Osamu Murata

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.

Morphology and distribution of blood fluke eggs and associated pathology in the gills of cultured Pacific bluefin tuna, Thunnus orientalis.

Infestations of blood flukes of the genus Cardicola have been observed in juvenile Pacific bluefin tuna (PBT) cultured in Japan. Infected fish harbor large numbers of parasite eggs in their gills. Although the link between blood fluke infection and juvenile mortality is not clear, accumulation of parasite eggs appears to be pathogenic to the fish. We investigated the origins, general morphology/distribution, and histopathology of these eggs in artificially produced 0 yr old PBT. Dead and live fish were sampled on several occasions from two culture facilities in Wakayama prefecture, Japan. The number of eggs in each gill filament was enumerated under a microscope. In addition, we estimated the total number of eggs by dissolving the gills in a weak NaOH solution. We observed two morphologically distinct egg types in the gill filaments, smaller, oval shaped eggs in the gill lamellae and larger, crescent shaped eggs that occurred primarily in the filamentary arteries. Based on the ITS2 sequence, the ovoid and crescent shaped eggs were identified as C. orientalis and C. opisthorchis, respectively. Eggs of the former species were more abundant (maximum: 6400 per filament) than the latter (maximum: 1400), but the number was highly variable among filaments. The eggs of the latter species were relatively evenly distributed among the filaments. In a heavily infected individual, we estimated a total of >4.5 million eggs were present in the gills on one side of the fish. The number of eggs from the two species was positively correlated to each other and the dead fish tended to harbor more eggs than the live fish. Histological observation revealed host responses around the eggs, including encapsulation by fibroblasts and nodule formation, as seen in response to other aporocotylid eggs. In addition, we observed widespread fusion of gill lamellae and blockage of the filamentary arteries in some instances. Our results provide information that can be used for routine diagnosis of Cardicola blood flukes in cultured tuna and suggest they represent a risk to juvenile PBT.

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.

Whole‐body heat transfer coefficient and body temperature change of juvenile Pacific bluefin tuna Thunnus orientalis according to growth

Bluefin tuna maintain a higher body temperature than ambient sea water. Body heat is derived mainly from metabolic heat to elevate and maintain regional body temperature that is higher than the ambient, while heat loss is caused by heat transfer throughout the whole body surface and gills. Retention of high body temperature is thought to differ at each growth stage, so that a larger body mass maintains a higher body temperature. We evaluated the whole-body heat transfer coefficient, thermal difference between each tissue and water temperature, and metabolic heat in tissues during swimming of juvenile bluefin tuna as a function of fork length (FL) using a small thermometer and a treadmill-type flow tank. A system for maintaining high body temperature was well developed in fish with FL greater than 20.0 cm. Whole-body heat transfer coefficient was fitted to a −0.695 power of mass. Juvenile bluefin tuna showed a transition speed of 3.0 FL/s at which they switched from aerobic to anaerobic motion.

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.

Construction of an expression vector containing a β-actin promoter region for gene transfer by microinjection in red sea bream Pagrus major

Transgenic technology has been widely applied to a variety of freshwater fish species. However there are few reports on the use of this technology in commercially important marine species. In this study, the construction of expression vectors containing the β-actin promoter region for use in the red sea bream Pagrus major, a species of considerable importance to the aquaculture industry in Japan is reported. The β-actin gene was cloned from a red sea bream genomic DNA library. Recombinant plasmids were constructed by linking the 5′ flanking region of the β-actin gene to the green fluorescent protein reporter gene, followed by the poly A signal sequence of simian virus 40 or the 3′ flanking region the β-actin gene. Expression of these constructs was examined following microinjection into zebrafish and red sea bream embryos, and compared to that of the expression vector pXI-GFP driven by the Xenopus elongation factor 1α. The results indicated that the construct consisting of the β-actin 5′-and 3′ flanking regions was the most efficacious. In future studies, it is planned to investigate the efficient condition for integration into chromosomes of the transgene.

Gonadal sex differentiation and sex control in red sea bream, Pagrus major

The gonadal sex differentiation in red sea bream, Pagrus major, which is one of the most important species for aquaculture in Japan, was revealed histologically. The suitable conditions for induction of all-male groups in the fish were investigated, and functional males were induced by the conditions of oral administration of 17α-methyltestosterone. The sex determination of this fish was also discussed.

Growth and Environmental Tolerances (Water Temperature and Low Salinity) of Hybrid Female Red Sea Bream Pagrus major × Male Black Sea Bream Acanthopagrus schregeli

The growth and tolerance for water temperature and salinity were compared among red sea bream Pagrus major (RSB) black sea bream Acanthopagrus schregeli (BSB) and their hybrid (), female RSB male BSB. The growth of the fish did not differ until 27 days after hatching (dAH), after which the most rapid growth was observed until approximately 300 dAH, followed by RSB and BSB. However, the RSB had out grown the fish by approximately 303 dAH. By 480 dAH, the RSB were largest, followed by the and BSB groups. The tolerances for high and low water temperature were significantly different for each species and growth stage. The largest tolerance spectrum was observed in the BSB group, and the tolerance spectrums gradually decreased with increasing final body weight. During the salinity tolerance trials, all of the species started to die following transfer into freshwater (0 psu). BSB showed the highest survival rate when kept in fresh water for an average of 29.9 hours, while the fish were more tolerant than the RSB fish. The fish were increasingly tolerant to fresh water and changes in water temperature as they grew, while the size of the temperature spectrum remained unchanged but shifted to a lower temperature range with growth. Our results demonstrate that performed well in terms of growth compared to parental fish, with higher temperature and salinity tolerances than RSB, and is thus suggested to be a suitable aquaculture species for Korea and northeast China.

Reproduction between Oplegnathus fasciatus and O. punctatus, and fertility of their interspecies

Striped beakfish Oplegnathus fasciatus and spotted knifejaw O. punctatus belong to the family Oplegnathidae, and are economically important fisheries resources in Japan and Korea. These species are distributed in the Pacific Ocean around southern parts of the Korean Peninsula, Japan, Taiwan, and Hawaii. These fish inhabit coastal rocky reefs, and feed on benthic animals. Mature females of these species spawn several times on the sandy/gravel bottom from April to July [1–3]. The ability to produce a hybrid, or to maintain reproductive isolation, is limited by two sets of reproductive barriers [4]. Prezygotic barriers include ecological isolation (e.g., geographical separation), temporal isolation (e.g., different mating seasons), behavioral isolation (e.g., incompatible courtship), mechanical isolation (e.g., incompatible genitalis), and gametic isolation (e.g., incompatible gametes). Postzygotic barriers include hybrid inviability, hybrid sterility, and hybrid breakdown [5]. Kamei and Takama [6] reported that O. fasciatus and O. punctatus would employ similar reproductive strategies (spawning ecology, such as spawning season, spawning time, spawning area, and spawning behavior), thus supporting the potential for hybridization. In the wild, fish with a similar body color pattern to artificially inseminated hybrids between O. fasciatus and O. punctatus [7, 8] were rarely found. Even though these two species have been speciated, why does the hybrid appear in the wild? To investigate fundamental information on the reproductive system of the hybrid between these two species, we explored possible prezygotic barriers to hybridization by comparing spontaneous spawning with artificial insemination. O. fasciatus, O. punctatus, and their F1 hybrids (O. fasciatus 9 O. punctatus) were reared in sea net cages (age 4–6 years) at Fisheries Laboratory, Kinki University, Wakayama, Japan. On 2–14 June 2007, we performed observation of spontaneous spawning in 3,000-L tanks in each cross group. The following four crosses were conducted: