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.

Differential modulation of digestive enzymes and energy reserves at different times after feeding in juveniles of the marine estuarine-dependent flatfish Paralichthys orbignyanus (Valenciennes, 1839).

Integrative studies are lacking on the responses of digestive enzymes and energy reserves in conjunction with morphological traits at distinct postprandial times in marine estuarine-dependent flatfishes of ecological and economic importance, such as Paralichthys orbignyanus. We determined total weight (TW), hepato-somatic index (IH), activities of digestive enzymes in the intestine, and the concentration of energy reserves in the liver and the muscle at 0, 24, 72, and 360 h after feeding in juveniles of P. orbignyanus. Amylase activity decreased at 72 h (about 30%). Maltase, sucrose, and lipase activities reached peak at 24 h (67%, 600%, and 35%, respectively). Trypsin and aminopeptidase-N activities at 24 and 72 h, respectively, were lower than those at t = 0 (53% and 30%). A peak increase in the concentration of glycogen and triglycerides in the liver (24 h) (86% and 89%, respectively) occurred. In muscle, glycogen and triglyceride concentrations were unchanged at 24 h and higher at 72 and 360 h (100% and 60%). No changes were found in TW, IH, free glucose in the liver and muscle, and protein in the liver. The protein concentration in the muscle sharply increased at 24 and 360 h after feeding (60%). The results indicate a distinct and specific response of central components of carbohydrate, lipid, and protein metabolism that could be adjustments at the biochemical level upon periods of irregular feeding and even of long-term food deprivation inside coastal lagoons or estuaries. The distinct responses of digestive enzymes in the intestine and energy reserves in the liver and muscle suggest the differential modulation of tissue-specific anabolic and catabolic pathways that would allow the maintenance of physical conditions.

Whole genome identification of olive flounder (Paralichthys olivaceus) cathepsin genes: Provides insights into its regulation on biotic and abiotic stresses response.

Cathepsins are major lysosomal enzymes involved in essential physiological processes, including protein degradation, tissue differentiation, and innate or adaptive responses. Several kinds of cathepsins have been reported in teleost fishes, but no characterization have been performed for the inflammatory response of cathepsin family in olive flounder until now. In our current study, a total of 17 cathepsins in olive flounder were systematically identified and characterized. Phylogenetic analysis clearly indicated that the cathepsin genes was highly conserved. Analysis of structure and motifs exhibited high sequence similarity of cathepsin genes in olive flounder. Expression profiles of cathepsin genes in different tissues and developmental stages showed that cathepsins were temporally and spatially specific. RNA-seq analysis of bacteria and temperature stresses revealed that members of cathepsin were involved in inflammatory responses. Collectively, our findings would provide a further reference for understanding the molecular mechanisms of cathepsins in olive flounder.

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.

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.

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.

Genome-wide DNA methylation mediates the resistance to vibriosis in Cynoglossus semilaevis.

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as "cAMP signaling pathway" and "NOD-like receptor signaling pathway". Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including "complement and coagulation cascades", and "cytokine-cytokine receptor interaction", which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture.

Toxic effects of combined exposure to cadmium and nitrate on intestinal morphology, immune response, and microbiota in juvenile Japanese flounder (Paralichthys olivaceus).

Cadmium (Cd2+) and nitrate (NO3-) are important environmental pollutants in the offshore marine ecological environment. However, limited research has explored their combined effects, particularly regarding their impact on the microbiota and intestinal health of marine fish. In this study, juvenile Japanese flounders (P. olivaceus) were immersed in seawater samples with different combinations of Cd2+ (0, 0.2, and 2 mg/L) and NO3- (0 and 80 mg/L NO3N) for 30 days to explore their toxic impacts on intestinal morphology, tight junction (TJ) barrier, immune response, and microbiota. Our results showed that Cd2+ or NO3- exposure alone led to histopathological damage of the gut, while their co-exposure aggravated intestinal damage. Moreover, co-exposure substantially decreased TJ-related gene expression, including occludin, claudin-10, and ZO-2, suggesting increased TJ permeability in the gut. Regarding the immune response, we observed upregulated expression of immune-related markers such as HSP40, IL-1ß, TNF-α, and MT, suggesting the onset of intestinal inflammation. Furthermore, Cd2+ and NO3- exposure led to changes in intestinal microflora, characterized by decreased the abundance of Sediminibacterium and NS3a_marine_group while increasing the prevalence of pathogens or opportunistic pathogens such as Ralstonia, Proteus, and Staphylococcus. This alteration in microbiota composition increased network complexity and α-diversity, ultimately causing dysbiosis in the fish gut. Additionally, combined exposure resulted in metabolic disorders that affected the predicted functions of the intestinal microbiota. Overall, our study demonstrates that Cd2+-NO3- co-exposure amplifies the deleterious effects compared to single exposure. These findings enhance our understanding of the ecological risks posed by Cd2+-NO3- co-exposure in marine ecosystems.

The cutaneous stress response system in three-spined stickleback and European flounder exposed to oxidative stress: Different mode of action.

In fish, the skin is directly exposed to multiple environmental stressors and provides the first line of defense against harmful external factors. It turned out that cortisol and melatonin (Mel) are involved in fish cutaneous stress response system (CSRS) similar to mammalian. This study investigates the mode of action of CSRS in two teleost species of different biology and skin characteristics, the three-spined stickleback and the European flounder, after exposure to oxidative stress induced by a potassium dichromate solution. The cutaneous stress response system presents different ways of action in two studied species: Mel concentration increases in the skin of both species, but cortisol concentration increases in the skin only in sticklebacks. Data suggest that stickleback skin cells can produce cortisol. However, cortisol is not involved in the response to oxidative stress in flounders. In stickleback skin, two genes encoding AANAT and ASMT/HIOMT (enzymes involved in Mel synthesis), aanat1a and asmt2, are expressed, but in flounder skin, only one, asmtl. Because gene expression does not change in stickleback skin after exposure to stress, the source of increased Mel is probably outside the skin. A lack of expression of the gene encoding AANAT in flounder skin strongly suggests that Mel is transported to the skin by the bloodstream from other sites of synthesis. Pigment dispersion in the skin after exposure to oxidative stress is found only in sticklebacks.

High-Resolution Magic Angle Spinning (HRMAS) NMR Identifies Oxidative Stress and Impairment of Energy Metabolism by Zearalenone in Embryonic Stages of Zebrafish (Danio rerio), Olive Flounder (Paralichthys olivaceus) and Yellowtail Snapper (Ocyurus chrysurus).

Zearalenone (ZEA) is a mycotoxin, commonly found in agricultural products, linked to adverse health impacts in humans and livestock. However, less is known regarding effects on fish as both ecological receptors and economically relevant "receptors" through contamination of aquaculture feeds. In the present study, a metabolomics approach utilizing high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was applied to intact embryos of zebrafish (Danio rerio), and two marine fish species, olive flounder (Paralichthys olivaceus) and yellowtail snapper (Ocyurus chrysurus), to investigate the biochemical pathways altered by ZEA exposure. Following the assessment of embryotoxicity, metabolic profiling of embryos exposed to sub-lethal concentrations showed significant overlap between the three species and, specifically, identified metabolites linked to hepatocytes, oxidative stress, membrane disruption, mitochondrial dysfunction, and impaired energy metabolism. These findings were further supported by analyses of tissue-specific production of reactive oxygen species (ROS) and lipidomics profiling and enabled an integrated model of ZEA toxicity in the early life stages of marine and freshwater fish species. The metabolic pathways and targets identified may, furthermore, serve as potential biomarkers for monitoring ZEA exposure and effects in fish in relation to ecotoxicology and aquaculture.

Expression characteristics of Hsd3b7 in the gonads of Paralichthys olivaceus.

Steroidogenesis is an important biological process for gonadal differentiation and development. In mammals, 3ß-hydroxysteroid dehydrogenase 7 (HSD3B7) could convert 3ß-hydroxy of 7α-hydroxycholesterol into a ketone and form 7α-hydroxy-4-cholesten-3-one, which may affect steroidogenesis. However, in fish, the study of Hsd3b7 is still lacking. In this study, Hsd3b7 was identified in the olive flounder Paralichthys olivaceus, an important mariculture fish. According to bioinformatics analysis, Hsd3b7 belongs to a Rossmann-fold NAD(P)(+)-binding protein and can interact in a predictable manner with Hsd17b2, -3, and - 4, which play a role in steroidogenesis. In the adult flounder, Hsd3b7 was expressed in various tissues, at particularly high level in male muscle. The expression levels of Hsd3b7 at gonadal development stages I-V initially increased and then decreased, with an inflection point in the ovary at stage III and in the testis at stage IV. At stage III, the expression level of Hsd3b7 was significantly higher in the ovary than in the testis (P < 0.01). The results of in situ hybridization (ISH) revealed that it was mainly expressed in oocytes of phases I-IV or around oocytes of phases IV-V in the ovaries and around spermatid lobules at stages IV-V in the testes. Three regulatory sites of SRY-box transcription factor 9 (Sox9), a transcription factor involved in steroidogenesis and gonadal differentiation, were predicted in the promoter of Hsd3b7. After intraperitoneal injection with the recombination flounder Sox9a, the expression of Hsd3b7 was significantly up-regulated (P < 0.01). During the flounder gonadal differentiation, 17ß-estradiol (E2, 5 µg/g feed) and 17α-methyltestosterone (T, 5 µg/g feed) were used to obtain the phenotypic female or male flounder, and the results showed that in the E2 group, Hsd3b7 expression was highest at 2 cm TL, the primordial gonad stage, which was significantly higher than that at 12 cm TL (P < 0.05). In the T group, Hsd3b7 expression level was also highest at 2 cm TL and significantly higher than at 10 and 12 cm TL (P < 0.05). Moreover, Hsd3b7 was detected to be localized mainly around oogonia and spermatogonia during the differentiated gonads with ISH. These findings first introduce the expression characteristics of Hsd3b7 and the effect of Sox9a on its expression, which contribute to our understanding of the function of Hsd3b7 in fish gonads.

Focal adhesion signaling pathway involved in skin immune response of tongue sole Cynoglossus semilaevis to Vibrio vulnificus infection.

Focal adhesion (FA) plays a key role in cell adhesion, migration and antibacterial immune, but it remained unclear in fish. In this study, half-smooth tongue sole Cynoglossus semilaevis were infected with Vibrio vulnificus, and then immune-related protein in the skin, especially for FA signaling pathway were screened and identified by iTRAQ analysis. Results showed that the differentially expressed proteins (DEPs) in skin immune response (eg., ITGA6, FN, COCH, AMBP, COL6A1, COL6A3, COL6A6, LAMB1, LAMC1, FLMNA) were firstly found in FA signaling pathway. Furthermore, the validation analysis of FA-related genes were basically consistent with the iTRAQ data at 36 hpi (r = 0.678, p < 0.01), and their spatio-temporal expressions were confirmed by qPCR analysis. The molecular characterization of vinculin of C. semilaevis was described. This study will provide a new perspective for understanding the molecular mechanism of FA signaling pathway in the skin immune response in marine fish.

Amh/Amhr2 Signaling Causes Masculinization by Inhibiting Estrogen Synthesis during Gonadal Sex Differentiation in Japanese Flounder (Paralichthys olivaceus).

The anti-Müllerian hormone (Amh) is a protein belonging to the TGF-ß superfamily, the function of which has been considered important for male sex differentiation in vertebrates. The Japanese flounder (Paralichthys olivaceus) is a teleost fish that has an XX/XY sex determination system and temperature-dependent sex determination. In this species, amh expression is up-regulated in genetic males and in temperature-induced masculinization during the sex differentiation period. However, to the best of our knowledge, no reports on the Amh receptor (Amhr2) in flounder have been published, and the details of Amh signaling remain unclear. In this study, we produced amhr2-deficient mutants using the CRISPR/Cas9 system and analyzed the gonadal phenotypes and sex-related genes. The results revealed that the gonads of genetically male amhr2 mutants featured typical ovaries, and the sex differentiation-related genes showed a female expression pattern. Thus, the loss of Amhr2 function causes male-to-female sex reversal in Japanese flounder. Moreover, the treatment of genetically male amhr2 mutants with an aromatase inhibitor fadrozole, which inhibits estrogen synthesis, resulted in testicular formation. These results strongly suggest that Amh/Amhr2 signaling causes masculinization by inhibiting estrogen synthesis during gonadal sex differentiation in the flounder.

Dynamic changes of urotensin II and its receptor during ovarian development of olive flounder Paralichthys olivaceus.

Urotensin II (UII) is a kind of fish somatostatins cyclic peptide, which was originally extracted from the caudal neurosecretory system (CNSS). The system of UII and UII receptor (UIIR) has been reported to have multiple physiological regulatory functions, such as cardiovascular control, osmoregulation, and lipid metabolism. However, the effect of UII and UIIR on the ovarian development has not been covered. This study investigated the expression pattern of UII and UIIR in the ovarian follicles and explored their impact on ovarian development in olive flounder Paralichthys olivaceus. The results showed that the highest UII and UIIR mRNA levels were observed at stage II and stage III follicles during ovarian development, respectively. In situ hybridization revealed that a strong signal of UII was expressed in the oocyte nuclei of stage II follicles, however, UIIR was found in the follicle cells and oocyte cytoplasm of stage II and stage III follicles. Similarly, immunohistochemistry found positive signal of UII was detected in the oocyte nuclei of stage II follicles. The results from in vitro culture of olive flounder follicles suggested the expression of UII and UIIR mRNA levels significantly increased by 10 IU/ml human chorionic gonadotropin (hCG) for 9 h. Furthermore, the transcriptional expression of UII and UIIR was not statistically significantly changed by 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP). These results firstly suggested that UII and UII receptor may play vital roles in regulating ovarian growth in olive flounder.

Expression of scp3 and dazl reveals the meiotic characteristics of the olive flounder Paralichthys olivaceus†.

Olive flounder Paralichthys olivaceus is an important cultured marine fish. We found that the meiosis marker scp3 and its intrinsic regulator dazl were mainly expressed in the gonads. During the ovarian differentiation, scp3 signal was detected first in pre-meiotic oogonia at 60-mm total length (TL) and then in primary oocytes at 80- and 100-mm TL, with a sharp increase in scp3 expression level observed at 80- and 100-mm TL. Dazl signal was detected in primordial germ cells at 30-mm TL and oogonia at 60-mm TL, but no significant change of expression was observed. During the testicular differentiation period, scp3 and dazl expression remained at low levels, and scp3 signal was weakly detected in spermatogonia at 80-mm TL, whereas dazl signal was not found. During the ovarian developmental stages, the highest expression levels of scp3 and dazl were detected at stages I and II, respectively, and strong signals of scp3 and dazl were detected in primary oocytes and oocytes at phases I and II. In the testis, the high expression of scp3 and dazl was detected at stages II-IV and II-III, respectively. Scp3 signal was weakly observed in pre-meiotic spermatogonia at stages I and II and strongly detected in primary spermatocytes at stages III-V. Dazl was detected in the nuclei of spermatogonia and spermatids at stages II-IV. Furthermore, scp3 expression in the ovary could be promoted by 17α-ethynylestradiol and tamoxifen, whereas dazl expression could be downregulated by tamoxifen.

Chromatic organization of retinal photoreceptors during eye migration of Atlantic halibut (Hippoglossus hippoglossus).

The retinas of fishes often have single and double cone photoreceptors that are organized in lattice-like mosaics. In flatfishes experiencing eye migration (i.e., the metamorphic process whereby one eye migrates to the other side of the head), the hexagonal lattice of single cones present in the larva undergoes major restructuring resulting in a dominant square mosaic postmetamorphosis consisting of four double cones surrounding each single cone. The expression of different opsin types during eye migration has not been examined despite its importance in understanding photoreceptor plasticity and whether cell fate (in terms of spectral phenotype) could influence square mosaic formation. Here, we probed the retina of Atlantic halibut undergoing eye migration for opsin expression using two antibodies, AHblue and AB5407, that labeled short wavelength sensitive 2 (SWS2) opsin and longer wavelength (predominantly middle wavelength sensitive, RH2) opsins, respectively. Throughout the retina, double and triple cones labeled with AB5407 exclusively, whereas the vast majority of single cones labeled with AHblue. A minority (<5%) of single cones in the square mosaic of the centroventral retina labeled with AB5407. In regions of mosaic transition and near peripheral growth zones, some single cones co-expressed at least two opsins as they labeled with both antibodies. Short wavelength (SWS2 expressing, or S) cones formed a nonrandom mosaic gradient from central to dorsal retina in a region dominated by the larval single cone mosaic. Our results demonstrate the expression of at least two opsins throughout the postmetamorphic retina and suggest opsin switching as a mechanism to create new cone spectral phenotypes. In addition, the S cone gradient at the onset of eye migration may underlie a plastic, cell induction mechanism by which a cone's phenotype determines that of its neighbors and the formation of the square mosaic.

Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein.

Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17ß-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens. Chg genes (ChgL and ChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9-250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE2). The results showed that 2 ng/L EE2 notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE2 induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments.