Yoshifumi Sawada

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.

Two types of tumor necrosis factor-α in bluefin tuna (Thunnus orientalis) genes: Molecular cloning and expression profile in response to several immunological stimulants

Tumor necrosis factor-alpha (TNF-alpha) is a key inflammatory mediator and has also the potential as a prominent biomarker of innate immunity. In this study, we identified and characterized TNF-alpha from bluefin tuna, which is an important cultured species. Two types of TNF-alpha were also cloned incidentally (TNF1 and TNF2). The open reading frame of TNF1 and TNF2 cDNA encoded 247 and 245 amino acids, respectively. The amino acid sequence identity among sea perch, red sea bream, and tiger puffer was 73, 70, 59% for TNF1 and 49, 51, 45% for TNF2, respectively. The identity between TNF1 and TNF2 amino acid sequences of the bluefin tuna was only 43%. The positions of cysteine residues, transmembrane sequence, and protease cleavage site in bluefin tuna TNFs were similar with other reported fish and mammalian TNF-alpha. In a phylogenetic analysis, TNF1 is grouped with other reported Perciformes TNF-alpha. On the other hand, TNF2 is grouped with ayu TNF and is quite distant from the fish TNF-alpha group and lymphotoxin-beta group. While TNF1 mRNA showed no significant difference in all tissues, TNF2 mRNA was expressed significantly higher in the blood than in the gill, intestine, head kidney, spleen, heart, and ovary. In peripheral blood leucocytes (PBL), expressions of TNF2 mRNA were significantly increased by stimulation with lipopolysaccharide, phytohemagglutinin, concanavalin A, pokeweed mitogen, phorbol myristate acetate in vitro, but those of TNF1 were not. Recombinant mature TNF1 and TNF2 proteins significantly enhanced phagocytic activity of PBL. Our results suggest that bluefin tuna possess two types of TNF-alpha homologue, and TNF2 is a potential biomarker for innate immunity.

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.

Kudoa prunusi n. sp. (Myxozoa: Multivalvulida) from the brain of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan.

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.

Ontogenetic changes in schooling behaviour during larval and early juvenile stages of Pacific bluefin tuna Thunnus orientalis.

Schooling was first observed at 25-27 days after hatching (26. 2-33. 8 mm, total length) in the Pacific bluefin tuna Thunnus orientalis. At this time, the mode of swimming changed from intermittent sprinting to continuous cruising, and this allowed the fish to adjust to an inertial hydrodynamic environment.

Electroretinographic Analysis of Night Vision in Juvenile Pacific Bluefin Tuna (Thunnus orientalis)

We used electroretinogram recordings to investigate visual function in the dark-adapted eyes of the juvenile scombrid fishes Pacific bluefin tuna (Thunnus orientalis) and chub mackerel (Scomber japonicus) and the carangid fish striped jack (Pseudocaranx dentex). Despite the fast swimming speed of the Pacific bluefin tuna, analysis of flicker electroretinograms showed that visual temporal resolution in this species was inferior to that in chub mackerel. Peak wavelengths of spectral sensitivity in Pacific bluefin tuna and striped jack were 479 and 512 nm, respectively. The light sensitivity of Pacific bluefin tuna was comparable to that of chub mackerel but lower than that of striped jack. The Pacific bluefin tuna may not need high-level visual function under dim light conditions in natural habitat because it is a diurnal fish. However, this low temporal resolution and light sensitivity probably explain the mass deaths from contact or collisions with net walls in cultured Pacific bluefin tuna.

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.

Induction of centrum defects in amberjack Seriola dumerili by exposure of embryos to hypoxia

Artificially hatched Seriola species have the problem of malformation, mainly in their vertebrae, head, and mouth parts. To clarify the cause of vertebral malformation, the effects of hypoxia during embryogenesis on the induction of centrum defects was investigated in artificially hatched amberjack Seriola dumerili. Firstly, 7-somite stage embryos were exposed to waters of 0, 12.5, 25, 50, and 100% dissolved oxygen (DO) for 0.5, 1, 2, 3, 4, and 5 h to confirm the effective dose (DO concentration and duration of exposure) of hypoxia that induces somitic disturbances in newly hatched larvae. Exposure of embryos to 12.5% DO concentration for longer than 0.5 h induced somitic disturbances. Following this result, centrum defects in juveniles were investigated by an induction experiment with embryos exposed to 12.5% DO for 2 h at the gastrula, 1- or 2-somite, 10-somite, 15-somite, or heart beating stage. This experiment revealed that centrum defects were induced only during somitogenesis, and somitic disturbances were the premonitory symptom of centrum defects. These results indicate hypoxia during somitogenesis as a possible cause of centrum defects in amberjack.

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.

Changes in the retinal cone density distribution and the retinal resolution during growth of juvenile Pacific bluefin tuna Thunnus orientalis

Tuna and marlin have well-developed vision. It has been suggested that the vision of the tuna is a major sensory element greatly affecting its behavior. In a behavioral experiment, Nakamura determined the visual acuity of adult skipjack tuna Katsuwonus pelamis. Kawamura et al. calculated the visual acuity of adult bluefin tuna Thunnus thynnus, histologically. Although there are developmental changes in the visual capability and increases in the visual acuity in many fish species with growth, juvenile Pacific bluefin tuna Thunnus orientalis has not been investigated. Furthermore, Fritsches et al. noted that the striped marlin Tetrapturus audax has different visual capabilities along different visual axes. The position of the area of high cell density, or the area centralis in the retina, is related to both habitat and the main visual axis of feeding behavior. Knowledge of developmental changes in retinal topography gives important clues to behavioral changes that occur with growth. To measure the distribution of retinal cone density at each growth stage, it is necessary to investigate specimens from each stage. Recently, full-cycle culture of the Pacific bluefin tuna Thunnus orientalis was achieved in the Fisheries Laboratory, Kinki University, Wakayama, Japan. Using full-cycle cultured specimens of this species, including juveniles, it was possible to investigate the cone density distribution at each stage in this species. In the present study, the authors determined the developmental changes in the cone density distribution, visual axis and minimum separable angle of cones with growth histologically. Specimens of Pacific bluefin tuna from six stages that were full-cycle cultured during 2003–2004 in the Fisheries Laboratory, Kinki University, were used: 30 (total length [TL] = 4.7 cm), 35 (TL = 6.2 cm), 41 (TL = 6.5 cm), 46 (TL = 11.1 cm), 80 (TL = 31.8 cm) days after hatching, and 1 yearold (TL = 102.6 cm). The eyes of the specimens were enucleated and fixed in Bouin’s solution for 24 h. Each retina was then divided into nine regions for 30to 46-day specimens, 25 regions for 80-day specimens and 33 regions for 1-year specimens, according to the eye size. After paraffin embedding, the retina was cut into sections parallel to the retinal surface using a microtome. The sections were stained with hematoxylin–eosin. For quantitative analysis, the sections were examined under light microscopy, photographed, and the number of cones in 0.01 mm of each region in the photomicrograph was counted. Photomicrographs of tangential sections of the retinal cones showed that twin cones form a regular mosaic of parallel rows, although there were occasional irregular single cones in each region from specimens at every stage. An example of a retinal tissue older than 1 year after hatching is shown in Figure 1. The total numbers of twin and single cones in 0.01 mm of each region were counted. Then, the density distributions of both cones determined from the right retinas of the specimens (30, 35, 41, 46, 80 days, and 1 year after hatching) were graded, drawn and displayed using contour lines at 25-cone intervals (Fig. 2). The specimens at even the youngest stage had already metamorphosed and were feeding on fish prey. The characteristics of the cone density distribution changed with each growth stage. In the specimen aged 30 days after hatching, the cone density in each peripheral area reached five times (668/132 = 5.03) that of the bottom region. Therewasnodefinitedirectionof acutevision, such as a visual axis, since no specialized region of maximum cone density existed. It seems reasonable to assume that the distribution at this stage *Corresponding author: Tel: 81-742-43-1511. Fax: 81-742-43-1316. Email: ns_torisawa@nara.kindai.ac.jp Received 10 November 2005. Accepted 27 March 2006. FISHERIES SCIENCE 2007; 73: 1202–1204