The effects of temperature and salinity on the egg hatching of the planktonic shrimp Acetes intermedius were studied. Four temperatures, 15, 20, 25 and 30°C, and eight salinities ranging from 0 to 35 at 5 ppt increments were employed to form the different combinations. Eggs failed to hatch at 15°C at all salinities examined and at salinity 0 to 10 ppt at all temperatures examined. Hatching success begins to decrease at salinities 15 ppt at 30°C and at salinity 20 ppt at both 25°C and 20°C. When salinity is not lower than salinity 25 ppt, the hatching success reaches nearly 90% on the average and is generally similar among temperatures tested. Hatching started at about 10, 14, and 29h after spawning at the incubation temperature of 30, 25, 20°C, respectively. Hatching time is the shortest at salinity 25 ppt at both 25 and 30°C, whereas it is similar among salinities at 20°C. The results suggest that the spawning ground of A. intermedius is likely located within the estuary where the salinity ranges from 25 to 30 ppt and the hatching time may vary seasonally in the southwestern coast of Taiwan.

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Studies were undertaken on long-term fluctuations of densities of different life stages (planktonic larvae, new settlers, and small and large bivalves) of three bivalve species (Ruditapes philippinarum, Musculista senhousia, Nuttallia olivaced) common on tidal flats, based on samples collected for 7 years from 1990 to 1996 in the Shitomo and Ano Rivers along the west coast of Ise Bay, central Japan. Seasonal and interannual variations in densities of different life stages for the three species were remarkable. In general, seasonal variations in densities of planktonic larvae corresponded to those of the benthic populations for each species, while interannual variations in densities of the larvae did not correspond to those of the benthic populations. However, ‘great’ peaks of densities of the larvae sometimes contributed to establish the benthic populations of each species.

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To determine the efficacy of daily rations for ayu Plecoglossus altivelis, we applied multiple regression analysis allocating the increment in ayu biomass as the response variable and such food items as rotifer Brachionusplicatilis, brine shrimp Artemiasalina nauplii and artificial edible compound (AEC), and ambient water temperature as the regressors. We examined the correlation between the parameters and the production rate of ayu cohort. In addition, we estimated the efficiency of the total food supply utilizing the “index of exhausted consumption” φ which measures the ratio of the discarded food against the utilized food.
The regression analysis was effective in 72 instances out of 91. The estimates of the regression coefficient which satisfies the non-zero test were counted to 2, 31, 34 and 56, respectively for water temperature, rotifer, brine shrimp and AEC. About a half the parameters for rotifer were negative. The production rate changed correlatively to AEC, but was not significantly affected by rotifer or brine shrimp. The influence of temperature was not clarified. The estimates of φ value for rotifer, brine shrimp and AEC were 167±15.2%, 49.0±3.42% and 39.3±3.06% (mean±S. E.), respectively. The indices correlated positively to the daily decreasing rate of individuals.

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A total of 2, 863 specimens of big eye Priacanthus macracanthus caught by bull trawl in the East China Sea and landed at Nagasaki Fish Market from May 1995 to February 1998, were examined to obtain information about their reproductive characteristics. From monthly changes in the gonadosomatic index, the spawning peak was estimated to be in May and June. From the distribution of oocyte diameters and histological observations of ovaries, the development process of oocytes was concluded to be of the group-synchronous oocyte development type. Maturation size was estimated to occur at 190mm FL from the relationship between the fork length and the frequency of mature specimens. Batch fecundity was calculated to be about 7 × 10⁴ at 190mm FL and 23 × 10⁴ at 250mm FL from the relationship between fork length and number of mature eggs.

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The structures of the siphons of the bivalve Nuttallia olivacea and changes of their states under extended conditions were examined. The inhalant and exhalant siphons had similar tissue arrangements: four circular muscle and six longitudinal muscle layers were arranged concentrically. The muscle fibers were held loosely in networks of connective tissue, which made the layers quite loosely packed, even under contracted conditions. Radial muscles divided the siphon walls into radial compartments. Both siphons had two wide dorsal and ventral hemolymph channels and had a distinct hemocoelic layer around the entire siphon wall. The inhalant and exhalant siphons had six and eight longitudinal nerve cords, respectively, that were fairly evenly spaced around the siphon wall and reached almost to the tips of the tentacles.
When the siphons were nearly fully extended, the densities of the longitudinal and circular muscle fibers decreased markedly and the spaces between these thin muscle fibers were filled with hemolymph. The longitudinal muscle fibers not only stretched and became very thin, but also slid out of place along each other longitudinally. The great extensibility of the siphons depends on such longitudinal muscle transformation, which probably makes the siphon highly vulnerable to amputation, even by small forces, such as biting by juvenile flounders.

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Changes in the number of immunoglobulin producing cells (IPC), immunoglobulin M (IgM) levels and leukocyte counts were investigated in rainbow trout reared under constant water temperature with special reference to gonadal maturation. During the spawning season, leucocytopenia, decreased number of antibody producing cells and low plasma IgM levels were observed. These reductions were apparently correlated with elevations in plasma cortisol (F), testosterone (T) and estradiol-17β in female and F, T and 11-ketotestosterone in males. In addition, decreased number of antibody producing cells in the skin lymphocytes and low mucus IgM levels were observed during the spawning season. These results suggest that endocrine changes related to gonadal maturation cause overall suppression of immune functions.

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The effects of steroid hormones on in vitro antibody producing cells from lymphocytes of the peripheral blood, head kidney, spleen and skin in juvenile rainbow trout were examined by ELISPOT assay. Cortisol, testosterone, estradiol-17β and 11-ketotestosterone reduced dose-dependently the number of cells producing both IgM and specific antibody against TNP-LPS. These steroid hormones also reduced IgM secretion from the head kidney and spleen lymphocytes. These results show that the low immunocompetence with the gonadal maturation is caused by the direct effects of steroid hormones on the antibody producing activity of lymphocytes. Moreover, the results also suggest that steroid hormones suppress the function of B lymphocytes, since TNP-LPS is thought to be thymus-independent antigen.

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Predation on intertidal sand-burrowing peracarid crustaceans by fish was investigated in a diel regime in the spring and summer of 1994 and 1995 in the surf-zone of a sandy beach in Otsuchi Bay, northeastern Japan. The mysid Archaeomysis kokuboi, the amphipod Haustorioides japonicus, and the isopod Excirolana chiltoni were the main peracarids preyed on by the surf-zone fish. The importance of each prey species was evaluated using indices of stomach fullness and the relative importance of each food item. The fish in the surf-zone exploited A. kokuboi predominately, and it was found in 60 and 71% of all the stomachs examined during the spring and summer, respectively. The mysid was preyed upon by SchlegePs black rockfish, the Japanese anchovy, the Japanese flounder, Ransonnet's surfperch and the Japanese opaleye, and this predation occurred exclusively at night. H. japonicus was found in 33 and 39% of the stomachs examined during the spring and summer, respectively. The amphipod was preyed upon by the green puffer, Ransonnet's surfperch, and the kite-fin dragonet. In contrast to the mysid, it was more important as prey during the daytime than at night. This study showed that a major part of the diet of surf-zone fishes consists of intertidal sand-burrowing peracarid crustaceans, suggesting that the surf-zone is a productive feeding ground for these fish.

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Oocyte and blood samples were taken from gravid female catfish Clarias macrocephalus at 4-h intervals to monitor the stage of oocyte development and serum steroid hormone profiles after injection of luteinizing hormone-releasing hormone analogue (LHRHa) and pimozide (PIM) during the off-season (February) and the peak of the natural breeding period (August). Results showed that the onset of final oocyte maturation (12h) and ovulation (16h), and levels of serum estradiol-17β (E₂) did not vary with season in LHRHa+PIM-injected fish. In February, ovulated eggs were stripped from three and two hormone-treated fish at 16h and 20h post-injection, respectively. In August, ovulation was observed in all hormone-treated females (n=5) at 16h post-injection but stripping of the eggs was possible only 4h thereafter. Serum E₂ levels were significantly different only with varying time post-injection; a marked increase occurred at 12h, but the elevation was higher in fish induced to ovulate during the peak (16.8ng/ml) than off-season (7.7ng/ml). Hormone-treated fish showed higher serum testosterone (T) levels during the peak season (17-23ng/ml) than those injected during the off-season (10-20ng/ml) at 4-12h post-injection. Serum 17α, 20β-dihydroxy-4-pregnene-3-one (DHP) levels of hormone-treated fish during the off-season were only about half the level (0.29 and 0.52 ng/ml) of those treated with the same hormones during the peak season (0.54 and 0.9ng/ml) at 8 and 12h postinjection, respectively. Development of oocytes and serum steroid hormone profiles after LHRHa+PIM-induced ovulation provide basic understanding of the processes that mediate final oocyte maturation and ovulation in captive C. macrocephalus.

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Specific antibodies were raised against Japanese eel gonadotropic hormone IIβ (GTH IIβ), thyroid-stimulating hormone β (TSHβ), and common α (GTH/TSHα) subunits using synthetic oligopeptides as antigens in order to examine GTH and TSH synthesis. The specificity of the antibodies was evaluated by Western blot and immunohistochemistry. By Western blot of eel pituitary, each antibody detected one specific band. The immunohistochemical results also indicate that GTH II and TSH can be reliably identified by these antibodies in eel. As details on the identify of eel GTH Iβ have not been published, we attempted to identify these cells by subtraction of GTH IIβ- and TSHβ cells from the entire population of GTH/TSHα positive cells. In chum salmon, the anti-GTH/TSHα positive cells which reacted with neither anti-salmon GTH IIβ nor anti-human TSHβ (β^--α⁺ cells) proved to be anti-salmon GTH Iβ positive in an adjacent section. In early vitellogenic Japanese eels and midvitellogenic New Zealand longfinned eels from the wild, many β^--α⁺ cells were observed. These results suggest that the β^--α⁺ cells in Japanese eels are probably GTH I cells. The development and validation of these antibodies will be significant for future studies on the role of GTHs and TSH in Japanese eels.

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Testes and blood samples were collected from males of the southern form short-finned pilot whale Globicephala macrorhynchus, caught by a small-type coastal whaling and the drive fishery off the Pacific coast of Japan in order to examine relationships of serum and testis concentrations of testosterone with testis weight, diameter of seminiferous tubules and density of reproductive cells. Serum and testis concentrations of testosterone ranged from 0.27 to 22.0ng/ml and from 14.4 to 287.1ng/g, respectively. Testis testosterone levels were more than 5 to 180 times higher than serum levels. Before the attainment of sexual maturity, both serum and testis testosterone levels were significantly correlated with testis weight and diameter of seminiferous tubules, but no significant relationship was shown after sexual maturity. There was no correlation between serum and testis concentrations of testosterone and density of reproductive cells in mature individuals.

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Temperature-dependent sex determination has been demonstrated in several species of fish. This study analyzes this phenomenon in barfin flounder Verasper moseri. The sex ratio was 1:1 when fish were maintained at 14°C from Day 34 to Day 95. However, acclimation to 18°C for 62 days resulted in an all male population. The proportion of males from 10.1mm up to 29.7mm in total length was affected by high temperature treatments. Survival rates were not affected by these treatments. Morphological differentiation of the gonad into either ovary or testis became distinguishable at 35mm in total length. This time corresponds to the end of the temperature-sensitive period. These results suggest that gonadal sex was determined by temperature prior to the onset of gonadal sex differentiation in this fish.

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The genetic diversity among three populations of ayu was analyzed by using fluorescence-based AFLP. Eight fish were collected from each of three locations representing native populations of the amphidromous form (AM), the landlocked form (LL), and the subspecific population from the Kawauchi River on Amami-oshima Island (RK). A total of 745 DNA fragments were scored using 19 arbitrarily chosen primer combinations, based on the restriction enzymes Msel and Eco RI with three and two base pair extensions, respectively. The proportions of polymorphic genes and the average heterozygosity were, respectively, 0.550 and 0.107 for AM, 0.521 and 0.095 for LL, and 0.135 and 0.022 for RK. The band-sharing coefficients were 0.839 for AM, 0.853 for LL, and 0.970 for RK. Genetic variation in each population varied directly with population size. The RK population, which is considered in danger of extinction, showed very low polymorphism. Nei's genetic distance was 0.017 between AM and LL, 0.410 between AM and RK, and 0.414 between LL and RK. These results suggest that AFLP markers provide useful data for the evaluation of genetic variability and divergence in fish.

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A feeding experiment was conducted to investigate the utilization of Ca from sea water by black sea bream Acanthopagrus schlegeli and the effect of dietary tricalcium phosphate on their growth and bone mineralization. Fish were fed the four casein based diets containing Ca-lactate, no supplement of Ca, and low and high levels of tricalcium phosphate for 10 weeks. The Ca unsupplemented diet gave growth and bone mineralization comparable to the diet with a Ca-lactate supplement. Low and high supplements of Ca from tricalcium phosphate appeared not to affect the growth and bone mineralization. The present study indicates that a dietary supplement of Ca may be dispensable in a black sea bream diet.

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Impacts of a weir on reproductive activities of introduced charrs, white-spotted charr Salvelinus leucomaenis and Dolly Varden S. malma, were investigated in a pond-associated stream system in south-western Hokkaido, Japan. Fish trapping during a breeding season revealed that only a few spawners of both species migrated upstream across the weir. White-spotted charr primarily spawned in two spawning grounds upstream and downstream of the weir. Length-distribution and scale annuli suggested that all the upstream spawners of the species were stream-residents, whereas downstream spawners were considered to include impoundment migrants. The sex ratio of the downstream spawners was about even, whereas that of the upstream spawners was strongly biased towards males. In contrast, Dolly Varden which had an even sex ratio, were observed to spawn only in the upstream ground. The original strain of white-spotted charr was anadromous form, in which most females generally descended to the sea, but that of Dolly Varden is stream-resident. The difference in strains could lead to the male-biased spawning group of white-spotted charr in the upstream reaches of the weir.

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Shipboard artificial fertilization experiments were carried out during November 1997 using four mature and mated females of Dosidicus gigas (320-407mm in mantle length) from Peruvian waters. A total of 167 hatchlings were obtained from about 3600 eggs which were kept at 18°C. Oviducal gland powder from the closely related species, Ommastrephes bartramii, was effective in expansion of chorion, which is essential for normal embryonic development. Hatching occurred 6-9 days after fertilization. Paralarvae survived up to 10 days after hatching without feeding. Mantle length was 0.9-1.3mm (mean 1.1mm) at hatching and increased to 1.1-1.5mm (mean 1.4mm) on the 7th day after hatching. Proboscis suckers were equal in size. Length of long axis of the statolith increased from ca. 40μm at hatching to ca. 60μm on the 4th day after hatching and remained around 63-67μm until the 10th day. Daily increments were indistinct in the statoliths.

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The relationships between guluronate content (G%) in alginates determined by ¹H-nuclear magnetic resonance (¹H-NMR) spectroscopy and their weight average molecular weight (WAMW) were examined to evaluate the reliability of the ¹H-NMR method. The partially hydrolyzed alginates with WAMW of 15k, 30k, 45k, 60k and 100k were prepared from alginates with WAMW of 150_??_200k by autoclaving. The G% in the partially hydrolyzed alginates determined by ¹H-NMR spectroscopy were independent on their WAMW, when the G% was less than 70%. The ¹H-NMR spectroscopic method using partially hydrolyzed alginates prepared by hydrolysis in an autoclave might be a convenient and reliable for the determination of G% in alginates.

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The effects of heat and acid treatments on eicosanoids concentration in the red alga, Gracilaria asiatica, were investigated. Leukotriene B₄ (LTB₄) and prostaglandin A₂ (PGA₂) increased but PGE₂ decreased when the alga was boiled with 4% acetic acid. Two isomers of LTB₄, which appeared in the HPLC of both the authentic LTB₄ and the extract of alga treated with acid, were identified as 6-trans-LTB₄ and 6-trans-12-epi-LTB₄. The concentration of LTB₄ increased when the alga was added with 4% acetic acid after boiling and incubated at 37°C at pH2 (pH similar to gastric fluid). Furthermore, the alga extract showed similar chemotactic activity to mice polymorphonuclear leukocytes as the authentic LTB₄. Mice orally administered with the algal extract containing 50μg of LTB₄ showed diarrhea, but it did not result in death.

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A prophenoloxidase (proPO) was purified from hemocyte of kuruma prawn Penaeus japonicus. An 134-fold purification of the enzyme with 25% yield was achieved by a combination of several chromatographies. The molecular mass of the native proPO was estimated to be 330 kDa by gel filtration. In SDS-PAGE analysis, two closely migrating subunits of 78 kDa and 72 kDa were detected under a reducing condition. ProPO was activated by SDS and methanol. Phenoloxidase (PO), the active form of proPO, had its optimum pH and temperature at approximately 9 and 37°C, respectively. The activity of PO depended on the concentrations of divalent cations, such as Mg²⁺ and Ca²⁺: the highest activity was obtained at the concentration of 50 mM for both ions. PO oxidized mono phenols and o-diphenol derivatives, but it was unable to oxidize the p-diphenol derivatives, suggesting PO from the prawn hemocyte catalyzes a consecutive reaction, hydroxylation of monophenols and deprotonation of o-diphenols.

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Phenoloxidase (PO) in crustaceans is believed to be derived from its precursor prophenoloxidase (proPO), however, the conversion mechanism remains unclear. Possible roles of β-l, 3-glucan, laminarin, of hemocyte lysate supernatant (HLS) from Penaeus japonicus in proPO activation were studied. PO activity was induced in HLS from Penaeus japonicus by treatment of laminarin, although laminarin had no influence on purified PO and proPO. p-APMSF and leupeptin, typical serine protease inhibitors, completely hampered this activation. Laminarin also enhanced activity of protease(s) in HLS, which specifically hydrolyzes synthetic peptides containing Arg in their P1 sites, while the activity was significantly suppressed by the serine protease inhibitors. These results indicate that the activation ofproPO in hemocyte of Penaeus japonicus may be achieved after at least three steps: that is the recognition of β-l, 3-glucan, activation of serine protease, and processing of proPO.

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The influence of adding starch on the elastic properties of fish-meat gel was studied by measuring its compression property and dynamic viscoelasticity. Samples with pregelatinized starch and with and without raw starch were used. Starch samples were used, as well. The results of the compression test were plotted using an equation obtained by substituting for volume changes as a function of β and μ directly into the original Mooney-Rivlin equation, ƒ=C₁(2λ-4βμ)+C₂(4β²λ-4β²μ-4β³μ), where ƒ is the stress, λ is the draw ratio, and C₁ and C₂ are constants. Fish-meat gels showed higher C₁ and C₂ values at 80°C than at 90°C, but the modulus of surimi showed a higher value at 90°C than at 80°C. Therefore we hypothesized that the gelatinization of starch was prevented by the surimi protein in the fish-meat gel at 90°C; the sample heated at 90°C showed lower packing effect than the sample heated at 80°C. And we also hypothesized the presence of the optimum heating temperature indicated a higher packing effect on the surimi protein.

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A κ-carrageenase-producing bacterium strain CA-1004 was isolated from the surface of a marine alga and identified to Vibrio sp. The strain produced a large amount of extracellular κ-carrageenase. A κ-carrageenase was purified 58-fold from cell-free culture fluid by ammonium sulfate precipitation and successive chromatographies. By SDS-PAGE the enzyme was ascertained to be homogeneous and had a molecular mass of 35 kDa. The enzyme exhibited maximal activity at pH 8.0 and 40°C. The pI and K_m of the enzyme were 9.2 and 3.3mg/ml, respectively. The enzyme activity was completely inhibited by Zn²⁺, Pb²⁺, Hg²⁺, and N-bromosuccinimide. The enzyme hydrolyzed κ-carrageenan and larger neocarraoligosaccharide sulfates than neocarrabiose sulfate to produce mainly neocarra-biose and-tetraose sulfates but did not act on neocarrabiose sulfate, p-nitrophenyl-α- and β-D-galactopyranosides, λ- and ι-carrageenans, agar, and porphyran.

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When assessed by ELISA using sera from five fish-sensitive subjects (1-5), the allergenicity of nine species of fish was found to vary not only among fish species but also among individual patients. In gel filtration on Sephadex G-75 of the crude extracts from Japanese eel and bigeye tuna, allergens were recognized in two fractions, i.e., Fr. I, near the void volume and Fr. II, in the later fractions. Subject 3 and 4 sera reacted to Fr. I, subject 1 and 2 sera reacted to Fr. II, and subject 5 serum reacted both Frs. I and II. The four allergens (Angj 1.01 and 1.02 from Japanese eel and Thu o 1.01 and 1.02 from bigeye tuna) for subjects 1 and 2 were isolated from Fr. II by reverse-phase HPLC on TSKgel ODS-120T. They were all identified as parvalbumins, based on their molecular weights, amino acid compositions, and positive reactions with a monoclonal anti-carp parvalbumin antibody. In conclusion, the major allergens in Japanese eel and bigeye tuna are parvalbumins and/or higher molecular weight substances, depending on patients.

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During artificial seawater acclimation in the laboratory of freshwater Japanese mitten crab Eriocheir japonicus, glycine and L-proline other than D- and L-alanine increased significantly in the muscle of the immature group, while no significant increase in glycine and L-proline was found in the mature group. Total inorganic ions in the muscle of the mature crabs increased significantly along with the salinity increments and reached the levels of estuarine specimens captured during downstream migration. In the immature group, inorganic ions also increased during salinity stress but the increase was far lower than those in the mature group. These data suggest that immature crabs cannot tolerate high ionic concentrations in muscle and have to increase glycine and L-proline during seawater acclimation. After adjustment of salinity tolerance, mature crabs incorporate inorganic ions in muscle and their cell volumes are likely regulated mainly by D- and L-alanine and inorganic ions.
Hemolymph ion concentrations and osmolalities increased largely during seawater acclimation for both immature and mature crabs and reached the same levels as those for estuarine and sea specimens in the natural environment. These data suggest that no special adjustment of hemolymph osmoregulatory capability occurs along with the maturation.

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The zebrafish Danio rerio proliferating cell nuclear antigen (PCNA) gene was cloned after screening of embryonic cDNA library of 48 to 72h after fertilization using random primer labelled human PCNA probe. Zebrafish PCNA gene cloned is 1247 bp including 5'-, 3'-untranslated regions and open reading frame (261 amino acids) and showed a high homology of amino acids to human (94.6%), mouse (92.7%), rat (93.5%), Xenopus (91.6%) and Drosophila (71.6%). It also expressed a transcript of approximately 1.3kb in various tissues (head, liver, intestine, ovary) and cultured fibroblast cell line. This report help in the understanding of the PCNA's role in fish at the replication initiation folk and elongation.

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