Program

Japanese dace Tribolodon hakonensis is one of the important fish species for Fukushima’s inland water fisheries. It is listed as a target species in 24 fishing areas licensed with common fishery rights by Fukushima Prefecture. However, at present, fishing for Japanese dace is prohibited in the water bodies within the exclusion zone, and in some rivers and lakes. In this study, ¹³⁷ Cs concentration data of Japanese dace inhabiting lakes and ponds in Fukushima Prefecture were analyzed to help predict future trend. Field samplings were conducted in 18 lakes and ponds in Fukushima Prefecture from 2012 to 2016. Japanese dace specimens were mainly collected by overnight gill-net fishing. ¹³⁷ Cs concentrations in their muscle tissues were measured by Germanium semiconductor detectors. The ecological half-life ( T_eco ), an indicator of future prediction, calculated from seven lakes and ponds where statistically significant relationship between days after the Fukushima accident and log-transformed ¹³⁷ Cs concentrations were found, ranged from 350 to 1,644 days (796 days in Lake Inawashiro ).This value was longer than the reported values (430 days) by Wada et al. (2016) which analyzed data from June 2011 to December 2014. This result suggested that T_eco is becoming longer in recent years in Lake Inawashiro.

Although six years have passed since the Fukushima Dai-ichi Nuclear Power Plant accident which was caused by the Great East Japan Earthquake and subsequent tsunami on 11 March 2011, seven wild fish species, including the Japanese dace Tribolodon hakonensis, which inhabit the radioactively contaminated rivers and lakes in Fukushima Prefecture, are still listed as restricted foodstuffs for shipment by the Japan Ministry of Health, Labour and Welfare. Japanese dace is widely distributed in Fukushima Prefecture and is a fish species subject to the Japanese type 5 common fishery right. Therefore, elucidating the uptake and elimination mechanisms of radiocaesium with this species will help predict future restorations of inland water fisheries. In this study, to address the bioaccumulation and elimination processes of radiocaesium in Japanese dace, feeding experiments under three different temperatures (15, 20 and 25℃) were conducted. All the fish were cultured using ¹³⁷Cs contaminated (81.8 Bq kg^-1) and non-contaminated pellets for 0-42 days and 43-182 days, respectively. Daily feeding rate was set as 1% body weight of them. Three samples were periodically taken from each temperature group, and ¹³⁷Cs concentrations in muscle tissues were measured by Germanium semiconductor detectors. Physical decay constant of ¹³⁷Cs was set as 0. Parameters related to uptake and elimination were estimated by applying the one-compartment model to ¹³⁷Cs concentration data sets. Results revealed that Japanese dace in 20℃ group showed the highest growth rate, although the fish in all temperature groups grew favorably. The fish in the higher-temperature group tended to show higher uptake and elimination rates of ¹³⁷Cs in the muscle tissue and, consequently, had a shorter biological half-life. The uptake and elimination speed parameters obtained in this study are expected to be useful to predict the behavior of ¹³⁷Cs radioactivity in this species in the field.

Radioactive substances derived from the TEPCO’s Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident were detected in marine products. Fukushima Prefecture started monitoring inspections immediately after the accident and has since reported radioactivity of seafood. Monitoring results revealed higher radiocesium (combined 134Cs and 137Cs) concentrations in marine products from shallow waters south of the FDNPP immediately after the accident. However, as time has passed, contamination levels in most samples have dropped below the detection limit (about 8.3 and 7.4 Bq/kg for 134Cs and 137Cs, respectively). The lower radiocesium concentrations in marine products are probably attributable to the following factors: rapid decrease of radiocesium concentrations in seawater, change of generations, and increased body mass through growth. Based on the cumulative monitoring results, pilot-scale trial fishing operations aimed at restoration of Fukushima’s coastal fisheries have expanded.

Fishing operations of Fukushima’s coastal fisheries have been restrained voluntarily because of the radioactive contamination of marine products after TEPCO’s Fukushima Dai-ichi Nuclear Power Plant accident, which occurred in the aftermath of the Great East Japan Earthquake and the subsequent tsunami. Changes in resource abundance of demersal fishes cannot be evaluated clearly through data obtained from pilot-scale bottom trawl fishing, which started on 22 June 2012 and which targeted specific species within the limited fishing grounds. We conducted bottom trawl investigations at depths of 100 m or deeper from the R/V Iwakimaru of the Fukushima Prefectural Fisheries Experimental Station. Then we compared the distribution densities (kg/km²) of demersal fishes in 2016 to those observed for 2008–2010. Results showed that the distribution densities of all important species increased by 1.3–7.5 times in 2016. Particularly, the increased densities of larger-sized adults were observed. Future fishery management measures must be established to use the increased demersal fish resources efficiently.

The tsunami caused by the 2011 Great East Japan Earthquake destroyed most bivalve aquaculture facilities along the Pacific coast of Tohoku, Japan. Subsequently, aquaculture has been recovering through farmers’ efforts and various supports, but some problems remain. One problem is the self-restriction on shipment of bivalves because of high levels of paralytic shellfish poisoning (PSP), which has occurred every year since the tsunami and inhibits the reconstruction of bivalve aquaculture. Such high levels of PSP are probably associated with changes in the seed population of the causative organism Alexandrium spp. Here we demonstrate changes in the density of Alexandrium spp. cysts in surface sediments in Sendai Bay just after the tsunami, and changes in their distribution during the subsequent several years. Cyst densities widely increased and reached a maximum density of 10 times that before the tsunami. After the tsunami, the area of relatively high cyst density shifted from the central part of the bay to the western part. The extreme increase in cyst density is considered to have been partly caused by the accumulation of Alexandrium cysts from deep layers in surface sediments due to the disturbance of sediments by the tsunami. Subsequently, the high density of cysts in the western part of the bay decreased in 2013 and 2014 and relative high densities were observed in surface sediments in the eastern offshore areas in 2015 and 2016. These results show that high-density areas for Alexandrium spp. have apparently moved, probably indicating horizontal transport of cysts by seawater currents as well as a supply of newly produced cysts.

Beach seines have been documented as being the most destructive gears affecting reef fish stocks in Kenya. The fishery was prohibited by the State Department for Fisheries and the Blue Economy in 2001. Comprehensive studies on the structure of the beach seine catches and effects on target species are limited. Therefore, this study determined size selectivity of the codends of 1’, 1.5’ and 1.75’ mesh sizes used in beach seine fishing for L. vaigiensis, S. sutor and L.lentjan from catch data collected in 2014 and 2015, by using SELECT (Share Each Length class Catch Total) method. The results indicated that for size selectivity, when 1’ codend was control (no size selectivity), 1.5’ and 1.75’ codends showed to retain larger sizes of fish. The 50 % retention lengths (L50) of 1.5’ and 1.75’ codends varied in the three most captured species. L50 values of 1.5’ was 14.0cm in 2014 but was 2.7cm in 2015, and 1.75’ was 14.1cm in 2014, 15.7cm in 2015 for L. vaigiensis. S. sutor values were only available in 2015 and for 1.5’ was 9.8 cm and 1.75’ was 9.1 cm. We did not observe a size selectivity for L. lentjan. A greater proportion above 50% of juveniles for the most abundant fish species were retained in 1’, 1.5’ and 1.75’ codends. This is the first information on the size selectivity of the beach seine fishery for commercially important fish that is also important for fisheries management in Kenya, but there is need for development of an experimental procedure to obtain data for robust selectivity analysis.

Fuel consumption in towed gear fisheries is typically attributed in towing period because the boat requires thrust to tow the gear. Hence one of fuel saving measure for towed gear fisheries is to use the low-drag gear. Therefore, the aim of this study is to propose a way to reduce the gear drag through examination of gear components to the gear drag.
1. Netting: Enlargement of mesh size and reduction in twine diameter are common options, but also we propose another way to reduce the gear drag, by adjusting the attack angle and the mesh opening (hanging ratio of the mesh) of the net panel. To confirm their effectiveness, we conducted the tank experiment by using the knotless netting (PE 50ply, 75mm). Six plain net panels (300 x 300mm, hanging ratio 3, 11, 20, 29, 50 and 76 %) were set on a square frame having 0, 15, 30, 45 and 90 degrees of attack angle in the flume tank (6 x 2 x 1.2m). Flows (from 0.25m/s to 1.0m/s) were given and drag force was measured. We observed that the drag force per unit net area increased as the attack angle increased, for panels with any hanging ratios. Drag force per unit net area for the panel with low hanging ratio (3 and 11%) was more than other panels of high hanging ratios when attack angle was more than 30 degree, but it was less on cases of low attack angle (0 and 15 degrees). Thus, smaller hanging ratio and attack angle can potentially reduce a drag of netting.

2. Floatation and ground rope: A review of current knowledge on their drag in towed gear is in progress and experiments to confirm/fill necessary information is also conducted.

Hippoid crabs fishery is a common artisanal fishery in some coast of southern Java, Indonesia. The crab were catched by local fishermen using a traditional gears, called “sorok”. Within 1 hour the gear could obtain 1 to 1.5 kg yield. When it is operated, the sorok could detect the presence of the crab 5 cm under the sediment and then it was catched by hand. Three crab species can be caught, i.e. Emerita emeritus, Hippa adactyla, and Albunea symmysta. In this study, we design and develop a fishing gear to collect the hippoid crab. Test result of the prototype showed that average forward speed was 1.2 m/s and traction wheel slip was 9.13%. Prototype performance was still not good yet, where the depth of catching device was up to 21 cm, so that pulling force was still too large. Trailer’s ground clearance was low so that it needed to lift-up it up to 16 cm from sediment surface. Traction wheel slip was small enough for distance of 5 m from beach edge, so that the prototype can be operated well, but for distance of 2 m from beach edge the prototype had loss its traction and caused sinkage of 30 cm. Screen separator mechanism was in good performance.

Recently machine learning methods have been increasingly applied in different research fields, as their competitive abilities of understanding data and prediction compared with traditional approaches have been recognized. The methods have also been applied to fishery data though comparison of estimation performance is still on the way. Here we attempt to evaluate the performance in prediction of the species density distribution of fishery species through simulation by considering the influencing factors such as biotic and environmental factors, using machine learning methods. As candidate methods of the machine learning methods, “Support Vector Machine”, “Lasso”, “Boosting”, “Random Forest”, and “Neural Network” were employed in addition to traditional approaches like “Generalized Additive Model”. Simulation data were repeatedly generated with some assumed scenarios and analyzed by these methods. To determine the best method that predicts the density distribution well, the MSE (Mean Squared Error) of the predicted results of each method was calculated and compared. The results showed that the performance of machine learning methods was likely better, especially Support Vector Machine and Random Forest, which suggested the competitive and potential abilities of understanding data and prediction compared with traditional methods. Further comparison of these methods will be conducted under different scenarios, and the results will be showed in the poster session.

Octopus is one of the fishery resources that is widespread in the waters of Indonesia. The region famous for its octopus fisheries is the spermonde islands in South Sulawesi. However, information of the technology and utilization of octopus in the region is still very limited. Japan International Cooperation Agency (JICA) project through Ajirushi Company in cooperation with Faculty of Fisheries and Marine Sciences Bogor Agricultural University are cooperate in the development of octopus industry in Makassar. The purposes of the research are to identify the fishing technology, fishing ground and to analyze the feasibility of the octopus fishery business in South Sulawesi. The survey was conducted for 4 days from 16-20 June 2016 in the Spermonde Islands by interviews with octopus fishermen, and collectors as well as coming to octopus landing centers. Based on the results the fishing technology used is still traditional methods by using spear fishing and octopus attractor. Octopus attractor is hand made by local fishermen based on local knowledge. Octopus attractor is constructed by 1.5 kg lead wire, 1 m hook wire, and 17 hook number 12. Fishing ground of octopus fishery is located in Lumu-lumu, Bone Tambu, Sabongko Island, Sagari Island. Peak season of octopus was observed in January-April with catch of 10-15 kg, middle season in June-August with catch of 3-5 kg, and low season in September-December with catch of 1-2 kg. An octopus fishing business is still prospective enough to be developed in the region. Development of fishing technology using pot octopus can be developed to increase the productivity and quality of octopus catch.

It has been recognized that hook shape affects fishing selectivity. Therefore commercial fishing hooks of various types and sizes are used. For this study, 3 types and 4 sizes of Japanese fishing hooks were used; 4 dimensions of a hook were measured to quantify its geometric character. Coastal bottom longlining using those hooks was done to ascertain differences of fish selectivity according to the hook shape and size. Three types of “Karei (flatfish) hook”, “Chinu (blackhead seabream) hook,” and “Mutsu (gnomefishs) hook” were used. Four dimensions were measured using software (Image J): a, total length; b, gape; c, bite of throat; and d, length from eye to gape line. In 8 experimental demersal longlining operations were conducted in the coastal area off the south east of Izu Peninsula in Japan. Around 80 hooks baited with cut squid or saury were used. In all, 143 fish of 20 species were caught. Moray eels Gymnothorax kidako were used as models for comparison with the following hook dimensions: a, 2.71 ± 0.65 cm; b, 1.40 ± 0.46 cm; c, 1.45 ± 0.49 cm; and d, 1.30 ± 0.57 cm. Although no correlation was found between the ratio of each dimension and total length, significant correlation (p=0.026) was found between the ratio of “a” to “d” and total length of fish using multiple regression analysis. Results suggest that caught moray eels would be maximized at around 80 cm when using a hook of which the “a” to “d” ratio is 0.4. Further analyses are expected to clarify the fish species selectivity, revealing various feeding habits and behaviors using fishing hooks of different types with demersal longlining.

The hatchery program has been the main tool for the management of chum salmon (Oncorhynchus keta) stocks in Japan. The hatchery program in Hokkaido had been developed and promoted by the Fisheries Agency until 1999. Thereafter, it has been conducted by non-profit salmon enhancement associations established in nine regions of Hokkaido, under the management of the Hokkaido Prefectural Government. The current system of conducting the hatchery program was established in the 1990s when chum salmon returns had reached a historic high; however, the number of chum salmon returning to Hokkaido has been decreasing since the mid-2000s. There are several emerging issues to be discussed and managed for the enhancement of salmon stocks of Hokkaido in future. For instance, 1) the lack of adult chum salmon for hatchery broodstock is a major issue. At present, fishermen remove a part of the set-nets to reduce fishing pressure and allow fish to enter the rivers when the number of adult chum salmon for the program is insufficient. Transplantation of eggs among regions is not conducted to prevent genetic disturbance, although transplants were often conducted until the early 1990s. 2) Running costs of the hatchery program are addressed by charging for commercial landings. Because chum salmon are migratory in the coastal areas, and commercial landings differ greatly among regions, the charges are pooled and redistributed to the nine salmon enhancement associations. Nevertheless, some salmon enhancement associations struggle financially in low return years. 3) In spite of the importance of recovering naturally spawning population for their sustainability, no official policy of managing them is present. Because the stock status of chum salmon in Hokkaido is less than the peak attained in the 1990s–2000s, discussion and consideration on future management should be prioritized, encouraged, and followed by fishermen and hatchery managers.

A fish pass is a structure on artificial barriers to facilitate diadromous fishes' natural migration. In this study, we propose a fish pass using baffle effects of water flow without baffle plates (hereinafter referred to as water flow fish pass, WFP).
Water flow fish pass is consist of fish pass and reservoir. A part of fish pass is mere channel without baffle plates. Parts of reservoir set up on both sides of mere channel as a fish pass. Water from the upstream flow into parts of reservoir. Weir plate is provided at the downstream end of reservoir, and water levels are equalized in reservoir, an upstream end of fish pass. In the practical application of water flow fish pass, the weir plate will be detachable. Earth and sand, driftwood piled up in reservoir, can be removed rapidly. Along the inclination of the fish pass channel bed, a plurality of orifice are provided in the reservoir side wall. As depth of reservoir is deeper in the downstream direction, and also runoff from orifice is stronger. Water flow from the orifices hits the main current of fish pass, thereby the power of fish pass flowing can be damping.
By the laboratory experiments, we confirmed that relatively slow velocity continuous flow occurred in cross-section central area of fish pass.

Coastal squid fisheries in Yamaguchi Prefecture typically operate small fishing boats under 5 tons, principally seeking swordtip squid (Uroteuthis edulis). Because the main fishing grounds are coastal areas, fuel consumption by fishing lamps during operation is higher than fuel consumption during cruising, accounting for around 60%. In this study, we first carried out experiments to gain data on the necessary light environment for attracting and catching squid, after which we designed LED fishing lamps and carried out catch tests.
We conducted experiments to gain an understanding of the necessary light environment for attracting and catching squid in the Susa coastal area of Hagi-city in Yamaguchi Prefecture on 6/8/2016, and 12/2/2016. In these experiments, we used digital scanning sonar (675 Hz) and ARIS acoustic camera (3 MHz) to measure marine organisms distributed below the ship. Additionally, we conducted catch tests using trial LED fishing lamps on 11/25/2016 and 1/25/2017.
In our experiment on 6/8, immediately after lighting the lamps at a comparatively low illumination level, we confirmed an echo reaction presumed to be swordtip squid intruding beneath the bottom of the ship from the bow direction. In addition, during our observations using an acoustic camera, at water depths of 15 meters or less we were unable to confirm any images presumed to be squid. Based on the results above, we prototyped an LED fishing lamp and carried out catch tests to ascertain the efficiency of the trial LED lamp. For the catch tests, on 11/25 we caught 22 squid with LED fishing lamps and 21 squid with conventional fishing lamps, while on 1/25 we caught 211 squid with LED fishing lamps and 310 squid with conventional fishing lamps. We will continue to collect data on the catch efficiency of the trial LED lamp.

Parapenaeus lanceolatus is widely distributed in the Indo-West Pacific region. This shrimp has long been commercially important in the Inland Sea of Japan and is an emergent fisheries resource in Kagoshima Bay, Japan. However, the population biology of this species has been insufficiently studied restraining its appropriate management and conservation procedures. Therefore, we provided detailed information on the reproduction of females and growth of P. lanceolatus using samples collected from Kagoshima Bay from January 2011 to February 2016. The ovarian maturation was studied by histological observation. The growth patterns and longevity were estimated based on the monthly length-frequency distributions. A series of component normal distributions was fitted to the length-frequency of each sample by sex, using a computer analysis based on Hasselblad’s maximum-likelihood method. Growth patterns for both sexes were modeled by fitting the von Bertalanffy, and Pauly and Gaschütz equations. The best fitted model was selected based on the Akaike’s information criterion. Parapenaeus lanceolatus showed asynchronous ovaries indicating multiple spawning in a single spawning season. Females with oocytes having cortical granules in their peripheral region were categorized as mature. The gonadosomatic index showed a steep relationship with ovarian maturation progression, and it could be used as a simple index of ovarian maturity. The size at sexual maturity of female P. lanceolatus was estimated to be 22 mm in carapace length (CL). The spawning season extended from July to April and peaked September-October. Both sexes were recruited during November to January with a modal size of around 10 mm in CL. The growth of P. lanceolatus was well described by the von Bertalanffy growth equation. Females had a higher growth rate and larger asymptotic length than those of males. The longevity was estimated to be approximately 27 months for both sexes.

Metapenaeopsis sibogae is distributed in the South Pacific and Southeast Asia and is gaining in commercial significance. However, studies on the population biology of M. sibogae are scant. Therefore, we presented the relative growth and size at sexual maturity of M. sibogae, using samples collected from Kagoshima Bay from January 2013 to December 2014, to aid its management protocols. The relationship between carapace length (CL) and body length (BL) was obtained by using the least squares method. Data for each sex were repeatedly partitioned into two size-delimited subsets, one for shrimps with a CL smaller than (early phage), and the other for shrimps with a CL equal or larger than (late phage) a hypothesized inflexion point. A separate regression function was calculated for each of the two subsets after each relocation of the hypothesized inflexion point and was performed iteratively with sequential relocation at 0.1 mm intervals. The transition point was selected based on the lowest combined sum of squared residuals in the resultant two subsets of data. A total of 2822 individuals were examined including1567 females (7.88-21.16 mm CL) and 1255 males (9.16-17.11 mm CL). The transition point of females, estimated as 15.0 mm CL, was similar to the 50% size at sexual maturity of female M. sibogae (Rahman & Ohtomi, unpublished), therefore, it might be associated with sexual maturation. The transition point of males (13.2 mm CL) could possibly be as a result of sexual maturity. BL increased with CL more steeply in early phased males than in early phased females. Positive allometric growth was found between body weight (BW) and CL in early phased males, whereas it was negative allometric in case of early phased females. However, after reaching transition point, the slope of the CL-BW relationship reduced more rapidly in males than that in females.

The Pacific saury Cololabis saira is widely distributed in the North Pacific, and their fishery is internationally managed under the North Pacific Fishery Commission (NPFC). The Pacific saury has been caught by several members of the NPFC including Japan. Currently, the amount of Japanese catch shows a decreasing trend, while those of other members tend to be increasing. Under this circumstance, to know the population dynamics in a better way, a formal stock assessment of this stock is required. We conducted a preliminary stock assessment using Bayesian state-space surplus production models to describe Pacific saury population dynamics based on time series of catch and Japanese abundance indices (both of fishery-independent and dependent). The models incorporated process and observation errors and were inferred using the Markov chain Monte Carlo simulation. Several model options were considered to compare the fitness of models. Furthermore, retrospective analyses were conducted for model checking and predictive evaluation of models. The models fitted to the data reasonably well, and no severe retrospective patterns were observed. However, the predictive ability might not have performed well due to noisy fishery-dependent data. Given estimated stock status and fishing intensity, we conclude that the current fishing effort is likely not harmful to the Pacific saury stock.

Smallscale blackfish Girella leonina is a commercially important fish inhabiting the Japanese coastal zone, but little is known about its spawning ground and season. We examined sexual maturation of adults and the distribution of settled juveniles in the upper reaches of the Kuroshio Current, in order to infer the reproductive ecology of G. leonina around the Japanese Archipelago. The maturation was analyzed for the adults caught off the northern coast of Taiwan. The distribution of the juveniles was examined by fish sampling in the intertidal zone of the Tanegashima island and the southeastern part of the Kyushu district. The species of the juveniles was genetically identified by PCR-RFLP of mtDNA. We histologically observed gonads of 101 G. leonina adults fished in winter and early spring during 2014–2017, and found mature oocytes from eight adults fished during late December to early January. However, gonadosomatic index (GSI) values were much lower than the GSI values reported for mature adults of Girella punctata. It is possible that the ovary of G. leonina may reach maturity in the neritic sea of the northern Taiwan and increase in size during the southward migration to the spawning ground. In mid March, we did not collect any juveniles of G. leonina in the southeastern Kyushu, but collected 30 juveniles of G. leonina (15.0–29.1 mm standard length) in the Tanegashima Island. It is likely that G. leonina juveniles born in the upper reaches of the Kuroshio Current during winter are transported to the coastal waters of the Tanegashima island and grow up there in early spring.

The village fishery project was the one of many activities that Department of Fishery of Thailand has been implemented since 1980s in order to solve the protein malnutrition problem over rural areas in Thailand. The village fishery project format is to apply public pond for fish culturing and also other agriculture utilizations which performed by people in the village and collaborating with the government officers. In this study, the interactive governance framework was applied to exploring and addressing the many dimensions of fisheries governance, especially to examine factors contributing to the implementation of village fishery project executed in Ban Nongmajub, Chiangmai province, the north of Thailand. Using key informant interviews and participant observation were examined and then assessed the characteristics of the natural and social systems associated with the village fishery project and of the governing system including of their interactions that provide the factors may contribute to the success of village fishery project implementation. The results showed that there was highly collaborative process in establishing the village fishery project with village leader and the committee. The high level of interactions among people in the village and government officers contributed positively to the successful of this project. On the other hand, the factors that struggled to maintain the village fishery project as a result of low collaboration from community due to distrust on village leader and too much diversity and complexity in social system. Moreover, lack of people’s collaboration in the village at the first step of the project.

Recently, a broad range of stock assessment models have been used according to the data richness/poorness and objectives of assessment. However, it is not straightforward to choose a plausible range of models objectively from several models if different data set are used because it is not possible to use model selection criteria like AIC in these situations. Given that the main objective of the stock assessment is to provide a better management advice for future fishery, we might be able to focus on the predictive ability when choosing a plausible one among candidate models. In fact, Kell et al. (2016) proposed a hindcasting approach, which is a predictive evaluation procedure via cross-validation approach. Here, as an attempt, we applied this model selection method with predictive evaluation to biomass index for the Indian Ocean bigeye tuna and compared variants of Bayesian state-space production models. Non-informative priors were used and posterior samples were generated using a Markov chains Monte Carlo (MCMC) method. This approach can also be applied to some age-structured models. More detail of results of the model selection and stock assessment will be presented onsite.

The Green sea turtle (Chelonia mydas) is reptile that lives widely in the tropics and subtropics. The species is categorized as “Endangered” in the IUCN (International Union for Conservation of Nature and Natural Resources) Red List . Ogasawara Islands are known as major spawning areas of this species. The population size in Ogasawara Island has been estimated based on surveys for the number of nests. However, formal statistical analyses have not been performed yet. In this study, we aimed at estimating abundance of mature females using mark-recapture methods. The data employed are obtained by spawning survey since 1975. We transformed the original observation to a specific format of data by considering remigration interval (spawning interval; 3~4 years) and appliedtwo models, Schunable and Schmacher-Eschmeyer methods, to the data. The results showed that the population size has been increasing since1980. The annual increasing rate was estimated as 6.82%/year. Although the driving information, number of nests, has been increasing with some fluctuations, the mark-recapture method successfully produced a smoothed abundance trend, which suggested usefulness of applying mark-recapture method to conduct the stock assessment of this species.

The bay of Balikpapan is one of bays directly flow into Makassar Strait and located around 90 km southwest of Mahakam Delta. Length of the bay is around 36 km, with mouth of bay wide (14 km) is wider than head of the bay (less then 1 km). More than 10 small rivers flow into the bay to contribute amount of freshwater mass. The deepest part of the bay is located at the middle part of the bay and close to the bay mouth, the depth is around 18 m. Economic activities in the bay are very intensive, for example in the east side of bay encountered oil refinery, steam power plants, petrochemical plants and several ports as supporting facilities. In this study used data from field observation, such as the results of measurements at 12 stations with the CTD sensor, tidal and current measurements. Other supporting data obtained from projects INDESO, Indonesian Navy and EMCWF. To understand the flow pattern within the bay, then the flow simulation model was conducted by using FM Module of MIKE 21 developed by DHI. The objective of this study is to obtain physical properties of the water mass of Balikpapan Bay and their water mass dynamics. In general stratification of water masses in the buy occurred, but very weak. The influence of the tidal waves that propagate into the bay fought to control the dynamics of water mass in the bay.

Key Words: Balikpapan Bay, stratification, water mass, CTD,

Aquaculture activities cannot be separated from the water quality parameters that support fish performance. Aquaculture releases organic compounds and nutrients (nitrogen, phosphorus, and other elements) that require treatment prior to disposal to the aquatic environment. Phytoremediation is one of the waste treatment methods that can be applied. This study was aimed to investigate the water quality characteristics of gourami cultivation wastewater remediated by romaine lettuce. Organic waste from gourami culture could be processed by phytoremediation using romaine lettuce. It can be seen that the lowest concentration of TAN, ammonium, nitrate, and orthophosphate was found on P2 (aquaponics system using 10 romaine lettuces) namely 1.74±0.76 mg/L, 1.70±0.74 mg/L, 1.59±0.81 mg/L and 0.59±0.61 mg/L, respectively. However, the lowest concentration of ammonia (0.02±0.02 mg/L) and nitrite (0.02±0.02 mg/L) was obtained in the treatment of P3 (aquaponics system using 20 romaine lettuces). The phenomenon above was supported by biplot analysis, although the highest wet weight of lettuce was found in the treatment of P2, yet the ammonia value was still relatively high with high closeness also. Meanwhile, the highest fish weight was produced in P3 which was accompanied by the lower ammonia concentration.

Offshore oil pollution is one of the main forms of marine pollution, carrying forward the study of public interest litigation about offshore oil pollution is helpful to promote the construction of ecological civilization in China. Nowadays, the quality of China's marine environmental is not satisfactory, and the marine pollution incidents often occur. In terms of the public interest litigation system about offshore oil pollution, there are a lot of problems, such as the right to identify the uncertainty; difficult to identify the scope of compensation; compensation funds are not in place and so on. Referencing advanced foreign public interest litigation concept and system, the paper suggested we can improve the public interest litigation system about offshore oil pollution in 1) mode architecture; 2) court accepted; 3) burden of proof; 4) costs of litigation.

The tsunami after the Great East Japan Earthquake caused a disturbance of the quality and quantity of chemical substances in the sediment. In addition to this disaster, there was also the accident of Fukushima Nuclear Power Plant. In response of these events, there were some concerns on what the change in chemical substances would have on the surrounding aquaculture environment. Therefore it was necessary to understand the status of water pollution in regards to chemical and radioactive substances in Onagawa Bay. From 2012 to 2016, we collected the surface layer (0-5 cm) of the bottom sediments at 8 points. The sediment was lyophilized and stored at -30°C until analysis. SpeedExtractor E 914 (Büchi) was used for extraction and n-hexane and hexane : acetone (1:1, v/v) were used as extraction solvents. For polycyclic aromatic hydrocarbons (PAHs), 16 types specified by EPA were analyzed by GC / MS (PAH Analyzer, Agilent). Radioactivity was analyzed for ¹³⁷Cs, ¹³⁴Cs and ¹³¹I using germanium semiconductor detector (ORTEC, GEM). Soon after disaster, the amount of n-hexane extract was more than the standard value for fishery (0.1% dry matter) at some points in Onagawa Bay, but gradually decreased. Normal hexane extract unequally distributed in the bay. higher at the inner bay and lower at the bay entrance. It was presumed that the current in the bay affected the distribution. In addition, as the particle size was smaller, the adsorption amount tended to be larger. Although the distribution of PAHs was also unequal, the distribution pattern was different from that of normal hexane extract Though radioactivity was detected in the sediment, it gradually decreased after the disaster.

【Introduction】The species of Epsilonproteobacteria have nutrient endosymbionts in the gills of Alviniconcha sp. or have episymbionts in the gills of Alvinocaris longirostris living in a deep-sea hydrothermal vent (Urakawa et al., 2005 and Tokuda et al., 2007). So far, deep-sea Epsilonproteobacteria have been well reported, however, there are few reports about the one of shallow-water invertebrates. Thus, we hypothesized that the gills of invertebrates in a shallow-water also have relationships with a variety of Epsilonproteobacteria as in the deep sea. The aim of this work was to investigate whether the bacterial group is present on the gills of shallow-water invertebrates or not. We selected the gills of abalone (Haliotis gigantean) and analyzed the bacterial community by using the next generation sequencer.
【Method】Abalone was collected at the Minami-Ise abalone farm (Japan) in December 2015. The gills of five abalones were individually excised, gathered into one tube and crushed by using a bead beater. DNA was extracted from the mixture by using the DNA purification kit. The PCR amplicon libraries were generated form sample (primer loci at V1‒V2 region) and subjected to Illumina Miseq. OTUs by aligning unique reads was created at a 3% genetic distance threshold using the Qiime.
【Result】Sequences related to Epsilonproteobacteria represented a significant proportion (41.2% of all reads). Six clusters were identified and affiliated to Epsilonproteobacteria such as Arcobacter sp. and Sulfurovum sp. and denovo 1546 that occupied 97% of annotated sequences of Epsilonproteobacteria was closely related to a sequence data from the gills of bivalve or gastropod inhabiting the depth of 200‒400 m sea (both of 96.7% similarity; Brissac et al., 2010 and Zdinden et al., 2010). Thus, it was revealed that a group of uncultured Epsilonproteobacteria inhabits the gills of abalone in a shallow-water.

The intestinal tracts of marine fish harbor members of the family Vibrionaceae at high densities while obligate anaerobes have not been detected as major components in fish intestines using by agar plate methods. However, sulfate-reducing bacteria (SRB), obligate anaerobes, are known to be widely distributed in estuarine and coastal sediments. This fact suggests that intestinal tracts of coastal fish may be populated by SRB to some extent. Therefore, we investigated the distribution of SRB in intestinal tracts of coastal fish and their environments by the MPN method, real-time PCR technology and clone library analysis.
Marine fish and sediments were collected along the coast of Kanagawa and Shizuoka Prefectures, Japan. DNA was extracted from microbial cells in each sample and purified using a FastDNA SPIN kit (MP Biomedicals). The density of SRB was determined by the MPN method and real-time PCR technology targeting the dissimilatory sulfite reductase gene (dsrA). Moreover, the dsrA gene in each sample were amplified by PCR, cloned into the pGEM-T vector (Promega) and transformed into Escherichia coli DH5α. Partial clone sequences were analyzed by the ABI 3130xl sequencer and compared with all sequence data in DDBJ/GenBank/EMBL databases using the BLAST algorithm.
As a result, SRB was detected in about 18% fish specimens consisting of various fish species. Members of the families Desulfobacteraceae, Desulfobulbaceae, Desulfomicrobiaceae and Desulfovibrionaceae were detected in fish intestines. Additionally, Desulfohalobiaceae, Desulfobacteraceae, Desulfovibrionaceae, Desulfobulbaceae, Desulfomicrobiaceae and Desulfonatronumaceae were detected in the libraries constructed from sandy sediments. These results suggested that intestinal tract of coastal fish was sometimes populated by diverse species of SRB.

It is important to elucidate the outbreak mechanism of harmful red tide for the problem of the discoloration of cultivated seaweed, Nori, in Japan. We focused on essential trace metals such as iron and examined the relationship between harmful algal bloom and the concentration distribution of trace metals in Harima-Nada, eastern part of Seto Inland Sea. Samplings were carried out monthly at two stations in Harima-Nada from May 2015 to May 2016. We investigated the phytoplankton species composition and the concentration of dissolved trace metals (Mn, Fe, Ni, Cu, Zn, Mo) in surface and bottom water samples at the northern coastal station (Stn. H30) and the offshore site (Stn. H7) in Harima-Nada. Dissolved iron (DFe) concentration was measured by a long-path liquid-waveguide spectrophotometry using PDTS (ferrozine) method. The concentration of other five trace metals was determined by an inductively coupled plasma optical emission spectrometry after pre-concentration using a polyamino polycarboxylic acid chelating resin. At the examined stations, Eucampia zodiacus which is suspected as a causative species for discoloration of Nori appeared from mid-December 2015 to late March 2016. There was a significant negative correlation between cell density of E. zodiacus and concentration of DMn, DFe, DZn (r = -0.39, -0.52, -0.45 in surface water, -0.51, -0.52, -0.48 in bottom water) at Stn. H30 in the cultivation period of Nori. Whereas, there was no relationship between them at Stn. H7 where E. zodiacus appearance was little. The bioactive trace metals such as Mn, Fe and Zn are necessary for the growth of E. zodiacus. Therefore, the results in this study suggest that trace metals were consumed by E. zodiacus and may play an important role in forming harmful algal bloom affected to Nori in Harima-Nada. This work was supported by JSPS KAKENHI Grant Number JP26440248.

[Introduction]
Interaction between planktonic microorganisms and viruses has been recognized to be one of the keys in ecosystem functioning and biogeochemial cycles in the ocean. However, cultured host-virus systems are still limited, which hampered to understand their dynamics. Recently we succeeded in founding of 1,352 complete viral genomes from marine environments including Osaka Bay. Further our metatranscriptome analysis suggests locality of host-viral community. Here, we present seasonal populational structures of host microbes for 2 years in Osaka Bay as the first step of comprehensive understanding of ecological dynamics of marine microorganisms-viruses.
[Materials and Methods]
Seawater samples in Osaka Bay were collected almost monthly at a 5-m depth at the entrance of Osaka Bay, Japan, from May 2015 to December 2016 and in the Japan Sea on 25th June 2016. Seawater was fractionated eukaryotic (≧3.0 μm) and prokaryotic (0.2-3.0 μm) fractions. The DNAs extracted from eukaryotic and prokaryotic fractions were subjected to PCR amplification using a 616f-1132r primer set and a Pro341F-Pro805R primer set, respectively. The amplicons were sequenced with MiSeq.
[Results and Discussion]
Major taxa of prokaryotes were Proteobacteria, Cyanobacteria and Bacteroidetes and their compositions were stable during a 2-year sampling period. Focusing on Bacteroidetes, which is well-known as the player of degradation of phytoplankton derived-polysaccharides in the ocean, we found that 4 OTUs (Operational Taxonomic Unit) belonging to Flavobacteria always dominantly occurred. No relationships were observed between these 4 OTUs dynamics and environmental parameters or eukaryotic dynamics. Of these 4　OTUs, two showed similar seasonal dynamics, suggesting their ecological or physiological relatedness. Thus, our future task is unveiling factors including their viruses that define dynamics of the major OTUs in each taxon.

In a fish-farming area, organic load is applied to water bodies in the form of residual food and excreta of cultured fish. Therefore, understanding the degradation process of the organic load in the fish-farming area becomes important to ensure a sustainable aquaculture. In this study, we focused on a hydrolysis process of environmental proteins originated from aquaculture activities in the seawater and bottom sediments of the fish-farming area.
From January 2015 to March 2017, field surveys were carried out every odd month in the fish-farming area (water depth=17 m) in Tanabe Bay, Wakayama. Seawater samples were obtained at a depth of 1 m (surface) and 1 m above the seafloor (bottom layer). Bottom sediment samples were collected from 0-1 cm surface layer of the seafloor. Hydrolytic ectoenzymatic activities, trypsin and leucine aminopeptidase (LAPase), in the collected environmental samples were determined using quenching fluorescent substrates. The measurements of trypsin and LAPase activities were carried out from July 2016 and January 2015 to March 2017, respectively.
The trypsin activity in the bottom layer water tended to rise during the low water temperature phase. In the low water temperature period (from November to March), the average values of trypsin activity of surface water, bottom layer water and bottom sediments are 0.0948±0.0519 nmol/cm³/h, 0.0723±0.0519 nmol/cm³/h and 51.0±15.3 nmol/cm³/h, respectively. In the high water temperature phase (from July and September), those average values of trypsin were 0.0711±0.0532 nmol/cm³/h, 0.0192±0.00290 nmol/cm³/h and 67.7±0 nmol/cm³/h, respectively. The LAPase activity showed the same seasonal variation trend as trypsin activity. The protein degradation process seems to be driven in winter.

Of dissolved phosphorus (P) compounds in coastal waters, organic P as well as orthophosphate (Pi) sometimes represents a high proportion. Some planktonic algae, for example diatoms, dinoflagellates, and others, are known to be able to utilize organic P compounds such as phosphate monoesters (PMEs); however, there is no report showing algal “preferable” P source. This study evaluated P utilization of the marine planktonic diatom Chaetoceros tenuissimus using our modified quantitative ³¹P-NMR, and thereby, discussed the ecological roles of dissolved organic P in dynamics of this diatom. A clonal axenic strain (2-10) of C. tenuissimus was first cultivated in P-replete and P-depleted media. Their cultures were inoculated into the media where Pi, PME, phosphate diester (PDE), and phosphonate (Phn) were all present. By using our ³¹P-NMR-based method, we analyzed P variations quantitatively in the cultures. The P-replete C. tenuissimus cultures utilized Pi as primary P source and switched to a PME compound after exhausting Pi. Phosphomonoesterase (PMEase) activity appeared to be induced when C. tenuissimus began to utilize PME. In contrast, the P-depleted C. tenuissimus cultures degraded PME and PDE compounds simultaneously, released Pi from those by using two phosphatase activities, and then utilized the released Pi and original Pi without discrimination. Our results suggest that C. tenuissimus in coastal waters may be changing P sources (Pi from/to phosphate esters) dramatically in respond to its P stress and P composition in coastal waters.
This research was supported by Grant-in-Aid for Scientific Research (26850117 and 16K14962).

Nutrients, especially inorganic nitrogen (N) and inorganic phosphorus (P), are typically deficient in the coastal surface waters of temperate regions during warm seasons, and then, likely limit the marine primary production. In such environments, the marine planktonic diatom Chaetoceros tenuissimus often forms blooms and its blooms decline dramatically. The present study revealed the resting stage of C. tenuissimus in culture experiments and discussed the algal bloom dynamics in coastal waters. We prepared the NP-replete, N-depleted, and P-depleted cultures of C. tenuissimus (clonal axenic strains, 2-6 and 2-10), cultivated them at 20°C under 0-h light:24-h dark (D) and 12-h light:12-h dark (LD) photo-cycles for over two months, and transferred them to NP-replete and the LD photo-cycles. The NP-replete and P-depleted cells of C. tenuissimus were dead under both D and LD conditions. Even when be transferred to LD and NP-replete conditions, no cells occurred. By contrast, the N-depleted cells, because having slight chlorophyll fluorescence, appeared to be alive in D conditions. These cells’ shape were, spherical and thick silicate-walled, different from vegetative cells’ that. The un-normal cells grew when be transferred to LD and NP-replete conditions. These results suggest that C. tenuissimus has a resting stage induced by N-depletion and light deficient. This resting stage is probably important in survival of C. tenuissimus in N deficient environments.
This research was supported by Kurita Water and Environment Found Education (16B043).

Coastal ecosystems in Chonburi province in the, eastern coast of the Gulf of Thailand have been threatened by the various anthropogenic activities such as fishing, human settlement, recreation and tourism that significantly contribute to the national economy of Thailand. Plastic pollution is one of serious environmental issues in these coasts and it negatively affects intertidal sessile biotic communities. Hence this study was focused on the detection and quantification of impacts on Rock Oyster, Saccostrea cucullata by plastic pollution in three selected beaches (Angsila, Bangsaen, and Samaesarn) in the eastern coast of Thailand. Rock Oyster samples were randomly collected from the littoral zone and purified samples were digested using concentrated nitric acid. Micro-Raman spectrometer was used to detect micro-plastic diversity in the samples and found that rock oysters were contaminated at an average rate of 0.37-0.57 particles/g in all 3 study sites. ANOVA revealed that the accumulation rate was significantly different among the three sites (p<0.05). Oyster samples collected from Angsila had the highest concentration of micro-plastic particles (0.57± 0.22) reflecting severe plastic pollution with high anthropogenic disturbances. Majority of detected micro-plastics were polyamide nylon (in AngSila-70.59% and Samaesarn-61.54%) and polyethylene particles (in Bangsaen -63.54% and Samaesarn-38.64%). Sessile invertebrates in intertidal habitats are highly susceptible to accumulation of micro-plastic particles during filter feeding. Ingested micro-plastic types and accumulation rate have strong correlation with severity of plastic pollution in these coastal habitats. Contamination levels of this invertebrate biotic group can be used as an indicator of health status of the respective coastal area. The need for immediate attention of plastic pollution management programmes focusing on site specific management priorities is emphasized in this study.

Marine debris, particularly plastic, forms a global environmental concern for coastal areas, but knowledge for remote areas is limited. Palau is located in the western Pacific Ocean, where virtually no information about plastic pollution is available. Here we present results from the first study to quantify the types, amounts and possible origins of marine debris, and the presence of microplastics on sandy beaches in Palau. Surveys were conducted from Sep. to Oct. 2016 on 7 sandy beach sites. We sampled a circular perimeter (radius:2.5m) at the high tide line (TL) and at the high strandline on the back shore (SL) of each site, and recorded the type and number of macro debris (> 5mm) in the sample perimeter. The country of manufacturing was also recorded if possible as a proxy for the origin of the item. We also sampled microplastics at the TL and SL of each site, by applying a 30 x 30 cm quadrat (20cm depth). Microplastics were identified using density separation, visual sorting under a binocular microscope and FT-IR. A total of 490 pieces of macrodebris were collected from 7sites, of which most (98%) were plastics, specifically, plastic bottles/cups/fragments (69%), polystyrene foam (25%) and resin sandals (4%). Types and numbers were higher at SL than TL in all sites. A total of 39 macrodebris, all of which were plastics, were considered to be from outside of Palau, with 30 of them from Indonesia. 192 microplastics were collected from 6 sites. Concentration varied from 0.33 to 29 l^-1 sand, and higher at SL than TL. Sites with higher macroplastic concentrations generally showed higher concentrations of microplastics. These preliminary results suggest that macroplastics could be used as a rough proxy for microplastics allowing for low cost monitoring of microplastics.

FXYD proteins are the regulators of sodium-potassium ATPase (NKA). In teleosts, NKA is a primary active pump that provides the driving force for driving many ion transport systems in osmoregulatory organs (e.g. intestines). The expression and functions of FXYD proteins in mammals and elasmobranches have been widely studied, and those of teleostean FXYD proteins were investigated in gills of limited species. Hence the purposes of the present study were to investigate the expression of the FXYD proteins and NKA α-subunit in intestines of two closely related medakas (Oryzias dancena and O. latipes) from different natural habitats to illustrate the association between NKA and FXYD proteins of two medaka species in response to salinity changes. The results showed that the fxyd12 mRNA level was the highest among all detected fxyd genes in intestines of both medakas. The association between FXYD12 and NKA was demonstrated via double immunofluorescent staining and co-immunoprecipitation. Upon salinity challenge, in addition, the expression profiles of intestinal FXYD12 and NKA (mRNA and protein levels) as well as NKA activity were similar. These results implied that FXYD12 may play a role in modulating NKA activity in intestines of the two medakas in response to salinity challenge for maintaining internal homeostasis. The present study further extends our understanding of the expression and potential role of FXYD12, the regulators of NKA, in vertebrates.

The physiological function of fish saccus vasculosus (SV) has long been unknown. Recently, very interesting findings have been reported regarding the masu salmon, showing that SV is a sensor of seasonal changes in photoperiods and has an important role in reproduction. However, there have been no reports on the physiological functions of SV in other fish species. In this study, to clarify the photoperiodic and physiological roles of SV in the Japanese eel, we examined the presence of rhodopsin family genes and key genes regulating seasonal reproduction in the SV. Genetic analysis using next-generation sequencing revealed that 14 different photoreceptor genes, but not long wavelength-sensitive opsin (lws) and short wavelength-sensitive opsin 1 (sws1) genes, are expressed in the brain. Using q-PCR, we found that photoreceptors, such as short wavelength-sensitive opsin 2 (sws2, blue sensitive), middle　wavelength-sensitive opsin (rh2, green sensitive), freshwater rhodopsin (fwo), deep-sea rhodopsin (dso), and neuropsin (opn5) were expressed, and among them sws2 was highly expressed in the SV. sws2 was abruptly upregulated in the entire brain and the SV of sexually matured female eels induced by hormonal treatments. Moreover, the expression of sws2 increased from summer to winter in male eels reared in the outside pond. In addition, histological analysis demonstrated that SV consists of two types of cells, coronet cells and supporting cells, in the epithelium. Immunocytochemical analysis revealed that both thyroid-stimulating hormone beta subunit (tshβ) and glycoprotein alpha subunit (GPα) were specifically expressed in the coronet cells. Moreover, we found that iodothyronine 5'-deiodinase (dio2), tshβ, and, gpα were expressed in the SV. These results indicate the possibility that the eel SV has a physiological role as a photoreceptive and photoperiodic organ.

Potassium ion (K+) is the most abundant cation in the body; however, little is known about the regulatory mechanisms of body K+ level in teleosts. Recently, it is reported that ROMKa expressed in branchial ionocytes is responsible for K+ excretion to cope with chronic K+ load under seawater conditions. Substantial branchial ROMKa gene expression was also detected in freshwater-acclimated fish, suggesting that ROMKa-mediated K+ excretion is also functional even in low K+ environments. Teleosts in freshwater conditions are likely to depend on feed rather than environmental water as a major K+ source due to very low K+ levels in freshwater. Therefore, freshwater fish have to deal with acute and temporal K+ load caused by food intake. In this study, we investigated changes of branchial ROMKa gene expression in response to feeding and its regulating factors in freshwater-acclimated Mozambique tilapia.
Time-course analysis in freshwater fish after feeding revealed that plasma K+ and branchial ROMKa transcript levels were promptly elevated at 3 h after feeding. The observed increase was lasted for 12 h, and then returned to basal levels at 24 h. In contrast, K+ concentration in the muscle was stable during the experimental period, suggesting that contribution of intracellular K+ capacity to plasma K+ regulation is not so significant.
Short-term (3 h) gill incubation in media with various K+ concentrations showed that extracellular high K+ (8 mM) evoked a significant ROMKa gene expression increase compared to normal K+ (4 mM), whereas low K+ (2 mM) did not affect its expression. These results suggest that ionocytes sense the increase of plasma K+, and stimulate ROMKa gene expression in a cell-autonomous manner. Since Cs+ shares the K+ excreting pathway, K+-supplemented feed is an effective countermeasure against potential problems related to radioactive cesium in freshwater aquaculture.

Petroleum residues from industrial and urban effluents, oil spills, and disasters represent the major sources of polycyclic aromatic hydrocarbons (PAHs) in estuaries and marine coastal areas. PAHs are composed of multiple benzene rings. For example, benzo(a)pyrene (5 rings) is a widely studied PAH known to be carcinogenic. Though light PAHs (2 and 3 rings) could also be toxic to fish, little is known about their effect on health of fish. In the present study, we examined stress- and redox-related biomarker expression in response to a dietary light PAH, anthracene (Ant), in coho salmon (Oncorhynchus kisutch). Fish received Ant orally (8 mg/BW 100 g/day) for two weeks. The percentage of liver to body weight (hepatosomatic index; HSI) and plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities showed no significant differences between the control group and Ant-treated group. Expression levels of heat shock protein 70 in liver and muscle of the group fed a diet containing Ant were observed to be significantly higher than those of the control group. The total glutathione levels in the gill and plasma of Ant-fed fish were higher than those in control fish. Plasma protein levels in Ant-fed fish were also higher than those in control fish, but there were no significant differences in plasma lipid (cholesterol, phospholipid and triglyceride) levels between the fish fed Ant and the control fish. Accordingly, Ant-induced stress might increase the metabolic mobilization of GSH due to consumption from scavenging oxidants generated by stress. These results concerning changing patterns of biomarkers suggest that Ant fed to coho salmon induces oxidative stress, which might result in damage to tissues. The present results also suggest that stress response to Ant in fish might be dependent upon tissues examined.

[Objective] To produce good quality eggs is a prerequisite for stable seed production. However, the molecular signature for and mechansims resulting in good egg quality are little understood. Among various maternal factors that accumulate in the eggs, maternal mRNA is an essential one for ensuring normal development. In this study, we aimed to reveal the relationship between maternal mRNA abundance, localization, and egg quality in artificially matured Japanese eel, Anguilla japonica.
[Method] High-throughput RNA sequencing was performed using good (n=3) and developing poor quality eggs (fertilizable but not developing normally, n=3). RPKM or FPKM values were calculated by mapping sequencing reads onto contigs obtained from de novo assembly or draft genome sequences, respectively, and the relationship between mRNA levels and obtained hatching rate were analyzed by multiple regression analysis. In addition, intra-egg localization of grip2, dazl and pou5f3 were investigated by in situ hybridization to assess the correlation with resulting fertilization and hatching rate.
[Results] Multiple regression analysis found that levels of 5 transcripts (gnpat, b4galnt1, acsl6, rtkn, trim24) correlated positively with hatching rate. In addition, in situ hybridization revealed that grip2 and dazl localized to the vegetal pole of the egg, whilst pou5f3 transcripts were seen tightly packed along the peripheral margin of the animal pole. Interestingly, when the distribution of these transcripts showed a broader distribution pattern, then the fertilization and hatching rates tended to be decreased, even though quantitative differences between good and poor quality eggs were not detected. These results suggest that the levels of a subset of 5 mRNA may correlate with hatchability. Furthermore, the localization pattern of certain mRNAs in the oocyte is possibly a cause the loss of egg quality.

[Objective] 11-Ketotestosterone (11KT) is the most potent androgen of fish. In females of some fish species, such as eels, 11KT is found at high concentrations in the serum and plays a role in lipid incorporation into the oocytes. However, the tissue responsible for the production of 11KT has remained unknown. This study aimed to identify whether the ovary possesses the ability to produce 11KT in Japanese eel. Furthermore, the potential synthetic pathway toward 11KT production was investigated.
[Method] Eel ovaries in late-vitellogenic and migratory nucleus stage were incubated with testosterone (T), 11β-hydroxyandrostenedione (11β-OHA4) and adrenosterone at 20 °C for 18 hours. After incubation, steroid metabolites extracted from the media were analyzed using liquid chromatography-mass spectrometry (LC/MS).
[Result] After incubation, 1.8% and 1.6% of T was conberted to 11β-OHA4 and 11β-hydroxytestosterone(11β-OHT), respectively,but 11KT was not detected. At the same time, 10% of T was converted to androstenedione, which suggests that the eel ovary has strong 17β-hydroxysteroid dehydrogenase activity for T. In incubations with 11β-OHA4, 1.4% of 11β-OHA4 was converted to adrenosterone but 11β-OHT was not detected. If adrenosterone was added to the incubation, the eel ovary converted 17% of adrenosterone to 11KT, suggesting strong 17-ketosteroid reductase activity for adrenosterone. This in vitro study suggested that eel ovary has the ability to produce 11KT. The pathway to reach 11KT through androstenedione, 11β-OHA4 and adrenosterone is evident, however its synthesis via testosterone and 11β-OHT has not been ruled out in this study.

Elasmobranchs display a wide variety of reproductive modes including viviparity and oviparity. Viviparity is further grouped into two types: a type of placental viviparity and a type of histrophy. Fetus of placental viviparity type is connected to an external yolk sac with a yolk stalk in early stages. During these stages yolk is transferred to the fetal intestine though the yolk stalk and is used for fetus growth. After the implantation the ductus vitellointestinails undergoes degeneration. Then the vitelline artery and vein develop and the yolk stalk forms an umbilical cord like structure. The function and the structure of the yolk stalk in the histrophy type, however, are still unclear. In this study we compared anatomical and histological structures of the yolk stalk in the placental viviparity and the histrophy types, using the Japanese stingray Dasyatis akajei and the Tiger shark Galeocerdo cuvier for the former type, and the Silvertip shark Carcharhinus albimarginatus for the latter. In the Silvertip shark, we also made a histological observation on the umbilical cord like structure. In the Silvertip shark fetus of preimplantational stages, the yolk stalk contained the vitelline artery, the vitelline vein, and the ductus vitellointestinails. The epithelia of the ductus vitellointestinails were columnar cell equipped with microvilli, and under the epithelia were the developed capillaries, suggesting an absorption function of yolk material. Since the structure of yolk stalk mentioned above was common to three different viviparous species, the Japanese stingray, the Tiger shark, and the Silvertip shark, it is likely that the structure is common in viviparous elasmobranch species. In contrast to the mammalian umbilical cord which contains two veins and one artery, only one vein and an artery were observed in the umbilical cord like structure of the fetus of Silvertip shark with developed placenta like organ (total length, 80cm).

【Objectives】The number of chum salmon (Oncorhynchus keta) returning to rivers in the Sanriku-region, northern Honshu, Japan has been decreasing since 1999. Marine environmental change including rise of water temperature associated with climate change and retention of coastal warm water mass is thought to be one of possible causal factors of such decrease. However, no information for molecular response of chum salmon against chronic thermal stress has been available so far. We investigated the changes in muscle protein abundance of juvenile chum salmon in response to thermal acclimation.
【Methods】Juvenile fish (8 months old) were acclimated at 10^oC and 18^oC (n=7, respectively) for 30-days. Soluble fraction of muscle proteins was separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins that displayed significantly different expression between acclimated groups were identified with LC-MS/MS analysis.
【Results】Comparative analysis of 2D-PAGE revealed 31 differentially abundant spots between two acclimated groups. Representative results are as follows. (1) The abundance of three proteins related to heat-shock response increased in the warm-acclimated group. As heat-shock protein consume ATP for chaperon activity, this appeared to rise the energy cost to maintain physiological function. (2) The expression of precerebellin-like protein that was one of acute phase proteins in teleost significantly increased in the warm-acclimated group. This acute phase protein may be induced by tissue injury with thermal stress. (3) The abundance of two glycolytic enzymes increased in the warm-acclimation group, which probably enhance an anaerobic ATP generation. On the other hand, electron transfer flavoprotein subunit α abundance increased in the moderate-temperature group, which may enhance energy production of lipid catabolism. These findings suggest an increased energy requirement in the 18^oC-acclimated fish, providing a clue to understand the molecular mechanism of thermal tolerance in chum salmon.

Vitellogenin (Vtg) is a main precursor of egg yolk proteins, which is synthesized in the liver of reproductive female by the stimulation of estradiol-17β (E2). Multiple Vtg proteins and/or transcripts have been identified in teleosts. They are typically classified into two or three subtypes. Insulin-like growth factor I (IGF-I) seems to regulate E2 synthesis in teleosts including salmonids, however the role of IGF-I for synthesis of multiple Vtg is unknown. The objective of this study is to measure mRNAs of dual Vtg subtypes (VtgAs and VtgC) in rainbow trout liver tissue exposed to E2 and/or IGF-I in vitro.
A cDNA encoding rainbow trout VtgC was cloned from the liver of E2-injected male. The sequence information of rainbow trout VtgAs was obtained from GenBank. qPCR for dual Vtgs were developed using KAPA SYBR FAST One Step qRT-PCR Kit. Following dissection of livers from mature male or immature female, they were cut and pre-incubated for 24 h in a culture medium. The incubation with E2 (10^-9 and 10^-8 M) and/or IGF-I (10^-8 M) were carried out in 24-well culture plates for 24 h at 10℃.
The levels of both Vtg subtypes in cultured immature female liver tended to decrease in groups of E2 alone or IGF-I alone comparing to the levels in a control group, while they tended to increase in E2 and IGF-I combination group comparing to the single administration groups. Both Vtg subtypes in cultured mature male liver tended to increase in single groups comparing to the control, while they decreased in combination group comparing to the single groups. These data suggested that transcriptional patterns of trout dual Vtgs seemed to be synchronous regardless of single or combination of hormone stimulation, while the responses were rather changed depending on gender and/or maturity.

Stranded coastal marine animals generally caused by a cold wind or a violent typhoon were reported in Japan. Although stranded tropical marine fishes and/or invertebrates were often found in Ryukyu Islands, southern Japan, during winter season, few reports were published in detail about such stranding phenomena. On 23–25 January 2016, Ryukyu Islands suffered extremely low temperatures by a serous cold wave coming from the Arctic, and a lot of fishes including many marine eels were stranded along the coasts of Ryukyu Islands (Amami-oshima, Okinawa, Miyako, Ishigaki and Iriomote islands). The present stranding phenomena may be caused by not strong winds but mainly extremely low temperatures on the bottom of the sea at the very low tide related to the spring tide because they could not be found frequently in the former reports caused by a cold wind or a violent typhoon. Additionally, the inference can be supported by that benthic marine eels and invertebrates dwelling sandy-bottom were stranded at the same time. Those stranded eels were found as very fresh conditions. From these facts, they were inferred to be stranded near their habitats. During this cold wave, we collected more than 117 individuals of stranded marine eels belonging to 21 species and four families. Three species are recognized as new records in Japanese water.

Myanmar estuaries are important area for fresh water fauna. We examine species compositions and abundance of freshwater shrimps in Myanmar estuary. Total 1290 shrimps were collected from river, canal and aquaculture pond around Yangon region and Ayeyarwaddy region. We have morphologically classified these shrimps that are called "Palaemonid Prawn".

The 90 samples on Too river, Ayeyarwaddy region in 2014/12/27
FAMILY: Palaemonidae
GENUS: Macrobrachium

Approximately 800 samples on aquaculture pond in 2014/12/27
FAMILY: Palaemonidae
GENUS: Palaemon or Macrobrachium

Approximately 300 samples on Twante Canal, Yangon region in 2016/1/16
FAMILY: Palaemonidae
GENUS: Palaemon or Macrobrachium

Haplotype analysis of the cytochrome oxidase subunit I (COI) of mitochondrial DNA was conducted to detect species using 5 individuals that were obtained on Twante Canal in 2014 and 2016.
A 680-bp segment of COI from 2 individuals was amplified and aligned. Both studied segment of COI was almost corresponded to the segment in Macrobrachium rosenbelgii (homology: 99%) using BLAST of DNA Data Bank of Japan (DDBJ) service.

In order to improve the efficiency of stock enhancement programs for the sea urchin Hemicentrotus pulcherrimus, information on food alga species, which affect growth and gonad production, is necessary. As the sea urchin breaks food macroalgae into small pieces by their teeth, macroalgal fragments in the guts are not informative to identify species with conventional microscopic observation. DNA barcoding method is a useful tool to identify species from a tiny macroalgal fragment, and we applied this method to investigate feeding habits of the sea urchin. We collected the sea urchin at random by skin diving in Mikuni and Sekumi, in Fukui Prefecture, Japan from 2013-2016, and investigated the composition of gut contents and gonad index (GI) of the specimens. Seaweed vegetation at the study sites was also recorded during the field sampling survey and in 2017. In addition, a laboratory experiment was conducted to examine the maximum GI value by feeding artificial diet for abalone aquaculture to the sea urchin. There were extensive Sargassum beds and no barren ground was observed in the study sites. Sargassacean species accounted for 19-60% in the guts of Sekumi specimens, but only a few sargassacean species were identified in the guts of Mikuni specimens. Sargassum hemiphyllum and Myagropsis myagroides were abundant in the guts of Sekumi, but S. hemiphyllum was not dominant in the study site. GI values of Sekumi specimens fluctuated from 6.3-15.4, and they were higher than those of Mikuni (GI: 6.5-10.7). GI value of the feeding experiment was up to 22.6 in a month. These results indicate that the sea urchin has a potential ability to increase their gonad, and food availability may affect gonad production in natural environment. Further studies on food availability will provide useful information to improve the efficiency of the stock enhancement.

A large number of Akoya pearl oysters are produced in the Uwa sea of Ehime, Japan, and their production is affected by environmental conditions. We investigated the effects of environmental conditions on the growth of pearl oyster cultured inshore and offshore in the same bay. The results showed that oysters cultured offshore grew faster than the ones inshore until they reach a year old. At over one year old, however, offshore cultured oysters grew slower than the ones inshore. Although there is no major differences in water temperature and dissolved oxygen between the areas throughout the experimental period, the offshore tide current is faster than that of inshore at all times. Also, the offshore chlorophyll a quantity is lower than that of inshore at some period.
To obtain a wide range of information on the environmental conditions, we confirmed the brightness temperature computed from TERRA/ASTER and Landsat-8/TIRS by measuring water temperature at 2 m depth in the same point of the Uwa Sea. The brightness temperature depends on the atmosphere and emissivity from the sea. The root mean square error (RMSE) of the ASTER data acquired on July 29, 2014, September 8, 2014, October 4, 2015 and March 28, 2016 were 0.842, 1.595, 0.246 and 1.578 ℃, respectively. The sea surface temperature was estimated from the data of bands 10 and 11 of TIRS by measuring water temperature. The RMSEs of the estimation derived from TIRS were within approximately 0.7 ℃. Hence, the estimation of sea surface temperature from the brightness temperature is crucial to practical ocean information system. Furthermore, the relationship between the ocean current and temperature will be analyzed. This can provide further information on ocean conditions which will be useful for pearl oyster culture.

The pearl culture using Japanese pearl oyster, Akoya, is one of the most important traditional marine industries in Japan. The characteristics of a cultured pearl are influenced by two kinds of pearl oysters: one is the donor pearl oyster, which provides a small piece of mantle to be transplanted, the other is the recipient pearl oyster, in which the pearl nucleus and a small piece of mantle are transplanted into the recipient’s gonad to produce a pearl. Therefore, it is expected that the sex of recipient’s pearl oyster and the quality of pearl they produced were greatly related. In our previous report, the sex of pearl oyster directly affected the quality of pearl.
To investigate sex change in Akoya pearl oyster, we examined the sex of pearl oyster. A small fragment of the gonads from 1-year-old pearl oysters was collected by biopsy. Using the collected part, the sex of the oysters was determined by microscopic observation, and then the remaining samples were stored for gene expression analyses. All pearl oysters were then cultured and checking of sex was performed every 4-months for 2 years. At the start of experiment, almost all pearl oysters were male, and the ratio of male decreases every survey. However, the male ratio increased past the spring, such as during breeding season. It is suggested that sex of pearl oyster was affected by seasons. The expression analysis of sex-related genes (<Dmrt2>, <Vgt>, <Zp>) indicated that all genes were expressed in all individuals and all period. These results suggested that Akoya pearl oysters were hermaphrodite, and females emerged at necessary time such as breeding season. We would like to take advantage of these characteristics and make more pearl culture efficient and produce high quality pearls.

Crassostrea angulata is the major commercial oyster species in Taiwan. The oyster is an inter-tidal filter feeding bivalve mollusk such as suspended particulate matters and phytoplankton in sea water. However, this species is cultured in western coast of Taiwan. The wild populations are distributed in middle to south coast. The effect of environmental condition upon life span in oyster can be pronounced. Evidently, its variations in the spawning period may constitute a decisive factor in spawning. The nearshore marine ecosystem of Taiwan are often severely affected by climate such as Typhoon, Mei-Yu(plum rain). This study surveyed the morphological pattern and physiological response to abiotic and biotic factors on seasonal dynamics of C. angulata from time series samples in coast area of Taiwan. The overall of individuals sex ratio showed no-significant variation in years. The oyster is gonochoristic, few individuals did not succeed in detecting sex determinant. The size-class distribution and gonadal development of the oyster displayed two spawn periods in April later spring and August to October. Based on histological analysis, the gonad of female oyster showed most of ovum had been rapid growth in two periods April and July and the gonad of female oyster C. angulata were characterized by the shrinkage of the follicles from which some of the ovum had already been discharged in October-November, after November, the gonad showed some cavity at the end of spawning in winter and small in size. In the study, some wild group of oysters which was kept in the inter-tide zone. The oyster influence by the flood-ebb tidal fluctuated was to permit them to spawn normally. The marine ecosystem often severely affected by climatic events such as Typhoon, Mei-Yu(plum rain). The driving force shaping the life history of oyster appears as a response to environmental variability.

Marine finfishes and crustaceans require highly unsaturated fatty acids (HUFAs) of the n-3 series such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) for normal growth and survival. In this research, we focused on jellyfishes which are recently prevailing and being raised as a popular aquarium animal.
Aurelia aurita s.l. is a common species in Japan and is easy to be reared. We carried out a series of experiments to examine its fatty acid requirement in laboratory.
Ephyrae of A. aurita were obtained and isolated from a stock culture of polyps. After releasing ephyrae it took about a month to rear young A. aurita. Artemia nauplii were nutritionally fortified by direct method using test oils containing different fatty acids and given to ephyrae every day. The change in bell diameter of test animal was measured under a stereomicroscope once every week. The enriched Artemia nauplii were analyzed using a gas chromatography after the completion of rearing experiment.
As results, DHA promoted growth of A. aurita, but linoleic acid and α-linoenic acid didn't affect to the growth. Also Artemia nauplii enriched by the oil containing EPA/DHA = 1 showed better growth than that of sole use of DHA group. As for survival, high rates were always noted in all treatments irrespective to fatty acids compositions.
These experimental results suggest that the fatty acids requirement of A. aurita is similar to that of ordinary marine fishes and require n-3 HUFAs. Without the presence of n-3 HUFA, A. aurita can survive for only 1 month from release and die.

Rational: Fishery production of the asari clam Ruditapes philippinarum has decreased severely in Ise Bay. Understanding the genetic structure of juvenile asari clam population would help manage the resources. Materials and Methods: The juvenile clams (shell length <10 mm) were sampled at 5 or 6 sites in Ise Bay and 1 site in adjacent Mikawa Bay in May and November 2013 and 2014. Total DNA was extracted from 24 individuals from each site at each sampling. The microsatellite analysis was performed on 5 loci (Asari16, Asari62, Asari64, KTp8 and RpN15). The analysis of molecular variance (AMOVA) were done using the software ARLEQUIN. Assignment analyses were done using the software STRUCTURE. Genetic relationship among the populations was estimated with the software PHYLIP using the neighbor-joining method. Results: Significant genetic differentiations were observed among the juveniles collected in November 2013 (global F_ST = 0.023, p = 0.00293) and those in November 2014 (global F_ST = 0.023, p = 0.00098). In contrast, genetic differentiation of juveniles collected in May were small (global F_ST = - 0.00029, p = 1 in 2013, global F_ST = - 0.00297, p = 1 in 2014). Assignment test estimated K = 3 as the most provable number for the populations in November 2013 and K = 1 in other samplings. In Ise Bay, the asari clam has two main spawning peaks: late spring to early summer and late autumn. The genetic structure revealed that the juveniles collected in May, born and settled in the previous autumn, were genetically more uniform among the populations than those collected in November, probably due to the dispersal of a single dominant population in autumn.
This study was supported in part by the Ministry of Agriculture, Forestry, and Fisheries of Japan and OKASAN-KATO FOUNDATION.

The regulatory mechanism controlling reproductive maturation in female crustaceans is complicated. It involves temporal and spatial coordination of various hormones and the consequential cellular events happened in the ovary. Due to the lack of comprehensive understanding of this mechanism, it remains a challenging task to rear female broodstock of economically important shrimp species, including Litopenaeus vannamei, under artificial conditions. In this study, the oligonucleotide microarray for transcriptomic analyses was employed to discover unidentified molecules participating in the ovarian growth in L. vannamei. A microarray with 29,918 probes on a single chip was constructed and the analyses were performed to compare the global gene expression profiles between immature and ripe ovary from adult L. vannamei. It was found that the transcript abundance of 418 genes was significantly increased (FDR: p < 0.05; FC: > 2), yet was considerably declined of 2,774 genes (FDR: p < 0.05; FC: < 0.5) in the ripe ovaries. Among those genes, Gamma interferon inducible lysosomal thiol reductase (GILTR) and Anti-lipopolysaccharide factor-like protein (ALF) showed exceedingly high expression levels while the expression levels of Neuroparsin 1 (NP1) and Thrombospondin (THS) were remarkably low. Consistent findings were observed from PCR-based analysis. RT-PCR reactions in which cDNA from immature or mature ovaries were used as templates showed that GILTR and ALF were only detectable in the ripe ovary but not in the immature ovary; on the contrast, NP1 was detected only in the immature ovary but not in the ripe ovary. Based on the combined results, GILTR, ALF and NP1 were found to be novel molecular markers for the evaluation of ovarian growth in L. vannamei. Future investigation of signaling cascades in which these proteins are involved may shade the lights of the reproductive regulation of ovaries and benefits raising female broodstock.

This study was performed to determine the efficacy of enrofloxacin in the treatment of bacteria in olive flounder. There is no analytical criteria that distinguishes the susceptibility among resistant strains of most fish pathogens including Streptococcus parauberis. Therefore, epidemiological cut-off (ECOFF) values for bacteria strains using normalized resistance interpretation (NRI) analysis was generated. Antibiotic susceptibility testing for enrofloxacin was conducted following CLSI guidelines (disk diffusion and broth) using six bacterial species (48 Streptococcus parauberis, 52 Edwardsiella tarda, 44 Photobacterium damselae, 54 Vibrio harveyi, 45 Streptococcus iniae and 40 Aeromonas salmonicida strains). Wild type cut off values for enrofloxacin in S. parauberis, E. tarda, P. damselae, V. harveyi, S. iniae, and A. salmonicida were ≥ 27, 16, 27, 14, 21, 37 mm, respectively. Minimum inhibitory concentration was determined only for 48 strains of S. parauberis. For efficacy of enrofloxacin against streptococcosis caused by S. parauberis, each group of 10 fish were intramuscularly injected with 0, 1, 5 and 10 mg enrofloxacin per kg fish body weight after fish were challenged with S. parauberis subcutaneously at a final concentration of 1×105 CFU mL-1. Antibiotic treatments were initiated at 1 day post challenge (dpc). Dead and moribund fish were removed and examined microbiologically for 3 weeks. Survived fish were used to check the presence of S. parauberis in the kidney and blood. As a result, the mortality of each group treated with 1, 5, and 10 mg kg-1 was 40, 10 and 0 %, respectively. Repeating experiments will be needed to determine precise effective dose of enrofloxacin for streptococcosis.

This study aimed to evaluate the effects of Pseudoalteromonas piscicida 1Ub and mannanoligosaccharide (MOS) supplementations through Artemia sp. on the growth performance and the health status of Pacific white shrimp (Litopenaeus vannamei) larvae. Bioencapsulation was performed by adding P. piscicida 1Ub at a concentration of 10⁶ CFU/mL, MOS at a concentration of 12 mg/L, and synbiotic (P. piscicida 1Ub at a concentration of 10⁶ CFU/mL and MOS at a concentration of 12 mg/L) to the rearing medium of Artemia sp. for 4 h. The administration of bioencapsulated Artemia sp. to shrimp larvae was conducted starting from Mysis3 to PL12. The results of this study demonstrated that daily growth rate (DGR), absolute length, RNA/DNA ratio, digestive enzymes activities (protease, lipase, amylase, and mannanase), survival, and total bacteria in shrimp larvae fed with bioencapsulated Artemia sp. were significantly higher (P<0.05) from those in control. Immune responses of Pacific white shrimp larvae (total haemocyte count, phenoloxydase, and respiratory burst) fed with bioencapsulated Artemia sp. were also higher (P<0.05) than those of control. The administration of synbiotic resulted the highest DGR (24.39±0.31 %/day), absolute length (13.00±0.50 mm), RNA/DNA ratio (0.6369±0.0094 ng//µl), digestive enzymes activities (protease 0.033±0.0007; lipase 0.047±0.0010; amylase 0.853±0.008; and mannanase 0.148±0.004 U/mL/minute), survival (92.67±1.26%), total bacteria (6.7 x 10⁷ CFU/larvae), total haemocyte count (7.6 x 10⁶ cells/mL), phenoloxydase (0.188±0.002 OD 490 nm), and respiratory burst (0.668±0.028 OD 630 nm). In conclusion, the supplementation of P. piscicida 1Ub, MOS, and synbiotic through Artemia sp. bioencapsulation could improve the growth performance and the health status of Pacific white shrimp larvae with the best results showed by the supplementation of synbiotic.

Outbreaks of viral and bacterial diseases along with high mortalities have threatened the aquaculture for years. To date, disease problems still impact the development of industry continuously. In order to seek an eco-friendly disease prevention strategy, the use of dietary supplements derived from plants has accelerated in recent years. Herein, the antiviral activity and bactericidal effect of two natural products, Rhamnan sulphate (RS) from Green laver (Monostroma latissimum) and Oregano essential oil from Origanum vulgare against important fish pathogens will analyze respectively. Groupers (Family Serranidae) are viewed as one of the important maricultured fish species in Asia-Pacific region. Therefore, grouper will use as a model fish in this study. First, the cytotoxicity effect of these two products was determined on grouper kidney (GK) and grouper fin (GF-1) cells, and appropriate concentration range was selected for following experiments. Subsequently, the antiviral activity of RS and Oregano against grouper viruses was confirmed by MTT tests and inhibition of GIV replication. However, antibacterial effect against Streptococcus agalactiae, S. iniae, Vibrio brasiliensis and V. parahaemolyticus varied of these two products.

【Objectives】The study investigated the effects of different levels of dietary vitamin C on growth and lysozyme activity of Nile tilapia (Oreochromis niloticus) in relation to nursing duration (short duration, normal juvenile (NJ) and prolonged stunted juvenile (PJ)) and sex.
【Materials and Methods】Semi-purified diets (40% protein) supplemented with four different levels vitamin C (0, 420, 840 and 4200 mg/kg) were prepared and fed to triplicate groups of tilapia (mean weight : 7.37 g and 11.31 g for NJ and PJ, respectively) for five weeks. Kidneys from the sacrificed fishes were sampled to determine the lysozyme activity following turbidimetric assay system.
【Results】On growth performances, vitamin C had a significant effect (p < 0.05) and the highest growth had been achieved in fish fed 420 mg/kg vitamin C, while prolonged nursing had no significant effect (p > 0.05); which might be associated to the short life cycle of tilapia as compared with other freshwater fish like carps. Supplementation of vitamin C had no significant effect on kidney lysozyme activity of both NJ and PJ, although NJ showed little increased lysozyme activity with increasing the dietary concentration of vitamin C. Nursing duration negatively affects kidney lysozyme activity (p < 0.05). Again, lysozyme activity significantly differs with sex, and female individuals showed higher activity compared with male counterparts. Although prolonged nursing of carp fry is a common practice in sub-tropics to achieve faster growth but this study revealed that this practice is not suitable for Nile tilapia and over dosages of vitamin C had no clear positive effects considering the growth and kidney lysozyme activity.

This investigation describes the potential of the phage for controlling experhmental 〈1〉A. veronii〈1/〉 infection in ranchu, a variety of goldfish 〈1〉Carassius auratus〈1/〉 and colored carp 〈1〉Cyprinus carpio〈1/〉. An 〈1〉A. veronii〈1/〉 strain was isolated from the kidney of a diseased ranchu, and the 〈1〉A. veronii〈1/〉 phage was isolated from the pond water collected at Fukuyama University were used in this study. The bacterial strain was cultured on trypto soy agar, and then the phage was propagated using trypto soy broth including the cultured strain. Ranchu weighing about 2 g and colored carp weighing about 16 g were maintained in aquarium at 23 ℃.
After intraperitoneal injection, oral administration or immersion of the phage to ranchu and colored carp, ten abdorminal scales of both fishes were removed, and they were challenged with the strain by immersion. Mortalities of both fishes injected with the phage intraperitoneally and control fishes were 0 and 100 %, respectively. By oral administration, respective mortalities of treatment fishes and control fisies were as follows: ranchu, 70, 100 %; colored carp, 20, 100 % (fish, treatment, control). Moreover, mortalities of both fishes immersed with the phage and control fishes were 100 and 100 %, respectively.
These findings indicate that intraperitoneally or orally administered the phage prevented ranchu and colored carp from experimental 〈1〉A. veronii〈1/〉 infection, and suggest that the potential use of phage for controlling the disease. However, the effect of phage immersion treatment was not found, further investigations are required to establish phage therapy of the disease.

By previous our study, Lactococcus lactis strain K-C2 showed antagonistic activities against various pathogens in aquaculture such as Edwardsiella tarda, Lactococcus garviae and Streptococcus iniae. Therefore, this strain is considered as novel probiotics to prevent disease in aquaculture. In this study, we tried to enrich L. lactis strain K-C2 in the feed organisms, Artemia sp. Commercial eggs of Artemia (Pacific Trading Co. Ltd., Fukuoka, Japan) were hatched in artificial seawater under the vigorous aeration and light irradiation. At 4 days after hatching, the Artemia was introduced to 80 ml of artificial seawater in a flask (26 individuals/ml). Strain K-C2 was added to the seawater containing Artemia at 3.2×10⁸ cfu/ml. As a control group, Artemia was cultured without addition of strain K-C2. After exposure to the cell suspensions of strain K-C2, Artemia was harvested and washed by sterile artificial seawater and homogenized. The homogenates was serially diluted, and each dilution was inoculated on a GYP agar plate medium containing calcium carbonate. The plates were incubated at 28^oC for 4 days, and viable colonies were counted to calculate colony forming unit (CFU). The colonies which degraded the calcium carbonate were randomly selected, and colony PCR was carried out using Lactococcus specific primer set (Forward primer, LacF 5’ GTACTTGTACCGACTGGAT 3’ and reverse primer, LacreR 5’ GGGATCATCTTTGAGTGAT 3’) to detect the strain K-C2. The selected colonies were estimated as L. lactis by PCR with specific primers. The number of L. lactis in Artemia was calculated to 4.2×10³ CFU/ 100 individuals. This study indicated that probiotic L. lactis strain K-C2 could be enriched in Artemia by exposure in seawater.

This study focused on the utilization of hot spring water (HSW) to eel culture system from the viewpoints of the environmental-friendly aquaculture. Hot spring water was collected in Saito city, Miyazaki, Japan. Japanese eel, Anguilla japonica was reared in tap water (TW) or HSW in a closed recirculating system for 26 days. The water temperature was maintained at 27^oC with a heater and a thermostat. After rearing for 26 days, the body weight of the HSW group was significantly higher than that of the TP group. In the fatty acid composition of eel edible portions, no significance was observed between groups. The total bacterial count in both groups was maintained at approximately 10⁴ colony forming unit/ ml during 26 days of rearing. The HSW slightly contained salts (salinity, 0.9 psu), and the total dissolved solids and conductivity were significantly higher than those of TW. At day 0 after rearing, the concentration of ammonia-nitrogen in the HSW group was significantly higher than that of the TW group, and no significant differences were observed throughout rearing period except for day 0. In case of nitrite-nitrogen, no significance was observed except for days 0 and 3. After 26 days of rearing, the fatty acid composition of eel body did not show significant differences between groups. After rearing trial, the eels in each group were challenged by Edwardsiella tarda. The cell suspension of E. tarda was intraperitoneally injected into the eels (10⁸ cells/ fish). After 14 days, the survival rate of eels of the HSW group was 87.5% and significantly higher than that of the TW group (62.5%). Thus, this preliminary experiment indicated that the growth of eel was improved, and the resistance of eel against Edwardsiellosis was stimulated by usage of HSP as rearing water.

In recent years, a number of researches involving the development of functional foods that promote increased food efficiency and growth rate of fish have been intensified. The additives as prebiotics and probiotics could be a viable option to pathogenic agent’s prevention. Therefore, this work aimed to select probiotic bacteria for freshwater fish, isolated from the intestine and skin of Nile tilapia (Oreochromis niloticus). Firstly, the intestine and skin samples of Nile tilapia were aseptically collected from 30 different fish farms, located in Southeast (São Paulo State), South (Paraná State) and Northeast (Pernambuco and Bahia State) regions. These samples were transported in sterilized tubes with brain heart infusion medium. For the microorganism selection, the Petri plates with tryptic soy agar were inoculated by a platinum loop (10µl) and then incubated in B.O.D stove for 24 hours at 30ºC. The microorganisms were then isolated. These colonies were collected and preserved in microtubes containing 500 µl of tryptic soy broth and glycerol (4:1). All samples were stocked at -20ºC. For probiotic microorganism selection, were applied pH and bile salts resistance test and inhibition test against Aeromonas hydrophila. Until this moment, were collected 1,402 microorganisms in which 189 samples were tested and 49 bacteria resist to pH and bile salt resistance tests and one bacteria inhibi the Aeromonas hydrophila growth. After these tests, growth performance and immunological test will be carried out with possible probiotic microorganisms to confirm its applicability in tilapia aquaculture.

Aeromonas hydrophila and Streptococcus agalactiae are major bacterial pathogens attacking tilapia during fry to adult stage. The manipulation of maternal immunity transfer via broodstock vaccination is one alternative technique to anticipate the high mortality in fry. This research aimed to analyze the efficacy of bivalent vaccine of A. hydrophila and S. agalactiae in tilapia broodstock as well as the protection of their offsprings through maternal transfer of immunity. Broodstocks were vaccinated with whole cell vaccine at a dose of 10⁸ CFU/fish with different ratio of A. hydrophila and S. agalactiae of (P1) 0:100, (P2) 25:75, (P3) 50:50, (P4), and 75:25, (P5) 100:0 % (^v/_v), a control without vaccination (C), was also applied. Challenge tests for fry from vaccinated broodstock were performed with A. hydrophila and S. agalactiae co-infection at a concentration of 10⁶ CFU/mL for 30 minutes at fry ages of 7, 14, 21, and 28 days after hatching, respectively. Antibody level was determined by an ELISA. The results showed that relative percent survival of treatment P2 was higher than other treatments. Relative percent survival of fry from treatment P2 at age of 7, 14, 21, and 28 days after hatching were 83.80%, 71.76%, 64.35%, and 52.31%, respectively. Also it showed that hight level of antibody against A. hydrophila and S. agalactiae in treatment P2 in broodstock, egg, and fry. As a conclusion, bivalent vaccine with 25:75% (^v/_v) of A. hydrophila and S. agalactiae on tilapia broodstock could improve immunity and protect the fry from pathogenic A. hydrophila and S. agalactiae infection.

Viral and bacterial diseases still smite to shrimp aquaculture industry and cause serious economic less. In the present study, recombinant proteins were mostly applied to develop vaccine against diseases and animals were always vaccinated by injection. However, injection method is difficult to apply to aquaculture. Therefore, the development of oral delivery system will be the main objective for the future. Recently, Listeria monocytogenes was applied to human cancer treatment. The pathogenicity of L. monocytogenes was deleted and this system can be attempted to aquaculture. In this study, the mutated L. monocytogenes was used to develop oral delivery system. The results indicated that the expression of green fluorescent protein gene (GFP) increased in the primary cultured shrimp hemocytes which were treated with heat-killed L. monocytogenes containing GFP. Besides, GFP was also detected in the shrimps fed with heat-killed L. monocytogenes. It indicated that the inactivated L. monocytogenes can enter shrimp cells. Our results show that the attenuated L. monocytogenes is a good candidate to develop the oral delivery system in shrimp aquaculture.

Herpesviral hematopoietic necrosis (HVHN) causes widespread mortality in goldfish Carassius auratus and Gibelio carp C. auratus gibelio aquaculture industry. The agent of this infection is Cyprinid Herpesvirus 2 (CyHV-2). A formalin inactivated vaccine was previously reported to have moderate effect in reducing mortality of infected fish. We previously reported that serum neutralizing antibodies are important in protecting fish from the virus infection. Therefore, the purpose of this study is to develop more effective measures against CyHV-2 infection. We focused on developing DNA vaccines as they have potential ability to induce both cell-mediated and humoral immunity in fish. In this study, we attempted to determine membrane proteins of CyHV-2 that can react with serum antibodies of surviving fish as candidate antigens for DNA vaccines. Predicted open reading frames (ORFs) encoding type 1 membrane proteins of CyHV-2 SaT-1 isolate were amplified and cloned into bacterial expression vectors pET-32a(+) by homologous recombination in competent E. coli strain ME9783. Histidine tag was at the C-terminus of the gene. Successful insertions of some genes were confirmed by colony PCR and sequencing. They were purified and transfected into E. coli strain BL21 for protein expression induced by 0.5mM IPTG. Successful expressions of pORF30, 136 and 153C were confirmed by Western blotting with anti-His antibodies. They were also tested the reaction with serum antibodies of surviving goldfish by Western blotting with anti-ginbuna IgM mouse monoclonal antibody (B12) and anti-mouse Ig goat IgG conjugated with HRP. A distinctive band was observed for pORF136 at the range of ≈32kDa. Previously, homologous protein ORF136 of koi herpesvirus (CyHV-3) was determined as one of the structural proteins. Thus, ORF136 of CyHV-2 is expected to be a structural protein. This study shows that fish antibodies recognize the expressed protein of CyHV-2 ORF136. Therefore, it can be a possible candidate for DNA vaccine against HVHN.

This work aimed to evaluate the commercial probiotic AQUA-PHOTO (Lactobacillus plantarum and Bacillus subtilis) added in feed of stressed Nile tilapia juveniles (Oreochromis niloticus), on TNF-α, HSP-70 and IL- β genes expression related to the immunology and stress condition responses. The experiment was conducted in completely randomized design with four treatments: 1-) probiotic addition in diet, 2-) probiotic addition in water, 3-) probiotic addition in diet and water 4-) control diet; and three replicates. 40 juveniles were distributed in three aquaria (60L) with a recirculation system composed by biological filter, aeration and heaters with thermostat adjusted to 26ºC. For the control diet group, the ultraviolet light lamp was used to avoid possible contaminations. The probiotic (Lactobacillus plantarum - 1,0x10⁶ UFC g^-1 and Bacillus subtilis - 1,0x107 UFC g-1) was added at proportion of 0,2 g.100g^-1 in diet and 0,104 g.L^-1 in water. During 30 days, the fish were fed until apparent satiation with experimental diets three times a day (10:00, 12:00, 14:00). At the end of the experiment, intestine samples were aseptically collected, stored in cryotubes with RNA later^® and transported to the Department of Microbiology, Faculty of Science, University of Malaga, Spain. The RT-qPCR analyses of TNF-α, HSP-70 and IL- β genes expression were proceed. According to the results, the TNF-α expression differed significantly among the treatments (p<0.05). The probiotic inclusion in diet and water provoked higher TNF-α gene expression than control group, demonstrating the capacity of these bacteria in stimulate the Nile tilapia immunology. The addition of commercial probiotic AQUA- PHOTO may be a suitable alternative to promote immune system and can reduce the antibiotics use in Nile tilapia production.

Lymphocystis Disease (LD) caused by infection of lymphocystis virus is a serious problem in aquaculture of Japanese flounder. Fish infected with LD lose commercial value due to tumor-like nodules. As a countermeasure for this disease, we have successfully produced LD-resistant (LD-R) Japanese flounder strain using Marker-assisted selection. However, the molecular mechanism underlying LD-R is still unclear. To identify the causative gene controlling LD-R by the association study, the reference sequence and SNP information within LD-R regions are needed. Recently, the reference genome of Japanese flounder has been published using short reads of Illumina platform (Changwei et al., 2017). In this study, we performed whole genome sequencing and assembly using Illumina short reads and PacBio long reads to construct more high-quality reference sequence of LD-R region. Moreover, we analyzed sequence variants in LD-R region by genome re-sequencing of 9 individuals.
The reference sequence of LD-R Japanese flounder (N=1) was obtained using Hiseq2500 and PacBio RSII. Short reads were acquired using Hiseq2500 and de novo assembled by Platanus. Long reads using PacBio RSII aligned to the scaffold by PBjelly.
Consensus sequence obtained from this assembly was used as the reference genome of LD-R in following analysis. Genome re-sequencing analysis of 9 individuals was performed using Hiseq2500. Sequence variants were detected using CLC Genomics Workbench.
We obtained 32.7x coverage of illumina reads and 22.6x coverage of PacBio reads. The reference sequence in LD-R region of Japanese flounder was constructed by hybrid assembly. Genome re-sequencing analysis generated 30.0x coverage of the Japanese flounder genome per individual. The variant detection showed 1,241 SNPs, 213 InDels, and 98 other sequence variants on average in LD-R region. These results will be important resources to perform the association study in LD-R region for identifying causative gene controlling the LD-R.

Bacterial coldwater disease (BCWD) causes serious damage to aquaculture and seed production of Ayu Plecoglossus altiveris. There is no approved vaccine against BCWD in ayu, so that development of vaccines for prevention is desired in the aquaculture industry. It has been reported that oil-adjuvanted formalin killed cells (Adj+FKC) of Flavobacterium psychrophilum were effective against BCWD in ayu. In this study, we investigated the removal of F. psychrophilum in spleen of Adj+FKC-vaccinated ayu after the bacterial challenge. Ayu were intraperitoneally injected with Adj+FKC and oil-adjuvanted PBS(-) (Adj+PBS), and were challenged with live F. psychrophilum (8.3×10⁸ CFU/ml) at 21 days after the vaccination. Naïve fish were also used as a negative control for the challenge test. Spleen was sampled at 1, 3 and 7 days after the challenge. The tissue sample was homogenized in modified cytophaga (MCY) broth, serially diluted and plated on MCY plate. The number of the bacteria in the spleen was measured by Miles and Misra method. In addition, DNA was extracted from the tissue sample and copy number of PPIC gene of F. psychrophilum was analyzed by real-time PCR. The bacterial colonies were detected in spleen of Naïve and Adj+PBS group at 1, 3 and 7 days after the challenge and the number of colonies were in a range of 5.0×10⁵ to 5.4×10⁷ CFU/ml. In contrast, the colonies were detected only from an individual at 1 day after the challenge in Adj+FKC group (6.7×10⁴ CFU/ml). Copy number of PPIC gene in Naïve and Adj+PBS fish was 23.8-fold and 23.9-fold higher than in Adj+FKC fish at 1 day after the challenge. These results suggest that F. psychrophilum were removed at extremely early stage of the infection in the spleen of Adj+FKC-vaccinated ayu.

Ayu has an approximately 1-year life span; the fish mature in autumn and die after the spawning. Infection rates of pathogens such as Flavobacterium psychrophilum in ayu increases during autumn. Down regulation of immune activity with sexual maturation is suggested to be a cause of the higher infection rates. In this study, we aimed to investigate maturation-associated changes in non-specific immune response against F. psychrophilum in ayu.
Ayu were collected at 8 time points during June to August 2015 and gonad somatic index (GSI) were recorded. The trunk kidney cells were collected and incubated with F. psychrophilum. The number of leukocytes with phagocytized bacteria was counted under microscope. F. psychrophilum were incubated with the serum and plated on modified cytophaga agar. The number of colony forming unit (CFU) was determined by Miles and Misra method. Furthermore, gene expression levels of interleukin (IL)-1β, IL-8, tumor necrosis factor (TNF) α and suppression of cytokine signaling (SOCS) 3 in the spleen were analyzed using real-time PCR at each time point.
The GSI increased from 16 July, and the maximum value was recorded on 26 August. The highest phagocytic rate (16.3 %) was observed on 16 July, and then significantly decreased to 6.3 % on 5 August (p<0.01). The CFU of the serum-treated bacteria was significantly higher on 12 and 26 August, compared with the values in June to July (p<0.01). Gene expression levels of IL-1β, IL-8 and TNFα in the spleen were not significantly changed during the period, while those of SOCS3 were significantly higher on 12 and 26 August, compared with 16 July (p<0.05). These results suggested that phagocytic and bactericidal activity against F. psychrophilum were decreased with sexual maturation, and SOCS3 may be related to the decrease of non-specific immune activity in ayu.

Bacterial Coldwater-Disease (BCWD) caused by Flabobacterium psychrophilum is a serious disease of ayu (Plecoglossus altivelis) in Japan. As a countermeasure for this disease, we have developed the microsatellite (MS) makers linked to the disease resistance trait and established resistant strains for BCWD by the Marker Assisted Selection (MAS). In this study, we analyzed the relationship between the markers linked to BCDW resistance and disease resistance trait in selective breeding strains by BCWD.
We used two selective breeding strains: Kiso river strain and hybrid strain (Kiso river strain x lake Biwa strain) in Gifu prefectural research Institute for fisheries and aquatic environments. The survival rate of Kiso river strain in BCWD challenge test for selective breeding program was approximately 50% in 2007 and 80% in 2016. The survival rate of hybrid strain was approximately 35% in 2007 and 80% in 2016. In order to detect an impact for selective breeding program in BCWD resistance locus, we have analyzed allele distributions of two strains using MS markers in both years (2007 and 2016). One MS marker associated with BCDW resistance and eight randomly selected markers located in other linkage groups were used. We compared the allele distributions on each strain in 2007 and 2016 by chi-square test.
In both strain, the allele distributions of several markers were significantly difference between 2007 and 2016, suggesting the genetic diversity was reduced due to breeding program. Especially, in MS marker linked to BCDW resistance, the difference of the allele distribution was highly significant, because of selection by challenge tests. These results suggest that the association study in BCDW resistance locus would be useful to identify a causative gene controlling the resistant to BCDW.

Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.

Yellowtail skin fluke disease is caused by Benedenia seriolae, which infect on yellowtail skin. Farmers treat skin flukes using fresh water bath and by changing nets regularly to remove attached eggs. However, these treatments are tedious for farmers and stress to the fish. Thus new protective method is required. Morphological and genetical classification, relationships with environmental conditions, and some treatment methods have been reported on B. seriolae. However, there are a few reports on genes of B. seriolae. Recently, proteases are focused because they play important roles in initial infection to mammals in Schistosoma, another Platyhelminthes. In this study, we measured B. seriolae protease activity and transcriptome analysis was carried out to identify various protease. B. seriolae were collected at Ehime prefecture in Japan. An infected yellowtail was bathed in fresh water for a few minutes, and detached B. seriolae were collected. To reveal the protease activity of B. seriolae, zymography assays were conducted using homogenate extracts of thirty individuals. Total RNA were extracted from a B. seriolae. cDNA was synthesized using the kit for next generation sequencing. MiSeq was used to determine the cDNA sequences, and determined reads were assembled by TRINITY. Assembled contigs were annotated by Blast2GO. Two protease bands were detected in zymography gels. Among four types of protease inhibitors (serine protease, cysteine protease, aspartic protease and metalloprotease inhibitor), a serine protease and metalloprotease inhibitor inhibited the activity, indicating that these proteases are serine protease and metalloprotease. These results indicated that B. seriolae abundantly produce these proteases. From NGS analysis, 2,307,736 reads, 27,374 contigs and 11,548 annotated contigs were obtained. Serine, cysteine, aspartic protease and metalloprotease were identified. At present, we are investigating the genes of proteases corresponding to the results of zymography from NGS results.

Antibody responses are one of the key features for adaptive immunity. To reduce the cost and time for development of antibody and detection of immune responses in many fish species, tools available for detection of antibody responses in various fish species are needed. Here, two synthetic peptides (fish IgH-1 and fish IgH-2) were designed based on the conserved sequence of fish immunoglobulin M heavy chain including Japanese flounder Paralichthys olivaceus, seabream Pagrus major, yellowtail Seriola quinqueradiata, carp Cyprinus carpio L., rainbow trout Oncorhynchus mykiss, hybrid sturgeon Huso huso x Acipenser ruthenus and banded houndshark Triakis scyllium. The synthetic peptides were used for peptide polyclonal antibodies (anti-fish IgH-1 and anti-fish IgH-2) production. The peptide antibody specificities were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Western blotting result showed that anti-fish IgH-1 antibody showed reactivity to IgM of all fish species tested except for banded houndshark under reducing and non-reducing condition. Anti-fish IgH-2 antibody reacted to IgM of seabream, yellowtail and rainbow trout under reducing condition. However, under non-reducing condition, anti-fish IgH-2 antibody solely reacted to IgM of rainbow trout. Attempts to use the peptide antibodies for detection of antibody titer were unsuccessful. These results indicate the application of anti-fish IgH-1 peptide antibody as a universal antibody for detection of immunoglobulins in various fish species by Wester blotting.

Immunoglobulin new antigen receptor (IgNAR) is an H-chain homodimer immunoglobulin found in cartilaginous fishes which does not associate with L chain. Each heavy chain comprises a single antigen binding variable (V) region, which contains a complementarity determining region (CDR) 1 and CDR3 loop. They are highly variable and critical for antigen binding. For these reasons, IgNAR V regions can be utilized for artificial antibody production. Banded houndshark Triakis scyllium has been used as a model fish for IgNAR V region library and recombinant Fab antibody. However, studies on antibody titer of banded houndshark IgNAR have not been conducted. In this study, to investigate antibody titer of IgNAR, anti-IgNAR rabbit IgG was produced from recombinant IgNAR consereved region. In western-blotting under reduced condition against the shark serum, anti-IgNAR rabbit IgG detected about 90 kDa protein corresponding to the estimated molecule weight of single IgNAR H-chain. In western-blotting without reduction of the shark serum, anti-IgNAR rabbit IgG detected about 500 kDa protein. These results suggested that IgNAR of banded houndshark forms polymers. In enzyme-linked immuno sorbent assay (ELISA) against Hs serum, anti-HsIgNAR rabbit IgG did not detect IgNAR. This result showed that anti-IgNAR rabbit IgG could not bind to the native form of IgNAR. In order to study more about IgNAR, anti-IgNAR antibody that can bind to native state IgNAR is required.

Recent studies have shown that mammalian adipose tissue (AT) exhibits immunological activity, and harbors many immune cells, especially macrophages. AT macrophages produce anti-inflammatory cytokines such as IL-10; however, obesity induces production of proinflammatory cytokines in AT, such as IL-6 and TNFα. Nevertheless, knowledge about the immunological functions of AT in fish species remains very limited. To understand the immunological properties of fish abdominal adipose tissue (AAT), we investigated the number of leukocytes present in AAT of the red sea bream(Pagrus major ), an aquaculture species with well-developed AAT. Microscopic observation of the paraffin sections of the AAT revealed a number of eosinophilic granulocytes distributed throughout the AAT. To examine the AAT leukocytes in greater detail, cells were isolated with a collagenase treatment. After centrifugation, non-adipocytes were recovered as precipitate and stained with May-Grünwald- Giemsa staining. Eosinophilic granulocytes comprised the majority (approximately 70%) of the non-adipocyte fraction. This evidently differs from the peripheral blood leukocytes of this species, in which more than 58% of leukocytes are lymphocytes. A small number of macrophages and lymphocytes were also observed in the AAT. Using reverse transcription PCR, we confirmed that leukocyte marker genes and immune-related genes, including CD4, CD8α, membrane and secretory forms of IgM, macrophage-mannose receptors, IL-1β, IL-8, and TNFα genes, were expressed in the AAT leukocytes. Moreover, when cinnabar particles were injected into the peritoneal cavity, a portion of the particles was taken up on the surface of the AAT cells. Taken together,these results suggest that AAT of P. major is an active immunological tissue, a fact that had not received much attention prior to this study.

Aquaculture development of some important farmed fish, which have great economic and cultural importance, is key due to the decline of wild populations. While in captivity, fry production relies on the spontaneous formation of male and female pairs. Sex determination of these types of fish is not possible based on morphologic criteria, however, and it is very difficult to optimize reproduction conditions and fry production in each pond, which seriously hampers the culture of this species. It is a proper way to developed sexing separation methodologies based on the measuring of 11-Ketotestosterone (11-KT) levels as an important androgenic sex hormone in maturing juveniles. Here we developed a label-free immunosensor system that detects 11-KT levels in fish with high sensitivity.
Detection of 11-KT using cyclic voltammetry (CV) is achieved by immobilizing anti-rabbit IgG antibody and anti-11-KT antibody on an Au electrode with self-assembled monolayer. The principle of the system is based on the maximum oxidation peak current change induced by the generation of a nonconductive antigen-antibody complex. The effects of temperature and pH on the CV oxidation peak current change were tested. We found that the highest current reduction (optimal immune response conditions) occurred at pH 7.0 and 25℃. The immunosensor showed a response to 11-KT levels, and the oxidation peak current linearly decreased in the range of 0.21 – 3.30 pmol ml^-1. Good correlation between the 11-KT concentration and sensor current change was found. The specificity of the label-free immunosensor system was investigated using other steroid hormones, such as testosterone. The specific detection of 11-KT was suggested by a minimal change in the anodic peak value of the other steroid hormones. Actual plasma samples collected from Nile tilapia (Oreochromis niloticus) were measured by the proposed system and compared with those concentration measured by ELISA, and good correlation was confirmed.

Control of infectious diseases is one of the crucial issues
in aquaculture production of crustacean species. However,
usage of antimicrobials has been limited and vaccines
have not been available since crustaceans are lacking in
acquired immune system. Here, we report Silkrose, which
contains an acidic polysaccharide of Silkmoth pupae
(Bombix mori) to activate innate immune system of
mammalian macrophages and exhibits protection for
infectious diseases of teleost fishes, is effective for
Vibriosis of Japanese tiger prawn (Marsupenaeus
japonicas).
To demonstrate efficacy of Silkrose for infectious disease
of Japanese tiger prawn, we performed challenge study
with a high virulent Vibrio penaecidae strain. Before
challenge, experimental prawns were fed with diets
containing 0.001, 0.01 and 0.1% Silkrose for two weeks,
and showed satisfactory growth. Survival rates for 28 days
post challenge were drastically improved in Silkrose
groups (73% in 0.001% Silkrose group, 77% in 0.01%
Silkrose group, 76% in 0.1% Silkrose group) in contrast to
control group in which all experimental prawns were dead.
Underlying cellular and molecular mechanisms of
immunization in crustacean remain elusive, but Silkrose is
expected to serve as a new tool to control diseases of
crustacean species in aquaculture production.
Keywords: Marsupenaeus japonicas, Vibriosis, Vibrio penaeicida,
innate immune system, disease control in aquaculture, Silkmoth

Recognition of lipopolysaccharide (LPS), the major component of outer membrane of Gram-negative bacteria, triggers innate immune responses in mammals. Two immunological pathways to recognize LPS have been understood in mouse and human as follows: recognition of extracellular LPS via toll-like receptor (TLR4), and recognition pathway of intracellular LPS mediated by caspase (Casp)-4/11 and inflammasome protein-complex consisting of NLR-PYD, ASC and Casp-1. However, the TLR4 gene does not exist or work on NF-κB activation in fish. In this study, we focused on identification of the inflammasome-associated genes such as NLR-PYD, ASC and Casp-1 in medaka (Oryzias latipes). From the results of comprehensive in silico genome data-mining, we could not identify Casp-4/11 homolog in fish and only Casp-1 gene was identified in the same subfamily (Casp-1 subfamily). NLR-PYD (i.e., NLRP12 homolog) gene belonging to an NLRC subfamily and the adaptor molecule ASC were also retrieved from the medaka genome database. The synteny of medaka Casp-1 and NLRP12 genes was not conserved to those of mammals and identities of their amino acid sequences were in low levels, although their domain structures were similar to that of other known vertebrates. Expression of the medaka Casp-1 and NLRP12 genes was significantly induced in head kidney and intestine stimulated with the formalin-killed Edwardsiella tarda. Further, in order to clarify the function of medaka Casp-1, cleavage of IL-1β by Casp-1 will be studied.

In the United States, colorful transgenic zebrafish (Danio rerio), tiger barb (Puntius tetrazona) and black tetra (Gymnocorymbus ternetzi), in which various fluorescent protein genes are introduced, are commercially available as GloFish. On the other hand, in Japan, the Japan Fisheries Research and Education Agency is authorized to conduct on-site inspections under the Law Concerning the Conservation and Sustainable Use of Biological Diversity through Regulation on the Use of Living Modified Organisms (LMOs) (Cartagena Protocol). Accordingly, we have developed the detection methods of LMOs such as GloFish, in order to monitor the occurrence of LMOs in Japan in case that they are illegally imported. Here we describe the detection methods of new GloFish fluorescent protein genes, i.e., Jred, Mcitrine, Venus, Emerald, CyPet and Cerulean.
The nucleotide sequence of each fluorescent protein gene was obtained from NCBI nucleotide database. The plasmid containing the positive control of each gene was synthesized by GenScript Co. Ltd. For PCR, 0.5pg of each plasmid was used. PCR primers were designed by the on-line software Primer3.
Twenty ng of zebrafish genome DNA was used as the negative control (non-transgenic). For the positive control, the length of the PCR products were designed to be 517bp for Jred, 506bp for Mcitrine, 710bp for Venus and Emerald, 557bp for CyPet, and 575bp for Cerulean.
The PCR product of each positive control was observed by agarose-gel electrophoresis as a single band at the expected DNA length. On the other hand, no PCR product was observed for the lane of negative control. From these results, we conclude that PCR detection systems of these fluorescent protein genes were established.

Crustacean does not posses specific immunity and hence non specific immunity play a major role in response to pathogen invasion by humoral factor such as antimicrobial peptide alongside cellular response. Crustins are one of the most reported AMP in crustacean. Crustins have fundamental function in antimicrobial activity and protease inhibition. Numerous isoform of this molecule has been reported in previous studies and in current study two new crustins isoform MjCRS8 and MjCRS9 has been identified and characterized from kuruma shrimp Marsupenaeus japonicas. The deduced amino acid sequence of a MjCRS8 consists of 197 amino acid residues and a signal peptide of 24 amino acid residues and deduced amino acid sequence of MjCRS9 consists of 152 amino acid residues and a signal peptide of 18 amino acid residues. Tissue distribution analysis revealed that both MjCRS8 and MjCRS9 are gill specific. Sequence analysis of both crustins showed 68 % identity with previously reported MjCRS. It was further shown that both crustin fall together with type II MjCRS in phylogenetic analysis. Together, these results suggested that there are more types of MjCRS isoform exist in M. japonicus which could possibly work as a multiple isoform protein complex in humoral immunity unlike single protein system of clotting and lysozyme. Further functional study may enhance our knowledge of crustin function which would unable us to understand the crustins isoform complex and how it work in innate immunity of shrimp.

Nacre formation is considered to be controlled by proteins secreted from mantle tissues. Therefore, quality of pearls is supposed to depend on protein profiles in nacre. There is little available information about what proteins determine the pearl quality. Our final objective is to find indicators to evaluate the quality of pearls. In the present study, we analyzed protein profiles in nacre to reveal its relationships with the pearl quality. Nacre was prepared by removing prismatic layer from Japanese pearl oyster shells using a disk sander. Nacre was decalcified with 0.5 M EDTA (pH 8.0) at room temperature for 5–14 days. The EDTA-soluble fraction was subjected to ultrafiltration to remove EDTA and replace the solvent with MilliQ water. After freeze-dried, the sample was dissolved with a minimal amount of MilliQ water. SDS-PAGE patterns revealed that the EDTA-soluble fraction was occupied mainly with nacrein and slightly with many kinds of proteins. The protein profiles seemed to depend on the pearl oysters. These findings indicate the possibility that the quality of nacre reflects them. We are analyzing more pearl oysters now.

To know more about the mystery of Japanese eel, Anguilla japonica with behavior, sex determination, reproduction and osmo-regulation on molecular level, there is a need to construct a completed genome as basis for omics studies. Then we can estimate the expression profiling under different spatiotemporal scenarios during their life cycle to realize the groups of genes that may regulate physiological phenomena. In past years, our team had built a transcriptome draft with rich annotations and a user-friendly interface using RNA-seq from a sample pool of fertilized eggs, preleptocephalus, leptocephalus and glass eel stages with 300M Pair reads, 58 billion bases (http://molas.iis.sinica.edu.tw/jpeel, PLoS ONE, 2015).
For accelerating assembly process and elevating the quality of genome from over 650-Gigabyte raw data, we developed a new algorithm named as QReadSelector to extract the raw reads with less error and shorten the time for assembling (https://github.com/moneycat/QReadSelector, BMC Genomic, 2015). Using the filtered reads, we have assembled the first version of Japanese genome as 1.06 Gb with 10,158 scaffolds, N50 over 1.5 Mb and coverage up to 122x from the blood of female yellow eel. By taking 1,211 scaffolds in length larger than 100Kb, these scaffolds can composite 92% of Japanese eel genome. According to transcriptome we assembled previously and those proteins of bony fishes from Uniprot, we construct the 27,350 gene models for Japanese Eel genome. After performing the similarity search with non-Redundancy database (nr), eighty-two percent of best hits can be found in Actinopterii. And the top3 species are Asian Arowana (Scleropages formosus, 15%)、Spotted gar (Lepisosteus oculatus, 13%), and Atlantic Salmon (Salmo salar,10%). Currently we have completed the web database for this first draft of Japanese eel genome with transcriptome (http://molas.iis.sinica.edu.tw/jpeel2016). We believe this world-leading Japanese eel multi-omic resource from this project will make great benefit to eel research community.

In past years, we have fulfilled most of goals we claimed including genome sequencing, de novo assembly and annotation for construction of bioinformatics core on giant grouper, Epinephelus lanceolatus . Over 20 samples from various developmental stages and tissue types were sequenced to estimate the expression profiling via this genome assembly. All of these omics have been integrated and implemented as web database/ applications and allowed our collaborators to perform their own analyses online directly.

According to most updated genome 3.0, only 338 scaffolds can assemble 99% of 1.06 Gb genome with N50 near 8.6 Mb. Among these 338 scaffolds, the number of those length larger than 1 Mb is around 167 which cover 93% of whole genome. Via gene prediction model constructed by those RNA-seq data and all the bonefish proteins in Swissport, there are 37,809 genes which can be translated for 38,226 proteins identified from genome. After annotation and comparison with other species, almost half of proteome can find their orthologs in large yellow croaker (Larimichthys crocea)、bicolor damselfish (Stegastes partitus) and Antarctic bullhead (Notothenia coriiceps). Over 80% of orthologs identified by translated proteins are belonged to Actinopteri. This well annotated genome of E. Lanceolatus will be model organism for all groupers on the comparative genomics and transcriptomics. Reference website: http://molas.iis.sinica.edu.tw/grouper2015/

The gold and cream colors of cultured Akoya pearls, as well as naturally yellow colors of the nacre of pearl oyster shells, are supposed to arise from yellow pigments. To date, two yellow components (E1 and E2), which may be related to the development of the yellow color in the nacres of Akoya pearls and oyster shells, have been identified. Fourier transform infrared spectroscopy analysis showed that the major E1 may be an iron-base inorganic compound, whereas the minor E2 may be an organic compound mainly consisting of polyamide. In this study, we report the distribution and the existence form of the E1 in the nacre of pearl oyster shells. E1, extracted with a HCl/MeOH solution from nacreous insoluble substances of the EDTA-decalcified yellow-colored nacre, was precipitated as the hydroxide using NaOH solution. Inductively Coupled Plasma-Mass Spectrometry analysis showed that the iron content of the E1-hydroxide was approximately 44 mass %, and the E1 solubilized with dilute-HCl showed positive reactions to KSCN and K₃[Fe(CN)₆] reagents, indicating that E1 exists as trivalent iron in the nacre. In order to investigate the iron distribution and existence form in the nacre, yellow-colored nacre from the pearl oyster shells was decalcified with EDTA, and the resulting sediment was treated with HCl/MeOH and SDS/DTT solutions. The SDS/DTT extract was further fractionated by ultrafiltration with different molecular weight cutoffs (100, 10, and 3 K). Although the EDTA and HCl/MeOH extracts and 3 K-fraction of the SDS/DTT extract, which turned conspicuous yellowish color, contained highly concentrated iron, the trivalent iron could be detected only in the HCl/MeOH extract. These results suggest that E1 related to the development of the yellow color exists in forms binding with nacreous water-soluble/insoluble molecules in the nacre of the pearl oyster shells.

In Japan, Pyropia yezoensis is one of the most important algal species used as food, and approximately 300,000-350,000 tons (wet weight) of the thalli are produced in Pyropia cultivation farms every year, most of which are processed to dried sheets of “nori”. However, the yield and quality of the cultivated Pyropia thalli are frequently affected by environmental factors. In particular, growth, development, and quality of Pyropia thalli are influenced by changing dissolved inorganic nitrogen (DIN) content, which consists of nitrate-N, nitrite-N, and ammonium-N. When the DIN in the cultivation area decreases, severe thallus discoloration (“iroochi”) occurs in the Pyropia cultivation farms, resulting in lower quality Pyropia thalli. However, recovery of discolored P. yezoensis thalli has been obtained by chemical fertilization using not only inorganic-N but also organic-N such as urea-N and amino acid-N. Thus, it is necessary for effective management of P. yezoensis cultivation to understand the molecular mechanisms and characteristics for inorganic and organic-N uptake and assimilation in P. yezoensis thalli. To date, three DUR3-like genes (PyDUR3.1, PyDUR3.2, and PyDUR3.3), which may be involved in uptake and transport of urea, have been identified in P. yezoensis. In this study, in order to understand the differences in physiological and molecular characteristics among the three PyDUR3 genes, PyDUR3.1, PyDUR3.2, and PyDUR3.3, their sequence similarities, genomic organization, and transcriptional regulation in P. yezoensis thalli grown under different N-conditions were investigated, and intracellular localization and functional complementation analyses of the PyDUR3 isoforms using the budding yeast expression system were performed.

Marine metagenomic DNA sequencing by using next generation sequencer (NGS) is a powerful tool that revealed the expansive biodiversity of microorganisms and clarified the relationship of their microbial communities. However, the majority of sequence by the metagenome analysis showed no homology with the sequence in the existing databases. Almost no sequence of a read of the NGS (100 bp) covers full length of protein coding genes. One the contrary, it covers coding sequence corresponding to some motifs. Moreover, it may be possible to regard motifs of proteins as kinds of minimum functional units rather than individual protein. Therefore, those may be utilized as index for characterization of environment. In this study, we tried a new metagenomic analysis method based on the information of the protein motifs. We utilized metagenome data (established by Gojobori group in CREST) of seawater samples in the Sendai Bay and the Sanriku offshore from February 2012 to May 2014. The nucleotide sequences were translated into amino-acid sequence using every 6 reading frames. The amino-acid sequences were analyzed using Pfam database and InterProScan software. Then, frequency of each motif was counted for each matagenome sample. The resulting matrix were analyzed by heat map, a cluster analysis, and principal component analysis (PCA). We found that these analyzes based on the frequency of motifs in each metagnomic sample can observe the difference in each condition such as season and pore-size of filters.

[Introduction]
Thanks to development of sequencing technologies, culture-independent methods are contributing to understand genomic information of uncultured microbes. However the importance of isolation is the same as before. Hyperthermophiles grow optimally over 80℃. Most of them are anaerobic because of low parcial pressure of oxygen in hot environments. Here we succeeded in isolation of characterization of a novel aerobic hyperthermophilic archaeon from a terrestrial hot spring.

[Material and Method]
Hot waters and sediments were collected from Atagawa hot spring, Shizuoka prefecture, Japan. Samples were inoculated to YP01 medium (0.1% yeast extract, 0.1% Bacto tryptone), at 90℃, in aerobic condition. Ten-fold dilution to extinction method was used for isolation of cells. The purity of an isolation was confirmed by microscopic observation and direct sequencing of partial 16S rRNA gene. Carbon utilization was determined by using modified medium which omitted Bacto tryptone and adding various substrates in concentration of 0.05, 0.1 and 0.2% (w/v).

[Results and Discussion]
After incubation at 90℃ for 4 days, growth of cells were observed. After 10-fold dilution to extinction, only cocci were observed. In direct sequencing, a partial 16S rRNA gene sequence was detected, indicating that clonal isolate was established. We designated this strain as AG1. Phylogenetic analysis of 16S rRNA gene showed that isolated cocci belonged to archaeal family Desulfurococcalesand most closely related to anaerobic archaea Ignisphaera aggregans with similarityof 95%. The cells were motile cocci (1.2 µm in diameter). Growth conditions of strain AG1 was as followed; 70-95℃ (optimally at 90℃), pH 6.0-8.5 (optimally at pH 6.5), salinity 0. The strain could grow without trace elements. Strain AG1 was heterotroph and utilized organic compounds. Since no terrestrial aerobic hyperthermophilic archaea in family Desulfurococcales has been reported, we proposed the strain AG1 as a representative of a novel genus.

Carbon monoxide (CO) occurs naturally from both biological and abiological sources in volcanic environments. So far, hydrogenogenic carboxydotrophic bacteria have been isolated from these environments. Of hydrothermal environments we focused on a non-volcanic hot spring where hot water is produced by the dehydration of rocks contained in oceanic plate.
A sample was collected from Miyuki-hot spring, located in Wakayama Prefecture, Japan. The DNA was extracted from each sample by using DNeasy® Blood & Tissue (QIAGEN). The V3-V4 region of the 16S rRNA genes were amplified by PCR, followed by the amplicon sequencing with MiSeq (Illumina) plat form. The same sample was used as inocula for isolation of carboxydotrophic bacteria. The sample was inoculated to modified medium Bfc (pH7.7) at 65°C under 100% CO in headspace. CO consumption and _H2 production in head space of media were analyzed using gas chromatography GC-2014 (Shimadzu). Spore formation was observed using malachite green staining.
No 16S rRNA gene fragments of carboxydotrophic bacteria were found in 39,759 sequenced reads, suggesting that the abundance of carboxydotrophic bacteria are very small in the environment. Using a serial 10-fold dilution to extinction method, we succeeded in purification of a thermophilic spore-forming bacterial strain designated SO3, producing _H2 . Cells of the new isolate were rod-shaped (1.7-2.8 µm in length and 0.8-1.0 µm in diameter). The phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SO3 belongs to the genus Carboxydocella which is composed of carboxydotrophic hydrogenogenic members. The 16S rRNA gene sequences of strain SO3 showed sequence similarity of 99% to Carboxydocella sp. JDF658 and Carboxydocella sp. ULO1 both of which we isolated from volcanic environments in Japan. We isolated strain SO3 from an environment where the abundance of the genus Carboxydocella was small, suggesting that strain SO3 has been survived by forming spores.

The aging mechanism of fish is different from that of mammals because of negligible senescence in fish. For example, most fish show postnatal muscle fiber formation throughout their lifespan (indeterminate growth). Regeneration of injured heart and nephrons is observed in fish even at the adult stage. Zebrafish (Danio rerio) is a well-known vertebrate model organism and shows above-mentioned negligible senescence phenomena. In zebrafish, the expression of telomere reverse transcriptase (TERT), an index of telomerase activity, has been shown in various tissues even in aged individuals. Thus, in order to survey the difference of age-related phenomena between mammals and fish, we conducted RNA-seq to examine age-related expression changes in telomere maintenance genes of zebrafish.
Total RNA was purified from 5 tissues including muscle, liver, gill, brain and heart at 4 growth stages (2-month-old, 7-month-old, 16-month-old and 39-month-old) and subjected to RNA-seq using an Illumina Hiseq2000 sequencer. Among known telomere maintenance genes in vertebrates, we chose 20 telomeric elongation promoting genes (“promoting genes”) and 37 suppressing genes (“suppressing genes”). Cluster analysis of these genes showed the high activity of promoting genes compared with suppressing genes in all examined tissues, well consisting with the high telomerase activity in fish. Principal component analysis of the expression pattern of promoting genes showed the clear segregation of tissues, suggesting the tissue-dependent manner in aging pattern. In the promoting genes, TERT expression was especially high in zebrafish skeletal muscle, suggesting the relationship between high plasticity of fish skeletal muscle and telomerase activity.

Polysaccharide-degrading enzymes are useful for producing oligosaccharides for biotechnological and medical applications. An agarolytic bacterium, Persicobacter sp. CCB-QB2, was isolated from a coastal area in Penang, Malaysia. It exhibits a diauxic growth and highly expresses an agarase, PdAgaC, during the second growth phase. A GH16 family agarase, PdAgaC is stable at 50 ºC and has a K_m of 5.15 mg·ml⁻¹ and a comparatively high k_cat of 2,213 s⁻¹. Interestingly, the activity is obviously enhanced by the adding of 0.1% SDS. The crystal structure of PdAgaC’s catalytic domain consists of a β-jelly roll fold. The substrate binding groove, however, is wider by ~1 Å than that of the other known agarase structures, which may possibly result in PdAgaC’s higher k_cat compared with that of those agarases. Persicobacter sp. CCB-QB2 also expresses an alginate lyase, AlyQ, from the polysaccharide lyase family 7 (PL7). While it cleaves both polygluturonate (poly-G) and polymannuronate (poly-M), AlyQ prefers poly-GM with a K_m and k_cat of 0.58 mg·ml⁻¹ and 32 s⁻¹, respectively. It has two additional carbohydrate-binding domains — while the function of the first domain remains unknown, the second domain is a family 32 carbohydrate-binding module (CBM32) that binds alginate termini. The crystal structure of a truncated version, which lacks the first domain, shows that both the CBM32 and catalytic domain do not interact with one another. The catalytic domain shares a conserved sugar-binding groove, but sugar recognition at subsite −1 differs completely from the presently known mechanism. Both PdAgaC and AlyQ may help Persicobacter sp. CCB-QB2, which probably feeds on seaweed, to degrade cell walls more efficiently for nutrient acquisition.

To yield good packaging film, the properties should be optimized for commercial applications. One of those is plasticization process. The plasticizer type and its concentration is important factor affecting the film properties, such as mechanical strength, good inhibiting ability, fragility prevention from high intermolecular strength. Our previous finding found that carrageenan concentration influenced the physico-chemical properties of the edible film. Thus, this study was aimed at producing a carrgeenan-based edible film and knowing the effect of plasticizer type on the physico-chemical properties.
Carrageenan meal was dissolved through heating at 70^oC while shaken, cooled down to 50^oC, and added with plasticizer (Glycerol, Sorbitol, Ethylene glycol, Polyethylene Glycol) of different concentrations, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 % v/v, and starch of 5 % b/v. The solution was then homogenized and warmed at 50^oC for 5 minutes, then added with fatty acid (bee wax) and set the pH to neutral, while warmed at 50^oC and stirred with a homogenizer. The solution was then poured into a 20 x 20 cm acrylic print, left in open air for 24 hours, and dried in the drying cupboard for 6 hours at 60^oC. Results found a thin edible film whose physical parameters are ready to test, thickness, water vapour permeability (WVP), tensile strength (TS), and elongation at break (EB).
Glycerol plastic-layered film reflected the highest EB among all plasticizers under the same concentration used, while Ethylene Glycol plastic-film showed the highest TS. Moreover, EB and WVP increase with plastisizer concentration. Further study will be focused on the optimal carrageenan concentration, platicizer type, and plasticizer concentration to yield the edible film for fisheries product application.

Effect of different washing solutions on chemical interactions and gel properties of silver carp (Hypophthalmichthys molitrix) surimi gels
Longteng Zhang, Qian Li, Yongkang Luo*
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, P.R. China
*Corresponding Author, E-mail: luoyongkang@cau.edu.cn
Abstract
【Objective】 This study aimed to investigate the effect of different washing solutions (distilled water, 0.1% and 0.2% CaCl2, 0.1% and 0.2% MgCl2) on chemical interactions and gel properties of surimi gels derived from silver carp (Hypophthalmichthys molitrix) fillets during setting and heating. 【Method】Two washing steps (distilled water followed by different washing solutions) were conducted. Actinmyosin sol was incubated at 40°C (setting) and 90°C (heating) at specific time intervals. Gel properties (breaking force, deformation, gel strength, whiteness, and expressible water content) and chemical interactions (non-covalent bonds, sulfhydryl groups, and disulfide bonds) were measured. Protein patterns were analyzed by SDS-PAGE, and correlations between gel properties and chemical interactions were conducted. 【Result】 Compared with distilled water and MgCl2 washing, 0.2% CaCl2 washing resulted in superior gel properties, which significantly (P<0.05) enhanced the whiteness, breaking force, and gel strength from 69.65, 117.00 g, and 647.43 g•mm to 72.29, 191.50 g, and 1288.00 g•mm, respectively. Besides, better water-holding capacity was obtained. Changes in chemical interactions also occurred during gelation process, CaCl2 washing enhanced the formation of hydrophobic bonds, while decreases in non-specific associations, ionic bonds, hydrogen bonds, sulfhydryl groups, and disulfide bonds were observed in all washing processes. Moreover, 0.2% CaCl2 accelerated the polymerization of myosin heavy chains, and hydrogen bonds were strongly correlated to gel properties.【Conclusion】 0.2% CaCl2 washing promoted the formation of hydrophobic bonds, which led to better gel properties.

This experiment were conducted to evaluate the effect of polyphenol on rheological properties of surimi gel

Many fish species are caught by the bottom trawling, but some of them are not used efficiently because of the size is small or the value is low in the market. These fish are called by-catch fish and not used efficiently as food products. Processing frozen surimi from by-catch fish is one of the effective utilization of fish resources, it is necessary to explore the gel properties for good use of it. In this study, it is investigated that the heat-induced gel properties of by-catch fish surimi containing more than 10 species fish meats and different preparation. Surimi was blended with 2.5% (w/w) of salt and heated at 85°C for 30 min to prepare the gel. Breaking strength of the gel without soaking in water is very low, but it was increased more than 1 times of soaking. Color of the gel made from surimi with skin and bone was dark. These tendency was shown even if the species and the percentage were different.

North Pacific krill Euphausia pacifica has a very low commercial value in Japan. To utilize the krill protein as a raw material for gelled product, the krill protein was recovered in our previous study. It was concluded that the NaCl solution treatment to recover the protein is useful method to obtain the protein with retaining its functionality, and that the autolysis of the protein during the recovering could be suppressed by inhibiting serine protease and metalloprotease. For the next step of the study, the gel-forming ability of the recovered protein in the presence of protease inhibitors was evaluated.
The krill was homogenized with NaCl solution (final conc. 4%) to solubilize the protein, centrifuged to remove its residue, and then the supernatant was diluted to 10 times (v/v) by cold water to obtain precipitated protein. The precipitate was dehydrated overnight in the presence of protease inhibitors (Benzamidine, SBTI, PMSF and EDTA), and collected as the recovered protein. The recovered protein was ground with 3% NaCl and heated at 40, 60 and 90°C for 30 min. After heating, the sample was cooled in ice water, and then physical properties and SDS-PAGE patterns were determined.
The thermal gel was not obtained when heating the recovered protein without protease inhibitors and in the presence of EDTA, except that very weak gels could be obtained when heated at 90°C. On the other hand, in the presence of serine protease inhibitors (Benzamidine, SBTI and PMSF), thermal gels were obtained when heated at 40, 60 and 90°C. Moreover, it was founded that the degradation of myosin heavy chain of those thermal gels was suppressed by the observation of SDS-PAGE pattern. These results suggest that weak gel-forming ability of the recovered North Pacific krill protein could be improved by using serine proteinase inhibitors.

The aged society has arrived in Japan. However, many kinds of sea food are difficult for elderly parsons to masticate and swallow. Though the mastication indicator of foods for elderly persons is established, swallowing one cannot provide the information whether they can swallow or not. Recently, tribological studies of food for smooth palate feeling have started. We are considering that tribology may be able to characterize the swallowing behavior. In this study, to establish the methods of tribological measurement and to characterize tribological properties of sea foods were aimed.
Agar gel and fish-meat gel were used as samples. Mastication behavior was observed to consider the sample condition for tribological measurement. Fish-meat gel was more salivation during mastication. Average particle size of bolus in agar gel was smaller. Samples for tribological measurement were prepared by cutting gels to have a similar particle size distribution of bolus and by adding thickening agent having the same rate of saliva in bolus to cut gels. Frictional force of samples showed typical Stribeck curves. Although the static friction coefficient of the surface of fish-meat gel was larger than those of agar gel, bolus of fish-meat gel was more lubricity than those of agar gel. Because solids in the bolus of fish-meat gel separated from the bolus during the measurement, the lubricity between the sensor and the thickening agent was detected. On the other hand, such separation was not observed in the bolus of agar gel. These phenomena could be reflected person having difficulty in swallowing. In the case of fish-meat gel, if they cannot move their own jaw well, bolus in the mouth will be separate into solid and liquid components. Therefore, fish-meat gel could be difficult for them to swallow.

Elasmobranches, such as sharks, retains large amount of urea in their body to maintain body fluids isosmotic. It’s an important osmoregulatory system for living in salt-rich environment and a gain of function in an evolutionary. Concentration of urea in shark body is 200 - 500 mM. It’s a good osmolyte, but urea is well known as a powerful protein denaturant. So, we wonder why muscle proteins of sharks are maintained even in the presence of urea in vivo. There are two assumptions of biosystem in quenching of urea, one is that an accumulation of large amount of trimethylamine-N-oxide (TMAO) suppresses protein denaturation by urea, the other is that muscle proteins of shark have a characteristic resistance against urea. We focused on a protection effect of ATP on fish muscle proteins, and demonstrated that ATP containing in muscle suppressed the thermal and cryodenaturation of muscle proteins, such as myosin and Ca-pump. Our goal is elucidation of the mechanism which maintains the function of muscle protein under the presence of urea. In this research, we studied the suppressive effect of ATP and TMAO on urea denaturation of shark myosin Ca-ATPase. Myosin was prepared from dorsal muscle of scalloped hammerhead ＜i＞Sphyrna lewini＜/i＞. For the study of the effect of ATP and TMAO on myosin denaturation by urea, myosin was incubated at various temperature in the presence of urea, ATP and TMAO under a Ca＜sup＞2+＜/sup＞-free condition. Then Ca-ATPase activity of the incubated myosin was assayed. High concentration of urea without ATP or TMAO induced a reduction of the myosin Ca-ATPase activity. In the case of myosin incubated with urea, ATP and/or TMAO, its Ca-ATPase activity was higher than incubated with urea. These results suggested that ATP and TMAO suppressed urea-denaturation of myosin Ca-ATPase. ATP may act as a safeguard of muscle protein against urea.

Trimethylamine-N-oxide (TMAO, (CH₃)₃NO) is a product of the oxidation of trimethylamine and found in aquatic organism, e. g., pisces fishes. TMAO has been known as a natural osmolyte. The amount of TMAO is higher with deep-sea fishes as cod and pollack. In postmortem changes in such species, large amount of formaldehyde is produced by demethylation of TMAO. TMAO demethylase (TMAOase, or trimethylamine-N-oxide formaldehyde-lyase, EC 4.1.2.32) has been proposed to be responsible for the production of formaldehyde. TMAOase is the enzyme which catalyzes the following reaction. (CH₃)₃NO → (CH₃)₂NH + HCOH
It has been reported that TMAOase, aspolin was detected and purified from the myofibrillar fraction of walleye pollack,. Aspolin mRNA was detected in skeletal muscle and kidney. Aspolin is the extremely aspartic acid rich protein.The implications of expression of aspolin in living tissue and physiological function of aspolin are still unclear.
We focused on aspolin in muscle of infected fish by parasites. We made Ichthyophonus hoferi (Opisthokonta, Ichthyosporea, Ichthyophonida) a target parasite. I. hoferi is a protozoan parasite which causes an internal infection of organs and muscles in walleye pollack Theragra chalcogramma, rainbow trout Oncorhynchus mykiss, yellowtail Seriola quinqeradiata and other Pacific fish. TMAOase activity was measured in determination of produced dimethylamine(DMA). The amount of TMAO in muscles from severe infected walleye pollack were reduced to a half amount of normal fish. In contrast, DMA amounts in the severe infected muscles were increased. Myofibrillar fractions from infected fish showed much higher TMAOase activity than that from normal fish. Furthermore, the expression volume of aspolin was increased in infected muscle by the measuring western blot with an antibody of poly-Asp. We discuss a host defense mechanism of aspolin induced by I. hoferi infection.

The objective of this research was to evaluate the quality changes of big-eye tuna (Thunnus obesus) with temperature fluctuation during logistic processes. Near infrared reflectance spectroscopy (NIRS) and Low-field nuclear magnetic resonance (LF-NMR) were used to determine the characteristic of water and protein. This characteristic were further evaluated by water holding capacity (WHC), water activity (a_w), Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Scanning Electron Microscope (SEM) during logistic processes. The results showed that WHC of samples were decreased more than 20% and the speed of protein degradation was increased with the rise of temperature during logistic processes. The absorbance value of NIRS was in downward trend. Relaxation time T₂ of 3 kinds of water, both the value and amplitude of immobilized water T₂₁ decreased while free water T₂₂ and its amplitude increased, and relaxation time T₁ did not change significantly. The results from NMR and NIRS indicated that the water distribution and protein aggregation was strongly affected by temperature fluctuation, which corresponded to the changes of WHC, SDS-PAGE and SEM, indicating the shelf-life of tuna was estimated to be 168 h.

Key words: big-eye tuna; LF-NMR; NIRS; logistic process; quality change

Metabolomics is a technology that provides comprehensive analysis of metabolites and is attracting attention as an emerging technology in food quality assessment. We proposed that a comprehensive analysis of such metabolites may provide insight into the changes in the freshness of fish meat. In this study, yellowtail muscle was used as the model for the storage test. The metabolites that changed during storage were analyzed to determine the suitability of this method as a novel freshness assessment tool.
The yellowtails were transported to the laboratory on ice within 9 hours of being killed (ikejime) at the market and then filleted. Fillets were separated into ordinary and dark muscle and then were minced using a food processor. The fish meat was stored at 0°C and 5°C for periods of between 0 and 14 days. A methanol-chloroform-water (2.5:1:1) solution was used to extract metabolites from the samples, and after oxime- trimethylsilyl (TMS) derivatization, the metabolites were analyzed using gas chromatography mass spectrometry (GC/MS). A list using the peak areas of the identified metabolites was created and subjected to multivariate analysis using SIMCA 14 software.
Orthogonal partial least squares (OPLS) analysis with Pareto scaling was conducted using the number of days in storage as the Y variable and the peak areas of each metabolite as the X variable. As a result, a model was developed that allowed the prediction of storage days at each of the different temperatures for both types of fish meat, ordinary and dark muscle. This model has the potential to be utilized as a new model in predicting the freshness of fish. In addition, various metabolites such as nucleic acid related compounds, amino acids, sugars and organic acids, contributed to this predictive model on fish freshness as crucial metabolite markers.

Evaluation of seafood quality by instrumental analysis will enable consumers and quality control managers objective measures for assessment. Taste plays an important role in determining of the food preference of consumers. The taste sensing system, SA-402B (Intelligent Sensor Technology Inc.), can estimate the intensity of taste, for example saltiness, umami and richness, in liquid state products, but not in solid state products. So, it is necessary to prepare extracts from the seafood in most cases. In the present study, we measured extracts related to taste from fish muscle, nori, and squid to test the applicability of the system for seafood assessments.
In the case of yellowtail, we prepared extracts from 3 different regions, dorsal ordinary muscle, ventral ordinary muscle, and dark muscle, and estimated the taste intensity of them. The intensity of richness in the extract from dorsal ordinary muscle was highest in order ventral ordinary muscle and dark muscle, resulting that the system could differentiate the 3 different extracts. In the case of tuna, similar results were also obtained.
In nori (sheets of dried laver seaweed), we prepared the extracts from 3 different grades of nori depending on the commercial price, excellent, high and middle class, and estimated the taste intensity of them. The intensity of umami in excellent grade of nori was higher than that of middle class. Principal component analysis using taste intensity was able to discriminate different grades.
In squid, we prepared extracts from 3 species of squid, Japanese common squid, purpleback flying squid, and neon flying squid, and estimated the taste intensity of them. The system could reliably differentiate the extracts from different species.
The system could differentiate the extracts from different region of fish muscle, different grade of nori and different species of squid, suggesting that the taste sensing system is applicable as an objective measure of seafood taste.

It is well known that freshness of fish decreases rapidly after their catch. The loss of freshness is irreversible and suppression of the reaction is always the most important subject for marine products. East Asian people prefer consuming fish as sushi and sashimi. In this case, freshness is the most important factor to determine their value.
Slurry ice is approximately -2.5 ºC sherbets like ice made from seawater. Slurry ice consists of micron order sphere ice crystals and brine solution. We studied the effect of slurry ice to quality preservation of fish.
Live Atka mackerel Pleurogrammus azonus was immersed in slurry ice. The fish died within 20 minutes and struggling was not observed. The energy related compounds of muscle were analyzed. Slurry ice kept ATP content high and suppressed lactic acid production. It is known that excessive exercise accelerates ATP consumption and lactic acid accumulation in muscle. Killing fish by slurry ice have an effect to keep the freshness high.
For a global distribution of fresh fish, we conducted transportation test of fresh fish from Japan to Taiwan. Pacific saury Cololabis saira caught off Hokkaido was chilled by slurry ice made by seawater on the fishing vessel just after catching and sent to Taiwan by airfreight in foamed polystyrene boxes filled with slurry ice. It took three days to be delivered to a Japanese restaurant in Taipei from its catch. Temperature of fish was kept constant at -1 ºC throughout the transportation. The freshness of the fish was sufficiently high for consuming as sushi. The K-value was 7%, which was lower than commercially distributed pacific saury in Japan (17%).
These results indicates that strict freshness control from fishing vessels to consumers by using slurry ice makes it possible to distribute high-value fresh fish in far area apart from fishing ground.

The purpose of this study was to determine the effect of antioxidant and antibacterial nanocapsule liquid smoke on a filet of catfish (Pangasius sp.). A combination of liquid smoke (corncob and coconut shell) processed into nanocapsule with a ratio of gum arabic, maltodextrin, and alginate each 1/6: 4/6: 1/6. Nanocapsuleliquid smoke is known to have a total content of phenols, carbonyl, and RSA respectively 3.682%; 3.439%; and 91.348%. Nanocapsule liquid smoke is applied to the catfish and stored at room temperature and cold temperature. Observations were made on days 0, 2, 4, 6, 8, and 10 to parameter PV, TBA, TVBN and TPC. The results showed that nanocapsule liquid smoke can effectively inhibit the oxidation of fat catfish showed with PV and TBA which are still acceptable, and capable of inhibiting the activity of microbes. It is indicated by the value TVBN and TPC which are still below standard at all temperatures and long storage time.

Postmortem autodigestion of juvenile body of sardines during cold storage is an obstacle to distribute raw juvenile sardines for eating it as raw fish. In this study, we investigated the changes in proteases activity and types of primary proteases in the whole body of juvenile sardines during cold storage and the effect of pressure of fishing on its autodigestion. The juvenile sardines pressed with plates by hand as a pressed fish model showed faster autodigestion during cold storage. The total proteases activity of body of juvenile sardines increased remarkably corresponding to temperature during cold storage. The result of study on proteases inhibitory spectrum suggested that the activation of total proteases activity was caused by the autodigestion of precursors of trypsin and chymotrypsin. The egg white also showed strong inhibitory activity for proteases of autodigestion of juvenile sardines.

[Objective] The specific spoilage organisms (SSO) in aerobically stored sturgeon fillets were qualitatively and quantitatively investigated, and the growth model of SSO was established to predict shelflife of sturgeon. [Methods] The combination of culture-independent (denaturing gradient gel electrophoresis, DGGE) and culture-dependent methods were used to analyze bacteria diversity in the sturgeon fillets stored aerobically on ice. The special spoilage organism (SSO) of aerobically stored sturgeon, was inoculated to sterile sturgeon fillets and stored aerobically. The models were developed based on the data of kinetic growth of SSO and sensory evaluation, pH, total volatile base nitrogen (TVBN) at the end of the shelf life of sturgeon. [Results] The results showed that the dominant spoilage bacteria were Shewanella sp. and Pseudomonas sp. The spoilage potential of Pseudomonas fluorescens, Pseudomonas mandelii and Shewanella putrefaciens was outstanding, and Pseudomonas fluorescens was the strongest spoiler. On the basis of the modified Gompertz equation, square root equations were used as the secondary model to construct growth and shelf life predictive models for Pseudomonas fluorescens and TVC. The results showed that the growth model for Pseudomonas fluorescens was better than that for TVC, and relative errors of predictive shelflives of sturgeon stored aerobically at 0~20℃ were -7.95% ~-3.28%. [Conclusion] SSOs are one of the most important factors to fish spoilage. The shelf life predictive models had great potential for application in ice storage sturgeon product.

In Nepal, the government has been increasing its effort to raise household food and economic security. This has recently been done through the promotion and support of household aquaculture activities. Aquaculture has been shown to significantly increase family protein and micronutrient consumption in addition to provide supplemental income. However, while aquaculture is growing rapidly in some regions of Nepal, other areas are not familiar with aquaculture practices and the support the government will provide. This can be attributed to lack of manpower in the government workforce and consequently community outreach and knowledge. This poster hopes to contribute to the expansion of aquaculture and consequently increased food and economic security in Nepal by: reviewing the importance and potential of the aquaculture industry in Nepal; describing the need for increased aquaculture specialists in the Nepali government workforce; and concisely explaining the current policies and processes involved for households that want to take part in government supported aquaculture. Essentially, the process can be identified in five steps: An individual decides within their VDC or municipality that they want to build a fish pond; 2) The individual visits a District Agriculture Office or Government Research Station and submits an application of interest; 3) A subject matter specialist will visit the proposed area of the individual and perform a feasibility study; 4) If the area is viable, the subject matter specialist will create a plan for the aquaculture system; 5) Lastly, the subject matter specialist will help connect the farmer to workshops and financial assistance to ensure implementation

The Research Center for Marine Education (RCME), a part of the Ocean Alliance, was founded at the University of Tokyo by the Nippon Foundation in April 2010. With the cooperation of school teachers, the RCME aims to promote marine/ocean education in Japanese elementary and secondary schools by curriculum development. Japanese children learn at school and at home that they live in a country surrounded by the ocean. However, children currently have limited opportunities to go to the ocean, and the available curriculum on ocean education is not enough. To expand this curriculum, the RCME surveyed the ocean literacy of school children all over Japan.
The survey consisted of an ocean knowledge test and questionnaires. The target groups were 6th-year elementary school students, 3rd-year junior high school students, and the head of the target schools. The survey period was August–November 2014. The survey method was based on stratified three-stage sampling. In 55 areas, 82 elementary schools and 78 junior high schools kindly agreed to participate in the survey. Overall, 4,574 students completed the survey.
The results of the “Questionnaires on learning and life” revealed that the percentages of children who like the ocean, eating fish, and visiting aquarium were 70% and higher. An example question in the “Ocean exam” involving fishery was “Which line in the graph of fish catch amount shows the recent annual change of Japanese ocean fishery?” The percentage of correct answers was very low. Thus, the knowledge of fishery in school children appears limited. This lack of knowledge may be due to a less interdisciplinary curriculum. Therefore, oceanic education in schools should be expanded using an interdisciplinary approach.