish oil and selenium levels.

BACKGROUND: Selenium (Se) functions through selenoproteins and is essential to growth and metabolism of vertebrates. The present study was conducted to identify twelve selenoproteins genes (selenoe, selenof, selenoh, selneoi, selenom, selenok, selneon, selenoo, selenot, selenos, selenou and msrb1) from yellow catfish. Their mRNA expression patterns, as well as their response to dietary oxidized fish oils and Se addition were explored. METHODS: We use 3'and 5' RACE PCR to clone full-length cDNA sequence of twelve selenoprotein genes from yellow catfish. Their mRNA expression patterns were assessed via quantitative real-time PCR. Yellow catfish were fed diet adequate Se+ fresh fish oil, adequate Se+ oxidized fish oil, high Se+ fresh fish oil and high Se+ oxidized fish oil, respectively, for 10 weeks. Their kidney, heart, brain and testis were used to assess the mRNA expression of twelve selenoprotein. RESULTS: Twelve selenoprotein genes had similar domains with mammals and the other fish. Their mRNAs were expressed widely in eleven tissues but varied with the tissues. Dietary oxidized fish oils and Se addition influenced their mRNA abundances of twelve selenoproteins in a tissue-dependent manner. CONCLUSION: Our study demonstrated the characterization and expression of twelve selenoproteins, and elucidated their responses in yellow catfish fed diets varying in oxidized fish oils and Se addition, which increased our knowledge into the biological function and regulatory mechanism of Se and selenoproteins in fish.

Molecular Characterization of Nine TRAF Genes in Yellow Catfish (Pelteobagrus fulvidraco) and Their Expression Profiling in Response to Edwardsiella ictaluri Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.

Hematological, immuno-antioxidant disruptions, and genes down-regulation induced by Aeromonas veronii challenge in Clarias gariepinus: The ameliorative role of silica nanoparticles.

Aeromonas veronii is a pathogenic bacterium associated with various diseases in aquaculture. However, few studies address the antibacterial activity using nanoparticles (NPs). Hence, the current study is innovative to evaluate the antibacterial efficacy of silica nanoparticles (SiNPs) against A. veronii infection in-vitro with a trial for treatment in-vivo. Primarily, we assessed the in-vitro antibacterial activity against A. veronii. Further, we investigated the hematological profile, immune-antioxidant response, and gene expression of African catfish (Clarias gariepinus) in response to SiNPs exposure and the A. veronii challenge. Fish (N = 120; weight: 90 ± 6.19 g) were distributed into four groups (30 fish/group) for a ten-days-treatment trial. The first (control) and second (SiNPs) groups were treated with 0 mg/L and 20 mg/L SiNPs in water, respectively. The third (A. veronii) and fourth (SiNPs + A. veronii) groups were treated with 0 mg/L and 20 mg/L SiNPs in water, respectively, and infected with A. veronii (1.5 × 107 CFU/mL). Results demonstrated that SiNPs displayed an in-vitro antibacterial activity against A. veronii with a 21 mm inhibitory zone. A. veronii infection caused a high mortality rate (56.67%) and substantial reductions in hematological indices and immune indicators [nitric oxide (NO) and immunoglobulin M (IgM)]. Additionally, marked decline in the level of antioxidants [superoxide dismutase (SOD), catalase (CAT), and reduced glutathione content (GSH)] as well as down-regulation in the immune-related genes [interleukins (IL-1ß and IL-8) and tumor necrosis factor-alpha (TNF-α)] and antioxidant-related genes [SOD1, glutathione peroxidase (GPx), and glutathione-S-transferase (GST)] were the consequences of A. veronii infection. Surprisingly, treatment of A. veronii-infected fish with SiNPs lessened the mortality rate, enhanced the blood picture, modulated the immune-antioxidant parameters, and resulted in gene up-regulation. Overall, this study encompasses the significant role of SiNPs, a new versatile tool for combating hematological, immuno-antioxidant alterations, and gene down-regulation induced by A. veronii infection and sustainable aquaculture production.

Gonadal transcriptomes reveal sex-biased expression genes associated with sex determination and differentiation in red-tail catfish (Hemibagrus wyckioides).

BACKGROUND: Red-tail catfish (Hemibagrus wyckioides) is an important commercially farmed catfish in southern China. Males of red-tail catfish grow faster than females, suggesting that all-male catfish will produce more significant economic benefits in aquaculture practice. However, little research has been reported on sex determination and gonadal development in red-tail catfish. RESULTS: In this study, we performed the first transcriptomic analysis of male and female gonads at four developmental stages at 10, 18, 30, and 48 days post hatching (dph) using RNA-seq technology. A total of 23,588 genes were screened in 24 sequenced samples, of which 28, 213, 636, and 1381 differentially expressed genes (DEGs) were detected at four developmental stages, respectively. Seven candidate genes of sex determination and differentiation were further identified. Real-time quantitative PCR (RT-qPCR) further confirmed that anti-Mullerian hormone (amh), growth differentiation factor 6a (gdf6a), testis-specific gene antigen 10 (tsga10), and cytochrome P450 family 17 subfamily A (cyp17a) were highly expressed mainly in the male, while cytochrome P450 family 19 subfamily A polypeptide 1b (cyp19a1b), forkhead box L2 (foxl2), and hydroxysteroid 17-beta dehydrogenase 1 (hsd17b1) were highly expressed in the female. The KEGG pathway enrichment data showed that these identified DEGs were mainly involved in steroid hormone biosynthesis and TGF-ß signaling pathways. CONCLUSIONS: Based on RNA-seq data of gonads at the early developmental stages, seven DEGs shared by the four developmental stages were identified, among which amh and gdf6a may be the male-biased expression genes, while foxl2, cyp19a1b and hsd17b1 may be the female-biased expression genes in red-tail catfish. Our study will provide crucial genetic information for the research on sex control in red-tail catfish, as well as for exploring the evolutionary processes of sex determination mechanisms in fish.

Yellow catfish RIO kinases (RIOKs) negatively regulate fish interferon-mediated antiviral response.

In mammals, right open reading frame kinases (RIOKs) are initially reported to participate in cancer cell proliferation, apoptosis, migration and invasion, and recently they have been related to host immune response. Little is known about the homologs of RIOKs in fish. In the current study, we cloned three homologous genes of RIOK family in yellow catfish (Pelteobagrus fulvidraco), termed Pfriok1, Pfriok2 and Pfriok3. Pfriok1, Pfriok2 and Pfriok3 were constitutively expressed at relatively high levels in yellow catfish tissues, and their mRNA levels were not changed under viral infection. Individual overexpression of PfRIOK1, PfRIOK2 and PfRIOK3 attenuated fish interferon (IFN) response, thereby promoting viral replication in fish cells. Mechanistically, yellow catfish RIOK proteins downregulated fish IFN response through attenuating TBK1 protein levels in cytoplasm. Our findings suggest that yellow catfish RIOK1, RIOK2 and RIOK3 are involved in downregulating fish IFN antiviral response.

Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.

The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a Km of 304.40 µM and GR activity with a Km of 58.91 µM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress.

Effects of Dietary Glycinin on Oxidative Damage, Apoptosis and Tight Junction in the Intestine of Juvenile Hybrid Yellow Catfish, Pelteobagrus fulvidraco ♀ × Pelteobaggrus vachelli ♂.

The objective of this study was to examine the influences of glycinin for growth and intestinal structural integrity related to oxidative damage, apoptosis and tight junction of juvenile hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × Pelteobaggrus vachelli ♂). Fish (initial weight, 1.02 ± 0.01 g) were fed diets containing five different levels of glycinin at 0%, 2%, 4%, 6%, and 8% for 8 weeks. The results demonstrated that dietary glycinin levels had a negative correlation with final weight, feed intake, protein efficiency ratio and survival rate of the experiment fish. When the level of dietary glycinin exceeded 4%, the structural integrity of the posterior intestine was observably impaired, characterized by disordered and exfoliated margin of intestinal villi, blurred and broken boundaries of tight junctions, damaged organelles and cell vacuolation. Levels of 4-8% dietary glycinin depressed the total antioxidant capacity and total superoxide dismutase activities of posterior intestine. Furthermore, a high level of dietary glycinin linearly and quadratically down-regulated the mRNA expressions of Claudin-1, Occludin and ZO-1, while it linearly and significantly up-regulated the mRNA expressions of Bax, Cyt C, Caspase 3, Caspase 9 and p53 in the posterior intestine. In conclusion, dietary 4-8% glycinin impaired the morphological structure of the posterior intestine by inducing oxidative stress and cell apoptosis, and eventually impeded the growth performance of juvenile hybrid yellow catfish.

Genome-wide identification of the traf gene family in yellow catfish (Pelteobagrus fulvidraco) and analysis of their expression in response to bacterial challenge.

Tumour necrosis factor (TNF) receptor-associated factor (TRAF) is a receptor protein that has important functions in the immune system. Nonetheless, there have been few reports of traf genes in teleost fishes. The present study aimed to identify the traf genes from the genomic information of yellow catfish (Pelteobagrus fulvidraco). Eight traf genes were identified and named, which are distributed on different chromosomes but have similar conserved protein domains. Phylogenetic and syntenic analyses demonstrated conservation of traf genes during evolution. In addition, yellow catfish has the relatively rare traf1 and traf5 genes. Gene structure and motif analysis revealed the homology and distribution diversity of the traf genes. Quantitative real-time reverse transcription PCR was used to study the expression patterns of traf genes in healthy fish tissues and after infection by Aeromonas hydrophila. The results demonstrated significant changes in traf gene expression, indicating a potential role in innate immunity.

The antibacterial activity of a novel NK-lysin homolog and its molecular characterization and expression in the striped catfish, Pangasianodon hypophthalmus.

Aeromonas hydrophila was a common bacterial pathogen in aquaculture resulting in considerable losses to the striped catfish aquaculture industry. As an emergent antimicrobial peptide (AMP), NK-lysin (NKL) had activity against various microorganisms. However, the antibacterial activity of NKL from striped catfish (Pangasianodon hypophthalmus) both in vitro and vivo remains unclear. In this study, the cDNA sequence of P. hypophthalmus NK-lysin gene (PhNK-lysin) was cloned and characterized. The amino acid sequence of PhNK-lysin contains a signal peptide sequence of 17 amino acid (aa) residues and a mature peptide composed of 130 aa. The saposin B domain of mature peptide comprised six conserved cysteines forming three putative disulfide bonds. Phylogenetic analysis revealed that the PhNK-lysin was most closely related to that of the channel catfish (Ictalurus punctatus) NK-lysin. The transcriptional levels of the PhNK-lysin were significantly upregulated in response to A. hydrophila infection in various tissues including heart, liver, spleen, head kidney, trunk kidney and gill. The synthetic PhNK-lysin-derived peptide consisting of 38aa showed antibacterial activity against Vibrio harveii, Aeromonas hydrophila and Escherichia coli. The MIC for V. harveii, A. hydrophila and E. coli were 15.625 µM, 250 µM and 31.25 µM respectively. Besides, the synthetic PhNK-lysin decreased the bacterial load of liver and trunk kidney in vivo as well as increased the survival rate of A. hydrophila infected striped catfish. Hence, these data suggest that PhNK-lysin had antimicrobial effect and protects the host from pathogenic infection.

An ancient truncated duplication of the anti-Müllerian hormone receptor type 2 gene is a potential conserved master sex determinant in the Pangasiidae catfish family.

The evolution of sex determination (SD) in teleosts is amazingly dynamic, as reflected by the variety of different master sex-determining genes identified. Pangasiids are economically important catfishes in South Asian countries, but little is known about their SD system. Here, we generated novel genomic resources for 12 Pangasiids and characterized their SD system. Based on a Pangasianodon hypophthalmus chromosome-scale genome assembly, we identified an anti-Müllerian hormone receptor type Ⅱ gene (amhr2) duplication, which was further characterized as being sex-linked in males and expressed only in testes. These results point to a Y chromosome male-specific duplication (amhr2by) of the autosomal amhr2a. Sequence annotation revealed that the P. hypophthalmus Amhr2by is truncated in its N-terminal domain, lacking the cysteine-rich extracellular part of the receptor that is crucial for ligand binding, suggesting a potential route for its neofunctionalization. Reference-guided assembly of 11 additional Pangasiids, along with sex-linkage studies, revealed that this truncated amhr2by duplication is a male-specific conserved gene in Pangasiids. Reconstructions of the amhr2 phylogeny suggested that amhr2by arose from an ancient duplication/insertion event at the root of the Siluroidei radiation that is dated to ~100 million years ago. Together these results bring multiple lines of evidence supporting that amhr2by is an ancient and conserved master sex-determining gene in Pangasiids, a finding that highlights the recurrent use of the transforming growth factor ß pathway, which is often used for the recruitment of teleost master SD genes, and provides another empirical case towards firther understanding of dynamics of SD systems.

Redeployment of odontode gene regulatory network underlies dermal denticle formation and evolution in suckermouth armored catfish.

Odontodes, i.e., teeth and tooth-like structures, consist of a pulp cavity and dentin covered by a mineralized cap. These structures first appeared on the outer surface of vertebrate ancestors and were repeatedly lost and gained across vertebrate clades; yet, the underlying genetic mechanisms and trajectories of this recurrent evolution remain long-standing mysteries. Here, we established suckermouth armored catfish (Ancistrus sp.; Loricariidae), which have reacquired dermal odontodes (dermal denticles) all over most of their body surface, as an experimental model animal amenable to genetic manipulation for studying odontode development. Our histological analysis showed that suckermouth armored catfish develop dermal denticles through the previously defined odontode developmental stages. De novo transcriptomic profiling identified the conserved odontode genetic regulatory network (oGRN) as well as expression of paired like homeodomain 2 (pitx2), previously known as an early regulator of oGRN in teeth but not in other dermal odontodes, in developing dermal denticles. The early onset of pitx2 expression in cranial dermal denticle placodes implies its function as one of the inducing factors of the cranial dermal denticles. By comprehensively identifying the genetic program for dermal odontode development in suckermouth armored catfish, this work illuminates how dermal odontodes might have evolved and diverged in distinct teleost lineages via redeployment of oGRN.

Extra-Fortification of Zinc Upsets Vitellogenin Gene Expression and Antioxidant Status in Female of Clarias magur brooders.

The present experiment was designed to evaluate the effect of graded level of zinc on Vitellogenin gene (Vtg) expression and antioxidant enzymes in threatened catfish, Clarias magur (C. magur). One hundred and eighty female C. magur with an average weight of 145 ± 5 g were allocated in twelve cemented tanks with dimension 4.5 × 2 × 1 m for a period of 60 days. Fish were distributed in four groups with three replicates following the completely randomised design. The first group treated as control (C) fed with basal diet contained normal zinc level, and remaining groups were fed with basal diets having 50, 200 and 300 mg/kg zinc acetate and treated as T1, T2 and T3 respectively. To evaluate the effect of dietary zinc supplementation on Vtg gene expression, three sampling were carried out, I sampling (April, before starting the experimental trail), II sampling (May, after 1 month of feeding trail) and III sampling (June before breeding season). In the present study, a dose-dependent relationship between Vtg gene expression and zinc inclusion in the diet of threatened catfish, C. magur, was reported. Vtg gene expression increased in all groups from I sampling to II sampling but the highest Vtg gene expression was found in T1 group and the lowest in T3 group at II sampling. Vtg gene expression among the treatments differs significantly (P < 0.05) in each sampling. Accumulation of zinc was measured by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) in C. magur and it was reported that the significantly higher (P < 0.05) zinc was accumulated in the liver and ovary of T3 group as compared to other groups. The antioxidant enzyme activities (superoxide dismutase, SOD, catalase and GST) were also measured in different tissues (liver, gill and ovary) to evaluate the effect of extra-supplementation of zinc on the antioxidant status. In T3 group, SOD, catalase and GST activities were significantly higher than those in other groups. In the current study, serum glucose level was also measured and it was found in increasing trend with inclusion of zinc in the diet of C. magur. In the present study, it can be concluded that the zinc exhibits beneficial effect only up to 50 mg/kg. Thus, it is concluded that supplementation of zinc at 200 mg/kg or more disrupts Vtg gene expression and antioxidant status.

In silico analysis of kiss2, expression studies and protein-protein interaction with gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHß) in Heteropneustes fossilis.

Kisspeptins, encoded by the kiss genes, are neuropeptides that regulate the onset of puberty, maturation of gonads, and fertility in higher vertebrates including fishes. The gene ontology suggests that kisspeptin plays an important role not only in reproduction but also in cell signaling, immune response and metabolic processes, and to decipher protein-protein interactions, computational approach has been favored. The present investigation focuses on the detailed structural analysis and molecular docking of kiss2 gene using in silico tools. A putative kiss2 protein of 113 amino acids was encoded by an open reading frame of 342 bp kiss2 gene. The protein is of 13.12 kDa with isoelectric point of 9.45. The secondary structure of the protein indicates more than 50% random coils, followed by 34% of alpha helix and 13% extended strand. The protein was found to be extracellular and secretory in nature. Since, protein-protein interactions play a very crucial role in every cellular process and due to unavailability of crystal structure of our protein of interest in fishes computational approach has been employed. The 3D PDB modeling and the molecular docking of kiss2, Gonadotropin-releasing hormone 2 (GnRH2) and luteinizing hormone beta (LHß) proteins in fishes have been demonstrated applying protein-docking approach. Molecular interactions of kiss2 protein were the highest with kisspeptin receptor 2 and lowest for the neuropeptide FF-amide peptide precursor protein. Expression of kiss2 transcripts, mainly in the brain and ovary of H. fossilis, supports its hypothalamic-pituitary-gonadal axis signaling and reproductive function. Further, changes in expression patterns of kiss2 mRNA during different developmental stages, indicate its potential role in embryonic development also. The present study conclusively reveals interaction of kiss2 with other neuropeptides. Prediction of binding structures and identification of key residues in protein-protein interaction illustrate direct interaction among these proteins, playing a cardinal role in neuroendocrine regulation of reproduction in catfish. Communicated by Ramaswamy H. Sarma.

Review of the armoured catfish genus Hypostomus (Siluriformes: Loricariidae) from the Parnaíba River basin, Northeastern Brazil, with description of a new species

The species of Hypostomus from the Parnaíba River basin were reviewed through molecular and morphological analysis. Five species were found in the basin, including a new species herein described. The distribution of H. pusarum was expanded to this basin, and a closely related species was recorded (H. aff. pusarum), also the presence of H. johnii and H. vaillanti was confirmed. The new species is distinguished from most congeners by its large number of premaxillary and dentary teeth, a wide dental angle of 115° to 135°, presence of a rounded dark spots on a lighter background and anteromedial region of the abdomen depleted of plaques (vs. anteromedial region of the abdomen covered by platelets and odontodes in H. johnii, H. pusarum, H. aff. pusarum and H. vaillanti). Furthermore, an identification key of the species from the Maranhão-Piauí ecoregion and maps with the geographic distribution of these species are presented. The species of Hypostomus in the Parnaíba River basin have different geographic distributions, suggesting different niches or geographical barriers, providing an opportunity for ecological and evolutionary studies.(AU)

As espécies de Hypostomus da bacia do rio Parnaíba foram revisadas por meio de análises moleculares e morfológicas. Cinco espécies foram encontradas na bacia, incluindo uma nova espécie aqui descrita. A distribuição de H. pusarum foi expandida para esta bacia, uma espécie intimamente relacionada foi registrada (H. aff. pusarum), e a presença de H. johnii e H. vaillanti foi confirmada. A nova espécie se distingue da maioria das congêneres por seu grande número de dentes nos pré-maxilares e dentários, um amplo ângulo do dentário de 115° a 135°, presença de manchas escuras arredondadas em um fundo mais claro e região anteromedial do abdômen sem placas (vs. região anteromedial do abdômen coberta por placas e odontódios em H. johnii, H. pusarum, H. aff. pusarum e H. vaillanti). Além disso, é apresentada uma chave de identificação das espécies da ecorregião Maranhão-Piauí e mapas com a distribuição geográfica dessas espécies. As espécies de Hypostomus na bacia do rio Parnaíba apresentam diferentes distribuições geográficas, sugerindo diferentes nichos ou barreiras geográficas, proporcionando oportunidade para estudos ecológicos e evolutivos.(AU)

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Seasonal expression and distribution of kisspeptin1 (kiss1) in the ovary and testis of freshwater catfish, Clarias batrachus: A putative role in steroidogenesis.

The central role of kisspeptin (kiss) in mammalian reproduction is well established; however, its intra-gonadal role is poorly addressed. Moreover, studies investigating intra-gonadal role of kiss in fish reproduction are scanty, contradictory and inconclusive. The expression of kiss1 mRNA has been detected in the fish brain, and functionally attributed to the regulation of reproduction, feeding and behavior. The kiss1 mRNA has also been demonstrated in tissues other than the brain in some studies, but its cellular distribution and role at the tissue level have not been adequately addressed in fish. Therefore, an attempt was made in the present study to localize kiss1 in gonadal cells of the freshwater catfish, Clarias batrachus. This study reports the presence of kiss1 in the theca cells and granulosa cells of the ovarian oocytes and interstitial cells in the testis of the catfish. The role of kiss1 in the ovary and testis of the catfish was also investigated using kiss1 receptor (kiss1r) antagonist (p234). The p234 treatment decreased the production of 17ß-estradiol in ovary and testosterone in the testis by lowering the activities of 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase under both, in vivo as well as in vitro conditions. The p234 treatment also arrested the progression of oogenesis, as evident from the low number of advancing/advanced oocytes in the treated ovary in comparison to the control ovary. It also reduced the area and perimeter of the seminiferous tubules in the treated catfish testis. Thus, our findings suggest that kiss is involved in the regulation of gonadal steroidogenesis, independent of known endocrine/ autocrine/ paracine regulators, and thereby it accelerates gametogenic processes in the freshwater catfish.