The excitatory effect of 5-HT1A and 5-HT2B receptors on the caudal neurosecretory system Dahlgren cells in olive flounder, Paralichthys olivaceus.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an essential role in the regulation of neural activity via multiple receptors. Here, we investigated the functional role of serotoninergic input on the Dahlgren cell population in the caudal neurosecretory system (CNSS) of olive flounder. In this study, the effect of 5-HT on the firing activity of Dahlgren cells was explored in terms of changes in firing frequency and firing pattern using multicellular recording electrophysiology ex vivo, and the role of several 5-HT receptor subtypes in the regulation was determined. The results revealed that 5-HT increased the firing frequency in a concentration-dependent manner and altered the firing pattern of Dahlgren cells. The effect of 5-HT on the firing activity of Dahlgren cells was mediated through the 5-HT1A and 5-HT2B receptors, selective agonists of both receptors effectively increased the firing frequency of Dahlgren cells, and selective receptor antagonists could also effectively inhibit the increase in firing frequency caused by 5-HT. In addition, the mRNA levels of major signaling pathway-related genes, ion channels, and major secretion hormone genes were significantly upregulated in CNSS after treatment with 5-HT. These findings demonstrate that 5-HT acts as an excitatory neuromodulator on Dahlgren cells and enhances neuroendocrine activity in CNSS.

Integration of environmental signatures and omics-based approaches on the European flounder to assist with health assessment of estuarine ecosystems in Brittany, France.

This study aimed to develop a multidisciplinary approach to assess the ecological status of six moderate-sized French estuaries. For each estuary, we gathered geographical information, hydrobiological data, chemistry of pollutants and fish biology, including integration of proteomics and transcriptomics data. This integrative study covered the entire hydrological system studied, from the watershed to the estuary, and considered all the anthropogenic factors that can impact this environment. To reach this goal, European flounder (Platichthys flesus) were collected from six estuaries in September, which ensures a minimum residence time of five months within an estuary. Geographical metrics are used to characterize land use in each watershed. The concentrations of nitrite, nitrate, organic pollutants, and trace elements were measured in water, sediments and biota. All of these environmental parameters allowed to set up a typology of estuaries. Classical fish biomarkers, coupled with molecular data from transcriptomics and shotgun proteomics, highlighted the flounder's responses to stressors in its environment. We analysed the protein abundances and gene expression levels in the liver of fish from the different estuaries. We showed clear positive deregulation of proteins associated with xenobiotic detoxification in a system characterized by a large population density and industrial activity, as well as in a predominantly agricultural catchment area (mostly cultures of vegetables and pig breeding) mainly impacted by pesticides. Fish from the latter estuary also displayed strong deregulation of the urea cycle, most probably related to high nitrogen load. Proteomic and transcriptomic data also revealed a deregulation of proteins and genes related to the response to hypoxia, and a probable endocrine disruption in some estuaries. Coupling these data allowed the precise identification of the main stressors interacting within each hydrosystem.

A first glimpse of larval ecology of halibut species in the Gulf of St. Lawrence, Canada.

Knowledge of the larval ecology of winter-spawning fish from the Estuary and Gulf of St. Lawrence, Canada, remains scarce due to the seasonal ice cover that prevents ichthyoplankton sampling using conventional methods. Two winter-spawning species, Atlantic halibut (AH, Hippoglossus hippoglossus) and Greenland halibut (GH, Reinhardtius hippoglossoides), support the most important groundfish fisheries of this area. In March 2020, the authors captured 10 halibut larvae ranging in size from 5 to 14 mm during an opportunistic survey in the GSL onboard an icebreaking vessel. Of these, eight were AH and two GH. Judging by their very small size, the larvae were only a few days old, suggesting that the spawning grounds are close to the capture sites. This effort constitutes a first step in validating the putative spawning areas for these two important GSL stocks. This knowledge is important for the conservation and sustainable management of these fisheries.

Inferring fish behaviour at the trawl mouth from escape location.

In this study, we used escape location underneath the trawl to understand groundfish herding behaviour at the trawl mouth. Three collecting bags (port, center, starboard) were mounted under the trawl and behind the footgear to collect escapees. The escape-at-length of species that escaped into the center bag were compared to the two wing bags to infer fish response behaviour, herding behaviour, and swimming capacity at the trawl mouth. For roundfish, smaller-sized individuals escaped more in the center for both Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus), <20 and 11 cm, respectively, indicating that larger-sized fish were to a greater extent seeking to escape under the trawl at the wings, vs small fish being herded to the center and likely overrun due to reduced swimming capacity. For flatfish and monkfish (Lophius piscatorius), results varied. European plaice (Pleuronectes platessa), American plaice (Hippoglossoides platessoides), and monkfish were caught most often in the wings, though not significantly for American plaice. Catches of dab (Limanda limanda) between 18 and 27 cm were significantly higher in the center, with no difference for smaller and larger individuals. The differences between fish escape location likely result from a combination of varying herding behaviour, size, and swimming capacity. Here, we were able to show how these size-dependent behaviours relate to fish response behaviour, escape behaviour, size, and likely swimming capacity.

Temporal trends of mercury levels in fish (dab, Limanda limanda) and sediment from the German Bight (North Sea) in the period 1995-2020.

As a toxic and harmful global pollutant, mercury (Hg) enters the marine environment through natural sources, and human activities. It bioaccumulates through the food chain and therefore, Hg is of great importance for environmental monitoring. This study aims to answer the question if Hg contamination in fish and sediment from the German Bight follows temporal trends. Therefore, 496 individual female dab (Limanda limanda) were analyzed. The Hg concentrations in the muscle of dab from the German Bight showed significant increase in function of time with an annual percental change of 1.4%, leading to a 41% increase in Hg contamination level within 25 years of monitoring. At the same time, Hg concentrations in sediment-analyzed in 86 samples-significantly decreased in the nearby North Sea environment. This surprising contradiction is shown in the present study and possible causes are discussed. It could be clearly shown that contamination in sediment and biota can follow completely different time courses and therefore, different environmental matrices should be considered in future monitoring studies. Age of the fish turned out to be a biological factor of particular importance for temporal trend analysis.

European flounder foraging movements in an estuarine nursery seascape inferred from otolith microchemistry and stable isotopes.

Despite the importance of estuarine nurseries in the regulation of many fish stocks, temporal and spatial movements and habitat use patterns of juvenile fish remain poorly understood. Overall, combining several movement metrics allowed us to characterize dispersal patterns of juvenile flounder, Platichthys flesus, along an estuarine seascape. Specifically, we investigated otolith microchemistry signatures (Sr:Ca and Ba:Ca ratios) and stable isotope ratios (δ13C and δ15N) in muscles of these juveniles, during three consecutive years to assess inter-annual fluctuations in their home range and isotopic niches. The morphological condition and lipid content of individuals were lower in years of high as compared to low dispersal along the estuarine gradient. We discuss these results in relation to the ecosystem productivity and intra- and inter-specific competition level, which in turn affects movements and foraging behaviors of juvenile flounders.

The effects of low pH and high water temperature on oxidative stress and cell damage in juvenile olive flounder Paralichthys olivaceus: comparison of single and combined environmental conditions.

The use of fossil fuels by anthropogenic activities causes ocean acidification and warming, and these changes in the marine environment can negatively affect the metabolism, growth, and survival of fish. In the present study, we evaluated the ability of olive flounder Paralichthys olivaceus to cope with future marine environmental changes by investigating the oxidative stress (cortisol, HSP70), antioxidant enzyme (superoxide dismutase; SOD, catalase; CAT) activity, and apoptosis (caspase-3) after exposure to control conditions (20 °C and pH 8.1), warming (30 °C) and acidification (pH 7.5) conditions, and a combined environment (30 °C and pH 7.5) for 28 days. Under warming conditions, increased oxidative stress, activity of antioxidant enzymes, and apoptosis were observed. Acidifying conditions showed negative effects at the beginning of exposure, but these effects were offset over time. Even in a combined environment of acidification and warming, negative effects were seen only at the beginning of exposure and were not sustained. In conclusion, the effects of acidification on oxidative stress, antioxidant response, and apoptosis in P. olivaceus did not exceed the effects of warming. These results suggest that P. olivaceus can cope with the predicted future acidifying environment.

Transcriptome profiles revealed high- and low-salinity water altered gill homeostasis in half-smooth tongue sole (Cynoglossus semilaevis).

Salinity is an important environmental factor that affects fish growth, development, and reproduction. As euryhaline fish, half-smooth tongue sole (Cynoglossus semilaevis) are a suitable species for deciphering the salinity adaptation mechanism of fish; however, the molecular mechanisms underlying low- and high-salinity responses remain unclear. In this study, RNA-seq was applied to characterize the genes and regulatory pathways involved in C. semilaevis gill responses to high- (32 ppt), low- (8 ppt), and control-salinity (24 ppt) water. Gills were rich in mitochondria-rich cells (MRCs) in high salinity. Compared with control, 2137 and 218 differentially expressed genes (DEGs) were identified in low and high salinity, respectively. The enriched functions of most DEGs were metabolism, ion transport, regulation of cell cycle, and immune response. The DEGs involved in oxidative phosphorylation, citrate cycle, and fatty acid metabolism were down-regulated in low salinity. For ion transport, high and low salinity significantly altered the expressions of prlr, ca12, and cftr. In cell cycle arrest and cellular repair, gadd45b, igfbp5, and igfbp2 were significantly upregulated in high and low salinity. For immune response, il10, il34, il12b, and crp increased in high and low salinity. Our findings suggested that alterations in material and energy metabolism, ions transport, cell cycle arrest, cellular repair, and immune response, are required to maintain C. semilaevis gill homeostasis under high and low salinity. This study provides insight into the divergence of C. semilaevis osmoregulation mechanisms acclimating to high and low salinity, which will serve as reference for the healthy culture of C. semilaevis.

Effect of low pH and salinity conditions on the antioxidant response and hepatocyte damage in juvenile olive flounder Paralichthys olivaceus.

Climate change due to increasing CO2 emissions results in the increase in water temperatures, which is accompanied by the decrease in pH and salinity levels of the ocean. Ocean acidification reflects the gradual pH reduction due to changes in the carbon chemistry, which is caused by the increase in anthropogenic CO2 emissions. The subsequent changes in the water temperatures and carbon chemistry of the oceans affect the survival and distribution of aquatic animals. In this study, we analyzed the levels of cortisol, superoxide dismutase, catalase, and caspase-3 in the plasma of juvenile olive flounder Paralichthys olivaceus under combined hyposalinity and acidification. To evaluate the physiological response to these changes, the superoxide dismutase activity and apoptosis were analyzed in the liver cells. Hyposalinity caused oxidative stress and cell damage, while also activating the antioxidant system. Environmental acidification affected the stress response and antioxidant mechanism of P. olivaceus in the early stage of acclimation but did not appear to exceed hyposalinity stress. These findings suggest that a hyposaline environment may be a stronger environmental stressor than an acidifying environment for P. olivaceus, and will help understand the capacity of P. olivaceus to cope with expected future ocean acidification.

Functional differentiation of two lhx8 paralogs and possible regulatory role of lhx8a in Japanese flounder (Paralichthys olivaceus).

Lhx8, belonging to the LIM-Homebox family, is involved in the tooth, nervous system, and primordial follicles development in mammals. However, little is known about the regulatory roles of lhx8 in teleosts. In this study, two lhx8 duplicates were identified in Paralichthys olivaceus, termed Polhx8a and Polhx8b, respectively. Bioinformatic analysis showed that Polhx8a was more likely to be a teleost-specific paralog. According to expression analysis, Polhx8a transcripts were almost exclusively concentrated in the oocytes, while Polhx8b was weakly expressed in the spleen, gill, and some facial organs, indicating sub-functionalization of this gene pair during evolution. Furthermore, Polhx8a mRNA level elevated from perinucleolar oocyte (PNO) stage to vitellogenic oocyte (VO) stage transition and changed after exogenous hormone stimulation, proving that Polhx8a was involved in the oocyte development and could be regulated by sex hormones. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) experiments captured the positive protein interactions between PoLhx8a and the other two oocyte-specific transcription factors: PoFigla and PoNobox. After knocking down lhx8a in embryos or adult ovaries in vivo, the expression of oocyte-associated genes was significantly down-regulated (P < 0.05). Our findings suggest the evolution and functional differentiation of lhx8 genes, and shed light on the potential role of lhx8a in protein interactions and gene regulation in teleosts.

The role of cholecystokinin and peptide YY in feed intake in Atlantic halibut (Hippoglossus hippoglossus) larvae.

Control of appetite and feed intake in fish larvae are still largely unexplored. Two of the key players in controlling vertebrate's feed intake are cholecystokinin (CCK) and peptide YY (PYY). Here we investigated the mRNA expression of pyy, cck and cck receptors (cckr) in the brain (head) and gut of Atlantic halibut larvae in response to three consecutive meals. We used Artemia nauplii cysts that are commonly ingested by halibut larvae when present as inert feed, and three water-soluble extracts as attractants to stimulate appetite. Cyst intake was not affected by the use of attractants and overall ingestion rate was low. Differences in mRNA expression of cck and pyy were observed between the halibut larvae that had eaten and those that had not despite readily available feed (cysts), supporting that mechanisms for control of feed intake are at least partly functional. All genes analysed were present in the brain and gut, however the different expression profiles between paralogues suggest potential divergent functions. In the gut, cck2 and pyyb mRNA expression was significantly higher in the larvae that ate cysts compared to larvae that decided to not eat, indicating that these genes play a satiety function in the halibut larvae similar to the general vertebrate scheme. However, cck2, cck2r1, and pyy mRNA expression in the brain were lower in the fed-filled larvae group compared to larvae before eating, which contrasts with the presumable anorectic function of these genes. Further research is required to fully evaluate how PYY and CCK affect the feeding biology in halibut larvae, contributing to formulate inert diets that can stimulate appetite and feed intake.

A Novel microRNA of Japanese Flounder Regulates Antimicrobial Immunity Involving a Bacteria-Binding CSF3.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse biological processes including immunity. In a previous high-throughput RNA sequencing study, a novel miRNA, pol-miR-novel_642, was identified from Japanese flounder (Paralichthys olivaceus), a farmed fish species with important economic value. In this study, we investigated the regulatory mechanism and the function of pol-miR-novel_642 and its target gene. We found that pol-miR-novel_642 targeted, in a sequence-specific manner, a flounder gene encoding an uncharacterized protein that is a structural homologue of murine granulocyte colony stimulating factor 3 (CSF3). The expression of pol-miR-novel_642 and its target gene (named PoCSF3-1) was regulated, in different manners, by the bacterial pathogen Edwardsiella tarda and the viral pathogen megalocytivirus. Overexpression of pol-miR-novel_642 or interference with PoCSF3-1 expression in flounder cells strongly potentiated E. tarda infection. Consistently, in vivo knockdown of PoCSF3-1 enhanced bacterial dissemination in flounder tissues but blocked viral replication, whereas in vivo overexpression of PoCSF3-1 inhibited bacterial dissemination and facilitated viral infection. Overexpression/knockdown of PoCSF3-1 and pol-miR-novel_642 also affected the activation of autophagy. Recombinant PoCSF3-1 (rPoCSF3-1) interacted with and inhibited the growth of Gram-negative bacteria in a manner relying on a PoCSF3-1-characteristic structural motif that is absent in mouse CSF3. rPoCSF3-1 also regulated the proliferation, inflammatory response, and immune defense of flounder head kidney leukocytes in a structure-dependent fashion. Together, these results reveal the function of a novel miRNA-CSF3 regulatory system of flounder, and add new insights into the role and mechanism of fish miRNA and CSF3 in antimicrobial immunity.

Diel rhythm of urotensin I mRNA expression and its involvement in the locomotor activity and appetite regulation in olive flounder Paralichthys olivaceus.

Urotensin I (UI), a member of the corticotropin-releasing hormone family of peptides, regulates a diverse array of physiological functions, including appetite regulation, defensive behavior and stress response. In this study, firstly, the tissue-specific distribution of UI mRNA in olive flounder (Paralichthys olivaceus) was characterized and we found that UI mRNA was highly expressed in caudal neurosecretory system (CNSS) tissue. Secondly, alignment analysis found that a conserved cAMP response binding (CREB) site and a TATA element were located in the proximal promoter of UI gene. In addition, treatment of forskolin activatated cAMP-CREB pathway and induced the up-regulation of UI mRNA in cultured CNSS, suggesting the role of CREB in regulating the UI mRNA expression. Furthermore, plasma UI concentration and UI mRNA in CNSS showed obvious daily rhythm, with higher values in the daytime while lower values in the nighttime. Thirdly, using bold personality (BP) and shy personality (SP) flounder as an animal model, we found that flounder exhibited significantly higher locomotor activity in the nighttime than in the daytime (P < 0.001), and BP flounder showed significantly higher locomotor activity (P < 0.001) compared with SP flounder both in the daytime and nighttime. Analysis of feeding behavior revealed that BP flounder showed a shorter latency to feed and more attacks to prey. Furthermore, the qPCR and immunohistochemistry results showed that BP flounder expressed significantly lower level of UI mRNA and protein in CNSS tissue. Collectively, our study suggested that the UI plays an important role in locomotor activity and appetite regulation, which provides a basis for understanding the mechanism of defensive behavior and animal personality in flounder.

Lagged recovery of fish spatial distributions following a cold-water perturbation.

Anomalous local temperature and extreme events (e.g. heat-waves) can cause rapid change and gradual recovery of local environmental conditions. However, few studies have tested whether species distribution can recover following returning environmental conditions. Here, we tested for change and recovery of the spatial distributions of two flatfish populations, American plaice (Hippoglossoides platessoides) and yellowtail flounder (Limanda ferruginea), in response to consecutive decreasing and increasing water temperature on the Grand Bank off Newfoundland, Canada from 1985 to 2018. Using a Vector Autoregressive Spatiotemporal model, we found the distributions of both species shifted southwards following a period when anomalous cold water covered the northern sections of the Grand Bank. After accounting for density-dependent effects, we observed that yellowtail flounder re-distributed northwards when water temperature returned and exceeded levels recorded before the cold period, while the spatial distribution of American plaice has not recovered. Our study demonstrates nonlinear effects of an environmental factor on species distribution, implying the possibility of irreversible (or hard-to-reverse) changes of species distribution following a rapid change and gradual recovery of environmental conditions.

Involvement of glycolysis activation in flatfish sexual size dimorphism: Insights from transcriptomic analyses of Platichthys stellatus and Cynoglossus semilaevis.

The starry flounder (Platichthys stellatus), a flatfish cultured at the margins of the North Pacific, displays an obvious female-biased growth advantage, similar to many other fish species. To reveal the molecular mechanism underlying sexual size dimorphism, a comparative transcriptomic analysis of the somatotropic and reproductive axes was conducted. In total, 156, 67, 3434, and 378 differentially expressed genes (DEGs) between female and male samples were obtained in the brain, liver, gonad, and muscle tissues (q < 0.05). These DEGs were significantly enriched for various GO terms, including ion channel activity, protein binding, lipid transporter activity, and glycolytic process. The significantly enriched KEGG pathways included insulin secretion, axon guidance, and glycolysis/gluconeogenesis. In a detailed analysis of DEGs in these significantly enriched pathways, 35 genes showed higher expression levels in female muscle tissues than in male muscle tissues. A protein-protein interaction network further revealed specific interactions involving the glycolysis related-protein enolase (ENO), triosephosphate isomerase (TPI), Bisphosphoglycerate mutase (BPGM), fructose-bisphosphate aldolase (ALDO), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, the role of glycolysis/gluconeogenesis was supported by an analysis of common DEGs between P. stellatus and Chinese tongue sole (Cynoglossus semilaevis). These results indicate that the activation of glycolysis in female muscle tissues contributes to flatfish sexual size dimorphism.

Visualization of Sox10-positive chromatoblasts by GFP fluorescence in flounder larvae and juveniles using electroporation.

Japanese flounder are left-right asymmetrical, with features, such as dark, ocular-side specific pigmentation. This pigmentation arises during metamorphic stages, along with the asymmetric differentiation of adult-type chromatophores. Additionally, among juveniles, tank-reared specimens commonly show ectopic pigmentation on their blind sides. In both cases, neural crest-derived Sox10-positive progenitor cells at the dorsal fin base are hypothesized to contribute to chromatophore development. Here, we developed a method to visualize Sox10-positive cells via green fluorescent protein (GFP) fluorescence to directly monitor their migration and differentiation into chromatophores in vivo. Electroporation was applied to introduce GFP reporter vectors into the dorsal fin base of larvae and juveniles. Cre-loxP system vectors were also tested to enable cell labeling even after a decrease in sox10 expression levels. In larvae, undifferentiated Sox10-positive progenitor cells were labeled in the dorsal fin base, whereas newly differentiated adult-type chromatophores were seen dispersed on the ocular side. In juveniles, Sox10-positive cells were identified in the connective tissue of the dorsal fin base and observed prominently in areas of ectopic pigmentation, including several labeled melanophores. Thus, it was suggested that during metamorphic stages, Sox10-positive cells at the dorsal fin base contribute to adult-type chromatophore development, whereas in juveniles, they persist as precursors in the connective tissue, which in response to stimuli migrate to generate ectopic pigmentation. These findings contribute to elucidating pigmentation mechanisms, as well as abnormalities seen in hatchery-reared flounders. The electroporation method may be adapted to diverse animals as an accessible gene transfer method in various research fields, including developmental and biomedical studies.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Single-nucleotide polymorphisms responsible for pseudo-albinism and hypermelanosis in Japanese flounder (Paralichthys olivaceus) and reveal two genes related to malpigmentation.

Paralichthys olivaceus is the kind of cold-water benthic marine fish. In the early stages of development, the symmetrical juveniles transform into an asymmetrical body shape through metamorphosis for adapting benthic life. After that, one side of the fish body is attached to the ground, and the eyes turn to the opposite side which is called ocular side. The body color also appears asymmetry. The skin on the ocular side is dark brown, and the skin on the blind side is white without pigmentation. Pseudo-albinism and hypermelanosis have been considered distinct body color disorders in flatfish. Pseudo-albinism and hypermelanosis in Paralichthys olivaceus are due to abnormal or uneven pigment distribution, due to the interaction of hereditary and environmental factors, rather than a single-nucleotide mutation of a specific gene. Here, we report three single-nucleotide polymorphisms (SNPs) responsible for both pseudo-albinism and hypermelanosis, which are located on two body color-related genes involved in melanogenesis-related pathways. c.2440C>A (P. V605I) and c.2271-96T>C are located on the Inositol 1,4,5-trisphosphate receptor type 2-like (ITPR2) (Gene ID: 109624047), they are located in exon 16 and the non-coding region, respectively, and c.2406C>A (P.H798N) is located in exon 13 of the adenylate cyclase type 6-like (AC6) gene(Gene ID: 109630770). ITPR2 and AC6 expression, which both participate in the thyroid hormone synthesis pathway associated with pseudo-albinism and hypermelanosis in P. olivaceus, were also investigated using qRT-PCR. In hypermelanotic fish, there were relatively higher levels of expression in ITPR2 and AC6 mRNA of hyper-pigmented skin of blind side than that of non-pigmented skin on the blind side and pigmented skin on the ocular side, while in pseudo-albino fish, expression level of ITPR2 and AC6 mRNA in pigmented skin of ocular side was significantly higher than that in non-pigmented skin both ocular and blind side. The results indicated that the expression of the two genes in abnormal parts of body color is positively correlated with pigmentation, suggesting that the influence of abnormal expression of two genes on the pigmentation in abnormal parts of body color deserves further study.

Expression of corticotropin releasing hormone in olive flounder (Paralichthys olivaceus) and its transcriptional regulation by c-Fos and the methylation of promoter.

Corticotropin-releasing hormone (CRH) has been implicated in multiple physiological processes, such as circadian rhythms, food intake and anxiety-like behavior. In telelost fishes, CRH is predominantly expressed in the caudal neurosecretory system (CNSS), a much low level in hypothalamus and gonads, while undetectable levels in other tissues. However, the mechanisms governing this tissue-specific expression remain unknown. In this study, firstly, we investigated the expression pattern of CRH mRNA in different tissues of olive flounder (Paralichthys olivaceus). Secondly, we found that flounder exhibited low locomotor activity in the daytime, concomitant with the highest CRH mRNA expression in CNSS at noon. Thirdly, we examined whether epigenetic mechanisms through DNA methylation are involved in tissue-specific expression of CRH mRNA. Promoter methylation of the CRH gene was assessed through bisulphate sequencing methods. In the proximal promoter, almost no methylation was detected in the muscle, hypothamus and CNSS. However, different methylation was detected in the distal promoter of CRH. The methylation was 63% in CNSS, while that in hypothalamus and muscle was 85% and 96%, respectively. Lastly, bioinformatics analysis revealed that a conserved AP-1 binding site (5-TCACTGA-3) was located in the proximal promoter of CRH gene. In vivo experiment, lipopolysaccharide (LPS) intraperitoneally injection in the flounder up-regulated c-Fos and CRH mRNA in CNSS (P < 0.01). In CNSS tissue culture experiment, forskolin treatment significantly induced the expression of c-Fos, c-Jun and CRH mRNA (P < 0.01). Collectively, our data provide the evidence that distal promoter methylation and c-Fos signal pathway are involved in transcriptional regulation of CRH expression in flounder.

Tissue-Specific Accumulation and Antioxidant Defenses in Flounder (Paralichthys olivaceus) Juveniles Experimentally Exposed to Methylmercury.

Methylmercury (MeHg) is the most toxic form of mercury and can accumulate in the cells of marine organisms, such as fish, causing adverse effects on various physiological functions. This study examined MeHg accumulation and its toxicological role in antioxidant defenses in tissues, including the liver, gills, and muscle of flounder (Paralichthys olivaceus) juveniles. After 30 d of MeHg exposure (0, 0.1, 1.0, 10.0, and 20.0 µg L-1), the accumulation of MeHg in the three tissues correlated positively with the concentration of MeHg and exhibited tissue specificity in the order of liver > gills > muscle. Among the antioxidant markers, the activities of SOD (superoxide dismutase) and GST (glutathione S-transferase) as well as the content of glutathione (GSH) in the liver and gills were induced at 0.1-10.0 µg L-1 but repressed at 20.0 µg L-1. The activities of SOD and GST and the content of GSH in the muscle significantly increased with increasing MeHg concentration. Catalase (CAT) activity in the liver was induced at 0.1-1.0 µg L-1 but inhibited at 10.0-20.0 µg L-1, whereas exposure to MeHg did not remarkably affect CAT activity in the gills and muscle. The levels of lipid peroxidation (LPO) increased dose dependently, showing tissue specificity with the highest level in the liver, then the gills, followed by muscles. Overall, higher sensitivity to oxidative stress induced by MeHg was detected in the liver than the gills and muscle. These findings improve our understanding of the tissue-specific accumulation of heavy metals and their roles in antioxidant responses in marine fish subjected to MeHg exposure.