The JSFS 85th
Anniversary-Commemorative International Symposium
“Fisheries Science for Future Generations”


01001 Abstract Proceedings

Mechanisms leading to the decline in Pacific oyster seedlings in Matsushima Bay, Japan

Shigeho Kakehi1, Takashi Kamiyama1, Hirokazu Abe2, Ryo Matsuura3, Akio Oshino3, Katsuaki Okabe4, Kazufumi Takayanagi4 and Katsumi Yokouchi1, 1Japan Fisheries Research and Education Agency, Japan, 2 Iwate Medical University, Japan, 3 Miyagi Prefecture Fisheries Technology Institute, Japan and 4Sanyo Techno Marine, Inc., Japan

In 2013, there was a significant decline in the wild seedling yield of the Pacific oyster Crassostrea gigas in Matsushima Bay, Japan. Although higher densities of early stage larvae occurred in 2013 compared with previous years, the pre-attachment stage larvae were rare indicating that the early stage larvae abruptly decreased before they reached the pre-attachment stage. We used hydrographic observational data obtained in 2011–2015 to determine whether hydrographic conditions explained the low yield and the absence of pre-attachment stage larvae. In August 2013, low salinity water was distributed at the surface resulting in the density difference between the surface and near the seafloor of the bay exceeding 10 kg m−3. This difference generated a strong outward driving force of 10.0 × 10−3 N m−3. We hypothesize that the oyster larvae were transferred out of the bay before they could reach the pre-attachment stage. The low salinity distribution resulted from both increased river discharge that flows into the bay and freshwater inputs derived from a river discharging outside a bay. The influence of freshwater from the outside river has likely increased in the bay as a result of topographical changes caused by the 2011 Tohoku earthquake.

01002 Abstract Proceedings

Comparison of the feeding environments of oyster in two bays in Miyagi Prefecture, and estimation of the suitable oyster culture density at Nagatsuraura Bay

Yutaka Okumura1, Hiroto Ota2, Yuichi Taniai3, Nana Takeyama3 and Motoyuki Hara4, 1Tohoku National Fisheries Research Institute, Japan, 2 Miyagi Prefecture Fisheries Technology Institute, Japan, 3 Miyagi Prefectural Government, Japan and 4Tohoku University, Japan

In Miyagi Prefecture, total oyster shipment since the earthquake off Tohoku in 2011 has been approximately 20,000 t/year (wet weight with shell), substantially lower than in preceding years (40,000–60,000 t/year). Although oyster culture by non-feeding is a major fishery, the industry has not fully recovered since 2011. Therefore, to aid recovery of oyster production while avoiding overcrowded farming, it was necessary to investigate the feeding environment and estimate a suitable stock density for the region. Specifically, we compared the feeding environments and growth of cultured oyster in Oginohama Bay and Nagatsuraura Bay. We speculate why the culture period is currently shorter at Nagatsuraura (one year) than at Oginohama (two years). Finally, based on our data, we estimated the most appropriate stock density for Nagatsuraura Bay.
We regularly measured the environmental conditions, particularly water temperature and chlorophyll-a (chl-a) concentration, and oyster growth in the culture areas of both bays. We also calculated the total filtration rate of oyster and the growth rate of phytoplankton in the culture areas. The suitable oyster culture quantity was estimated from the phytoplankton quantity in the culture areas minus the total filtration of phytoplankton by oyster, resulting in a value greater than zero.
Maximum chl-a concentration was approximately 100 and 9 µg/L in Nagatsuraura Bay and Oginohama Bay, respectively. Oyster at Nagatsuraura grew faster than at Oginohama. The more rapid growth rate at Nagatsuraura was attributed to higher phytoplankton abundance, because the difference in water temperatures between the two bays was smaller than the difference in chl-a concentrations. By current estimations, we believe that a hanging-culture quantity of 98–120 rafts × 300 ropes × 12.5 masters × 14 ind./master would not constitute overcrowding. We aim to improve the precision of our estimation by increasing the study parameters in future research.

01003 Abstract Proceedings

Recovery of coastal fauna after the 2011 tsunami in Japan revealed by bimonthly underwater visual censuses conducted over six years

Reiji Masuda1, Makoto Hatakeyama2, Mizuki Ogata1, Katsuhide Yokoyama3 and Masaru Tanaka4, 1Kyoto University, Japan, 2 NPO Mori-umi, Japan, 3 Tokyo Metropolitan University, Japan and 4Moune Institute for Forest-Sato-Sea Studies, Japan

Massive tsunamis induce catastrophic disturbance in marine ecosystems, yet they can provide unique opportunities to observe the process of ecological succession. Here we report the recovery of fauna after the 2011 tsunami in northeast Japan based on underwater visual censuses performed every two months over six years. The censuses were conducted at four locations in and around the Moune-Bay, Kesennuma, and species, size and abundance of fishes and conspicuous invertebrates were recorded. Both total fish abundance and species richness increased from the first to the second year after the tsunami followed by stabilization in the following years. Short-lived fish, such as the banded goby Pterogobius elapoides, were abundant in the first two years, whereas long-lived species, such as the black rockfish Sebastes cheni, increased in the third year or later. Tropical fish species were recorded only in the second and third years after the tsunami. The body size of long-lived fish increased during the survey period resulting in a gradual increase of total fish biomass. The recovery of fish assemblages was slow at one site located in the inner bay, where the impact of the tsunami was the strongest. Apart from fish, blooms of the moon jellyfish Aurelia sp. occurred only in the first two years after the tsunami, whereas the abundances of sea cucumber Apostichopus japonicus and abalone Haliotis discus hannai increased after the second and fourth years, respectively. We conclude that it takes approximately three years for coastal fish assemblages to recover from a heavy disturbance such as tsunami and that the recovery is dependent on species-specific life span and habitat. These findings provide some insights for the management of fisheries resources, particularly for designing marine protected areas and also for planning rotational harvest.

01004 Abstract Proceedings

Legal and constitutional system of very large sea wall construction of recovery affairs from Great East Japan Earthquake disaster and decision making of fishery villages

Satoquo Seino1 and Masayoshi Tanishita2, 1Kyushu University, Japan and 2Chuo University, Japan

Very large sea wall construction was executed for recovery from Great East Japan Earthquake disaster along the coasts of the Pacific in Tohoku District of Japan. The affairs aimed at to protect the margin of national land and disaster risk reduction of human habitat.
Social and legal systems of these coastal construction recovery affairs were analysed. Sea Coast Law, Acts related to Land Use, Disaster Recovery Special Law and real estate management social customs were very complicated in usual times. When the disaster recovery, governmental systems were collapsed and degraded, then all sectors could not solve and change these latent problems.
Fishery villages were changed totally by big construction project to move human habitat to the upper hills and covered with concrete protection facilities.
Originally fishery people judge their circumstances for fishery activities by various senses. Then, shutting the visual openings of the fishery villages is very serious problem for their personal and professional life.
Cases of fishery villages in Miyagi and Iwate prefecture were analysed. Especially, comparative studies the process of decision making Kesen-numa City revealed that participatory process in decision making of the villages was critical. Administration stratum and sectionalism were obstacle to make the residential design freely.
Many lessons learned should be utilized to prevent next recovery project in the world.

02001 Abstract Proceedings

The use of bridle line on the bottom gill nets as the criteria of environmentally friendly fishing gear

Isrojaty J. Paransa, Tokyo University of Marine Science and Technology, Japan

Indonesia has natural resources abundant marine and coastal waters with an area of 6.32 million km2 that it contains coastal resources such as coral reefs, fish, mangroves and other marine life. These resources are the main target of the activities of coastal communities to catch fish with bottom gill net. To catch demersal fish, fishermen normally enable transfer surface gill nets to bottom gill net, by adding weights to the foot line. This additional weights function, to sink the net as a overall, increase the speed of sinking and maintain the shape of the net, so that the net can have a shape expected or rectangular while operating in bottom waters. Operation nets are usually done on coral reefs, because fishermen believe when the net is operated on a coral reef then catches obtained better. But otherwise, the operation on a coral reef can damage the coral reef substrate, due to the lower net direct contact with the seabed, even other organisms which symbiosis the reefs will upraised with the catch. If the operation continues could give a negative impact on the ecosystem can bottom waters. Although the bottom gill net fishing gear has been developing states for a long time, but the efficiency and selectivity still has potential for development, to fulfill the criteria of environmentally friendly fishing gear and sustainable. The bridle line mounted at the bottom of the net is used as a parameter to measure the damage caused by the operation of the bottom gill net and to lift the foot line and the weight of the bottom waters. However, in this study the shorter the size of the bridle line will increase the number of coral rubble and use bridle line can reduce catches decreased significan with a length of bridle line that is used.

02002 Abstract Proceedings

Time domain analysis of a coupled system with trawler and trawl gear using a numerical method

Chun-Woo Lee1, Jihoon Lee2 and Subong Park1, 1Pukyong National University, Korea and 2Chonnam National University, Korea

Trawling is one of the very important fishing methods used in fisheries. It involves a coupled system, with the fishing vessel as an elastic structure and the fishing gear as a flexible structure. While towing a gear of large size, the trawler is subjected to changes in course, speed, and wave action, and its mobility is restricted. In addition, the gear towed by the trawler undergoes changes in shape and behavior according to the behavior of the trawler, especially in the cases when the trawl is operated under harsh sea conditions. Therefore, understanding of the interaction between the fishing vessel and the fishing gear is important for proper handling of the gear to increase fishing efficiency and improve the safety of the vessel. This study presents an interaction model using a coupled system comprising the vessel and the gear to reflect the influence of oceanic factors such as currents and waves; the model can also be analyzed in a time domain. The effect of fishing gear on the fishing vessel is analyzed using a numerical method to obtain the decrement in vessel speed during operation. Furthermore, the mutual interference between the stern of the trawler and trawl doors is also analyzed considering the amplitude of vertical oscillation under different conditions of wave height, wave length, and wave periods. The results will be helpful in understanding the fishing gear behavior from the point of view of gear handling and safety of the vessel.

02003 Abstract Proceedings

Bottom feeding and high density stocking improve yield and profitability of milkfish culture in marine cages

Crispino Saclauso1 and Joebert Toledo2, 1University of the Philippines Visayas, Philippines and 2Feedmix Specialists Inc II, Philippines

High density stocking often results in slow growth and low survival in aquaculture. This is attributed to diminished space, inequitable access to food, and aggressive behavior as a consequence of crowding and deterioration of water quality at high population densities.Under crowded conditions, aggression depresses growth that results in the deaths of subordinate conspecifics. In non-aggressive species like milkfish, crowding significantly affects size variance hypothesized to have been caused by reduced food access in high density culture where fish are fed from the top.
The study aims to improve yield and profitability of milkfish production in high density culture through bottom feeding. With increased density, yield per unit volume is also expected to increase thus diminish the area required in generating equivalent production at lower stock densities.
Milkfish at densities of 50 pieces m-3, 75 pieces m-3 and 100 pieces m-3 were reared for 120 days in replicated 5mx5mx5m floating net cages in a marine cove with a depth of 10 m and a flow rate of 8 cm sec -1. Fish were fed floating pellets through top feeding and hydro-kinetic bottom feeder.
Bottom feeding increased production of milkfish cultured at very high density. Net yield (kg m-3) increased by 75% with the doubling of stocking density from 50 m-3 to 100 m-3. Size variance was reduced and condition index (K) and feed conversion ratio (FCR) were improved. Bottom feeding optimizes food utilization through equitable and better access to food hence, reduces feed wastage. Bottom feeding and high density stocking enhances production efficiency without compromising environmental integrity.

02004 Abstract Proceedings

Anatomical and microscopic structure of thoracic adhesive apparatus in hill stream catfish genus Glyptothorax from the northern of Thailand

Tosapon Chamnivikaipong and Apinun Suvarnaraksha, Maejo University, Thailand

A Hill stream catfish genus Glyptothorax were found in fast flowing streams. Due to the resistance offered by the fishes against high speed water currents, an adhesive apparatus (ADH) are developed to help the fishes maintain in the stationary position in high speed water currents. This present study is a first study of ADH of Glyptothorax from the northern of Thailand. An external morphology was studied by camera and scanning electron microscope (SEM), which shown a difference in structure and arrangement of unculi or hooklet from ADH surface. An internal morphology was studied by histological method, which shown a minute structure of tissue layer on the under surface.

02005 Abstract Proceedings

An overview of purse seine fisheries management in Malaysia

Ledhyane Ika Harlyan and Takashi Matsuishi, Hokkaido University, Japan

Malaysian fisheries management has become a model for developing countries in Asia, yet it is comparatively unknown outside Asian region. During coupled decades, purse seine noticed as one of the most effective fishing gear contributing more landings and played significant role in Malaysian fisheries along with its catches. A set of conservation and management measures have been formulated and applied since mid-1980s, however the characteristics of Southeast Asian fisheries (i.e. as a multi-species, multi-gear, many fishers, landing sites, and data-poor situation) seemed to complicate its implementations. This study gives an overview of the existing management system and highlights some of its challenges. The most prominent measures are technical measures (i.e. closed fishing area, fishing zones, conservation of marine habitat), input controls (i.e. control on fishing effort and fishing units, registration of fishers) and community based fisheries management. Some valuable progresses have been accomplished, but the limitations of these measures remain, since they do not directly constrain the fleets from targeting and diminishing a certain individual stock in the mixed fishery. Therefore, a set of integrated adaptive measures are considerably needed to deal with these situations.

02006 Abstract Proceedings

Importance of piscivory for growth and survival in early life stages of Pacific bluefin tuna observed by rearing experiments

Yosuke Tanaka, Kazunori Kumon, Kentaro Higuchi, Takeshi Eba, Akefumi Nishi, Hideki Nikaido and Satoshi Shiozawa, Japan Fisheries Research and Education Agency, Japan

Survival in early life stages of fishes is considered to be the main cause of inter-annual variability in their recruitment. Growth of larvae and juveniles is one of the most important factors for their survival. Rearing techniques for Pacific bluefin tuna (PBT) larvae have been remarkably developed for the last decade and enable us to investigate the detailed eco-physiological traits of PBT larvae. Although hatchery-reared larval PBT shows the shift of feeding habit from zooplanktivory to piscivory, there are no reports about piscivory of wild PBT larvae in fields. The objective of the present study is to elucidate the importance of piscivory for growth and survival in early life stages of PBT by rearing experiments. Firstly, we examined the influence of piscivory on growth of hatchery-reared PBT larvae. Rearing experiments revealed that the tuna larvae after onset of piscivory showed significantly faster growth than the tuna in zooplanktovious stage, which were supported by very high activity of the protein synthesis. Secondly, relationship between prey utilization and growth variation in hatchery-reared PBT larvae were investigated by nitrogen stable isotope and otolith analyses since marked growth variations of PBT larvae were observed in mass culture tanks. Nitrogen stable isotope analysis revealed that the larger fish rapidly shifted their prey to fish larvae, but smaller fish depended more on rotifers as the main prey item. Otolith analysis revealed that small growth variations during the early larval stage of PBT could induce subsequent large growth variations in the late-larval and juvenile stages through changes in their feeding habit. Our findings suggest that the piscivory is the key factor for growth of larval PBT and the growth variations induced by piscivory have the potential to lead to the inter-annual variations in recruitment success via growth selective survival process.

02007 Abstract Proceedings

Aspects of the reproductive biology of brook trout Neolissochilus stracheyi (Day, 1871) in Northern, Thailand

Thapanee Pholdee and Apinun Suvarnaraksha, Maejo University, Thailand

Reproductive biology of Brook trout, Neolissochilus stracheyi (Day, 1871), were sampled in the Northern of Thailand during October 2010 - September 2011 from fisherman, using gillnet of various mesh sizes. For the study of relationships between total length and body weight, spawning seasons, sex ratio and fecundity. At least 66 fish samples were used every month. The result showed that the total 821 Brook trout had 7.4-61(23.32±5.91 cm). There were 353 males that had fish samples there were 8.5-45.1 cm of TL (22.36±5.91cm) and 468 females that had 7.4±61 cm of TL (24.04±7.09 cm). The relationships equation between total length and weight was W = 0.008TL3.064 (r2 = 0.940) for total fish, W=0.007TL3.107 (r2 = 0.946) for females and W=0.010TL3.003 (r2 = 0.930) for males. The relative condition factor (K) of male and female was 1.090±0.067 and 1.116±0.102 respectively. The max of GSI was in June for male and female. The spawning season was found all year round with 3 peaks: January, June and August. Estimated fecundity of mature ovaries ranged from 220 to 3,500 eggs. The estimated average fecundity was 1404.55 ±651.51 eggs. The relationships between fecundity and total length revealed F= 121.1TL-2100 and relationships between fecundity and weight revealed F= 4.535 + 128.78. The results from studies can be further applied for the economic culture, conservation and management of the Brook trout in Northern, Thailand.

02008 Abstract Proceedings

Governing of village fishery project in Chiangmai, Thailand

Daracha Thiammueang, Maejo University, Thailand

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

02009 Abstract Proceedings

Formation of pseudo-bands on the otoliths of walleye pollock Gadus chalcogrammus juveniles at high water-temperature

Yuuho Yamashita, Takashi Yokota, Toru Nakagawa, Hiroshige Tanaka, Masayuki Chimura and Mitsuhiro Ishino, Japan Fisheries Research and Education Agency, Japan

Translucent annual bands normally form in the otoliths of walleye pollock Gadus chalcogrammus in winter, but similar translucent zones occasionally appear in other seasons during the juvenile phase (pseudo-bands). Because pseudo-bands could hinder precise age determination in this species, we examined the formation of translucent bands in different water-temperatures regimes. Fertilized eggs were collected from the bloodstock tank in Akkeshi Laboratory, Japan Fisheries Research and Education Agency in January, 2016, and larvae were reared to juvenile stage. Two 4000-L tanks were prepared, and 1500 juveniles were added to each tank on June 9. The juveniles were fed to satiation with formula food until October 27 and the two tanks were exposed to different temperature regimes: 1) natural temperature fluctuation (NT, not artificially controlled, 19.8 °C on September 8 and 12 °C on October 27); and 2) controlled temperature (CT, maintained at 12 °C). Approximately 20 juveniles were sampled from each tank at 1- or 2-week intervals to observe their growth and otoliths. Survival rates and condition factors (body weight/body length3) were not different between the tanks during the experiment. In contrast, the body lengths, body weights, and otolith lengths of the juveniles in the CT tank were larger than those in the NT tank (p <0.01). Under a full-feeding regime, high temperature is thought to be a limiting factor for growth but not for health condition. On September 15 and 29, a translucent band was observed at the exterior edge of otoliths removed only from the individuals in the NT tank. An opaque band was observed outside this translucent band when the experiment was terminated. These observations suggest that the pseudo-bands are formed under high water-temperature conditions.

02010 Abstract Proceedings

Possibility of myoglobin as a molecular marker for phylogenetic relationship of fish

Ming-Chih Huang1, Yoshihiro Ochiai2 and Shugo Watabe3, 1National University of Tainan, Taiwan, 2 Tohoku University, Japan and 3Kitasato University, Japan

[Introduction] Fish show a large biodiversity in the strategies to adapt to respective inhabiting environments. In order to know the genetic relationship among fish species, many attempts have been made based on various molecular markers such as mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit I (COI). Not all of them are, however, successful for this elucidation. In the previous study, we found the availability of muscle tropomyosin (TM) as a good molecular marker. In this study, myoglobin (Mb) was evaluated as a possible alternative molecular marker for the elucidation of phylogenetic relationship of fish.
[Methods] Publicly available data of the DNA and deduced amino acid sequences of Mb, cytb and COI were obtained for longtooth grouper (Epinephelus bruneus), medaka (Oryzias latipes), whale shark (Rhincodon typus), bluefin tuna (Thunnus thynnus), green spotted puffer (Tetraodon nigroviridis), torafugu (Takifugu rubripes), zebrafish (Danio rerio), Atlantic salmon (Salmo salar) and tilapia (Oreochromis mossambicus). The phylogenetic analyses were performed based on the different methods namely, maximum likelihood, neighbor joining and UPGMA.
[Results and discussion] The phylogenetic trees depicted based on Mb sequences were similar to those based on the traditional classification markers. The primary, secondary, and the modelled 3-D structures of Mbs were similar among Mbs from different fish species, but were clearly distinguishable among the nine species. Such differences in the structures would be responsible for adaptation of Mb molecules to the physiological conditions of each species. These results suggest that Mb can be a molecular marker for the phylogenesis of fish, but is slightly inferior to TM.

02011 Abstract Proceedings

Flipper beat frequency and body acceleration of loggerhead turtles Caretta caretta swimming in a set net

Maika Shiozawa1, Daisuke Shiode1, Keiichi Uchida1, Seiji Akiyama1, Fuxiang Hu1 and Yoshio Hirai2, 1Tokyo University of Marine Science and Technology, Japan and 2Nitto Seimo Co., Ltd., Japan

Turtle releasing system (TRS) is a method to allow sea turtles to escape out from a submerged bag net of a set net. This system consists of a turtle releasing device (TRD) and a quadratic-prism shaped sloping upper panel of the bag net. Turtles start pushing their heads up continuously (defined as pushing up) to the surface to breathe with the lapse of time. The sloping upper panel guides turtles to the TRD installed at the apex. In this study, flipper beat frequency and body acceleration of loggerhead turtles in the bag net were examined to assess pushing up behavior and desire for breathing.
Experiments were conducted in August and November 2013, June 2014, July 2015, and September 2016 in a set net in Mie pref., Japan. Fifteen wild loggerhead turtles (SCL: 63.3~89.6cm) were used in the experiments. A video camera (HDR-AS100V, Sony Inc.), an acceleration data logger (W1000-3MPD3GT/ W380-PD3GT, Little Leonardo Co.), and a depth logger (DEFI-D20, JFE Advantech Co., Ltd.) were attached on the carapace of the turtle, and the turtle was put into the submerged bag net. Flipper beat frequency was obtained from video images, and ODBA (overall dynamic body acceleration), an index of body motion, and water temperature were obtained from an acceleration data logger, respectively.
Flipper beat frequency of the loggerhead turtles swimming in the bag net varied between approximately 0.2 to 1.5 Hz during thirty minutes’ observations, and it tended to increase in ascent leading to pushing up unlike in natural environment. Flipper beat frequency had a positive correlation with water temperature (P<0.0001). Loggerheads alternated series of pushing up and swimming with the lapse of time. Average of ODBA in the series of pushing up increased as the number of the series proceeded, that is, pushing up was strengthened with time elapsing without breathing.

02013 Abstract Proceedings

Garlic extract product enhancing growth performance, digestive and immune system in Nile tilapia (Oreochromis niloticus)

Mallika Supa-aksorn1, Krisna Rungruangsak-Torrissen2, Sudaporn Tongsiri1 and Jiraporn Rojtinnakorn1, 1Maejo University, Thailand and 2Institute of Marine Research, Ecosystem Processes Research Group, Matre Research Station, Norway

One of the most important problems of Nile tilapia commercial aquaculture is epidemic diseases. The farmers usually use drugs and chemicals to prevent this serious problem, and this effects a residual drug and spreading out of drug-resistant bacteria in pond and environment. Many herbs are considered for alternative antimicrobial agents for prevention and treatment the diseases. For this study, our garlic extract product (GEP) was developed that containing high allicin, S-allylcysteine, and S-allylmercaptocysteine, the major biocompound in garlic. Nile tilapia juveniles were fed with GEP 0.5 and 1.0 %(w/w) added feed (GEP 0.5 and GEP 1.0) that compared to normal feed (control). Blood and digestive tract were collected at 8 weeks after feeding. Immune activities were identified by blood composition (haematocrit, red blood cell count, white blood cell count) and humoral immune responses (lysozyme analysis). The specific activities of 4 digestive enzymes, i.e. amylase, lipase, trypsin and chymotrypsin were determined. Histology of foregut, midgut, and hindgut was investigated and villus height was measured. The growth performances, i.e. weight gain (WG), feed efficiency (FE) and specific growth rate (SGR), were measured. It results revealed that all immune responses, the digestive enzyme activities and villus height in GEP 0.5 and GEP 1.0 groups were significant higher than control group. However, these values were not significant between two garlic extract supplementation groups. Interestingly, group with higher blood parameters, higher digestive enzyme activities and longer intestine villi related to higher growth parameters and trypsin/chymotrypsin (T/C) ratio. It was obviously that GEP showed trend to enhance growth performance in Nile tilapia. Therefore, GEP might be benefited as a special feed additive in other fish culture instead of antibiotics that will be high advantage for aquaculture safety.

02014 Abstract Proceedings

Effective gene delivery of chitosan nanoparticles in giant freshwater prawn (Macrobrachium rosenbergii)

Pichai Phongudom1, Suwalee Ma-anan1, Chuphong Phakpum1, Chalermchail Pilapong2 and Jiraporn Rojtinnakorn1, 1Maejo University, Thailand and 2Chiang Mai University, Thailand

Chitosan nanoparticles (CS-NPs) are solid colloidal particles, which have special properties such as biocompatibility, biodegradability and mucosal delivery activity. In human, CS-NPs has been applied as carrier, adhesive and active molecules to target organs, and enhance performance of drugs. There were few reports of CS-NPs in fish and shrimp. Moreover, there is no any research involving toxicity of CS-NPs in aquatic animals. In this study, chitosan nanoparticles were synthesized by ionotropic gelation technique using commercial chitosan and tripolyphosphate (TPP) as crosslinker. Physical and chemical characteristics of nanoparticles were identified by SEM, nanosizing and zeta potential. It indicated that CS-NPs had size of 274±73 nm with zeta potential range of -11.16±0.467 mV. For in vivo toxicity test, giant freshwater prawn were fed with CS-NPs 0.1, 0.5, 1 and 5 %(w/w) added feed. Histopathology of interested tissues, i.e. heart, gill, stomach, intestine, liver and pancreas were followed at 2 and 4 weeks of feeding. It showed that heart, gill, stomach and, intestine of groups showed normal cells and distinctly structure. Liver and pancreas tissues of all high dose groups showed abnormal cells In liver tissue, hepatopancreatic tubularis separating from reserve inclusion cells were observed. Whereas in low dose group, all tissue was normal. For in vivo gene expression test, pGFP encapsulated in chitosan nanoparticles (CS-NPs- pGFP) was used. Giant freshwater prawn was fed with CS-NPs- pGFP 0.1 % (w/w) (1 μg per prawn) for one day. The expression was analyzed under the fluorescent microscope at 1, 3, 5 and 7 days after feeding, It showed green fluorescence in all organs such as gill, stomach, intestine, heart, brain and pancreas beginning at 1 day after feeding and increasing till 7 days. We concluded that our chitosan nanoparticles well carried gene for tissue expression with low toxicity in shrimp. Thus it can be continued available as gene delivery system by feed additive for shrimp and other aquatic animals.

02015 Abstract Proceedings

Fish pass using baffle effects of water flow without plates

Masaaki Ishikawa and Jyunji Shirai, National Institute of Technology, Kisarazu College, Japan

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

03001 Abstract Proceedings

Abundance and diversity macrozoobenthos in Karang Ranjang beach, Ujung Kulon National Park, Banten, West Java

Febri Ramadhan, Fauzia yudhanti, Nisrina R. Aisy, Dini Rosyana, Muhammad Rizky Halawi, Anastasia Kenes and Bagus Chandra, Institut Pertanian Bogor, Indonesia

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.

03002 Abstract Proceedings

The effect of underwater ship noise on humpback whales in the waters of Ogasawara (Bonin) Islands, Japan

Kyoichi Mori1, Ryosuke Okamoto2, Koki Tsujii2,5, Tomonari Akamatsu3, Naoya Umeda4, Yoko Mitani5, Rina Yamada1 and Takahiro Kijima6, 1Teikyo University of Science, Japan, 2 Ogasawara Whale Watching Association, Japan, 3 Fisheries Research Agency, Japan, 4 Osaka University, Japan, 5 Hokkaido University, Japan and 6Ministry of Land, Infrastructure, Transport and Tourism, Japan

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.

03003 Abstract Proceedings

Impact of temporal change of salinity to biodefense mechanism of Haliotis gigantea

Novi Arisman, Nadia Istiqomah and Takao Yoshimatsu, Mie University, Japan

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.

03004 Abstract Proceedings

Vitamin B12 effect to reduce turbidity stress on Moina macrocopa

Nadia Istiqomah, Novi Arisman and Takao Yoshimatsu, Mie University, Japan

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.

04001 Abstract Proceedings

Feel good in hypoxia? – From microbes to whales, diverse life forms subsist on the “dead zone” in an enclosed bay (Omura Bay, Nagasaki) –

Minoru Wada1, Ryuji Kondo2, Yu Umezawa1, Motohiro Shimanaga3, Dongsung Kim4, Toshikazu Suzuki1, Yuuki Kawabata1, Yoshiki Matsushita1, Atsushi Ishimatsu1, Takashi Aoshima1, Hiroyuki Takasu1, Takuro Nakamura5 and Masao Amano1, 1Nagasaki University, Japan, 2 Fukui Prefectural University, Japan, 3 Kumamoto University, Japan, 4 Korea Institute of Ocean Science and Technology, Korea and 5UMIDAYORI Diving Service, Japan

A “dead zone” represents an uninhabitable aquatic area where dissolved oxygen (DO) availability is below the limit of tolerance in aerobic organisms. As spreading of this life-threating zone has become evident in many coastal areas around the globe, it is urgently needed to assess responses of aquatic biota to hypoxia in order to help predict the ecosystem consequences. Omura Bay in Nagasaki is a typical enclosed bay that develops strong seasonal hypoxia (dead zone) every summer, yet the bay still holds rich marine resources and active fisheries. Aiming at (1) clarifying microbiological mechanisms by which dead zones are formed, and (2) obtaining quantitative understanding on how diverse types of inhabitants respond and adapt to the low oxygen condition, we began analyses of community structure and functions of micro-organisms, and monitoring distributional and behavioral patterns of macro-organisms in relation to hypoxia at the center of the bay. Major findings include (1) a uni-modal relationship between DO and sediment bacterial richness occurred with a peak at suboxic condition, which may reflect a transition where both strict anaerobic and aerobic bacteria can co-exist; (2) hypoxic condition favored a group of meiofauna (nematode) that mostly fed on bacteria, suggesting greater importance of organic matter transfer from bacteria through nematode in hypoxia than in normoxia; (3) a school of small pelagic fishes kept migrating into near-bottom water soon after DO declined to less than 4 mg/L; and (4) finless porpoises in Omura Bay also seemed to come often in and/or around the hypoxic water mass in the center of the bay. These results suggest that the hypoxic water mass in Omura Bay is not simply a “dead zone”, but it serves as an ephemeral “dining zone” for diverse life forms.

04002 Abstract Proceedings

Wastewater of gourami (Osphronemus goramy) cultivation treatment by romaine lettuce (Lactuca sativa L. var. longifolia)

Hefni Effendi, Yusrianti Purwandari and Yusli Wardiatno, Bogor Agricultural University, Indonesia

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

04003 Abstract Proceedings

On the perfection of public interest litigation system of China's offshore oil pollution

Zhen Lin, Yi Tang and Huajie Liu, Shanghai Ocean University, China

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

04004 Abstract Proceedings

Protein hydrolysis activity in fish-farming area of Tanabe Bay, Wakayama

Hiroki Iguchi, Keiya Yamazaki, Keitaro Kato, Gentoku Nakase, Toshinori Suzuki, Akito Taniguchi and Mitsuru Eguchi, Kindai University, Japan

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

04005 Abstract Proceedings

Environmental DNA metabarcoding with MiFish primer reveals marine fish fauna of Tsushima Island, Nagasaki for establishing a marine protected area

Mitsuhiro Aizu1, Satoquo Seino1, Tetsuya Sado2 and Masaki Miya2, 1Kyushu University, Japan and 2Natural History Museum and Institute, Chiba, Japan

A local government has promoted the establishment of a marine protected area (MPA) for sustainable management of the marine ecosystem along the coast of Tsushima Island, Nagasaki Prefecture, Japan. Current and comprehensive information on the marine ecosystem should be utilized to establish an MPA; however, conventional methods of inventory surveying involve long time periods, incur significant costs, and require taxonomic expertise. In addition, conventional methods generally cannot cover an entire ecosystem for various reasons; for example, small fishes may remain concealed in shore reefs. This is where environmental DNA (eDNA) metabarcoding can be helpful, and the technique has been applied to reveal aquatic communities. In particular, MiFish primer is as a good tool to identify local fish fauna. For this study, water samples were collected along coastal area of Tsushima Island in October 2016 to assess the current marine fish fauna. The study area was separated into six water sampling areas to elucidate differences in fish fauna. Total DNA was extracted from each water sample and metabarcoding was conducted using MiFish primer in MiSeq sequencing. As a result, over 150 species or similar species were identified. Some species were found in the study area that previous studies using conventional methods had not found. Clustering analysis using hierarchical methods revealed different fish fauna in northern and southern coastal areas of Tsushima Island, suggesting that each area may have different environmental factors, such as bottom conditions or the existence/absence of seaweed. This study suggests that eDNA metabarcoding with MiFish primer offers sufficient resolution for inventory surveys and could potentially provide valuable information to more accurately understand the marine ecosystem near Tsushima Island. Continuous surveying is recommended in order to provide crucial information for establishment of an effective MPA.

04006 Abstract Proceedings

Distribution and behavior of harmful algae and trace metals in Harima-Nada, Japan

Nodoka Kai1, Kanako Naito1, Saeko Mito2, Kazutaka Miyahara3 and Setsuko Sakamoto4, 1Prefectural University of Hiroshima, Japan, 2 Research Institute of Innovative Technology for the Earth, Japan, 3 Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries, Japan and 4National Research Institute of Fisheries and Environment of Inland Sea, Japan

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

04007 Abstract Proceedings

The transition of chemical substances from marine sediments in Onagawa Bay after Great East Japan Earthquake

Toshiyasu Yamaguchi, Takuma Nanba, Azuki Seki, Akiko Yamauchi, Toshiki Nakano, Kenji Kaneko, Minoru Sato and Yoshihiro Ochiai, Tohoku University, Japan

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

05001 Abstract Proceedings

Fertilizer application to enhance the growth of raft-cultured oysters

Tamiji Yamamoto1, Shigma Ishida1, Shinya Nakahara2, Kiyonori Hiraoka2, Yuhei Omichi2 and Hidemi Mutsuda1, 1Hiroshima University, Japan and 2Hiroshima Environment and Health Association, Japan

Oligotrophication is one of the serious problems in the Seto Inland Sea, Japan, due to rapid reduction measures of nutrient from the land. The effects are reported not only on discoloration of cultured Nori but also cultured bivalves. Although the oyster farming in Hiroshima Bay is a top production in Japan, it has been decreasing to 2/3 as low as that of the peak production in 1980s. Water transparency is usually 2 times high in the oyster culture raft compared to those surrounding area, which indicates high filtering activities by oysters, and implies that feed phytoplankton abundance may not be sufficient for oysters in the rafts. We applied fertilizer containing a proper elemental ratio to enhance oyster growth through enhancing phytoplankton growth. Individual oyster weight at which 600 g packed fertilizer was added per 20 individuals was 20% higher than those of control site without fertilizer. Three-dimensional numerical simulation in diffusion of eluted nutrients showed almost no influence to the surrounding area. We also propose an appropriate amount of fertilizer to be applied to enhance the oyster growth in harmonizing with environmental conditions at the presentation.

05002 Abstract Proceedings

Creation of fish habitats with thinning timbers in Mitsu Bay - a case study of ICZM

Jamaluddin Fitrah Alam1, Tamiji Yamamoto1, Haruka Nakamura1, Tetsuya Umino1, Shinya Nakahara2, Kiyonori Hiraoka2 and Hideyuki Ohtoshi3, 1Hiroshima University, Japan, 2 Hiroshima Environment and Health Association, Japan and 3Higashi-Hiroshima City, Japan

In Japan, the forest rate is quite high (ca. 70%) in developed countries and the fisheries production has been breaking down proved by low self-sufficiency rate (ca. 60%). Artificial reef is a gear to enhance fishery production by providing hiding space for juvenile fish and feed animals for fish. Three types of artificial timber reefs (ATRs): simple ATRs, ATRs with oyster shells (ATRsOS) and ATRs with leaves and branches (ATRsLB) were deployed at 2 sites (Kazahaya and Kidani) in Mitsu Bay, Hiroshima, Japan, and their performance was evaluated by field observations from 2015 to 2017. Attached organisms, benthos, and fish at the sites were compared with those at control sites with no ATRs and existing artificial concrete reef (ACRs) sites. The results in Kazahaya and Kidani showed a successional processes of producers followed by higher trophic level animals. Attached organisms were abundant at ATRsLB compared to ATRs and ATRsOS. The mean abundance and wet weight of benthos and fish at all the ATRs were higher compared to those at the control site but still below those at ACRs at the last observation. Among 18 fish species observed, 5 dominant species and some other fish appeared are those economically valuable species. It is concluded that ATRs stuffed with leaves and branches are effective to enhance fishery production by which may perform as shelters and provide feed. Using thinning timbers along with leaves and branches for artificial fish reefs is a good practice to link forests and seas in terms of integrated coastal zone management in Japan where high forest rate and low fishery production.

05003 Abstract Proceedings

Quorum sensing suggesting activities of algicidal bacteria that kill red tide raphidophyte Heterosigma akashiwo and Chattonella antiqua

Isamu Kodama1, Nobuharu Inaba2 and Ichiro Imai1, 1Hokkaido University, Japan and 2Japan Fisheries Research and Education Agency, Japan

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.

05004 Abstract Proceedings

Diatom resting stage cells and benthic diatoms in the sediment of the Japanese coastal waters

Nami Ako1, Kazuyosi Miyamura2, Hideki Iwano2, Natsuko Nakayama3, Takeshi Masuda4 and Ichiro Imai1, 1Hokkaido University, Japan, 2 Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Japan, 3 Japan Fisheries Research and Education Agency, Japan and 4Mie Prefecture Fisheries Research Institute, Japan

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.

05005 Abstract Proceedings

Evaluation of the environmental condition of Submarine Groundwater Discharge (SGD) by using nitrate stable isotope ratio recorded in sea lettuce(Ulva sp.)

Takeuchi Masaru1, Ryo Sugimoto1, Katsuhiro Kitagawa1, Shiho Kobayashi2, Kengo Ikuta2, Jun Shoji3, Makoto Yamada4, Hisami Honda4, Makoto Taniguchi4 and Osamu Tominaga1, 1Fukui Prefectural University, Japan, 2 Kyoto University, Japan, 3 Hiroshima University, Japan and 4Research Institute for Humanity and Nature Center, Japan

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 δ15NNO3- 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 δ15NNO3- 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 δ15NNO3- 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 δ15NNO3- 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..

05006 Abstract Proceedings

Contribution of the Submarine Groundwater Discharge (SGD) to sessile bivalve production; estimate by the carbon stable isotope ratio recorded in the shell

Osamu Tominaga1, Ryo Sugimoto1, Masaru Takeuchi1, Makoto Yamada2, Hisami Honda2, Shiho Kobayashi3, Jun Shoji4 and Makoto Taniguchi2, 1Fukui Prefectural University, Japan, 2 Research Institute for Humanity and Nature, Japan, 3 Kyoto University, Japan and 4Hiroshima University, Japan

Submarine Groundwater Discharge (SGD) has been shown to be an important pathway between land and sea for the transport of terrestrial materials. SGD results in various biogeochemical changes in the coastal environments, such as coastal primary production, eutrophication, and benthic production. Therefore, it is important to investigate the ecological significance of SGD. In the present study we investigated two subjects 1) whether the δ13 C of ambient water reflect the proportion of fresh water flow rate in SGD, 2) whether the δ13 C recorded in the shell of the sessile bivalve reflect that of the ambient water. The SGD rate and the proportion of fresh water flow rate in SGD were measured by the seepage meter at Kamaiso beach along the Mt. Chokai volcanic coast in June and September 2016. The rock-oyster was also sampled at Kamaiso Beach and adjacent 3 areas (Mega, Takinoura, Torisaki). There was a positive significant relationship between salinity and the δ13 C of ambient water (p<0.01). The δ13 C of the shell was high at high SGD flow rate sites and the positive significant correlation was found between the δ13 C of the shell and that of ambient water. In order to estimate the proportion of fresh water flow rate in SGD from the δ13 C of the rock-oyster shell, we used the two end member mixing model including DIC concentration of both sea water and fresh underground water. The estimated average fresh water flow rate in SGD ranged from 4 % (Torisaki) to 35% (Mega). These values are higher than those estimated from the δ13 C of ambient water (1.3% Torisaki and 18.1% Mega). The difference of these results are seems to be the difference of local SGD environment around rock-oyster.

05007 Abstract Proceedings

In situ bottle experiments demonstrating the feasibility of diatom resting stage cells in sediments to prevent red tide occurrences of noxious flagellates

Ichiro Imai1, Koya Morita1, Yumi Imai1, Kazuyoshi Miyamura2, Makoto Noda2 and Ken-Ichiro Ishii3, 1Hokkaido University, Japan, 2 Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Japan and 3Kyoto University, Japan

It is empirically known that HABs by noxious flagellates such as Chattonella and Karenia have occurred when diatoms are scarce in water columns. Diatoms produce resting stage cells under nutrient-depleted conditions, and rapidly sink to sea bottom and disappear from the water columns. Giving enough light to the abundant diatom resting stage cells is considered to enhance their germination and resultant vegetative cells are expected to rapidly proliferate in the surface water and to overwhelm harmful flagellate populations by the exhaustion of inorganic nutrients (N and P). Simulating these situations, we made bottle experiments containing small amounts of bottom sediments and noxious flagellates of C. antiqua and K. mikimotoi in Saiki Bay of Oita Prefecture, Japan in 2014. Sediments containing diatom resting stage cells stored in a refrigerator were added into 2L volume transparent pet bottle at concentrations of 0.1g L-1 and 0.01g L-1. K. mikimotoi was inoculated at a concentration of 20 cells mL-1 and C. antiqua 50 cells mL-1. The 1/100 strength of SWM3 medium was added as nutrient source for phytoplankton in the bottles. These bottles were hanged at surface and the depth of 5m, and samplings were made for cell enumeration of flagellates and diatoms, and for nutrient measurements. Diatoms grew well in all the bottles examined, though faster growths were observed in the bottles at 0m. C. antiqua was easily overwhelmed by proliferated diatoms after the incubation period. K. mikimotoi slightly increased to 70 cells mL-1 at most, and then decreased. It is suggested that sediment-lift is a possible strategy to prevent harmful flagellate red tides. The most abundantly appeared diatoms were belonging to the genus Skeletonema.

06001 Abstract Proceedings

Manipulating the Hemigrapsus life-cycle: On-demand prey for octopus mariculture?

Leo J.-H. Che and Ian G. Gleadall, Tohoku University, Japan

The common East Asian octopus, Octopus sinensis d’Orbigny, 1841 (‘madako’), is very attractive for development as a commercially farmed species because of its highly prized taste and rapid growth rate, but its planktonic paralarval phase and subsequent settling stage are currently barriers to closing its life cycle for successful mariculture. Hemigrapsus is a brachyuran crustacean genus including small crabs (‘isogani,’ ‘iwagani’) endemic to Japanese coasts. Attention recently has been drawn particularly to H. sanguineus (de Haan, 1835) and H. takanoi Asakura & Watanabe, 2005, because in the last few decades they have become highly successful invasive species in both Europe and North America. Their effects are devastating on both endemic edible crab species and on mussel beds: they apparently out-compete much larger species such as Cancer pagurus; and they feed actively on coastal molluscs such as Mytilus spp. It is judged that Hemigrapsus species are a potential source of crab larvae and juvenile crabs to supply as feed to paralarvae and recently settled juveniles of O. sinensis. Reliable production of crab larvae on a year-round basis has appeal as a prey source superior to the use of organisms such as Artemia (on which octopus paralarvae will feed, but with which there seems to be major nutritional mis-match). Advantages of Hemigrapsus species as a crustacean feed source for octopuses include a lack of competition as target items for human seafood because of their small size, and their presumed hardiness in being able to out-compete larger species of crab. In addition, research on species in this genus may uncover ways to mitigate their invasive prowess. We here report on experiments (1) to raise them in captivity; (2) to extend their spawning season; and (3) to test their suitability as prey organisms for O. sinensis mariculture.

06001 Abstract Proceedings

Manipulating the Hemigrapsus life-cycle: On-demand prey for octopus mariculture?

Leo J.-H. Che and Ian G. Gleadall, Tohoku University, Japan

The common East Asian octopus, Octopus sinensis d’Orbigny, 1841 (‘madako’), is very attractive for development as a commercially farmed species because of its highly prized taste and rapid growth rate, but its planktonic paralarval phase and subsequent settling stage are currently barriers to closing its life cycle for successful mariculture. Hemigrapsus is a brachyuran crustacean genus including small crabs (‘isogani,’ ‘iwagani’) endemic to Japanese coasts. Attention recently has been drawn particularly to H. sanguineus (de Haan, 1835) and H. takanoi Asakura & Watanabe, 2005, because in the last few decades they have become highly successful invasive species in both Europe and North America. Their effects are devastating on both endemic edible crab species and on mussel beds: they apparently out-compete much larger species such as Cancer pagurus; and they feed actively on coastal molluscs such as Mytilus spp. It is judged that Hemigrapsus species are a potential source of crab larvae and juvenile crabs to supply as feed to paralarvae and recently settled juveniles of O. sinensis. Reliable production of crab larvae on a year-round basis has appeal as a prey source superior to the use of organisms such as Artemia (on which octopus paralarvae will feed, but with which there seems to be major nutritional mis-match). Advantages of Hemigrapsus species as a crustacean feed source for octopuses include a lack of competition as target items for human seafood because of their small size, and their presumed hardiness in being able to out-compete larger species of crab. In addition, research on species in this genus may uncover ways to mitigate their invasive prowess. We here report on experiments (1) to raise them in captivity; (2) to extend their spawning season; and (3) to test their suitability as prey organisms for O. sinensis mariculture.

06002 Abstract Proceedings

Net-chasing training improves the behavioral characteristics of fish and crustaceans

Kohji Takahashi1 and Reiji Masuda2, 1Nagasaki University (JSPS PD), Japan and 2Kyoto University, Japan

Hatchery-reared seedlings for stock enhancement often have inferior behavioral characteristics unsuited for natural environments. Such behavioral characteristics are formed by rearing in safe and comfortable tank environments, and thus the rearing environment of seedlings should be altered to improve them. Repeated net-chasing in a rearing tank would allow them to learn avoidance responses for quasi-predator attacks and when faced with threatening situations. We investigated the effect of a net-chasing treatment to improve behavioral characteristics of fish (Japanese flounder Paralichthys olivaceus and red sea bream Pagrus major) and a crustacean (Indian bait prawn Palaemon pacificus). For Japanese flounder, we tried to improve maladaptive off-bottom swimming during foraging of juveniles by net-chasing treatment (two weeks). When fed at the surface, the off-bottom swimming of net-chased fish was repressed, whereas control fish showed an increase of off-bottom swimming. For red sea bream, we investigated the change in their behavioral characteristics by a net-chasing treatment (three weeks), and then a predator (marbled rockfish Sebastiscus marmoratus) exposure test was conducted. Net-chased fish exhibited higher tolerance to stress induced by environmental change and better alertness to an unknown object than did control fish. Furthermore, net-chased fish also exhibited a better survival rate than control fish in the predator exposure test. For Indian bait prawn, we investigated the effect of net-chasing treatment for crustaceans. Net-chased (eight day treatment) prawns entered a shelter more quickly, and showed higher avoidance rate to a contact stimulus than control. These studies showed that net-chasing treatment will be useful for behavioral improvement of released fish and crustaceans during the rearing period. Compared to the previous training, e.g., learning specific predator information, the net-chasing treatment may have wide applicability for the training of seedlings before release to improve survival traits of the fish.

06003 Abstract Proceedings

Expressions of dual vitellogenin mRNAs in rainbow trout liver tissue exposed to estradiol-17β and/or insulin-like growth factor I in vitro

Haruna Amano, Masahiro Nomura, Ryo Ohgushi, Miho Yokoyama, Kazuya Kawashima and Shunsuke Moriyama, Kitasato University, Japan

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

06004 Abstract Proceedings

Distribution of freshwater shrimps in Myanmar estuary

Tomonori Usami1, Than Than Lwin2, Masashi Yokota1 and Yo Usami1, 1Tokyo University of Marine Science and Technology, Japan and 2Ministry of Agriculture, Livestock and Irrigation, Myanmar

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

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

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

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

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

06005 Abstract Proceedings

Fatty acid requirement of a jellyfish Aurelia aurita s.l.

Haruka Onishi, Takashi Mishima and Takao Yoshimatsu, Mie University, Japan

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

06006 Abstract Proceedings

Lysozyme from Placozoa, an early diverging metazoan, lyses both gram-positive and negative bacteria

Munetsugu Bam1 and Hiroaki Nakano2, 1University of Yamanashi, Japan and 2University of Tsukuba, Japan

Lysozyme is an enzyme that hydrolyzes the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan of bacterial cell walls. The enzyme is ubiquitously present in nature, being contained in secretions and body fluids of various organisms to prevent bacterial invasion. It has been used in food preservatives and pharmaceuticals as a natural antibacterial agent. However, hen egg-white lysozyme (HEWL), the most widely used lysozyme, shows little activity against Gram-negative bacteria, probably due to the dense outer membrane surrounding the cell membrane present in the bacteria. This is a large drawback as many food poisoning bacteria and pathogens are Gram-negative bacteria. Placozoans are flat amoeba-like marine invertebrate animals, about 1-2 mm, that can be found across wide areas in Japan. They lack neurons and muscle cells, but are not sessile or parasitic, making them the simplest extant free-living animals. Their phylogenetic position implies they are important for uncovering the origins of animals. We have previously observed placozoans feeding on Spirulina sp. in lab cultures. Since Spirulina are Gram-negative bacteria, we predicted that placozoan lysozyme can act against Gram-negative bacteria, and investigated the biochemical properties of the enzyme. We isolated the lysozyme gene from Placozoa and performed analyses on its optimum reaction temperature and pH, the influence of salt to its lytic activity, and thermal stability. Placozoan lysozyme showed lytic activity against both Gram-negative and Gram-positive bacteria. Moreover, it had higher lytic activity and greater thermal stability when compared to HEWL. These results indicate that placozoans lysozyme could be applied in various fields as a natural antibacterial agent, more efficient than the presently used HEWL, and acting against both Gram-negative and Gram-positive bacteria.

06007 Abstract Proceedings

Major harmful shell-boring species of polydorids from cultured Akoya oysters in Ago Bay, Japan

Keita Higuchi, Naoki Hashimoto and Kiyohito Nagai, K. MIKIMOTO & CO., LTD., Japan

Polydorids are one of the most common shell-boring worms in molluscan aquaculture. A report in 1964 identified the main harmful species affecting Akoya oysters (Pinctada fucata martensii) on cultured-pearl farms in Ago Bay, Mie Prefecture, Japan, as Polydora ciliata, and described a technique for its control. In 2014, damage to Akoya oysters in Ago Bay from polydorids was unusually severe. Effective polydorid control involves immersing oysters in saturated brine during the period when larval polydorids settle onto shells and metamorphose into juveniles. The effect of this treatment could be maximized if the peak settlement period for polydorid larvae were known. Therefore, we conducted a study to identify the major polydorid species in Ago Bay and their ecology. From May 2015 to September 2016, 147 living polydorids were collected from Akoya oysters. Morphologic data and 18S rRNA sequences revealed 1 genus, 4 species, and several unidentifiable species. Polydora haswelli was the most abundant species with 96 individuals (65.3%); P. ciliata was not found. It is unclear if this result reflects a change in local polydorid species, or the reported confusion with classification. We confirmed that P. haswelli was the major harmful species on cultured Akoya oysters in Ago Bay; it was reproducing, and using Akoya shell as a calcareous substrate. Also, results from rearing its pelagic larvae established the planktonic period at about one month. Akoya shell placed in a container with competent larvae supported metamorphosed juveniles four days later. A survey focusing on seasonal variations in P. haswelli larvae could lead to an effective program for polydorid control.

06008 Abstract Proceedings

Long periods with closed shells observed from winter to spring in Akoya oysters (Pinctada fucata martensii)

Sho Okano1, Jyoji Go1, Kiyohito Nagai1, Susumu Segawa2 and Tsuneo Honjo3, 1K. MIKIMOTO & CO., LTD., Japan, 2 Tokyo University of Agriculture, Japan and 3Kagawa University, Japan

The shell movements of Akoya oysters in Ago Bay, Mie Prefecture, Japan, are continuously monitored throughout the year with the “Shell-Lingual” marine environmental monitoring system. To date, Shell-Lingual has captured the abnormal frequency and specific pattern of closing and opening shells of Akoya oysters during the period of red tide and low dissolved oxygen in summer, and these warning-like movements and pattern are used as indicators to mitigate damage to cultured Akoya oysters. This series of measurements also shows that Akoya oysters frequently keep their shells closed for periods exceeding an hour every winter. We investigated the relationship between continuously closed shells and water temperature using data from December 2013 to April 2017. The closed-shell behavior in Akoya oysters appeared at water temperatures above about 10 °C, and many closed-shell periods of 4 to 8 hours were observed at temperatures from 10 to 13 °C. Furthermore, the length of time with closed shells tended to be shorter as water temperatures increased over 13 °C. Almost 4 years of observations reveal that the shortening of the closed-shell period with the increase in water temperature tended to be delayed in years when the winter period with water temperatures below 11 °C was long. Thus, because the shortening of the closed-shell period with temperature increase is regarded as an indicator of Akoya oysters becoming active, we infer that low-water temperatures in winter delay the return of oyster activity. This suggests that the shortening of closed-shell periods with water temperature increase is an indicator of recovery from damage to Akoya oysters from low water temperatures. We expect this data to be utilized for spring culture management in pearl farming.

06009 Abstract Proceedings

In vitro 11-ketotestosterone production by the ovary from Japanese eel, Anguilla japonica

Yuya Hasegawa1, Mark P. Lokman2, Shigeho Ijiri1 and Shinji Adachi1, 1Hokkaido University, Japan and 2University of Otago, New Zealand

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

06010 Abstract Proceedings

Some artificially applied stressors have a positive effect on fish fitness

Toshiki Nakano1, Haiyun Wu2, Takafumi Arimoto2, Hideaki Endo2, Toshiyasu Yamaguchi1 and Yoshihiro Ochiai1, 1Tohoku University, Japan and 2Tokyo University of Marine Science and Technology, Japan

Fish are exposed to various environmental stressors, such as pollutants and acute changes in temperature, and the chances of succumbing to infectious diseases may be increased as a result. Exposure of organisms to stressors may result in a series of biochemical and physiological changes in the body. At the organismal level, these changes are mediated by the neuroendocrine system. There is also a cellular stress response which is a characteristic feature of the induction of a family of special proteins following exposure to stressful situations. Although maintaining homeostasis should be a key process for coping with stress, changes in physiological set points would be an important aspect of the adaptive response, especially as a mechanism for coping with stressors. In the course of studies on the fish health in response to a stress, we found that stress caused by handling or chemicals regulates the expression of growth-related genes, such as growth hormone receptor (ghr) and insulin-like growth factor-1 (igf1) genes, in fish. In addition to these stress trials, we examined the redox state, such as levels of glutathione and lipid hydroperoxide, in response to heat shock or high doses of a dietary antibiotic (oxytetracycline, OTC). The results concerning the changing patterns of stress-related biomarkers indicate that both heat shock and dietary OTC induce oxidative stress, which might enhance oxidation in the body. Hence, antioxidative dietary supplements could suppress environmental stimuli-induced oxidative stress and improve the redox state in aquacultured fish. Furthermore, manipulation of positive stress (good stress or desirable stimuli, such as mild physiological or thermal treatment, osmotic stress, chemical stress, rearing density and light condition), might be useful to control fish fitness in terms of health, growth, the immune system, etc. The possibilities of using positive stress and avoiding negative stress (bad stress) for fish will also be discussed.

07001 Abstract Proceedings

The development of a new generation of functional commercial tilapia feeds to reduce the use of harmful antibiotics in intensive aquaculture systems

Wing-Keong Ng, Chik-Boon Koh and Chaiw-Yee Teoh, Universiti Sains Malaysia, Malaysia

The rapid expansion of the global tilapia production is due in large part to the intensification of culture systems made possible with the use of commercial pelleted feeds. Intensification of culture systems often leads to increased incidence of disease. Functional feeds are the new generation of aquafeeds with additional function, usually with health-promoting properties beyond their nutritional value. Our research into the use of organic acids as a functional feed additive in tilapia feeds as an alternative to the use of harmful antibiotics will be presented.

Four 20-meter diameter plastic circular floating cages (2 replicates per treatment) in a tilapia farm were used in the study. All male Nile tilapia fingerlings (~25 g) were stocked at a density of 60,000 fish per cage. The tilapia feeds, containing no added organic acids or 2% of a prototype organic acids blend were produced and purchased from Cargill Ltd., Malaysia. With the exception of the added organic acids, both feeds were similarly formulated. Two to four weeks before the final harvest, 85 large fish were randomly sampled using a dip net from each cage. All fish were dissected, tissues excised and fecal samples extracted from the terminal segment of the tilapia gut.

Results indicated that dietary organic acids did not significantly (P>0.05) affect growth and biological indices but a general trend of improved nutrient utilization efficiency was observed. Reduced antibiotic use was registered during the culture period when fish were fed organic acid-added feeds. Decreased total viable bacteria and increased lactic acid bacteria counts in the gut of tilapia fed organic acids-added feeds was observed. Tilapia fed organic acids-added feeds had significantly (P<0.05) enhanced serum lysozyme activity. Functional feed additives such as organic acids can be used as an immuno-stimulant to offer a more environmentally friendly strategy for intensive tilapia farming.

07002 Abstract Proceedings

Novel biosubstances, α-mangostin and gartanin, from mangosteen (Garcinia mangostana L.) candidate for anti-saprolegniasis agent

Chanon Jaichuen1, Jiraporn Rojtinnakorn1 and Kashio Hatai2, 1Maejo University, Thailand and 2Nippon Veterinary and Life Science University, Japan

The two biosubstances of α-mangostin (a natural xanthonoid) and gartanin (a naturally-occurring derivative of xanthone) from mangosteen (Garcinia mangostana L.) have been reported as the novel bioactive compounds in human as food supplement, herbal cosmetics and pharmaceuticals. In this study, we were extracted from mangosteen pericarp and purified by HPLC fractionation. We investigated for their in vitro antifungal activities against saprolegniasis fungal species; i.e. Aphanomyces invadans, Achlya bisexualis and Saprolegnia diclina. Fungistatic effect was determined by agar disc diffusion and microscopic techniques. Fungicidal effect of hyphae was determined by immersion and observing for the presence of fungal growth. It resulted that α-mangostin and gartanin exhibited fungistatic effects on mycelium growth of A. invadans at concentrations of 125 and 250 ppm respectively, and S. diclina at concentrations of 125 ppm. Whereas A. bisexualis was inhibited at concentrations of 250 ppm. It also showed that inhibition efficacy gradually increased upon higher concentration. The abnormal hypha growth, with short branch and dense, was observed at the mycelium edge of inhibition zone. Fungicidal effect of zoospore was also investigated at concentrations of 125, 250, 500, 1000 ppm. Zoospore growth was observed by agar plate culture and SEM technique. Therefore, α-mangostin and gartanin are to be candidate a novel effective antifungal bio-substance that instead of antibiotics in Saprolegniasis fungi treatment. The pharmaceutical form of α-mangostin and gartanin for aquatic animals will be developed. Our result will be been high impact for safety and organic aquaculture.

07003 Abstract Proceedings

Effects of biofloc in snakeskin gourami (Trichogaster pectoralis Regan) culture system on growth performance and fillet quality

Orapint Jintasataporn1 and Srinoy Srinoy Chumkam2, 1Kasetsart University, Thailand and 2Valaya Alongkorn Rajabhat University, Thailand

The study on effects of biofloc in snakeskin gourami (trichogaster pectoralis regan) culture system on growth performance and fillet quality was conducted by focusing in intensive closed culture system with and without biofloc. The study was assigned in CRD with four treatments and four replicates. Two protein level diets composed by high digestibility materials were applied to snakeskin gourami in intensive system with and without biofloc. The treatments were T1:High nutrient density diet of 38%CP without biofloc, T2: High nutrient density diet of 38%CP with biofloc, T3:Normal nutrient density diet of 28%CP without biofloc, T4: Normal nutrient density diet of 28%CP with biofloc. Fish were culture in 1000Ltank at the density of 30ind./m2 and fed the treatment diet for 12 weeks. The results showed that snakeskin gourami fed 38% crude protein diet with biofloc exhibited the highest growth performance(p<0.05) and low feed conversion ratio (FCR; p<0.05). The high protein diet (38%) demonstrated the high serum protein and hematocrit (p<0.05). The biofloc in culture system both high and normal protein diet groups improved fillet yield (p<0.05) and fillet water holding capacity by reducing drip loss at 72 hr. (p<0.05). Otherwise, the biofloc can increase (p<0.05) the whiteness(L*) of snakeskin gourami skin in fresh fish and dry salted fish. The proximate composition of whole body snakeskin gourami showed the low moisture, high protein and ash (p<0.05) in group of fish fed high protein diet (38%). The high lipid oxidation in dry salted fish after chill at 4oC for 72 hr (p<0.05) exhibited in snakeskin gourami fed high protein diet with biofloc. Therefore, feeding snakeskin gourami with high nutrient diet incorporation with biofloc in intensive culture system can promote growth performance, feed utilisation and fillet quality.

07004 Abstract Proceedings

Effect of high concentration of dissolved oxygen in the rearing water on the rearing performance and stress response in red sea bream and Japanese eel

Daisuke Katayama1, Kiyoshi Nakajima2, Takehiro Ichinose2, Tomokazu Fujita2 and Tomonari Kotani1, 1Kagoshima University, Japan and 2Sanso Electric Co. Ltd., Japan

A lower level of dissolved oxygen in the rearing water affected on the rearing performance of aquatic organisms. Therefore, the saturation level of dissolved oxygen (DO) is usually kept as high up to 100% as possible. On the other hand, it is unknown what effect the high DO concentration gives for reared finfish. This study aimed to investigate the effect of high DO concentration in the rearing water on the rearing performance and stress response in red sea bream and Japanese eel.
In order to increase the DO concentration over saturation, the equipment for production of high DO concentration water was used. The freshwater with 300% DO saturation and the seawater with 380 % DO saturation were produced by that equipment. The DO saturation level of rearing water was controlled at >200% (HW), 130-140% (MW) and 100% (NW). The growth of body weight of experimental fishes was approximately 100g. The experimental rearing of Japanese eel was also conducted with the same protocol as red sea bream. In both rearings, the growth of body weight was measured. In the last day of rearing, the blood was sampled from experimental fishes and the cortisol concentration in blood was analyzed.
In the rearing of red sea bream, the growth of body weight was highest in MW and lowest in HW (P<0.05). On the other hand, the cortisol concentration in blood was the highest in MW. In the rearing of Japanese eel, the growth of body weight in MW and HW was superior to NW and the cortisol concentration was higher in MW and HW than in NW. As a result, high DO level beyond saturation gave some stress to fishes and that stress adoption stimulated the feeding behavior.

07005 Abstract Proceedings

Response to mass selection for correlation on growth traits in climbing perch, Anabas testudineus (Bloch,1792)

Jomsuda Duangwongsa1, Sanga Leesanga2, Suchat Jul-a-dung2, Metta Tipbunpot2 and Pradchayaporn Akaboot3, 1Maejo University, Thailand, 2 Department of Fisheries, Thailand and 3Prince of Songkla University, Thailand

The study of mass selection responses was conducted to compare the correlation between growth characteristics and their relation in order to obtain small head and large body in Climbing Perch, Anabas testudineus. Two populations from Thailand: pre-selected population from the best six sources growing groups (25 males and 25 females) and fourth generations post-selected populations(100 males and 100 females) were used. The correlations among mixed sexes, female and male of eight growth traits including weight(W), total length(TL), head length(HL), head width(HW), head depth(HD),body length(BL), body width(BW) and body depth(BD) showed 0.35 to 0.99 for pre-selected and 0.30 to 0.99 for post-selected, respectively. The correlation of HL and BL showed 0.62, 0.89 and 0.45 for pre-selected and 0.55,0.46 and 0.62 for post-selected, respectively. The correlation of BW and BL showed 0.77,0.98 and 0.78 for pre-selected and 0.50, 0.32 and 0.77 for post-selected, respectively. The correlation of BD and BL showed 0.57,0.72 and 0.56 for pre-selected and 0.49, 0.36 and 0.57for post-selected, respectively. The results suggests that mass selection has effects on the use of correlation between growth and the proportion of the shape desired Climbing Perch.

07006 Abstract Proceedings

Effects of different levels of vitamin C and prolonged nursing on growth and innate immunity of Nile tilapia

Debasish Saha and Takao Yoshimatsu, Mie University, Japan

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

07007 Abstract Proceedings

Verification of the flow field in rearing rectangular tank using flow visualization

Tetsuya Sumida1, Wataru Yamazaki2 and Shigeaki Shiotani3, 1National Institute of Technology, Oshima College, Japan, 2 Nagaoka University of Technology, Japan and 3Osaka University of Economics and Law, Japan

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.

07008 Abstract Proceedings

Land-based aquaculture of salmon trout using spring water from undersea tunnel in north Japan

Kosuke Suzuki1, Satoru Fukuda2, Hikari Izumi2, Yutaka Maeda3, Koichi Okamura1 and Naotsune Saga2, 1Okamura Foods Co., Ltd., Japan, 2 Hirosaki University, Japan and 3Aomori Prefectural Industrial Technology Research Center, Japan

[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).

07009 Abstract Proceedings

Difference in fatty acid composition of Rhodomonas sp. at each growth phase

Yudai Aritaki, Satoshi Yamamoto, Takashi Mishima and Takao Yoshimatsu, Mie University, Japan

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.

07010 Abstract Proceedings

Silkrose is effective for vibriosis of Japanese tiger prawn (Marsupenaeus japonicas): evidence from challenge study

Indri Afriani Yasin1, Takashi Ohta1, Atsushi Hashizume1, Atsushi Ido1, Chiemi Miura1,2 and Takeshi Miura1, 1Ehime University, Japan and 2Hiroshima Institute of Technology, Japan

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

07011 Abstract Proceedings

Enrichment of probiotic Lactococcus lactis strain K-C2 in Artemia

Yuki Hayami, Nguyen Thi Hue Linh and Yousuke Taoka, University of Miyazaki, Japan

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

07012 Abstract Proceedings

Rearing trial of eel, Anguilla japonica, in a closed recirculating system using hot spring water welled up in Saito City, Miyazaki, Japan

Seika Okane, Kazuho Umetsu, Mizuki Horoiwa, Yukie Kamata, Yuki Hayami, Setsuko Nagai, Noriko Nagasaka, Kenya Horii, Nguyen Thi Hue Lihn, Ryusuke Tanaka, Katsuhisa Uchida and Yousuke Taoka, University of Miyazaki, Japan

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

08001 Abstract Proceedings

Determination of immunoglobulin novel antigen receptor (IgNAR) in vivo affinity maturation in brownbanded bamboo shark (Chiloscyllium punctatum)

D.P.N. De Silva1, 2, S. Hosoya1, N. Mizuno1, S. Kinoshita1, M. Totsuka1 and S. Asakawa1, 1The University of Tokyo, Japan and 2Uva Wellassa University, Sri Lanka

Immunoglobulin novel antigen receptor (IgNAR) is a unique antibody found only in cartilaginous fish. It is a heavy chain homodimer with smallest variable region (around 12 kDa) identified to date. Affinity maturation of IgNAR was tested in few shark species but the results were not always promising. Therefore current study aimed to determine IgNAR in vivo affinity maturation against specific antigen in brownbanded bamboo shark (Chiloscyllium punctatum). Mature bamboo sharks were immunized with hen egg lysozyme antigen for four times at monthly intervals. Collection of around 10 ml of blood from the caudal vein was performed under general anaesthesia prior to each immunization. Whole blood was centrifuged and plasma samples were separated for the analysis of IgNAR antibody using enzyme linked immunosorbant assay (ELISA). SDS-page and western blotting analysis was also performed to determine the plasma protein content. Anti-horn shark IgNAR (rabbit polyclonal) and anti-rabbit IgG antibodies were used as primary and secondary antibodies respectively. According to western blot analysis presence of IgNAR antibody in both unimmunized and immunized shark plasma was identified. ELISA results indicated an increase of antigen specific IgNAR titer upon antigen exposure by fourth immunization. Therefore ELISA revealed affinity matured IgNAR from brownbanded bamboo shark. In conclusion, C. punctatum develops antigen specific IgNAR upon immunization, which can be used for future immunotherapeutics.

08002 Abstract Proceedings

Changes in functional traits and diversity of bacterial communities associated with oyster aquaculture farms in Ofunato Bay

Md. Shaheed Reza1, Atsushi Kobiyama1, Jonaira Rashid1, Yuichiro Yamada1, Yuri Ikeda1, Daisuke Ikeda1, Nanami Mizusawa1, Kazuho Ikeo1, Shigeru Sato1, Takehiko Ogata1, Mitsuru Jimbo1, Toshiaki Kudo1, Shinnosuke Kaga2, Shiho Watanabe3, Kimiaki Naiki3, Yoshimasa Kaga3, Katsuhiko Mineta4, Vladimir Bajic4, Takashi Gojobori4 and Shugo Watabe1, 1Kitasato University, Japan, 2 Iwate Prefectural Government, Japan, 3 Iwate Fisheries Technology Center, Japan and 4King Abdullah University of Science and Technology, Saudi Arabia

Ofunato Bay in Iwate Prefecture, Japan is the home of important aquaculture activities. Chlorophyll a (chl-a) concentration has been reported to be high in the bay that attracted buoy-and-rope-type oyster aquaculture facilities for Pacific giant oyster (Crassostrea gigas). Since oysters filter suspended material and excrete organic matters into the seawater, the bacterial communities residing in its vicinity may show dynamic seasonal changes. Accordingly we are studying bacterial composition and their functional traits in the seawater near oyster aquaculture facilities from September 2014 employing shotgun metagenomic technique and correlating them with water quality parameters including temperature, salinity, pH, DO, NO2+3-N, NH4-N, and chl-a. We previously reported that during 2015, diverse communities of free-living bacteria in seawater near the oyster aquaculture facilities exhibited intense inter-seasonal differences. It was also observed that during the same period the distribution and abundance of several functional traits including “amino acid metabolism”, “energy metabolism”, “metabolism of cofactors” and “translation” revealed by KEGG Ontology (KO) analysis were significantly different (p<0.05) between surface (1m) and deep (10m) waters near oyster aquaculture facilities. Functional abundance was also significantly different when second tier KO categories between seawater near oyster aquaculture facilities were compared to those far from there in Ofunato Bay. We are currently analyzing such changes of bacterial communities in 2016, and interested in identifying their temporal variations in relation to physical and biological environmental factors for two consecutive years.

08003 Abstract Proceedings

Metagenomic analyses on the microbial communities in the Ofunato Bay: seasonal changes and site-specific differences

Shugo Watabe1, Md. Shaheed Reza1, Atsushi Kobiyama1, Kazuho Ikeo1, Jonaira Rashid1, Yuichiro Yamada1, Yuri Ikeda1, Daisuke Ikeda1, Nanami Mizusawa1, Shigeru Sato1, Takehiko Ogata1, Mitsuru Jimbo1, Toshiaki Kudo1, Shinnosuke Kaga2, Shiho Watanabe3, Kimiaki Naiki3, Yoshimasa Kaga3, Katsuhiko Mineta4, Vladimir Bajic4 and Takashi Gojobori4, 1Kitasato University, Japan, 2 Iwate Prefectural Government, Japan, 3 Iwate Fisheries Technology Center, Japan and 4King Abdullah University of Science and Technology (KAUST), Saudi Arabia

The Ofunato Bay is a type of an enclosed bay in the Sanriku Rias Coast, where seawater enviroments rich in nutrients are affected by both warm and cold ocean currents along with river, providing good aquaculture fields for marine bivalves. We started the metagenomic survey on microbial communities in the Ofunato Bay as a KAUST project since 2014 and here we summarize our obtained results. Sampling stations are KSt. 1 at the innermost, KSt. 2 at the center and KSt. 3 at the bay entrance. Seawaters were collected every month at 1 m and 8 or 10 m depths from the three stations. Water quality was monitored with a profiler for several parameters including temperature and chlorophyll a concentrations. Collected seawaters were filtered sequentially through 5, 0.8 and 0.22-μm filters, while DNA was extracted only from the 0.22 μm-filters mainly targeting free-living bacteria and subsequently applied to a MiSeq sequencer to obtain the whole genome sequencing reads. Most abundant bacteria belonged to Alphaproteobacteria which consist of Planktomarina, Canditatus Pelagibacter, and Bartonella, irrespective of the three stations and two seawater depths. Discrimination analysis with ROC-AUC, targeting the two depth, three stations and 12 months from January to December 2015 for seasonality (12 months to adjacent continuous 2 seasons) revealed that the period of July to August were significantly different from other months. The responsible genera responsible for this discrimination were unclassified Verrucomicrobia, unclassified Proteobacteria, and Coraliomargarita. This discrimination was further confirmed by the hierarchal cluster dendrogram. ROC-AUC for local combination clearly discriminated KSt. 2 from KSt. 1 plus KSt. 2 at the KEGG matrix, whereas the bacterial community in KSt. 3 at 1 m depth was different from others as revealed by PCA on the relative abundances of bacterial genera. Further analysis is under progress on the effects of environmental factors.

08004 Abstract Proceedings

Seasonal changes in the microbial communities of the Tama River at suburban Tokyo revealed by metagenomic analysis

Nanami Mizusawa, Md. Shaheed Reza, Chiharu Oikawa, Daisuke Ouchi, Atsushi Kobiyama, Yuichiro Yamada, Yuri Ikeda, Daisuke Ikeda, Kazuho Ikeo, Shigeru Sato, Toshiaki Kudo, Ko Yasumoto, Mitsuru Jimbo and Shugo Watabe, Kitasato University, Japan

Metagenomic studies have enabled us to identify taxonomic profiles of bacterioplankton and also elucidate ecological functions of the residing microbial communities. As the Tama River is regarded as a life line of urban Tokyo, we examined seasonal differences of the bacterial communities in a non-tidal, urban-impacted part of this river to better understand their ecological functions. Water samples (~2L) were collected every month from September 2015 to December 2016 with sterile beakers from the surface to 12.5 cm depth at a middle reach of the Tama River in Tama City, following the guidelines of the Bureau of Sewage, Tokyo Metropolitan Government (2012). Of the collected water, 500 ml were filtered sequentially through Isopore™ membranes with a 45 mm diameter each of 5, 0.8 and 0.2-µm pore sizes. As we targeted free-living bacterial fraction, DNA was extracted only from 0.2-µm filter membranes and we prepared shotgun metanogemic libraries using Nextera XT library preparation kit (Illumina). The libraries were then sequenced with an Illumina MiSeq using MiSeq Reagent Kit v3. Acquired Illumina reads were assigned to their taxonomic affiliations by BLASTN comparisons to the GenBank nonredundant nucleotide database. Betaproteobacteria and Flavobacteriia were the most abundant groups in the Tama River. The relative abundance of Flavobacteriia decreased from 51.4 % in May to 1.9 % in December, while that of Gammaproteobacteria increased gradually from 1.1 % in August to 12.1 % in November. A marked increase of Bacteroidia was observed in February. Negative correlations against water temperature were observed with the relative abundances of Gammmaproteobacteria, Bacilli, Mollicutes, Chlamydiia and Chlorobia. A positive correlation against dissolved oxygen was observed with Flavobacteriia, while a negative correlation observed with Gammaproteobacteria. These results suggest that seasonal changes in environmental variables contribute to the dynamic changes in the bacterial communities in the Tama River.

08005 Abstract Proceedings

Development of the detection methods of the new fluorescent protein genes that are introduced in GloFish

Ichiro Oohara1, Nobuhiko Ojima1, Miyuki Mekuchi1, Motoshige Yasuike1 and Masahiro Kobayashi2, 1Japan Fisheries Research and Education Agency, Japan and 2Fisheries Agency, Japan

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

08006 Abstract Proceedings

Rapid 16S rRNA metagenomic analysis on the microbial communities in salted and fermented squid

Daisuke Ouchi1, Nanami Mizusawa1, Yoshihiro Takahashi1, Ko Yasumoto1, Mitsuru Jimbo1, Atsuko Nagaoka2, Nobuhiko Ueki2, Yoko Matsuoka2, Jianrong Wan2 and Shugo Watabe1, 1Kitasato University, Japan and 2Suzuhiro Kamaboko Honten Co. Ltd., Japan

Salted and fermented squid is called “shiokara” in Japanese, which is one of the most famous traditional seafood products in Japan. While various factors affect the maturation of these products during fermentation process, the microbial community is believed to be associated with the production of taste-active compounds such as free amino acids and peptides. Thus it is considered that the microbial communities play important roles in the quality control of shiokara. In this study, as a first step to determine parameters for monitoring the quality of shiokara, microbial communities were investigated by the 16S rRNA metagenomic analysis. Two types of commercially available shiokara products, akadukuri and namakouji, were used as starting materials. Samples were collected periodically to analyze the changes in the microbial communities during the fermentation process. We extracted DNA using CTAB buffer from the liquid part of the products and purified by using AMpure XP beads (Beckman Coulter). To analyze the bacterial communities in shiokara, the V1-V3 region of the 16S rRNA gene was amplified from the purified DNA. The 16S rRNA amplicon products were sequenced with a Miseq next generation sequencer (Illumina). Acquired Illumina reads were joined by overlapping forward and reverse reads using the FLASH command and analyzed by SILVAngs pipelines. We found that the major bacteria in both types of the product belonged to Vibrio, while this bacterial community was not changed dramatically during the fermentation process. Furthermore, the major bacteria in the squid muscle used as the materials for shiokara production were also Vibrio. These results suggest that the microbial communities in the starting materials play the major roles during the shiokara fermentation.

08007 Abstract Proceedings

The total genome analysis of wakame (Undaria pinnarida) and related colocalization bacteria

Yoshihiro Takahashi1, Md. Shaheed Reza1, Nanami Mizusawa1, Ko Yasumoto1, Misturu Jimbo1, Nobuyoshi Nanba1, Hiroki Koyama2, Kazutoshi Yoshitake3, Takanori Kobayashi4, Yoichi Sato5 and Shugo Watabe1, 1Kitasato University, Japan, 2 Hiroshima University, Japan, 3 The University of Tokyo, Japan, 4 Japan Fisheries Research and Education Agency, Japan and 5Riken Food Co. Ltd., Japan

Wakame has been traditionally consumed in Japan and known to contribute to the improvement of skin and hair, while it helps reduce high blood pressure and cholesterol levels. Iwate Prefecture is the top producer of wakame in Japan as an important marine bioresource in this Prefecture. The Great East Japan Earthquake in March 11th, 2011, hit Iwate Prefecture and subsequent tsunami brought about enormous damages around the coastal area. Wakame culture was also damaged by this earthquake and it is important for people engaged in wakame production to reinforce the brand strength for recovery from such disaster. The present study was undertaken based on the above mentioned background.
Wakame gametophytes were prepared from alive specimens of matured wakame from Taro, Iwate Prefecture, and Hayama, Kanagawa Prefecture as a reference, by a conventional method. DNA extraction and purification were performed using ISOPLANT (Nippon Gene) with alginate lyase (Nippon Gene) and GM quicker 2 (Nippon Gene). Shotgun libraries were constructed using Nextera XT DNA preparation kit (Illumina). The 16S rRNA gene was amplified by PCR with specific primers to construct amplicon libraries for investigating bacteria colocalized in the wakame gametophytes.
The whole genome sequences were assembled using CLC Assembly Cell™ version 8.0 (QIAGEN). The total contig lengths for the genomes of Taro male, Taro female, Hayama male and Hayama female gametophytes were 0.36, 0.36, 0.5 and 0.25 Gbp, respectively, with N50 of 960, 872, 1,507 and 718 bp, respectively, indicating that the genome sequences were still fragmented. SILVAngs analyses on the 16S rRNA gene libraries revealed that the total sequences were 0. 4 M, 0.5 M, 0.6 M and 0.5 M, respectively, in the above gametophyte order, while OTUs were 19,901, 17,995, 25,958 and 16,404, respectively. The major bacteria were composed of Marinobacter and Sphingorhabdus.

09001 Abstract Proceedings

Desialylation of gangliosides regulates triglyceride accumulation in fish liver

Kazuki Oishi, Mina Miyazaki, Wataru Nishimura, Masaharu Komatsu and Kazuhiro Shiozaki, Kagoshima University, Japan

【Objective】 Regulation of muscle lipid content is an important concern in aquaculture fish in terms of fish meat quality. Triglyceride (TG) is synthesized in the liver and then transported to muscles. Recently, it is reported that gangliosides are involved in TG accumulation in mouse liver. However, roles of gangliosides in TG accumulation are still not clear in vertebrates. In this study, the influence of desialylation of ganglioside on lipid metabolism was examined with fish hepatocyte. Especially, we focused on sialidase Neu3 which is a ganglioside specific sialidase.
【Materials and Methods】 mRNA levels of neu3a and lipid accumulation related genes in the liver of fasting medaka were analyzed by real-time PCR. To examine Neu3a influences toward lipid metabolism, neu3a-stable transfectant cells were established with fish hepatic Hepa-T1 cell. After the cells were exposed to oleic acid, lipid droplet formations were observed by Oil Red O staining. Alteration of ganglioside composition in stable transfectants were assessed by thin layer chromatography (TLC).
【Results and Discussion】 Hepatic neu3a gene expression was up-regulated under deficient energy conditions, accompanied with pparg and lpl1 genes expression. By bioinformatics analysis, several binding sites of lipid metabolism-related transcription factors were predicted in the upstream of neu3a transcription start site. After oleic acid exposure, newly synthesized TG was detected as lipid droplet in the cell. Bigger size of lipid droplets were observed in neu3a-stable transfectant cells compared with vector. By TLC analysis, decrease of ganglioside GM3 and increase of lactosylceramide were detected in neu3a-stable transfectant cells. These results suggested that alteration of ganglioside composition by Neu3a would regulate TG accumulation in fish liver.

09002 Abstract Proceedings

Efficacy of fibrolytic enzymes on digestibility of some local raw materials for aquafeed

Sittichai Hatachote, Orapin Jintasataporn and Srinoy Chumkam, Kasetsart University, Thailand

The efficacy of fibrolytic enzymes on digestibility of some local raw materials for aquafeed were investigated by focusing on four local raw materials for aquafeed including soybean meal, rice bran, cassava pulp and cassava skin. Four fibrolytic enzymes including Xylanase at 24,000 U/Kg(T1) , Cellulase at 10 U/Kg(T2), Mannanase at 750 U/Kg(T3) and a combination of Cellulase and Mannanase(T4) at the same propotion (0.5: 0.5 of T2 and T3) were assigned for evaluated the efficacy on digesting these four materials. The experiment was conducted by using 4x4 factorial in completely randomize design. Aquafeed materials were incubate with phosphate buffer at the optimum pH for each ezyme from each treatment. Results of in vitro digestibility showed that cellulases exhibited the highest enzyme efficacy (P<0.05) to digest fiber from these four material follow by mananase, combination (T4) and xylanase, respectively. For the aquafeed materials in this study, the results showed that fiber from soybean meal in term of cellulose was easy to digest than rice bran, cassava skin and cassava pulp, respectively. Mannan in soybean meal was easy to digest than cassava skin and cassava pulp, respectively. The mannan in rice bran seem to be very low so it is difficult to evaluate the efficacy of enzyme. Xylan in soybean meal also was easy to digest than rice bran, cassava pulp and cassava skin, respectively. The sum of sugars (glucose, xylose, maltose and mannose) form soybean meal by T1, T2, T3,and T4 enzyme activity were 0.279±0.019, 0.213±0.015, 0.383±0.026 and 0.098±0.013 U/ml, respectively. Sugars from rice bran were 0.184±0.019, 0.141±0.015, 0.253±0.026 and 0.034±0.013 U/ml, respectively. Sugars from cassava pulp were 0.113±0.019, 0.087±0.015, 0.156±0.026 and 0.027±0.013 U/ml, respectively. Sugars from cassava skin were 0.155±0.019, 0.119±0.015, 0.213±0.026 and 0.040±0.013 U/ml, respectively.

09003 Abstract Proceedings

The effects of dietary combined use of soybean extract and plant oil mixture on growth and whole body and tissue essential and non-essential amino acid and fatty acid compositions in juvenile Nile Tilapia, Oreochromis niloticus L.

Kenan Engin and Almila Uysal, Mersin University, Turkey

Diets in which 50 % of the fish meal crude protein was replaced by soybean extract along with 60, 80 and 100 % of fish oil replacement by mixture of soy ,canola and linseed oils (1:1:1) were used in the study. Four isonitrogenous and isoenergetic diets were fed to juvenile Nile tilapia, Oreochromis niloticus (16,2 g AIWW) in triplicate (25 fish per tank) twice in equal portions at a fixed rate of 4 % BW.d-1 for 90 days. Fish doubled its initial weight and no statistical difference found among dietary treatments in terms of measured growth parameters. Diets supplemented with soybean extract and increasing amount of plant oil mixture proportionally increased the crude protein and significantly decreased (P<0,05) the crude ash levels in fish whole body samples compared to that of fish fed fish meal and oil control diet. Fish fed diets supplemented with soybean extract and increasing amount of plant oil mixture tended to increase the tissue essential and non-essential amino acid levels indicating that L-lysine and DL-methionine used in these diets were effectively used for growth in juvenile Nile tilapia. Whole body and tissue fatty acid compositions demonstrated that DHA was probably deposited whereas EPA was used for energy production in fish fed diets supplemented with soybean extract and increasing amount of plant oil mixture. Significantly higher (P<0,05) intermediate metabolites of the n-6 LC HUFA biosynthesis in whole body and tissue samples compared to that of fish fed the control diet also implicated that Nile tilapia might have activated the Δ5 and Δ6 elongation and desaturation enzymes. It was concluded that the combined replacement of dietary fishmeal and oil by soybean extract and plant oil mixture did not compromise growth rates, whole body and tissue essential and non-essential amino acid compositions, and n-3/n-6 ratios in this species.

09004 Abstract Proceedings

Growth performance of adult yellowtail Seriola quinqueradiata fed extruded low-fish meal diet in commercial aquaculture; potential for cost reduction

Noriko Ishida1, Hitoshi Matsuo2, Kazuki Matsukura3, Tomohiko Koshiisi4, Soetsu Yanagi5 and Shuichi Satoh6, 1Japan Fisheries Research and Education Agency, Japan, 2 Azuma-Cho Fishery Cooporative, Japan, 3 Nagasaki Prefectural Institute of Fisheries, Japan, 4 Marubeni Nisshin Feed Co., Ltd., Japan, 5 Kagoshima Prefectural Fisheries Technology and Development Center, Japan and 6Tokyo University of Marine Science and Technology, Japan

In Japanese fish aquaculture, about 70% of production costs are used on fish feed which contains fish meal ≥ 50% in diets. Recent increases in fish meal price emphasize the need for alternative low-fish meal diets to maintain aquacultural production. Another consideration is food palatability as fish readily ingest high-fish meal (≥ 50%) diets, but not low-fish meal (<35%) diets.
To examine performance of low-fish meal diets, 6 month feeding experiments were conducted at two commercial farms in Kagoshima prefecture each rearing 4000 adult fish in net cages (10m x 10m x 10m). The fish meal content in test diet was 30% and that of the control diet was 50%. Total calorie and protein contents in the experimental diet were set higher than the control diet.
In the first year, the experimental diet was designed using plant ingredients as a partial replacement and experimental fish growth was comparative to control fish. Growth performance and feed efficiency (Control diet; 2.99, Experimental diet; 2.83) were similar.
In the second year, chicken meal was mainly used as a replacement protein for comparison of cost reduction. When the weight approached 6 kg, fish growth rate markedly reduced. Furthermore, growth performance and feed efficiency of the experimental diet were slightly inferior to the control.
Cost reduction was further examined using four net cages (5m x 5m x 5m). One was for the control diet; other three were fed low-fish meal diets. One experimental (low-fish meal) diet cage was switched to the high-fish meal diet at November and in another cage switched at December and in the last cage the experimental diet was continued. Feed efficiencies were 2.51in control; 2.41 in November switched cage; 2.36 in December switched cage , and 2.60 in continued cage. Feeding efficiency cost based on the control as 100%, was 83.5% in November switched cage, 86.2% in December switched cage, and 92.8% in continued cage. Therefore, the switching method is considered effective for cost reduction.

09005 Abstract Proceedings

Molecular cloning of novel chitinase genes from two species of crustaceans, red king crab Paralithodes camtschaticus and snow crab Chionoecetes opilio

Kakeru Fujimori, Momo Kanai, Ryo Nishino, Hiromi Kakizaki, Hideto Fukushima and Masahiro Matsumiya, Nihon University, Japan

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.

09006 Abstract Proceedings

Characterization and primary structure of chitinase from the hepatopancreas of red snow crab Chionoecetes japonicus

Ryuichi Houya, Hiromi Kakizaki, Ryo Nishino, Hideto Fukushima and Masahiro Matsumiya, Nihon University, Japan

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.

09007 Abstract Proceedings

Characterization of tilapia sialidases and its significance in bacterial infection

Akinobu Honda1, Petros Kingstone Chigwechokha2, Rena Wakamatsu1, Yuko Kamada1, Masaharu Komatsu1 and Kazuhiro Shiozaki1, 1Kagoshima University, Japan and 2Mzuzu University, Malawi

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.

09008 Abstract Proceedings

Chitinolytic enzyme of Japanese sardine (Sardinops melanostictus): distribution of the organs and molecular cloning of a novel chitinase gene from the kidney

Miku Watanabe, Satoshi Kawashima, Hiromi Kakizaki, Hideto Fukushima and Masahiro Matsumiya, Nihon University, Japan

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.

09009 Abstract Proceedings

Condition optimization of trypsin and chymotrypsin activities in Lates calcarifer

Winyoo Boonprasert, Prachaub Chaibu, Chanagun Chitmanat, Jongkon Promya and Kriangsak Mengamphan, Maejo University, Thailand

The Asian sea bass is one of the most economically important food fish cultured in the tropical and subtropical regions in the Asia–Pacific. Because of its relatively high market value, it has become an attractive commodity of both large to small-scale aquaculture enterprises. The development of suitable feed is required for this fish to make sure if its ingredients are properly digested. In addition, the digestive enzymes play a role in the growth and survival of aquatic animals. Trypsin and chymotrypsin are the 2 proteases that play the essential role in protein digestion and involving in growth rate. In Lates calcarifer, these two enzymes are indicated as growth parameters by feed. Therefore, enzyme assay at optimal condition is significant for its accuracy activity determination. Digestive enzyme distribution and activity in the digestive tracts of the Lates calcarifer. Characterization of digestive enzymes from digestive tracts of Lates calcarifer showed that Trypsin had optimum conditions at pH 8.0 temperature 60 °C while Chymotrypsin had optimum conditions at pH 5.0 temperature 60 °C.

09010 Abstract Proceedings

Growth and digestive enzyme activity of Nile tilapia fed with diet containing fermented grass from cow stomach

Worawit Chukwannuan and Sudaporn Tongsiri, Maejo University, Thailand

Although Nile tilapia is one of the most economically important freshwater fish, the cost of feed is the main obstacle factor to be considered for their cultivation. The culturing process should be improved by low-cost feed development from farming by-products. The grass fermentation in cow stomach is the low-cost raw material which could be used for feed ingredient. In this experiment, grass fermentation in cow stomach was added in the feed at a ratio percentage of 0 (control), 5 (GF5), 10(GF10), and 15(GF15). All feeds consisted of 30 percent proteins. The characteristics of digestive enzymes in the Nile Tilapia fed with the experimental feeds were also investigated. The results of growth were found that the final weight, the average increased weights, and average daily weight gain of fish in treatment GF10 were highest and significantly different (P<0.05). The results of digestive enzyme, the specific proteinase activity, was found to be highest among the extract enzyme from the intestine in the fish that was fed with GF10 at a pH value of 10 and 12. In conclusion, the diet containing 10 percentage of grass fermentation in cow stomach was suitable for culturing Nile tilapia.

09011 Abstract Proceedings

Garlic (Allium sativum) extract product enhancing digestive physiology and growth performance in marble goby (Oxyleotris marmorata) juvenile

Jaturong Matidtor, Maejo University, Thailand

Marble goby is one of high price commercial freshwater fish. For its aquaculture, low growth rate and survival rate have being particular problem in nursery. This study investigated efficacy of garlic extract, which well known as high benefit for digestive system, as feed additive in marble goby juvenile. The fishes with size of 3.12±0.72 g were fed with natural food (blood worm, N), commercial diet (control feed, C), commercial diet mixed garlic aqueous extract (GAE) and ethanol extract (GEE) at concentration 0.5% (w/w) for 12 weeks. Growth parameters, digestive enzymes activities and histology were analyzed at 4, 8 and 12 weeks. For growth parameters, weight gain (WG), specific growth rate (SGR) and feed efficiency (FE), were calculated. For digestive enzyme activities, amylase, lipase, trypsin and chymotrypsin activities, which play major roles as sources of metabolic energy for growth, were determined. For histology, major digestive organs (esophagus, stomach, midgut and hindgut) were collected, prepared for H&E staining permanent slide and observed by compound microscope. It results that amylase activity showed significant different (p < 0.05) at 4 and 8 weeks from N and C groups. Whereas lipase activity of GAE showed significant different (p < 0.05) at 12 weeks. For histology analysis, GAE and GEE showed that villi heights were longer and tissue structure were more complete than N and C groups. Midgut villi, the important role for nutrient absorption, were patently increased in GAE and GEE. Interestingly, all growth parameters of GAE and GEE were significantly different (p ˂ 0.05). We concluded that garlic extracts could be effective for protein digestive enzymes, nutrition absorption, and growth performance in marble goby juvenile. Our research will be high useful to develop artificial diet formula for marble goby nursery that expect to helpful for increasing production.

10001 Abstract Proceedings

Simultaneous determination of freshness and histamine of fish flesh

Minoru Sato, Yorihiko Shiba, Michiko Sasaki, Shinya Ito, Kenichiro Kurashima, Toshiyasu Yamaguchi, Toshiki Nakano and Yoshihiro Ochiai, Tohoku University, Japan

Fish is an important nutritive component of the human diet as it provides protein, energy and many other functional components and thus helps sustain health. Concerning fish quality, fish deteriorate more quickly post-mortem than meat derived from terrestrial animals, quick and simple methods are required to confirm fish freshness and quality. Concerning fish safety, about more than 50% of food intoxication caused by fish is occurred by histamine. Histamine is well known as the substance causing allergy-like food poisoning after ingestion of Scombroid fish that are not chilled adequately between harvest and consumption. The authors propose that fish should be confirmed the freshness and histamine contents to take in a good and safe fish, and to produce safety processing food.
Simultaneous determination freshness and histamine have done as followed. First step, freshness of fish flesh is measured by Freshness Checker (QS-SOLUTION), which is based on the paper electrophoresis method. After taking freshness data of fish on the electrophoresis paper by UV detection, histamine is developed by spraying histamine developing reagents on the same paper. By these quick and simple procedure, it can be avoided sea food intoxication and fish can be ingested and used for seafood processing in good and safely.

10002 Abstract Proceedings

Fluorescence fingerprint approach for monitoring the post-mortem changes in frozen horse mackerel fillets

Md. Mizanur Rahman, Mario Shibata, Naho Nakazawa, Tomoaki Hagiwara, Kazufumi Osako and Emiko Okazaki, Tokyo University of Marine Science and Technology, Japan

Frozen fish consumed as ‘Sushi’ and ‘Sashimi’ is required high freshness quality due to hygienic and good properties of texture, color, flavor, and taste. However, the traders can’t know the exact initial condition of frozen food instantly without thawing them. Moreover, the chemical methods for tracking the changes in the fish body are destructive and time-consuming. So, the present study was aimed to propose a noninvasive method using the fluorescence fingerprints (FFs) for monitoring the fish freshness frozen at the early stage after death. The study was especially focused on the changes of nicotinamide adenine dinucleotides (NAD and NADH) which are deeply involved in the progress of glycolysis and expected to be an indicator of the state of rigor mortis.
Fifty-six horse mackerel (Trachurus japonicus) were sacrificed and kept on ice for 14 different periods (0-48 hour) to prepare samples with different freshness, then filleted and frozen. FFs of the frozen samples were then acquired using fluorescence spectrophotometer (F-7000) aided with an external fiber optic probe at -30°C. Then, the NAD and NADH content of the same frozen samples were determined using relevant biochemical methods. The FFs were masked and partial least square (PLS) regression models were built by comparing with the chemical data.
By chemical analyses, it was confirmed that the NAD content decreased and NADH content increased gradually during the post-mortem changes. The PLS models of NAD and NADH content were observed with coefficient of determination (R2) of 0.71 and 0.88, respectively with 8 latent factors (LF). These fluorescent intrinsic compounds revealed a good correlation with the FF data. Thus, FFs coupled with the chemometrics could be a rapid approach for monitoring the post-mortem changes in frozen fish muscle nondestructively.

10003 Abstract Proceedings

Study on the thawing method for frozen spotted mackerel with high freshness

Yuki Fuchiyama, Naho Nakazawa, Kaihei Okumura, Kazufumi Osako and Emiko Okazaki, Tokyo University of Marine Science and Technology, Japan

Recently, studies have investigated the production of frozen mackerel with high freshness for sashimi production in Japan. pH of mackerel meat with high freshness tends to decrease markedly after thawing due to the progression of glycolysis. As it is assumed that pH is correlated with the quality deterioration of thawed meat, the development of a suitable thawing method for frozen mackerel without pH decrease is required to improve the quality of thawed meat. Temperature control before thawing (TCBT), which is the method of storage at -20 to -2°C for several days before thawing, has been proposed as one of the effective methods to prevent a decrease in the pH of frozen fish meat. Although it is presumed that glycolysis is suppressed by using TCBT, the detailed mechanisms of TCBT have yet to be clarified. The objective of this study was accordingly to elucidate the biochemical changes in frozen spotted mackerel meat with high freshness during TCBT.
Frozen spotted mackerel caught in Kamaishi Bay and cultivated in a fish tank were used as samples. They were sacrificed instantly and blood was removed using the “Kubiore” procedure. The fish were subsequently frozen and maintained at -60°C until use. After TCBT at -10°C, the frozen meat was thawed at 5°C for 18 h. pH, ATP, NAD, and lactic acid in both frozen and thawed meats were measured.
In frozen meat without TCBT, pH decreased to a value of 6.0 after thawing. In contrast, in frozen meat subjected to TCBT at -10°C over 3 days, the pH was maintained at approximately 6.4 after thawing. Little lactic acid in the meat was produced during the thawing process and NAD in frozen meat was decreased during TCBT. It thus appears that glycolysis in the meat is suppressed during the thawing process as a consequence of TCBT.

10004 Abstract Proceedings

Effect of the processing/freezing method on properties of frozen surimi gels

Yi Lun Tung1, Yoshinobu Hiraoka2, Ru Jia1, Naho Nakazawa1, Kazufumi Osako1 and Emiko Okazaki1, 1Tokyo University of Marine Science and Technology, Japan and 2Ehime Institute of Invention and Innovation, Japan

【Objective】In our research conducted to elucidate the factors that affect the quality deterioration of frozen surimi-based products, it has been shown that various factors affected the quality of heat-induced surimi gels upon freezing. On the other hand, Hiraoka et al. (2011) have reported that the quality of Jakotempura, which is a traditional fish paste product, prepared by Method B (setting → freezing → heating), was better than that of Jakotempura prepared by Method A (setting → heating → freezing), in terms of showing low thawing drip loss. However, the mechanism underlying the differences is still unclear. Therefore, this study aimed to elucidate this phenomenon by using heat-induced surimi gels as a model.
【Methods】Frozen surimi was thawed and ground with 30% water and 3% NaCl. After grinding with with/without 5% potato starch, the surimi paste was shaped and processed by Method A or Method B. For freezing, surimi gels were vacuum-packed, subjected to either quick freezing or slow freezing, and stored at −20 or −40 °C for 1 day or 4 weeks. Microscopic observations, the water-holding capacity and physical properties of thawed gels were evaluated.
【Results】For heat-induced surimi gels without starch, Method B was not more effective than Method A. On the other hand, in the case of heat-induced surimi gels with starch, Method B was significantly more effective than Method A, with lower thawing drip loss. Microscopic observations confirmed that the starch granules in the gel processed by Method A were destroyed after slow freezing. On the other hand, starch granules in samples processed by Method B maintained their shape. It suggested that the higher water-holding capacity obtained by Method B was due to the fact that starch granules were still intact after setting and freezing, which provided tolerance to freezing.

10005 Abstract Proceedings

Quality evaluation method for frozen surimi by suwari speed and activation energy

Koki Yamada, Hiroo Fuchimoto, Akane Konishi, Ryousuke Takahashi, Hideto Fukushima and Masahiro Matsumiya, Nihon University, Japan

[Objectives] Frozen surimi are made from various fish species and several grades are on the market. However, their quality standards are not fully established. Suwari (sitting) treatment is important one in the manufacturing process of fish gel products. Furthermore, physical properties of sitting gel are quit different among various frozen surimi. Therefore, we focused the speed and the activation energy in the sitting reaction around 30 °C in this study. And it was examinated whether these values are suitable for quality evaluation or not.
[Methods] Frozen surimi of Alaska pollock (SA and RA grade), Blue grenadier (FA and KA grade) and Threadfin bream (SA and KA grade) were used in this study. According to general methods, meat pastes were prepared. Sitting reaction of meat paste was proceeded in three temperature ranges between 16 to 30 °C. The sitting speed (K s-1) was obtained from the breaking strength. And then, the activation energy (Ea) of the sitting reaction was calculated from the sitting speed of each temperatures.
[Results] In comparison with each Ea, Alaska pollock one was the highest value and Blue grenadier and Threadfin bream ones were almost same. Therefore, it is thought that the latter two surimi are easier to sit than that of Alaska pollock. On the other hand, in the case of different grades from the same fish, the sitting speed was clearly lower in the low grade surimi. From the these results, it was suggested that the Ea is good indicator for the quality among fish species. In addition, the sitting speed should also be good one, in particular among grades of the same fish.

10006 Abstract Proceedings

Application of emulsifying fish oil into commercial surimi-based product

Liqiong Niu1, Yoshihisa Suzuki2, Hideto Fukushima3, Gao Yuanpei1, Naho Nakazawa1, Kazufumi Osako1 and Emiko Okazaki1, 1Tokyo University of Marine Science and Technology, Japan, 2 Maruyo Suzuki Company, Japan and 3Nihon University, Japan

Fish oil is an excellent source of long-chain polyunsaturated fatty acids‎, such as ‎icosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3). To increase the value of food with high functionality, new products containing added fish oil have been developed in recent years. ‎Previously, we investigated the effects of emulsifying fish oil into heat-induced surimi gels and found that positive effects on quality characteristics, such as color, water-holding capacity, and gel-forming ability and on the prevention of quality deterioration by freezing. To apply this technology in the surimi-based product industry, this study was aimed to clarify the effect of ‎emulsifying fish oil on the quality of ‎the commercial product sasa-kamaboko.
Frozen surimi was ground with water, salt, and other seasonings to obtain surimi paste. Fish oil (0 (control), 2.5, and 5.0%) was emulsified by vigorous mixing under a vacuum. Emulsified surimi paste was shaped and heated by far infrared rays and baked to obtain sasa-kamaboko. The products were cooled, individually vacuum-packed, and frozen both rapidly and slowly. Measurement of water-holding ‎capacity, oil particle sizes, physical properties, and whiteness before and after frozen storage was conducted to evaluate the products. Microscopic observation and sensory evaluation were also performed.
The emulsified product displayed higher water-holding capacity and whiteness compared to the control. The texture of the emulsified product showed high scores in sensory evaluation. Microscopic observation revealed that the sizes of ice crystals in the frozen emulsified products were smaller and frozen damage of thawed products was lower compared to in control products. These results showed that the emulsification of fish oil positively affects not only the quality of commercial surimi-based products, but also on the quality changes during frozen storage.

10007 Abstract Proceedings

cDNA cloning of paramyosin from several kinds of squid mantle muscle

Takayuki Kajita, Yoshiko Takeda, Saki Yoshida, Hiromi Kakizaki, Hideto Fukushima and Masahiro Matsumiya, Nihon University, Japan

[Objectives] The gel product made from squid muscle shows unique texture, which is different of kamaboko made from fish muscle. It is suggested that the difference between these two products is caused of paramyosin specifically existing in invertebrate muscle. Nevertheless, it has not been reported the primary structure of squid paramyosin. Therefore, in this study, the primary structures of paramyosin from several kinds of squids were determined by cDNA cloning and the structural characteristics were investigated.
[Materials and Methods] Freeze mantle muscle from several kinds of squid Ommastrephes bartramii, Dosidicus gigas, Sepia esculenta and Gonatus onyx were used in this study. Firstly, total RNA was prepared from each mantle muscles, and cDNA was synthesized by using a reverse transcriptase. Primers were designed from the conserved sequences of several kinds of mollusks previously reported. PCR, 3’ RACE and 5’ RACE method was performed to obtain a DNA fragment, and the fragment was sequenced. The deduced amino acid sequences were aligned using ClustalW. And then, the phylogenetic tree was constructed using the neighbor-joining method.
[Results] Three cDNAs encoding paramyosin from the O. bartramii, D. gigas and G. onyx were cloned. The O. bartramii paramyosin cDNA was cloned up to 2,605 bp and contains an ORF of 2,574 bp encoding 858 amino acids. The D. gigas one was cloned up to 2,691 bp and contains an ORF of 2,640 bp encoding 880 amino acids. The G. onyx one was cloned up to 2,609 bp and contains an ORF of 2,574 bp encoding 858 amino acids. The phylogenetic analysis including three paramyosins obtained in this study, showed that three paramyosins formed a group, which was near to that of octopus.

10008 Abstract Proceedings

pH-dependence of coiled-coil structural parameters of shrimp tropomyosin studied by molecular dynamics simulation

Hideo Ozawa1, Koji Umezawa2, Mitsunori Takano3, Shoichiro Ishizaki4, Shugo Watabe5 and Yoshihiro Ochiai6, 1Kanagawa Institiute of Technology, Japan, 2 Shinshu University, Japan, 3 Waseda University, Japan, 4 Tokyo University of Marine Science and Technology, Japan, 5 Kitasato Univerisity, Japan and 6Tohoku University, Japan

Ingestion of marine invertebrate species, including shrimp, squid and abalone, causes food allergy. The major allergen in invertebrate foods is tropomyosin (TM), which consists of the α-helical coiled coil structure throughout almost the whole molecule. TM, like other ordinary allergens, shows higher resistance against digestion in gastric than intestine. The reason of such difference in digestibility is unknown. We therefore performed constant-pH molecular dynamics simulation at pH 1 and pH 7 (10 ns × 3) for TM of kuruma shrimp Marsupenaeus japonicus. The trajectories were then analyzed by TWISTER to obtain the coiled-coil radius and phase per residue. As a result, the average values of the coiled-coil radius at pH 1 and pH 7 were 4.83 Å and 4.80 Å, respectively. The differences in the radius were observed around the acidic cores: the radius around Asp137 was larger at pH 7 than at pH 1, while the radius around Glu218 was lower at pH 7 than at pH 1. The average values of the coiled-coil phase per residue at pH 1 and 7 were -3.38° and -3.18°, respectively. The coiled-coil structure around Asp137 at pH 7 was less twisted than that at pH 1. In addition, the phases around Glu218 at pH 7 were more fluctuated than those in other region. These differences between pH 1 and pH 7 were mainly found at the two acidic cores, even though longer simulations are needed to confirm the observed difference. The pH-dependence of the overall coiled-coil structure or its rigidity was little, except those found at the two acidic cores, and thus it seems to be difficult to explain the difference in the digestibility between the stomach and small intestine by the pH-dependence of overall structural properties of the coiled-coil.

10009 Abstract Proceedings

The anti-allergic activities of polysaccharides from Chondrus verrucosus (Rhodophyta, Gigartinales)

Xiaolu He, Akiko Yamauchi, Toshiki Nakano, Toshiyasu Yamaguchi and Yoshihiro Ochiai, Tohoku University, Japan

[Objective] Many researches have been done on health functional properties of red algae-derived substances, such as flavonoids and steroids. In this study, polysaccharides were extracted from red algae (Chondrus verrucosus) and the anti-allergic activities were investigated.

[Materials and Methods] (1) Purification of polysaccharides: The specimens were collected on the coast of Kesennuma, Miyagi, Japan in May, 2016. Polysaccharide was extracted with 3 volumes of 0.17 N hydrochloric acid and was further purified by anion-exchange column chromatography with a sodium chloride concentration linear gradient of 0 to 2 M.
(2) The anti-allergic activities: Hyaluronidase inhibitory activity, and mast cell degranulation release effect on rat basophilic leukemia (RBL-2H3) cultured cell line by the antigen and A23187 (calcium Ionophore) stimulation were investigated.
(3) The percentages of surface molecules: The Muse® Human CD4 T Cell Assay, and human Lymphocyte CD69 Assay were performed using the Muse® Cell Analyzer.

[Results and Discussion] The polysaccharide was fractionated into the three major fractions which were eluted at around 0.69 M, 0.96 M and 1.69 M NaCl, respectively. The sulfate group content in the 0.69 M (34.5%) and 0.96 M (42.0%) fractions were higher than in the 1.69 M (12.5%) fraction. Each fraction showed the ability to inhibit cell proliferation and changed the ratio of CD4 T cells, and CD69 expression. For release inhibition of β-hexosaminidase, the 0.69 M (59.1%) and 0.96 M (62.9%) fractions showed higher degranulation inhibiting activity than the 1.69M (49.8%) fraction. From these results, it was inferred that the polysaccharides derived from the red algae C. verrucosus have the ability to suppress allergies.

10010 Abstract Proceedings

Species identification by DNA barcoding and sarcoplasmic protein profiles of underutilized Indonesian freshwater fish

Asya Fathya Nur Zakiah1, Mala Nurilmala1, Agoes Mardiono Jacoeb1 and Yoshihiro Ochiai2, 1Bogor Agricultural University, Indonesia and 2Tohoku University, Japan

[Objective] Freshwater fish are becoming the important source of animal protein along with the worldwide shifting of protein needs from red meat (livestock) to white meat (fish). Freshwater fish have high quality of protein and amino acid composition. Several species of local freshwater fish have not been characterized, even though they have a high potential as a source of healthy food based on local wisdom information. Thus, the research aimed to identify underutilized freshwater species based on morphology and DNA barcoding, together with the protein profiles.
[Materials and methods] Fresh specimens of freshwater (five species) were stored at -800C as a whole until used. The morphological identification of the species was carried out by referring to Kottelat (1993). DNA barcoding was performed using cytochrome oxidase subunit 1 (COI) as a molecular marker. DNA was isolated from the ordinary muscles according to manufacturer’s protocol, then the amplification was carried out by PCR. Sarcoplasmic protein fractions were analyzed by SDS-PAGE with 12.5% gel. The molecular weight was determined by Photocapt and ImageJ software.
[Results and discussion] By morphological identification and DNA barcoding, five species were identified as snakehead (Channa striata), striped snakehead (Channa micropeltes), marble goby (Oxyeleotris marmorata), Asian redtail catfish (Hemibagrus nemurus), and soro brook carp (Tor sp.). Specific primers successfully amplified the fragments of 500-600 bp. Obtained sequence of striped snakehead has been submitted to GenBank with the accession number KU204858. Protein profiles of the five species showed similiarity with 15-18 bands at 6.9-156.3 kDa. The major proteins in snakehead were 15.6 kDa and 38.3 kDa components, 11.3-16.4 kDa and 45.7-59.6 kDa components for striped snakehead, 6.9-15.1 kDa components for marble goby, 6.9 kDa and 39.2 kDa components for Asian redtail catfish, and 6.9 kDa and 34.3-58.5 kDa components for soro brook carp.

10011 Abstract Proceedings

Effect of Sargassum hystrix on blood biochemistry brofile of stress-snduced Wistar Rats

Amir Husni, Radipta Lailatussifa and Alim Isnansetyo, Universitas Gadjah Mada, Indonesia

Sargassum hystrix is a kind of brown algae which have pharmacology effects from its metabolic activities. This study aims to know the influence of using S.hystrix powder as an alternative functional food to treat stress-induced Wistar Rats by measure cortisol hormone, the profile of glucose, triacylglycerol, total cholesterol, and histopathology of the liver. Standard pellet diet of rats, mixed of S.hystrix powder and standard pellet diet, S.hystrix powder each 450 mg/kg were compared with diazepam for anti-stress activities using swimming restraint animal stress model 5 minutes/days and fasting for 10 days. All treatment groups administered orally at dosages 450 mg/kg, while Diazepam as the standard drug of anti-stress was used at 0,18 mg/kg p.o. Both treatment of S.hystrix powder and mixed of S.hystrix powder with standard pellet diet of rats 450 mg/kg p.o showed good anti-stress effect. They can improve and balance all of parameters profile.

10012 Abstract Proceedings

Extraction and characterization of acid- and pepsin-soluble collagen from scales of marine and freshwater fishes caught from temperate and sub-tropical countries

Sheik Md. Moniruzzaman, Kigen Takahashi, Emiko Okazaki and Kazufumi Osako, Tokyo University of Marine Science and Technology, Japan

Food, pharmaceutical and cosmetic industries around the world are observing a growing demand for collagen. The widely-used mammalian collagen (porcine and bovine) has significant limitations due to socio-cultural, religious and health-related concerns. Moreover, intense competition exists between manufacturers for acquisition of mammalian sources, which led to increased demand and excessive costs. These urged scientists to find and develop alternatives to mammalian collagen. The fish collagen is not associated with BSE and is acceptable for Islam. Furthermore, the sources are discarded wastes (i.e., scales, skin etc.) of fish processing industries. Therefore, production and application of fish collagen pleases the needs of human consumption as well as reduces pollution. It is assumed that habitat temperature affects the properties of collagen and collagen from warm water fish has higher thermal stability. However, no information exists for acid- and pepsin-soluble collagen (ASC and PSC) from scales of temperate and sub-tropical country fishes. Considering the above-mentioned phenomena, the aim was to clarify the effect of environmental temperature on collagen properties from scales of marine lizardfish (Japan and Vietnam) and freshwater carp (Japan and Bangladesh) fishes.
After pre-treatment, ASC was extracted with 0.5 M acetic acid. For undissolved residue after acid extraction, 1% pepsin was used. The yield of collagen was determined by hydroxyproline content in scales and collagen. Amino acid composition was analyzed by HPLC. For SDS-PAGE, both collagens were loaded onto polyacrylamide gel (7.5%) and high molecular weight markers was used to estimate the molecular weight of proteins. Denaturation temperature was analyzed using DSC.
The crude protein and ash contents were higher in carp than lizardfish scales. The yield of PSC was higher compared to ASC. Both collagen contained major amino acids and glycine was the dominant amino acid found. The SDS–PAGE pattern revealed that both ASCs and PSCs were type I collagens.

10013 Abstract Proceedings

Study on the fatty acid compositions of deep-sea isopod Bathynomus doederleinii

Takayuki Sawasaki, Kigen Takahashi, Kouichi Kurose, Emiko Okazaki and Kazufumi Osako, Tokyo University of Marine Science and Technology, Japan

Deep-sea isopod Bathynomus doederleinii is a crustacean inhabiting deep sea and commonly found at the sea around Japan. Deep-sea isopod is usually caught as a bycatch of basket trap fishing, and discarded at the sea because it has no commercial value. Therefore, to utilize the deep-sea isopod as a food resource, its basic nutritional information of the muscle and whole body was examined in our previous study. In the previous study, both muscle and whole body of the deep-sea isopod contained higher amount of crude total lipids than other edible crustacean’s meat did. In the present study, the lipid class and its fatty acid composition of the deep-sea isopod were analyzed.
Deep-sea isopod was caught in the East China Sea by basket trap fishing. Crude total lipids of their muscle and whole body were extracted according to Folch et al. procedure. A silica gel column was used for separating the crude total lipids into the classes, including cholesteryl esters, triacylglycerols (TAG), cholesterols, diacylglycerols, free fatty acids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC). TAG, PE and PC thus obtained were converted into fatty acid methyl esters and individual FAME was quantified by using GC-FID. To qualify the FAME by using GC-MS, DMOX derivatives were prepared from the FAME of crude total lipids.
TAG was the dominant lipid class in the crude total lipids of muscle and whole body of the deep-sea isopod, and it contained high amount of monounsaturated fatty acids (MUFA), such as 18:1n-9, 16:1n-7 and 20:1n-9. The lipids in whole body contained high amount of phospholipids, including PE and PC. The phospholipids contained higher levels of polyunsaturated fatty acids (PUFA), including 20:4n-6, 20:5n-3 and 22:6n-3 than TAG did. These results suggest that deep-sea isopod is MUFA and PUFA-rich marine species and can be utilized as these fatty acids resource.

10014 Abstract Proceedings

Effect of spawning on endogenous proteinases in abdominal muscle of threadfin bream Nemipterus virgatus

Jin-yang Liu1, Asami Yoshida1, Kazuya Shirota2, Yasuhiko Shiina2 and Kiyoshi Osatomi1, 1Nagasaki University, Japan and 2Maruha Nichiro Corporation, Japan

Threadfin bream (Nemipterus virgatus) is one of the most important commercial fishery species in Japan and Southeast Asia, and it has been widely used as a raw material for surimi productions. However, the surimi of threadfin bream is susceptible to modori phenomenon which is due to the presence of the endogenous proteases. Furthermore, the activity of the endogenous proteases is always affected by spawning. In this study, we investigated the effect of spawning on endogenous proteases in the muscle of threadfin bream.
Threadfin bream was sampled in the spawning period of July and the November, respectively. The characterization of autolysis was investigated using various protease inhibitors. The protease activities of sarcoplasmic and myofibrillar fractions were measured using various fluorogenic synthetic substrates. The gelatinolytic activity of sarcoplasmic fraction was determined by gelatin zymography. The results were concluded as follows: (1) Threadfin bream possessed a higher autolysis activity in abdominal muscle during spawning period. (2) The main working protease in the autolysis process was a serine protease. (3) It was confirmed by substrate specificity and inhibitory properties that the main working protease in sarcoplasmic fraction was trypsin-type serine proteinase. (4) The serine proteinase of 50 kDa in sarcoplasmic fraction possessed an especially high gelatinolytic activity. (5) The activity of myofibril-bound serine proteinase (MBSP) was detected in myofibrillar fraction.

10015 Abstract Proceedings

The removal of fishy odor from tilapia skin using for gelatin extraction

Patwarin Tohmadlea and Jirapa Hinsui, Kasetsart University, Thailand

Commercial gelatin production was extracted from bovine and porcine, but fish skin was not for commercial production because of fishy odor. Tilapia production is growing worldwide and to better utilize wastes from the processing industry, one important is production of high quality fish gelatin to meet the need of markets that made value for Tilapia skin being material for commercial gelatin production. Fish skin was used for only laboratory gelatin extraction because of fishy odor in tilapia skin. The objective of this research was to remove fishy odor from Tilapia Skin using for gelatin extraction. tilapia (Oreochromis niloticus) skin was treated with 3 treatments; alkaline-acid (NaOH-citric acid-sulfuric acid), NaCl and NaCl and alkaline-acid treatment at room temperature. Then the skin was washed until washing water was neutral. The gelatin was extracted by hot water at 50 °C for 3 h then gelatin were evaporated by rotary evaporator at 50 °C for 3 h and dried by hot-air oven at 50 °C for 24 h. The gelatin yield, gel strength, quality and quantity of volatile compounds were analyzed by using GC-MS and sensory evaluation analysis. The best treatment was NaCl and alkaline-acid treatment that shown high yield, high gel strength and was accepted by the panelists in the appearance, color, and fishy odor. NaCl and alkaline-acid treatment get rid of fishy odor from tilapia skin that made value for tilapia skin being material for commercial gelatin production.

10016 Abstract Proceedings

The effect of initial freshness on the quality change of tuna meat during frozen storage

Naho Nakazawa1, Ritsuko Wada2, Hideto Fukushima3, Shinji Kono4, Ryusuke Tanaka5 and Emiko Okazaki1, 1Tokyo University of Marine Science and Technology (TUMSAT), Japan, 2 National Fisheries University, Japan, 3 Nihon University, Japan, 4 Mayekawa Mfg. Co. Ltd., Japan and 5Miyazaki University, Japan

Tuna is one of the most valuable fish for production “Sashimi” and “Sushi” in Japan. The color of tuna meat, which is one of the most important quality factors, deteriorates due to the formation of metmyoglobin (metMb) caused by autoxidization during frozen storage. Additionally, the texture of meat deteriorates due to frozen denaturation of myofibrillar protein. Recently, ATP, the most important initial freshness indicator of fish meat, has been shown to suppress the formation of metMb, and frozen denaturation of myofibrillar protein. However, the initial freshness of frozen tuna meat can differ significantly, because tuna may be either alive or dead when the catch is landed. Therefore, it has been suggested that the initial freshness of tuna meat affects the quality changes during frozen storage. Accordingly, in this study we aimed to elucidate the relationships between the initial freshness of frozen tuna meat, quality changes during frozen storage, and frozen storage temperature.
We used frozen bigeye tuna, for which the initial freshness (pH and ATP content) had been determined. The frozen meat was sliced, packed in a plastic bag and maintained at -20, -35, -40, -45, or -60°C for 14 months. We measured the metMb ratio and Ca2+-ATPase activity during frozen storage.
The increase in the ratio of metMb in the high-freshness meat was lower than that in low-freshness meat at -40 to -20°C. The decrease in the ratio of myofibrillar Ca2+-ATPase activity of the high-freshness meat was higher than that of the low-freshness meat. However, no obvious difference was found in the changes of these indices at temperatures below -45°C, irrespective of the initial freshness of the tuna meat. Therefore, we suggest that the initial freshness of tuna meat affects quality changes during frozen storage, and that these changes can be suppressed at temperatures lower than -45°C.

10017 Abstract Proceedings

Effect of salting and frozen storage on the quality of tuna meat

Qingqing Jiang, Naho Nakazawa, Kazufumi Osako and Emiko Okazaki, Tokyo University of Marine Science and Technology, Japan

【Background and Purpose】Lightly salted fish products have become increasingly popular. Because these products have high water content and low salt content, they need to be kept in frozen storage to have a long shelf-life. Unfortunately, little information is available on the quality changes of salted fish meat during frozen storage. The water holding capacity, color, and texture are decisive quality attributes for the consumer purchasing decisions. Therefore, the purpose of this study was to clarify the effect of salting and frozen storage on the quality changes of tuna meat.
【Material and Methods】Frozen bigeye tuna meat was thawed and cut into slices (3 cm×2 cm×0.5 cm). After soaking in different concentrations of NaCl solution, the samples were stored at -20°C for 1 w. The quality of the meat was evaluated by product yield, thawing loss, centrifuging loss, color characteristics, and texture profile.
【Results】The highest yield was obtained in 0.5 M and 1 M salted samples after both salting and frozen storage. The centrifuging loss decreased in 1 M or higher solutions salted samples after salting, but increased in the 2 M and 3 M salted samples after storage. After salting with 0.5 M or higher solutions, the samples became glossy and the hug angle of 1 M salted samples was the lowest and the most stable after frozen storage. The springiness, adhesiveness, and cohesiveness increased after salting with 0.5 M or higher solutions, indicating that salted fish products have a characteristic mouthfeel and texture.

10018 Abstract Proceedings

Influence of the protease inhibitors on the gel-forming ability of recovered North Pacific krill Euphausia pacifica protein using NaCl solution treatment

Kayo Amano, Kigen Takahashi, Emiko Okazaki and Kazufumi Osako, Tokyo University of Marine Science and Technology, Japan

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

10019 Abstract Proceedings

Tribology of agar and fish meat gel

Yuri Tashiro1, Chikako Tomohiro2 and Hiroo Ogawa2, 1Kyoto Prefectural University, Japan and 2Tokyo University of Marine Science and Technology, Japan

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

10020 Abstract Proceedings

A new method for assessing the freshness of fish meat based on metabolome analysis technology

Ryota Mabuchi, Huiqing Zhao, Rumi Kondo and Shota Tanimoto, Prefectural University of Hiroshima, Japan

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

10021 Abstract Proceedings

Applicability of a taste sensing system to objectively assess taste of seafood

Ken Touhata1, Masaaki Habara2, Hidekazu Ikezazki2 and Noriko Ishida1, 1Japan Fisheries Research and Education Agency, Japan and 2Intelligent Sensor Technology, Inc., Japan

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

10022 Abstract Proceedings

Chemical characterization of polysaccharides from three seaweed species and their potential biological activities

Xiaolu He, Shenjing Liu, Akiko Yamauchi, Toshiki Nakano, Toshiyasu Yamaguchi and Yoshihiro Ochiai, Tohoku University, Japan

[Objective] Algal polysaccharides exhibit structural features such as sulfate groups, which distinguish them from polysaccharides of terrestrial plants. In the present study, polysaccharides were isolated from the three seaweed species, including one red algae (Chondrus verrucosus) and two brown alga (Saccharina japonica and Undaria pinnatifida), and their chemical characteristics and potential biological activities were compared.

[Materials and Methods] (1) Polysaccharides preparation: Extracted with 3 volumes of 0.17 N hydrochloric acid and was further purified by anion-exchange column chromatography with a sodium chloride concentration linear gradient of 0 to 2 M.
(2) Chemical analysis: Total sugar content of the polysaccharides was determined by the phenol-sulfuric acid method. The monosaccharide composition was analyzed by GLC. The sulfate content was determined by using the BaCl2-gelatin turbidimetry method.
(3) Biological activities: Hyaluronidase inhibitory activity and β-hexosaminidase inhibitory activity were measured.

[Results and Discussion] Polysaccharides from the marine algae showed different chemical characteristics depending on the species. Firstly, total yield of polysaccharides from dried algae varied from 5.8% (S. japonica), 4.6% (U. pinnatifida) to 45.2%(C. verrucosus), indicating that C. verrucosus was richer in polysaccharides than the two brown species. Secondly, C. verrucosus showed a higher sugar content in the ranges of 47.4%~76.8% than the brown seaweeds (26.7%~40.1%), and a higher sulfate content(12.5%~34.5%)than the two brown seaweeds (7%~30.3%). Some of the variations can be attributed to the differences in species and inhabiting environment. Finally, C. verrucosus with a higher sulfate content showed the higher hyaluronidase inhibitory activity and degranulation inhibiting activity than the two brown species.

10023 Abstract Proceedings

Utilization of the sawdust from frozen tuna processing – Composition analysis and quality evaluation of trial products

Emi Shinohara1, Naoko Yoshikawa2 and Yoshihiro Ochiai3, 1Tokai University, Japan, 2 Shizuoka Institute of Science and Technology, Japan and 3Tohoku University, Japan

[Introduction] During cutting process of frozen tuna with a chain saw, a lot of sawdust is unintentionally produced. There has been, however, no effective use of the dust. In the present study, attempts were made to explore a way of utilization by referring to the functional components in the dust.
[Materials and methods] The sawdust of frozen bigeye tuna was obtained on the spot at a tuna processing company, and was kept at -80℃ until used. Free amino acid composition were determined by HPLC method. pH values and lactate content were also measured. The color stimulus values and histamine content was measured during storage at 4℃ after thawing. Organoleptic evaluation was carried out on the trial products (seasoned broth and dumpling). The breaking strength was measured for the dumpling.
[Results and discussion] Free amino acids were effectively extracted by boiling from the sawdust. The major components were anserine (ca. 520 mg/100 g) and taurine (ca. 100 mg/100 g). The L* value slightly increased 31.0 to 35.1, while the a* value greatly decreased from 21.4 to 6.5 during three days of chilled storage after thawing. The organoleptic scores for the broth exceeded those for skipjack one as a control. Curry powder was useful in masking a slight fishy odor. The gel strength of the dumpling was improved from about 115 g to 150 g by the addition of gelation improver consisting of transglutaminase, and such an increment successfully improved the organoleptic scores. Histamine concentration increased from 2.3 ppm at the start up to as low as 4.4 ppm after 3 days. It is suggested the sawdust can be effectively utilized for the materials for delicacies equipped the health promoting functions by using the fresh one and processing quickly. Further attention, however, should be paid against metal pieces from chainsaw edges.

10024 Abstract Proceedings

Characterization of composition and lipid in various deep sea fish species from Suruga Bay

Jun Howard1, Akiko Yamauchi2, Toshiyasu Yamaguchi3, Toshiki Nakano4 and Yoshihiro Ochiai5, Tohoku University, Japan

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.

10025 Abstract Proceedings

Pesticide residues control in foods imported into Japan; HACCP-oriented approach to ensure regulatory compliance

Chiaki Miura1, Goichiro Yukawa1, Naoko Hamada- Sato1 and Naoki Shinoda2, 1Tokyo University of Marine Science and Technology, Japan and 2Cargill Japan Ltd., Japan

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.

10026 Abstract Proceedings

The structural fluctuation of the histamine oxidase by molecular dynamics simulation

Hideo Ozawa1, Yoshihiro Ochiai2 and Akira Yamamura1, 1Kanagawa Institiute of Technology, Japan and 2Tohoku University, Japan

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.

10027 Abstract Proceedings

Effect of starch grain size on changes in quality of heat-induced surimi gels upon freezing

Ru Jia1, Toyohiko Katano2, Yuki Watanabe1, Naho Nakazawa1, Kazufumi Osako1 and Emiko Okazaki1, 1Tokyo University of Marine Science and Technology, Japan and 2Nihon Starch Co., LTD., Japan

Surimi-based products such as kamaboko tend to deteriorate upon freezing. In our previous study, we found both positive and negative effects of addition of various kinds of starch on quality changes of surimi gels upon freezing. In the present study, starches with different grain sizes were prepared, and their effects were determined on the changes in the quality of surimi gels after freezing.
Frozen surimi was thawed and chopped with NaCl and starch, including potato starch and wheat starch with a normal or micro grain size, then heated at 90 °C for 30 min and frozen by quick or slow freezing. After 1 week of frozen storage at -20 °C, the water-holding capacity (WHC), microscopic observation, physical properties by puncture test and texture profile analysis (TPA) were determined.
For WHC, there was no significant difference between surimi gels with the two sizes of starch grains before freezing. After freezing, surimi gels with large-size grain starch showed a higher drip loss, especially in the case of slow freezing. The results of microscopic observations corresponded to those of WHC. Regarding the breaking strength, there was no significant difference between surimi gels containing two types of potato starch. However, surimi gels containing wheat starch with a micro grain size showed a significantly lower breaking strength than those containing normal grain size, both before and after freezing. The hardness, determined by texture profile analysis, showed the same tendency as the breaking strength. It was considered that the physical changes were related to different retrogradation degrees of potato starch and wheat starch.
Above results revealed that the changes in the quality of starch-containing surimi gels, were affected not only by freezing conditions but also by starch properties such as the grain size, retrogradation, and structural destruction.

10028 Abstract Proceedings

Study on the methods for the concentration determination of collagens from calf and fish

Xin Chen, Jingjing Chen, Junjie Zhang and Rui Duan, HuaiHai Institute of Technology, China

The concentration of collagen determined by the biuret method is usually lower than its true value, due to the collagen’s triple helix structure. In order to obtain more accurate measurement results, the conditions for the determination of collagen concentration by biuret method were studied. The collagen extracted from the calf tendon was employed as the raw material. The collagen solutions were heated at 40℃ for 5min, 15min and 30min, respectively. The same operations were also performed at 60℃ and 80℃, respectively. Then the absorbance of the samples was measured by using biuret method. The results showed the sample heated at 40℃ for 5min had the highest absorbance value, which increased 50% than that of the unheated one. With the increase of heating temperature and extension of heating time, the absorbance values gradually decreased. It was in accordance with the degradation of collagen molecules showed on SDS-PAGE pattern. The collagen concentrations were also determined by the Lowery method. The results indicated that the Lowery method was more accurate for determination of collagen from calf tendon compared to the biuret method. However, there was little difference in accuracy between the Lowery and the modified biuret methods for the determination of collagen from fish skin. Therefore, the modified biuret method was more applicable for fish collagen due to its simplicity of operation.

10029 Abstract Proceedings

Removing fish scale by using tuna protease

Jirapa Hinsui, Thongpipat Luangthong, Supannika Thikakun, Nichaphat Detkamhaeng and Natechanok Mahachoo, Kasetsart University, Thailand

Fish fillet and whole fish with skin on, but without scales, are important in the market of fish processing industries. Descaling by hand is a very labour-intensive operation and although descaling machines are available, they have not proven to be satisfactory. In many cases, the machine-descaling process must be repeated, but even then remaining scale requires manual removal. Therefore, much of the flesh and skin are lost, and the process appears to be uneconomical. Mechanical descaling of some fish species such as canned sardine (Sardinella gibbosa) is a difficult task due to high liability of soft muscle damage. An enzymatic process has been developed to remove the scales in a very gentle manner in order to yield a product of superior quality compared to the corresponding mechanically descaled product. The objective of this research was to remove fish scale by using tuna protease. The protease was extracted from tuna viscera by stirring with 0.2 M acetic acid at 4ºC for 3 h, then centrifuge at 6,000 xg for 30 min. The crude extract was protease activity and soluble protein determination and enzyme characterization. The protease specific activity was 0.84 U/mg protein. Temperature optimum and stability of protease were 50 and 0-10ºC, respectively, where as pH optimum and stability of protease were 2.0. Crude extraction was applied to 1 inch square, whole fish skin and whole fish for 6 h at 4 and 25 ºC. Degree of hydrolysis, enzyme efficiency and sensory evaluation were determined. Descaling by tuna protease at 25 ºC was faster than 4 ºC. The result shown in satisfactory with no remaining scale for 2 h crude extract treatment at 25 ºC and the flesh was accepted by panelists. This protease can applied with various fish species in fish processing industries.

10030 Abstract Proceedings

Towards the effective utilization of deep sea fish resources

Yoshihiro Ochiai1, Yuki Terada2, Mizuki Matsumoto3 and Naoko Yoshikawa3, 1Tohoku University, Japan, 2 Tokai University, Japan and 3Shizuoka Institute of Science and Technology, Japan

[Objective] Around 2400 fish species are estimated to inhabit the bathyal zone, which is defined as the one deeper than 200 m. There seems be a tremendous amount of underutilized fish species, which could be used as food, pharmaceuticals, animal feeds, and so on. In order to estimate the availability of these species for human consumption, it is essential to characterize their nutritional components and functional substances for more species.
[Materials & methods] More than thirty species were collected from the catch of drag net fishing in Suruga Bay, Shizuoka Pref. The specimens were transported to the laboratory in ice water, and stored at -80C after cleaning and filleting. Edible parts (ordinary muscle in most species) were the major targets for this study. Inedible parts were weighed to estimate the yield. Proximate composition was measured by the official methods. Free amino acid composition was determined by the conventional chromatography techniques. Protein components of muscle water-soluble fractions were analyzed electrophoretically.
[Results & discussion] The yield for the edible part varied from 37 to 72%. While the water content tended to be high (>80% for a few species) for most species, it varied greatly depending on the species. This was also true for the protein (>10% for some species) and lipid content (<10% for several species). Some oily species contain not a small amount of wax ester which would cause keriorrhea when ingested beyond the acceptable amount of flesh. Some species were rich in taurine and anserine, both of which would show health promoting functions. Protein patterns were quite species-specific, and degradation was recognized in some species, indicative of quality deterioration during storage. These data suggest that not a few species are available for human consumption. The safety and possibility of utilization other than food and pharmaceuticals will also be discussed.

10031 Abstract Proceedings

Development of a new thawing method applying electromagnetic wave irradiation

Minoru Sato1, Toshiyasu Yamaguchi1, Akiko Yamauchi1, Toshiki Nakano1 and Yoshihiro Ochiai1, Tohoku University, Japan

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.

10032 Abstract Proceedings

Species identification by DNA barcoding and bioprospecting of Indonesian seahorses

Mala Nurilmala1, Evi Maya Sari1, Asadatun Abdullah1, Agus Oman Sudrajat1 and Yoshihiro Ochiai2, 1Bogor Agricultural University, Indonesia and 2Tohoku University, Japan

[Objective] Seahorse (Hippocampus spp.) is one of the marine organisms belonging to the family Syngnathidae. They are unique organisms exhibiting male pregnancy. Seahorse is harvested from the wild throughout the year in Indonesia, then the dried products are traded internationally. However, there have been a few reports on Indonesian seahorse. Thus, the exploration of seahorse was carried out in this study with molecular approach using DNA barcoding for identification of seahorse species accurately. The proximate composition and amino acid profiles were also determined, and the secondary metabolites were further investigated.
[Materials and methods] The specimens of seahorse were collected from the coasts of Sumatera and Seribu Islands, Indonesia. The proximate analysis of water, protein, lipid, and ash was carried out based on AOAC methods. The species identification was carried out by a DNA barcoding method using mitochondrial cytochrome oxidase subunit 1 (COI) as a molecular marker. The amplification of isolated DNA was carried out by PCR, then the sequences were obtained for further bioinformatical analysis. The ethanol extract from the whole body of seahorse was subjected to phytochemical analysis to determine its secondary metabolites.
[Results and discussion] The DNA was isolated and amplified successfully using the primers as designed from COI gene sequences. BLAST analysis showed that the species of seahorse were H. kuda and H. comes. The results of chemical composition showed that the seahorse (H. comes) consisted of 66.16 ± 0.33% moisture; 22.73 ± 0.17% protein; 1.18 ± 0.23% lipid; and 9.55 ± 0.15 ash. Hydrolysates of seahorse proteins contained the amino acids such as glutamic acid, aspartic acid, arginine, and alanine as the major components. In addition, the results of the qualitative phytochemical analysis showed that the ethanol extract contained flavonoids, triterpenoids, steroids, saponins, and phenol hydroquinone.

10033 Abstract Proceedings

Extraction and characterization of freshwater and marine fish skin collagen as Halal gelatin rources

Mala Nurilmala1, Agoes Mardiono Jacoeb1, Heny Suryamarevita1 and Yoshihiro Ochiai2, 1Bogor Agricultural University, Indonesia and 2Tohoku University, Japan

[Objective] Gelatin, protein polymer from collagen conversion, is a multifunctional ingredient used in foods and non-food industries such as pharmaceuticals and cosmetics. Generally, commercial gelatin comes from bovine and pig sources threatening health problems (BSE) and religious sentiment. The alternative source is necessary to meet the requirement of “halal” for Moslem society. In addition, Jewish need it for kosher food. Finding good raw materials as alternative sources derived from fish skin waste for industrial fish-processing is important. Thus, the aim of this study was to extract and characterize fish skin gelatin from various fish species.
[Materials and methods] Fish skins from pangasius (Pangasius sp.), red tilapia (Oreochromis niloticus), red snapper (Lutjanus sanguineus ) and parrotfish (Scarus ghobban) were collected. They were rinsed in 1% limewater, then soaked in 1% citric acid solution for 12 hours. Washing process was performed for 5 times, then the skins were extracted in distilled water at 65oC for 6 hours, followed by drying with an evaporator. The obtained gelatins were subjected to characterization including yield, gel strength, viscosity, pH, color, amino acid composition, and SDS-PAGE. In addition, all the fish skins were prepared to investigate the light microscopic structure of collagen by dying with haematoxylin-eosin and Masson's trichrome.
[Results and discussion] Gelatins from marine fish had relatively higher rheological characteristics than those from freshwater species. Gelatin from parrotfish showed the most favorable properties with the yield of 24.7%, gel strength of 118.4 gf, viscosity of 22.0 cP, pH 4.59. The main amino acids were glycine and proline. SDS-PAGE showed that the gelatins consisted of β, α1, and α2 chains as the gelatins from other sources. Microscopic observation revealed that fish skin of marine fish contained more collagen than freshwater fish did. In conclusion, fish skin gelatins can be good sources of halal gelatin.

11001 Abstract Proceedings

Haptenic properties of tetrodotoxin conjugated to carrier proteins by using dithiol reagents

Suzuka Takaishi, Ko Yasumoto, Atsushi Kobiyama and Shigeru Sato, Kitasato University, Japan

Yotsu-Yamashita et al. (2005) reported that 4,9-anhydro-tetrodotoxin (4,9anh-TTX), a derivative of TTX found in puffer fish, reacts with biological thiols such as cystein (Cys) to form 4-S-Cys adduct of TTX. Not only Cys, various thiol compounds couple to 4,9anh-TTX. This finding enable us to design a new haptenic antigen essential to develop immuno-detection of TTX. Sulfhydryl moiety was introduced to tetrodotoxin (TTX) by a reaction between 4,9anh-TTX and (+) dithiothreitol (DTT). A new haptenic antigen was obtained from the mixture of a bifunctional coupling reagent (GMBS)-treated bovine serum albumin (BSA) and the DTT –TTX adduct. The ratio of TTX coupled to the carrier protein was estimated to be 14 % (w/w) by fluorescent intensity after sodium hydroxide treatment. In addition, 4,9anh-TTX was treated with 1,2-ethanedithiol (EDT) and resulting EDT-TTX adduct was introduced to GMBS-treated keyhole limpet hemocyanin (KLH). These two antigens (BSA-DTT-TTX and KLH-EDT-TTX) were inoculated biweekly to rabbits. High titer of anti-serum was observed within 4 months from the rabbits inoculated with KLH-EDT-TTX. The antiserum reacts well, not only with TTX, but also with 4epi-TTX, 11oxo-TTX as well as deoxy-TTX analogues which often found in TTX-bearing aquatic organisms such as puffer fish as well as toxic crab and gastropods. Separately, biotin-labeled TTX was prepared by a combination of maleimide-PEG2-biotin and DTT-TTX. By using the antiserum and biotin-labeled TTX, a new ELISA kit for TTX and its derivatives was designed.

12001 Abstract Proceedings

Bromophenols as antioxidants and tyrosinase inhibitors

Md. Reazul Islam, Daisuke Mikami and Hideyuki Kurihara, Hokkaido University, Japan

[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.

12002 Abstract Proceedings

Decreasing mechanism of lipoxygenase-mediated peroxides by seaweed constituents

Hideyuki Kurihara and Kenta Yachiyama, Hokkaido University, Japan

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.

12003 Abstract Proceedings

Juvenile Acropora tenuis attracts Symbiodinium by using GlcNAc-binding lectin

Ryota Takeuchi1, Mitsuru Jimbo1, Fumika Tanimoto1, Nami Kuniya1, Hiroshi Yamashita2, Go Suzuki2, Ko Yasumoto1 and Shugo Watabe1, 1Kitasato University, Japan and 2Japan Fisheries Research and Education Agency, Japan

Most reef-building corals acquire symbiotic dinoflagellates, Symbiodinium spp., from surrounding environments and establish symbiosis with Symbiodinium. Since Symbiodinium occur in low densities in the water column, corals would attract symbionts using chemotactic compounds to acquire them. We found a coral Acropora tenuis crude water extract attracted specific Symbiodinium strains, including strains NBRC102920 (clade A) and CCMP2556 (clade D), but not strains GTP-A6-Sy (clade A), CCMP421 (clade E) and CS-156 (clade F). The chemotactic activity in crude A. tenuis water extract remained after dialysis, but lost completely after heat treatment, indicating that the chemotactic compounds are water-soluble, heat-labile macromolecules. As juvenile A. tenuis tend to acquire the strains which attracted by the crude water extract and an factor involved in the acquisition was a carbohydrate-binding protein, lectin, chemoattraction seems to be inhibited by carbohydrates. As expected, N-acetyl-glucosamine (GlcNAc) inhibited the chemotactic activity. The GlcNAc-bining lectin purified from crude water extract, ActL, demonstrated 70% of the chemotactic activity in the crude water extract, and this chemoattraction was inhibited by addition of anti-ActL antibody, suggesting that ActL is a major factor for attraction. ActL and crude water extract attracted same strains which were acquired by juvenile A. tenuis. Moreover, rearing medium of 10 juvenile A. tenuis contained 34 ng/ml of ActL. It estimated that a polyp releases ActL at 3.4 ng/ml/juvenile polyp/day, and ActL at 3.4 ng/ml showed the chemotactic activity in vitro. These results suggest that ActL was released from juvenile A. tenuis and selectively attract some specific Symbiodinium.

12004 Abstract Proceedings

Effective extraction of carotenoprotein from Pacific white shrimp (Litopenaeus vannamei) shells using albacore tuna (Thunnus alalunga) spleen trypsin

Tanchanok Poonsin1, Benjamin K. Simpson2, Soottawat Benjakul3, Wonnop Visessanguan4, Asami Yoshida5 and Sappasith Klomklao1, 1Thaksin University, Thailand, 2 McGill University, Canada, 3 Prince of Songkla University, Thailand, 4 National Science and Technology Development Agency, Thailand and 5Nagasaki University, Japan

The effect of partitioned trypsin from albacore tuna (Thunnus alalunga) spleen using an aqueous two-phase system on the recovery and properties of carotenoprotein from Pacific white shrimp (Litopenaeus vannamei) shells was investigated. The partitioned trypsin from albacore tuna spleen showed an efficient hydrolysis and recovery of carotenoprotein from Pacific white shrimp shells. The recovery of carotenoprotein was maximized by the hydrolysis of shrimp shells using 0.8 trypsin units/g shrimp shells for 45 min at 25oC and shrimp shells/buffer ratio of 1:2 (w/v). Carotenoprotein consisted of 72.37% protein, 18.79% lipid, 7.14% ash, 1.61% chitin, and 73.25 µg total astaxanthin/g sample. It was rich in essential amino acid and Asp/Asn and Glu/Gln were found as dominant amino acids. When hydrolytic activities of albacore tuna and bovine trypsins toward carotenoprotein in Pacific white shrimp shells were compared, the recovery efficacy of protein and pigment by albacore tuna trypsin was similar to that achieved by bovine trypsin. Therefore, carotenoprotein from Pacific white shrimp shells could be successfully extracted using partitioned trypsin from albacore tuna spleen and could serve as the value-added nutritive food ingredients.

13001 Abstract Proceedings

Current supporting activities and the future issues in the Southeast Asia region by South East Asia Fisheries Development Center

Kaoru Ishii, Southeast Asian Fisheries Development Center (SEAFDEC), Thailand

Southeast Asia region is the one of the most blessed fishery areas which have produced various kinds of fishery products and distributed them to the world-wide. South East Asia Fisheries Development Center (SEAFDEC), which was established in 1967 to promote sustainable fisheries in the Southeast Asia and is joined by the ten Southeast Asian countries and Japan as the member countries, has developed fisheries technologies and disseminated them to fisheries communities in this region. For those activities, SEAFDEC has introduced some international cooperation tools such as Japan Trust Fund (JTF). The activities by SEADEC with JTF mainly classify into four components: to enhance the capability for sustainable utilization of fisheries resources, to strengthen the promotion of sustainable fisheries, to promote sustainable aquaculture and enhance fishery resources and to promote sustainable development in inland fisheries. SEAFDEC, in addition, is encountering newly emerging technical issues in this region which SEAFDEC are internationally expected to deal with: further dissemination of measures for combating the IUU fishing after effectuation of the FAO Port State Measure Agreement, improvement of the appropriate management scheme of world-wide concerned fishery resources such as eels and sharks, research on fisheries affected by the climate change, rehabilitation of the critical habitat and fishing grounds and dissemination and development of post harvest methodologies, etc. Regarding the rehabilitation of the critical habitat and fishing grounds as one of newly emerging issues, the team of the Training Department of SEAFDEC is now conducting the project named “Promotion of sustainable fisheries resources enhancement measures in critical habitats/fishing grounds in Southeast Asia” This project, considered with the characteristics of the target sites, introduces the appropriate resource enhancement tools such as voluntary management rules/guidelines and facilities such as artificial reefs with capacity building of fishers and local management staff.

13002 Abstract Proceedings

Role of SEAFDFEC/MFRDMD toward food security in the Southeast Asian region

Raja Bidin Raja Hassan, Southeast Asian Fisheries Development Center (SEAFDEC), Malaysia

The Marine Fishery Resources Development and Management Department (MFRDMD) is the 4th department under the Southeast Asian Fisheries Development Center. Located on the east coast of Peninsular Malaysia, facing to the South China Sea, one of the richest marine ecosystem in the world. Marine resources especially fishes need proper conservation and management regimes in order to sustain their exploitation for food security. Therefore SEAFDEC/MFRDMD is responsible to provide management guidelines and recommendations to ASEAN Member States (AMS) through workshops, regional consultations, meetings and bilateral dialogues. Furthermore in many cases, especially for shared stocks, a single country fish resources management is unable to handle migratory stock effectively. MFRDMD as accorded by its mandate and mission will facilitate AMS in collaboration with other local and international organizations to minimise impact that threaten food security. With increasing number of population in this region, a greater challenge need to be addressed and will remain the ultimate focus for SEAFDEC/MFRDMD in future. Expansion of MFRDMD roles are inevitable especially when dealing with marine fishery resources development and management program.

13003 Abstract Proceedings

Fish less, earn more: An experience of Japanese cooperation in Senegal

Naohiko Watanuki, OAFIC Co., Ltd., Japan

Since 2003, the Japanese Technical Cooperation has been working on the management of octopus fisheries in Senegal under the slogan of “Fish Less, Earn More” taking into consideration the current state of health of the octopus resources. The most challenging situation is the unavailability of price difference between good and bad quality octopus. A Senegalese octopus is somewhat poor in quality because of the improper handling after they are caught and are seldom exported to Japan where sashimi and sushi are abundant. The sources of octopus in Japan are usually from Morocco and Mauritania. Europe buys Senegalese octopus where they are being cooked in various ways. The behaviors of Senegalese fishers and fish dealers are unchangeable because of the unavailability of price difference. Therefore, to change the traditional way of pricing, add value to octopus, and diversify its export market, we conducted technical demonstration with the fisheries administration and the fishing company. One of the methods used was comparing the octopus caught by pot fishing and handled carefully using ice and plastic bags with an ordinary octopus. The study shows that there was a noticeable difference between the two octopuses in terms of quality as well as in prices offered by the company. We therefore assume that fishers and fish dealers will be able to earn more by improving the quality of octopus. Diversifying export markets is an important strategy since Senegalese economy depends heavily on fishing industry.

13004 Abstract Proceedings

Quantitative evaluations of competitive races to fish in fisheries under output controls

Yudai Hanzawa1 and Kazumi Sakuramoto2, 1Fisheries Research & Education Agency, Japan and 2Tokyo University of Marine Science and Technology, Japan

As one of output controls which restrict the number of catches, TAC (Total Allowable catch) system has been used since 1997 in Japan. This system, however, is often criticized because setting upper catch limits can cause competitive races for fish. Besides, as catches quotas are allocated to each area based on the past records in Japan, another type of competitions, those for catch quotas in the future can happen. It is difficult to quantify the effects of these two types of competitions because it is unclear what causes them. In this research, we firstly defined the condition where each type of competition would happen. Then, we tried to quantify their effects on catches separately by using Bayesian state-space model, which predicts the Japanese Sardine monthly catches caught by large- and medium-scale purse seine fishery. In this model, these competitions are regarded as reactions to catch quotas, and considered in these models in addition to stock and seasonal variation. Moreover, we also consider the effects of the Great East Japan Earthquake. All these variables vary according to the number of stock or time series and are analyzed at the same time by Bayesian estimation. The results indicated the competitions to upper catch limits were not found in any period. The other type of competitions, the reactions to catch quotas in the future contributed some of catches (3,368 tons in 2009, and 2,097 tons in 2010). It is important because if there were no these reactions, catch quotas for next three years would decrease 1.5 % on average. That means more than 15,000 tons decrease as a whole. These reactions are reasonable for all fishers and can happen not only in TAC system, but also in other output control such as IQ (Individual Quota) management.

13005 Abstract Proceedings

Fishing quotas can be justified under common price of shared resources?

Takeshi Ogawa, Senshu University, Japan

Using differential games, this paper analyzes output controls of internationally shared renewable resources such as transboundary natural forests. A two-country, two-good (that is, shared-resource and nonresource based goods) general equilibrium trading model is employed for this purpose. Each country noncooperatively decides its extent of utilization of shared natural forests via welfare maximization of the country considering the other country’s utilization and the shared stock of natural forests. In this context, economists may have hitherto posited that both countries will choose incomplete specialization; that is, both countries not only use shared natural forests but also manufacture nonresource based goods. However, even if the solution concept is chosen by not only open-loop type but also Feedback Nash solution which is economically suitable but difficult to derive some result, this paper shows that it cannot occur that both countries choose incomplete specialization along the transition process. This means that at least one country chooses complete specialization. If an incomplete specialization equilibrium occurs in both country contexts, at least one country does not follow noncooperative welfare maximization. The key conditions are that the shared resource good’s price and market is common, that each country focuses on price, and that each good cannot be retained. Therefore, the results apply in the context of another shared resource good, that is, shared fisheries, for instance, tuna or eel. This presentation shows whether this analysis has robustness or not, in repeated games, in the formulation of increasing return to scale production/harvest functions, for introducing various kinds of tariff/tax/subsidy to break internationally common price.

13006 Abstract Proceedings

Economical feasibility of chlorella and dried artemia-based artificial feed for ornamental koi fish (Cyprinus carpio haematopterus) larviculture: case in Indonesia

Fahma Fiqhiyyah Nur Azizah and Gede Suantika, Institut Teknologi Bandung, Indonesia

Feed is an important component in aquaculture. One of the factors that influence high cost of feed is the price of its raw materials, such as fishmeal. The alternative complements were microalgae and dried-Artemia. Based on previous research, artificial feed based Chlorella and Artemia has better performance for koi carp larvae compared to commercial feed with survival rate of 72,11% and 70.22%, respectively. In order to meet industry sector, feed must be carried out its economic feasibility. Therefore, research on the economic feasibility of artificial feed based on Chlorella and Artemia for koi ornamental fish larvae was done.
Economic feasibility included calculation of investment cost, operational cost, raw material cost and cost of goods sold. All prices were taken from market survey. The calculation was used for the analysis of PBP, NPV, IRR, and B/C Ratio, both for control and experiment feed. For control, operational costs were assumed to be 40% of sale price. Production capacity was assumed 2000 kg/day for small scale industry.
Based on the calculations, the overall Chlorella production cost from cultivation to powder reached US$0.60/kg, dried-Artemia’s price was US$22.45/kg. Other raw materials contributed about US$0.41/kg of feed. Feed control (commercial) was purchased for US$4.10/kg. The investment cost included land, plan building, and feed equipment reached US$69,403.57, the operational cost included labor, management fee, and depreciation reached US$17,277.87/month. Cost of goods sold of feed was US$1.86/kg/kg, and the sale price was assumed 150% of COGS. From the calculation of profit and loss and cash flow, it was calculated that NPV for 10 years was US$3,703,642.62, PBP occurred at 3.08 years, IRR with 10% discount rate was 28.59%, and mean B/C ratio for 10 years was 2.61. Theoretically, it can be concluded that artificial feed based on Chlorella and Artemia for koi carp larvae was economically feasible to be produced at industrial scale.

13007 Abstract Proceedings

SH"U"N Project: A science outreach to fisheries sector and consumer

Mitsutaku Makino, Hiroki Wakamatsu, Shingo Watari, Masashi Kiyota, Shiroh Yonezaki, Juri Hori, Hiroshi Zenitani, Toyomitu Horii, Shinji Uehara, Hiroyuki Shimada, Masaki Kananiwa, Keiichi Mito, Tatsu Kishida and Yoshioki Oozeki, Japan Fisheries Research and Education Agency, Japan

Japan Fisheries Research and Education Agency launched a science outreach activity named SH”U”N Project (Sustainable, Healthy and “Umai” Nippon seafood project). Based on the scientific information on 1) stock status, 2) ecosystem impact, 3) fisheries management, and 4) regional sustainability, as well as 5) health and seafood safety information, the sustainabilities of the Japanese fisheries resources are assessed. The results are reviewed by the External Review Committee, and the public comments are gathered to validate the results. All the data and results of above processes are open to public, and we hope this information will be utilized by fishers’ organization or local government officers to apply to fisheries certification schemes (e.g., MEL-Japan or MSC, etc.) or to develop local fisheries management strategies. For the consumers, a smartphone application software is developed to show the recommendation list of fish species for todays’ dish. Also, consumers can search the fish species, and store their purchase/consumption records. Such data will be utilized by the Agency to understand the research needs and to develop the Agency’s research strategy.

13008 Abstract Proceedings

Visualising characteristics of knowledge and experiences for fishery management activities in Japan

Shion Takemura and Mitsutaku Makino, Japan Fisheries Research & Education Agency, Japan

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.

13009 Abstract Proceedings

Where do risks in shrimp farming come from? Empirical result from small-scale farmers in East Java, Indonesia

Riski A. Lestariadi and Yamao Masahiro, Hiroshima University, Japan

This study was conducted to analyze the farmer's perception of risk and risk management strategies in small-scale shrimp farming. Data were obtained in 2016 from interviews with 166 shrimp farmers in the northern and southern coastal of East Java, Indonesia. The factor analysis was used in data reduction to identify a small number of the factor related to sources of risk and risk management strategies in this study. The results found 32 sources of risk. Shrimp price volatility and high mortality due to shrimp diseases were the most important risk sources in study areas. Regarding risk management strategies, the result showed that small-scale shrimp farmers’ have practiced 35 strategies to cope with the risk in their farms. The top five strategies in small-scale shrimp farming included strict management of water quality, strict feeding management, applying better management practices, preventing shrimp diseases by regular checking, and reducing brackish water pond size. The exploratory factor analysis showed that the risks in small-scale shrimp farming derived from 8 factors consisted of input and pond preparation; finance and credit access; production; personal; harvesting and marketing; weather and environment; policy and institutional; and business environment. These factors explained 73.1% of the observed variation. Furthermore, nine factors sorted for risk management strategies which included diseases prevention; education and technology improvement; the input of production; farm management; government support; risk sharing and insurance; financial; household adjustment; and alternative income sources. To conclude, identifying such risk sources and risk management strategies could contribute to a better understanding of the nature of risk and uncertainty in small-scale shrimp farming. The research findings are expected to contribute for policymakers to design policies and regulations to support the sustainability of shrimp industry, especially in small-scale level.

13010 Abstract Proceedings

The activities of IFRDMD

Arif Wibowo, Southeast Asian Fisheries Development Center (SEAFDEC), Indonesia

Inland fishery is one of the important components of the economies in the Southeast Asian region and its contributions in rural communities are particularly important in poverty alleviation, creating employment, food security, livelihoods, nutritional and societal well-being. Considering the importance of inland fisheries, the SEAFDEC (consists of 10 ASEAN member states (AMS and Japan) established a regional center for inland fisheries. IFRDMD was established to serve as a centre for providing guidelines for the proper development and management of inland fishery resources of AMS, which could consist of freshwater, saline water and mixture of both, and distributed throughout the land such as rivers, lakes, floodplains, reservoirs, wetlands, estuaries, and inland saline systems. The role of the department includes contributing to sustainable management of important commercial species under international concerns within Southeast Asian region. The SEAFDEC/IFRDMD collaborated closely with the Indonesian research institute for inland fisheries (RIFF). The institute provide financial support for management and operations department, supplying human resources, access to a variety of multidisciplinary laboratory facilities and earmarking of corresponding research.
Under the support of the Japanese Government through the trust fund (JTF) thrust 1, developing and promoting responsible fisheries for poverty alleviation and food security, IFRDMD carries out research program on promotion of responsible utilization of inland fisheries in Southeast Asia for improved management of inland fisheries in the region. IFRDMD emphasized the need to undertake two areas of activities, which are improvement of data collection, and enhancement of governance through the application of ecosystem approach to fisheries and co-management.

14001 Abstract Proceedings

25 years of PICES: a trajectory of the international cooperation on marine science in the North Pacific Ocean

Tokio Wada and Hiroya Sugisaki, Japan Fisheries Research and Education Agency, Japan

PICES, the North Pacific Marine Science Organization, was established in 1992. The purpose is to promote the marine science in the North Pacific Ocean. Since the establishment, sustainable use of marine living resources and conservation of marine ecosystems have been common interests to the contracting parties; Canada, China, Japan, Korea, Russia, and USA.
Over the past quarter century, PICES has provided many scientific accomplishments through inter-disciplinary collaboration by scientists. Description and comparison of various marine ecosystems, evaluation of population fluctuation of commercial fish stocks under climate change, and development of a numerical model forecasting ecological response to climate change are the typical examples. These accomplishments have stimulated marine science in the contracting parties and contributed their marine policies.
Capacity building for postgraduate students and early carrier scientists is also an important role of PICES. Several approaches, such as summer schools, training for professionals, and internships in the secretariat, have been conducted under the cooperation with the contracting parties and other scientific organizations.
Japanese government and scientists have contributed the scientific accomplishments and international cooperation. Two PICES projects on enhance the capability for harmful algal blooms and fisheries management in developing Pacific Rim countries were funded by Japanese government. At the time of the Great East Japan Earthquake disaster, Japan support the collaborative studies on the distribution and diffusion of radioactive chemicals and tsunami debris and their ecological impacts by providing information and funds. Since the global environmental changes become the menace for human life, it is necessary to establish a mutual support relationship between marine ecosystems and human well-beings. Japanese traditional approaches, such as “Sato-umi”, are good examples of human activities harmonized with marine ecosystems, which should be more emphasized through the discussions in PICES.

14002 Abstract Proceedings

Fishery cooperation in the South China Sea under the the strategy of blue growth

Peng Wei, ShangHai Ocean University, China

Blue growth theory has played an important role in the development of marine industry in Europe, but also provides a theoretical basis and guidance for the sustainable development of marine industry in the world. The South China Sea, as one of the largest sea areas in the world with the largest number of disputed areas, is facing the threat of fishery habitat destruction and fishery resource decline. China is actively pursuing fishery cooperation with the ASEAN countries in the South China Sea in accordance with the Blue Growth strategy, to protect the ecological environment and conserve fishery resources, in order to realize the sustainable development of the South China Sea fishery and other industries. This paper systematically expounds the theory of Blue Growth, analyzes the basis of the fishery cooperation of the countries in the South China Sea, and puts forward the idea of the South China Sea fishery cooperation under the strategy of Blue Growth.

14003 Abstract Proceedings

Activity of the Research Center for Marine Education (RCME), the University of Tokyo

Kaoru Kubokawa, Yoshihiro Niwa, Koudai Taguchi, Mitsumasa Hioki and Daiki Kato, The Tokyo University, Japan

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

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