Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole ( Cynoglossus semilaevis).

Chinese tongue sole ( Cynoglossus semilaevis) is an economically important marine fish species with a ZZ/ZW sex determination mechanism, which can be influenced by temperature. Alternative splicing (AS) is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation, but has rarely been reported in fish. In this study, to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation, we combined isoform and RNA sequencing (Iso-Seq and RNA-Seq) to perform transcriptome profiling of male and female gonads in C. semilaevis. In total, 81 883 and 32 341 full-length transcripts were obtained in males and females, respectively. A total of 8 279 AS genes were identified, including 2 639 genes showing differential AS (DAS) between males and females. Many intersecting DAS genes and differentially expressed genes (DEGs) were enriched in the meiotic cell cycle pathway, and genes related to gonadal differentiation, such as esrrb and wt1a, were found to have sex-specific isoforms. Thus, this study revealed AS events in the gonadal transcriptomes of male and female C. semilaevis, described the characteristics of active transcription in the testes, and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.

Dietary gamma-aminobutyric acid ameliorates growth impairment and intestinal dysfunction in turbot (Scophthalmus maximus L.) fed a high soybean meal diet.

Over-substitution of fishmeal with soybean meal (SBM) commonly induces inferior growth and intestinal dysfunction in fish. This study aims to evaluate whether dietary gamma-aminobutyric acid (GABA) could ameliorate the adverse effects in turbot fed a high-SBM diet (HSD). Two hundred and seventy turbots were randomly divided into three treatment groups including turbots fed on a control diet (CNT, containing 60% fishmeal), an HSD (with 45% fishmeal protein replaced by SBM), and an HSD supplemented with GABA (160 mg kg-1) for 53 days. The growth and feed utilization parameters were calculated and the intestinal antioxidant status, inflammation, apoptosis, and microbiota were evaluated using assay kits, histological analysis, qRT-PCR, high throughput sequencing, and bioinformatics analysis. The results showed that GABA ameliorated HSD-induced growth impairment and enhanced feed intake of turbot. GABA ameliorated HSD-induced intestinal oxidative stress and apoptosis by restoring the MDA content, CAT and T-AOC activities, and apoptosis-related gene (Bcl-2, Bax, Bid, and Caspase-3) expressions to similar levels to those in the CNT group. GABA also alleviated HSD-induced intestinal inflammation through down-regulating the expressions of TNF-α, IL-1ß, and NF-κB p65 and up-regulating the expression of TGF-ß1. Furthermore, GABA reversed HSD-induced microbiota dysbiosis through regulating the overall bacterial richness and dominative bacterial population. Spearman's correlation analysis indicated that the altered microbiota was closely associated with growth and intestinal function. Collectively, GABA could ameliorate HSD-induced intestinal dysfunction via relieving oxidative stress, inflammation, apoptosis and microbiota dysbiosis, and these findings would contribute to a better understanding of the function of GABA in the fish intestine.

The linkage of cell cycle and DNA replication with growth difference in female Chinese tongue sole (Cynoglossus semilaevis): Analysis from transcriptomic study and WGCNA.

In addition to the typical sexual size dimorphism, considerable size differences within the female population of the Chinese tongue sole (Cynoglossus semilaevis) have become a further bottleneck of the improvement of sole aquaculture. To identify the internal mechanism, transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) were employed simultaneously. Transcriptomic analyses of brain, pituitary gland, liver, gonad, and muscle tissues from two female groups with size differences identified 109, 698, 1325, 2299, and 2141 differentially expressed genes (DEGs), respectively. The results of these enrichment analyses suggest that the up-regulation of neuroactive ligand-receptor interaction, cell cycle, DNA replication, and MAPK signaling pathway in the group with larger females may be involved in the regulation of the observed growth differences. WGCNA of DEGs showed that cell cycle and DNA replication might be crucial pathways for accelerating cell growth in the groups with larger females. Finally, a series of hub genes including 6-phosphofructokinase type C (pfkp), ribosome biogenesis protein (wdr12), bleomycin hydrolase (blmh), and semaphorin-3A (sema3a) were recognized by the illustrated network map of modules. The linkage of cell cycle, DNA replication, and hub genes in the growth regulation of C. semilaevis provides further information for a better understanding of growth differences in fish.

Molecular cloning and immune characterization of CIITA in artificially challenged Chinese tongue sole (Cynoglossus semilaevis) with Vibrio harveyi.

In mammals, Class II, major histocompatibility complex (MHC II) transactivator (CIITA) recognizes microbial pathogens and triggers immune responses. In Chinese tongue sole Cynoglossus semilaevis, Cs-CIITA was prevalently expressed in various tissues. Cs-CIITA, Cs-MHC IIA and Cs-MHC IIB were expressed significantly higher in skin in susceptible families infected with Vibrio harveyi, while higher expression of Cs-CIITA and Cs-MHC IIB was examined in liver in resistant families. In addition, the three genes were up-regulated in gill, skin, intestine, liver, spleen and kidney at 48 h or 72 h after V. harveyi infection. Furthermore, the three genes were co-expressed in the epithelial mucous cells of gill, skin, and intestine. Knockdown of Cs-CIITA regulates the expression of other inflammation-related genes, including CD40, IL-1ß, IL-8, RelB, NFκB, and Myd88. These results suggest that CIITA functions in the inflammatory responses of C. semilaevis against V. harveyi, via MHC II transcriptional regulation.

Mild Effects of Sunscreen Agents on a Marine Flatfish: Oxidative Stress, Energetic Profiles, Neurotoxicity and Behaviour in Response to Titanium Dioxide Nanoparticles and Oxybenzone.

UV filters are potentially harmful to marine organisms. Given their worldwide dissemination and the scarcity of studies on marine fish, we evaluated the toxicity of an organic (oxybenzone) and an inorganic (titanium dioxide nanoparticles) UV filter, individually and in a binary mixture, in the turbot (Scophthalmus maximus). Fish were intraperitoneally injected and a multi-level assessment was carried out 3 and 7 days later. Oxybenzone and titanium dioxide nanoparticles induced mild effects on turbot, both isolated and in mixture. Neither oxidative stress (intestine, liver and kidney) nor neurotoxicity (brain) was found. However, liver metabolic function was altered after 7 days, suggesting the impairment of the aerobic metabolism. An increased motility rate in oxybenzone treatment was the only behavioural alteration (day 7). The intestine and liver were preferentially targeted, while kidney and brain were unaffected. Both infra- and supra-additive interactions were perceived, with a toxicodynamic nature, resulting either in favourable or unfavourable toxicological outcomes, which were markedly dependent on the organ, parameter and post-injection time. The combined exposure to the UV filters did not show a consistent increment in toxicity in comparison with the isolated exposures, which is an ecologically relevant finding providing key information towards the formulation of environmentally safe sunscreen products.

Investigating the effect of nitrate on juvenile turbot (Scophthalmus maximus) growth performance, health status, and endocrine function in marine recirculation aquaculture systems.

Nitrate (NO3-), a potential toxic nitrogenous compound to aquatic animals, is distributed in aquatic ecosystems worldwide. The aim of this study was to investigate the effects of different NO3- levels on growth performance, health status, and endocrine function of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems (RAS). Fish were exposed to 0 mg/L (control, CK), 50 mg/L (low nitrate, LN), 200 mg/L (medium nitrate, MN), and 400 mg/L (high nitrate, HN) NO3-N for 60 d in experimental RAS. Cumulative survival (CS) was significantly decreased with increasing NO3- levels in LN, MN, and HN. The lowest CS was 35% in the HN group. Growth parameters, including absolute growth rate, specific growth rate, and feed conversion rate, were significantly different in HN compared with that in the CK. Histological survey of gills and liver revealed dose-dependent histopathological damage induced by NO3- exposure and significant differences in glutamate pyruvate transaminase and glutamate oxalate transaminase in MN and HN compared with that in the CK. The hepatosomatic index in HN was significantly higher than that in the CK. Additionally, NO3- significantly increased bioaccumulation in plasma in LN, MN, and HN compared to that in the CK. Significant decreases in hemoglobin and increases in methemoglobin levels indicated reduced oxygen-carrying capacity in HN. Additionally, qRT-PCR and enzyme-linked immunosorbent assay (ELISA) were developed to investigate key biomarkers involved in the GH/IGF-1, HPT, and HPI axes. Compared with that in the CK, the abundance of GH, GHRb, and IGF-1 was significantly lower in HN, whereas GHRa did not differ between treatments. The plasma T3 level significantly decreased in LN, MN, and HN and T4 significantly decreased in HN. The CRH, ACTH, and plasma cortisol levels were significantly upregulated in HN compared with that in the CK. We conclude that elevated NO3- exposure leads to growth retardation, impaired health status, and endocrine disorders in turbot and the NO3- level for juvenile turbot culture should not exceed 50 mg/L NO3-N in RAS. Our findings indicate that endocrine dysfunction of the GH/IGF-1, HPT, and HPI axes might be responsible for growth inhibition induced by NO3- exposure.

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.

lncRNA DMRT2-AS acts as a transcriptional regulator of dmrt2 involving in sex differentiation in the Chinese tongue sole (Cynoglossus semilaevis).

Long non-coding RNAs (lncRNAs) contribute to various biological processes, including sexual development. As a member of the DMRT family, dmrt2 plays a very important role in sex determination and differentiation. In this study, we cloned and characterized the lncRNA DMRT2-AS (referred to as dmrt2 antisense) associated with dmrt2 from the gonads of the Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of DMRT2-AS was 537 bp. Based on a sequence alignment, DMRT2-AS overlapped with dmrt2 in reverse on exon 4 and intron 3, with a region of overlap of 221 bp on exon 4. RT-qPCR showed that DMRT2-AS was highly expressed in the testis of Chinese tongue sole. In addition, the expression of DMRT2-AS increased continuously during male gonadal development. In vitro experiments and bioinformatics predictions showed that DMRT2-AS promoted the expression of dmrt2 at the transcriptional level. These results suggest that DMRT2-AS acts as a transcriptional regulator of dmrt2 and plays an important role in the gonadal differentiation of male.

Cloning and functional analysis of c/ebpα as negative regulator of dmrt1 in Chinese tongue sole (Cynoglossus semilaevis).

c/ebpα is a member of the C/EBP family of transcription factors, which are involved in cell growth and differentiation and have a conserved basic leucine zipper (bZIP) domain. However, little is known about its function in sex determination and differentiation. In the present study, c/ebpα was cloned from the gonads of Chinese tongue sole (Cynoglossus semilaevis). The full-length cDNA of c/ebpα was 1583 bp, with a 198-bp 5' UTR, a 446-bp 3' UTR, and a 939-bp open reading frame encoding a 312-amino acid peptide. qRT-PCR revealed that c/ebpα was predominantly expressed in undifferentiated gonads of male C. semilaevis at 30 dpf and 60 dpf and peaked at 60 dpf. Expression levels of c/ebpα in the testis were constantly higher than those in ovaries at all developmental stages. Moreover, a dual-luciferase assay revealed that c/ebpα could negatively regulate the male-determining gene dmrt1 in vitro. These results provide fundamental information indicating that C. semilaevis c/ebpa might be involved in early gonadal differentiation and functions as a negative regulator of dmrt1 by repressing its transcription.

The Assessment of Diet Contaminated with Aflatoxin B1 in Juvenile Turbot (Scophthalmus maximus) and the Evaluation of the Efficacy of Mitigation of a Yeast Cell Wall Extract.

This study aimed to investigate the effects of dietary AFB1 on growth performance, health, intestinal microbiota communities and AFB1 tissue residues of turbot and evaluate the mitigation efficacy of yeast cell wall extract, Mycosorb® (YCWE) toward AFB1 contaminated dietary treatments. Nine experimental diets were formulated: Diet 1 (control): AFB1 free; Diets 2-5 or Diets 6-9: 20 µg AFB1/kg diet or 500 µg AFB1/kg diet + 0%, 0.1%, 0.2%, or 0.4% YCWE, respectively). The results showed that Diet 6 significantly decreased the concentrations of TP, GLB, C3, C4, T-CHO, TG but increased the activities of AST, ALT in serum, decreased the expressions of CAT, SOD, GPx, CYP1A but increased the expressions of CYP3A, GST-ζ1, p53 in liver. Diet 6 increased the AFB1 residues in serum and muscle, altered the intestinal microbiota composition, decreased the bacterial community diversity and the abundance of some potential probiotics. However, Diet 8 and Diet 9 restored the immune response, relieved adverse effects in liver, lowered the AFB1 residues in turbot tissues, promoted intestinal microbiota diversity and lowered the abundance of potentially pathogens. In conclusion, YCWE supplementation decreased the health effects of AFB1 on turbot, restoring biomarkers closer to the mycotoxin-free control diet.

Microalgal extracts induce larval programming and modify growth and the immune response to bioactive treatments and LCDV in Senegalese sole post-larvae.

Immunostimulants are key molecules in aquaculture since they heighten defensive responses and protection against pathogens. The present study investigated the treatment of Senegalese sole larvae with a whole-cell crude extract of the microalgae Nannochloropsis gaditana (Nanno) and programming of growth and the immune system. Larvae at hatch were treated with the Nanno extracts for 2 h and thereafter were cultivated for 32 days post-hatch (dph) in parallel with an untreated control group (CN). Dry weight and length at 21 days post-hatch (dph) were higher in post-larvae of the Nanno than CN group. These differences in weight were later confirmed at 32 dph. To evaluate changes in the immune response associated with Nanno-programming treatments, the Nanno and CN post-larvae were supplied with two bioactive compounds yeast ß-glucan (Y) and a microalga extract from the diatom Phaeodactylum tricornutum (MAe). The bioactive treatments were administrated to the treatment groups through the live prey (artemia metanauplii, 200 artemia mL-1) enriched for 30 min with MAe or Y (at 2 mg mL-1 SW) or untreated prey in the case of the negative control (SW). The effect of the treatments was assessed by monitoring gene expression, enzyme activity and mortality over 48 h. The post-larvae sole supplied with the bioactive compounds Y and MAe had increased mortality at 48 h compared to the SW group. Moreover, mortality was higher in Nanno-programmed than CN post-larvae. Lysozyme and total anti-protease enzymatic activities at 6 and 24 h after the start of the trial were significantly higher in the Nanno and MAe supplied post-larvae compared to their corresponding control (CN and SW, respectively). Immune gene transcripts revealed that il1b, cxc10 and mx mRNAs were significantly different between Nanno and CN post-larvae at 6 and 24 h. Moreover, the expression of il1b, tnfa, cxc10, irf3, irf7 and mx was modified by bioactive treatments but with temporal differences. At 48 h after bioactive treatments, Y and SW post-larvae were challenged with the lymphocystis disease virus (LCDV). No difference existed in viral copy number between programming or bioactive treatment groups at 3, 6 and 24 h after LCDV challenge although the total number of copies reduced with time. Gene expression profiles in the LCDV-challenged group indicated that post-larvae triggered a wide defensive response compared to SWC 24 h after challenge, which was modulated by programming and bioactive compound treatments. Cluster analysis of expressed genes separated the SW and Y groups indicating long-lasting effects of yeast ß-glucan treatment in larvae. A noteworthy interaction between Nanno-programming and Y-treatment on the regulation of antiviral genes was observed. Overall, the data demonstrate the capacity of microalgal crude extracts to modify sole larval plasticity with long-term effects on larval growth and the immune responses.

Gene clusters related to metamorphosis in Solea senegalensis are highly conserved.

The flatfish, Solea senegalensis has considerable scientific interest and commercial value. The metamorphosis in this species occurs between 12 and 19 days after hatching and it takes about 1 week to complete. Eleven Bacterial Artificial Chromosomes (BAC) clones containing the various candidate genes involved in the process of metamorphosis: thyroxine 5 deiodinase 3 (dio3); forkhead box protein E4 (foxe4); melatonin receptor type 1C (mel1c); calsequestrin 1b (casq1b); thyrotropin subunit beta (tshß); thyrotropin-releasing hormone receptor 1, 2, and 3 (trhr1, trhr2, trhr3); thyroid hormone receptor α a and b (thrαa, thrαb); and thyroid hormone receptor beta (thrß) were analyzed by multiple Fluorescence in situ Hybridization (mFISH) and Next Generation Sequencing (NGS) techniques. The mFISH technique localized the 11 BAC clones on 12 different chromosome pairs because three of them, specifically the trhr1a, trhr2 and thrß BAC clones, showed double signals. This signal duplication indicates a duplication of the genomic region inserted within the BAC clone, which provides evidence for the Teleost-Specific Whole Genome Duplication (TS-WGD). Micro-synteny and phylogenetic analysis showed that Cynoglossus semilaevis is the nearest species to S. senegalensis and that Danio rerio is the most distant one. The tshß BAC clone was highly conserved as the genes belonging to this BAC were located on a single chromosome in all the species studied. These genes participate in proliferation, migration and cell-death, which are key processes during metamorphosis. Overall, micro-synteny analysis showed that most candidate genes are found in conserved genomic surroundings.

A combination of genome-wide association study screening and SNaPshot for detecting sex-related SNPs and genes in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) males and females exhibit great differences in growth rate and appearance. The species is heterogametic (ZW/ZZ) and has sex-reversed "pseudomales" that are genetically female and physiologically male. In this study, we identified eight sex-specific single nucleotide polymorphism (SNP) markers for the sex identification of C. semilaevis by using a combination of genome-wide association study (GWAS) screening and SnaPshot validation. Candidate SNPs were screened using genotyping by sequencing to perform GWAS of the differential SNPs between the sexes of C. semilaevis. The SNP loci were amplified using a multiplex PCR system and detected via SNaPshot, which enables multiplexing of up to 30-40 SNPs in a single assay and ensures high accuracy of the results. The molecular markers detected in our study were used to successfully identify normal males and pseudomales from 45 caught and 40 cultured C. semilaevis specimens. Linkage disequilibrium analysis showed that the eight SNP loci were related to each other, with a strong linkage. Moreover, we investigated the expression of prdm6 mRNA containing a missense SNP and confirmed that the gene is differentially expressed in the gonads of the different sexes of C. semilaevis; the expression of prdm6 mRNA was significantly higher in the males than in the females and pseudomales. This means prdm6 may be related to sex differentiation in C. semilaevis.

The intestine of artificially bred larval turbot (Scophthalmus maximus) contains a stable core group of microbiota.

Generally speaking, fish intestinal microbiota is easily affected by food or water environment, and it may be dynamically changed along with body growth. However, it remains unclear whether fish gut microbiota can be affected under any conditions. In the present study, we focused on cultured larval turbot (Scophthalmus maximus) and tracked its artificial breeding process from eggs to larvae in two farms located in different regions of China. Through continuous sampling, we analyzed and compared characteristics of intestinal microbiota in turbot larvae and its correlation with the bacteria in water and food at different developmental stages. The results showed that there was a steady group of microbiota in larval gut, and the highest relative abundance of strain was same between the two farms. This microbiota was established soon after hatching of fertilized eggs. Particularly, the structure of this microbiota was nearly not changeable afterward 3-4 months of development. The bacteria carried by fertilized eggs might play an important role during the formation of this microbiota. In conclusion, our findings suggested that there was a core microbiota represented by Lactococcus sp. in gut of artificially bred turbot larvae. The relative proportion of such strain in gut was higher than 30% at the initial stage of turbot life.

New Insights on Vitamin K Metabolism in Senegalese sole (Solea senegalensis) Based on Ontogenetic and Tissue-Specific Vitamin K Epoxide Reductase Molecular Data.

Vitamin K (VK) is a key nutrient for several biological processes (e.g., blood clotting and bone metabolism). To fulfill VK nutritional requirements, VK action as an activator of pregnane X receptor (Pxr) signaling pathway, and as a co-factor of γ-glutamyl carboxylase enzyme, should be considered. In this regard, VK recycling through vitamin K epoxide reductases (Vkors) is essential and should be better understood. Here, the expression patterns of vitamin K epoxide reductase complex subunit 1 (vkorc1) and vkorc1 like 1 (vkorc1l1) were determined during the larval ontogeny of Senegalese sole (Solea senegalensis), and in early juveniles cultured under different physiological conditions. Full-length transcripts for ssvkorc1 and ssvkorc1l1 were determined and peptide sequences were found to be evolutionarily conserved. During larval development, expression of ssvkorc1 showed a slight increase during absence or low feed intake. Expression of ssvkorc1l1 continuously decreased until 24 h post-fertilization, and remained constant afterwards. Both ssvkors were ubiquitously expressed in adult tissues, and highest expression was found in liver for ssvkorc1, and ovary and brain for ssvkorc1l1. Expression of ssvkorc1 and ssvkorc1l1 was differentially regulated under physiological conditions related to fasting and re-feeding, but also under VK dietary supplementation and induced deficiency. The present work provides new and basic molecular clues evidencing how VK metabolism in marine fish is sensitive to nutritional and environmental conditions.

Changes in the Microbiome of Mariculture Feed Organisms after Treatment with a Potentially Probiotic Strain of Phaeobacter inhibens.

The Phaeobacter genus has been explored as probiotics in mariculture as a sustainable strategy for the prevention of bacterial infections. Its antagonistic effect against common fish pathogens is predominantly due to the production of the antibacterial compound tropodithietic acid (TDA), and TDA-producing strains have repeatedly been isolated from mariculture environments. Despite many in vitro trials targeting pathogens, little is known about its impact on host-associated microbiomes in mariculture. Hence, the purpose of this study was to investigate how the addition of a TDA-producing Phaeobacter inhibens strain affects the microbiomes of live feed organisms and fish larvae. We used 16S rRNA gene sequencing to characterize the bacterial diversity associated with live feed microalgae (Tetraselmis suecica), live feed copepod nauplii (Acartia tonsa), and turbot (Scophthalmus maximus) eggs/larvae. The microbial communities were unique to the three organisms investigated, and the addition of the probiotic bacterium had various effects on the diversity and richness of the microbiomes. The structure of the live feed microbiomes was significantly changed, while no effect was seen on the community structure associated with turbot larvae. The changes were seen primarily in particular taxa. The Rhodobacterales order was indigenous to all three microbiomes and decreased in relative abundance when P. inhibens was introduced in the copepod and turbot microbiomes, while it was unaffected in the microalgal microbiome. Altogether, the study demonstrates that the addition of P. inhibens in higher concentrations, as part of a probiotic regime, does not appear to cause major imbalances in the microbiome, but the effects were specific to closely related taxa.IMPORTANCE This work is an essential part of the risk assessment of the application of roseobacters as probiotics in mariculture. It provides insights into the impact of TDA-producing Phaeobacter inhibens on the commensal bacteria related to mariculture live feed and fish larvae. Also, the study provides a sequencing-based characterization of the microbiomes related to mariculture-relevant microalga, copepods, and turbot larvae.

Temperature-independent genome-wide DNA methylation profile in turbot post-embryonic development.

The morphological and biological characteristics of ectothermic vertebrates are known to be strongly influenced by environmental conditions, particularly temperature. Epigenetic mechanisms such as DNA methylation have been reported to contribute to the phenotypic plasticity observed in vertebrates in response to environmental changes. Additionally, DNA methylation is a dynamic process that occurs throughout vertebrate ontogeny and it has been associated with the activation and silencing of gene expression during post-embryonic development and metamorphosis. In this study, we investigated genome-wide DNA methylation profiles during turbot metamorphosis, as well as the epigenetic effects of temperature on turbot post-embryonic development. Fish growth and rates of development were greatly affected by rearing temperature. Thus, turbot raised at ambient temperature (18 °C) achieved greater body weights and progressed through development more quickly than those reared at a colder temperature (14 °C). Genome-wide DNA methylation dynamics analyzed via a methylation-sensitive amplified polymorphism (MSAP) technique were not significantly different between animals reared within the two different thermal environments. Furthermore, comparisons between phenotypically similar fish revealed that genome-wide DNA methylation profiles do not necessarily correlate with specific developmental stages in turbot.

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.

Repeatability of flatfish reflex impairment assessments based on video recordings.

Using measures of reflex impairment and injury to quantify an aquatic organism's vitality have gained popularity as survival predictors of discarded non-target fisheries catch. To evaluate the robustness of this method with respect to 'rater' subjectivity, we tested inter- and intra-rater repeatability and the role of 'expectation bias'. From video clips, multiple raters determined impairment levels of four reflexes of beam-trawled common sole (Solea solea) intended for discard. Raters had a range of technical experience, including veterinary students, practicing veterinarians, and fisheries scientists. Expectation bias was evaluated by first assessing a rater's assumption about the effect of air exposure on vitality, then comparing their reflex ratings of the same fish, once when the true air exposure duration was indicated and once when the time was exaggerated (by either 15 or 30 min). Inter-rater repeatability was assessed by having multiple raters evaluate those clips with true air exposure information; and intra- and inter-rater repeatability was determined by having individual raters evaluate a series of duplicated clips, all with true air exposure. Results indicate that inter- and intra-rater repeatability were high (intra-class correlation coefficients of 74% for both), and were not significantly affected by background type nor expectation bias related to assumed impact from prolonged air exposure. This suggests that reflex impairment as a metric for predicting fish survival is robust to involving multiple raters with diverse backgrounds. Bias is potentially more likely to be introduced through subjective reflexes than raters, given that consistency in scoring differed for some reflexes based on rater experience type. This study highlights the need to provide ample training for raters, and that no prior experience is needed to become a reliable rater. Moreover, before implementing reflexes in a vitality study, it is important to evaluate whether the determination of presence/absence is subjective.

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.