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During the induction of Japanese eel maturation, administering maturation-inducing steroids (MIS) or their precursors at an inappropriate maturational status is a major cause of poor egg quality. In this study, we investigated the feasibility of controlling oocyte maturation progress by rearing in cold and warm water to manipulate the timing of MIS administration. Mature females with oocytes at the migratory nucleus stage were reared for two terms (3 days to 1 day and 1 day to 0 days before MIS administration) at 20/20 °C, 20/15 °C, 15/20 °C, or 15/15 °C, and the maturational status was monitored based on their lipid droplet morphology and oocyte diameter. Oocytes matured faster at 20 °C than at 15 °C in either term. Next, the mature females were reared at 15 or 20 °C depending on the maturational status of each female 3 days and 1 day before MIS administration; the immature females were reared at 20 °C to accelerate their maturation. Consequently, the maturational status of most females was similar at MIS administration. After improvement, this method would lead most females to the optimum maturational status at MIS administration by properly rearing in cold and warm water.

Organogenesis of Japanese eels (Anguilla japonica) was investigated histologically from the late leptocephalus to the yellow eel stages. Early organogenesis, such as the formation of inner ears and the appearance of round blood cells that might be larval erythrocytes, had already begun at the late leptocephalus stage. During the first developmental phase (M1-M3 stages) of metamorphosing into early glass eels (G1 stage), the formation of gills and lateral muscles progressed conspicuously with a drastic body shape change from leaf-like to eel-like. In contrast, obvious regression in esophageal muscle and pancreas occurred during metamorphosis. Formation of lateral line canals advanced continuously until the yellow eel stage. When second developmental phase was initiated at the G1 stage, cone photoreceptor cells appeared, and formation of esophageal, stomach and intestinal muscles was initiated. Differentiation of gastric glands began at one week after metamorphosis. Erythrocytes increased continuously in density in glass eels and elvers (G1-E2 stages), and the morphological features of cone cells and olfactory epidermal cells became clearer with stage progression. In early elvers (E1 stage), the swim bladder initiated inflation, the stomach fully expanded and the rectal longitudinal fold changed to a circular one. Swim bladder gas glands appeared in late elvers (E2 stage). In the yellow eels (juvenile stage), almost all organ structures were formed. These observations indicate that the organogenesis of A. japonica is still ongoing after metamorphosis into glass eels, and the M1 to E2 stages are considered to be a homologous phase to first metamorphosis that is a transformation from larval to juvenile stage in other teleosts. In comparison to conger eels, the completion of the body shape change to eel-like occurs at the G1 stage, when organogenesis is still in progress, being followed by a prolonged duration of the G1-E2 stages before reaching the yellow eel juvenile stage, which may be a unique characteristic that is related to the early migratory life history of A. japonica. This article is protected by copyright. All rights reserved.

The aim of this study was to develop sperm cryopreservation protocols for large-scale fertilization in Japanese eel (Anguilla japonica), by testing the feasibility of combining large-volume straws and a low sperm dilution ratio. Experiments were conducted using both 0.25-ml straws and 2.5- and 5-ml straws (macrotubes) for sperm cryopreservation. Cooling rates in macrotubes were measured at three internal positions and at different heights above the surface of liquid nitrogen (LN). There was no significant difference in the cooling rate among internal positions in the straws. The cooling rate could be adjusted to the appropriate range for Japanese eel sperm (6.3 °C–8.6 °C min−1) by positioning the 2.5- and 5.0-ml straw 0.5–5.0 cm above the surface of LN. We succeeded in scaling up the storage volume from 0.25 to 4.5 ml (18-fold) without decreasing the motility using a 5.0-ml straw. The number of storable motile spermatozoa was the highest at a range of dilution ratios (sperm to extender, v/v) from 1:1 to 1:5. Although frozen-thawed sperm showed a significant decrease in the proportion of motile sperm to egg compared with than fresh sperm, there was no significant difference in the fertilization rate, and fertilized eggs with cryopreserved sperm did not affect the hatching rate or survival rate at 7 days after hatching. Using a segmented linear analysis, we found that 10,180 ± 3077 motile spermatozoa per egg are required to maximize fertilization success. We estimated that spermatozoa stored in a 2.5- and 5.0-ml straw can fertilize approximately 184,185 and 331,532 eggs, respectively. Furthermore, our investigation of the effect of long-term storage in LN on the sperm quality showed that the storage period does not affect the post-thaw motility rate even after 2 years of storage. Our results demonstrate that cryopreservation techniques for Japanese eel sperm using 2.5- and 5.0-ml straws could be used for large-scale fertilization programs.

The Japanese eel (Anguilla japonica) is one of the most important aquaculture fish species in Eastern Asia. A major bottleneck in the mass production of glass eels is the long larval period that they undergo in captivity. In the present study, 1689 glass eels, originating from a partly factorial cross (43 sires × 32 dams), were used to investigate the potential value of genomic selection for 10 traits related to the timing of metamorphosis and the body size at that time. Genotyping by random amplicon sequencing-direct (GRAS-Di®) sequencing was used to identify and genotype single nucleotide polymorphism (SNP) markers, which were used to assign parentage and as a basis for genomic selection. The genomic heritabilities of the 10 metamorphic traits obtained by genomic best linear unbiased prediction (GBLUP) models were moderate (range 0.21–0.38), and the standard errors were smaller than those determined by the pedigree-based best linear unbiased prediction (ABLUP) models. The genomic prediction of breeding values outperformed the pedigree-based prediction, resulting in a 3%–19% improvement in the prediction accuracy. The impact of reduced SNP densities on the predictive ability of GBLUP was tested using a linkage disequilibrium-based pruning and random thinning method to reduce 17,017 SNPs to 2117 SNPs. An SNP set of 8000 was considered to be sufficient to achieve predictive ability comparable to that obtained using higher SNP densities for both methods. The selective genotyping of the top individuals in the training population led to a severe reduction in the predictive ability compared with the random genotyping of the same number of samples. These results will be useful for decision-making in establishing best-practice breeding programs when applied to genomic selection for metamorphic traits for shortening the larval period and improving seed quality in Japanese eels.

In the present study, methods for the cryopreservation of Anguilla japonica spermatozoa were examined. Spermatozoa were collected from artificially matured males and incubated in Japanese eel K30 artificial seminal plasma (K30 ASP) before experiments. Sperm motility was investigated using computer-assisted sperm analysis (CASA). 10% and 15% MeOH as cryoprotectant was the most successful cryoprotectant with percentage of the initial motility of 59.7 ± 12.1%; the combination of 5% MeOH and 5% DMA was also viable. DMSO was unsuitable as a cryoprotectant with K30 ASP as it showed no cryopreservation properties and was toxic to sperm, causing sperm to be immotile immediately after dilution. Japanese eel spermatozoa had a narrow range of optimal cooling rates (6.3–28.6 °C min− 1) and immersion temperatures of − 40 to − 70 °C were effective. The different fetal bovine serum (FBS) concentration in extender, temperature of milt before cooling, dilution rates (3–100 times) and equilibrium time showed no significant difference. The results indicate that the type of extender media greatly influenced the suitability of cryoprotectant. The establishment of a protocol to cryopreserved Japanese eel sperm will benefit the artificial seed production of Japanese eel and provide an important tool for genetic and breeding studies. Statement of relevance The development of the sperm cryopreservation protocol for Japanese eel will ensure a stable supply of high quality seed for experimental and commercial purposes.

Pacific bluefin tuna (Thunnus orientalis) is one of the most important species for aquaculture in Japan. In recent years, the reduction in muscle fat content during sexual maturation in farmed T. orientalis has become a serious problem. Herein, as a first step toward sterilization to avoid the deterioration of flesh quality associated with sexual maturation, we examined cold-shock conditions for inducing triploidy in T. orientalis. In this study, three treatment variables (initiation timing, duration, and water temperature of the cold-shock treatment) were considered, using the fertilized eggs collected immediately after spontaneous spawning in a land-based tank. We found that the optimal cold-shock conditions for T. orientalis were 5 °C for 15 min starting from 5 min after fertilization. Under these optimal conditions, we obtained high triploidization (96.6 ± 3.3%) and total hatching (64.5 ± 8.7%) rates. Therefore, using eggs obtained by spontaneous spawning in a land-based tank and the triploidization method established in this study, it is possible to repeatedly and efficiently produce triploid T. orientalis. Statement of relevance In recent years, deterioration of flesh quality associated with sexual maturation in aquaculture-produced Thunnus orientalis has become a serious problem. To avoid this problem by sterilization technique, in this study, we have developed an efficient method for the induction of triploidy in T. orientalis, using the fertilized eggs collected immediately after the spontaneous spawning in a land-based tank. We believed that the results of present study are a valuable first step toward the sterilization of T. orientalis.

The Japanese eel (Anguilla japonica) is among the most important aquaculture fish species in Eastern Asia. The present study aimed to identify the genetic parameters underlying body size and the timing at metamorphosis from leptocephali to glass eels in captive-bred Japanese eels, with the intent to foster sustainable development. Larvae from a partly factorial cross (14 sires × 11 dams) were reared until the point of metamorphosis into glass eels. In these organisms, we observed moderate heritability and mild genetic correlations among traits related to body size (h2 = 0.16-0.33) and timing at metamorphosis (h2 = 0.36-0.41). In an F1 full-sib family, quantitative trait loci (QTL) mapping for these traits identified one significant (genome-wide P < 0.05) and five suggestive QTLs (chromosome-wide P < 0.05). These results suggest that in the Japanese eel, metamorphic traits exhibit a polygenic genetic structure comprising many QTLs with small effects. In addition, we updated the genetic linkage map for the Japanese eel and integrated it with our newly constructed de novo genome assembly. The information and tools generated from this study will contribute to the development of freshwater eel genetics and genomics.

Bacteria with lethal effect on eel larvae were isolated from moribund captive-bred eel larvae and their 16S rRNA gene sequences were analysed. Nucleotide sequence of 10 isolates showed highest similarity with Lacinutrix algicola, Crocinitomix catalasitica and Pseudoalteromonas rubra. Age-dependent changes in the susceptibility of eel larvae were observed in response to challenge by a highly lethal isolate. Compared with 10 and 11 days after hatching (DAH), larval susceptibility to the highly lethal isolate was lower at 18 DAH. We found that the bacterial isolates have lethal effect on the captive-bred eel larvae, especially at the early developmental stage. These results will be useful to establish appropriate culture practices for eel larvae that will improve the success of mass production of glass eels for aquaculture.

Shark eggs-based diet is the only diet by which eel larvae can grow to glass eels in captivity. However, the high level of lipids in the diet is suggested to negatively affect eel larvae. This paper examines the effect of defatted shark eggs (DSE) and hen egg yolk (HY) on growth and survival of larvae of Japanese eel Anguilla japonica. Lyophilized shark egg and commercial HY were defatted with n-hexane, and four experimental diets were prepared using both defatted and untreated shark eggs and HY. Larvae were reared for 3 weeks by feeding the experimental diets. The highest survival rate was observed in the larvae fed DSE, and larvae fed HY showed the lowest survival rate. The best growth was found in larvae fed DSE, followed by shark eggs and defatted HY, and the worst growth was in HY-fed larvae. These results show that decreasing dietary lipids improves the nutritional value of both shark eggs and HY for eel larvae. Regulation of the dietary lipid level may positively affect the larval performance of eels by combination of ingredients with a low lipid content.

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Our objective was to evaluate the effects of hormonal treatments on the milt quality during the induction of spontaneous spawning in male eels. We measured the changes in milt characteristics (sperm motility, milt pH, seminal plasma ionic concentration) during the course of hormonal treatments intended to induce spontaneous spawning, and before and after spawning. Male eels received weekly injections of hCG (1 IU/g BW/wk) to maintain spermiation. To induce spontaneous spawning, males received another priming injection of hCG (1 IU/g BW) two days before spawning, followed 24 h later by an injection of 17α-hydroxyprogesterone (OHP: 1 μg/g BW). Three males, treated as described above, and one female, that had received hormonal injections to induce vitellogenesis and final maturation, were transferred to a tank to spawn. In Experiment 1, we measured the characteristics of milt (1) before administering the priming dose (1–2 days after the weekly injection of hCG), (2) before injection with OHP, (3) after spawning (24 h after the OHP injection), and (4) before weekly injections with hCG (4–5 days after OHP injection). To evaluate the effects of hormonal treatment on the characteristics of milt we administered the same treatments but did not allow spawning (Experiment 2). In both experiments, the percent motility remained high prior to OHP injection, decreased significantly 24 h after OHP injection, then recovered by the beginning of weekly hCG injection. The changes in potassium ion concentration were similar to those in sperm motility (%) but there was no change in pH and sodium ion concentrations. When the results were analyzed by individual in Experiment 1 (n = 34), the relationship between sperm motility (%) and milt pH showed significant correlations without 24 h after the OHP injection. The relationship between sperm motility (%) and potassium ion concentration showed a highly significant correlation 24 h after the OHP injection. Thus, both pH and potassium ion concentration regulate motility in Japanese eel spermatozoa during hormonal treatment. Furthermore, administration of OHP into spermiating males reduced motility by decreasing the potassium ion concentration in the seminal plasma.

Spontaneous polyploids and mosaics have often been observed in artificially propagated larvae of the Japanese eel, Anguilla japonica. However, the mechanisms responsible for such unusual cytotypes are unclear. In this study, we examined the relationship of such polyploidization and mosaicisms in larvae resulting from artificial propagation to egg quality (fertilization rate and hatching rate) and viability of larvae, and then clarified the inducing factors and the mechanism for occurrence of such phenomena. Eggs stripped from females after induced maturation were artificially inseminated with sperm pre-cultured with artificial seminal plasma. Ploidy was determined by measuring the relative DNA content of the nuclei with flow cytometry. Of 968 embryos from 32 full-sib families, 9.1% were determined to be abnormal, most of which were triploids (86.5% of abnormal embryos); others were haploids (1.1%), aneuploids (2.3%), and mosaics (10.1%). The percentage of normal diploids from each family varied between 56.3% and 100% (90.9±11.7%, n=32). A significant positive correlation was found between the fertilization rate (P

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption.

The pancreatic digestive enzymes, trypsin, chymotrypsin, lipase and amylase were partially characterized, and changes in their activities were examined during the initial ontogeny of Japanese eel Anguilla japonica larvae from 5 to 34 days post-hatching (dph). The pH optima of the eel larval enzymes were narrower than those other fish species; trypsin activity was highest at pH 9, chymotrypsin and amylase activities were highest at pH 7 and 8, and lipase activity was highest at pH 8 and 9. In an analysis of thermal profiles, the larval pancreatic enzymes had a high optimal temperature and high thermal stability, which are typical of fish from the tropics. At 12 and 13 dph, lipase activity and gene expression levels of trypsin (-a and -b), lipase and amylase decreased markedly, suggesting a marked change in larval metabolism at that time. These data could be useful in the development of artificial larval diets in Japanese eel.

The quality of eggs obtained from maturation-induced Japanese eel, Anguilla japonica , is unstable. One of the causes for low quality eggs is that females are artificially induced to ovulate at an inappropriate maturational status. Here we investigated the feasibility to use the morphological changes of lipid droplets in oocytes as a biomarker to show the optimum timing for injections of salmon pituitary extract (SPE) for priming and maturation-inducing steroid (MIS) for final oocyte maturation and ovulation. Various sizes of oocytes (700 to 1000 μm) were collected by cannulation from maturation-induced female eels during five days until ovulation. Morphology of the lipid droplets in these oocytes were classified into 10 stages mainly on the basis of their diameter, which increased with the progress of maturation as the droplets fused together. To assess the maturational status of each female, the lipid droplet stage was determined for 10 oocytes randomly chosen among the most advanced mode of developing oocytes and the median value was defined as the stage for the individual. Relationship between the lipid droplet stage at induction of ovulation and hatchability of the ovulated eggs was examined in 23 females, in which the hatching rate markedly fluctuated. Stages of females from which good quality eggs were obtained converged around stage 4 (five largest droplets, 55 to 70 μm) at priming with SPE, stage 6 (90 to 110 μm) at injection with MIS, and stage 7 (110 to 130 μm) at ovulation, while those of females from which low quality eggs were obtained showed distributions over broader ranges. Furthermore, 466 females were induced to mature and the lipid droplet stage at induction of ovulation was examined in 13 females from which high quality eggs (hatching rate >80%) were obtained. Most of the 13 females were at stage 3 (40 to 55 μm) or 4 when primed with SPE, at stage 6 when injected with MIS, and at stage 7 when they ovulated. These results suggest that the optimum stages for SPE priming, MIS injection, and ovulation are stages 3 to 4, 6, and 7, respectively, though the optimum stage for priming will be variable if the temperature and/or the time between priming and MIS injection is varied. We conclude that the lipid droplet stage can be used as a sensitive biomarker to show the optimum timing for inducing ovulation by hormonal treatments.

The first genetic linkage map of the Japanese eel (Anguilla japonica) was constructed based upon Amplified Fragment Length Polymorphism (AFLP) and microsatellite (STR) markers with an F1 pseudo-testcross strategy. The segregation of 106 microsatellite loci, which included 74 newly developed, and 463 polymorphic loci identified from 56 AFLP primer combinations was studied in 46 F1 individuals derived from a single female and a male. A total of 319 markers (99 STRs + 220 AFLP) are placed on the female map and 314 markers (91 STRs + 223 AFLP) on the male map. The female map spans 1732.4 cM (Kosambi) in 22 linkage groups with an average spacing of 7.2 cM; the male map spans 964.9 cM in 22 linkage groups with an average spacing of 6.3 cM. The average ratio of male:female recombination rates was 1:2.11. The estimated coverage of the genome was 82.4% for the female map and 74.0% for the male map. This map is the first linkage map in the order Anguilliformes and provides a basis for mapping quantitative trait loci (QTL) and for breeding applications.

During 2008 and 2009, a total of 12 adult Japanese eels Anguilla japonica were captured in the southern part of the West Mariana Ridge, the presumed spawning area. We compared the sta- ble carbon and nitrogen isotope ratios (δ 13 C and δ 15 N) between the 'Mariana silvers' (terminal phase) and those of yellow and silver eels caught in rivers, lakes and coastal areas of Japan (initial phase). Profiles of stable isotope signatures between the initial and terminal phases were similar; both char- acteristically had a wide range for δ 13 C (-24.9 to -12.0‰ and -20.5 to -11.3‰ for the initial and terminal phases, respectively) and δ 15 N (6.5 to 18.4‰ and 9.0 to 18.1‰, respectively). Mesopelagic fishes, including several other anguillid species caught near the West Mariana Ridge, characteristi- cally had a very narrow range of δ 13 C (-16.9 to -15.3‰) and a wide but lower range of δ 15 N (5.3 to 11.1‰) than the Japanese eels. The very similar profiles in stable isotopic signatures between the initial and terminal phase eels, distinct from those of Mariana mesopelagic fishes, indicate that Japanese eels do not assimilate nutrition from the marine environment during long (ca. 6 mo) spawn- ing migration and retain the initial isotopic values of where they ceased feeding.

The occurrence of morphological deformities under different incubation salinities (24, 30, 33, 36 and 42 practical salinity units [psu]) was examined in Japanese eel larvae. Fertilized eggs hatched at all salinity treatments. The survival rate until yolk resorption decreased at higher than 36 psu. The proportions of deformed larvae reared at 36 psu were significantly lower than at other treatments. Pericardial edema and abnormal lower jaw increased at lower than 33 psu and spinal curvature occurred at high salinity (42 psu). In contrast, the incubation salinity did not significantly affect the relative frequency of abnormal neurocranium. These results imply that the optimal salinities for rearing Japanese eel eggs and embryos are 34–35 psu from the viewpoints of survival and deformity. Taken together with previous study [Kurokawa, T., Okamoto, T., Gen, K., Uji, S., Murashita, K., Unuma, T., Nomura, K., Matsubara, H., Kim, H.S., Ohta, H., Tanaka, H., in press. Influence of Water Temperature on Morphological Deformities in Cultured Larvae of Japanese Eel, Anguilla japonica , at Completion of Yolk Resorption. J. World Aqua. Soc.], the rearing of eel eggs and embryos at 25 °C and 34–35 psu probably decreases the mortality and deformity rate of eel larvae at yolk resorption stage.

This paper describes the relationship between the egg vitamin concentrations and the egg quality in the Japanese eel Anguilla japonica. No notable relation was found between any vitamin and the fertilization rate. Hatching and survival rates of larvae, however, significantly increased with an elevated level of egg vitamin C (VC). In contrast to VC, the relation between vitamins E (VE) and A (VA) concentrations and survival rate showed a clear peak, with a reduced survival rate at both higher and lower vitamin concentrations. The ratio of VE to lipid or highly unsaturated fatty acid (HUFA) in eggs positively correlated with hatching and survival rates of larvae. High-quality (HQ) eggs were determined as eggs that produced larvae having a survival rate higher than 80% at 8 days post hatch, and low-quality (LQ) eggs were determined as eggs that did not hatch. The level of VC of HQ was significantly higher than LQ. The results of this study suggest that HQ eggs, which produce larvae having a high survival rate, must have high levels of VC and VE/HUFA ratio and contain optimum levels of VA and VE in Japanese eel.

The occurrence of morphological deformities under different rearing water temperatures (18, 20, 22, 24, 26, 28, and 30 C) was examined in Japanese eel larvae. The rates of hatching and survival until yolk resorption at 22–26 C were higher than those at other water temperatures. Fertilized eggs never hatched at 18 and 30 C. The rates of occurrence of abnormal larvae reared at the water temperatures 24–28 C were lower than those at 20 or 22 C. Pericardial edema and lower jaw deformities occurred most frequently at lower temperatures (20 and 22 C). In contrast, the incubation temperature did not significantly affect the relative frequency of some neurocranial deformities and of spinal curvature. These results imply that the optimal temperatures for rearing Japanese eel eggs and embryos are 24–26 C from the viewpoints of survival and deformity.

To elucidate the utilization of major yolk nutrients in eggs and yolk-sac larvae of Japanese eel Anguilla japonica, contents of free amino acids (FAA), 380 kDa lipovitellin (oLv; the major yolk protein in ovulated eggs), and lipids were measured. All larvae hatched by the 2nd day after fertilization at a water temperature of 23 °C, and the hatched larvae absorbed almost all of the yolk mass by the 10th day. The FAA composition in ovulated eggs is unique in that glutamine (Gln) is markedly high (32% of total FAA) in comparison to other pelagic eggs of marine teleosts suggesting the importance of Gln for early development of the Japanese eel. Total FAA content decreased to 45% of the initial level by the 4th day after fertilization. Gln content showed a rapid decrease to 5% of the initial level within 2 days after fertilization. oLv content, measured by enzyme-linked immunosorbent assay using an antiserum against lipovitellin, decreased until the 4th day and was, thereafter, undetected. Total content of triacylglycerol (TG) was stable until the 2nd day, and then decreased until the 8th day. On the other hand, the content of phospholipids (PL) gradually decreased until the end of yolk sac absorption. From these data, we divided the pattern of nutrient utilization in embryos and larvae of Japanese eel into two phases: first, utilization of FAA (especially Gln) and oLv from the 1st day, followed by TG utilization from the 2nd to 4th day by which time more than 30% of the FAA and the oLv stock have been utilized. In addition, PL was utilized as a subsidiary energy source throughout development. Almost all the yolk nutrient were absorbed by the 8th day after fertilization, and it is considered that the larvae need to start feeding before this period.

The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder.

Development of the thyroid gland of the Japanese eel (Anguilla japonica) was studied with the use of tank-reared fish. A single thyroid follicle was first found in larvae at 29 days post hatching (dph), total length (TL) 12 mm. Until reaching 25 mm in TL (100 dph), leptocephali had one or two follicles per individual. The inner colloid of the follicles was weakly immuno-positive against the anti-thyroxine (T4) antibody. The number of thyroid follicles and the immunoreactivity later increased as the larvae grew. Thyroid hormones (TH) T4 and triiodothyronine (T3) were not detected in premetamorphic larvae by radioimmunoassays, but became detectable during metamorphosis. The maximum level of T4 was seen in fish at the end of metamorphosis and in just-metamorphosed juveniles, whereas T3 reached the highest level during metamorphosis and declined toward the end of metamorphosis. The results indicated that the thyroid gland first became active during metamorphosis in the development of eel.

Eggs of Japanese eel Anguilla japonica were obtained by artificial induction of maturation. Fertilization and hatching rates, and survival rates of normal larvae were examined. The levels of total lipids and polar lipids (PL) of high quality (HQ; >80% survival rate at 8 days post-hatch) eggs were significantly lower than low quality (LQ; hatching and survival rates were zero) eggs. The docosahexaenoic acid (DHA) level in PL of HQ eggs, however, was significantly higher than in LQ eggs. In contrast to DHA, arachidonic acid level was negatively correlated with egg quality variables and it was lower in HQ than in LQ eggs. Most of the egg lipid and fatty acid components showed larger variation in LQ than in HQ eggs.

Genetic improvement of the Japanese eel ( Anguilla japonica ) can be achieved by artificially controlling its life cycle using recent advances in reproductive biology. In this study, we developed 43 microsatellite loci to confirm Mendelian inheritance at 10 of them as well at 16 previously reported in two full-sib families produced by artificial insemination. In order to establish a base for aquaculture genetics of this species in the near future, these microsatellite loci were mapped in relation to the centromere by half-tetrad analysis using four artificially induced triploid families. The second division segregation frequency ( y ) of the microsatellite loci ranged from 0.008 to 0.968 (mean ± SD = 0.645 ± 0.298). These results suggest the presence of strong chiasma interference in the eel. Significant differences were observed for the map distances of microsatellite loci between the two isolation procedures. Microsatellites isolated using the enrichment procedure were mapped to various sites starting from the centromere to the telomere, whereas those from the conventional size-selected library showed a tendency to be distributed in the telomeric region.

A method was developed for assessing fertility, hatchability and larval survival in the Japanese eel, Anguilla japonica. Fertilized eggs were stocked in 24-, 48-, and 96-well plates at one egg to one well and maintained at 23 °C without removing dead larvae, changing the water or feeding. Upon hatching, the larvae were often trapped on the surface of the water where they died (surface death). The remaining larvae then showed rather stable survival in the three types of plates beyond the yolk-sac stage, which was completed by day 8 after hatching (DAH). Polyethylene glycol 6000 (PEG 6000) added to the rearing water at 1 or 10 μg/ml markedly suppressed surface death without apparent effect on hatchability, the survival profile or abnormality. These results indicated that the survival rate can be determined easily by stocking the fertilized eggs individually in the wells of microplates with water containing PEG 6000 and by rearing the hatched larvae until the completion of yolk absorption, with little management. By this method, a series of parameters for evaluating the egg quality, rates of fertilization, hatching, survival and abnormality were determined in the artificially maturation-induced Japanese eel: unstable fertility and frequent abnormal development were revealed to be impediments to constant production of larvae at first feeding.

Artificial reproduction and subsequent improved culture of the Japanese eel Anguilla japonica have not yet been achieved, but are expected in the near future. To allow the development of aquaculture activities, chromosome manipulation techniques are necessary for the genetic improvement of the eel. In the present paper suitable conditions were determined for heat shock treatment for the induction of triploidy. This was done using fertilized eggs obtained from gametes of cultured eels that were induced to mature by repeated injections of salmon pituitary extract, followed by an injection of 17α,20β-dihydroxy-4-pregnen-3-one to induce ovulation in the female eels and by repeated injections of human chorionic gonadotropin in the male eels. Three variables (i.e. timing after fertilization, temperature, and duration of the shock) were varied to maximize the yield of triploid larvae by examining hatching and triploid rates of treated groups. Ploidy was determined by DNA content flow cytometry and/or chromosome counting. The triploid yield was the highest (70%), when a 37°C heat shock was applied for 3 min, 10 min after fertilization. Under such conditions the rate of triploidy reached 70–100% and the rate of hatching was 5.2–24.6% when the control hatching rate was 28.5% on average.

Weekly injections of salmon pituitary extracts (SPE) were administered to female Japanese eel, Anguilla japonica at a dose of 20 mg/fish. This induced vitellogenesis and caused oocytes to reach the migratory nucleus stage. Later, a majority of the females that received an injection of SPE at a priming dose, followed 24 h later by 17,20β-dihydroxy-4-pregnen-3-one (DHP), ovulated 15 to 18 h after the final injection. In cultivated males, repeated injections of human chorionic gonadotropin (hCG) at a dose of 1 IU/g BW/week induced spermatogenesis and spermiation. Since potassium ions were revealed to be an essential constituent for the maintenance of motility in the eel spermatozoa, artificial seminal plasma containing KCl was designed as a diluent of milt, and enabled the preservation of milt for several weeks in refrigeration. As a result, artificial fertilization performed immediately after ovulation with pre-diluted and stocked milt consistently resulted in the production of high-quality gametes. Recently, a slurry-type diet made from shark egg yolk has been found to be a suitable feed for captive-bred eel larvae. Although preleptocephalus larvae can be reared with this diet beyond the depletion of their yolk and oil droplet stores, it remains inadequate because larvae reared under this way cannot be raised to the following stage. Therefore, the diet was improved by supplements of krill hydrolysate, soybean peptide, vitamins and minerals. Larvae fed on this new diet have grown to 50 to 60 mm in total length (TL), and have begun to metamorphose into glass eels approximately 250 days after hatching.

Hyaluronans (HAs) are glycosaminoglycans produced in the bodies of Anguilliform and Elopiform leptocephali, and play a role in metabolic energy. In mammals, HA synthesis-promoting substances (HASPS) up-regulate the expression of HA synthase (HAS) and increase the amount of HA in the body. In this study, Japanese eel leptocephali were fed a HASPS containing diet. We analyzed HAS1s and HAS2 expression, HA content, and their influence on growth. HASPS extracted from Grifola frondosa promoted HAS1s and HAS2 mRNA and HA content. Other than mammals, these results are first reported in vertebrate. Moreover, HASPS extracted from G. frondosa promoted leptocephalus growth. The relationship between growth and HA in the leptocephali is not yet clear. However, based on our results we hypothesize that HA is involved in the storage of energy, which is metabolized to sugars when needed for metabolic energy.

We studied the profiles of thyroid hormone receptors (TRs) in Japanese eels (Anguilla japonica) during development from hatched larvae to juveniles. Two TRαs (TRαA and TRαB) and one TRβ (TRβA) cDNA clones were generated by RACE. The TRαA, TRαB and TRβA cDNAs encoded 416, 407 and 397 amino acid proteins with much higher homologies to the Japanese conger eel (Conger myriaster) TRs than to other fish TRs. In a transiently transfected Japanese eel cell line, Hepa-E1, the TRs showed thyroid hormone (TH)-dependent activation of transcription from the TH-responsive promoter. Four TR cDNA clones, including TRβB reported in a previous study, were analyzed by real-time RT-PCR. The TR mRNA levels in hatched larvae were determined. The two TRβ mRNAs were present at low levels but there was a peak in the TRαs during the larval stage before metamorphosis. During metamorphosis, the two TRαs both exhibited peaks and expression of the two TRβs was higher than during the early growth stage. This expression pattern is similar to that of the Japanese conger eel. It is possible that thyroid hormones control the early development of Japanese eels and Japanese conger eels through TRs. This is the first analysis of the expression sequence of TRs during early larval stages of Anguilliformes.

Hatchery produced leptocephalus larvae of the Japanese eel (Anguilla japonica ) were reared on a research vessel and fed natural particulate organic matter (POM) collected in the Western North Pacific. A small net (36 cm diameter, 60 µm mesh) was vertically towed from 200 m depth to surface in 2013, and POM-enriched water (POMEW) filtered through 350 µm mesh was fed to the leptocephali for 11 days. Although the swollen gut corroborated the active ingestion of POM by the leptocephali, low survival rate and heavily melanized gut followed by necrosis in the mid-hindgut region of the leptocephali were observed. A large net (1.14 m diameter, 30 µm mesh) was used in 2014 and 2015, which was horizontally drifted subsurface (100–175 m). POMEWs filtered through 53 or 25 µm meshes were fed to the leptocephali for 5–18 days. Neither melanized gut nor necrosis occurred, but considerably low survival rate and little growth comparable with that in a starved condition were observed. No apparent shift in stable carbon and nitrogen isotope ratios was observed in the reared leptocephali. These indicated that the ingested POMs were not assimilated by the leptocephali and suggested that smaller particles may be important for the leptocephali.

Primordial germ cells (PGCs) are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4) are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica) embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio) for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders).

Pepsinogen is the precursor form of the gastric-specific digestive enzyme, pepsin. Ghrelin is a representative gastric hormone with multiple functions in vertebrates, including the regulation of growth hormone release, stimulation of food intake and gastrointestinal motility function. We investigated chronological changes in the distribution of pepsinogen-expressing cells by in situ hybridization and ghrelin-immunoreactive cells by immunohistochemistry in the Japanese eel (Anguilla japonica) during metamorphosis from the leptocephalus sage to the elver stage. The ghrelin-producing cells first appeared in the gastric cecum and pyloric portion of the stomach in the late phase of metamorphosing leptocephali, whereas the pepsinogen-producing cells were first detected in the early phase of the glass-eel stage. These suggest that endocrine cells differentiated earlier than exocrine cells in the eel stomach. Accompanying eel development, the distribution of ghrelin-producing cells spread to the esophagus and other regions of the stomach, but not to the intestine. These results may be related to the changes in dietary habits during metamorphosis in the Japanese eel.

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150–200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success., Little is known about the reproductive ecology of freshwater eels. In this article, the authors describe the capture of two species of eels together with eggs and newly hatched larvae, and suggest that spawning takes place during the new moon at shallower depths than previously thought.

Hyaluronan (HA) is a linear polysaccharide of high molecular weight that exists as a component of the extracellular matrix. The larvae (leptocephali) of the Japanese conger eel (Anguilliformes:Conger myriaster) have high levels of hyaluronan (HA) which is thought to help control body water content. We isolated glycosaminoglycans (GAGs) from Japanese conger eel leptocephali and measured the changes in tissue HA content during metamorphosis. HA content decreased during metamorphosis. In contrast, neutral sugar content increased during metamorphosis. We hypothesize that the leptocephali utilize a metabolic pathway that converts HA to glucose during metamorphosis. Glucose may then be metabolized to glycogen and stored in the juvenile life-history stage.

The growth rate of fish shows extensive plasticity in response to various environments. Metabolic responses of fish to excessive nutritional shortages such as starvation have been reported, but the effects of moderate nutrient shortage remain unclear. We examined expression levels of some genes related to ATP metabolism and to myogenesis, the RNA/DNA ratio, and the protein/DNA ratio of fish under different feeding conditions: a diet of 212–432% (frequent feeding, FR) or 32–82% (restricted feeding, RE) of initial body weight per week was supplied. The expression levels of nucleoside diphosphate kinase (NDK) -Z2 , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and myogenin genes of RE fish were higher than those of FR fish, although the RNA/DNA ratio and the protein/DNA ratio were unaffected by the feeding amount. Moreover, expression levels of NDK -Z2 and GAPDH were upregulated to a greater extent than those for myogenin and myostatin 1 under restricted feeding. Together, our results show that gene expression is more sensitive to nutrient conditions of fish than traditional indicators such as the RNA/DNA ratio. The ATP metabolic system is more sensitive to moderate nutrient shortages than the myogenic system.

The nuclear DNA contents of the Japanese eel larvae were measured to examine the genetic soundness of the eggs obtained by artificial induction of maturation and fertilization. Of the 12.4% abnormal polyploid larvae, 77.9% were triploid and the rest were haploid, tetraploid, pentaploid, or aneuploid.

The eel has long been esteemed as an important food fish in the world, especially in Japan, and has been used as an experimental fish for many fields of fish physiology. However, the decreases in eel resources have been a serious concern in recent years. The catches of glass eels as seedlings for aquaculture have shown a long-term decrease in both Europe and East Asia. To increase eel resources, the development of techniques for artificial induction of maturation and spawning and rearing their larvae have been eagerly desired. Recent progress of reproductive physiology of fish, especially mechanisms of oocyte maturation and ovulation in female and of spermatozoa maturation in male, facilitate to establish techniques for hormonal induction of maturation and spawning in sexually immature eels. With persistent effort to development of rearing techniques of larvae, we have first succeeded to produce glass eel. These applied techniques are may contribute to understand the basic reproductive physiology of the eel.