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.

The effects of a phase-shifted photoperiod regime to advance maturation and spawning were examined in 3-year- old Pacific bluefin tuna Thunnus orientalis. . Fish in three broodstock groups reared in sea cages were separately transferred at age 2 years to land-based indoor tanks, in June or July, in each of the three trial years; they were then exposed to a 2- or 2.5-month advanced photoperiod cycle. In all three trials, when the fish were 3 years old, the spawning was initiated 2-3 months before the normal spawning season. During the continuous spawning, the fish were maintained under a constant long photoperiod of 15 h light and water temperature of 24-25 degrees C. In the 2019/2020 trial, 114 spawning days were recorded between March and July 2020 in a group of 28-30 females and 23-28 males; in the 2020/2021 trial, 100 days were recorded between April and July 2021 in a group of 30-32 females and 20-22 males; and in the 2021/2022 trial, 138 days were recorded between April and September 2022 in a group of 6-8 females and 7-10 males. The number of eggs produced daily peaked 1-2 months after the onset of spawning and thereafter decreased. Egg diameter showed a negative relationship with days elapsed from the onset of spawning. The analysis of parentage assignment of the larvae in the 2019/2020 trial showed that at least 50% of females and 75% of males contributed to the spawning, whereas histological observation of gonads in the 2020/2021 trial showed that 33% of females completed vitellogenesis and 85% of males reached spermiation. The analysis of parentage assignment also showed that the spawning interval of females declined between March and June, and the mean number of eggs produced daily per female peaked in April and decreased until June. Our observations demonstrate that manipulation of a phase-shifted photoperiod regime is a powerful technique for advancing maturation and spawning in the Pacific bluefin tuna at age 3 years.

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.

Genome editing by manipulating the embryos of Pacific bluefin tuna (PBT) was recently proposed for improving the breeding and aquaculture production of PBT. However, the yield of genome-edited eggs is limited due to the narrow timing of genome editing of embryos and the labor-intensive process. Therefore, the development of a small-scale larviculture method is necessary for efficient evaluation of the phenotype and traits of genome-edited PBT larvae. The plankton-kreisel tank can form a vertical rotating flow that may prevent the sinking syndrome of PBT larvae. In this study, we applied a plankton-kreisel tank (8-L) for PBT larviculture up to 10 days post-hatch (dph). We compared the survival rate and growth of PBT larvae reared in the 8-L plankton-kreisel tank and an 8-L cylindrical tank (CT). The survival rate in the plankton-kreisel tank at 10 dph (58.9 ± 4.8

In this study, we examined the effects of a long photoperiod on the reproduction of Pacific bluefin tuna (Thunnus orientalis) in a land-based tank. Three-year-old fish were shifted from a short (10.5 L:13.5D) to long (15 L:9D) photoperiod in December. The broodstocks exhibited gonadal development after this photoperiod change and spawned spontaneously the following March, approximately three months earlier than the normal spawning season. Although the results were based on data from a small sample size of fish, the study suggests that a long photoperiod induces the onset of gonadal development in Pacific bluefin tuna, thereby accelerating their spawning time.

飼育水温の違いがクロマグロ人工種苗の生残と成長に及ぼす影響を調べた。異なる水温（17, 20, 23, および26℃）での飼育試験の結果，生残率は低水温区で高くなる傾向が認められた。体サイズは低水温区で小型化する傾向にあるものの，23℃区の成長は26℃区と同等であり，飼料効率は23℃区で最も高い値が得られた。本研究の結果から，クロマグロ人工種苗は17℃以上の海域で育成可能であり，23℃付近が成長により適した水温であると示唆された。

Female fishes allocate a certain quantity of lipids for egg production once mature. In aquaculture, such physiological changes often result in the deterioration of flesh quality. In the study reported here, we aimed to characterize the seasonal changes in the lipid contents in aquaculture-produced female Pacific bluefin tuna (PBT) Thunnus orientalis, which is one of the most commercially important species for aquaculture worldwide. We analyzed the seasonal changes in the lipid content and weight of tissues and organs responsible for lipid allocation towards egg production, including the muscles, liver, mesenteric fat (perigonadal fat), and ovaries. During the spawning season, the total lipid content of the muscles and liver of the mature females decreased, whereas that of the ovaries increased; similarly, the weight of perigonadal fat decreased. We hypothesized that muscle, liver, and perigonadal fats contribute significantly to the accumulation of lipids during the maturation of female PBT. This is the first study to report an association between a reduction in muscle lipid content and sexual maturation in aquaculture-produced female PBT. These findings provide important insights regarding efficient aquaculture operations for this economically important fish. In addition, this study is the first to provide data on the association between the lipid dynamics of female PBT and sexual maturation in aquaculture, further enhancing our understanding of the energy allocation strategy during maturation of female bluefin tuna.

To understand the physiological mechanisms by which pituitary-derived gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) regulate asynchronous oocyte development, we investigated the function and expression of Fsh and Lh receptors (Fshr and Lhr, respectively) in Pacific bluefin tuna (PBT, Thunnus orientalis). As a first, we cloned the full-length cDNAs encoding PBT Fshr and Lhr. Recombinant PBT Fsh and Lh single-chain proteins were produced in abundance using stable CHO-DG44 cell lines and were subsequently purified from the culture medium, culminating in their yields being 87.0 and 88.2%, respectively. An in vitro reporter assay using homologous recombinant Gths revealed that PBT Fshr and Lhr responded strongly to their corresponding ligands in a dose-dependent manner, with no cross-activation over a wide range of concentrations. Moreover, quantitative expression analysis of Fshr and Lhr at the follicle level showed that fshr gene expression was highly upregulated in the ovarian follicles through vitellogenesis, while lhr expression was significantly upregulated and peaked in fully vitellogenic ovarian follicles. These findings suggest that asynchronous-type oocyte development is primarily attributed to the differential function and expression of Gthrs, rather than the ligand, in PBT.

Triploidization influences various biological characteristics of fish, which is associated with reductions in the number of multiple cell types in different tissues/organs. Our behavioral analyses revealed that triploid Pacific bluefin tuna (Thunnus orientalis) larvae exhibit lower sensitivity to light compared to diploids. Furthermore, histological analyses revealed a reduction in the number of ganglion cells and an increase in their size in the retinas of triploid T. orientalis larvae. Our findings provide the first evidence indicating that triploidization reduces sensory perception during the larval stage of fish.

High mortality of age-0 hatchery-reared Pacific bluefin tuna (PBT, Thunnus orientalis) frequently occurs in sea cages during the winter. Because the main spawning season of captive PBT broodstocks is delayed by approximately 2 months compared with that of wild populations, hatchery-reared PBT juveniles are usually smaller than wild-captured fingerlings, suggesting that body size is related to survival during winter. To produce large fingerlings with thermal tolerance to cold water and prevent mass mortality of age-0 hatchery-reared fish during winter, we developed an advanced spawning technique in PBT broodstocks by manipulating environmental cues such as water temperature and day length in a land-based tank. In November 2017, fifty-eight 2-year-old PBT were transferred to a land-based tank (diameter, 20 m; depth, 6 m; volume, 1880 m3). After the broodstocks experienced first spawning between June and August 2018 induced by normal photothermal conditions, uniformly shifted photothermal conditions corresponding to December 20 (winter solstice condition) to October 20 were applied to induce spawning between April and June 2019. The application of 2-month advanced photothermal cycles allowed PBT to spawn approximately 2 months earlier than the normal spawning season of the previous year. Moreover, parentage analyses revealed that at least two females and five males spawning at 3 years of age participated in advanced spawning events and produced viable larvae at 4 years of age. These results indicate that uniformly shifted photothermal conditions starting at winter solstice conditions are effective in accelerating the sexual maturation and spawning of PBT broodstock.

Tunas (genus Thunnus) are one of the most ecologically and commercially important fish worldwide. To establish a biological basis for reproduction in this globally essential species, we have recently studied crucial reproductive aspects of the Pacific bluefin tuna (T. orientalis; PBT), as a model of tuna species, based on our closed-cycle aquaculture technology. In this study, we clarified the global expression profile of the genes regulating gonadal sex differentiation in PBT, as this developmental process is vital to sexual reproduction. Based on the results of our comparative (RNA-sequencing) and temporal (qRT-PCR) transcriptome analyses using the updated genome dataset, we propose the molecular mechanisms of gonadal sex differentiation in PBT. In female gonads, foxl2 and cyp19a1a (coding aromatase) are expressed at the onset of sex differentiation. Active aromatase-mediated estrogen biosynthesis, which includes positive regulation of cyp19a1a expression by Foxl2, induces ovarian differentiation. By contrast, dmrt1 and gsdf are upregulated in differentiating male gonads lacking active estrogen synthesis. Dmrt1 and Gsdf would mainly promote testicular differentiation. Furthermore, androgen biosynthesis is upregulated in differentiating male gonad. Endogenous androgens may also be vital to testicular differentiation. This study provides the first comprehensive data clarifying the molecular basis for gonadal sex differentiation in tunas.

Abstract Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture worldwide. For the conservation of this species and sustainable development of the tuna farming industry, production based on closed‐cycle aquaculture should be promoted. In this study, to develop an efficient method for transporting PBT eggs, we simulated egg transportation to optimize stocking density and shipping duration, which are the two variables critical to the transportation procedure of PBT. Based on the findings of this study, we concluded that PBT eggs should be transported at a density of 1 × 104 eggs/L within 12 h in the field. Furthermore, we showed that collisions between eggs during transportation negatively affected the normal hatching rates of eggs, and polyethylene glycol (PEG) treatment (at 1000 μg/mL for 12 h) could reduce such damage. Our results suggest that PEG treatment is a practical approach that contributes to the stable transportation of PBT eggs, particularly in cases where the effects of physical shock damage is considerably increased.

Tuna (genus Thunnus), particularly Pacific bluefin tuna (T. orientalis; PBT), are commercially important fish in the aquaculture industry worldwide. The objective of this study was to investigate sexual dimorphism in the growth performance of aquaculture-produced PBT and develop techniques for its sex manipulation, for the first time in tuna. A comparison of the body size between sexes revealed that male-cultured PBTs were larger than females at harvest. We also confirmed that cyp19a1a (encoding a gonadal aromatase) expression increased specifically in the genotypic female PBT gonads during sex differentiation. This suggested that aromatase plays an important role in ovarian differentiation and that the suppression of aromatase activity may effectively induce masculinization in genotypic females. Therefore, we administered letrozole—an aromatase inhibitor (AI)—into sexually undifferentiated PBT through the oral route. AI administration resulted in a 100% sex reversal of genotypic females into phenotypic males at the molecular level. Our results provide the basis for future studies on the establishment of mono-sex male production technology in PBT, which would help improve the productivity of closed-cycle PBT aquaculture. Furthermore, this study offers important insights into the understanding of the sex-wise growth of tuna species in aquacultural settings, and developing sex manipulation techniques.

In the aquaculture of Pacific bluefin tuna (PBT, Thunnus orientalis), low survival rates during the larval stage remain a significant issue for the stable supply of seedlings reared from eggs. Egg quality is an important factor affecting seedling production success. If egg quality can be evaluated before hatching, the efficiency of seedling production will be improved by selectively cultivating the seedlings with a high survival rate. In our previous study, we developed a system using a convolutional neural network to evaluate PBT egg quality by estimating the normal hatching rate. The system used an image containing one PBT egg as the input. To further improve the efficiency of the egg quality estimation system, we updated the system that estimates the egg quality of a bulk sample (approximately 30 eggs). Our results indicated that the proposed system can estimate the normal hatching rate of the bulk sample with higher accuracy than the visual inspections of three field experts and visualize the normal hatching rate of each egg in the bulk sample. The proposed system will serve as a foundation for assessing the quality of PBT eggs and increasing the efficiency of seedling production.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Fish species have a variety of sex determination systems. Tunas (genus Thunnus) have an XY genetic sex determination system. However, the Y chromosome or responsible locus has not yet been identified in males. In a previous study, a female genome of Pacific bluefin tuna (T. orientalis) was sequenced, and candidates for sex-associated DNA polymorphisms were identified by a genome-wide association study using resequencing data. In the present study, we sequenced a male genome of Pacific bluefin tuna by long-read and linked-read sequencing technologies and explored male-specific loci through a comparison with the female genome. As a result, we found a unique region carrying the male-specific haplotype, where a homolog of estrogen sulfotransferase gene was predicted to be encoded. The genome-wide mapping of previously resequenced data indicated that, among the functionally annotated genes, only this gene, named sult1st6y, was paternally inherited in the males of Pacific bluefin tuna. We reviewed the RNA-seq data of southern bluefin tuna (T. maccoyii) in the public database and found that sult1st6y of southern bluefin tuna was expressed in all male testes, but absent or suppressed in the female ovary. Since estrogen sulfotransferase is responsible for the inactivation of estrogens, it is reasonable to assume that the expression of sult1st6y in gonad cells may inhibit female development, thereby inducing the individuals to become males. Thus, our results raise a promising hypothesis that sult1st6y is the sex determination gene in Thunnus fishes or at least functions at a crucial point in the sex-differentiation cascade.

Closed-cycle aquaculture using hatchery produced seed stocks is vital to the sustainability of endangered species such as Pacific bluefin tuna (Thunnus orientalis) because this aquaculture system does not depend on aquaculture seeds collected from the wild. High egg quality promotes efficient aquaculture production by improving hatch rates and subsequent growth and survival of hatched larvae. In this study, we investigate the possibility of a simple, low-cost, and accurate egg quality prediction system based only on photographic images using deep neural networks. We photographed individual eggs immediately after spawning and assessed their qualities, i.e., whether they hatched normally and how many days larvae survived without feeding. The proposed system predicted normally hatching eggs with higher accuracy than human experts. It was also successful in predicting which eggs would produce longer-surviving larvae. We also analyzed the image aspects that contributed to the prediction to discover important egg features. Our results suggest the applicability of deep learning techniques to efficient egg quality prediction, and analysis of early developmental stages of development.