The existence and distribution patterns of microsatellites in the tiger puffer genome were evaluated utilizing DNA databank entries. Microsatellite regions amounted to only 0.96% of the 22.5 million bp included in the tiger puffer database. Di-nucleotide repeats were most common (90.1% of all microsatellites). Twelve microsatellite loci were isolated from the entries. Analysis of genetic polymorphism at these loci in the wild tiger puffer (n=50) revealed a high degree of length polymorphisms, in which the number of alleles per locus ranged from 11 to 34. The expected heterozygosity was between 0.869 and 0.953. Mendelian inheritance was demonstrated in all but two loci (which seemed to include null alleles) by analysis of genotype ratios in the offspring of a single captive. Probability of identity of all loci ranged from 0.007 to 0.028. Band sharing index was low (0.119) in wild tiger puffer and high in the offspring of the captive (0.525). These results indicate the usefulness of the microsatellite markers identified for future population analyzes in tiger puffer.

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In order to establish criteria with which to evaluate the nutritional condition of wild first-feeding larvae of Japanese anchovy Engraulis japonicus, the whole body RNA:DNA ratio and daily protein growth rate of reared larvae were individually determined. Anchovy larvae were reared for up to 9 days after the first-feeding day at 20°C and 25°C under various feeding conditions. Percent survival of the group starved for 2 days after the first-feeding day was extremely lower than that of those fed from the first-feeding day and 1 day starved groups at the two temperatures, even if they were supplied with food thereafter. Therefore it was concluded that the point of no return (PNR) was between 1 and 2 days after the first-feeding day within this temperature range. The RNA:DNA ratio of the PNR criterion was determined. Also, a 0% growth formula was established representing the relationship between the daily growth rate in protein, RNA:DNA ratio and temperature for first-feeding larvae.

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The oriental river prawn and the striped prawn were compared to identify characteristics of the external shape of their pleopods in terms of swimming ability. All data obtained were compared using mean values. The aspect ratio AR (length of pleopod²/area of pleopod) of the striped prawn was larger than that of the oriental river prawn. There was no difference between male and female prawns of the same species. Sexual dimorphism was found in the R ratio (ratio of seta length to pleopod length) of the striped prawn but not in the oriental river prawn. During paddling, the drag on the pleopods at the recovery stroke was relatively larger in the oriental river prawn than in that of the striped prawn. These results suggest that hydraulic efficiency during one paddling of the pleopod is greater for the striped prawn than for the oriental river prawn.

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The distribution of snow crab, Chionoecetes spp., larvae was studied by horizontal plankton net tows using a multiple opening/closing nets and environmental sensing system from the surface to the bottom off Wakasa Bay (approx. 500 m) in the Sea of Japan. The hatching season was concluded to occur between early February and late April with a peak occurring in March. First zoeae occurred most densely in March, second zoeae in April, and megalopas between April and June within the Tsushima Warm Current (0-200 m). The temperature of the strata at which more than 88% of each stage of larvae were distributed varied from 8.4 to 15.0°C. As the larval stage progressed, they gradually moved to deeper water strata. In and after June, megalopas observed in the premolt stage started a downward migration beyond the thermocline to the cold bottom water (water proper to the Sea of Japan, <5°C all year round). Therefore, the settlement of megalopas and metamorphosis to the first benthic stage was concluded to occur in June. The second zoeae and megalopas underwent diurnal vertical migrations. The durations of the first and second zoeal stages and the megalopal stage were estimated at approximately 20 days, 20 days, and 60 days, respectively.

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A new myxozoan parasite was found from net-pen-cultured Chinese sea bass, Lateolabrax sp. in Japan. Diseased fish exhibited chronic mortalities with clinical signs of anemic gills and enlarged bulbus arteriosus. Irregular-shaped plasmodia developed in the bulbus arteriosus, with released mature spores accumulating in the lumen. Massive influx of spores into the gills caused local occlusion and congestion of gill capillaries, lamellar hypertrophy, and degeneration of the gill epithelium. Spores were ovoid with two equal caudal appendages. Average spore size was 10.7 μm in length, 7.5 μm in width and 6.2 μm in thickness. Average caudal appendage length was 37.7 μm. A new species name, Henneguya lateolabracis, is proposed as the causative agent of cardiac henneguyosis in Chinese sea bass.

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Feeding habits of the two asteroids dominant in Ise Bay, Luidia quinaria and Astropecten scoparius, were examined as regards their temporal and spatial distributions and also the oxygen-poor water in the bay, based on megabenthos samples collected in the bay from November 1996 to November 1997. Luidia quinaria was distributed widely in the whole part of the bay while A. scoparius was mainly found in the southern part around the bay mouth area. Analysis of the stomach contents of these asteroids demonstrated that L. quinaria preferred to ingest echinoderm, particularly an ophiuroid, Ophiura kinbergi, that occupied approximately 60% of the percentage total food items; while the stomach contents of A. scoparius were dominated by molluscs, a minute gastropod Voorwindia paludinoides and a bivalve Alvenius ojianus, which occupied approximately 59% and 33% of the percentage of total food items, respectively. The differences in the feeding habits of these asteroids may be due to their distribution pattern, which may be determined by the differences in sediment characteristics. Distribution and feeding habits of L. quinaria in summer may be affected by the occurrence of oxygen-poor water.

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Surveys of the estrogenic activity in coastal areas of Japan were performed by using two serum vitellogenins (Vg-530 and Vg-320) of male Japanese common goby Acanthogobius flavimanus as biomarkers. To evaluate the relationship between the Vg concentrations in the serum of male fish and the concentrations of environmental estrogens in seawater, 3 week exposure experiments were performed using estradiol (E₂), nonylphenol (NP) and bisphenol A (BPA). From the results of the E₂ exposure, mean concentrations of the Vg and the ratio of Vg-positive fish increased from actual concentration at 10.5 ng/L E₂ treatment. Exposure of NP and BPA showed that the 19.0 μg/L NP treatment induced the Vg but 133.7 μg/L BPA did not. As for the survey of the estrogenic activity in coastal areas, the mean Vg concentrations and ratio of Vg-positive fish in some urban areas were similar to values of the 10.5 ng/L E₂ treatment. Although E₂ and NP were detected in the seawater from urban areas, the concentrations were concluded as not sufficient to independently induce the Vg. Considering these results, it is concluded that the total estrogenic activity in some urban areas is dependent not only on E₂ but synergistically with other environmental estrogens.

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To enhance heterotrophic activities for decomposing organic matter in a eutrophic bottom environment, two promising bacterial strains isolated from the study site, named Enterobacter sp. 9410-O and Pseudomonas sp. W-4 were introduced by absorbance onto porous substrates in the sediment-bottom water complex system. Strain 9410-O grew well at >20°C, considered as mesophilic, while the other strain W-4 grew at a wide rage of temperatures from 5°C to 30°C and was psychrotolerant. Addition of 9410-O stimulated net regeneration of inorganic nitrogen (DIN) and inorganic phosphorus (DIP) from the sediment by approximately four-fivefold at a high temperature of 26°C, but some stimulation was noticed at the lower temperature of 13°C. However, strain W-4 stimulated DIN and DIP net regeneration both at low (13°C; approx. threefold) and high temperatures (26°C; approx. four-fivefold). These results suggest that the application of such effective bacteria as described here would be promising for the stimulation of self-purification in the field of eutrophic bottom environments.

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Body color intensity of red-colored koi carp Cyprinus carpio and goldfish Carassius auratus auratus varieties were measured to evaluate the effect of paprika used as a feed additive. Digital photos of the experimental fish were processed and analyzed by using special software. The red, green and blue (RGB) values of images were recorded and grayscale values of R, G and B were analyzed. The RGB values seem to play different roles in the development of the visible ‘redness’ of the two species. In most cases the B values decreased continuously during the administration of the paprika as a carotenoid feed additive, which seemed to have no effect on this process. The G values remained unchanged or decreased slightly as redness increased due to paprika feeding. The R values had a tendency to increase due to paprika feeding but significant differences can be expected only after 4 weeks at the feeding conditions applied here. Both the initial rate of redness and genetic background are thought to influence the rate of red color intensity change, which was observed to be different in the two fish species studied here.

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The Ryukyu-ayu, Plecoglossus altivelis ryukyuensis, is an amphidromous fish that is endemic to Amami-oshima Island in southernmost Japan. Its abundance, however, has been appreciably reduced during the last two decades such that the subspecies is now considered to be endangered. The variation of the mitochondrial DNA control region was investigated among specimens of the extant populations in the eastern and western parts of Amami-oshima Island (Sumiyo Bay and Yakeuchi Bay areas, respectively), using restriction fragment length polymorphism analysis in order to estimate the extent of gene flow between the two areas. Of a total of 165 fish including temporally different samples, four haplotypes were detected and each area possessed the two haplotypes. However, the common haplotypes shared between these areas were not observed, which indicates that recent gene flow has not occurred between these populations. The nucleotide divergence between populations was much higher than the nucleotide diversity within each population, and the neighbor-joining phylogram among haplotypes showed that the haplotypes are associated with their geographic area. These results suggest that the two populations of Ryukyu-ayu on Amami-oshima Island have been historically formed.

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The authors propose a new bycatch reduction device for trawling with a simply constructed TREND (trawl flow regulative ecological friendly netting device). TREND is a cylinder-shaped netting device with a rectangular-shaped mesh. We examined a selective mechanism from the viewpoint of the flow inside and around TREND. A series of model experiments was conducted and the flow speeds were measured at a flume tank. The ratio of flow speed decreased at points close to the netting and increased slightly at other points in all sections. The angles of tuft at the top netting in the rear area were almost twice the value in the front sections. As the exhaustion of flow and reduction of speed occurred simultaneously, these tendencies were remarkable at higher speed. This flow towards the outside of TREND was considered to be acting as a bycatch reduction device for trawling.

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Long-diving marine mammals, such as sperm whales and Baird's beaked whales are frequently missed in line transect sampling even though they are on the trackline. The methods so far developed to estimate the proportion of animals missed on the trackline are complicated or sensitive to model assumptions. A simpler model is developed in the present study. The model requires an estimate of mean surfacing rate that is independent of the line transect survey and does not require any complicated survey design and non-robust parameters, such as scan rate of the binoculars of observers. In addition, it can flexibly take account of systematic heterogeneity using covariates. The abundance is estimated using the Horvitz-Thompson estimator. The simulation test showed that the proposed method is promising.

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Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were fortified at a level of 1.5% in a composed diet. The effects were confirmed in 0-year black sea bream Acanthopagrus schlegeli in terms of lipid metabolism and physiological activity. The EPA group was high in EPA in muscle, liver, intraperitoneal fat body (IPF), eye and brain. The levels of DHA in liver, eye, brain and heart were also high in the EPA group, suggesting that conversion of EPA to DHA occurred in those organs. Fortification of DHA increased the levels of DHA in organs except the eye, but did not affect EPA levels. Both the EPA and DHA groups showed smaller adipocytes or lower levels of lipid content than the control group. The starvation followed by feeding experiment caused marked body weight loss in the control group by consumption of muscle protein and lipids in IPF. Fortifications of EPA and DHA induced less mobilization of muscle protein and IPF lipids as energy. Liver function and resistance to air-dipping were improved by both EPA and DHA fortifications. The present results implied conversion of EPA to DHA in the fish with regard to parameters, such as lipid metabolism and physiological vitality.

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An enzyme biosensor system was developed for rapid and continuous determination of total cholesterol in fish plasma. The system was constructed with an immobilized enzyme membrane, Clark-type oxygen electrode, flow system and personal computer. The enzyme membrane was prepared from cholesterol esterase, cholesterol oxidase, photosensitive polymer and an ultra-thin dialysis membrane. The optimum assay conditions of the sensor system were established as follows: pH 6.8, temperature 25°C and flow rate 0.2 mL/min. The calibration curves were linear in the range of 15-80 mg/dL for yellowtail, 17-42 mg/dL for amberjack and 20-70 mg/dL for rainbow trout. One assay was completed within 5 min and it was possible to determine the sample continuously. The sensor system was applied to the determination of total cholesterol in yellowtail plasma. A good correlation was observed between the values determined by the sensor method and the conventional method (correlation coefficient was 0.90).

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To clarify the annual testicular maturation cycle in the helmet crab Telmessus cheiragonus, seasonal histological changes of the testis and occurrence of grasping pairs and recently copulated females with fresh sperm plugs were investigated. The testis was composed of seminiferous ducts with many testicular lobules. Development of the lobules was divided into six stages (spermatogonia proliferation, first spermatocyte, reduction division, spermatid, spermiogenesis and free sperm stage) based on the spermatogenic phase of the germ cells in each lobule. The mean percentage of the lobules during each stage was calculated every 1-3 months. Developed lobules occurred with a high percentage throughout the year except in June when developing lobules predominated. Grasping pairs and females with fresh sperm plugs were collected during April-July with a peak in May-June. These results suggest that the males have testis with many developing lobules in June when they are active for mating. Newly formed spermatozoa may be stored in the testis and the vas deferens until the next mating season after June, as the spermatozoa may not have an opportunity to fertilize eggs until the next season and there is no sign of spermatozoa absorption in the testis.

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We assessed the ability to discriminate local stocks of walleye pollock Theragra chalcogramma based on data from five elements (Na, K, P, Ca and Sr) using electron probe micro analyzer (EPMA) from the otoliths of 26 adult fish (standard length 339-576 mm) caught at four sites (Rausu, Kushiro, Minami-kayabe and Musashi Bank) around Hokkaido, Japan in 1998 and 1999. To compare the elemental compositions, the otolith formation periods were arranged based on the annual rings to allow analysis of comparative winter periods. Elemental concentrations of Na and K remained at a stable level throughout the otolith development. The pattern of Sr concentration differed for each individual. The 1999 winter samples collected from Kushiro and Minami-kayabe distributed similarly and were discriminated at a near-significant level from samples at Rausu. These results indicate that we might be able to discriminate the local Nemuro and Pacific stocks of walleye pollock spawned around Hokkaido using their otolith elements by comparing portions of otoliths formed in the same year.

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Stress-induced apoptosis was characterized in larval embryos of Japanese flounder using a caspase-3-like activity assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining of whole mounts. Severe stress conditions, such as heat shock by temperature shift from 16°C to 28°C or 29°C for 1 h, and ultraviolet (UV) irradiation at 20-100 mJ/cm², induced extensive apoptosis in embryos. Following the induction of stress, many TUNEL-positive apoptotic cells were found in the yolk sac, heart, and larval fin. Caspase-3-like activity against a fluorogenic substrate, acetyl-Asp-Glu-Val-Asp-α-4-methylcoumaryl-7-amide, increased in a dose-dependent manner during stress-induced apoptosis. In addition, C2-ceramide treatment (5-10 μM) induced a similar apoptosis pattern to stress treatment. These findings indicate that a pro-apoptotic pathway, which is caspase-3 mediated and acts through ceramide signaling, plays a major role in induction of apoptosis under stress conditions.

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Toxicity of puffer fish Lagocephalus lunaris and Lagocephalus spadiuceus landed and sold in Samut Sakorn fish landing in Thailand were studied monthly for one year from August 2000 to August 2001. Lagocephalus spadiceus was not toxic while L. lunaris was toxic, although the toxicity levels varied greatly among the different fish and the organs. Marked seasonal variation was also observed in L. lunaris toxicity. They were toxic for 9 months from March to November and the highest ratio of toxic specimens (100%) and the highest toxic scores in all the organs were found in August. The maximum toxicity found was 148 MU/g in the skin, 243 in the muscle, 813 in the liver, 529 in the ovaries and 2920 in the testes. The gonad was consistently found to have the highest toxicity and the testes were more toxic than the ovaries. High-performane liquid chromatography (HPLC) analyses of toxic specimens showed the presence of tetrodotoxin, 4-epitetrodotoxin and 4, 9-anhydrotetrodotoxin, but not paralytic shellfish toxins. Quantity of tetrodotoxin detected by HPLC well explained the toxicity observed in the mouse bioassay.

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Pacific whiting protein solubility was significantly affected as the pH shifted away from the isoelectric point (pH 5.5). The highest breaking force of gels was measured for fish proteins treated at pH 11, while high deformation values were obtained at pH 2 and 11. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that fish proteins were highly degraded by acid or alkali treatment. High activity of cathepsin B-like enzyme was detected from acid-aided fish proteins. Strong cathepsin L-like activity was found in fish proteins treated at pH 10.5, corresponding well to the lower breaking force and deformation. Disulfide bonds were thought to contribute to the high texture value of fish proteins treated at pH 11.

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L-amino acid oxidase (LAAO) activity, as well as mechanisms of antimicrobial action of aplysianin A, a sea hare-derived 340 kDa homotetrameric protein, was determined. Spectrophotometric and High-performance liquid chromatography analyses of aplysianin A indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. Aplysianin A can specifically catalyze oxidation of basic amino acids (L-arginine and L-lysine), and is the first protein from marine invertebrate animals with LAAO activity. Substrate specificity of aplysianin A is markedly different from that of commonly known LAAO, such as snake venom LAAO, which prefer hydrophobic amino acids. Km value of aplysianin A was the smallest of those for all known LAAO reported. In the presence of catalase, the antibacterial activity of aplysianin A was inhibited as expected, indicating that the antibacterial action of aplysianin A results from hydrogen peroxide production during the reaction with substrates. Interestingly, aplysianin A acted as an antibacterial agent even in the presence of excess catalase. Antibacterial assays in various media suggested that this phenomenon was simply attributed to the consumption of amino acids required for bacterial growth in the media by aplysianin A.

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The non-destructive determinations of the fat content in glazed and thawed bigeye tuna using a portable near infrared (NIR) spectrophotometer were performed. For frozen bigeye tuna, a sufficient and accurate calibration equation could not be obtained using the NIR method under glazed conditions. The most accurate section for the NIR measurement of fat content in bigeye tuna was the upper abdomen section near the anus. The accuracy of the calibration equation increased by removing the glaze. In addition, the accuracy became even higher with thawed tuna. For frozen bigeye tuna, the temperature compensation type calibration equation was efficient for the fat determination after removing the glaze.

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For the purpose of producing fish sauce without any addition of salt, an autolytic technique under pressurization combined with warming was investigated. While rancidity caused by the autolysis could be inhibited by pressurization at higher than 50 MPa combined with warming at 50°C, the effect of pressurization on the autolysis was not observed in the range of 50-250 MPa. The optimum temperature and treatment time for the autolysis under pressurization were 50°C and 48 h, respectively. The extract of anchovy produced under the autolysis conditions under 60 MPa at 50°C for 48 h contained 2.6% total nitrogen, 1.4% formol nitrogen, and 11.6% free amino acids by weight. These contents were higher than those of commercially available fish sauce and soy sauce. The sensory test revealed that saltiness was hardly perceived, and odors like ‘cheesy’ and ‘rancid’ were not as strong as in fish sauce. Thus, this autolytic technique under pressurization combined with warming could be a new method for producing a good-quality autolytic extract similar to fish sauce without any addition of salt in an extremely short period of time.

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We have previously cloned three distinct isoforms of group IB phospholipase A₂ (PLA₂), hepatopancreas DE-1 and DE-2 PLA₂ and gill G-3 PLA₂, from the red sea bream Chrysophrys major and found that they are expressed in a tissue-specific manner in the red sea bream. To understand the function of three group IB PLA₂ isoforms, we constructed a bacterial expression system for DE-1, DE-2 and G-3 PLA₂. The cDNAs encoding for mature PLA₂ were amplified by polymerase chain reaction (PCR) and subcloned in-frame with a glutathione S-transferase (GST) encoded by the vector pGEX-4T-1. The resulting plasmids were used to transform Escherichia coli BL21 (DE3) cells. Three recombinant PLA₂ were expressed as a fusion protein with GST in E. coli cells by induction of Isopropyl-1-thio-β-D-galactopyranoside. The bacterial cells were lyzed with strong alkaline solution and the fusion proteins were recovered as a soluble form. The fusion proteins were purified with affinity chromatography and cleaved by trypsin. Then, the recombinant DE-1 and G-3PLA₂ were purified to near homogeneity by reversed-phase high-performance liquid chromatography (HPLC), and the recombinant DE-2 PLA₂ by ion exchange chromatography and reversed-phase HPLC. Enzyme properties of purified recombinant DE-1, DE-2 and G-3 PLA₂ were found to be essentially the same as those of native PLA₂.

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Fish protein hydrolysate (FPH) was prepared from fish scrap of five marine species by enzymatic treatment for the effective utilization of fish wastes. The major components of the FPH were peptide (82.3-85.8%) with other nitrogenous compounds (11.5-16.3%). Lizard fish Saurida wanieso surimi containing FPH (5.0%, dried weight/wet weight) was stored at -25°C and the suppressive effects of FPH on the decrease in the gel-forming ability, unfrozen water content and Ca-ATPase activity were determined during the frozen storage. FPH maintained a high gel-forming ability and Ca-ATPase activity were determined during the frozen storage. FPH also suppressed the decrease of unfrozen water content of surimi. These results suggest that FPH can be used as a cryoprotectant to suppress the denaturation of muscle protein of lizard fish surimi during frozen storage.

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We studied the potential utilization of rabbit fish (Siganus fuscescens) meat in textured products. We examined the gel-forming ability of rabbit fish caught between July 2001 and August 2002 off the coast of Nagasaki. Fish obtained from approximately July to September (during the spawning season and immediately after), had low gel-forming ability. Fish caught in winter or spring showed high elastic gel formation. Washing of meat with fresh water was found to be important for gel formation. It was concluded that rabbit fish meat has a high gel-forming ability and, therefore, that rabbit fish meat is suitable for textured food production except during the spawning season.

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The glass transition state of Katsuobushi (boiled and smoke-dried bonito) was studied by differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). DSC and DMA data proved Katsuobushi as a glassy material. The glass transition temperature (Tg) measured by DSC was about 33°C in 14.8% moisture. In general, moisture content of Katsuobushi on the market is approximately 12-15% and the Tg value of Katsuobushi containing such moisture was approximately 10-30°C and this was within the room temperature range. Furthermore, the Tg of Katsuobushi showed strong dependence on moisture content, and the Tg value varied from 11 to 165°C with moisture levels from 18.04 to 0%.

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