The purpose of the present paper was to examine the size relationship between the juvenile stone flounder Platichthys bicoloratus and the bivalve Nuttallia olivacea, the siphon of which is important prey for the juvenile flounder. Juvenile stone flounder feed mainly on tips of the inhalant siphon of the bivalve. The maximum width of siphon tips in the stomach contents of age-0 fish could not reach that of age-1 fish, although the siphons in age-0 fish became larger as they grew. This size discrepancy indicated a limitation in the size of bivalves available to juveniles. The proportion of total cropping frequency for the siphon of the bivalve by juveniles was estimated according to the bivalve size class. Most bivalves that had cropped siphon tips ranged from 5 to 30 mm in shell length. The total cropping frequency per bivalve was particularly intense on bivalves of 10-25 mm shell length in spite of their small proportion of 24.9% of the total. This frequency intensity indicated that the size of bivalves with cropped siphons by juvenile stone flounder might depend on the ability of juveniles rather than the size composition of the bivalves.

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Effects of hypoxia on the principal prey and growth of flathead flounder Hippoglossoides dubius were studied in Funka Bay. Of the three dominant year-classes that occurred in recent years, the 1995 year-class was small in total length at age≥3 and low in condition factor at age≥2. Ophiuroids (almost Ophiura sarsi), which were the dominant prey in the 1980s, were of little importance and instead, crustaceans such as mysids, natant decapods and pelagic amphipods, bivalves and fish were important prey items for H. dubius in 2000-2001. In addition, the feeding intensity of H. dubius in 2000-2001 was lower than that in the 1980s. These facts are closely related to a reduction of prey abundance, particularly ophiuroids. It seems that the hypoxia that occurred in the central part of the bay during the summer and autumn of 1995-1997 caused the poor food supply and low growth rate at ages 2-4 of the 1995 year-class.

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The purpose of the present paper was to reveal the migration pattern of shirauo Salangichthys microdon Bleeker, in the Ishikari River system and its adjacent coastal sea areas. To this end, the strontium-calcium (Sr:Ca) ratios of the otoliths of 98 individuals were examined. Profiles of the Sr:Ca ratios during the life history after hatching in the estuary of the Ishikari River fell into two different patterns. One was characterized by a high (slightly fluctuating) Sr:Ca ratio throughout the life history; the other was characterized by an initial high Sr:Ca ratio followed by a lower value later in life. The former pattern was considered to be that of estuary-sea residents and the latter that of upstream migrants. Most upstream migrants migrated upstream once, although several did it twice. Back-calculated body lengths during the upstream and seaward migrations of the upstream migrants varied widely from 6.4 mm to 39.8 mm and from 13.5 mm to 79.4 mm, respectively. These findings suggest that estuary-sea residents and upstream migrants co-occurred spatiotemporally, and suggest a wide variation in migration patterns in the shirauo's life history.

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Fishing experiments were carried out in Yeongil Bay, Korea using an encircling gill net with four different mesh sizes and two different hanging ratios to measure the mesh selectivity for gizzard shad Konosirus punctatus. Twenty-six trials were conducted giving a total catch of 485 gizzard shad. The ‘share each length's catch total’ (SELECT) analysis with maximum likelihood method was used to fit the different functional models, the normal, lognormal, and bi-normal model, for a selectivity curve to the catch data. In addition, two cases in which the relative fishing intensity was either estimated or fixed were compared. The bi-normal model with the fixed relative fishing intensity was found to fit the data best. For the selectivity curve on the bi-normal model, the relative length (the ratio of fish length to mesh size) with the maximum efficiency was obtained as 3.70. From this, the focal fish lengths in the commercial encircling gill net, with mesh size ranging from 5.0 to 6.0 cm, were inferred to be 18.5-22.2 cm.

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Among all of the decapods that have been studied, only one kind of gland, the androgenic gland, has been found attached to the subterminal ejaculatory region of the vas deferens. In the present study of the red swamp crayfish, it was found that two kinds of gland-like tissues were attached to the subterminal ejaculatory region of the vas deferens. One of the tissues, which is referred to as TIB, is located inside the body cavity, whereas the other tissue, which is referred to as TIC, is inside the coxa. The characteristics of TIB resemble those of the androgenic gland of other malacostracans. The TIC cells, however, are smaller and more basophilic due to the small size and high density of their cytoplasm and nuclei. Moreover, black fibers, similar to the nerve fibers of the ganglion, were detected among the TIC cells after Bodian's stain. The conclusion is that TIC may be a newly found male sexual characteristic and further research is needed to confirm its functions.

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The purpose of the present paper was to examine the genetic population structure of Buccinum tsubai in the Japan Sea. Mitochondrial 16SrRNA gene sequence analysis was performed with specimens from various stations in the Japan Sea. Mitochondrial lineages were clearly patterned geographically in four separate areas: the Hokkaido area, the Yamagata-Toyama area, the Yamato Bank area, and the San'in area. The main distribution depth range of B. tsubai is between 200 m and 1000 m isodepths, and the horizontal distance between the 200 m and 1000 m isodepth lines represents the specific spatial scale of the habitat (SSSH). These four areas were separated either by the complete discontinuity of the SSSH area or by its narrow spatial extension. Genetic distances between the main haplotypes of each area were calculated as Jukes-Cantor distances, the value of which ranged between 0.012 and 0.017. This value seemed to be unrelated to the geographic distance. There was no tendency for clustering according to depth. In future, the morphological characters of the four lineages of B. tsubai should be compared in detail in order to elucidate significant genetic differences among them.

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The feeding habits of albacore Thunnus alalunga (fork length: 48.9-76.2 cm, n=132) were examined from late spring to early autumn in relation to its northward migration in the transition region between the subtropical and subarctic fronts in the central North Pacific. Samples were collected at night using surface gill nets or during daytime pole-and-line surveys in 2001 and 2002. During May and June, albacore fed mainly on Japanese anchovy Engraulis japonicus, which accounted for 27.2%, 67.0%, and 45.5% of the total stomach contents by number (Cn), wet weight (WW), and frequency of occurrence (F), respectively, and secondarily on the subarctic gonatid squid Gonatopsis borealis (Cn, 15.8%; WW, 10.8%; F, 28.8%). From July to September, albacore continued to depend on Japanese anchovy (Cn, 48.2-52.8%; WW, 79.9-95.2%; F, 27.8-85.4%). These results corresponded well with the remarkable rebound of the Japanese anchovy stock since the 1990s. Gonatopsis borealis, the main squid prey from May to June, almost disappeared from the stomachs of albacore from July to September, probably due to the northward migration of this squid to subarctic waters in summer. The feeding impact of albacore on the Japanese anchovy stock in the transition region was conservatively estimated to be from 1400 to 2100 tons per day from late spring to early autumn.

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Two experiments were conducted to evaluate feed quality and body phosphorus (P) and nitrogen (N) retention by rainbow trout fed test diets with low amounts of fish meal content and alternative low-P protein sources. A fish meal (FM) diet was used as control. Fish weighing 2.0 g and 134.7 g on average were reared with the experimental diets for 30 weeks and 15 weeks, respectively. The experimental diets had a good growth rate and feed utilization. In the first experiment the P retention was higher in the group of fish fed test diets (56 and 69%) compared to the FM-based diet (36%); N retention rates were similar for all groups. In the second experiment, fish were fed the test diet that had the best P retention in the earlier experiment. The P retention was lower than the values obtained in the first experiment (smaller fish), but still higher in the group of fish fed the low FM diet in comparison with the control group (36.0% and 22.2%, respectively). This represents a P loading into the water of 5.9 kg/t and 12.8 kg/t production for the test and the control diet, respectively. Therefore, low-P-loading diets for rainbow trout can be developed through the appropriate combination of alternative protein sources.

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This study examines the toxicity of three dispersants and heavy fuel oil to a marine fish, red sea bream (Pagrus major). Also studied was the toxicity of a mixture of heavy fuel oil and the least toxic of the three dispersants. The 24-h LC50 of all three dispersants were at least 1500 mg/L; these dispersants appeared relatively less toxic to marine fish than others studied in the past. The mean lethal oil concentration of the water-accommodated oil fraction was 325 μg/L. Mixtures of oil and dispersant were more toxic than dispersant or oil alone. Large amounts of dispersant decreased the toxicity of the mixture for the marine fish. Use of a dispersant-to-oil percentage of 20%, which is recommended by the manufacturer because of its efficiency in oil emulsification and dispersion, yielded higher 24-h oil concentrations and resulted in a higher mortality rate than did the use of higher percentages of dispersant. The application of dispersant to oil in coastal areas, especially with higher activities of fisheries and aquaculture like Japan, must be considered carefully in the context of the benefits versus environmental cost.

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To investigate the mechanisms of calcium carbonate formation of teleost otoliths, using the double labeling technique of ⁴⁵Ca²⁺ and H¹⁴CO₃^-, the effects of tissue elimination, a metabolic inhibitor (cyanide) and acidity on the incorporation of calcium and inorganic carbon into endolymph and otoliths were examined. Incorporation of calcium and inorganic carbon into otoliths was decreased to 36% and 45%, respectively, by removing the sacculus. In contrast, cyanide inhibited incorporation of inorganic carbon and calcium into endolymph and otoliths in a concentration-dependent manner. Acidity in the incubation medium decreased the accumulation of inorganic carbon within the endolymph and otoliths. Calcium incorporation was not affected by variation of pH in the medium. These results indicate that calcium and bicarbonate are actively transported to endolymph via the sacculus, and it is suggested that the transport is involved in the oversaturation state for calcium carbonate in the endolymph and regulates subsequent calcification of otoliths.

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Shell growth and age determination of Corbicula japonica were investigated from samples collected monthly at two sites and field growth experiments from November 1999 to November 2000 in Lake Shinji. Both specimens did not grow in winter when the water temperature was below 11°C, but grew rapidly from spring to early summer when the water temperature rose from 15°C to 30°C, before further growing slowly from summer to autumn after the main breeding period. The monthly marginal growth distribution of the samples indicated that the concentric groove on the shell surface was an annual growth ring. It is concluded that the age of C. japonica in the i-ring group when the shell growth resumed in spring was i+0.5 (i=1, 2, …), because the first true growth ring was formed at 0-2 mm in shell length, which then faded with growth. Lee's phenomenon was observed in the age determination results, and thus the growth coefficient and the asymptotic shell length of the von Bertalanffy growth equation were not estimated from the samples. However, these parameters were estimated from the annual shell length increments of the experimental individuals, which were 0.331/year and 28.2 mm, respectively.

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A feeding trial was conducted with two newly developed microparticle diets (MD-Q and MD-V), which differed in the mixing ratios of two types of casein hydrolysates (C700 and C800) as the protein source. The ratio of C700 and C800 were adjusted to 7:3 in MD-Q and 6:4 in MD-V, respectively. Japanese flounder larvae were fed from 7 days after hatching (d. a. h.) to 32 d. a. h. on live food (LF), MD solely, MD+1/3 LF (either of the MD and one-third quantity of the live food) and 1/3 LF (one-third quantity of live food alone), respectively. They were then switched to Kyowa diet B from 33 d. a. h. until 40 d. a. h. The larvae fed on MD-Q exclusively had a higher survival rate (36.4%) than those fed on MD-V (24.2%) by 22 d. a. h. Unfed larvae could survive up to only 12 d. a. h. Larvae fed on MD-Q+1/3 LF also had a significantly higher survival rate (34.2%) than those on MD-V+1/3 LF (16.3%) and 1/3 LF solely (15.4%) at 32 d. a. h. These results suggest the potential of MD-Q as a part replacement for LF from the early developmental stage in the seed production of Japanese flounder.

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The search for dinucleotide repeat microsatellites within scaffolds 1-25 of genome database JGI Fugu v3.0 for the pufferfish Takifugu rubripes revealed that 80% of microsatellite loci consisted of five to 13-fold repeats with locus-specific differences in density. Eleven out of 15 microsatellite loci isolated from the database with which genotyping using wild pufferfish was successfully performed showed polymorphism; that is, the means of the number of alleles and expected and observed heterozygosities at these 11 loci were 21.8, 0.915 and 0.829, respectively. It was confirmed that eight out of the 11 polymorphic loci were inherited through the Mendelian law and one pair of microsatellite loci derived from the same scaffold was linked. These results demonstrated that these loci are useful for constructing a linkage map in the pufferfish as DNA markers.

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One of the biggest and long-standing difficulties in investigation of larval ecology in the field is species-level identification. In the present study, we developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis based on the large subunit (LSU) rRNA gene (rDNA) D1/D2/D3 region for identification of multiple species of bivalve larvae using 14 species of bivalve collected from Maizuru Bay. The LSU rDNA D1/D2/D3 region of all analyzed species could be amplified by PCR using bvD1f/bvD3r primers, and RFLP analysis by HaeIII digestion on the PCR products showed species-specific fragment patterns. Furthermore, this analysis applied to single bivalve larvae in Maizuru Bay revealed efficient amplification of the target region and the species-specific pattern from 80% of the larvae, 75% of which showed a pattern that matched a certain pattern of the adult bivalves. In addition, the analysis of inter- and intraspecies variation of the LSU rDNA D1/D2/D3 region using the sequence data of the genus Crassostrea from the DDBJ database showed high applicability of this RFLP analysis on closely related species. Because of the wide applicability and technical simplicity, this method can become the standard for the identification of bivalve larvae species once the sequence data of the LSU rDNA D1/D2/D3 region of many bivalve species have been accumulated.

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In order to assess a daily change of genetic variability during spawning season, hatched larvae of red sea bream sampled on different dates were assayed by polymorphic markers such as microsatellite DNA (msDNA) and mitochondria DNA (mtDNA) control region. Based on the microsatellite loci, the average number of alleles per locus ranged between 13.7 and 18.3. The expected heterozygosities ranged between 0.843 and 0.919. A total of 23 mtDNA haplotypes were detected via digestion of mtDNA D-loop sequences with five endonucleases: Taq l, Alu l, Mbo l, Rsa l and Hinf l. Significant fluctuation of genetic variability during spawning season was detected by both types of DNA markers. It was suggested that the genetic variability was maintained by pooling the seed fish collected on different spawning dates in a hatchery.

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Green fluorescent protein (GFP) and red fluorescent protein (RFP) genes regulated by the medaka skeletal muscle actin promoter were microinjected into fertilized d-rR medaka eggs to establish transgenic medaka lines. Intense fluorescence was detected in skeletal muscle. During development, GFP and RFP became detectable in anterior somites at the 12- and 30-somete stages, respectively. After hatching, intense fluorescence in skeletal muscle enabled individual fish to be identified under normal lighting without fluorescent microscopy. Fluorescence was also observed in the gills and esophagus of the adult fish. These data indicated that medaka lines are convenient not only for the study of skeletal muscle but also for the identification of cells or individuals in various studies.

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The present study was conducted to investigate the effect of eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) on the survival and the occurrence of molting failure to megalops of mud crab Scylla serrata larvae fed enriched Artemia. Survival rate, intermolt period, carapace width at the first crab stage, ratio of chela to carapace length at the fifth zoeal stage and the occurrence of molting failure to megalops were observed. Mud crab larvae were reared in 1-L plastic beakers and fed with Artemia enriched at five different levels of EPA (0.31% to 1.36% EPA; referred to as E25, E50, E75 and E100) or four different levels of DHA (0.75-0.95% EPA and 0.49-1.38% DHA; referred to as D25, D50, D75 and D100). As a negative control, larvae were fed Artemia enriched with oleic acid (OA). Mud crab larvae fed Artemia containing low (0.41-0.45% EPA and trace DHA) or high (1.36% and 0.95% EPA and 0.16% and 1.38% DHA) amounts of EPA and DHA showed a significantly lower survival rate and prolonged intermolt period (P<0.05). Moreover, a high frequency of molting failure to the megalops stage (34 and 33%) occurs coincident with a high chela to carapace length ratio (43 and 44%) in mud crab larvae fed high amounts of EPA and DHA (E100 and D100), suggesting that both of these treatments contain EPA and DHA in excess. These results indicate that during Artemia feeding, EPA and DHA content should be adjusted to 0.71-0.87% and 0.49-0.72% for maintaining a high survival, accelerating the intermolt period, and producing larger carapace width in the first crab stage.

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Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas. The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.

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Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7-98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.

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The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus, and six strains of Bifidobacterium were evaluated. In vitro, the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C. perfringens. The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.

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We have cloned a cDNA encoding the matrix metalloproteinase (MMP) from oyster Crassostrea gigas. The clone contains a 1797 bp open reading frame encoding a protein of 599 amino acids. Oyster MMP (Cg-MMP) has a transmembrane domain at its C-terminal and a furin/prohormone convertase cleavage site at the end of a propeptide domain, which are commonly observed in membrane-type MMPs (MT-MMPs). This suggests that Cg-MMP is an MT-MMP. The deduced amino acid sequence of oyster MMP shares approximately 30% identity with human MT4-MMP and MMPs from fruit fly and hydra. Cg-MMP mRNA was detected in the gill, mantle and adductor muscle, and more intense signals in the northern blot analysis were recognized in the gill and adductor muscle. Similar tissue distribution was observed for tissue inhibitor of metalloproteinase (Cg-TIMP) in oyster. In response to hypoxic stress, the abundance of Cg-MMP mRNA was elevated in the gill, while that of Cg-TIMP mRNA remained almost constant. These findings suggest that promotion of collagen metabolism may be implicated in the hypoxic adaptation in oyster.

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β-galactosidase of the intestine of Tilapia nilotica was purified by ammonium sulfate precipitation, followed by PAPTG-Sepharose 4B affinity chromatography, ethylenediamineetetraacetic acid ion-exchange chromatography, polyexchanger PBE 94 chromatofocusing, and Sephadex G-100 gel filtration. β-galactosidase was found to be a single band when examined by poly-acrylamide gel electrophoresis. The purifications of β-galactosidase were 27-fold from the crude extract. β-galactosidase showed optimum activity at pH 5.0 at 40°C, and was specifically found to be able to hydrolyze p-nitrophenyl β-galactopyranoside. It degrades galactan and agarose, and produces galactose. β-galactosidase was strongly inhibited by Hg²⁺ and PCMB. β-galactosidase is considered to be secreted by the upper and middle parts of the intestine and most of the activity was detected in the intestinal juice.

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Arsenobetaine, the major arsenic compound in marine animals, is substantially non-toxic. It is, however, possible that arsenobetaine undergoes bacterial transformation during manufacturing of fermented fishery products. In the present study, therefore, three types of fish sauce were examined for arsenic concentrations and species compared with those in the raw materials (sardine, Japanese sandfish and Japanese common squid). Arsenic concentrations of the three types of fish sauce were almost equivalent to those in their raw materials, suggesting no accumulation of arsenic during fermentation. Arsenic speciation was performed by a combination of cation-exchange liquid chromatography (LC) and electrospray ionization (ESI)-single quadrupole mass spectrometry (MS). Optimal conditions of LC/ESI-MS were established to analyze seven arsenic compounds (arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, trimethylarsine oxide, arsenocholine and tetramethylarsonium ion) found in biological samples. When analyzed by LC/ESI-MS, the major arsenic compound in the raw materials was arsenobetaine as expected, while not arsenobetaine but dimethylarsinic acid was identified as the major arsenic compound in the three types of fish sauce. These results suggest that arsenobetaine in the raw materials is converted to dimethylarsinic acid but not to arsenate with high toxicity by bacterial actions during manufacturing of fish sauce.

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In this paper, the detection of type I collagen degradation during the softening phenomenon of yellowtail muscle, was examined. Acid soluble collagen was isolated from dorsal ordinary muscle at death and after 24-h chilled storage. In the abundant ratio of subunit components, an increase in β12 chain (5.4 points) and a decrease in components with molecular weights larger than γ chain (7.0 points) after 24-h chilled storage, was found. Type I collagen was detected in the alkali-soluble fraction by SDS-PAGE. Its amount calculated from hydroxyproline contents in alkali-soluble fraction was increased from 0.097 mg/g muscle to 0.155 mg/g muscle during 24-h storage. The increased alkali-soluble collagen (0.058 mg/g muscle) was about 1.4% of whole collagen. These results suggest that a slight decomposition of type I collagen of yellowtail muscle may occur and subsequently becomes alkali-soluble corresponding to postmortem softening.

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