Role of Endogenous Cathepsin L in Muscle Protein Degradation in Olive Flounder (Paralichthys olivaceus) Surimi Gel.

We investigated the effect of endogenous cathepsin L on surimi gel produced from olive flounder (Paralichthys olivaceus). The amino acid sequences of six proteins predicted or identified as cathepsin L were obtained from the olive flounder genome database, and a phylogenetic analysis was conducted. Next, cathepsin L activity toward N-α-benzyloxycarbonyl-l-phenylalanyl-l-arginine-(7-amino-4-methylcoumarin) (Z-F-R-AMC) was detected in crude olive flounder extract and a crude enzyme preparation. A considerable decrease in the level of myosin heavy chain (MHC) in surimi occurred during autolysis at 60 °C. In contrast, the levels of actin, troponin-T, and tropomyosin decreased only slightly. To prevent protein degradation by cathepsin L, a protease inhibitor was added to surimi. In the presence of 1.0% protease inhibitor, the autolysis of olive flounder surimi at 60 °C was inhibited by 12.2%; the degree of inhibition increased to 44.2% as the inhibitor concentration increased to 3.0%. In addition, the deformation and hardness of modori gel increased as the inhibitor concentration increased to 2.0%. Therefore, cathepsin L plays an important role in protein degradation in surimi, and the quality of surimi gel could be enhanced by inhibiting its activity.

Flatfish (Pleuronectiformes) species richness and depth distribution in the Gulf of Nicoya, Golfo Dulce, and two adjacent areas on the Pacific coast of Costa Rica / Riqueza de especies de lenguados (Pleuronectiformes) y distribución por profundidad en el Golfo de Nicoya, Golfo Dulce y dos áreas adyacentes en la costa del Pacifico de Costa Rica

Introduction: Information on the number of flatfish species and their depth distributions is scarce for the Eastern Tropical Pacific. Historical data is important to evaluate changes in ecosystems due to local, regional and global stressors. Objective: To provide information on the presence, depth distributions and lengths of flatfish species collected by trawl nets. Methods: Fish studies were conducted by trawling at four sites along the Pacific coast of Costa Rica by the survey vessels Skimmer (1979-1980), Nishin Maru (1987-1988) and Victor Hensen (1993-1994). The published lists of species were consulted, museum specimens were accessed, and an updated list of flatfish species assembled. Results: A total of 36 species were found over a depth range of 8-359 m. The family Paralichthyidae included 13 species followed by the Cynoglossidae with 12 species, Achiridae with six and Bothidae with five. Thirty-five species were collected at depths from 10-235 m in the Gulf of Nicoya and Golfo Dulce. Monolene asadeae was collected offshore at a depth range of 254-359 m. The Gulf of Nicoya estuary yielded 27 species during the Skimmer expedition over a depth range of 8-60 m, while 31 species were found during the V. Hensen survey at depths from 10-228 m. The V. Hensen survey in Golfo Dulce (20-235 m) collected 19 species, and 17 species in Coronado Bay (21-187 m). Off the Nicoya Peninsula (56-359 m) 13 species were collected by the Nishin Maru shrimp trawler. A total of 21 species (58 %) were found at depths greater than 100 m which were probably exposed to low oxygen concentrations. During the V. Hensen survey Symphurus chabanaudi and S. elongatus were collected more frequently in the Gulf of Nicoya, while S. leei was collected more frequently in Golfo Dulce. Cluster analyses based on presence-absence data for the Gulf of Nicoya and Golfo Dulce revealed low station similarity indicating possible habitat partitioning among species. The size (Total length) of 33 species measured from both the Gulf of Nicoya and Golfo Dulce ranged from 8 cm (Syacium cf longidorsale, Trinectes xanthurus) to 50 cm (S. ovale). Only 12 species were found with lengths over 20 cm. Data on flatfish landings by the semi-industrial fishing fleet for the period 2000-2016 indicates that this group represents less than 1 % of the total landings (shrimp and bycatch), with a minimum of 365 kg in 2001 and a maximum of 13 414 kg in 2013. Conclusions: The number of flatfish species of the Pacific coast of Costa Rica appears relatively high but comparable to the numbers found in other tropical regions. A reduced fishing impact on the populations together with this updated list of flatfish provide a good baseline for a new survey of fish populations. Both are important for a future updating of the trophic models available the Gulf of Nicoya and Golfo and their use as tools for better management of the ecosystems.

Introducción: Es escasa la información sobre el número de especies y la distribución batimétrica de los lenguados del Pacífico Este Tropical. Esos datos son importantes para evaluar cambios en los ecosistemas debidos a tensores locales, regionales y globales. Objetivo: Proveer información de la presencia, distribución batimétrica y longitudes de especies de lenguados recolectados por red de arrastre. Métodos: Evaluaciones de los peces mediante redes de arrastre fueron conducidas en cuatro sitios a lo largo de la costa del Pacífico de Costa Rica por los buques de investigación Skimmer (1979-1980), Nishin Maru (1987-1988) y Victor Hensen (1993-1994).Se consultó las listas publicadas de especies, se revisó ejemplares depositados en el museo y se integró una lista actualizada de especies de lenguados. Resultados: Un total de 36 especies de lenguados fueron encontrados en un ámbito de profundidad de 8 a 359 m. La familia Paralichthyidae incluyó 13 especies seguida por los Cynoglossidae con 12 especies, Achiridae tuvo seis y Bothidae cinco especies. Treinta y cinco especies fueron recolectadas entre 10 y 235 m en el Golfo de Nicoya y Golfo Dulce. Monolene asadeae fue recolectada aguas afuera en un ámbito de profundidad de 254-359 m. El Golfo de Nicoya produjo 27 especies durante la expedición del Skimmer y en un ámbito de profundidad de 8 a 60 m, mientras que 31 especies fueron encontradas durante el muestreo del V. Hensen en profundidades entre los 10 y 228 m. El muestreo del V. Hensen en el Golfo Dulce (20 a 235 m) produjo 19 especies y 17 en Bahía de Coronado (21-187 m). Afuera de la península de Nicoya (56-359 m) 13 especies fueron recolectadas por el camaronero Nishin Maru. Un total de 21 especies (58 %) fueron encontradas a profundidades mayores de 100 m y posiblemente expuestas a las bajas concentraciones de oxígeno. Durante el muestreo del V. Hensen, Symphurus chabanaudi y S. elongatus fueron capturados más frecuentemente en el Golfo de Nicoya, mientras S. leei lo fue en Golfo Dulce. El tamaño (Longitud Total) de 33 especies medidas en ambos Golfo de Nicoya y Golfo Dulce oscilaron entre 8 cm (Syacium cf longidorsale, Trinectes xanthurus) y 50 cm (S. ovale). Solo 12 especies fueron encontradas con longitudes mayores de 20 cm. Datos de desembarcos de lenguados por la flota semi-industrial para el periodo 2000-2016 indican que este grupo representa menos del 1 % de los desembarcos totales (camarones y fauna acompañante), con un mínimo de 365 kg en 2001 y un máximo de 13 414 kg en 2013. Conclusiones: Los análisis de conglomerados basados en datos de presencia-ausencia para el Golfo de Nicoya y Golfo Dulce revelaron baja similitud de estaciones indicando partición del hábitat entre las especies. La diversidad de la fauna de lenguados de la costa del Pacífico de Costa Rica aparece relativamente alta pero comparable con la encontrada en otras regiones tropicales. El número de buques arrastreros ha disminuido significativamente en el Pacífico de Costa Rica después del 2014. Esta reducción del impacto en las poblaciones y la lista actualizada de lenguados proveen una buena base para un nuevo estudio de las poblaciones de peces. Ambas son importantes para una futura actualización de los modelos tróficos disponibles para el Golfo de Nicoya y Golfo Dulce y su utilidad como herramientas para un mejor manejo de los ecosistemas.

Acanthocephalan parasites of the flounder species Paralichthys isosceles, Paralichthys patagonicus and Xystreurys rasile from Brazil / Acantocéfalos parasitos de espécies de linguados Paralichthys isosceles, Paralichthys patagonicus e Xystreurys rasile no Brasil

Abstract Flounders are commercially and economically important fish. A total of 120 specimens of flounders (60 Paralichthys isosceles, 30 Paralichthys patagonicus and 30 Xystreurys rasile) were collected off the coast of the state of Rio de Janeiro, Brazil. The fish were measured, necropsied and filleted, and then had their organs investigated for acanthocephalans. Taxonomic identification of the parasites was based on morphological, morphometric and genetic characters. Paralichthys isosceles and P. patagonicus were parasitized by juveniles of Serrasentis sagittifer, Bolbosoma turbinella, Corynosoma australe and C. cetaceum; Xystreurys rasile was parasitized by C. australe. Genetic characterization confirmed the identification of specimens of Bolbosoma turbinella and Corynosoma australe, as demonstrated by phylogenetic analyses using both ITS and cox1 molecular targets. Parasite indices of prevalence, intensity, mean intensity, abundance, mean abundance, and range of infection, as well as infection site, were evaluated for each parasite species. This is the first report of S. sagittifer parasitizing P. isosceles and P. patagonicus, and B. turbinella parasitizing P. patagonicus.

Resumo Os linguados são peixes comercial e economicamente importantes. Um total de 120 espécimes de linguados (60 Paralichthys isosceles, 30 P. patagonicus e 30 Xystreurys rasile) foram coletados no litoral do estado do Rio de Janeiro, Brasil. Os peixes foram medidos, necropsiados, filetados e tiveram seus órgãos investigados para a presença de acantocéfalos. A identificação taxonômica foi baseada em caracteres morfológicos, morfométricos e genéticos. Paralichthys isosceles e P. patagonicus estavam parasitados por acantocéfalos juvenis de Serrasentis sagittifer, Bolbosoma turbinella, Corynosoma australe e C. cetaceum; Xystreurys rasile estava parasitado com C. australe. A caracterização genética confirmou a identificação dos espécimes de Bolbosoma turbinella e Corynosoma australe, como demonstrado por análises filogenéticas usando ambos marcadores moleculares ITS e cox1. Foram analisados os índices parasitários: prevalência, intensidade, intensidade média, abundância, abundância média, amplitude de variação da infecção e sítio de infecção de cada espécie de parasito. Este é o primeiro registro de S. sagittifer parasitando P. isosceles e P. patagonicus, e de B. turbinella parasitando P. patagonicus.

Acanthocephalan parasites of the flounder species Paralichthys isosceles, Paralichthys patagonicus and Xystreurys rasile from Brazil.

Flounders are commercially and economically important fish. A total of 120 specimens of flounders (60 Paralichthys isosceles, 30 Paralichthys patagonicus and 30 Xystreurys rasile) were collected off the coast of the state of Rio de Janeiro, Brazil. The fish were measured, necropsied and filleted, and then had their organs investigated for acanthocephalans. Taxonomic identification of the parasites was based on morphological, morphometric and genetic characters. Paralichthys isosceles and P. patagonicus were parasitized by juveniles of Serrasentis sagittifer, Bolbosoma turbinella, Corynosoma australe and C. cetaceum; Xystreurys rasile was parasitized by C. australe. Genetic characterization confirmed the identification of specimens of Bolbosoma turbinella and Corynosoma australe, as demonstrated by phylogenetic analyses using both ITS and cox1 molecular targets. Parasite indices of prevalence, intensity, mean intensity, abundance, mean abundance, and range of infection, as well as infection site, were evaluated for each parasite species. This is the first report of S. sagittifer parasitizing P. isosceles and P. patagonicus, and B. turbinella parasitizing P. patagonicus.

Revised classification of the righteye flounders (Teleostei: Pleuronectidae) based on multilocus phylogeny with complete taxon sampling.

Members of the family Pleuronectidae are common representatives of the marine benthic fauna inhabiting northern regions of the Atlantic and Pacific oceans. The most recent comprehensive classification of the family, based entirely on morphological synapomorphies, recognized five subfamilies, 23 genera, and 61 extant species. However, several subsequent molecular studies have shown that many synapomorphic characters discovered in the morphological study might represent homoplasies, thereby questioning the reliance on these characters with the warning that they may provide misleading information for testing other morphology-based evolutionary hypotheses. In the present study, we propose a comprehensive taxonomic reassessment of the family Pleuronectidae based on the molecular phylogeny reconstructed from four nuclear and three mitochondrial loci and represented by complete taxon sampling of all but one valid species currently assigned to this family. To check for robustness of the phylogenetic hypothesis, we analyzed the effect of base compositional heterogeneity on phylogenetic signal for each locus and compared six different gene partitioning schemes. The final dataset, comprising 14 partitions and 154 individuals, was used to reconstruct phylogenetic trees in RAxML, MrBayes and BEAST2. Alternative topologies for several questionable nodes were compared using Bayes factors. The topology with the highest marginal likelihood was selected as the final phylogenetic tree for inferring pleuronectid relationships and character evolution. Based on our results, we recognize the Pleuronectidae comprising five subfamilies, 24 genera and 59 species. Our new phylogeny comprises five major monophyletic groups within the family, which we define as the subfamilies within the family: Atheresthinae, Pleuronichthyinae, Microstominae, Hippoglossinae and Pleuronectinae. Taxonomic composition of most of these subfamilies is different from that proposed in previous classifications. We also re-assess hypotheses proposed in earlier studies regarding intra-relationships of species of each lineage. Results of the current study contribute to better understanding of the evolutionary relationships of pleuronectid flatfishes based on molecular evidence, and they also provide the framework towards future comprehensive morphological revision of constituent lineages within the family Pleuronectidae.

Roles of Two Sox9 Genes during Gonadal Development in Japanese Flounder: Sex Differentiation, Spermatogenesis and Gonadal Function Maintenance.

The transcription factor sox9 has been implicated in cartilage formation and testis determination in mammals. Here, two duplicates of sox9 were found in Japanese flounder (Paralichthys olivaceus) named Posox9a and Posox9b, respectively. Phylogenetic and gene structure analyses revealed that Posox9a and Posox9b were homologous to that of teleosts and tetrapods. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that both Posox9a and Posox9b expressed higher in testis than in ovary of adult tissues. The in situ hybridization (ISH) analysis of gonads showed that Posox9a and Posox9b mRNA were both detected in oocytes, Sertoli cells and spermatocytes. During sex differentiation, the expression of Posox9a exhibited obvious sexual dimorphic expression from 60 days after hatch (dah) with higher expression in male preferred individuals than female preferred individuals and increased gradually from 30 to 100 dah. A similar pattern was detected in Posox9b expression. After injection of androgen (17α-methyltestosterone) of different concentrations, the expression level of Posox9b increased significantly, whereas Posox9a did not change obviously. These results indicated that the two sox9 genes of Japanese flounder had converse functions in sex differentiation, whereas their differences in 17α-methyltestosterone administration were obvious and worthwhile for exploring evolutionary and adaptive significance. This study provided a foundation for further exploration of the roles of Posox9 genes during the sex determination and differentiation, spermatogenesis and gonadal function maintenance of Japanese flounder.

Cloning and functional characterization of fads2 desaturase and elovl5 elongase from Japanese flounder Paralichthys olivaceus.

Japanese flounder Paralichthys olivaceus has an essential requirement for long-chain polyunsaturated fatty acids (LC-PUFA), particularly docosahexaenoic acid and eicosapentaenoic acid, but the enzymes involved in LC-PUFA biosynthesis are thought to be absent or to have low activity. Teleost fish, in particular, have quite diversified substrate preference of these enzymes even among closely related species, implying that each species could have different LC-PUFA biosynthetic capabilities. Therefore, in the present study, we characterized Japanese flounder fatty acid desaturase 2 (Fads2) and elongation of very long-chain fatty acids protein 5 (Elovl5) in order to precisely characterize the LC-PUFA biosynthesis pathway. Fads2 has Δ6 and Δ8 desaturase activity and Elovl5 has elongase activity toward C18 and C20 PUFA, suggesting that Japanese flounder is capable of synthesizing 20:4n-3 and 20:3n-6 from 18:3n-3 and 18:2n-6, respectively. Expression analysis showed that the fads2 was highly expressed in the brain and eye, while the elovl5 was highly expressed in the eye and pyloric caeca. This information will be beneficial for developing an ideal feed to support the aquaculture of Japanese flounder.

Population status of Greenland halibut Reinhardtius hippoglossoides (Walbaum, 1793) of the Laptev Sea.

This is the first study to perform a comparative genetic analysis of Greenland halibut in the samples from the Atlantic (waters of west and east of Greenland), Arctic (Laptev Sea), and Pacific (the western part of the Bering Sea) ocean basins using seven microsatellite loci. The obtained data clearly demonstrate that the Greenland halibut population in the Laptev Sea belongs to the groups of the Atlantic Ocean basin. Apparently, the Greenland halibut of the Laptev Sea is represented by a dependent population, which is replenished due to the drift of immatures from the spawning grounds in the Barents Sea with the transformed Atlantic water flow along the continental slope. In addition, the Arctic population can be partially replenished due to the breeding of the halibut in local spawning grounds.

The complete mitochondrial genome of the marbled flounder Pseudopleuronectes yokohamae (Pleuronectiformes, Pleuronectidae).

We sequenced the complete mitochondrial genome of the marbled flounder Pseudopleuronectes yokohamae collected from the Yellow Sea off China. The mitogenome comprised a 16 864-bp circular DNA molecule containing 37 genes and an AT-rich control region known as the D-loop. Phylogenetic analysis based on the mitochondrial genomes of marbled flounder indicated that P. yokohamae and Verasper variegatus are the most closely related species, which strongly supports their close phylogenetic affinity.

Molecular Cloning, Promoter Analysis and Expression Profiles of the sox3 Gene in Japanese Flounder, Paralichthys olivaceus.

Sox3, which belongs to the SoxB1 subgroup, plays major roles in neural and gonadal development. In the present study, Japanese flounder Paralichthys olivaceus sox3 gene (Posox3) and its promoter sequence were isolated and characterized. The deduced PoSox3 protein contained 298 amino acids with a characteristic HMG-box domain. Alignment and phylogenetic analyses indicated that PoSox3 shares highly identical sequence with Sox3 homologues from different species. The promoter region of Posox3 has many potential transcription factor (TF) binding sites. The expression profiles of Posox3 in different developmental stages and diverse adult tissues were analyzed by quantitative real-time RT-PCR (qRT-PCR). Posox3 mRNA was maternally inherited, and maintained at a considerably high expression level between the blastula stage and the hatching stage during embryonic development. Posox3 was abundantly expressed in the adult brain and showed sexually dimorphic expression pattern. In situ hybridization (ISH) was carried out to investigate the cellular distribution of Posox3 in the ovary, and results showed the uniform distribution of Posox3 throughout the cytoplasm of oogonia and stage I-III oocytes. These results indicate that Posox3 has potentially vital roles in embryonic and neural development and may be involved in the oogenesis process. Our work provides a fundamental understanding of the structure and potential functions of Sox3 in Paralichthys olivaceus.

Identification and Characterization of a PRDM14 Homolog in Japanese Flounder (Paralichthys olivaceus).

PRDM14 is a PR (PRDI-BF1-RIZ1 homologous) domain protein with six zinc fingers and essential roles in genome-wide epigenetic reprogramming. This protein is required for the establishment of germ cells and the maintenance of the embryonic stem cell ground state. In this study, we cloned the full-length cDNA and genomic DNA of the Paralichthys olivaceus prdm14 (Po-prdm14) gene and isolated the 5' regulatory region of Po-prdm14 by whole-genome sequencing. Peptide sequence alignment, gene structure analysis, and phylogenetic analysis revealed that Po-PRDM14 was homologous to mammalian PRDM14. Results of real-time quantitative polymerase chain reaction amplification (RT-qPCR) and in situ hybridization (ISH) in embryos demonstrated that Po-prdm14 was highly expressed between the morula and late gastrula stages, with its expression peaking in the early gastrula stage. Relatively low expression of Po-prdm14 was observed in the other developmental stages. ISH of gonadal tissues revealed that the transcripts were located in the nucleus of the oocytes in the ovaries but only in the spermatogonia and not the spermatocytes in the testes. We also presume that the Po-prdm14 transcription factor binding sites and their conserved binding region among vertebrates. The combined results suggest that Po-PRDM14 has a conserved function in teleosts and mammals.

Locus number estimation of MHC class II B in stone flounder and Japanese flounder.

Members of major histocompatibility complex (MHC) family are important in immune systems. Great efforts have been made to reveal their complicated gene structures. But many existing studies focus on partial sequences of MHC genes. In this study, by gene cloning and sequencing, we identified cDNA sequences and DNA sequences of the MHC class II B in two flatfishes, stone flounder (Kareius bicoloratus) and homozygous diploid Japanese flounder (Paralichthys olivaceus). Eleven cDNA sequences were acquired from eight stone flounder individuals, and most of the polymorphic sites distributed in exons 2 and 3. Twenty-eight alleles were identified from the DNA fragments in these eight individuals. It could be deduced from their Bayesian inference phylogenetic tree that at least four loci of MHC class II B exist in stone flounder. The detailed whole-length DNA sequences in one individual were analyzed, revealing that the intron length varied among different loci. Four different cDNA sequences were identified from one homozygous diploid Japanese flounder individual, implying the existence of at least four loci. Comparison of the cDNA sequences to the DNA sequence confirmed that six exons existed in this gene of Japanese flounder, which was a common feature shared by Pleuronectiformes fishes. Our results proved the multi-locus feature of MHC class II B. The sequences we obtained would provide detailed and systematic data for further research.

Characterization of novel precursor miRNAs using next generation sequencing and prediction of miRNA targets in Atlantic halibut.

BACKGROUND: microRNAs (miRNAs) are implicated in regulation of many cellular processes. miRNAs are processed to their mature functional form in a step-wise manner by multiple proteins and cofactors in the nucleus and cytoplasm. Many miRNAs are conserved across vertebrates. Mature miRNAs have recently been characterized in Atlantic halibut (Hippoglossus hippoglossus L.). The aim of this study was to identify and characterize precursor miRNA (pre-miRNAs) and miRNA targets in this non-model flatfish. Discovery of miRNA precursor forms and targets in non-model organisms is difficult because of limited source information available. Therefore, we have developed a methodology to overcome this limitation. METHODS: Genomic DNA and small transcriptome of Atlantic halibut were sequenced using Roche 454 pyrosequencing and SOLiD next generation sequencing (NGS), respectively. Identified pre- miRNAs were further validated with reverse-transcription PCR. miRNA targets were identified using miRanda and RNAhybrid target prediction tools using sequences from public databases. Some of miRNA targets were also identified using RACE-PCR. miRNA binding sites were validated with luciferase assay using the RTS34st cell line. RESULTS: We obtained more than 1.3 M and 92 M sequence reads from 454 genomic DNA sequencing and SOLiD small RNA sequencing, respectively. We identified 34 known and 9 novel pre-miRNAs. We predicted a number of miRNA target genes involved in various biological pathways. miR-24 binding to kisspeptin 1 receptor-2 (kiss1-r2) was confirmed using luciferase assay. CONCLUSION: This study demonstrates that identification of conserved and novel pre-miRNAs in a non-model vertebrate lacking substantial genomic resources can be performed by combining different next generation sequencing technologies. Our results indicate a wide conservation of miRNA precursors and involvement of miRNA in multiple regulatory pathways, and provide resources for further research on miRNA in non-model animals.

PoCRIP1, Paralichthys olivaceus cysteine-rich intestinal protein 1: molecular characterization, expression analysis upon Edwardsiella tarda challenge and a possible role in the immune regulation.

Cysteine-rich intestinal protein (CRIP) is a LIM domain protein containing a zinc-finger motif and plays a role in the regulation of the inflammatory immune response. In the present study, we isolated a CRIP1 cDNA, designated PoCRIP1, from an olive flounder Paralichthys olivaceus intestine cDNA library by EST analysis. The PoCRIP cDNA consists of 421 bp with a polyadenylation signal sequence, AATAAA, and a poly(A) tail; it encodes a polypeptide of 76 amino acids containing a double zinc-finger motif (Cys(2)HisCys and Cys(4) sequences). The deduced amino acid sequence of PoCRIP1 showed 75.3-94.7% homology with CRIP1s of other species, including mammals. The PoCRIP1 transcript was highly expressed in the intestine and pyloric ceca and moderately expressed in the gill, heart, kidney, liver, muscle, spleen, skin, and stomach of normal conditioned flounder. Inducible expression of the PoCRIP1 transcript was observed in flounder challenged with Edwardsiella tarda, an economically important pathogen for aquaculture of flounder. Over-expression of PoCRIP1 augmented p65-driven flounder IL-6 promoter activity in HINAE cells. These results suggest that PoCRIP1 may function in the immune response of the flounder through the regulation of cytokine expression.

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus / Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

Este estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.

The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15).

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif "LRLRGG". Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.

Demographic history and population structure of blackfin flounder (Glyptocephalus stelleri) in Japan revealed by mitochondrial control region sequences.

The demographic history and population genetic structure of the blackfin flounder (Glyptocephalus stelleri) along coastal regions of Japan were investigated. Genetic variation in DNA sequences was examined from the first hypervariable region of the mitochondrial DNA control region. A high level of haplotypic diversity (h = 0.99 +/- 0.004) was detected, indicating a high level of intrapopulation genetic diversity. The starburst structure of the minimum spanning tree suggested a very recent origin for most haplotypes. The demographic history of G. stelleri was examined using neutrality tests and mismatch distribution analysis, which also indicated a Pleistocene population expansion at about 124,100-413,400 years ago. Hierarchical molecular variance analysis and conventional population Fst comparisons revealed no significant genetic differentiation throughout the range examined.

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus.

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6-36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.

Growth differentiation factor 15, a novel acute phase response gene in Japanese flounder, Paralichthys olivaceus.

The acute phase response, an important aspect of innate immunity, leads to the production of acute phase proteins (APPs) in the liver which would consequently help restore homeostasis to the body. Here, we identified a novel cytokine, growth differentiation factor 15 (GDF15) from Japanese flounder. Three out of the 384 EST sequences derived from liver of Japanese flounder treated with formalin-killed Edwardsiella tarda showed significant homology with GDF of various species. After obtaining the full-length cDNA, the deduced amino acid sequence exhibited low identity (<30%) with GDF15s of higher vertebrates. The predicted ORF of JFGDF15 revealed a signaling peptide at the N terminal, a TGFbeta propeptide domain and a TGFbeta domain. The mature peptide domain of JFGDF15 contains an RXXR motif, a furin cleavage site, required for the release of the mature peptide and conserved amino acids, which are signature features of TGFbeta superfamily proteins. JFGDF15 mRNA transcripts were detected in fish, 6h post-injection with PBS. The transcripts were highly up-regulated in liver at 6h post-injection with formalin-killed E. tarda. Moreover, up-regulation of the transcripts was also observed at 12h post-injection with formalin-killed Streptococcus iniae.

Hepatic transcriptomic profiles of European flounder (Platichthys flesus) from field sites and computational approaches to predict site from stress gene responses following exposure to model toxicants.

Genomic technologies offer opportunities to gain a more global assessment of the health status of an organism through an understanding of the functional pathways that are responding to pollutant exposure. We have developed a 13,000 clone cDNA toxicogenomics microarray for Platichthys flesus, the European flounder (EU-GENIPOL Project). We aimed to distinguish the origins of flounder taken from six sampling sites of different pollution status in Northern Europe according to their hepatic gene expression profile using bioinformatic approaches. To determine which gene expression differences may relate to pollutant impact, we have completed complementary laboratory exposures of flounder to selected toxicants and determined the associated gene expression profiles. Using multivariate variable selection coupled with a statistical modelling procedure (GALGO) we can predict geographical site but the accuracy is limited to specific sites. The search space for a combination of genes that effectively predicts class membership is very large, however, by combining the signatures derived from acute laboratory exposure to individual chemicals to limit the search space, a very accurate model for classification of all the different environmental sites was achieved. The final model utilised the expression profiles of 16 clones and validation with a qPCR array comprising these genes correctly assigned the site of origin for fish obtained from three of the sites in an independent sampling. These data would imply that the gene expression fingerprints obtained with these arrays are primarily attributable to variations in chemical pollutant responses at the different sites, indicating their potential utility in environmental impact assessment.