Physiological and molecular responses to thermal stress in red cusk-eel (Genypterus chilensis) juveniles reveals atrophy and oxidative damage in skeletal muscle.

The red cusk-eel (Genypterus chilensis) is a native species with strong potential to support Chilean aquaculture diversification. Environmental stressors, such as temperature, may generate important effects in fish physiology with negative impact. However, no information exists on the effects of thermal stress in Genypterus species or how this stressor affects the skeletal muscle. The present study evaluated for the first time the effect of high temperature stress in red cusk-eel juveniles to determine changes in plasmatic markers of stress (cortisol, glucose and lactate dehydrogenase (LDH)), the transcriptional effect in skeletal muscle genes related to (i) heat shock protein response (hsp60 and hsp70), (ii) muscle atrophy and growth (foxo1, foxo3, fbxo32, murf-1, myod1 and ddit4), and (iii) oxidative stress (cat, sod1 and gpx1), and evaluate the DNA damage (AP sites) and peroxidative damage (lipid peroxidation (HNE proteins)) in this tissue. Thermal stress generates a significant increase in plasmatic levels of cortisol, glucose and LDH activity and induced heat shock protein transcripts in muscle. We also observed an upregulation of atrophy-related genes (foxo1, foxo3 and fbxo32) and a significant modulation of growth-related genes (myod1 and ddit4). Thermal stress induced oxidative stress in skeletal muscle, as represented by the upregulation of antioxidant genes (cat and sod1) and a significant increase in DNA damage and lipid peroxidation. The present study provides the first physiological and molecular information of the effects of thermal stress on skeletal muscle in a Genypterus species, which should be considered in a climate change scenario.

Transcriptome analysis of blood for the discovery of sex-related genes in ricefield eel Monopterus albus.

The blood acts as a transfer channel for a variety of factors in the whole body. The ricefield eel (Monopterus albus) is a protogynous hermaphrodite vertebrate. Until now, no research has reported an analysis of the blood transcriptome during the process of sexual development in the ricefield eel. In this study, the transcriptome sequencing of blood samples from male and female ricefield eels was completed with a total of 34.70 Gb clean data. The clean data of each sample all reached 5.23 GB, and the percent of the Q30 basic group was 88.62% and above. A total of 106,369 unigenes were obtained after assembly, including 13,296 unigenes with a length of more than 1 kb. Further functional annotation analysis showed that there are 28,522 unigenes that can be annotated. The annotations of genes with differential expression revealed that there were 563 genes with significant differential expression in the blood of male and female ricefield eels, including 91 upregulated genes and 472 downregulated genes. Among which, 14 genes may be closely related to sex differentiation, the qPCR was used to confirmed the expression pattern of those genes and result shown that 11 genes were downregulated and 3 genes were upregulated, consistent with the results of our RNA-Seq analysis. This blood transcript dataset will open future research avenues on ricefield eel sex development and differentiation.

Doping of transition metal dichalcogenides in molecularly imprinted conductive polymers for the ultrasensitive determination of 17ß-estradiol in eel serum.

Molecularly imprinted polymers (MIPs) have been developed to replace antibodies for the recognition of target molecules (such as antigens), and have been integrated into electrochemical sensing approaches by polymerization onto an electrode. Electrochemical sensing is inexpensive and flexible, and has demonstrated utility in point-of-care devices. In this work, several 2D (conductive) materials were employed to improve the performance of MIP sensors. Screen-printed electrodes were coated by the electropolymerization of aniline and metanilic acid, commingled with target molecules and various 2D materials. Tungsten disulfide (WS2) with an average particle size of 2 µm was found to increase the sensitivity of detection of molecularly imprinted conductive polymer-coated electrodes to 17ß-estradiol. As estradiol concentrations are important to eel aquaculture, we screened eel serum samples to determine their 17ß-estradiol concentrations, which were found to be in the range 28.2 ± 3.6 to 73.0 ± 11.6 pg/mL after dilution. These results were in agreement with measurements using commercial immunoanalysis.

The Influence of Sex, Parasitism, and Ontogeny on the Physiological Response of European Eels (Anguilla anguilla) to an Abiotic Stressor.

Migration of adult European eels (Anguilla anguilla) from freshwater feeding grounds to oceanic spawning grounds is an energetically demanding process and is accompanied by dramatic physiological and behavioral changes. Humans have altered the aquatic environment (e.g., dams) and made an inherently challenging migration even more difficult; human activity is regarded as the primary driver of the collapse in eel populations. The neuroendocrine stress response is central in coping with these challenging conditions, yet little is known about how various biotic factors such as sex, parasites, and ontogeny influence (singly and via interactions) the stress response of eels. In this study, mixed-effects and linear models were used to quantify the influence of sex, parasitism (Anguillicola crassus), life stage (yellow and silver eels), and silvering stage on the stress response of eels when exposed to a standardized handling stressor. The physiological response of eels to a standardized abiotic stressor (netting confinement in air) was quantified through measurements of blood glucose and plasma cortisol. The relationships between biotic factors and the activity of gill Na+/K+-ATPase was also examined. Analyses revealed that in some instances a biotic factor acted alone while in other cases several factors interacted to influence the stress response. Blood glucose concentrations increased after exposure to the standardized stressor and remained elevated after 4 h. Variation in plasma cortisol concentrations after exposure to the stressor were found to be time dependent, which was exacerbated by life stage and parasitism condition. Males and nonparasitized silver eels had the highest Na+/K+-ATPase activity. Silvering stage was strongly positively correlated with Na+/K+-ATPase activity in female eels. Collectively, these findings confirm that the factors mediating stress responsiveness in fish are complicated and that aspects of inherent biotic variation cannot be ignored.

Pharmacokinetics and residue depletion of praziquantel in rice field eels Monopterus albus.

We investigated the pharmacokinetic characteristics of praziquantel (PZQ) in rice field eels Monopterus albus. Pharmacokinetic parameters were determined following a single intravenous administration (5 mg kg(-1) body weight [bw]) and a single oral administration (10 mg kg(-1) bw) at 22.0 ± 0.7°C. We also evaluated residue depletion in tissues following daily administration of PZQ (10 mg kg(-1) bw) that was given orally for 3 consecutive days at 22.0 ± 0.7°C. Following intravenous treatment, the plasma concentration-time curve was best described by a 3-compartment open model, with distribution half-life (t(1/2α)), elimination half-life (t(1/2ß)), and area under the concentration-time curve (AUC) of 0.54 h, 17.10 h, and 14505.12 h µg l(-1), respectively. After oral administration, the plasma concentration-time curve was best described by a 1-compartment open model with first-order absorption, with absorption half-life (t(1/2Ka)), elimination half-life (t(1/2Ke)), peak concentration (C(max)), time-to-peak concentration (T(max)), and AUC estimated to be 2.28 h, 6.66 h, 361.29 µg l(-1), 5.36 h, and 6065.46 h µg l(-1), respectively. The oral bioavailability (F) was 20.9%. With respect to residue depletion of PZQ, the t(1/2ß) values of muscle, skin, liver, and kidney were 20.2, 28.4, 14.9, and 54.1 h, respectively. Our results indicated rapid absorption, rapid elimination, and low bioavailability of PZQ in rice field eels at the tested dosing conditions.

Triacylglyceride physiology in the short-finned eel, Anguilla australis­changes throughout early oogenesis.

During certain stages in an animal's life cycle, energy requirements may exceed energy intake from the diet. The spawning migration of temperate eels is a textbook example of negative energy balance, forcing these fish to rely on stored fats (triacylglycerides) to provide their muscles with energy for swimming and their growing oocytes with the nutrients needed to develop and support healthy offspring. We predicted broad implications of this great need for endogenous triacylglycerides in terms of their packaging, transport, and ovarian uptake. To test this, serum lipid concentrations and transcript abundances of intestinal and hepatic triacylglyceride packagers and ovarian triacylglyceride modifiers and receivers were investigated throughout previtellogenesis (feeding phase) and into early vitellogenesis (fasting phase) in short-finned eels. A switch from exogenous to endogenous triacylglyceride packaging was seen as the liver upregulated transcript levels of apolipoprotein B and microsomal triacylglyceride transport protein and downregulated those of apolipoprotein E and lipoprotein lipase. In the intestine, the reverse response was observed. Furthermore, ovarian transcript abundances of triacylglyceride modifiers and receivers increased (apolipoprotein E, lipoprotein lipase, and vitellogenin receptor), indicative of increased triacylglyceride uptake during previtellogenesis. We propose that increased hepatic apolipoprotein B production is a conserved vertebrate response to prolonged periods of negative energy balance.

Novel host-specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus.

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host-specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron-regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad-host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid-encoded fish-specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish-farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.

Looking for protein expression signatures in European eel peripheral blood mononuclear cells after in vivo exposure to perfluorooctane sulfonate and a real world field study.

The decline of European eel population can be attributed to many factors such as pollution by xenobiotics present in domestic and industrial effluents. Perfluorooctane sulfonate (PFOS) is a ubiquitous compound of a particular concern in Europe. PFOS can reach high concentrations in tissues of organisms and many toxic effects have been reported in fish. This study aimed at evaluating the toxicological effects of PFOS in European eel peripheral blood mononuclear cells (PBMCs) at the protein expression level. To identify proteins whose expression was modified by PFOS, we performed a proteomic analysis on the post-nuclear fraction of PBMCs after a chronic exposure (28 days) of yellow eels to zero, 1 or 10 µg/L PFOS. This in vivo study was completed by a proteomic field study on eels sampled in Belgian rivers presenting different PFOS pollution degrees. Proteins were separated by two-dimensional in-gel electrophoresis (2D-DIGE) to compare the post-nuclear fraction of PBMCs from the reference group with cells from fish exposed to the pollutant of interest. On the 28 spots that were significantly (p < 0.05; ANOVA followed by a Dunnett post-hoc test) affected by PFOS in the in vivo experiment, a total of 17 different proteins were identified using nano-LC ESI-MS/MS and the Peptide and Protein Prophet of Scaffold software. In the field experiment, 18 significantly (p < 0.05; ANOVA followed by Dunnett's test) affected spots conducted to the identification of 16 different proteins. Interestingly, only three proteins were found in common between in vivo and in situ experiments: plastin-2, alpha-enolase and glyceraldehyde 3-phosphate dehydrogenase. Comparing the results with a previous study, plastin-2 and alpha-enolase were also been found to be affected after in vitro exposure of PBMCs during 48 h to either 10 µg or 1 mg PFOS/L. Potential use of these proteins as biomarkers of PFOS exposure in European eel could indicate early warning signals.

The hemoglobin system of the serpent eel Ophisurus serpens: structural and functional characterization.

The hemoglobin system of the serpent eel Ophisurus serpens was structurally and functionally characterized with the aim of comparing it to the hemoglobin system of other fish species, as oxygen loading under the severe habitat conditions experienced by O. serpens could have necessitated specific adaptation mechanisms during evolution. The hemoglobin system of O. serpens includes one cathodic and four anodic components. The molecular mass of the α and ß chains of the cathodic component as well as the 2 α and 4 ß of the anodic components were determined. Analysis of the intact α and ß chains from cathodic hemoglobin and their proteolytic digestion products by high-resolution MS and MS/MS experiments resulted in 92 and 95 % sequence coverage of the α and ß globins, respectively. The oxygen binding properties of both hemoglobin components were analyzed with respect to their interactions with their physiological effectors. Stripped cathodic hemoglobin displayed the highest oxygen affinity among Anguilliformes with no significant effect of pH on O2-affinity. In the presence of both chloride and organic phosphates, O2-affinity was strongly reduced, and cooperativity was enhanced; moreover, cathodic hemoglobin contains two indistinguishable GTP-binding sites. Stripped anodic hemoglobins exhibited both low O2-affinity and low cooperativity and a larger Bohr effect than cathodic hemoglobin. The cathodic hemoglobin of O. serpens and the corresponding component of Conger conger share the greatest structural and functional similarity among hemoglobin systems of Anguilliformes studied to date, consistent with their phylogenetic relationship.

Simultaneous quantification of Pacific ciguatoxins in fish blood using liquid chromatography-tandem mass spectrometry.

Ciguatera fish poisoning (CFP) is a food intoxication caused by exposure to ciguatoxins (CTXs) in coral reef fish. Rapid analytical methods have been developed recently to quantify Pacific-CTX-1 (P-CTX-1) in fish muscle, but it is destructive and can cause harm to valuable live coral reef fish. Also fish muscle extract was complex making CTX quantification challenging. Not only P-CTX-1, but also P-CTX-2 and P-CTX-3 could be present in fish, contributing to ciguatoxicity. Therefore, an analytical method for simultaneous quantification of P-CTX-1, P-CTX-2, and P-CTX-3 in whole blood of marketed coral reef fish using sonication, solid-phase extraction (SPE), and liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The optimized method gave acceptable recoveries of P-CTXs (74-103 %) in fish blood. Matrix effects (6-26 %) in blood extracts were found to be significantly reduced compared with those in muscle extracts (suppressed by 34-75 % as reported in other studies), thereby minimizing potential for false negative results. The target P-CTXs were detectable in whole blood from four coral reef fish species collected in a CFP-endemic region. Similar trends in total P-CTX levels and patterns of P-CTX composition profiles in blood and muscle of these fish were observed, suggesting a relationship between blood and muscle levels of P-CTXs. This optimized method provides an essential tool for studies of P-CTX pharmacokinetics and pharmacodynamics in fish, which are needed for establishing the use of fish blood as a reliable sample for the assessment and control of CFP.

Changes in plasma angiotensin subtypes in Japanese eel acclimated to various salinities from deionized water to double-strength seawater.

Our knowledge of complexity of the renin-angiotensin system (RAS) has grown in recent years and various angiotensin peptides including Ang II, Ang III, Ang IV, and Ang (1-7) were found to have specific functions. Using a combination of HPLC and radioimmunoassay (RIA), we established a high resolution method to quantify various angiotensin subtypes in the plasma of eel acclimated to deionized water (dW), freshwater (FW), seawater (SW), and double-strength seawater (DSW). [Asn(1), Val(5)]-Ang II, [Asp(1), Val(5)]-Ang II, [Val(4)]-Ang III, and [Val(3)]-Ang IV are all present in the circulation and both Ang II subtypes were significantly higher in DSW eel. When the eel was transferred from FW to SW, plasma immunoreactive (ir) Ang II concentration increased and its levels were highly correlated to plasma osmolality, suggesting that the elevated plasma osmolality is the major stimulus for activating the RAS during high salinity transfer. To examine the conversion of [Asn(1)] to [Asp(1)] residue in vivo and in vitro, synthetic [Asn(1), Val(5)]-Ang II was injected into the circulation or incubated with plasma, but the production of [Asp(1), Val(5)]-Ang II was insignificant, which implies that the conversion may occur at the angiotensinogen level. An asparaginase assay was further developed for measuring asparaginase activity and the highest activity was in liver in both FW and SW eel. This new method of analysis can be extended to study the endogenous angiotensin ligands in the local RAS. The potential significance of [Asn(1)] to [Asp(1)] conversion on Ang II metabolism and function is discussed.

Plasma biochemistry values of recently wild-caught purple mouth moray eels (Gymnothorax vicinus).

The primary purpose of this study was to establish plasma biochemistry parameters for healthy recently wild-caught purple mouth moray eels (Gymnothorax vicinus) to provide a baseline of data for improved medical care in an aquarium or zoologic setting and for wild health assessments. Thirty-one clinically healthy purple mouth moray eels of unknown age and sex were caught from the wild, and were anesthetized 50 days following capture for blood collection from the ventral coccygeal vein. The median plasma biochemistry values were as follows: hematocrit = 21%, creatinine kinase = 2,100 U/L, lactate dehydrogenase = 97 U/L, aspartate aminotransferase = 88 U/L, alanine aminotransferase = 51 U/L, alkaline phosphatase 3,939 U/L, gamma-glutamyl transpeptidase = 1 U/L, amylase = 40 U/L, blood urea nitrogen = < 11 mg/dl, glucose = 21 mg/dl, calcium = 12.5 mg/dl, triglyceride = 206 mg/dl, creatinine = 0.1 mg/dl, cholesterol = 334 mg/dl, total bilirubin = < 0.1 mg/dl, phosphorus = 6.5 mg/dl, total protein = 4.2 g/dl, albumin = 1.5 g/dl, globulin = 2.7 g/dl, albumin/ globulin ratio = 0.6, sodium = 185 mmol/L, potassium = 3.7 mmol/L, and chloride = 175 mmol/L. Alkaline phosphatase isoenzyme results indicate that the majority of the plasma alkaline phosphatase is the liver isoenzyme. The data acquired in this study also provide baseline values for cholesterol and triglycerides in recently wild-caught moray eels to aid in monitoring elevations to these values in an aquarium setting over time so adjustments to the dietary regime may be utilized to prevent or improve conditions such as lipid keratopathy.

Swimming suppresses hepatic vitellogenesis in European female silver eels as shown by expression of the estrogen receptor 1, vitellogenin1 and vitellogenin2 in the liver.

BACKGROUND: When European silver eels (Anguilla anguilla) venture into the Atlantic Ocean for their 6,000 km semelparous spawning run to the Sargasso Sea, they are still in a prepubertal stage. Further sexual development appears to be blocked by dopaminergic inhibition of hypothalamus and pituitary activity. Recently, we found that swimming for several weeks in freshwater stimulated the incorporation of fat droplets in the oocytes. So, it was hypothesized that long term swimming in seawater would release the inhibition further and would also stimulate the production of vitellogenin by the liver. METHODS: For this study a swim-flume was constructed to allow simulated migration of migratory female silver eels for 3 months (1,420 km) in natural seawater at 20 degrees C. Primers were designed for polymerase chain reactions to measure the mRNA expression of estrogen receptor 1 (esr1), vitellogenin1 (vtg1) and vitellogenin2 (vtg2) genes in the liver of European female silver eels. RESULTS: In comparison to resting eels, swimming eels showed a diminished expression of esr1, vtg1 and vtg2 in the liver. They also had lower plasma calcium (Ca; indicative of vitellogenin) levels in their blood. This showed that vitellogenesis is more strongly suppressed in swimming than in resting eels. However, when eels were subsequently stimulated by 3 weekly carp pituitary extract injections, the expression of the same genes and plasma levels of Ca strongly increased in both groups to similar levels, thus equalizing the initial differences between resting and swimming. CONCLUSIONS: It is concluded that vitellogenesis remains suppressed during resting and even more during swimming. The fact that swimming stimulates fat deposition in the oocytes but suppresses vitellogenesis indicates that these events are separated in nature and occur sequentially. Swimming-suppressed vitellogenesis may imply that in nature eels undergo vitellogenesis and final maturation near or at the spawning grounds.

Homologue of mammalian apolipoprotein A-II in non-mammalian vertebrates.

Although apolipoprotein with molecular weight 14 kDa (apo-14 kDa) is associated with fish plasma high-density lipoproteins (HDLs), it remains to be determined whether apo-14 kDa is the homologue of mammalian apoA-II. We have obtained the full cDNA sequences that encode Japanese eel and rainbow trout apo-14 kDa. Homologues of Japanese eel apo-14 kDa sequence could be found in 14 fish species deposited in the DDBJ/EMBL/GenBank or TGI database. Fish apo-14 kDa lacks propeptide and contains more internal repeats than mammalian apoA-II. Nevertheless, phylogenetic analysis allowed fish apo-14 kDa to be the homologue of mammalian apoA-II. In addition, in silico cloning of the TGI, Ensembl, or NCBI database revealed apoA-IIs in dog, chicken, green anole lizard, and African clawed frog whose sequences had not so far been available, suggesting both apoA-I and apoA-II as fundamental constituents of vertebrate HDLs.

Cortisol mobilizes mineral stores from vertebral skeleton in the European eel: an ancestral origin for glucocorticoid-induced osteoporosis?

Endogenous excess cortisol and glucocorticoid (GC) therapy are a major cause of secondary osteoporosis in humans. Intense bone resorption can also be observed in other vertebrates such as migratory teleost fish at the time of reproductive migration and during fasting when large amounts of calcium and phosphate are required. Using a primitive teleost, the European eel, as a model, we investigated whether cortisol could play an ancestral role in the induction of vertebral skeleton demineralization. Different histological and histomorphometric methods were performed on vertebral samples of control and cortisol-treated eels. We demonstrated that cortisol induced a significant bone demineralization of eel vertebrae, as shown by significant decreases of the mineral ratio measured by incineration, and the degree of mineralization measured by quantitative microradiography of vertebral sections. Histology and image analysis of ultrathin microradiographs showed the induction by cortisol of different mechanisms of bone resorption, including periosteocytic osteolysis and osteoclastic resorption. Specificity of cortisol action was investigated by comparison with the effects of sex steroids. Whereas, testosterone had no effect, estradiol induced vertebral skeleton demineralization, an effect related to the stimulated synthesis of vitellogenin (Vg), an oviparous specific phospho-calcio-lipoprotein. By contrast, the cortisol demineralization effect was not related to any stimulation of Vg. This study demonstrates GC-induced bone demineralization in an adult non-mammalian vertebrate, which undergoes natural bone resorption during its life cycle. Our data suggest that the stimulatory action of cortisol on bone loss may represent an ancestral and conserved endocrine regulation in vertebrates.

Ganglioside from eel serum high density lipoprotein (HDL) and its role as a ligand for HDL binding protein.

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.

Identification of eel ghrelin in plasma and stomach by radioimmunoassay and histochemistry.

The structure of ghrelin has been determined in the Japanese eel, Anguilla japonica. In this study, we identified immunoreactive ghrelin in extracts from plasma and stomach of the eel by radioimmunoassay (RIA) using an antiserum against octanoylated rat ghrelin [1-11]. Using the antiserum, we examined localization of ghrelin-immunopositive cells in the eel stomach. Detection of ghrelin mRNA-expressing cells was also attempted in the eel stomach using a cRNA probe specific for the eel ghrelin gene. Furthermore, we examined ghrelin expression patterns in plasma and stomach after transfer of freshwater (FW) eels to seawater (SW). Multiple types of immunoreactive ghrelin were detected using RIA. These were octanoylated eel ghrelin and other ghrelins that may have different fatty acid modifications, suggesting that this RIA can detect acylated ghrelin of eels as seen previously in the case of rat. Ghrelin-immunopositive cells were observed in the mucosal layer of the stomach, especially in the neck of the fundic gland. Ghrelin mRNA-expressing cells showed similar distribution and characteristics to the immunopositive cells. Plasma ghrelin levels in FW eels starved for one week before experimentation were approximately 40 fmol/ml. Plasma ghrelin levels in control-transferred FW eels did not change for 7 days, but significantly increased on day 14. Plasma ghrelin levels transiently increased fivefold 6h after SW transfer and then declined to the FW level by 24h after transfer. Ghrelin content and ghrelin mRNA levels in the stomach did not change after SW transfer, except for a transient decrease in ghrelin content seen 24h after transfer. The present results suggest that ghrelin may participate in osmoregulation in eels.

Pharmacokinetics of ciprofloxacin in eels by high-performance liquid chromatography with fluorescence detection.

The plasma kinetics of ciprofloxacin (CF) were investigated in the eels after administration by oral gavage and bath treatment. Plasma concentrations of CF were determined by high-performance liquid chromatography with fluorescence detection. The mean concentration time data after oral gavage of a single dose (10.0 mg/kg CF) and after bath treatment by exposure (10 microg/ml CF) to medicated water for 48 h were both best fitted by a one-compartment model. After oral gavage in eels, the half-time of absorption (T1/2Ka) was 0.10 h, the half-time of elimination (T1/2Ke) was 51.87 h, and the maximum plasma concentration (Cmax) was 0.4552 microg/ml at Tmax 0.88 h. After bath treatment, the (T1/2Ka) was 0.02 h, the (T1/2Ke) was 15.46 h, and the Cmax was 0.1175 microg/mL at Tmax 0.22 h.

Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater.

Sulfate is required for proper cell growth and development of all organisms. We have shown that the renal sulfate transport system has dual roles in euryhaline eel, namely, maintenance of sulfate homeostasis and osmoregulation of body fluids. To clarify the physiological roles of sulfate transporters in teleost fish, we cloned orthologs of the mammalian renal sulfate transporters Slc13a1 (NaSi-1) and Slc26a1 (Sat-1) from eel (Anguilla japonica) and assessed their functional characteristics, tissue localization, and regulated expression. Full-length cDNAs coding for ajSlc13a1 and ajSlc26a1 were isolated from a freshwater eel kidney cDNA library. Functional expression in Xenopus oocytes revealed the expected sulfate transport characteristics; furthermore, both transporters were inhibited by mercuric chloride. Northern blot analysis, in situ hybridization, and immunohistochemistry demonstrated robust apical and basolateral expression of ajSlc13a1 and ajSlc26a1, respectively, within the proximal tubule of freshwater eel kidney. Expression was dramatically reduced after the transfer of eels from freshwater to seawater; the circulating sulfate concentration in eels was in turn markedly elevated in freshwater compared with seawater conditions (19 mM vs. 1 mM). The reabsorption of sulfate via the apical ajSlc13a1 and basolateral ajSlc26a1 transporters may thus contribute to freshwater osmoregulation in euryhaline eels, via the regulation of circulating sulfate concentration.

Hematology patterns of migrating European eels and the role of EVEX virus.

We show that European eels infected with the rhabdovirus EVEX (Eel Virus European X) virus, developed hemorrhage and anemia during simulated migration in large swim tunnels, and died after 1000-1500 km. In contrast, virus-negative animals swam 5500 km, the estimated distance to the spawning ground of the European eel in the Sargasso Sea. Virus-positive eels showed a decline in hematocrit, which was related to the swim distance. Virus-negative eels showed a slightly increased hematocrit. Observed changes in plasma lactate dehydrogenase (LDH), total protein and aspartate aminotransferase (AAT) are indicative of a serious viral infection. Based on these observations, we conclude that eel virus infections may adversely affect the spawning migration of eels, and could be a contributing factor to the worldwide decline of eel.