The freshwater eel Anguilla japonica is rapidly decreasing in number and has not yet been successfully mass produced. This may be at least partially attributable to the unique and long early life history of the eel. Therefore, we investigated its ontogeny of morphometry and growth pattern in larval stages to provide baseline information for understanding the early life history and improving seed rearing technology. This study was conducted for 200 days after hatching (DAH) and analyzed morphometry and allometry for eel larvae. The following cultured eel larval stages were identified: the yolk sac larvae stage (0–6 DAH, 3.23–6.85 mm total length (TL)), the pre-leptocephalus stage (7–30 DAH, 6.85–15.31 mm TL), and the leptocephalus stage (50–200 DAH, 15.31–60.06 mm TL). Cultured and wild eel larvae could be divided into characteristic larval stages at similar sizes. However, compared to wild eels, cultured eels had a slower growth rate and fewer preanal myomeres. Meanwhile, cultured eel larvae rarely had a mixed feeding period as the absorption of endogenous reserves was completed by 7 DAH. The lower jaw of eel larvae was significantly longer than the upper jaw from 50 DAH. In the pre-leptocephalus and leptocephalus stages, eel larvae showed continuous positive allometric growth at trunk height and tail muscle height with change to the willow leaf-like form. These growth characteristics may be the result of adaptation to the migration over long distances and to a diel vertical migration. The inflection point in the body parts growth patterns showed only before 30 DAH, and mass mortality appeared at this period. Therefore, to improve the growth and survival rates of cultured eel seed, it is necessary to focus on improving the feeding and rearing protocol until 30 DAH.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) β-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG β-subunit CTP region (amino acids 115–149) was inserted between the β-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000–5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34–40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39–46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC₅₀ value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG β-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32–34 and 34–36 kDa, respectively, and that of LH-M and FSH-M was 40–43 and 42–45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8–10 kDa. The EC₅₀ value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

Beneficial microorganisms can increase nutrient digestion and absorption in farmed fish. This study investigates the effects of supplemental feeding of Bacillus species isolated from the intestines of wild glass eels on the growth, survival, and gene expression of farm-raised eel larvae for 30 days after hatching. Three species of Bacillus (B. velezensis, AJBV; B. subtilis, AJBS; B. licheniformis, AJBL) without hemolytic activity were isolated, and an experiment compared the growth of eel larvae fed an artificial diet supplemented with each Bacillus species. There were no significant differences in the total length and body depth of eel larvae at 30 days after hatching in all groups. During the feeding period, 149 eels survived from the initial 1000 in the control group. On the other hand, 240, 178, and 141 eels survived in the AJBV, AJBS, and AJBL groups, respectively. However, there were no significant differences in survival rates despite the difference in the number of surviving eel larvae among the groups. In the comparison of gene expression of genes involved with growth (growth hormone, growth hormone receptor 1, insulin-like growth factor II-2) and those involved with digestive enzymes (amylase, trypsin, lipase), there were also no significant differences among the groups. Our results confirm that dietary supplementation with each of the three host-associated Bacillus does not affect the growth and survival rates of eel larvae reared on an artificial diet up to the first 30 days after hatching, nor does it significantly affect related gene expression.

We produced a recombinant eel luteinizing hormone (rec-eel LH) analog with high potency in Chinese hamster ovary DG44 (CHO DG44) cells. The tethered eel LH mutant (LH-M), which had a linker comprising the equine chorionic gonadotropin (eLH/CG) β-subunit carboxyl-terminal peptide (CTP) region (amino acids 115 to 149), was inserted between the β-subunit and α-subunit of wild-type tethered eel LH (LH-wt). Monoclonal cells transfected with the tethered eel LH-wt and eel LH-M plasmids were isolated from five to nine clones of CHO DG44 cells, respectively. The secreted quantities abruptly increased on day 3, with peak levels of 5000–7500 ng/mL on day 9. The molecular weight of tethered rec-eel LH-wt was 32–36 kDa, while that of tethered rec-eel LH-M increased to approximately 38–44 kDa, indicating the detection of two bands. Treatment with the peptide N-glycanase F decreased the molecular weight by approximately 8 kDa. The oligosaccharides at the eCG β-subunit O-linked glycosylation sites were appropriately modified post-translation. The EC₅₀ value and maximal responsiveness of eel LH-M increased by approximately 2.90- and 1.29-fold, respectively, indicating that the mutant exhibited more potent biological activity than eel LH-wt. Phosphorylated extracellular regulated kinase (pERK1/2) activation resulted in a sharp peak 5 min after agonist treatment, with a rapid decrease thereafter. These results indicate that the new tethered rec-eel LH analog had more potent activity in cAMP response than the tethered eel LH-wt in vitro. Taken together, this new eel LH analog can be produced in large quantities using a stable CHO DG44 cell system.