Comparative analyses of the inheritance patterns of transgene transmission and expression for up to F₅ progenies were carried out in different lines of transgenic mud loach Misgurnus mizolepis containing the chloramphenicol acetyltransferase (CAT) reporter gene. Based on Southern blot hybridization, the analyses revealed different integration sites of transgenes in host chromosomes as well as different transmission modes among transgenic lines, including stable inheritance of integrated transgenic constructs, co-transmission of integrated and extrachromosomally persistant copies, and transgene rearrangement through successive generations. CAT-enzyme-linked immunosorbent assay expression analysis found three different major patterns of inherited expression of the transgene: (i) stable persistence of consistent level of expression; (ii) sudden loss of transgene expression following passage through the germline; and (iii) different levels of reduction in the expression in successive generations.

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In recent years, there has been debate from various perspectives on the question of whether the mitten crabs Eriocheir japonica and E. sinensis should be considered as two different species. It has been reported that the chromosome numbers of these two crabs differ from each other; for E. japonica it is 2n=148, n=74, whereas for E. sinensis it is 2n=146, n=73. These data suggest that E. japonica and E. sinensis are different species. Nevertheless, the chromosome number of E. japonica was obtained in 1937 by the method of paraffin section. This classical gonad (testis) sectioning method is now known as an unreliable method for determining the number of chromosomes. In the present study the number of chromosomes in E. japonica and E. sinensis is re-examined using the air-drying method. The results show that E. japonica and E. sinensis have the same number of chromosomes (2n=146, n=73), which means that the number of chromosomes cannot be used as a criterion to distinguish these two species. Even though observation of the chromosomes under the scanning electron microscope was attempted, it was not possible to perform karyotype analysis in the two species because centromeres could not be identified in some of the chromosomes.

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Feeding and growth of the Manila clam Ruditapes philippinarum fed on diatom Thalassiosira sp. were examined in the laboratory. The specific clearance rate (CR) relative to soft-body dry weight (w) of clams (shell length: 8-28 mm) followed a power function of w with an exponent of -0.32. The CR for the juvenile clams (shell length: 9-12 mm; w: approx. 10 mg) increased from 8 to 20 L/g per h in the temperature range 12-21°C, and then decreased gradually in the range 21-30°C. Growth rates (G) of juveniles (shell length: 10-12 mm) in terms of ash-free dry weight of the soft body were measured at 9°C and 20°C and increased linearly with weight-specific daily ration (Q) at both temperatures. G values were higher at 9°C than at 20°C when Q was fixed, probably owing to the lower catabolic rate at 9°C. The slope and y-intercept of G against Q gave an assimilation efficiency (0.62) and daily loss of body weight by catabolic processes (1.4%/day) at 20°C.

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A newly designed frame trawl with a 5 m² mouth area was constructed for quantitative sampling of larval and juvenile pelagic fishes and its performance was tested in the ocean. Towing depth of the trawl was easily controlled by adjusting the warp length, and the stability at depth increased with decreasing weight of the net in water. Larval and juvenile fishes were sampled using the trawl at stations in the Kuroshio-Oyashio transition region from 29 May to 24 June 1998. A total of 44 oblique tows were conducted and towing depths of the frame trawl had a high degree of stability despite the various sea conditions and the fluctuations in the ship speed. Larvae and juveniles of Japanese anchovy Engraulis japonicus and Myctophidae made up the majority of the specimens. It was concluded that the newly constructed trawl was appropriate for the quantitative sampling of pelagic fish larvae and juveniles.

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The sea urchin Anthocidaris crassispina (A. Agassiz) is a dominant herbivore on rocky shores in the warm temperate region of Japan. To clarify the relationship between macroalgal community and A. crassispina on rocky shores, A. crassispina collected in the Sargassum area and neighboring Corallina area were compared with respect to their density, growth and reproduction. Density of A. crassispina was higher in the Corallina area than in the Sargassum area. A. crassispina in the Sargassum area reached a larger size and had higher gonad indices than those in the Corallina area throughout the year. The annual reproductive cycles were almost the same in the two different habitats. These results indicate that Sargassum spp. support better growth and reproduction of A. crassispina.

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The territorial behavior of juvenile red sea bream Pagrus major and crimson sea bream Evynnis japonica was examined at Morode Cove, Ehime Prefecture. Four exclusive types of behavior (attack, mutual attack, head down threat and mutual display) and one escaping behavior (fleeing) were observed in both species. Juvenile of the two sea bream could discriminate between each other and change their territorial behavior depending on the presence of conspecifics or heterospecifics. The territorial behavior toward conspecifics was more aggressive than toward heterospecifics. Interrelationships between the two sea bream were more aggressive than in other heterospecific cases. The two sea breams performed following behaviors toward solo and schooled mullid species. E. japonica ascended the water column more frequently than P. major in high frequency. The former joined conspecific aggregations foraging for planktonic animals around their territories. These observations suggest that territorial individuals of the two sea bream could discriminate intruders utilizing similar food resources in order to maintain their territories.

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Artificial reproduction and subsequent improved culture of the Japanese eel Anguilla japonica have not yet been achieved, but are expected in the near future. To allow the development of aquaculture activities, chromosome manipulation techniques are necessary for the genetic improvement of the eel. In the present paper suitable conditions were determined for heat shock treatment for the induction of triploidy. This was done using fertilized eggs obtained from gametes of cultured eels that were induced to mature by repeated injections of salmon pituitary extract, followed by an injection of 17α, 20β-dihydroxy-4-pregnen-3-one to induce ovulation in the female eels and by repeated injections of human chorionic gonadotropin in the male eels. Three variables (i.e. timing after fertilization, temperature, and duration of the shock) were varied to maximize the yield of triploid larvae by examining hatching and triploid rates of treated groups. Ploidy was determined by DNA content flow cytometry and/or chromosome counting. The triploid yield was the highest (70%), when a 37°C heat shock was applied for 3 min, 10 min after fertilization. Under such conditions the rate of triploidy reached 70-100% and the rate of hatching was 5.2-24.6% when the control hatching rate was 28.5% on average.

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The reproductive performance of 941 female specimens of black rockfish Sebastes inermis collected from four sites (from north to south, Ayukawa, Shichigahama, Yuriage and Haragama) along Sendai Bay, northern Japan from October 2000 to March 2001, was analyzed. Mature female S. inermis ranged from 2 to 9 years in age, from 15 to 29 cm in total length and from 80 to 496 g in total weight. Mean gonadosomatic index ranged from 0.42 to 14.6, showing a similar tendency in all locations, increasing from October, peaking around early December, and decreasing thereafter. The same pattern was observed in ovary weight, ranging from 0.72 to 33.74 g. Seasonal fluctuation in the percentage of occurrence of four development stages of ovaries (ripe, fertilized, eyed and spent) indicated that parturition season extended from late December to early March in Sendai Bay. However, parturition shifted on both a temporal and spatial scale, that is, the extrusion began and finished later, the more northerly was the site. There were significant differences in the age of spawners as extrusion season progressed, with older female S. inermis (approx. 5 years old) occurring at the beginning and younger ones (approx. 2 years old) occurring at the end of the season. A similar tendency was also observed in size, weight and ovary weight in most ovarian developmental stages.

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The nucleotide sequences of Japanese flounder Paralichthys olivaceus, major histocompatibility complex (MHC) cDNA, classical MHC class Iα, non-classical MHC class Iβ, MHC class IIα and IIβ, were determined. The domain structures and antigen binding motifs of vertebrate MHC are conserved in the Japanese flounder MHC. A phylogenetic analysis supports the classification of these genes into class I and class II MHC. Classical MHC class Iα was ubiquitously expressed, whereas the non-classical MHC class Iβ was expressed mainly in lymphoid organs, gills, intestine and stomach. The MHC classes IIα and IIβ were also ubiquitously expressed.

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Tenacibaculum sp., the causative bacterium of spotting disease of sea urchin Strongylocentroutus intermedius, has been known to enter into the viable but non-culturable (VBNC) state in 75% artificial seawater (ASW) at 5°C. From this fact it is suspected that this bacterium probably resuscitates from the VBNC state in summer. To examine whether non-culturable cells of Tenacibaculum sp. are able to regain the culturable state, VBNC cells in 75% ASW and 75% ASW containing homogenated sea urchin (HSU) were treated with temperature upshift (5°C to 25°C) and addition of iron chloride. Resuscitation from VBNC cells with temperature upshift was observed only in 75% ASW containing HSU for 3 days incubation after entry into the VBNC state. Resuscitation of VBNC cells treated with iron chloride was observed in each microcosm. Resuscitation from the VBNC state was confirmed by dilution experiments and measurements of generation time. Resuscitated cells regained original characteristics, agglutination against anti-Tenacibaculum sp. strain F-2, pathogenicity to sea urchin and morphology.

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Anthocidaris crassispina collected from the Sargassum area and neighboring Corallina area were examined for their diet, and the stable isotope ratios of gonad and gut contents. Sargassum spp. were primary constituents of gut contents in both habitats, the percentages of them in the Corallina area were less than those in the Sargassum area and the other constituents were almost all calcareous algae. In the stable isotope analyses, gut contents were significantly different, but gonads were not significantly different between two habitats. It is suggested that there were abundant food sources in the Sargassum area, in contrast to the Corallina area where drifting seaweeds compensate the shortage of food supply from this area.

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The present study investigated the thermal stability and denaturation rate of myoglobin (Mb) from migratory, demersal, aquaculture and elasmobranch fish species using thermal analysis and dynamic analysis of Mb denaturation. The concentration of Mb was 1.86-2.80 mg/g muscle in the migratory fish, and the tilapia had the lowest Mb concentration of 0.94 mg/g muscle. By analyzing the phase transition temperature (T_m) and denaturation rate of Mb, dolphinfish and horse mackerel Mb showed lower T_m at 46.4 and 51.8°C, respectively. The main endothermic peak was around 71.8°C for spotted shark Mb. Although the Mb denaturation behaved as biphasic first-order reaction for all the Mb examined, spotted shark Mb showed the highest thermal stability and was followed by chub mackerel and tilapia Mb. The horse mackerel Mb showed the lowest thermal stability.

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The extracellular production of a thermolysin inhibitor by 534 wild-type strains isolated from marine sediments and related-type strains was examined. The inhibition of thermolysin activity by strain T-177 was observed on a casein agar medium. According to our detailed taxonomic study, strain T-177 is related to Brevibacillus laterosporus. Further screening for a thermolysin inhibitor was performed using strain T-177 and nine other taxonomically related Brevibacillus type strains, and the ability to produce the inhibitor was observed in five strains. Among these strains, B. reuszeri IFO 15719^T exhibited the highest thermolysin inhibitory activity in broth media, and thus was used for the purification and characterization of the inhibitor. Chromatographic analyses suggested that this substance is a monomeric protein with a molecular mass of 60 kDa.

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The changes in viscoelasticity of invertebrate muscle natural actomyosin, including paramyosin, during thermal gelation were investigated by dynamic viscoelasticity measurements. For the thermal gelation of invertebrate actomyosin, an increase in the storage modulus was observed at relatively high temperature ranges. Paramyosin alters the characteristic texture of invertebrate gel products considerably; the cooked gels become more elastic and have greater cohesiveness with the addition of small amounts of paramyosin. Therefore, the characteristic feature of invertebrate meat gel is considered to be a reflection of the properties of paramyosin.

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The oil of Erabu sea snake Laticauda semifasciata, a marine reptile, is used as a health promoting food in Japan. The aims of the present study were to determine the effect of Erabu sea snake oil on the learning ability in mice. Male mice (6 months old) were fed a diet containing 5% lard, 5% Erabu sea snake oil, or 5% fish oil mixture, containing the same percentages (12.8%) of docosahexaenoic acid as the sea snake oil. Maze-learning ability was assessed 4 months after the start of the experiment and the brain fatty acid composition was measured after 4.5 months. In the third trial run of the maze-learning task, the number of times that a mouse strayed into blind alleys in the maze was significantly lower in the sea snake and fish oil mixture groups than in lard group. These results suggest that sea snake oil exerts an equivalent influence on the learning ability in mice to that of fish oil.

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Dried sea cucumber must first be soaked in water, then heated and finally left at 20°C for 24 h, this procedure being repeated several times in preparation for cooking. In the case of dried sea cucumber soaked in distilled water, it gradually increased in weight. After full-scale soaking twice, its weight increased to 7.6-fold the original weight, which softened it thoroughly. The water content after this soaking process was 94.5%, which is almost the same as that of raw sea cucumber. During the soaking process, not only was water absorbed, but components of the ingredients of sea cucumber were eluted into the soaking water. Among these components, ash was eluted fastest, next was glycosaminoglycan and last was collagen. In addition to distilled water, rice-washing water, a coarse-tea-infused solution and a potassium carbonate solution were used as the soaking solutions. Sea cucumber absorbed water and swelled in the potassium carbonate solution faster than in the other three solutions. Textural measurements and a sensory evaluation clarified that the softest sea cucumber resulted from the rice-washing water, and the hardest sea cucumber from the coarse tea.

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Eel muscle contains lipid as much as 15-20% of wet weight of muscle. However, it is uncertain whether lipid is synthesized in muscle or transported from another tissue such as liver. We prepared isolated muscle cells to investigate the synthetic ability of lipid by muscle cell itself and studied lipoprotein metabolism in coculture with eel hepatocytes. Isolated eel muscle cells were prepared by collagenase digestion of skeletal muscle and their length and width were approximately 2.2 mm and 5-20 μm, respectively. The length corresponded to that of myotome between myocommata. Cultured muscle cells synthesized lipid and protein from ¹⁴C-acetate, but the ability of lipid synthesis by muscle cells was below 1/100 that of eel hepatocytes. In the absence of lipoprotein lipase (LPL), the highest component among ¹⁴C-lipid changed from ¹⁴C-phospholipid in muscle cells cultured alone to ¹⁴C-triacylglycerol (¹⁴C-TG) in cocultured muscle cells. These results suggest that cocultured muscle cells incorporate the very low-density lipoprotein (VLDL)-like lipoprotein secreted by cocultured hepatocytes, of which the main lipid is ¹⁴C-TG. In the presence of LPL in the coculture system, ¹⁴C-lipid in muscle cells increased 1.6-fold during 48 h incubation compared to that without LPL. ¹⁴C-Free fatty acids to total ¹⁴C-lipid in muscle cells increased from 1.1 to 32.6% by addition of 6 U/mL of LPL. Furthermore, the secreted VLDL-like lipoprotein was decreased and a gel filtration column separated formed high-density lipoprotein (HDL)-like lipoprotein. These results suggest that ¹⁴C-free fatty acids produced by LPL digestion of ¹⁴C-VLDL-like lipoprotein were transported to muscle cells. We concluded that there are two metabolisms of the secreted lipoprotein dependent or independent on LPL.

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