Degradation of outer membrane proteins of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis>

To examine the possibility that outer membrane proteins (OMP) of Synechococcus sp. remain in seawater, we investigated the stability of OMPs in vitro and in situ. Some fractions prepared from Synechococcus sp. CSIRO-94 were treated with trypsin and proteinase K. Four tightly bound OMPs were separated from Synechococcus. We designated the two major OMPs of 52 kDa and 48 kDa as Omp52Sy and Omp48Sy, respectively. Degradation of the OMP in natural seawater was monitored in microcosms to which intact Synechococcus cells and outer membrane (OM) were added. Omp52Sy and Omp48Sy were the most stable against trypsin and proteinase K among the OMPs when they were embedded in the OM. However, in the microcosm experiment using intact cells, Omp52Sy and Omp48Sy were detected in the particulate fraction only during the first 4 days, after which they could not longer be detected. Omp52Sy and Omp48Sy were the most stable proteins among the Synechococcus OMPs in vitro, but they might be degraded in situ. This indicates that stability of Synechococcus porin differs depending on complex formation with other membrane molecules, which might cause different preservation of microbial membrane proteins in the dissolved protein pool in the ocean. This study suggests that Gram negative bacterial OM with thin peptidoglycan forms a lipid bilayer that proptects OMP, but Synechococcus OM with thick peptidoglycan cannot form a lipid bilayer. The incomplete bilayer might not be able to protect from protease attack in the natural environment.     

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.     

Genetic diversity and population structure of three dominant myctophid fishes (<Emphasis Type="Italic">Diaphus theta,Stenobrachius leucopsarus</Emphasis>, and <Emphasis Type="Italic">S. nannochir</Emphasis>) in the North Pacific Ocean

We compared the genetic diversity of three dominant myctophid fishes in the North Pacific Ocean that have different diel vertical migration patterns on the basis of the nucleotide sequences of the mitochondrial gene for cytochrome b. No genetic structure was detected for each of these three species. The genetic diversity progressively increased for Diaphus theta, a diel migrant species showing clear diel vertical migration; Stenobrachius leucopsarus, a semi-diel migrant, in which only part of the population migrates vertically; S. nannochir, a non-diel migrant. All three species were suggested to have experienced a recent, sudden population expansion. Interspecific differences in genetic diversity might be attributable to differences in the degree of population size reduction during the glacial periods; this degree in turn corresponds to the energy demand of the fishes.     

Ultimate Lateral Capacity of Rigid Pile in <Emphasis Type="Italic">c</Emphasis>–<Emphasis Type="Italic">φ</Emphasis> Soil

To date no analytical solution of the pile ultimate lateral capacity for the general c–φ soil has been obtained. In the present study, a new dimensionless embedded ratio was proposed and the analytical solutions of ultimate lateral capacity and rotation center of rigid pile in c–φ soils were obtained. The results showed that both the dimensionless ultimate lateral capacity and dimensionless rotation center were the univariate functions of the embedded ratio. Also, the ultimate lateral capacity in the c–φ soil was the combination of the ultimate lateral capacity (f _c ) in the clay, and the ultimate lateral capacity (f _φ ) in the sand. Therefore, the Broms chart for clay, solution for clay (φ=0) put forward by Poulos and Davis, solution for sand (c=0) obtained by Petrasovits and Awad, and Kondner’s ultimate bending moment were all proven to be the special cases of the general solution in the present study. A comparison of the field and laboratory tests in 93 cases showed that the average ratios of the theoretical values to the experimental value ranged from 0.85 to 1.15. Also, the theoretical values displayed a good agreement with the test values.     

Species diversity of the genus <Emphasis Type="Italic">Skeletonema</Emphasis> (Bacillariophyceae) in the industrial harbor Dokai Bay,Japan

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.     

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.     

Numerical experiment on <Emphasis Type="Italic">Kyucho</Emphasis> around the Tango Peninsula induced by Typhoon 0406

A numerical experiment using a three dimensional level model was performed to clarify the mechanism generating a strong coastal current, Kyucho, induced by the passage of Typhoon 0406 around the tip of the Tango Peninsula, Japan in June 2004. Wind stress accompanied by Typhoon 0406 was applied to the model ocean with realistic bottom topography and stratification condition. The model well reproduced the characteristics of Kyucho observed by Kumaki et al. (2005), i.e., the strong alongshore current with maximum velocity of 53 cm s⁻¹ and its propagation along the peninsula with propagation speed of about 0.6 m s⁻¹ one half-day after the typhoon’s passage. Coastal-trapped waves (CTW) accompanied by downwelling were induced along the northwest coast of the peninsula by the alongshore wind stress. The energy density flux due to the CTW flowed eastward along the coast, and indicated scattering of the CTW around the eastern coast of the peninsula. In addition, significant near-inertial internal gravity waves were also caused in the offshore region from the west of the Noto Peninsula to the north of the Tango Peninsula by the typhoon’s passage. The energy flux density of the near-inertial fluctuations flowed southward off the Fukui coast, and part of the energy flux was trapped on the tip of the Tango Peninsula, flowing with the coast on its right. It was found that the strong current, Kyucho, at the northeastern tip of the Tango Peninsula was generated by superposition of the near-inertial internal gravity waves and subinertial CTW.     

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.     

Bottom fauna associated with <Emphasis Type="Italic">Bathymodiolus azoricus</Emphasis> (Mytilidae) mussel beds in the hydrothermal fields of the Mid-Atlantic Ridge

The structure of the assemblages associated with the mussel aggregations of Bathymodiolus azoricus was investigated. The mussel beds were found on the hydrothermal vent fields on the Mid-Atlantic Ridge (the Menez Gwen, Lucky Strike, and Rainbow areas) at the depths of 850–2400 m. The community structure of the mussel bed assemblages varied between the studied areas. Large number of species was unique to Menez Gwen mussel beds; the most observed taxa were not specialized hydrothermal species. All the other, nonunique species were found for the Lucky Strike region. The lowest mussel assemblage structure evenness was observed in the shallowest area, the Menez Gwen area (850 m depth). We assume that two types of mussel assemblages—nematode-dominated and copepod-dominated ones—exist in the Lucky Strike field. The assemblages of B. azoricus differ significantly from the assemblages of B. thermophilus inhabiting the Pacific hydrothermal vents.     

Dietary shift and feeding intensity of <Emphasis Type="Italic">Stenobrachius leucopsarus</Emphasis> in the Bering Sea

The food habits of the dominant myctophid Stenobrachius leucopsarus were examined in the central basin of the Bering Sea in relation to oceanographic conditions, in summer 2002 and 2003 and spring 2006. S. leucopsarus exhibited an ontogenetic and seasonal dietary shift. In spring, small fish (≤40 mm) preyed mainly on Neocalanus flemingeri/plumchrus whereas large fish fed mainly on Neocalanus cristatus. In summer, small fish preyed mainly on Metridia pacifica whereas large fish fed mainly on euphausiids (Thysanoessa spp.). In the summer of 2003, when water temperature in the epipelagic layer (≤100 m) was warmer, reflecting the prevalence of the Alaskan Stream, small-sized S. leucopsarus showed a higher stomach content index, perhaps reflecting the greater abundance of M. pacifica. Thus, the present study shows that the physical variability in the epipelagic layer affects not only diets but also feeding performance of micronekton.     

Degradation and utilization of protein derived from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> by marine microbial community

Microbial degradation and utilization of proteins derived from bacterial detritus were investigated in a microcosm experiment using Pseudomonas aeruginosa detritus as a substrate. To assess the effects of natural marine microbial communities on degradation and utilization of protein derived from P. aeruginosa cells, four microcosms were prepared: natural seawater (containing the natural microbial community) with P. aeruginosa detritus (N+Pa), autoclaved seawater with P. aeruginosa detritus (A+Pa), natural seawater (N) and autoclaved seawater (A) without adding anything as a control. The numbers of total and growing bacterial cells, protease activity, and transition of P. aeruginosa proteins were monitored in the four microcosms. Changes in the numbers of total and growing bacterial cells and protease activities indicated that bacterial detritus significantly stimulated the microbial community in the microcosms. Both the surviving P. aeruginosa in A+Pa and natural microbial community in N+Pa microcosms were able to degrade and utilize P. aeruginosa detritus; however, the community in N+Pa including various microbes maintained high activity longer, indicating that diversity is an important factor in keeping the community active. Even under the very high protease activity in N+Pa, 39-kDa and 48-kDa proteins from P. aeruginosa remained in the microcosm during the entire experiment (150 days). Immunoblotting suggested the 48-kDa protein was an intact molecule of OprP, which had been detected from the dissolved fraction of natural seawater in previous studies. This result suggests that the protein molecules that had been detected from natural seawater actually had a high tolerance to microbial degradation.     

The role of gametes of the macroalgae <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> (L.) Le Jolis and <Emphasis Type="Italic">Fucus vesiculosus</Emphasis> L. (Fucales,Phaeophyceae) in summer nanoplankton of the White Sea coastal waters

Studies of macrophytes in the coastal zone of the Artic Seas, including the White Sea, have shown the essential role of these algae in the activity of the coastal half-latitude ecosystems. In summer, during the macrophyte reproduction period, a great number of reproduction products are released into the water. For a short time, this considerably affects the ratio of the nanoplankton in the populations that inhibit the vast and shallow coastal areas. At different coastal sites in Chernorechenskaya Inlet, Kadalaksha Bay, during the period of intensive reproduction of Ascophyllum nodosum and Fucus vesiculosus, 42 plankton samples were collected in 2005. During this period the concentration of antherozoids in the water reached 55000 cells/ml (216 mg C/m³). The number of eggs was within the range of 0.05–0.7 cells/ml. The proportion of antherozoids in the total biomass of nanoplankton varied at different coastal sites from 0.37 to 99%, with a mean of 46% for the reproduction period of A. nodosum, and only 7% for the reproduction period of F. vesiculosus. As was shown by counts of F. vesiculosus female gametes in sedimentation traps, 1 m² of the macrophyte bed (assuming 100% coverage) produces 18000–108000 eggs per day (0.33–2 mg C). The calculated flux of the reproductive material from the brown algae beds to the coastal water shows good agreement with the sample counts.     

Excretion and respiration rates of the scyphomedusa <Emphasis Type="Italic">Aurelia aurita</Emphasis> from the Inland Sea of Japan

We measured the ammonium excretion, phosphate excretion and respiration rates of the scyphomedusa Aurelia aurita from Ondo Strait, in the central part of the Inland Sea of Japan, at 28 and 20°C. The rates measured at 28°C were converted to those at 20°C using the Q₁₀ values, i.e. 1.56, 1.57 and 2.80, for ammonium excretion, phosphate excretion and respiration rates, respectively. The composite relationships between metabolic rates and wet weight of a medusa (WW, g, range 11–1330 g) at 20°C were expressed by the following allometric equations. For ammonium excretion rate (N, μmoles N medusa⁻¹d⁻¹): N = 0.497WW ^1.09, phosphate excretion rate (P, μmoles P medusa⁻¹d⁻¹): P = 0.453WW ^0.84, and respiration rate (R, μmoles O₂ medusa⁻¹d⁻¹): R = 96.9WW ^1.06. Mean O:N ratios (i.e. atomic ratios of 16.9 and 11.0 at 28 and 20°C, respectively) indicated that the metabolism of A. aurita medusae was protein-dominated. These metabolic parameters enabled us to estimate the nitrogen and phosphorus regeneration rates of an A. aurita medusa population typical of early summer in the Ondo Strait (means of water temperature, medusa individual weight and population biomass: 20°C, 200 g WW and 50.8 g WW m⁻³, respectively). Regenerated nitrogen and phosphorus were equivalent to 10.0 and 21.6% of phytoplankton uptake rates, respectively, nearly twice that estimated for mesozooplankton, demonstrating that A. aurita medusae are key components of the plankton community, influencing the trophic and nutrient dynamics in the Ondo Strait during early summer.     

Reproductive ecology of the dominant dinoflagellate, <Emphasis Type="Italic">Ceratium fusus</Emphasis>, in coastal area of Sagami Bay,Japan

The seasonal abundance of the dominant dinoflagellate, Ceratium fusus, was investigated from January 2000 to December 2003 in a coastal region of Sagami Bay, Japan. The growth of this species was also examined under laboratory conditions. In Sagami Bay, C. fusus increased significantly from April to September, and decreased from November to February, though it was found at all times through out the observation period. C. fusus increased markedly in September 2001 and August 2003 after heavy rainfalls that produced pycnoclines. Rapid growth was observed over a salinity range of 24 to 30, with the highest specific rate of 0.59 d⁻¹ measured under the following conditions: salinity 27, temperature 24°C, photon irradiance 600 μmol m⁻²s⁻¹. The growth rate of C. fusus increased with increasing irradiance from 58 to 216 μmol m⁻²s⁻¹, plateauing between 216 and 796 μmol m⁻²s⁻¹ under all temperature and salinity treatments (except at a temperature of 12°C). Both field and laboratory experiments indicated that C. fusus has the ability to grow under wide ranges of water temperatures (14–28°C), salinities (20–34), and photon irradiance (50–800 μmol m⁻²s⁻¹); it is also able to grow at low nutrient concentrations. This physiological flexibility ensures that populations persist when bloom conditions come to an end.     

Direct examination of the dietary preference of the copepod<Emphasis Type="Italic">calanus helgolandicus</Emphasis> using the colorimetric approach

The food selectivity of tethered females of the copepodCalanus helgolandicus was examined by using the colorimetric approach. First, feeding behavior of the copepod did not show any differences between the red-color stained with neutral red and non-stained diets using the diatomCoscinodiscus curvatulus. Then, the copepods were fed a mixtures of two diets, the diatomC. curvatulus, stained with neutral red, and the dinoflagellateGymnodinium sanguineum for 14~60 minutes of feeding duration. The foregut colors of females were examined using a stereo-microscope and a video monitor. The foreguts of animals fed with a high density of diatoms in mixed diets showed a dark red color, whereas those fed with a high density of dinoflagellate in mixed diets were a dark yellow. The results suggest that this species of copepod may not selectively feed either one of the diets used for this study. Their feeding activity may be more likely related to the density of available prey in their environment. Therefore, this quick and easy colorimetric approach could provide very useful information, like the pre-screening procedure before designing and conducting the time-consuming and complex feeding experiments to understand the feeding ecology of copepods.     

Intercellular localization of nitrogenase in a non-heterocystous cyanobacterium (cyanophyte), <Emphasis Type="Italic">Trichodesmium</Emphasis> sp. NIBB1067

The nitrogenase activities of cyanobacteria of the genus Trichodesmium are strictly light-dependent, although they do not develop heterocysts. Recently, the development of heterocyst-equivalent cells (diazocytes) was suggested in Trichodesmium spp. However, no cells with a similar appearance to diazocytes could be found in Trichodesmium sp. NIBB1067. An immunocytochemical analysis of nitrogenase in individual cells was performed using polyclonal antibodies generated from DNA fragments of genes encoding the Fe-protein and the α-and the β-subunit of the MoFe-protein of nitrogenase of Trichodesmium sp. NIBB1067. Visualization of the antibody binding was carried out using a horseradish peroxidase-conjugated secondary antibody and a chromogenic substrate, 3,3′-diaminobenzidine, to avoid the masking effects caused by the bright autofluorescence emitted by Trichodesmium cells. Nitrogenase proteins were detected in almost all the cells (always higher than 95% of total cells) grown under nitrogen-limited conditions. These results indicate that Trichodesmium sp. NIBB1067 does not differentiate heterocyst-equivalent cells.     

Estimation of the <Emphasis Type="Italic">in situ</Emphasis> Ultraviolet-A absorption of seawater by a simple irradiance inversion model

An irradiance inversion model to estimate the in situ absorption coefficient of seawater has been developed for the Ultraviolet-A (UVA) wavelength domain. Input parameters are sun angle and the up-and downward planar irradiances measured for at least two depths. The present method does not require seawater to be sampled, and is a discrete wavelength method which returns the absorption coefficient at a given wavelength from the irradiances measured at that wavelength without assuming a spectral shape of any optical properties a priori. Comparison between the model results and spectrophotometric measurements shows that the model is practically useful when cloud cover in the atmosphere is ≤ 50%. According to the present method, measurements of the irradiances enable simultaneous observation of the in situ underwater UVA radiation level and the absorption capacity of bulk seawater using a radiometer.     

Evidence for the 8,200 a <Emphasis Type="SmallCaps">b</Emphasis>.<Emphasis Type="SmallCaps">p</Emphasis>. cooling event in the middle Okinawa Trough

Based on new and existing data on oxygen isotopes, alkenone-surface seawater temperature trends, planktonic foraminifers, lithology, and clay mineral composition of piston cores, a distinct cooling event has been identified around 8,200 cal a b.p. in the middle Okinawa Trough, northwest Pacific. This corresponds to the 8,200 a b.p. cooling event recorded in many places of the Northern Hemisphere. During this event, the local temperature decreased by 1°C, and the δ¹⁸O value increased by 0.6‰. A strengthened Asian winter monsoon is the most probable cause for this event, which thus adds further credibility to the contention that we are dealing here with a global phenomenon.     

Distribution of the bacteria <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in the western part of the Sea of Okhotsk

The Amur River’s influence on the distribution of the opportunistic bacteria Listeria monocytogenes in the western part of the Sea of Okhotsk is discussed. The presence of Listeria in the seawater, sea ice, and sediments on the northeastern Sakhalin shelf and slope supports the idea of its connection with the Amur River discharge. The hypothesis of the allochtonic parentage of L. monocytogenes in the sea’s development is proved.