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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 FROM AUTHOR]

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 °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. • A phase-shifted photoperiod advanced spawning of 3-year-old Pacific bluefin tuna. • Spawning was initiated in March or April and then recorded over 100 spawning events. • There was a negative relationship between egg size and days from onset of spawning. • The spawning interval of females declined between March and June. • The number of eggs produced daily per female peaked in April and decreased until June. [ABSTRACT FROM AUTHOR]

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. • We induced advanced spawning of Pacific bluefin tuna in a land-based tank. • Uniformly shifted photothermal cycles accelerated the time of sexual maturity. • The mean normal hatching rate during advanced spawning period was 85.0%. • Individual spawning performances were revealed by parentage assignment using SNPs. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species. [ABSTRACT FROM AUTHOR]

We examined the annual reproductive cycle of both female and male hatchery-produced Pacific bluefin tuna (PBT, Thunnus orientalis) broodstock at 3 years of age, including the associated changes in plasma levels of sex steroid hormones and the gene expression of key endocrine factors. The females progressed to vitellogenesis in April, and approximately 50% of the females were observed in late vitellogenesis between June and July at water temperatures between 24.1 °C and 29.0 °C. Approximately 90% of the males were in spermiation for at least 7 months between April and October, which included the period when vitellogenic females were present. Furthermore, the gonadosomatic index of both females and males peaked between June and July. These results indicate that the reproductive season of hatchery-produced PBT peaks between June and July, which is in agreement with the reproductive season reported for PBT broodstock originating from wild-caught juveniles. Furthermore, the percentage of hatchery-reared females that reached late vitellogenesis in the present study was higher than that in wild-caught captive-reared females reported in previous literature. Analysis of plasma sex steroid levels and gene expression profiles suggested that oocyte and ovarian development were associated with 17β-estradiol (E2) control, whereas spermatogonial differentiation towards meiosis seemed to be mediated by 11-ketotestosterone (11-KT). Furthermore, the production of E2 and 11-KT is likely regulated by luteinizing hormone and follicle-stimulating hormone, respectively, via their related receptors. The findings of this study are expected to contribute to improvements of technologies for obtaining fertilized eggs from hatchery-produced PBT broodstock. • We examined the reproductive cycles of hatchery-produced Pacific bluefin tuna. • 50% of females in full vitellogenesis were observed between June and July. • Most males reached full spermiation between April and October. • Plasma sex steroid profiles were associated with gonad development. • Pituitary gene expression of fshb and lhb was related to sex steroid production. [ABSTRACT FROM AUTHOR]

This study examined the biochemical characteristics of key digestive enzymes in artificially produced Pacific bluefin tuna juveniles and revealed details of the digestive physiology of juveniles fed a commercial pellet diet and raw fish over long-term (225 days) and short-term (response to feeding) periods. pH and thermal profiles of key digestive enzymes of pepsin, trypsin, chymotrypsin, lipase and amylase were examined in juvenile bluefin tuna and the characteristics differed from those previously reported for larval and adult tuna. Long-term feeding (225 days) of the commercial diet markedly increased stomach and pyloric caeca size as well as storage/synthesis levels of the digestive enzymes. Highly increased gene expression levels of cholecystokinin (cck) and its receptor (cck1r) were also found in juveniles fed the commercial diet, suggesting cck might be involved in the gastrointestinal hypertrophy and digestion enhancement seen in the present study. In the short-term experiment (response to feeding), juveniles fed the commercial diet displayed faster gastric emptying, higher pH in the intestinal content and increased digestive enzymes storage/synthesis whereas the enzymes secretion was similar with those of juveniles fed raw fish. These indicate that juvenile bluefin tuna morphologically and physiologically adapted their digestive system to the artificial diet, however the adaptation was not sufficient to obtain a comparable performance as tuna fed raw fish feed. Biochemical characteristics of the digestive enzymes and the observed effects of artificial diet on digestive physiology could be good indicators to improve feeding strategies such as diet composition, feeding ration and frequency in juvenile tuna. • Biochemical characteristics of key digestive enzymes were revealed in juvenile Pacific bluefin tuna and the characteristics differed from those previously reported for larval and adult tuna. • Long-term feeding (225 days) of the commercial diet markedly increased stomach and pyloric caeca size as well as storage/synthesis levels of digestive enzymes compared with juveniles fed the raw fish feed. • Juveniles fed the commercial diet displayed faster gastric emptying, higher intestinal content pH and increased digestive enzymes storage/synthesis after feeding whereas the enzyme secretion levels were similar with juveniles fed the raw fish feed. • Juvenile tuna morphologically and physiologically adapted their digestive system to the artificial diet, while the adaptation was not sufficient to enable comparable growth with the raw fish feed. [ABSTRACT FROM AUTHOR]