The present experiment was undertaken to establish the dietary manganese (Mn) requirement in Atlantic salmon fry and to examine the effect of Mn intake and status on disease resistance. Salmon fry were divided into six triplicate groups and fed a casein/gelatine diet (containing 1.1 mg Mn/kg) supplemented with 0, 3, 6, 9, 12 or 24 mg Mn/kg (as MnSO₄) for 12 weeks. Growth, mortality and whole body Mn were recorded every 4 weeks. Concentrations of calcium, phosphorus, zinc and iron in whole fish and total hepatic superoxide dismutase activity were measured after 12 weeks of feeding. After the feeding experiment, the fish were challenged with Vibrio anguillarum. Dietary Mn levels did not affect growth and mortality. Whole body Mn concentration responded readily to dietary Mn supplementation and reached a plateau in fish receiving diets supplemented with more than 6 mg Mn/kg. Regression analysis showed that whole body Mn concentration was maintained at dietary Mn concentrations of 7.5-10.5 mg/kg dry feed. Supplementation of only 3 mg Mn/kg diet was enough to saturate the total hepatic superoxide dismutase activity. The dietary Mn levels did not affect the mortality after bacterial bath challenge with a virulent strain of V. anguillarum. In conclusion, a minimum Mn requirement of 7.5-10.5 mg/kg dry diet was needed to maintain Mn status in Atlantic salmon during start feeding.

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The magnitude of intraspecific and interspecific genetic differentiation in Pagrus major collected from two Japanese areas, the East China Sea (ECS) and the South China Sea (SCS), and Pagrus auratus, collected from Australia (AUS) and New Zealand (NZ), was estimated using restriction fragment length polymorphism (RFLP) analysis and DNA direct sequencing of the mtDNA control region. The RFLP haplotypic diversities in P. major samples were high (0.88-0.93); in contrast, these diversities were relatively lower (0.58-0.65) in P. auratus samples. The relative relationships among samples that resulted from RFLP analysis were almost the same as those from DNA direct sequencing, except that values from the former were less sensitive and were one-third to one-fifth lower than those from the latter. A significant heterogeneity was observed in the distribution of RFLP haplotypes between samples from P. auratus and P. major, and between samples from AUS and NZ. The difference of the nucleotide substitution by direct sequencing in the control region between P. auratus and P. major was 3.48%. Based on the substitution rate, the division time between samples from P. auratus and P. major was assumed to be 2-6 million years ago. With regard to morphological aspects, there was a significant difference in the bump between NZ and ECS samples, although there were no other significant external morphological differences. From these results, we suggest that the relationship between these ‘species’ is at the level of a subspecies. Accordingly, P. major might be renamed P. auratus major and P. auratus renamed P. auratus auratus.

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A total of 325 pueruli of the spiny lobster Panulirus japonicus was successfully reared in the laboratory at Minami-Iku Station of Japan Sea-Farming Association, Shizuoka, Japan, during 1989-1997. Of these pueruli, 136 individuals metamorphosed into the first juvenile stage. The duration of the phyllosoma stage ranged from 231 to 417 days (mean 319.4 days), and it has a tendency to extend as the increase of water volume in the rearing tanks. The number of molting in the phyllosoma stage was 20-31. The body length of the last-stage phyllosoma ranged from 27.9 to 34.2 mm and the duration of the last stage was 11-26 days. The carapace length of the puerulus stage was 6.0-8.0 mm and the duration was 9-26 days. The present data and those of previous studies suggest that the body size and the duration of phyllosoma stage in captivity are affected by environment as in the field. The duration of the puerulus stage is considered to be controlled basically by water temperature and nutritional conditions in the phyllosoma.

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The characteristics of lipid deposition in juveniles of red sea bream Pagrus major, yellowtail Seriola quinqueradiata, and Japanese flounder Paralichthys olivaceus, were investigated by feeding diets with three lipid levels (approximately 11, 16 and 20%). In red sea bream, the viscera contributed 40-50% to the whole body lipid store and deposited 35-39% of dietary lipid intake (body lipid gain/lipid intake: lipid deposition ratio), while the carcass contributed 40% and deposited 30-37%. In yellowtail, 50% of the whole body lipid store was found and 25% of dietary lipid intake was deposited in the carcass, while less than 10% was found and about 3% was deposited in the viscera. In Japanese flounder, although 80% of the whole body lipid store was located in the carcass, the lipid deposition ratio of the carcass decreased from 33% down to 19% as the increase of lipid intake, and the viscera represented around 10% of whole body lipid store and deposited 3% of lipid intake. Thus, the juveniles showed different traits of body lipid deposition in their responses to changes in dietary lipid level.

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Diets containing 40% of either starch or glucose were fed to triplicate groups of tilapia Oreochromis niloticus × O. aureus (initial weight: 0.80±0.01 g) for 8 weeks, followed by a fasting of 1, 2 and 3 weeks. In starvation, fish previously fed the diet containing glucose showed significant (P<0.05) weight loss compared with fish fed the diet containing starch. Liver glycogen was higher in fish fed a starch diet compared with fish fed a glucose diet during both feeding and fasting periods. Starvation also resulted in decreased liver glycogen in fish as the fasting period increased. Plasma glucose was higher in starch-fed fish during feeding and 1 week after fasting compared with glucose-fed fish. Plasma glucose of both dietary groups was lower in starvation than in the feeding period. Elevated plasma glucose was found 3 weeks and 2 weeks after fasting in fish prefed with starch and glucose diets, respectively. Plasma triglyceride increased as the fasting period increased in both groups. In starvation, plasma triglyceride were higher in fish prefed the starch diet than fish fed the glucose diet. Relative to the glucose diet, body lipid content was higher in fish fed the starch diet prior to fasting and 1 week after fasting. These data suggest that the physiological responses of tilapia during starvation are affected by prefasting diets containing different carbohydrates.

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Eighteen samples of virus-like growth suppression agents against the marine phytoplankton Alexandrium catenella, Gymnodinium mikimotoi and Tetraselmis sp., obtained from the coastal waters at the mouth of Funka Bay, Hokkaido, Japan, from 1993 to 1995, were characterized on size estimation, heat stability, nuclease sensitivity, proteinase K sensitivity, stability under acidic condition, titration, diethyl ether sensitivity, and ultraviolet (UV) sensitivity. All agents were affected by heating at 50°C for 30 min, exposure to acidic conditions below pH 5.0, passing through a 0.05 μm filter, RNase treatment, and irradiation of UV dosage at 5 × 10⁴μW/s per cm². The growth suppression effects of 11 agents from Tetraselmis sp. and A. catenella disappeared after proteinase K treatment, however, seven agents from G. mikimotoi were unaffected by this treatment. Furthermore, 11 agents against Tetraselmis sp. and A. catenella were collected from the bottom fraction by ultracentrifugation, while seven agents against G. mikimotoi were collected from the upper fraction, not from the precipitated fraction. These results suggest that at least two types exist in the virus-like agents showing growth suppression for phytoplankton.

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We studied microbial consortia with nonylphenol (NP)-degrading activities from various aquatic environments in Tokyo. The microbes with notable activity were observed in two locations: Tokyo Bay and waste water of a sewage treatment plant. For the first sample's microbes, about 70% of NP was degraded in 45 days' incubation at 25°C in the medium containing NP (1000 ppm) as a sole carbon source. For the second sample's microbes, NP was almost completely degraded in 30 days and succeeded several times with the increase of the degrading activities. In another medium in which the NP/glucose mole ratio was 1, the degrading activities were not affected by glucose.

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A complementary DNA (cDNA) encoding salmon type gonadotropin-releasing hormone (sGnRH) has already been isolated and characterized in the goldfish Carassius auratus. In the present study, we isolated and characterized a second sGnRH cDNA which was different from the reported sGnRH cDNA from the brain of goldfish by rapid amplification of cDNA ends. This cDNA included an open reading frame of 282 bp encoding the 94 amino acid residues of sGnRH precursor. Amino acids identity between the second sGnRH precursor and the previously reported sGnRH precursor was 86.2%. Genomic Southern blot analysis showed that the goldfish harbors these two distinct sGnRH genes in its genome. Expressions of the two sGnRH genes were observed in the brain, pituitary, and gonad of goldfish by reverse transcription-polymerase chain reaction analysis. Moreover, the levels of two sGnRH mRNA in these tissues were different depending on sex and maturity of the fish. The differential expressions of two sGnRH genes suggest the existence of the differential regulatory mechanism of the two sGnRH genes and the multiple physiological functions of sGnRH.

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A stable homozygous line of transgenic mud loach (Misgurnus mizolepis) carrying a CAT reporter construct was developed. The founder transgenic mud loach was produced by microinjection of a CAT reporter gene driven by a carp beta-actin promoter and regulatory region (pFV4CAT) into fertilized eggs. The presumptive homozygous transgenic fish were generated by sister-brother mating between two heterozygous transgenic F₂ siblings. Out of 28 transgenic individuals tested, five fish transferred the transgene into their progeny at a rate of 100%, four fish at 80-91%, and remaining 19 individuals inherited transgenic construct at a rate of approximately 50%. The level of expressed CAT enzyme in tissues (liver and spleen) from homozygous transgenic fish was higher than in those from heterozygous transgenic fish, although the increased amounts of CAT expression were slightly variable among homozygous individuals. The average level of CAT expression in homozygous fish was 1.8-fold (for liver) and 1.6-fold (for spleen), that of heterozygous fish.

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The present study investigates the dynamics of oocyte and embryonic development between two successive parturitions (about 22-day interval) under 16 Light: 8 Dark photoperiod at 25°C, in order to develop criteria for the ovarian cycle in the viviparous mosquitofish Gambusia affinis. On day 0 after parturition, the ovaries contained oocytes of various stages. Vitellogenic oocytes belonged mostly to a basal group and prominent group, each with two peaks and ranging 0.2 to 1.4 mm in diameter. The prominent group of oocytes increased in diameter by day 5, and the hepatosomatic index reached a peak on day 3, indicating active vitellogenesis. The oocyte maturation and fertilization did not occur simultaneously in an individual, but in order of oocyte size (oocytes over 1.7 mm in diameter were fertilized) between days 2 and 5. Mature oocytes were fertilized without ovulation, and then continued through embryonic development in the follicle. Many vitellogenic oocytes that did not reach fertilization degenerated and disappeared by day 10. Oil-droplet stage oocytes seem to be recruited by this time. Yolk accumulation for the next gestation started from day 10, and advanced slowly after that.

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In order to investigate the process of otolith-matrix production and deposition, ultrastructural immunolocalization of an EDTA-soluble fraction of otolith matrix (otolith soluble-matrix, OSM) was studied in the sacculus of rainbow trout, Oncorhynchus mykiss, just-hatched fry. In the transitional epithelial cells of a cylindrical type, OSM was packed in the vesicles whose diameter was 100-250 nm. In the transitional epithelial cells of a cuboidal type and squamous epithelial cells, OSM was packed into the vesicles whose diameter was 0.5-1.5 μm. Sensory epithelium was immunonegative. Exocytosis of small vesicles, dilations of translucent vesicles, and extrusions of cytoplasm were frequently associated with the apical surface of the transitional and squamous epithelial cells. The OSM-immunopositive fibrous substance existed in the endolymph and was deposited onto the otolith surface. Otolith matrix was an OSM-immunopositive fibrous substance arranged into the meshwork. Densely packed layers and less densely packed layers of the matrix were deposited alternatively. The present results indicate that OSM is produced in the transitional and squamous epithelial cells, is stored in the two types of secretory granules, is secreted into the endolymph, and is deposited onto the otolith surface.

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In order to determine the accumulation performance of differing set-net designs, catch analysis was conducted upon small scale set-nets of both the single-trap and double-trap type. Total catch per haul was significantly higher after the design change to the double-trap net. The ratio of catch per intermittent haul to successive haul was defined as the accumulation index, which was used as an index to evaluate set-net accumulation performance. The accumulation index was calculated as 1.10 for the single-trap net and 1.52 for the double-trap net for the total catch. For the dominant species such as jack mackerel Trachurus japonicus, total catch increased after the design change, as did the accumulation index. In contrast, for species such as Japanese barracuda Sphyraena japonica, total catch decreased after the design change, as did the accumulation index. For any dominant species, the catch per haul and the accumulation index tended to increase or decrease in a similar manner. These results suggest that for each dominant species, catch differences between set-nets of different designs can be attributed to changes in their accumulation performances.

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Marine labyrinthulid isolates produced algal lytic zones on the double-layer agar with living cells of diatom, whereas they formed thin film-like colonies on agar media containing bacterial cell extracts or dead cell debris. Phospholipid fraction extracted from bacterial cells such as Vibrio parahaemolyticus stimulated the colony formation of labyrinthulid isolates. The molecular phylogenetic analysis using SSU rDNA sequences indicated that labyrinthulid isolates made a clade with some representatives belonging to the Labyrinthulida. Docosahexaenoic acid (DHA) was found to be the fatty acid with the highest content in cellular lipids of a representative strain L95-2 increasing up to 80% of total fatty acids dependent on culture conditions.

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Effects of ultraviolet (UV) irradiation on genetical inactivation and morphological features of sperm were examined in the Pacific oyster. Ultraviolet light (254 nm) was effective for inactivating sperm chromosomes. Irradiation for 60 s at a UV intensity of 72 erg/mm² per s was the optimum dose to achieve haploid gynogenesis. The rates of the fertilization and the development of D-shaped larvae decreased with increasing irradiation time, and the development of the eggs fertilized with the genetically inactivated sperms terminated before reaching the D-shaped stage. Electron microscopy showed clear destruction of the sperm acrosome and flagellum in the UV-irradiated sperms. As the duration of irradiation increased, the acrosome of sperms tended to suffer greater damage until the sperms eventually lost their flagella. Abnormalities in these structures have appeared to account, at least in part, for the decline of the fertilization rate of eggs inseminated with UV-irradiated sperms.

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To determine the mesh selectivity of a sweeping trammel net for Japanese whiting Sillago japonica, experimental fishing operations using three different inner net mesh lengths (27.5, 31.0 and 33.5 mm) were carried out in Tateyama Bay, Chiba Prefecture, Japan from August to November 1996 and from April to May 1997. The length mode of captured fish increased in proportion to the mesh length from 150-160 mm of the smallest mesh to 190-200 mm total length of the largest mesh. The master curve of selectivity in terms of length ratio to the mesh length (l/m) showed a wide selection range of 50% relative efficiency compared to the mesh selectivity curve of gill net. The curve had a peak at l/m of 6.3 with the selection range from 5.4 to 7.4. This suggested that the trammel net caught a large number of entangled or pocketed fish in comparison to the gilled fish due to the effect of inner net slackness. In terms of body girth ratio to mesh perimeter (G/P), the curve had a peak at G/P of 1.31, which reflected that relative efficiency of capture reached its maximum when the body girth of fish was 1.31 times that of the mesh perimeter.

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The complete amino acid sequence of phospholipase A₂ (PLA₂) from the pyloric ceca of the starfish Asterina pectinifera was determined by automated Edman degradation. The A. pectinifera PLA₂ (APLA₂) consists of 137 amino acids with an unblocked N-terminus and its molecular weight is calculated to be 15 300.1. The enzyme contains 14 cysteine (Cys) residues at the corresponding positions of the same residues which have been shown to be involved in intramolecular disulfide bonds in mammalian pancreatic PLA₂. The region involving an active site and a Ca²⁺-binding loop shows fairly high sequence homology (75%) between the APLA₂ and porcine pancreatic PLA₂. The APLA₂ conserved the amino acid sequence of the loop portion of the porcine pancreatic PLA₂ except for the deletion of two amino acids. These features indicate that the APLA₂ can be classified into the group 1 type PLA₂. In contrast, the homology between the APLA₂ and porcine pancreatic PLA₂ was calculated to be 47% in the whole region. Further, the insertion of sixteen residues and the deletion of three residues were required in the sequence of the APLA₂ to align the corresponding region to the β-wing of porcine pancreatic PLA₂. These differences in amino acid sequence of the APLA₂ may account for its specific properties such as the higher activity and the characteristic substrate specificity.

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Crude 5'-adenylic acid deaminase (5'-AMP deaminase), extracted from hoshi-nori (dried nori) and yaki-nori (toasted nori), was investigated. Substantial activity of 5'-AMP deaminase was found in the extracts of both dried and toasted nori, but the activity of the latter was lower than the former. The properties of the enzyme were as follows. The optimum pH and optimum temperature were observed in the ranges of pH 7.0-8.0 and 30-50°C, respectively. Metal ions Ca²⁺ and Sr²⁺ were most effective on the activation of enzyme, while Cu²⁺ and Fe²⁺ did not influence the activity. Ca²⁺ was also effective for thermal stability of the enzyme. The enzyme activity was comparatively stable even at 50°C in the presence of 0.1 M Ca²⁺, while it was completely inactivated at 50°C in the absence of Ca²⁺. The enzyme was specific only to 5'-AMP. The other adenylic derivatives such as 3'-AMP, 5'-ADP, and 5'-ATP were not reacted. The K_m value for 5'-AMP was estimated to be 2.77 mM. Addition of p-CMB, an inhibitor of SH-enzyme, to the reaction mixture inhibited the enzyme activity. Half inhibition was observed with 5 mM p-CMB concentration. The properties of the enzyme were not essentially different between dried and toasted nori. The activity of the latter was lower than that of the former, and excess toasting (180°C 15 s) caused a remarkable loss of activity.

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Effects of osmotic stress on protein synthesis in a carp epidermal cell line, EPC, were investigated by means of two-dimensional polyacrylamide gel electrophoresis. Hypoosmotic stress (150 mOsm/kg H₂O) induced the increased synthesis of six proteins, P1 (124 kDa, isoelectric point (pI) 6.1), P2 (69 kDa, pI 5.4), P3 (68 kDa, pI 5.5), P4 (61 kDa, pI 6.3), P5 (58 kDa, pI 5.4), and P6 (49 kDa, pI 6.2). The increase of the synthesis of P1, P2, P4, P5, and P6 occurred within the initial 3 h of hypoosmotic stress and continued thereafter, while that of P3 was transient. Hypoosmotic stress also induced the decreased synthesis of a protein P7 (60 kDa, pI 6.5-6.8). The decrease of the synthesis of P7 occurred from 12 h of hypoosmotic stress and continued thereafter. Hyperosmotic stress (450 mOsm/kg H₂O) transiently induced the increased synthesis of three proteins, P1, P3, and P8 (60 kDa, pI 5.8-6.1). Subcellular distribution analysis revealed that P2 and P6 were recovered in both the cytosolic and the membranous fractions, while P1, P3, P4, P5, P7, and P8 were recovered only in the cytosolic fraction. These changes of protein synthesis are discussed in view of physiological response of cells to osmotic stress.

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Carboxyl groups of myosin rod were chemically modified by means of carbodiimide-mediated amidation using glycine ethyl ether as an amino donor. Mobility of the rod on urea-polyacrylamide gel electrophoresis was reduced on modification, which indicates a reduced negative charge of rod. The modification significantly promoted the filament formation of rod especially at alkaline pH. Modified rod required neither lowering pH (pH 6.0) nor Mg²⁺ addition for its filament formation. Amino groups of rod were also modified using the same principle of carbodiimide-mediated modification using sodium formate as a donor. This modification led to an opposite effect on the filament formation of rod. Modification rather inhibited the self-assembly of rod. Thus, it was concluded that surface negative charges of the rod play an important role in filament formation. Factors that decrease the negative charge of rod such as lowering pH, adding Mg²⁺, or blocking carboxyl groups led to the promotion of the filament formation similarly.

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In order to evaluate the potential of using frozen mackerel muscle proteins as meat binders, freeze-thawed mackerel (Scomber australasicus) was ground with 0.1% reducing agents (cysteine, sodium bisulfite (NaHSO₃), or a mixture of both) and freeze-dried. The extractability, Ca-ATPase, reactive and total sulfhydryl of actomyosin increased with the addition of reducing agents. When 2.5% or more (w/w) of the meat binder was mixed with pork chunks and set at 40°C for 60 min, the tensile strength of the restructured pork stick was significantly higher than that without the meat binder. According to the data obtained, the binding ability of the binder could be greatly improved by the reducing agents which could recover the native protein.

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The activity of bacterial extracts to transform gonyautoxins to saxitoxins was found to be due to glutathione. This activity was also found in another thiol compound, 2-mercaptoethanol. These thiol compounds did not reduce 1-N-OH of gonyautoxin 1 and 4. Interestingly, the stable intermediate conjugates of paralytic shellfish toxins and thiol compounds were formed in the course of the reaction, which demonstrates that this is a two-step reaction consisting of formation of intermediate and degradation of the intermediate to saxitoxins.

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The accuracy in the differentiation between Micrococcus and Staphylococcus with three culture methods has been evaluated, using bacterial strains isolated from ika-shiokara, a traditional Japanese fermented squid meat. A total of 530 strains of Gram-positive, catalase-positive cocci isolated from ika-shiokara as well as the type strains of Micrococcus (eight strains) and Staphylococcus (26 strains) was differentiated with three culture methods using furazolidone-peptone (FP) agar, Schleifer-Krämer (SK) agar, and oxidation-fermentation (OF) test of glucose. All the type strains of both genera used were clearly differentiated on FP agar, and well differentiated on SK agar with some exceptions, while only 46% of Staphylococcus could be identified correctly on the OF test. Among the 530 strains of Gram-positive, catalase-positive coccal isolates, 491 strains were identified as Staphylococcus on the FP & SK agar, while only 73 strains were identified as Staphylococcus on the OF test. To obtain detailed accuracy, 55 strains were randomly selected from the 530 strains and were further examined by DNA base composition (mol GC%) assay. The results on the FP & SK agar revealed 100% similarity with the results obtained by mol GC% assay, while the OF test of glucose revealed only 24% similarity with them. These results confirmed that OF test of glucose such as Baird-Parker's method is misleading method, which is still of relatively common use for differentiation of these two genera in food microbiological study. The simultaneous use of FP & SK agar was suggested to be useful in routine identification system in food microbiological study.

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In order to clarify the influence of the interposition of pink muscle fiber into the dorsal ordinary muscle on the post-mortem temporal change of K-value, using carp, the dorsal muscle was divided into three muscle parts toward depth with the naked eye, as follows: dark muscle part, intermediate muscle part, and deepest ordinary muscle part. The muscle fiber types in these parts were discriminated by the inactivation of actomyosin ATPase activity for both acid and alkaline pre-incubations, and the temporal changes of ATP related compounds in these parts were then measured at a kept temperature of 32°C. Three muscle parts were organized from the muscle fiber types as follows: dark muscle part was organized from only red muscle fiber type, intermediate part from only pink muscle fiber type in a thin layer and from two muscle fiber types of not only pink muscle fiber but also white muscle fiber of the IIa or IIb subtype in a region of mosaic pattern, and deepest ordinary muscle part from two muscle fiber types of white muscle fiber IIa or IIb. The temporal change of K-value among muscle fiber types was considerably different and remarkably faster in the order of red muscle fiber, pink muscle fiber, and white muscle fiber. From these results, it was considered that the interposition of pink muscle fiber into the dorsal ordinary muscle might accelerate the temporal change of K-value.

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Applicability of a coagulation test was evaluated for the determination of the previous heat condition of heated blue marlin meat. Meat blocks were heated at different temperatures between 50 and 70°C. Proteins were extracted with different concentrations of NaCl, then subsequently subjected to the coagulation test. The coagulation method was able to determine the end-point temperature (EPT) of heated blue marlin meat within a range of 1-2°C, up to 67°C. The best correspondence between the actual EPT and coagulation temperature of the filtrates was found when proteins were extracted with 0.9% saline solution. Within the range of 50-67°C, sample preparation methods and holding times had no significant effect on the coagulation temperature of the filtrates. Protein bands of the filtrates on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel gradually disappeared with increasing temperature. One band with a molecular weight of about 31 kDa was detected in 67°C-, but not in 70°C-heated meat. This protein component was responsible for coagulation at 67°C and was found to be lactate dehydrogenase from the analyses by gel filtration, SDS-PAGE, and enzyme activity.

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A freshness index for fish muscle (K-value) was measured for six species of fish using freshness testing paper (FTP) and column chromatography. The correlation coefficient between these K-values determined by both methods was 0.917, although K-values of reddish muscle of big-eyed tuna, flying fish and common horse mackerel were evaluated lower by FTP than by column chromatography. The changes of K-values which were determined at regular intervals by FTP indicated that the decrease in freshness of amberjack and big-eyed tuna muscles was due to a first-order reaction. The kinetic parameters for the freshness-lowering, activation energy and frequency factor were obtained from Arrnhenius' plots of K-values at the storage temperatures between -1 and +20°C, and then it was clarified that the quality of amberjack and big-eyed tuna with the different history of storage temperature should be predictable on the basis of these kinetic parameters.

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