Recent advances in studies on osmoregulation in early life stages of fish are reviewed with special reference to extrabranchial chloride cells. Numerous chloride cells are present in the yolk-sac membrane of Mozambique tilapia embryos and larvae adapted to fresh water (FW) and seawater (SW). Chloride cells in SW often form multicellular complexes together with adjacent accessory cells, whereas chloride cells exist individually in FW. Chloride testing and X-ray microanalysis have shown that the SW-type, chloride cell complexes have the definitive function of chloride secretion. According to in vivo sequential observations on chloride cells in the yolk-sac membrane of tilapia, single FW-type chloride cells are transformed into multicellular SW-type cells in response to SW transfer, suggesting plasticity in the ion-transporting functions of chloride cells. A unique in vitro experimental model, named a ‘yolk-ball’ incubation system, has been established recently, in which the yolk sac is separated from the embryonic body and subjected to in vitro incubation. In the yolk balls prepared from FW tilapia embryos, chloride cells form multicellular complexes after SW transfer, indicating that chloride cells are equipped with an autonomous mechanism of functional differentiation that is independent of embryonic endocrine and nerve systems. The yolk-ball incubation system definitely serves as an excellent experimental model for further studies on chloride cell differentiation and functions. As chloride cells are commonly observed in the yolk-sac membrane and other body surfaces of fish embryos and larvae, it could be generalized that extrabranchial chloride cells play central roles in osmoregulation until gill chloride cells become functional.

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Between September 1996 and March 1999, a total of 135 fish species in 43 families were recorded from the mangrove estuary of Sikao Creek, Trang Province, Thailand, using two sizes of beach seine and a bag net. A checklist of the species is given, with preliminary descriptions of their assemblage structure. In terms of the number of species per family, Gobiidae was the most diverse (28 species), followed by Leiognathidae (11 species) and Engraulidae (10 species). In terms of individual numbers, Engraulidae, Leiognathidae and Ambassidae were the most dominant, whereby the 20 most abundant species comprised 88.5% of the total number of individuals collected. The fish assemblage structure was compared with published accounts of other tropical Indo-West Pacific mangrove estuaries, and found to be similar to those of tropical Australia. Although a grater number of species were recorded from Sikao Creek than in comparable studies in other geographic regions, all of the studies were similar in that they have relatively few species that are clearly dominant in abundance.

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The ayu Plecoglossus altivelis altivelis, including two ecological forms (amphidoromous and landlocked), is a common and commercially important freshwater fish in Japan, whereas the endangered subspecies P. a. ryukyuensis (Ryukyu-ayu) inhabits a limited area of the Ryukyu Islands in southern Japan. The efficiency of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis containing the entire D-loop region was examined to evaluate genetic variability and divergence in ayu populations, including the endemic two populations of the Ryukyu-ayu on the same island, and was compared with genetic data obtained from nuclear markers presented previously. The haplotype diversities (h) of the two populations of Ryukyu-ayu (0.251 and 0) were far smaller than those of the two common forms (0.766-0.928), suggesting that strong founder and/or bottleneck effects occurred in the subspecies populations. A common haplotype was not observed between the two populations of Ryukyu-ayu, suggesting little or no gene flow between them. The net nucleotide sequence divergence value (d_A) between the two populations of Ryukyu-ayu (0.276%) was almost same as that between the two forms of the former subspecies (0.265%). These results almost coincided with the results derived from nuclear markers and suggest that PCR-RFLP analysis of the mitochondrial DNA D-loop region is useful for evaluating genetic variability and divergence among ayu populations.

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By means of biochemical analyses on an individual basis, developmental changes in nutritional status during metamorphosis and settlement were examined in laboratory-reared Japanese flounder Paralichthys olivaceus. Although whole-body DNA content increased gradually throughout metamorphosis and settlement, the RNA content generally remained constant during the late metamorphosing stage and postmetamorphic phase. Subsequently, the RNA:DNA ratio decreased drastically from the late metamorphosing stage to the postmetamorphic phase following a continuous elevation in the ratio during early and mid-metamorphosing stages. The changes in protein content coincided well with RNA content. The protein : DNA ratio also peaked at the postmetamorphic phase and decreased for several days thereafter, suggesting the occurrence of hypertrophy until the postmetamorphic phase, followed by hyperplasia. It is speculated that the nutritional status of Japanese flounder becomes lower when associated with entry to the final phase of metamorphosis and/or settlement at which feeding habits drastically change. Poor nutritional status of the newly settled juveniles may increase their vulnerability to predators in the nursery ground.

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In order to develop a food chain (phytoplankton-fish) in a closed ecological recirculating aquaculture system (CERAS), unialgal cultures of Spirulina platensis were fed to tilapia Oreochromis niloticus (bodyweight; 1.59 ± 0.26 g, standard length; 3.4 ± 0.17 cm) for 9 weeks, using a commercial diet as the control treatment. Water temperature was 28 ± 0.5°C. Juvenile tilapia grew efficiently [specific growth rate (SGR)=3.14, feed efficiency (FE)=1.22] and showed a high survival rate (95%) for the duration of the trial without any nutrient supplements, although the fish fed the commercial diet had a slightly higher growth rate. A proximate composition analysis showed that fish fed Spirulina had a higher protein content, a higher polar lipid content and a lower ash content than fish fed the commercial diet. The abundance of linoleic acid and γ-linolenic acid (GLA) in the Spirulina was reflected in the fatty acid profile of the fish. Σn-6 highly unsaturated fatty acid (20:3n-6, 20:4n-6, 22:4n-6, 22:5n-6), which seemed to be metabolized from linoleic acid and GLA, increased and were more pronounced in the polar lipid fraction. On the contrary, Σn-3 fatty acids, such as α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid, decreased. No significant differences were observed in the mineral and essential amino acid contents in the fish at the experiment's completion, even though there were some differences between raw Spirulina and the commercial diet. The results suggest that it is feasible to construct a food chain using raw Spirulina to feed transition-stage tilapia in a CERAS.

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Intact follicles of the bambooleaf wrasse were incubated with different radioactively labeled steroid precursors during final oocyte maturation (FOM), and the maturation-inducing hormone (MIH) of this species was examined to compare the activities of the steroid metabolites produced on germinal vesicle breakdown (GVBD) in vitro. Of the metabolites obtained by thin layer chromatography, two steroids, 17, 20β-dihydroxy-4-pregnen-3-one (17, 20β-P) and 17, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S), both had the greatest effect on GVBD in vitro. During FOM, a shift in steroidogenic enzymes, a decrease in C17, 20-lyase activity, and an increase in 20β-HSD activity were found. In addition to this shift, continuous high 21-hydroxylase activity throughout all the oocyte developmental stages was observed, and, in all likelihood, this 21-hydroxylase activity enables ovarian follicles to synthesize enough 17, 21-P. Consequently, activated 20β-hydroxysteroid dehydrogenase converted 17-P and 17, 21-P to 17, 20β-P and 20β-S, respectively, during FOM. Thus, the present study not only provides evidence on the physiological role of 17, 20β-P and 20β-S as MIH in the bambooleaf wrasse, but also details the enzymatic kinetics of 17, 20β-P and 20β-S biosynthesis.

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Thirteen-day-old tilapia larvae (mean weight: 0.01 g), at the onset of exogenous feeding, were stocked at five fish per liter in rearing tanks. They were fed unialgal cultures of Spirulina platensis from varying ages [0, 12, 17, 21, and 28 days after the onset of exogenous feeding with a standard length (SL) of 0.8 cm, 1.8 cm, 2.0 cm, 2.5 cm, and 3.0 cm, respectively]. Feeding experiments on larvae fed different amounts of raw Spirulina were also designed. Acceptability tended to improve with an increase in the initial SL of the experimental larvae, and larval tilapia from 2.0 cm SL showed significantly rapid growth when fed raw Spirulina. Larval tilapia that were fed solely raw Spirulina at a feeding rate of 30% (on a dry basis) of bodyweight in the first 3 weeks, 10% in weeks 4-6, and 3% in weeks 7-10, kept growing without any abnormality. Final weight and SL reached 3.2 ± 0.4 g and 4.5 ± 0.2 cm (n=20), respectively, demonstrating that raw Spirulina was accepted by tilapia even just after the onset of exogenous feeding. Growth and survival rate of larvae fed Spirulina at different feeding rates in the first 3 weeks just after the onset of exogenous feeding showed a significant difference. Larval tilapia fed to satiation had the best growth performance throughout the 10-week rearing period, whereas those fed at a feeding rate of 10% stopped growing eventually and stunting was found. The availability of an abundant supply of Spirulina during the early stages seemed to be important for the normal growth and development of tilapia. Raw Spirulina appears to be an effective uni-feed for larval tilapia when developing a food chain of phytoplankton-fish for a closed ecological recirculating aquaculture system (CERAS).

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The ability of newly hatched larvae of Japanese Spanish mackerel Scomberomorus niphonius to resist starvation was studied in rearing experiments. Because Japanese Spanish mackerel larvae exhibit almost complete piscivory from the first feeding stage and begin to cannibalize under lack of piscine prey, they were isolated from one another during the experiments. Newly hatched larvae commenced feeding on fish prey (red sea bream Pagrus major larvae) on day 6 (after hatching) and completed yolk sac absorption by day 9 at 18.5°C. All the starved larvae had died by day 10. More than 90% of fish fed red sea bream larvae survived until day 10, whereas all fish fed rotifer (Brachionus plicatilis) had died by the same day, indicating that rotifer is useless as a prey for the survival of Japanese Spanish mackerel larvae at the first feeding stage. A clear point-of-no-return (PNR), 0-1 day of recoverable starvation, was found at the post-first feeding stage. Delayed feeding experiments demonstrated that the percentage survival on day 10 was 100% for larvae fed from day 5, 91.6% from day 6, 16.7% from day 7, 8.3% from day 8, and 0% for those fed from day 9. Fish first fed from day 7 or 8 showed significantly retarded growth during the following adequately fed period compared with fish fed from the first feeding (day 6). These results show that Japanese Spanish mackerel larvae are highly vulnerable to fasting and that the larvae's first feeding success might be critical for growth and survival in the period that follows.

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A method is presented for determining the equilibrium configuration and tensions of submerged rope systems in a uniform current based on a non-linear finite element formulation. A standard straight rope element is adopted to model rope segments of the system. Rope stretch is included in the formulation, and a hydrodynamic model is introduced for the rope. The structural and hydrodynamic models are coupled in order to account for the interaction between the structural response and hydrodynamic load acting on the rope itself. The Newton-Raphson method is employed in the formulation, ensuring the global convergence of the iteration to the correct response (and hence to the correct equilibrium configuration and tension of the rope system) from any set of initially estimated responses. Good agreement between this numerical simulation and experimental behavior was revealed in an application of this method to a long-line model.

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Body shape transformation of flyingfish Parexocoetus mento mento was examined to relate its functional importance in flight evolution and ecology using morphometrics. With growth, snout elongation and head depth decreased relative to other anterior body proportions. Associated with the decrease in head depth were a decrease in eye diameter and shortening of postorbital length, resulting in a small head. The lengths of the trunk and caudal regions increased disproportionately relative to their depth, resulting in an overall elongated body shape. The distance from the last dorsal fin ray to the base of the first upper caudal fin ray increased at a greater rate than the distance from the last anal fin ray to the base of the first lower caudal fin ray, resulting in a ventrally inclined caudal fin. Ontogenetic changes in the shape of the head and body are considered to improve streamlining, while the downward inclination of the tail acts to maximize the extended hypocaudal tail lobe, which remains in the water or close to the water's surface during those times when the body is out of the water.

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The seasonal bloom of the toxic dinoflagellate Alexandrium tamarense, with reference to the ambient oceanographic conditions in Hiroshima Bay, Seto Inland Sea, Japan is described. Long-term observations on the vegetative cells of A. tamarense were conducted biweekly to monthly at one fixed station in northern Hiroshima Bay, where recurrent paralytic shellfish poisoning (PSP) incidents have been occurring since 1992. Over the 5-year study period, from April 1994 to December 1998, vegetative cells of A. tamarense were detected each year within the period from January to June. Observed annual maximum cell densities of A. tamarense reached 10³-10⁴ cells/L, and mostly peaked at a depth layer of 5 m at the sampling station in April or May. Oceanographic conditions during the bloom period were as follows: water temperatures ranged from 10.2°C to 20.2°C, and thermal stratification gradually developed around April. Inorganic nutrient concentrations were markedly low throughout the bloom period. Particularly, the annual lowermost concentration of SiO₂-Si was observed each year during this period. These environmental features indicate that the occurrence of vegetative cells of A. tamarense seems to be explained by temperature and nutrient conditions and that the A. tamarense bloom developed subsequently to or concomitantly with the diatom spring bloom.

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To estimate the natural and fishing mortality of whelk, Neptunea arthritica, a multiple tagging experiment was carried out in Shiriuchi, Hokkaido between August 1991 and March 1994. Whelks collected by trapping surveys were used in tagging experiments. All recaptured whelks were released back again so that a statistical model based on binomial distribution could be developed. Maximum likelihood estimator of the natural mortality coefficient and that of exploitation rate were determined numerically. Analysis of the recovery data show: (i) no size-dependent mortality was observed in whelks larger than 40 mm in shell height; (ii) the daily natural mortality coefficient was 1.438×10^-3, thus annual natural mortality rate was 41%; and (iii) the exploitation rate of commercial fishing in 1994 was approximately 77.3%. At least 53% of the total mortality was attributable to fishing in the exploited area.

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An intra-annual analysis of the intermediate fecundity, batch fecundity and oocyte size of ripening ovaries of Pacific sardine (Sardinops sagax) in northern Chile was carried out over an annual cycle from April 1992 to March 1993. Both the shorter and larger oocyte size classes of ripening ovaries were used to estimate the intermediate fecundity and batch fecundity, respectively. The relationship between both fecundates and their respective mean oocyte sizes were studied as well. Clear seasonal variations of the intermediate and batch fecundity were observed, which follows a similar trend reported in previous studies for the reproductive cycle of S. sagax. Two peaks were found with two periods of lower production of oocytes between them: the first peak occurred around winter and the second around summer. Mean oocyte diameters of the intermediate mode (ODIM) and mean oocyte diameters of the most advanced mode (ODAM) were found to have the same seasonal traits; that is, bigger oocytes in winter than in summer. The highest decrease in the oocyte diameter occurred during those months with lower reproductive activity. Possible factors influencing the fecundity variations as reproductive season progressed are discussed.

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The predation pressure on the siphons of the bivalve Nuttallia olivacea by the juvenile stone flounder Platichthys bicoloratus in the Natori River estuary, Miyagi, was estimated from both sides of the predator-prey relationship to analyze the specific relationship in which predators remove a part of the prey. Feeding experiments determined the feeding rate required to achieve the growth of juvenile stone flounders in the field. A juvenile cropped siphon tips at a mean of 56 per day from March to early July. The siphon tips that a juvenile consumed in a season amounted to 6375 pieces (6.2 g). In terms of predation, the duration since the last cropping in the field from the regeneration of cropped siphons was analyzed. The mean cropping rate of 0.211 times a day per clam in an area of intensive predation indicated that a clam was cropped 25.8 times (22.1 mg) in a season by fishes. The mean cropping rate by juvenile stone flounders was estimated to be 0.160 times a day. Of the 25.8 croppings, 19.5 (16.9 of the 22.1 mg) could be attributed to juvenile stone flounders. The results proved that a juvenile stone flounder preyed on approximately 370 clams in a season.

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The effects of temperature and salinity on the metamorphosis of nauplii into protozoea I of a planktonic shrimp Acetes intermedius were studied. Eggs or nauplii I, hatched under optimal conditions (30°C, 30 ppt), were incubated under 15 different combinations of three temperatures (20°C, 25°C, and 30°C) and five salinities (15 ppt, 20 ppt, 25 ppt, 30 ppt, and 35 ppt) until reaching protozoea I stage. At salinities of 20-35 ppt, eggs required 28 h, 45 h and nearly 4-5 days to develop into protozoea I at 30°C, 25°C and 20°C, respectively. Nauplii metamorphosed into protozoea I at salinities of 25 ppt and 30 ppt earlier than at 20 ppt and 35 ppt at temperatures of 25°C and 30°C, whereas no regularity in time length existed among salinities at 20°C. Irrespective of hatching conditions, the rate of metamorphosis reached approximately 90-100% at temperatures of 25°C and 30°C at salinities of 20-30 ppt, but was significantly reduced at 20°C for all salinities examined. The metamorphosis rate of nauplii was generally higher at salinities of 20-30 ppt at all temperatures. Multiple linear regression analysis revealed that the temperature experienced by nauplii accounted for most of the variation in the metamorphosis rate.

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Primary cultures of tilapia (Oreochromis mossambicus) hepatocytes were developed and effects of several hormones on in vitro production of female-specific proteins (vitellogenin; VTG) were investigated. The hepatocytes were maintained for more than 2 weeks in Leibovitz-15 medium without the addition of any serum components. Absence of insulin in the culture media did not affect either cell attachment or monolayer formation. When VTG levels in the culture media were measured by an enzyme-linked immunosorbent assay (ELISA) using antibody against tilapia egg homogenate (a-E), VTG was detected in the medium for at least 8 days from hepatocytes isolated from a vitellogenic female but not from a male fish. However, the addition of estradiol-17β (E₂) to the media of hepatocyte cultures from the male tilapia resulted in the elevation of VTG. Insulin and ovine growth hormone treatments did not have a significant effect on VTG induction. These results suggest that E₂ is a major steroid hormone to induce VTG synthesis in the tilapia hepatocytes and that involvement of other hormones in the vitellogenic process is different among teleost species.

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Telomerase is a ribonucleoprotein that synthesizes telomeric DNA onto chromosome termini and maintains the telomere length, and is expressed in immortal cells, stem cells, and germ cells. The objectives of the present investigation were to detect telomerase activity in the eyed embryos of rainbow trout Oncorhynchus mykiss using stretch polymerase chain reaction (PCR) assay and to estimate the relative telomerase activity and compare it with the value found in HeLa cells. High levels of telomerase activity could be detected quantitatively in the embryos using stretch PCR assay. Telomerase activity in embryos completely vanished using pretreatments with heating and RNase A. Relative telomerase activity per cell in embryos was 19.3-50.7-fold higher than that in HeLa cells. Conversely, relative activity on the basis of the protein amount showed no significant difference between the embryos and HeLa cells. This finding supports previous results in which high level expressions of telomerase are demonstrated in the normal cells and organs of fish species.

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Landlocked ayu stocks in the Biwa Lake system were investigated using a mitochondrial DNA marker to evaluate their genetic structure. Samples examined consisted of spring migrants (o-ayu), fall migrants (ko-ayu), and juvenile residents in the lake at a predifferentiated stage. Compared to amphidromous fish, the landlocked fish, overall, were characterized by a lower genetic diversity that has resulted from lower haplotype diversity and a higher similarity in nucleotide sequences between haplotypes. Some nucleotide diversities that were unique to the samples showed that the landlocked ayu are not genetically homogeneous, suggesting the occurrence of a single metapopulation throughout the Biwa Lake system. The pairwise fixation index inferred different respective local populations for the o-ayu and ko-ayu ascending the same stream in the same year class, as well as the isolation by distance among local populations of ko-ayu. For conservation practices during transplantation with fish from non-native stocks, it should be taken into account that the Biwa Lake stocks have a high probability of not being genetically uniform.

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Growth rate and photosynthesis of Microcystis aeruginosa, Scenedesmus quadricauda and Aulacoseira granulata exposed to different concentrations of the agricultural pesticides CNP (p-nitrophenyl 2, 4, 6-trichlorophenyl ether), MEP [O, O-dimethyl O-(3-methyl-4-nitrophenyl) thiophosphate], ISP [isoprothiolane (C₁₂H₁₈O₄S₂)], and TBT (tri-n-butyltin chloride) were determined. The effective concentration (EC₅₀) for growth and photosynthesis in each species of microalga was then calculated. Inhibition of growth and photosynthesis in the three microalgae was greatest when exposed to CNP and TBT. Microcystis aeruginosa and A. granulata showed a higher tolerance, whereas S. quadricauda showed a higher sensitivity. Except for MEP, the EC₅₀ values for growth obtained in the three microalgae were higher than those for photosynthesis. The growth-photosynthesis response relationship showed that, for CNP and TBT, growth of the three organisms tested were less inhibited than their photosynthesis at a lower exposure (0.001-0.05 μg/L). At a higher exposure (0.10-1.0 μg/L), the response between relative growth rates and relative photosynthesis was proportional. For MEP and ISP, a proportional response existed between relative growth rates and relative photosynthesis in all test organisms. These results suggest that the inhibition of growth and photosynthesis by agricultural pesticides differs for the three microalgae. The differences can be explained in terms of the physico-chemical properties of the four pesticides and the physiological and morphological properties of the three microalgae.

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The otolith microstructure and microchemistry of a tropical eel, Anguilla marmorata, were examined in glass eels collected from Taiwanese and Japanese coasts. Ages at metamorphosis (onset of metamorphosis) and recruitment, respectively, were 114 ± 13.8 (mean ± SD) days and 144 ± 15.7 days in Taiwanese stock, and 123 ± 13.9 days and 154 ± 17.3 days in Japanese stock. No significant differences were found in ages between the two stocks. The duration of metamorphosis estimated from otolith microstructure was 10-24 days, which is the same in the different stocks. For both stocks examined, close linear relationships were found between ages at metamorphosis and recruitment, suggesting that individuals that metamorphosed earlier were recruited to the coasts at a younger age.

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The optimum dietary protein level for the growth of young Japanese flounder was investigated. Three replicate groups of fish (average weight 22.7 g) were fed six isocaloric diets containing various protein levels from 40% to 65% for 9 weeks. Casein and white fishmeal were used as the dietary protein sources. Weight gain and feed efficiency of fish fed diets with 45% and 50% protein levels were significantly higher than the other groups (P < 0.05). Protein efficiency ratio of fish fed diets with 55%, 60%, and 65% protein levels was significantly lower than that of other groups (P < 0.05). Plasma total protein concentration increased with increasing dietary protein level up to 50% and glucose concentration decreased with increased dietary protein, probably as a result of the dietary protein and carbohydrate contents. Proximate composition of dorsal muscle was not significantly affected by dietary protein level (P > 0.05), but moisture, protein and lipid contents of liver were significantly affected (P < 0.05). The study's results indicate that a dietary protein level of 45% can be recommended for optimum growth and efficient protein utilization of flounder growing between 22.7 g and 110 g.

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Sensory evaluations were conducted on kamaboko gels prepared from silver carp (SC) surimi and from walleye pollack (WP) surimi, and were then compared. Different kamaboko gel specimens, produced with or without the extract from silver carp muscle (Esc) or walleye pollack muscle (Ewp), were tested using a panel of university students for their odor, flavor, texture, whiteness, and overall acceptability. According to the sensory evaluation results done in Kyoto, there were significant differences (P < 0.005) between SC kamaboko gel and WP kamaboko gel for all sensory evaluation indices. When compared with WP kamaboko gel, kamaboko gel derived from SC surimi was unacceptable to the Japanese students especially because of its odor. When the same investigations were carried out in Shanghai (coastal area) and Wuhan (inland area) of China, the former showed the same pattern as that observed for Kyoto and its difference was smaller than that for Kyoto; the latter exhibited a different pattern as that for Kyoto and there were fewer differences among all of the test specimens. Marine fish meat gels and freshwater fish meat gels had the same acceptability by students from Wuhan. A slight increase in their sensory scorings of kamaboko gels occurred when Ewp was added, especially when scoring the odor of SC kamaboko gels. The results indicated that there were some differences in the acceptability of SC kamaboko gels, which were apparently influenced by the area and tradition of fish consumption.

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The effects of the deacetylation degree (DD) and preparation methods for chitosan on antimicrobial activity were evaluated. Chemically prepared chitin (CH-chitin) and microbiologically prepared chitin (MO-chitin) were obtained from shrimp shells. The CH-chitin and MO-chitin were further chemically deacetylated to obtain various chitosan products of which their DD ranged from low (47-53%) through medium DD (74-76%) to high (95-98%). In addition, MO-chitin was deacetylated also by various proteases. The antimicrobial activities of these products were evaluated in medium with pH 6.0. Neither the CH-chitin, MO-chitin nor protease-deacetylated chitinous products showed any antimicrobial activity. For chitosan, antimicrobial activity increased with increasing DD, and was stronger against bacteria than against fungi. The minimal lethal concentrations (MLC) of chitosan with a high DD against Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholerae, and V. parahaemolyticus were all in the range of 50-200 p.p.m., whereas the MLC against Candida albicans and Fusarium oxysporum were 200 p.p.m. and 500 p.p.m., respectively. No antifungal activity was found at 2000 p.p.m. against Aspergillus fumigatus or A. parasiticus. Pretreatment of fish fillets (Oncorhynchus nereka) with 1% chitosan solution (high DD) for 3 h retarded the increase in the volatile basic nitrogen content, as well as the counts for mesophiles, psychrotrophs, coliforms, Aeromonas spp., and Vibrio spp. The shelf life was consequently extended from 5 days to 9 days.

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The effect of bisphenol A and nonylphenol on carp Cyprinus carpio phagocytic cells was analyzed in vitro. Head kidney leukocytes of carp were exposed to 10 nM of bisphenol A or nonylphenol, separately. Subsequently, the production of superoxide anions was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 0.1 nM up to 10 nM of bisphenol showed an increase in the production of superoxide anions in comparison with the control macrophages, which were not treated with bisphenol A. Nonylphenol also induced the production of superoxide anions in phagocytic cells prepared from carp kidney. Phagocytic cells treated with both bisphenol A and nonylphenol decreased phagocytic activity. However, the production of nitric oxide in carp macrophages did not change when treated only with either bisphenol A or nonylphenol. These results indicate that bisphenol A and nonylphenol affected the function of phagocytic cells in vitro.

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The tri-monthly changes in the gonad index (GI), the frequency of bitter gonads, and the contents of a novel bitter amino acid, pulcherrimine of green sea urchin Hemicentrotus pulcherrimus caught off Iwaki, are described. The mean GI values of mature male and female individuals were lowest in May 1999. Immature individuals were found in May, August, and November 1999, and their percentages were 20%, 60%, and 4%, respectively. However, many mature sea urchins were found in all the months examined. More than 95% of mature ovaries had a bitter taste in November 1998, February 1999, and November 1999; whereas in May and August 1999, only 60% of the mature ovaries had a bitter taste. In August 1999, 20% of the immature gonads tasted bitter. Individuals with a low pulcherrimine content in their ovaries increased from February to August 1999 and decreased from August to November 1999. These results suggest that the amount of pulcherrimine in the ovaries of the green sea urchin is related to seasonal changes.

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Plasma protein concentrate (PPC) was added as a gelation aid material to horse mackerel surimi, and changes in the physical properties of surimi during heating and reheating in a soy sauce-based liquid seasoning; this is, stewing, were investigated. The rigidity of 2% PPC gel was lower than 2 kPa, and yet the addition of PPC increased the surimi rigidity markedly. Differential scanning calorimetry analysis showed that the existence of PPC promoted protein molecular aggregation. During the stewing process, the decrease in water content and water activity of kamaboko sections with PPC added was slower than that of kamaboko without PPC. The gradually hardened matrix resulted in a higher cutting force, cohesiveness (chewiness) and reduced deformation in stewed products.

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Effect of Na-gluconate on physical properties of preheated and two-step heated gel prepared from walleye pollack surimi was investigated in connection with the progress of the cross-linking reaction of myosin heavy chain (HC). The salt-ground surimi to which either 8% sorbitol or Na-gluconate was added, was preheated at 25°C or 40°C. Increases in the breaking strength and the breaking strain, together with the cross-linking reaction of HC, of the salt-ground surimi with Na-gluconate proceeded more slowly than in that with sorbitol during the preheating period. The subsequent heating at 90°C for 30 min enhanced the breaking strength of the preheated gel but slightly decreased the breaking strain irrespective of the type of additive used. Gel stiffness of the two-step heated gels formed with Na-gluconate was higher than that formed with sorbitol at the same breaking strength, suggesting that the two-step heated gels with Na-gluconate were harder but not elastic. This characteristic of the two-step heated gels formed with Na-gluconate was mainly developed due to the subsequent heating. The cross-linking reaction of HC largely proceeded during the preheating period but hardly took place during the subsequent heating period. These results strongly suggest that Na-gluconate contributed to the production of the two-step heated gel with higher gel stiffness by affecting the formation of non-covalent bonds, such as the hydrophobic interaction between myofibrillar proteins.

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The shelf life of fish salads in modified atmosphere was studied and compared with those of air-packaged (control) products. Samples were evaluated at 0 days, 7 days, and 14 days at 4°C, respectively. The sensory quality of modified-atmosphere packaging (MAP) groups was significantly (P≤ 0.05) higher than the control group. Control packages were below the limit of acceptability at the seventh day of storage. However, MAP samples (O₂ : CO₂ : N₂=1 : 7 : 12 and CO₂ : N₂=3 : 7) were not rejected until the 14th day of storage. Modified-atmosphere packaging decreased the microbiological activity and the sensory score changes, as well as extending the shelf life of samples by approximately 50%.

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The effect of potassium lactate treatment on the shelf life of vacuum-packaged fish was studied. Chub mackerel was treated with 2% and 4% potassium lactate solutions, and their shelf lives were compared with those of control (not treated) samples. Samples were stored at 4°C and sensory evaluation, and chemical and microbiological analyses were done every three days. Control group spoiled after six days of storage. The addition of potassium lactate decreased microbiological activity and treated samples remained in good quality until the ninth day of storage. There was no significant difference between the shelf life of samples treated with either 2% or 4% potassium lactate. These results indicate that treatment with potassium lactate extends the shelf life of vacuum-packaged and refrigerated chub mackerel.

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Effects of sorbitol on the autolysis of mantle muscle of squid was investigated by changing the NaCl concentration and the incubation temperatures. When myofibrils were incubated at 25°C, sorbitol altered the NaCl concentration-dependent profile of autolysis. Sorbitol shifted the autolysis peak toward lower NaCl concentrations, leading to an increase in the autolysis rate at approximately 0.2 M NaCl. It was considered that this rate increase was caused by the solubilization of myofibrils with sorbitol, which exposed the cleavage site of the heavy meromyosin-light meromyosin junction to the enzyme. Sorbitol suppressed autolysis at concentrations above 0.3 M NaCl. When myofibrils were incubated at 35°C, sorbitol markedly enhanced autolysis even in medium containing 0.5 M NaCl. It was considered that this peculiar enhancement in the presence of sorbitol is caused by the stabilization of the proteolytic enzyme itself by sorbitol. Thus, the complicated feature of autolysis in the presence of sorbitol was the result of the combined effects of the following three different functions of sorbitol: (i) suppression of proteolytic activity; (ii) promotion of autolysis by solubilization of the myofibrils; and (ii) stabilization of the proteolytic enzyme.

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Adenosine triphosphatase (ATPase) inactivation of squid myosin subfragment-1 (S-1) was strongly suppressed upon addition of adenylimidophosphate (AMPPNP), an analogous compound of adenosine triphosphate (ATP); whereas suppression by adenosine diphosphate (ADP) and pyrophosphate (PPi) was not as effective. Adenylimidophosphate did not stabilize myofibrils in which myosin is stabilized by F-actin. Thus, the stabilization effect by F-actin and AMPPNP is not additive. Pyrophosphate markedly accelerated the inactivation of myofibrils through the loss of protection by F-actin. The suppressive effect of F-actin on myosin ATPase inactivation was much greater than that by AMPPNP. The stabilization effects of AMPPNP and F-actin on S-1 were almost completely lost when the medium contained approximately 1 M KCl.

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