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Pacific bluefin tuna (PBT), Thunnus orientalis , is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production. • To develop technologies for producing sterile fish, detailed and reliable data on gonadal development are needed. • We demonstrated the morphological changes and timing of gonadal sex differentiation in Pacific bluefin tuna (PBT). • We also showed the developmental process of the ovary and testis during the immature stages of PBT. • This study will provide the necessary basis for future studies on sterile PBT production. [ABSTRACT FROM AUTHOR]

Pacific bluefin tuna ( Thunnus orientalis ) has two spawning grounds, and each is used at different times: May–June in the Nansei Islands of western North Pacific Ocean and July–August in the Sea of Japan. Although previous studies developed body-length based methods to differentiate between individuals originating in each spawning ground, these methods are only applicable to the young-of-the-year (YOY). In this study, we evaluate a simple technique for discerning the natal grounds of Pacific bluefin tuna greater than one year in age by observing the first annulus on the vertebrae of fish caught around Japan. We found a significant linear relationship between fork length and vertebral radius, and determined that the first annulus forms in boreal winter. Both the back-calculated fork length at the time of first annulus formation and the measured fork length of YOY in February and March had a bimodal frequency distribution, and the peaks of the two frequency distributions had similar means. The larger fork length individuals appear to have originated near the Nansei Islands, whereas the smaller fork length individuals appear to have originated in the Sea of Japan. This is because PBF hatched around the Nansei Islands will have longer growth time for the YOY to develop the first annulus at longer distance from the vertebrae center. Similarly, we observed bimodal distributions for the vertebral first annulus radius of mature Pacific bluefin tuna (>120 cm) in both spawning grounds. Our technique offers improved sample treatment efficiency and lower costs. In addition, this method may help reveal the contribution rate of each spawning ground. [ABSTRACT FROM AUTHOR]

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. • Postovulatory follicles were retained up to 38.5 h after ovulation. • The spawning fraction, estimated by Day 0 spawners, may have been overestimated. • The revisited total spawning fraction throughout the Sea of Japan was 0.43. • The estimated spawning fraction depended on the fishing area and fishing date. [ABSTRACT FROM AUTHOR]

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. • LAB isolated from jeotgal prevented discoloration of tuna meat. • Two strains showed superior antioxidant activity and metmyoglobin conversion. • LAB cell-free supernatant treatment to maintain redness and to prevent discoloration. [ABSTRACT FROM AUTHOR]

Farming of Pacific bluefin tuna (PBT), Thunnus orientalis , is a rapidly growing industry in Japan. Aporocotylid blood flukes of the genus Cardicola comprising C. orientalis, C . opisthorchis and C . forsteri are parasites of economic importance for PBT farming. Recently, terebellid polychaetes have been identified as the intermediate hosts for all these parasites. We collected infected polychaetes, Terebella sp., the intermediate host of C . opisthorchis , from ropes and floats attached to tuna cages in Tsushima, Nagasaki Prefecture, Japan. Also, Neoamphitrite vigintipes (formerly as Amphitrite sp. sensu Shirakashi et al., 2016), the intermediate host of C . forsteri , were collected from culture cages in Kushimoto, Wakayama Prefecture, Japan. The terebellid intermediate hosts harbored the sporocysts and cercariae in their body cavity. Developmental stages of these blood flukes were molecularly identified using species specific PCR primers. In this paper, we describe the cercaria and sporocyst stages of C . opisthorchis and C . forsteri and compare their morphological characteristics among three Cardicola blood flukes infecting PBT. We also discuss phylogenetic relations of the six genera of the terebellid intermediate hosts ( Artacama , Lanassa , Longicarpus , Terebella , Nicolea and Neoamphitrite ) of blood flukes infecting marine fishes, based on their morphological characters. [ABSTRACT FROM AUTHOR]

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri . This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis , C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts. [ABSTRACT FROM AUTHOR]

Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis , has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production. [ABSTRACT FROM AUTHOR]

We present a numerical approach that, in conjunction with a fully set up Dynamic Energy Budget (DEB) model, aims at consistently approximating the feeding history of cultivated fish from the commonly measured aquaculture data (body length, body mass, or the condition factor). We demonstrate the usefulness of the approach by performing validation of a DEB-based model for Pacific bluefin tuna ( Thunnus orientalis ) on an independent dataset and exploring the implied bioenergetics of this species in captivity. In the context of validation, the results indicate that the model successfully accounts for more than 75% of the variance in actual fish feed. At the 5% significance level, predictions do not underestimate nor overestimate observations and there is no bias. The overall model accuracy of 87.6% is satisfactory. In the context of tuna bioenergetics, we offer an explanation as to why the first reproduction in the examined case occurred only after the fish reached seven years of age, whereas it takes five years in the wild and sometimes as little as three years in captivity. Finally, we calculate energy conversion efficiencies and the supply stress throughout the entire lifetime to theoretically underpin the relatively low contribution of growth to aerobic metabolism implied by respirometry and high feed conversion ratio observed in bluefin tuna aquaculture. [ABSTRACT FROM AUTHOR]

Abstract: Since Kudoa septempunctata in olive flounder (Paralichthys olivaceus) was indicated to cause food poisoning in humans, other Kudoa species are suspected to have pathogenic potential. Recently, a myxosporean possibly associated with food poisoning in humans consuming raw Pacific bluefin tuna, Thunnus orientalis, was identified as Kudoa neothunni. This is a known causative myxosporean of post-harvest myoliquefaction in yellowfin tuna Thunnus albacares. Regardless of the significant differences in the 28S rDNA sequence and the pathological character (with/without myoliquefaction) between the two T. orientalis and T. albacares isolates, they were considered intraspecific variants of K. neothunni. However, the light and low-vacuum electron microscopic observations in the present study revealed that there were two morphotypes; pointed- and round-type spores, which were significantly differentiated by the ratio of suture width to spore width. Furthermore, the two morphotypes were genetically distinguishable by the 28S rDNA sequence analysis. This morphological and molecular evidence validates that the two Kudoa types are separate species, and thus the pointed- and round-types are referred to as K. neothunni and Kudoa hexapunctata n. sp., respectively. K. neothunni was detected solely from T. albacares, whereas K. hexapunctata n. sp. was found not only from T. orientalis but also from T. albacares. [Copyright &y& Elsevier]

Abstract: We found aporocotylid larval stages (sporocysts and cercariae) from five individuals of terebellid polychaete Terebella sp., which were collected from seabed substrate and ropes and floats attached to tuna cages in a tuna farm on the coast of Tsushima Island, Nagasaki, Japan. Nucleotide sequences of the regions of internal transcribed spacer 2 ribosomal DNA and 28S ribosomal DNA from these larval stages were 100% identical to those of Cardicola opisthorchis registered in GenBank. C. opisthorchis is a pathogen causing blood fluke infection of Pacific bluefin tuna Thunnus orientalis, which is considered to have a significant impact on the Japanese Pacific bluefin tuna aquaculture industry. This is the first description of the intermediate host of C. opisthorchis. This indicates that the life cycle of C. opisthorchis is completed within tuna farms in this area. [Copyright &y& Elsevier]

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. • System developed for estimating normal hatching rate for bulk PBT egg samples. • System leverages deep learning to estimate normal hatching rates from bulk images. • Estimated normal hatching rate of each egg in a bulk sample is visualized. • Estimation of proposed system is more accurate than experts' visual inspections. [ABSTRACT FROM AUTHOR]

Abstract: Although full-cycle aquaculture of Pacific bluefin tuna, Thunnus orientalis, has been established by Kinki University, Japan, most juvenile die after transfer to net cages offshore due to the stress associated with fluctuations in environmental conditions. We examined the effect of environmental conditions on mortality of juvenile using a generalized liner model (GLM). Juveniles were reared for more than 300days in a net cage and number of dead fish was examined over time and in relation to water temperature, salinity, moon phase, and water transparency. Mortalities soon after transferring the fish from the indoor rearing tank to the net cage occurred independently of environmental factors and were considered to be due to the handling stress and lack of acclimation. In subsequent periods, fluctuations in water temperature were the most critical factor affecting juvenile survival. Our results showed that optimal water temperatures for juvenile bluefin tuna aquaculture are considered to range between 15 and 25°C. Salinity and lunar irradiance (half moon to full moon) fluctuations had less marked effects on juvenile survival. Juveniles in net cages cannot move to avoid those unfavorable environmental fluctuations and this inability to locate areas of optimal temperature may complicate their body temperature maintenance because the capacity for endothermy in juveniles is undeveloped. We propose that sites selected for culturing juvenile Pacific bluefin tuna have water temperatures ranging from 15 to 25°C with constant temperatures that avoid large fluctuations. [Copyright &y& Elsevier]

Abstract: A new aporocotylid blood fluke is described, based on specimens from the ventricle of the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), cultured in Wakayama and Nagasaki Prefectures, Japan. The new species is morphologically similar to the members of the genus Cardicola Short, 1953, but shows distinct differences in the body form, location of the testis and the orientation of the ootype. The body of the new species is long and slender, whereas other Cardicola species are small and generally lanceolate. The testis is mostly located posterior to the caeca and anterior to the ovary, occupying 31–45% of body length, in contrast to the known Cardicola species, whose testis is typically intercaecal. The ootype is oriented anteriorly, while in most congeners, it is directed posteriorly or horizontally. Phylogenetic analyses of this aporocotylid, together with Cardicola orientalis Ogawa, Tanaka, Sugihara et Takami, 2010 from the same host, were conducted based on DNA sequences of the ITS2 rDNA and the 28S region of ribosomal RNA. The analyses revealed that the new blood fluke belongs to the genus Cardicola despite the marked morphological differences. Thus, this aporocotylid is named Cardicola opisthorchis n. sp. and the generic diagnosis is emended in this paper. In addition, 100% identity among the ITS2 sequences from the present species, Cardicola sp. from T. orientalis in Mexico and Cardicola sp. from the northern bluefin tuna, Thunnus thynnus (Linnaeus) in Spain suggests that C. opisthorchis n. sp. has a broad geographical distribution and that it infects both the Pacific and northern bluefin tuna. [Copyright &y& Elsevier]

Abstract: We reviewed research on the broodstock management and larviculture of the Pacific northern bluefin tuna (PBT) Thunnus orientalis in Japan. Japan has been at the forefront of PBT research since 1970 due to the participation by federal and prefectural governments and universities in a national project aimed to optimize productivity of the sea around Japan. In 1979, scientists at Kinki University succeeded in the first spontaneous spawning in captivity by the broodstock of 5year-old PBT. Successful spawning was also performed in 1980 and 1982, but no spawning then occurred until 1993, when Maruha Nichiro Holdings, Inc. (MNH) and Nippon Formula Feed Manufacturing Company, Ltd. (NFFMC) became involved in tuna farming and succeeded in the spawning of four-year-old broodstock. Since then, successful spawning of PBT in captivity has been reported from several sites as well as spawning in Kinki Univ. since 1994. With the successful spawning of PBT, the Fisheries Research Agency (FRA; formerly, Japan Sea Farming Association) of Kinki University, in conjunction with MNH, NFFMC, and Takuyo Ltd., has actively carried out research on and development of tuna larviculture technology. Thus, knowledge about broodstock management and larviculture has accumulated in Japan, but technical problems with larviculture still remain to be solved. There are 9 sites of successful spawning in net pens in Japan so far. At 4 of these sites in regions around Amami Island, yearly spawning has stably occurred. We have been accumulating data about the period of maturation, environmental key factors triggering the spawning, the age of onset of spawning, and the pattern of spawning through measurement of the ambient environment, gonad morphometry, endocrinology, mitochondrial DNA analysis, and daily careful observation of broodstock. Research on PBT larviculture at Kinki University, FRA, MNH, NFFMC, and Takuyo have succeeded in producing tens of thousands of hatchery-raised juveniles. As a result, Kinki University succeeded in establishing the full life cycle of PBT in captivity, and also achieved its aquaculture life cycle. However, solutions are needed for the remaining technological issues of PBT larviculture, including sinking syndrome, where larvae die on the bottom of the tank during the early phase of larviculture, the search for appropriate food (species, size, and nutrition) around the transition stage from larva to juvenile, cannibalism and collision against walls in the juvenile, and malformation and viral diseases in the young stage. [Copyright &y& Elsevier]

Abstract: Kudoa prunusi n. sp. (Myxozoa; Multivalvulida) is described from the brain of Pacific bluefin tuna Thunnus orientalis cultured in Japan. Numerous white cysts, up to 0.5mm in size, were found on and in the brain. Spores having typically five spore valves and five polar capsules resembled a five-petal cherry blossom in apical view and were conical shape with a round bottom in side view. Average spore size was 9.63 (8.5–10.3)μm in width and 7.50 (6.7–8.6)μm in length. The spore dimensions of K. prunusi overlapped with those of Kudoa yasunagai ex Sillago ciliata having five to six spore valves, but they were clearly distinct in spore shape, 18S rDNA and 28S rDNA sequences (0.3% and 1.7% differences, respectively). Phylogenetic analysis of 18S rDNA revealed that K. prunusi grouped with the brain-infecting multivalvulid species, K. yasunagai, K. chaetodoni, K. lethrini and K. neurophila, rather than five-valved Kudoa spp. Combined with morphological, molecular and biological differences, K. prunusi was proven to be a new species. [ABSTRACT FROM AUTHOR]

Abstract: Schooling behavior traits during the process of retinomotor response from scotopic to photopic vision were examined in cultivated juvenile Pacific bluefin tuna (PBT) at 3 different ages. After a sudden change in illumination from darkness to 300lx, retinal adaptations changed from scotopic to photopic vision. Retinomotor and schooling indices showed strong agreement, with juvenile PBTs forming polarized schools upon complete retinal adaptation to photopic vision. The behavioral and retinal adaptation to sudden illumination took 20, 15, and 10min after illumination in PBT 25, 40, and 55days after hatching (dah). At 40dah, PBT took a longer time to adapt than fish aged 55dah and showed the highest swimming speed, including momentary bursts of swimming immediately after illumination. This suggested that these fish were swimming at high speed under poor visibility conditions. In contrast, PBT at 55dah showed a gradual increase in swimming speed that correlated with their retinal adaptation. Therefore, behavioral and retinal adaptation traits changed during growth, suggesting that the high mortality in PBT around 40dah, due to collisions with the tank and net walls at dawn, may be because these adapt more slowly than fish at 55dah and were swimming at a relatively high speed under conditions of poor visibility. [Copyright &y& Elsevier]

Abstract: A new sanguinicolid blood fluke, Cardicola orientalis n. sp., is described from the afferent branchial artery and heart of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel) cultured in Mie and Nagasaki Prefectures, Japan. The new species is most similar to C. ambrosioi Braicovich, Etchegoin, Timi et Sardella, 2006 from the Brazilian flathead, Percophis brasiliensis Quoy & Gaimard, but can be differentiated by the position of the female genital pore (in midline or slightly sinistral in C. orientalis vs. sinistral in C. ambrosioi) and much longer distance between male and female genital pore (101μm vs. 27μm). In wet mount preparations of infected fish, eggs were accumulated in great numbers in the gill lamellae and afferent filament arteries. Importance of this blood fluke infection of cultured Pacific bluefin tuna in Japan is discussed. [Copyright &y& Elsevier]

Parasitic copepods infecting large scombrid fishes have been known for a long time because their hosts are economically important. Most studies, however, have focused on their morphology or their infection status in aquaculture from pathological viewpoints, and very few quantitative surveys have been conducted under conditions in the wild. This study therefore investigated the prevalence of Euryphorus brachypterus (Caligidae) in wild Pacific bluefin tuna (PBF). Results of sampling from August to September 2014 at the western area of the Tsugaru Strait, Japan showed that 13.2% of the PBF individuals (n = 1978) were infected with this copepod. The prevalence of infections was highest in larger fish but varied among landing dates, which were classified into three clusters and in all smaller fish, the prevalence of infections was zero. This suggests that E. brachypterus mainly uses the larger PBF, which becomes sources of further infections in other seas, and that at least two host populations with different infection statuses at the strait. [ABSTRACT FROM AUTHOR]

Abstract: Age determination of wild captured Pacific bluefin tuna, Thunnus orientalis, was conducted using sagittal otoliths of 806 specimens (47–260cm in fork length) caught in the waters off Japan and Taiwan. Otoliths were transversely sectioned and the opaque and translucent zones were analyzed. Opaque zones mainly appeared on the otolith edge from April to July, indicating that the opaque zone is formed annually. The opaque zones formed during later life (age 10+) were more distinct than the earlier zones. The estimated ages of specimens ranged from 1 to 26 years. Parameters of the von Bertalanffy growth function were estimated to be 249.6cm, 0.173, and −0.254 years for L ∞ , k, and t 0, respectively. Growth of younger fish was rapid up to 5 years old attaining about 150cm, and then growth rate decreased. After that, fish attained about 200cm at 9 years old and about 225cm (90% of L ∞ ) at 13 years old (50% of maximum age). This paper updates the biological information on length at age with a large size range to support stock assessment model analyses for this commercially valuable species. [Copyright &y& Elsevier]

Abstract: The amino acid sequences of three pepsinogens (PG1, PG2 and PG3) of Pacific bluefin tuna (Thunnus orientalis) were deduced by cloning and nucleotide sequencing of the corresponding cDNAs. The amino acid sequences of the pre-forms of PG1, PG2 and PG3 were composed of a signal peptide (16 residues each), a propeptide (41, 37 and 35 residues, respectively) and a pepsin moiety (321, 323 and 332 residues, respectively). Amino acid sequence comparison and phylogenetic analysis indicated that PG1 and PG2 belong to the pepsinogen A family and PG3 to the pepsinogen C family. Homology modeling of the three-dimensional structure suggested that the remarkably high specific activity of PG2 toward hemoglobin, which had been found previously, was partly due to a characteristic deletion of several residues in the S1′-loop region that widens the space of the active site cleft region so as to accommodate protein and larger polypeptide substrates more efficiently. Including the tuna and all other fish pepsinogen sequences available to date, the molecular phylogenetic comparison was made with reference to evolution of fish pepsinogens. It was suggested that functional divergences of pepsinogens (pepsins) occurring in fishes as well as in mammals, correlated with differences in various aspects of fish physiology. [Copyright &y& Elsevier]

Abstract: Previous studies have shown that feeding enriched Artemia induces growth failure in Pacific bluefin tuna (PBT) Thunnus orientalis larvae. This growth failure cannot be improved even if the docosahexaenoic acid (DHA) content in enriched Artemia is elevated to the same level as that in yolk-sac larvae, an ideal live feed for PBT larviculture. This might be caused by the differences in the DHA in the live feeds; i.e., yolk-sac larvae of marine fish have a high level of DHA in the larval phospholipids (PLs) but enriched Artemia store DHA in their neutral lipids (NLs). To test this hypothesis two experiments were conducted to evaluate the effect of dietary PL rich in DHA on growth and survival of PBT larvae (Experiment 1) and juveniles (Experiment 2). Three isoproteic and isolipidic artificial test diets (Diets 1, 2 and 3) and two reference live feeds (Diets 4 and 5) were prepared. Diets 1 and 3 were supplemented with NL and PL fractions of commercial salmon Oncorhynchus gorbuscha roe lipid (SRL) as the lipid source, respectively, whereas Diet 2 was supplemented with a mixture of both lipid fractions. Diets 4 and 5 were enriched Artemia and yolk-sac larvae of Japanese parrot fish Oplegnathus fasciatus, respectively. Diets 1, 3, 4 and 5 were given to PBT larvae for 10 days, whereas Diets 1, 2 and 3 were fed to PBT juveniles for 10 days. In Experiment 1, the growth and survival of PBT larvae fed Diet 3 (high PL fraction) were significantly improved when compared with larvae fed Diets 1 and 4, although the best growth and survival were obtained in larvae fed Diet 5. Similar results were obtained in Experiment 2; i.e., growth and survival were significantly improved in PBT juveniles fed Diets 2 and 3. In both experiments, fish fed Diets 2, 3 and/or 5 had higher total lipid contents, TAG levels, n-3 HUFA and/or DHA levels in NL fraction of the body when compared with fish fed Diets 1 or 4, while such a difference was not observed in the PL fraction. In juveniles over 90% of deaths were caused by collisions with the tank walls and the significant difference in mortality between treatments implies diet also affects behavior. SRL PL is concluded to enhance the growth and survival of PBT larvae and juveniles along with the accumulation of storage lipid and DHA in the body. [Copyright &y& Elsevier]

Abstract: We followed three patches of larval Pacific bluefin tuna (Thunnus orientalis) using Lagrangian drifters in 2004 and 2005 from May to June and determined the age-specific larval instantaneous mortality rates, which were estimated to be between 0.06 and 2.75day−1. The mortality rates differed significantly between patches and between ages within the same patch, which were higher than most of the estimations for other marine fish larvae. The results indicate that for the purpose of understanding the process of the larval survival, the use of larval patches is appropriate as a unit of observation. Evidence of the occurrence for net avoidance is presented and also it is concluded that net extrusion was observed for the larvae. Length-specific catch probability of the larvae was estimated to correct for length distribution biases derived from the net avoidance and net extrusion. [Copyright &y& Elsevier]

Abstract: Feeding enriched Artemia induces growth failure in Pacific bluefin tuna (PBT) Thunnus orientalis larvae; however, feeding of yolk-sac larvae of marine fish promotes larval growth. It is considered that this growth failure partly results from dietary docosahexaenoic acid (22:6n-3, DHA) deficiency. Therefore, we examined the effect of DHA contents in enriched Artemia on the growth of PBT larvae. Artemia nauplii were enriched with graded levels of DHA ethyl ester, and fed to PBT larvae for 9 days. Yolk-sac larvae of Japanese parrotfish Oplegnathus fasciatus were used as a reference diet. The DHA contents in Artemia increased with the enrichment from 0 mg g−1 dry weight basis (DW) to 25 mg g−1 DW, while the content in the reference diet was 21 mg g−1 DW. Feeding of enriched Artemia significantly improved the growth of PBT larvae. However, this improvement was negligible when compared with the excellent growth of the larvae that were fed the reference diet. PBT larvae that were fed the reference diet accumulated approximately twice or much higher levels of DHA in the neutral and polar lipids in the body when compared with the larvae that were fed enriched Artemia. These results show that PBT larval growth cannot be promoted by feeding enriched Artemia even if the DHA contents in Artemia are elevated to the same levels as those of yolk-sac larvae. The incorporation of dietary DHA into phospholipids in the fish body may be desirable for the normal growth of PBT larvae. [Copyright &y& Elsevier]

Abstract: Prey–predator interactions among planktonic organisms in larval tanks of bluefin tuna and red sea bream were investigated quantitatively, and carbon flow models were constructed from the results of feeding experiments. Two different carbon pathways were uncovered in the rearing water of both fish species. One was from autotrophic nano-plankton (ANP) to rotifers to fish larvae (artificial food chain) and the other was from bacteria to heterotrophic nano-plankton (HNP) to heterotrophic micro-plankton (HMP) (a microbial loop). Surprisingly, bluefin tuna selectively consumed HMP, especially dinoflagellates, throughout the experiment. In the bluefin tuna tank, the microbial loop was linked to the artificial food chain. On the other hand, red sea bream larvae did not consume HMP and the pathway in the red sea bream tank was more straightforward than in the bluefin tuna tank. Energy and nutrients transferred to red sea bream larvae via the artificial food chain only, except for 3 days after hatching. Biomass of micro-protozoa were high (3.2–158.4 ng C ml− 1) in the bluefin tuna tank with higher water temperature (26.0–27.6 °C) and low (3.0–4.9 ng C ml−1) in the red sea bream tank with lower temperature (19.8–20.1 °C). The study suggests that difference in water temperature is a significant factor for the microbial loop in rearing tanks of fish larvae. This is the first report suggesting that a microbial loop, established naturally, contributes energy and nutrient gain to bluefin tuna larvae reared in an artificially controlled environment. [Copyright &y& Elsevier]

Blood fluke infection is one of the main causes of mortality in cultured juvenile Pacific bluefin tuna Thunnus orientalis (PBT) in Japan. We investigated 532 wild juvenile PBT of four different fish groups caught for tuna culture seed stock from off the Goto Islands, Nagasaki, Japan between 2011 and 2014, to determine whether they were infected with blood flukes. We detected the blood flukes, Cardicola orientalis and C. opisthorchis , from all four fish groups investigated. From this, it is suggested that bluefin tuna blood flukes are brought into tuna farms with the infected wild seed stock. [ABSTRACT FROM AUTHOR]

Highlights: [•] The juvenile PBT suffers high mortality after stocking in sea net cages. [•] Half the number of juveniles had died within the first 4 days. [•] One-third of the dead fish had ingested inorganic matter such as wood or Styrofoam waste during the first 7 days, and the dead fish showed poor growth. [•] Improvement in feeding method can increase the survival rate of juveniles in sea net cages. [Copyright &y& Elsevier]

The market for Pacific bluefin tuna (Thunnus orientalis) is expanding as global consumption increases. Meeting this demand has led to overfishing and consequent depletion of wild populations, resulting in a focus on aquaculture as an alternative to fishing. However, under the crowded conditions typical of tuna aquaculture settings, infectious diseases are a common problem associated with considerable economic losses, especially with regard to Pacific bluefin tuna, which is a high-priced species. Studying diseases in Pacific bluefin tuna requires techniques that enable experimental infection; however, no promising methods are available due to the difficulty of handling these fish, including transportation, retention, and injection. In the present study, therefore, we developed a method for experimental infection of juvenile Pacific bluefin tuna with red seabream iridovirus (RSIV), which causes an acute and highly contagious disease in this species. The route of experimental infection was validated using Pacific bluefin tuna and greater amberjack (Seriola dumerili), which is used as a model species susceptible to the disease. Oral infection with the virus was found to be suitable for reproducing red seabream iridoviral disease (RSIVD). A high copy number of the RSIV genome was detected in Pacific bluefin tuna that died following RSIV experimental infection, indicating that the virus efficiently replicated in the infected fish. In addition to virus detection, a histopathologic abnormality specific to RSIV infection was detected in the infected fish. These results indicate that experimental infection via the oral route is effective for reproducing RSIVD in Pacific bluefin tuna. • The experimental infection route for red seabream iridovirus (RSIV) was determined. • Oral infection was optimal for reproducing RSIV disease in Pacific bluefin tuna. • RSIV efficiently replicated in RSIV-infected Pacific bluefin tuna. • Infected tuna exhibited RSIVD-specific histopathologic abnormalities. [ABSTRACT FROM AUTHOR]

Pacific bluefin tuna (Thunnus orientalis) is one of the most important species for aquaculture in Japan. In recent years, the reduction in muscle fat content during sexual maturation in farmed T. orientalis has become a serious problem. Herein, as a first step toward sterilization to avoid the deterioration of flesh quality associated with sexual maturation, we examined cold-shock conditions for inducing triploidy in T. orientalis. In this study, three treatment variables (initiation timing, duration, and water temperature of the cold-shock treatment) were considered, using the fertilized eggs collected immediately after spontaneous spawning in a land-based tank. We found that the optimal cold-shock conditions for T. orientalis were 5 °C for 15 min starting from 5 min after fertilization. Under these optimal conditions, we obtained high triploidization (96.6 ± 3.3%) and total hatching (64.5 ± 8.7%) rates. Therefore, using eggs obtained by spontaneous spawning in a land-based tank and the triploidization method established in this study, it is possible to repeatedly and efficiently produce triploid T. orientalis. In recent years, deterioration of flesh quality associated with sexual maturation in aquaculture-produced Thunnus orientalis has become a serious problem. To avoid this problem by sterilization technique, in this study, we have developed an efficient method for the induction of triploidy in T. orientalis , using the fertilized eggs collected immediately after the spontaneous spawning in a land-based tank. We believed that the results of present study are a valuable first step toward the sterilization of T. orientalis. • We established a technique for the mass production of triploid Pacific blue tuna. • The optimal condition was 5 °C for 15 min starting from 5 min after fertilization. • High triploidization (96.6%) and hatching rates (64.5%) were obtained. [ABSTRACT FROM AUTHOR]