Effects of chronic nitrate exposure on the intestinal morphology, immune status, barrier function, and microbiota of juvenile turbot (Scophthalmus maximus).

Coming along with high water reuse in sustainable and intensive recirculating aquaculture systems (RASs), the waste products of fish in rearing water is continuously accumulated. Nitrate, the final product of biological nitrification processes, which may cause aquatic toxicity to fish in different degrees when exposed for a long time. Therefore, the present study was conducted to evaluate the impact of chronic nitrate exposure on intestinal morphology, immune status, barrier function, and microbiota of juvenile turbot. For that, groups of juvenile turbot were exposed to 0 (control check, CK), 50 (low nitrate, L), 200 (medium nitrate, M), and 400 (high nitrate, H) mg L-1 nitrate-N in small-sized recirculating aquaculture systems. After the 60-day experiment period, we found that exposure to a high concentration of nitrate-N caused obvious pathological damages to the intestine; for instance, atrophy of intestinal microvilli and necrosis in the lamina propria. Quantitative real-time PCR analysis revealed a significant downregulation of the barrier forming tight junction genes like occludin, claudin-like etc. under H treatment (P < 0.05). Intestinal MUC-2 expression also decreased significantly in the nitrate treatment groups compared to that in the control (P < 0.05). Additionally, the expression of HSP70 and HSP90 heat-shock proteins, toll-like receptor-3 (TLR-3), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) significantly increased (P < 0.05), whereas that of transforming growth factor-ß (TGF-ß), lysozyme (LYS), and insulin-like growth factor-I (IGF-I) significantly decreased with H treatment (P < 0.05). The results also revealed that intestinal microbial community was changed following nitrate exposure and could alter the α-diversity and ß-diversity. Specifically, the proportion of intrinsic flora decreased, whereas that of the potential pathogens significantly increased with M and H treatments (P < 0.05). In conclusion, chronic nitrate exposure could weaken the barrier function and disturb the composition of intestinal microbiota in marine teleosts, thereby harming their health condition.

Dietary Astragalus polysaccharides ameliorates the growth performance, antioxidant capacity and immune responses in turbot (Scophthalmus maximus L.).

Supplying immunostimulants to aquatic feed has been an effective way to enhance the health of aquatic animals and substitute for antibiotics. In the present study, the potential effects of Astragalus polysaccharides (APS) were evaluated in turbot, Scophthalmus maximus. Two levels of APS (50 and 150 mg/kg) were added to the basal diet (CON) and a 63-day growth trial (initial weight 10.13 ± 0.04 g) was conducted. As the results showed, significant improvement on growth performance in the APS groups were observed. In addition, dietary 150 mg/kg APS significantly increased the total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX) and lysozyme activities in liver. Meanwhile, APS diets induced the mRNA expression of toll-like receptors (TLRs) such as tlr5α, tlr5ß, tlr8 and tlr21, while reduced the expression of tlr3 and tlr22. The expression of inflammatory genes myeloid differentiation factor 88 and nuclear factor kappa b p65 and pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1ß were up-regulated in APS groups while the expression of anti-inflammatory cytokine transforming growth factor beta was inhibited. Taken together, the present study indicated that Astragalus polysaccharides could remarkably enhance the growth performance, antioxidant activity and maintain an active immune response in turbot.

Reference gene validation for quantification of gene expression during ovarian development of turbot (Scophthalmus maximus).

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a powerful and sensitive method used in gene expression analysis. Suitable reference genes, which are stable under all experimental circumstances and tissues significantly improve the accuracy of qRT-PCR data. In this study, the stability of six genes, namely, 18S ribosomal RNA (18s), beta-actin (actb), elongation factor 1-alpha (ef1α), glyceraldehyde-3-phosphate-dehydrogenase (gapdh), cathepsin D (ctsd), and beta-2-microglobulin (b2m) were evaluated as potential references for qRT-PCR analysis. The genes were examined in the hypothalamus-pituitary-ovary-liver (HPOL) axis throughout turbot ovarian development via using the geNorm, NormFinder and BestKeeper algorithms. Results showed that the most stable reference genes were ef1α, actb, and ctsd in the hypothalamus, pituitary, ovary and liver, respectively. The best-suited gene combinations for normalization were 18s, ef1α, and ctsd in the hypothalamus; actb, ctsd, and 18s in the pituitary; actb, and ctsd in the ovary; gapdh and ctsd in the liver. Moreover, the expression profile of estrogen receptor α (erα) manifested no significant difference normalization to the aforementioned best-suited gene during turbot ovarian development. However, no single gene or pair of genes is suitable as an internal control and account for the amplification differences among the four tissues during ovarian development. In summary, these results provide a basic data for the optimal reference gene selection and obtain highly accurate normalization of qRT-PCR data in HPOL axis-related gene expression analysis during turbot ovarian development.

Beneficial influences of dietary Aspergillus awamori fermented soybean meal on oxidative homoeostasis and inflammatory response in turbot (Scophthalmus maximus L.).

High levels of soybean meal (SBM) in aquafeed leads to detrimental inflammatory response and oxidative stress in fish. In the present study, fermentation with Aspergillus awamori was conducted to explore the potential effects on improving the nutritional quality of soybean meal and the health status of turbot. A 63-day feeding trial (initial weight 8.53 ±â€¯0.11 g) was carried out to evaluate the utilization of fermented soybean meal (FSM) by juvenile turbot. 0% (FM, control), 30% (S30, F30), 45% (S45, F45), and 60% (S60, F60) of fish meal were replaced with SBM or FSM, respectively. As the results showed, fermentation significantly reduced the contents of anti-nutritional factors in SBM, including raffinose (-98.8%), glycinin (-98.5%), ß-conglycinin (-97.4%), trypsin inhibitors (-80%) and stachyose (-80%). A depression of fish growth performance and activities of superoxide dismutase and lysozyme were observed in S45 and S60 groups, while these inferiorities were only observed in F60 group. Meanwhile, fermentation also improved the heights of enterocytes and microvillus significantly in the F45 and F60 groups compared with those in SBM. An induced expression of anti-inflammatory cytokine transforming growth factor-ß and depression of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1ß in the distal intestine were observed in the F45 and F60 groups. Taken together, this study indicated that fermentation with Aspergillus awamori could improve the replacement level with soybean meal from 30% to 45% in turbot.

Resveratrol attenuates oxidative stress and inflammatory response in turbot fed with soybean meal based diet.

Adding immunopotentiators to plant protein based diets has been a feasible way to improve fish growth performance and healthy status. In this study, an 8-week trial was carried out to explore the effects of resveratrol, a natural polyphenolic compound, on growth performance, anti-oxidative capacity and immune responses in turbot fed soybean meal based diet. As the results showed, replacement 45% fish meal with soybean meal (SBM) significantly depressed the fish growth, feed utilization and the heights of villi and microvilli in distal intestine. The mRNA levels of hepatic antioxidant enzymes, including superoxide dismutase (sod), glutathione peroxidase (gsh-px) and peroxiredoxin 6 (prx 6), were highly inhibited in SBM group. The inflammation related genes in intestine were also responsive to soybean meal. Supplying resveratrol showed no effects on fish growth performance but significantly restored the intestinal morphology and improved the mRNA levels of hepatic antioxidant enzymes as well as the activity of SOD. Meanwhile, resveratrol significantly improved the mRNA levels of anti-inflammatory cytokine transforming growth factor-ß and inhibited the expression of pro-inflammatory cytokines tumor necrosis factor-α (tnf-ɑ), interleukin-1ß (il-1ß) and interleukin-8 (il-8). The results indicate that resveratrol could attenuate the oxidative stress and inflammatory response induced by soybean meal in turbot. This study shows resveratrol is an effective immunopotentiator to carnivorous fishes fed plant protein sources.

Effects of the isoflavone daidzein in Senegalese sole, Solea senegalensis: Modulation of the oestrogen receptor-ß, apoptosis and enzymatic signalling pathways.

Phytochemicals are widely present in the aquatic environment and they are derived from many anthropogenic activities. The isoflavone daidzein is a natural compound that is found in the soya products used as habitual constituents of aquafeeds. Nevertheless, this isoflavone possesses oestrogenic and apoptotic properties. The present study determined the effects of daidzein (at 20 mg/L) during the first month and a half of life (from 7 to 44 days post-hatching -dph-) of the flatfish Senegalese sole, Solea senegalensis, focusing at the metamorphosis. We have analysed different gene expression levels and immunohistochemical protein patterns implicated in some oestrogenic, apoptosis and enzymatic pathways. In general, the oestrogen receptor (ERß) and stimulating apoptosis death receptor factor (Fas) transcript levels showed similar baseline patterns and transcriptional responses induced by daidzein. Both ERß and Fas were up-regulated by this isoflavone at the pre-metamorphosis and metamorphosis, and they were down-regulated in post-metamorphosed stages. The expression pattern of the apoptotic effector caspase (Casp6) was exclusively up-regulated at the pre-metamorphic phase. The Birc5 transcripts (i.e. anti-apoptosis, Survivin) were down-regulated by daidzein during certain metamorphic and post-metamorphosed stages. Besides, daidzein showed an up-regulating effect on both enzymatic complexes, the haemoprotein CYP1A and the acetylcholinesterase (AChE), except for a temporary AChE down-regulation in some post-metamorphosed stages. Immunostaining analysis only showed increased CYP1A signals in the liver of daidzein exposed fish. Overall, a majority of the transcriptional oestrogenic and apoptotic imbalances could be gradually and/or temporarily stabilised. Most controls and exposed larvae (70-80%) developed and grew following normal ontogenetic developmental patterns.

Expression analysis and characterization of zglp1 in the Chinese tongue sole (Cynoglossus semilaevis).

Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein that regulates the interaction between somatic cells and germ cells during gonad developmental process in mammals. In this study, the zglp1 of Chinese tongue sole, Cynoglossus semilaevis (cysezglp1), was cloned and characterized for the first time in fish. Cysezglp1 had an open reading frame with five exons and was located to chromosome 9. The open reading frame of cysezglp1 consisted of 1692 nucleotides and encoded a 583 amino acid polypeptide. The predicted protein contained two zinc finger structures (Znf1 and Znf2), one of which was highly homologous to the GATA-type zinc finger domain. Multiple sequence alignment showed that Znf1 was conserved across different species while Znf2 was more divergent. Through quantitative Real-time PCR (qRT-PCR), we found that cysezglp1 was predominantly expressed in gonads, and the expression level of the ovary was significantly higher than that of the testis. We compared expression level in different embryonic stages and found that cysezglp1 mRNAs were mainly expressed in the fertilized egg to the cleavage stage, subsequently declining in the blastula stage. Cysezglp1 expression was not detected from the gastrulation stage onward. In the ovary, cysezglp1 expression was detected at 120 days after hatching and expression gradually increased with the maturation of the ovary. In situ hybridization showed that the cysezglp1 was mainly expressed in oocytes. Taken together, our results suggest that cysezglp1 may play an important role in the process of oogenesis in Chinese tongue sole.

Examining the role of estrogenic activity and ocean temperature on declines of a coastal demersal flatfish population near the municipal wastewater outfall of Orange County, California, USA.

Wastewater treatment plant effluent introduces a mixture of pollutants into marine environments; however, the impacts of chronic sublethal exposures on populations are often unclear. Presence of estrogenic agents in sediments and uptake of these compounds by demersal flatfishes has been reported at the Orange County Sanitation District (OCSD) wastewater outfall. Furthermore, estrogenic activity has been identified in male flatfish in the area, potentially contributing to observed population declines in the OCSD region. Rising ocean temperatures may further contribute to flatfish declines as relationships between temperature and abundance have been reported in the Southern California Bight. To investigate declines, sex ratios, condition factor, organ health indices, hormones, and vitellogenin were quantified in flatfish collected at OCSD outfall and reference sites. Additionally, historical temperature data was examined for trends with population abundances. Rather than being linked to estrogenic activity, results indicated that population declines were more correlated to increases in ocean temperature.

Synergistic effects of dietary Bacillus sp. SJ-10 plus ß-glucooligosaccharides as a synbiotic on growth performance, innate immunity and streptococcosis resistance in olive flounder (Paralichthys olivaceus).

Bacillus sp. SJ-10 (BSJ-10) was identified from traditional Korean fermented fish, the previously recognized prebiotic ß-glucooligosaccharides (BGO), and their combination as a synbiotic were prepared to evaluate their individual and synergistic effects in olive flounder (Paralichthys olivaceus). Four diets (one control and three treatments) were formulated containing neither BSJ-10 nor BGO (control), 1 × 108 CFU g -1 BSJ-10 (BSJ-10), 0.1% BGO (BGO), and 1 × 108 CFU g-1 BSJ-10 + 0.1% BGO (BSJ-10 + BGO). Triplicates of 15 fish (weight 10 ± 0.25 g) were randomly allocated to the four diet groups and fed one of the diets for 8 weeks. At the end of the experiment, fish weight gain (WG), specific growth rate (SGR), feed conversion ratio, and protein efficiency ratio in BSJ-10, BGO and BSJ-10 + BGO diets were positively modulated (P < 0.05) compared with control. Specially, WG and SGR were significantly (P < 0.05) higher in BSJ-10 + BGO than that of BSJ-10 and BGO (individual component). The innate immune parameters such as respiratory burst, superoxide dismutase, and lysozyme activity (LSZ) of fish fed BSJ-10 and BSJ-10 + BGO (both groups) were significantly (P < 0.05) higher than the control. Moreover, myeloperoxidase activity (MPO) and LSZ of fish fed BSJ-10 + BGO were significantly higher compared with individual component. Compared with control, intestinal BSJ-10 content, expression of interleukin (IL)-1ß in liver and kidney, and tumor necrosis factor (TNF)-α in liver were higher in both groups, but microvillus length was increased (P < 0.05) only in BSJ-10 + BGO. During in vivo challenge experiment with Streptococcus iniae (1 × 108 CFU ml-1), survival rate of fish was significantly higher in all treatment groups versus control. Moreover, in BSJ-10 + BGO, protection against S. iniae infection and transcription of TNF-α and IL-6 in gill were significantly (P < 0.05) higher than the individual component. Collectively, an improved WG, SGR, MPO, LSZ, transcription of IL-6 and TNF-α, and cumulative survival rate against streptococcosis clearly demonstrates a synergistic outcome of diet BSJ-10 + BGO as synbiotic in olive flounder.

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.

The anti-androgenic effect of chronic exposure to semicarbazide on male Japanese flounder (Paralichthys olivaceus) and its potential mechanisms.

Semicarbazide (SMC), a new marine pollutant, has anti-estrogenic effects on female Japanese flounder (Paralichthys olivaceus). However, whether SMC also affects the reproductive endocrine system of male marine organisms is currently unclear. In this study, Japanese flounder embryos were exposed to 1, 10, and 100 µg/L SMC for 130 days. Plasma testosterone (T) and 17ß-estradiol (E2) concentrations were significantly decreased in male flounders after SMC exposure. The expression of genes involved in T and E2 synthesis, including steroidogenic acute regulatory protein, cytochrome P450 11A1, 17α-hydroxylase, 17ß-hydroxysteroid dehydrogenase and cytochrome P450 19A, was down-regulated in the gonads, which may explain the decrease in plasma sex hormones levels. Moreover, SMC-mediated changes in the transcription of these steroidogenic genes were associated with reduced levels of follicle-stimulating hormone beta subunit (fshß), luteinizing hormone beta subunit (lhß), follicle-stimulating hormone receptor (fshr) and luteinizing hormone receptor (lhr) mRNA. In addition, down-regulated transcription of fshß and lhß in the SMC exposure groups was affected by reduced mRNA levels of seabream gonadotropin-releasing hormone (sbgnrh), g-protein-coupled receptor 54 (gpr54) in the kisspeptin/gpr54 system, as well as the gamma-aminobutyric acid (GABA) synthesis enzyme glutamic acid decarboxylase (gad). Overall, our results showed that environmentally relevant concentrations of SMC exerted anti-androgenic effects in male flounders via impacting HPG axis, kiss/gpr54 system and GABA synthesis, providing theoretical support for investigating reproductive toxicity of environmental pollutants that interfere with the neuroendocrine system.

Expression pattern and functional analysis of R-spondin1 in tongue sole Cynoglossus semilaevis.

R-spondin 1 (Rspo1) is a potential female-determining gene in mammals that could regulate the Wnt/ß-catenin signaling pathway. The deletion of Rspo1 causes sex reversal in females. To investigate sexual determination and differentiation, we cloned and analyzed the Rspo1 gene in Cynoglossus semilaevis. Phylogenetic and gene structure analyses revealed that Rspo1 gene exhibited high sequence conservation and contained an N-terminal signal peptide, two furin-like cysteine-rich domains (FU1 and FU2), a thrombospondin type 1 repeat, and a C-terminal region enriched with basic charged amino acids. qRT-PCR revealed that Rspo1 expressed sexual dimorphism in gonad, with higher expression levels in the ovary than in the testis, thus, suggesting the involvement of Rspo1 in gonad differentiation. In situ hybridization results demonstrated that Rspo1 was expressed in premature germ cells, including spermatogonia and spermatocytes in the testis and stage II and stage III oocytes in the ovary. The methylation levels in two CpG sites of Rspo1 promoter significantly differed among females, males, and pseudomales. After 30days of exposure to high temperature, the expression of Rspo1 significantly decreased in female individuals, some of which were prone to males. However, no difference of Rspo1 gene expression was observed between the control group and high-temperature group in males. These preliminary findings suggested that Rspo1 played a crucial role in sex determination and development. This study laid the groundwork for further sex control breeding techniques in C. semilaevis.

Phylogeny, expression patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea senegalensis.

BACKGROUND: The identification of DNA methyltransferases (Dnmt) expression patterns during development and their regulation is important to understand the epigenetic mechanisms that modulate larval plasticity in marine fish. In this study, dnmt1 and dnmt3 paralogs were identified in the flatfish Solea senegalensis and expression patterns in early developmental stages and juveniles were determined. Additionally, the regulation of Dnmt transcription by a specific inhibitor (5-aza-2'-deoxycytidine) and temperature was evaluated. RESULTS: Five paralog genes of dnmt3, namely dnmt3aa, dnmt3ab, dnmt3ba, dnmt3bb.1 and dnmt3bb.2 and one gene for dnmt1 were identified. Phylogenetic analysis revealed that the dnmt gene family was highly conserved in teleosts and three fish-specific genes, dnmt3aa, dnmt3ba and dnmt3bb.2 have evolved. The spatio-temporal expression patterns of four dnmts (dnmt1, dnmt3aa, dnmt3ab and dnmt3bb.1) were different in early larval stages although all of them reduced expression with the age and were detected in neural organs and dnmt3aa appeared specific to somites. In juveniles, the four dnmt genes were expressed in brain and hematopoietic tissues such as kidney, spleen and gills. Treatment of sole embryos with 5-aza-2'-deoxycytidine down-regulated dntm1 and up-regulated dntm3aa. Moreover, in lecithotrophic larval stages, dnmt3aa and dnmt3ab were temperature sensitive and their expression was higher in larvae incubated at 16 °C relative to 20 °C. CONCLUSION: Five dnmt3 and one dnmt1 paralog were identified in sole and their distinct developmental and tissue-specific expression patterns indicate that they may have different roles during development. The inhibitor 5-aza-2'-deoxycytidine modified the transcript abundance of dntm1 and dntm3aa in embryos, which suggests that a regulatory feedback mechanism exists for these genes. The impact of thermal regime on expression levels of dnmt3aa and dnmt3ab in lecithotrophic larval stages suggests that these paralogs might be involved in thermal programing.

Effects of the soya isoflavone genistein in early life stages of the Senegalese sole, Solea senegalensis: Thyroid, estrogenic and metabolic biomarkers.

This study examines the effects induced by environmentally relevant concentrations of the isoflavone genistein (3mg/L and 10mg/L) during early life stages of the Senegalese sole. Throughout the hypothalamus-pituitary-thyroid (HPT) axis, several neurohormonal regulatory thyroid signalling patterns (thyroglobulin/Tg, thyroid peroxidase/TPO, transthyretin/TTR, thyroid receptors/TRß, and iodothrynonine deiodinases, Dio2 and Dio3) were analysed. Furthermore, the expression patterns of estrogen receptor ERß and haemoprotein Cyp1a were also evaluated. In the control larvae, progressive increases of constitutive hormonal signalling pathways have been evidenced from the pre-metamorphosis phase onwards, reaching the highest expression basal levels at the metamorphosis (Tg, TPO, Dio2) and/or during post-metamorphosis (TTR, TRß, ERß). When the early larvae were exposed to both genistein concentrations (3mg/L and 10mg/L), a statistically significant down-regulation of TPO, TTR and Tg mRNA levels was clearly detected at the metamorphic stages. In addition, the Dio2 and Dio3 transcript expression levels were also down and up-regulated when exposed to both genistein concentrations. In the larvae exposed to genistein, no statistically significant responses were recorded for the TRß expression patterns. Nevertheless, the ERß and Cyp1a transcript levels were up-regulated at the middle metamorphic stage (S2, at 16 dph) in the larvae exposed to high genistein concentrations and, only the ERß was down-regulated (S1, at 12dph) at the lower doses. Finally, all these pointed out imbalances were only temporarily disrupted by exposure to genistein, since most of the modulated transcriptional signals (i.e. up or down-regulation) were quickly restored to the baseline levels. Additionally, the control and genistein-exposed Senegalese sole specimens showed characteristic ontogenetic patterns and completely suitable for an optimal development, metamorphosis, and growth.

Total substitution of dietary fish oil by vegetable oils stimulates muscle hypertrophic growth in Senegalese sole and the upregulation of fgf6.

The long term effects of fish oil (FO) substitution by increasing the levels of vegetable oils (VO), 0% (CTR), 50% (VO50) and 100% (VO100), in diets for Senegalese sole were evaluated in terms of skeletal muscle cellularity and expression of related genes. After 140 days of feeding, all fish had similar body weight and length. The inclusion of 50% VO did not result in differences in muscle cellularity, but dorsal muscle cross-sectional area and fast-twitch fibre diameter increased in fish fed total FO substitution, whilst fibre density was reduced (P < 0.05) in relation to CTR. The total number of fibres was similar in all treatments. FO substitution did not affect the transcript levels of myogenic genes (myf5, mrf4, myog, myod1, myod2), but resulted in a two-fold increase of fgf6 transcript levels compared to CTR (P < 0.05). The relative expression of igf-I was higher in VO100 than in VO50, but was similar to CTR. FO substitution resulted in cellularity changes related to the stimulation of muscle hypertrophic growth, but not hyperplastic growth, and associated with a nutritional modulation of fgf6 by dietary VO. This study indicates that 50% VO does not affect the muscle phenotype, but total FO substitution stimulates muscle hypertrophy.

Gonadogenesis analysis and sex differentiation in cultured turbot (Scophthalmus maximus).

As a flatfish, the turbot (Scophthalmus maximus) is one of the most important farmed fish species with great commercial value, which has a strong sexual dimorphism on growth rate and sexual maturity. In this study, using histology, the basic information on proliferation and migration of germ cells and early gonadal development during sex differentiation in turbot were described in detail. There were six to nine individual primordial germ cells (PGCs) with large nuclei until 15 days post-hatching (dph). The PGCs located under the mesonephric ducts undergo migration along the dorsal mesentery toward the region of the genital ridge from 0 to 15 dph. During migration, the number of PGCs was constant, and the expression of vasa had no significant changes. At 20 dph, the aggregation of somatic cells at genital ridge indicated the formation of primary gonad. Furthermore, the number of PGCs was increased to 60 and the expression of vasa was upregulated for the first time. The undifferentiated gonads with no morphological indications of sex differentiation grew larger with the increase in germ cells and somatic cells number/size from 20 to 35 dph. During 36-52 dph, cytological gonadal differentiation was observed. In presumptive testes of type I gonadal tissue (with a lance shape), the number of germ cells increased steadily and the germ cells had the same characteristics as before. Meanwhile, in presumptive ovaries of type II gonadal tissue (with a club-like shape), the germ cells proliferated and induced in two different populations of germ cells. One type had the morphological characteristics as undifferentiated germ cells, while the other type of germ cells underwent mitosis exhibiting smaller size and mottled nuclei. At 60 dph, ovarian cavity was present in the gonad of type II, which would develop into ovaries. However, spermatogonial cysts were not noticed in the gonad of type I until 90 dph, which indicated the formation of the testes.

Molecular characterization and developmental expression patterns of apolipoprotein A-I in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-I (ApoA-I) is an essential component of the high density lipoproteins (HDL). In this study, the cDNA and genomic sequences of this apolipoprotein were characterized for first time in Solea senegalensis. The predicted polypeptide revealed conserved structural features including ten repeats in the lipid-binding domain and some residues involved in cholesterol interaction and binding. The gene structure analysis identified four exons and three introns. Moreover, the synteny analysis revealed that apoA-I did not localize with other apolipoproteins indicating a divergent evolution with respect to the apoA-IV and apoE cluster. The phylogenetic analyses identified two distinct apoA-I paralogs in Ostariophysi (referred to as Ia and Ib) and only one (Ib) in Acanthopterygii. Whole-mount in situ hybridization located the apoA-I signal mainly in the yolk syncytial layer in lecitotrophic larval stages. Later at mouth opening, the mRNA signals were detected mainly in liver and intestine compatible with its role in the HDL formation. Moreover, a clear signal was detected in some regions of the brain, retina and neural cord suggesting a role in local regulation of cholesterol homeostasis. After metamorphosis, apoA-I was also detected in other tissues such as gills, head kidney and spleen suggesting a putative role in immunity. Expression analyses in larvae fed two diets with different triacylglycerol levels indicated that apoA-I mRNA levels were more associated to larval size and development than dietary lipid levels. Finally, qPCR analyses of immature and mature transcripts revealed distinct expression profiles suggesting a posttranscriptional regulatory mechanism.

Dietary indispensable amino acids profile affects protein utilization and growth of Senegalese sole larvae.

In diet formulation for fish, it is critical to assure that all the indispensable amino acids (IAA) are available in the right quantities and ratios. This will allow minimizing dietary AA imbalances that will result in unavoidable AA losses for energy dissipation rather than for protein synthesis and growth. The supplementation with crystalline amino acids (CAA) is a possible solution to correct the dietary amino acid (AA) profile that has shown positive results for larvae of some fish species. This study tested the effect of supplementing a practical microdiet with encapsulated CAA as to balance the dietary IAA profile and to improve the capacity of Senegalese sole larvae to utilize AA and maximize growth potential. Larvae were reared at 19 °C under a co-feeding regime from mouth opening. Two microdiets were formulated and processed as to have as much as possible the same ingredients and proximate composition. The control diet (CTRL) formulation was based on commonly used protein sources. A balanced diet (BAL) was formulated as to meet the ideal IAA profile defined for Senegalese sole: the dietary AA profile was corrected by replacing 4 % of encapsulated protein hydrolysate by CAA. The in vivo method of controlled tube-feeding was used to assess the effect on the larvae capacity to utilize protein, during key developmental stages. Growth was monitored until 51 DAH. The supplementation of microdiets with CAA in order to balance the dietary AA had a positive short-term effect on the Senegalese sole larvae capacity to retain protein. However, that did not translate into increased growth. On the contrary, larvae fed a more imbalanced (CTRL group) diet attained a better performance. Further studies are needed to ascertain whether this was due to an effect on the voluntary feed intake as a compensatory response to the dietary IAA imbalance in the CTRL diet or due to the higher content of tryptophan in the BAL diet.

Tumour necrosis factor-α inhibits hepatic lipid deposition through GSK-3ß/ß-catenin signaling in juvenile turbot (Scophthalmus maximus L.).

In this study, the mechanism that TNFα inhibits lipid deposition through GSK-3ß/ß-catenin signaling was investigated in the liver of juvenile turbot (Scophthalmus maximus L.) by injection of TNFα or TNFα inhibitor pomalidomide (POM). It was found that TNFα inhibited the expression of GSK-3ß and induced ß-catenin expression. TNFα inhibited the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα), as well as the activity of lipoprotein lipase (LPL) and fatty acid synthetase (FAS). In addition, the level of triglyceride (TG), total cholesterol (TC), nonesterified fatty acid (NEFA), and glycerol was decreased by TNFα treatment in the liver. In the plasma, the level of TG, TC, low density lipoprotein cholesterol (LDL-C), NEFA, and glycerol was decreased, but high density lipoprotein cholesterol (HDL-C) was increased by TNFα treatment. However, compared to TNFα, POM had the opposite effect on the biochemical indexes and genes related to lipid deposition in the liver. The results indicated that TNFα may regulate hepatic lipid metabolism and fat distribution through GSK-3ß/ß-catenin signaling as well as transcription factors PPARγ and C/EBPα in juvenile turbot.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.