The testis histology of artificially maturated European eel (Anguilla anguilla L.) at the end of sexual maturation, and spermatozoa ultrastructure in freshwater rearing. Short communication

The artificial induction of sexual maturation of European eel males was carried out by using weekly hCG administrations. Histological pictures showed that the testis tissues developed and regressed naturally and no pathological changes took place under the conditions of artificial rearing in freshwater. According to light and electron microscopic investigations the morphology and motility of the spermatozoa of males kept in freshwater proved to be similar to those in seawater. The authors suppose that freshwater rearing of males is not a barrier factor in the artificial propagation of European eels.     

Decline in non-native freshwater eels in Japan: ecology and future perspectives

The occurrence, distribution, and biological characteristics of non-native freshwater eels were analyzed using 5524 eels collected from 16 sites in Japan between 1997 and 2005. Three hundred seventy-four fishes (6.8%) were identified as non-native European eels, Anguilla anguilla, while the remainder (93.2%) were native Japanese eels, A. japonica. The European eel was found at 7 sites (44%), including 3 rivers, 2 freshwater lakes, one brackish lake, and one sea bay, suggesting a wide rage of habitat use. This variability of habitat use was also evidenced by the otolith microchemistry, which showed that they had lived in not only freshwater but also in seawater habitats. The sites with European eel were localized within the vicinity of southern Japan where a number of these eels were cultivated in the early 1970’s, suggesting that some had escaped from the culture ponds or were released intentionally into nearby natural waters. The large body size (mean total length: 803 mm), pigmented skin, enlarged eyes, and relatively matured gonads (mean gonad somatic index: 1.9) found in non-native European eels indicated that most had metamorphosed into the migratory silver phase, suggesting their ability to initiate spawning migration. However, the proportion of European eels in Mikawa Bay in 1997 was more than 12%, which decreased markedly to less than 2% after 2001, corresponding to the recent decline in import of European glass eels for aquaculture. This suggests that the population of European eels will decrease in Japanese waters in the future.     

Maturation patterns in female European eel: age and size at the silver eel stage

The maturation pattern in the female European eel Anguilla anguilla was studied by investigating age and size patterns of silver eels in different aquatic environments in Sweden, covering limnic, brackish and marine waters. The results neither supported the hypothesis that there is a critical size or age when eels enter the silvery stage, nor that size and age at maturity are positively related. Age at maturity, however, was observed to be negatively related to growth rate in all localities, i.e. the female reproductive tactic apparently is to become sexually mature at the earliest possible opportunity. Furthermore, it was recognized that a significant amount of variation was due to habitat differences, since the female eel maturation pattern deviated systematically between sampling sites, as it did also when the effect of growth rate was eliminated. Thus, the ability of the female eel to adjust maturation to an optimal size and age can be questioned, because the panmictic nature of the eel means local adaptations are unlikely Growth rate dependent differences suggest that variations in maturation patterns between eel environments are linked more to the opportunity for nutrient accumulation than to other aspects of growth.     

Expression of sodium/hydrogen exchanger 3 and cation-chloride cotransporters in the kidney of Japanese eel acclimated to a wide range of salinities

Reabsorption of monovalent ions in the kidney is essential for adaptation to freshwater and seawater in teleosts. To assess a possible role of Na⁺/H⁺ exchanger 3 (NHE3) in renal osmoregulation, we first identified a partial sequence of cDNA encoding NHE3 from the Japanese eel kidney. For comparison, we also identified cDNAs encoding kidney specific Na⁺–K⁺–2Cl^? cotransporter 2 (NKCC2α) and Na⁺–Cl^? cotransporter (NCCα). In eels acclimated to a wide range of salinities from deionized freshwater to full-strength seawater, the expression of NHE3 in the kidney was the highest in eel acclimated to full-strength seawater. Meanwhile, the NCCα expression exhibited a tendency to increase as the environmental salinity decreased, whereas the NKCC2α expression was not significantly different among the experimental groups. Immunohistochemical studies showed that NHE3 was localized to the apical membrane of epithelial cells composing the second segments of the proximal renal tubule in seawater-acclimated eel. Meanwhile, the apical membranes of epithelial cells in the distal renal tubule and collecting duct showed more intense immunoreactions of NKCC2α and NCCα, respectively, in freshwater eel than in seawater eel. These findings suggest that renal monovalent-ion reabsorption is mainly mediated by NKCC2α and NCCα in freshwater eel and by NHE3 in seawater eel.     

Sea resident Japanese eel collected from Kozushima Island,Japan

A Japanese eel Anguilla japonica was collected off Kozushima Island, one of the volcanic Izu Islands in Japan. The external morphology corresponded to a silvering stage (Silvering Index: S1) with a melanized body coloration of the head, and the tip of the lower jaw to the anus. However, the value of the gonado-somatic index of the female eel was low, and the oocytes were not matured (peri-nucleolus stage). Otolith analyses indicated that the individual had exclusively inhabited the sea for five years without entering freshwater. This record is the first collection of silver-phased Japanese eel in the Kuroshio region, which could be on the spawning migration route of the species, although it was not determined if the eel was settling around the Izu Islands or migrating to the spawning ground.     

RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica.

An RBCC (RING finger, B-box, and coiled-coil) protein was identified that belongs to the superfamily of zinc-binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater-specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.     

Changes in osmotic water permeability of the eel gills during seawater and freshwater adaptation

Summary Changes in osmotic water permeability of the isolated gills of the Japanese eel,Anguilla japonica were studied during transfer to seawater or to fresh water. The water permeability increased gradually during the course of seawater transfer and attained a maximal level after 2 weeks. The water permeability of the freshwater eel gills was reduced when calcium ions were added to the incubation medium at a concentration of 1 mM, where-as no effect of the ion was observed on the gills of the seawater-adapted eel even at a higher concentration (10 mM). In contrast to seawater transfer, the water permeability decreased to a low level almost immediately (3 h) after transfer from seawater to fresh water. The acute reduction of the water permeability was also seen in the gills of the hypophysectomized eel after transfer to fresh water.The gradual increase in the gill water permeability during seawater transfer is correlated with an increase in the number of chloride cells. In scanning electron microscopy, chloride cells of seawater-adapted eel gills exhibit a pit-like structure, which was larger than in the freshwater eel. On transfer from seawater to fresh water, the pit diameter became smaller within 6 h. Hypophysectomy did not affect the change in gill surface structures during transfer to fresh water. The junctions between the chloride cells of seawater eel gills are reported to be of the leaky type. The parallel change in osmotic water permeability and in pit size of the chloride cells during seawater or freshwater transfer or after hypophysectomy suggests that these cells could provide a major route of water as well as ion movement.This paper is a portion of a thesis presented to Hokkaido University by t. Ogasawara in partial fulfilment of the requirements for Doctor of Fisheries     

高温季节控温诱导池养鳗鲡性腺发育的研究

在高温季节(6～9月),采用人工控温的方法,通过注射外源激素hCG和CPE,对日本鳗鲡进行了催熟试验.结果表明,在人工控制温度(18±2℃)条件下,雌性和雄性日本鳗鲡均可达到性成熟,诱导成熟率分别为73.33%和67.77%,而常温条件下(25～32℃)的诱导成熟率分别为0和33.33%.同时对控温条件下诱导成熟的部分雌性鳗鲡进行了催产,其排卵率为70%,受精率为32%,均接近或达到了低温季节相应的指标(平均分别为62.1%和3.2%).对胚胎发育过程也进行了观察,受精卵能正常发育,在22和2℃下,分别经过39 h 15 min和3 h 9 min孵化出膜.试验证明,在高温季节采用人工控温的方法诱导日本鳗鲡性成熟是可能的.     

Histological and enzyme histochemical changes found in the renal tubules of eels transferred from freshwater to seawater

Renal tubules of the eel,Anguilla japonica, transferred from freshwater to seawater were examined histologically and histochemically. The epithelial cells of renal tubules in eels adapted to seawater for 10 days were less interdigitated in comparison with those in freshwater. The poor interdigitation of epithelial cells seems to be a structure relating to high water permeability in the renal tubules of eels in seawater. Newly formed nephrons and degenerated ones were especially recognized in eels adapted to seawater for 2 and 4 days. These findings indicate that some of the nephrons equipped with poorly interdigitated epithelial cells are produced newly within a few days after transfer from freshwater to seawater. No significant difference was encountered in the activities and localizations of all enzymes examined between the renal tubules of freshwater and seawater eels. The enzyme activities and localizations did not reflect the physiological changes in the renal tubules of eels adapted from freshwater to seawater.     

Different prevalences of Renibacterium salmoninarum detected by ELISA in Alaskan chinook salmon Oncorhynchus tshawytscha spawned from freshwater and seawater

Soluble antigen of Renibacterium salmoninarum (Rs) was detected by a polyclonal enzyme-linked immunosorbent assay (ELISA) at significantly higher prevalences in adult chinook salmon Oncorhynchus tshawytscha that matured in freshwater than in the same cohort of fish spawned after maturation in seawater. The cumulative results were consistent during 4 yr of comparison at the Little Port Walter Hatchery on Baranof Island, Alaska, USA. Possible causes for this difference are discussed. Maturation of chinook salmon broodstock in seawater has become a practical strategy at this hatchery to reduce the prevalence of Rs-positive parent fish and the numbers of culled eggs.     

Dietary effects on fatty acid composition in muscle tissue of juvenile European eel, <Emphasis Type="Italic">Anguilla anguilla</Emphasis> (L.)

The role of intracontinental migration patterns of European eel (Anguilla anguilla) receives more and more recognition in both ecological studies of the European eel and possible management measures, but small-scale patterns proved to be challenging to study. We experimentally investigated the suitability of fatty acid trophic markers to elucidate the utilization of feeding habitats. Eight groups of juvenile European eels were fed on eight different diets in a freshwater recirculation system at 20°C for 56 days. Three groups were fed on freshwater diets (Rutilus rutilus, Chironomidae larvae, and Gammarus pulex) and four groups were reared on diets of a marine origin (Clupea harengus, Crangon crangon, Mysis spec., and Euphausia superba) and one on commercial pellets used in eel aquaculture. Fatty acid composition (FAC) of diets differed significantly with habitat. FAC of eel muscle tissue seemed to be rather insensitive to fatty acids supplied with diet, but the general pattern of lower n3:n6 and EPA:ARA ratios in freshwater prey organisms could be traced in the respective eels. Multivariate statistics of the fatty acid composition of the eels resulted in two distinct groups representing freshwater and marine treatments. Results further indicate the capability of selectively restraining certain fatty acids in eel, as e.g. the n3:n6 ratio in all treatments was <4, regardless of dietary n3:n6. In future studies on wild eel, these measures can be used to elucidate the utilization of feeding habitats of individual European eel.     

Does a 5500-km swim trial stimulate early sexual maturation in the European eel (Anguilla anguilla L.)?

The catadromous European eel (Anguilla anguilla L.) undertakes a 6000-km spawning migration from its freshwater habitats to the Sargasso Sea. In large Blazka swim tunnels of 127 l, the physiological effect of such a prolonged swimming performance on sexual maturation in adult female eels was investigated. Two groups of eels were placed in swim tunnels for 173 days, one group was able to swim at 0.5 body lengths/second (Swim group) covering a distance of c. 5500-km over the experimental period, and one group kept in static (End Control group). A control group was sampled at the start of the experiment in order to determine the initial stage of reproductive development (Initial Control group). At the end of the swim trial, the maturation parameters 11-ketotestosterone, pituitary levels of LH and plasma levels of estradiol were higher (although not significantly) in the Swim compared to the End Control group. In addition, no significant differences were observed in most measured morphometric and reproductive parameters, including eye-index, gonadosomatic index, hepatosomatic index, and plasma levels of vitellogenin, cortisol and melanophore-stimulating hormone (MSH). Also, pituitary levels of both MSH, and adrenocorticotropic hormone (ACTH) were unaffected. In contrast, the oocyte diameter was found to be significantly higher in the Swim compared to the End Control group. Based on these observations we conclude that a period of prolonged swimming might be a physiological stimulus necessary for the onset of maturation in the European eel.     

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.     

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.     

Cost of transport and optimal swimming speed in farmed and wild European silver eels (Anguilla anguilla)

A swimming speed of 0.4 meters per second (m s(-1)) is the minimal speed for European female silver eels to reach the spawning sites in the Sargasso Sea in time. As silver eels cease feeding when they start their oceanic migration, the cost of transport (COT) should be minimised and the swimming speed optimised to attain the highest energetic efficiency. In this study, we have investigated the optimal swimming speed (U(opt)) of silver eels since U(opt) may be higher than the minimal swimming speed and is more likely to resemble the actual cruise speed. A variety of swimming tests were performed to compare endurance swimming between farmed eels and wild eels, both in freshwater and in seawater. The swimming tests were run with 101 silver female eels (60-96 cm, 400-1500 g) in 22 Blazka-type swim tunnels in a climatised room at 18 degrees C with running freshwater or seawater. Tests were run at 0.5-1.0 m s(-1) with increments of 0.1 m s(-1), and either 2 h or 12 h intervals. Remarkably, both tests revealed no changes in oxygen consumption (M O2) and COT over time. U(opt) values ranged between 0.61 and 0.68 m s(-1) (0.74-1.02 BL s(-1)) for the different groups and were thus 53-70% higher than the minimal speed. At U(opt), the COT was 37-50 mg O2 kg(-1) km(-1). These relatively very low values confirm our earlier observations. COT values in seawater were about 20% higher than in freshwater. Assuming that migrating female silver eels cruise at their U(opt), they will be able to cover the distance to the Sargasso Sea in 3-4 months, leaving ample time for final maturation and finding mates.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

Factors influencing otolith strontium/calcium ratios in Anguilla japonica elvers

The effects of temperature and salinity on the concentration ratios of strontium (Sr) to calcium (Ca) within the sagittal otoliths of elvers of the Japanese eel, Anguilla japonica, were studied by spot analysis using a wavelength dispersive X-ray electron microprobe. A total of 340 elvers were used: 100 elvers were reared for 15 days under various salinity conditions (freshwater, one-third seawater, two-thirds seawater and pure seawater at 22 °C; 240 elvers were reared for 58 days under various water temperature conditions (12, 17, 22, 27 °C) in either freshwater or pure seawater. Otolith Sr/Ca ratios were found to be positively correlated with water salinity. On the other hand, the Sr/Ca ratios were not found to be significantly different among the various temperature groups. The above results strongly suggest that the physiological mechanism of incorporation of Sr and Ca within the otolith of an eurythermal fish, Japanese eel, does not change within this range of temperatures (12–27 °C).     

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation

Background

The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males.

Results

Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity.

Conclusions

This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-799) contains supplementary material, which is available to authorized users.     

Colonisation of freshwater habitats by the European eel Anguilla anguilla

1. The spatial distribution of European eels in 18 U.K. rivers was related to distance from tidal limit using a negative exponential model. This function accounted for between 19 and 90% of the variation in eel density where quantitative data was available. For semiquantitative data the negative exponential function was a significant predictor of eel densities in only six out of 10 cases, although all rivers showed a consistent decline in abundance with distance upstream from the tidal limit. 2. The spatial distribution of different age groups of European eel in River Severn showed an initial rapid dispersion into freshwater followed by a much slower dispersion rate. Movement of the population upstream by a wave‐form migration process does not occur in this system. Instead colonisation of freshwaters can be seen as a two‐phase dispersion. Phase‐1 is a rapid dispersion upstream driven by density at the point source. Phase‐2 commences once the eels become yellow eels and is equivalent to random diffusion of particles. 3. These processes have important implications for the penetration of freshwaters with reduced numbers of eel larvae arriving on the coast of Europe and North America. Eel abundance will decrease more in freshwaters in an upstream direction whilst it may remain stable or decrease to a lesser extent in estuaries. They are also able to explain the demography of eels migrating upstream over weirs and the observations of varying sex ratios within catchments. We conclude that a dispersion model dependent on age, temperature, difficulty of migration, habitat quality and density of eels should be an important part of freshwater eel management.