The substitution of dietary docosahexaenoic acid (DHA) with eicosapentaenoic acid (EPA) reduces larval growth in gilthead sea bream. However, the value of EPA when dietary DHA is able to meet the requirements of the larvae has not been sufficiently studied. Dietary phosphoacylgliceride levels also affect fish growth and it has been suggested that they enhance lipid transport in developing larvae. The present experiment was carried out to further study the effect of dietary lecithin and eicosapentaenoic acid on growth, survival, stress resistance, larval fatty acid composition and lipid transport, when DHA is present in the microdiets of gilthead sea bream. Eighteen thousand gilthead sea bream larvae of 4.99±0.53mm total length were fed three microdiets tested by triplicate: a control diet [2% soybean lecithin (SBL) and 2.89% EPA], a low EPA diet (2% SBL and 1.63% EPA) and a no SBL diet (0% SBL and 2.71% EPA). Handling, temperature and salinity tests determined larval resistance to stress. The results show that when dietary DHA levels are high, but dietary arachidonic acid (ARA) levels are about 0.2%, EPA is necessary to improve larval growth, and survival. Larval EPA content, but not DHA or ARA, was affected by dietary EPA levels. Increased dietary EPA improved larval stress resistance to handling and temperature tests, which could be related to its possible role as a regulator of cortisol production, whereas it did not affect stress resistance after salinity shock. Larvae fed the no SBL diet showed a lower lipid content characterized by a low proportion of saturated and monounsaturated fatty acids, together with a significant reduction in the appearance of lipoprotein particles in the lamina propria and in the size of such particles, denoting a critical reduction in dietary lipid transport and utilization, and lower larval growth and survival rates.

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The release of zoospores from zoosporangia in Laminaria japonica mainly occurred at night. The maximum swimming speed of the zoospores was observed immediately after release (160 μm/s), and then decreased with time. The zoospores exhibited clear chemotaxis to high concentrations of nitrate-N and phosphate-P. Only half of the zoospores were observed to swim against seawater with a flow velocity of 124 μm/s. The zoospores became floating spores after they stopped swimming in the seawater. Low light and low water temperature conditions prolonged the swimming period of the zoospores. However, the photosynthesis of the zoospores was suppressed in low water temperatures, and was also limited under high light irradiance. These results suggest that photosynthesis in zoospores does not prolong their swimming period, and that when their swimming energy is exhausted, floating spores appear.

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Although most members of the genus Enteromorpha are important edible green algae, some species are also potentially economically valuable crops. Samples of E. prolifera were obtained from the Yoshino River estuary, Tokushima, Japan and cultured in laboratory conditions at 10°C, a salinity of 20 psu, under white light with 12 h light: 12 h dark cycle and at a photon fluence rate (PFR) of 40 μmol/m² per s for 30 days. In the present study, the effect of salinity and PFR on the induction of reproductive cells and rhizoid formation were investigated. Synchronous formation of swarmers by thalli was induced in excised disks of 1.2 mm diameter after 2-5 days incubation. The optimum salinity for maturation of reproductive cells was between 5.0 and 52.0 psu, and between 13.2 and 45.3 psu for swarmer release, although the lower limit for swarmer release was 5.0 psu. Maturation of reproductive cells and swarmer release required a PFR higher than 16 μmol/m² per s. The minimum PFR for swarmer release was 8 μmol/m² per s. Many rhizoids were formed between 1.6 and 52.0 psu and photon fluence rates between 8 and 320 μmol/m² per s. Rhizoids were formed in a polarized manner.

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The black rockfish Sebastes inermis is one of the most important fishery species along the coast from southern Hokkaido to Kyushu, Japan and is often found in rocks and Zostera areas. We conducted biotelemetry using coded ultrasonic transmitters to clarify the movement of the black rockfish that inhabited the seawall of the Kansai International Airport. We released 25 black rockfish at two points. One was the airport seawall and the other was side shallows off the Sensyu district. Seventeen black rockfish returned to their capture site after release. We used the V-test to determine whether the direction of movement was random or orientated. The black rockfish moved at random along the seawall within some hours after release (P>0.05). Four hours after release, they moved to their home site intentionally (P<0.0025).

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The treatment of exogenous gonadotropin or gonadotropin-releasing hormone analog (GnRHa) is able to solve some problems that are caused by small milt volume or viscous milt in artificial insemination of flatfish species. However, the mechanism of the action of the exogenous hormones on milt production has not been well understood. Therefore, experiments were carried out to study the effects of GnRHa on the milt production and characteristics of seminal plasma in starry flounder Platichthys stellatus. Males were implanted with GnRHa pellets at three different dosages (50, 100 or 200 μg/kg body weight) during their natural spawning season. Milt volume and sperm production increased in a dose-dependent manner after GnRHa pellet treatments. Spermatocrit and sperm concentration were significantly lower in GnRHa treated groups than in the control group. The sperm motility index was high from day 4 to day 49 in all experimental groups including the control group, and it declined after day 49 in all groups except the 200 μg treated group in which it remained high until day 85. During the experimental period, osmolality, K⁺ and total protein tended to be higher in the control group than in the GnRHa treated groups. The results suggested that GnRHa treatment increased the milt volume and sperm production ability in starry flounder. The increase of milt volume was twice as rapid as the increase of sperm production. No significant differences in physicochemical composition of blood plasma and seminal plasma were found according to GnRHa dosages, but there were significant differences between GnRHa treated groups and the control group.

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We carried out mail and access point surveys to estimate the catch numbers, angling effort of recreational angling and the population of sockeye salmon Oncorhynchus nerka in Lake Toya in 1998 and 1999. The mail survey questions in questionnaires distributed to anglers with a recreational angling license included date of angling, caught fish species and size, and catch number. In the access point survey we also asked anglers if they had a license. We measured the fork length and weight of caught fish. The catch rate (catch number in a day by each angler) from both surveys showed no significant difference, suggesting no bias for the catch rate between surveys. The estimated total angling effort was 1760 people (1998), and 1516 people (1999). The estimated recreational catch was 28 889 (1998) and 5455 (1999), that is, two or three times larger than the commercial catch. The fish population was estimated by using the DeLury method as 60 262 (1998) and 10 806 (1999). The total exploitation rate was 62% (1998) and 78% (1999). The size of caught fish was much larger in 1999 than 1998 because of the difference in age composition.

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Striped bonito Sarda orientalis larvae and juveniles were reared in a laboratory from hatching to 14 days post hatching, and the development of their digestive system was investigated histologically. On day 2 (first feeding day), the larval-type digestive system was differentiated. On day 3, a blind sac with densely distributed gastric glands developed and pyloric ceca appeared, suggesting that the digestive system was attaining adult-type structure. The yolk was completely absorbed on day 4. Bonito larvae preyed on rotifers and/or Artemia nauplii on day 2, but almost the whole gut was occupied by fish larvae from day 4. In the striped bonito, the adult-type digestive system was established right after first feeding, and this precocious development was well synchronized with the appearance of piscivory.

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Japanese conger Conger myriaster has well-developed splenic ellipsoids (sheath structure) that are thought to participate in the elimination of foreign substances and effete hemocytes from circulating blood. After injection of fluorescent latex beads (LB, 0.5 or 2.0 μm in diameter) through the aortic bulb of the Japanese conger, the spleens and kidneys were examined by light and electron microscopy. The splenic ellipsoids trapped many LB of 0.5 μm in diameter but none of 2.0 μm. This suggests that the LB were filtered by ellipsoids depending on size. Latex beads of 0.5 μm were ingested by macrophages beneath the endothelium of the arteriole in ellipsoids but not by endothelial cells. At 24 h, many 0.5 μm LB were found in the ellipsoids and splenic cord, but there were few in the melanomacrophage centers (MMC). However, most of the 0.5 μm LB had accumulated in the MMC within 48 h. Many macrophages laden with the microspheres were found around MMC. The 2.0 μm LB were detected in the splenic cord and MMC 24 h and 48 h after injection. This suggests that the macrophages take up 0.5 μm LB in the ellipsoids and migrate through the splenic cord to MMC. The process of entrapment and accumulation of particles in the kidney was also examined and discussed.

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The phenomenon of mass mortality of larval mud crab Scylla serrata is described during the metamorphosis to megalops due to abnormal molting by morphologically advanced last stage zoeas (fifth stage) in the seed production. Seven seed production trials were carried out using 100 kL concrete tanks. The larvae were reared on rotifers and after the third or fourth zoeal stage with Artemia nauplii. Minced mysid was also given from the megalopal stage. The seven trials were based on the addition of different concentrations of the marine phytoplankton Nannochloropsis or of the freshwater Chlorella to the larval rearing water. The survival rate, morphological features of the fifth stage zoeas, and frequency of abnormal molting during the metamorphosis to megalops were examined. The mass mortality occurred during the metamorphosis to megalops in tanks containing high concentrations of Nannochloropsis. The fifth stage zoeas in these tanks were equipped with some advanced morphological features similar to those of megalops, such as, long endopods of the second antennae, large chelipeds and pleopods with plumose setae. The frequency of abnormal molting tended to increase depending on the degree of megalopal features in the fifth zoeal stage. We propose that zoeal morphogenesis is accelerated by the nutritional conditions of zoeas due to the supplementary effect of Nannochloropsis, which contains eicosapentaenoic acid; an essential fatty acid for larval mud crab.

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The aim of this paper is to examine from a statistical viewpoint the model and the estimation performance for covered-codend experiments in which fish escape from the covernet. The logistic function was assumed for the selectivity curves of the codend and covernet. First, we evaluated the efficiency of the maximum likelihood (ML) estimation based on the model that considers fish escape from the covernet compared with the conventional model that does not consider the effect of fish escape. Second, we assessed the biases of the ML estimators of parameters in the codend selectivity curve by using the conventional model when fish escape occurred. Finally, we examined the performance of model selection based on the Akaike information criterion through Monte Carlo simulations. It is concluded that the model considering fish escape is effective for the analysis of selectivity. In addition, the estimator involving the model selection performed better than estimators based on fixed models. The interpretation of these results and topics of future research are also discussed.

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The mitochondrial 16S rRNA gene was amplified by polymerase chain reaction using total DNA as a template from muscle tissues of alfonsino and related fish species of alfonsino belonging to the genus Beryx, B. splendens, B. mollis and B. decadactylus. While the sequence of the 16S rRNA gene consisted of 492 nucleotides irrespective of the three species, three, nine and eight interspecific variations were shown between B. splendens and B. mollis, between B. splendens and B. decadactylus, and between B. mollis and B. decadactylus, respectively. The pelagic eggs, which were collected off Izu Islands and showed morphological characteristics typical to the genus Beryx were all identified as those of B. splendens from the nucleotide sequence of the mitochondrial 16S rRNA gene.

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Although the notion of growth coefficient is important in growth analysis, its meaning is not necessarily pointed out clearly, at least analytically. The present paper, based on the von Bertalanffy model, gives an extended quantity of it and investigates its properties for logistic and Gompertz models. It is age dependent and converges to the coefficient of age in the models, growth coefficient in the traditional sense. The graphs of the quantity and the length show similarity for the logistic model, but not for the Gompertz model, although they show the same type of fluctuation. Moreover, the analysis indicates that the newly defined quantity has a property suitable to be called a growth density of the growth of length. Our discussion also shows the importance of the notion of growth remainder.

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Young-of-the-year (YOY) Sebastes inermis uses seagrass beds as a temporary nursery ground after completing the planktonic stage in offshore waters. In the present study, the otolith microstructure was used to reveal the early life history of this species in a Zostera marina bed in Matsushima Bay, northern Japan. In 1998 and 1999, the settlement season began in early spring and catch per unit effort (CPUE) of YOY decreased markedly during the summer months. The smallest YOY S. inermis collected were 20 and 21 mm total length (TL) at 70-80 days after extrusion in 1998 and 1999, respectively. In both years, YOY that were extruded earlier (mostly in January) first appeared in the Zostera bed, and they were the first to leave, whereas those YOY extruded later (mainly in February) tended to stay beyond the summer in the Zostera belt. Birth dates of YOY showed two distinct cohorts, January and February, which showed a similar pattern of growth in both years; rapid growth after settlement peaking in May in 1998 (mean=0.73mm/day) and in April in 1999 (mean=0.61 mm/day), decreasing during the summer months, with the lowest mean values in August in both years (mean=0.40 mm/day for 1998 and 0.30 mm/day for 1999). In both years, the decrease in both CPUE and growth rates in summer months synchronized with a decline in biomass, density, leaf length and shoot weight of Z. marina. Total CPUE was higher in 1999 than in 1998, whereas the recent growth rate (i.e. 1 week before the fish were collected) was significantly higher in 1998 than in 1999, suggesting a density-dependent mechanism.

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The number of halophilic bacteria in 17 kinds of salted fish products made in the Loochoo Islands, Okinawa (in the subtropical zone) or the Noto Peninsula, Ishikawa (in the temperate zone, facing the Sea of Japan) was examined with media containing 0-20% (w/v) NaCl. In four of five samples of ‘suku-garasu’, salted fries of Siganus fuscescens made in Okinawa containing 21-28% NaCl, the bacterial number was 10³ c.f.u./g or lower. For the other ‘suku-garasu’ that contained 16% NaCl, there were 10⁷ c.f.u./g of halophilic lactococci with high levels of lactic acid and water-soluble proteins, free amino acids and volatile basic nitrogen, as indicators of fermentation in protein-rich foods. In salted mackerel made in the Noto Peninsula, the number of halophilic lactococci was 10⁵ c.f.u./g, which was higher than that in salted squid. Two salted mackerel samples that contained 22-24% NaCl had a high level of lactic acid. However, the lactic acid concentration was very low in a sample of salted mackerel containing 28% NaCl. Characteristics of the isolated halophilic lactococci were similar with those of genus Tetragenococcus. Effects of NaCl concentration and temperature on the growth of halophilic lactococci were also examined.

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In Miyagi Prefecture, the cultured brown alga Wakame (Undaria pinnatifida) is boiled and then salted for human consumption. The stipes are discarded. To explore a use for the discarded stipes, we studied their dietary value on the growth of the juvenile sea urchin Strongylocentrotus nudus and the gonadal enhancement of adults and compared them with that of fresh stipes, blades and sporophyll in the rearing experiment. Rapid growth in juveniles was most effectively promoted by the intake of fresh blades, with consumption of boiled stipes being next in value. The intake of boiled and salted stipe was higher than fresh stipe and sporophylls; however, sporophylls promoted the best gonadal enhancement in adults. Despite the low carbohydrate and crude protein content in boiled stipe, we estimated that boiled stipe could enhance the gonadal index by as much as 18%, the minimum level for market, after 5 months of feeding. The results suggest that boiled stipe would be an effective deterrent to the grazing intensity of the wild sea urchin within coralline flats where marine afforestation is being attempted.

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Effects of water flow on the growth, smoltification, immune function and swimming performance were studied in cultured 1-year-old masu salmon Oncorhynchus masou masou. The experimental fish were exposed to 2, 13 or 23cm/s flow for 11 months. Fish exposed to 13 and 23cm/s flows had larger body size after 11 months and higher condition factor after 2 months than those exposed to 2cm/s flow. Blood levels of growth hormone and insulin-like growth factor-I were unaffected by flow conditions. The higher flow delayed progress of smoltification, whereas there was no clear difference in blood levels of thyroxine between the three flow groups. Superoxide anion produced by leukocytes was the highest in fish receiving 23cm/s flow, whereas no difference was detected in the plasma immunoglobulin M concentration between the three flow groups after 11 months. Increased flow resulted in higher swimming performance during the rearing period. Correlations between the relative swimming speed and the specific growth rate (SGR) were observed. The swimming speed with the maximum SGR was estimated to be 1.04-fold the fork length per second. These results indicate that water-flow conditions can affect growth and fitness of juvenile masu salmon, which are desirable for both release programs and aquaculture of this species.

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Two experiments were conducted to determine a suitable feeding schedule and the optimal density of Artemia nauplii on the survival and development of mud crab Scylla serrata larvae. The first experiment consisted of 10 treatments, where rotifers were shifted to Artemia nauplii at the zoea (Z) 1, 2, 3, 4 and 5 stages. Five other treatments were similar to these first five; however, rotifers were continuously added together with Artemia nauplii. The second experiment consisted of five treatments that were carried out to determine the optimal Artemia nauplii density. Four treatments fed different Artemia densities (0.5, 1, 1.5, 4individuals (ind.)/mL) and in the fifth treatment the density of Artemia nauplii was increased with the developing zoeal stage. The survival rate until the Z5 stage was higher when Artemia were supplied from the Z3 stage (P<0.05). Mortality caused by cannibalism was observed either when Artemia feeding was delayed or when fed at a low density (0.5 ind./mL). In contrast, feeding Artemia from the early zoeal stage or at a high prey density (4ind./mL) accelerated metamorphosis and caused the acceleration of morphological characteristics, such as a greater chela length, carapace length and setae bearing pleopods at the Z5 stage. The chela length at the Z5 stage decreased proportionally with the delay of Artemia feeding or a decreasing prey density. The chela to carapace length ratio (ChL/CL ratio, %) could be used as an indicator to predict the success of survival to megalops. A ChL/CL ratio over 45% produced a high percentage of megalopal molting failure. Therefore, to avoid cannibalism or excessive feeding in the larval mud crab, Artemia should be given from the Z3 stage at an optimal level of 1.5ind./mL or increasing density according to the zoeal stage.

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The nitrogen budget of polychaete Perinereis nuntia vallata fed on Japanese flounder feces was estimated at 20°C. Worms of 0.2-0.6 g body weight ingested 4.9-25.7mg/individual per day of flounder feces and excreted 1.2-5.5 mg/individual per day (dry weight) of fecal pellets corresponding to 12.2-44.1% (mean 22.4%) of fed flounder feces. Mean ammonia excretion rate of the worms fed the feces was 0.1 mg/individual per day. Based on the ingestion and excretion data and nitrogen content of flounder feces, worm fecal pellets and worm bodies, it was estimated that the worms assimilate 62.8% of nitrogen intake. In 3 weeks rearing with the flounder feces, commercial diets for flounder and polychaetes, and nitrogen assimilation rates for the worms were 46.3%, 47.4% and 53.7%, respectively. From these results, about half of the nitrogen ingested by the worms is converted into worm body tissue.

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A new cohort of the Manila clam Ruditapes philippinarum was established in Sanbanse (the shallow coastal area of Tokyo Bay) during October-November 1998. Growth of clams within this cohort was monitored monthly or bimonthly at five stations along an inshore-offshore transect in Sanbanse through November 1999. Mean shell length was about 0.4 mm in November 1998 and increased gradually to about 1 mm in March 1999. The clams then grew rapidly during the warm season and mean shell length reached 25-29 mm in November 1999 at each station. Comparisons of growth among stations in terms of shell length and soft-body dry weight suggest that the growth of R. philippinarum was faster at offshore than inshore stations during warm periods.

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We amplified three kinds of cDNA fragments encoding stress proteins from the rotifer Brachionus plicatilis by polymerase chain reaction in order to investigate the molecular mechanisms underlying mediation of its lifespan. The stress proteins were heat shock protein 70 (HSP70), glucose regulated protein 94 (GRP94) and ubiquitin-conjugating enzyme, which have been suggested to extend the lifespans of fruitfly and yeast. The isolated clones consisted of 579, 776 and 257 bp in the above order, respectively, and their deduced amino acid sequences showed 81, 59 and 48% identities, respectively, with corresponding sequences from the nematode Caenorhabditis elegans. As rotifers in the stationary phase can extend their lifespan we subsequently performed northern blot analysis on rotifers both in the exponential and stationary phases. The mRNA levels of HSP70 and GRP94 in the exponential growth phase were 2.5 and 1.6 times higher, respectively, than those in the stationary phase, whereas those of ubiquitin-conjugating enzyme did not differ between rotifers in the two phases. These results suggest that stress proteins are not directly responsible for the extension of rotifer lifespan, but another factor (s) is possibly involved. Our results, however, demonstrated that these genes are useful as molecular markers for monitoring the population growth of rotifer.

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We investigated the protective effects of a fermented vegetable product (FVP) against the hemolysis and lipid peroxidation of Japanese flounder (Paralichthys olivaceus) erythrocytes in vitro. 2, 2'-Azobis (2-aminopropane) dihydrochloride (AAPH) and tert-butyl hydroperoxide were used as model compounds that cause oxidative stress through the generation of free radicals and induction of lipid peroxidation, respectively, in erythrocytes. The hemolysis and lipid peroxidation of erythrocytes induced with 125mM AAPH and 25mM tert-butyl hydroperoxide, respectively, were suppressed by the administration of 1-2mg/mL FVP (P<0.01). These results suggested that the FVP reduces oxidative stress in fish and may be useful for the rearing of cultured fish.

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In the present study, in order to enable effective utilization of scallop shells, the in vitro activities of components that were extracted from scallop shells were investigated. In particular, the scallop shell's useful ability to protect skin was evaluated. The following were found: (i) scallop shell extract inhibited generation of the superoxide anion, which was generated by xanthine and xanthine oxidase; (ii) when the scallop shell extract was supplied to culture medium for skin fibroblast cells, the cell growth rate was increased; and (iii) the scallop shell extract showed strong inhibitory activities for elastase. In the present paper, we describe the possibility of the effective utilization of scallop shells as cosmetics.

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Chub mackerel from the Bungo Channel, Oita Prefecture, Japan, receives a high market price. This high price is due to the tough texture that is not present in chub mackerel sold in public markets. The purpose of the present study was to resolve the effect of an inhabited sea area (the area where the a fish population lives: Bungo Channel or Kumanonada sea in the present paper) on meat firmness and the post-mortem change in chub mackerel during chilled storage. Chub mackerel were obtained alive from the Kumanonada sea in Mie Prefecture, Japan, in March 2000. They were killed instantly by decapitation as were those collected from the Bungo Channel in Oita Prefecture. Dorsal muscle was collected every 4h and measurements of its breaking strength and histological observation of its structure were carried out. The breaking strength of chub mackerel from the Kumanonada sea was lower than that of the chub mackerel from the Bungo Channel. In contrast, the softening rate of meat was almost the same after 16-20 h storage. Using light microscopy, separation between muscle cells was observed from 4h after death, suggesting weakening of the endomysium. Additionally, transmission electron microscopy showed that collagen fibrils existing near muscle cells tended to disintegrate predominantly. According to these results, it was clarified that chub mackerel from the Bungo Channel has tougher meat than that from the Kumanonada sea, and the difference in meat texture was speculated to be due to the difference in exercise intensity between these two sea areas.

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Tuna cooking juice containing approximately 4% solubilized proteins was collected and hydrolyzed by two commercial enzymes, Orientase (O-R) and Protease XIII (A-O). To separate and concentrate the protein hydrolysate, ultrafiltration (UF) and reverse osmosis (RO) were used. Results indicated that the active enzyme A-O displayed greater quantities V_max/K_m than O-R. After 5h of incubation at 50°C with a ratio of substrate: enzyme=25: 1 v/v the degree of protein hydrolysis in tuna cooking juice was approximately 32%, and total free amino acid contents of the cooking juice hydrolysates were 6.80 (O-R) and 8.33 (A-O) mmol/100 mL. After UF via an AF-30-1812-T cartridge equipped with a molecular weight cut-off (MWCO) of 10 kDa, the enzyme active residues in retentate were 20.59 and 19.89%, and the total free and essential amino acids of the permeate were 7.01, 3.75 and 6.77, 3.27 mmol/100 mL for O-R and A-O, respectively. The hydrolysates mainly consisted of peptides less than 2kDa according to gel filtration. Sensory evaluation of concentrated hydrolysates through RO confirmed that aroma, umami taste and overall quality of the hydrolysates were suitable as a condiment.

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Three L-rhamnose-binding lectins, named CSL1, CSL2, and CSL3, were isolated from eggs of chum salmon (Oncorhynchus keta) by affinity chromatography and reversed-phase high-performance liquid chromatography. The yields of CSL1, CSL2, and CSL3 from 2kg eggs were 2.7, 1.7 and 29.6 mg, respectively. The subunit of CSL1 was composed of 286 amino acid residues with three tandemly repeated domains, while the subunits of both CSL2 and CSL3 were composed of 195 amino acid residues with two tandemly repeated domains. The amino acid sequence homologies among CSL showed 42-52% identities. CSL showed 94-97% sequence identities to three corresponding L-rhamnose-binding lectins, named STL1, STL2 and STL3, from steelhead trout (Oncorhynchus mykiss) eggs. Each CSL showed different hemagglutinating activities against rabbit and human erythrocytes. The lectins also showed different inhibition patterns in the hemagglutination towards rabbit erythrocytes by lipopolysaccharides from Gram-negative bacteria including a fish pathogen, Aeromonas salmonicida. CSL agglutinated Escherichia coli and Bacillus subtilis. These results indicate that CSL could be useful biochemical tools for recognizing specific molecules either in soluble forms or on cell surfaces.

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A trypsin inhibitor was purified from the egg of skipjack tuna Katsuwonus pelamis by ammonium sulfate precipitation, gel filtration on a Sephadex G-100 column, ion-exchange chromatography on diethylaminoethyl-Sephacel, rechromatography on a Sephadex G-100 column and reversed-phase high-performance liquid chromatography on a C₁₈ column. The molecular mass of the purified trypsin inhibitor was approximately 78 kDa as estimated by gel filtration and 39 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis. The purified trypsin inhibitor was stable in the pH range from 4.0 to 10.0 and at temperatures below 40°C. The purified inhibitor was rich in Gly, Glu, Ser, Asp and Lys, but poor in Cys, Tyr, Phe, Ile and His. In addition, the activity of the purified trypsin inhibitor was increased in the presence of metal ions such as K⁺, Na⁺, Mg²⁺ and Ca²⁺.

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Nile tilapia were exposed to severe hypoxic conditions. The respiratory frequency (RF) of the fish reached a maximum level at approximately 10 mmHg PO₂ and decreased gradually thereafter. The fish sank to the bottom and subsequently their respiration began to stop. The hypoxic conditions increased the hematocrit value. As RF decreased, plasma cortisol and glucose levels climbed drastically. Just before respiratory arrest, the ATP and the total adenylate (TA) concentrations were significantly depressed in the liver, kidney and ordinary muscle but not in the heart or gill. As RF decreased, ATP and TA in both the liver and kidney decreased rapidly. Cytochrome oxidase activity increased significantly in the brain, heart, gill and ordinary muscle until respiratory frequency peaked. However, from the peak RF, this activity decreased in the liver and kidney. Lactic acid levels in both ordinary muscle and liver increased markedly when the fish sank. These results indicate that the stress response to hypoxic conditions is induced markedly with the decreased RF. It is also suggested that the decreased energy status in the liver and kidney of tilapia occurs at the same stage as the metabolic depression in the whole body, without increases in the aerobic and anaerobic metabolisms.

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