Program

The sand lance is perciformes fish that lives on the coast of Japan. Sand lance is one of commercially valuable fishery populations in Sanriku area. The peak spawning season is an early period in calendar year, and then the fishery by dip nets starts in February for adult sand lances, which continues until June, and that for juvenile sand lances is operated from March to May. As an atypical ecological life history of this species, they have an aestivation for juveniles and adults from July to December. Until December, a part of juveniles become mature, and they produce juveniles in the next year. Fortunately, independent catch-par-unit effort (CPUE) data are available, which motivate to develop stage-based models for this population. Here, the objective in this research is to reveal dynamics of the stock by using catch and effort data of this stock. Firstly, nominal CPUE series from 1994 to 2015 were standardized using generalized linear models to draw year-trend of abundance. Then stage-based models were constructed to account for monthly survival probabilities as well as a reproductive process. For stock-recruitment relationship, different functions (linear, Ricker, Beverton-Holt and Hockey-stick) were assumed and tested. The maximum likelihood method is employed for estimating parameters in the models although some of parameters such as maturity rate for juveniles, were assumed to be known. As a result, Ricker stock-recruitment model was selected as the best model. Regarding the biomass levels, those both for juveniles and adults tend to be increasing in recent years. The results did not take into account any predation effects, which might be influential to the population dynamics. Note that the analysis here didn’t specifically account for the impact of the 2011 East Japan great earthquake.

Purpose:
The greater amberjack Seriola dumerili is an important species for aquaculture in Japan. Juveniles of S. dumerili in the South China Sea (SCS) or East China Sea (ECS) are captured for the seedlings. However, the information of the population structure of S. dumerili from SCS to ECS is very limited. In this study, we investigated the population structure of S. dumerili using samples from three different areas in ECS by mitochondrial DNA (mtDNA) sequencing.
Materials and methods:
Larvae, juveniles and adults of S. dumerili were collected around the coast of Kyushu, Japan, southern part of ECS (SECS) and Taiwan. From the proper tissues of 66, 21, and 78 fish for each group, DNA was extracted using the DNeasy Blood & Tissue kit. The mtDNA control region of S. dumerili was amplified by PCR and PCR products were sequenced directly. The multiple alignments of obtained sequences and phylogenetic tree construction were performed using MEGA. The haplotype divergence was calculated using DnaSP. Pairwise Fst value was calculated using Arlequin.
Results:
We obtained the mtDNA control region (840 bp) sequences from 165 fish. The number of haplotypes were 58 (Kyushu), and 20 (SECS) and 66 (Taiwan), respectively. High values of haplotype divergence (0.99 for each) were detected among three population. Pairwise Fst tests revealed no significant differences among three groups. The neighbor-joining tree for all fish represented the mixed distribution of three groups. These results postulate the possibility that S. dumerili in the East China Sea is mainly derived from one population.

The ayu Plecoglossus altivelis has a wide range of distribution over the Japanese Archipelago including subtropical islands on the southernmost. Latitudinal variation in the environmental conditions possibly affects the life history schedule of ayu in association with spawning or upstream migration. On one hand, ecological difference in ayu within a narrow range of latitude is less well known. In this study, we focused on geographical variation in the early growth of ayu comparing between habitats with different latitude, and those faced with Japan Sea and Pacific Ocean. The growth pattern of ayu was estimated using the otolith chemical analysis combined with the otolith increment analysis. A total of 75 individuals collected in 2001 from 15 rivers were divided into 3 groups by sampling localities: low latitude group (LLG) between N 31° and N 35°; middle latitude group (MLG) between N 36° and N 40°; high latitude group (HLG) between N 41° and N 45°. Additionally, MLG was examined to compare the growth pattern between east-west gradient of Japan Archipelago as divided into 2 group; Japan Sea group (JSG) and Pacific Ocean group (POG). Between 3 latitudinal groups, HLG showed lower growth rates during juvenile stage than LLG. LLG experienced higher temperature than HLG during juvenile period that may influence the growth rates of juveniles. Further, JSG showed lower growth rates than POG, although JSG experienced almost same temperature with POG. The tidal difference is larger in Pacific Ocean than in Japan Sea. Tidal condition can have an influence on the abundance of plankton in the surf zone and consequently on the prey availability of juvenile ayu. It is clear that complex factors were involved in the early growth of ayu.

OBJECTIVE
Since the interests and demands for ecosystem modelling have been growing recently, there have been many case studies related to it. The ecosystem modelling requires a lot of data like time series of catch and abundance as well as information on inter-specific relationship. In practice, however, it is hard to obtain all the necessary data, hence in most cases statistical modelling and estimation tend to be conducted with lack of data although the impact of lack of data on the analyses has not been well addressed. Here, we aim at evaluating how the impact is crucial in prediction of the ecosystem dynamics and management and then seeking possible ways to overcome this problem through a simulation study.

MATERIALS AND METHODS
Firstly, as operating models to generate simulation data, a family of models are constructed and conditioned by mimicking an ecosystem in of the Barents Sea. Then, among several potential issues in the ecosystem modelling such as existence of ghost populations, limited data availability, and time series bias of data, we focused on the issue of ghost populations. Finally, simulated data were analyzed by using an assessment model, EwE, which is commonly used as ecosystem models, and the effect of lack of data was evaluated by comparing the estimated population dynamics by the model with the true dynamics.

RESULTS AND DISCUSSION
In the scenario of ghost populations, it was suggested that their impact depended on their ecological niche. When we chose a species having small niche as ghost species, the difference in dynamics between the true and estimated populations was ignorable. On the other hand, when the ghost species have large niche, there were significant differences overall. Results of other issues will be shown in the poster session.

Heart rate (HR) is one index expressing not only the metabolic rate but also autonomic nervous activities using time series analysis, where spectral characteristics are predictors of physiological conditions of fish. So far, fish electrocardiography (ECG) can record for several days, even under an unrestrained condition, but complicated processes must be used to detect HR from the ECGs because data logger loaded on a fish records only data points of heart electric potentials. It is necessary to inspect a series of consecutive HR manually to confirm correct detection of QRS complexes, even when using a software program to detect R-R intervals automatically. In this study, it was recorded ECG of fish (Pagrus major and Seriola quinqueradiata), which are major industrial fish species in Japan, reared in a net pen for several days. A cepstrum, which has been used for vocal processing and which has recently been applied for analysis of human ECG, was applied to express changes of HR. Results were compared with those obtained using typical algorithms to detect assent points accompanied with QRS in the heart electric potentials. The quefrency, which was calculated in a cepstral calculation process, expressed the changes of consecutive HR with no filtering or inspection procedures, even using unclear ECG records. Peaks of HR distribution obtained from conventional algorithm and quefrency were concordant. Fish HR in the net pen changed according to the time of day and past days. In addition to detection of HR changes, the cepstrum obtained using raw ECG data showed a slope in the spectrum, indicating fluctuation in the HR variation.

Masu salmon, Oncorhynchus masou, endemic to the Far East, is an important fisheries resources in Russian, Japan and Korean coasts surrounding the Sea of Japan and the Sea of Okhotsk. Understanding of the genetic characteristics in masu salmon is important for their conservation and fisheries management for sustainable use. We examined the genetic population structure of masu salmon in the Sanriku-region, Pacific coast of northern Honshu, Japan, using about 500 individuals of 11 collections, i.e. 6 rivers and 5 coastal areas in Iwate prefecture, with mitochondrial and microsatellite DNA markers. Masu salmon in Sanriku were genetically differentiated from those of Hokkaido and other regions in Honshu. Pairwise population FST estimates favored distinct genetic differentiation among the Sanriku river samples. However, no genetic differentiation between coastal and river samples and within coastal samples were inferred from the FST estimation. These results suggest that masu collections examined herein are reproduced in the Sanriku-region, randomly distributing or homogeneously migrating along the Sanriku coast, and that they contribute as the region’s fisheries resources under little hatchery activities.

LED lighting technology is well developed and is used in fishing activities such as lifnet, purse seine and squid jigging. LED is also used to reduce turtle bycatch on the gillnet and longline fisheries. The use of green LED in gillnet fisheries can reduce turtle bycath by 60-75% without reducing the target catch. However, information of turtle behavior response to LED light is still very limited. The objective of research is to analyze the response of green sea turtle (Chelonia mydas) behavior to green LED light. The behavioral response of turtle (n = 11, Curve Carapace Length (CCL) size of 45-50 cm, aged 12-27 days) to the green LED lights (Electrolume green-single color , NOAA) was observed using infrared video camera setup 245 cm above the experimental tank. Behavioral response of green turtle was observed by comparing the movement activity in the dark condition (without light) as a control and green LED light. Movement activity dan behavior response was analyzed using frame by frame video analysis and scoring of the turtle movement activity as an index . The results showed the use of green LED lights was significantly affected the turtle behavior. The turtle showed a very active response to the green LED light with the index range of 3.71 - 3.76, whereas the control showed a passive response with an index range of 1.10 to 1.64. Active response of turtle to green LED showed a high intensity in entering the shelter compared to the control.

Arctic Pacific is experiencing profound ecosystem changes. Warming and reduction of summer sea-ice cover have large implications for the ecosystem functioning. In this sense, Chukchi Sea is a hotspot where such changes in the system are already visible, such as shifts in the phenology, species composition and northward faunal range expansions indicate a changing system. Tanner crab, Chionoecetes bairdi, is a commercially important species distributed on the continental shelf of the North Pacific Ocean and Bering Sea from Kamchatka to Oregon. At its northern distribution limit in the SE Bering Sea it has sustained a historical lucrative fishery, but has been shown signs of overfishing since 1975. In this paper, using plankton samples collected in the Pacific Subarctic/Arctic sector, we describe the distribution, abundance, and larval stage composition of C. bairdi during summer of two contrasting periods: 1991-1992 (sea-ice), 2007-2008 (sea-ice retreat). A significant decrease in abundance from cold to warm period was found, that may be related with the stock decline. Interestingly, intrusion of zoea II larvae of C. bairdi were observed for the first time in the Chukchi Sea during 1992. We suggest that the long planktonic phase (3-5 months), in combination with the oceanographic circulation, may facilitate eventual long-distance transport. Recent trawling surveys have not detected the presence of adults suggesting that these larval transfers have been abortive. However, if the environmental changes continue it may favor the establishment of permanent populations of C. bairdi, that can change unpredictably the function of benthic ecosystems in the Arctic.

The Indo-Pacific bottlenose dolphin (Tursiops aduncus) occurs around Mikura Island. Since its population size is small around 140, an individual identification survey is possible and has been conducted since 1994 for this population. In this survey, researchers dived and observed scars on the dolphins’ body surface and chips of their fin as clues for individual identification. Through this survey, observed information was recorded almost daily for each dolphin and can be regarded as mark re-sighting data. To take advantage of this rich amount of information about individual and observation time, we developed a state-space model to describe the daily live/death status as a hidden Markov process and the detection/missed-detection as an observation process, where the parameters of interest are the survival and detection probabilities. We also assume the time-varying survival probabilities and examined whether the survival probability depends on life-stage. To estimate those parameters and deal with many latent variables, we used a Bayesian method with Markov chain Monte Carlo simulation. The result indicated that the annual survival probability might have been almost homogeneous over the study period and above 95% except for neonate (age younger than or equal to 1). The survival probability for neonate tends to be lower than that for other elder groups and has been changing dynamically. More details of the result will be introduced on the poster. As a future work, based on the findings of this study, we will investigate the relationship between the population dynamics and the survival probability, which might contribute to the construction of comprehensive model for this population.

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.

[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.

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.

Germline stem cells (GSCs), which can differentiate into eggs and sperm, are thought to be key players in the fish gonadal sexual plasticity. To understand the molecular and cellular mechanisms of GSCs, we focused on the pou5f1(Oct4) gene, one of the major gene involved in the stem cell state in vertebrates, and compared its expression in gonad between the bamboo leaf wrasse Pseudolabrus sieboldi (hermaphroditism) and the Japanese anchovy Engraulis japonicus (gonochorism). Using degenerate PCR followed by 5’- and 3’- RACE, the cDNA sequences containing the complete open reading frame (ORF) of pou5f1 genes were isolated from the bambooleaf wrasse and the Japanese anchovy. In both species, the real-time PCR analyses showed higher expression levels in adult ovaries than adult testis. In situ hybridization (ISH) also revealed that the pou5f1 mRNA expressed in the oogonia and oocytes of the ovaries, as well as in spermatogonia of the testis. Our analysis demonstrated similar pattern of pou5f1 expression in the gonads of both the hermaphrodite and gonochoristic species. Additionally, adult female and male Japanese anchovy were treated with aromatase inhibitor (AI) and estrogen, respectively, and the effect of sex steroids on pou5f1 expressions was analyzed. Gonadal pou5f1 expression level dropped sharply by 7 days of AI treatment in female, while E2 treatment upregulated the expression in male. In the Japanese anchovy, sex-specific expression was modulated by sex steroids, thus implicating that the GSCs of the adult Japanese anchovy are capable of differentiating into female and male, under the influence of the sex steroids secreted from gonadal somatic cells.

The family Sillaginidae are widely distributed across Indian and Western Pacific Oceans. As a hotspot of research, taxonomy and diversity of Sillaginidae is still chaotic due to the similarity of morphological characteristics and color patterns. Based on the elaborate descriptions about the taxonomy of Sillaginidae by Mckay (1992), many researchers gave contributions to the studies on this family successively. At present, 35 species of 3 genus have been reported, four of which are new species found in recent years. Moreover, the cryptic diversity which is found existing widely in Sillago sihama is crucial to the systematization of Sillaginidae. However, systematic studies of Sillaginidae in the coast of China are still lacking.
To expose the biodiversity and taxonomy of Sillaginidae in China, we checked more than 7000 individuals collected from the coast of Chinese waters based on morphological features. As a result, a total of 12 species belonging to genera Sillago were reviewed. S. aeolus, S. sihama, S. japonica, S. asiatica, S. sinica and S. ingenuua were common, S. chondropus and S. parvisquamis were rare in China. S. shaoi was new species which we reported in 2016. S. microps was endemic species in Taiwan. S. megacephalus and S. boutani were controversial, because of the limited samples and ambiguous original descriptions. In addition, three suspected cryptic species need further studies. We provided detailed morphological descriptions as accurate criterion for identifying Sillaginidae species in China.

Coral reefs are heavily influenced by the human activities through fishing activities, pollution and habitat loss throughout the world, including Indonesia, which could affect the associated organisms like rabbitfish (Siganidae). Genetic diversity also can provide some information about long-term condition in aquatic organism. The study was conducted to assess genetic diversity of rabbitfish (Siganus canaliculatus) accross environmental gradient in Jakarta Bay and Kepulauan Seribu. A total of 31 individuals was collected in study sites. A molecular markers mitochondrial Cytochrom oxidase 1 (CO1) was used to amplify DNA. The result of phylogenetic tree formed 2 major clade is clade 1 and clade 2. AMOVA analysis showed a high Fst value 0.38, this proves that the population of Siganus canaliculatus in north zone (National Park) and south zone Seribu Island is different significantly and both of populations was closed geneticly. The genetic diversity ranged 0.007-0.062 and was including low category. Value of genetic diversity (Hd) of S. canaliculatus contained in Pulau Semak Daun and Pulau Damar is equal to 0.50 and nucleotide diversity ( π ) is the highest for 0.104. Genetic diversity of S. canaliculatus categorized as low 0.076 to moderate 0.500. Environment and humans seem affect the value of genetic diversity.

The Bigeyed Greeneyes(Bigeyed G.) Chlorophthalmus albatrossis (Aome-eso in Japanese) belonging of　Genus Chlorophthalmides、Family Chlorophthalmidae, Order Aulopiformes distributed at the Pacific Ocean from Suruga Bay of Shizuoka Prefecture to south coast of Japan and to Palau Submarine Ridge and East China Sea. Suruga Bay is the one of the largest and the deepest Ocean Bay. Entrance of Suruga Bay is about 56 km, the plot of 60 km long from front to back and the depth from 5 km coast is 1,000 m and entrance depth is 2,800 m. A lot of fish and invertebrate migrate with Japan (Kuroshio) current to Suruga Bay. Suruga Bay developed shallow fisheries anciently and also deep sea trawl are developed of 150 to 400m depth, and also deep sea basket of 800m depth are developed. Deep sea trawl nets at Suruga Bay are permitted from May to September (150n days). Raw Bigeyed G. often to sell at the local Fish market and supermarket etc. at Shizuoka Prefecture.
Present study, investigated EPA, DHA and total fat contents of Bigeyed G. skeletal muscle were examined.
Materials of Bigeyed G. were collected at the Suruga Bay by the deep sea trawl on (1)2014-Dec.,(2) 2015-Jan.,(3)-Feb.,(4) -March and (5) -April. Each month materials were １lot 5 or 6 specimens, and 5 or 6 lots each month of (1) to (5)..
Total skeletal fat contents of Bigeyed G. indicated the highest month of January. December 2014 was the lowest in these 5 months. The highest January total fat content decrease from February to April gradually. This decrease of Total fat value to April 2015 seems to be related with breeding Season.
In the case of EPA and DHA contents of Bigeyed G. in Dec, 2014 to April 2015 seems to be 2 gaps of Dec.2014 and March 2015. These two gaps seems to be related with the 2 spawning seasons of Bigeyed G..

The recruitment of walleye pollock Gadus chalcogrammus Japanese Pacific stock (JPS) exhibits large annual fluctuations. A high growth rate of the 2008-2011 year classes of JPS larvae in the yolk-sac stage resulted in a high larval abundance in the main spawning ground at Funka Bay. Here, we examined the influence of egg size on the survival rate of larvae (maternal effect) by measuring hatch check diameters (HCD) of the sagittal otoliths. We collected eggs and larvae of JPS from 2013 to 2015 using a plankton net (80cm diameter). We measured water temperature, salinity and counted the copepod nauplii of the prey collected by a Van-Dorn bottle. We then measured the egg diameters, embryonic notochord lengths, yolk-sac volumes, and otolith diameters in eggs in the final stage of development and HCD and otolith daily increment in the larvae. We found that larvae derived from large eggs tend to hatch with notochord lengths, yolk-sac volumes, and sagittal otoliths becoming larger in all three years, and HCD could be used back calculate egg diameter of larvae after hatching. There was also a significant positive relationship between HCD and larval age in January to March 2014 (r² = 0.42, p < 0.001) and 2015 (r² = 0.24, p < 0.01), with no significant difference in slope between the 2 years (p = 0.76) but a significantly greater intercept in 2015 (p < 0.001). The mean egg diameter gradually increased from 2013 to 2015 with increased age of the parental fish stock. There was almost no difference in environmental conditions sampling periods, with the exception of March 2015, which was particularly cold. In 2015, it was mainly older parent fish (≥ 6 years old) and repeat spawners that produced large eggs, indicated that egg size may contribute to survival of larval JPS.

The outbreak process of the bacterial cold-water disease among ayu Plecoglossus altivelis altivelis in the Tama River watershed was examined to infer the mechanism of the outbreak in the natural ecosystems. We conducted the epidemiological survey on the pathogen of the disease, Flavobacterium psychrophilum, and the analyses of carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N), for the ayu reared in the seed production facility and the riverine ayu collected in the watershed. The pathogen was frequently detected in both the seed-production ayu and the riverine ayu collected in a tributary where the recreational fishing of ayu is popular, suggesting that the release of the infected seed-production ayu was a main source of the infection among the ayu in the watershed. Based on the stable isotopic distribution, the riverine ayu collected in the tributary was distinctly divided into two groups. The isotopic distribution of one group, characterized by depletion in ¹³C and enrichment in ¹⁵N, was almost identical to the distribution of the seed-production ayu. Therefore, this group was identified as the seed-production ayu just after the release in the watershed, and its isotopic distribution likely reflects the formula feed in the rearing facility. By contrast, the isotopic distribution of the other group, characterized by enrichment in ¹³C and depletion in ¹⁵N, was close to the distributions of epilithic organic matter and aquatic insect collected in the tributary. It is likely that the isotopic distribution of this group reflects the natural diets through the feeding behavior in the tributary. The outbreak of the disease was found not only for the latter group but also for the former, thus suggesting that the infected seed-production ayu became ill just after the release in the river because of the environmental stress.

Stable isotope (SI) analysis has been used for studies on aquatic ecosystems. A small toothed whale, narrow-ridged finless porpoise Neophocaena asiaorientalis, is a top predator of coastal marine ecosystems in Japanese Archipelago, and the species is protected by Japanese law. SI analysis of tissue samples from the carcasses of stranded dead porpoises can provide useful dietary, regional and trophic information in their habitats. However, such samples are under various conditions with an unknown period of time after death, and have a risk to misunderstand SI values by deterioration such as decomposition and/or autolysis. To investigate the effects of decomposition on SI values in muscle tissues of the stranded finless porpoises, we measured carbon and nitrogen contents of the muscle samples by elemental analyzer, and compared the contents between fresh bycatch samples (n=47) and stranding samples (n=197) collected from individuals from 1994 to 2017 in Ise-Mikawa Bay, central Japan. Mean±SE (CV) of carbon and nitrogen contents per 1 mg of muscles in bycatch individuals were 0.462±0.0016 mg (0.024) and 0.147±0.0005 mg (0.022), respectively. Variations of carbon and nitrogen contents were extremely small. They were not significantly different by sex and month and uncorrelated with body length. These indicate that muscle carbon and nitrogen contents are constant in freshly collected samples. On the contrary, corresponding values for stranding samples were 0.442±0.0036 mg (0.116) and 0.134±0.0013 mg (0.135), respectively, and CVs are 5-6 times higher than bycatch samples. This discrepancy between two different sample sources is considered to be caused by deterioration after death. It suggests that contents of carbon and nitrogen in muscle tissues may be used to exclude inappropriate deteriorated samples with unknown levels for SI analysis of necropsies from stranded finless porpoises.

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.

Sexual reproduction is an important biological phenomenon required for establishing coral populations. To date, however, the molecular and cellular mechanisms underlying coral sexual reproduction remain largely unknown. We then performed a differential screen (suppression subtractive hybridization) and a transcriptome analysis to identify genes related to gametogenesis in the stony coral Euphyllia ancora. As the oogenesis-related gene, we identified a clone encoding a novel red fluorescent protein cDNA of E. ancora (named EaRFP). Microscopic observation and quantitative RT-PCR revealed that EaRFP is almost exclusively expressed in the ovary of the adult coral. The combination of the ovarian-cell separation method and the RT-PCR analysis revealed that the oocytes, but not the ovarian somatic cells, are the cells expressing EaRFP. Immunohistochemical analysis revealed that the expression of EaRFP starts in the early stage of the oocyte and continues until the maturation period. Furthermore, recombinant EaRFP was shown to possess an H₂O₂ degradation activity. These results raise the possibility that EaRFP plays a role in protecting the oocytes from oxidative stress from the early to late stages of oogenesis. By contrast, in male colony, green fluorescent was observed in the testis. RT-PCR analysis demonstrated that testis expressed a novel green fluorescent protein (named EaGFP). The combination of the testicular-cell separation method and immunohistochemical analyses revealed that the testicular somatic cells, but not the germ cells, are the cells expressing EaGFP. Previous studies have shown that GFP in corals plays a role in photoprotection of symbiotic algae. However, since only a few symbiotic algae are present in the testis of E. ancora, EaGFP is unlikely to be involved in the photoprotection of symbiotic algae. EaGFP might have other functions in the testis. The present study provides the first evidences for the potential involvement of FPs in coral gametogenesis.

Neuropeptides act as modulator and hormones, and play important roles in various biological processes of animals. To date, the presence and the functions of neuropeptides, including the distribution of neuronal cells in coral body are still largely unknown. To get a better understanding of the physiological functions of neuropeptide in stony corals, we established a transcriptome database of the stony coral Euphyllia ancora, and explored the genes possibly encoding the precursor proteins of neuropeptides. Through the course of data mining, we identified a gene encoding Glycine-Leucine-Tryptophan-amide family neuropeptides (GLWamides) that have been shown to induce muscle contraction (myoactivity) and larval metamorphosis in several cnidarians. By 5’ and 3’ RACE-PCR, we successfully elucidated the full-length of GLWamide precursor cDNA in E. ancora (named EaGLW precursor). Tissue distribution of EaGLW precursor by quantitative-RT-PCR analysis revealed that EaGLW precursor transcripts were highly expressed in the mouth and the tentacle in the polyp of both sexes. Immunodetection with anti-GLWamide antibody demonstrated that GLWamide-positive cells (GLWamide neurons) were mainly distributed in the ectodermal regions of the mouth and the tentacle. GLWamide-neurons were also detected in the gonads. Furthermore, GLWamide treatment was shown to induce the tentacle and the mouth contraction of adult E. ancora polyp. These results strongly suggested that GLWamide is, at least, involved in myoactivity in tentacle and mouth of the adult coral. To the best of our knowledge, this study is the first to show the presences of a gene encoding a precursor protein of a neuropeptide, neuronal cells, and its possible role in stony corals.

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.

The color of the brown alga Undaria pinnatifida after boiling is an important factor determining its marketable value. Our previous laboratory study showed that decreased nutrient availability and elevated irradiance in growth environments resulted in increases of lightness and yellowness (i.e. discoloration) of this alga. However, little is known about the optimal levels of nutrient and irradiance required to decrease these color values and the combined effects of these factors and boiling. Therefore, we conducted two culture experiments to test the effects of four nutrient levels (non-enriched and 1.25, 5, and 25% PESI enriched treatments), four irradiance levels (0, 10, 30, and 180 µmol m^–2 s^–1), and boiling on lightness L*, redness a*, and yellowness b* of this alga. L* and b* did not differ between non-enriched and 1.25–5% PESI treatments, but were lower in the 25% PESI treatment. L* and b* were lowest at 0–10 µmol m^–2 s^–1, although negative growth occurred at 0 µmol m^–2 s^–1. Interaction between irradiance and boiling on a* indicated that decreased irradiance had a positive or little effect on a* before boiling, but had a negative effect after boiling. These results suggest that 25% PESI and 10 µmol m^–2 s^–1 were the optimal nutrient and irradiance levels, respectively, to decrease the three color values of this alga after boiling.

Photosynthetic responses to temperature and irradiance were investigated on two life history stages [macroscopic sporophyte (SPO) and microscopic gametophyte (GAM)] of an edible brown alga, Cladosiphon okamuranus from Japan. Measurements were carried out by using optical dissolved oxygen sensors and pulse-amplitude modulated (PAM) fluorometer. Photosynthesis-irradiance (P–E) curves at 28 ºC revealed higher saturation irradiance of GAM (E_k = 342 µmol photons m^-2 s^-1) than of SPO (E_k = 260 µmol photons m^-2 s^-1). This can be attributed to the higher maximum net photosynthetic rate of GAM (NP_max = 25.01 µg O₂ g_fw^-1 min^-1) than of SPO (8.57), given their relatively similar initial slopes [α = 0.08 µg O₂ g_fw^-1 min^-1 (µmol photons m^-2 s^-1)^-1, GAM; 0.03, SPO]. Results of oxygenic gross photosynthesis and dark respiration experiments over a temperature range of 8–40 °C revealed similar trend in temperature responses, as well as in temperature optima for both stages (29.5 ºC, SPO; 29.7 ºC, GAM), which further support their perennial occurrence. Maximum quantum yields (F_v/F_m) after 48 h of exposure over a temperature range of 8–40 ºC were also assessed. C. okamuranus sporophyte F_v/F_m began to drop at 16 ºC, and further declined to nil at 36 ºC. Gametophyte F_v/F_m barely decreased up to 28 ºC, and yet with high values at 36 ºC. These suggest the greater tolerance of the gametophytes to high temperature, which could give this microscopic stage an advantage of survival in relation to possible increase in seawater temperature due to ocean warming.

Seagrasses often form dense assemblages and provide a number of functions within the coral reef ecosystem. In the present study, effects of irradiance and temperature on the photosynthesis of a tropical seagrass, Halodule uninervis (Cymodoceaceae) were determined by using the plants from Amami-Oshima Island, Ryukyu Archipelago, Japan, as its marginal region of northern distribution. Measurements were carried out by using optical dissolved oxygen sensors and pulse-amplitude modulated (PAM) fluorometer. Oxygenic net photosynthesis-irradiance (P–E) curves at 15 and 24 ºC revealed that there was no characteristic photoinhibition at 1000 µmol photons m^-2 s^-1. The response of oxygenic gross photosynthesis (GP) rates over a temperature range of 8–40 °C showed a gradual increase with rising temperature up to 32 ºC, and a decrease thereafter. Maximum quantum yields (F_v/F_m) after 72 h of exposure to a similar temperature range were relatively stable at temperatures between 8–28 ºC; however, values gradually declined above 30 ºC. Results of the photoinhibition-recovery experiments at 300 and 1000 µmol photons m^-2 s^-1, and at 15 and 24 ºC revealed that the effective quantum yields (Φ_PSII) were clearly depressed during PAR exposures at 15 ºC, with failure to recover in samples under 1000 µmol photons m^-2 s^-1 even after the 12-h dark acclimation, suggesting the possibility of photodamage brought about by the combined stress. This species is considered to be well-adapted to the current environment in Amami-Oshima Island. Its distribution at higher latitudes is restricted by the cold water temperature and strong irradiance in winter.

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.

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.

Heat shock response is essential for the viability of all living organisms. One of the most important heat shock response is induction of heat shock proteins (HSPs) which assist protein folding and prevent protein denaturation, and these expression is mainly regulated by heat shock transcription factor 1 (HSF1).
In vertebrates, sex is normally determined by genotype. However, in poikilothermic vertebrates, including reptiles, amphibians, and fishes, sex determination is sometimes influenced by environmental factors, such as temperature. Little is known about the molecular mechanisms underlying environmental sex determination. Medaka (Oryzias latipes) is a teleost fish with an XX/XY sex determination system. Previously, it was reported that high water temperature (HT) treatment during the sex differentiation period causes masculinization of XX medaka and rapidly induces the expression of hsp70 and hsp27 in XX fish. However, the roles of HSPs in environmental sex determination remains unclear. In this study, we generated HSF1 knockout medaka and then analyzed the phenotypes of the fry reared at normal water temperature (26℃) or HT(33℃). In the knockout medaka, HT did not induce the expression of hsp70 and hsp27 and the survival rate decreased compared with wild-type fish. Moreover, RNA-seq analysis revealed the expression of many genes changes in the knockout fish. In future, we will focus on the detail phenotypes of the knockout medaka.

Regeneration is a common phenomenon among animals. Members of the phylum Arthropoda, comprising over 80% of total animal species, exhibit strong capacities for regeneration, but little is known about the molecular mechanisms mediating this process. In this study, we investigated the role of the activin signaling pathway in limb regeneration in the decapod crustacean Procambarus fallax f. virginalis. We identified and cloned a downstream transcription factor in the activin pathway, Smox. The Smox gene showed 3 splicing variants, but only one of them encoded a complete Smox transcription factor. Gene knockdown of Smox by RNAi induced formation of smaller limb buds and regeneration of complete but smaller pereopods after autotomy. This indicates that activin signaling via Smox functions in regulation of pereopod size. The expression levels of both Smox and the activin receptor babo were closely correlated with molting. The expression level of Smox during the molting cycle increased when the receptor babo was knocked down by RNAi, indicating that Smox and babo transcription are linked. Our study suggests that the Babo-Smox system in activin signaling is conserved in decapods, and supports an evolutionary conservation of this aspect of molecular signaling during regeneration between protostomes and deuterostomes.

Increasing concentrations of nitrate and nitrite in surface and groundwater are prevalent worldwide and pose an environmental concern. Previously we have shown that nitrate and nitrite act as an endocrine disruptor to affect dopaminergic and serotonergic neurons during early development of zebrafish. In this study we extended our study to examine the effects of nitrate and nitrite on visual nervous system in zebrafish. To analyze effects in early development, fertilized eggs were exposed to NaNO₃ and NaNO₂ at 1, 5 and 10 mg/L of NO₃-N and NO₂-N, respectively. Measurement of eye size to body length ratio and acridine orange staining to detect apoptosis were conducted at 48 h post-fertilization (hpf). Optomotor response (OMR) test to assess visual function was carried out at 168 hpf. Nitrate at 10 mg/L and nitrite at 5 and 10 mg/L induced a significant reduction in eye to body length ratio. Apoptosis in the eye was significantly increased in the embryos exposed to nitrate at 5 and 10 mg/L and nitrite at 1, 5 and 10 mg/L. The OMR test showed that the visual function of the larvae was significantly decreased in nitrate at 10 mg/L and nitrite at 5 and 10 mg/L. To analyze the effect on retinal tissue, adult fish were exposed to nitrate and nitrite at 10 mg/L for 7 days, and the eyes were histologically analyzed by H&E staining. Nitrate and nitrite exposure significantly decreased the thickness of retinal layers including outer segment of photoreceptor (OS), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Taken together, these data demonstrated that nitrate and nitrite have negative effect on development of the eye, suggesting that they may pose a deleterious effect on visual system in aquatic organisms.

Objective: The vasa gene plays a crucial role in the development of germ cell lineage and is a most reliable molecular marker for studies on germ cell development in vertebrates and invertebrates. However, little is known about the expression pattern and function of vasa in pacific abalone.
Methods: The full-length cDNA of vasa was isolated and characterized by PCR and multiple sequence alignment. Spatial and temporal expression in different tissues and developmental stage of embryos were examined by RT-PCR and in situ hybridization. Finally, RNAi effects in oocytes and early embryos were assessed by real-time PCR.
Results: The cDNA length of vasa was 3,866 base pairs and encoded 801 amino acid residues. It was mainly expressed in gonads and unfertilized eggs, and its mRNA was predominantly localized in oogonia and stage 1-3 oocytes of ovary, in spermatogonia and primary spermatocytes of testis. In unfertilized egg, vasa was detected in animal hemisphere. During embryogenesis, it was strongly expressed in the irregular patches of animal portion of 32-cell stage. Later on, it was restricted to cells of ventral paraxial bands in trochophore and the fields of visceral mass in veliger. To evaluate effects of vasa knockdown, the dispersion and inhibitor effect of vasa dsRNA in ovary were examined. After 12 hours post-injection, dsRNA with vasa DIG-labelled delivered to full-grown oocytes of female. Expression levels of vasa in RNAi group using 3 different dose of dsRNA were significantly lower than control group at 7 days post-injection (dpi) and 14 dpi. Similar inhibitor effects were observed in eggs and 8-cell stage of embryos obtained from treated females using 100 g vasa dsRNA. The results revealed that RNAi of target gene can be effectively utilized in oocytes and early embryos of pacific abalone to reduce amount of mRNA level.

Objective: Pigment–binding proteins derived from crustacean shells play a major role in color change during cooking. Due to the lack information of pigment–binding protein derived from Litopenaeus vannamei shell, we isolated and characterized the red color–related pigment–binding protein derived from L. vannamei shell in this study.
Methods: The protein was extracted from shrimp shell, fractionated with ammonium sulfate precipitation, and purified by gel filtration and anion exchange HPLC. Color changing ability was investigated by heating the protein solution up to 100 ºC for 10 min and monitored by a colorimeter. Identification of the protein was performed by peptide mass fingerprint analysis by MALDI–TOF–MS, and secondary structure change was determined by a CD spectropolarimeter.
Results: A homogeneous monomer, termed LvPBP75, with a molecular mass around 75 kDa was purified from shrimp L. vannamei shell. Peptide mass fingerprint analysis by MALDI–TOF–MS showed that LvPBP75 consists of hemocyanin. Meanwhile, the absorption spectrum of acetone extract from the precipitate of heated LvPBP75 is typical of astaxanthin with absorption maxima at 481 nm. LvPBP75 gave initial color change at 30 ºC (p < 0.05), but no significantly changes (p > 0.05) at 30~60 ºC. Another significant color change was detected when heated up to 80 ºC. CD spectra showed a main α–helix structure of LvPBP75, and the disappeared α–helices are transformed dominantly to β–sheet (r² = 0.9787 ) and turn (r² =0.9407) during heat treatment. The results suggest a novel function of the hemocyanin as binding with pigment, and its involvement in L. vannamei shell color change. Comparative investigation on the pigment–binding protein among shrimp species is now in progress.

The milkfish (Chanos chanos) is an important aquaculture species, but intolerant to cold environments. Fluctuation of environmental temperatures affects physiological responses, behavior, and survival rate of fish. The “warm-temperature-acclimation associated 65-kDa protein” (Wap65) of teleosts was identified under heat shock and had two isoforms. The two isoforms of teleostean Wap65 were both found to be related to the induction of immune responses in fish and were highly conserved to mammalian hemopexin in sequences showing high affinity with heme to neutralize free-heme for transport to the liver. Both the two isoforms of wap65 genes were found to be related to the induction of immune responses in fish. In this study, we have isolated and characterized two isoforms of wap65 genes (Ccwap65-1 and Ccwap65-2) from milkfish livers. The expression of Ccwap65-1 was down-regulated in livers of the seawater (SW) milkfish, while no response of the freshwater (FW) group. Compared to those of the normal temperature (28°C) group, the expression levels of Ccwap65-2 in livers of SW and FW milkfish were up-regulated after exposure to low temperature (18°C) for 12 h and 96 h, respectively. After intraperitoneal injection of LPS, the expression of Ccwap65-2 elevated in both SW and FW milkfish, while Ccwap65-1 transcript of either SW or FW group did not respond to LPS treatment. This study has identified a novel immune biomarker, Ccwap65-2, in milkfish livers under hypothermal stress. Acute increase of hepatic Ccwap65-2 expression in response to more pathogen infection indicated by elevated expression of some immune-response genes may lead to better cold tolerance of SW milkfish rather than FW individuals upon cold challenge.

The Vp_PirAB-like toxin is identified as the virulence factor of acute hepatopancreatic necrosis disease (AHPND), an outbreak disease in shrimp farms. The Vp_PirAB-like toxin compose of Vp_PirA-like and Vp_PirB-like proteins, which was firstly discovered in the plasmid DNA of Vibrio parahamolyticus. By protein domain prediction, Vp_PirAB-like toxin showed similarity to delta-endotoxin, an insecticidal protein. The study of delta-endotoxin revealed that the mutation of its receptors cause reducing of toxin binding and resulting in insect resistance. It is possible that Vp_PirAB-like toxin receptor shares amino acid sequence identity to the receptor of delta-endotoxin. Previous study suggested that Vp_PirAB-like protein is heat stabile and formalin-resistant, therefore, the formalin-killed cells (FKC) V. parahaemolyticus AHPND strain was used for the selection of shrimp that are resistant to Vp-PirAB-like toxins.
To identify genes involved in toxins resistance, total RNA were isolated from hepatopancreas and stomach of shrimp that survived after FKC feeding challenge. RNA sequencing was performed using next-generation sequencing together with cDNA libraries of non-treated group and AHPND-immersed (24h) group. RNA sequences were de novo assembled and the homologs of delta-endotoxin receptors (cadherin, alkaline phosphatase, aminopeptidase-N and ATP-binding cassette C) were searched using local blast. The RNA sequences of each receptor homolog from Vp_PirAB-like toxin resistant group were compared with the other two groups.
About sixteen transcripts of delta-endotoxin receptors homologs were found in L. vannamei transcriptome. Among the toxin-resistant group, eleven homologs showed difference in amino acid sequence with the other groups. The genotype of the selected homologs will be further analyzed among individual shrimp to elucidate the association of sequence differentiation with Vp_PirAB-like toxin resistance.

Bacillus spp. are used as probiotics in shrimp farming, where they are known to enhance growth and disease resistance in shrimp. Although the mechanism of the effects of probiotics in shrimp is still unclear, it is known that Bacillus spp. produce antimicrobial substances that help fight harmful microorganisms. In this study, Bacillus amyloliquefaciens TOA5001 (Toa Pharmaceutical Co., Ltd.) was evaluated, where its antimicrobial activity against Fusarium sp. and Vibrio parahaemolyticus (EMS/AHPND strain) was assessed using the cross-streak method on heart infusion media with 3% NaCl. Results showed that the growth of Fusarium sp. and V. parahaemolyticus were inhibited by B. amyloliquefaciens. Filtered cell-free supernatant of the culture solution of this strain also showed antimicrobial activity. Proteins in the cell-free supernatant were fractionated by using Amicon Ultra, and only the fraction constituted of more than 100 kDa proteins showed antimicrobial activity. Spores of B. amyloliquefaciens were then incorporated to shrimp feed and was fed to Litopenaeus vannamei shrimp for the duration of two and four weeks. After feeding, challenge tests with Fusarium sp., V. parahaemolyticus and WSSV were conducted. Shrimp fed with B. amyloliquefaciens showed significantly higher survival rate after challenging with V. parahaemolyticus and a slower decrease of survival rate after challenging with Fusarium sp. compared to the control groups. However, in the case of WSSV challenge, no significant difference was seen after the treatment. Our results may suggest that B. amyloliquefaciens TOA5001 and the antimicrobial substances it releases in the water, may have affected V. parahaemolyticus impairing its infection. Further studies are needed to identify these antimicrobial substances and their mode of action, and to examine the best conditions on using this strain to help eliminate shrimp pathogens.

Antimicrobial peptides (AMPs) act as robust weapons in innate immunity and play a major role in protecting fish from opportunistic pathogens. Piscidins which belong to fish AMPs are crucial effectors of the fish innate immune response. Typically, piscidins exhibit broad-spectrum antimicrobial activity and modulate the immune response. In this study, we discovered two novel rock bream piscidin isoforms (RP4 and RP5) and investigated their gene expression patterns and antimicrobial and cytotoxic activities. Their open reading frames (ORF) consist of 240 and 249 base pairs (bp), encoding 80 and 83 amino acid residues, respectively, and possess a conserved AMP 12 domain. The predicted tertiary structure of the AMP 12 domain of RP4 and RP5 is an amphipathic alpha-helix structure. RP4 and RP5 mRNAs are ubiquitously expressed in healthy fish. After pathogen infection, RP4 expression was increased relative to that of RP5. However, RP5 exhibited stronger antimicrobial and haemolytic activity than RP4. Because RP5 can greatly inhibit bacteria, a small amount of RP5 can protect the host from pathogens. Additionally, because RP4 is highly up-regulated after pathogen infection, it may have other immune response function in rock bream in addition to pathogen killing.

CD2 is expressed on virtually all T cells and natural killer (NK) cells as a surface molecule of the about 50 kDa molecular size in a mammal. CD2 is known as receptor of CD48 (SLAM 2) and CD58 (LFA-3) in humans and rodents, respectively. In this study, we identified a rock bream CD2 (RbCD2) gene and investigated its gene expression in rock bream. And using flow cytometry, we confirmed the distribution of RbCD2 positive cell in the peripheral blood leukocytes (PBLs) and leukocytes of kidney, spleen and gill. The ORF of RbCD2 (1011 bp) encoded 337 amino acids, and it contained a proline rich portion of the cytoplasmic tail which interacts with the SH3 and GYF domains of CD2AP and CD2BP2. Quantitative real-time PCR analysis revealed that RbCD2 was highly expressed in PBLs, head kidney and trunk kidney. After Edwardsiella tarda and red seabream iridovirus (RSIV) infection, RbCD2 gene expression was increased in the head kidney and gill, respectively. Especially, Streptococcus iniae infection could induce RbCD2 gene expression in all studied tissues. In healthy rock bream, the flow cytometry results that RbCD2 positive cells was 70% to 80% and distributed in the lymphocyte area. But RbCD2 positive cell distribution was not substantially less than 1% in the granulocyte area. These results may be helpful to provide the possibility as a marker of CD2 positive cell and to study the function of blood cell and the cell-mediated immunity in teleost.

Circadian rhythms are physical, mental and behavioral changes that follow a roughly 24-h cycle, responding primarily to light and darkness in an organism's environment. These changes occur in most living organisms, including animals, plants and many microbes. Circadian rhythm oscillations are the daily changes in physiology and are due to involvement of numerous genes whose expression peaks and declines approximately 12 h apart to undergo a full cycle within 24 h. These circadian rhythm controlling genes are called as clock genes that include brain and muscle ARNT-like-1 (Bmal1), Clock, Period and Cryptochrome genes in mammals. In recent years, it has been known that clock genes regulate cytokine gene expression. But there is little or no information on clock genes and their functions in fish. Therefore, cloning and structural analysis of medaka, Oryzias latipes bmal1 gene was conducted. Moreover, expression pattern of the gene and some inflammatory cytokine genes in light and dark periods was assessed in this study. Results revealed that the Bmal1 was composed of 630 amino acid residues, which contained functional domains such as HLH and PAS. High sequence conservation was confirmed in the regions coding these domains among vertebrate Bmal1. Comparison of the medaka and human genomes showed that some synteny exists around the bmal1 gene. A circadian oscillation of bmal1 gene was observed in the brain during light and dark periods (LD12:12). And the rhythmic expression of cytokine genes such as IL-1b, IL-17A/F1 and TNF-a was confirmed in the same sample. Moreover, the induction level of IL-17A/F1 and TNF-a gene expression by LPS was different at zeitgeber time (ZT) 2 and ZT14. Therefore, it was suggested that fish immune response has circadian rhythm similar to that of mammalian species.

Three Nile tilapia hepatic antimicrobial peptide hepcidin/HAMP cDNAs and genes, encoding 87-a.a. HAMP1, 90-a.a. HAMP2 and 83-a.a. HAMP3, were identified from transcriptome of Taiwan Nile tilapia NT1 strain infected by virulent Streptococcus iniae. The secreted mature peptide sequences of 22-a.a. HAMP1 and 26-a.a. HAMP2 of Nile tilapia with four disulfide bonds are identical to previously identified strong AMP TH1-5 and TH2-3 of Mozambique tilapia, respectively but 19-a.a. HAMP3 is a novel HAMP composed of three disulfide bonds. Three Nile tilapia HAMP genes were differentially activated not only in the liver but also in head kidney, spleen and gill by S. iniae infection in dose of LD50 (1.5x10⁵ CFU/g BW) to contribute for defense against S. iniae infection. Due to their liver-abundant and pathogen-inducible expression, 2.6 kb promoters of two HAMP3 genes, HAMP3a and HAMP3b were obtained from Nile tilapia NT1 strain. Putative binding sites of liver-enriched and immune-related transcription factors including HNF1α、HNF3β、HNF4α、C/EBPα、NFκB、IRFs and STAT3 were predicted in both HAMP3 promoters but one NFkB binding site was lost at 5’-end of 2.6 kb promoter and a deletion in 5’-UTR of HAMP3b gene. Especially, two HAMP3 genes encoding identical HAMP3 protein composed of three disulfide bonds in 19-a.a. mature HAMP3 peptide but under control of different promoter regulation were firstly identified in tilapia. The 19-a.a. HAMP3 mature peptide was chemically synthesized to test its antimicrobial activity against critical pathogens S. iniae and S. agalactiae for tilapia. The 19-a.a. HAMP3 synthetic peptide exhibited inhibitory activity on the growth of both S. iniae (MIC= 100 uM, 100% inhibition) and S. agalactiae (100 uM HAMP3: 66% inhibition) whereas showed little inhibitory effect on Vibrio vunificus. The novel antimicrobial peptide HAMP3 can be applied for treatment of Streptococcosis in aquaculture industry of tilapia.

Infectious hematopoietic necrosis (IHN) virus is an important pathogen of farmed salmonid fish in Japan. In the previously study, we have demonstrated that a diet supplemented with high-concentration (5,000 mg/kg diets) ascorbic acid (AsA) can prevent several disease including IHN in salmonid fish. However, detailed knowledge regarding the effective mechanism of AsA supplementation related to virus infection is limited. Therefore, we focused on the effects of AsA supplementation on antiviral factors production during virus infection. The present study investigated the expression pattern of interferon (IFN) and IFN-related genes in rainbow trout injected with poly (I:C) after feeding of diet supplemented with or without high-concentration AsA. All feeding with or without AsA were conducted for 7 days, and fish in each group were injected intraperitoneally with poly (I:C) or PBS at the end of feeding period. Five fish per group were sampled at pre-injection, 12 and 24h post-injection, and gill, spleen, head kidney, kidney, liver and intestine tissues were collected for gene expression analysis by qRT-PCR. In AsA/poly (I:C) group, most of the IFNs and ISGs expression levels significantly up-regulated in all tissues (except for gill), compared with AsA/PBS and normal/PBS groups. Meanwhile, the expression levels in AsA/poly (I:C) group at 12h post-injection were less than those in normal/poly (I:C) group. These results suggest that enough IFNs have already been produced by AsA feeding for 7 days or high-concentration AsA supplementation activated other immune system in rainbow trout.

This study aimed to evaluate Nodulisporium sp. KT29 metabolites to increased growth performance and immune response of vaname shrimp in marine culture. This study used completely randomized design with three treatments and three replications. The treatments were additional of Nodulisporium sp. KT29 metabolites with doses 0 (control), 10 and 20 mLkg^-1 in the diets. Vaname shrimp (PL-10) were cultured for 30 days in cage (1×1x1.8 m³) with stocking density of 500 shrimpm³. The shrimp were fed five times a day with feeding rate 50-35% of body weight. This study was conducted in cage of sea farming PKSPL-IPB, Semak Daun Island, Thousand Island, Indonesia. The results showed that Nodulisporium sp. KT29 metabolite caused damage of Vibrio harveyi cell. Addition of Nodulisporium sp. KT29 with doses 10 and 20 mLkg^-1 in the feed increased biomassa (379.47±0.04g and 347.75±0.05 respectively), daily growth rate (17.66±0.27% and 17.60±0.41%, respectively), and decreased FCR (1.22±0.11 and 1.23±0.13, respectively) of shrimp which culture in cage culture. Beside that, addition of Nodulisporium sp. KT29 metabolite also increased survival rate, phenoloxidase and respiratory burst activity after challenge test with white spot syndrome virus (WSSV) and V.harveyi co-infection. Therefore, it concluded that metabolite Nodulisporium sp. KT29 with dose 10 mLkg increase growth and immune respons of vaname shrimp after challenge test with WSSV and V.harveyi.

Lancefield group C pathogenic Streptococcus dysgalactiae (GCSD) causes serious　diseases in several farmed fish species, such as amberjack Seriola dumerili and yellowtail S. quinqueradiata, and mastitis and endocarditis in animals.
The emm-like gene that encodes M-like protein is widely used for typing the human pathogen S. pyogenes. Multilocus sequence analysis (MLSA) has also been used to examine the genetic relationships among several Streptococcus species. In this study, 86 strains isolated from fish and five S. dysgalactiae strains isolated from animals were used to examine the genetic relationship by emm typing and MLSA (map, pfl, ppaC, pyk, rpoB, sodA, and tuf).
Results revealed that the emm type of S. dysgalactiae isolated from fish was stL1929, which was the same as that of group C streptococci isolated from dogs and humans. MLSA revealed that all the strains isolated from fish made a clade, which was included in the clade of β-hemolytic S. dysgalactiae subsp. dysgalactiae, and had the closest relationship with the CCUG48477 strain isolated from a dog (stL1929, S. dysgalactiae subsp. dysgalactiae). Therefore, fish pathogenic S. dysgalactiae isolates could be identified as S. dysgalactiae subsp. dysgalactiae. This is the first study to report the emm-like gene sequence typing of the fish pathogen S. dysgalactiae.

Lactococcus garvieae serotype II strains have been isolated from yellowtail cultured in several fish farms since 2012. Because these strains did not agglutinate with a routine diagnostic antiserum raised against capsulated L. garvieae cells (KG- phenotype cells, serotype Ia), they were proposed to be newly emerging L. garvieae serotype II strains. These strains have incessantly caused infections in fish farms, which could not be prevented using an L. garvieae serotype I vaccine. The antibiotic resistance of L. garvieae serotype II strains is also unknown. Therefore, an epidemiological study of L. garvieae serotype II strains is required to prevent L. garvieae serotype II infections in fish farms. A total of 66 L. garvieae strains isolated from Seriola quinqueradiata and S. dumerili collected 2012–2016 were used in this study. An agglutination test using anti L. garvieae serotype Ia rabbit serum and anti L. garvieae serotype II rabbit serum was performed to confirm the serotype. The genotype was determined by performing biased sinusoidal field gel electrophoresis (BSFGE) using all serotype II strains and Sma I, a restriction enzyme. Based on the complete genome sequences of L. garvieae serotypes Ia (Lg2) and II (122061) strains, a PCR primer set was designed to distinguish L. garvieae serotype I strains from serotype II strains. The minimum inhibitory concentrations of several antibiotics for these strains were also determined. BSFGE revealed that all L. garvieae serotype II strains were homologous. A PCR assay was successfully developed for effective discrimination between serotypes I and II strains based on amplified fragments with different sizes (1285 bp for serotype II strains and 285 bp for serotype I strains). All the L. garvieae serotype II strains were sensitive to antibiotics tested, but the rate of resistance to lincomycin was relatively high (36/66 strains) and this rate increased with time.

An outbreak of ulcer disease of orange-spotted grouper (Epinephelus coioides) occurred in southern Taiwan in November 2015. Several bacteria were isolated from moribund fish and characterized by its morphological, physiological and biochemical studies. The most dominant one among the five isolated bacteria was designated as TN1. The result of 16S rDNA gene sequence analysis indicated that TN1 is close to Vibrio brasiliensis with sequence similarities of approximately 99%. Furthermore, V. brasiliensis showed specific immune responses with serum collected from survival fish. Subsequently, attempt was made to screen vaccine candidate antigens of TN1 by immunoprecipitation approach with magnetic beads. According to the result of SDS-PAGE analysis, at least eight proteins were detected. These proteins were then eluted for protein identification by mass spectrometer.

Edwardsiella species are the most important bacterial fish pathogens in various fish species causing huge economic losses in aquaculture industry worldwide. Its pathogenicity was confirmed in olive flounder by oral intubation at dose of 1.04 x 10 ⁸ CFU/fish, as cumulated mortality was approximately 50%. The strain showed multi-drug resistance against oxytetracycline, nalidixic acid, florfenicol, chloramphenicol, sulfamethoxazole and sulfadiazine. Whole genome sequencing was conducted using de novo Illumina/PacBio hybrid assembly and genome prediction and annotation process based on Prokka framework. As a result, the genome contains one chromosome of 4,199,472 base pairs comprising 3,722 predicted protein coding sequences, and a single plasmid of 140,272 base pairs harboring multi-drug resistant genes. Average nucleotide identity analysis of the genome sequence of several strains belonging to Edwardsiella was performed and the result showed that our isolate is most closely related to E. anguillarum (Identity=99.48%), which is newly recognized pathogenic species in eels. Phylogenetic tree using core genes of hitherto sequenced E. anguillarum strains and other species showed that EET61 was clustered well with E. anguillarum clade, and distinct from other species. We also identified this strain harbors type III, IV and VI secretion systems, as well as flagella and pili related genes that are involved in virulence. Our study revealed that E. anguillarum species have open pan-genome and newly sequenced genome could bring 95 new genes on average so far. In addition, this strain contained multi-drug resistance genes based on a web-based tool, "Resistance Gene Identifier". In conclusion, E. anguillarum, can affect eel farms located throughout the country, and the genome sequence and comparative analysis will contribute to better understanding of the pathogenicity of and development of control measures for this pathogen in the future.

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of herpesviral hematopoietic necrosis in goldfish Carassius auratus and gibelio carp C. auratus gibelio. In general, it is thought that fish surviving virus infection can be virus carrier by permitting the virus to harbor inside persistently or latently and may be the source of infection by shedding the virus after reactivation. In this study, the presence of CyHV-2 genome was investigated in the tissues of asymptomatic surviving goldfish to determine whether the virus reactivation occurs in the tissues of the fish in vitro and in vivo. PCR specific to CyHV-2 was done on the tissues including spleen, kidney, heart, brain, gill and fin immediately after being dissected from the fish and on those after being incubated in Medium 199 at 25°C for 5 days. Although there were no virus DNA detected in each tissue freshly dissected from 5 asymptomatic surviving fish, 4 of 5 fish turned positive for the PCR after the in vitro incubation. In addition, the presence of CyHV-2 genome was also examined in the surviving fish which has been subjected to stress by injection with anti-ginbuna IFN-γs rabbit antibodies (anti-IFN) or dexamethasone and by treatment of water temperature fluctuation. The virus detection rate and number of positive tissues of the fish in the anti-IFN and dexamethasone injected groups were higher than those in the corresponding control groups. In contrast, the presence of virus was not so different between the temperature fluctuation and constant groups. These results showed that the asymptomatic surviving fish was a carrier and the immune depression increased the virus in fish tissues, suggesting that the asymptomatic surviving fish become the potential infection source of CyHV-2.

Lates calcarifer or known as Asian sea bass is a marine fish commodity with high economical value. Asian seabass cultured infected with diseases cause mass mortality in seeds in Indonesia. The vaccination on fish juvenile prior to grow out culture in the cage in open sea, the mortality, however, remains high. In this context, reisolation and reidentification of disease-causative agents are needed to confirm the homology between pathogens in order to generate an effective vaccine against diseases causative agents in seabass. The purpose of this study was to reisolate and identify the disease-causing agents in both bacterial and viral diseases that infect juvenile stadia in seabass. Seabass juveniles used as the tested samples were the fish that showed clinical symptoms of infectious diseases, inactive movement, slow response, no appetite, bleeding in the abdomen and the base of the fin, alteration in body color and moribund. Bacterial isolation was performed using nutrient agar, marine agar, and thiosulphate citrate bile salts sucrose (TCBS) agar. Bacterial identification was performed according to Barrow & Feltham method. Isolation of iridovirus was done by using DNAeasy kit (Qiagen, USA). Identification of iridovirus by polymerase chain reaction (PCR) method using one primer set according to OIE, ie 1-F (5'-CTC-AAA-CAC-TCT-GGC-TCA-TC-3 ') and reverse primer 1-R (5 '-GCA-CCA-ACA-CAT-CTC-CTA-TC-3') which produces a PCR product of 570 bp. The results showed that seabass fry were infected by both bacterial and viral agents. The bacteria found in the fish was identified as Bacillus sp., Pseudomonas sp., Vibrio parahaemolyticus, and Vibrio alginolyticus, while the viral disease identified was Iridovirus.

Japanese eel (Anguilla japonica) is highly valued species for aquaculture in East Asian countries, but natural stocks of eels have decreased.
Mass production of glass eels is one of the effective ways to solve this problem. For establishment of mass production of glass eels, selective breeding will gain importance. In a coordinated breeding program, parentage assignment is essential. In this study, we try to parentage assignment of Japanese eel in multiple breeding method using tri-, tetra-, penta- nucleotide microsatellite of 8 loci. Genotype was performed by Gene Mapper® and parentage assignment was performed by PARFEX v1.0.Two sets of mass spawning were performed. In first set, five female and 15 male eels were used and 490 offspring consists were sampled for parentage assignment. In second set, three female and 16 male eels were used and 371 offspring were sampled. In first sets family, all progenies were assigned to parental couple. In second sets family, almost all progeny (98.9%) were also assigned to parental couple. The efficiency of parentage assignments is very high and our method might be a useful tool for breeding program. High contribution rate in the next generation was detected in some parents.This result suggests that some parents might have the effective trait for mass production of glass eels.

Tilapia are among the world's most important aquaculture finfish. In recent years, Streptococcus is recognized as major infectious disease causing significant economic loss in aquaculture in various country. The hepatic antimicrobial peptide hepcidin/HAMP had been reported to defend against various bacterial pathogens and viruses.We had identified three Nile tilapia HAMP genes, HAMP1, HAMP2 and HAMP3, which are differentially induced in the liver, spleen, head kidney and gill by Streptococcus iniae infection. According to newly released genome assembly of Nile tilapia, 18 hepcidin genes including 12 HAMP1 genes, 1 HAMP2 gene, 1 HAMP3 gene and 4 novel HAMP4 genes were found in LG11 and two separate contig825 and contig1099 of Nile tilapia genome. Identification of DNA markers associated with disease resistance may facilitate the acceleration of the breeding selection for disease resistance. This study presents the preliminary investigation into the association of genotype of microsatellites/SSRs related with hepcidin genes and disease resistance of hybrid Nile tilapia (NT1 x NT2 strains) in National Taiwan Ocean University. We discovered 17 hepcidin-related SSRs and designed SSR-specific PCR primer sets by WebSat to detect these Type I microsatellite DNA markers. Four fluorescent labels with 2 to 3 sizes of PCR products were used for multiple SSRs genotyping of 95 tilapia samples at the same time by ABI 3730XL Genetic analyzer. Disease resistance and genotype of SSRs related with hepcidin genes of tilapia will be compared to establish useful molecular markers applied in marker-assisted selection of disease-resistance Nile tilapia for sustainable and profitable tilapia aquaculture.

Following the rising trend of fishmeal price, development and practical application of low fishmeal diet (LFD) is needed for finfish aquaculture. Rainbow trout (Oncorhynchus mykiss) is globally cultured, omnivorous species relatively weak to LFD feeding. Our goal is to breed rainbow trout which grows faster under LFD feeding.
We've crossed two strains of rainbow trout known to have a different tolerance against LFD. Individually-tagged F2 progeny was reared under experimental LFD feeding for over a year, with occasional weighing for phenotyping.
Then, using extracted DNA from above F2 progeny and F1 parents (N=88), we constructed reduced-representation library (RRL: modified from dg-GBS method of Peterson et al., 2014) for DNA sequencing, then sequenced it in a single lane of HiSeq4000 (illumina).
After mapping the obtained sequence reads to a draft genome assembly of rainbow trout, SNPs were called for genetic linkage mapping. Then, linkage analysis was done searching for quantitative trait loci (QTL) against observed phenotypes.
In consequence, we constructed genetic linkage map consists of 30 linkage groups, 920 SNP markers from 82 individuals of F2 progeny. From linkage analysis, we found a single QTL associated with growth under LFD feeding. This QTL was located on Chr15, showing high LOD value exceeding the genome-wise significance threshold of P=0.01.
In addition, we compared these results to that from another analysis pipeline (sequence reads mapped to Atlantic salmon Salmo salar genome, high-quality assembly of phylogenetically close species).
From these results, we concluded, by using SNPs generated by RRL sequencing, the practical linkage map of rainbow trout can be made by relatively low cost. Further, marker-assisted selection of LFD-tolerant rainbow trout can be expected.

We previously established a germ cell transplantation technology that would　provide a powerful tool for the conservation of endangered species and the efficient production of commercially valuable seeds. Use of in vitro amplified germ cells would supply donor cells simpler and easier compared with those prepared from live fish. In this study, in order to produce live fish derived from in vitro cultured cells, we first cultured the rainbow trout germ cells in vitro and then transplanted the resulting cells into recipient fish. vasa-GFP transgenic and dominant albino rainbow trout were used as materials for germ cell culture. The initial germ cells were enriched by differential plating from whole testicular cells. These germ cells (mainly type-A spermatogonia labeled with GFP) were cultured in DMEM/F12 media supplemented with 1% rainbow trout blood plasma, 10% fetal bovine serum and some additional factors on mitomycin C-treated Sertoli cells derived from rainbow trout testis as feeder cells. Whenever the germ cell cultures reached 80% confluence, subcultures were passaged at a dilution rate of 1:2. After a month, the amplified cells were collected and intraperitoneally transplanted into wild-type, triploid rainbow trout hatchlings.
After a month, we observed that cultured cells displayed a clear green fluorescence and proliferated with high efficiency. Furthermore, the transplanted germ cells were incorporated into the recipient gonads and resumed gametogenesis. One year after the transplantation, some recipient males matured. By performing a mating study, we confirmed that the offspring carrying the donor-derived phenotype (albino and GFP-positive) were produced. Thus we could successfully establish a culture condition of trout germ cells in vitro for at least a month without losing the potential to differentiate into functional sperm. Currently, we have been raising female recipients in order to obtain cultured cell-derived eggs.

[Purpose] Yellow drum (Nibea albiflora) is an important commercial marine fish species in China, which exhibits significant sexual size dimorphism, with female growing faster than the males. An alternative approach to all-female production involves the induction of gynogenesis and the masculinization to produce genotypic female which are of male phenotype. This study was to optimize the 17α-methyltestosterone (MT) treatment to induce masculinization of sexually undifferentiated of gynogenetic yellow drum by investigating different concentrations on immersion treatments.
[Materials and Methods] The gynogenotes of yellow drum at 30 days post hatching (dph) were subdivided into 5 groups. Each group was immersed for a period of 2 h per day in a bath of 17α-MT at a concentration of 0, 0.2, 2, 10 and 50 ppm. The gynogenetic fish were subjected to immersion for 60 days. The effects of 17α-MT on growth, sex ratios and gonadal development were investigated at 45, 60, 70, 90, 120 dph by body size measurement and gonadal histology. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detected cyp19a1a and dmrt1 expression in gonads of groups at 120 dph.
[Results] Growth suppression was observed at high concentration groups (2, 10, 50 ppm) compared to that in control groups and 0.2 ppm treated groups. Control groups yielded 100% female and 0.2 ppm treated groups yielded 100% male. Spermatozoa were first observed at 60 dph in 0.2 ppm groups. Sterile gonads and ovotestis were observed at high concentration of 17α-MT treated groups. RT-PCR revealed that male specific-gene dmrt1 was expressed only in treatment groups, while female specific-gene cyp19a1a was specifically expressed in control group. The present data revealed that treatment of 30 dph gynogenetic fry with 17ɑ-MT produced 100% sex reversed fish by 2-h immersion at a dose of 0.2 ppm.

The occurrence of disease in Indian major carps was studied for two consecutive years in Jammu and Kashmir. The samples for bacteriological analysis were collected under active surveillance in nutrient broth. A total 120 samples (5 fish per pool) were collected and screened for Aeromonas hydrophila by biochemical tests and polymerase chain reaction. The culture and biochemical identification for Aeromonas hydrophila was done on Rimler-Shotts media base, Indole, citrate, Voges-proskauer were positive and methyl red tested negative. The screened samples tested positive were subjected to polymerase chain reaction. PCR result showed positive using A. hydrophila extracellular aer A fragment 683bp and 597bp fragment of Hyl H region of the aerolysin gene and haemolysin gene).

Concurrent resistance to antibiotics of different structural classes has arisen in a multitude of bacterial species and may complicate the therapeutic management for both human and animal. The objective of the study was to determine the Multiple Antibiotic Resistances (MAR) and MAR index of isolated tetracycline resistance bacteria from aquaculture sources. 42 tetracycline resistance bacteria which were previously isolated from aquaculture farms were tested against Oxytetrcyline (OTC), Tetracycline (TET), Amphicillin (AMP), Amoxicillin (AMX), Sulfamethaxasol (SMX), Trimethoprim (TMP), Erythromycin (ERM), Cloxacillin (CLOX) and Ciprofloxacin (CIP). The MAR was determined by using Triphenyl Tetrazolium Chloride (TTC) assay and an agar dilution method following CLSI guidelines and then MAR index was calculated. Of these isolates 64.3% (27of 42) were resistant to three or more antibiotics and considered as multiple antibiotic resistant according to WHO and CLSI. Among the MAR isolates, 93% were resistant to OTC, 90% tor TET, 34% to AMX, 14% to AMP, 13% to ERM, 11% to SMX-TMP and 2 % were resistant to CLOX were detected. None of bacteria were resistant to CIP. In this study, MAR index was ranged from 0.03 to 0.42 for the isolated bacteria and the highest MAR index was recorded by Staphylococcus sp. (0.42) while the lowest was shown by Enterobacter sp. (0.03). The results of the study indicate that overuse and misuse of antibiotics led to emergence of MAR bacteria and it compromise effectiveness of antibacterial therapy. As a result infectious microorganisms are becoming resistant against most of antibiotics. Thus, education, antibiotic control measures, fundamental epidemiological and scientific research advocated as important preventive measures of development of MAR.

Key words: Multiple Antibiotic Resistance (MAR), Tetracycline, Antibiotic, Multiple Antibiotic Resistance Index

Digestive tract microbial communities have recently become a topic of increasing interest, especially in terms of the host microbiota and pathogenic bacteria interactions. Microbial balance is believed to be important for human health and diseases. These studies reveal cause-and-effect relationshipswhich suggest that the microbiome influences, not only colonization by pathogenic bacteria, but also affects their growth and enhances their virulence. Acute hepatopancreatic necrosis disease (AHPND) is an emerging (since 2009) disease in Southeast Asia. It is generally accepted that this disease is caused by Vibrio parahaemolyticus (VP) with extra-chromosomal elements producing a virulent toxin, causing damage to shrimp hepatopancreas. In the case of AHPND-causing VP strains which replicate in the shrimp stomach, whether stomach microbiome change during the course of AHPND, which bacterial taxa are affected and the role of the environmental microbiome in the shrimp stomach microbiome remains unaddressed. We applied a standardized approach to monitor shrimp grow-out pond from the 21st day after stocking with postlarvae that ultimately succumbed to an outbreak of AHPND. We collecting 10 shrimp samples per day, all of which were tested for disease markers individually. We used a culture-independent metagenomics approach with next generation sequencing technology (NGS) to characterize the AHPND-associated microbiome. Our multivariate analysis examined the individual microbiomes from 37 shrimp and 25 culture pond water samples and determined a total of 4576 OTUs. The samples were clustered into two groups on PCA, with a cutoff date at 21 ^st Sept., whereas the taxonomic richness of bacteria was decreased at the late time points in disease marker positive shrimp. Instead of VP abundance was the critical factor for AHPND pathogenesis, there were also third party microbiota that were potential AHPND biomarkers. Key microbial community contributions to differences in this study may be a critical factor in controlling AHPND.

Taiwan is one of grouper aquaculture technology center and the bridge of information transfer for Asia. However, the fast growth in genetic breeding is not established well and limits the development of grouper industry. In this study, we conducted cDNA subtraction library screening on the growth rate variety of grouper. After reviewing significant growth-related gene group, we outlined the main molecule markers involved in insulin signaling pathway. Furthermore, we determined and identified the key molecule, insulin-like growth factor 2 (IGF2) from a series of candidate growth-related genes. The genetic database to generate fast-growth grouper strain profile is further established by the evaluation of IGF2 expression levels in dragon grouper (Epinephelus lanceolatus) and tiger grouper (Epinephelus fuscoguttatus). Based on the genetic database profile, it can further help grouper farmers in production of dragon and tiger grouper broodstock to match and control breeding of dragon and tiger grouper broodstock strains more precisely. This method can significantly shorten the time required for traditional breeding by morphology and improve the breeding management system of dragon and tiger grouper hybrid strains. Taken together, these results can facilitate understanding the molecular mechanisms of growth rates in grouper and accelerate the molecular selection of the specific broodstock with superior growth.

PURPOSE: In the coastal areas of southern Japan, food poisonings have been caused by eating parrot fish (Scarus ovifrons) (Taniyama, 2002). It is thought that a toxin causing the poisonings is derived from prey organisms of the parrot fish. Therefore, it is important to determine prey species of parrot fish. In this study, we aimed to determine the prey species of the parrot fish.
METHODS: Twenty specimens of Scarus ovifrons were collected in Kochi, Tokushima and Wakayama Prefectures. Specimens of Calotomus japonicus as a control were also collected from these prefectures. DNA was extracted from the gut contents. The 18S rDNA V1-V3 region of the samples was PCR-amplified with a primer set of 18S rDNA and a blocking primer that specifically binds to 18S rDNA of the fish. The PCR products were sequenced using the Roche GS junior.
RESULTS: In a case that PCR products amplified from the parrot fish without the blocking primer were analyzed by a 18S metabarcoding analysis, the proportion of the parrot fish reads in the total reads was 62.3%. When those amplified with the blocking primer were analyzed, the proportion was decreased to 17.2%. A total of 33,190 reads was obtained from 20 specimens of Scarus ovifrons. Among them, the top three families showing the highest numbers of reads were Dinophyta, Heterokontophyta and Annelida. In contrast, the top three prey families of Calotomus japonicus were Heterokontophyta, Chlorophyta and Mollusca.
DISCUSSION: The blocking primer was useful for suppressing amplification of sequences derived from parrot fish. When prey species of Scarus ovifrons and Calotomus japonicus were compared, it was suggested that composition of the prey species was greatly different from each other. In the future, it is expected to search the prey species of S. ovifrons for an organism that produces a toxin causing the food poisoning.

The sustainable growth of the shrimp industry continues to be threatened by emerging infectious diseases, where acute hepatopancreatic necrosis disease (AHPND) was recently identified. The disease is caused by toxins identified to be as VP_PirAB toxin encoded in a plasmid of a specific strain of Vibrio parahaemolyticus. With the emergence of antibiotic-resistant bacteria, alternative strategies should be developed. Although shrimp are not capable of producing their own antibodies against pathogens, IgY antibodies procured from immunized hen egg can render passive immunization against a specific pathogen. Compared with other antibody production method, IgY production is easy, cost-effective, and less invasive for animal models.

The key to an effective passive-immunization is to identify immunogenic target antigens. In this study, we produced five different types of IgY antibodies through immunization with Toxin A, Toxin B, Toxin A & B injected separately (AB), a mixture of Toxin A & B (AB mix), and with the formalin killed cells (FKC) of Vibrio parahaemolyticus strain. The produced IgY was collected from egg yolk powder and incorporated to the shrimp feeds. The generated feeds (A, B, AB, ABmix, and FKC) were then administered separately to five treatment groups, where shrimp fed with the commercial feed was used as control. After a week of feeding, the effect of treatments was assessed by challenge test with the AHPND strain V. parahaemolyticus. Results showed the least mortality (%) in shrimp treated with A, AB and FKC, which showed 13%, 17% and 0% mortality, respectively. On the other hand, treatment with B, AB mix, together with the control showed 86%, 82%, and 100%, respectively.

Although further optimization of the feed formulation and feeding program is needed, this results demonstrates the use of passive immunization of shrimp with IgY as a promising disease control method against AHPND.

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.

It is known that corals made their skeletons in the extracellular fluids called subcalicoblastic calcifying medium (SCM) which are located between calicoblastic cells and carbonate skeletons. It is also known that there is an alkalization of the calcifying fluids in the calcification processes of corals. Venn et al. (2011) and Ohno et al. (2017) reported that pH of SCM in hard corals was about 0.5~1.0 higher than that of ambient seawaters. Additionally, Allemand et al. (2011) suggested that Ca²⁺-ATPase, which is located in the membranes of calicoblastic epithelial cells surrounding the SCM, contributes to the alkalization of the calcifying fluids. However, the mechanisms involved in the alkalization of the calcifying fluids are largely unknown. We reported that biogenic polyamines were able to react with carbon dioxide (CO₂) and accelerate calcium carbonate (CaCO₃) formation in seawater (Yasumoto et al., 2014). Thus, we hypothesized that the biogenic polyamines, which are grouped to the most common type of intracellular amines, play important roles in the alkalization of the calcifying fluids. To ascertain such hypothesis, we investigated whether or not there would be a local pH increase in the calcifying fluids in the primary polyps of Acroporid coral by using pH indicator reagent SNARF-1. When the primary polyps were treated with 50 µM of SNARF-1 on 1 day post metamorphosis, the calcifying fluids of polyps were pH 9.0~10.0, which were about 1.0~2.0 higher than that of ambient seawaters. Moreover, the pH increase was specifically observed in the region close to the CaCO₃ skeletons. Our data propose the possibility that some basic substances are involved in the alkalization of the calcifying fluids in primary coral polyps.

It is inferred that presence-absence pattern of protein motifs of all of the proteins in an organism is similar among evolutionally close species, but few researches have been presented about this issue. To address this issue, we generated phylogenetic tree of 325 bacteria based on presence-absence pattern of protein motifs. In this tree, species evolutionally close tended to form clusters.
For comparison, we also generated the phylogenetic trees based on DNA sequence of 16S ribosomal RNA, gene and amino acid sequence of DNA gyrase B subunit. In order to evaluate the similarity of trees between the two methods, we examined distances of all combinations of two species in both trees. Then we examined correlation between the distances by protein motifs and the distances by DNA or amino acid sequences. They showed moderate correlations, indicating that both trees have moderate correlation.

It is well known that the anthropogenic eutrophication enriched with various substances including phosphate in coastal waters has resulted in coral degradation. However, to the best of our knowledge, the phosphate threshold value to inhibit the coral calcification has been unclear, due to the unknown mechanisms involved in the inhibition of the calcification by phosphate. In this study, to elucidate the inhibition mechanisms for phosphate against coral calcification, we examined its effect on the bottom skeleton formation in primary polyps of Acropora digitifera by using the fluorescence derivatizing reagent having phosphate group (FITC-AA). When the primary polyps were treated with 50 µM of FITC-AA, the bottom skeleton of the primary polyps showed the fluorescence from FITC-AA within a few minutes, suggesting the phosphate binding. Furthermore, when the polyps were treated with 10 µM of FITC-AA, irregular patterns of the elongated skeleton were observed. These results led us to conclude that phosphate is transported via a paracellular pathway to the subcalicoblastic extracellular calcifying medium. In island regions, groundwater is one of the most important clues to transport the nutrients contained in livestock or agricultural wastewaters. However, the actual conditions of coastal pollution with such nutrients have not been understood because of unperceived submarine groundwater discharge. We thus investigated the conditions of corals and the concentrations of nutrients around the Yoron Island. As a result, the coastal submarine groundwater discharge was found to contain 1 to 2 µM of phosphate as much as the concentration in the coastal ground water under agricultural land. Moreover, the amount of phosphate contained in the surface layers of bottom calcareous sands close to the region of submarine groundwater discharge were about 5 µmol/g. These results indicate that the phosphate adsorbed to the bottom sand may also inhibit coral skeleton formation.

The nacreous protein sheets, the organic matrices obtained by decalcifying nacre, contain various proteins that might control the nacre formation of shells and pearls. They, considered to interact with other components in the sheets, can be extracted by boiling with detergents under reductive conditions (the boiling method). This treatment possibly promotes degradation of the proteins. In the present study, we have developed a method to extract the proteins electrically from the nacreous protein sheets under non-reducing and cooling conditions (the electrical method) and compared the extracts with those obtained by the boiling method. Nacre was prepared by removing prismatic layer from Japanese pearl oyster shells using a disk sander. The nacreous protein sheets were prepared by decalcifying nacre with 0.5 M EDTA. Proteins in the sheets were extracted by the electrical method—the sheets were treated at 100 V for 3 hours in the SDS running buffer (0.3%SDS, 1.44% glycine, 0.1% SDS); by the boiling method—the sheets were boiled for 2 min, sonicated for 10 min, and then boiled for 2 min, in the 2 x SDS-Sample buffer (20 mM Tris-HCl, 2% SDS, 2% 2-mercaptoethanol, 40% glycerol, pH 6.8). SDS-PAGE for the sample by the electrical method gave more bands than that by the boiling method, implying that the electrical method is capable of extracting more proteins from the nacreous protein sheets. Electrophoresis for the sample by the electrical method under non-reducing conditions revealed that some proteins in nacre form intermolecular disulfide bonds. These findings indicate that the electrical method is more useful to analyze proteins in nacre.

Invertebrate muscle contraction is controlled by a myosin-linked regulatory system, where a direct Ca2+ binding to myosin activates myosin to interact with actin. Although calponin and troponin—they are involved in an actin-linked regulatory system in vertebrate muscles—are expressed in bivalve muscles, their function there are unknown. In the present study, we characterized tropomyosin, troponin, and calponin of Japanese pearl oyster adductor muscle and analyzed their interaction in order to reveal the actin-linked regulatory system in molluscan muscles. We constructed their expression vectors using gene synthesis services. Alanine and serine were added to the N-terminus of tropomyosin to mimic its acetylation. Troponin and calponin were expressed as His-tagged proteins. All of the proteins were successfully purified by a series of chromatography. The purified proteins were subjected to interaction analyses by isothermal titration calorimetry (ITC). ITC detected no interaction of troponin and calponin with Ca2+. Interaction of tropomyosin with calponin was detected by a solid-phase binding assay, but ITC were unable to detect their interaction due to their insufficient concentrations. These findings indicate that tropomyosin, troponin, and calponin of bivalve adductor muscles might have different functions from those in vertebrate muscles.

In shrimp farming, biological environment of culture pond is considered critical on the productivity. It is, however, still obscure what biological composition is suitable for shrimp culture. Since most organisms are hard to cultivate in artificial media, it is impossible to grasp whole biological community by the cultivation methods. Therefore, we focused on metagenome analysis. In this study, we compared the planktonic and bacterial community between geologically different shrimp culture pond waters in Japan and Thailand, as a trial application of metagenome analysis.
Pond waters were sampled from 2 farms (2014) and 1 farm (2015) of kuruma shrimp Marsupenaeus japonicus in Kumamoto, Japan and from 3 farms (2015) and 1 farm (2016; same as 2015) of whiteleg shrimp Litopenaeus vannamei in Suratthani, Thailand. Organisms including planktons and bacteria were collected on polycarbonate membrane filters using a filtration system. After DNAs were extracted from the filters, partial sequences of 18S rRNA and 16S rRNA gene were amplified by PCR. Then, DNA sequences of the amplicons were obtained using Illumina MiSeq. Claident (http://www.claident.org) was used to perform bioinformatic analyses. Using beta-diversity and non-metric multidimensional scaling (NMDS) for composition similarity, samples were plotted in table. For bacterial community, kuruma shrimp samples made a group different from whiteleg shrimp samples in the table. For planktonic community, the data could not be plotted in a table because of great variety among the samples. In kuruma shrimp pond, dominant planktons were diatom and dinoflagellate in almost samples. In whiteleg shrimp pond, component was extremely different in each sample and dominant planktons were green algae, zoosporic organism and diatom.

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.

Pinctada fucata is a well-known pearl oyster which produces high quality Akoya pearls. Color is one of the most important factors of pearl quality. Selective breeding has shown that at least yellow coloration is genetic trait among various pearl colors. Here, we conducted comparative RNA-seq analysis between pearl sacs producing pearls with different yellow color tone to screen genes involved in determination of yellow coloration of pearl.
We used two pearl oyster strains with shell nacre at clearly different yellow color tones. The mantle piece was dissected each from two strains and the two pieces were transplanted together into one mother oyster of another strain. In three months after transplantation, pearls and pearl sacs were collected. The yellow color tones of the two pearls were obviously different even if they were produced in the same mother oyster, and well coincided with those of the shell nacre of the donor pearl oyster. Then, mRNA was purified from the pearl sac and subjected to RNA-seq using an IonProton sequencer. In the cluster analysis based on the entire expressed genes, the expression patterns were not well discriminated by the levels of yellow color tones of the pearl produced. Actually only one gene was differentially expressed gene (q <0.05) between the two pearl sacs with different yellow color levels. The number of genes associated with the yellow color was considered to be quite small. Alternatively the structural difference in the genes may be involved in the yellow pearl coloration. The gene expression pattern of the pearl sac would be influenced by the mother oyster, suggesting the importance of the mother oyster in the pearl formation.

Sharks including banded dogfish Triakis scyllium accumulate urea—a protein denaturant—in their bodies, meaning that their proteins are urea-resistant. How have those proteins acquired urea-resistance? We have been studying on a relationship between the primary structures of the proteins—especially myosins, major muscle proteins—and their urea-resistance. Light meromyosin (LMM), a part of the myosin molecule, forms a coiled-coil structure throughout its length. We compared the primary structure of Triakis LMM with that of carp LMM, which is not urea-resistant, and found several amino acid substitutions in the hydrophobic core of the coiled-coil structure. In the present study, we constructed Triakis LMM variants (TS-LMMs), where amino acids in the hydrophobic core were replaced with counterparts of carp LMM (CC-LMMs); and evaluated the effects of substitutions on the urea-resistance of TS-LMMs with ultraviolet absorption spectral analysis. Ultraviolet absorption spectra (200–230nm) of TS-LMMs were measured under various urea concentrations (0–1 M). Peaks of the spectra of all TS-LMMs shifted to longer wavelengths in accordance with an increase of urea concentrations. The absorbance of all TS-LMMs increased between 0–0.5 M urea, but decreased subsequently after 0.6 M urea. There were differences in the degree of change in the peak of the spectra and absorbance of each TS-LMMs. These findings indicate that the amino acids in the hydrophobic core of the coiled-coil structure might contribute to the urea-resistance.

The ayu / sweetfish, Plecoglossus altivelis, is a one-year lifespan fish belong to the genus Plecoglossus and family Plecoglossidae. Ayu is economically important fish for food and recreational fishing in Japan. However, only few genomic resources are available in ayu. In this study, we constructed draft genome sequence of ayu by whole genome shotgun method using next generation sequencing.
Genomic DNA was extracted from fin of male ayu reared at Yoshida Station (Shizuoka), Tokyo University of Marine Science and Technology. Shotgun sequencing library was constructed with insert sizes of approximately 300 bp. Jumping libraries were constructed with insert sizes of 3 kb, 8 kb, 20 kb and 40 kb. Short read DNA sequencing was carried out illumina HiSeq2000 platform using 100 bp paired-end sequencing. Genomic DNA was also sequenced on PacBio RSII for long read sequencing. Short reads were assembled de novo using Platanus. PacBio Long reads were used for gap filling and to upgrade short read assembly using PBJelly pipeline.
Approx. 250x read coverage for short reads and 15x coverage for long reads (estimated genome size of ayu: 450 Mb) were obtained by shotgun sequencing. Assembly of Illumina and Pacbio sequencing reads yielded 1,136 scaffolds larger than 5,000 bp in length. Totals size of assembly was 460,100,202 bp which well coincident with estimated genome size of ayu. Longest scaffold was 17.19 Mp and the assembly had an N50 value of 4.44 Mb. 70% of scaffolds were anchored to the linkage map of ayu using microsatellites marker (510 markers). Our draft genome sequence is expected to accelerate establishment of economically valued strain of ayu using genomic breeding.

Much attention has been attracted on food and feed functions of carotenoids. In this study, we overexpressed endogenous 1-deoxy-D-xylulose 5-phosphate synthase (DXS), reductase (DXR), and Orange protein (OR) in the green alga of Chlamydomonas reinhardtii to evaluate the importance of these proteins in Chlorophyceae carotenoid biosynthesis. DXS and DXR are thought to play important roles in the non-mevalonate (MEP) pathway to IPP synthesis. OR protein is a DnaJ-like chaperone and thought to support phytoene synthase, a key enzyme in plant carotenoid synthesis. The constructed protein expression vectors include dual promoters of heat-shock protein 70A (HSP70A) and ribulose bisphosphate carboxylase small chain 2 (RBCS2). These promoters control the zeocin resistant marker gene of ble and target CDS connected by foot and mouth disease virus (FMDV) 2A peptide.
Nuclear transformations of the protein expression vectors including CDS for OR, DXS, and DXR were conducted. A OR transformant produced lutein and β-carotene contents higher than those of the parental strain. DXS transformants produced lutein content per-medium higher than those of the parental strain. The DXR transformant of CrDXR#4 produced lutein and β-carotene contents per-cell higher than those of parental strain; however, this transformant produced lutein and β-carotene contents per-medium lower than those of parental strain. Considering these results, overexpression of OR protein could enhance carotenoid synthesis in C. reinhardtii. The OR protein in Arabidopsis thaliana is previously reported as the chief posttranscriptional regulator of phytoene synthase (PSY) in controlling carotenoid biosynthesis. Similarly, the present OR protein in C. reinhardtii could control carotenoid biosynthesis in cooperation with PSY.

Milkfish Chanos chanos is the national fish in the Philippines. The production volume in 2015 is the second highest in the world. Feed cost occupies a considerable part of the management expenses for milkfish production. Therefore, reduction in the feed cost is important to improve aquaculture management. Development of low fish meal feed is one of the methods to cut feed cost. To clear the availability of poultry by-product meal for alternative fish meal source in milkfish feed, the effect of partial replacement of dietary fish meal using poultry by-product meal on growth and body compositions were investigated.
Juvenile milkfish of 48.0 g were fed for 12 weeks on 2 experimental feeds containing different poultry by-product meal (8 and 12%) and fish meal (10 and 5%) levels respectively, and for the control feed of no poultry by-product meal, high fish meal (20%) was used. The contents of crude protein, crude fat and crude starch content in all feeds were around 27, 10 and 31%, respectively. The feeding trial was carried out using 6 net cages (5 m x 5m x 4m) with duplication for each dietary treatment. Weight gain, specific growth rate, feed conversion ratio and condition factor were not affected by the inclusion levels of PM in the feeds. Plasma glucose, triglyceride, cholesterol, phospholipid and total protein concentrations were not different among the dietary groups. Also, whole body moisture, crude protein, crude fat and ash contents were not influenced by the dietary treatments. Furthermore, hepatic moisture, crude protein, crude fat, glycogen and ash contents as well as hepatosomatic index were not different among the dietary groups. These results indicate that milkfish can utilize effectively the poultry by-product meal, hence fish meal content for milkfish feed could be 75% replaced by the poultry by-product meal without retarding the growth performance.

In general, the demonstration trial is essential to evaluate the feed performance. The trial should be carried out on the real scale of aquaculture field (i.e. stocking density, net cage size, feeding amount and so on). Juvenile milkfish stocking density rates in the Philippines are quite high, from 20 fish to 50 fish / m³. Therefore, it requires more expenses to conduct a demonstration trial for juvenile milkfish. It is necessary to reduce the cost as much as possible without losing the meaning of a demonstration trial. To cut the cost of a demonstration trial, the influence of stocking density on growth and biochemical compositions were researched in juvenile milkfish Chanos chanos under the near real scale of aquaculture grow-out net cages in the Philippines.
Juvenile milkfish of 78.0 g was reared in a 5m x 5 m x 4 m cages at, 30 fish / m³ (control density group; CDG) and 15 fish / m³ (low density group; LDG) and were fed for 84 days on commercial feed. The feeding trial was carried out in duplication for each stocking density. Weight gain, specific growth rate, feed conversion ratio and condition factor were not different between CDG and LDG. Also, survival rates were not different among stocking groups. Plasma glucose, triglyceride, cholesterol, phospholipid and total protein concentrations were not influenced by the stocking density. Also, whole body moisture, crude protein, crude fat and ash contents were not different between CDG and LDG. Furthermore, hepatic moisture, crude protein, crude fat, glycogen and ash contents as well as hepatosomatic index were not different both stocking groups. These findings indicate that the stocking density could be 50% reduced without affecting the growth and the biochemical compositions of the milkfish.

【Introduction】Yellowtail (Seriola quinqueradiata) meat contains high amount of docosahexaenoic acid (DHA), is known to have various health benefits for human. Previous study using tuna oil or algal meal (All-G Rich: Schizochytrium sp.) as dietary DHA source showed that fillet DHA level increased by increasing dietary DHA level and feeding frequency. However, the influence of different dietary All-G Rich levels on DHA accumulation has not yet been elucidated. In this study, All-G Rich was used as the dietary DHA source to increase fillet DHA level, and the effect on DHA accumulation by various dietary DHA level was investigated.
【Materials and methods】Six iso-nitrogenous and iso-lipidic diets were prepared by substituting 0, 25, 50, 75, 100, or 125 g of 150 g pollock oil/kg diet with All-G (Control, All-G25, All-G50, All-G75, All-G100, and All-G125, respectively). Triplicate groups of fish were fed by hand to apparent satiation once daily. The fish were counted and weighed at 4, 8, and 12 weeks after the feeding trial was started. At 12 weeks, fillets were collected from three fish per tank and pooled for analysis of proximate and fatty acid composition. Further, six diets were subjected to the same analysis as that for the fillet.
【Results】Dietary DHA level (%) in crude lipid ranged from 14.1% (Control) to 24.5% (All-G125). Growth was not adversely affected by dietary DHA level (%). Fillet DHA level (%) in crude lipid increased with increasing dietary DHA level (%). The fillet DHA (%)/dietary DHA (%) ratio was similar among all dietary groups. In conclusion, All-G Rich was a suitable DHA source for DHA-rich yellowtail production, and DHA accumulation in fillet ensured constant fillet DHA (%)/dietary DHA (%) ratio.

The lipid and fatty acid compositions of seven edible macroalgae, Monostroma nitidum, Undaria pinnatifida, Undaria undariodes, Eisenia bicyclis, Gelidium elegans, Mazzaella japonica, and Campylaephora hypnaeoides, in the sea off Japanese coast were examined. Two glycolipids (monogalactosyldiacylglycerol and digalactosyldiacylglycerol) were observed as major components in their polar lipids, while the depot triacylglycerols was mostly found in their neutral lipids. The major polyunsaturated fatty acids (PUFA) in the green algal lipid consisted of various kinds of shorter chain (C16 and C18) n-3 PUFA, such as 16:3n-3, 16:4n-3, 18:3n-3, and 18:4n-3, while those in the brown and red algae included high levels of longer chain (LC) n-3 and n-6 PUFA, such as 20:4n-6 (arachidonic acid, ARA) and icosapentaenoic acid (EPA, 20:5n-3). The high levels of ARA and EPA in the glycolipids of brown and red algae suggest a biosynthetic ability of these LC-PUFA differed from that of the green alga, which is able to make mostly shorter chain PUFA. Similar to other macroalgae, significant levels of various n-3 and n-6 PUFA indicate that all these edible macroalgae are healthful marine foods including markedly high levels of n-3 and n-6 PUFA.

Marine teleosts are generally unable to synthesize docosahexaenoic acid (DHA; 22:6n-3) from α-linolenic acid (18:3n-3) because of the lack of elongase activity toward C22 fatty acids, in addition to some desaturase activities among Δ6, Δ5, and Δ4 desaturations. Therefore, it is essential to receive DHA by a diet for a healthy growth. However, a lineage of Achiridae, including “freshwater soles,” expanded its habitat from marine to freshwater, which is very poor in DHA. To elucidate this nutritionally paradoxical migration, here we examined the DHA biosynthetic pathways of three freshwater soles Trinectes maculatus, Hypoclinemus mentalis, and Apionichthys finis. We isolated desaturase and elongase genes from each species and analyzed their functions by heterologous expression in yeast. The results indicated that the DHA biosynthetic pathways of these freshwater soles were complemented by different mechanisms. The desaturase of T. maculatus alone performed Δ6, Δ5, and Δ4 desaturations, steps which were sufficient for DHA synthesis. On the other hand, two desaturase genes were isolated from H. mentalis. They had 98% homology in amino acid sequences but showed different activities as Δ5Δ6 and Δ4Δ5, respectively. However, their elongases had the ability to convert mainly C18-C20 substrates; therefore, these two freshwater soles were capable of synthesizing DHA via Δ4 pathway, not requiring the elongation of C22 substrate. On the other hand, the desaturase of A. finis exhibited Δ5Δ6 activity but negligible Δ4 activity. However, its Δ6 activity toward the C24 substrate was relatively high, and its elongase could provide C24 fatty acid by its ability to elongate the C22 substrate efficiently, in addition to C18-C20 substrates. Therefore, A. finis appears to be able to synthesize DHA by reacquiring the Sprecher pathway. Our data suggested that functional diversification of fatty acid-metabolizing enzymes enables the increase of fitness in freshwater environment.

The aqua cultural production of the rainbow trout in Shizuoka prefecture is the highest in Japan, and is important for the local industries. Thus far, the trout of approximately 150 g in size were typically cultivated. Recently, however, large trout more than 2 kg have been preferred for merchandise as Sashimi. The lipid information of cultured trout is of more importance from the standpoints of nutrition and their commodity value.
It has been known that the fatty acid (FA) composition of fish body depends largely on the fish feed. Especially, omega-3 polyunsaturated FA such as docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3), rich in fish oil, has variety of health benefits. Not only their contents in lipids but also their lipid forms, including the positional distribution of the constituent FAs in lipids, have known to influence their functions and absorbability. The positional distribution of FAs in the acyl glycerols of aqua cultured trout, however, has not yet been investigated in detail. In this study, the FA composition and distribution in triacylglycerols (TAG) of trout, cultivated in Shizuoka, were investigated.
The content of DHA in fish body TAG was 11±2%, and was similar to that of the fish feed. 18:1n-9 content, 26±2%, was significantly higher than that in the feed (17%). In contrast, the contents of 18:2n-6 (8%) and EPA (2%) were lower than the feed (13% and 6%, respectively). The positional distribution of FAs was different in the feed and TAG of the fish body. The contents of 16:0 and 18:0 distributed in the sn-2 position were significantly lower than the fish feed lipids, whereas that of 18:1n-9 and DHA distributed in the sn-2 position were higher. TAG obtained from different body parts showed similar profiles in the FA composition and distribution.

The lipid and fatty acid compositions of two tropical gastropods (Strombus luhuanus and Turbo marmorata) with a bivalve (Pinctada margaritifera) in the coral reef were examined. Two phospholipids (phosphatidylethanolamine and phosphatidylcholine) were observed as major components in their muscle lipids while the depot triacylglycerols was mostly found in their gonadal and digestive gland lipids. The major polyunsaturated fatty acids (PUFA) in the gastropod tissue lipids consisted of various kinds of n-3 and n-6 PUFA: arachidonic acid (ARA, 20:4n-6), docosatetraenoic acid (n-6 DTA, 22:4n-6), and docosapentaenoic acid (n-3 DPA, 22:5n-3), while those in the bivalve included high levels of docosahexaenoic acid (DHA, 22:6n-3). The significant levels of ARA and EPA in the triacylglycerols of all specimens in-dicate an influence of the dietary algal lipids. Similar to other herbivorous gastropods, the low levels of DHA with significant levels of various n-3 and n-6 PUFA in the gastropods also indicate their assimilation of reef algal lipids. S. luhuanus and T. marmorata are healthful marine foods including markedly high levels of various kinds of n-3 and n-6 PUFA, such as ARA and n-3 DPA, which inhibits platelet aggregation.

Protein and other certain nutrients have been reported to play an important role in reproductive performances of fish. The aim of this study was to evaluate the squid liver meal (SLM) as the major protein source for prematuration diet of rabbitfish. Two prematuration diets containing either fishmeal (SL0) or squid liver meal (SL1) as the major protein source were formulated to accomodate the five months feeding trial. The two tested diets were iso-nitrogenous (40%) and iso-lipidic (14%) and supplemented with several micronutrients specifically for gonadal development. Initial weight of rabbitfish used were 352.6±45.0 g and stocked into four of 2x2x2.5 m3 cages with density of 25 fish per cage. The test diets were fed to the stocks twice a day in the morning and in the afternoon. Variables observed were growth, gonadosomatic index (GSI) and amino acid profiles of gonads and fillet of both male and female stock. The weight gains (WG) of broodstock fed the two diets were nearly similar in which SL0 had WG of 40.1±2.2% and 42.8±2.0% for SL1. The GSI of male fed SL1 diet was relatively higher than that of fed with SL0 which was 7.5±0.2% and 8.1±0.3%, respectively. The GSI of female was 5.6±0.1% much lower than GSI of female fed SL1 which was 11.4±0.5%. Furthermore, total amino acid (TAA) in gonad of male stock was positively influenced by dietary SLI indicated by its higher TAA (62.4%) compared to TAA content of SL0 group (46.1%). The TAA content in oocite of female stock was slightly improved when fed SL1 rather than SL0 (49.3% vs 30.1%, respectively). The dietary squid liver meal seemed not affect the TAA content in the fillet demonstrated by similar TAA content in the two groups which was 55.6% for SL0 and 54.1% for SL1.

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.

Fish meal (FM) is a major protein source in fish feed and is made from small wild-caught fishes. The dietary inclusion levels of FM are around 30–70%; therefore, FM quality is a very important determinant of feed quality, and its precise evaluation is important to predict feed performance. FM quality is currently graded based on its chemical composition. However, the evaluated FM grade is sometimes not reflected in fish growth. To resolve these issues, we aimed to evaluate FM quality using biological response of yellowtail (Seriola quinqueradiata). Appetite-related hormones (neuropeptide Y; NPY and cholecystokinin; CCK) and a digestive hormone (CCK) were assessed for their suitability as indicators of feed performance.
[Injection test] Nine kinds of FM were dissolved in phosphate-buffered saline at a concentration of 13.8 g/100 mL and were orally administered to juvenile yellowtails at 1.5%/g body weight. The hypothalamus and pyloric caeca were collected 40 minutes after administration, following which the expressions of genes encoding the candidate hormones was measured using quantitative real-time PCR. [Growth trial] Nine iso-nitrogenous and iso-energetic diets were prepared by using the 9 FM types used in the injection test and use in two growth trials for juvenile yellowtails to evaluate the quality of the fish diets. Duplicate group of fish were each fed by hand to apparent satiation once daily. The correlation between gene expression levels from the injection test and growth performance from the growth trial were evaluated.
The gene expression of appetite-related hormones did not show significant correlations with growth performance under these experimental conditions. However, the digestive hormone cck was significantly correlated with feed efficiency in two growth trials. These results suggest that the gene expression levels of cck in pyloric caeca could be a good indicator of FM quality.

A feeding trial was conducted to evaluate the utility of Zonocerus variegatus meal (VGM) in the practical diet of hybrid catfish (Heteroclarias) fingerlings through their growth performance and nutrient utilization for 70 days. Six isonitrogenous diets were formulated in which VGM was included in the diet at different inclusion levels - D1 (0%), D2 (10%), D3 (20%), D4 (30%), D5 (40%) and D6 (50%). Each treatment had three replicates, eighteen net hapa (0.5 × 0.5 × 1m) were suspended in three outdoor concrete tanks (8m×5×1.5m). The concrete tanks were filled to 5/6 of its volume (40m3) with filtered and dechlorinated tap water, 20 fish with the initial average weight of 2.17±0.14 were stocked in each hapa. Water temperature and other water quality parameters were monitored daily. Except for the specific growth rate (SGR) where fish fed D3 and D4 were not significantly different (P>0.05), the result also showed that fish fed with D4 (30% inclusion) had the highest value in other growth performance indices measured (P< 0.05), while fish fed D6 (50% inclusion) gave the lowest value. There were no significant differences among fish fed D1, D2, and D3. However, they were higher than fish fed D5. Fish fed D4 had the highest value in all nutrient utilization parameters recorded and was significant (P<0.05) except for protein efficiency (PE) value which was not different with D2. Although D6 had the lowest total feed intake (TFI), there was no significant difference (P>0.05) between D6 and D5. Fish fed D4 had the highest apparent digestibility co-efficient of crude protein and was significantly different from others, except for D3. D6 had the lowest significant value but was not significantly different from D5. The proximate composition result revealed that carcass lipid increased with an increase in the inclusion level of VGM in the diet. It could be concluded that 30% inclusion of VGM can improves growth performance and nutrient utilization of hybrid catfish without any adverse effect health status, suggesting that VGM could be a suitable ingredient for hybrid catfish.

To understand the energy source at embryonic developmental stages as well as newly hatched larvae, proximate composition, fatty acids, and amino acids were quantified in developing eggs and yolk-sac larvae (until 36 hours after hatching) of the Pacific bluefin tuna (PBT), Thunnus orientalis.
Total 3 lots of naturally fertilized PBT eggs were obtained from the broodstocks culturing at net cage. The eggs were incubated at 26℃ with filtered sea water and samples were collected at the following stages: early cleavage (EC), appearance of embryo (E), appearance of Kupffer’s vesicle (K), heart beat initiation (H), just before hatching (BH), and larvae at 12 h (12h L) and 36h L after hatch (36 h L).
The hatching rates of 3 lots were 86.9%, 80.7%, and 73.4%. There were no significant differences in dry weight, protein, lipid, ash, and energy contents per egg (dry basis) during embryonic development, but all parameters were significantly decreased after hatching. There was no correlation between hatching rate and changes in nutritional contents in eggs and hatched larvae. Proteinogenic amino acids didn’t change during embryonic development, but significantly decreased after hatching. However, almost all free amino acids showed gradual decreasing trend from E to 12h L except glutamic acid, which suddenly fall from EC to E and stabled thereafter. Although C14:0, C16:0 and C16:1 significantly decreased from EC to E, other fatty acids and lipid content didn’t show remarkable variation at embryonic developmental stages but reduced significantly after hatching. The results revealed that free amino acids were used as main energy source during embryonic developmental stages, while fatty acids were used after hatching in PBT.

Undaria undariodes, one of the phaeophyceae, is distributed locally in Japan. The alga is classified as a similar group of U. pinnatifida, a popular edible alga in east Asia. For few years, cultivation of U.undariodes has been carried out in Southern part of Mie Prefecture. The alga is desired as one of the local specialties for contribution to regional activation through getting popularity by branding performances. In this study, we intended to contribute the performance, biologically active potentials of the constituents in U. undariodes were surveyed. In the present, we focused on fucoxanthin as an anti-metabolic syndrome reagent, and on scavengers against reactive oxygen species as antioxidants. Undaria undariodes and U. pinnatifida were collected from several places in southern part of Mie Prefecture. 1) Each of the wet algae was homogenized and extracted with 100% acetone. The extract was concentrated and performed to normal phase column chromatography on silica gel for collecting fucoxanthin fraction. Make to constant volume of the fraction with 100% hexane resulted in calculation of the fucoxanthin content by absorbance measurement of lambda max. It is suggested that the content of fucoxanthin produced by U. undariodes was almost equal to that of U. pinnatifida. 2) 30 g of wet U. undariodes was homogenized and extracted with 100% EtOH. The extract was dried and partitioned with water and ethyl acetate. The residue was re-extracted with 50% EtOH. The extract was dried and partitioned with water and 1-butanol. Antioxdant properties of these fractions were examined by recording of scavenging activities against hydroxyl radicals, one of the typical reactive oxygen species, using ESR spectroscopic technique. Especially, aqueous fraction was found to exhibit a significant potency as an antioxidant. These results suggest that U. undariodes was found to be a food and a cosmetic material with high quality for human health.

[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.

【Objective】
Red seaweed, dulse contains high concentrations of water-soluble phycobiliproteins (PP), and thermolysin-digested PP showed anti-inflammatory activity in vitro and in vivo. PP was easily extracted from lyophilized leaves, whereas the extraction from frozen leaves was impaired in the presence of cell wall. The aim of this study is to develop an efficient method for extraction of PP from frozen dulse by degradation of cell wall using carbohydrase.

【Methods】
Frozen pulverized leaves were suspended in distilled water, phosphate buffer (pH 6-8), and acetate buffer (pH 4-6), and cellulase (from Aspergillus niger) was added to the suspension to degrade cell wall. The supernatants of the cellulase-treated extract (CE) were subjected to the following experiments: (a) The optimum condition of cellulase treatment was investigated by varying reaction temperature, pH, reaction time and cellulase concentration, and the yield of CE at the optimum condition was compared with the water extract from the lyophilized leaves (WE). The protein components of the both extracts were also examined. (b) CE was digested by thermolysin at 70 ºC and pH 8.0 (dCE) after concentration of PP using ammonium sulfate precipitation.
Anti-inflammatory activity of the resulting dCE was assessed by measuring productions of inflammatory mediators (nitric oxide and TNF-α) in lipopolysaccharide-stimulated RAW 264.7 murine macrophages.

【Results】
(1) The most effective extraction of CE from frozen leaves was achieved at 25 ºC, pH 7-8 for 90 min with 0.1%(w/v) cellulase concentration. There were no marked differences in the amount of the extracted PP and the extracted protein components between CE and WE. (2) The secretions of inflammatory mediators in RAW 264.7 cells were significantly inhibited by dCE as the same as WE. These results indicate that cell wall degradation using carbohydrase is a useful method for effective extraction of PP from raw leaves.

Abalone production in Fujian, China has reached up to 101 thousand tons by 2015. Along with the change in dietary habits of consumers, there is a trend of utilizing abalone to prepare canned abalone and dried abalone. As a result, a large amount of abalone viscera are generated in the abalone processing factories, due to about 25% viscera in the abalone weights. Abalone viscera is rich in protein, but has not been effectively utilized except as raw materials of condiment.In this study, abalone viscera hydrolysates was fractionated into three fractions (FA: >10 kDa; FB: 1-10 kDa; FC: 0.2-1 kDa) by ultrafiltration and nanofiltration (UF/NF) membranes, and the chemical composition and antioxidant properties of the fractions were investigated. The carbohydrate and protein content of FA were 55.12% and 21.49%, respectively. On the other hand, the protein contents of FB and FC rose to about 75%, while the carbohydrate contents fell to around 2%. Furthermore, the contents of K and Na in the FA were higher than the others, but the highest contents of P and hydrophobic amino acid were found in the FC. The FC showed the highest DPPH and hydroxyl radical scavenging activities with IC₅₀ of 3.07 mg/mL and 1.48 mg/mL respectively, and the most excellent inhibiting ability of linoleic acid oxidation. After FC was subjected to a Sephadex G-15 gel filtration chromatography, two fractions (FC-I and FC-II) were obtained, and the antioxidant activities of FC-II were higher than those of FC-I. The FC-II was dominated by Tyr and Phe, and the molecular weight was around 360 Da.

【Objectives】
Lifestyle diseases, which are diseases caused by habitual behaviors in daily life, such as eating, drinking, smoking, and lack of exercise, have recently become a major concern. People with lifestyle diseases are often initially asymptomatic and therefore go untreated. Hence, preventive measures, specifically the ingestion of functional foods should be established. In this study, we aimed to develop a new functional food material with improved bioactivity and antihypertensive effects by fermenting low-grade (i.e., low utility value) Saccharina angustata with lactic acid bacteria.
【Materials & Methods】
We evaluated the following health functionality activities of Saccharina angustata fermented by 13 species of Lactobacillus spp.: α-glucosidase inhibitory activity, angiotensin I-converting enzyme (ACE) inhibitory activity, pancreatic lipase inhibitory activity, and DPPH radical scavenging activity. For fermented Saccharina angustata that showed significant differences in these activities compared to unfermented Saccharina angustata, we conducted animal experiments to examine their effects in vivo.
【Results & Findings】
Prior to fermentation, the ACE activity of Saccharina angustata was not inhibited; however, fermentation significantly inhibited the ACE activity. In the animal experiments, both single administration and long-term administration of fermented Saccharina angustata significantly decreased systolic blood pressure compared to the control group. These results indicate that fermented Saccharina angustata has an antihypertensive effect both in vitro and in vivo.
【Conclusion】
Saccharina angustata fermented by lactic acid bacteria has an antihypertensive effect and is thus a potential functional food material that could combat lifestyle diseases. Further validation of low-grade Saccharina angustata is expected.

[Objective] Sturgeon cartilage contains rich chondroitin sulfate which has many biological functions，such as immunity improvement, anti-coagulation, and so on. The present study was designed to evaluate the sturgeon chondroitin sulfate on colorectal cancer cell growth inhibition, and determine the main factors influencing the growth of colorectal cancer. [Methods] We chose three representative colon cancer cells Caco-2, HCT-116, SW480, CCK-8 method was used to determine the cell growth curve, chondroitin sulfate on the proliferation of cancer cells inhibition. To determine the Pro apoptotic ability of the sturgeon chondroitin sulfate on colorectal cancer and the role of way, we used the cell line HCT-116 as a model. The cycle and apoptosis were determined by flow cytometry before and after treatment. The cell morphology was observed by cell staining method. And the enzyme activity was determined by Caspase-3 apoptosis. [Results] Our study indicated that the proliferation of sturgeon chondroitin sulfate on colorectal cancer cells had certain inhibition. The suppression of high dose group was respectively 70.94%, 90% and 75%, significantly higher than the positive control group. After treatment, the percentage of G1/G0 phase of cell cycle was increased to 88.56%. The proportion of S phase was shortened to 4.47%, and the G1/G0 phase of cell block was prolonged. Meanwhile, the high dose of sturgeon chondroitin sulfate treated cells, cell morphological changes, nuclear pyknosis and cell fragmentation, apoptotic enzyme activity mainly increased significantly, the enzyme activity reached 1645IU/ μg，the apoptosis rate was 63.73%. Results showed that sturgeon chondroitin sulfate significantly inhibited the proliferation activities of colon canner cells in a dose dependent manner, and induced apoptosis in colon canner cells. [Conclusion] Our study indicated that sturgeon chondroitin sulfate has the anticancer activity, which lays the theoretical foundation for the development of functional food of sturgeon.

Extracellular protease production from Pseudomonas sp. PD14 isolated from a fish gut has been investigated using fish processing wastes as nitrogen source. Among the waste preparations tested, Japanese halfbeak (Hyporhampus sajori) viscera (HV) flour (1.0%) supported the maximum production of 365.0 U.mg protein-1. Effect of media components on protease production in the optimized concentration of fish processing waste revealed that the enzyme can be optimally produced using maltose (0.50%) as carbon source, at pH 8.0, and incubated for 48 h at 40°C. Partial characterization of the enzymatic properties showed that the pH and temperature optimum is at 8.0 and 50°C, respectively. Furthermore, the enzyme exhibited thermostability retaining 50% activity at 60°C and fairly stable up to pH 11.0. Considering the high costs of industrial enzyme production, the use of fish processing wastes for microbial enzyme production offers a promising future for enzyme biotechnology.

In Japan, Pyropia yezoensis is one of the most important algal species used as food, and approximately 300,000−350,000 tons (wet weight) of the thalli are produced in Pyropia cultivation farms every year. Pyropia yezoensis thalli contain many nutritionally and physiologically functional constituents. However, quality and yield of cultivated P. yezoensis thalli are frequently affected by outbreaks of thallus discoloration called “iroochi”, resulting in lower quality P. yezoensis thalli. Recently, glycerol galactoside (GG), which provides some useful bioactivities for human health, has been found to be enriched in low quality P. yezoensis thalli. In this study, we investigated the bioactive potential of constituents in low quality P. yezoensis thalli. A P. yezoensis “iroochi” thallus sample with no market value, collected from the coast of the Ariake Sea in Saga Prefecture, contained approximately 38 and 3% (dry weight) total carbon and nitrogen, respectively. Lyophilized powder of the discolored P. yezoensis thalli was homogenized in H₂O or 75% EtOH. After complex treatments by deproteinization, delipidation, ultrafiltration, or deionization, antioxidant properties of the extracts were examined using the electron spin resonance (ESR) technique. The results showed that the extracts exhibited significant potency in scavenging hydroxyl radicals. When human skin fibroblasts were cultured in presence of the extracts, mRNA levels of COL1A1 and HAS2 genes involved in maintaining healthy skin were significantly increased. Component analysis by HPLC showed that the extracts included a high amount of GG with some minor components. These results suggest that low quality P. yezoensis thalli could be an effective resource for applications in the food and cosmetic fields. This research was supported by grants from the Project of the NARO Biooriented Technology Research Advancement Institution (the special scheme project on regional developing strategy).

【Objectives】Collagen, a major component of connective tissue, has a characteristic triple helical structure with 338 uninterrupted Gly-X-Y repeats, where X and Y are frequently proline (Pro) and 4-hydroxyproline (Hyp), respectively. Generally, water temperature of fish habitats and denaturation temperature of collagen are correlated, indicating that biochemical properties of collagen adapt to the water temperature. However, few reports have examined this. Here, we purified type I collagen from seven fish species from various habitat temperatures to investigate collagen adaptation to water temperature.
【Materials and Methods】Acid-soluble collagen (ASC) of Common dolphinfish, Roughear scad, Blue mackerel, Common wolf eelpout, Giant grenadier, Okhotsk atka mackerel, and Pacific grenadier was purified from skin by standard methods at 4ºC. Denaturation temperature was determined by circular dichroism analysis (J-720, Jasco) by observing the dissipation of the 221-nm peak specific to the triple helical structure of collagen. Amino acid composition was analyzed using amino acid analyzer L-8500A (Hitachi).
【Results】Denaturation temperature of type I collagen for the seven different species was recorded as 28.8, 30.4, 27.5, 20.2, 19.2, 18.1, and 18.3 ºC, respectively. Amino acid composition revealed that Gly, Pro, and Hyp content were 32–36%, 7–12%, and 4–8%, respectively, which was typical for type I collagen. Common dolphinfish, found at the highest temperature, had the highest Pro and Hyp content, and the lowest Ser content. In contrast, Pacific grenadier, found at the lowest temperature, showed a reverse tendency. These results, combined with those of previous reports (23 species in total) show that the correlation coefficient of denaturation temperature and the Pro, Hyp, and Ser content were 0.66, 0.85, and 0.80 (p<0.01), respectively. This suggests that fish collagens adapt to lower water temperature by changing their amino acid composition, especially by replacement of Pro or Hyp by Ser.

[Background] Since red seabream meat is usually eaten raw as “Sashimi”, the meat texture is the most important food attribute. Touhata et al. (2000) reported that the meat texture (breaking strength) was positively correlated with the collagen content of red seabream meat. In this study, we investigated the relation of the expression of collagen metabolism-related genes to collagen content and texture of red seabream meat in order to develop a novel method to assess the meat texture using quantitative PCR system (qPCR).
[Materials and Methods] Fish specimens were obtained in Jul., Sep., Dec., 2014, May and Nov., 2016. The meat breaking strength was measured using a cylindrical plunger. The meat hydroxyproline (Hyp) content was measured as an estimate of collagen content. In qPCR, we targeted the following collagen metabolism-related genes; prolyl 4-hydroxylase α(I) (P4H), matrix metalloproteinase-9 (MMP9) and tissue inhibitor metalloproteinase-2 (TIMP2). β-actin was used as an internal control.
[Results] The mean individual breaking strength value was ranged from 38.0 (May 2016) to 59.3 gw (Jul., 2014). The mean individual Hyp content was ranged from 1.0 (May 2016) to 1.8 µmol/g meat (Sep., 2014). The expression of P4H, MMP9 and TIMP2 genes varied with season, and the highest value of expression of these genes was observed in May 2016. The breaking strength value was positively correlated with the Hyp content as previously reported. On the other hand, the breaking strength value was negatively correlated with the expression of genes (P4H, r = -0.59; MMP9, r = -0.43; TIMP2, r = -0.61) (p < 0.01). Also, the Hyp content was negatively related to the expression of genes (P4H, r = -0.46; MMP9, r = -0.43; TIMP2, r = -0.58) (p < 0.01). These results suggested that the meat texture can be assessed by analyzing expression of collagen metabolism-related genes using qPCR.

[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.

[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.

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.

The common fishing method used by fishermen in Central Java Province, Indonesia is by using “cantrang” (seine net) device forbids the use trawl. Ministry of Marine and Fisheries Affairs has issued ministerial regulations PERMEN KP No. 2, 2015 that forbids the use trawl and seine net devices after 31st December 2016. This regulation did not receive good responses from all stakeholders. It was was deemed necessary to conduct this research to identify and evaluate the economic and social impacts both directly and indirectly. There are few sub-sectors and businesses that have been severely affected especially fishermen, local governments and authorities, fish processors, duck raisers, makers of “selambar” rope, grocery store owners and vegetable traders. Prohibition of the use of “cantrang” will cause huge economic losses, thus it is not feasible. Total economic impact (loss of income) of all stakeholders is US$ 146,819.137.43 impacting mostly fishermen (78.33%) using “cantrang” device. Whilst, total social impact (loss of employment) is US$ 112,723,134.54 (1 US$= IDR 13,000) involving 66.621 manpower with 80.11% of them are fishermen.

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.

Indonesia is the world’s largest archipelagic state with some 17.508 island, and 54.716 km of coast line. Indonesia fisheries production hit 5,8 millions tonnes in 2012, with the industry valued at Rp 79,4 trillion. Illegal, Unregulated and Reported fishing is recognized globally as a threat to the management and conservation of marine resources and ecosystems, and in particular to sustainable fisheries. According to FAO Fisheries and Aquaculture Department, illegal fishing caused losses estimated at 23 US$ billion per year with about 30 % of illegal fishing in the world occurring in Indonesia. An effort to stop the illegal fishing in Indonesia, Indonesia declared the mission on Indonesia waters agendas is sovereignty, security, and prosperity. Otherwise, based on the Law No. 45/2009 on Fisheries of Indonesia and Exclusively Economic Zone, the Government legally punishes actor of the illegal fishing by burning and sinking their ships to assert the sovereignty of Indonesia.

Squid jigging is one of the most important fisheries for Japanese common squid, Todarodes pacificus, in the Japanese waters. Although the dominant stocks divided by the spawning seasons are managed by the TACs and the fishing licenses in the Japanese EEZ, the stock sizes have been fluctuated decadally related to long-term climate changes. In Sanriku district, northeastern Japan, small-scale squid jigging for Japanese common squid is an important gear for the capture fisheries operated by individual households in the coastal waters and the fluctuations of squid stock may influence on the sustainability of regional fisheries. This study was carried out to evaluate long-term fluctuations in the small-scale squid jigging fishery operated off Sanriku and to discuss the relationship with stock and socio-economic factors based on the statistics from 1970 to 2008 on catch, fishing effort and price in Iwate Prefecture and oil price for vessel fuel. The present study represented four groups consisting of the early to mid-1970s, the late 1970s to 1980, the 1980s to 1991 and the early 1990s and later based on the cluster analysis. The results showed a decreased phase until 1980 and a low abundant period from the 1980s to1991 in catch, related to the decadal fluctuations in the winter stock. Although the winter stock was assessed abundant since the 1990s, the recent groups since 1980s were equally characterized by much lesser than the 1970s in catch and fishing efforts. The groups since the 1990s and during the 1980s were characterized by lower squid price and higher oil price, respectively. Therefore, the present results suggest that the sustainable operation has been difficult in the small-scale squid jigging for Japanese squid in Sanriku since the winter stock decrease in the 1980s due to economic reasons such as lower squid price and higher oil price.