Swimming performance and energetics of the Japanese eel (Anguilla japonica) across growth and early migratory phases remain poorly quantified. We used swim-tunnel respirometry to compare selected environmental and developmental contrasts in yellow and early silver eels from the Tone River estuary. Critical swimming speed (Ucrit), optimal swimming speed (Uopt), minimum cost of transport (minCOT), oxygen consumption at Uopt, and low-flow MO2 measured at 0.1 m s−1 were evaluated in sequential freshwater trials at 25 and 18 °C, an exploratory Y2 freshwater–seawater comparison at 18 °C, and a Y2–S1 comparison in seawater at 18 °C. Ucrit and low-flow MO2 were numerically higher under FW25 than FW18, but this contrast was confounded with fixed trial order and fasting history. In Y2 eels, SW18 was associated with higher low-flow MO2 than FW18, whereas active-speed-derived variables were limited by sample size. Under SW18, no clear stage-associated differences were detected between Y2 and S1 eels. Laboratory-derived Uopt and Ucrit exceeded field-estimated active horizontal swimming speeds, but this comparison should be interpreted cautiously.

In the artificial seed production of Japanese eels, Anguilla japonica, the frequent occurrence of glass eels with jaw abnormalities has become a serious problem. To gain fundamental insights into this issue, we evaluated the effects of lower jaw abnormalities on survival during the glass eel stage and revealed that these abnormalities are lethal. Bone staining of glass eels and cartilage staining of leptocephali larvae showed that the abnormality was caused by mandibular dislocation in both stages. Focusing on the jaw opening angle (OA), we examined the relationship between OA and the occurrence of mandibular dislocation during the glass eel stage using logistic regression and receiver operating characteristic (ROC) analyses. The results indicated that the probability of mandibular dislocation increases when OA exceeds 69.3°. Therefore, individuals with an OA of 70° or greater were defined as having mandibular dislocation, and their appearance timing and body size were investigated. As a result, individuals with an OA of 70° or greater appeared when the total length reached approximately 35 mm, and their occurrence increased with age. These findings provide important insights into the mechanisms underlying mandibular dislocation and may contribute to its prevention in artificial eel seed production.

Recently, our group (Japan Fisheries Research and Education Agency, FRA) developed a new tank for the mass production of eel seedlings capable of producing 1,000 glass eels per tank. However, the development of eel seedling production technology has been undertaken only by a limited number of research institutions, and research using the new tank has been conducted solely at the FRA. Therefore, it remains unclear whether these technologies are equally applicable to other research institutions, i.e., whether they possess reproducibility and versatility. Accordingly, this study aimed to evaluate the reproducibility and practicality of the developed technology. For this purpose, technology transfer was implemented at the Miyazaki Prefectural Fisheries Research Institute (Miyazaki), the Kagoshima Prefectural Fisheries Technology and Development Center (Kagoshima), and Yamada Suisan Co., Ltd. (Yamada). As a result, all institutions successfully produced juvenile eels: 560 individuals at Miyazaki, 2,269 individuals at Kagoshima, and 1,347 juveniles at Yamada. Since two of the three institutions succeeded in producing over 1,000 seedlings from a single tank, it was confirmed that the technology using this tank is highly versatile.

A reliable system for the stable supply of high-quality hatched larvae is essential to ensure mass production of hatchery-reared glass eels for aquaculture. However, the conditions for large-scale larval incubation until the onset of the first feeding (that is, 7 days post hatching [dph]) remain suboptimal. This can lead to high mortality rates. Microbial interference is a critical factor influencing egg hatching success and larval survival in several marine fish species. In this study, we aimed to determine whether the high mortality observed during large-scale incubation of Japanese eel larvae at the preleptocephalus stage is attributable to microbial interference, and to optimize the incubation protocol through microbial management using alternatives to antibiotics. The survival rate of eel larvae at 6 dph was substantially improved by the addition of antibiotics to 200-L half-pipe tanks. This suggests that the high mortality observed during large-scale larval incubation was caused by bacterial activity in the rearing environment. Larval survival tended to be higher following treatment with silver nitrate, likely due to the antibacterial effects of silver ions. Daily treatment with silver ions at concentrations between 225 and 461 mu g/L provides suitable conditions for incubating up to 2200 hatched larvae per liter until the onset of the first feeding stage. Silver ions are acceptable alternatives to antibiotics owing to their established use as antibacterial agents across a wide range of applications, including human healthcare and various industrial processes. Therefore, microbial control using silver ions is a promising approach for the large-scale incubation of eel preleptocephali.

To determine suitable conditions for incubating Japanese eel Anguilla japonica eggs in 100 L tanks, we investigated the effects of different aeration and seawater exchange rates, and stocking densities of eggs on the hatching and survival of hatched larvae 6 days post-hatching (dph). The optimal aeration and seawater exchange rates required to be adjusted to 0.2 L/min and 0-2,880%/day, respectively. Aeration rates > 1.0 L/ min significantly decreased and increased the percentage of hatching eggs and hatched larvae with skin injuries, respectively, suggesting that high aeration rates would cause the death of hatched larvae by physical contact with each other or the tank walls. Furthermore, no difference was observed in hatching and survival of hatched larvae 6 dph between low (approximately 5x10(4) eggs) and high stocking densities (approximately 45x10(4) eggs) in tanks with seawater exchange rates of 720 and 2,880%/day. These results suggest that aeration and seawater exchange rates of 0.2 L/min and 720-2,880%/day, respectively, are suitable conditions for incubating approximately 50x10(4) eggs stocked in a 100 L tank.

Variations in stable oxygen isotopes ( δ 18 O) in the body water of animals reflect changes in δ 18 O values in environmental water and the animals' physiological processes. Euryhaline/anadromous/catadromous fish species move across salinity gradients; changes in δ 18 O values in environmental water thus lead to changes in δ 18 O values in body water. Few studies have systematically evaluated the effect of salinity on δ 18 O values in fish body water. Accordingly, this study demonstrated a well‐established approach to investigating the relationship between salinity and δ 18 O values in fish body water (blood water and muscle water). This study comprised two experimental designs. First, we implemented continual changes in salinity levels to assess the corresponding variations in δ 18 O values in the blood water of Japanese eels under controlled laboratory conditions. Second, field (river and estuary) studies were conducted for multiple fish species on Japan's Amami Island. Samples of fish blood water and muscle water were analysed in accordance with a general headspace protocol to estimate δ 18 O values; this estimation was conducted using isotope ratio mass spectrometry, which involved an equilibration method for sample preparation. In the laboratory experiment involving changes in salinity, the difference in δ 18 O values in fish blood water and ambient water was less than 1‰ within 2–3 days after fish were moved to a new environment. However, the difference varied with salinity and metabolism. In the field, obvious differences in δ 18 O values between fish blood water and environmental water were observed in the estuary area, implying that fish frequently move across salinity gradients. In the laboratory experiment and field observations, δ 18 O values were higher in muscle water than in blood water. Analysing δ 18 O values in environmental water, blood water and muscle water is useful for monitoring the behaviours of regional migratory fish, such as temporary feeding in estuaries. Our analytical method is ideal for measuring δ 18 O values in fish body water because it enables analysing various organic tissues by using small amounts of body fluids, facilitating the examination of the ecology of aquatic animals.

The Japanese eel, Anguilla japonica, is an economically important aquaculture fish, but the mass production of artificial seedlings is still in the developmental stage. One of the most important issues for producing more eel seedlings is to increase tank size. In this context, we investigated the effect of tank diameter and axis length. We found that it was possible to increase tank size by extending the axis of the tanks, and achieved rearing performance comparable to that of small experimental tanks. Based on this result, a new cylindrical tank prototype with an axis length of 1500 mm and diameter of 400 mm was manufactured, with subsequent rearing data showing that larvae reared in this new tank showed better growth than those in the previous large-scale tank. The tank structure was then refined and changed to a semi-cylindrical design. In addition, tank components were changed to use fiber-reinforced plastics (FRP), with the aim to produce low-cost tanks on a large scale. This enabled long-term rearing experiments to be conducted in the new FRP tanks, the results of which demonstrated that approximately 1000 glass eels could be produced in each tank, with survival rates of around 4

Intensive efforts have been ongoing to establish the reliable method for rearing large numbers of leptocephali for mass production of glass eel seedlings for aquaculture. Nevertheless, massive mortality was observed to occur due to filamentous bacteria, and strain Aur-1 was isolated as the causative bacterium. The new isolate was identified as Aureispira anguillae based on taxonomic characterization. The isolated Aur-1 strain caused mortality to 111 days old leptocephali at an LD 50 of 2.6 × 104 MPN/mL at 2 d, indicating that the isolated strain was pathogenic to eel leptocephali and the development of a control method for this bacterium is necessary for mass production of eel leptocephali. In order to investigate methods for controlling this bacterium, the effectiveness of several antibacterial substances was examined. The strain showed high sensitivity to silver ions, which inhibited its growth at more than 90 μg/L of silver nitrate. The addition of silver ions to rearing water at concentrations greater than 100 μg/L was clearly effective in inhibiting the mortality of leptocephali infected with Aur-1 in challenge trials. Even at 50 μg/L of silver ions, infective mortality of the leptocephali was reduced. Silver ions did not affect the survival of leptocephali during the experimental period at doses of less than 500 μg/L. This suggested that the infectious disease caused by Aur-1 can be controlled by adding silver ions to the water when rearing Japanese eel leptocephali.

Background Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing. Results During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the "most characteristic genes" during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel. Conclusion Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

The only diet that has been capable of rearing eel larvae to the glass eel stage in captivity is the shark egg (SE)-based diet (SE). This study investigated the potential of alternative dietary components, namely hen egg yolk (HEY), milk proteins, and fish protein hydrolysate (FPH), on the growth and survival of eel larvae. In the first experiment, a diet containing HEY and skimmed milk powder (HS) was compared to SE. There were no significant differences in growth and survival rate between the two diets except in the early part of the experiment period. In the second experiment, HS was modified by the addition of FPH and casein (FC), and larval performance was evaluated for three dietary regimens (SE, HS, and FC). The performance of larvae fed HS and FC was found to be comparable or superior to those fed SE. It was observed that larvae fed the alternative diets to SE were able to progress to glass eels. However, more skeletal abnormalities were observed in HS in experiment 1. The results of this study indicate that a combination of HEY and milk proteins is suitable as a larval eel diet, and that it has the potential to replace SE.

Visual implant elastomer (VIE) marking was evaluated as a tool for individual tagging of larval Japanese eels, Anguilla japonica , during rearing conditions. Combinations of eight colors of VIE tag were used to individually mark leptocephalus larvae (104 -110 days post hatched) by injecting them with either a mixture of a silicone color material and a curing agent, or only color material. Injections were at three locations on the dorso-lateral body surface for individual identification, and a sham -operated control group was also prepared. Tag retention was 100% with or without curing agent, and individuals could be tracked until larvae completely metamorphosed into glass eels. After some initial mortality, the survival and growth rates were not different between each treatment group. These results suggested that VIE tagging could provide good individual larva identification method for research during rearing conditions.

To evaluate the quality of glass eels that were derived from late-stage Anguilla japonica leptocephali raised on an old-type of shark egg-based diet (SE) or a new-type of fish meal-based diet (FSY), specific gravity (SG), feeding tests, and body compositional and RNA-seq gene-expression analyses were conducted. The largest content difference between the two dietary groups was in lipids, with the body-compositional index (BCI) being 29.2% lower in FSY-leptocephali and 43.1% lower in FSY-glass eels at day 3 post-metamorphosis, but the difference between groups narrowed to 21.6% at post-metamorphosis first feeding. FSY-leptocephali had heavier SG, a larger number of differentially expressed genes (DEGs) related to muscle development and/or movement, and higher creatine kinase expression, suggesting that they settled easily, and were forced to swim more against fast bottom currents in the tanks while consuming more energy. It was found that the days until first feeding in FSYglass eels was significantly shorter than in SE glass eels, with lower variability among eels. The number of DEGs was larger in FSY-glass eels, with the largest difference of 3.6 times occurring at day 10 post-metamorphosis, especially in the metabolism category, but at first feeding, they decreased drastically in both groups narrowing the difference. In the FSY-glass eels, the number of DEGs related to lipid, amino acid, and protein metabolisms were larger at days 3-20 after metamorphosis. Total enzyme expression levels for acylglycerol degradation, ketone body biosynthesis and citric acid cycle pathways also tended to be higher, suggesting that they obtained energy aerobically using lipids and other materials. Based on the patterns of up- and down-regulation of expression levels, growth hormone, ghrelin/obestatin prepropeptide and pro-melanin-concentrating hormonelike were detected as possible orexigenic hormones, and proopiomelanocortin and nucleobindin-2 as possible anorexigenic hormones. These results indicate that diet quality affects not only SG and other characteristics of leptocephali, but even body composition, metabolism and time to first feeding in the post-metamorphic glass eels for use as seedlings for aquaculture.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15–30 °C, pH 7–9, and seawater concentrations of 60–150

Temporal changes of feeding incidences, digestive organ tissues, and mRNA expression of digestive enzymes were investigated in artificially reared Anguilla japonica glass eels. Incidences of daily feeding and cumulative first feeding exceeded 50% at day 40 and 33, respectively, but tended to increase gradually. Time until first feeding varied from day 11-61 among individuals. Glass eels without food after metamorphosis showed notable developmental changes in their digestive system that included increases in esophagus goblet cells and blood vessel diameters, elongation of the stomach, differentiation and increased number of gastric glands, and in-creases in gall bladder sizes. A distinct qualitative change, which was gastric gland differentiation, occurred in all eels until week 2 after metamorphosis. All quantitative character values showed trends of gradual changes in their averages and had high coefficients of variation, especially for goblet cell numbers. Few deaths and no histological features related to severe starvation such as notably more hepatocyte necrosis or desquamation of intestinal epithelial cells were observed even after 9 weeks. Hepatocyte-vacuole stored glycogens and pancreatic zymogen granules were also found, suggesting that they were not expressing symptoms of starvation. Relative expressions of five digestive enzymes were lowest at week 0 and increased gradually in their averages with the passage of weeks, along with large variations among fish. This study found that half of the post-metamorphic glass eels began feeding after 4 weeks, but that there were large individual variations in the timing of feeding onset, the degree of developmental of their digestive organs, and in digestive enzyme expressions.

Growth hormone (Gh) regulates somatic growth in fishes, particularly through the Gh -insulin-like growth factor-I (Igf-I) axis. In this study, recombinant Japanese eel Ghs with or without C-terminal peptides of human chorionic gonadotropin (CTP), which are known to prolong the half-life, were produced using the HEK 293 and CHO expression system. The effect of recombinant Gh administration to eel larvae on their somatic growth was investigated in short-term feeding experiments, and it was found that three types of recombinant Ghs with CTP (CTP-reGh, reGh-CTP and reGh-CTP x 2) were more effective in promoting somatic growth in eel larvae than recombinant Ghs without CTP. Among the three recombinant Ghs with CTP, reGh-CTP x 2 had the highest growth-promoting effects, however only when provided in the short term. After long-term administration of reGh-CTP x 2, there was no difference in growth between the Gh administrated group and the control group. The survival rate of eel larvae were not affected by recombinant Ghs. In addition, the mRNA expression of gh, Gh receptors, Igf-I and IGF-II were measured by quantitative real-time PCR, and significant reductions in the expression of gh, Gh receptors and Igf-I were observed. These findings provide useful tools to study the mechanisms of somatic growth and increase understanding of Gh regulation in anguillid eel larvae.

Anguillid eels are the iconic example of catadromous fishes, because of their long-distance offshore spawning migrations. They are also a good model for research on the onset mechanisms of migrations to breeding areas, because the migrations begin in inland waters. When eels transform from yellow eels to silver eels, it is called silvering. Silver eels show various synchronous external and internal changes during silvering, that include coloration changes, eye-size increases, and gonadal development, which appear to be pre-adaptations to the oceanic environment and for reproductive maturation. A strong gonadotropic axis activation occurs during silvering, whereas somatotropic and thyrotropic axes are not activated. Among various hormones, 11-ketotestosterone (11-KT) drastically increases during spawning migration onset. Gradual water temperature decreases simulating the autumn migratory season, inducing 11-KT increases. Administration of 11-KT appeared to cause changes related to silvering, such as early-stage oocyte growth and eye enlargement. Moreover, 11-KT may be an endogenous factor that elevates the migratory drive needed for the spawning migration onset. These findings suggested that water temperature decreases cause 11-KT to increase in autumn and this induces silvering and increases migratory drive. In addition, we newly report that 11-KT is associated with a corticotropin-releasing hormone that influences migratory behavior of salmonids. This evidence that 11-KT might be among the most important factors in the spawning migration onset of anguillid eels can help provide useful knowledge for understanding endocrinological mechanisms of the initiation of spawning migrations.

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.

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 oesophageal muscle and pancreas occurred during metamorphosis. Formation of lateral line canals advanced continuously until the yellow eel stage. When the second developmental phase was initiated at the G1 stage, cone photoreceptor cells appeared, and the formation of oesophageal, stomach and intestinal muscles was initiated. Differentiation of gastric glands began at 1 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 swimbladder initiated inflation, the stomach fully expanded and the rectal longitudinal fold changed to a circle. Swimbladder 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 ongoing after metamorphosis into glass eels, and the M1-E2 stages are considered to be a homologous phase to first metamorphosis, which is a transformation from the larval to the juvenile stages 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.

Whole-genome duplication and genome compaction are thought to have played important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis , belongs to the superorder Stomiati, order Osmeriformes. Stomiati is phylogenetically classified as sister taxa of Neoteleostei. Thus, ayu holds an important position in the fish tree of life. Although ayu is economically important for the food industry and recreational fishing in Japan, few genomic resources are available for this species. To address this problem, we produced a draft genome sequence of ayu by whole-genome shotgun sequencing and constructed linkage maps using a genotyping-by-sequencing approach. Syntenic analyses of ayu and other teleost fish provided information about chromosomal rearrangements during the divergence of Stomiati, Protacanthopterygii and Neoteleostei. The size of the ayu genome indicates that genome compaction occurred after the divergence of the family Osmeridae. Ayu has an XX/XY sex-determination system for which we identified sex-associated loci by a genome-wide association study by genotyping-by-sequencing and whole-genome resequencing using wild populations. Genome-wide association mapping using wild ayu populations revealed three sex-linked scaffolds (total, 2.03 Mb). Comparison of whole-genome resequencing mapping coverage between males and females identified male-specific regions in sex-linked scaffolds. A duplicate copy of the anti-Müllerian hormone type-II receptor gene ( amhr2bY ) was found within these male-specific regions, distinct from the autosomal copy of amhr2 . Expression of the Y-linked amhr2 gene was male-specific in sox9b -positive somatic cells surrounding germ cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells in sexually undifferentiated gonads of both genetic males and females. Loss-of-function mutation for amhr2bY induced male to female sex reversal. Taken together with the known role of Amh and Amhr2 in sex differentiation, these results indicate that the paralog of amhr2 on the ayu Y chromosome determines genetic sex, and the male-specific amh-amhr2 pathway is critical for testicular differentiation in ayu.