Shigeru Miyashita

Status of Bluefin Tuna Farming, Broodstock Management, Breeding and Fingerling Production in Japan

Development of bluefin tuna farming in Japan has a 30-year history. This paper reviews recent developments as well as the current status and problems of farming, broodstock management, and fingerling production in Japan. Farmed bluefin tuna by wild seed-stock comprises approximately 15–20% (2,400 tons) of the annual catch of bluefin in Japan. Comparisons among farms in Japan indicate that bluefin tuna growth is positively correlated with annual water temperature. Broodstock commence spawning at approximately 3–5 years of age, and eggs are collected from within the cages. The number of bluefin tuna eggs collected in Japan has varied greatly—between zero and about 500 million—with variance between farms and years. Current larval rearing techniques produce tens of thousands of fingerlings of 35–50 mm per season in Japan, but survival rates are 0.01–4.5%. Although there are the bottlenecks in egg collection and larval rearing under the present technology, the approaches for enhancing bluefin resources and the replacement of wild seed-stock by artificial fingerling to tuna farms have firmly been improving in Japan.

Growth and morphological development of larval and juvenileepinephelus bruneus (perciformes: Serranidae)

The growth and morphological development of larval and juvenileEpinephelus bruneus were examined in a hatchery-reared series. Average body length (BL) of newly-hatched larvae was 1.99 mm, the larvae growing to an average of 3.96 mm by day 10, 6.97 mm by day 20, 12.8 mm by day 30, 22.1 mm by day 40 and 24.7 mm by day 45 after hatching. Newly-hatched larvae had many mucous cells in the entire body epidermis. By about 4 mm BL, the larvae had developed pigment patterns peculiar to epinepheline fishes, including melanophores on the dorsal part of the gut, on the tips of the second dorsal and pelvic fin spines, and in a cluster on the ventral surface of the tail. Spinelets on the second dorsal and pelvic fin spines, the preopercular angle spine and the supraocular spine, had started to develop by about 6 mm BL. The notochord tip was in the process of flexion in larvae of 6–8 mm BL, by which time major spines, pigments and jaw teeth had started to appear. Fin ray counts had attained the adult complement at 10 mm BL. After larvae reached 17 mm BL, elements of juvenile coloration in the form of more or less densely-pigmented patches started to appear on the body. Squamation started at 20 mm BL. Major head spines had disappeared or became relatively smaller and lost their serrations by 20–25 mm BL.

Production of cloned red sea bream, Pagrus major, by chromosome manipulation

Red sea bream, Pagrus major, is one of the most important fish cultured in Japan. Two clones of red sea bream were produced. Eggs from a mitotic gynogenetic diploid (mitotic-G2N) red sea bream were inseminated either with sperm from a mitotic-G2N male to produce a heterozygous clone (hetero-clone), or with UV-irradiated sperm of Japanese parrot fish (Oplegnathus fasciatus) and the second meiotic division suppressed by cold shock to produce a homozygous clone (homo-clone). Normal diploids were also produced from one male and female as a control. The clonal status of the fish was confirmed by multilocus DNA fingerprinting. The fingerprinting patterns differed between individuals within the normal diploids. However, there was no variation between individuals within hetero- or homo-clones. The patterns of the homo-clones and the mother were identical, and all the bands of homo-clones were also observed in hetero-clones. Thus, the clonal status of homo- and hetero-clones was confirmed and the production of clones from the broodstock of mitotic-G2N was achieved. The hatching rates, survival rates and growth of the hetero- and homo-clones were recorded for a brief comparison with results of diploid controls.

Ontogenetic changes in RNA, DNA and protein contents of laboratory-reared Pacific bluefin tuna Thunnus orientalis

The ontogenetic changes in the growth potential of larval and juvenile laboratoryreared Pacific bluefin tuna were examined based on RNA-DNA and protein-DNA ratios. Experimental fish were reared at the Ohshima Experiment Station of Kinki University Fisheries Laboratory in August 2002. Samples were taken from 13 to 35 days after hatching (DAH). Metamorphosis from larva to the juvenile stage was observed around 23 DAH. Somatic growth of Pacific bluefin tuna was accelerated after metamorphosis. The value of the RNA-DNA ratio from 13 to 19 DAH increased slightly from 3.77±0.58 (mean±SD) to 7.28±2.23. After that, the ratio markedly increased from 13.89±3.71 on 21 DAH to 19.11±4.27 on 23 DAH, which was the end of the metamorphic period. After 25 DAH, the ratio remained at a high level of 15–20. The protein-DNA ratio showed a similar tendency to the RNA-DNA ratio. These results suggest that the rapid increase in the RNA-DNA ratio in the metamorphic period supports the consequent rapid somatic growth in the juvenile stage. The high ratio after the metamorphic period could be because of the species-specific traits large prey exhibit for their survival and because of the tuna’s fast-growth after the juvenile stage.

Association between bacterial community structures and mortality of fish larvae in intensive rearing systems

Bacterial community structures were analyzed in water used for rearing fish larvae by fluorescence in situ hybridization. In Experiment 1, red sea bream Pagrus major larvae were reared in two commercial seed production tanks. The survival rate in Tank 1 was higher than in Tank 2, even though phytoplankton, Nannochloropsis sp., was added to both tanks. In Tank 2, γ-proteobacteria became dominant (∼70% of total bacteria) on day 13, there after heavy larval mortalities occurred. In Tank 1, however, α-proteobacteria and the Cytophaga-Flavobacterium cluster were predominant from day − 1 until day 13; no significant mortality was recorded. In Experiment 2, marble goby Oxyeleotris marmoratus larvae were cultured with or without Nannochloropsis sp. At the end of the experiment, larval survival rates in aquaria with Nannochloropsis sp. were significantly (P <0.05) higher than those without. In rearing water without Nannochloropsis sp., γ-proteobacteria increased during rearing. In rearing water with Nannochloropsis sp., α-prote obacteria and the Cytophaga-Flavobacterium cluster were predominant at the beginning of the experiments and the relative abundance of γ-proteobacteria was maintained at a lower level throughout the experiments. The predominance of α-proteobacteria and the Cytophaga-Flavobacterium cluster appears to be a good indicator of successful larval production.

Characterization of transthyretin in the Pacific bluefin tuna, Thunnus orientalis.

Abstract A cDNA encoding transthyretin was cloned from the Pacific bluefin tuna (Thunnus orientalis). This cDNA contains a complete open reading frame encoding 151 amino acid residues. The deduced amino acid sequence is 81% and 55% identical to the gilthead seabream and common carp forms, respectively, and 33–39% to mammalian, reptilian, and amphibian forms. A 1.0-kb transcript was found in the the liver and ovary; the liver is the main source of this protein. Analysis of triiodo-L-thyronine (T3) and L-thyroxine (T4) binding demonstrated that both T3 and T4 bind to bluefin transthyretin. The binding activity of T3 for bluefin transthyretin is higher than that of T4. These results indicate that bluefin transthyretin acts as a transporter of thyroid hormones (THs) in the plasma, and plays an important role in the function of THs in target cells.

Testes maturation of reared Pacific bluefin tuna Thunnus orientalis at two-plus years old

Stable reproduction is essential for supplying artificially hatched fish to tuna aquaculture. We observed testes maturation in reared Pacific bluefin tuna (PBT) Thunnus orientalis at 2+ years of age. The incidence of males with mature testes was 25.0%, and 40% of the males had developing testes that contain spermatozoa, while oocytes of the same aged females were not mature. These fish were wild-caught at 0+ years old in August 1997 and the gonads were examined in October 1998 and January–February 2000. Therefore, the age at examination in 2000 was estimated to be 2 years and 7–10 months old considering the spawning season of the wild PBT and the size when captured. Histological examination of thematured and developing testes showed that they contained spermatozoa, spermatids, spermatocytes, and spermatogonia. All the spermatozoa were observed to be motile in sea water under light microscopy. From the results of this and previous studies, matured males are probably fertile for at least 5 months a year in Kushimoto. The testes maturation observed at young age in captivity is considered promising to reduce the cost of brood-stock maintenance for the juvenile production of PBT, especially if the sperm are cryopreserved.

Infection dynamics of Kudoa yasunagai (Myxozoa: Multivalvulida) infecting brain of cultured yellowtail Seriola quinqueradiata in Japan

We monitored infection by a brain-infecting myxozoan Kudoa yasunagai in hatchery-reared juvenile yellowtail Seriola quinqueradiata at a culturing site in Japan. Infection was detected by PCR and microscopic observation once every 1 to 4 wk during 2010 and 2011. In both years, we detected first infection in mid-July by PCR. Prevalence increased rapidly after the onset of infection, peaking at 100% within 4 wk. Parasites required less than 10 d to reach the brain after invasion. Development of plasmodia and formation of cysts took 4 to 8 wk. Infection did not reach a plateau and number of cysts tended to decline over time, suggesting possible recovery from the infection. A drastic decline in infection prevalence was observed during the season of highest water temperature (>30°C) in 2010. To understand this phenomenon, we conducted a laboratory experiment to compare infection prevalence and cyst formation in fish kept at 25°C and 30°C. However, we could not detect obvious differences between the treatment groups during the 4 wk of the experiment. There was no apparent pathology associated with the infection. These results suggest that pathological effects of K. yasunagai may differ between fish species or that other factors are important in the development of infectious signs.

Effect of fasting on physical/chemical properties of ordinary muscles in full-cycle cultured Pacific bluefin tuna Thunnus orientalis during chilled storage

Using the full-cycle cultured (FC) Pacific bluefin tuna [body weight 16.3±1.9 kg (pre-fasting group, pre-FG), 14.2±0.9 kg (post-fasting group, post-FG)], changes in the physical/chemical properties of the cephalal parts of dorsal (D) and ventral (V) ordinary muscles (OM) by fasting (6 days) during chilled storage (4°C) were investigated. Condition factors were 26.7 (pre-FG) and 20.3 (post-FG, P < 0.05). Fasting changed the liver color to green. Fasting also decreased the amount of protein and lipid contents of the DOM and VOM of FC tuna. The breaking strength and pH of the DOM and VOM of post-FG tuna were higher (P <0.05) than for pre-FG tuna during storage. In contrast, the glycogen contents of DOM and VOM of post-FG tuna were lower than for pre-FG tuna. The color values (L*, a* and b*) of DOM of post-FG tuna were lower than for pre-FG tuna throughout the storage period. In addition, the metmyoglobin (metMb) content of DOM of post-FG tuna was lower (P <0.05) than that of pre-FG tuna, and the metMb content of VOM of post-FG tuna remained low after fasting. These results indicate that fasting suppresses deterioration (especially meat color) of FC tuna muscles during chilled storage.

Developing 23 new polymorphic microsatellite markers and simulating parentage assignment in the Pacific bluefin tuna, Thunnus orientalis

Twenty‐three new polymorphic microsatellite markers were isolated in the Pacific bluefin tuna, Thunnus orientalis. Each locus comprised three to 34 alleles. The expected and observed heterozygosities ranged between 0.46 and 0.96 and between 0.44 and 0.97, respectively. The Kto9, Kto11, and Kto42 markers demonstrated significant deviation from Hardy–Weinberg equilibrium; high null allele frequencies (0.08–0.14) were observed in the deviating group. From the results of simulation of parentage assignment, a combination of four loci (i.e. Kto15, Kto23, Kto38, and Kto39) was considered the best for parentage assignment.