New polymorphic microsatellite markers derived from hemocyte cDNA library of Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> challenged by the protozoan parasite <Emphasis Type="Italic">Perkinsus olseni</Emphasis>

Manila clam Ruditapes philippinarum is one of the most important benthic animals in the coastal north Pacific region, where clam populations have been mixed genetically through trade and aquaculture activities. Accordingly, identification of the genetically different clam populations has become one of the most important issues to manage interbreeding of the local and introduced clam populations. To identify genetically different populations of clam populations, we developed 11 expressed sequence tag (EST)-microsatellite loci (i.e., simple sequence repeat, SSR) from 1,128 clam hemocyte cDNA clones challenged by the protozoan parasite Perkinsus olseni. Genotype analysis using the markers developed in this study demonstrated that clams from a tidal flat on the west coast contained 6 to 19 alleles per locus, and a population from Jeju Island had 4 to 20 alleles per locus. The expected heterozygosity of the 2 clam populations ranged from 0.472 to 0.919 for clams from the west coast, and 0.494 to 0.919 for clams from Jeju Island, respectively. Among the 11 loci discovered in this study, 7 loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction. The 5 loci developed in this study also successfully amplified the SSRs of R. variegatus, a clam species taxonomically very close to R. philippinarum, from Hong Kong and Jeju Island. We believe that the 11 novel polymorphic SSR developed in this study can be utilized successfully in Manila clam genetic diversity analysis, as well as in genetic discrimination of different clam populations.     

Distribution of ephyrae and polyps of jellyfish <Emphasis Type="Italic">Aurelia</Emphasis><Emphasis Type="Italic">aurita</Emphasis> (Linnaeus 1758) <Emphasis Type="Italic">sensu lato</Emphasis> in Mikawa Bay,Japan

We surveyed the distribution of colonies of polyps of Aurelia aurita sensu lato (s.l.) in Mikawa Bay, Japan. First, we surveyed the distribution of ephyrae of A. aurita s.l. at 75 stations encompassing the whole of Mikawa Bay in early 2008. A total of 37 ephyrae were sampled mostly from fishing ports. Ephyrae were most abundant around the islands located near the mouth of the bay, and decreased from the western part to the eastern part of Mikawa Bay. Next, we selected five fishing ports in Mikawa Bay where ephyrae occurred and surveyed the underside of floating piers and underwater overhangs of wharfs. We found dense colonies of polyps of A. aurita s.l. under nearly all of the floating piers at the two islands located near the mouth of the bay. Fitting a logistic regression model to the dataset showed that the percentage coverage of Aurelia polyps was significantly greater at the two islands compared with the other locations. In addition, the coverage of Aurelia polyps was greater when the coverage of other fouling organisms was in the range of 65–90%, and the coverage of Aurelia polyps was lower on floating piers with a vinyl surface and on concrete wharfs. The combined distribution of polyp colonies of A. aurita s.l. in Ise Bay and Mikawa Bay suggested that A. aurita s.l. in the two bays probably forms a single population and shoals of medusae mainly originate from protected harbors along the mouth-part of the bays.     

<Emphasis Type="Italic">Zostera marina</Emphasis> seed burial can be enhanced by Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis>: A microcosm study

Seagrass seed bank plays a key role in the regeneration of new vegetation when seagrasses are removed by the natural or man-made disaster. Various factors may affect the development of sediment seed bank. We conducted a microcosm experiment to test the effects of burrowing and feeding activities of Manila clam, Ruditapes philippinarum on the burial of Zostera marina seeds in sediments. The effects of lasting time (3-hour, 1-day, 3-day, 7-day, 14-day and 28-day), clam density (0, 2, 4 and 8 clams with shell length of 3 cm in each microcosm) and clam size (shell length of 2, 3 and 4 cm at 4-clam density) on seed burial were examined in plastic microcosm cores (30 cm high × 10 in inner diameter) in a 28-day period. Results showed that the seed burial depth significantly increased with time, the density and the size of clams. No seeds were buried in the sediment in the cores without clams during the whole experiment period. For the 3-cm clams, about 91.61% of the seeds were buried in the sediment at the end of the experiment in the high-density treatment (8 clams at each core); while in the medium and low-density treatments (4 and 2 clams in each core, respectively), about 76.93% and 60.61% of the seeds were buried in the sediment, respectively. For the size treatments, large (4 cm) clams buried 89.56% of the seeds at the end of the experiment, much more than those of medium (3 cm, 76.93%) and small (2 cm, 61.50%) size clams. During the whole experiment period, nearly all of the buried seeds were at a depth of from 0 cm to 5 cm. These results suggested that Manila clam Ruditapes philippinarum may play an important positive role in seagrass seed bank dynamics in the field.     

Effects of temperature on growth,photophysiology, Rubisco gene expression in <Emphasis Type="Italic">Prorocentrum donghaiense</Emphasis> and <Emphasis Type="Italic">Karenia mikimotoi</Emphasis>

To explore the effects of temperature changes on dinoflagellate bloom succession in the coastal waters of the East China Sea, changes in the growth, photophysiology, and Rubisco gene expression of Prorocentrum donghaiense and Karenia mikimotoi, two harmful algal species, were investigated at different temperatures (16 to 28°C). The maximal specific growth rate and the maximal mRNA expression of Rubisco gene in P. donghaiense and K. mikimotoi occurred at 20 and 24°C, respectively. The photosynthetic activity of P. donghaiense was generally stable, but K. mikimotoi photosynthesis increased when temperatures rose from 16 to 28°C. The effective photochemical efficiency (F _q ^′ /F _m ^′ ) and the maximal relative electron transfer rate (rETR_max) of K. mikimotoi increased significantly with increasing temperature, and the lowest and highest values occurred at 16 and 28°C, respectively. It seems that P. donghaiense has higher photosynthetic capacity than K. mikimotoi due to its higher F _q ^′ /F _m ^′ , rETR_max, and photosynthetic efficiency (α). However, K. mikimotoi has a higher growth rate than P. donghaiense. These results suggest that the photosynthetic activity and genetic responses of dinoflagellates are species-dependent. It is likely that temperature changes affect species composition during blooms, leading to the observed patterns of bloom succession.     

Molecular phylogeny of the genus <Emphasis Type="Italic">Chondracanthus</Emphasis> (Rhodophyta), focusing on the resurrection of <Emphasis Type="Italic">C. okamurae</Emphasis> and the description of <Emphasis Type="Italic">C. cincinnus</Emphasis> sp. nov.

Determining the taxonomic status of the red algal genus Chondracanthus based on morphological characters is challenging due to the similarity and high degree of plasticity of the thallus. Since the taxonomic history of several Chondracanthus species remains unclear, we analyzed the plastid rbcL and mitochondrial COI genes of the specimens from Korea and Japan, in combination with morphological observations, to examine their phylogenetic relationships. Our results confirmed the distinction of C. okamurae, which is separated from C. intermedius, and identified a novel species, C. cincinnus sp. nov. Three species (C. okamurae, C. intermedius and C. cincinnus) formed a monophyletic clade with C. tenellus. C. okamurae is distinguished by linear, narrow, cylindrical to compressed, slightly recurved axes, and a high-intertidal to subtidal distribution. It was collected from Korea and Japan, while C. intermedius was identified from Japan only. A new species, Chondracanthus cincinnus sp. nov., is characterized by linear, compressed, strongly recurved axes, and a low-intertidal to subtidal distiribution. Based on the molecular phylogeny using rbcL and COI data, we herein resurrect C. okamurae as a distinct species and identify C. cincinnus as a new species.     

Phylogenetic relationship between symbionts of tubeworm <Emphasis Type="Italic">Lamellibrachia satsuma</Emphasis> and the sediment microbial community in Kagoshima Bay

Vestimentiferan tubeworms acquire their symbionts through horizontal transmission from the surrounding environment. In the present study, we constructed a 16S rRNA gene clone library to investigate the phylogenetic relationship between diverse microbes in the sediment and symbiotic bacteria in the trophosome of the tubeworm, Lamellibrachia satsuma, from Kagoshima Bay, Japan. Two symbiotic bacterial phylotypes belonging to the classes γ- and ε-Proteobacteria were found from this tubeworm trophosome. They were very closely related to the symbionts of several other marine invertebrates. The most predominant bacteria in the sediment were ε-Proteobacteria. A broad diversity of bacteria belonged to non-proteobacterial phyla such as Planctomycetes, Acidobacteria, and Chloroflexi was observed. The presence of sulfur oxidizers (i.e., ε-Proteobacteria and γ-Proteobacteria) and sulfur reducers (i.e., δ-Proteobacteria) may play a significant role in the sulfur cycle in these habitats and provide multiple sources of nutrition to the cold-seep communities. Closely related clones of ε-Proteobacteria symbiont in the species level and of γ-Proteobacteria symbiont in the genus level were found in the surrounding sediment. The similarity of symbiont clones of L. satsuma with other symbionts and free-living bacteria suggests the possibility of opportunistic symbiosis in ε-Proteobacteria and the co-evolution of γ-Proteobacteria having occurred after symbiosis with the tubeworms.     

Molecular ecological study of <Emphasis Type="Italic">Siganus spinus</Emphasis> and <Emphasis Type="Italic">S. guttatus</Emphasis> from Okinawan waters based on mitochondrial DNA control region sequences

The mitochondrial DNA (mtDNA) control region was sequenced to examine the genetic variability and gene flow of juvenile Siganus spinus and S. guttatus. In total, 461 nucleotide sequences were obtained from 69 specimens of juvenile S. spinus from Okinawa Island and Ishigaki Island, in which 17 variable sites and 22 haplotypes were identified. Haplotype diversity (h) was high (0.9244 in Okinawa and 0.8984 in Ishigaki), whereas nucleotide diversity (π) was low (0.0063 in Okinawa and 0.0059 in Ishigaki). The two populations were not genetically distinct. Siganus guttatus, which do not form large schools at recruitment, in contrast S. spinus, were also analyzed by studying 152 individuals collected off Okinawa Island, Miyako Island, and Ishigaki Island. Of 476 nucleotide sequences, 50 were variable, and 42 haplotypes were identified. Genetic variability values were high (h = 0.8766 and π = 0.0151 in Okinawa; h = 0.9640 and π = 0.0192 in Miyako; h = 0.9161 and π = 0.0199 in Ishigaki). The Okinawa population was genetically isolated from the Miyako and Ishigaki populations. As a result, genetic diversity was high for each of these siganid populations despite their being target species for fisheries; however, the degree of inter-population gene flow was higher for S. spinus than S. guttatus, suggesting that these species exhibit different dispersal strategies.     

High mortality and poor growth of green mussels, <Emphasis Type="Italic">Perna viridis</Emphasis>, in high chlorophyll-<Emphasis Type="Italic">a</Emphasis> environment

The current study was carried out from May 2014 to April 2015 to estimate the stock status of P. viridis in Marudu Bay. The gonad development was monitored by histological examination, while the population parameters including asymptotic length (L_∞), growth coefficient (K), mortality rate (Z, F and M), exploitation level (E) and recruitment of P. viridis were estimated using the lengthfrequency data. Results of the current study demonstrated that P. viridis in Marudu Bay spawned throughout the year with two major peaks, one in April to May and another one in October to December. The recruitment pattern was continuous with the peak in May to June 2014, which corresponded to the first spawning peak in April. However, no significant recruitment was observed from the second spawning peak due to the difference in spawning timing between male and female populations. The estimated asymptotic length (L_∞), growth coefficient (K), total mortality (Z), natural mortality (M), fishing mortality (F) and growth performance (ф) of P. viridis in Marudu Bay were estimate to be 117 mm, 0.97 yr^-1, 4.39 yr^-1, 1.23 yr^-1, 3.16 yr^-1 and 4.123, respectively. The exponent b of the lengthweight relationship was 2.4 and exploitation level (E) was 0.72. The high mortality, low condition indices and negative allometric of P. viridis in Marudu Bay is caused by a lack of suitable food in the surrounding water.     

Behavioral patterns and <Emphasis Type="Italic">in-situ</Emphasis> target strength of the hairtail (<Emphasis Type="Italic">Trichiurus lepturus</Emphasis>) via coupling of scientific echosounder and acoustic camera data

The present study focuses on the influence of target strength (TS) changes in the swimming angle of the hairtail (Trichiurus lepturus). We measured in-situ TS at 38 and 120 kHz with luring lamps at a fishing ground for jigging boats near the coastal waters of Jeju-do in Korea. Swimming angle and size of hairtails were measured using an acoustic camera. Results showed that mean preanal length was estimated to be 13.5 cm (SD = 2.7 cm) and mean swimming tilt angle was estimated to be 43.9° (SD = 17.6°). The mean TS values were ?35.7 and ?41.2 dB at 38 and 120 kHz, respectively. The results will assist in understanding the influence of swimming angle on the TS of hairtails and, thus, improve the accuracy of biomass estimates.     

Encystment and excystment of <Emphasis Type="Italic">Gyrodinium instriatum</Emphasis> Freudenthal et Lee

In the present study, we have investigated the conditions influencing encystment and excystment in the dinoflagellate Gyrodinium instriatum under laboratory conditions. We incubated G. instriatum in modified whole SWM-3 culture medium and in versions of modified SWM-3 from which NO₃ ⁻, PO₄ ³⁻, NO₃ ⁻ + PO₄ ³⁻, or Si had been omitted and observed encystment. Percentage encystment was high in the media without N and without P, while the percentage encystment in the medium lacking both N and P was highest. Moreover, to investigate N or P concentration which induced the encystment, Gyrodinium instriatum was also incubated in media with different concentrations of inorganic N and P; the concentrations of NO₂ ⁻ + NO₃ ⁻ and PO₄ ³⁻ were measured over time. The precursors of cysts appeared within 2 or 3 days of a decrease in NO₂ ⁻ + NO₃ ⁻ or PO₄ ³⁻ concentration to values lower than 1 μM or 0.2 μM, respectively. When cysts produced in the laboratory were incubated, we observed excystment after 8–37 days, without a mandatory period of darkness or low temperature. We incubated cysts collected from nature at different temperatures or in the dark or light and observed excystments. Natural cysts excysted at temperatures from 10 to 30°C, in both light and dark, but excystment was delayed at low temperatures. These studies indicate that G. instriatum encysts in low N or P concentration and excysts over a wide temperature range, regardless of light conditions, after short dormancy periods.     

Vertical Distributions of Larvae of the Clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> and the Striped Horse Mussel <Emphasis Type="Italic">Musculista senhousia</Emphasis> in Eastern Ariake Bay,Southern Japan

We have examined the vertical distributions of planktonic bivalve larvae, particularly the clam Ruditapes philippinarum and the mussel Musculista senhousia which are common and abundant on tidal flats of eastern Ariake Bay, southern Japan. Submersible pumping gear was used to take samples at 2 stations every 2 hours during the spring tide over a whole tidal cycle and/or through daytime and nighttime. Water samples were pumped up from 3 to 5 depths from the surface to sea bottom. Regardless of tidal cycles, D-shaped larvae were concentrated near the surface, while umbo larvae were found at the surface to intermediate depths. On average, these larval densities were significantly higher at the station close to the shore than the offshore station, with no significant difference between daytime and nighttime and between flood and ebb tides at each station. The velocity and direction of water movement at both stations revealed remarkable difference between the surface and bottom waters. The larvae at the surface may quickly disperse and be transported elsewhere, while those in water close to the sea bottom may tend to be retained.     

Total Lipid and Fatty Acid Classes in Decomposing Mangrove Leaves of <Emphasis Type="Italic">Bruguiera gymnorrhiza</Emphasis> and <Emphasis Type="Italic">Kandelia candel</Emphasis>: Significance with respect to Lipid Input

Changes in the concentration of total lipid and fatty acids (FAs) during the decomposition of mangrove leaves were investigated by field experiments using yellow leaves of Bruguiera gymnorrhiza (L.) Lamk. and Kandelia candel (L.) Druce, in order to quantify mangrove contribution to lipid and fatty acid inputs to marine sediments. Total lipid and total FA in the fresh (green and yellow) and decomposing leaves of both species were significantly higher during winter than summer. During decomposition, total lipid content and FA concentration, in particular branched chain fatty acids (BrFAs) and bacterial fatty acids (BFAs), increased to a maximum concentration in 45 days during winter and in 17 days during summer. Lipids were lost faster in K. candel leaf detritus than in B. gymnorrhiza leaf detritus in which >90% of the total lipid original weight was lost during the summer experiment and <60% during the winter experiment. The changes in the concentrations of total lipids and FAs in the decomposing leaves also indicate that mangrove leaves are significant sources of fatty acids and probably other lipid compounds to estuarine ecosystems and that tidal waters transport the lipids and FAs adsorbed to particulate matter from mangroves to adjacent estuarine sediments and the ocean.     

Temporal changes in the geographic distribution of two clam species <Emphasis Type="Italic">Meretrix lusoria</Emphasis> and <Emphasis Type="Italic">M. petechialis</Emphasis> along the coast of Japan and South Korea

The objectives of this study were to distinguish fossil specimens of Meretrix (Bivalvia, Veneridae) using discriminant score and multivariate analysis, and to examine temporal changes in the geographical distributions of M. lusoria and M. petechialis in Japan and South Korea. Fossil shells of Meretrix were collected from Miyagi, Fukui, Aichi and Kumamoto in Japan, and Gimhae, Seosan, Siheung and Ganghwa-do in South Korea. The outside of the right valve was photographed with a digital camera, and ten characteristics of the shell morphology, such as shell height and shell length, were measured using image analysis software and a digital slide caliper. The discriminant score and multivariate analysis of the shell morphology identified all of the examined fossils as M. lusoria, while living individuals collected from the western coast of South Korea were identified as M. petechialis. These results suggest that the fossil shells excavated from the western coast of South Korea are a different species from the living individuals collected from the same areas. Radioactive carbon isotopic results revealed that the ages of the four fossil shells collected from tidal flats in Ganghwa-do ranged between 3,270 ± 30 and 1,830 ± 30 Cal BP. These results suggest that M. lusoria inhabited the western coast of South Korea until at least 2,000 years ago, but was replaced by M. petechialis during the last 2,000 years. Therefore, we propose that either M. petechialis spats were introduced artificially, or planktonic larvae migrated naturally from China to the western coast of South Korea during the last 2,000 years.     

Interactions between the pathogenic bacterium <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and red-tide dinoflagellates

Vibrio parahaemolyticus is a common pathogenic bacterium in marine and estuarine waters. To investigate interactions between V. parahaemolyticus and co-occurring redtide dinoflagellates, we monitored the daily abundance of 5 common red tide dinoflagellates in laboratory culture; Amphidinium carterae, Cochlodinium ploykrikoides, Gymnodinium impudicum, Prorocentrum micans, and P. minimum. Additionally, we measured the ingestion rate of each dinoflagellate on V. parahaemolyticus as a function of prey concentration. Each of the dinoflagellates responded differently to the abundance of V. parahaemolyticus. The abundances of A. carterae and P. micans were not lowered by V. parahaemolyticus, whereas that of C. polykrikodes was lowered considerably. The harmful effect depended on bacterial concentration and incubation time. Most C. polykrikoides cells died after 1 hour incubation when the V. parahaemolyticus concentration was 1.4×10⁷ cells ml⁻¹, while cells died within 2 days of incubation when the bacterial concentration was 1.5×10⁶ cells ml⁻¹. With increasing V. parahaemolyticus concentration, ingestion rates of P. micans, P. minimum, and A. carterae on the prey increased, whereas that on C. polykrikoides decreased. The maximum or highest ingestion rates of P. micans, P. minimum, and A. carterae on V. parahaemolyticus were 55, 5, and 2 cells alga⁻¹ h⁻¹, respectively. The results of the present study suggest that V. parahaemolyticus can be both the killer and prey for some red tide dinoflagellates.     

Winter thermal inversion and <Emphasis Type="Italic">Trichodesmium</Emphasis> dominance in north-western Bay of Bengal

Clear thermal inversion was observed with cold surface waters (< 24°C) overlying the warm (> 26°C) subsurface water in the coastal waters of the northwestern Bay of Bengal during winter (January 2015). Simultaneously, preponderance of the cyanobacteria Trichodesmium erythraeum was observed dominating the phytoplankton community with > 90% of total population, reaching maximum density of 9.8 × 10⁵ filaments/L. Further, the Trichodesmium predominance was associated with low water temperature (< 24°C).     

Physiological and biochemical responses of <Emphasis Type="Italic">Prorocentrum minimum</Emphasis> to high light stress

Prorocentrum minimum is a common bloomforming photosynthetic dinoflagellate found along the southern coast of Korea. To investigate the adaptive responses of P. minimum to high light stress, we measured growth rate, and generation of reactive oxidative species (ROS), superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) in cultures exposed to normal (NL) and high light levels (HL). The results showed that HL (800 μmol m^?2 s^?1) inhibited growth of P. minimum, with maximal inhibition after 7–9 days. HL also increased the amount of ROS and MDA, suggesting that HL stress leads to oxidative damage and lipid peroxidation in this species. Under HL, we first detected superoxide on day 4 and H₂O₂ on day 5. We also detected SOD activity on day 5 and CAT activity on day 6. The level of lipid peroxidation, an indicator of cell death, was high on day 8. Addition of diphenyleneiodonium (DPI), an NAD(P)H inhibitor, decreased the levels of superoxide generation and lipid peroxidation. Our results indicate that the production of ROS which results from HL stress in P. minimum also induces antioxidative enzymes that counteract oxidative damage and allow P. minimum to survive.     

Age and Growth of <Emphasis Type="Italic">Sillago aeolus</Emphasis> in Okinawa Island,Japan

In a study of the age and growth of Sillago aeolus, a total of 414 fish were collected from December 2000 to March 2002, around Okinawa Island. Ages of 403 specimens were determined by otoliths. Monthly changes in the percentage of otoliths with an opaque zone in the outer margin indicated that an opaque ring formed once a year, occurring between February and May. The rings can therefore be defined as annual rings. Ages of females were estimated to be 0+ to 4+, while those of males were 0+ to 2+. The von Bertalanffy growth curves were well fitted to age-standard length data of males and females, as follows: Male: L_t = 209.6 [1 − exp{−0.70 (t + 0.58)}]. Female: L_t = 297.7 [1 − exp{−0.42 (t + 0.61)}].