Post-settlement dynamics of Japanese flounder Paralichthys olivaceus in Tango Bay: Seasonal patterns in growth, mortality, and recruitment potential.

Japanese flounder Paralichthys olivaceus is one of the most valuable coastal flatfish species in East Asia. To investigate post-settlement growth and mortality, juveniles were sampled in Tango Bay (Japan) weekly throughout the settlement period in 2007 and 2008. Otolith (lapillus) microstructure analysis enabled the categorization of juveniles into six biweekly cohorts each year. Later cohorts exhibited higher growth rates possibly because of higher water temperatures. A key observation was the direct relationship between high mortality and high density in mid-season cohorts in both years, pointing to density-dependent mortality. This increased mortality may be attributed to predation, including cannibalism by earlier cohorts. Furthermore, growth-selective mortality was evident soon after settlement, underscoring the vulnerability of slow growers to predation during the early juvenile stage. Although earlier and later cohorts were less abundant but showed promising recruitment potential, the prospective contribution of mid-season cohorts to the adult population remained uncertain. The results clearly highlight the importance of density-dependent mortality in population regulation in post-settlement Japanese flounder.

The incorporation of strontium and barium into the otoliths of the flounder Paralichthys olivaceus at early life stages demonstrates resilience to ocean acidification.

Ocean acidification could modify the bioavailability and chemical properties of trace elements in seawater, which could affect their incorporation into the calcareous structures of marine organisms. Fish otoliths, biomineralized ear stones made by aragonite, are suspended within the endolymph fluid of teleosts, indicating that the elemental incorporation of otoliths might also be susceptible to ocean acidification. In this study, we evaluated the combined effects of CO2-induced ocean acidification (pH 8.10, 7.70, and 7.30, corresponding to ocean acidification scenarios under the representative concentration pathway 8.5 model as projected by the Intergovernmental Panel on Climate Change) and water elemental concentrations of strontium (Sr) and barium (Ba; low, medium, and high) on elemental incorporation into otoliths of the flounder Paralichthys olivaceus at early life stages. Our results revealed that the elemental incorporation of Sr and Ba into otoliths was principally dependent on the corresponding water elemental concentrations rather than on ocean acidification. Moreover, the partition coefficients (DMe) of Sr and Ba may stabilize after dynamic equilibrium is reached as the water elemental concentration increases, but are not affected by ocean acidification. Therefore, the incorporation of Sr and Ba into otoliths of the flounder at early life stages may not serve as an effective indicator of ocean acidification. In other words, the findings suggest that ocean acidification does not impact the incorporation of Sr and Ba incorporation into otoliths when tracing the temperature or salinity experiences of the flounder. Our findings will provide new knowledge for understanding the potential ecological effects of ocean acidification on the recruitment dynamics of fish species.

Importance of experienced thermal history: Effect of acclimation temperatures on the high-temperature tolerance and growth performance of juvenile marbled flounder.

Experienced thermal history often affects the temperature tolerance of fish; however, the effect of thermal history on growth performance is unclear. To contribute to effective stocking (release of hatchery-reared juveniles in the field), we conducted four laboratory experiments using juvenile marbled flounder (Pseudopleuronectes yokohamae, around 30 mm standard length and 0.3 g body wet weight) acclimated at 12 °C and 24 °C for approximately 2 weeks to investigate the effects of acclimation temperature on high-temperature tolerance, food consumption, and growth performance. The acclimation to 24 °C increased tolerance to high temperatures, as shown in a 24-h exposure experiment and in a temperature elevation experiment. The 50% lethal temperature (upper incipient lethal temperature) was estimated to be 25.9 °C and 29.0 °C for the 12 °C and 24 °C acclimation groups, respectively. In subsequent experiments, we tested the effects of high and low temperature acclimation on the food consumption and growth performance of two size groups of juveniles (28.7 ± 2.0 and 34.5 ± 2.9 mm, mean ± SD), that were reared at temperatures ranging from 14 °C to 23 °C. The optimal temperature for growth was 20 °C and did not differ between the acclimation temperatures or between the size groups. However, food consumption and growth performance were suppressed by acute temperature changes. Specifically, feeding and growth were lower in the 24 °C-acclimated group than in the 12 °C-acclimated group when exposed to 14 °C, which is close to the natural water temperature at release in the field. These results suggest that experienced thermal history does not affect the optimal temperature but can affect the growth performance of juveniles. To maximize the post-release growth of hatchery-reared juveniles, the influence of thermal history should be taken into consideration and acute thermal changes before release should be avoided.

Analyses of mRNA-seq and miRNA-seq of the brain reveal the sex differences of gene expression and regulation before and during gonadal differentiation in 17ß-estradiol or 17α-methyltestosterone-induced olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is a commercially important flatfish species cultured in East Asia. Female flounders generally grow more rapidly than males, therefore control of the sex ratio seems to be a proposed way to increase production. However, the sex determination gene and sex determination mechanism have yet been elucidated. The brain is an important organ that is involved in gonadal development. To explore the sex differences of gene expression in the brain before and during the flounder gonadal differentiation, we used messenger RNA (mRNA)-seq technology to investigate transcriptomes of male and female brains. Between female and male brains, 103 genes were differentially expressed before ovarian differentiation, 16 genes were differentially expressed before testicular differentiation, and 64 genes were differentially expressed during gonadal differentiation. According to annotation and Kyoto Encyclopedia of Genes and Genomes information, the differentially expressed genes (DEGs) were involved in circadian rhythm, circadian rhythm-fly, circadian entrainment, dopaminergic synapse, calcium signaling, glutamatergic synapse, taste transduction, herpes simplex infection, long-term depression, retrograde endocannabinoid signaling, and the synaptic vesicle cycle pathways. MicroRNA (miRNA)-seq was performed during the gonadal differentiation and the target genes of miRNAs were predicted. Integrated analysis of mRNA-seq and miRNA-seq showed that 29 of the 64 DEGs were regulated by the differentially expressed miRNAs during the gonadal differentiation. Our study provides a basis for further studies of brain sex differentiation and the molecular mechanism of sex determination in olive flounder.

Probiotic effects of mixture of Groenewaldozyma salmanticensis and Gluconacetobacter liquefaciens on growth and immune responses in Paralichthys olivaceus.

This study was performed to evaluate the effects of dietary probiotics on growth, non-specific immune responses and disease resistance in olive flounder, Paralichthys olivaceus. During 8 weeks, the fish were fed the five experimental diets such as a basal commercial diet (CON), oxytetracycline (OTC) and three basal diets containing Bacillus subtilis (BS), a commercial microbial product (CES) and a mixture of yeast and bacterium (PI), respectively. Fish fed all the probiotics diets and OTC showed a significantly higher growth than fish-fed CON (P < 0·05). Fish-fed PI had a significantly higher nitroblue tetrazolium activity, whereas fish-fed CES showed a higher lysozyme level (P < 0·05). A 7-day challenge test also showed that fish-fed PI had a cumulative survival rate equivalent to that of fish-fed OTC (P < 0·05). Moreover, the diet (PI) appeared to increase the diversity of microbial community in the fish. All these results suggest that the probiotics diet could function as a potential antibiotic replacer in the olive flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique in revealing that a diet mixture of yeast, Groenewaldozyma salmanticensis and bacterium Gluconacetobacter liquefaciens can enhance growth, innate immunity and diversity of microbial community including dominant species in the olive flounder. All these indicate that the diet mixture could function as a potential antibiotic replacer in one of the most commercially important fisheries in South Korea.

A comprehensive description of oocyte developmental stages in Pacific halibut, Hippoglossus stenolepis.

Accurate characterization of oocyte development is essential to understanding foundational aspects of reproductive biology and successful management of Pacific halibut (Hippoglossus stenolepis). Here this study provides complete histological descriptions for eight oocyte developmental stages in addition to postovulatory follicles and demonstrates the potential for oocyte size frequency distribution to act as a proxy for ovarian developmental stage and future maturity assessments. Importantly, it provides the first histological evidence that Pacific halibut have a group-synchronous ovarian developmental pattern with determinate fecundity and support for their batch-spawning strategy.

An hypoxia-tolerant flatfish: consequences of sustained stress on the slender sole Lyopsetta exilis (Pleuronectidae) in the context of a changing ocean.

Slender sole Lyopsetta exilis is an abundant groundfish on the continental shelf and inner waters of British Columbia, Canada, where it reaches a maximum standard length of 44 cm. Benthic image surveys coupled with oxygen measurements in Saanich Inlet document a dense population in bottom conditions near anoxia (0.03 ml l-1 oxygen) where diel migrating zooplankton intersect the bottom; we confirm this species is a planktivore, which limits its depth range to the base of the migration layer. In a comparison with slender sole from a nearby well-oxygenated habitat, several probable effects of living in severe hypoxia emerge: both sexes are significantly smaller in Saanich and the sex ratio is male-skewed. Otoliths from the Saanich fish were difficult to read due to many checks, but both sexes were smaller at age with the largest female (20 cm) from the hypoxia zone registering 17 years. Hypoxia appears to have a direct consequence on growth despite good food supply in this productive basin. Hyperventilation, a low metabolic rate and a very low critical oxygen tension help this fish regulate oxygen uptake in severely hypoxic conditions; it will be particularly resilient as the incidence of hypoxia increases on the continental shelf. Data from small-mesh bottom-trawl surveys over four decades reveal an increase in mean annual catch per unit effort in southern regions of the province, including the outer shelf and the Strait of Georgia. The California Cooperative Oceanic Fisheries Investigations (CalCOFI) ichthyoplankton database records a general decline in fish larvae on the Oregon-California shelf since 1990, but slender sole larvae are increasing there, as they are in the Strait of Georgia. We project that the slender sole will gain relative benefits in the future warming, deoxygenated northeast Pacific Ocean.

Novel breeding approach for Japanese flounder using atmosphere and room temperature plasma mutagenesis tool.

BACKGROUND: Artificial induction of mutagenesis is effective for genetic resource innovation and breeding. However, the traditional mutation methods for fish breeding are not convenient or safe for daily use. Hence, development of a simple, safe and effective mutagenesis method with a high mutation rate and applicability to multiple fish species, is needed. RESULTS: We reported the first successful mutagenesis in a marine aquaculture fish species, Japanese flounder, Paralichthys olivaceus, using a novel atmosphere and room temperature plasma (ARTP) mutagenesis tool. ARTP treatment time was optimized for the fertilized eggs and sperm, respectively. Eggs fertilized for 60 min were treated by ARTP with a radio-frequency power input of 120 W, and the ARTP treatment time was 25 min. Under an ARTP radio-frequency power input of 200 W, the optimal treatment time for sperm diluted with Ringer's solution by 1:40 v/v was 10 min. The ARTP-treated group presented differences in morphological traits such as body height, total length among individuals at day 90 after hatching. Whole-genome sequencing was used to reveal the mutation features of ARTP-treated individuals collected at day 120 after hatching. In total, 69.25Gb clean data were obtained from three controls and eight randomly selected ARTP-treated individuals, revealing 240,722 to 322,978 SNPs and 82,149 to 86,798 InDels located in 17,394~18,457 and 12,907~13,333 genes, respectively. The average mutation rate reached 0.064% at the genome level. Gene ontology clustering indicated that genes associated with cell components, binding function, catalytic activity, cellular process, metabolic process and biological regulation processes had higher mutation rates. CONCLUSIONS: ARTP mutagenesis is a useful method for breeding of fish species to accelerate the selection of economically important traits that would benefit the aquaculture industry, given the variety of mutations detected.

The evolution and possible role of two Sox8 genes during sex differentiation in Japanese flounder (Paralichthys olivaceus).

Sox8 genes, as members of the Sox family, have been studied widely in mammals. However, regulation of sox8 genes in teleosts has rarely been studied, and functional analysis of these genes in teleosts has rarely been performed. Here, two duplicates of sox8 genes were identified in Japanese flounder, Posox8a and Posox8b. The analysis of expression showed that Posox8a and Posox8b were expressed in Sertoli cells of the testis, indicating that they play important roles in development and functional maintenance of the testis. Positive selection and phylogenetic analysis found that both Posox8a and Posox8b underwent the purification selection during evolutionary and that sox8 was most likely to be the ancestor sox8a. These results suggested that both Posox8a and Posox8b had important biological functions after generation from three rounds of whole-genome duplication in Japanese flounder. The functional differentiation of Posox8a and Posox8b was verified using cell transfection and dual-luciferase reporter assays; Posox8a overexpression-promoted 3ß-hydroxysteroid dehydrogenase expression and Posox8b overexpression-promoted cytochrome P450 aromatase (cyp19a1; P450arom) expression. Finally, combined with Posox8a and Posox8b expression analysis from 30 to 100 days after hatch, we speculated that Posox8a and Posox8b might participate in the process of sex differentiation and gonadogenesis by regulating sex hormone biosynthesis in the Japanese flounder. Our study is the first to demonstrate the possible mechanism of Posox8a and Posox8b in Japanese flounder sex differentiation and gonadogenesis, laying a solid foundation for functional studies of sox8 genes in teleosts.

A novel ladder-like lectin relates to sites of mucosal immunity in Atlantic halibut (Hippoglossus hippoglossus L.).

A novel 27 kDa ladder-lectin-like protein, showing a multimeric structure under non-reducing conditions, was isolated from halibut serum by binding to N-acetyl glucosamine. Mass-spectrometry analysis did not show significant homology with known proteins. Specific antibodies were produced and used in immunohistochemistry on tissue sections of early halibut ontogeny from 119 until 1050 °d post hatching. A strong positive response was detected in the mucosal cells of the skin, gills and gut, indicating a role in the mucosal immune defence at these sites. Further immunopositivity was detected in liver, myeloma of kidney and the brain at different developmental stages but predominant expression was found in mucosal surfaces at later stages of development tested (1050 °d). It is still uncertain whether this ladder-like lectin forms part of the complement pathway, as a lectin or ficolin, or if it belongs to galectins. A strong detection in mucosal surfaces on skin, gills and gut, show similar patterns of expression as both mucosal lectins and galectins in other fish. Detection in neuronal tissue may indicate putative roles in tissue remodelling of brain and in ongoing neurogenesis in the fish eye.

Heat-killed Bacillus sp. SJ-10 probiotic acts as a growth and humoral innate immunity response enhancer in olive flounder (Paralichthys olivaceus).

Investigations were carried out to evaluate and quantify the effects of dietary supplementation with heat-killed (HK) Bacillus sp. SJ-10 (BSJ-10) probiotic (1 × 108 CFU g-1) on the growth and immunity of olive flounder (Paralichthys olivaceus). Flounder (averagely 9.64 g) were divided into two groups, and fed control and HK BSJ-10 (HKBSJ-10)-inoculated diets for 8 weeks. Investigations were carried out on growth and feed utilizations, innate immunity, serum biochemical parameters, microvilli length, and pro- and anti-inflammatory cytokine gene (tumor necrosis factor [TNF]-α, interleukin [IL]-1ß, IL-6, and IL-10) transcriptions. Compared to control, HKBSJ-10 diet significantly (P < 0.05) enhanced weight gain and protein efficiency ratio, 1.17 and 1.11 folds respectively. Humoral innate immune parameters, lysozyme and superoxide dismutase in treatment group were also elevated by 1.34 and 1.16 folds. Similarly, an increased (P < 0.05) relative expressions of TNF-α, IL-1ß, IL-6 were recorded in liver (2.71, 3.38, and 4.12 folds respectively), and gill (2.08, 1.98, and 1.81 folds respectively) than that of controls. Moreover, after challenge with Streptococcus iniae (1 × 108 CFU mL-1), the HKBSJ-10-fed group exhibited significantly higher protection (P < 0.05) against streptococcosis compared to controls, validating the observed changes in immune parameters and induction on the cytokine-encoding genes. Therefore, HKBSJ-10 increases growth, modulates innate immune parameters, and protects olive flounders against streptococcosis.

Characterization of a Bacillus sp. isolated from fermented food and its synbiotic effect with barley ß-glucan as a biocontrol agent in the aquaculture industry.

The purpose of this study was to evaluate Bacillus sp. SJ-10, isolated from traditional Korean seafood, as a probiotic. Strain SJ-10 was demonstrated to be safe, on the basis of in vitro tests confirming the absence of cytotoxicity, hemolysis, and genes with toxigenic potential, and was susceptible to antibiotics. It met the probiotic prerequisites of a spore count that remained almost constant, acid and bile tolerance under simulated gastrointestinal conditions, and significant adhesion to Caco-2 cells. Moreover, SJ-10 demonstrated beneficial properties as a probiotic: broad-spectrum antimicrobial activity, hydroxyl radical, antioxidant activity, production of functional enzymes such as ß-galactosidase and phytase, and selective growth via ß-glucan fermentation. The fish-feeding trial demonstrated that olive flounder fed diets containing SJ-10 alone or in combination with ß-glucan exhibited significantly higher growth performance and pathogenic disease resistance compared with those fed diets containing ß-glucan alone, indicating that SJ-10 diets exerted a beneficial effect as an antibiotic replacer in terms of growth performance and disease resistance in olive flounder.

Methylation status and expression patterns of myomaker gene play important roles in postnatal development in the Japanese flounder (Paralichthys olivaceus).

Myomaker is a membrane protein that plays a crucial role in the fusion of myoblasts during muscle growth. DNA methylation, a significant factor, regulates gene expression. The aim of this study was to examine the methylation and mRNA expression patterns of the myomaker gene during 8 different postnatal developmental stages in the Japanese flounder (L: 7 days post hatch (dph); M1: 21 dph; M2: 28 dph; M3: 35 dph; J1: 90 dph; J2: 180 dph; A1: 24 months; A2: 36 months). Muscle tissue samples were taken from Japanese flounder at different postnatal development stages to measure the extent of DNA methylation and gene expression. Methylation level in the promoter and exon 1 of myomaker was measured using bisulfite sequencing, and the relative expression of myomaker during each developmental stage was measured by quantitative PCR. The relative expression levels of myomaker were up-regulated from stages L to M2, M3 to J2, and methylation of myomaker was negatively correlated with mRNA expression. Furthermore, the CpG site located at -26 bp in the promoter was the lowest methylated region in all developmental stages. These results offer a basis for understanding the mechanism by which myomaker regulates muscle formation during postnatal development.

Effects of green light on the growth of spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus, and on the endocrine system of spotted halibut at different water temperatures.

We have previously shown that the somatic growth of barfin flounder, Verasper moseri, was promoted by green light. The present study was undertaken to elucidate whether growth-promoting effect of green light can be observed in other flatfishes and to understand the roles of endocrine systems in green light-induced growth. Herein, we demonstrated facilitation of growth by green light in the spotted halibut, Verasper variegatus, and Japanese flounder, Paralichthys olivaceus. Blue and blue-green light showed potencies that were similar to that of green light, while the potencies of red and white light were equivalent to that of ambient light (control). We also examined the effects of green light on growth and endocrine systems of V. variegatus at various water temperatures. Growth of the fish was facilitated by green light at four different water temperatures examined; the fish were reared for 31 days at 12 and 21 °C, and 30 days at 15 and 18 °C. Increase in condition factor was observed at 15 and 18 °C. Among the genes encoding hypothalamic hormones, expression levels of melanin-concentrating hormone 1 (mch1) were enhanced by green light at the four water temperatures. Expression levels of other genes including mch2 increased at certain water temperatures. No difference was observed in the expression levels of pituitary hormone genes, including those of growth hormone and members of proopiomelanocortin family, and in plasma levels of members of the insulin family. The results suggest that green light may generally stimulate growth of flatfishes. Moreover, it is conceivable that MCH, production of which is stimulated by green light, is a key hormone; it augments food intake, which is intimately coupled with somatic growth.

Skeletal development of the chondrocranium in the tongue sole Cynoglossus semilaevis (Pleuronectiformes: Cynoglossidae).

This study provides a comprehensive description of chondrocranial development before, during and after larval metamorphosis in the tongue sole Cynoglossus semilaevis, a commercially valuable flatfish in China. Samples were collected at regular intervals ranging from 1 to 23 days post hatching (dph). Based on observations of cleared and double-stained specimens and images from sections stained with safranin O-fast green, major morphological events during early development were described. No cartilaginous structure was visible at hatching. From 2 dph onwards, cartilaginous structures such as the trabecular bar and some elements of the mandibular, hyoid and branchial arches appeared. At this time also, cartilaginous structures of the neurocranium started to form. Hypertrophic chondrocytes could be observed in many splanchnocranium elements at 5 dph. The start of ossification was indicated by alizarin red stain visible at 14 dph. At 17 dph, most of the cartilaginous skeleton was ossified. Soon after, the right eye started to migrate and pass through a slit beneath the dorsal-fin base and above the skull. Metamorphosis was complete at 20 dph, at which time the dorsal-fin base cartilage extended onto the anterior region of the head. Meanwhile, extremities of the hyoid and branchial arch elements remained cartilaginous. At 23 dph, endochondral ossification of the splanchnocranium was nearly complete. Unlike previous observations of other Pleuronectiformes, our study indicates that endochondral ossification of C. semilaevis skull cartilage occurs before metamorphosis.

Proteomic variation in metamorphosing Paralichthys olivaceus induced by exogenous thyroid hormone.

Thyroid hormone (TH) is essential for Paralichthys olivaceus metamorphosis. Exogenous TH treatment induces premature metamorphosis in P. olivaceus larvae and a series of studies have been conducted to identify thyroid hormone-regulated functional genes and microRNAs involved in the metamorphosis of P. olivaceus; however, the proteins involved in this process remain to be fully clarified. In this study, the differential proteomic responses of P. olivaceus larvae to exogenous TH treatment were examined using tandem mass tags (TMT) for quantitation labeling followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The expression levels of 629 cellular proteins were identified to be significantly affected by TH treatment. The reliability of our TMT-labeled LC-MS/MS analysis was verified by examining the mRNA and protein levels of four selected proteins using quantitative real-time reverse-transcription PCR and western blot analyses. The possible biological significance of these proteins was further investigated by Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction analyses. Notably, we identified and described five groups of proteins involved in different important life events that were significantly regulated by exogenous TH treatment. Our study provides an improved understanding of the molecular mechanisms by which TH regulates the metamorphosis of P. olivaceus.

Identification of Sox10-positive cells at the dorsal fin base of juvenile flounder that are correlated with blind-side skin ectopic pigmentation.

Flounder develop left-right asymmetric body color, with a dark ocular side and white blind side. However, ectopic pigmentation often occurs on the blind side when juveniles are reared in tanks. To examine the developmental mechanism underlying ectopic pigmentation, we first examined the pigmentation process on the blind side and the localization of chromatoblasts during spontaneous and regeneration-stimulated ectopic pigmentation. Wild-caught juveniles that had completed metamorphosis in a natural environment were reared in tanks, where they exhibited ectopic pigmentation on the blind side that was initiated at the base of the dorsal and anal fins, with chromatoblasts appearing at the edges of scales and melanophores spreading on the scale papilla beneath the epidermis. During tissue regeneration at the base of the dorsal fin in juvenile before ectopic pigmentation, melanophores and chromatoblasts newly appeared on regenerated blind-side skin, resulting in rapid pigmentation at the wounded site. During regeneration-stimulated pigmentation, gch2-positive chromatoblasts were detected only under the regenerated epidermis. Next, we found that Sox10-positive cells were localized in connective tissue at the base of the dorsal fin and that when connective tissue was labeled with DiO, DiO-labeled melanophores appeared in regenerated skin of the blind side after wounding. Therefore, we conclude that in flounder juveniles, Sox10-positive progenitors of pigment cell lineage reside at the base of the dorsal fin and start migrating to the blind-side skin in response to specific stimuli, resulting in ectopic pigmentation. Ectopic pigmentation in flounder could be a good model for examining the flexibility of pigment cell differentiation.

Structural and functional maturation of skin during metamorphosis in the Atlantic halibut (Hippoglossus hippoglossus).

To establish if the developmental changes in the primary barrier and osmoregulatory capacity of Atlantic halibut skin are modified during metamorphosis, histological, histochemical, gene expression and electrophysiological measurements were made. The morphology of the ocular and abocular skin started to diverge during the metamorphic climax and ocular skin appeared thicker and more stratified. Neutral mucins were the main glycoproteins produced by the goblet cells in skin during metamorphosis. Moreover, the number of goblet cells producing neutral mucins increased during metamorphosis and asymmetry in their abundance was observed between ocular and abocular skin. The increase in goblet cell number and their asymmetric abundance in skin was concomitant with the period that thyroid hormones (THs) increase and suggests that they may be under the control of these hormones. Several mucin transcripts were identified in metamorphosing halibut transcriptomes and Muc18 and Muc5AC were characteristic of the body skin. Na+, K+-ATPase positive (NKA) cells were observed in skin of all metamorphic stages but their number significantly decreased with the onset of metamorphosis. No asymmetry was observed between ocular and abocular skin in NKA cells. The morphological changes observed were linked to modified skin barrier function as revealed by modifications in its electrophysiological properties. However, the maturation of the skin functional characteristics preceded structural maturation and occurred at stage 8 prior to the metamorphic climax. Treatment of Atlantic halibut with the THs disrupter methimazole (MMI) affected the number of goblet cells producing neutral mucins and the NKA cells. The present study reveals that the asymmetric development of the skin in Atlantic halibut is TH sensitive and is associated with metamorphosis and that this barrier's functional properties mature earlier and are independent of metamorphosis.

Chronic stress inhibits growth and induces proteolytic mechanisms through two different nonoverlapping pathways in the skeletal muscle of a teleost fish.

Chronic stress detrimentally affects animal health and homeostasis, with somatic growth, and thus skeletal muscle, being particularly affected. A detailed understanding of the underlying endocrine and molecular mechanisms of how chronic stress affects skeletal muscle growth remains lacking. To address this issue, the present study assessed primary (plasma cortisol), secondary (key components of the GH/IGF system, muscular proteolytic pathways, and apoptosis), and tertiary (growth performance) stress responses in fine flounder ( Paralichthys adspersus) exposed to crowding chronic stress. Levels of plasma cortisol, glucocorticoid receptor 2 ( gr2), and its target genes ( klf15 and redd1) mRNA increased significantly only at 4 wk of crowding ( P < 0.05). The components of the GH/IGF system, including ligands, receptors, and their signaling pathways, were significantly downregulated at 7 wk of crowding ( P < 0.05). Interestingly, chronic stress upregulated the ubiquitin-proteasome pathway and the intrinsic apoptosis pathways at 4wk ( P < 0.01), whereas autophagy was only significantly activated at 7 wk ( P < 0.05), and meanwhile the ubiquitin-proteasome and the apoptosis pathways returned to control levels. Overall growth was inhibited in fish in the 7-wk chronic stress trial ( P < 0.05). In conclusion, chronic stress directly affects muscle growth and downregulates the GH/IGF system, an action through which muscular catabolic mechanisms are promoted by two different and nonoverlapping proteolytic pathways. These findings provide new information on molecular mechanisms involved in the negative effects that chronic stress has on muscle anabolic/catabolic signaling balance.

Effects of food availability on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus).

Unfavorable environmental conditions and inappropriate culture practices have increased the vulnerability of cultured fish to disease infection. Up to date many studies have aimed to determine a feeding regimen to maximize productivity; however, very little information on immune responses of cultured fish in response to underfeeding or overfeeding is available. Therefore, a preliminary study was conducted to evaluate effects of graded feeding levels (i.e., food availability) on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus). Six different feeding rates including 1, 4, 7, 10, 13, and 16% body weight per day (BW/d) were randomly assigned to three replicate tanks stocking 150 fish (average initial body weight: 0.27 ±â€¯0.02 g; mean ±â€¯SD) per tank. A feeding trial lasted for two weeks. Based on the results of the weight gain, nutrient gain, and whole-body compositions and energy content, the feeding rate of 10%, 13%, and 16% BW/d resulted in high nutritional status, whereas the feeding rate of 1% and 4% BW/d resulted in low nutritional status. Intermediate nutritional status was observed at the feeding rate of 7% BW/d. In the given rearing conditions the optimum feeding rate resulting in the maximum growth was estimated to be 11.9% BW/d based on the quadratic broken-line regression model, chosen as the best-fit model among the tested models. Expression of immune-related genes including IL-8 and IgM was significantly down-regulated in the flounder fed at 1% BW/d in comparison to those fed at 7% BW/d. Interestingly, expression of these genes in the flounder fed at 10%, 13%, and 16% BW/d was relatively down-regulated in comparison to that of the flounder fed at 7% BW/d. Although no statistical difference was detected, overall response patterns of other immune-related genes, including TLR3, polymeric Ig receptor, lysozyme C-type, GPx, SOD, and Trx followed what IL-8 and IgM exhibited in response to the various feeding rates. Given the current challenges in aquaculture of the flounder our findings suggest to prohibit underfeeding or overfeeding (i.e., ad-libitum feeding) when culturing the young flounder.