Year-to-year fluctuations in the recruitment of the juvenile sea urchin Strongylocentrotus nudus were monitored between 1980 and 1995 off the coasts of Okushiri Island and Shimamaki on the Sea of Japan coast, southwestern Hokkaido. High recruitment levels (14.1 ind./m²) of juveniles in 1984 and a continuous recruitment after 1990 were observed at the survey areas. The relationship between juvenile densities and average water temperature in September of the previous year was statistically significant. We suggest that high water temperature in September contributes to shortening of the larval period. The dominant 1983 year-class population contributed to an increase in commercial catch at the survey areas, and this was also found in the sub-prefectures of Shiribeshi and Hiyama.

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An ecological survey on the causative bacterium, Flexibacter sp. of spotting disease of sea urchin Strongylocentrotus intermedius was carried out for 14-months at monthly intervals from September, 1993 at Shakotan-Cho Fisheries Breeding Center and at coastal area near the Breeding Center in Bikuni, Hokkaido, Japan. The causative bacterium of spotting disease was not detected in any samples from winter season to early summer season until seawater temperature reached at 20°C. But the bacterium was detected in various samples from the Breeding Center and of wild sea urchins and seaweed (Codium fragile) at stations 1 and 2 in the mid August when seawater temperature was 20°C or higher. Spotting disease occurred within a few days after the first detection of the bacterium. After ceasing of the disease the bacterium was continuously detected from some samples in the Breeding Center from September to December when the seawater temperature was declining.
From these results, it is speculated that the causative bacterium proliferated in natural seawater at above 20°C and entered into rearing tanks through supplied seawater and then the disease was caused by bacterial attaching and proliferating on surfaces of sea urchins.

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Effects of the mother's environmental salinity on seawater tolerance of the offspring were examined in the guppy. Seawater tolerance of newborn guppies was expressed as a survival rate 24 h after transfer to 35 ppt seawater at birth. Newborn guppies in the seawater-adapted F22-SW(SW) strain showed a higher survival rate in seawater than those in the freshwater-adapted F22 and F22-SW(FW) strains. When gravid females of the seawater-adapted F22-SW(SW) strain yielded their offspring more than 5 days after acclimation to fresh water, the survival rate of the newborn guppies in seawater decreased to a level comparable to that of the freshwater-adapted F22 and F22-SW(FW) strains. On the other hand, when gravid females of the freshwater-adapted F22 strain yielded their offspring after acclimation to seawater, survival rates of their newborn guppies in seawater increased and most of them were able to survive in seawater. These results indicate that the mother's environmental salinity enhances the seawater tolerance of the offspring and contributes to successful reproduction of the guppy in seawater.

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We studied age, growth, and maturity of the oceanic whitetip shark Carcharhinus longimanus in the North Pacific, and the reproduction in the North and South Pacific, captured with tuna longline from Nov. 1967 to Oct. 1995. Vertebral rings were examined from 111 males (precaudal length: 54-172cm) and 114 females (precaudal length: 50-195cm). Minimum and maximum numbers of translucent rings were 0 and 11. Marginal increment analysis suggested annualus deposition occurs during spring. A growth difference between sexes was not found. A von Bertalanfly growth equation combining both sexes was as follows;
L_t=244.58×{1-e^{-0.103×(t+2.698)}}
L_t was expressed as precaudal length in cm at age t.
The reproductive condition was examined from 136 males and 85 females. Maturation was determined to be when precaudal length was between 125 and 135cm for both sexes (4-5 years). The parturition period is extended over a long duration. The birth size was 45-55cm and litter size varied from 1-14 with a mean of approximately six in the North Pacific.

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Age, growth and maturity of the bagrid fish Pseudobagrus aurantiacus from the middle reaches of the Chikugo River, Kyushu Island, Japan were studied by examination of vertebral centrum and histological observations. Marginal increments of vertebral centrum formed annual rings from March to April. The growth of this fish was expressed by von Bertalanffy's equation as L_t=340.4[1-exp{-0.131(t+0.759)}] for males and L_t=266.8[1-exp{-0.146(t+0.920)}] for females, where L_t is standard length (SL) in mm and t is age in years. Males grow faster and live longer than females.
The spawning period of this population was considered to take place from July to August (mainly July). The minimum size at maturity was 77.6mm SL for males and 90.0mm SL for females. The age at which 50% of the fish reached maturity was 1-year-old for males and 2-year-old for females. The earlier maturity of males may be related to their faster growth.

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The distribution of ganglion cells was examined in the retina of three cetacean species: the false killer whale, the pacific white-sided dolphin and the beluga. Three parameters of vision were estimated from the distribution of ganglion cells, i. e., the retinal resolving power (visual acuity), the field of best vision, and the direction of visual axis. In these three species, the retinal resolving power was 2.6-3.3 cycles per degree, the width of field of best vision was 12.8-15.4°, and the direction of visual axis was 51-68° from the geometric center of the retinal hemisphere. Interspecific differences are small, and these parameters are similar among most marine cetaceans. However, there are many differences in these parameters between marine cetaceans and terrestrial mammals.

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A combination of electrophoretic and heat denaturation techniques was used to identify the gene loci and alleles of the isozymes in goldfish and crucian carp. Heat denaturation experiments revealed different heat stabilities among three loci (Gpi-1^*, Gpi-2^*, and Gpi-3^*) encoding glucosephosphate isomerase (GPI) in goldfish and crucian carp. The heat stability differences were found in all the three loci examined. Cross experiments indicated that the observed differences among alleles at each locus in heat stability are controlled by a single incomplete dominant gene. Moreover, the heat stability alleles demonstrated a divergence in Gpi-1^* and Gpi-2^* loci between goldfish and crucian carp. The heat denaturation technique was thus useful for the revelation of hidden genetic variations of isozymes.

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RAPD (Random Amplified Polymorphic DNA) analysis was utilized to identify six sturgeon species. Amplifications with eight different primers gave reproducible electrophoretic patterns which could be regarded as a data-set consisting of monomorphic and polymorphic characters. Some of these characters were species-specific. Band-sharing analysis and numerical taxonomy methods allowed us to generate a phenetic tree. Our results provided evidence that RAPD markers can be used to discriminate species of sturgeons and indicated new possible systematic considerations within the examined taxa, in agreement with their geographical distribution and their inhabitancy system.

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Two groups of 10- and 11-year-old striped jack Pseudocaranx dentex (Bloch et Schneider) of 4.8kg average weight were used to determine whether these fish were able to spawn normally after being fed with dry pellets. Four months prior to spawning, they were divided into a test group and a control group and fed a commercial soft-dry pellet (cSDP) and a raw fish mix (RF), respectively, three times per week, in floating cages. For the spawning manipulation, the fish were transferred to indoor spawning tanks where they were fed once a day, for six days a week.
The RF group spawned eggs on 37 days during a 66-day period, whilst the cSDP group spawned on only 27 days during a 40-day period, the former therefore producing about three times the amount of eggs produced by the cSDP group. The eggs from the RF and cSDP groups had buoyancy rates of 83.2% and 71.0%, and fertilization rates among buoyant eggs of 79.6% and 78.2%, respectively. The percentage of normal larvae obtained from the total eggs produced was also higher for the RF group, 60.3% compared to 45.9% of the cSDP group. Despite this, the egg diameters and survival activity index of larvae were identical in the two groups. These results show that broodstock striped jack can also be cultivated by feeding cSDP prior to spawning.

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Annual changes in oocyte development in the ovary and serum vitellogenin, an egg yolk precursor, were investigated in the rabbitfish, Siganus canaliculatus, in Okinawa, southern Japan. The progression of oocyte development was histologically divided into eight stages; the chromatin-nucleolus, perinucleolus, oil-droplet, primary yolk, secondary yolk, tertiary yolk, migratory nucleus and maturation stages. The oocytes laden with yolk first appeared in March. From April through June, the stage composition of oocytes was characterized by a high percentage of oocytes advanced beyond the primary yolk stage and was correlated with the increase of gonado-somatic index (GSI). Post-ovulatory follicles could be observed in the ovaries in May. The GSI values gradually decreased after June and were maintained at low levels from October through February. During these months, the ovaries were occupied by the immature oocytes at the chromatin-and peri-nucleolus stages. Vitellogenin, which was measured immunologically with rabbit anti-rabbitfish vitellogenin antiserum, changed in accordance well with the monthly fluctuation of GSI as well as development of oocytes laden with yolk. These results suggest that S. canaliculatus starts vitellogenesis in March and spawns several times in the following three months in Okinawa.

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Salinity tolerance was examined in 14 strains of the guppy. Salinity tolerance was measured as LD₅₀ 24 h after transfer to serial concentrations of salinity. The LD₅₀ showed high values at birth and significantly decreased within 5 days after birth. The LD₅₀ was higher at birth than at 10 days after birth and at an adult stage in all of the strains. Strain differences were observed in the LD₅₀ at birth, 10 days after birth, and at an adult stage. Positive correlation was not observed between the strain differences at birth and at the adult stage but was observed between 10 days after birth and at the adult stage, suggesting that genetic control for salinity tolerance at birth differs from that of specimens over 10 days after birth. Cross experiments revealed that survival rates of F₁ hybrids in 35 ppt seawater at birth showed a positive correlation with rates of female parents but not with male parents. This indicated a maternal effect on salinity tolerance of newborn guppies at birth. The results of backcross suggested that the salinity tolerance of newborn guppies is not controlled by cytoplasmic genetic factors but by the mother's genotypes.

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The effects of addition of powdered protein (egg white, milk protein, soybean protein and bovine plasma protein) to kamaboko and ham on the growth of bacteria were studied. P. fluorescens IFO3081 could not grow on the surface of a model kamaboko prepared from surimi of codfish, salt, sugar, potato starch and water but was able to grow on that containing egg white, milk protein or soybean protein. Since these powdered proteins are rich in low-molecular-weight nitrogen sources utilizable by microorganisms which are unable to digest high-molecular-weight proteins, the strain grew on the kamaboko and ham containing these powdered proteins. On the other hand, bovine plasma protein was found to have activity to suppress proliferation of the strain. From these results, the use of powdered protein for kamaboko and ham has both advantages and disadvantages in terms of food preservation.

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Bacteriophages of Lactococcus garvieae, designated as PLgW and PLgS, were isolated from sea water and sediment samples by an enrichment method. Morphological and genomic features of these phages were in agreement with those of the L. garvieae phage, designated as PLgY, belonging to the family Siphoviridae that was detected in an L. garvieae strain isolated from diseased yellowtail in the previous study. One hundred and eleven strains of L. garvieae examined were divided into 14 phage types (A_??_N) by using the phage isolates which were differentiated from each other in the infectivity, with a major phage type (type A) containing 73 strains. One phage type (type N) consisting of 9 bacterial strains was insensitive to any of the phages used. However, there were no apparent correlations between the phage types and the geographical sources of the bacterial strains or between phage types and the antigenic forms (KG- and KG+).

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Needle-like filamentous bacteria have been isolated commonly from the cultures of three marine microalgae including Chattonella marina, Heterosigma akashiwo and Tetraselmis sp. All filamentous strains showed good growth in the peptone-yeast extract medium with artificial sea water, but failed to grow in the medium with 3% NaCl. A major group of filamentous bacteria showed neither flagellar nor gliding motilities and formed whitish pink and slightly rough colonies on the agar medium. A major pigment extracted from cells of the whitsh pink-colored filamentous strains (WP strains) was considered to be flexixanthin-like carotenoid according to its absorption spectrum. All of WP strains and one greenish orange (OG) strain among filamentous isolates had mole% of GC contents of DNA from 58.7 to 60.0%. DNA probes prepared from the representative strains of WP filamentous bacteria were hybridized with DNAs from all of WP strains and the OG strain but not with those from other filamentous strains and Cytophaga-Flexibacter reference strains.

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The forestomach contents of 114 minke whales Balaenoptera acutorostrata randomly sampled in the northwestern part of the North Pacific in the summer of 1994 and 1995 were analyzed. Twelve prey species consisting of 7 fishes, 4 euphausiids and 1 copepod were identified. Pacific saury Cololabis saira was the most important prey species, occurring in 77.2% of the stomachs and composing 80.6% of the weight ingested, with some spatial and temporal variation. Japanese anchovy Engraulis japonicus was the next most important prey species, occurring in 14.0% of the stomachs and composing 6.7% of the weight ingested. Diurnal changes in the mean forestomach content weight and in the relative freshness of the forestomach contents showed little change between 06:00-18:00 h. The minke whales fed mainly on pelagic schooling fishes at the surface in the daytime.

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The effects of rearing period in seawater on induced maturation by hormonal treatments were examined in the cultured female Japanese eels Anguilla japonica. The oocytes of female eel (30 month old) kept in seawater for 3 months completed vitellogeneisis by significantly smaller number of injection of salmon pituitary extracts than those of female kept in seawater for one week. Fertility and hatching rates were not significantly different between the oocytes of the two experimental groups. Similar results were obtained in the experiment which was performed using 6-month older females. These results indicate that long term rearing in seawater increased responsiveness to salmon pituitary extract in ovarian maturation. Histological observation revealed that oocytes of females kept in seawater for 3 months became large and attained at the primary yolk globule stage, whereas oocytes of the females kept in seawater for one week were at the oil drop stage. Electron microscopic observation showed that gonadotrophs became active in the pituitary of females kept in seawater for 3 months. These results are the first findings showing that vitellogenesis is triggered under artificial rearing condition without any hormone treatments in cultured immature eels. These morphological results indicate that long term rearing in seawater induces vitellogneisis in the females, resulting in the shortened period for compeletion of vitellogeneisis by the injection of salmon pituitary extract.

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Histochemical characteristics of lateral muscle fiber types in the Japanese sardine Sardinops melanostictus were confirmed and their developmental process investigated. In 102 and 106mm young specimens in standard length (SL), four muscle fiber types, i. e., red, pink, white, and tonic-like fibers were recognized. Pink fibers mainly occurred in the anterior trunk region. Two types of tonic-like fibers were recognized around the red triangular wedge along the horizontal septum. Newly-hatched larvae (free embryos) had a single layer of superficial fibers containing a few myofibrils and inner white fibers. Histochemical reactions for NADHD and lipid in the superficial fibers gradually increased and became strong enough to be classified as red fibers by the first-feeding stage. Beyond about 20mm SL, stratification of the red fibers, differentiation of the tonic-like fibers, and mosaic appearance of the white fibers occurred. By 30mm SL, pink fibers and two types of tonic-like fibers were differentiated. The structure of the lateral muscle in a 37mm SL juvenile was complete and did not differ from those of young specimens, 102 and 106mm SL.

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Pressurized and heated carp muscles were examined by electron microscopy to clarify the effects of high pressure upon their fine structures, in relation to the changes of their physical properties. At pressures over 300 MPa, the striated structure of the myofibrils disappeared. At 500 MPa the contractile elements of the muscle fibers disintegrated, thus resulting in an appearance of irregular electron-opaque bodies. Heated muscle fibers, on the other hand, showed that the myofibrils completely lost their striations and changed into homogeneous materials. Although high pressure and heat affected the structural profiles of such cytoplasmic membrane structures as the mitochondria and endoplasmic reticulum, no membrane disruption was observed in those structures. The appearance of glycogen granules was not affected by either high pressure or heating. In addition, the texture values of carp muscles rose with an increase in pressure, and the viscoelasticity was higher in the pressurized muscles than in the heated muscles.

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The incorporation of dansyl glutamine (DG) into fish muscle protein mediated by transglutaminase (TGase) was examined in order to get new information on the behavior of the lysine residues of the protein during setting fish flesh sol. Muscle TGase-free minces from various fish and poultries were ground with NaCl, Alaska pollack muscle TGase extract, and DG and incubated at 30°C. With the prolongation of incubation, the amount of DG incorporated increased. The increment of its amount was larger in the order of Alaska pollack>Pacific mackerel>horse mackerel_??_chicken_??_beef_??_pork, whose order was in agreement with that of the increment of the breaking force of suwari gel formed from each species without DG, with that of the increment of cross-linked myosin heavy chain and the decrement of myosin heavy chain in the gel, and with that of the increment of the amount of monodansyl cadaverine incorporated. In addition, myosin heavy chain and cross-linked myosin heavy chain were labeled with DG during setting muscle TGase-free Alaska pollack sol at 30°C for 3 h in the presence of the muscle TGase extract from its surimi.

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This study was conducted to clarify the energy requirements for maintenance of body weight and activity, and for maximum growth in carp and rainbow trout, specifically examining the effect of feed preparation methods on dietary energy budgets and nitrogen balance. Carp (31g, 17°C), rainbow trout juvenile (31g, 9 and 16°C), and rainbow trout fingerlings (5.7g, 16°C) were fed isocontent ordinary dry pellets (DP) produced by a pellet mill and extruded pellets (EP) by a twin-screw extruder.
Carp fed EP to satiation showed markedly reduced feeding rate, low percent gain and feed efficiency compared to those fed DP to satiation. In rainbow trout EP tended to be generally lower in the feeding rate and higher in the percent gain and feed efficiency than DP at the satiating levels. This tendency was conspicuous for the growth of fingerling and the feed efficiency at a low temperature. The energy digestibility of EP increased from that of DP in rainbow trout, in relation to the gelatinization of starch.
The energy budget in fish fed enough amounts of energy required for maximum growth was determined. The fecal energy loss in DP was relatively large among all the tests, and the digestible and metabolizable energy values of EP were elevated in relation to the fecal energy values. The non-fecal energy losses in rainbow trout were somewhat higher in EP than those in DP. The heat increment of feeding was high in DP and the proportions of productive energy were high in EP in all the experiments.

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The changes in extractive components and glycogen in hard clam meat during storage at 0, 5, 10, 20 and 30°C were investigated. The initial levels of ATP-related compounds, glycogen, succinic acid and free amino acids in hard clam were 3.34±0.19μmol/g, 1201±52mg/100g, 9.44±2.55μmol/g, and 1226±106mg/100g, respectively. ATP in all samples decreased markedly during storage, while the accumulation of ADP, AMP, inosine, and hypoxanthine varied depending on the 5 storage temperatures. The decrease in glycogen was common for all the samples. In contrast, succinic acid increased with time of storage, and the increase rate was faster for the samples stored at 5 and 10°C. The changes in levels of free amino acids in hard clam during storage at different temperatures were inconsistent. The samples stored at 5 and 10°C showed a relatively high increase in the amounts of free amino acids, particularly in alanine, taurine, and arginine. The enrichment of free amino acids and succinic acid might potentially improve the flavor quality of hard clam stored at 5 and 10°C.

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The thermal behavior of basic amino acid residues of muscle proteins during incubating fish and poultry minces was investigated. For the transglutaminase-free muscle minces from Alaska pollack, Pacific mackerel, and horse mackerel, their acidic dye-binding ability was increased upon their incubation at 30°C for 5 h and simultaneously, the rise of pH was suppressed in their titration with 0.1_N NaOH. The increment of the dye-binding ability and the suppression of pH rise, however, were not observed for the minces from beef, chicken, and pork.
Based on the above results, it was presumed that the basic amino acid residues are easy-to-expose on the molecular surface of fish proteins during the incubation but hard-to-expose in the case of poultry proteins.

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The changes of glycogen, glucose and several other glycolytic intermediates as well as muscle pH were examined in the adductor muscle of noble scallop during storage at 0°C and 5°C. Phosphorylase, hexokinase (HK), and phosphofructokinase (PFK) activities were also examined in relation to pH and temperature.
Glycogen content immediately after death was 3.6g/100g and dropped rapidly during early storage. The initial contents of glucose and other glycolytic intermediates were low. Glucose and frucotose-6-phosphate accumulated to a lesser extent compared with glucose-6-phosphate. Glucose-1-phosphate, fructose-1, 6-bisphosphate, and α-glycerol phosphate increased during strage, then decreased as the storage progressed. On the other hand, phosphoenolpyruvate and pyruvate increased continuously. The initial lactic acid content was 0.82μmol/g, consisting of 85% L-Lac. There were no substantial concentration changes of L-Lac during storage. On the other hand, D-Lac increased and reached 3.2μmol/g. The initial pH value was 6.88 and dropped faster at 5°C than at 0°C. Phosphorylase and PFK activities decreased as the incubation pH decreased, forming a sigmoid flexure. On the other hand, HK activity increased markedly as the pH dropped lower than 6.2. When the incubation temperature was lowered, PFK activity decreased faster than HK and phosphorylase activities.

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The cDNA of the estrogen receptor (ER) has been isolated from red seabream liver and sequenced. The cDNA contains a complete open reading frame encoding 581 amino acid residues. The structural homology indicated that the red seabream ER (rsER) lacks region A in region A/B (trans-activation domain) as well as tilapia, medaka and rainbow trout ERs. In region B, amino acids were conserved also in rsER around a serine residue that is putatively phosphorylated by a mitogen-activated protein kinase. In region C (DNA binding domain), rsER shows a high degree of sequence conservation with other fish ERs, particularly eight cysteins in two zinc-fingers and the P-box were completely conserved. Region E (hormone binding domain) was also highly conserved. On the other hand, in regions D and F, low homology was revealed.
The trans-activation function of this clone was verified by transfection into HeLa cells, which lack endogenous ER. A CMV-based expression vector consisting of rsER cDNA was cotransfected with a chimeric reporter plasmid consisting of an estrogen responsive element fused to the thymidine kinase promoter and the chloramphenicol acetyl transferase (CAT) gene. In the presence of estradiol 17-β, the CAT activity was stimulated by up to 10-fold. This showed that this clone encoded the functional receptor.

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Gas-liquid chromatography (GLC) analysis revealed a high content of very long chain fatty acids in the marine sponge Halichondria panicea from the coast of south Hokkaido, Japan. Three major components of the fatty acids were concentrated by argentation thin-layer chromatography (TLC) and reversed phase TLC. They were identified as demospongic acids, 5, 9-hexacosadienoic (26:2), 5, 9, 19-hexacosatrienoic (26:3), and 5, 9, 20-heptacosatrienoic acids (27:3) by gas chromatographic-mass spectrometry analysis of their picolinyl esters and GLC analysis of the oxidative ozonolysis products. The proportion of 5, 9, 20-27:3 (25.4%) in total fatty acids was much higher than that had been observed for other sponges. The contents of 5, 9-26:2 (10.0%) and 5, 9, 19-26:3 (7.2%) were comparable to those reported. All three demospongic acids were abunduntly found in the lipid classes of phosphatidylserines and phosphatidylethanolamines.

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We cloned and analyzed a complement regulatory protein cDNA (designated HCRF) from the Japanese flounder Paralichthys olivaceus. The length of the cDNA obtained was 1662 bp. The predicted translation product represents a protein of 453 amino acids which displays a putative hydrophobic leader sequence. There were 7 short consensus repeats (SCRs) which are closely related to those of the previously described SBP1 and SBCFR-1 of complement regulatory plasma proteins of barred sand bass Paralabrax neblifer. The SCRs of HCRF, SBP1, and SBCFR-1 constitute 7, 17, and 3 domains, respectively. SCR domains Nos. 2 to 7 of HCRF resembled domains Nos. 12 to 17 of SBP1. SCR domains Nos. 6 and 7 of HCRF also closely resembled domains Nos. 2 and 3 of SBCFR-1, respectively.
The expression of the complement regulatory protein-like mRNA in flounder liver was demonstrated by Northern blot hybridization.

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To elucidate the cysteine metabolism in rainbow trout, the tissue levels of metabolites derived from dietary cyst(e)ine were examined. First, the distribution of hypotaurine among tissues was examined for the rainbow trout fed a commercial diet. Hypotaurine content was highest in the liver (1.2μmol per g tissue), followed by the kidney (0.3μmol per g tissue) but it was not detectable in the brain or the blood. The value obtained in the liver was far higher than the values that have been reported for fish so far. Effects of dietary administration of excess cystine on the changes in the tissue levels of metabolites were examined. The excess cystine administration increased the level of hypotaurine in the tissues. The level of cysteine sulfmate also tended to increase, but the quantity was very small compared with that of hypotaurine. Although cysteine sulflnate may play an important role also in cysteine metabolism in rainbow trout as well as mammals, cysteine sulfinate seems to be quickly metabolized into hypotaurine.

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The influence of Ca²⁺ concentration around myofibrillar Mg²⁺-ATPase on the speed and pattern of rigor mortis in fish species or cultured and wild fish was studied. The change of contraction in pillar form muscle with the lapse of storage time reflected very well the temporal change of gram of tension in muscle of cultured red sea bream. This result showed that the contractile percentage of muscle is very useful on the progress of rigor mortis. The progress of muscular contraction differed among fish species and between cultured and wild fish of the same species, and which were seen in four parameters of rigor mortis: the progressive speed, pattern, and the time and level of peak reached. These four parameters were closely related to one another. The ranking among fishes in myofibrillar Mg²⁺-ATPase activity was not consistent with the difference of speed of progression of muscular contraction. The progress of superprecipitation reaction of actomyosin in muscle of cultured red sea bream was changed remarkably in four parameters by a very small difference of Ca²⁺ concentration, coinciding very well with the mutual relation among the four parameters on the progress of muscular contraction. Therefore, it was considered that the difference of the progress of muscular contraction among fish species and between cultured and wild fish can be mainly caused by the difference of Ca²⁺ concentration around myofibrils.

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In addition to the two protease inhibitors (AXPI-I and II) previously characterized, another inhibitor (AXPI-III) has been isolated from the aqueous extract of the sea anemone Anthopleura aff. xanthogrammica by acetone precipitation, gel filtration, cation-exchange FPLC, and reverse-phase HPLC. Like AXPI-I and II, AXPI-III is a basic polypeptide and its amino acid composition is characterized by the presence of 6 half-Cys residues and the absence of Met and Trp. AXPI-III is potently inhibitory against trypsin and also considerably inhibitory against α-chymotrypsin. Both AXPI-II and III did not inhibit the binding of ¹²⁵I-α-dendrotoxin to rat synaptosomal membranes, suggesting that they are not blockers of voltage-sensitive potassium channels. Analyses of the N-terminal portion and one asparaginylendopeptidase-digested fragment established the complete amino acid sequence of AXPI-III comprising 61 residues. The overall sequence homology and the conserved location of half-Cys residues confirmed that AXPI-III belongs to the Kunitz-type family.

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The ordinary muscle of fresh milkfish was ground with different concentrations of alkaline reagent (sodium carbonate), acid (malic and citric acid), and glucono-δ-lactone), and processed into kamaboko gel through setting and heating. In the presence of sodium carbonate, the whiteness of kamaboko was decreased dependent on the concentration, though the gel-strength was not much affected. The addition of malic or citric acid could improve the whiteness of kamaboko, but the gel-strength was greatly deteriorated. On the other hand, glucono-δ-lactone, when added up to 0.4%, lightened the kamaboko without deteriorating the quality, and was thus considered to be an effective whiteness improver for milkfish kamaboko.

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