Skeletonema is a dominant diatom, especially in coastal waters. However, species identification and quantification in the natural environment has not yet been established, as species identification can be nearly impossible with light microscopy. In the present study, we developed primer sets for multiplex and real-time PCR to identify and enumerate the 10 Skeletonema species, and applied this technique to samples obtained from the tidal zone of the Chikugo River and its adjacent waters of the Ariake Sea from spring to summer. In total, eight Skeletonema species were detected during the investigation. Among the eight, the S. marinoi–dohrnii complex was detected at all three stations. Skeletonema potamos was the second most frequently detected, mainly in the Chikugo River. The overall number of detected species was low at the upper tidal zone of the Chikugo River, where salinity was < 2. These results reveal the diverse Skeletonema community in the brackish environment, and the primer sets designed in the present study are useful for analyzing species composition of Skeletonema.

We investigated water quality and sessile animals in Dokai Bay, Japan, during periods of heavy eutrophication （ 1991-1992 ） and improved conditions （ 2010-2011 ） to evaluate the usefulness of sessile animals as environmental indicators. Hypoxia disappeared and total nitrogen and phosphorus decreased following the control of industrial discharges into the bay. Reduced eutrophication led to the spread of oxygenated water to the inner bay, accompanied by expanded distributions of marine sponges and bryozoans. In addition, the improvement of water quality appears to have added 40 new species to the community of gastropod mollusks （ snails ） . The Shannon diversity index for mollusks increased, and the total number of sessile animal species increased to 1.6 times that before water quality improvement. However, the total wet weight of two species of bivalve mollusks, mussels and Japanese oysters, decreased to 1.2% that before the improvement; statistics confirmed that this drastic decrease was a result of increasing water temperature. These results show that the community and population of sessile animals can be used as indicators of eutrophication and increasing water temperature, because of their dependence on environmental factors.

The species composition of the genus Skeletonema ( Bacillariophyceae) was investigated in five Japanese coastal waters by analyzing large subunit rDNA D1-D3 sequences and the fine morphological structures of vegetative and germinated cells. The predominant species were S. grevillei from Main Okinawa Island Midwaters in the subtropical zone and S. dohrnii from the Ariake Sound, Toyama Bay, and Onagawa Bay in the temperate zone, and Funka Bay in the subarctic zone. S. japonicum was found with S. dohrnii in the latter four bodies of water. During the warm season, S. costatum, which can acclimate to low salinity, was found predominantly in the Ariake Sound and Toyama Bay, where the salinity was low, despite the fact that S. costatum is able to germinate from sediments over the temperature range of 10-30 degrees C. The absence of S. costatum vegetative cells in the winter was due to lower specific growth rates of S. costatum than those of S. dohrnii and S. japonicum. During warm periods, S. grevillei and S. pseudocostatum occurred as the dominant species in Onagawa Bay and Funka Bay respectively, where salinities were usually > 30. The species composition and seasonal succession of Skeletonema were closely related to temperature and salinity.

SUMMARYSmall subunit (SSU) and large subunit (LSU) rDNA sequences have been commonly used to delineate the taxonomy and biogeography of the planktonic diatom genus Skeletonema, but the genes occur as multiple copies and are therefore not suitable for barcoding purposes. Here, we analyzed phylogenetic relationships of Skeletonema using the mitochondrial‐encoded cytochrome c oxidase I gene (cox1), as well as partial LSU rDNA (D1–D3) and SSU rDNA, to identify the factors that define species and to evaluate the utility of these three markers for this taxon. Twelve Skeletonema species were divided into six clades, I–VI, each of which comprised the same species by the three markers: clades I (S. japonicum, S. grethae, S. pseudocostatum, and S. tropicum), II (S. menzelii), III (S. dohrnii and S. marinoi), IV (S. costatum, S. potamos, and S. subsalsum), V (S. grevillei), and VI (S. ardens). However, the branching order among these clades was incongruent among the markers. In clade III, six S. marinoi strains had identical cox1 sequences. These S. marinoi strains branched along with S. dohrnii, except for strains from the Gulf of Naples, with high support in cox1. Species delimitation between S. dohrnii and S. marinoi was therefore not supported. In clade IV, S. costatum and S. subsalsum were robustly clustered, with S. potamos as a sister clade in the cox1 tree, not in the LSU and SSU trees. In clade II, cox1 also confirmed that S. menzelii includes three subclades potentially distinguishable from each other by morphological features. Cox1 proved to be the most useful marker for the identification of Skeletonema species because it gave a tree with highly supported clades, has sufficient variation within and among species, encodes a protein in a single copy, and requires relatively few primers.

The estuarine planktonic calanoid copepod Eurytemora americana is described for the first time from Japan based on specimens from Dokai Bay, a small inlet of northern Kyushu in 1975, when the species was exclusively dominant in the innermost bay. This is the southernmost record of the species in the Northern Hemisphere. Morphological descriptions are provided, especially on characters different from or overlooked in previous descriptions. Compared with the recent description of the species from Korea, a distinct morphology is found in the spinule number on the male fifth leg, suggesting inter-population variation and therefore no gene flow between the Japanese and Korean populations. Eurytemora americana in Dokai Bay is regarded as an invasive alien species introduced via ship ballast water. Despite the dominance in 1975, the copepod was not collected from brackish waters in the innermost part of the bay in 2008. Global warming in spring and/or winter periods after the 1970s is a possible reason for the disappearance of this cold water species.

s of Poster Presentations 1: Marine microalga Rhodomonas sp. as a novel live food for bivalves Satoshi Yamamoto, Masanori Okauchi and Takao Yoshimatsu *1 Graduate School of Bioresources, Mie University, Tsu, Mie, Japan; *2 National Research Institute of Aquaculture, Fisheries Research Agency, Minamiise, Mie, Japan E-mail: takaoyos@bio.mie-u.ac.jp Recently, many species of bivalves, including the asari clam have become more popular as seafood allover the world. In spite of the increasing demands of bivalves, the natural resources are becoming scarce year by year. Therefore, stable aquaculture production of bivalves is highly required to substitute the natural resources. Marine microalga Rhodomonas sp. is widely distributed in Japan’s coastal waters. This species has a high content of highly unsaturated fatty acids (HUFAs) that enhance the survival and growth of bivalves. For this reason, Rhodomonas sp. is expected to play an important role as feed for bivalve aquaculture. In this study, suitable conditions for mass culture of Rhodomonas sp. in terms of temperature, salinity and light intensity were investigated. A series of rearing experiments was conducted with one of the three factors altered at a time. In temperature, higher cell increase rate was observed between 16 and 24°C, and the maximum cell density (435.8 × 10 cells ml) was observed at 24°C. In salinity, higher cell increase rate was observed between 14 and 35 psu, and the maximum cell density (373.9 × 10 cells ml) was observed at 21 psu. In light intensity, higher cell increase rate was observed between 35 and 80 μ mol m s, and the maximum cell density (360.1 × 10 cells ml) was observed at 80 μmol m s. 2: Observation and monitoring of asari clam predators in a population-collapsed habitat using Field Server Naoaki Tezuka, Naoto Kajihara, Tokihiro Fukatsu, Kazumasa Oguri and Takehisa Yamakita *1 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hatsukaichi, Hiroshima, Japan; *2 National Agriculture and Food Research Organization Agricultural Research Center, Tsukuba, Japan; *3 Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan E-mail: tezukan9@fra.affrc.go.jp The Nakatsu tidal flat, located in western Seto Inland Sea, Japan, is known as an asari clam, Ruditapes philippinarum, population-collapsed habitat. A recent study observed low clam survival rates, < 15%/yr, from post-settlement until fishable size in this flat. Based on previous netting/caging experiments, predation has been suggested as a major cause of clam mortality. However, we do not have enough information on predator species composition or when and how often the predators appear on the flat. We employed the Field Server, originally developed for agricultural field monitoring equipped with a web-camera and sensors, to observe the asari clam predators on the Nakatsu tidal flat. Field Server observations, comprised web-camera images retrieved every 30 min via the internet, revealed that ducks (Anas acuta) often appear on the monitoring site during low tide in winter. These results suggest that predation by ducks may be a significant cause of asari clam mortality during winter in the Nakatsu tidal flat. This study was supported by the Ministry of Agriculture, Forestry and Fisheries of Japan. 水 研 機 構 研 報, 第 42 号,79 104, 平 成 28 年 Bull. Jap. Fish. Res. Edu. Agen. No. 42,79-104,2016

Perforatus perforatus was originally distributed from England to the Mediterranean and West Africa. It was first reported from the East Asia following its discovery in three ports near Ulsan Port, Korea, on 24 June 2006 and some other ports along the southeast coast of Korea. In Japan, this species was first reported on the basis of specimens collected at 2012 from buoys for the cultivation of rock-oysters, and thereafter was found to be widely distributed in northern Honshu. Here we re-identify as Perforatus perforatus a Japanese specimen that had been collected on 11 May 1992 from the Dokai Bay of Kitakyushu in northern Kyushu. We also provide photographs of its calcareous plates. This specimen represents the oldest record of P. perforatus from the East Asia and thereby provides an excellent example of the importance of the preservation of historical collections and preserved in the Kitakyushu Museum of Natural History and Human History, Kitakyushu, Fukuoka, Japan (KMNH IvR 500844).

Species compositions of planktonic cells and benthic resting-stage cells of the marine diatom Skeletonema were investigated in Osaka and Tokyo Bays. Seven to 11 strains were isolated from each of the monthly water samples for species identification by molecular analysis. The species frequency of the strains indicated that S. sp. cf. marinoi-dohrnii complex was dominant throughout the year in Osaka Bay, whereas in Tokyo Bay the dominant species was replaced by S. japonicum during the cold season. The other species identified were S. costatum, S. japonicum, S. pseudocostatum, and S. tropicum in Osaka Bay, and S. menzelii and S. tropicum in Tokyo Bay. For species identification of resting-stage cells, sediments in each bay were sampled at 2–3 stations and stored in the dark for ≥85 days. Eight to 12 strains were isolated from each of the diluted sediments incubated at five temperatures (10–30°C). S. costatum, S. japonicum and S. sp. cf. marinoi-dohrnii complex were identified in these stains; the resting stage of S. japonicum was newly recognized. The dominant species of resting-stage cells in each bay largely agreed with those of planktonic cells on a yearly basis. S. sp. cf. marinoi-dohrnii complex in the sediment could germinate at all the temperatures but S. japonicum did not occur at 30°C. The results of seasonal occurrences and incubation experiments indicate that S. sp. cf. marinoi-dohrnii complex is eurythermal and S. japonicum prefers lower temperatures. Possible factors for the difference in the community of Skeletonema between Osaka and Tokyo Bays are discussed.

The genus Skeletonema includes phytoplankton species that are important primary producers in marine food chains. Brackish waters have been reported to be one of the important habitats of some species of Skeletonema. To elucidate the species diversity of Skeletonema in brackish waters, we investigated three Japanese brackish bodies of water: the coastal waters of Toyama Bay, a tidal area of the Chikugo River, and a constructed reservoir in Isahaya Bay. We used molecular analysis based on large subunit rDNA and fine morphological structure to identify species. Skeletonema costatum s.s. (sensu stricto) was isolated at salinities as low as 0.6, but Skeletonema dohrnii, Skeletonema subsalsum, and Skeletonema tropicum were not found at salinities below 11.0. S. costatum s.s. could survive transfer from a medium with a salinity of 15 to a salinity of 2, but S. dohrnii did not survive in the same experiment. Only S. costatum s.s. germinated from the sediment of a reservoir in which the salinity was 0.1–1.4; incubation conditions included temperatures of 10, 15, 20, 25, and 30 °C and salinities of 5 and 30. Skeletonema costatum s.s. was identified as the species most adaptable to low-level salinity variations throughout its lifecycle.

The effect of temperature on the growth of Skeletonema ardens, S. costatum sensu stricto, the S. marinoi-dohrnii complex, S. japonicum, S. menzelii, S. pseudocostatum and S. tropicum isolated from Dokai Bay in southern Japan were examined under five to seven different temperatures and an irradiance of 150 mu mol m-2 s-1. The effect of irradiance on the growth of the seven Skeletonema species was also examined under a wide range of irradiances ranging from 7-700 mu mol m-2 s-1 at 20 degrees C. All Skeletonema species were able to grow at temperatures ranging from 15 to 25 degrees C. Intra-species differences in specific growth rates of four strains for S. menzelii, and five strains for S. ardens, the S. marinoi-dohrnii complex, S. japonicum and S. tropicum were not significant (Kruskal-Wallis test, P 0.05). Significant inter-species differences in specific growth rates were observed at 10, 15, 25 and 30 degrees C (Kruskal-Wallis test, P 0.01; Steel-Dwass test, P 0.01). The S. marinoi-dohrnii complex and S. japonicum grew faster than other species at the lower temperatures of 10 and 15 degrees C, and S. ardens and S. menzelii grew at the highest temperature of 35 degrees C. The maximum specific growth rates (max) from growth-irradiance curves ranged from 1.50 to 3.44 d-1. Threshold values of irradiance (I0) and saturation irradiance (S) for growth ranged from 3.9 to 7.6 mu mol m-2 s-1, and from 250 to 740 mu mol m-2 s-1, respectively. In Dokai Bay, our results suggested that the occurrence of Skeletonema species should be affected mainly by temperature and less by irradiance. In particular, only two species, the S. marinoi-dohrnii complex and S. japonicum could maintain their populations in the surface water during the cold season, whereas during other periods, all seven species could maintain their population under the strong influence of estuarine circulation, which rapidly flushed the surface water out of Dokai Bay. Temperature and irradiance dependent growth values were in good agreement with their geographical distributions. The S. marinoi-dohrnii complex and S. japonicum were capable of growing in cold regions, but our results suggested that S. ardens and S. menzelii will tend to prefer tropical regions.

Water quality and red tide occurrence were monitored monthly at Dokai Bay, Kitakyushu City, Japan, for 28 years (1980 similar to 2007). Although total nitrogen (TN) and total phosphorus (TP) concentrations decreased significantly over this period, red tides formed during the warm season every year. A comparison of the community of red-tide organisms between periods of high concentrations of TN (9.1 mg L-1) and TP (0.170 mg L-1) (1988 similar to 1992) and low concentrations of TN (2.0 mg L-1) and TP (0.121 mg L-1) (2003 similar to 2007) showed that the diatoms Skeletonema spp. were dominant during both periods, but their cell density and frequency of forming red tides were significantly lower during the period of low TN and TP. Furthermore, small-sized diatoms, including Thalassiosira spp., Chaetoceros spp., Cyclotella sp., and the raphidophyte Heterosigma akashiwo, increased their predominance of red tide component organisms and occurrence frequency of their cell densities to form monospecific red tides during the low TN and low TP period. These findings suggest that the decrease in TN and TP concentrations in Dokai Bay did not affect the seasonality or the frequency of red tide formation, but did result in increased species diversity of red-tide organisms.

Biologically important nutrient concentrations in Dokai Bay have declined as a result of reductions in anthropogenic inputs of total nitrogen and total phosphorus. A decrease in nutrient concentrations affects phytoplankton growth, thereby changing the biochemical characteristics of autochthonous particulate matter. We therefore investigated changes in the C/N/P molar ratio of suspended particulate matter (SPM) in the summer, when phytoplankton growth is vigorous, before environmental quality standards (EQSs) were attained (1995–1998) and afterward (2006–2009). We found that the ratio of particulate organic nitrogen (PON) to particulate phosphorus (PP) changed in conjunction with changes in the ratio of dissolved inorganic nitrogen to dissolved inorganic phosphorus (DIP) that resulted from reductions in nutrient loading. Furthermore, we suggest that because the DIP concentration in seawater was high before EQSs were attained, inorganic phosphorus was possibly adsorbed onto SPM. After the attainment of EQSs, however, the DIP concentration fell, and PON/PP was high. Phosphorus limitation of phytoplankton growth in the mouth of the bay may explain the high PON/PP ratios after EQS attainment.

Seven Skeletonema species were identified at one station in the industrial harbor Dokai Bay, Japan, in October 2007 and then monthly from January 2008 to December 2009 by morphological scanning electron microscopy observations and molecular analyses of mainly the large subunit (LSU) rDNA. We refer to one species identified as S. dohrnii using LSU rDNA, but as S. marinoi using the small subunit (SSU) rDNA and consequently we use the term S. marinoi-dohrnii complex. This is the first time that S. ardens , S. costatum s.s., and S. pseudocostatum have been recorded in Japan and that S. menzelii and S. tropicum have been identified by molecular methods in Japan. The S. marinoi-dohrnii complex was isolated with high frequency all year-round, and S. japonicum was also isolated with high frequency but not so in summer. S. Tropicum was isolated from September to December, but S. ardens and S. costatum s.s. were isolated only when the water temperature exceeded 20°C. S. pseudocostatum bloomed just once, in summer, but S. menzelii was isolated in May and again in October. The continuous year-round occurrence of S. costatum s.l. in the bay is supported by the succession of these seven species. Six of the species, except S. menzelii , were important components of algal blooms in the bay. One to four species of Skeletonema were isolated every month. Monthly species diversity was higher when S. ardens, S. costatum s.s., and S. tropicum that are usually associated with tropical or subtropical waters, were isolated. In general, species diversity of the genus Skeletonema was very high in Dokai Bay.

The importance of the nitrogen source for phytoplankton growth in a highly eutrophic embayment, Dokai Bay, was investigated. The DIN concentration often exceeded 100 µM of which 40–70% was NH 4 + . During two incubation experiments, the natural assemblage of mainly diatoms took up NH 4 + instead of NO 3 − . The growth of two Skeletonema species isolated in Dokai Bay were significantly faster on NH 4 + (1.86 and 1.27 div. d −1 respectively) than on NO 3 − (1.55 and 1.04 div. d −1 respectively). Our results indicated that these diatoms could grow faster by using NH 4 + compared to NO 3 − in this eutrophic bay.

熱帯・亜熱帯性海産珪藻のSkeletonena tropicum栄養細胞についてモニタリング調査を16 年間(1991~2006年)にわたり温帯海域の洞海湾で行った結果, S. tropicumは7月から10 月の夏季から初秋を中心に出現し, 冬季から春季には出現しないという規則的な季節的消長を示した。生存に不適な低水温期には, S. tropicumは休眠期細胞となって海底堆積物中で越冬した。S.tropicumの休眠期細胞は夏季に2.9 mg g-1という高濃度の硫化物(AVS)に眼された後にも発芽が可能で, 有機汚濁に対して耐性が認められた。また発芽は, 高水温で, 光照射がある場合に可能であった。栄養細胞はNH4-Nが711 μMと他の植物プランクトンが増殖阻害を受けるような水質にもよく適応し, 濃密な赤潮を形成した。S. tropicumは上記のような生存戦略をとることにより, 温帯海域の富栄養化の進行した洞海湾に定着していると考えられる。