Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r² ≥ 0.995) were established between C_t and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve.     

Physical processes leading to the development of an anomalously large Cochlodinium polykrikoides bloom in the East sea/Japan sea

An anomalously large Harmful Algal Bloom (HAB) was observed in the southwest coast of the East/Japan Sea (hereafter the East Sea) during the summer of 2013. During this time period, the presence of Cochlodinium polykrikoides (C. polykrikoides) was detected by the Geostationary Ocean Color Imager (GOCI) and validated by in-situ observations. GOCI observations have been available since 2011, thus allowingto examine various stages of the physical condition of the developing C. polykrikoides bloom, thereby other multi-satellite and buoy measurements obtained between 2011 and 2013. Research results indicate that this HAB is related to four processes: the transport of C. polykrikoides from the south coast of Korea to the HAB area; a relatively high insolation; continuous coastal upwelling; and a favorable Sea Surface Temperature (SST) for C.polykrikoide growth. In examination of the main transport mechanisms, geostrophic current measurements were used to estimate the flow trajectories, showing water from the south coast to the HAB area off the southeast coast of Korea. Result shows that ninety percent of the water from the south coast reached the HAB area in 2013. Furthermore, to examine the insolation mechanism, the Photosynthetically available radiation (PAR) value was derived from the Moderate Resolution Imaging Spectoradiometer (MODIS), showing that PAR values were relatively high in the HAB area during HAB period (47 Ein m⁻¹ day⁻¹). Moreover, Upwelling age (UA) was calculated in order to investigate the strength of coastal upwelling events, which were found to support relatively high UA values during the HAB period. The mean UA value during the HAB period was 1.01, higher than those in 2011 and 2012 which were 0.61 and 0.76, respectively. Finally, SST in the HAB area was also analyzed to examine which conditions were most favorable for HAB growth. Therefore, the results of this study suggest that the four mechanisms can explain the relative contributions of the anomalously HAB development observed off the southeast coast of Korea.     

Differential effects of typhoons on ichthyotoxic Cochlodinium polykrikoides red tides in the South Sea of Korea during 2012–2014

The harmful dinoflagellate Cochlodinium polykrikoides is known to cause fish death by gill-clogging when its abundance exceeds approximately 1000 cells ml⁻¹. Thus, red tides of this dinoflagellate have caused considerable loss in the aquaculture industry worldwide. Typhoons carrying strong winds and heavy rains may alter the process of red tide events. To investigate the effects of typhoons on C. polykrikoides red tides, daily variations in the abundance of C. polykrikoides, and wind speeds in three study areas in the South Sea of Korea were analyzed during the periods of C. polykrikoides red tides and the passage of 14 typhoons during 2012–2014. The typhoons differentially affected Cochlodinium red tides during the study period, and the daily maximum wind speed generated by the typhoon was critical. Four typhoons with daily maximum wind speeds of >14 m s⁻¹ eliminated Cochlodinium red tides, while three typhoons with daily maximum wind speed of 5–14 m s⁻¹ only lowered the abundance. However, other typhoons with daily maximum wind speeds of <5 m s⁻¹ had no marked effect on the Cochlodinium abundance. Therefore, typhoons may sometimes eliminate C. polykrikoides red tide events, or reduce cell abundances to a level that is not harmful to caged fish cultivated in aquaculture industries. Thus, typhoons should be considered when compiling red tide dynamics and fish-kill models.     

Control of ichthyotoxic Cochlodinium polykrikoides using the mixotrophic dinoflagellate Alexandrium pohangense: A potential effective sustainable method

Red tides dominated by Cochlodinium polykrikoides often lead to great economic losses and some methods of controlling these red tides have been developed. However, due to possible adverse effects and the short persistence of their control actions, safer and more effective sustainable methods should be developed. The non-toxic dinoflagellate Alexandrium pohangense is known to grow well mixotrophically feeding on C. polykrikoides, and populations are also maintained by photosynthesis. Thus, compared with other methods, the use of mass-cultured A. pohangense is safer and the effects can be maintained in the long term. To develop an effective method, the concentrations of A. pohangense cells and culture filtrate resulting in the death of C. polykrikoides cells were determined by adding the cells or filtrates to cultured and natural populations of C. polykrikoides. Cultures containing 800 A. pohangense cells ml⁻¹ eliminated almost all cultured C. polykrikoides cells at a concentration of 1000 cells ml⁻¹ within 24 h. Furthermore, the addition of A. pohangense cultures at a concentration of 800 cells ml⁻¹ to C. polykrikoides populations from a red-tide patch resulted in the death of most C. polykrikoides cells (99.8%) within 24 h. This addition of A. pohangense cells also lowered the abundances of total phototrophic dinoflagellates excluding C. polykrikoides, but did not lower the abundance of total diatoms. Filtrate from 800 cells ml⁻¹ A. pohangense cultures reduced the population of cultured C. polykrikoides by 80% within 48 h. This suggests that A. pohangense cells eliminate C. polykrikoides by feeding and releasing extracellular compounds. Over time, A. pohangense concentrations gradually increased when incubated with C. polykrikoides. Thus, an increase in the concentration of A. pohangense by feeding may lead to A. pohangense cells eliminating more C. polykrikoides cells in larger volumes. Based on the results of this study, a 1 m³ stock culture of A. pohangense at 4000 cells ml⁻¹ is calculated to remove all C. polykrikoides cells in ca. 200 m³ within 6 days. Furthermore, maintenance of A. pohangense populations through photosynthesis prepared A. pohangense to eliminate C. polykrikoides cells in future red-tide patches. Moreover, incubation of A. pohangense at 2000 cells ml⁻¹ with juvenile olive flounder Paralichthys olivaceus for 3 days did not result in the death of fish. Therefore, the method developed in this study is a safe and effective way of controlling C. polykrikoides populations and can be easily applied to aqua-tanks on land.     

Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes

The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928 cells mL^?1, while G. impudicum was present at low abundances (below 25 cells mL^?1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment.     

Characterization of the toxicity of Cochlodinium polykrikoides isolates from Northeast US estuaries to finfish and shellfish

Harmful algal blooms caused by Cochlodinium polykrikoides are annual occurrences in coastal systems around the world. In New York (NY), USA, estuaries, bloom densities range from 10³ to 10⁵ mL^?1 with higher densities (≥10⁴ cells mL^?1) being acutely toxic to multiple fish and shellfish species. Here, we report on the toxicity of C. polykrikoides strains recently isolated from New York and Massachusetts (USA) estuaries to juvenile fish (Cyprinodon variegates) and bay scallops (Argopecten irradians), as well as on potential mechanisms of toxicity. Cultures of C. polykrikoides exhibited dramatically more potent ichthyotoxicity than raw bloom water with 100% fish mortality occurring within ～1 h at densities as low as 3.3 × 10² cells mL^?1. More potent toxicity in culture was also observed in bioassays using juvenile bay scallops, which experienced 100% mortality during 3 days exposure to cultures at cell densities an order of magnitude lower than raw bloom water (～3 × 10³ cells mL^?1). The toxic activity per C. polykrikoides cell was dependent on the growth stages of cultures with early exponential growth cultures being more potent than cultures in late-exponential or stationary phases. The ichthyotoxicity of cultures was also dependent on both cell density and fish size, as a hyperbolic relationship between the death time of fish and the ratio of algal cell density to length of fish was found (～10³ cells mL^?1 cm^?1 yielded 100% fish mortality in 24 h). Simultaneous exposure of fish to C. polykrikoides and a second algal species (Rhodomonas salina or Prorocentrum minimum) increased survival time of fish, and decreased the fish mortality suggesting additional cellular biomass mitigated the ichthyotoxicity. Frozen and thawed-, sonicated-, or heat-killed-, C. polykrikoides cultures did not cause fish mortality. In contrast, cell-free culture medium connected to an active culture through a 5 μm nylon membrane caused complete mortality in fish, although the time required to kill fish was significantly longer than direct exposure to the whole culture. These results indicate that ichthyotoxicity of C. polykrikoides isolates is dependent on viability of cells and that direct physical contact between fish and cells is not required to cause mortality. The ability of the enzymes peroxidase and catalase to significantly reduce the toxicity of live cultures and the inability of hydrogen peroxide to mimic the ichthyotoxicity of C. polykrikoides isolates suggests that the toxicity could be caused by non-hydrogen peroxide, highly reactive, labile toxins such as ROS-like chemicals.     

Gymnodinium litoralis sp. nov. (Dinophyceae), a newly identified bloom-forming dinoflagellate from the NW Mediterranean Sea

Recurrent high-biomass blooms of a gymnodinioid species have been periodically recorded at different sites in the NW Mediterranean Sea (Catalan and Sardinian coast), causing intense discolorations of the water. In this study, several strains of the causative organism were isolated and subsequently studied with respect to the morphology of the vegetative cells and different life cycle stages, pigments profile, and molecular phylogeny. Based on phylogenetic analyses, the strains were placed within the Gymnodinium sensu stricto clade. The species possessed a horseshoe-shaped apical groove running anticlockwise around the apex and the major accessory pigment was identified as peridinin. These characteristics place the organism within the Gymnodinium genus, as defined today, although some other characteristics, such as vesicular chambers in the nuclear envelope and a nuclear fibrous connective were not observed. Morphologically, the isolates highly resemble Gyrodinium vorax (Biecheler) but major differences with the latter suggest that they comprise a new species, Gymnodinium litoralis sp. nov. The resting cyst of this species is described herein from field samples of the Catalan and Sardinian coast; pellicle cysts were observed in field samples and also in cultures. This species recurrently produces high biomass blooms (>10⁶ cell L⁻¹) in summer along several beaches and coastal lagoons in the NW Mediterranean Sea (L’Estartit, La Muga River mouth, and Corru S’Ittiri). Knowledge about its geographic distribution is limited, since the precise identification of G. litoralis from the field or fixed samples can be difficult. Therefore we expect that molecular studies will reveal a much wider distribution of the species.     

Morphology,phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.     

Alteration of plankton communities and biogeochemical cycles by harmful Cochlodinium polykrikoides (Dinophyceae) blooms

Cochlodinium polykrikoides is a globally distributed, ichthyotoxic, bloom-forming dinoflagellate. Blooms of C. polykrikoides manifest themselves as large (many km²) and distinct patches with cell densities exceeding 10³ ml⁻¹ while water adjacent to these patches can have low cell densities (<100 cells ml⁻¹). While the effect of these blooms on fish and shellfish is well-known, their impacts on microbial communities and biogeochemical cycles are poorly understood. Here, we investigated plankton communities and the cycling of carbon, nitrogen, and B-vitamins within blooms of C. polykrikoides and compared them to areas in close proximity (<100 m) with low C. polykrikoides densities. Within blooms, C. polykrikoides represented more than 90% of microplankton (>20 μm) cells, and there were significantly more heterotrophic bacteria and picoeukaryotic phytoplankton but fewer Synechococcus. Terminal restriction fragment length polymorphism analysis of 16S and 18S rRNA genes revealed significant differences in community composition between bloom and non-bloom samples. Inside the bloom patches, concentrations of vitamin B₁₂ were significantly lower while concentrations of dissolved oxygen were significantly higher. Carbon fixation and nitrogen uptake rates were up to ten times higher within C. polykrikoides bloom patches. Ammonium was a more important source of nitrogen, relative to nitrate and urea, for microplankton within bloom patches compared to non-bloom communities. While uptake rates of vitamin B₁ were similar in bloom and non-bloom samples, vitamin B₁₂ was taken up at rates five-fold higher (>100 pmol⁻¹ L⁻¹ d⁻¹) in bloom samples, resulting in turn-over times of hours during blooms. This high vitamin demand likely led to the vitamin B₁₂ limitation of C. polykrikoides observed during nutrient amendment experiments conducted with bloom water. Collectively, this study revealed that C. polykrikoides blooms fundamentally change microbial communities and accelerate the cycling of carbon, some nutrients, and vitamin B₁₂.     

Controls on the initiation and development of blooms of the dinoflagellate Cochlodinium polykrikoides Margalef in lower Chesapeake Bay and its tributaries

Massive blooms of the dinoflagellate Cochlodinium polykrikoides occur annually in the Chesapeake Bay and its tributaries. The initiation of blooms and their physical transport has been documented and the location of bloom initiation was identified during the 2007 and 2008 blooms. In the present study we combined daily sampling of nutrient concentrations and phytoplankton abundance at a fixed station to determine physical and chemical controls on bloom formation and enhanced underway water quality monitoring (DATAFLOW) during periods when blooms are known to occur. While C. polykrikoides did not reach bloom concentrations until late June during 2009, vegetative cells were present at low concentrations in the Elizabeth River (4 cells ml⁻¹) as early as May 27. Subsequent samples collected from the Lafayette River documented the increase in C. polykrikoides abundance in the upper branches of the Lafayette River from mid-June to early July, when discolored waters were first observed. The 2009 C. polykrikoides bloom began in the Lafayette River when water temperatures were consistently above 25 °C and during a period of calm winds, neap tides, high positive tidal residuals, low nutrient concentrations, and a low dissolved inorganic nitrogen (DIN) to dissolved inorganic phosphorous (DIP) ratio. The pulsing of nutrients associated with intense but highly localized storm activity during the summer months when water temperatures are above 25 °C may play a role in the initiation of C. polykrikoides blooms. The upper Lafayette River appears to be an important area for initiation of algal blooms that then spread to other connected waterways.     

Seed populations of a harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef in the East China Sea

An unarmored dinoflagellate Cochlodinium polykrikoides has formed red tides responsible for fish mass mortalities especially in coastal areas of western Japan and southern Korea almost every summer to autumn. In laboratory culture, the optimum temperature for growth of the species is ca. 27 °C. Since the species cannot survive in water of temperatures of less than 10 °C, it was considered to over-winter in some certain regions as a motile form or resting cyst, and expand its distribution after the temperature increases to a level tolerable for growth. To determine the over-wintering regions and migration pattern of C. polykrikoides, occurrences of the motile cells were surveyed in the coastal and offshore areas of western Kyushu, Japan and south coast of the Korean Peninsula from April 2006 to August 2008. Cells of C. polykrikoides were found at 14 sites during the investigated period. Motile cells occurred throughout the year in Usuka Bay, Hirado of West Japan. From offshore regions of the Goto Islands and off Shin-Nagasaki Fishing Port, motile cells of C. polykrikoides were first detected from late May, and continuously occurred until February in Nama Bay of the Kami-Goto Islands. This first appearance was before red tides of C. polykrikoides reported at coastal areas in western Kyushu. In Korea, this species was first observed in May and disappeared after October in 2007. These occurrence patterns imply that Usuka Bay in Hirado is one of the over-wintering regions in western Kyushu, and also this species is possibly transported into the northern part of the East China Sea by the Tsushima Warm Current every year.     

Harmful algal bloom (HAB) in the East Sea identified by the Geostationary Ocean Color Imager (GOCI)

Harmful Cochlodinium polykrikoides blooms have frequently appeared and caused fatal harm to aquaculture in Korean coastal waters since 1995. We investigated the applicability of GOCI, the world's first Geostationary Ocean Color Imager, in monitoring the distribution and temporal movement of a harmful algal bloom (HAB) that was discovered in the East Sea near the Korean peninsula in August 2013. We identified the existence of C. polykrikoides at a maximum cell abundance of over 6000 cells/mL and a chlorophyll a concentration of over 400 mg/m³. In areas of C. polykrikoides blooms, GOCI remote sensing reflectance (R_rs) spectra demonstrated the typical radiometric features of a HAB, and from the diurnal variations using GOCI-derived chlorophyll concentration images, we were able to identify the vertical migration of the red tide species. We also found that the formation and propagation of the HAB had relations with cold water mass in the coastal region. GOCI can be effectively applied to the monitoring of short-term and long-term movements of red tides.     

Cryptic diversity within the harmful dinoflagellate Akashiwo sanguinea in coastal Chinese waters is related to differentiated ecological niches

Blooms of the harmful dinoflagellate Akashiwo sanguinea are responsible for the mass mortality of fish and invertebrates in coastal waters. This cosmopolitan species includes several genetically differentiated clades. Four clonal cultures were established by isolating single cells from Xiamen Harbour (the East China Sea) for morphological and genetic analyses. The cultures displayed identical morphology but were genetically different, thus revealing presence of cryptic diversity in the study area. New details of the apical structure complex of Akashiwo sanguinea were also found. To investigate whether the observed cryptic diversity was related to environmental differentiation, 634 cells were obtained from seasonal water samples collected from 2008 to 2012. These cells were sequenced by single-cell PCR. For comparison with Chinese material, additional large subunit ribosomal DNA sequences were obtained for three established strains from Malaysian and French waters. To examine potential ecological differentiation of the distinct genotypes, growth responses of the studied strains were tested under laboratory conditions at temperatures of 12 °C to 33 °C. These experiments showed four distinct ribotypes of A. sanguinea globally, with the ribotypes A and B co-occuring in Xiamen Harbour. Ribotype A of A. sanguinea was present year-round in Xiamen Harbour, but it only bloomed in the winter and spring, thus corresponding to the winter type. In contrast, A. sanguinea ribotype B bloomed only in the summer, corresponding to the summer type. This differentiation supports the temperature optimum conditions that were established for these two ribotypes in the laboratory. Ribotype A grew better at lower temperatures compared to ribotype B which preferred higher temperatures. These findings support the idea that various ribotypes of A. sanguinea correspond to distinct ecotypes and allopatric speciation occurred in different climatic regions followed by dispersal.     

Improved real-time PCR method for quantification of the abundance of all known ribotypes of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides by comparing 4 different preparation methods

Red tides by the ichthyotoxic dinoflagellate Cochlodinium polykrikoides have caused large scaled mortality of fish and great loss in aquaculture industry in many countries. Detecting and quantifying the abundance of this species are the most critical step in minimizing the loss. The conventional quantitative real-time PCR (qPCR) method has been used for quantifying the abundance of this species. However, when analyzing > 500 samples collected during huge C. polykrikoides red tides in South Sea of Korea in 2014, this conventional method and the previously developed specific primer and probe set for C. polykrikoides did not give reasonable abundances when compared with cell counting data. Thus improved qPCR methods and a new specific primer and probe set reflecting recent discovery of 2 new ribotypes have to be developed. A new species-specific primer and probe set for detecting all 3 ribotypes of C. polykrikoides was developed and provided in this study. Furthermore, because the standard curve between cell abundance and threshold cycle value (Ct) is critical, the efficiencies of 4 different preparation methods used to determine standard curves were comparatively evaluated. The standard curves were determined by using the following 4 different preparations: (1) extraction of DNA from a dense culture of C. polykrikoides followed by serial dilution of the extracted DNA (CDD method), (2) extraction of DNA from each of the serially diluted cultures with different concentrations of C. polykrikoides cultures (CCD method), (3) extraction of DNA from a dense field sample of C. polykrikoides collected from natural seawater and then dilution of the extracted DNA in serial (FDD method), and (4) extraction of DNA from each of the serially diluted field samples having different concentrations of C. polykrikoides (FCD method). These 4 methods yielded different results. The abundances of C. polykrikoides in the samples collected from the coastal waters of South Sea, Korea, in 2014–2015, obtained using the standard curves determined by the CCD and the FCD methods, were the most similar (0.93–1.03 times) and the second closest (1.16–1.33 times) to the actual cell abundances obtained by enumeration of cells. Thus, our results suggest that the CCD method is a more effective tool to quantify the abundance of C. polykrikoides than the conventional method, CDD, and the FDD and FCD methods.     

Bloom forming Alexandrium ostenfeldii (Dinophyceae) in shallow waters of the Åland Archipelago,Northern Baltic Sea

In the past years, late summer blooms of the bioluminescent dinoflagellate Alexandrium ostenfeldii have become a recurrent phenomenon in coastal waters of the central and Northern Baltic Sea. This paper reports exceptionally high cell concentrations (10⁵ to 10⁶ cells L^?1) of the species found during bioluminescent blooms in 2003 and 2004 in a shallow embayment of the Åland archipelago at the SW coast of Finland. Clonal cultures were established for morphological, molecular, toxicological and ecophysiological investigations to characterize the Finnish populations and compare them to other global A. ostenfeldii isolates. The Finnish isolates exhibited typical morphological features of A. ostenfeldii such as large size, a prominent ventral pore and an orthogonally bent first apical plate. However, unambiguous differentiation from closely related Alexandrium peruvianum was difficult due to considerable variation of sulcal anterior plate shapes. The Finnish strains were genetically distinct from other isolates of the species, but phylogenetic analyses revealed a close relationship to isolates from southern England and an A. peruvianum morphotype from the Spanish Mediterranean. Together these isolates formed a distinct clade which was separated from a clade containing other Northern European, North American and New Zealand populations. Toxin analyses confirmed the presence of the PSP toxins GTX2, GTX3 and STX in both Finnish isolates with GTX3 being the dominant toxin. Total relative PSP toxin contents were moderate, ranging from approximately 6 to 15 fmol cell^?1 at local salinities of 5 and 10 psu, respectively. Spirolides were not detected. Salinity tolerance experiments showed that the Finnish isolates were well adapted to grow at the low salinities of the Baltic Sea. With a salinity range of approximately 6 to 20–25 psu, Baltic populations are physiologically distinct from their marine relatives. Vigorous production of different cyst types in the cultures suggest that cysts may play a crucial role in the survival and retainment of A. ostenfeldii populations in the Baltic Sea.     

Seasonal changes of the introduced Caulerpa racemosa var. cylindracea (Caulerpales,Chlorophyta) at the northwest limit of its Mediterranean range

A study of the meadows of the invasive Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque was carried out over one year at Marseilles (Provence, France) where the alga is thriving, probably since 1994, in the cold waters of the north western Mediterranean Sea. At an early phase of colonisation, the C. racemosa meadow is characterized by a patchy distribution pattern. Several years are necessary to obtain a dense and continuous meadow. In one area colonized for more than 4 years, C. racemosa has developed a continuous meadow with wide seasonal variations. Maximum development was reached in autumn (biomass: 82 ± 3 g DW m⁻²; length of stolons: 1162 ± 86 m m⁻²; number of apices: 8360 ± 405 m⁻²; number of erect axes: 20955 ± 1499 m⁻²) and the minimum from winter to early spring (respectively, 0.3 ± 0.1 g DW m⁻²; 3 ± 1 m m⁻²; 220 ± 55 apices m⁻²; 35 ± 15 erect axes m⁻²). Seasonal variations in the growth rate were highly significant. The season of high growth lasted from June to October. The apical growth rate of a stolon reached a maximum of 7.5 ± 0.3 mm day⁻¹ in early October, then began to decrease significantly from the end of October to December, before becoming nearly nil from January to early May. Annual net production rate expressed in terms of stolon length and biomass was estimated as 5801 m m⁻² a⁻¹ and 612 g DW m⁻² a⁻¹, respectively. During the growth period, the turnover rate of the C. racemosa stolons was estimated at from 25 to 46 days. The growth rate was closely correlated to the seawater temperature (R² = 0.83), whereas no significant correlation was found between growth and irradiance. During the growth period, a decrease in temperature rapidly affects the growth rate, which soon recovers its earlier level when the temperature rises again. In winter, the growth rate decreased rapidly with the seasonal drop in the seawater temperature. Grazing by fish (Sarpa salpa and Boops boops) can also affect the growth rate from September to December by consumption of the erect axes and stolon apices, enhancing the ramification of stolons. Seasonal changes at Marseilles are much sharper than those reported for warmer Mediterranean localities (French Riviera, Italy, Croatia): in winter and early spring C. racemosa meadows decreased and locally disappeared, leaving a barren substrate. C. racemosa survives the lower winter seawater temperatures of the north-western Mediterranean Sea probably in the form of zygotes and/or small fragments (rhizoids, stolons, propagules).     

Abundance and distribution of Ulva microscopic propagules associated with a green tide in the southern coast of the Yellow Sea

The presence of Ulva microscopic propagules may play an important role in the rapid development of high-biomass blooms of green algae in the Yellow Sea. Six cruises were conducted, to determine the abundance and distribution of Ulva microscopic propagules associated with a green tide that developed in the southern coastal waters of the Yellow Sea from April to August, 2012. Results indicated that Ulva microscopic propagules were widespread in these waters, with the highest density being up to 4800 ind. L⁻¹, prior to the appearance of the green tide in April. High densities were also widely distributed along the coast during May and June, after the appearance of the floating green tide. The quantity of Ulva microscopic propagules significantly decreased when the floating green tide declined in July, reaching densities of up to 162 ind. L⁻¹, following the disappearance of the floating green tide in August. Quantitative studies on the distribution patterns of Ulva microscopic propagules along the southern coast of the Yellow Sea indicated a significant correlation between density and salinity, turbidity and nutrient concentrations. Temporal and geographical distribution patterns of Ulva microscopic propagules were also significantly affected by the presence of a large biomass of attached, or floating, Ulva species algae.     

Genetic structure of Octopus vulgaris (Cephalopoda,Octopodidae) in the central Mediterranean Sea inferred from the mitochondrial COIII gene

The polymorphism of the mitochondrial gene cytochrome oxidase III was studied in the Mediterranean octopus, Octopus vulgaris Cuvier, 1797. A total of 202 specimens from seven sampling sites were analysed with the aim of elucidating patterns of genetic structure in the central Mediterranean Sea and to give an insight into the phylogeny of the Octopus genus. Phylogenetic analyses showed that individuals from the central Mediterranean belong to the O. vulgaris species whose limits should nevertheless be clarified. Concerning genetic structure, two high-frequency haplotypes were present in all locations. The overall genetic divergence (Φ_ST = 0.05, P < 0.05) indicated a significant genetic structuring in the study area and an AMOVA highlighted a significant break between western and eastern Mediterranean basins (Φ_CT = 0.094, P < 0.05). Possible explanations for the observed patterns of genetic structuring are discussed with reference to their relevance for fisheries management.     

Growth of eight Gambierdiscus (Dinophyceae) species: Effects of temperature,salinity and irradiance

Little is known about how the growth of individual Gambierdiscus species responds to environmental factors. This study examined the effects of temperature (15–34 °C), salinity (15–41) and irradiance (2–664 μmol photons m⁻² s⁻¹) on growth of Gambierdiscus: G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus and G. ruetzleri and one putative new species, Gambierdiscus ribotype 2. Depending on species, temperatures where maximum growth occurred varied between 26.5 and 31.1 °C. The upper and lower thermal limits for all species were between 31–34 °C and 15–21 °C, respectively. The shapes of the temperature vs. growth curves indicated that even small differences of 1–2 °C notably affected growth potentials. Salinities where maximum growth occurred varied between 24.7 and 35, while the lowest salinities supporting growth ranged from <14 to 20.9. These data indicated that Gambierdiscus species are more tolerant of lower salinities than is generally appreciated. Growth of all species began to decline markedly as salinities exceed 35.1–39.4. The highest salinity tested in this study (41), however, was lethal to only one species, Gambierdiscus ribotype 2. The combined salinity data indicated that differences in salinity regimes may affect relative species abundances and distributions, particularly when salinities are <20 and >35. All eight Gambierdiscus species were adapted to relatively low light conditions, exhibiting growth maxima at 50–230 μmol photons m⁻² s⁻¹ and requiring only 6–17 μmol photons m⁻² s⁻¹ to maintain growth. These low light requirements indicate that Gambierdiscus growth can occur up to 150 m depth in tropical waters, with optimal light regimes often extending to 75 m. The combined temperature, salinity and light requirements of Gambierdiscus can be used to define latitudinal ranges and species-specific habitats, as well as to inform predictive models.