The expression profiles of cyp19a1, sf-1, esrs and gths in the brain-pituitary during gonadal sex differentiation in juvenile Japanese eels.

Eels are gonochoristic species whose gonadal differentiation initiates at the yellow eel stage and is influenced by environmental factors. We revealed some sex-related genes were sex dimorphically expressed in gonads during gonadal sex differentiation of Japanese eel (Anguilla japonica); however, the expression of sex-related genes in the brain-pituitary during gonadal sex differentiation in eels is still unclear. This study aimed to investigate the sex-related gene expressions in the brain-pituitary and tried to clarify their roles in the brain and gonads during gonadal sex differentiation. Based on our previous histological study, the control eels developed as males, and estradiol-17ß (E2) was used for feminization. Our results showed that during testicular differentiation, the brain cyp19a1 transcripts and aromatase proteins were increased significantly; moreover, the cyp19a1, sf-1, foxl2s, and esrs (except gperb) transcripts in the midbrain/pituitary also were increased significantly. Forebrain gnrh1 transcripts increased slightly during gonadal differentiation of both sexes, but the gnrhr1b and gnrhr2 transcripts in the midbrain/pituitary were stable during gonadal differentiation. The expression levels of gths and gh in the midbrain/pituitary were significantly increased during testicular differentiation and were much higher in males than in E2-feminized females. These results implied that endogenous estrogens might play essential roles in the brain/pituitary during testicular differentiation, sf-1, foxl2s, and esrs may have roles in cyp19a1 regulation in the midbrain/pituitary of Japanese eels. For the GnRH-GTH axis, gths, especially fshb, may be regulated by esrs and involved in regulating testicular differentiation and development in Japanese eels.

Effects of dopamine and melatonin treatment on the expression of the genes associated with artificially induced sexual maturation in Japanese eel, Anguilla japonica.

Japanese eel (Anguilla japonica) is a commercially important fish species in Asia. Understanding factors like photoperiod, temperature, and lunar cycles is crucial for successful aquaculture and managing its reproduction. Melatonin and dopamine (DA) are essential for regulating reproduction in vertebrates, including fish. This study investigated the effects of melatonin and DA on the reproductive system of mature male Japanese eels to better understand reproductive regulation in fish. To clarify the effects of these hormones on sexual maturation in eels, a critical stage in the reproductive process, sexual maturation was induced by injecting human chorionic gonadotropin, which stimulates the production of sex hormones. To check the effect of melatonin and DA on sexual maturation, DA, melatonin, and DA + domperidone were intraperitoneally injected into fish from each group (six per treatment) at a dose of 1 mg/kg body weight. The fish were then examined using quantitative RT-PCR by comparing the messenger RNA level of reproduction-related genes (gonadotropin releasing hormone 1; gnrh1, gonadotropin releasing hormone 2; gnrh2, follicle stimulating hormone; fshß, luteinizing hormone; lhß and DA receptor 2b; d2b), involved in the gonadotropic axis in eels, to those that received a control injection. The results indicate significant differences in the expression levels of gnrh1, gnrh2 and d2b in the brain and d2b, fshß, lhß in the pituitary at different stages of sexual maturation. Melatonin appears to enhance the production of sex gonadotropins, whereas DA inhibits them. These findings suggest an interaction between melatonin and DA in regulating reproduction in Japanese eels.

Tissue-specific promoters regulate the transcription of cyp19a1 in the brain-pituitary-gonad axis of Japanese eel (Anguilla japonica).

Aromatase is a key enzyme that catalyzes the biosynthesis of estrogens. Previous study indicated that putative tissue-specific promoters of the one aromatase gene (cyp19a1) may drive the differential regulatory mechanisms of cyp19a1 expression in Anguilla japonica. In the present study, for elucidating the transcription characteristics and the function of putative tissue-specific promoters of cyp19a1 in the brain-pituitary-gonad (BPG) axis during vitellogenesis, we investigated the transcriptional regulation of cyp19a1 by 17ß-estrogen (E2), testosterone (T), or human chorionic gonadotropin (HCG) in A. japonica. The expression of estrogen receptor (esra), androgen receptor (ara), or luteinizing hormone receptor (lhr) was up-regulated as cyp19a1 in response to E2, T, or HCG, respectively in the telencephalon, diencephalon, and pituitary. The expression of cyp19a1 was also upregulated in the ovary by HCG or T in a dose-dependent manner. Unlike in the brain and pituitary, the expression of esra and lhr, rather than ara, was upregulated by T in the ovary. Subsequently, four primary subtypes of 5'-untranslated terminal regions of cyp19a1 transcripts and the corresponding two 5' flanking regions (promoter P.I and P.II) were identified. The P.II existed in all BPG axis tissues, whereas the P.I with strong transcriptional activity was brain- and pituitary-specific. Furthermore, the transcriptional activity of promoters, the core promoter region, and the three putative hormone receptor response elements were validated. The transcriptional activity did not change when the HEK291T cells co-transfected with P.II and ar vector were exposed to T. These results suggested that the expression of cyp19a1 was upregulated indirectly through esra and lhr rather than ara by T in the ovary, whereas the expression of cyp19a1 was upregulated directly through androgen receptor and the downstream androgen response element of tissue-specific P.I in the brain and pituitary. The results of the study reveal the regulatory mechanisms of estrogen biosynthesis and provide a reference for optimizing the technology of artificially induced maturation in eels.

The influence of dietary Coenzyme Q10 on growth performance, antioxidant capacity and resistance against Aeromonas hydrophila of juvenile European eel (Anguilla anguilla).

The present study was conducted to investigate the effects of dietary Coenzyme Q10 (CoQ10) on the growth performance, body composition, digestive enzyme activity, antioxidant capacity, intestinal histology, immune-antioxidant gene expression and disease resistance of juvenile European eel (Anguilla anguilla). Fish were fed a diet supplemented with CoQ10 at concentrations of 0, 40, 80 and 120 mg/kg for 56 days. The results indicated that dietary CoQ10 supplementation did not significantly affect final body weight (FBW), survival rate (SR), weight gain (WG), feed rate (FR), viscerosomatic index (VSI) or hepatosomatic index (HSI) among all experimental groups. However, the highest FBW, WG and SR were found in the 120 mg/kg CoQ10 group. Dietary 120 mg/kg CoQ10 markedly improved feed efficiency (FE) and the protein efficiency ratio (PER). The crude lipid in the body and triglycerides (TG) and total cholesterol (TC) in serum were obviously lower in the 120 mg/kg CoQ10 group than in the control group. For digestive enzymes, protease activity in the intestine was markedly boosted in the 120 mg/kg CoQ10 group. The serum activities of SOD, CAT and GST in the 120 mg/kg CoQ10 group were significantly higher than those in the control group. Dietary 120 mg/kg CoQ10 efficiently enhanced superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities in the liver, while the malondialdehyde (MDA) content was significantly decreased. No significant histological changes in the liver were identified in any group. Dietary supplementation with 120 mg/kg CoQ10 improved antioxidant capacity and immunity by upregulating the expression of cyp1a, sod, gst, lysC, igma1, igmb1 and irf3 in the liver. Furthermore, the cumulative survival rate of juvenile European eel against challenge with Aeromonas hydrophila was significantly elevated in the 80 and 120 mg/kg CoQ10 supplemented groups. Conclusively, our study suggested that supplementing the diet of juvenile European eel with CoQ10 at a concentration of 120 mg/kg could promote their feed utilization, fat reduction, antioxidant capacity, digestibility, immune-antioxidant gene expression and resistance to Aeromonas hydrophila without negative effects on fish health status.

Purification and characterization of the dipeptidyl peptidase-IV inhibitory peptides from eel (Anguilla rostrata) scraps enzymatic hydrolysate for the treatment of type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a serious threat to human health. Owing to the action of dipeptidyl peptidase-IV (DPP-IV), the half-life of entero-insulin hormone after secretion is extremely short, causing insufficient insulin secretion in diabetic patients. Dipeptidyl peptidase-IV inhibitors can be used as a new treatment for T2DM. In this study, the proteins of eel (Anguilla rostrata) scraps hydrolyzed using Protamex protease (EPHs) were found to have strong DPP-IV inhibitory activity. The study also provided research ideas for the development and utilization of A. rostrata scraps. RESULTS: The median inhibition concentration (IC50 ) value of EPHs was 5.455 ± 0.24 mg mL-1 . The peptide fractions with the highest DPP-IV inhibitory activity were sequentially separated by ultrafiltration, gel filtration chromatography (GFC), and reversed-phase high performance liquid chromatography (RP-HPLC) in a continuous hierarchical manner and analyzed using matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF/TOF MS/MS). Three peptides that revealed significant inhibitory activity were screened among the identified sequences, with sequences of Phe-Pro-Arg (IC50  = 62.14 ± 1.47 µM), Tyr-Pro-Pro-Ser-Phe-Ser (IC50  = 102.65 ± 4.57 µM), and Tyr-Pro-Tyr-Pro-Ala-Ser (IC50  = 68.30 ± 3.85 µM). Molecular docking simulations revealed that their inhibitory effect was mainly due to the formation of hydrogen bonds with amino acid residues in the active sites of DPP-IV. Analysis of the inhibition patterns of the synthetic peptides displayed that Phe-Pro-Arg and Tyr-Pro-Pro-Ser-Phe-Ser displayed competitive inhibition, whereas Tyr-Pro-Tyr-Pro-Ala-Ser showed mixed competitive/non-competitive inhibition. CONCLUSIONS: The protein hydrolysates isolated from eel scraps are potential functional food ingredients for the treatment of T2DM. © 2023 Society of Chemical Industry.

Oxygen consumption and acid secretion in isolated gas gland cells of the European eel Anguilla anguilla.

Swimbladder gas gland cells are known to produce lactic acid required for the acidification of swimbladder blood and decreasing the oxygen carrying capacity of swimbladder blood, i.e., the onset of the Root effect. Gas gland cells have also been shown to metabolize glucose via the pentose phosphate shunt, but the role of the pentose phosphate shunt for acid secretion has not yet been evaluated. Similarly, aerobic metabolism of gas gland cells has been largely neglected so far. In the present study, we therefore simultaneously assessed the role of glycolysis and of the pentose phosphate shunt for acid secretion and recorded oxygen consumption of isolated swimbladder gas gland cells of the European eel. Presence of glucose was essential for acid secretion, and at glucose concentrations of about 1.5 mmol l-1 acid secretion of gas gland cells reached a maximum, indicating that glucose concentrations in swimbladder blood should not be limiting acid production and secretion under physiological conditions. The data revealed that most of the acid was produced in the glycolytic pathway, but a significant fraction was also contributed by the pentose phosphate shunt. Addition of glucose to gas gland cells incubated in a glucose-free medium resulted in a reduction of oxygen uptake. Inhibition of mitochondrial respiration significantly reduced oxygen consumption, but a fraction of mitochondria-independent respiration remained in presence of rotenone and antimycin A. In the presence of glucose, application of either iodo-acetate inhibiting glycolysis or 6-AN inhibiting the pentose phosphate shunt did not significantly affect oxygen uptake, indicating an independent regulation of oxidative phosphorylation and of acid production. Inhibition of the muscarinic acetylcholine receptor caused a slight elevation in acid secretion, while forskolin caused a concentration-dependent reduction in acid secretion, indicating muscarinic and c-AMP-dependent control of acid secretion in gas gland cells.

Molecular insight, expression profile and subcellular localization of two STAT family members, STAT1a and STAT2, from Japanese eel, Anguilla japonica.

Signal transducer and activator of transcription 1 (STAT1) and STAT2 are critical components of type I and type II IFNs signaling. To date, seven STAT family proteins have been identified from mammals. However, the information on STAT genes in teleost fish is still limited. In the present study, two STAT family genes (STAT1a and STAT2) were identified from Japanese eel, Anguilla japonica and designated as AjSTAT1a and AjSTAT2. The open reading frames of AjSTAT1a and AjSTAT2 are 2244 bp and 2421 bp, encoding for polypeptides of 747 aa and 806 aa, respectively. Both AjSTAT1a and AjSTAT2 contain the conserved domains of STAT proteins. Phylogenetic analysis was performed based on the STATs protein sequences, and showed that AjSTAT1a and AjSTAT2 shared the closest relationship with Oncorhynchus mykiss. Quantitative real-time PCR analysis revealed that AjSTAT1a and AjSTAT2 were expressed in most examined tissues, with the highest expression both in blood. Significantly up-regulated transcripts of AjSTAT1a and AjSTAT2 were detected in response to poly I:C stimulation, and Edwardsiella tarda induced increase in the expression of AjSTAT1a and AjSTAT2 genes. Subcellular localization analysis showed that in both IFNγ-stimulated and unstimulated EPC cells AjSTAT1a and AjSTAT2 were mainly distributed in the cytoplasm, but few AjSTAT1a was distributed in the nucleus. All these results suggested that AjSTAT1a and AjSTAT2 may be critical for regulating the host innate immune defense against pathogens invasion.

Macrophage-stimulating activity of European eel (Anguilla anguilla) peptides in RAW264.7 cells mediated via NF-κB and MAPK signaling pathways.

European eel (Anguilla anguilla) is considered to be a vital commercial fish species. In this study, the effect and molecular mechanism of bioactive peptides from European eel on macrophage-stimulating activity in RAW264.7 cells were investigated. Eel peptide (EP) markedly induced NO and iNOS production and promoted TNF-α and IL-6 secretion in a concentration-dependent manner. Moreover, EP dose-dependently activated NF-κB and MAPK signaling pathways in RAW264.7 cells. In addition, EP was purified using a Sephadex A-25 column and a Bio-Gel P-6 column, and the fraction (Fr-1-1) showing the strongest NO-inducing activity was obtained. Then, the molecular weights of the components in Fr-1-1 were analyzed by LC-MS/MS and found to range from 700 to 1900 Da for the majority of components, which suggested that Fr-1-1 mainly consisted of peptides containing 8-20 amino acid residues. Overall, our results indicated that EP from Anguilla anguilla activated macrophages and could be used as a potential nutraceutical or pharmaceutical.

Seawater transfer down-regulates C-type natriuretic peptide-3 expression in prolactin-producing cells of Japanese eel: Negative correlation with plasma chloride concentration.

In euryhaline fishes, atrial and B-type natriuretic peptides are important hormones in hypo-osmoregulation, whereas osmoregulatory functions of C-type natriuretic peptides (CNPs) remain to be investigated. Although four CNP isoforms (CNP1-4) are mainly expressed in the brain, multiorgan expression of CNP3 was found in euryhaline Japanese eel, Anguilla japonica. Here we identified the CNP3-expressing cells and examined their response to osmotic stress in eel. CNP3 was expressed in several endocrine cells: prolactin-producing cells (pituitary), glucagon-producing cells (pancreas), and cardiomyocytes (heart). Pituitary CNP3 expression was the highest among organs and was decreased following seawater transfer, followed by a decrease in the freshwater-adaptating (hyper-osmoregulatory) hormone prolactin. We also showed the negative correlation between CNP3/prolactin expression in the pituitary and plasma Cl- concentration, but not for plasma Na+ concentration. These results suggest that CNP3 in the pituitary (and pancreas) plays a critical role in freshwater adaptation of euryhaline eel together with prolactin.

Genome-wide analysis of MicroRNA-messenger RNA interactome in ex-vivo gill filaments, Anguilla japonica.

BACKGROUND: Gills of euryhaline fishes possess great physiological and structural plasticity to adapt to large changes in external osmolality and to participate in ion uptake/excretion, which is essential for the re-establishment of fluid and electrolyte homeostasis. The osmoregulatory plasticity of gills provides an excellent model to study the role of microRNAs (miRs) in adaptive osmotic responses. The present study is to characterize an ex-vivo gill filament culture and using omics approach, to decipher the interaction between tonicity-responsive miRs and gene targets, in orchestrating the osmotic stress-induced responses. RESULTS: Ex-vivo gill filament culture was exposed to Leibovitz's L-15 medium (300 mOsmol l- 1) or the medium with an adjusted osmolality of 600 mOsmol l- 1 for 4, 8 and 24 h. Hypertonic responsive genes, including osmotic stress transcriptional factor, Na+/Cl--taurine transporter, Na+/H+ exchange regulatory cofactor, cystic fibrosis transmembrane regulator, inward rectifying K+ channel, Na+/K+-ATPase, and calcium-transporting ATPase were significantly upregulated, while the hypo-osmotic gene, V-type proton ATPase was downregulated. The data illustrated that the ex-vivo gill filament culture exhibited distinctive responses to hyperosmotic challenge. In the hyperosmotic treatment, four key factors (i.e. drosha RNase III endonuclease, exportin-5, dicer ribonuclease III and argonaute-2) involved in miR biogenesis were dysregulated (P < 0.05). Transcriptome and miR-sequencing of gill filament samples at 4 and 8 h were conducted and two downregulated miRs, miR-29b-3p and miR-200b-3p were identified. An inhibition of miR-29b-3p and miR-200b-3p in primary gill cell culture led to an upregulation of 100 and 93 gene transcripts, respectively. Commonly upregulated gene transcripts from the hyperosmotic experiments and miR-inhibition studies, were overlaid, in which two miR-29b-3p target-genes [Krueppel-like factor 4 (klf4), Homeobox protein Meis2] and one miR-200b-3p target-gene (slc17a5) were identified. Integrated miR-mRNA-omics analysis revealed the specific binding of miR-29b-3p on Klf4 and miR-200b-3p on slc17a5. The target-genes are known to regulate differentiation of gill ionocytes and cellular osmolality. CONCLUSIONS: In this study, we have characterized the hypo-osmoregulatory responses and unraveled the modulation of miR-biogenesis factors/the dysregulation of miRs, using ex-vivo gill filament culture. MicroRNA-messenger RNA interactome analysis of miR-29b-3p and miR-200b-3p revealed the gene targets are essential for osmotic stress responses.

Involvement of Estrogen and Its Receptors in Morphological Changes in the Eyes of the Japanese Eel, Anguilla japonica, in the Process of Artificially-Induced Maturation.

During the long migration from river habitats to the spawning ground, the Japanese eel undergoes sexual maturation. This spawning migration occurs concurrently with morphological changes, such as increases in eye size; however, the mechanisms by which sex steroids and their receptors influence these changes in peripheral tissues remain unclear. The aim of this study was to investigate changes in the eyes of female Japanese eels during sexual maturation, and our research focused on estrogen receptor (ER)α and ERß transcripts. During ovarian development, the gonadosomatic index increased and yolk-laden oocytes developed rapidly. These changes occurred in conjunction with a steady increase in plasma levels of estradiol-17ß (E2). Concomitant increases in transcript levels of ERα and ERß in eye, brain, pituitary, and ovary were also observed. Fluorescence in-situ hybridization analyses revealed that ERα and ERß transcripts were present in the choriocapillary layer and photoreceptor layer of the eyes, and the analysis also revealed that their signals in these layers became stronger in mature females compared to those observed in immature females, suggesting that under the influence of gonadotropins, morphological changes in the eyes are regulated by E2 through the activation of its receptors. In conclusion, E2 plays a crucial role in physiological adaptations that occur in peripheral tissues during the spawning migration.

Does silvering or 11-ketotestosterone affect osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis)?

Silvering has been associated with advancing osmoregulatory ability. Given the demonstrated role of 11-ketotestosterone (11KT) in mediating many of the silvering-related changes, we investigated the role of 11KT in driving this advanced osmoregulatory ability in the New Zealand short-finned eel (Anguilla australis). Yellow (non-migratory) eels with or without 11KT implants and blank-implanted silver (migratory) eels, either held in freshwater or subjected to seawater challenge, were sampled to determine serum [Na+] and [Cl-], pituitary prolactin mRNA levels, gill Na+/K+-ATPase activity and gill mRNA levels for Na+/K+-ATPase-α1 subunit and for Na+/K+/2Cl- co-transporter-1α-subunit. Developmental stage and 11KT treatment advanced the eels' osmoregulatory ability. Thus, serum [Na+] and [Cl-] were affected by developmental stage and 11KT treatment upon seawater challenge. However, seawater challenge, not 11KT treatment or developmental stage, produced the strongest and the most consistent effects on A. australis osmoregulatory processes, inducing significant effects in all the relevant parameters we measured. In light of our results and in view of the eel's marine ancestry, we contend that A. australis, or freshwater eels in general, are highly tolerant and able to adapt quickly to changing salinities even at the yellow stage, which may preclude a critical need for an advanced osmoregulatory ability at silvering.

Gonadal development and expression of sex-specific genes during sex differentiation in the Japanese eel.

The process of gonadal development and mechanism involved in sex differentiation in eels are still unclear. The objectives were to investigate the gonadal development and expression pattern of sex-related genes during sex differentiation in the Japanese eel, Anguilla japonica. For control group, the elvers of 8-10cm were reared for 8months; and for feminization, estradiol-17ß (E2) was orally administered to the elvers of 8-10cm for 6months. Only males were found in the control group, suggesting a possible role of environmental factors in eel sex determination. In contrast, all differentiated eels in E2-treated group were female. Gonad histology revealed that control male eels seem to differentiate through an intersexual stage, while female eels (E2-treated) would differentiate directly from an undifferentiated gonad. Tissue distribution and sex-related genes expression during gonadal development were analyzed by qPCR. The vasa, figla and sox3 transcripts in gonads were significantly increased during sex differentiation. High vasa expression occurred in males; figla and sox3 were related to ovarian differentiation. The transcripts of dmrt1 and sox9a were significantly increased in males during testicular differentiation and development. The cyp19a1 transcripts were significantly increased in differentiating and differentiated gonads, but did not show a differential expression between the control and E2-treated eels. This suggests that cyp19a1 is involved both in testicular differentiation and development in control males, and in the early stage of ovarian differentiation in E2-treated eels. Importantly, these results also reveal that cyp19a1 is not a direct target for E2 during gonad differentiation in the eel.

Biotransformation, antioxidant and histopathological biomarker responses to contaminants in European and American yellow eels from the Gironde and St. Lawrence estuaries.

Since the early 1980s, populations of American (Anguilla rostrata) and European eels (Anguilla anguilla) have suffered a sharp decline. The causes of their decline are likely multifactorial and include chemical pollution. A field study was conducted in eight sites varying in organic and metal contamination along the St. Lawrence (Eastern Canada) and Gironde (France) systems to investigate the relationships among contaminants, biological characteristics and biotransformation, antioxidant and histopathological biomarkers in eels from both species. For A. rostrata, no major influences of persistent organic contaminants on biomarkers were identified. For A. anguilla, eels from the most contaminated site expressed higher surface of MelanoMacrophage Centers (MMCs) and eels from another contaminated site expressed higher amount of spleen lipofuscin pigment. These two histopathological biomarkers were also associated with aging. Compared to eels from the cleanest French site, higher hepatic catalase activity and density of MMC in eels from contaminated sites was related to higher concentration of organic (DDT and metabolites, sum of PCBs, sum of PBDEs) and inorganic (Hg and Cd) contaminants. In both species, a higher deposition of spleen hemosiderin pigment was measured in eels from the most brackish sites compared to eels living in freshwater environments. Our results suggest an association between higher hemosiderin pigment and metal contamination (As for A. anguilla and Pb for A. rostrata). Parasitism by A. crassus was observed in European eels from freshwater sites but not in eels from brackish habitats. Overall, contamination may pose a greater risk for the health of European compared to American eels.

Na+/K+-ATPase response to salinity change and its correlation with FXYD11 expression in Anguilla marmorata.

The Na+/K+-ATPase (NKA) is a primary electrogenic protein that promotes ion transport in teleosts. FXYD11 is a putative regulatory subunit of the NKA pump. The regulation of Na +/K + -ATPase and FXYD11 is of critical importance for osmotic homeostasis. To investigate the changes of the two genes under different salinity environments, we first identified NKA (AmNKAα1) and FXYD11 (AmFXYD11) in Anguilla marmorata, and then evaluated the mRNA levels of NKA and FXYD11 as well as the activity of NKA in the gill and kidney at different timepoints (0, 1, 3, 6, 12, 24, 48, 72, 96, and 360 h) under three salinity conditions-0‰ (fresh water: FW), 10‰ (brackish water: BW), and 25‰ (seawater: SW). In the gill, the mRNA levels of AmNKAα1 and AmFXYD11 and the enzyme activity of AmNKAα1 were higher in BW and SW than in FW; the protein abundance was positively correlated with the specific activity of NKA in BW/SW. However, in the kidney, the mRNA level of AmNKAα1 in the BW group was higher than that in the FW group. In addition, AmFXYD mRNA levels in both BW and SW groups were significantly lower than that in the FW control group. These results suggested that AmFXYD11 was tissue specific in response to different salinity environment. Our results clearly demonstrated the important roles of AmNKAα1 and AmFXYD11 in osmotic homeostasis of juvenile A. marmorata under saline environment.

Radionuclides (210Po and 210Pb) and Some Heavy Metals in Fish and Sediments in Lake Bafa, Turkey, and the Contribution of 210Po to the Radiation Dose.

The pollution level of Lake Bafa was investigated by collecting fish samples {Dicentrarchus labrax (sea bass), Liza ramada (mullet) and Anguilla anguilla (eel)}, surface sediment, and core samples. In all these samples, 210Po and 210Pb concentrations were estimated, and total annual dose rates were obtained for each species. Some heavy metal (Cr, Ni, Pb, Cd, Mn, Fe, and Zn) concentration levels were obtained for the fish and a core sample. The sediment mass accumulation rate was found to be 3.27 g·m-2·day-1 (0.119 g·cm-2·y-1) from a core sample. The heavy metal concentrations in the vertical profile of samples from the core were also observed. The measured concentration of Zn, Pb, Cd, and Cr were between the ERL (effects range low) and ERM (effects range median) limits, while Ni concentrations were higher than the ERM limit. The observed concentrations of Cd, Pb, and Zn in fish samples did not exceed the limits in accordance with Turkish Food Regulations. Further, the maximum effective dose equivalent of 210Po in the area was found to be 1.169 µSv·y-1.

Production and characterization of monoclonal antibodies against recombinant tethered follicle-stimulating hormone from Japanese eel Anguilla japonica.

We prepared monoclonal antibodies (mAbs) against a recombinant tethered follicle-stimulating hormone (rec-FSH) from Japanese eel Anguilla japonica that was produced in Escherichia coli. Positive hybridomas (clones eFA-C5, eFA-C10, eFA-C11, eFA-C12, eFA-C13, and eFB-C14) were selected by using the eel FSH antigen in ELISA, and anti-eel FSH mAbs were purified from culture supernatants by performing affinity chromatography. Three of the 6mAbs were characterized and their isotypes were identified as IgG2b (eFA-C5 and eFA-C11) and IgG1 (eFB-C14). In western blotting assays, the mAbs recognized the antigen as a 24.3-kDa band, and further detected bands of 34 and 32kDa in the supernatants of CHO cells transfected with cDNA encoding tethered eel FSHß/α and LHß/α, respectively. PNase F-mediated deglycosylation of the recombinant proteins resulted in a drastic reduction in their molecular weight, to 7-9kDa. The mAbs eFA-C5 and eFA-C11 recognized the eel FSHα-subunit that is commonly encoded among glycoprotein hormones, whereas eFB-C14 recognized the eel FSHß-subunit, and immunohistochemical analysis revealed that the staining by these mAbs was specifically localized in the eel pituitary. We also established an ELISA system for detecting rec-tethered FSHß/α and LHß/α produced from CHO cell lines. Measurement of biological activities in vitro revealed that only weak activity of rec-FSHß/α was detected. The activity of rec-LHß/α was found to be increased in a dose-dependent manner for eel oocyte maturation.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

Phagocytic cell responses to silica-coated dithiocarbamate-functionalized iron oxide nanoparticles and mercury co-exposures in Anguilla anguilla L.

Immune system responses in fish are considered as suitable and sensitive biomarkers for monitoring aquatic pollution. However, a clear knowledge gap persists in the literture on the immunotoxic potential of engineered nanoparticles toward aquatic organisms such as fish. Employing major enzymatic- (glutathione reductase, GR; glutathione peroxidase, GPX; glutathione sulfo-transferase, GST; catalase, CAT) and thiol- (non-protein thiols, NP-SH; total glutathione, TGSH)-based defense biomarkers, this study assessed the response of phagocytes isolated from peritoneum (P-phagocytes), gill (G-phagocytes), head kidney (HK-phagocytes), and spleen (S-phagocytes) of European eel (Anguilla anguilla L.) to silica-coated magnetite particles (Fe3O4@SiO2/SiDTC, hereafter called IONP; size range: 82 ± 21 to 100 ± 30 nm; 2.5 mg L(-1)) alone and IONP and mercury (Hg; 50 µg L(-1)) concomitant exposures. Responses of previous biomarkers were studied in P-phagocytes, G-phagocytes, HK-phagocytes, and S-phagocytes collected during 0, 2, 4, 8, 16, 24, 48, and 72 h of exposures. Contingent to hour of exposure to IONP, Hg, and IONP + Hg GST, GPX, CAT, NP-SH, and TGSH exhibited their differential responses in all the phagocytic cells considered. In particular, under IONP exposure, the potential occurrence of the GSH-independent antioxidant defense was indicated by the observed herein inhibition in the enzymatic- and thiol-based defense in A. anguilla phagocytes. In contrast, the response of P-, G-, HK-, and S-phagocytes to the increasing Hg exposure period reflected an increased detoxification activity. Notably, the occurrence of an antagonism between IONP and Hg was depicted during late hours (72 h) under IONP + Hg concomitant exposure, where elevations in the defense biomarkers were depicted. Overall, the P-, G-, HK-, and S-phagocytic cells exhibited a differential induction in the studied enzymes and thiols to counteract impacts of IONP, Hg, and IONP + Hg concomitant exposures. Future studies on the fish immunotoxicity responses to IONP exposure in multi-pollution conditions can be benefited with the major outcomes of the present study.

Anguilla japonica lectin 1 delivery through adenovirus vector induces apoptotic cancer cell death through interaction with PRMT5.

BACKGROUND: Our previous studies have demonstrated that, through adenovirus mediated gene delivery, various exogenously expressed lectins elicited cytotoxicity to cancer cells, utilizing protein arginine methyltransferase 5 (PRMT5) as a common binding target. METHODS: In the present study, a FLAG tagged Anguilla japonica lectin 1 (AJL1) expression cassette was genetically harbored in a replication-defective adenovirus genome, forming Ad.FLAG-AJL1. The exogenous AJL1-induced cytotoxicity and the underlying mechanisms were analyzed. RESULTS: The exogenous AJL1 suppressed the proliferation of a variety of human cancer cells by inducing apoptosis. Caspase 9, Bcl-2, X-linked inhibitor of apoptosis protein, mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase were found to be responsible for the exogenous AJL1-induced cytotoxicity. AJL1 was further suggested to regulate PRMT5-E2F-1 pathway, a pathway shared by previously reported marine lectins Dicentrarchus labrax fucose binding lectin and Strongylocentrotus purpuratus rhamnose binding lectin. A localization study revealed that exogenous AJL1 widely distributed in the cell membrane and cytoplasm. CONCLUSIONS: The results of the present study suggest that the PRMT5-E2F-1 pathway may act as a common target for exogenous lectins including AJL1, and the cellular response to exogenous AJL1 may suggest a novel agent for cancer gene therapy. Copyright © 2016 John Wiley & Sons, Ltd.