Program

We investigated the community diversity of macrobenthos in different habitats of Shupaisha Wetland in Oujiang River estuary in three habitat forms, i.e.,mangrove habitat, Spartina alterniflora habitat, and naked beach habitat from October 2014 to June 2015. In total, 48 species were recorded, belonging to 40 genus, 31 families, 15 classes,and 5 phyla. The average inhabitant density and biomass were the highest in mangrove habitat, followed by S. alterniflora habitat and naked beach habitat.In addition, we studied the community diversity of macrobenthos in different habitats using species diversity index and G-F diversity.The mangrove habitat showed higher diversity than those of the other habitats.One-way ANOVA analysis revealed no significant difference in season in species number, inhabitant density, biomass, and species diversity, but in habitat in species number, Shannon-Wiener index, and Margalef species richness. Authors believe that artificial restoration of mangrove will enhance the diversity of macrobenthos community.

Drupella, a genus of sea snails is famous corallivorous muricid gastropod as well as crown-of-thorns starfish, Acanthaster planci. The gastropods usually form groups consisting of 1-10 on the coral. However, sometimes the gastropods cause outbreaks and damage to the coral communities.
Coral damage has occurred in the sea area of Ashizuri-Uwakai national park of Japan due to the outbreak of Drupella fragum. The reason of outbreaks of D. fragum has been unclear, and it is suspected that rising seawater temperature and change in salinity (Lam and Shin, 2006).
To protect the corals from the damage caused by the gastropods, the extermination activity is also important, and it is actually done in these areas. Nevertheless, there is a limit to the manual extermination by human, and efficiency improvement is demanded. We thought that the outbreak could be predicted by measuring the number of larvae floating in the sea water. However, identification of gastropod larvae based on morphology is difficult. Therefore, we produced specific polyclonal antibodies against the larvae of D. fragum and the closely related species, D. cornus. Using the immunohistochemically staining, anti-D. fragum larvae antibody specifically recognized the larvae of D. fragum, which were hatched in the closed water tank. Similarly, anti-D. cornus larvae antibody recognized specifically the larvae of D. cornus. These antibodies could not cross-react each other, and it became possible to specifically stain each species. In the future, using these antibodies, we will investigate the number of these gastropod larvae in environmental seawater, and we predict the outbreak occurred mainly in the summer. By prediction of outbreaks, it is possible to efficiently exterminate the corallivorous muricid gastropod and we believe that our method can contribute to conservation of corals.

Ayu (Plecoglossus altivelius altivelius ）is a commercially important resource, a fish with a longevity of about 1 year. It hatches in rivers in the autumn and then swims downstream to the sea. The larvae and juveniles feed on zooplankton in coastal waters. Upstream migration begins in spring, during which it switches its food from zooplankton to benthic microalgae. Growth variation in the Ayu was investigated in the Hirose River, in Miyagi Prefecture, Northeastern Japan. Ayu were collected using a casting net at three stations in 2016. Standard length and body weight were measured. The hatching date of individuals was estimated based on daily otolith rings. To analyze food source utilization, carbon stable isotope ratio (δ13C) measurements were taken from muscle samples around the caudal fin. In general, δ13C reflects that of the diet. There is a difference in δ13C between marine zooplankton (δ13C around -20‰), and freshwater benthic microalgae (δ13C around -16‰). Hatching date composition of Ayu shows a mode within September and October. Mean values of standard length of Ayu increase during upstream migration. The results show a weak positive correlation between standard length and age in days. However, fishes showed large variations in body size (there was differnence in up to 60mm in standard length between the largest and smallest fish) among individuals of the same age collected at the same station and the same sampling time. The larger fishes had higher δ13C values than their smaller siblings. These results suggested that individuals which switch to benthic microalgae earlier take a larger amount of benthic microalgae consequently undergo faster growth.

The whale shark (Rhincodon typus) is found seasonally in Probolinggo waters from December/January until April/May throughout of the year. It has been discovered that the appearance this largest shark are for the reasons of food and protection comfortable oceanographic conditions, and presumably protection. High organic contents and suitable physical factors trigger high production of phytoplankton followed by zooplankton that fed by many early life stages of marine animals including crustaceans, molluscan, and fishes. Those are the main food item for the whale shark. It remains unknown the origin of the arrival and destination of the whale shark after spend several months in Probolinggo waters. Since they are considered as world’s species, it is important to do demographic studies for the whale shark, from which the ID-data may internationally integrated eg. wild-book for whale shark which enable for better conservation and management. For ID identification purpose, 2 snorkelers were swim around the shark and carefully take photo using GoPro 5+. The important object is the area inside the top the fifth gills-the bottom of the fifth gills-the edge of left pectoral fin, also the anal fin where males and females could be distinguished from the presence of claspers. We also recorded the position using GPS, estimating the size, and noticed any wound on their body. ID-photos is determined by the spot pattern on left pectoral fin using I3S and the photos was processed by Paint.NET. The results found approximately 70 individual of whale sharks consisting of 30 females and 40 males. The size ranged between 1,5 – 6 m suggesting that the study area is an important nursery ground. Sex ratio was much higher compared to other whale shark habitat in the National Park of Cenderawasih Bay, Papua. The results of the research implies for better conservation and management.

Ujung kulon National park is a conservation area in Indonesia that holding important role in Maintaining sustainability of biological resources and balancing ecosystem. Intertidal zone is the habitat of varios biotic organism. One of biotic who lives in this region are macrozoobenthos. As an organism who lives in bottom, macrozoobenthos have relatively fixed habitat. Causing by Its behaviour, every changes of water quality and substrate of his habitat, not only affect the composition, but also the abundance. The composition and abundance of macrozoobenthos depends on the tolerance or sensitivity to environmental changes. Difficult access to the site can be one factor of bad monitoring in coastal areas such as Karang Ranjang Beach in Ujung Kulon National Park. Retrieval of data and observations about diversity of makrozoobentos in the intertidal zone of Karang Ranjang beach was held on July, 28-29 2015 by using the quadratic transect method. As the result, this observations indicate that there are 16 species of macrozoobenthos in Karang Ranjang beach. Index of Heterogeneity (H ') obtained fairly low at 1.58, with index of evenness (E) is also fairly low by 0.3 and the Simpsons-Dominance Index is 0.4 due to there is one type whose dominate, it is the Sea Anemone.

The spotted seal and the harbor seal are relative species. They have several similarities and dissimilarities in morphology and ethology, such as they have similar body size and diet while they breed in different environmental areas, season and mating systems. The aim of this study was to examine differences of their skull’s shapes in both species and ages using landmark method.
In this study, we used the skulls from 116 spotted seals and 207 harbor seals. Total 71 landmarks on 19 points of maxilla from dorsal site, 27 points of ventral site, 13 points of left lateral site, and 12 points of mandible from left site were digitized on photographs using the tpsDig software. And each point was measured angle and distance from the standard point. First, we estimate the age of each species which the skull’s shape were consistent figure (similar figure) by hockey stick regression method. Second, we separated the groups of similar figure ages and before this ages on each species and discriminated species each group using random forests and examined important discriminated points.
As results, it demonstrated that the similar figure ages of spotted seals are 7 years and harbor seals 5 years or more. In the group of similar figures, it discriminated species in 98% by angles of two points. Two points were located on external auditory meatus and premolar point of maxilla. Difference in those points between species showed that harbor seal has external auditory meatus on the posterior position and fatter rostrum rather than spotted seal as skull’s shape. Because males of harbor seal compete against the other males by biting for getting females in breeding season, so it would be associated with developed rostrum.

　Recently, the number of harbor seals (Phoca vitulina stejnegeri ) in Daikoku island which main haul-out site of eastern Hokkaido, JAPAN has been increased. Consequently, the fishery damage caused by the seals has been increased. However, because the seals leave no trace on nets after they fed, it is unclear how much seals actually feeding on the fish and what is the impact on the fisheries.
In this study, we aimed to clarify where and how seals use at the haul-out sites or coastal areas. We attached satellite transmitters to nine harbor seals; two adult females, one adult male, three subadults and three yearlings, and clarified the frequency of haul-out times, behavioral ranges and diving behaviors at each growth class and season.
Eight of nine seals were using Daikoku island as main haul-out site. The haul-out frequency of adults increased in breeding season. In contrast, the haul-out frequency in same season of subadults and yearlings is low, and increased in molting season.
　The behavioral ranges was wide in yearlings and subadults, and tended to narrow with growth. Also, the adults were no longer to leave the haul-out sites in the breeding season. In addition, yearlings showed a tendency to move from Daikoku island to another eastern haul-out sites since autumn.
　In the diving behaviors, before and in breeding season, all of the seals were doing shallow diving. However, in molting season, subadults and yearlings had more deep diving than in breeding season.
　In summary, the behavioral ranges and diving behaviors of yearlings are widely, deeper and far away from their main haul-out site, but change to be narrower, shallower and closer with growth. It seemed that adults had the fixed feeding spot, but subadults and yearlings had widly feeding areas.

Recently, the number of spotted seals which visit Hokkaido has been increasing, their distribution spreads southward, and the period of staying in Hokkaido becomes longer. With this, fishery damage becomes more serious. But quantification of this is difficult.
In this study, we aimed to understand that stomach contents of the spotted seals has relation to fishing yields statistics and change of food habits to past, and consider impact to ecosystem. In order to compare food habits between present and past, we collected and examined 146 stomach contents of 3 different areas (Rebun island;25, Lake Saroma;45, Rausu;76) in spring.
In Rebun island, Cottidae ssp, and Theragra chalcogramma were most important foods in 1999, but in 2011 and 2017, cephalopods were. This change might have relation with declination of resources of Theragra chalcogramma by Miyake et al (2005).
In Lake Saroma, Osmeridae ssp, and Clupea pallasii were most important foods in 2014 and 2017. Osmeridae ssp, and Clupea pallasii had been caught for catch target species from long ago, so there was possibility spotted seals had used this species for important foods from long ago.
In Rausu, Theragra chalcogramma was overwhelmingly most important food in 1982 and 1999, but in 2007, 2010 and 2014, Theragra chalcogramma, Ammodytes personatus, and Eleginus gracilis were. The fishing yields statistics showed that catch of Theragra chalcogramma had declined and the peek was later than 1980s. So there was possibility not to be enough resources of this specie in season when the spotted seals stayed and the spotted seals additionally used other species for foods.
This study pointed out some change in times and each area. It was demonstrated that the spotted seals used food item which was easy to eat, and they changed food habits with food item composition in each area.

Irrawaddy dolphin (Orcaella brevirostris) is known an euryhaline dolphin species. In Thai water, it can be found along the coasts of Andaman Sea and the Gulf of Thailand including the inner and middle part of Songkhla Lake. 12 and 21 tissue samples of fisheries-caught or stranded dolphins from Songkhla Lake and the upper Gulf of Thailand were collected, respectively. The genetic variation of the samples were investigated using partial DNA sequences of Cytochrome b (671 bp) and control region genes (417 bp) in mitochondrial genome. 5 variable sites and 6 haplotypes and 7 variable sites and 5 haplotypes were obtained from Cytochrome b gene and control region sequences, respectively. 3 haplotypes of Cytochrome b gene sequences (H_Cytb1, H_Cytb2, and H_Cytb3) and 2 haplotypes (H_CR1 and H_CR2) were restricted to Songkhla Lake samples. There was no sharing haplotype between the samples of the two areas. This result indicated that genetic variation of O. brevirostris in both populations was low and the Songkhla Lake’s population trends to be restricted to the area and separated from the outside populations

Fishery stock enhancement by release is a process in which large number of organisms such as fish, shrimps, crabs, shell fish etc. are reared under artificial conditions and subsequently released into the natural environment where the natural fishery resources are depleted. In order to rebuild the Chinese coastal fish stock, large-scale releases of fishery resource species were started in the mid of 1980s, and Chinese shrimp Fenneropenaeus chinensis was the main target species for restoration. Since 1990s, fish, shellfish and other aquatic animals were also released for enhancement. In 2005, Action Plan on the Enhancement of Fishery Resources of Shandong Province was started, and a more large-scale release program was initiated. The number of marine species released in Shandong Province increased from 11 in 2005 to 26 in 2015, and crustacean was the mostly released species. Due to the stock enhancement program, a remarkable ecological, social and economic effectiveness has been achieved. With the increasing amount of the releases, scientific assessment methods for the stock enhancement programs need to be developed. This study successfully applied a physical tagging in Japanese flounder Paralichthys olivaceus and molecular genetic markers in the Chinese shrimp Fenneropenaeus chinensis to assess the effectiveness of the stock enhancement programs for the two species in Yellow Sea and Bohai Sea. In addition, the genetic tagging method was first successfully introduced in the assessment of the fish stock enhancement program in China, providing a basic theoretical framework for the effectiveness and ecological risk assessment of the fish and crustacean stock enhancement by release and a theoretical basis for the decision making in the fishery stock enhancement.

Five seagrass species (Zostera marina, Z. caespitosa, Z. japonica,Phyllospadix iwatensis and Phyllospadix japonicus) are found in the coastal areas of Shandong Peninsula, in which the dominant species is Zostera marina. Many seagrass habitats around this area have been completely destroyed or are now in rapid decline due to both natural and anthropogenic disturbances. In order to restore the eelgrass resources, a series of transplanting experiments are explored with the staple, TERFS and sandwiched method in the Swan Lake and Shuangdao Bay located on the coast of the Shandong Peninsula. Survival rates of Zostera marina shoots were over 65% and the survived eelgrass transplants have become habitats for some marine animals. In the meantime, we conducted Zostera marina seed planting experiments using burlap bags under field settings to assess seedling establishment. Rate of seedling establishment attained 25.7% at sixth month after planting. Average shoot density reached 78.0 shoots/m² after 22 months of planting. These results will provide valuable guidance for future restoration efforts in this area.

In Thailand, the occurrence and impact of marine non-indigenous species (NIS) had not been well studied. However, recently, UNESCO/IOC Sub-Commission for the Western Pacific (WESTPAC) program on the “Marine Biodiversity and Conservation” had published a report on current status of marine non-indigenous species in the Western Pacific region. This report aimed to consolidate the available information on the current status and list of marine NIS in nine countries; China, Indonesia, Japan, Republic of Korea, Malaysia, Philippines, Singapore, Thailand, and Vietnam. In Thailand, pathways of introduction of marine NIS were divided into 3 ways: intentional introduction (28.57%), unintentional introduction (28.57%), and unknown (42.86%). The intentional introduction included aquaculture and aquarium trade pathways. However, little is known about the impact of those marine NIS on marine ecosystems. So far, only effects of the escape cultured white shrimp, Litopenaeus vannamei (Boone, 1931) had been studied. In the laboratory study, the feeding behavior of L. vannamei was examined and compared with that of five native shrimps and a native crab species to determine the behavioral interactions and potential for food competition. The results showed that L. vannamei was non-selective, consuming the first piece of meat encountered. In addition, L. vannamei was much more aggressive in competing for food than the native shrimp species. Thus, L. vannamei may pose a serious threat to native shrimp species if it fully establishes in marine waters of Thailand.

In coastal waters, extensive water traffic creates underwater sound, which potentially affect marine fauna including cetaceans. A Japanese team launched a new project since 2015 to observe possible effects of underwater ship noise on humpback whales in the breeding grounds of Ogasawara (Bonin) Islands which is located 1000 km south of Tokyo, Japan. An advantage of this field site is no other noise contamination of large ships because of less frequent presence of vessels in this remote water except for the subject liner. Land-based visual observers recorded humpback whales by a theodolite during 30 minutes before and 1 hour after the liner passing across the study area to locate surface position and their surface behavior: blowing, fluke up/down diving, breaching and so on. Total research effort was 32 days for 92 groups of humpback whales in 2016 and 24 days for 51 groups in 2017. For example, average surface time was 87.3 sec and diving time was 339.6 sec at the presence of the liner (n=2), and surface time was 57.3 sec and diving time was 268.5 sec at the absence of the liner (n=2). Radiated noise from the liner was measured in a deep water separately and the position and operational conditions of the liner were precisely monitored onboard. Two autonomous stereo recording systems were deployed horizontally at 20 m in depth under the study area. We will analyze not only direct behavioral reaction of humpback whales but also acoustic reaction of this animal and added the result of the simulated ship noise propagation of the liner. This project would continue one more year to provide a scientific evidence of the minimum sound exposure level to elicit behavioral reaction of the humpback whales. This project is supported by the Japan Ship Technology Research Association (JSTRA) and the Nippon Foundation.

The cumulative events of hyposaline stress due to extreme precipitations and freshwater run of trigger by global climate change is threatening the coastal marine eco-system. In the present study, the effect of short change in salinity on survival and bio-defence responces: lysozyme activity and phenol oxidase activity, of giant abalone Haliotis gigantea were investigated. The test animals were exposed to a temporary periods (3 h per day) of hyposaline stress (26 and 20 psu), followed by recovery period mimic conditions typical for culture site experiencing heavy freshwater input, with a quick return to initial salinity (32 psu). Haemolymph of the abalone were collected for checking the lysozyme activity and phenol oxidase activity. Results showed that means of lysozyme activity and phenol oxidase activity of abalone exposed to temporary salinity of 26 and 20 psu were lower than control (32 psu). Mean Survival rate of abalane kept on 32 (control), 26, and 20 psu salinity were 87%, 77%, and 80%, respectively. Even though the results were not significant different (p > 0.05) amongs treatments, this study indicate that under temporary hyposaline stress, giant abalone might be vulnerable by treat of deasease due to lower lysozyme activity and phenol oxidase activity.

Raising concern on alteration of the amount, intensity and frequency of precipitation which is lead to extreme weather, were inevitably recently. The sudden change of environmental condition, such as heavy rainfall, may cause high runoff that have adverse effect and beneficial consequences for zooplankton as filter feeder. To overcome the detrimental effect of sudden environmental change, the present study assessed the effect of dissolved vitamin B₁₂ in water on the fitness of Moina macrocopa. We exposed neonates of Moina macrocopa (<24 hour) individually using microplate to different level of turbidity (0, 15, 50, 100, 600 NTU) for 3 hours/day during their lifespan. The highest turbidity was decided based upon the field data after heavy rainfall occurrence. After short exposure, the animals then fed by Chlorella sp (free of vitamin B₁₂) followed by adding different doses of vitamin B₁₂ (0, 5, 15, 25 µg/L). Turbidity exposure, additional of vitamin B₁₂ and its interaction was significantly (p<0.05) affect the survivorship and fecundity of Moina macrocopa. The highest fecundity were recorded in the highest levels of vitamin B₁₂ (p<0.05). In addition, delay on maturity was recorded in the test animals that exposed to turbidity without addition of vitamin B₁₂. Moreover, the animal exposed to 600 NTU without additional of vitamin B₁₂ showed 100% mortality before reproduction time. In case of neonates size, there is no significant different among the group treatments. The findings showed that presence of dissolved vitamin B₁₂ allow the animal to deal with turbidity exposure. Thus, this study enabled us to consider water-soluble vitamin as one of potential component for reducing impact of sudden environmental change.

ABSTRACT
The objective of this study was to assess the community structure of mangroves and the presence of current carbon stock condition in Manalu Bay, Sangihe Islands, Indonesia. Mangrove forest plays important role for living organisms, including human, through either ecological or economic function. One of the ecological roles is as carbon absorber in terms of climate change mitigation. Manalu Bay was selected as study site since it had uniqueness with 109.13 ha of natural mangrove forest which connected 2 islands in the area. Besides, Manalu Bay is a tourism development, mariculture, and gold mining exploitation area. Overexploitation of the area could result in environmental destruction. This study was done in October 2016. Mangrove data collection used a 125 m-transect with 10 x 10 m quadrat placed at 5 points. Mangroves covered in transects were identified, and recorded the diameter of tree, sapling, and seedling. Biomass was also measured to obtain information on nutrients and carbon storage in all mangroves or certain species. Carbon stock potential was calculated using allometric equation. The sediment carbon storage data were analyzed using CNS Analyzer. Results showed that Rhizophora mucronata, Avicennia alba and Bruguiera gymnorrizha dominated the mangrove ecosystem in Manalu Bay. Carbon storage of the mangrove ecosystem in Manalu Bay was found 19.5-20.9 tons C/ha and 106.43 – 170.64 MgC/Ha in the sediment.
Keywords: Carbon, Stock Mangroves, Manalu Bay, Sangihe Islands

PURPOSE: The primary causative agent of CFP is the benthic dinoflagellate genus Gambierdiscus. Species composition of Gambierdiscus spp. at shallow waters (0-3 m depth) in southern coastal areas of Japan was previously surveyed and revealed the existence of five Gambierdiscus species in the areas (Nishimura et al., 2013).In contrast, the species composition at deeper waters (10-30 m depths) has not been elucidated. In this study, we investigated species composition of Gambierdiscus spp. at various depths (3-30 m) in the areas and aimed to reveal the horizontal/ vertical species composition of Gambierdiscus spp.
METHOD: Between May, 2014 and May, 2016, we collected macroalgal specimens at 3-30 m depths at Kochi and Okinawa Prefectures. Microalgal fractions were prepared from the specimens and the genomic DNA was extracted. Species compositions of Gambierdiscus spp. were estimated by using qPCR which was developed for of various Gambierdiscus species (Nishimura et al., 2016). Then, we compared the horizontal/ vertical species compositions at the two prefectures.
RESULT AND DISSCUSION: During the investigation period, five species (Gambierdiscus australes, G. cf. silvae, G. scabrosus, Gambierdiscus spp. type 2 and type 3) were detected from both areas. Gambierdiscus sp. type 2 was dominant at Kochi Prefecture, whereas G. cf. silvae was dominant at Okinawa Prefecture. In terms of the vertical species composition at Kochi, Gambierdiscus sp. type 2 was dominant at shallow waters (3 m depth), whereas Gambierdiscus sp. type 3 was dominant at deeper waters (15-30 m depths). At Okinawa, G. scabrosus was dominant at shallow waters, whereas G. cf. silvae was dominant at deeper waters. Considering these results, the horizontal/ vertical species compositions of Gambierdiscus spp. were different between the depths and the areas.

Algicidal bacteria (AB) and growth–inhibiting bacteria (GIB) that kill microalgae and grow using organic matter derived from killed microalgae appear to be promising control agents for harmful algal blooms (HABs). This study reports an interesting characteristics of algicidal or growth inhibition activities of those bacteria that isolated from the biofilms on surface of the seagrass Zostera marina in Puget Sound, WA, USA and the coast of Hinase Islands, Okayama Prefecture, Japan. Co–culture experiments were made using these bacteria and the red tide raphidophyte Heterosigma akashiwo and Chattonella antiqua. The bacterial strain of Pseudoalteromonas sp. entirely killed H. akashiwo when bacterial cells were added as aggregation of cells (colony cake). However, this bacterial strain showed no killing and growth inhibition of H. akashiwo when bacterial cells were inoculated as a cell suspension (liquid culture). And further, other bacterial strains (Phaeobacter spp.) showed strong growth inhibition activities against C. antiqua when bacterial cells were added as small colony cake of cells as compared to the addition as a cell suspension. These results strongly suggest that a quorum sensing mechanism is involved in the killing mechanism of microalgae by algicidal bacteria. Some bacterial strains showed no algicidal or growth inhibition activities even when bacterial cells were added as a colony cake or a cell suspension although these were isolated as AB or GIB. These results indicate that algicidal or growth inhibition activities of AB or GIB were lost through culturing and cryopreservation.

Most of planktonic diatoms form resting stage cells against environments unsuitable for growth, and survive in bottom sediments. However, there is a paucity of findings on resting stage cells of benthic diatoms.The abundance and taxonomic composition of viable resting stage cells of centric diatoms and vegetative cells of benthic diatoms were investigated in 4 sea areas of western part of Japan. The bottom sediments were collected at 5 stations in Suo–Nada and at 11 stations in Saiki Bay, Oita Prefecture on June 3, 2016. Samplings were made at one station in Tajiri Port, Hiroshima Prefecture, in May–August, 2016, and in Ago Bay, Mie Prefecture in July–October, 2016. Resting stage cells and benthic diatoms in bottom sediments were enumerated by the most probable number method (MPN method) and direct count method within 5 days after the each sampling. The diatom resting and vegetative cells in bottom sediments were distributed on the orders of 10² to 10⁵ based on both countings. High densities of resting stage cells were detected throughout this study period, suggesting that resting stage cells in the bottom sediments well function as a seed population. Benthic diatoms were generally more abundant at shallow water stations than deep water in Suo–Nada and Saiki Bay because the light reached the bottom sediments as compared with deep sea area. However, in shallow bottom sediments at Tajiri Port and Ago Bay, the abundance of benthic diatoms drastically decreased when the dissolved oxygen level showed extremely low values (anoxia) water masses at bottom layer. This result suggest that the abundance of benthic diatoms were significantly influenced by the light intensity and oxygen concentrations at the bottom layer.

Submarine groundwater is nutritionally rich and stable in temperature throughout the year, indicating positive effects on fishery production under a variety of changes in environmental conditions including global warming. A recent study indicated that juvenile marbled flounder Pseudopleuronectes yokohamae were abundant around the area with high submarine groundwater discharge in the Seto Inland Sea, southwestern Japan. In order to examine effects of submarine groundwater on feeding and growth of juvenile marbled flounder, physical and biological surveys and on-site cage experiments were conducted in coastal waters of Seto Inland Sea. Fish sampling was conducted by the use of a push-net (0.3 x 1.5 m, 2 mm mesh). Invertebrates (crustaceans and polychaetes, as prey for the fishes) were collected with a bottom plankton net (0.3 x 0.4 m, 1 mm mesh) and a sediment sampler (10 x 10 cm circular cylinder). On-site experiments were made using cages (0.9 x 0.9 x 0.3 m, 5 mm mesh) with cultured juvenile marbled flounder inside. Biweekly sampling from March to May showed high juvenile abundance of in April with elevation of juvenile abundance around the area of high submarine groundwater discharge. The dependence of food webs on organic matters of terrestrial origin was examined by stomach contents and stable isotope analyses. Stomach contents of the juveniles were dominated by gammarids and polychaetes. Cage experiments showed that recent juvenile growth rates back-calculated from the otolith daily rings were higher in area with high submarine groundwater discharge.

Submarine groundwater discharge (SGD) supplies high concentration of nutrients and increases biological productivity in the coastal areas. On the other hand, SGD causes eutrophication. Therefore, it is important to understand an ecological role of SGD. In this study, we estimated how much SGD contributes to biological production in the coastal area where fresh groundwater inputs, by using nitrogen stable isotope ratio of Ulva sp..
We analyzed salinity, the concentration of dissolved inorganic nitrogen (DIN) and radon (²²²Rn) and δ¹⁵N^NO3- of ambient water at 5 sites (Kamaiso, Torisaki, Takinoura, Mega, Nishihama) in Yuza Yamagata in June 2015 and 2016. We also collected Ulva sp. in the study are and analyzed δ¹³C and δ¹⁵N.
Kamaiso and Mega were high SGD areas and Torisaki and Nishihama were low SGD areas judging from the ²²²Rn concentration. The δ¹⁵N^NO3- values of groundwater collected at land ranged from 1.7 to 2.5‰ and those of ambient water in high SGD areas and low SGD areas ranged from 2.2 to 4.5‰ and 5.6 to 8.6‰, respectively. The δ¹⁵N of Ulva sp. in high SGD areas ranged from -0.3 to 1.5‰, and those in low SGD areas ranged 1.6 to 1.9‰. In order to evaluate the contribution of SGD to Ulva production, we used two-source model with δ¹⁵N^NO3- and nitrate concentration of groundwater and sea water. The contribution rates of SGD estimated by δ¹⁵N of Ulva sp. ranged from 15.5 to 42.8% in high SGD areas, and 12.8% in low SGD areas. The contribution rates estimated by δ¹⁵N^NO3- of ambient water was substantially similar as that by Ulva sp.. This study showed that it was possible to estimate the contribution rate of SGD to the biological production by using the nitrogen stable ratio recorded in Ulva sp..

In order to investigate the influence of the watershed on coastal biota, we collected the snail Batillaria multiformis from tidal flats at river mouths in Kunisaki Peninsula, Kyushu, Japan and nearby region and determined the isotopic compositions (δ¹³C, δ¹⁵N). As the habitat of B. multiformis shifted seaward along two transects at the mouths of Iroha and Katsura rivers, the δ¹³C of the snail decreased. A Bayesian mixing model (SIAR) showed that the most important food sources were seagrasses and/or a saltmarsh plant, followed by benthic microalgae and marine phytoplankton and that the importance of marine phytoplankton increased at more seaward stations. Observations on the tidal flat and gut content analysis, however, suggested that benthic microalgae formed the major component of the diet. Regarding the δ¹⁵N, for both rivers there were no clear trends along the river-sea direction, but we found that there was a significant difference between the two tidal flats. Therefore, we collected B. multiformis from a further 23 tidal flats and examined the relationship between its δ15N and the watershed characteristics. Among 37 parameters, there were significant negative relationships between the δ¹⁵N and the average slope, the ratios of volcanic rock and forested land, and significant positive relationships between the δ¹⁵N and the ratios of paddy fields and agricultural land. Enriched δ¹⁵N of the snail is considered to have resulted from denitrification under stagnant water in the catchment areas.

Japan is a country of rich forests surrounded by sea. Ecological connectivity of forests, rivers and seas is an important factor affecting the biological productivity in rivers and coastal waters. However, the effect may be different according to the different types of land usage and river ecosystem, so that the effects of the connectivity on the biological production are varied and have not been fully studied. We therefore compared the land-use structure, nutrient supply, and riverine production between two rivers, the Katsura River and Iroha River, in Oita Prefecture. The two rivers have different land-use patterns: the ratios of forests and farmlands in the former are 77% and 17%, while those in the latter are 64% and 29%, respectively. The concentration of dissolved inorganic nitrogen that was thought to have originated from forests was higher in the Katsura River. Carbon and nitrogen stable isotope analyses revealed that both rivers had similar food web structures, which composed of Japanese eel (Anguilla japonica) as the top predator and benthic microalgae as the primary producer. However, the biomass and growth rate of eels were significantly higher in the Katsura River than those in the Iroha River. In addition, aquatic animals, which were available prey items for eel inhabiting the Katsura River, were more abundant than those inhabiting the Iroha River. The biomass of benthic microalgae was also higher in the Katsura River than that in the Iroha River. Our results suggest that rich forests can support rich aquatic production through the abundant supply of nutrients.

Seaweed and seagrass beds are considered as important places for marine organisms, and various investigations are performed there. Although quantitative surveys using seine-net have been conducted at sandy and muddy areas, especially Zostera marina beds, the methods of biological collecting survey have not yet been established at rocky areas like Sargassum beds. The aim of this study was to consider a method that can collect fish and shrimps by using a seine-net at the rocky area, and to verify that the method is useful for collecting surveys. Sampling was conducted at two different shallow subtidal zone that were a Sargassum meadow and an unvegetated area at Koinoura coast (Fukutsu City, Fukuoka Prefecture, Japan). A seine-net (1.45 m deep × 11 m long, 1.0 × 1.5 mm mesh size) was tested under different patterns of deployment at both zone that included a pattern was a new method of enclosing the target area in a circle with the net and then raising the lower end after closing it. This method at Sarugassum meadow was conducted in May - December, 2016. The other pattern was a standard method that was to drag the net about 3 meters so that area of the survey was the same as the new method. The comparative surveys were conducted from October to December. Through the entire periods, 33 species of fish, shrimps and mysids (6,365 individuals in total) were collected. The result of the comparative surveys at Sarugassum meadow showed some differences; 16 individuals (2 fishes) were collected by the new method and 4 individuals (3 fishes) by dragging. As a result, although it was difficult to conclude that the new method is useful for quantitative investigation, the new collection method also has several advantages for fish collecting surveys in these habitats.

The purpose of this study is to build data mining models to predict fish occurrence. Numbers of data in this study are more than 1500. These data were sampled at different site and time. We select Random Forest (RF), Logistic Regression (LR) and Boosting Trees (BT) models which are commonly used in binary classification. In this study, we also evaluate the extrapolation ability of models. In addition, we apply Variable Importance (VI) and Partial Dependency Plot (PDP) to analyze simulation mechanism of models. In results, performance of RF and BT is good in most of the simulations. We consider that RF and BT are more appropriate to predict fish occurrence than LR. And these two models have great extrapolation ability. We finally emphasize the importance of data variety in prediction through our VI and PDP analysis.

The ability to identify the time of ovulation can improve efficient seed production in aquaculture. Ovulation is difficult to detect, however, due to environmental and physiologic factors that influence fertility and ovulation rates. In fish plasma, however, the levels of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) rapidly increase just before ovulation. Therefore, monitoring DHP levels is an effective way to predict ovulation. We developed a biosensor system to detect DHP by analyzing the rate of decrease in the output current of an enzyme reaction using the amperometric method as the immunoreaction causes electrochemical changes of the electrode surface.
To prepare the immunosensor, an anti-DHP antibody and enzyme (glucose oxidase) were immobilized on Au electrode surface (3.0 mm diameter) modified with a self-assembled monolayer. The sensor was immersed into a DHP standard solution. We used the amperometric method to quantify DHP levels by analyzing the rate of decrease in output current due to the immunoreaction generating a non-conductive antigen-antibody complex that inhibited the enzyme producting electron .
Evaluation of the reaction conditions of this sensor, revealed an optimal temperature of 30 ℃,and optimal pH of 6.5. The immunosensor was specific DHP. The specific detection of DHP was indicated by the minimal change in the anodic peak value when the sensor was exposed to other steroid hormones, and there was a high linear correlation (correlation coefficient : 0.9812) between the rate of decrease in output current of the sensor and the DHP concentration in the range of 7.81~500 pg ml^-1. These findings suggest that the proposed sensor system can be used to analyze DHP levels, and can be used to construct a system with little load on organisms and environment.

It is known that there are two kinds of stress: Eustress and Distress. Eustress motivates continued efforts and promotes strength. When the stress is no longer tolerable and manageable, the distress comes. It is said that blue light stimulation enhances fish feeding efficiency. Thus, adequate and appropriate amounts of stress might be beneficial for fish. To explore this possibility, we monitored the responses to different levels of stress in fish using our proposed wireless biosensor system that monitors blood glucose levels as an indicator of stress in fish.
In this study, Nile tilapia (Oreochromis niloticus) was adopted as test fish. Variation in environmental salinity was selected as a stress factor that might be considered beneficial for fish. To investigate the influence of rearing conditions with different concentrations of salinity on fish stress responses, the recovery rate following exposure to a higher level of stress was used as the evaluation index of eustress. At first, test fish were immobilized by the wireless biosensor after acclimatization to the breeding water prepared at a variety of salt concentrations for 3 days as a stress factor. Then, test fish were exposed to air for 10 minutes, and the stress recovery rates were monitored after the test fish was returned to the breeding water.
Evaluation of the influence of breeding conditions with different salinity concentrations on the test fish revealed that variations induced changes in the glucose levels over time. Salt bathing does not seem to have a positive effect on fish stress recovery. Other fish species that are not euryhaline like Nile tilapia, however, may respond differently to stress resulting from changes in the environmental salinity.

We developed a biosensor system to measure the glucose concentration in fish as an indicator of fish stress. Uniform immobilization of the enzyme on the electrode surface was difficult, however, and thus the sensor response was often reduced in the high glucose concentration range observed during stress monitoring. Therefore, we attempted to enhance the sensor response by immobilizing the enzymes using a self-assembled monolayer (SAM) in which molecules can spontaneously assemble to form a regular array.
The oxidation current is measured by applying voltage to the hydrogen peroxide produced by the enzymatic reaction of glucose oxidase (GOx) and glucose. The glucose concentration is obtained based on the output current. For preparation of the biosensor, GOx was immobilized on the working electrode modified with a SAM. After immersing the sensor in a phosphate buffer solution (0.1 M, pH 7.8), standard glucose solution was added.
Optimization of the conditions revealed that the optimal pH for the sensor was 7.4-7.8. The proposed biosensor showed a good correlation between the output current and a glucose concentration range of 10-3500 mg dl^-1. Compared with the dynamic measurement range of the most responsive conventional biosensor (10-100 mg dl^-1), the dynamic measurement range of this newly developed sensor was significantly improved in the high concentration range. The uniformity of the enzyme array on the electrode by the immobilization of GOx using a SAM led to an efficient enzymatic reaction. In addition, the output current and glucose concentration were well correlated in an actual fish sample.

Gonadal development demands high consumption of energy which reduces growth rate of fish and make fish susceptible to diseases. Therefore, method to interfere with the gonadal development might have an impact on production of fish aquaculture. Follicle stimulating hormone (FSH) is one of gonadotropins and regulates the gonadal maturity. It was shown that autoantibodies against FSH β-subunit causes infertility in human female.
In the present study, juvenile rainbow trout (Oncorhynchus mykiss) were injected with recombinant FSH β-subunit proteins (rFSH) mixed with freund’s complete adjuvant to induce anti-rFSH antibodies which can interfere with FSH functions. After injection, serum was taken from fish at 0, 14 and 28 days after the injection and antibody titers were measured by ELISA. The serum of rFSH-injected fish showed significantly higher antibody titer against rFSH than the before injection. The increase had been kept for 4 weeks. In addition, anti-rFSH antibodies were detected in the rFSH-injected fish serum by Western-blot assay. These results showed the immune system had recognized rFSH proteins as antigens and produced anti-rFSH antibodies in the rainbow trout. However, antibody titers showed individual differences, which might have been caused by non-specific antibody bindings. In next step, tissue section analysis will be analyzed to observe the actual change in gonadal tissue development.

Artificial and natural hybridization of Dojo loach Misgurnus anguillicaudatus (2N=50, DD for short) and large scale loach Paramisgurnus dabryanus (2N=48, PP for short) are well-grown. However, these hybrid loaches have not yet been examined for fertility and ploidy of gametes. Histological observations, artificial propagation, observations of embryo development and larvae morphology, and ploidy analyses were conducted to determine the fertility and ploidy of gametes of allodiploid (DP for short) and allotriploid (DDP for short) loaches, which produced by diploid M. anguillicaudatus females × P. dabryanus males and induced from fertilized eggs of diploid M. anguillicaudatus females × P. dabryanus males by cold shock to prevent the second polar body release, respectively. Ovaries of DP and DDP were of few eggs, while Ⅲ and Ⅳ stage of ovum were observed. Testes of these two loaches were delayed-developed without spermatid or mature sperm. Results obtained here showed that DP and DDP were female weakened fertile and male sterile. Moreover, DP females and DDP females could respectively produce few viable haploid eggs, and few viable haploid and diploid eggs. This study will provide valuable information for fish hybrids researches.

This study was conducted to evaluate how spermiation male or ovulating female could act as an inducer of ovulation in another females of tiger barb. In the first experiment, males and/or females of tiger barb as inducer fish were stimulated to spawn using 0.01 mL ovaprim per fish and then placed inside spawning net in the aquaria. A group of un-injected males and females were used as control. Then ten mature females of tiger barb for each treatment were placed into aquaria outside the spawning net. After the inducer fish had spawned, ten females outside the spawning nest were manually stripped to examine the occurrence of ovulating females. In the second experiment, a group of tiger barb were placed into spawning net in five different aquaria: (1) control (un-induced males and females), (2) Only females were artificially induced or not, without males existence, (3) only males were artificially induced or not, without females existence. Then ten mature females of tiger barb for each treatment were placed outside the spawning net in the aquaria. After six hours, the ten females were manually stripped to examine the occurrence of ovulating females.
In the first experiment, it was shown that artificially induced spawning fish could stimulate another fish outside spawning net to ovulate. However, it was not clear whether the stimulation came from spawning males or females. In the second experiment, it was shown that artificially induced males without females were able and more potent in stimulating another females outside spawning net to ovulate

Keyword: induction ovulation, spermiation males, Puntius tetrazona, tiger barb.

Introduction: There have been few studies on the influence of the wavelength and quantity of ultraviolet (UV) light in natural sea water on marine fish. In this study, we investigated the influence of various wavelengths and quantities of UV light on the survival, feeding, and growth rate of eggs and larvae of Pacific bluefin tuna.
Materials and Methods: Experiment I: On the basis of the UV level at Cape Hedo, Okinawa, Japan, from April to August, we established high, medium, and low UV irradiation groups. Eggs were irradiated and the normal hatching rate was measured. Experiment II: We established UV1 and UV2 groups in which the UVA and B irradiation amounts were the average intensities at water depths of 10 to 20 m. In each test group, eggs were stocked in a 200-L water tank, irradiated with UV together with white light, and reared for 10 days. Experiment III: For comparison with Experiment II, we established UVA only and UVAB test groups in which UVA and B were set to correspond to the UV intensities at depths of approximately 5 to 8 m.
Results and Discussion: Experiment I: There was no significant difference in hatching rate between the groups. Experiment II: UV radiation tended to increase feeding and growth rates. In addition, the number of intestinal rotifers and total length of larvae in the UV2 test group were significantly higher compared with the control group. Experiment III: The number of intestinal rotifers increased in both UVA and UVAB test groups, and larval growth in the UVAB test group was superior to that in the control group. These results indicate that natural UV irradiation does not adversely affect the eggs and larvae of Pacific bluefin tuna, and indeed promotes feeding and growth within the range of their distribution depth.

Introduction
Mass mortality by sinking deaths of Pacific bluefin tuna (PBT) larvae is one of the major problems inhibiting the large-scale culture. In this study, the effects of the tank shape, water current control and rearing density on the growth performance were examined.
Materials and methods
Experiment-I: A flat- or round-bottom 200-L tank equipped with an aeration or the pump system were employed. For 10 days, the water current in the tank as well as the feeding, growth, and survival rate of the larvae were compared. Experiment-II: The effects of water pump flow rate (1.5, 3.0, 4.5 and 6.0 L/min) on the water current of tank and on the growth performance of the larvae were examined in the round-bottom tank. Experiment-III: The effects of rearing density (3, 10, 30 and 100 ×1000 larvae/kL) on the growth performance of the larvae were examined.
Results and Discussion
Experiment-I: The feeding, growth, and survival rate of the larvae and the water current near the tank bottom were highest in the round-bottom tank equipped with a pump. Experiment-II: Based on these results, the survival rate of larvae and the water current near the tank bottom were higher in the experimental groups for which a flow rate of 4.5 and 6 L/min was used. Experiment-III: There were no differences of growth performance of larvae in all group at days 6. Although the ultra-high density group (100 ×1000 larvae / kL) of larvae indicated the low growth compared to low density group, the amount of larvae were highest in ultra-high density group up to 10 dph. These results suggest that the optimal flow velocity should be within the range which a flow rate of 4.5 and 6 L/min, and the larval PBT were able to produce with ultra-high density of 100 ×1000 larvae / kL.

The flow structures in the rearing tank were generated due to interference between water and air bubbles released from airstone. The survival rate of larval fishes is considered to be sensitive to the flow structure in the rearing tank. It has been reported that optimize aeration rate for rearing larvae fishes was existed. However, few studies estimating flow field in the rearing tank have been conducted. Thus, it is significant to study the flow field in rearing tank in detail.
In relation to past studies, the rearing experiments of seven-band grouper and devil stinger with circular tanks of three different proportions under constant water volume was performed. The result indicated that survival rate for both fish larvae became to be greatest in the most a long thin tank. We have performed numerical simulation and flow visualization in a circular tank. The flow patterns in vertical cross-section changed from a single-pair vortex system to two-pair vortex system as the value of AR (the ratio of water depth to tank inner radius) changed from 1.0 and 2.0. We speculate that between survival rates and flow structures have a correlation.
In general, the geometry of rearing tanks has been used to those of circular, rectangular and octagon. Recently, it is reported that survival rates are different in the rearing experiments for circular and rectangular tanks. Therefore, it is significant to study the flow field in the rectangular tank. The flow pattern in rectangular tank may change to that of circular tank.
We have guessed the flow pattern by numerical simulation, and then verified the guessed flow pattern using flow visualization. The visualized flow patterns were approximately consisted with those of numerical simulation.

Introduction
Although the development of seedling production technology is required, the mass production of Longtooth grouper (LTG) larvae is problematics. We reported that round-bottomed tanks are more effective at reducing sinking deaths than general flat-bottomed tanks. In this study, we evaluated the most appropriate water-current control systems for rearing larval LTG and examined the optimum flow speed for each condition and developmental stage.
Materials and Methods
Experiment I: Larvae were reared for 10 days in triplicate in four round-bottom tanks with pump flow rates of 500, 1000, 1500, and 2000mL/min. Experiment II: Larvae were reared for 10 days in three experimental groups, set up in triplicate, in round-bottomed tanks with air flow rates of 40，200, and 400 mL/min. Experiment III: Larvae were reared for 10 days in triplicate in four round-bottomed tanks with pump flow rates of 1500mL/min and air flow rates of 200 mL/min. The flow rate rearing systems were changed from pumps to aeration on days 3 and 6.
Results and Discussion
Experiment I: Optimal survival rates and total lengths were obtained at the 1500 mL/min water-pump flow rate. Experiment II: The average survival rate was approximately 67% at the 40 and 200 mL/min aeration rates, which was significantly higher than that obtained at the 400 mL/min aeration rate. Experiment III: The survival rate of the aeration group was significantly higher than that of the pump groups. However, the survival rate for the group that had the flow system changed from pump to aeration on days 3 was approximately 80%, which was the highest. These results suggest that the optimum pump rate for LTG larva rearing is 1500 mL /min for the first 3 days and that the optimum air flow rate is 200 mL/min for the subsequent 3 to 10 days using semi-spherical round-bottomed tanks.

Recirculating aquaculture systems are the effective means for environmentally friendly and high productivity of aquaculture production. This study evaluated the efficacy of aeration in closed systems on the growth of pacific white shrimp. The experiment was designed in to 3 groups; air stone (control), air stone + wet and dry central drain system (T1) and air stone + wet and dry central drain system + Thalassiosira weissflogii (T2) 3 replications of each treatment were perform. Shrimps were culture into cement tank consisted 4 tons of water for 45 days. Dissolved oxygen (DO) during culture period was 7.1, 7.3 and 7.5 ppm in control, T1 and T2 respectively. At the end of the study, the harvest results showed that shrimp weight and survival rate in treatment groups were significantly (P 0.05) higher than control group. Consequently, increasing the shrimp growth and survival without water exchange, in wet and dry central drain system + Thalassiosira weissflogii system.

Introduction Although ayu are amphidromous fish, the effects of rearing water salinity on the growth performance of juveniles are unclear. In this study, juvenile ayu were reared in water with different salinities, and survival, growth rate, and stress tolerance were examined.
Materials and Methods Experiment I: The plasma osmotic pressure of juveniles reared in freshwater was measured. The salinity of the freshwater was increased from 0 to 11, 22, and 34 psu in 11-psu daily increments, and survival rate for 6 days without feeding was examined. Additionally, juvenile fish reared in freshwater were stocked directly in rearing water of 9, 13, 22, 28, and 34 psu, and survival rates were measured the following day. Experiment II: Twenty juveniles were stocked in the 500L tank of a recirculating aquaculture system, fed with formula diet, and reared for 30 days. Three experimental groups of 0, 11 and 34 psu were established in duplicate and growth performances were compared. At the end of the experiment, plasma osmotic pressure, stress tolerance, skin rupture strength, and meat quality were compared.
Results and Discussion Experiment I: The plasma osmotic pressure of juveniles was 330±12.2 mOsm/kg, which is equivalent to 11 psu seawater. The survival rates of all fish gradually or directly exposed to water of different salinities were 100%. Experiment II: The weight gain at 0 and 11 psu was significantly higher than at 34 psu. The plasma osmotic pressure of fish at 34 psu was significantly higher than that at the others salinities. Handling stress tolerance at 0 and 11 psu was significantly higher than at 34 psu, and that at 11 psu was the highest among the three groups. These results indicate that juvenile ayu readily adapt in freshwater and seawater and grow well at salinities from 0 to 11 psu.

Aquaponics is a modern dual-production system that creates a symbiotic relationship between aquaculture and crops grown in hydroponic systems. Studies have shown that aquaponics play significant roles in food production. However, such studies are relatively new and, have not been completely explored in Kenya. The aim of this study was to develop a low-tech aquaponics system for small and medium scale farmers with a view of improving food security in Kenya. This study assessed the growth and physiology of Nile Tilapia (Oreochromis niloticus) and an African indigenous vegetable, amaranth (Amaranthus cruentus). The efficacy of the hydroponics system as a bio-filter of total ammonium nitrogen (TAN), total organic carbon (TOC) and total suspended solids (TSS) from the fish culture tanks was also evaluated. The results showed a higher turnover (biomass and grain yield) of crops grown in the aquaponics system than those grown in the field under all treatments. The total fish weight increased by 4.3% and length increased by 1.7% while the grain yield for the amaranth increased by 8.7% compared to those grown in the field. There was no significant difference in growth of fish grown in the aquaponic system and those reared in earthen ponds. The TAN and TOC were reduced by 6.2% and 7.1%, respectively by the aquaponic system, while the TSS was reduced by 12.8%. The hydroponics system appears to be an efficient aquaculture waste reduction technique that also increases production yield, thus appropriate for food production in the developing countries like Kenya. Further studies are recommended to identify suitable substrates for use in the hydroponics systems, and cropping density as well as energy reduction strategies.

The culture of two species of microalgae, Chaetoceros gracilis and Tetraselmis tetrathele with wastewater discharged from the recirculating aquaculture system with kuruma shrimp and their seed production using the microalgae as initial live feeds has been established in our previous study. In this study, the effect of manganese supplementation to aquacultural wastewater as liquid medium for the culture of two species of microalgae were defined from point of view of the feed value for seed production of kuruma shrimp. Feeding experiment of the larvae during the nauplius to post-larval stage with 2L-Erlenmeyer flasks was conducted with feeding two species of microalgae cultured with F medium as a control, the wastewater, and that enriched with manganese, respectively. The initial density of the larvae and water exchange were set to 50ind./L and 10% exchange every 2days. The water temperature, salinity and aeration were maintained at 28ºC, 32psu and 0.5L/min. Eicosapentaenoic and linoleic acid compositions of C. gracilis cultured with wastewater were less than half than other two groups, while the fatty acid compositions of T. tetrathele were not significant difference among the groups. The survival rate of kuruma shrimp larvae fed on two microalgae cultured with waste water enriched with manganese chloride was similar to control, while, that cultured with only waste water was lower than other two groups. The survival rate of each group fed both two microalgae thought the feeding period was superior compared with that fed on C. gracilis during earlier zoea stage and T. tetrathele during later zoea to mysis stage, separately. These results reveal that the manganese content and fatty acid composition of C. gracilis, the manganese content of T. tetrathele, and also survival rate of kuruma shrimp larvae were improved by waste water enriched manganese as algal culture medium, and feeding method.

[Objective] Salmon trout, large rainbow trout Oncorhynchus mykiss farmed in seawater, is one of the most popular seafood, and stable commercial production of the species is required in Japan. Year-round seawater farming can provide stable supply of salmon trout. However, period of salmon farming at the sea surrounding Japan is limited, because the seawater temperature during summer is too high for salmon trout. In Aomori Pref., around 1.2 million liters of seawater per hour are pumped from the undersea tunnel (the Seikan tunnel) and released on ground. Utilization of the spring water pumped from the undersea tunnel for land-based aquaculture can provide year-round seawater farming of salmon trout in Japan at a low utility cost. In this study, we tried rearing salmon trout during all seasons using the spring water pumped from the undersea tunnel.
[Methods] Donaldson strain, which has been cultivated in Japan for long period, was used. Immature fish (body weight: 441 g, n = 140) were reared in 10 m³ tank using the spring water pumped from the undersea tunnel (flow rate: 150–200 L/min, salinity: 26–28‰) from May, 2016 to March, 2017.
[Results] The temperature of the spring water was lower than that of surface of the sea surrounding Aomori Pref. in summertime (highest temperature: 21.9°C), in contrast higher than that in wintertime (lowest temperature: 11.5°C). The mortality rate in summer was 16.9% and highest among all seasons. The growth rate kept low value after summer and the feed conversion rate decreased in autumn. After experimental period, 73% of the fish survived and average body weight of those reached optimal size for shipment. These results suggest the possibility of year-round seawater farming of salmon trout in Japan. This research was supported by grants from the BRAIN (the special scheme project on regional developing strategy).

To elucidate the factors that lead to deformities in hatchery-reared red-spotted grouper Epinephelus akaara, we conducted two experiments in relation to feed. In Exp. 1, larvae were reared under 4 different feed combinations (Artemia and rotifers (Ar), formula feed and rotifers (Fo), formula feed soaked with water and rotifers (SF), rotifers (Ro)) from 15 to 30 days after hatching (DAH) to investigate whether feed affects their deformity rates. Furthermore, effects of swim bladder inflation of larvae on their deformity rates were examined. In Exp. 2, the effect of different Artemia feeding regimes (Artemia feeding started on 11 DAH, 16 DAH, 21 DAH, 26 DAH, and only rotifers (Ro)) on deformity rates were examined from 11 to 31 DAH. Results of EXP. 1 showed that the centrum deformity (CD) rate and spinal deformity (SD) rate in the abdominal areas were highest in Fo (CD was 43.4% and SD was 45.9%), second highest in SF (19.4% and 24.3%), and low in Ar (3.1% and 11.2%) and Ro (5.9% and 9.8%). While, there were no significant difference in the 1st to 5th pterygiophore of dorsal fin deformity (PD) rates among the feed combinations. Opercular deformity (OD) rate and shortening of the lower jaw (SLJ) rate in individuals with un-inflated swim bladders were significantly higher compared to those with inflated swim bladders, regardless of the feed combinations. In EXP. 2, no significant difference in OD, SLJ, SD, PD were observed among the different Artemia feeding regimes. The present results suggest that: 1) observed OD, CD and SD in the abdominal areas may be attributed to physical effects such as the expansion of formula feed in the stomach. 2) Failure of initial swim bladder inflation would potentially be one of the factors that cause OD and SLJ. 3) PD is initiated before 11 DAH.

The demand for rotifers such as Brachionus rotundiformis, which is considered indispensable for larviculture, has increased considerably in the tropical countries. Fresh microalga is the common diet, but culture protocols are laborious and costly, thus limit sufficient production of rotifers and, disrupt fish seedling production programs in microalgae-based hatcheries. This study developed cheap and stable fishwaste diet (FWD) as an alternative to microalgae. The diets were: FWD1 (fishwastes only), FWD2 (FWD1 + starch) and control (Chlorella vulgaris only). The FWD was used to determine the population density, specific growth rate and nutritive value of the rotifer, B. rotundiformis (SS-type) in 30 l tanks of sea water (22 ppt.), in which 20 rotifers ml^-1 were stoked and semi-continuously cultured for 18 days at 28±1^oC without aeration. Coefficient of variation (CV %) was calculated to determine the stability of the cultures. The culture medium and rotifer gut were screened for bacteria in sterile conditions and, Zobell marine agar was used to make agar solution for plating. The bacterial colonies were aseptically isolated for biochemical identification according to the Bergey’s manual. The rotifers were harvested and analyzed for total lipids. FWD2 produced significantly higher rotifer density than FWD1 and control diet, and up to 1,188±69.7 rotifers ml^-1 were obtained between 8-13 days with FWD2. FWD did not affect CV, which were 11.43±5.18, 7.47±1.68 and 8.01±3.72% for FWD1, FWD2 and control, respectively. The FWD-fed rotifers ingested bacterial species such as Pseudomonas sp., Bacillus sp., Thiocapsa sp., and Shewanella sp., while the control-rotifers mostly ingested Micrococcus sp. About 0.35 and 0.39 mg g^-1 of DHA and EPA, respectively was obtained in the FWD-fed rotifers and 0.0 mg g^-1 of DHA and EPA in the control-rotifers. The FWD may have containd essential nutrients and probiotics, which explain the high rotifer growth in the FWD cultures.

The marine copepod Tigriopus japonicus can be mass-cultured, as well as shows higher nutritional value and wide size distribution associated with developmental stages. In spite of these advantages, their meiobenthic habitat remains an obstacle for utilization in fish larviculture. This study investigated the phototactic behavior of T. japonicus under various light conditions with an aim of regulating their distribution by light.
Phototactic behavior was determined at two developmental stages: nauplius and adult (including males and females). Their light sensor, eyespot was firstly characterized by area and absorbance. Secondly, phototactic behavior i.e., phototaxis was estimated with the following methods. Fifteen individuals were dark-adapted for 5 minutes before 15-minute lighting on a side of experimental vessel (15×3×3 cm). Light conditions were set-up by the combination of four light wavelengths (white peaks at 460, 570 nm, blue at 470 nm, green at 525 nm, red at 660 nm), and five intensities (0.5, 2.0, 3.5, 5.0, 15.0 W/m²) using LEDs (light emitting diodes). The distribution of individuals under each treatment was compared with those under total darkness (control).
The eyespot efficiently absorbed the light wavelength range from 400 to 550 nm regardless of developmental stages and sexes. Phototaxis showed different patterns between nauplii and adults; no differences between the sexes. The nauplii showed negative phototaxis with all tested light wavelengths at 0.5-5.0 W/m², while they lost phototactic movement at 15 W/m² except the red light induced negative phototaxis. On the other hand, the adults showed negative phototaxis at 0.5 and 3.5 W/m² and positive phototaxis at 2.0 W/m² under the all light wavelengths. The obtained results elucidate that T. japonicus showed different patterns of phototaxis related to developmental stages and the potential of regulating their distribution in a water column with light irradiation.

Highly unsaturated fatty acids (HUFAs) such as Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) are essential for the growth and survival of marine animals. Rhodomonas sp. is used in this study because it has high levels of HUFAs. Fatty acid composition in microalgal cells vary by changes in the culture environment. In this study, Rhodomonas sp. was cultured at different growth phases (log-phase of growth, transitional phase, stationary phase), incubation temperatures (16, 20, 24 ℃) and photon flux densities (PFD: 50, 200, 550 µmol m⁻² s⁻¹). As the culture period increased with differences in growth phases, there noted a decrease in fatty acids with long carbon chains and an increase in fatty acids with short carbon chains. It was found that HUFAs such as EPA and DHA reached to their highest peaks in the growth phase (10.2%, 6.2%) and then decreased as the culture progresed. Regarding the temperature, fatty acids such as myristic acid and palmitic acid increased according to the rise in temperature. EPA and DHA reached maximum levels at 20 °C (12.2%, 5.3%). With accordance to PFD, myristic acid and palmitic acid increased as light intensity became stronger, and EPA and DHA remarkably decreased when reaching a PFD of 550 µmol m⁻² s⁻¹. There was no significant change in HUFA amount at PFD, 50 μmol m⁻² s⁻¹ and 200 µmol m⁻² s⁻¹, but the growth rate was lower at PFD, 50 µmol m⁻² s⁻¹. For this reason, it is considered that a PFD of 200 µmol m⁻² s⁻¹ is best suited for producing Rhodomonas sp. as live feed for marine animals in aquaculture.

Chitin, a β-1, 4 linked polymer of N-acetyl-D-glucosamine, is the main component of exoskeleton of crustaceas and cell walls of fungi. Chitinases are enzyme that hydrolyzes β-1, 4 glycosidic bond of chitin, which are widely distributed in animals, plants and bacteria. In crustaceans, chitinases play important physiological role such as digestion of diets, ecdysis and protection against viral pathogens in vivo. Although several studies have been investigated for the chitinase genes from some crabs and shrimps, there have been no reports on chitinase genes from hermit crab. In this study, therefore we tried the cloning of chitinase cDNA from the midgut gland of a hermit crab Paralithodes camtschaticus and a crab Chionoecetes opilio.
Total RNA was extracted from midgut gland by using RNA extraction reagent (ISOGEN). First strand cDNA was synthesized using total RNA and reverse transcriptase. Internal fragment of chitinase gene from crabs were amplified using the synthesized cDNA, DNA polymerase and degenerate primers which were designed based on the conserved region of amino sequences of GH family 18 chitinases from several species. The chitinase gene fragment of 480 bp was obtained from midgut gland of P. camtschaticus. The deduced amino acid sequences of DNA sequences showed the similarity of 73% with the chitinase of Eriocheir sinensis. Amplitied cDNA fragment from midgut gland of C. opilio is currently analyzed. Moreover, the phylogenetic analysis revealed that P. camtschaticus chitinase didn’t belong with the group of crab chitinase and shrimp chitinase. The possibility that a new group of chitinase is constituted if a study of the hermit crab chitinase progressed was suggested. Presently, we attempt to determination of 3’ and 5’ site of P. camtschaticus chitinase by the RACE method.

Chitin, a β -1, 4-linked polymer of N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant biomasses in the earth, second only to cellulose, and is now regarded as a renewable resource. Chitinase, endo-type chitinolytic enzyme, is known to decompose chitin to N-acetylchitooligosaccharides ((GlcNAc)_n) and to be distributed widely in organisms. In crustacean, chitinase is distributed not only in digestive organs but also in the integument, in which chitinase would relate to molting. Red snow crab Chionoecetes japonicus is an important fisheries resources and its shell is the natural sources of chitin. However, little has been reported on chitinase of C. japonicus. This study describes the characterization of the C. japonicus chitinase and the cloning of its corresponding cDNA.
C. japonicus chitinase (Cjchi) was fractionated from crude enzyme solution of the hepatopancreas by using hydrophobic interaction chromatography (HIC) method. Chitinase activity depending on pH was measured using pNP-(GlcNAc)_n (n=2, 3) at 37 ◦C for 20 min in the range of pH 2.5 to 8.0. Total RNA was also extracted from the hepatopancreas of C. japonicus with ISOGEN. Internal sequences of cDNA (CjChi) from the hepatopancreas of C. japonicus chitinase were amplified using the primers designed from the conserved amino acids region of GH family 18 chitinase from several arthropods.
Chitinase activity showed pH depending manner and was the highest around pH 5.5 against pNP-(GlcNAc)₂. In contrast, its activity was not detected against pNP-(GlcNAc)₃. Namely, Cjchi has strong substrate selectivity not to pNP-(GlcNAc)₃ but to pNP-(GlcNAc)₂. About 500 bp nucleotide sequence was obtained and determined as internal sequence of Cjchi. This sequence showed 73% similarity to the chitinase from the hepatopancreas of swimming crab Portunus trituberculatus. Now, we are trying to highly purify this chitinase and clone full-length chitinase cDNA.

Tilapia is one of the major aquacultured fish around the world, especially in South-East Asia and Africa. To increase volume of tilapia landing, identification and utilization of genes involved in fish growth, embryogenesis and cell differentiation are essential. On the other hand, tilapia is well suited for molecular biological study owing to its revealed whole genomic sequences. Here, we focused on tilapia sialidase enzyme as a target molecule.
Sialidases remove sialic acids from the terminal end of sugar chains, associated with biological process such as embryogenesis, bacterial infection, cyto-differentiation and cell proliferation in mammals. Regulation of these functions can be applicable to stable aquaculture of tilapia. However, characters of tilapia sialidases were not fully understood. First, to clarify desialylation mechanism, novel tilapia sialidase genes, two neu1 genes (neu1a and neu1b), neu3 and neu4 were cloned. Their enzymatic profiles, including optimal pH and substrate specificity, were quite different from each other, suggesting the different physiological functions of tilapia sialidases. Next, subcellular localizations of tilapia sialidases were investigated by indirectly immunofluorescence staining. Tilapia Neu1a and Neu1b were located at lysosome and Neu3 was at plasma membrane. On the other hand, tilapia Neu4 was localized to nuclear and its localization was altered by cell differentiation.
To know the tilapia sialidase functions, influences of tilapia sialidases on Edwardsiella tarda infection was examined since E. tarda utilizes sugar chains on host cell surface at cell attachment. After the establishment of tilapia sialidase overexpressing cells, E. tarda was infected for the transfected cells. As a result, Neu1a and Neu4 sialidase induced the increment of bacterial infection, while Neu3 significantly suppressed the infections. These results suggest that tilapia sialidases could be useful as an effective bio-marker in aquaculture.

Our laboratory reported two types of chitinase (acidic fish chitinase-1, acidic fish chitinase-2) that are active at acidic pH and involved in digestion in the stomach of Actinopterygii. In addition, we suggested the existence of the new fish chitinase which probably has a different function from the digestion. In this study, we used Sardinops melanostictus in which it has been reported the characteristic and cDNA cloning of chitinase isozymes (SmeChi-1, SmeChi-2) in the stomach. At first, we examined the body distribution of chitinase (endo-type) and β-N-acetylhexosaminidase (Hex, exo-type). Next, we tried to clone the novel chitinase cDNA from the kidney.
Chitinase and Hex activities were measured using pNP-(GlcNA)_n, (n=2, 3) and pNP-GlcNAc as substrates, respectively. Total RNA was extracted from the kidney. RT-PCR was performed to obtain chitinase cDNA fragment using reverse transcriptase with a oligo dT primer. RACE method was performed to obtain sequences of the upstream (5ʹ) and downstream (3ʹ) regions. The full-length chitinase cDNA was confirmed by using PrimeSTAR Max DNA polymerase with proofreading activity.
From the results of distribution of enzymatic activities, the chitinase activity was observed in the kidney, spleen and heart except the digestive organs. On the other hand, Hex activity was detected with all organs. This result is consistent with the report that Hex is related to bodywide metabolism. Full-length cDNA of novel chitinase was obtained from the kidney of S. melanostictus (SmeChi-3, 1,531 bp), which contained 1,482 bp open reading frames. The domain structure of SmeChi-3 was presumed according to those of SmeChi-1 and SmeChi-2. SmeChi-1 and SmeChi-2 have a serine and glycine-rich linker region, which is charastristic in AMCase. In contrast, SmeChi-3 contained no apparent sequence in the linker region. Phylogenetic analysis revealed of the existence of new chitinase group, which was named FCase-3, differed from AFCase-1 and AFCase-2.

The degradation of body wall proteins is considered as a major factor of autolysis during sea cucumber storage and processing, which is induced by endogenous proteinases. Non-collagenous protein played an important role on the body wall intergrity. However, little information is available for non-collagenous proteins in body wall associated with autolysis.In this study, the major non-collagen protein from body wall was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies, resulting in a single band with molecular mass of 400 kDa as a disulfide-linked dimer under non-reducing and 200 kDa under reducing condition on SDS-PAGE. Peptide mass fingerprinting (PMF) obtained 24 fragments with 268 amino acid residues, which were identical to major yolk protein 1 (MYP) from sea cucumber (Apostichopus japonicus) coelomic fluid. The results of PAS staining and β-elimination indicated MYP is a glycoprotein. Circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) analysis showed that denaturation temperature of MYP was 58.00°C and its secondary structure was severely dissociated until 90.37 °C. The main protein conformation of MYP was β-sheets by CD and fourier transform infrared spectroscopy (FTIR) spectroscopy. Meanwhile, the degradation of MYP by intestinal tract proteinases of sea cucumber was occurred at acidic condition from 4.0-6.0 and inhibited by leupeptin and chymostatin, suggesting the thermal stable MYP could be hydrolyzed by endogenous proteinases such as cathepsins from intestinal tract during autolysis processing.

There is the negative trend of depleting marine resources, and large scale fisheries has contributed to a dangerous level of population stock. In this instance, researches on deep sea bio-resource will increase and can act as a good additional resources, while marine regulations, technology, and awareness are developing. In this study, marine fatty acid profiles and other biochemical components were analyzed among various species of deep fish, namely, escolar Lepidocybium flavobrunneum, oilfish Ruvettus pretiosus, and rattail fish Coryphaenoides marginatus were sampled from Suruga Bay and several other points which contain deep sea zone. Only the ordinary muscle of the specimens were analyzed. In particular, the viability of a particular biochemical compound known as wax-ester was also studied upon. The analytical procedures used include proximate analysis and lipid analysis(fatty acid composition) using gas chromatography. The results displayed a high amount of mono-unsaturated as well as poly-unsaturated fatty acids in oilfish and escolar compared to rattail fish. In regards of some specific and other chemical components, these deep sea species possessed nutritional components in the form of crude protein and fatty acids(oleic acid, DPA, and DHA) which were comparable concentration-wise to some commercially distributed shallow water species such as salmon, amberjack, sardine,etc. Overall data of the analysis showed the availability of the lipid for a wide range of variety of uses, i.e., lubricant, fish-feed, health supplements, etc. In regards for further continuation of the research, the actual availability and practical application require other scientific information such as population stock, species or individual and seasonal variations. Other parts of the body and organs are also considered to be of analytical interest.

Freezing is one of the popular methods to preserve food. This technique, however, causes significant damages on food materials sometimes. Sea urchins deteriorate easily and melt rapidly. To prevent this, alum has been used for keeping the meat from melting but it causes bitter taste. Therefore, many investigations have been carried out on freeze preservation of sea urchins so far but no established method is available. In this study, effects of freezing or freeze-drying were investigated on the quality of sea urchin with special care on the pretreatments before freezing.
Sea urchin Strongylocentrotus nudus from Iwate and Hemicentrotus pulcherrimus from Nemuro were used. In freezing, three samples were used with different pretreatments: (1) whole shell with no treatment; (2) sample with shell mouth opened; (3) meat sample removed from the shell. After freezing at -40 oC and thawing, only the sample (1) without pretreatment showed the best quality in shape not much different from the fresh sample.
In freeze-drying, two different pretreatments were applied: (1) sample with shell mouth opened; (2) meat sample removed from the shell. After freeze-drying, both samples kept good shape not much different from the fresh sample after rehydration although freeze-drying time is much different between the two samples.
Dielectric property was measured to the samples after freezing or freeze-drying in the frequency range from 50 Hz to 3MHz. After freezing, the electrical impedance slightly reduced especially at the higher frequency range. A similar result was obtained in freeze-drying after rehydration. These results show that the cell structure of sea urchin may not be destroyed seriously in freezing or freeze-drying although a substantial drip removal was observed in some cases after freezing-thawing.

In order to investigate the effect of freezing methods on the volatile flavor of grass carp meat, three different freezing methods, the quick-freezing, ethanol immersion freezing and ordinary refrigerator freezing were used to pretreat the fresh dorsal meat and red meat. In this study, a new type of material (MonoTrap) was applied as the adsorbent to extract the volatile compounds in dorsal meat and red meat, The volatile flavor components of dorsal meat and red meat at different freezing methods were analyzed and identified by electronic nose and gas chromatography-mass spectrometry(GC-MS).The results indicated that the freezing rate of quick-freezing exhibited 6.4 times higher than that of ethanol immersion freezing, and 16.7 times higher than that of ordinary refrigerator freezing, and the fastest was quick-freezing. The odor between fresh grass carp meat and three different freezing methods of grass carp meat could be well discriminated by electronic nose. Principal component analysis (PCA)showed that the odor components of ordinary refrigerator freezing and fresh meat have the maximum differences. The results of GC-MS analysis showed that there are 45, 33, 28 and 22 volatile compounds were detected in fresh, quick-freezing, ethanol immersion freezing and ordinary refrigerator freezing dorsal meat respectively, and there are 49、38、33 and 25 volatile compounds were detected in fresh, quick-freezing, ethanol immersion freezing and ordinary refrigerator freezing red meat respectively ,the contents of major volatile compounds were aldehydes and alcohols. Research shows that pentanal,hexanal,heptanal,octanal,2-octenal,nonanal,2-nonenal,decanal,2-decenal,undecanal,2,4-decadienal,hexanol,2-Octen-1-ol,1-octen-3-ol,xylene had important contribution to the formation of the overall flavor grass carp meat. Among the three kinds of freezing methods, the volatile flavor compounds types and contents of the fresh meat were closest to quick-freezing meat. The quick-freezing was more beneficial for maintaining the quality of the freezing grass carp meat.

Fish has been subjected to processing such as drying, salting and other techniques in order to improve preservability. Recently, long-term preservation has become possible with the development of cryopreservation technology. In addition, various freezing methods have been developed for maintaining quality. Since the thawing process also influence the quality of product, improvement of thawing method was required. In this research, electromagnetic wave irradiation device was developed and thawing test was performed on frozen fish meat. Physical properties of the thawed fish meat were measured with a creep meter. Freshness of the meat was determined for the composition of nucleic acid-related compounds. Metmyoglobin conversion rate of the thawed fish meat was analyzed by HPLC. We tried this thawing method for various frozen food materials including fish meat and also applied to composite materials such as sushi. As a result of thawing test at various frequencies, 100 MHz irradiation proved to be most effective for thawing. Though heating capacity of 100 MHz irradiation was inferior to that of 2,450 MHz irradiation, permeability of 100 MHz irradiation was superior to 2,450 MHz. The frozen fish meat could be quickly and uniformly thawed by the irradiation frequency at 100 MHz. Consequently it was shown that the surface temperature at the end of thawing was kept low. The texture of the thawed product was closer to fresh fish than the product thawing in refrigerator. The amount of drip generation was reduces during the thawing process. The freshness of the product was retained and deterioration of the color was also suppressed. The 100 MHz high frequency irradiation is proposed as an effective thawing method. This thawing method is also valuable for maintaining the quality of frozen foods.

With the aim to investigated water dynamics effects on drying methods in Sur clam during dehydration and rehydration, using the non-destructive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) techniques. Three water populations were observed in clam samples led into different directions when the clams were dried by hot air at 60 oC for 8 h versus sun-dried for 53 h. Close correlations were observed between the A2 parameter of NMR obtain through moisture ratio, and the Page model exhibited the maximum coefficient R2 value with the minimum root mean squared error. Similarly, the rehydration of hot-air dried and sun-dried clams was also assessed by NMR and MRI revealing that the absorbed water was mainly the immobilized and free water, and the proper rehydration time was 180 and 120 min, respectively, for the hot-air and sun dried clams. Remarkably, the NMR and MRI characterization showed that the water release of hot-air dried clams and sun-dried samples were clearly classification combinewith the principal component analysis (PCA) of NMR relaxation data. The rehydrated clams could be distinguished from the boiled samples before dehydration process through the PCA of NMR relaxation data, but undistinguishable between the rehydrated hot-air dried and sun-dried clams. NMR and MRI could be effective methods to evaluate dehydration and rehydration processes of Sur clams non-destructively.

An effective method to remove cadmium (Cd) from scallop by-product enzymatic hydrolysate (SBEH) was developed, and the Cd bonding sites were studied. Cation-exchange resin, anion-exchange resin, activated charcoal, and zeolite were tested. The result showed that strong acid polystyrene cation-exchange resin was the most suitable sorption material for the removal of Cd. The optimal conditions were as follows: SBEH concentration at 17.07 mg/mL, pH value at 4.92, and adsorption time with 11.73 min, while 4 g resin was added in 50 mL solution at 30℃. The experimentally measured removal rate was 95.38% under these conditions. The Cd element has an intense bonding pattern with macromolecules, and more than half of the Cd element exists as positively charged ion in the SBEH. The cation-exchange resin removed Cd by dumping the macromolecules associated with the Cd element. Infrared (IR) spectra suggested that the sulfate ester groups and carboxyl groups in the macromolecules were binding sites of the Cd element in SBEH. These results provide a useful method for Cd removal from SBEH that potentially can be used for industrial processing.

To comply with the Food Sanitation Act in Japan, we used Hazard Analysis and Critical Control Point (HACCP) procedures in the importing business. Generally, prerequisite programs (PRPs) and HACCP are applied to food manufacturing processes but not to food importation procedures.
We identified imported food items which have high number of violations by analyzing 3,018 violations that occurred in Japan (2012-2015). As a result, the category of “Agricultural chemical residues” was one of the most frequent issues (736 violations). The item with the largest number of violations was shrimp, however the violations decreased after the Maximum Residue Limit (MRL) amendment for Ethoxyquin in 2014. Taking the situation above into consideration, we identified food items which show the 2nd to 5th large number of violations and compared MRLs of the items between the EU and Japan. Since the major destination of the items is the EU, 71% of violations for cocoa bean, 72% for sesame seed, 50% for pepper and 31% for tea leaf arose from the difference between EU and Japanese MRL regulations. The result indicates a hypothesis that producers make efforts to control their products in compliance with EU regulations and not those stipulated under Japanese law.
Based on this hypothesis, we used a general HACCP worksheet to assess the importing process of cocoa bean. We applied control measures from the guidelines for hygiene control of importing processed foods issued by the Ministry of Health, Labor and Welfare. “Contract arrangement to ensure the product compliance with Japanese regulations” and “pre-shipment confirmation of test results to meet Japanese MRLs” were determined as two CCPs with 2 criteria, “importer’s direct responsibilities for respective process management” and “further steps to mitigate violation risk”.
The HACCP-oriented approach and PRPs using the guidelines can optimize risk control procedures for food importers to ensure compliance.

Krill oils (KOs) were prepared by using different extraction methods (hexane and supercritical carbon
dioxide), different materials (heat-dried krill powder, freeze-dried krill powder and freeze-dried krill tail
meat powder) and different standing times during hexane extraction. The fluoride concentrations in
these KOs were positively correlated with the protein concentrations. Absorbents were employed to
remove fluoride from KO. Activated clay achieved the highest fluoride removal rate (70.66%), followed by
calcium oxide (14.49%), activated carbon (11.97%), calcium chloride (9.32%), activated alumina
(0.94%) and chitosan (0.52%). Meanwhile, the protein removal rates of the above absorbents were
positively correlated with the fluoride removal rates.

Histamine causes food poisoning for red-fleshed fish such as tuna, mackerel and so on. We have developed the sensor of histamine, useful for the quality control of fish meat. This sensor contains a histamine oxidase (HOD) from a bacterium Arthrobacter cystallopoietes KAIT-B-007, which converts histamine to imidazole acetaldehyde, and releases H₂O₂ and NH₃. For the elongation of the expiration date of this sensor, a mutant enzyme with higher stability is expected. Before the determination of target locations of mutation, the regional flexibility of HOD should be understood. For this purpose, molecular dynamics simulation would be the best tool. For the molecular dynamics simulation, the 3D structure as an initial structure is essential. However, there is no depository of HOD in the protein data bank (PDB). Thus, we tried to model the 3D structure of HOD by homology modeling with Swiss-model, where amine oxidase (PDB: ID 1w4n) was used as a template and the resultant model of HOD was started with Pro15. HOD binds topa quinone (TPQ) at 388th residue. Thus, the parameter of TPQ was constructed with Amber tools by TIP.cif in PDB. Then, we performed molecular dynamics simulations (100 ns × 5) with Amber 14. The last 50 ns for each simulation was used for the calculation of root mean square fluctuation (RMSF) of the main chain Cα, C and N, as the indicators for the flexibility. The average of RMSF was 1.14 Å. The residues whose RMSF were over 3 Å were His57 (outer loop region), Ala214-Thr230 (outer region), Thr362-Asn369 (inner β sheet), Pro465-Asn474 (outer loop region), Thr628 and Leu629 (C-terminus). These region(s) could be the candidates for the more stabilized mutant.

The aim of this study was to conduct a long-term investigation of the biological parameters and individual responses to abiotic, PAHs and biotic factors on seasonal fluctuations of Crassostrea angulata abundance from time series samples along the west coast of Taiwan. This study would help us to determine how we should use PAH concentration as bioindicators for biomonitoring studies and for the evaluation of pollution origins. We sampled three sampling stations each month from 2010 to the present in terms of reproduction variation of C. angulata with geographical area and time along the west coast of Taiwan (Fangyuan, Taihsi, and Dongshih). The PAH concentrations in 5 g of oyster samples were obtained through extraction, separation and quantification. Total concentration of PAHs in this study varied during 2010-2016. ∑PAHs and BaP exhibited negative correlation r=0.599, p=0.040 (2013), confirming a specific pollution from unburned pollution events. Relationships between contamination by PAHs and physiological status indicated that C. angulata was more susceptible to adverse effects of these pollutants. PAH ratios are traditionally used to assess the importance of pyrogenic and petrogenic origin in indicate organisms. Compositions and relative abundance of individual PAH concentration in C. angulata were no similar, where the 2+3 ring and 4ring PAH were predominant. The result suggest that the PAH contamination in study sites show a mixed pollution origin from both petrogenic and pyrolytic origins during sampling. The datas are in accordance with environmental parameter reproductive periods of oysters, ΣPAHs and molecular ratio of PAHs analysis, which suggest that pyrolytic and petrogenic pollution in three major aquaculture area in Taiwan. These results are important in biomonitoring of coastal environment for oysters aquaculture, and is associated with the safety for marine organism and ecology.

Pink prawns are often eaten raw. Therefore, the quality and time limit for consumption of raw pink prawns needs precise evaluation. Seafood quality can be comprehensively evaluated by measuring changes in parameters such as K-value, oxidation, pH, color, and bacterial count. The K-value is increased by enzymatic reactions and hence, is often used as a parameter to evaluate fish quality. However, K-value measurement is difficult and time-consuming. Therefore, a tool for quality visualization by analyzing enzymatic reaction-induced changes in color—biothermometer (BTM)—has been developed. As BTM coloring is correlated to the K-value in several types of fish, the time limit for raw consumption can be evaluated by color. We investigated the quality and time limit for consumption of pink prawns and the utility of BTM for pink prawn quality evaluation.
For the quality evaluation, the K-value, pH, color, bacterial count, and sensory characteristics were investigated for 6 days. The BTM coloring was also measured for 6 days by storing the BTM with pink prawns.
During this test period, the K-value increased from 1.9 % to 52 %, the pH varied between 7.0 and 7.4, the L^* value showed a small increase, and the a^* and b^* values were unchanged. The viable bacteria count was less than 10³ CFU/g, but the marine bacteria count showed a slight increase from 10³ CFU/g to 10⁴ CFU/g. The sensory evaluation tests indicated that less than 50 % of the test subjects could eat the raw prawns on day 4. A high correlation was confirmed between the K-value and BTM coloring (R² = 0.97). Thus, the K-value can be used for evaluation of pink prawn quality and the prawns can be consumed raw for up to 3 days after being caught. Moreover, the pink prawn quality could be visualized using BTM.

Seafood mislabeling is a common worldwide problem in international and domestic fish markets. In Japan, the law requiring traders to describe the species of the materials/foodstuffs and their place of origin with labels was strengthened by the Ministry of Agriculture, Forestry and Fisheries of Japan in response to consumer demand. Here, we performed PCR-RFLP analysis for species identification of salmonid fishes in commercial seafood sold as “salmon” to assess the extent of seafood mislabeling in Japanese markets. A total of 55 samples were collected from fish shops, supermarkets, sushi restaurants and convenience stores in Shizuoka Prefecture, Japan. Fish fillets were accurately labeled by traders with the correct species’ names in 17 samples that included 5 salmonid species: Atlantic salmon Salmo salar, silver salmon Oncorhynchus kisutch, sockeye salmon Oncorhynchus nerka, king salmon Oncorhynchus tshawytscha and rainbow trout Oncorhynchus mykiss. On the other hand, processed food products labeled “salmon” contained various salmonid species. Three salmonid species (O. mykiss, O. kisutch and S. salar) were detected in samples of “salmon” sushi; two species (O. mykiss and O. nerka) were detected in samples of smoked “salmon”; and chum salmon Oncorhynchus keta and O. mykiss were detected in samples of dried “salmon”. Only O. mykiss was detected in samples of “salmon” flakes. Various salmonid species (O. keta, O. kisutch, O. mykiss, O. tshawytscha and O. nerka) were detected in samples of grilled or fried “salmon” in bento lunch boxes.

In view of the extreme climate-changing impact, today we should maintain the environment friendly production and enhance the added value of aquatic products. The accreditation Body (AB) will monitor each brand and product standard that fits international production and marketing standards and in order to promote these products and history-traceable aquaculture products. Through established Taiwan's good agricultural practices (TGAP) operating benchmarks, AB can produce safe, traceable aquatic products, through international certification bodies to verify the "production and marketing history of agricultural products logo" which allow consumer to distinguish and purchase these products by using relevant signs, and then these action can help establishing marketing segments in aquaculture products, enhance marketing value of products.
In order to promote the domestic aquatic products towards high quality, safety, exquisite. Taiwan CAS branded an award excellent aquatic products to domestic high-quality aquatic raw materials mainly critically inspect raw materials, production, processing to storage and transportation in order to fit established HACCP control system. This system can enhance aquatic products manufacturing technically or self-management capacity during manufacture process, it also can improve the quality of aquatic products both health and safety.

The effects of different cooking on the flavor quality of white shrimp (Penaeus vannamei) were investigated by using electronic nose (e-nose) and electronic tongue (e-tongue) combining with the taste activity value (TAV) and the equivalent umami concentration (EUC).The taste components, flavor nucleotides and free amino acids were determined by mino acid analyzer and high performance liquid chromatography (HPLC). The results showed that e-tongue and e-nose can effectively distinguish 4 kinds meat of different cooked shrimps (raw shrimp, cooked shrimp, cooked shrimp for placing at 4 degree centigrade for 12h, and cooked for the second time after storing). Compared with raw shrimp meat, the contents of IMP and AMP decreased in cooked shrimp meat. The contents of ATP, ADP, IMP and AMP in the secondary cooked shrimp meat were significantly increased comparison with the cooked shrimp meat. And the contents of flavor nucleotide in the shrimp meat treated with placing at 4 degree centigrade for 12h was significantly higher (P <0.05). The total amounts of fresh sweet amino acids in raw shrimp and cooked shrimp were relatively high, while the total amounts of bitter amino acids and free amino acids were reduced. The EUC of second cooked shrimp meat treatment group was only half compared to the meat of cooked shrimp. However, there was no significant difference in the TAV value of shrimp treated by firstly and secondly cooked treatments.

In November 2015, one patient presented with symptoms of toxicity (including numbness of the lips, weakness of the lower limbs, coma, and vomiting), after eating whole boiled samples of the scavenging gastropod Nassarius (Alectrion) glans “Kinshibai” in Nagasaki, Japan. This food poisoning case was the third recorded in Japan.
Investigation of this case involved evaluation of the toxin profile of this gastropod (determined from two food residue samples), and monitoring of tetrodotoxin (TTX) levels in serum and urine.
Both food residue samples (Food 1 and 2) contained high concentrations of the toxin; the toxicity scores were 595 MU/g (Food 1) and 5035 MU/g (Food 2), based on the TTX bioassay. The toxin was analyzed both by liquid chromatography with triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS) and quadrupole time-of-flight tandem mass spectrometry (LC-qTOF-MS/MS), which revealed the presence of TTX (0.38 mg/body for Food 1 and 2.45 mg/individual for Food 2), and related TTX compounds.
Serum and urine specimens were pre-treated using an ultrafiltration spin column and analyzed by LC-QqQ-MS/MS for TTX. Maximum TTX concentrations were 42.8 ng/mL in serum on Day 1 and 1400 ng/mL in urine collected from Day 0 to 1. The patient regained consciousness on Day 6, when serum TTX concentration fell below 1 ng/mL.
From the results of measured TTX concentrations, ingestion of lethal dose was estimated in this case.
Moreover, the toxin content of this gastropod was determined to consist primarily of TTX, which accounted for approximately 59% (Food 1) and 67% (Food 2) of total toxicity. The remaining toxicity was thought to be due to 11-oxo-TTX, which related TTX compounds.
As with past poisoning cases, it was concluded that this gastropod, is a highly dangerous species with regard to food poisoning.

Diarrhetic shellfish toxins (DST) are defined as Okadaic acid (OA) group and regulatory limit of DST is total OA equivalent of 0.16 mg per kg of edible tissue in CODEX STAN 292-2008. Testing method of DST using LC/MS/MS has been officially authorized from 2015 in Japan.
Biodistribution and individual differences of DST were investigated in scallops collected in Mutsu Bay Aomori. The 14-20 individuals of the scallops collected from Nonai station were divided into digestive glands, gonads, mantles, gills, adductor muscles, and the concentrations of OA group in each tissue were quantified by LC/MS/MS after hydrolysis. Dominant toxin in the scallops was DTX1. More than 97 % in total amounts of DTX1 was detected in the digestive glands. DSTs were hardly detected in the adductor muscles.
Scallops and blue mussels were collected from Nonai station, and the concentration of DTX1 contained in the digestive glands was quantified for each individual of 30 samples. The average value was different between blue mussel and scallop, and the mussels showed higher DTX1 than the scallop. The numbers of individuals required to correctly reflect the DTX1 content of a sample group was estimated by the bootstrap analysis. Using the data of 30 individuals of scallops or blue mussels, values that can be taken by the average value of samples 5 to 25 were calculated repeatedly by 10,000 times with random sampling without duplication. In the scallop, using 10 individuals fell within ± 20% of 30 individual’s average with a probability of 99.8%. On the other hand, in the blue mussel, average of 19 individuals and 15 individuals fell within ± 20% of 30 individual’s average with 98% and 90% probability, respectively.
Although a series of studies gave us useful information for improving DST risk management, further investigation will be required.

【Purpose】Tetrodotoxin (TTX) poisoning occurs annually in Japan and many other countries. TTX is predominantly excreted into the urine from the body; however, the detailed urinary excretion pathway has not well understood. The purpose of this study is to examine the transport characteristics of urinary excretion of TTX using the porcine renal proximal tubule epithelial cell line LLC-PK1 monolayers.
【Methods】LLC-PK1 cell monolayers were grown in 24-well transwell inserts at 7 days after seeding. Time course study was started by replacing the buffer (pH7.4) in apical or basolateral side by the buffer containing 50 µM TTX. Inhibition study was performed using typical transporter inhibitors that dissolved into the buffer containing 50 µM TTX to final concentrations of 5 mM. At the end of incubation, the buffer in receiver chamber was rapidly collected and subjected to determination of TTX by LC-MS/MS.
【Results】Time course of the transport of TTX in the direction of urinary excretion and renal reabsorption at 37 °C showed that the amount of TTX transported increased linearly for up to 60 min. Although the amount of TTX transported also increased linearly at 4 °C, the value significantly decreased to about 20% of the value at 37 °C. These results indicated that the transport is carrier-mediated. In the inhibition study, where the cell monolayers were incubated at 37 °C for 30 min, the transport in the direction of urinary excretion was significantly reduced by probenecid, TEA, _L-carnitine, and cimetidine, and slightly reduced by PAH, whereas it was not affected by MATEs inhibitors. The transport in the direction of renal reabsorption was slightly reduced by PAH and probenecid but not by TEA and _L-carnitine. These findings indicated that TTX was mainly urinary excreted by OCTs and OCTNs, and partially transported by OATs and MRPs.

Pufferfish belonging to the genus Takifugu possess a potent toxin, tetrodotoxin (TTX), specific to voltage-gated sodium channels of excitable membranes of muscle and nerve tissues. TTX has been detected in various disparate taxa including starfish, gastropods, crustaceans, flatworms and ribbonworms, and TTX-producing bacteria have been isolated from these TTX-bearing animals. Although TTX in marine pufferfish has thought to accumulate in the pufferfish via the food web starting with marine bacteria, details in the toxification process in pufferfish still remains unclear. In this study, we demonstrate that TTX in the larval and adult planocerids contribute to toxification of the pufferfish through the life history. Species-specific PCR method was developed against the flatworm Planocera multitentaculata, and the planocerid-specific DNA fragment was detected in the intestinal contents of the wild pufferfish Takifugu niphobles. Predation experiments using the larval pufferfish as a predator and larval flatworm as a prey demonstrated that almost of larval flatworms were fed by the larval pufferfish, and the flatworm-fed pufferfish were rapidly toxified. Additionally, predation experiments using the young pufferfish as a predator and adult flatworm as a prey also showed that the flatworm was fed by the young pufferfish, and after two days of the feeding, the toxin was detected in skin, liver and intestine of the predatory pufferfish. These results suggest that the pufferfish are rapidly toxified by feeding on the toxic flatworms.

In the present study, we conducted two tetrodotoxin (TTX) administration experiments using non-toxic cultured Takifugu rubripes of different ages to investigate the effects of growth (organ development) on TTX dynamics in the pufferfish body. Firstly, TTX-containing feed homogenate was administered to 6-month-old (body weight 53.5±6.9 g) and 15-month-old (328±36 g) fish at a dose of 0.4 µg/g body weight by oral gavage. After 24 h, these specimens were killed and the skin, muscle, and liver were separated. TTX concentration (µg/g) in each tissue, determined by LC-MS, revealed that the TTX distribution profile, particularly the TTX concentration of the liver, greatly differed between the two ages; the TTX score of 15-month-old fish (3.3 µg/g) was nearly 5-fold that of 6-month-old fish (0.68 µg/g). The TTX accumulation ratio (ratio (%) of accumulated TTX in each tissue (µg/tissue) to the administered dose (µg/individual)) of the liver was less than 10% in 6-month-old fish, but that of 15-month-old fish reached 70%. Secondly, 3-month-old (4.9±0.3 g) and 8-month-old (142±23 g) fish were administered TTX at several doses (0.2-5.3 µg/g body weight) into the dorsal muscle to investigate the dose-dependency of TTX accumulation. LC-MS or HPLC-FLD analyses revealed that the TTX accumulation ratio of the liver in the two ages at 24 h after the toxin administration were almost constant irrespective of the dose, but the scores of the 8-month-old fish (~20%) were higher than that of the 3-month-old fish (~10%). These experiments reveal that the higher the age, the higher the TTX accumulation ratio and hepatosomatic index (2.7-10) of the test fish, suggesting that the TTX dynamics in T. rubripes are linked to the development of the liver.

Tetrodotoxin (TTX) known as pufferfish toxin, has been detected from taxonomically diverse organisms including amphibians, fish, cephalopods, gastropods, crustaceans, starfishes, flatworms and ribbonworms. The ecology of TTX-bearing organisms at lower trophic levels had not been investigated except for several investigations. Recently, we found that the flatworm Planocera multitentaculata would contribute to the toxification of the pufferfish Takifugu niphobles. Therefore, in this study, we investigated the TTX dynamics in the flatworm body in association with their sexual maturation. The flatworm specimens were monthly collected from an intertidal zone in Kanagawa Prefecture, Japan. The TTX content of the flatworm was measured using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The toxicity was 280 ± 232 µg/g and 142 ± 63 µg/g in the specimens with mature ovaries and mature testes, respectively, during their spawning period (April - July), whereas that was 134 ± 45 µg/g in those during late spawning, significant differences were observed among these values (P < 0.05). In the peak of the spawning (May), the toxicity of the specimens with mature ovaries was 372 ± 285 µg/g, whereas those with immature ovaries was 106 ± 35 µg/g with significant difference (P < 0.05). Additionally, before spawning period (January - March), the specimens with mature ovaries were not observed, and no significant difference was observed between specimens with mature testes and immature specimens (P > 0.05).

The antimicrobial peptide epinecidin-1 (Epi) was identified from Epinephelus coioides and ameliorates antibiotic-resistant bacteria-induced sepsis in mice via its bactericidal activity. However, it is unclear whether Epi suppressed polymicrobial sepsis in mice. Lipopolysaccharide (LPS) is a key inducer of inflammation during bacterial infection and sepsis. Whether Epi antagonizes LPS activity in vitro and in vivo is not fully understood. The protective effects of Epi against polymicrobial sepsis-induced inflammation and lung injury in cecal ligation and puncture were examined in mice. The LPS-antagonizing activity and mechanism of Epi were tested in LPS-induced endotoxemia mice and LPS-treated Raw264.7 mouse macrophage cells. Epi markedly rescued mice from endotoxemia and polymicrobial sepsis with delayed administration and was associated with the suppression of lung and systemic inflammatory responses and amelioration of histological lung injury. In vitro studies revealed that Epi suppressed LPS-induced inflammatory cytokine production. Mechanistically, Epi disrupted the interaction between LPS and LPS binding protein, competed with LPS for binding on the cell surface, and inhibited Toll-like receptor 4 endocytosis, resulting in inhibition of LPS-induced reactive oxygen species /p38/Akt/nuclear factor κ B signaling and subsequent cytokine production. In this study, we demonstrated that Epi is a promising therapeutic agent for endotoxemia and polymicrobial sepsis.

Chronic wound infection is a leading cause of mortality in injured patients around the world. Presently, available treatments with systemic antibiotics have the lacunae of antibiotics’ insufficient tissue penetration ability and development of antibiotic resistant pathogens. Identification and novel alternative therapeutic agents with wound healing and antimicrobial function are necessary to heal the wound as well as cure infections. This report shows that the antimicrobial peptide (AMP) Epinecidin-1 (Epi-1) efficiently heals MRSA infected heat burn injury and protection from infection in pig models. The presence of optimal level of Epi-1 induced cell proliferation promotes cell cycles by increasing S-phase cells. It also proliferates and covers wounded region under in vitro cell proliferation assay in immortalized human epithelial HaCaT cells. Next, the in vivo wound healing efficiency of Epi-1 was tested in heat burn pig skin infected with MRSA under in vivo conditions. When six hours post infection the injury was treated with Epi-1 for 1h, the wound was completely healed in 25 days. Whereas in untreated control the injury was not healed even 25 days after infection. Histological staining of wound sections with H&E showed that Epi-1 enhances the vascularization and increased epithelial activities in the wound region. Neutrophil recruitment to the wounded region in Epi-1 treated sections have been visualized by Giemsa staining. In addition, Epi-1 mediated enhanced extra cellular compound collagen formation has been confirmed by Masson’s trichrome staining of wound sections. The induction of sepsis associated blood c reactive protein (CRP) and pro-inflammatory cytokine IL-6 in response to MRSA infection also suppressed in pigs received Epi-1. Taken together, the biomaterial Epi-1 heals the wound through increasing epithelial cell proliferation, vascularization, formation of collagen to heal the wound region, and it controls the MRSA infection mediated sepsis in pigs.

[Objectives] Bromophenols are distributed in Rhodomelaceae red algae. They have displayed various functions such as enzyme inhibition. Two novel bromophenols along with eight known ones were isolated from the two species Odonthalia corymbifera and Neorhodomela aculeata, Rhodomelaceae. They exhibited almost same antioxidant activity and tyrosinase inhibition, compared to the respective standards BHA and kojic acid.
[Methods] All the bromophenols were isolated from algal methanol extracts by using various chromatographic techniques. Structural elucidation was done by consideration of NMR and MS data. Radical-scavenging (DPPH & ABTS), metal-reducing (CUPRAC & FRAP) and copper-chelating assays were employed for assessment of antioxidant activity. Mushroom tyrosinase inhibition was examined by colorimetric method using tyrosine as a substrate.
[Results] The two new bromophenols were identified as a trimer of 2,3-dibromo-4,5-dihydroxybenzyl units, “bromophenol units,” and a complex of the unit and a 4-cyclopentene-1,3-dione moiety. The known bromophenols were also identified as two bromophenol unit monomers, two symmetric unit dimers and four asymmetric dimers. Bromophenol unit dimers showed stronger antioxidant and tyrosinase inhibitory activity than corresponding unit monomers and trimers. The symmetric unit dimers exhibited higher functionality than asymmetric unit dimers. Bromophenol consisting of two 2,3-dibromo-4,5-dihydroxybenzyl moieties showed the highest inhibition against tyrosinase among the bromophenols isolated. It was revealed to be a noncompetitive inhibitor from the results of kinetic study.

[About the author]Md. Reazul Islam has graduated in Fisheries Science from Bangladesh Agricultural University in 2008. He is studying in Hokkaido University as a Ph.D candidate, supervised by Dr. Hideyuki Kurihara.

The objective of this study was to identify peptides, based on active components of the red algae seaweed Pyropia yezoensis, that inhibit generation of reactive oxygen species (ROS), a key property associated with anti-aging and anti-oxidative activities. Phycobilin, specific to red algae, covalently binds with water-soluble proteins. Pigment bound proteins, known as phycobiliprotein (PBP), are of three types, phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). We identified PBPs previously reported to have antioxidant activities in P. yezoensis and, based on these data, synthesized several peptides (PBP 1-13) and examined their inhibition of ROS generation. The existence of PBPs of each type, PE, PC, and APC, was established in P. yezoensis and all were analyzed. In addition, PBP 1-2, 7-9 peptides from PE were synthesized and showed antioxidant activities in HepG2 cells. In HepG2 cells, treatment of PBP 2 diminished H2O2-mediated oxidative stress and restored the expression of certain antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Furthermore, phosphorylated nuclear factor erythroid-derived 2-like 2 (p-Nrf2) was elevated by PBP 2 treatment. Overall, these results suggest that Nrf2-SOD/CAT/GPx pathways might be involved in the PBP 2-mediated antioxidant effects. In this study, we identified several candidate peptides of P. yezoensis with promising antioxidant and, potentially, anti-aging properties.

【Purpose】Japanese shore crab Gaetice depressus is reported to be tolerant to administration of paralytic shellfish toxins (PSTs), even though the shore crab does not bear PSTs. It is suggested that the crab has unknown detoxification mechanism against PSTs. In order to clarify the underlying mechanism of G. depressus, we examined neutralizing effect against PSTs of the shore crab hemolymph.
【Methods】Specimens of the shore crab G. depressus were captured in Sagami Bay and Tokyo Bay, Kanagawa Prefecture. The hemolymph was withdrawn from the hemocoel of live specimens, diluted with MilliQ water, and centrifuged. The resulting supernatant was used as a hemolymph sample. The sample was mixed with PST solution and allowed to stand at 4℃ for 1 hour. After incubation, the toxins were extracted with 0.1 M HCl by heating and subjected to toxicity assay by mouse assay and analysis for toxin component by LC-MS/MS method. Other 8 species of crabs were also used as specimens.
【Results】The hemolymph sample of G. depressus significantly decreased in toxicity of PST solution including saxitoxin (STX) and decarbamoyl STX. The toxicity of a mixture of the PST solution and physiological saline was 7.56 mouse unit (MU)/mL, while that of a mixture of the PST solution and the hemolymph sample was 4.53 MU/mL. In LC-MS/MS analysis, toxin composition of the PST solution mixed with the hemolymph markedly changed. STX almost disappeared and decarbamoyl STX largely increased. However, toxin composition of the PST solution mixed with physiological saline was unchanged. On the other hand, the hemolymph sample did not affect gonyautoxins. In addition, hemolymph samples of other 8 crab species did not transform STX into decarbamoyl STX. These findings suggest that the hemolymph of G. depressus has specific bio-transformation of PST exclusively STX to neutralize PST toxicity.

Seventeen species of seaweeds (macroalgae) involving nine Rhodophyta, six Phaeophyta and two Chlorophyta were collected from the coast of Nagasaki prefecture, Japan and their methanol extracts were screened for algicidal activity against the red-tide phytoplankton Chattonella antiqua. Among the macroalgae tested, the red alga Tricleocarpa cylindrical, the brown alga Undaria pinnatifida and the green alga Codium cylindricum showed the lysis of C. antiqua at the concentration of 0.1 mg/mL. Here, we describe isolation of the algicidal principles of T. cylindrical. The methanol extract of T. cylindrical was fractionated by solvent-solvent partitioning using hexane/80% methanol and then water/ethyl acetate. The ethyl acetate extract was further purified by chromatography using HP20, RP-18 and Silica gel 60 to afford an active fraction, which showed 100% mortality to C. antiqua at a concentration of 20 ug/mL. Spectroscopic analysis (NMR, IR and MS) suggested that the active fraction consisted of a mixture of (E)-8-hydroxy-9-octadecenoic acid and (E)-11-hydroxy-9-octadecenoic acids.

Several compounds derived from seaweed extracts showed decreasing activity of peroxide production mediated with lipoxygenase. They were identified as fatty acid methyl ester mixture (FAMEM), carotenoid (fucoxanthin, FX), chlorophyll-related compound (pheophytin a, PPA), and bromophenol (2,3-dibromo-4,5-dihydroxybenzyl methyl ether, BP). In this study, we investigated their decreasing mechanisms, along with nordihydroguaiaretic acid (NDGA) as a positive control, of peroxide production using various lipoxygenase activity assays. Lipoxygenase activity was assessed by indamine dye method converted from lipid peroxide using soybean lipoxygenase and linoleic acid as a substrate. This assay consisted of lipoxygenase reaction (LR), indamine dye formation (IDF), and absorbance measurement (AM) steps. When all tested compounds were added before the AM step, all the resulting absorbance values were same to the value without adding the test compound. All the compounds did not affected the formed indamine dye. FAMEM, BP and NDGA decreased the resulting absorbance values with adding them before the LR step while they did not decrease the values before the IDF step. FAMEM, BP and NDGA were decided to be lipoxygenase inhibitors. FX decreased indamine dye formation comparably in both experiments added before the LR and the IDF steps. These results suggested that FX was a reductant for peroxide, not a lipoxygenase inhibitor. Although PPA decreased indamine dye formation in both the experiments, addition before the LR step decreased highly compared with addition before the IDF step. These results suggested that PPA had both characteristics of lipoxygenase inhibitor and reductant of peroxide. Thus this study revealed decreasing mechanisms of seaweed constituents against lipoxygenase reaction products.

Mutagenesis is one method used to increase the biomass of microalga for further use. This mutagenesis research was conducted chemically by adding ethyl methane sulfonate (EMS) into media of 3 microalgae species (Nannochloropsis oculata, Dunaliella salina, and Tetraselmis chuii). The results showed that the highest density of N. oculata was occurred in 0.5 M EMS, while on D. salina and T. chuii were at 0.1 M EMS. The highest specific growth rate on N. oculata is in EMS 0.1 M, while on D. salina and T. chuii are in control. Microalgae cell size at N. oculata on the concentration of 0.1 M EMS had a cell size three times larger than the control and had a higher percentage of fatty acids as well. While at D. salina had twice and half times than the control, and at T. chuii had twice times than control as well. The highest ratio of RNA/DNA on three microalgae species were at the treatment of 0.5 M EMS while the control is the lowest. For dry biomass, the highest value on the stationary and death phase is in 0.1 M EMS treatment. EMS treatment can improve lipid concentration three times at exponential phase and stationary phase and protein, but no effect in carbohydrates.
Keywords: microalgae, random, mutan, Nannochloropsis oculata,
Dunaliella salina and Tetraselmis chuii

Polyamines are involved in a wide range of plant cellular processes, including cell division, morphogenesis and stress responses. However, their exact roles are not enough understood. Recently, we discovered that polyamines were able to react with CO2 dissolved in water. Thus, the resultant carbamate anions shift gradually to more stable bicarbonate/carbonate anions in the aqueous solution. The ability of polyamines to capture CO2 from atmosphere led us to examine the roles of polyamines in photosynthesis. Consequently, we supposed that the atmospheric CO2 captured by polyamines could be a substrate for carboxylation reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is an enzyme involved in the first major step of carbon fixation in photosynthesis. In this study, we investigated the effects of polyamine solutions retaining CO2 on photosynthesis of cyanobacteria Synechocystis sp. ATCC27184. To introduce atmospheric CO2 into polyamine solutions, those containing polyamines (putrescine and spermidine) each at 0.1 M were added to multidishes of 24 wells and allowed to stand for up to 48 hours at room temperature. Then, dissolved O2 was measured in a 1 ml reaction chamber containing the cyanobacteria and the above-mentioned polyamine solutions. As a result, a spermidine solution retaining CO2 slightly increased the O2 generation in the cyanobacteria. In contrast, a putrescine solution retaining CO2 strongly inhibited the O2 generation in the cyanobacteria. We additionally examined the contribution of polyamine transporters to proliferation and photosynthesis of the cyanobacteria using polyamine transporter inhibitors, which were synthesized by the previously reported method (Burns et al, 2009). The polyamine transporter inhibitors interfered with the proliferation and O2 generation in the cyanobacteria. These results suggest that spermidine, which is the most common type among polyamines in the cyanobacteria, along with its transporter, at least partly contribute to the photosynthesis of the cyanobacteria.

Benefit of fish consumption has been associated with reduced risk of developing various life-style diseases, such as cardiovascular disease, high blood pressure, stroke, some cancers, type 2 diabetes, rheumatoid arthritis and other inflammatory diseases. There is no doubt that seafood is an excellent source of nutrients, supplying people with polyunsaturated fatty acids (PUFAs), vitamins, selenium, iodine, and proteins, which are essential for healthy eating habits. Since a biologically active form of organic selenium, selenoneine, has been identified in tuna and other fishes, we focus on the health benefit of selenium redox functions by fish consumption. Selenium is an essential nutrient for human health. The benefits of selenium intake are many and various. The antioxidant activity of selenium plays a protective role in 50 human diseases, including prostate, lung, and intestine/colon cancer, immunodeficiency, and heart diseases. Selenium deficiency is also linked to animal diseases such as myopathy, exudative diathesis, and pancreatic degeneration in domestic animals and birds. Recent epidemiological studies suggested that fish consumption is associated with obesity and inflammation, and chronic inflammation is closely related to metabolic syndrome, cardiovascular disease, stroke, cancer, and type 2 diabetes. Among many nutrients present in fish, PUFAs and selenium are suggested to be effective to anti-inflammatory function. The contents of both nutrients in the fish meats were closely correlated. Blood PUFA levels of the fish eating population in Japan were associated with selenium levels. Therefore, selenium and/or PUFAs are important in health promotion by seafood intake, and differential roles of selenium and PUFAs on health benefit should be characterized.

Blood is made up of different types of cells such as red, and white blood cells, as well as platelet. Although these cells have discrete functions, they all come from cells called blood stem cells. It differentiates upon exposure to hematopoietic cytokines including erythropoietin (EPO), granulocyte colony-stimulating factor, (G-CSF), and thrombopoietin (TPO). These cytokines differentiate the stem cell into red, white blood cell and to platelet, respectively. Dysfunction in hematopoiesis lead to anemia related symptoms. Therefore, molecules that modulate cytokine-mediated signal transductions are of great interest regarding biomedical and therapeutic points of view. We screened 756 marine extracts in our library in search for such molecules and found that several extracts showed TPO-like function. One that showed the most potent activation was a water extract from a sponge collected in Chuuk Island. We purified the active compound guided by a cell based assay using a murine proB cell derived BaF3-cMpl cell that expressed TPO receptor gene cmpl. The cells grow depending of a presence of TPO or TPO-like agonists. The active principal, named as Thrombocorticin (ThC), is a 14kDa novel protein, and was stable over wide range of pH and temperature. ThC displayed concentration-dependent proliferation of BaF3-cMpl cells, and had stronger activity than Eltrombopag, a small molecule drug used to treat thrombocytopenia. ThC, however did not show any activities for cells with other hematopoietic cytokine receptors including IL-3, EPO and G-CSF. These data indicated that ThC significantly binds to Mpl receptor, though the size and shape differs largely from TPO. Here we present isolation, characterization and biological activity of THC.

Marine natural products are remarkable source for neuroactive compounds. Tetrodotoxin (TTX), a selective blocker of Na+ channel, for example is a potent neurotoxin as well as indispensable tool in neuroscience research. We have isolated novel excitatory amino acid, dysiherbaine, a potent and selective agonist for a subtype of ionotropic glutamate receptors (iGluRs), from a marine sponge Lendenfeldia chondrodes showing capability of marine invertebrate as interesting sources of novel bioactive molecules. We have recently isolated new brominated beta-carboline alkaloids irenecarbolines A (1) and B (2), N-methyl-beta-carbolinium chloride (3), 1, 3, 9-trimethyl-8-oxoisoguanine (4) from a solitary ascidian, Cnemidocarpa irene as acetylcholinesterase (AChE) inhibitors. Moreover, we found that this tunicate contained highly diverse aromatic secondary metabolites, and some of them including 1 and 3 were contained in large amount in the blood of tunicate, thus the blood was fluorescent. Therefore the biosynthetic machinery and physiological functions of these compounds are of particular interest. Besides the enzyme inhibition activity, we searched further for bioactive compounds in this tunicate using mouse behavioral assay and found some fractions upon separation by Sephadex LH-20 had activities to modulate behaviors of mice. One of those active fractions induced in mice unusual behavioral activity such as loss of posture and ataxia motions. These profiles were similar to those observed for alfa-Amino-3-hydroxy-5-methyl-isoxazolepropionic acid receptor (AMPAR) antagonist including 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX). Presence of AMPA antagonists in nature is rare, and only an example is kaitocephalin (KCP). The active fraction therefore was further separated guided by the behavioral activity and resulted in isolation of 5. Isolation, structure determination and biological activities for aromatics from this tunicate will be presented.

Japanese seaweeds have been utilized as industrial resources for foods, medicine and fertilizers. However, seaweeds are not used enough. Industrial materials derived from Japanese seaweeds are less than land-based products. There should be still an unused ingredient. Seaweeds are expected to have bioactive substances which land plants cannot produce, because marine environment is different from land environment. On the other hand, hemagglutinins are expected to use as medicine, healthful foods and reagents, because hemagglutinins have specific carbohydrate binding abilities. Fifteen species of edible seaweeds were surveyed for hemagglutinins. Rabbit and sheep erythrocytes were used as standard erythrocytes. The efficiency of five fractionation method were compared in order to select a suitable extraction procedure for the screening. Red algae Gracilaria sp., Gracilariopsis sp. and Gelidium sp. had strong hemagglutinationg activities. Their hemagglutinating activities were not inhibited by any of the monosaccharides tested, but were inhibited by glycoproteins such as fetuin and asialofetuin. Their hemagglutinating activities also had no need for any additives such as metal ions or 2-mercaptoethanol. When their hemagglutinins are applied in industry, these properties will be of great advantage. The results suggest that their hemagglutinating activities were different from that of land plant hemagglutinins. Thus, such Japanese edible seaweeds seem to be useful resources of hemagglutinins.

Chondropsin class of macrolide lactams is one of the largest secondary metabolites biosynthesized by a hybridization of polyketide synthase and non-ribosomal peptide synthase. They were isolated from taxonomically diverse marine sponges and found to show potent antitumor activity by inhibiting vacuolar H⁺-ATPases (V-ATPases). In our search for bioactive compounds using cell-based assay, poecillastrins, the same family as chondropsins, were isolated from sponges collected in deep-sea near Japan. Structural analysis of them will be reported.

Abstract: The immune system is important in preventing and controlling various infectious diseases. Immunoactive peptides derived from food proteins could exert immunomodulatory activity without side effects. Paphia undulata is a good source of protein with low fat, it is suitable for the production of immunoactive peptides. The objective of this study was to obtain an immune hydrolysate from Paphia undulata via enzymatic hydrolysate approach and characterize the immunoactive peptides. The hydrolysate conditions of alkaline protease were optimized as followed: temperature at 51°C; time of 3.14 h; E/S of 4,321U/g−1 (Paphia undulata meat). The hydrolysate obtained under the optimum conditions was by the ethanol precipitation method. The ultrafiltration fraction with molecular of the resulted product showed strong Immunoactive activity in vivo, such as lymphocyte proliferation activity, macrophage phagocytosis, promoting the secretion of IL-6, etc. The <5 kDa fraction was then filtrated in a Sephadex G-25 column. After a two-step isolation, the isolated fractions (P2 and P3) including the peptides Pro-His-Thr-Cys, Val-Gly-Try-Thr, Glu-Phe, Leu-Phe, and Glu-Gly-Ala-Lys, Trp-Ile or Trp-Leu, respectively, could significantly enhance lymphocyte proliferation ability. These results showed that the isolated fractions of Paphia undulata hydrolysates are potential ingredients for development of immune health food.

Oysters (Ostrea plicatula Gmelin) are a good source of high quality easily digestible protein and essential amino acids of high amino acid score and hence quite beneficial for human health. Therefore it is quiet reasonable to assume that they could be ideal initiating materials for the production protein derived bioactive peptides. In the present study, soft tissue from cultured farm fresh oyster was subjected to enzymatic peptide extraction procedure using neutral proteinase. The crude hydrolysates obtained were partially concentrated by ultrafiltration and fractionated into 3 sizes namely, above 10, 4-10 and <4 kDa according to the molecular size. Antioxidant capacities and antifatigue activities of 3 fractions were evaluated by DPPH free radical scavenging assay and mice swimming exhaustion model, respectively. Of the three fractions collected by ultrafiltration, fraction <4 kDa that showed the highest antioxidant activity. The intragastric administration of fraction <4 kDa prolonged 78.2% of the swimming time to exhaustion in mice, decreased 24.8% of the levels of serum lactic acid and 11.2% of the level of blood urea nitrogen in mice. Finally, it increased 1.00-fold of the levels of liver glycogen, and 55.6% of the level of gastrocnemius muscle glycogen in mice. It is reasonable to assume that fraction <4 kDa could accelerate free radical scavenging and alleviate injury from radicals generated during exercise. The further purification and structures analysis of the bioactive peptide is now in progress. The whole study may contribute to the ongoing research on antifatigue peptides from marine sources and their application in food and medicine.

The endogenous protease is abundant in Litopenaeus vannamei's head. But UV-C irradiation could activate the endogenous enzymes in the body of many aquatic animals and induced its autolysis reaction. The autolysis kinetics of shrimp head after UV-C irradiation stress was investigated. During the first 5 hours of autolysis, the release of the autolysis products was in line with the first-order equations of the reaction rate: Y = 37.681e−0.173t, Pe = −1.769Y + 74.156, and TP = −1.5117Y + 60.866. A good linear correlation was founded between the release of total protein and that of products with molecular weight of 3000 Da after these products associated with residual total protein were autolyzed. The autolysis speed accelerated in the initial 3 h and decelerated in the next 2 h. The autolysis rate of shrimp head was affected by temperature, pH, and substrate concentration. In contrast to the inconsistent effect of substrate concentration on autolysis rate constants, the effects of pH and temperature on the autolysis rate constants of shrimp head showed a consistent pattern. An Arrhenius equation (lnKa = 8090.2/T − 26.497) was established to validate the proposed autolysis kinetic equations. The autolysis rate of products with molecular weight < 3000 Da increased rapidly from 0 h to 3 h. In the next 2 h, the autolysis rate of these products slowed down. Furthermore, the aliphatic amino acids showed a higher amount release than that of other amino acids during the autolysis. The amounts of released heterocyclic amino acids (Trp and His) were also much more than that of other amino acids.

We looked for antibiotics from deep-sea marine sponges. We examined antimicrobial and antifungal activities of small individual sponges and found significant activity in one sponge. The extract of the sponge was fractionated to afford six nitrogen-containing metabolites. their structure elucidation is currently underway.

The cyanobacteria are recognized as promising producers of many secondary metabolites of which have structural diversity and potent bioactive properties. Chemical studies on the marine cyanobacterial constituents have been studied on Japanese cyanobacteria. However, most of these studies have been done for the cyanobacteria collected mainly from the sub-tropical area especially from Okinawa prefecture. So far, the bioactivity of the cyanobacteria from Tokyo Bay coastal area has not been well estimated. Therefore, we initiated the bioactive screening of the cyanobacteria collected from Tokyo Bay area. The sample collections were done during the summer of 2016 at the three points in Tokyo Bay coastal area. The collected samples were taxonomically identified under the microscope. About the sample from the two points around Oi Central Seaside Park, 80% Lyngbya aestuarii and 20% Pleurocapsa fuliginosa were observed. About the sample from Tateyama Station of Tokyo University of Marine Science and Technology, 90% Lyngbya semiplena and 10% Scytonematopsis pilosa were observed. The sample was extracted with organic solvents. The extracts were applied to the liquid-liquid partition. Hexane, ethyl acetate, 1-butanol and water layers were obtained for each sample. The bioactivities of the ethyl acetate layer of each sample were examined with L1210 cell cytotoxicity test and the diatom growth inhibiting activity test. Every ethyl acetate fractions from these collections revealed potent bioactivity. Now we are trying to isolate the bioactive compounds from these samples. We will report the precise bioactivity of the samples and the separation trial of the bioactive compounds from the cyanobacteria from the Tokyo Bay coastal area.

Genome mining is a powerful method for finding novel secondary metabolites. In our study on the biosynthetic gene cluster for the cyclic octapeptides surugamides A–E (inhibitors of cathepsin B), we found a putative gene cluster consisting of four successive non-ribosomal peptide synthetase (NRPS) genes, surA, surB, surC, and surD. Prediction of amino acid sequence based on the NRPSs and gene inactivation revealed that surugamides A–E are produced by two NRPS genes, surA and surD, which were separated by two NRPS genes, surB and surC. The latter genes are responsible for the biosynthesis of an unrelated peptide, surugamide F. The pattern of intercalation observed in the sur genes is unprecedented. The structure of surugamide F, a linear decapeptide containing one 3-amino-2-methylpropionic acid (AMPA) residue, was determined by spectroscopic methods and was confirmed by solid-phase peptide synthesis.

It is well-known that pH shifting method is a good protein recovery method that can effectively remove bone spurs, skin, fat and other impurities. Alkaline (ALPI) and acid (ACPI) protein isolates exhibited higher protein yields (87.6%, 76.3%, respectively). In addition to the high recovery of myofibrillar protein, a portion of the water-soluble proteins were also recovered. This study details the effect of pH-shift on the conformation of myosin extracted from carp (Cyprinus Carpio L.) muscle by measuring ATPase activity, Circular dichroism spectroscopy analysis and fluorescence analysis. Native myosin extracted from carp was treated at extreme acid-Alkali conditions (pH 2.5, 3.5, 11.5, 12.5 - 0.05 mol/L KCl). For all treated samples, The Ca2+-ATPase activities were completely lost. However circular dichroism spectroscopy analysis showed that there were no changes in the content of α-helix by different treatments. As Ca2+-ATPase activity is a very important index showing the structure change of myosin head portion, and the content of α-helix indicates the changes in rod tail portion, it suggested that pH-shift treatment destructed the myosin head portion, however there was no effect on the myosin tail structure. We also found that the tryptophan fluorescence of myosin was quenched, while the surface hydrophobicity was increased by pH treatment. Furthermore, the sulfhydryl content was decreased and cleavage sites were changed detected by chymotrypsin digestion. To sum up, the above results indicated that pH shifting method affected the tertiary structure of myosin.

It has been known that cultured cells exhibit characteristic morphological change depending on the mode of action of drugs. Thus, morphology guided cell-based assay is a powerful approach to detect bioactive compounds. In our laboratory, approximately 70 purified natural products, with or without known mode of action, have been profiled by observation of their influence on morphological change of several cell lines. By using this profiling, we found some of actin-target natural products showed the common morphological changes in cells. This led us to isolate actin-targeting natural products, jaspisamide A and its analog, jaspisamide C from marine sponge extracts. Jaspisamide A showed unique bioactivity compared with other trisoxazole macrolides. Therefore we focused on its substructure and tried to elucidate the active site by actin-binding-assisted structure conversion from other trisoxazole macrolide.

Marine sponges content a variety of bioactive secondary metabolites. By resent researches, it was revealed that there are unique symbiotic bacteria in sponges and they are producers of active compounds. It was reported that the content of dysiherbaine, a cytotoxic amino acid found in marine sponges, in the sponge Lendenfeldia chondrodes collected at the same region varied among individual sponge colonies. The content was dependent on whether there was the cyanobacteria, the producer of dysiherbaine, was in the sponge. From this report, we hypothesized that the metabolic profiles of marine sponges were difference by individual colonies because of the difference of bacterial composition in the sponge colonies. In most previous researches, conspecific sponges collected at the same region were extracted together and no attention was payed to their individual differences. In this research, to utilize bacterial resources in sponges more efficiently, we compared metabolic profiles by individual sponge colonies and searched for the compound which was contained by only several colonies.
We extracted 16 colonies of Theonella swinhoei with yellow interior collected at Hachijo Island by MeOH and analyzed these extracts by LC-MS. As a result, we found a compound which could be detected in only 7 out of 16 colony extracts. We isolated the “colony dependent” compound, determined the structure, and examined bioactivity.
The molecular formula of the compound was determined as C₇₄H₉₅BrN₁₆O₂₆ by HR-ESIMS. Interpretation of 1D and 2D NMR spectra revealed that the compound was a new analogue of theonellamide. The structure of the compound lacked a methyl group of p-bromo-β-methyl-phenyl-alanine in theonegramide. Although the absolute configuration of the isoserine residue in theonegramide had been assigned as R, that of our compound was assigned as S. The compound exhibited cytotoxicity against HeLa cells with an IC₅₀ value of 1.9 μM.

In Japan, sea cucumbers are mainly processed to dried or salted products, and exported abroad, while domestic consumption of them are relatively low. For the promotion of domestic consumption and effective utilization of sea cucumber resources, we have embarked on fundamental investigation on protein components in dermis of sea cucumber, and found a unique glycoprotein with molecular weight of 400K (400K-GP). Recent works on 400K-GP were reviewed here, mainly focusing on our data for the blue population of Japanese common seacucumber Apostichopus japonicus. The 400K-GP is one of the major proteins in the dermis, occupying about 30% of its total protein and quite soluble in water or neutral buffer solution with low ionic strength. Immunohistochemical study clarified its distribution in gap region between collagen fibers as well as around collagen fibers. These results suggest the 400K-GP may exist in dissolved form in body fluid. On SDS-PAGE, it showed appropriate molecular weight of 400K under non-reducing condition but its band shifted to 200K in the presence of reducing agent, 2-mercaptoethanol. In addition, the components of 400K and 200K were both reactive for periodic acid - Schiff (PAS) reaction. It was therefore suggested to be a glycoprotein consisting of two 200kDa subunits linked each other by disulfide bonds. The 400K-GP was quite rich in Asp and Glu (more than 100 residues / 1,000 residues) in amino acid composition, and estimated to have a neutral sugar content of about 9.2% by phenol-sulfuric acid method. Moreover, the 400K-GP was suggested to have inhibiting activity of cell spreading for baby hamster kidney cell in contrast to bovine fibronection (as a control) with very high cell spreading activity. Further studies are now in progress on the physiological functions of this protein, aiming at its industrial use.

[Objectives] In order to avoid atopic and inflammatory diseases, natural resources have been developed and applied to food and medicines. Therefore, this study was carried out to verify the anti-inflammatory and anti-atopic effects of Sargassum miyabei ethanol extract(SMEE).

[Methods] The murine macrophages Raw264.7 and splenocyte were used in this study and MTT assay was performed to measure the cell viability. The production of cytokines and the expression of mediators were examined by ELISA and immunoblotting. In addition, DNCB and croton oil applied mouse models were used to verify anti-atopic and anti-inflammatory activity, respectively, in vivo test.

[Results] In anti-inflammatory activity, the inhibition of NO, pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and mediators were observed. The ear edema and the number of infiltrated mast cells in mouse model were also reduced effectively after SMEE treatment. In anti-atopic activity, the production of IL-4 and IgE in serum and IFN- γ, IL-4 and IL-5 in splenocytes were significantly reduced by SMEE. The reduction of dorsal skin edema and the number of infiltrated mast cells were shown in photomicrograph. In conclusion, it is suggested that SMEE have a potent anti-inflammatory and anti-atopic effects and be applied to a therapeutic agent.

[Objectives] Seaweeds contain lots of polysaccharides, which have various biological functions such as antiviral, antitumor and anti-inflammatory activities. The alginic acid, which is one of the polysaccharides, has been limited in their industrial applications because of its high viscosity in manufacture process. This study was conducted to investigate the characterization and immunoregulatory activity of enzymatic extract of Gracilaria textorii using polysaccharides-degrading enzyme.

[Methods] The alginate oligosaccharide produced by crude enzyme from S. oneidensis PKA 1008 was used in this study. The mixture of the Gracilaria textorii and crude enzyme from Shewanella oneidensis PKA 1008 was incubated at ratio of 1:1 (v/v) for 0-48 h to obtain the optimum degrading conditions for the Gracilaria textorii. The changes in pH, reducing sugar, viscosity, and color of enzymatic extract were measured.

[Results] The optimal conditions of crude enzyme for alginate-degrading activity was pH 9, 30℃, 7% alginate, and 48 h. The alginate oligosaccharides were confirmed by using thin-layer chromatography. These results suggest that alginate oligosaccharides of Gracilaria textorii produced by crude enzyme from S. oneidensis PKA 1008 exhibit potent immunoregulatory activity.

This presentation illustrates the state of aging fishers and their contribution to fishery production. The issue of aging is clarified through the data analysis based on the Fishery Census. Their contribution is based on the results of interviews and data analysis. The aging problem started as the fishery labor force of the cohort of the birth years between 1925 and 1934, or the so-called First Decade Showa Generation, became elderly because they composed the largest portion of the labor force. The next cohort, 1934-1945, and the succeeding five years, of which the latter is called the Baby Boomers, also accounts for a significant population and all are already over 65 years old. Since these cohorts are in the process of retiring, the number of aged fishers has been decreasing since 2005. It should be noted that the percentage of aged fishers out of total fishers, which is currently 36%, is increasing due to the fact that younger generations do not opt to be fishers: the number of new entrants is approximately 2,000 per year. We observe such characteristics as the fishers who engage in less profitable fishing methods, such as angling, gillnet and hand-picking are more likely to be aged fishers. Nevertheless, aged fishers have important roles as post-harvest labor after landing and as advisors for beginners. Therefore, the fact of the decreasing number of aged fishers is apparently a loss to Japanese fishery. In order to mitigate the negative effect, we suggest that policy makers take actions to amend fishery infrastructure so that aged fishers can continue working, to incorporate support programs for aged fishers into the facilitation of fishery labor supply, and to deregulate new fishing methods to adjust for the smaller number of fishers. Above all, cooperation with younger generations will generate a synergy in fishery production.

Anthropogenic Activities and Waste Disposal Practices in the Ports and Fish Landing Areas of Camotes Islands, Cebu, Philippines
Serapion N. Tanduyan, Jonita V. Literatus, Eva P. Muaña, Eddele M. Condrillon, Jonar D. Gonzales, DharelMiro, JocelNagsuban, Homer Garciano, Jeziel Nudalo
Cebu Technological University, San Francisco, Campus
Tel:( +63 -032) 3188163/ (+63-032) 3188164; e-mail: sntanduyan@yahoo.com

ABSTRACT

Ports and fish landing areas are the busiest parts of the aquatic environments because of transportation services. . Actual interview and a field visits were made using an interview guide to gather data at the municipalities of San Francisco, Poro,Tudela and Pilar.
Results shows that anthropogenic activities include fishing, fish vending, business like entrepreneurship (sari-sari store).San Francisco topped among the number of fishermen ,fish vending and business activities followed by Poro, then Tudela and Pilar.In transportation services include motorcycle and multicab driving and stevedoring services. Then there is beauty and wellness. For the entertainment these include billiard, videoke , computer games, tour guiding and sports fishing.San Francisco, topped also among these activities followed by Poro, Tudela and Pilar Among all the activities, fishing and fish vending were the most common with the frequency 158 (94.04%) over other activities.
San Francisco topped among all the municipalities(98%) on proper waste segregation , then by Poro (75%),Tudela (65%) and Pilar (62%). Plastics ,bottles and fish offals were the common wastes which brings Camotes Sea clean and free from pollution.
Fish vendors are the fishermen themselves (60%) and real o vendors is (40%) in San Francisco. In Poro it is (40%) and (60%);Tudela is (55%) and (45%) and Pilar is (60%) and (40%) in that order.
Environmental problems like bad weather conditions ranked first followed by financial problem, social and technical problem the workers in the ports and fish landing areas.

Keywords: Anthropogenic, Ports, Camotes Islands

Recreational fishing (RF) is a popular activity around the globe. It is mainly a fun-seeking activity. By its name, RF could take place on vessels, at shoreline, or at docks. In this study, RF refers to the activity that fishers take tourists on board fishing vessels out to sea to go fishing.
In Taiwan, RF was a derivative of the fisheries transformation policy which was initiated in early 1990s when fishery authorities were dedicated to diversify traditional fisheries into tourism in order to address socio-economic problems facing declining coastal fisheries. RF has become a major outcome of this policy, along with other recreational activities such as whalewatching, boating. This activity involves taking of fisheries resources. However, with a growing number of anglers participating in this activity, a lack of harvest statistics from RF was noted. This missing information has retarded the ability to understand the magnitude of the RF sector and its contribution to fish mortality relative to commercial fisheries, and to take precautionary measures to avoid unsustainable fishing. In particular, while commercial fishing has been repeatedly blamed for the declines in fish populations, the RF sector also has the potential to negatively affect fish and fisheries. In this regard, monitoring and compiling of harvest from RF are key processes for science and research as well as for management.
There are currently around 150 fishing vessels engaged in RF. This research aimed to evaluate the magnitude of RF and compares it with that of commercial fishing. A survey of harvest and catch species from sampled operators was conducted. The estimation of harvest was done based on the size of angling population, catch amount per angler per trip and other relevant information. The harvest from RF was further compared to that of commercial harvest and relevant management implications were inferred.

Japan is one of the world’s largest fish-eating countries with a long history, but recently stable supply of marine products and sustainable development of fishing villages are urgent issues due to various changes in both natural environment and society. In order to solve these problems, a lot of good practices for fisheries resource management have been accumulated throughout Japan. Thus, new analytical methodology is necessitated to identify characteristics from amount of data and to learn from various good practices’ information. The aim of this study is to develop a method to identify characteristics of knowledge and experience from good practices, and to compare characteristics between different fishing villages’ activities.
A text database of communities’ fisheries management activities was constructed from documents, which provided by National Federation of Fisheries Co-operative Associations in Japan. Then, semantic network analysis was conducted for this text database. Finally, we compare characteristics of semantic network in different climatic zones (e.g. Hokkaido and Kyushu region). Semantic network analysis is newly proposed analytical methodology that extracts important categories and their network (semantic network) in several text data using natural language processing.
Results of semantic network analysis showed that semantic networks of both Hokkaido and Kyushu region consist of categories related to “fisheries resources” and “resource management activities”. Particular fish species are emphasized in Hokkaido region (e.g. sea cucumber, salmon, scallop, kombu, sea urchin etc), while Kyusyu region emphasized fishing gears and methods (e.g. fixed shore net, pole and line, gill net, small trawl net etc).
These results indicate that semantic network analysis allow us to identify commonness and differences among good practices in Japan. We will conduct further analysis to compare semantic networks in different fish species, time period, and fishing gears.

Anchovy (Stolephorus sp) is one of the well-known marine fishery commodities in Indonesia. The fish species is commonly caught in Saamaake coastal water, East Halmahera, North Mollucas Province. These activities had been conducted for 7 months, from June 2016 to January 2017 aiming to evaluate the management of community empowerment by improving the quality of anchovy product and initiation of market development. The methods utilized in this study were training and mentoring, while the targets were the fishermen (15 fisherman groups or 60 persons in total). The program implementation was conducted based on mapping result which was evaluated at the end of the program. Some activities have been performed to improve the product quality (construction of new drying facility and storage rehabilitation), to produce product packaging and labeling, and to initiate a market development. As a result of the observations, the community has gained adequate knowledge and technology related to the fishery product handling and processing. The dried anchovy can be marketed and labeled in small sizes (100 g, 200 g, 500 g, and 1000 g), and can also be marketed to broader areas e.g. Antam Ltd (company) office in Ternate, Antam Ltd. Cooperation in Buli, and Ternate Airport of North Mollucas Province. In accordance with the data analysis, it is revealed that the program is suitable for the community (100%), including in gaining skill development (92%). The program is also considered to be suitable for the fisherman (92%). As through this program, they gain capability in running their business more independently.

Keywords: Management, community empowerment, Anchocvy quality, market, Saramaake, North Molucas, Indonesia.

Overall planning of land and sea demonstrates the firm belief of China's peaceful development achievements of maritime power, as a strategic option. Coastal areas become space vector, with its special geographical location, superior resources conditions and developed economic level. Access to relevant literature, to understand current research, combing the development process of overall planning of land and sea, including how the concept proposed, how it content rich, how it ultimately up to the national level and how it becoming China's marine development strategy. Further research is needed in terms of overall planning of land and sea mechanism framework and evaluation system.

PRESENT STATUS SEAWEED CULTIVATION AND PROSPECT APPLICATION OF SATO UMI CONCEPT IN INDONESIA
By : Wisnu Sujatmiko, Ratu Siti Aliah, Deddy Yaniharto, Suhendar I. Sachoemar
Indonesia is one of the world's largest seaweed producers and exporters with the main products being Eucheuma and Gracilaria. Eucheuma includes Eucheuma cottonii / Kappaphycus alvarezii, Eucheuma spinosum, Eucheuma striatum cultivated in the sea. Gracilaria include (Gracilaria verrucosa, Gracilaria sp.) cultivated in brackish water ponds. The number of workers in seaweed cultivation reaches 855,400 more people.
Seaweed production increased significantly, Eucheuma 3,399,436 tons (2010) rose to 8,971,463 tons (2014) increased 28.2% and Gracilaria production 515,581 (2010) rose to 1,105,529 tons (2014) increased by 21.09% . Tatal production in 2014 amounted to 10,076,992 tons and in 2015 10,355,000 tons rose by 0.0275%. The very low percentage increase in 2015 is likely to be caused by declining environmental quality of cultivation, global warming, over capacity of planting, low knowledge or skills of farmers, the presence of disease or decreasing the quality of seaweed seeds.
To overcome the decrease of seaweed production is needed to repair one of them by applying Sato Umi concept which has been applied in Japan. The concept of Sato Umi in general is to involve the community actively to maintain a harmonious interaction between seaweed farming activities with environment and human resources in a sustainable manner. Activities that can be done include monitoring the quality of the cultivation environment, developing seaweed cultivation in accordance with the carrying capacity of the land, developing a seaweed seedling, conducting education and training for farmers to improve and maintain the productivity of seaweed. The pilot project of Sato Umi concept is being implemented in Bantaeng Regency of South of Sulawesi.
Keyword :
Seaweed, Productifity, Sato Umi, Bantaeng

Islas de Gigantes is a group of island lying in the Visayan Sea, central Philippines. It is composed of four barangays (Villages) situated in two major islands, Gigantes norte (Asluman and Granada) and Gigantes sur (Gabi and Lantangan). It is significant both ecologically and economically. It is one of the only 11 sites in the Philippines considered as zero extinction sites (here are only 587 such sites in the world). Economically, the island is a traditional fishing community. However, ecotourism is also gaining importance as a major contributor to the local economy with the government’s thrust of promoting tourism in the area. Both activities are reliant on the islands’ coastal resources. While assessments of the coastal resources have been conducted, there is scant information on the users these resources. Thus, this study was conducted to profile the coastal resource users in the area.
The study used structured questionnaires and focus group discussion. It covered all Villages in the island. A typical coastal resource user has completed basic education (91%), owns a house (100%) of mixed (45%) or light (38%) material and which sits on a land which is mostly either owned (29%) or as informal settler (29%). Further, only more than half of the households have toilets (54%). Fishing is the major source of household income (75%). Most are engaged in squid fishing (36%), fish (33%) and crab (18%). The dominance of squid may indicate stressed environment while crabs are also targeted as there is a processing plant in the area. Mean monthly household income is PhP 6,936+3,546 which are spent on food (46%) and education (19%). Also, insufficient income (79%) dominates family related problem while sanitation (lack of waste management, 27% and toilet, 12%) and illegal and dynamite fishing (12%) are considered as community problems.