Pacific Bluefin Research Articles

Mercury Monitoring in Farmed Pacific Bluefin Tuna (Thunnus orientalis) using Liquid Asymmetric-electrode Plasma Optical Emission Spectroscopy: Ventricle Tissue as a Potential Indicator

Tuna muscle is known to contain high levels of mercury, necessitating the monitoring of mercury concentrations in farmed fish at production sites. There is currently no straightforward in on-site method for testing whether the mercury levels in farmed tuna exceed regulatory limits. This study investigated the potential application of liquid asymmetric electrode plasma optical emission spectroscopy (LAEP-OES) to estimate mercury concentrations in the tissues of cultured Pacific bluefin tuna at aquaculture farms. No significant differences in mercury concentrations were observed between the ventricle tissue and biopsied tissue from tuna bleeding site (TTBS), which can be harvested without reducing the market value of farmed tuna (Hg concentration of the ventricle tissue and the TTBS, median: 0.41 and 0.40 mg/kg, respectively). In addition, the relationship between mercury concentrations in the ventricle tissue and in the edible portions of three tuna fish was examined. The findings showed that that total mercury level in the edible tissues varied, with the ratio of the mercury level in edible tissues to that in the ventricle ranging from 0.39 to 1.08. A positive correlation was observed between the average total mercury levels in edible tissues and ventricle tissue. These results suggest that determining the total mercury level in the ventricle, which is typically discarded at processing sites, may serve as a convenient proxy for quality control and monitoring purposes using the LAEP-OES method. However, larger sample sizes are needed to validate the proposed approach more robustly.

Identification of cymothoid isopods probably responsible for mass mortality of hatchery-reared Pacific bluefin tuna Thunnus orientalis juveniles after transfer to sea cages

Identification of cymothoid isopods probably responsible for mass mortality of hatchery-reared Pacific bluefin tuna Thunnus orientalis juveniles after transfer to sea cages

Molecular characterization and stage-dependent gene expression of gonadotropin receptors in Pacific bluefin tuna, Thunnus orientalis, ovarian follicles

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.

Bluefin tuna sperm production is hastened by surrogacy in small Euthynnus

Pacific bluefin tuna (Thunnus orientalis) remains heavily depleted due to overexploitation. Aquaculture and stock enhancement based on artificial seedlings could be effective solutions to this problem. However, widespread adoption of seedling production is limited because spawning in captivity of bluefin tuna, a large pelagic top predator, requires much space, time, cost, and labour. To downsize and shorten their generation time, here, we show that xenogeneic germ cell transplantation enables a surrogate production of functional bluefin tuna sperm in hybrid little tuna (genus: Euthynnus), which attain sexual maturity at a smaller body size and shorter time. Intraperitonially transplanted bluefin tuna germ cells migrate towards and are incorporated into the gonads of little tuna larvae. These recipients produce functional donor-derived bluefin tuna sperm at a mere 8 months of age and 1 kg of body size. This result represents an advance toward compact, low-cost, and time-efficient seedling production that will improve the efficiency of bluefin tuna aquaculture.

Effects of triploidization on light sensitivity in larval stage of Pacific bluefin tuna Thunnus orientalis.

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.

An in situ and real-time analytical method for detection of freeze-thew cycles in tuna via IKnife rapid evaporative ionization mass spectrometry

Freezing is one of the most commonly used preservation methods for Bluefin tuna (Thunnus orientalis). However, repeated freezing and thawing would inevitably occur due to the temperature fluctuation, leading to the microstructure damage, lipid oxidation and protein integrity decline of tuna muscle without notable visual appearance change. In this study, we used a rapid evaporative ionization mass spectrometry (REIMS) technique for the real-time determination of the extent of repeated freezing and thawing cycles in tuna fillets. We found significant variance in the relative abundance of fatty acids between bluefin tuna and its fresh counterpart following freeze-thaw cycles. Meanwhile, the difference is statistically significant (p < 0.05). The quality of tuna remains largely unaffected by a single freeze-thaw cycle but significantly deteriorates after freeze-thaw cycles (freeze-thaw count ≥2), and the relative fatty acid content of the ionized aerosol analysis in the REIMS system positively correlated with the number of freeze-thaw cycles. Notably, palmitic acid (C 16:0, m/z 255.23), oleic acid (C 18:1, m/z 281.24), and docosahexaenoic acid (C 22:6, m/z 327.23) displayed the most pronounced changes within the spectrum of fatty acid groups.

Spawning performance of 3-year-old Pacific bluefin tuna Thunnus orientalis exposed to a phase-shifted photoperiod regime

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.

Molecular Identification of an “Abnormal Tuna” Caught in the Taiwan Strait off Southwestern Taiwan

A female tuna of odd appearance, with the size of a Pacific bluefin tuna (Thunnus orientalis Temminck and Schlegel, 1844), anal fin count of bigeye tuna (Thunnus obesus Lowe, 1839), pectoral fins of yellowfin tuna (Thunnus albacares Bonnaterre, 1788), caught in waters west of the island of Xiaoliuqiu, Pingtung, Taiwan, received considerable media attention after its landing at Tungkang fishing port on 14 December 2020. This tuna has now been identified by molecular systematic means. Fishermen and fish merchants suggested it was a hybrid form, unique in living memory; however, it had matured ovaries and developing ova. To confirm whether this fish was a hybrid, we determined its parental species by analysing the cytochrome b gene (cyt b) and cytochrome c oxidase subunit 1 (COI) sequences of the mitochondrial DNA for maternal inheritance, and the internal transcribed spacer 1 (ITS1) gene sequence from the nuclear DNA to confirm both parents’ lineages. Genomic DNA was isolated from fast-skeletal muscle, and primers were designed based on the known sequences of conserved regions among tunas. According to cyt b and COI, the mother of the peculiar tuna was a Pacific bluefin tuna (T. orientalis), and the ITS1 sequence showed that both parents were of this species. We therefore conclude that despite the mixed morphological appearance, this abnormal tuna was a Pacific bluefin tuna, not a hybrid.

Validation of a New Liquid Asymmetric-Electrode Plasma Optical Emission Spectroscopy (LAEP-OES) Method for Measurement of Total Mercury in Tuna.

Mercury intake is caused by eating seafood, such as tuna and other predatory fish species. To reduce the health risks of mercury intake, it is necessary to continuously measure and monitor mercury concentrations at fish farms and markets. We have developed a compact system that can detect multiple heavy metals by liquid asymmetric-electrode plasma optical emission spectroscopy (LAEP-OES). The validity of the LAEP-OES method for total mercury levels was evaluated using standard solutions, certified substances, and specimens of bluefin tuna and other fish species. All specimens were dissolved in 4 M lithium hydroxide solution and then dispensed into a sample reservoir well of the single-use measurement reagent pack. Total mercury levels were automatically measured within 15 min of placement into the dedicated equipment. A total of 102 fish specimens, classified into 10 fish species, were evaluated using the new method and the results were compared to those obtained from validated analytical methods. LOD (0.02 mg/kg), LOQ (0.07 mg/kg), repeatability (4.0%), intermediate precision (9.8%), and trueness (recoveries 107%) of the proposed method were within satisfactory limits for total mercury levels in fish. Additionally, when using various fish species, the method had a strong positive correlation with the results of cold-vapor atomic absorption spectrometry (CV-AAS, the official method) with Spearman rs = 0.984. The LAEP-OES method can be used for measuring total mercury levels in bluefin tuna. Total mercury measurement using this new method has the potential to be applied to other fish species. Total mercury levels in fish were measured using our unique analysis system. Pacific bluefin tuna, southern bluefin tuna, and Atlantic bluefin tuna distributed in the Japanese market were analyzed for total mercury in their wild and farmed fish varieties.

Examining the emerging quota transfer system for Japanese Pacific bluefin tuna fisheries through social network analysis

Examining the emerging quota transfer system for Japanese Pacific bluefin tuna fisheries through social network analysis

Effect of a long photoperiod on the timing of spawning in the Pacific bluefin tuna, Thunnus orientalis

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.

Distinct natal origins based on vertebral ring analysis corroborate the migration pattern of Pacific bluefin tuna in the North Pacific Ocean

Distinct natal origins based on vertebral ring analysis corroborate the migration pattern of Pacific bluefin tuna in the North Pacific Ocean

Lateral bone ridge expansion and internal tissue replacement for vertebral body growth in Pacific bluefin tuna Thunnus orientalis.

Vertebral growth is an essential developmental process to support the expansion of the vertebrate body. In teleosts, the lateral side of the vertebral bodies develops to form different structures among species in the late stages of vertebral growth, although lateral structures are not apparent in the early stages. Lateral structures are one of the structural features that determine the diversity of teleost vertebrae. However, explanations for the formation of lateral structures are conflicting because few reports have investigated the growth of teleost vertebral bodies. To clarify the growth process, we analyzed the morphological changes in the vertebral body of Pacific bluefin tuna Thunnus orientalis at different developmental stages using micro-computed tomography(CT) scans. The micro-CT scans showed that the vertebral centrum formed a plate-like ridge on the lateral side along the cranial-caudal direction and extended laterally with increasing thickness. Simultaneously, the proximal region of the lateral ridges became porous as the vertebrae grew to form bone marrow cavities. Furthermore, we used histological observations to describe the relationship between these morphological changes and osteoblast and osteoclast activities. Osteoblasts accumulated on the distal edges of the lateral ridges, whereas osteoclasts were distributed in the bone marrow cavities. These observations suggest that bone resorption occurs proximally to form bone marrow cavities in addition to bone synthesis at the edges of the lateral ridges. The bone marrow cavities were occupied by blood vessels, extracellular matrix, and adipocytes, and the internal tissue composition changed to increase the area of adipose tissue. Because the ratio of bone volume decreases in large vertebrae, bone formation and resorption are regulated to separate the external cortical and internal trabecular bones to support the vertebrae. This study is the first to report the formation of lateral structures and can be applied to similar lateral structures in the vertebrae of other teleost species.

Nighttime Lighting Influences on the Plankton Feeding and Growth of Juvenile Pacific Bluefin Tuna, Thunnus orientalis.

During fingerling production of Pacific bluefin tuna (PBF) Thunnus orientalis, heavy mortality can occur immediately after juveniles are transferred from nursery tanks to sea cages; however, nighttime lighting can moderate this mortality. Additionally, various live prey aggregate due to nighttime lighting in practical sea cage culture of PBF. Here, we investigated whether the growth and viability of PBF juveniles could be improved through promoting feeding on live prey that aggregate under nighttime lighting. Two treatment groups were established using land-based tanks under constant environmental conditions, one in which the juveniles were fed live prey at night (night-feed in four replicate tanks) and the other in which juveniles were not fed during the night (control in four replicate tanks). Although the survival rate did not differ significantly between the two groups, growth was significantly improved in the night-feed group, in which 69-78% of the juveniles showed evidence of feeding during the night. Thus, nighttime lighting plays a vital role in the aggregation of various live prey. PBF juveniles consume these prey in sea cages, which promotes their growth. This may partially serve as a countermeasure against the heavy mortality observed in sea cages.

The reproductive potential of Pacific bluefin tuna (Thunnus orientalis) farmed in sea cages in South Korea

AbstractIdentifying environmental effects on the reproduction cycle and sex hormones is essential for ensuring the sustainable productivity of Pacific bluefin tuna (PBT, Thunnus orientalis) aquaculture. In the present study, 70 PBTs, commercially farmed in sea cages near Yokjido Island, South Korea, were investigated from April 2020 to March 2021. Environmental data, including water temperature and photoperiod duration, were collected. The gonadosomatic index (GSI) was calculated, and male and female maturity stages were classified monthly in proportions based on histological analysis of the gonads. Male and female gonadal developmental stages were classified into four major stages. Mature male and female PBTs were observed from May to August and June to August, respectively. Monthly changes in sex steroid hormone levels in PBT plasma were evaluated. The GSI and sex steroid hormones of males and females increased in May, peaked in June, and decreased thereafter. Our results confirmed the reproductive potential of farmed PBT and verified that reproductive maturity is reached in July. This study, to the best of our knowledge, is the first to investigate the reproductive biology of PBT in South Korea, and our results constitute a reference for future research on PBT aquaculture.

Pre-treatment methods for stable oxygen and carbon isotope analyses of structural carbonates of bones in marine teleost fishes.

Although the proportion of structural carbonates in vertebrate bones is low, the values of isotopes, namely stable oxygen (δ18 O) and carbon (δ13 C), in structural carbonates provide environmental and physiological information, which can be beneficial for estimating the palaeontological and ecological parameters of vertebrates. However, a few studies have analysed the isotopes of structural carbonates in modern teleost fishes, and a well-developed protocol for sample preparation is lacking. We examined different pre-treatment methods of preparing bone samples of three marine teleost fishes (Japanese flounder, Pacific bluefin tuna and yellowtail) and investigated the effects of the cleaning methods on the stable isotope values of structural carbonates among vertebrae in the same individual. Isotope values were analysed using isotope ratio mass spectrometry. Physical cleaning was the most promising pre-treatment method and resulted in δ18 O values that were comparable to those of otoliths. Chemical treatments with NaOH and H2 O2 changed the percentage of structural carbonates in the bone and affected δ18 O and δ13 C values. High-temperature treatments, such as boiling and roasting, altered δ18 O values due to the exchange of oxygen with environmental water or vapour. Our results suggest that chemical cleaning methods used to prepare bone phosphate or collagen samples for isotope analyses are not suitable for structural carbonates. Physical cleaning is the appropriate pre-treatment method for analysing the isotopes of structural carbonates. Also, we emphasise that standardising the vertebral number is necessary to make δ13 C values comparable between specimens in the same species.

Transcriptome characterization of gonadal sex differentiation in Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel)

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.

Revisiting the spawning fraction of Pacific bluefin tuna (Thunnus orientalis) caught in the Sea of Japan based on diurnal changes in spawning markers

The spawning fraction (SF) is an important reproductive trait used to assess the reproductive potential of indeterminate spawning fishes. To estimate the SF more accurately, the durations of spawning markers in ovaries need to be determined. The SF of the Pacific bluefin tuna Thunnus orientalis (PBF) in the Sea of Japan (SOJ) is higher than the SFs in its other main spawning grounds. The present study aimed to revisit the SF of the PBF in the SOJ by focusing on diurnal changes in spawning markers and estimating the duration of spawning markers in PBF ovaries. According to diurnal changes in the postovulatory follicle (POF) degeneration process and final maturation process of oocytes, POFs were observed in ovaries up to 38.5 h after ovulation. The revisited total SF throughout the spawning season was 0.34–0.43 in Area 1 and 0.50–0.64 in Area 2; these values are smaller than the SF previously estimated in the SOJ. The estimated SF of the PBF in the present study was equal to or slightly higher than that in the water between the Nansei Islands, Japan, and the northeast of the Philippines and higher than that in the Kuroshio-Oyashio transition area.

Potential application of lactic acid bacteria for controlling discoloration in tuna (Thunnus orientalis)

Pacific bluefin tuna (Thunnus orientalis) is a highly valued seafood consumed globally. The red color of tuna meat is considered a crucial indicator of its freshness and value, influencing consumer purchasing decisions. Therefore, the present study aimed to investigate the effect of lactic acid bacteria (LAB) isolated from traditional Korean fermented seafood (jeotgal) on preventing the discoloration of tuna meat during storage. Thirty-six strains of LAB (M1–M36) were isolated and examined for superior antioxidant and metmyoglobin conversion activities. Two strains, M1 (Lactococcus lactics) and M8 (Leuconostoc citreum), were selected. Subsequently, the tuna meat was treated with the cell-free supernatant (CFS) of the selected LAB strains and stored at 4 °C. Treatment with CFS maintained a significantly high level of redness (a*) in the tuna meat for up to 48 h; furthermore, this tendency was consistent with the relative quantification of myoglobin derivatives. The oxymyoglobin content in CFS-treated tuna meat remained above 60% for up to 60 h. Conversely, the metmyoglobin content in the untreated group reached over 60% only after 48 h, resulting in complete discoloration. Conclusively, this study demonstrated that LAB-CFS can prevent tuna meat discoloration during refrigerated storage, indicating its potential for maintaining the quality and color of tuna meat.

Optimization of stocking density and shipping duration for transportation of Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), eggs, and experimental verification of polyethylene glycol treatment to reduce collision damage

AbstractPacific 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.