Induction of spermatogenesis in Japanese eel by recombinant goldfish gonadotropins

We previously demonstrated the biological activities of single-chain recombinant gonadotropins (scGTHs) of goldfish Carrassius auratus follicle-stimulating hormone (scFSH) and luteinizing hormone (scLH), produced by a baculovirus–silkworm larvae system, by using in vivo bioassays with some fishes including Japanese eel Anguilla japonica. Among the bioassays, we succeeded in induction of spermatogenesis of sexually immature male Japanese eels by both scFSH and scLH, especially resulting in the occurrence of spermatozoa in scLH-administered males. However, those recombinant hormones did not induce enlargement of testes. In order to further confirm the potency of recombinant GTHs for use in aquaculture species, we administered scFSH and scLH to males of Japanese eel at higher dosage and frequency (eight times with 2–5 days interval) than those of the previous study (five or six times with 7 days intervals), including combination of scFSH and scLH administration (scFSH–scLH). Gonadosomatic indices (GSI) of scLH- and scFSH–scLH-administered males were larger than those of initial control males and of control males that were injected with saline. Enlargement of testes was also confirmed by measurement of testicular lobe size in scFSH-, scLH-, and scFSH–scLH-administered males. By histological observation, occurrence of spermatozoa was confirmed in scLH- and scFSH–scLH-administered eels. Although milt production was not induced, higher dosage and frequency of scGTH administration was effective in promoting testicular development of immature eels. Thus, single-chain fish GTHs produced by the baculovirus–silkworm larvae system could be a useful tool for promotion of gonadal maturation in aquaculture fishes.     

Occurrence of estuarine and sea eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and a migrating silver eel <Emphasis Type="Italic">Anguilla anguilla</Emphasis> in the Tokyo Bay area,Japan

Otolith microchemical analyses of the strontium (Sr) and calcium (Ca) concentrations in the eels Anguilla japonica and A. anguilla caught in Tokyo Bay were undertaken to reconstruct the eels’ migratory histories. A. japonica in the yellow stage (immature stage) were caught in a bay without any adjacent rivers or streams. A. anguilla was in the silver stage (early maturing stage), and the eel was confirmed to have just begun spawning migration to the open ocean from Tokyo Bay based on the otolith Sr:Ca ratios, which showed a typical catadromous life history with low Sr:Ca ratio values throughout the eel’s life after recruitment. The mean Sr:Ca ratios in A. japonica from the elver mark to the otolith edge indicated the eels belonged to several general categories of migratory histories, including sea eels (average Sr:Ca ratio ≥6.0 × 10⁻³) and estuarine eels (average Sr:Ca ratio 2.5 to 6.0 × 10⁻³) based on the criteria reported previously in A. japonica. All eels had a certain freshwater life period, although the period was highly variable among fish. These results indicate that A. japonica has a flexible pattern of migration, with the ability to adapt to various habitats and salinities.     

Video observation of an eel in the Anguilla japonica spawning area along the West Mariana Ridge

Some anguillid spawning areas are known based on collections of small larvae, but recently for the Japanese eel Anguilla japonica, adult spawners have been caught in trawls and their eggs and preleptocephali collected. The spawning area of A. japonica is located along the western side of the West Mariana Ridge, but the natural spawning behavior of this species or that of any other anguillid species has never been observed. This study reports on the first effort to observe spawning aggregations of anguillid eels that was conducted by the R/V Yokosuka using the Shinkai 6500 submersible and a Deep-Tow camera system in the A. japonica spawning area in July 2012. The submersible was deployed mostly at 200–800 m during daytime and the Deep-Tow was deployed mostly at 130–250 m during nighttime, both in multiple oblique depth tracks along linear transects. Various fishes and invertebrates were seen in the pelagic environment during day and night, but no spawning aggregations were observed. One eel was briefly recorded by a Deep-Tow camera at 20:13 on 17 July (2 days before new moon) at a depth of 179 m. The eel was recorded for <1 s as it passed in front of the camera. Its anterior body and head shape were consistent with a male A. japonica, or possibly a Derichthys serpentinus eel, but not with other mesopelagic eels. Because the tail region of the eel was not visible, species identification was not possible.     

Growth differences of Japanese eels Anguilla japonica between fresh and brackish water habitats in relation to annual food consumption in the Kojima Bay‐Asahi River system,Japan

To learn about the relationships between feeding and growth of temperate eels in freshwater and brackish water habitats, we analysed 533 yellow‐phase Japanese eels Anguilla japonica collected in both types of habitats in southeastern Japan. Because male eels were very rare in each habitat (FW, N = 1; BW, N = 20), characteristics of female eels were compared between the different habitats. Annual food consumption was evaluated with the consideration of instantaneous food consumption and annual activity period. Stomach fullness index (stomach content weight/body weight) was used as an indicator of instantaneous food consumption. The ratios of number of months with eel catch to those when eel sampling was conducted were used as an indicator of activity period. Female yellow eels tended to be older and slower growing in fresh water (N = 78; age, mean ± SD = 7.9 ± 2.4 years; growth rate, 59.8 ± 14.0 mm year^?1) than in brackish water (N = 229; age, 5.5 ± 1.8 years; growth rate, 90.1 ± 24.4 mm year^?1). Irrespective of sex, yellow eels in brackish water had a higher stomach fullness index and a greater ratio of months with eel catches, indicating greater annual food consumption by brackish water eels. These results indicate that greater annual food consumption contributes to the greater growth rates of Japanese eels in brackish water habitats.     

Genetic identification and morphology of naturally spawned eggs of the Japanese eel Anguilla japonica collected in the western North Pacific

Eggs of the Japanese eel Anguilla japonica collected in the western North Pacific were identified by onboard species-specific polymerase chain reaction (PCR) and DNA nucleotide sequencing after the cruise. Fish eggs of various species were collected by large plankton net tows at 12 stations along the southern part of the West Mariana Ridge on 19–25 May 2009. A total of 43 fish eggs were distinguished morphologically as possibly being of A. japonica. Thirty-one of those were analyzed by PCR, which included 15 eggs collected at 12°50–55′N, 141°15–20′E (in 5 tows) that showed positive results. The 16S ribosomal RNA (rRNA) gene sequences of eggs determined after the cruise indicated that 31 A. japonica eggs had been collected. The remaining eggs were of mesopelagic eel species (Serrivomeridae and Derichthyidae), or unidentified species. The morphological characteristics of the A. japonica eggs were consistent with those of artificially spawned eggs, except they had a slightly larger diameter. The egg diameter range did not overlap with those of mesopelagic eels of the Serrivomeridae, which often spawn in the same area as A. japonica. These results suggest that egg diameter and embryo shape can be used to morphologically identify naturally spawned A. japonica eggs.     

Decreasing dietary lipids improves larval survival and growth of Japanese eel Anguilla japonica

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.     

Freshwater eels: A symbol of the effects of global change

Temperate eels Anguilla anguilla (European eel), A. rostrata (American eel) and A. japonica (Japanese eel) are three catadromous species which have been declining since the 1970s/1980s despite their remarkable adaptive capacity. Because of their specific life cycles, which share distant oceanic spawning grounds and continental growth stage, eels are affected by five components of the global change: (a) climate change affecting larval survival and drift, (b) an increase in pollution leading to high levels of contamination exacerbated by their high lipid levels, (c) increasing fragmentation and habitat loss that reduce dramatically the amount of available habitats and induce increased spawner mortality, (d) the appearance of Anguillicola crassus a parasitic alien nematode that impairs spawning success, and (e) the impact of commercial and recreational fisheries for all life stages of eel. In this context, the rapid increases of pressures during the “Great Acceleration” have surpassed the adaptive capacity of eels. This illustrates that cumulative effects of global change can lead to the collapse of species, even in species that have amazingly high adaptive capacities.     

Development of an in vitro culture system for producing eel larvae from immature ovarian follicles in Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis>

To standardize conditions during the final maturation and ovulation of ovarian follicles from Japanese eel, we have developed a culture system for the production of fertilizable eggs from post-vitellogenic ovarian follicles in vitro. Post-vitellogenic ovarian follicles were incubated in culture medium supplemented with 17α,20β-dihydroxy-4-pregnen-3-one (DHP) with or without bovine serum albumin (BSA) to assess the effects of protein concentration. Eggs that ovulated during incubation were fertilized, and the remaining follicles were incubated in prostaglandin F_2α (PGF_2α) for a further 3 or 6 h before fertilization. Male eels were injected repeatedly with human chorionic gonadotropin. The quality of eggs obtained under the different culture conditions was evaluated after artificial fertilization in terms of hatching success. Hatching rates tended to decrease with increasing concentrations of BSA in the incubation medium in a dose-dependent manner. The addition of PGF_2α drastically increased the number of eggs that ovulated, but the rate of hatching was greatly decreased compared with eggs obtained earlier by DHP incubation alone. The larvae obtained from artificially fertilized eggs produced in vitro survived for 14 days without feeding. We conclude that in vitro culture systems thus have a great potential for the acquisition of good quality eggs under tightly controlled artificial conditions, culminating in the production of eel larvae.     

Late arrival of Anguilla japonica glass eels at the Sagami River estuary in two recent consecutive year classes: ecology and socio-economic impacts

To determine the actual status of the recruiting glass eel stock of Anguilla japonica, we conducted a continuous monthly monitoring program for 2?years between November 2009 and October 2011 at the Sagami River estuary, Japan. A total of 114 and 372 A. japonica glass eels were observed in the 2009?C2010 and 2010?C2011 seasons, respectively. Recruitment patterns were the same in both years, starting in early winter (December in 2009 and November in 2010), increasing slightly until March, decreasing in April, but attaining the largest peak in June, after which no glass eels were observed during August?COctober. The Japanese eel has been known to spawn mainly in the summer, and glass eels recruit to their freshwater growth habitats during the winter to early spring. Our results clearly demonstrate an unexpected late arrival of glass eels in the early summer for two recent consecutive year classes. The summer recruitment found in our study indicates the unusual phenology of the Japanese eel, which may be a possible response to recent climate change.     

Virus infections of the European Eel in North Rhine Westphalian rivers

Viral infections have been suggested to play a role in the decline of the panmictic population of the European eel (Anguilla anguilla). However, despite the importance of knowledge about pathogenic eel viruses, little is known about their spread in the wild European eel population and only a few eel pathogenic viruses have been described so far. In this study, we aimed to investigate the health status of the A. anguilla stock in North Rhine Westphalia (NRW) State of Germany. For this purpose, we examined tissue samples of 16 elvers, 100 yellow eels and 6 silver eels, sampled from the rivers Rhine, Lippe and Ems. Virus detection was performed via a combination of cell culture and PCR. Next to the detection of frequently encountered pathogenic eel viruses (anguillid herpesvirus 1 and eel virus European X (EVEX)), we isolated the eel picornavirus 1 (EPV-1) from tissue of yellow eels and elvers and demonstrate the distribution of EPV-1 in wild eel population in NRW.     

Predation on glass eels of Japanese eel <Emphasis Type="Italic">Anguilla japonica</Emphasis> in the Tone River Estuary,Japan

Aquaculture of Japanese eel Anguilla japonica relies upon the natural recruitment of their glass eels (juveniles); however, predation that could influence glass eel recruitment remains unknown. In the present study, we aimed to elucidate the proportion of predation on A. japonica glass eels through stomach content analysis of predatory fishes collected in the estuary region of the Tone River system and its vicinity in Japan. Species of the predated glass eels were identified by DNA barcoding. A total of 270 predatory fishes of 15 taxa was collected over 2 years. The overall proportion of predation on glass eels, genetically identified as Japanese eel, was 0.7%, but this rose to 2.0% when data were limited to fishes caught during the peak months of glass eel recruitment. A glass eel was found in the stomach contents of a channel catfish Ictalurus punctatus, an invasive species in this river system, and a blackfin sea bass Lateolabrax latus. These fishes are therefore considered potential predators of A. japonica glass eels. However, as the proportion of predation was low, and the glass eels represented only small proportions of predator stomach contents, further investigation is needed for a better understanding of predation on A. japonica glass eels, and its effects on the early life stages of this endangered species.     

Application of ELISA-based kit for detecting AOZ and determining its clearance in eel tissues

Furazolidone, an antibacterial drug that was once widely used in the livestock industry and aquaculture, is now prohibited in numerous countries. It is difficult to detect residual furazolidone because it is readily metabolized in animal tissues but, by using and liquid chromatography coupled with tandem mass spectrometry, its metabolite, 3-amino-2-oxazolidinone (AOZ) can be detected. Here we describe the validity of an enzyme-linked immunosorbent assay (ELISA) kit to detect AOZ in Japanese eel Anguilla japonica tissue. ELISA is capable of detecting AOZ at 1.0 μg/kg in an eel sample with excellent accuracy and precision. Our results show that ELISA is suitable for regulatory purposes and for studying the fate of AOZ residues in eel treated with furazolidone. To measure the persistence of AOZ in eel tissues, eels (1.4–6.5g) were immersed in tanks containing 2 and 10 mg furazolidone/L for 3 h, and then maintained in a tank supplying well water for the next 160 days. The half-lives of AOZ, calculated from the linear terminal part of the excretion curve, were 25.0 days in muscle and 21.6 days in liver from fish exposed to 2 mg/L furazolidone. In the eels treated with 10 mg/L furazolidone, by contrast, high levels of AOZ were detected in liver and muscle, but the half-lives of AOZ were similar to those in fish treated with 2 mg/L furazolidone. The half-lives of AOZ in eel tissues were prolonged by the condition of low water temperature.     

Compositions and functions of the hatching froth from ricefield eel (<Emphasis Type="Italic">Monopterus albus</Emphasis> Zuiew)

The ricefield eel (Monopterus albus) is an economic fish species in China. To improve its artificial reproduction, we studied the compositions and functions of the hatching froth secreted by ricefield eels. The froth showed a viscosity of 2.72 mPaS, composed of glycoproteins with a sugar content of 0.156 mg/ml and a protein content of 0.250 mg/ml at a ratio of 1.60. The froth proteins were identified by SDS-PAGE as four proteins with the molecular weights of 42.5, 58.6, 65.3, and 98.2 kDa respectively. Their total amino acid content was 201.88 mg/100 ml, consisting of 16 kinds of amino acids. Filed observation revealed three beneficial effects of the froth on the hatching of the eel eggs. First, the froth increased the hatching rates of fertilized eggs at a range of water temperatures. Second, the froth decreased the rates of infection of the fertilized eggs by the water mold. Finally, the froth accelerated velum breakage in the fertilized eggs. Of the different hatching methods, froth incubation achieved the highest hatching rate and larvae survival rate. These results demonstrate the indispensable function of the froth of ricefield eels, and offer practical reasons for protecting the froth during the breeding season.     

Exotic silver eels Anguilla anguilla in Japanese waters: seaward migration and environmental factors

To understand the ecology of the exotic silver European eel Anguilla anguilla introduced into Japanese waters, the migratory behavior of 106 specimens captured in the coastal waters of Japan between April 1997 and March 2002 was analyzed. Their migratory behavior was apparently correlated with various environmental factors, particularly photoperiod, water temperature, lunar phase, and passage of atmospheric depressions, and was similar to the behavior of the species in European waters. These findings suggest that transplanted European eels retain their ability to respond to environmental cues for seaward migration in similar temperate habitats. The timing of the migration of silver European eels coincided with that of the native Japanese eels A. japonica, suggesting that the silver European eel was synchronized physiologically with the native eel by the same environmental factors.     

Evidence of niche segregation between freshwater eels and conger eels in Kojima Bay, Japan

The aim of this study was to examine the possible interspecific interactions between the freshwater eel Anguilla japonica and the marine conger eel Conger myriaster. Spatial distribution, foraging time, and diet were compared between 380 A. japonica and 221 C. myriaster juveniles in a large brackish water bay and a tributary river estuary. Almost no C. myriaster were caught in the estuary, but both species coexisted in the bay at similar densities. For both species, percentages of eels with stomach contents were greater in the morning than in the afternoon, indicating they forage at night. Both species mainly fed on benthic crustaceans in the bay, but the size of the main prey species, mud shrimp Upogebia major, was significantly larger in A. japonica. The difference in prey size appears to be related to the eel body size (A. japonica: 559.5 ± 88.0 mm, C. myriaster: 356.4 ± 56.0 mm), which probably reflects the differences in the life history-related movements of these eels, with A. japonica using the bay at large sizes and C. myriaster using the bay as a nursery area before moving to deeper water. The differences in their migratory and movement patterns may result in niche segregation in the brackish bay where both species coexist.     

Insulin-like growth factor I of Japanese eel, Anguilla japonica: cDNA cloning, tissue distribution, and expression after treatment with growth hormone and seawater acclimation

In an attempt to understand growth regulation in the Japanese eel, Anguilla japonica, we cloned insulin-like growth factor-I (IGF-I) cDNAs and examined their mRNA expression in several tissues. Two eel IGF-I (eIGF-I) cDNAs encoding preprohormones, eIGF-I-Ea1and eIGF-I-Ea2, were cloned from the liver by polymerase chain reaction (PCR). The preproIGF-Is were identical in signal peptide and mature IGF-I, but different in the E domain—eIGF-I-Ea2 mRNA was 36 bp longer than eIGF-I-Ea1 mRNA. Eel IGF-I was 83–94% identical with that of teleosts, 71% identical with that of dogfish, 87% identical with that of bullfrog and chicken, and 83% identical with that of humans. In both males and females the highest eIGF-I-Ea1 mRNA levels were observed in the liver, with detectable levels also found in the gills, heart, stomach, spleen, kidney, intestine, swim-bladder, muscle, and gonads. eIGF-I-Ea1 mRNA levels in the liver were higher in females than in males whereas in the intestine they were lower than in males. eIGF-I-Ea2 mRNA was detected in all the tissues examined and at similar levels in males and females. In this experiment higher eIGF-I-Ea1 mRNA levels were observed in the liver of larger glass eels than in those of smaller fish. eIGF-I-Ea2 mRNA levels were also higher in larger eels, although they were lower than IGF-I-Ea1 mRNA levels. Both eIGF-I mRNA levels in liver were positively correlated with the body size of the␣glass eels. Intraperitoneal injection of recombinant eel GH (reGH), 0.25 μg g⁻¹ body weight, into glass eels resulted in a significant increase in both eIGF-I mRNAs in the liver 1 day after injection compared with control fish, but no elevation was observed 2 days after injection. Incubation of liver slices with reGH at concentrations of 10, 100, and 1,000 ng mL⁻¹ for 24 h resulted in a significant concentration-dependent increase in the levels of both eIGF-I mRNAs. Higher levels of eIGF-I-Ea1 and Ea2 mRNA were observed in the gills ofseawater-reared eels than in those of freshwater-reared fish, but no differenceswere observed in the whole kidney. These results suggest that IGF-I is involved in the regulation of somatic growth and also in adaptation of the Japanese eel to seawater.     

Evaluation of the pigmentation stages and body proportions from the glass eel to yellow eel in Anguilla japonica

The pigmentation stages of Anguilla japonica were classified from the glass eel to yellow eel stage using 412 wild-caught eels from Hamana Lake and detailed developmental observations of 10 laboratory-reared eels. The sequential appearance of pigment was similar to that in A. anguilla, except for delayed pigment formation on the nerve cord of A. japonica. The general classification of pigmentation stages (V_A–VI_B) in A. anguilla was applicable to A. japonica, but the V_B stage could be separated into two stages (V_B1, V_B2) in A. japonica. The completion of guanine deposition on the intra-abdominal membrane may be a trait to discriminate the yellow eel stage (VII) from the VI_B stage. In natural conditions, wild-caught 0-age eels (n = 3,298) did not increase in size and retained a slender body form until VI_A4, and then became thicker during VI_B. The transition between body forms and growth may correspond to their ecological change from using passive transport in glass eels with tidal flow into rivers, to their settlement and further movements of elvers upstream. Considering this behavioral transition and pigmentation progression, it is proposed that the terms "glass eel" and "elver" could be defined as stages V_A–VI_A4 and VI_B, respectively.     

Long‐term records (1781–2013) of European eel (Anguilla anguilla L.) production in the Comacchio Lagoon (Italy): evaluation of local and global factors as causes of the population collapse

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日本鳗鲡产卵后生存及其繁育

为证明日本鳗鲡(Anguilla japonica)生活史最后一步一产后鳗的命运,本研究模拟产后的日本鳗鲡继续在海水中养殖,观察其存活率及繁育情况.结果表明,产后鳗在海水中停食约18 d后,体能得到恢复,部分亲鱼开始出现摄食,1个月左右全部恢复摄食,经244 d养殖,雌、雄鳗体质量增加,存活率达94.6%.随后,给产后鳗注射外源性促性腺激素(鲤鱼脑垂体匀浆CPE和人绒毛膜促性腺激素HCG)后激发其退化的性腺(卵巢和精巢)重新发育(与当年银鳗作对照).通过性腺组织切片观察产后鳗和对照鳗性腺发育成熟的全过程及其差异,发现产后鳗起初性腺发育比当年银鳗差,但经多次注射激素后,产后鳗性腺成熟与当年银鳗同步,证明产后鳗生殖细胞对激素的敏感件高.应用17α,20β-双羟孕酮和促性腺激素释放激素类似物(GnRH-A3)使催熟的产后鳗和对照鳗均产卵和排精,并孵化出仔鱼,从而有力地证明,鳗鲡产后虽体质弱,但待体能恢复后能够继续生存和繁殖.本研究旨在探讨利用产后鳗作为今后鳗鲡人工繁殖亲鳗的可行性.     

Several possible spawning sites of the Japanese eel determined from collections of their eggs and preleptocephali

The spawning area of the Japanese eel is located at the southern part of the West Mariana Ridge in the western North Pacific, but their spawning events have not been observed. To further understand Japanese eel spawning ecology, an interdisciplinary research survey by the R/V NATSUSHIMA (NT14-09, 14 May–4 June 2014) was conducted to detect spawning sites based on the seamount, salinity front, new moon and third quadrant (spawning south of front, west of ridge) hypotheses. Attempts were made to film spawning events with underwater camera systems and to consider if eels might be detected in hydroacoustic observations. Although no Japanese eels or spawning events were video-recorded and no eel aggregations could be clearly identified acoustically, three eggs were collected at two stations in the third quadrant region at or just south of 13° N on 26 and 27 May. Three or four days later, newly hatched preleptocephali were collected at two stations far to the south, including 224 at a station > 160 km southwest of the egg catches, and a few preleptocephali were caught at two stations closer to the egg stations. The eggs and southern preleptocephali were from discrete spawning events, which indicated that at least two spawning sites occurred in May 2014.