Toxic dinoflagellate Centrodinium punctatum (Cleve) F.J.R. Taylor: An examination on the responses in growth and toxin contents to drastic changes of temperature and salinity.

To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15-40). C. punctatum grew over a temperature range of 15-25 °C, with an optimum of 20 °C., and over a salinity range of 25-40, with optimum salinities of 30-35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum.

Effect of wastewater from the in-water cleaning of ship hulls on attached and unattached microalgae.

Environmental spills of in-water hull cleaning wastewater (HCW) containing heavy metals and biocides is inevitable, and the effects of HCW on microalgae are unknown. To investigate this, we conducted microcosm experiments by adding HCW to natural seawater. HCW samples were obtained from two different cleaning methods (soft: sponge, hard: brush), and 5 % or 10 % were added to natural seawater as treatments. Dissolved Cu concentrations were 5 to 10 times higher in the treatments than those in the control. There were significant differences in growth of unattached microalgae depending on HCW dose (chlorophyll a: 34.1 ± 0.8 µg L-1 in control vs. 12.6 ± 4.3 µg L-1 in treatments). Conversely, the biomass of attached microalgae increased with HCW dose, which was associated with most of the nutrient reduction later in the experiment, rather than unattached microalgae. Our findings suggest that HCW can significantly impact microalgal community, especially depending on spill volume.

Alexandrium catenella (Group I) and A. pacificum (Group IV) cyst germination, distribution, and toxicity in Jinhae-Masan Bay, Korea.

To better understand the outbreaks of paralytic shellfish poisoning and bloom dynamics caused by Alexandrium species in Jinhae-Masan Bay, Korea, the germination and distributions of ellipsoidal Alexandrium cysts were investigated, and paralytic shellfish toxins (PSTs) profiles and contents were determined using strains established from germling cells. The phylogeny and morphological observations revealed that the germinated vegetative cells from ellipsoidal cysts collected from the surface sediments in Jinhae-Masan Bay belong to Alexandrium catenella (Group I) and A. pacificum (Group IV) nested within A. tamarense species complex. Cyst germinations of A. catenella (Group I) were observed at only 10 °C, whereas cysts of A. pacificum (Group IV) could germinate at temperature ranges of 10 to 25 °C. Maximum germination success (85%) for isolated cysts occurred at 15 °C, and the germling cells were A. pacificum (Group IV). The results indicate that the variation in water temperature in Jinhae-Masan Bay can control the seasonal variations in germination of cysts of A. catenella (Group I) and A. pacificum (Group IV). The germination rates of ellipsoidal Alexandrium cysts were different among sampling sites in Jinhae-Masan Bay, probably because of differences in distribution and abundance of A. catenella (Group I) and A. pacificum (Group IV) in the sediments. The ellipsoidal Alexandrium cyst concentrations were much higher in February than in August, however the distributions were similar. Gonyautoxins 3 and 4 (GTX-3 and GTX-4) contributed a large proportion (>90%) of the toxins produced by strains A. catenella (Group I) and A. pacificum (Group IV) established from germling cells, and the total cellular contents were higher in A. catenella (Group I) than in A. pacificum (Group IV).

A new potentially toxic dinoflagellate Fukuyoa koreansis sp. nov. (Gonyaulacales, Dinophyceae) from Korean coastal waters: Morphology, phylogeny, and effects of temperature and salinity on growth.

To clarify an unspecified toxic Gambierdiscus-like species isolated from seawaters off Jeju Island, Korea, its morphology and molecular phylogeny based on the small subunit (SSU) and partial large subunit (LSU) rRNA gene sequences were examined. Cells were narrow in ventral view and broad in lateral view with a smooth surface. The round thecal pores were evenly distributed, with an average diameter of 0.41 µm. Cell depth, width and height were 51.7 ± 4.5 µm, 43.0 ± 4.2 µm and 55.0 ± 4.7 µm, respectively, and depth-to-width (D/W) and height-to-width (H/W) ratios were 1.1 ± 0.2 µm and 1.3 ± 0.02 µm, respectively. The nucleus was located in the hypotheca. Scanning electron microscope observations revealed that the cells displayed a plate formula of Po, 4', 6'', 6c, 6s, 5''' and 2''', and transmission electron microscope observation demonstrated that the cells contained crystal-like particles. Morphological features indicated that the unspecified Korean isolate belonged to the genus Fukuyoa, and based on the H/W and D/W ratios, the apical pore H/W ratio and thecal pore size, it could be differentiated from other Fukuyoa species. The phylogenetic analyses based on the SSU and LSU rRNA sequences revealed that the Korean isolate was nested within the genus Fukuyoa with high support, and it grouped with F. cf. yasumotoi isolated from Japan. Based on the morpho-molecular data, a new species, Fukuyoa koreansis sp. nov. is proposed. The maximum growth rate (0.254 d-1) of F. koreansis was observed at 25°C and a salinity of 25. The required levels of temperature and salinity for growth distinguished Fukuyoa koreansis from Gambierdiscus species.

Dynamics of marine bacterial biofouling communities after initial Alteromonas genovensis biofilm attachment to anti-fouling paint substrates.

To determine how bacterial communities succeed after the initial attachment of the bacterial biofilm adhesion using 16S rDNA meta-barcoding in plates coated with copper-based anti-fouling (AF) and non-AF (control) coatings as well as ambient seawater, coated plates were submerged in a marine environment in situ. Alteromonas genovensis (Gammaproteobacteria) in AF coating and Pacificibacter sp. (Alphaproteobacteria) in the control plate were initially abundant. In the AF coating, the abundance of A. genovensis decreased rapidly, whereas that of genus Phaeobacter (Alphaproteobacteria), Serratia (Gammaproteobacteria) and Cupriavidus (Betaproteobacteria) increased. Bacterial community in the control plate had a strong connection to pathogenic Vibrio spp. associated with the growth of invertebrates. Therefore, in the in situ AF coating experiment, A. genovensis accumulation was initially and intensively increased, and the bacteria responded to chemical antagonism, induced the proliferation of specific biofilm bacteria and influenced the interactions and recruitment of additional bacterial communities.

The genome of the giant Nomura's jellyfish sheds light on the early evolution of active predation.

BACKGROUND: Unique among cnidarians, jellyfish have remarkable morphological and biochemical innovations that allow them to actively hunt in the water column and were some of the first animals to become free-swimming. The class Scyphozoa, or true jellyfish, are characterized by a predominant medusa life-stage consisting of a bell and venomous tentacles used for hunting and defense, as well as using pulsed jet propulsion for mobility. Here, we present the genome of the giant Nomura's jellyfish (Nemopilema nomurai) to understand the genetic basis of these key innovations. RESULTS: We sequenced the genome and transcriptomes of the bell and tentacles of the giant Nomura's jellyfish as well as transcriptomes across tissues and developmental stages of the Sanderia malayensis jellyfish. Analyses of the Nemopilema and other cnidarian genomes revealed adaptations associated with swimming, marked by codon bias in muscle contraction and expansion of neurotransmitter genes, along with expanded Myosin type II family and venom domains, possibly contributing to jellyfish mobility and active predation. We also identified gene family expansions of Wnt and posterior Hox genes and discovered the important role of retinoic acid signaling in this ancient lineage of metazoans, which together may be related to the unique jellyfish body plan (medusa formation). CONCLUSIONS: Taken together, the Nemopilema jellyfish genome and transcriptomes genetically confirm their unique morphological and physiological traits, which may have contributed to the success of jellyfish as early multi-cellular predators.

cDNA and gene structures of two phospholipase A2 isoforms, acidic PLA2 PA4 and PLA2 PA3A/PA3B/PA5, in Nemopilema nomurai jellyfish venom.

We have shown that Nemopilema nomurai jellyfish venom (NnV) contains various kinds of proteolytic enzyme activities, including phospholipase (PLA), metalloproteinase (MP) and hyaluronidase activities. In this study, we reported the full-length cDNA and gene sequences of two PLA2 isoforms: acidic PLA2 PA4 and PLA2 PA3A/PA3B/PA5. The full-length cDNA of acidic PLA2 PA4 contains 483 nucleotides (nt), which encode 160 amino acids (and the stop codon), including a signal peptide, six cysteine residues that form disulfide bonds, and metal-binding and catalytic active sites. The gene sequence of the acidic PLA2 PA4 is 1667 base pairs (bp) long and encodes three exons and two introns. The 5' donor (GT) and 3' acceptor (AG) splice sites are highly conserved. The PLA2 PA3A/PA3B/PA5 gene contains 1366 bp, and the 498 nt of the mature mRNA encode 165 amino acids (and the stop codon). The protein includes a signal peptide, six cysteine residues that form disulfide bonds, and metal-binding and catalytic active sites. The three exons and two introns also have highly conserved donor and acceptor splice sites. InterProScan predicted PLA2 activity domains in both isoforms. These results extend our understanding of the PLA2 venom of the N. nomurai jellyfish and will facilitate further research.

Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

Effects of temperature and nutrients on changes in genetic diversity of bacterioplankton communities in a semi-closed bay, South Korea.

Bacterioplankton communities in a semi-closed bay (Jangmok Bay, South Korea) were analysed using a 16S rDNA multiplex 454 pyrosequencing approach. Diversity and operational taxonomic units of bacterioplankton communities in the Jangmok Bay are highest in cold water seasons and lowest in warm water ones. During cold seasons, α-proteobacteria respond rapidly to pulses of the concentration of inorganic nutrients, while γ-proteobacteria during warm water seasons are the most active type of bacterioplankton resent in the prevailing conditions, which include high dissolved organic carbon, chemical oxygen demand and primary production. Cyanobacteria, a minor group constituting 4.58% of the total bacterioplankton, are more abundant at low temperature. Flavobacteria are more abundant in nutrient-rich conditions and the abundance of this group also demonstrated a delayed decline following summer phytoplankton blooms. The pronounced seasonal oscillations in phosphorus concentration and temperature exert strong selection pressure on bacterioplankton communities.

Enhancing the efficacy of electrolytic chlorination for ballast water treatment by adding carbon dioxide.

We examined the synergistic effects of CO2 injection on electro-chlorination in disinfection of plankton and bacteria in simulated ballast water. Chlorination was performed at dosages of 4 and 6ppm with and without CO2 injection on electro-chlorination. Testing was performed in both seawater and brackish water quality as defined by IMO G8 guidelines. CO2 injection notably decreased from the control the number of Artemia franciscana, a brine shrimp, surviving during a 5-day post-treatment incubation (1.8 and 2.3 log10 reduction in seawater and brackish water, respectively at 6ppm TRO+CO2) compared with water electro-chlorinated only (1.2 and 1.3 log10 reduction in seawater and brackish water, respectively at 6ppm TRO). The phytoplankton Tetraselmis suecica, was completely disinfected with no live cell found at >4ppm TRO with and without CO2 addition. The effects of CO2 addition on heterotrophic bacterial growth was not different from electro-chlorination only. Total residual oxidant concentration (TRO) more rapidly declined in electro-chlorination of both marine and brackish waters compared to chlorine+CO2 treated waters, with significantly higher amount of TRO being left in waters treated with the CO2 addition. Total concentration of trihalomethanes (THMs) and haloacetic acids (HAAs) measured at day 0 in brackish water test were found to be 2- to 3-fold higher in 6ppm TRO+CO2-treated water than in 6ppm TRO treated water. The addition of CO2 to electro-chlorination may improve the efficiency of this sterilizing treatment of ballast water, yet the increased production of some disinfection byproducts needs further study.

Effects of the algicide, thiazolidinedione derivative TD49, on microbial communities in a mesocosm experiment.

We investigated the effects of the algicide thiazolidinedione derivative TD49 on microbial community in mesocosm experiments. The TD49 concentration exponentially decreased over time, with half-life of 3.5 h, following addition in the seawater (R2=0.98, P<0.001). Among microbial communities, heterotrophic bacteria and heterotrophic nanoflagellates (HNFs) grew well in all treatments following the addition of TD49. The abundance of HNFs lagged behind the increase in heterotrophic bacteria by 24 h in the 0.2 and 0.4 µM TD49 concentrations (R2=0.28, P<0.05), and by 48 h in the 0.6 and 1.0 µM TD49 concentrations (R2=0.30, P<0.05). This implies a strong concentration-dependent top-down effect of TD49 on microbial communities, with indications that the degradation of planktonic organisms, including the target alga, led to high heterotrophic bacteria concentrations, which in turn stimulated the population growth of predatory HNF. However, total ciliate numbers remained relatively low in the TD49 treatments relative to the control and blank groups, suggesting limited carbon flow from bacteria to these grazers even though the abundance of aloricate ciliates gradually increased toward the end of the experimental period, particularly at the high TD49 concentrations. TD49 appears to provide an environmentally safe approach to the control of harmful algal blooms (HABs) in aquatic ecosystems.

Direct linkage between dimethyl sulfide production and microzooplankton grazing, resulting from prey composition change under high partial pressure of carbon dioxide conditions.

Oceanic dimethyl sulfide (DMS) is the enzymatic cleavage product of the algal metabolite dimethylsulfoniopropionate (DMSP) and is the most abundant form of sulfur released into the atmosphere. To investigate the effects of two emerging environmental threats (ocean acidification and warming) on marine DMS production, we performed a large-scale perturbation experiment in a coastal environment. At both ambient temperature and ∼ 2 °C warmer, an increase in partial pressure of carbon dioxide (pCO2) in seawater (160-830 ppmv pCO2) favored the growth of large diatoms, which outcompeted other phytoplankton species in a natural phytoplankton assemblage and reduced the growth rate of smaller, DMSP-rich phototrophic dinoflagellates. This decreased the grazing rate of heterotrophic dinoflagellates (ubiquitous micrograzers), resulting in reduced DMS production via grazing activity. Both the magnitude and sign of the effect of pCO2 on possible future oceanic DMS production were strongly linked to pCO2-induced alterations to the phytoplankton community and the cellular DMSP content of the dominant species and its association with micrograzers.

Improved method for minimizing sulfur loss in analysis of particulate organic sulfur.

The global sulfur cycle depends primarily on the metabolism of marine microorganisms, which release sulfur gas into the atmosphere and thus affect the redistribution of sulfur globally as well as the earth's climate system. To better quantify sulfur release from the ocean, analysis of the production and distribution of organic sulfur in the ocean is necessary. This report describes a wet-based method for accurate analysis of particulate organic sulfur (POS) in the marine environment. The proposed method overcomes the considerable loss of sulfur (up to 80%) that occurs during analysis using conventional methods involving drying. Use of the wet-based POS extraction procedure in conjunction with a sensitive sulfur analyzer enabled accurate measurements of cellular POS. Data obtained using this method will enable accurate assessment of how rapidly sulfur can transfer among pools. Such information will improve understanding of the role of POS in the oceanic sulfur cycle.

Inter-layer charge disproportionation in the dual-layer organic metal (tTTF-I)2ClO4 with unsymmetrical I···O halogen bond interactions.

A mixed-valence salt of tTTF-I with ClO4(-), formulated as (tTTF-I)2ClO4, is characterized by the presence of two crystallographically independent donor molecules, segregated in different layers and linked together through I···O interactions with the ClO4(-) anion disordered at room temperature. The tTTF-I donor molecule was prepared by metallation of tTTF (trimethylene tetrathiafulvalene) followed by reaction with iodine to afford the mono and diiodo derivatives tTTF-I and tTTFI2, respectively. The crystal structure of the latter neutral tTTFI2 shows the occurrence of strong type II, II halogen bond interactions. Band structure calculations of the dual-layer structure of the 2 : 1 salt (tTTF-I)2ClO4 show co-existence of both 1D open and 2D closed Fermi surfaces. The salt undergoes a metal-insulator phase transition at T(MI) = 90 K, associated with an electronic dimensionality decrease, since already at 100 K, the 2D part of the Fermi surface transforms into 1D corrugated planes. High resolution X-ray investigations performed at 100 K, combined with multipolar refinements, indicate an approximately equivalent +0.5e charge in both donor molecules, as also deduced from the intramolecular bond distances. On the other hand, Raman spectroscopic investigations show that at ambient temperature the charge is actually distributed non-uniformly in conducting layers of tTTF-I molecules, with the identification of molecules with charges +1, +0.5, 0e, while at low temperature the charge distribution becomes essentially uniform (+0.5e), as confirmed from the X-ray high resolution data. These apparently contradictory behaviors are actually a consequence of a partial electron transfer between the two independent slabs to reach a common Fermi level in the metallic phase.

Complete mitochondrial genome of the jellyfish, Chrysaora quinquecirrha (Cnidaria, Scyphozoa).

We sequenced 16,775 bp of the linear mitochondrial DNA of the jellyfish Chrysaora quinquecirrha and characterized them. C. quinquecirrha has 13 protein-coding genes (PCGs), 16S rRNA and 12S rRNA with 3 tRNAs (tRNA-Leu, tRNA-Ser(TGA), tRNA-Met) as shown in Aurelia sp. nov. Both have another two PCGs such as helicase and orf363 with telomeres at both ends. The PCGs of C. quinquecirrha shows anti-G bias on 2nd and 3rd positions of PCGs as well as anti-C bias on 1st and 3rd positions of PCGs.

Evaluation of disinfection efficacy and chemical formation using MPUV ballast water treatment system (GloEn-Patrol).

This study was undertaken to evaluate the efficacy of inactivation of several indigenous marine species and the formation of oxidants and other by-products using medium-pressure ultraviolet (MPUV) ballast water treatment. The ballast water treatment system (BWTS) used in this study was composed of filtration modules as a pretreatment process, followed by a UV irradiation process equipped with a polychromatic MPUV lamp. The experiments were performed on seawater (Busan, >32 PSU) and brackish water (Nakdong River, 20-22 PSU) with flow rates of 50 and 250 m(3)/h. The disinfection efficacy of the system was evaluated using indigenous species (>50 microm and 10-50 microm) and surrogate microorganisms (E. coli and Enterococci group). The test results successfully met the D-2 regulation of the IMO (International Marine Organization). In addition, oxidants, such as H202, total residual oxidants (TRO) and OH radicals, and potential halogenated by-products, such as haloacetic acids, trihalomethanes and total organic halides, that had potentially formed after MPUV treatment, were measured. In conclusion, the ballast water treatment system employing the MPUV physical process not only effectively eliminated indigenous species in ballast water but also generated no harmful by-products.

Enhanced production of oceanic dimethylsulfide resulting from CO2-induced grazing activity in a high CO2 world.

Oceanic dimethylsulfide (DMS) released to the atmosphere affects the Earth's radiation budget through the production and growth of cloud condensation nuclei over the oceans. However, it is not yet known whether this negative climate feedback mechanism will intensify or weaken in oceans characterized by high CO(2) levels and warm temperatures. To investigate the effects of two emerging environmental threats (ocean acidification and warming) on marine DMS production, we performed a perturbation experiment in a coastal environment. Two sets of CO(2) and temperature conditions (a pCO(2) of ∼900 ppmv at ambient temperature conditions, and a pCO(2) of ∼900 ppmv at a temperature ∼3 °C warmer than ambient) significantly stimulated the grazing rate and the growth rate of heterotrophic dinoflagellates (ubiquitous marine microzooplankton). The increased grazing rate resulted in considerable DMS production. Our results indicate that increased grazing-induced DMS production may occur in high CO(2) oceans in the future.

Phytoplankton viability in ballast water from international commercial ships berthed at ports in Korea.

We investigated the viability of phytoplankton from ballast water of international commercial ships berthed at the ports of Ulsan and Onsan, Korea. The incubation conditions used were temperatures of 13 degrees C to represent ambient water and 20 degrees C to represent the thermal optimum, as well as nutrients in ballast water, shipside water, and F/2 medium. Phytoplankton in new (approximately 7 days) and old (20 and 2 days) ballast water survived when incubated under the nutrients typical of shipside water and F/2 medium at 13 degrees C and 20 degrees C. Colonization process was mostly dominated by Skeletonema costatum, Cylindrotheca closterium and pennate diatoms (<10 microm in diameter). S. costatum and C. closterium were persistent during incubation time, whereas pennate diatoms dominated in the three types of media from doubling to last phase of growth. This study showed that bloom-forming and pennate diatoms appear to be the species most likely to become successfully established in the two ports.

Why did the copepod Calanus sinicus increase during the 1990s in the Yellow Sea?

Recent field and retrospective data were combined to investigate variations from 1984 to 1998 in the spring abundance of dominant copepods in the Yellow Sea. A calanoid copepod, Calanus sinicus, was chosen to assess the long-term changes in abundance associated with temperature, salinity, chlorophyll-a and predator abundance. Average anomalies of sea surface temperature (SST), salinity, and the abundance of C. sinicus were positive in the 1990s and negative in the 1980s. The average abundances of C. sinicus in the study area in the 1990s were also significantly higher than those in the 1980s (p<0.01). Catches of the anchovy Engraulis japonicus, a predator of C. sinicus, showed a decreasing trend during the study period. The higher abundances of C. sinicus in the 1990s may have been affected by an increase in water temperature and a decrease in predators, without distinctive changes in chlorophyll-a concentrations during the study period.