DNA Methylation Mediates Sperm Quality via piwil1 and piwil2 Regulation in Japanese Flounder (Paralichthys olivaceus).

DNA methylation is an important way to regulate gene expression in eukaryotes. In order to reveal the role of DNA methylation in the regulation of germ cell-specific piwi gene expression during spermatogenesis of Japanese flounder (Paralichthys olivaceus), the expression profiles of piwil1 (piwi-like 1) and piwil2 (piwi-like 2) genes in the gonads of female, male, and sex-reversed pseudo-male P. olivaceus were analyzed, and the dynamic of DNA methylation was investigated. As a result, piwil1 and piwil2 genes were highly expressed in the testis of both male and pseudo-male P. olivaceus, with significant variation among male individuals. The DNA methylation levels in the promoter regions of both piwil1 and piwil2 were negatively correlated with their expression levels, which may contribute to the transcriptional regulation of piwi genes during spermatogenesis. There was also sperm quality variation among male P. olivaceus, and the sperm curvilinear velocity was positively correlated with the expression of both piwil1 and piwil2 genes. These results indicated that the DNA methylation in piwil1 and piwil2 promoter regions may affect the initiation of piwi gene transcription, thereby regulating gene expression and further affecting the spermatogenesis process and gamete quality in P. olivaceus.

Estimation of accumulation potential for tritium in olive flounder on exposure of treated water derived from Fukushima Daiichi Nuclear Power Station: Tritium transfer from seawater and food chain into organically bound tritium in the targeted fish.

This study estimated the accumulation potential of tritium, a major radionuclide released from the Fukushima Daiichi Nuclear Power Station (FDNPS), into the olive flounder as organically bound tritium (OBT) using a computer simulation model. In this estimation, two transfer pathways into the OBT were assumed: formation from tritiated water (HTO) in the tricarboxylic acid (TCA) cycle, and ingestion of OBT through the food chain (from phytoplankton, small fish, to the flounder). The food chain structure was reconstructed based on fish growth model. The OBT concentration in the flounder was estimated on three scenarios: Tritium was supplied to the flounder as only HTO in seawater (Scenario 1), as HTO in seawater and OBT formed from HTO in the small fish (Scenario 2), and as HTO in seawater and OBT accumulated in the small fish through the formation and ingestion of OBT in phytoplankton (Scenario 3). The estimated OBT concentrations in the flounder were in the following order: Scenario 3 > 2 > 1. The ratio of the estimated concentration in Scenario 1 to that in Scenario 3 reached a certain value (66 % after a year from the start of HTO exposure), indicating that the tritium transfer from the seawater into the flounder more significantly contributed to this concentration than ingestions of the small fish and the phytoplankton. Additionally, the difference between the estimations of Scenarios 1 and 2 is significantly larger than that between Scenarios 2 and 3. This suggests that phytoplankton contributed weakly to the OBT concentration in the flounder compared to the small fish.

Olive Flounder By-Product Prozyme2000P Hydrolysate Ameliorates Age-Related Kidney Decline by Inhibiting Ferroptosis.

This study explores olive flounder by-product Prozyme2000P (OFBP) hydrolysate as a potential treatment for age-related kidney decline. Ferroptosis, a form of cell death linked to iron overload and oxidative stress, is increasingly implicated in aging kidneys. We investigated whether OFBP could inhibit ferroptosis and improve kidney health. Using TCMK-1 cells, we found that OFBP treatment protected cells from ferroptosis induced by sodium iodate (SI). OFBP also preserved the mitochondria health and influenced molecules involved in ferroptosis regulation. In aging mice, oral administration of OFBP significantly improved kidney health markers. Microscopic examination revealed reduced thickening and scarring in the kidney's filtering units, a hallmark of aging. These findings suggest that OFBP hydrolysate may be a promising therapeutic candidate for age-related kidney decline. By inhibiting ferroptosis, OFBP treatment appears to improve both cellular and structural markers of kidney health. Further research is needed to understand how OFBP works fully and test its effectiveness in more complex models.

Association Between Muscle Growth and Transcription of a Mutant MSTN Gene in Olive Flounder (Paralichthys olivaceus).

Myostatin (MSTN, also known as growth differentiation factor-8 (GDF-8)), a member of the transforming growth factor ß (TGF-ß) superfamily, functions as a negative regulator of skeletal muscle development and growth. However, it is also expressed in a wide range of tissues in fish and thus may have more diverse roles in this group than in mammals. In this study, we assessed the genome-wide transcriptional expression pattern associated with the CRISPR/Cas9-mutated MSTN gene in the olive flounder (Paralichthys olivaceus) in association with changes in cell proliferation and transportation processes. There were no differences in the hepatosomatic index, and the growth of male and female fish increased in the F1 progeny of the MSTN mutants. Furthermore, the histopathological analysis showed that myostatin editing resulted in a 41.24% increase in back muscle growth and 46.92% increase in belly muscle growth in male flounder compared with normal flounder, and a 16.01% increase in back muscle growth and 14.26% increase in belly muscle growth in female flounder compared with normal flounder. This study demonstrates that editing of the myostatin gene enhances muscle growth in olive flounder, with a notably more pronounced effect observed in males. Consequently, myostatin-edited male flounder could represent a valuable asset for the flounder aquaculture industry.

Identification and functional characterization of annexin A2 in half-smooth tongue sole (Cynoglossus semilaevis).

Annexin A2 (AnxA2), belonging to the annexin family, plays a crucial role in immune responses. In this study, the cDNA of the AnxA2 gene was identified in half-smooth tongue sole, Cynoglossus semilaevis. The transcript of AnxA2 gene in C. semilaevis (CsAnxA2) showed broad tissue distribution, with the highest expression level observed in the gut. CsAnxA2 expression was significantly up-regulated in the intestine, spleen, and kidney tissues following exposure to Shewanella algae. Immunohistochemical staining revealed that CsAnxA2 was predominantly expressed in epithelial cells and significantly elevated after S. algae challenge. Subcellular localization showed that CsAnxA2 was primarily localized in the cytoplasmic compartment. Moreover, proinflammatory cytokines (IL-6, IL-8 and IL-1ß) exhibited significant upregulation after CsAnxA2 was overexpressed in vivo. One hundred and fifty-eight CsAnxA2-interacting proteins were captured in the intestinal tissue, showing the top two normalized abundance observed for actin beta (ACTB) and protein S100-A10 (p11). Fifty-four high abundance CsAnxA2-interacting proteins (HIPs) were primary enriched in ten pathways, with the top three significantly enriched pathways being Salmonella infection, glycolysis/gluconeogenesis, and peroxisome proliferator-activated receptor (PPAR) signaling pathway. These results provide valuable information for further investigation into the functional mechanism of AnxA2 in C. semilaevis.

Identification and functional analysis of circpdlim5a generated from pdlim5a gene splicing in the skeletal muscle of Japanese flounder (Paralichthys olivaceus).

Circular RNAs (circRNAs) are non-coding RNAs with endogenous regulatory functions, including regulating skeletal muscle development. However, its role in the development of skeletal muscle in Japanese flounder (Paralichthys olivaceus) is not clear. Therefore we screened a candidate circpdlim5a, which is derived from the gene pdlim5a, from the skeletal muscle transcriptome of Japanese flounder. We characterized circpdlim5a, which was more stable compared to the linear RNA pdlim5a. Distributional characterization of circpdlim5a showed that circpdlim5a was predominantly distributed in the nucleus and was highly expressed in the skeletal muscle of adult Japanese flounder (24 months). When we further studied the circpdlim5a function, we found that it inhibited the expression of proliferation and differentiation genes according to the over-expression experiment of circpdlim5a in myoblasts. We concluded that circpdlim5a may inhibit the proliferation and differentiation of myoblasts and thereby inhibit skeletal muscle development in Japanese flounder. This experiment provides information for the study of circRNAs by identifying circpdlim5a and exploring its function, and offers clues for molecular breeding from an epigenetic perspective.

Relevance of flounder caging and proteomics to explore the impact of a major industrial accident caused by fire on the Seine estuarine water quality.

On September 26th 2019, a major fire occurred in the Lubrizol factory located near the Seine estuary, in Rouen-France. Juvenile flounders were captured in the Canche estuary (a reference system) and caged one month in the Canche and in the Seine downstream the accident site. No significant increases of PAHs, PCBs and PFAS was detected in Seine vs Canche sediments after the accident, but a significant increase of dioxins and furans was observed in water and sewage sludge in the Rouen wastewater treatment plant. The proteomics approach highlighted a dysregulation of proteins associated with cholesterol synthesis and lipid metabolism, in fish caged in the Seine. The overall results suggested that the fire produced air borne dioxins and furans that got deposited on soil and subsequently entered in the Seine estuarine waters via runoff; thus contaminating fish preys and caged flounders in the Seine estuary.

Isotopes and otolith chemistry provide insight into the biogeochemical history of mercury in southern flounder across a salinity gradient.

Methylmercury (MeHg) continues to pose a significant global health risk to wildlife and humans through fish consumption. Despite numerous advancements in understanding the mercury (Hg) cycle, questions remain about MeHg sources that accumulate in fish, particularly across transitional coastal areas, where harvest is prominent and Hg sources are numerous. Here we used a unique combination of Hg and nutrient isotopes, and otolith chemistry to trace the biogeochemical history of Hg and identify Hg sources that accumulated in an economically important fish species across Mobile Bay, Alabama (USA). Fish tissue Hg in our samples primarily originated from wet deposition within the watershed, and partly reflected legacy industrial Hg. Results also suggest that little Hg was lost through photochemical processes (<10% of fish tissue Hg underwent photochemical processes). Of the small amount that did occur, photodegradation of the organic form, MeHg, was not the dominant process. Biotic transformation processes were estimated to have been a primary driver of Hg fractionation (∼93%), with isotope results indicating methylation as the primary biotic fractionation process prior to Hg entering the foodweb. On a finer scale, individual lifetime estuarine habitat use influenced Hg sources that accumulated in fish and fish Hg concentrations, with runoff from terrestrial Hg sources having a larger influence on fish in freshwater regions of the estuary compared to estuarine regions. Overall, results suggest increases in Hg inputs to the Mobile Bay watershed from wet deposition, turnover of legacy sources, and runoff are likely to translate into increased uptake into the foodweb.

Paradoxical effects on ice nucleation are intrinsic to a small winter flounder antifreeze protein.

Antifreeze proteins (AFPs) bind to ice in solutions, resulting in non-colligative freezing point depression; however, their effects on ice nucleation are not well understood. The predominant plasma AFP of winter flounder (Pseudopleuronectes americanus) is AFP6, which is an amphiphilic alpha helix. In this study, AFP6 and modified constructs were produced as fusion proteins in Escherichia coli, subjected to proteolysis when required and purified prior to use. AFP6 and its recombinant fusion precursor generated similar thermal hysteresis and bipyramidal ice crystals, whereas an inactive mutant AFP6 produced hexagonal crystals and no hysteresis. Circular dichroism spectra of the wild-type and mutant AFP6 were consistent with an alpha helix. The effects of these proteins on ice nucleation were investigated alongside non-AFP proteins using a standard droplet freezing assay. In the presence of nucleating AgI, modest reductions in the nucleation temperature occurred with the addition of mutant AFP6, and several non-AFPs, suggesting non-specific inhibition of AgI-induced ice nucleation. In these experiments, both AFP6 and its recombinant precursor resulted in lower nucleation temperatures, consistent with an additional inhibitory effect. Conversely, in the absence of AgI, AFP6 induced ice nucleation, with no other proteins showing this effect. Nucleation by AFP6 was dose-dependent, reaching a maximum at 1.5 mM protein. Nucleation by AFP6 also required an ice-binding site, as the inactive mutant had no effect. Furthermore, the absence of nucleation by the recombinant precursor protein suggested that the fusion moiety was interfering with the formation of a surface capable of nucleating ice.

Molecular characterization and ontogenetic expression profiles of LPXRFa and its receptor in Japanese flounder (Paralichthys olivaceus).

Investigations concerning the LPXRFa system are rarely conducted in flatfish species. Here, we first identified and characterized lpxrfa and its cognate receptor lpxrfa-r genes in the Japanese flounder (Paralichthys olivaceus). The coding DNA sequence of lpxrfa was 579 bp in length, wich encoded a 192-aa preprohormone that can produce three mature LPXRFa peptides. The open reading frame (ORF) of lpxrfa-r was 1446 bp in size, and encoded a 481-aa LPXRFa-R protein that encompassed seven hydrophobic transmembrane domains. Subsequently, tissue distribution expression profiles of lpxrfa and lpxrfa-r transcripts were assayed by quantitative real-time PCR. The results indicated that expressions of lpxrfa transcripts were detected at the highest levels in the brain of both females and males, however, lpxrfa-r transcripts were remarkablely expressed in the brain tissue of female fish and in the testis tissue of male fish. Furthermore, transcript levels of lpxrfa and lpxrfa-r genes were investigated during early ontogenetic development, with the maximum expression levels at 30 days post-hatching. Overall, these data contribute to providing preliminary proof for the existence and structure of the LPXRFa system in Japanese flounder, and the study is just the foundation for researching physiological function of LPXRFa system in this species.

Endocrine patterns associated with ovarian development in female Pacific halibut (Hippoglossus stenolepis).

The Pacific halibut (Hippoglossus stenolepis) is a large migratory demersal flatfish species that occupies a top trophic role in the North Pacific Ocean and Bering Sea ecosystems, where it also supports various fisheries. As a first attempt to characterize the endocrine mechanisms driving sexual maturation in this important species, we collected pituitary, ovarian and blood samples from Pacific halibut females captured in the wild that were classified histologically into various female developmental stages. We conducted gene expression analyses of gonadotropin beta subunits in the pituitary and observed that mRNA expression levels of fshb gradually increased throughout vitellogenesis, remained elevated until before ovulation and declined after spawning. In contrast, the mRNA expression levels of lhb markedly increased during oocyte maturation and remained elevated until after spawning. Ovarian mRNA expression levels of the gonadotropin receptor genes fshr and lhr peaked during oocyte maturation and before spawning, respectively, immediately following the developmental stage at which pituitary fshb and lhb mRNA expression first reached maximum levels. The ovarian gene expression patterns of steroidogenic enzyme genes cyp19a1 and hsd20b2 paralleled those of fshr and lhr, respectively. Testosterone and 17ß-estradiol (E2) plasma levels increased concomitantly with fshr and cyp19a1 mRNA expression levels, and vitellogenin plasma levels increased throughout vitellogenesis and reached maximum levels prior to spawning. These results are consistent with the notion that in female Pacific halibut, as in other teleosts, vitellogenesis and oocyte maturation and ovulation are likely under the control of pituitary gonadotropic hormones Fsh and Lh, respectively.

LncRNA pol-lnc78 as a ceRNA regulates antibacterial responses via suppression of pol-miR-n199-3p-mediated SARM down-regulation in Paralichthys olivaceus.

Long non-coding RNAs (lncRNAs) function as key modulators in mammalian immunity, particularly due to their involvement in lncRNA-mediated competitive endogenous RNA (ceRNA) crosstalk. Despite their recognized significance in mammals, research on lncRNAs in lower vertebrates remains limited. In the present study, we characterized the first immune-related lncRNA (pol-lnc78) in the teleost Japanese flounder ( Paralichthys olivaceus). Results indicated that pol-lnc78 acted as a ceRNA for pol-miR-n199-3p to target the sterile alpha and armadillo motif-containing protein (SARM), the fifth discovered member of the Toll/interleukin 1 (IL-1) receptor (TIR) adaptor family. This ceRNA network regulated the antibacterial responses of flounder via the Toll-like receptor (TLR) signaling pathway. Specifically, SARM acted as a negative regulator and exacerbated bacterial infection by inhibiting the expression of inflammatory cytokines IL-1ß and tumor necrosis factor-α (TNF-α). Pol-miR-n199-3p reduced SARM expression by specifically interacting with the 3' untranslated region (UTR), thereby promoting SARM-dependent inflammatory cytokine expression and protecting the host against bacterial dissemination. Furthermore, pol-lnc78 sponged pol-miR-n199-3p to ameliorate the inhibition of SARM expression. During infection, the negative regulators pol-lnc78 and SARM were significantly down-regulated, while pol-miR-n199-3p was significantly up-regulated, thus favoring host antibacterial defense. These findings provide novel insights into the mechanisms underlying fish immunity and open new horizons to better understand ceRNA crosstalk in lower vertebrates.