Adenylate kinase 1 overexpression increases locomotor activity in medaka fish.

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

The pink salmon genome: Uncovering the genomic consequences of a two-year life cycle.

Pink salmon (Oncorhynchus gorbuscha) adults are the smallest of the five Pacific salmon native to the western Pacific Ocean. Pink salmon are also the most abundant of these species and account for a large proportion of the commercial value of the salmon fishery worldwide. A two-year life history of pink salmon generates temporally isolated populations that spawn either in even-years or odd-years. To uncover the influence of this genetic isolation, reference genome assemblies were generated for each year-class and whole genome re-sequencing data was collected from salmon of both year-classes. The salmon were sampled from six Canadian rivers and one Japanese river. At multiple centromeres we identified peaks of Fst between year-classes that were millions of base-pairs long. The largest Fst peak was also associated with a million base-pair chromosomal polymorphism found in the odd-year genome near a centromere. These Fst peaks may be the result of a centromere drive or a combination of reduced recombination and genetic drift, and they could influence speciation. Other regions of the genome influenced by odd-year and even-year temporal isolation and tentatively under selection were mostly associated with genes related to immune function, organ development/maintenance, and behaviour.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus.

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3-10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3-10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3-10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3-10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3-10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio alginolyticus infection.

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.

Fish TNF and TNF receptors.

Tumor necrosis factors (TNFs) are a group of cytokines that play critical roles in regulating a diverse range of physiological processes in vertebrates. TNFs function by activating a large number of structurally related receptors, leading to TNF mediated biological processes which are evolutionarily conserved. Fish have a much diversified TNF family, partly due to the whole genome duplication events which have occurred in this lineage, providing an excellent model to investigate the neo- and sub-functionalised properties of TNF superfamily. Fish possess most of the TNFs and receptors found in mammals and also some homologues exclusively present in fish. It seems that TNFSF4 (OX40), TNFSF7 (CD27) and TNFSF8 (CD30) and their cognate receptors are absent in teleosts. It has been shown that fish viruses are able to produce TNFR homologues to establish infection by manipulating the host immune system. Understanding the roles of TNFSFs in fish immune defence and the pathogenesis of fish diseases will provide insights into the functions of TNFSFs from an evolutionary perspective and better strategies for improving fish health and welfare in aquaculture. This review summarises recent advances in the study offish TNF biology and focuses on the molecular properties and immunological functions of the TNF and TNFR superfamily.

Alternative Fish Species for Nutritional Management of Children with Fish-FPIES-A Clinical Approach.

In the Mediterranean region, fish is a common cause of food protein-induced enterocolitis syndrome (FPIES) in children. No laboratory tests specific to FPIES are available, and oral food challenge (OFC) is the gold standard for its diagnosis and testing for achievement of tolerance. Children with FPIES to fish are usually advised to avoid all fish, regardless of the species. Fish are typically classified into bony and cartilaginous, which are phylogenetically distant species and therefore contain less cross-reacting allergens. The protein ß-parvalbumin, considered a pan-allergenic, is found in bony fish, while the non-allergenic α-parvalbumin is commonly found in cartilaginous fish. Based on this difference, as a first step in the therapeutic process of children with FPIES caused by a certain fish in the bony fish category (i.e., hake, cod, perch, sardine, gilthead sea bream, red mullet, sole, megrim, sea bass, anchovy, tuna, swordfish, trout, etc.), an OFC to an alternative from the category of cartilaginous fish is suggested (i.e., blue shark, tope shark, dogfish, monkfish, skate, and ray) and vice versa. Regarding the increased mercury content in some sharks and other large species, the maximum limit imposed by the European Food Safety Authority (EFSA) for weekly mercury intake must be considered. An algorithm for the management of fish-FPIES, including alternative fish species, is proposed.

The evolution and functional characterization of CXC chemokines and receptors in lamprey.

Chemokines are a large family of soluble peptides guiding cell migration in development and immune defense. They interact with chemokine receptors and are essential for the coordination of cell migration in diverse physiological processes. The CXC subfamily is one of the largest groups in the chemokine family and consists of multiple members. In this study, we identified homologues of three chemokine ligands (CXCL8, CXCL_F5 and CXCL12) and two CXC receptor like molecules (CXCR_L1 and CXCR_L2) in lamprey. Sequence analysis revealed that they share the same genomic organization with their counterparts in jawed vertebrates but synteny was not conserved. Lamprey CXCL8 and CXCL12 have four conserved cysteine residues whilst the CXCL_F5 has two additional cysteine residues. In addition, CXCL_F5 is evolutionarily related to the fish specific CXC chemokine groups previously identified and contains multiple cationic aa residues in the extended C- terminal region. The two CXCRs possess seven transmembrane domains and conserved structural elements for receptor activation and signaling, including the DRYXXI(V)Y motif in TM2, the disulphide bond connecting ECL2 and TM3, the WXP motif in TM6 and NPXXY motif in TM7. The identified CXC chemokines and receptors were constitutively expressed in tissues including the liver, kidney, intestine, heart, gills, supraneural body and primary leukocytes, but exhibited distinct expression patterns. Relatively high expression was detected in the gills for CXCL8, CXCL_F5 and CXCR_L1 and in the supraneural body for CXCL12 and CXCR_L2. All the genes except CXCL12 were upregulated by stimulation with LPS, pokeweed and bacterial infection, and the CXCL8 and CXCL_F5 was induced by poly (I:C). Functional analysis showed that the CXCL8 and CXCL_F5 specifically interacted with CXCR_L1 and CXCR_L2, respectively. Our results demonstrate that the CXC chemokine system had diversified in jawless fish.

Glutathione-S-transferase alpha-4 in Hippocampus abdominalis (big-belly seahorse): Molecular characterization, antioxidant properties, and its potent immune response.

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST_C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 °C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO4, CdCl2, and ZnCl2. Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H2O2; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism.

Grass carp Mre11A activates IFN 1 response by targeting STING to defend against GCRV infection.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.

Plasma Proteome Responses in Salmonid Fish Following Immunization.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.

Fingerprinting of hatchery haplotypes and acquisition of genetic information by whole-mitogenome sequencing of masu salmon, Oncorhynchus masou masou, in the Kase River system, Japan.

Stocking hatchery fish can lead to disturbance and extinction of the local indigenous population. Masu salmon Oncorhynchus masou masou, which is endemic across Japan, is a commonly stocked fish for recreational fishing in Japan. To conserve the indigenous resource, their genetic information is required, however, especially on Kyushu Island, the paucity of genetic information for this species has hindered proper resource management. Here, to identify hatchery mitogenome haplotypes of this species, stocked in the Kase River system, Kyushu Island, Japan, and to provide mitogenomic information for the resource management of this species, we analyzed the whole-mitogenome of masu salmon in this river system and several hatcheries potentially used for stocking. Whole-mitogenome sequencing clearly identified hatchery haplotypes, like fingerprints: among the 21 whole-mitogenome haplotypes obtained, six were determined to be hatchery haplotypes. These hatchery haplotypes were distributed in 13 out of 17 sites, suggesting that informal stocking of O. m. masou has been performed widely across this river system. The population of no hatchery haplotypes mainly belonged to clade I, a clade not found in Hokkaido Island in previous studies. Sites without hatchery haplotypes, and the non-hatchery haplotypes in clade I might be candidates for conservation as putative indigenous resources. The whole-mitogenome haplotype analysis also clarified that the same reared strain was used in multiple hatcheries. Analysis of molecular variance suggested that stocked hatchery haplotypes reduce the genetic variation among populations in this river system. It will be necessary to pay attention to genetic fluctuations so that the resources of this river system will not deteriorate further. The single nucleotide polymorphism data obtained here could be used for resource management in this and other rivers: e.g., for monitoring of informal stocking and stocked hatchery fishes, and/or putative indigenous resources.

Comparative Characterization of Two cxcl8 Homologs in Oplegnathus fasciatus: Genomic, Transcriptional and Functional Analyses.

CXCL8 (interleukin-8, IL-8) is a CXC family chemokine that recruits specific target cells and mediates inflammation and wound healing. This study reports the identification and characterization of two cxcl8 homologs from rock bream, Oplegnathus fasciatus. Investigation of molecular signature, homology, phylogeny, and gene structure suggested that they belonged to lineages 1 (L1) and 3 (L3), and designated Ofcxcl8-L1 and Ofcxcl8-L3. While Ofcxcl8-L1 and Ofcxcl8-L3 revealed quadripartite and tripartite organization, in place of the mammalian ELR (Glu-Leu-Arg) motif, their peptides harbored EMH (Glu-Met-His) and NSH (Asn-Ser-His) motifs, respectively. Transcripts of Ofcxcl8s were constitutively detected by Quantitative Real-Time PCR (qPCR) in 11 tissues examined, however, at different levels. Ofcxcl8-L1 transcript robustly responded to treatments with stimulants, such as flagellin, concanavalin A, lipopolysaccharide, and poly(I:C), and pathogens, including Edwardsiella tarda, Streptococcus iniae, and rock bream iridovirus, when compared with Ofcxcl8-L3 mRNA. The differences in the putative promoter features may partly explain the differential transcriptional modulation of Ofcxcl8s. Purified recombinant OfCXCL8 (rOfCXCL8) proteins were used in in vitro chemotaxis and proliferation assays. Despite the lack of ELR motif, both rOfCXCL8s exhibited leukocyte chemotactic and proliferative functions, where the potency of rOfCXCL8-L1 was robust and significant compared to that of rOfCXCL8-L3. The results, taken together, are indicative of the crucial importance of Ofcxcl8s in inflammatory responses and immunoregulatory roles in rock bream immunity.

The Evolution of Duplicated Genes of the Cpi-17/Phi-1 (ppp1r14) Family of Protein Phosphatase 1 Inhibitors in Teleosts.

The Cpi-17 (ppp1r14) gene family is an evolutionarily conserved, vertebrate specific group of protein phosphatase 1 (PP1) inhibitors. When phosphorylated, Cpi-17 is a potent inhibitor of myosin phosphatase (MP), a holoenzyme complex of the regulatory subunit Mypt1 and the catalytic subunit PP1. Myosin phosphatase dephosphorylates the regulatory myosin light chain (Mlc2) and promotes actomyosin relaxation, which in turn, regulates numerous cellular processes including smooth muscle contraction, cytokinesis, cell motility, and tumor cell invasion. We analyzed zebrafish homologs of the Cpi-17 family, to better understand the mechanisms of myosin phosphatase regulation. We found single homologs of both Kepi (ppp1r14c) and Gbpi (ppp1r14d) in silico, but we detected no expression of these genes during early embryonic development. Cpi-17 (ppp1r14a) and Phi-1 (ppp1r14b) each had two duplicate paralogs, (ppp1r14aa and ppp1r14ab) and (ppp1r14ba and ppp1r14bb), which were each expressed during early development. The spatial expression pattern of these genes has diverged, with ppp1r14aa and ppp1r14bb expressed primarily in smooth muscle and skeletal muscle, respectively, while ppp1r14ab and ppp1r14ba are primarily expressed in neural tissue. We observed that, in in vitro and heterologous cellular systems, the Cpi-17 paralogs both acted as potent myosin phosphatase inhibitors, and were indistinguishable from one another. In contrast, the two Phi-1 paralogs displayed weak myosin phosphatase inhibitory activity in vitro, and did not alter myosin phosphorylation in cells. Through deletion and chimeric analysis, we identified that the difference in specificity for myosin phosphatase between Cpi-17 and Phi-1 was encoded by the highly conserved PHIN (phosphatase holoenzyme inhibitory) domain, and not the more divergent N- and C- termini. We also showed that either Cpi-17 paralog can rescue the knockdown phenotype, but neither Phi-1 paralog could do so. Thus, we provide new evidence about the biochemical and developmental distinctions of the zebrafish Cpi-17 protein family.

Identification of peptide biomarkers for authentication of Atlantic salmon and rainbow trout with untargeted and targeted proteomics approaches and quantitative detection of adulteration.

Atlantic salmon is often adulterated or substituted by rainbow trout with much lower price and quality. However, it is extremely difficult to distinguish Atlantic salmon and rainbow trout due to their similar appearance and close relationship in species. In the present work, untargeted and targeted proteomics approaches were both implemented to identify species-specific peptide biomarkers of Atlantic salmon and rainbow trout. Potential peptide biomarkers were obtained through matching HRMS data with UniProt database, screened by BLAST and then verified with real samples. Five peptide biomarkers were identified each for Atlantic salmon and rainbow trout. MRM method was established for quantitative measurement of rainbow trout Adulteration in Atlantic salmon, showing high sensitivity and repeatability. The biomarker peptide GDPGPGGPQGEQGVVGPAGISGDK was used for quantification. The limit of the detection (LOD) of adulteration of rainbow trout is 0.19%, and the limit of quantitation (LOQ) is 0.62%. Furthermore, this method was successfully applied to analyze a number of Atlantic salmon and Rainbow trout samples from different regions and different batches, as well as commercially available processed products.

Effects of Yersinia ruckeri invasion on the proteome of the Chinook salmon cell line CHSE-214.

Yersinia ruckeri is an important bacterial pathogen of fish, in particular salmonids, it has been associated with systemic infections worldwide and, like many enteric bacteria, it is a facultative intracellular pathogen. However, the effect of Y. ruckeri's interactions with the host at the cellular level have received little investigation. In the present study, a culture of Chinook Salmon Embryo (CHSE) cell line was exposed to Y. ruckeri. Afterwards, the proteins were investigated and identified by mass spectrometry and compared to the content of unexposed cultures. The results of this comparison showed that 4.7% of the identified proteins were found at significantly altered concentrations following infection. Interestingly, infection with Y. ruckeri was associated with significant changes in the concentration of surface adhesion proteins, including a significantly decreased presence of ß-integrins. These surface adhesion molecules are known to be the target for several adhesion molecules of Yersiniaceae. The concentration of several anti-apoptotic regulators (HSP90 and two DNAj molecules) appeared similarly downregulated. Taken together, these findings suggest that Y. ruckeri affects the proteome of infected cells in a notable manner and our results shed some light on the interaction between this important bacterial pathogen and its host.

Black carp RIPK1 negatively regulates MAVS-mediated antiviral signaling during the innate immune activation.

Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that black carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.

Cbx2, a PcG Family Gene, Plays a Regulatory Role in Medaka Gonadal Development.

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish.

Isolation and characterization of hypoxia inducible heme oxygenase 1 (HMOX1) gene in Labeo rohita.

The HMOX1 gene plays role in several biological processes and is also responsive to hypoxia stress. Freshwater carp fish, Labeo rohita, is reported as hypoxia sensitive, but the information of annotated hypoxia genes in public domain is very scanty for this species. Here, an attempt was made to isolate and characterize HMOX1 gene in L. rohita using information from zebrafish. HMOX1 gene was obtained by mapping HMOX1 protein of zebrafish over assembled genome of L. rohita. Aligned region was used for designing primers for HMOX1 amplification. Eight overlapping sets of primers were designed for amplifying ~540 bp long successive overlapping fragments. Splicing of overlapping amplicons generated 3715 bp fragment that was confirmed as HMOX1 gene having full coding region with 6 exons between 184 and 2156 bp positions. HMOX1 characterization is an initiative for L. rohita genes annotation to support the characterization of new genes in the important species.

A Comprehensive Integrated Genetic Map of the Complete Karyotype of Solea senegalensis (Kaup 1858).

Solea senegalensis aquaculture production has experienced a great increase in the last decade and, consequently, the genome knowledge of the species is gaining attention. In this sense, obtaining a high-density genome mapping of the species could offer clues to the aquaculture improvement in those aspects not resolved so far. In the present article, a review and new processed data have allowed to obtain a high-density BAC-based cytogenetic map of S. senegalensis beside the analysis of the sequences of such BAC clones to achieve integrative data. A total of 93 BAC clones were used to localize the chromosome complement of the species and 588 genes were annotated, thus almost reaching the 2.5% of the S. senegalensis genome sequences. As a result, important data about its genome organization and evolution were obtained, such as the lesser gene density of the large metacentric pair compared with the other metacentric chromosomes, which supports the theory of a sex proto-chromosome pair. In addition, chromosomes with a high number of linked genes that are conserved, even in distant species, were detected. This kind of result widens the knowledge of this species' chromosome dynamics and evolution.