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.

Soy Isoflavones Induce Feminization of Japanese Eel (Anguilla japonica).

Under aquaculture conditions, Japanese eels (Anguilla japonica) produce a high percentage of males. However, females gain higher body weight and have better commercial value than males, and, therefore, a high female ratio is required in eel aquaculture. In this study, we examined the effects of isoflavones, genistein, and daidzein on sex differentiation and sex-specific genes of eels. To investigate the effects of these phytoestrogens on the gonadal sex, we explored the feminizing effects of soy isoflavones, genistein, and daidzein in a dose-dependent manner. The results showed that genistein induced feminization more efficiently than daidzein. To identify the molecular mechanisms of sex-specific genes, we performed a comprehensive expression analysis by quantitative real-time PCR and RNA sequencing. Phenotypic males and females were produced by feeding elvers a normal diet or an estradiol-17ß- or genistein-treated diet for 45 days. The results showed that female-specific genes were up-regulated and male-specific genes were down-regulated in the gonads, suggesting that genistein induces feminization by altering the molecular pathways responsible for eel sex differentiation.

Grape seed proanthocyanidin extract regulates lipid metabolism of the American eel (Anguilla rostrata).

The current study was aimed to examine the effect of grape seed proanthocyanidin extract (GSPE) on regulating lipid metabolism of American eels. A total of six cement tanks of fish were randomly divided into a control group fed with a commercial diet and a GSPE group fed with a commercial diet supplemented 400 mg/kg GSPE. There were three replicates in each group. Results suggested that GSPE could decrease the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and increase the high-density lipoprotein cholesterol level in serum. GSPE might regulate lipid metabolism through upregulating linoleic acid metabolism and arachidonic acid metabolism along with downregulating metabolisms of phenylalanine, tyrosine, and tryptophan biosynthesis and valine, leucine, and isoleucine biosynthesis.

Bone resorption and body reorganization during maturation induce maternal transfer of toxic metals in anguillid eels.

During their once-in-a-lifetime transoceanic spawning migration, anguillid eels do not feed, instead rely on energy stores to fuel the demands of locomotion and reproduction while they reorganize their bodies by depleting body reserves and building up gonadal tissue. Here we show how the European eel (Anguilla anguilla) breaks down its skeleton to redistribute phosphorus and calcium from hard to soft tissues during its sexual development. Using multiple analytical and imaging techniques, we characterize the spatial and temporal degradation of the skeletal framework from initial to final gonadal maturation and use elemental mass ratios in bone, muscle, liver, and gonadal tissue to determine the fluxes and fates of selected minerals and metals in the eels' bodies. We find that bone loss is more pronounced in females than in males and eventually may reach a point at which the mechanical stability of the skeleton is challenged. P and Ca are released and translocated from skeletal tissues to muscle and gonads, leaving both elements in constant proportion in remaining bone structures. The depletion of internal stores from hard and soft tissues during maturation-induced body reorganization is accompanied by the recirculation, translocation, and maternal transfer of potentially toxic metals from bone and muscle to the ovaries in gravid females, which may have direct deleterious effects on health and hinder the reproductive success of individuals of this critically endangered species.

Effect of biological additives on Japanese eel (Anguilla japonica) growth performance, digestive enzymes activity and immunology.

Japanese eel (Anguilla japonica) has become a commercially important fish species all over the world. High-density aquaculture has led to congestion and contributed to bacterial infection outbreaks that have caused high mortality. Therefore a 56-days feeding trial was conducted to determine the effects of dietary Bacillus amyloliquefaciens (GB-9) and Yarrowia lipolytica lipase2 (YLL2) on growth performance, digestive enzymes activity, innate immunity and resistance to pathogens of A. japonica. Fish growth performance was significantly affected by dietary YLL2 supplementation but not by GB-9. Fish fed diets with YLL2 at 2.0 g/kg diet in combination of high and low levels of GB-9 (5.0 g/kg and 2.0 g/kg) produced the highest growth. For digestive enzyme, lipase and trypsin activities was promoted by dietary containing YLL2, while amylase activities was increased by dietary containing YLL2, GB-9 single or combination. For innate immunity, the mucus lysozyme activity, leukocytes phagocytosis activity and reactive oxygen species level of skin, peroxidase and lysozyme activity of serum were enhanced in fish fed with GB-9 compared to those in control group (p < 0.05). The highest resistance to Vibrio anguillarum and Aeromonas hydrophila was determined in fish fed with 5.0 g kg-1 GB-9 + 2.0 g/kg YLL2. This study demonstrated that GB-9 and YLL2 enhanced non-specific immune defense system of A. japonica, providing them with higher resistance to pathogens. The present results suggested that the combination of these supplements could be considered as potential biological additives for aquaculture farmed fish.

Characterization and expression of cDNAs encoding P450c17-II (cyp17a2) in Japanese eel during induced ovarian development.

Estradiol-17ß (E2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17α,20ß-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17α-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cyp17a2-which encodes P450c17-II-was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I-V) encoding P450c17-II were identified that had minor sequence variations. HEK293T cells transfected with all but type II P450c17-II converted exogenous progesterone to 17α-hydroxyprogesterone (17α-P), providing evidence for 17α-hydroxylase activity; however, a failure to convert 17α-P to androstenedione indicated that C17-20 lyase activity was absent. Cyp17a2 mRNA was expressed mainly in the head kidney, ovary, and testis, and quantitative PCR analysis demonstrated that expression in the ovary increased during induced vitellogenesis and oocyte maturation/ovulation. In contrast, P450c17-I showed both 17α-hydroxylase and C17-20 lyase activities, and cyp17a1 expression increased until the mid-vitellogenic stage and remained high thereafter. Considering the high level of cyp17a2 transcript in the eel ovary at the migratory nucleus stage together with our previous report demonstrating that eel ovaries have strong 17α-P-to-DHP conversion activity, the failure of artificially maturing eels to produce the maturation-inducing DHP may be explained by a deficiency in 17α-P production due to the persistence of cyp17a1 expression after the completion of vitellogenesis.

Subcellular partitioning of non-essential trace metals (Ag, As, Cd, Ni, Pb, and Tl) in livers of American (Anguilla rostrata) and European (Anguilla anguilla) yellow eels.

We determined the intracellular compartmentalization of the trace metals Ag, As, Cd, Ni, Pb, and Tl in the livers of yellow eels collected from the Saint Lawrence River system in Canada (Anguilla rostrata) and in the area of the Gironde estuary in France (Anguilla anguilla). Differential centrifugation, NaOH digestion and thermal shock were used to separate eel livers into putative "sensitive" fractions (heat-denatured proteins, mitochondria and microsomes+lysosomes) and detoxified metal fractions (heat-stable peptides/proteins and granules). The cytosolic heat-stable fraction (HSP) was consistently involved in the detoxification of all trace metals. In addition, granule-like structures played a complementary role in the detoxification of Ni, Pb, and Tl in both eel species. However, these detoxification mechanisms were not completely effective because increasing trace metal concentrations in whole livers were accompanied by significant increases in the concentrations of most trace metals in "sensitive" subcellular fractions, that is, mitochondria, heat-denatured cytosolic proteins and microsomes+lysosomes. Among these "sensitive" fractions, mitochondria were the major binding sites for As, Cd, Pb, and Tl. This accumulation of non-essential metals in "sensitive" fractions likely represents a health risk for eels inhabiting the Saint Lawrence and Gironde environments.

Structural and functional characterization of neuropeptide Y in a primitive teleost, the Japanese eel (Anguilla japonica).

In the present study, the first full-length cDNA encoding Neuropeptide Y (NPY) was cloned from the brain of Japanese eel (Anguilla japonica). The open reading frame of Japanese eel NPY gene is 294 bp in length, encoding a precursor protein of 97 amino acids, which contains a 36-amino-acid mature peptide. Sequence analysis showed that the Japanese eel NPY peptide is similar to that of other species. Real-time PCR revealed that NPY in Japanese eel is mainly expressed in the brain, especially in the hypothalamus and the optic tectum thalamus. The effect of a negative energy balance on NPY gene expression was examined subsequently. The mRNA level of NPY in the hypothalamus and the optic tectum thalamus showed a pronounced increase after 4 days of food deprivation. The biological activities of Japanese eel NPY were further investigated in vivo and in vitro. Intraperitoneal injection of the NPY peptide into Japanese eel could potently elevate the expression of the mammalian gonadotropin-releasing hormone (mGnRH) in hypothalamus and the follicle-stimulating hormone beta (FSHß), the luteinizing hormone beta (LHß) and growth hormone (GH) in pituitary. In static incubation studies, the stimulatory effects of NPY on mGnRH expression in hypothalamic fragments and on FSHß, LHß and GH expression in pituitary cells were also observed. However, in vivo and in vitro studies showed that NPY exhibits an inhibitory action on the expression of thyroid-stimulating hormone beta (TSHß) in pituitary. The results indicate that NPY is involved in the regulation of multiple physiological processes in Japanese eel.

Effects of crude oil and oil fractions on the liver P450-dependent monooxygenase activities and antioxidant defence system of different freshwater fish species.

The effects of crude oil (Szeged-Algyo, Hungary) and oil fractions (F1: rich in aromatics; F2 fraction: free from aromatics) were investigated on liver CYP1A isoenzymes and antioxidant defence system following their i.p. injection into different freshwater fish species: carp (Cyprinus carpio L.), silver carp (Hyphothalmichtys molitrix V.), and European eel (Anquilla anquilla). A dose of 2 mL kg -1 crude oil enhanced EROD activity 8-fold in carp and only 5-fold in eel after 3 days. Oil fraction F1 caused only a 2-fold induction in EROD activity only in carp, while F2 fraction caused significant inhibition in all three investigated fish species. The antioxidant parameters [lipid peroxidation (LP), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH)] were measured following the treatment. A decrease of 50% in CAT activity was observed after oil treatment. The GSH level enhanced, resulting the protective effects against LP. The same dose of crude oil but a longer duration time resulted in lower CYP1A induction in carp and antioxidant parameters had returned close to control. In all treatments the EROD isoenzymes proved to be more sensitive and the effects of oil treatment showed species to be different. Carp proved to be more sensitive than eel or silver carp.

A quantitative real-time RT-PCR assay for European eel tyrosine hydroxylase.

Dopamine (DA) plays a key inhibitory role in pubertal development of the European eel, but how DAergic neuronal activity is regulated is not known in this species. In order to investigate the regulation of DA inhibition at the molecular level, we developed a quantitative real-time RT-PCR (qrtRT-PCR) assay, using the Light Cycler system, for the expression of eel tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. Two different reference genes were compared: the previously cloned eel cytochrome b, and eel acidic ribosomal phosphoprotein P0, the latter of which we cloned and partly sequenced. To further validate the assay, different methods of total RNA extraction were tested and compared. When applied to cDNA extracted from dissected brains of juvenile eels, the expression of TH was highest in the olfactory bulb, followed by the telencephalon including preoptic area, and the di-/mesencephalic areas excluding the optic lobes. TH expression in the optic lobes and in the medulla oblongata was low, whereas no expression could be detected in corpus cerebellum. This distribution pattern is in agreement with earlier studies on TH in the eel using immunohistochemistry, RT-PCR, and Northern blotting. The developed qrtRT-PCR assay provides a new tool for understanding the mechanisms regulating central DA inhibition of puberty in juvenile eels.

Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel (Anguilla anguilla).

To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the European eel gonad (EeCYP19) contains an open reading frame of 1539 bp, encoding a deduced protein of 513 residues. The predicted amino acid sequence shows 97% identity with that of the Japanese eel, and 59-69% of identity with those of the CYP19 gonadal and brain forms of other teleost fish. Two potential initiation sites (ATG) were found downstream of the first ATG codon. A fluorescent-based method of real-time PCR was developed to quantify EeCYP19 expression. The expression levels of EeCYP19 in the gonads of adult males were approximately 12- and 30-fold lower than the levels in adult females and juvenile eels previously treated with E2, respectively. Expression of aromatase was found only in a single specimen in the control group. In contrast, no difference was found among sexes in the aromatase expression in the brain. Treatment with aromatase inhibitor (AI) of juvenile eel resulted in the total loss of aromatase expression in the gonads and brains. The results of this work revealed that AI treatment not only reduces the synthesis of estradiol, but reduces the expression levels of EeCYP19 as well. No evidence for the presence of a distinct extra-gonadal (brain) form of aromatase in the European eel could be provided.

Molecular cloning and characterization of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase cDNAs from Japanese eel ovary.

3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) is a crucial steroidogenic enzyme which catalyzes an essential step in the biosynthesis of all classes of steroid hormones. Two closely related cDNAs, encoding Japanese eel ovarian types I and II 3beta-HSD, were cloned and characterized. Both cDNAs putatively encoded 375 amino acid residues sharing high sequence homology with those of rainbow trout (71%) and mammalian (approximately 45-50%) 3beta-HSD. Transient expression of types I and II 3beta-HSD in COS-7 cells revealed that both proteins possess 3beta-hydroxysteroid dehydrogenase as well as Delta(5)-Delta(4) isomerase activity for both pregnenolone and dehydroepiandrosterone, with the preference of pregnenolone over dehydroepiandrosterone as substrate, although the type I protein is more active than the type II. By northern blot analysis, a single band of the 3beta-HSD transcript of approximately 1.5kb in length was observed in ovarian tissue and the total transcript abundance of both 3beta-HSDs remained constant throughout ovarian development artificially induced by gonadotropin-rich salmon pituitary homogenate. This lack of change in 3beta-HSD transcript abundance during ovarian development did not correlate with the fluctuation of its enzymatic activity reported previously, which may suggest that changes in 3beta-HSD activity during ovarian development may be, in part, post-transcriptionally regulated in the Japanese eel ovary.

Characterization of a cDNA encoding P-450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development.

A cDNA encoding P450 aromatase (CYP19) was isolated from a Japanese eel (Anguilla japonica) ovarian cDNA library. This cDNA contains a complete open reading frame encoding 511 amino acid residues. The deduced amino acid sequence is 59% and 65% identical to the catfish and rainbow trout forms, respectively, and 52-54% to mammalian and chicken forms. Non-steroidogenic COS-7 cells transfected with the eel CYP19 cDNA converted exogenous androstenedione to estrone, thus verifying its identity. Northern blot analysis indicated that there was a single 2.1 kb transcript in the ovary. A 2.1 kb transcript was also found in the brain but not in the spleen, head kidney, kidney, or liver. Throughout ovarian development induced by weekly injections of salmon pituitary homogenate (SPH, 20 microg/g body weight), the 2.1 kb transcript was barely or not detectable in the ovaries. However, signals greatly increased in intensity in oocytes in the migratory nucleus stage and then decreased slightly in the post-ovulatory ovary. These changes in transcript levels are consistent with the changes in aromatase activity of ovarian follicles, suggesting that aromatase activity in ovarian follicles is mainly regulated at the transcriptional level. In addition, fadrozole was found to significantly inhibit aromatase activity in a heterologous expression system using COS-7 cells, which indicates that fadrozole treatment could be useful to control E(2) production during artificial maturation of eels.

Two isoforms of the Na+/K+/2Cl- cotransporter are expressed in the European eel (Anguilla anguilla).

Two cDNA isoforms of the NKCC1 secretory cotransporter have been isolated from the European eel. The NKCC1a isoform exhibited mRNA expression in a wide range of tissues in a similar fashion to mammals, whereas NKCC1b was expressed primarily in the brain. The effect of freshwater (FW) to seawater (SW) transfer on NKCC1a expression was dependent on the developmental stage. In non-migratory yellow eels, NKCC1a mRNA expression in the gill was transiently up-regulated 4.3-fold after 2 days but also subsequently by 2.5-6-fold 3 weeks after SW transfer. Gill NKCC1a expression was localised mainly in branchial chloride cells of SW acclimated yellow eels. In contrast to yellow eels, NKCC1a mRNA abundance was not significantly different following SW acclimation in silver eel gill. NKCC1a mRNA abundance decreased in the kidney following SW acclimation and this may correlate with lower tubular ion/fluid secretion and urine flow rates in SW teleosts. Kidney NKCC1a mRNA expression in silver eels was also significantly lower than in yellow eels, suggesting some pre-acclimation of mRNA levels. NKCC1a mRNA was expressed at similar low levels in the middle intestine of FW- and SW-acclimated yellow or silver eels, suggesting the presence of an ion secretory mechanism in this gut segment.

Chemical behaviour of three radionuclides (cesium, americium and technetium) and their uptake at the cytosolic level in aquatic organisms.

In previous papers, we observed that there were great differences in the subcellular accumulation and elimination of 137Cs, 99Tc and 241Am in different marine animals. In this study, we analysed and compared the behaviour of these radionuclides at the cytosolic level. We found great similarities in the cytosolic distribution of a given radionuclide in the various organisms studied. The chemical characteristics of the radionuclides seem to play a major role in the biochemical affinity and metabolic behaviour in different aquatic organisms.

Capacity of the European eel (Anguilla anguilla) to elongate and desaturate dietary linoleic acid.

The ability of eels (Anguilla anguilla) to further desaturate and chain elongate linoleic acid, 18:2n-6, was studied by feeding diets containing either corn oil or a fish oil to groups of elvers for 12 wk and analyzing proportions of fatty acids in tissue lipids. Over the 12-wk period elvers given both dietary treatments increased in weight by fourfold. The proportion of arachidonic acid, 20:4n-6, present in polar lipids of elvers fed the diet containing corn oil increased from an initial value of 5% by weight to 12% by weight; the corresponding value for elvers given a diet containing fish oil, with little linoleic acid in it, was less than 4% by weight. The capacity of this fish to modify dietary linoleic acid metabolically was confirmed by examining the metabolic fate of radioactive carbon after giving [1-14C]linoleic acid orally to eels. Seven days after administration of the linoleic acid approximately 10% of the radioactivity recovered in liver fatty acids was present in trienes and tetraenes with about 4% occurring in arachidonic acid. Compositional analyses from the feeding experiment also indicated that dietary docosaenoic acid, 22:1n-11, is preferentially oxidized by eels.

Further observations on the distribution and properties of teleost melanin concentrating hormone.

The distribution of melanocyte concentrating hormone (MCH) bioactivity was mapped in the trout brain from cryostat sections cut in several planes. Most of the bioactivity occurred in the ventral third of the hypothalamus, with about 30% of the activity in the dorsal hypothalamus. The bioactivity was rapidly lost if the hypothalami were extracted in dilute acid, with a final extraction pH of 5.2. This loss, which can be avoided if the extract is heated, is presumed to be the result of hypothalamic enzyme activity. Preliminary chemical characterisation indicates that the molecule is a small basic peptide, of less than 2000 daltons (Da) and with an isoelectric point greater than 9.5. MCH bioactivity was also found in the hypothalamus but not the pituitary of Lampetra, Rana, Xenopus, and the rat. The activity from Xenopus and Lampetra had a similar Rf value to MCH from trout during polyacrylamide gel electrophoresis. Partially purified MCH of trout origin, free from MSH bioactivity, induced melanin concentration in eel melanophores but Xenopus melanophores failed to respond.

Response of prolactin cells to environmental calcium in the eel (Anguilla anguilla L.).

Prolactin (PRL) cell activity was investigated in eels kept in fresh water (FW), deionized water (DW) supplemented or not with Ca (2 mM), in Ca-enriched FW (10 mM), in normal (Ca 3.4 mM) or Ca-free 1/3 sea water (SW), and in SW (Ca 10.2 mM) or Ca-free SW (Ca 0.15 mM). Light-microscopic studies, including measurement of the nuclear area and cell height, showed that PRL cell activity, reduced in DW, is not affected by Ca supplementation. Activity is reduced in Ca-enriched FW, in 1/3 SW and in SW, conditions inducing an increase in the plasma sodium level. The lack of calcium in saline environments partly suppresses the nuclear atrophy occurring in SW. There is no significant correlation between external or total plasma calcium concentration and PRL cell activity. In artificial Ca-free SW, eels show a rapid increase in plasma osmolarity and sodium levels; there is a significant negative correlation between these two plasma values and the nuclear area or cell height of PRL cells. As in some other teleosts, plasma osmolarity and plasma sodium seem to play a more important role than external or internal calcium in controlling PRL secretion. This correlation is not apparent in eels kept in SW, having unstimulated PRL cells but active calcium-sensitive (Ca-s) cells in the pars intermedia.

Seasonal variations in circadian rhythm of hypothalamic catecholamine content in the eel (Anguilla anguilla L.).

1. Changes in catecholamine content (CA) in hypothalamus of male and female eel (Anguilla anguilla L.) were defined using the Falck-Hillarp histochemical fluorescence method for a 24-hr period during the summer (L:D = 16:8) and winter (L:D = 8:16). 2. A circadian rhythm in the catecholamine content was found in the hypothalamus. CA content was lower by night than by day. 3. A longer light day (summer period) caused the shifting of rhythm phase in CA content. 4. Sex of eels does not affect CA content in the hypothalamus.