For development of vaccine against pseudotuberculosis in cultured yellowtail, Seriola quin-queradiata, efficacy of experimental vaccination was investigated.
Three kinds of vaccine used in this study were prepared from formalin-killed pasteurella piscicida cells by the following methods: vaccine I, cells suspended in FREUND'S complete acjuvant; vaccine II, cells suspended in saline; and vaccine III, cells mixed with minced meat of sand launce, Ammodytes personatus, and CMC.
These vaccines were administered by different methods and the respective experimental groups of each method on fish were established as follows: Injection method, fish were immunized with vaccine I by intraperitoneal injection one time (group A). Spray method, fish were sprayed with vaccine II for 5-7 s by a sand blasting gun (group B). Immersion methods, a) fish were immersed in vaccine II for 3 min (group C) and b) fish were immersed in the hypertonic solution for 2 min then in vaccine II for 3 min (group D). Oral methods, fish were fed with a) vaccine III (group E) and b) vaccine III mixed with 1.5% NaHCO₈ (group F). Both methods were administered daily for a week. The artifical challenge with virulent P. piscicida was carried out three weeks after vaccination.
Survival rations for a week after challenge are: 100% for group A and B, 95% (group F), 88% (group E), 73% (group D), and 60% (group C) in comparison with 0% survival in the control. All vaccinations were highly effective. Agglutinating antibody titer in serum determined 3 weeks after vaccination in group A was 1:32-1:128, in group B and F were 1:16≥, in group C, D and E were 1:4≥ and in the control was 1:4>. That of the skin mucus in group B, C and D were 1:32, in group F was 1:4 and in group A, E and the control were 1:4>. This suggests that agglutinating antibody is produced at the tissue stimulated with antigen or transferred to the tissue, or both.

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The cultured seedings of Japanese black abalone Haliotis discus discus were transplanted to a rocky coastal area at Asakawa in Tokushima Prefecture, southern Japan, during May 1975 and 1976. About 100 individuals ranging from 8mm to 18mm in shell length were released at four releasing positions each year. The abalone were released in a cage made of netting (5mm in mesh size) at station 1b, in order to prevent animals from intruding. These abalone were observed under water by skin diving for 7 days in 1975 and 8 days in 1976.
The rate of survival in both large and small tide pools was very low ranging from 0.0% to 4.0% but was 51.6% at station 1b. In the channel, the released abalone were observed under spines of sea urchin Anthocidaris crasspina and the rate of survival was 23% and 28%. About half of the empty shells were broken around the margin, 7.1-55.4% of the empty shells were broken in pieces.
Further experiments on predation were conducted in laboratory tanks, using animals which were collected at the releasing positions. The small crab Gaetice depressus could prey upon young abalone smaller than 15mm in shell length. The shells of abalone killed by this crab were either damaged or not damaged. The crab Charybdis japonica ate those smaller than 27mm in shell length. All of the shells of abalone killed by this crab were broken in pieces.

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The observations and analyses of eye movements on three groupers, Epinephelus moara, Epinephelus sp., and E. septemfasciatus which were resting motionless on the bottom of anaquarium revealed that these fishes move their eyes vertically only with saccadic movement, and the maximum degree of their vertical saccade was measured at 65°, 50°, and 30° for respective species. Four restrained fishes, Cyprinus carpio, Carassius auratus langsdorfi, Sarotherodon mossambicus. and Lepomis machrochirus did not show any vertical pursuit eye movement to vertically moving horizontal stripes in their visual field. The results were considered to suggest that it is difficult to stabilize the retinal image of vertically moving object in these fishes, or a posibility of some nerve system which specially analyzes a vertically moving image.

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Effect of digested night soil, marine plankton and light on the viable count of intestinal bacteria in the seawater were examined in order to estimate the sanitary problems of ocean fertilization with night soil.
One hundred ml each of aged seawater with or without digested night soil (1%), phyto-plankton Nitzschia closterium, Phaeodactyllum tricornutum, Dunaliella tertiolecta, Brachiomonas sp., Chlamydomonas sp., Platymonas sp., Nannochloris sp. and Chlorella sp. and zooplankton Tigriopus japonicus, were inoculated wiht 10⁸ cells per ml each of Escherichia coli, Salmonella typhimurium or Vibrio parahaemolyticus, and rotary shaken with or without illumination (ca. 6, 000lx) for several days at 20°C. Bacterial cells were counted every day by using selective media. In the dark and clean seawater, E. coli and S. typhimurium survived more than 6 and 15 days, respectively, and V. haemolyticus rather propagated. On the other hand, the former two species and the latter one died off within 1 and 3 days, respectively, in the illuminated seawater containing digested night soil and marine plankton.

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The mineralization rate in the marine sediments was estimated in the coastal areas of Beppu Bay, Osaka Bay and Hiuchi Nada in the Seto Inland Sea by the kinetic approach method described by M. J. HARRISON et al.
The highest mineralization rate of sediments was measured on the surface in Hiuchi Nada, the second in Osaka Bay and the lowest in Beppu Bay. Vilable counts of heterotrophic bacteria on the surface of sediments of Osaka Bay and Hiuchi Nada were larger than those of Beppu Bay. Since the C/N ratio in Beppu Bay was larger than those in Hiuchi Nada and Osaka Bay, the activity of microorganisms was lower in Beppu Bay than that in the other two areas.
As the mineralization rate and the number of heterotrophs decrease significantly with the column depth of sediments, the surface of the sediments is important for mineralization of organic materials. Comparing the mineralization rate on the surface of the sediments with that in the overlaying water, the percentage of the mineralization in the shallow waters like Osaka Bay was larger than that at the relatively deep waters of Beppu Bay.

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In order to investigate the participation of the hydrophobic groups into muscle protein on its gelation on heating at low temperature, viz. Setting, the elasticity was measured on the flesh sol from dolphinfish, Coryphaena hippurus, containing p-toluenesulfonyl chloride (TSCI) or α-naph-thalenesulfonyl chloride (NSCI). Washed mince was ground for 25min with TSCI or NSCI (0.3% of the mince) together with NaCI (3%) and NaHCO₃ (0.15%). With the addition of TSCI or NSCI, a slight increase in the breaking stress and the decrease in pH by about 0.2 was observed in the flesh sol. On heating at 40°C or by standing at 4°C, the flesh sol changed to gel and a remarkable increase in the breaking stree was observed, but sucrose (10%) diminished the effect of TSCI or NSCI. In the flesh sol without TSCI or NSCI, however, the gelation was delayed even at 40°C and the breaking stress of the resulting gel was low.
The preheating of the flesh sol containing TSCI or NSCI at 40°C caused to increase the breaking stress of the fish gel obtained by further heating the sol at 90°C for 25min.
Form the above results, the dolphinfish flesh sol, which is classified into a hard-to-set fish, seems to come to behave like the flesh sol from the easy-to-set fish, on the addition of TSCI or NSCI.

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Enumeration of heterotrophic bacteria in river waters of differing degree of eutrophica-tion has been done by using agar media containing different concentrations of organic nutrients. The higger counts of bacteria in any station at any season were obtained by low nutrient agar medium (0.5g of polypeptone, 0.25g of yeast extract, 0.1g of glucose, and 15g of agar in 1, 000ml of distilled water), in comparison with the counts of bacteria on high nutrient media. It was suggested from these results that the low nutrient medium is superior to recover the heterotrophic bacteria inhabiting in river waters. Furthermore, it has been found that bacteria isolated from less polluted waters were more sensitive to high nutrient concentrations than those from highly polluted waters in experiments on the growth response of bacteria to the concentrations of organic nutrients.
Since the higher counts of bacteria in river waters were obtained at about 35°C in summer and about 20°C in winter, it appears that incubation temperature for counting must be selected in accordance with water temperature in river.

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The amount of nitrogen excretion as well as activities of amino acid-degrading enzymes in the hepatopancreas and protein efficiency ratio in carp, Cyprinus carpio, were investigated. These fish were raised individually in aquariums using a closed-cirulating system at 20°C, and fed on a high-carbohydrate control diet containing 44% of protein and 29% of carbohydrate for a 14 day conditioning period. The fish averageing 51g each were divided into three groups of 5 fishes. One group was fed on a high protein diet replacing the carbohydrate of the control diet with protein, another group on a high fat diet replacing some of the protein of the control diet with fat, and the remaining group on the control diet, this was done continuously during the 20 day experimental period. The amount of excretion of ammonia and total nitrogen in the control group was constant during the experimental period. On the other hand, the amount of nitrogen excretion increased strikingly in the high protein group, and gradually decreased in the high fat group. There was a significant difference in the amount of nitrogen excretion of the three groups after the 6th day of the dietary change. Compared with the high protein group, the control and the high fat groups; excreting smaller amounts of nitrogen showed higher protein efficiency ratios and lower activities of hepatopancreatic arginase and alanine aminotransferase at the end of the experiment. The findings suggest that dietary carbohydrate and fat cause a reduction in the activities of amino acid-degrading enzymes in the hepatopancreas, resulting in a low nitrogen excretion rate and a high protein efficiency ratio.

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In order to reveal the binding manners of Cu²⁺ in insolubilized DNA, two kinds of Cu-DNAs, prepared by the electrolysis method [Cu-DNA(E)] and by the reaction of DNA with a copper sulfate [Cu-DNA(S)] were studied respectively for their reaction with sodium chloride and protamine.
The binding site of Cu²⁺ in Cu-DNA(E) seemed to be between the phosphate group and base, while that of Cu-DNA(S) seemed to move from between the phosphate group and base to between bases after the cleavage of a hydrogen bond between the bases.
Cu²⁺ bound between the phoshpate group and base seemed to be substituted reversibly by Na⁺. On the other hand, when protamine bound with Cu-DNA(S), the Cu²⁺ binding between bases seemed to be wrapped into DNA-protamine.
Before these experiments, the insolubilization of DNA by copper sulfate was investigated, and the conditions for maximum yield as well as some properties of Cu-DNA(S) were elucidated.

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The present work was undertaken to investigate the intracellular distribution of the enzymes such as cytochrome c oxidase (the mitochondrial marker), β-glucronidase, β-N-acetylglucosamini-dase, acid phosphatase and cathepsin D (the lysosomal markers), catalase (the peroxisomal marker) and alkaline phosphatase (the microsomal marker) in ordinary and dark muscles of carp according to differential centrifugation. Some particle properties of both muscle lysosomes were also examined.
In the ordinary muscle, fractionation of total homogenate showed that all of the enzymes have the highest relative specific activity of cytochrome c oxidase, the four lysosomal enzymes and catalase and alkaline phosphatase were found in the mitochondrial, lysosomal and microsomal fractions, respectively.
The buoyant densities of ordinary and dark muscle lysosomes were about 1.03%g/ml in iso-osmotic self-generating gradients composed of modified colloidal silica (PERCOLL), but a slight amount of lysosomes with higher buoyant density, about 1.04%g/ml, was found in dark muscle.
The maximum stability of the muscle lysosomes was observed in sucrose over 0.2M and at pH 7. Temperature of incubation of dark muscle lysosomes in 0.25_M sucrose had a slight effect on lysosomal-enzyme releases as compared to those from ordinary muscle. Carp muscle lysosomes were less stable than those from mammalian and chicken muscles when exposed to incubation at 37°C.

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Comparison of genetic data of serum esterase and transferrin systems of the skipjack tuna, Katsuwonus pelamis Linnaeus, collected from the Atlantic, Indian, and Pacific Oceans together with the results reported earlier, (1) disclosed that fish from the Indian Ocean are distinguishable from those collected both in the Atlantic Ocean and the western Pacific Ocean and (2) suggested that the skipjack tuna, now inhabiting in the worlds major oceans, first appeared in the worlds major oceans, first appeared in the Indian Ocean and then some of the members of the population emigrated to the other oceans, resulting in genetic diversification into the geographical populations, called the Atlantic Subpopulation, Indian Subpopulation, Western Pacific Subpopulation, and Central-eastern Pacific Subpopulation (s), after reproductive isolation.
Regarding the east-west difference of genetic compositions within the Pacific Ocean, analyses of our data on some 500 samples from more than 24, 000 fish indicated the “step structuure” with a gap between 160°E and 175°E more disinctly in the southern hemisphere. Apparent differences in allele frequencies of genetic systems, found recently in 6-phosphogluconate dehydrogenase and phosphoglucose isomerase, between the waters of Japan and Papua New Guinea suggested the necessity of further accumulation of the data before any conclusive statement on population structure in the regions.

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Effects of added chemicals such as SH-group blocking agents, oxidizing agents and reducing agents on the production of hydrogen sulfide in retorted Kamaboko were examined. The addition of disodium maleate or N-ethylmaleimide at 0.01M level reduced the concentration of hydrogen sulfide in retorted Kamaboko by about 60 and 70%, respectively. However, disodium fumarate was not effective.
Among the oxidizing agents, both of potassium bromate and potassium ferricyanide at 0.01M level inhibited the production of hydrogen sulfide almost completely, but hydrogen peroxide at the same concentration level did not inhibited. However, potassium bromate at use level promitted Japanese Sanitation Law was not effective.
The addition of sodium sulfite, sodium bisulfite or sodium hydrosulfite at 0.01M level inhibited the production of hydrogen sulfide. On the other hand, those reducing agents such as sodium ascorbate and sodium nitrite increased the concentration of hydrogen sulfide to about 210 and 180%, respectively.

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In order to examine the extent of myxosporidian infestation of hake in the Peruvian waters, about 6, 500 fish caught by factory trawlers operating off Salaverry and about 2, 400 fish caught by small coastal trawlers off Paita were examined during July to September 1977. Two detection methods were applied; the visual observation of cysts in the raw flesh and the textural examination of softening in the slowly cooked flesh. The latter method was found to be more effective.
More than 40% of hake off Paita were infested, while 14.4% of hake off Salaverry were infested by myxosporidian parasites. Higher incidence of the infestation was observed for hake of bigger size. There was no clear relationship between the incidence of the infestation and the coefficient of corpulence, sex of fish, or flesh color. Cysts with yellow and/or black color seemed to be more active enzymatically than white ones. The anterior part of the fillet was the most severely infested.

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In the previous paper, it was shown that the partially frozen horse mackerels Trachurus japonicus spoiled after three months' storage at -4°C and that the majority of the isolates from the spoiled fish consisted of Pseudomonas I/II.
The present study was undertaken to find any differences in the spoilage rates of thawed fish and unfrozen fish, and in the bacterial flora of the spoilages.
The thawed fishes, partially frozen at -3°C for 15 and 37 days, spoiled after 6 and 3 days' storage at 0°C, respectively, and their bacterial flora in the spoilage mainly consisted or Pseudomonas III/IV-H and Moraxella in the former and of Pseudomonas I/II in the latter. The other thawed fishes, frozen at -20°C for 15 and 67 days, spoiled after 12 and 13 days' storage at 0°C, respectively, and their flora in both spoilages chiefly consisted of Pseudomonas I/II and Moraxella. Since unfrozen controls spoiled after 11 days' storage at 0°C, it was shown that thawed fish, partially frozen, seem more perishable than unfrozen fresh ones.

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The ability of white-spotted char liver homogenate to convert chlorinated nitrobiphenyl ethers and their nitroso and amino derivatives to mutagens was investigated by using Salmonella/microsome test.
p-Nitrobiphenyl ether (p-NO₂), 3-chlor p-nitrobiphenyl ether (3-CNO₂), 4-chloro p-nitrobiphenyl ether (4-CNO₂) and all the nitroso derivatives used induced mutations in Salmonella typhimurium without liver homogenate. Mutagenicities of these compounds were closely related to the bacterial reduction process. While all the amino derivatives used showed no mutagenic activity without liver homogenate.
With liver homogenate, the mutagenic activities of p-NO₂, 3-CNO₂and 4-CNO₂ slightly increased while those of the nitroso derivatives greatly increased, and paminobiphenyl ether and 4-chloro p-aminobiphenyl ether among the amino derivatives showed the mutagenic activities. The results obtained reflected that white-spotted char liver homogenate had the ability to convert these compounds to mutagens.
The mutagenic activity of these compounds was affected by not only the number of chlorine but also by its position on phenoxy group in the molecules.

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Rishiri-konbu Laminaria japonica var. ochotensis and gagome-konbu Kjellmaniella crassiforia, both of which were collected at four places in Hokkaido, were assayed for arsenic contents in their various parts, and the following results were obtained.
1. The arsenic content was highest at the base and lowest at the top of frond, the approximate proportions being 2:1. The content tended to be higher at the center than at the marginal part, and this tendency was clear at the base of frond.
2. The arsenic content was significantly higher at the cortex part than at the marrow part of frond. The proportions were 1:2.2 at the top and 1:2.5 at the base. The arsenic content at the marrow did not differ so widely from the top to the base.
3. The above results revealed that arsenic is distributed more densely in meristematic tissues, suggesting that some arsenic compounds may be concerned with the metabolism of those tissues.

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The characterization and purification of the agglutinin in the Aplysia eggs capable of agglutinating vertebrate erythrocytes and marine bacteria were investigated. The agglutinin prepared from A. kurodai was specific to B cells as to human erythrocytes, while that of A. juliana was non-specific. They were independent upon divalent cations for hemagglutinating activity, though slight enhanced activity was observed by addition of Ca²⁺. The activity remained unchanged after the digestion with proteolytic enzymes, but was reduced partially by the treatment with 2-mercaptoethanol or periodate. The hemagglutination was inhibited by D-galacturonic acid, but not by D-glucuronic acid. The agglutinin was purified from A. kurodai eggs by affinity chromatography followed by gel-filtration. The purified preparation reacted with B cells and rabbit blood cells as low as 0.06μg/ml. The molecular weight of egg agglutinin was estimated to be 70, 000 daltons. The ratio of subunit (13, 000) to a native agglutinin was very close to 6.

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Radioisotope tracer experiments on binding of radionuclides to proteins in fish were carried out in order to gain further information on biochemical behavior of radionuclides in marine fish. The radionuclides, which were bound to proteins in fish through sea water or food, were extracted with a Tris-acetate buffer solution and separated by gel filtration on Sephadex G-75.
Most of ¹³⁷Cs in the fish liver were bound only to a peptide with a molecular weight of 1, 100-1, 300. The most remarkable feature of ⁶⁰Co in the profiles of the gel filtration was the presence of two clear radioactivity peaks and the radioactivity appeared to transfer from a low molecular weight protein to a high molecular weight protein in the uptake, and the reverse phenomenon was observed in the excertion. Therefore, this suggested that these proteins had each inherent turnover rate for ⁶⁰Co. The profiles of the gel filtration of ⁶⁵Zn varied widely among species of fish, tissues or organs even in the same fish and pathways of the uptake.
Thus, it was considered that there existed some different rules peculiar to each radionuclide in the binding of radionuclides to proteins in fish.

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Alkaline phosphatase (EC 3.1.3.1) from various tissues of skipjack, Katsuwonus pelamis, and also from hepatic tissues of several marine animals were examined for some enzymatic properties. The total activity of the enzymes from skipjack tissues was high in the pyloric caecum, kidney, and liver, but low in the ordinary and cardiac muscles, and bone. The enzyme activeity of fish livers was higher than that of invertebrate hepatopancreas. The optimal pH values of skipjack enzymes significantly differed from the kind of tissues, rangin from 7.3 to 10.2 The hepatic enzymes from most marine animals, however, showed similar optimal values around pH 10, except those from skipjack (pH 8.8) and prawn (pH 8.4). Michaelis constants of the enzymes were found between 0.02 and 0.1 m_M against p-nitrophenyl phosphate. The inhibitory effects of several effectors, such as p-chloromercuribenzoate, L-tryptophan, and L-phenylalanine, on the enzymes depended on the tissues and on the species. The skipjack enzymes seemed to be divided into two groups as to their heat stability. On cellulose acetate electrophoresis, the skipjack enzymes showed different isoenzyme patterns depending on the tissues.
It was concluded from the above data that the tissue-specificity of alkaline phosphatase is much higher than its species-specificity.

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Skipjack liver alkaline phosphatase (EC 3.1.3.1) and nucleotide pyrophosphatase (EC 3.6.1.9) were compared in respect to some physico-chemical and enzymatic properties. Neuraminidase storgly inactivated both enzymes in a similar fashion, whereas muramidase inactivated alkaline phosphatase more slowly than the other enzyme. Nucleotide pyrophosphatase was also inactivated by several proteolytic enzymes more easily than alkaline phosphatase. Sodium lauryl sulfate, urea, and guanidine hydrochloride inactivated both enzymes in a similar manner, and the protective effect of MgCl₂ was also similar. In the presence of MgCl₂, alkaline phosphatase showed the optimum temperature at 35°C and nucleotide pyrophosphatase at 45°C. In the absence of MgCl₂, however, both enzymes showed the identical optimal temperature at 45°C. All the substrates common to both enzymes inhibited them competitively. Nucleotide pyrophosphatase was inhibited by all the substrates specific to alkaline phosphatase but the inhibition types were different for both enzymes with some substrates. All the substrates for nucleotide pyrophosphatase also inhibited alkaline phosphatase by the competitive or the mixed type manner. These results suggested that the two skipjack enzymes are closely similar to each other in their primary or higherdimentional structure.

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The lipid compositions of the different tissues and gut content of deep-sea shark, Dalatias licha, were determined.
In this species, the liver contained 83.4% lipids, which was much larger than that of muscle, ovary and gut content, ranging from 17.3% to 0.8%. The liver and ovary lipids were characterized by high percentages of squalene (40.1%, 20.3%) and diacyl glyceryl ethers (27.8%, 35.7%). On the contrary, the lipids of muscle and gut content contained small amounts of the above types of lipids.
Glyceryl ethers of diacyl glyceryl ethers of liver and ovary were very similar in composition: the predominant constituents were 18:1 (52.7%-50.8%) and 20:1 (27.1%-24.7%). In both liver and ovary, the component fatty acids of diacyl glyceryl ethers and triglycerides were also similar in composition, predominantly consisting of monoenes (74.8%-81.3%).
Based on the characteristic lipid composition and/or the component glyceryl ethers or fatty acids of diacyl glyceryl ethers and triglycerides, it is suggested that the ovary tissue has directly accumulated the liver lipids for egg development.

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