Genetic Mechanism of Tissue-Specific Expression of PPAR Genes in Turbot (Scophthalmus maximus) at Different Temperatures.

In this study, we used PCR to measure the levels of the peroxisome proliferator activated receptor genes PPARα1, PPARα2, PPARß, and PPARγ in the intestine, liver, gill, heart, kidney, brain, muscle, spleen, skin, and stomach of turbot (Scophthalmus maximus) cultured under different temperature conditions (14, 20, 23, 25, and 28 °C). We used split-split-plot (SSP) analysis of variance, additive main effects and multiplicative interaction (AMMI) analysis, and genotype main effects and genotype × environment interaction (GGE) biplot analysis to evaluate the genotype × tissue interaction effects on gene expression. The results of the SSP analysis of variance showed that temperature and tissue × gene have highly significant (p < 0.01) effect on the expression of S. maximus PPAR genes. The AMMI analysis results revealed that the expression of PPAR genes at the appropriate temperature (14 °C) mainly depended on genotype × tissue interaction and tissue effects. Under stress temperatures, genotype effects, tissue effects, and genotype × tissue interaction, all had significant effects on the expression of PPAR genes. The contribution of the genotype effect slowly increased with increasing temperature; it increased faster at 20 °C and then slowly declined at 25 °C. The contribution of the tissue effect slowly increased from 14 to 20 °C, where it sharply decreased, and then it stabilized after a slight fluctuation. The contribution of the genotype × tissue interaction effect showed a fluctuating upward trend throughout the experiment, and it had a significant impact on PPAR gene expression. The key temperature at which the three effects changed was 20 °C, indicating that it is the limit temperature for active lipid metabolism under high-temperature stress. The GGE biplot analysis results showed that under suitable water temperature, the expression difference of PPAR genes in the liver was the largest; at 20 and 23 °C, the expression difference in the gill was the largest; and at 25 and 28 °C, the expression difference in the brain was the largest. Overall, our results suggest that the mechanism responsible for PPAR gene expression under the three high temperatures (23, 25, and 28 °C) was relatively consistent, but it differed from that at 20 °C.

Molecular characterization of ubiquitin-conjugating enzyme gene ube2h and siRNA-mediated regulation on targeting p53 in turbot, Scophthalmus maximus.

Ubiquitin-conjugating enzymes are key factors in the ubiquitin proteasome pathway (UPP), which play key roles in ubiquitination. These enzymes affect the efficiency of UPP during stress conditions. P53 has important control of cell cycle arrest and apoptosis in response to cellular stress; these modifications are critical for the stability and transcriptional activity of p53 as the protein activates downstream target genes that dictate the cellular response. However, few studies have investigated the effects of thermal stress in turbot (Scophthalmus maximus), specifically the UPP signaling pathway, and the crosstalk between the ube2h and p53. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot UBE2H gene (Sm-ube2h) and perform bioinformatics analysis. Our results showed that the cDNA of the Sm-ube2h was 718 bp in length, encoding a 189 amino acid protein, with a theoretical isoelectric point of 4.77. It also contained a catalytic (UBCc) domain. Expression of Sm-ube2h in different tissues was detected and quantified by qPCR, which was highest in the spleen and lowest in the liver. We also investigated the Sm-ube2h expression profiles in the liver and heart after thermal stress, and changes in Sm-ube2h and p53 under thermal stress, upon RNA interference. Our data speculated that Sm-ube2h and p53 exhibited antagonistic effects under normal temperature conditions after ube2h interference, but displayed synergistic effects under thermal stress, suggesting the crosstalk between UPP and p53 signaling pathway. Our results improved our understanding of the underlying molecular mechanism of thermal tolerance in turbot.

Genome-wide identification of NOD-like receptors and their expression profiling in mucosal tissues of turbot (Scophthalmus maximus L.) upon bacteria challenge.

The innate immune system plays an important role in host defense against pathogenic infections. In the innate immune system, several families of innate pattern recognition receptors, including Toll-like receptors, RIG-I-like receptors, NOD-like receptors (NLRs), and DNA receptors (cytosolic sensors for DNA), are known to play vital roles in detecting and responding to various pathogens. In this study, we identified 29 NLRs in turbot including 4 NLRs from subfamily A: NOD1, NOD2, CIITA, NLRC5, 1 NLR from subfamily B: NLRB1, 21 NLRs from subfamily C: NLR-C3.1∼NLRC3.21, 1 from NLRX subfamily, and two that do not fall within these subfamilies: APAF1, NWD1. Phylogenetic analysis showed that these NLR genes were clearly divided into five subfamilies. Protein-protein interaction network analysis showed that some of these NLR genes shared same interacting genes and might participate in signal transductions associated with immunity. The evolutionary pressure selection analysis showed that the Ka/Ks ratios for all detected NLR genes were much less than one, implying more synonymous changes than non-synonymous changes. In addition, tissue expression analysis showed that the relative higher expression levels were observed in gill, skin and intestine. Meanwhile, NLR genes expression after bacterial infection results showed that most NLR genes participated in the process of defense of V. anguillarum and A. salmonicida infections in mucosal tissues. Taken together, identification and expression profiling analysis of NLR genes can provide valuable information for further functional characterization of these genes in turbot.

Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018.

BackgroundThe increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment.AimTo evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon.MethodsFrom 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae.ResultsNo zoonotic parasites were found in any of the fish investigated.ConclusionsThe risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation.

Transcriptome analysis provides insights into the effects of myo-inositol on the turbot Scophthalmus maximus.

Myo-inositol is an essential vitamin for most animals, and it can modulate multiple physiological functions. In this study, we performed transcriptome gene expression profiling of gill tissue from turbot Scophthalmus maximus fed different concentrations of myo-inositol (0, 300, 600, 900, 1200 mg/kg). Results of expression tendency analysis, Weighted Gene Co-Expression Network Analysis (WGCNA), integrated transcriptome analyses, and KEGG annotation analysis of all differentially expressed genes (DEGs) demonstrated that the cytokine-cytokine receptor interaction played a core role in effects of myo-inositol on turbot, which was followed by the Jak-STAT signaling pathway. The results of qRT-PCR also showed myo-inositol mediated the gene expression of the cytokine-cytokine receptor interaction and the Jak-STAT signaling pathway in turbot. The ELISA assay indicated that myo-inositol affected the concentration change of interleukins (IL-2 and IL-10). Consequently, the interleukins associated with immune functions in the cytokine-cytokine receptor interaction played a core role in the effects of myo-inositol on turbot, which was followed by the Jak-STAT signaling pathway. Additionally, 10 hub genes associated with myo-inositol-traits were identified via WGCNA.

Molecular cloning, characterization and expression analysis of p53 from turbot Scophthalmus maximus and its response to thermal stress.

The tumor suppressor protein, p53 plays a crucial role in protecting genetic integrity. Once activated by diverse cell stresses, p53 reversibly activates downstream target genes to regulate cell cycle and apoptosis. However, few studies have investigated the effects of thermal stress in turbot, specifically the p53 signaling pathway. In this study, the rapid amplification of cDNA ends was used to obtain a full-length cDNA of the turbot p53 gene (Sm-p53) and perform bioinformatics analysis. The results showed that the cDNA of the Sm-p53 gene was 2928 bp in length, encoded a 381 amino acid protein, with a theoretical isoelectric point of 6.73. It was composed of a DNA binding and a tetramerization domain. Expression of Sm-p53 in different tissues was detected and quantified by qRT-PCR, and was highest in the liver. We also investigated the expression profiles of Sm-p53 in different tissue and TK cells after thermal stress. These result suggested that Sm-p53 plays a key role, and provides a theoretical basis for Sm-p53 changes in environmental stress responses in the turbot.

Molecular function of gonadotrophins and their receptors in the ovarian development of turbot (Scophthalmus maximus).

Gonadotropins (GtHs) and their receptors (follicle-stimulating hormone receptor, FSHR; luteinizing hormone receptor, LHR) are involved in the regulation of gametogenesis and play important roles during the reproductive cycles in vertebrate species, including fish. This minireview focuses on the molecular characterization and quantification of GtHs (common glycoprotein α subunit CGα, FSHß, and LHß) and their receptors (FSHR and LHR) throughout the reproductive cycle of female turbot Scophthalmus maximus. Information about GtHs, FSHR, LHR as well as other ligand-receptors interaction from different teleosts are also included in this review for the implications they may have on the functions of GtHs, FSHR and LHR in the reproductive development of turbot. These findings may enhance our understanding of the physiological roles of the GtHs, FSHR and LHR in controlling of flatfish ovarian development during the reproductive cycle and contributing to the improvement of management strategies for turbots in captivity.

Involvement and expression of growth hormone/insulin-like growth factor member mRNAs in the ovarian development of turbot (Scophthalmus maximus).

Accumulating evidence suggests that the growth hormone (GH)/insulin-like growth factor (IGF) system participates in fish reproduction. To understand the physiological functions of the GH/IGF system, the mRNA expression profiles of all known members of the GH/IGF system, including hepatic and ovarian gh, GH receptor (ghr), IGFs (igf-i, igf-ii), IGF-I receptor (igf-ir) and IGF binding protein (igfbp1, igfbp2), pituitary gh, and hepatic vitellogenin (vtg) were investigated during ovarian development in turbot Scophthalmus maximus. Results showed that ghr, igf-i, igf-ii, igf-ir, and igfbp2 were expressed in the liver and ovary, whereas igfbp1 and gh were undetected. The hepatosomatic index (HSI) and gonadosomatic index (GSI) gradually increased and peaked during the late vitellogenesis (Latvtg) and migratory nucleus (Mig-nucl) stages, respectively. The mRNA expression profiles of ovarian ghr, igf-ii, hepatic igf-ir, vtg, and pituitary gh were similar to the HSI; ovarian igf-i and igf-ir expression was close to the GSI. However, the hepatic mRNA levels of ghr, igf-i, and igf-ii peaked at the early vitellogenesis (Evtg) stage, and then drastically declined during ovarian development. The mRNA expression of hepatic igfbp2 decreased and reached the lowest at the atresia (Atre) stage, whereas that of ovarian igfbp2 increased and peaked at Latvtg stage. Furthermore, significant correlations between pituitary gh, ovarian ghr, igf-i, and igf-ii, and hepatic ghr, igf-i, igf-ir, and igf-ii were observed, respectively. These results suggest that GH/IGF members appear to play distinct roles in the regulation of ovarian development in turbot and will be valuable for fish reproduction and broodstock management of aqua-cultured fish species.

Molecular cloning and expression analyses of immunoglobulin tau heavy chain (IgT) in turbot, Scophthalmus maximus.

Immunoglobulins (Igs) are humoral mediators playing the prevailing role in the innate and adaptive immunities of jawed vertebrates and provide obligatory duties to protect the organism from a wide variety of pathogens. In the present study, the membrane-bound and secretory immunoglobulin T (mIgT and sIgT) genes of turbot (Scophthalmus maximus) were cloned for the first time with the intention of better understanding the IgT functions. The mIgT cDNA is 2, 049 bp in length including a leader region, a variable region, four constant regions and a transmembrane region (TM), while the 1, 932 bp sIgT cDNA lacks the transmembrane region. In healthy turbot, the total IgT was highly expressed in gill, spleen and liver followed by peripheral blood leucocytes (PBL), skin and hindgut, and then in stomach, head kidney, trunk kidney, midgut and foregut. The expression levels of sIgT in PBL, gill, skin and spleen were much higher than mIgT. Furthermore, the expression profiles of turbot mIgT and sIgT were investigated post vaccination with formalin-inactivated Vibrio anguillarum via intraperitoneal injection and immersion, and the results showed that the expressions of mIgT and sIgT were both significantly induced by two administration routes, whereas intraperitoneal injection mostly induced mIgT expression in systematic organs including head kidney, spleen and trunk kidney, and the immersion vaccination elicited a much stronger response of sIgT in mucosa-associated tissues including gills, liver, hindgut and skin. Taken together, these results demonstrated that mIgT and sIgT were differentially expressed in different tissues and both responded positively to the vaccinations in turbot, and indicated that IgT-secreting plasma cells are abundant in mucosa-associated tissues and played important roles in mucosal immunity of turbot.

Expression profile and localization of vitellogenin mRNA and protein during ovarian development in turbot (Scophthalmus maximus).

Egg yolk generation is a common physiological process in oviparous animals. To understand oogenesis and reproductive capacity, it is necessary to characterize vitellogenins (Vtgs), which are the precursors of major egg yolk proteins (Yps). Therefore, to improve our understanding of the entire process of egg yolk generation in female turbot (Scophthalmus maximus), we obtained full-length cDNAs of vtg genes, examined gene expression in the female liver and ovary, and analyzed Vtg synthesis in the ovary. Three distinct complete polypeptide sequences were identified and designated as VtgAa, VtgAb, and VtgC, which confirmed the multiplicity of the vtg gene in turbot and showed that it follows a "three vtg model". The expression of these three vtg genes in the female liver was far higher than that in other tissues, including the ovary. The expression of all three vtg genes was extremely low before vitellogenesis, and then increased and was maintained at a high level until the degradation stage, which was in accordance with changes in the concentration of estradiol-17ß (E2) and the gonadosomatic index. Compared with the liver, the ovary had a higher E2 level and lower vtg expression, suggesting that some other factors limit high vtg expression in the ovary of turbot. Transcripts of vtgAb and the Yps derived from them were both detected in oogonia and primary oocytes, which showed that these might possess the ability to perform autosynthesis of yolk. These findings add to our understanding of the reproductive physiology of Vtg synthesis in turbot.

Piwil1 gene is regulated by hypothalamic-pituitary-gonadal axis in turbot (Scophthalmus maximus): A different effect in ovaries and testes.

As constituent factors of Piwi-interacting RNA (piRNA) pathways, Piwi proteins are essential for germline maintenance and gonadal development. Previous studies show that Piwi-piRNA pathways could be regulated by hypothalamic-pituitary-gonadal (HPG) axis, however, related studies have not been reported in marine species. Here we reported the identification of turbot (Scophthalmus maximus) piwil1 gene, which was abundantly expressed in testis and ovary in a tissue-specific manner. Phylogenetic and genomic structure analyses revealed that piwil1 was conserved in its sequence and function during vertebrate evolution. We also investigated the effects of HPG axis hormones, including human chorionic gonadotropin (hCG), estradiol-17ß (E2) and 17α-methyltestosterone (MT), on gonadal piwil1 expression via in vivo and in vitro approaches. In ovary, hCG and E2 suppressed piwil1 expression both in vivo and in vitro, and MT increased piwil1 expression in vivo. In testis, hCG had upregulating effects on piwil1 expression in vivo and in vitro, and MT also increased piwil1 expression in vitro. In addition, E2 suppressed expression of piwil1 in vivo. These results indicated that the decreased or increased expression of piwil1 regulated by hormones might play a crucial role during gonadal differentiation and development in S. maximus.

Roles of insulin-like growth factors in metamorphic development of turbot (Scophthalmus maximus).

Larval turbot (Scophthalmus maximus) undergo metamorphosis, a late post-embryonic developmental event that precedes juvenile transition. Insulin-like growth factors (IGFs) are important endocrine/autocrine/paracrine factors that provide essential signals to control of the embryonic and postnatal development of vertebrate species, including fish. Accumulating evidence suggests that IGFs are involved in regulating the metamorphic development of flatfish. This mini review focus on the functions of all known IGFs (IGF-I and IGF-II) during the metamorphic development of turbot. Information about IGFs and insulin-like growth factors binding proteins (IGFBPs) from other teleosts is also included in this review to provide an overview of IGFs functions in the metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGFs system in controlling of flatfish metamorphosis and contributing to the improvement of broodstock management strategies for larval turbot.

Molecular characterization and quantification of estrogen receptors in turbot (Scophthalmus maximus).

Estrogens regulate various reproductive processes via estrogen receptor (ER)-mediated signaling pathway in vertebrates. In this study, full-length sequences coding for ERα, ERß1 and ERß2 were isolated from female turbot (Scophthalmus maximus) by homology cloning and a strategy based on rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR). The nucleotide and amino acid sequences of turbot ERs showed high homologies with the corresponding sequences of other fish species and significant homology with the Japanese flounder (Paralichthys olivaceus) and the European sea bass (Dicentrarchus labrax). Turbot ERs contained six typical nuclear receptor-characteristic domains and exhibited high evolutionary conservation in the functional domains. Quantitative real-time polymerase chain reaction analysis revealed that the erα and erß (ß1, ß2) mRNAs were abundant in the liver and ovary, respectively. Furthermore, hepatic mRNA levels of erα and vitellogenin (vtg) were found increased gradually from pre-vitellogenesis to late-vitellogenesis stages, with the highest values observed at the late-vitellogenesis stage, and then decreased from migratory-nucleus to atresia stages. However, mRNA levels of erα in the ovary remained unchanged during ovarian development. Hepatosomatic index, gonadosomatic index, serum estradiol-17ß and the mRNA levels of erß1 and erß2 in the ovary manifested results similar to the expression of erα mRNAs in the liver. These findings indicated that ERα is mainly involved in hepatic vitellogenesis, and ERßs may play crucial roles to regulate ovarian development in turbot. Overall, this study improves understanding of the physiological functions of turbot ERs, which will be valuable for fish reproduction and broodstock management.

Nutrient sensing and metabolic changes after methionine deprivation in primary muscle cells of turbot (Scophthalmus maximus L.).

The low methionine content in plant-based diets is a major limiting factor for feed utilization by animals. However, the molecular consequences triggered by methionine deficiency have not been well characterized, especially in fish species, whose metabolism is unique in many aspects and important for aquaculture industry. In the present study, the primary muscle cells of turbot (Scophthalmus maximus L.) were isolated and treated with or without methionine for 12 h in culture. The responses of nutrient sensing pathways, the proteomic profiling of metabolic processes, and the expressions of key metabolic molecules were systematically examined. Methionine deprivation (MD) suppressed target of rapamycin (TOR) signaling, activated AMP-activated protein kinase (AMPK) and amino acid response (AAR) pathways. Reduced cellular protein synthesis and increased protein degradation by MD led to increased intracellular free amino acid levels and degradations. MD also reduced glycolysis and lipogenesis while stimulated lipolysis, thus resulted in decreased intracellular lipid pool. MD significantly enhanced energy expenditure through stimulated tricarboxylic acid (TCA) cycle and oxidative phosphorylation. Collectively, our results identified a comprehensive set of transcriptional, proteomic, and signaling responses generated by MD and provided the molecular insight into the integration of cell homeostasis and metabolic controls in fish species.

Effect of Initiation Time of Hydrostatic Pressure Shock on Chromosome Set Doubling of Tetraploidization in Turbot Scophthalmus maximus.

The objective of the study was to clarify the effects of initiation time on chromosome set doubling induced by hydrostatic pressure shock through nuclear phase fluorescent microscopy in turbot Scophthalmus maximus. The ratio of developmentally delayed embryo and chromosome counting was used to assess induction efficiency. For the embryos subjected to a pressure of 67.5 MPa for 6 min at prometaphase (A group), chromosomes recovered to the pre-treatment condition after 11-min recovering. The first nuclear division and cytokinesis proceeded normally. During the second cell cycle, chromosomes did not enter into metaphase after prometaphase, but spread around for about 13 min, then assembled together and formed a large nucleus without anaphase separation; the second nuclear division and cytokinesis was inhibited. The ratio of developmentally delayed embryo showed that the second mitosis of 78% A group embryo was inhibited. The result of chromosome counting showed that the tetraploidization rate of A group was 72%. For the embryos subjected to a pressure of 67.5 MPa for 6 min at anaphase (B group), chromosomes recovered to the pre-treatment condition after about 31-min recovering. Afterwards, one telophase nucleus formed without anaphase separation; the first nuclear division was inhibited. The time of the first cleavage furrow occurrence of B group embryos delayed 27 min compared with that of A group embryos. With the first cytokinesis proceeding normally, 81.3% B group embryos were at two-cell stage around the middle of the second cell cycle after treatment. Those embryos were one of the two blastomeres containing DNA and the other without DNA. The first nuclear division of those embryos was inhibited. During the third cell cycle after treatment, 65.2% of those abovementioned embryos were at four-cell stage, cytokinesis occurred in both blastomeres, and nuclear division only occurred in the blastomere containing DNA. Of those abovementioned embryos, 14.0% were at three-cell stage and cytokinesis only occurred in the blastomere containing DNA. The result of chromosome counting showed that the tetraploidization rate of B group was only 7%. To summarize what had been mentioned above, mechanisms on chromosome set doubling of tetraploid induction would be different with different initiation time of hydrostatic pressure treatment. Chromosome set doubling was mainly due to inhibition of the second mitosis when hydrostatic pressure treatment was performed at prometaphase. Otherwise, chromosome set doubling was mainly due to inhibition of the first nuclear division when hydrostatic pressure treatment was performed at anaphase. Induction efficiency of tetraploidization resulted from inhibition of the second cleavage was higher than which resulted from inhibition of the first nuclear division. This study was the first to reveal biological mechanisms on the two viewpoints of chromosome set doubling through effect of initiation time of hydrostatic pressure treatment on chromosome set doubling in tetraploid induction.

Influence of immunostimulant polysaccharides, nucleic acids, and Bacillus strains on the innate immune and acute stress response in turbots (Scophthalmus maximus) fed soy bean- and wheat-based diets.

Immunostimulants are widely applied in aquaculture practice and may have beneficial effects on the immune system and physical functions allowing higher tolerance to stress. In the current study, the impact of four (i-iv) dietary active ingredients on the immune and stress response of turbot was examined in two experiments (I and II). A basal low fish meal (FM; 32%) diet was formulated and supplemented with (i) yeast ß-glucan and mannan oligosaccharide (GM), (ii) alginic acid (AC), (iii) yeast nucleotides and RNA (NR), or (iv) Bacillus strains (BS). The basal diet (C-LF) and a high FM (59%) control (C-HF) were maintained. All six diets were fed to juvenile turbots for 84 days in experiment I and for additional 28 days prior to experiment II. Immunological and hematological parameters were determined in experiment I. In experiment II, physical stress response to a typical short-term (<1 day) aquaculture handling procedure (combination of capture, netting/transfer, and crowding) was investigated. For this, turbot blood was sampled before and at 0.5, 1, 4, and 24 h post stress. Plasma lysozyme activity, neutrophil reactive oxygen species (ROS) production, and total plasma protein levels did not significantly differ between treatment groups; however, plasma cholesterol increased significantly in fish fed GM, AC, NR, and C-HF compared to C-LF (I). A significant increase in plasma glucose and triglyceride was observed in GM and NR treatments, while glucose levels were significantly higher in C-HF compared to C-LF. Moreover, the immunostimulant-supplemented diets exhibited significantly lower cortisol levels compared to controls C-LF (at 0.5 h) and C-HF (at 1 h) post stress, respectively (II). According to our findings, FM substitution did not modulate the innate immune response but was associated with reduced levels of cholesterol. Dietary immunostimulants were not effective enough to boost the immune response, but we believe they might be helpful to trigger metabolic advantages during stressful handling events on fish farms.

Identification and expression of piwil2 in turbot Scophthalmus maximus, with implications of the involvement in embryonic and gonadal development.

Piwil2, a member of the Argonaute family, is involved in the biogenesis of PIWI-interacting RNAs (piRNAs) and plays an important role in regulating gametogenesis. In the present study, we identified turbot Scophthalmus maximus piwil2 gene, named Smpiwil2, which contained a PAZ domain and a PIWI domain. Sequence comparison, genomic structure and phylogenetic analyses showed that Smpiwil2 is homologous to that of teleosts and tetrapods. The Smpiwil2 transcript showed higher expression in the ovary than in the testis, demonstrating a sexually dimorphic gene expression pattern. In situ hybridization (ISH) showed that Smpiwil2 was expressed in the oogonia and all the stages of oocytes in the ovary as well as in spermatogonia and spermatocytes in the testis. Embryonic expression profile revealed that Smpiwil2 was maternally inherited, and its level was higher from the zygote to the blastula stage and subsequently decreased until hatching. Moreover, a CpG island was predicted to locate in the 5'-flanking region of Smpiwil2 gene, and its methylation levels detected by sodium bisulfite sequencing showed significant disparity between females and males, implying that the sexually dimorphic expression of Smpiwil2 might be regulated by methylation. These results indicated that Smpiwil2 had potentially vital functions in embryonic and gonadal development in this species. In addition, the temporal and sex differences in Smpiwil2 expression indicated that this gene may play different roles in gonadal development of different sexes.

Dietary gossypol suppressed postprandial TOR signaling and elevated ER stress pathways in turbot (Scophthalmus maximus L.).

Gossypol is known to be a polyphenolic compound toxic to animals. However, its molecular targets are far from fully characterized. To evaluate the physiological and molecular effects of gossypol, we chose turbot (Scophthalmus maximus L.), a carnivorous fish, as our model species. Juvenile turbots (7.83 ± 0.02 g) were fed diets containing gradient levels of gossypol at 0 (G0), 600 (G1), and 1,200 (G2) mg/kg diets for 11 wk. After the feeding trial, fish growth, body protein, and fat contents were significantly reduced in the G2 group compared with those of the G0 group (P < 0.05). Gossypol had little impact on digestive enzyme activities and intestine morphology. However, gossypol caused liver fibrosis and stimulated chemokine and proinflammatory cytokine secretions. More importantly, gossypol suppressed target of rapamycin (TOR) signaling and induced endoplasmic reticulum (ER) stress pathway in both the feeding experiment and cell cultures. Our results demonstrated that gossypol inhibited TOR signaling and elevated ER stress pathways both in vivo and in vitro, thus providing new mechanism of action of gossypol in nutritional physiology.

Expression of insulin-like growth factors at mRNA levels during the metamorphic development of turbot (Scophthalmus maximus).

Insulin-like growth factors I and II (IGF-I and IGF-II) are important regulators of vertebrate growth and development. This study characterized the mRNA expressions of igf-i and igf-ii during turbot (Scophthalmus maximus) metamorphosis to elucidate the possible regulatory role of the IGF system in flatfish metamorphosis. Results showed that the mRNA levels of igf-i significantly increased at the early-metamorphosis stage and then gradually decreased until metamorphosis was completed. By contrast, mRNA levels of igf-ii significantly increased at the pre-metamorphosis stage and then substantially decreased during metamorphosis. Meanwhile, the whole-body thyroxine (T4) levels varied during larval metamorphosis, and the highest value was observed in the climax-metamorphosis. The mRNA levels of igf-i significantly increased and decreased by T4 and thiourea (TU, inhibitor of endogenous thyroid hormone) during metamorphosis, respectively. Conversely, the mRNA levels of igf-ii remained unchanged. Furthermore, TU significantly inhibited the T4-induced mRNA up-regulation of igf-i during metamorphosis. The whole-body thyroxine (T4) levels were significantly increased and decreased by T4 and TU during metamorphosis, respectively. These results suggested that igf-i and igf-ii may play different functional roles in larval development stages, and igf-i may have a crucial function in regulating the early metamorphic development of turbot. These findings may enhance our understanding of the potential roles of the IGF system to control flatfish metamorphosis and contribute to the improvement of broodstock management for larvae.

FAST Real Time PCR for control of intra-species recycling in aquaculture feed, focused to the most relevant fish species farmed in Europe.

Recent regulations in animal feed composition prohibit intra-species recycling, the recycling of one given animal species to the same species, in order to avoid potential safety risks to human and animal health. These regulations have generated the need of their control in aquaculture by effective and specific analytical techniques. To date, most studies of species identification and detection in feedstuffs are focused on land species, but few studies are focused on species composition in fish feed. The present work describes five methodologies based in Real Time PCR for detection of the most relevant fish species farmed in Europe: gilthead sea bream (Sparus aurata); sea bass (Dicentrarchus labrax); turbot (Scophthalmus maximus); rainbow trout (Onchorynchus mykiss); and salmon (Salmo salar), in order to guarantee the intra-species recycling regulation in aquaculture feedstuffs.