The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21 °C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24 h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.     

Mapping the Distribution of Cysts from the Toxic Dinoflagellate Cochlodinium polykrikoides in Bloom-Prone Estuaries by a Novel Fluorescence In Situ Hybridization Assay

Cochlodinium polykrikoides is a cosmopolitan dinoflagellate that is notorious for causing fish-killing harmful algal blooms (HABs) across North America and Asia. While recent laboratory and ecosystem studies have definitively demonstrated that Cochlodinium forms resting cysts that may play a key role in the dynamics of its HABs, uncertainties regarding cyst morphology and detection have prohibited even a rudimentary understanding of the distribution of C. polykrikoides cysts in coastal ecosystems. Here, we report on the development of a fluorescence in situ hybridization (FISH) assay using oligonucleotide probes specific for the large subunit (LSU) ribosomal DNA (rDNA) of C. polykrikoides. The LSU rDNA-targeted FISH assay was used with epifluorescence microscopy and was iteratively refined to maximize the fluorescent reaction with C. polykrikoides and minimize cross-reactivity. The final LSU rDNA-targeted FISH assay was found to quantitatively recover cysts made by North American isolates of C. polykrikoides but not cysts formed by other common cyst-forming dinoflagellates. The method was then applied to identify and map C. polykrikoides cysts across bloom-prone estuaries. Annual cyst and vegetative cell surveys revealed that elevated densities of C. polykrikoides cysts (>100 cm⁻³) during the spring of a given year were spatially consistent with regions of dense blooms the prior summer. The identity of cysts in sediments was confirmed via independent amplification of C. polykrikoides rDNA. This study mapped C. polykrikoides cysts in a natural marine setting and indicates that the excystment of cysts formed by this harmful alga may play a key role in the development of HABs of this species.     

Dynamics of bacterial community structure during blooms of Cochlodinium polykrikoides (Gymnodiniales,Dinophyceae) in Korean coastal waters

Recent studies of dinoflagellates have reported that blooms can be closely related to the characteristics of the associated bacteria, but studies of the correlation between the toxic dinoflagellate, Cochlodinium polykrikoides and their associated bacterial community composition has not been explored. To understand this correlation, changes in bacterial community structure through the evolution of a C. polykrikoides bloom in Korean coastal waters via clone library analysis were investigated. Although there were no apparent changes in physio-chemical factors during the onset of the C. polykrikoides bloom, the abundance of bacteria bourgeoned in parallel with C. polykrikoides densities. Alpha-, gamma-proteobacteria and Flavobacteria were found to be dominant phyletic groups during C. polykrikoides blooms. The proportion of gamma-proteobacteria was lower (11.8%) during peak of the bloom period compared to the post-bloom period (26.2%). In contrast, alpha-proteobacteria increased in dominance during blooms. Among the alpha-proteobacteria, members of Rhodobacterales abruptly increased from 38% of the alpha-proteobacteria before the bloom to 74% and 56% during the early bloom and peak bloom stages, respectively. Moreover, multiple sites concurrently hosting C. polykrikoides blooms also contained high portions of Rhodobacterales and principal component analysis (PCA) demonstrated that Rhodobacterales had a positive, significant correlation with C. polykrikoides abundances (p ≤ 0.01, Pearson correlation coefficients). Collectively, this study reveals the specific clades of bacteria that increase (Rhodobacterales) and decrease (gamma-proteobacteria) in abundance C. polykrikoides during blooms.     

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.     

Satellite detection of harmful algal bloom occurrences in Korean waters

Cochlodinium polykrikoides (p) is a planktonic dinoflagellate known to produce red tides responsible for massive fish kills and thereby serious economic loss in Korean coastal waters, particularly during summer and fall seasons. The present study involved analyzing chlorophyll-a (Chl-a) from SeaWiFS ocean color imagery collected over the period 1998–2002 to understand the spatial and temporal aspects of C. polykrikoides blooms that occurred in the enclosed and semi-enclosed bays of the Korean Southeast Sea. NOAA-AVHRR data were used to derive Sea Surface Temperature (SST) to elucidate physical factors affecting the spatial distribution and abundance of C. polykrikoides blooms. The time series of SeaWiFS-derived Chl-a gave an impression that recent red tide events with higher concentrations appeared to span more than 8 weeks during summer and fall seasons and were widespread in most of the Korean Southeast Sea coastal bays and neighboring oceanic waters. Coupled eutrophication and certain oceanic processes were thought to give rise to the formation of massive C. polykrikoides blooms with cell abundances ranging from 1000 to 30,000 cells ml⁻¹, causing heavy mortalities of aquaculture fish and other marine organisms in these areas. Our analysis indicated that Chl-a estimates from SeaWiFS ocean color imagery appeared to be useful in demarcating the locality, spatial extent and distribution of these blooms, but unique identification of C. polykrikoides from non-bloom and sediment dominated waters remains unsuccessful with this data alone. Thus, the classical spectral enhancement and classification techniques such as Forward Principal Component Analysis (FPCA) and Minimum Spectral Distance (MSD) to uniquely identify and better understand C. polykrikoides blooms characteristics from other optical water types were attempted on both low spatial resolution SeaWiFS ocean color imagery and high spatial resolution Landsat-7 ETM+ imagery. Application of these techniques could capture intricate and striking patterns of C. polykrikoides blooms from surrounding non-bloom and sediment dominated waters, providing improved capability of detecting, predicting and monitoring C. polykrikoides bloom in such optically complex waters. The result obtained from MSD classification showed that retrieval of C. polykrikoides bloom from the mixed phase of this bloom with turbid waters was not feasible with the SeaWiFS ocean color imagery, but feasible with Landsat-7 ETM+ imagery that provided more accurate and comparable spatial C. polykrikoides patterns consistent with in situ observations. The dense phase of the bloom estimated from these imageries occupied an area of more than 25 km² around the coastal bays and the mixed phase extended over several hundreds kilometers towards the Southeast Sea offshore due to exchange of water masses caused by coastal and oceanic processes. Sea surface temperature analyzed from AVHRR infrared data captured the northeastward flow of Tsushima Warm Current (TWC) waters that provided favorable environmental conditions for the rapid growth and subsequent southward initiation of C. polykrikoides blooms in hydrodynamically active regions in the Korean Southeast Sea offshore.     

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.     

Grazers and vitamins shape chain formation in a bloom-forming dinoflagellate, Cochlodinium polykrikoides

Predators influence the phenotype of prey through both natural selection and induction. We investigated the effects of grazers and nutrients on chain formation in a dinoflagellate, Cochlodinium polykrikoides, which forms dense blooms and has deleterious effects on marine ecosystems around the world. Field populations of C. polykrikoides formed longer chains than laboratory cultures without grazers. In the field, chain length of C. polykrikoides was positively correlated with the abundance of the copepod Acartia tonsa. Chain length of C. polykrikoides increased when exposed to live females of A. tonsa or its fresh (<24 h post-isolation) exudates for 48 h. These results suggest that dissolved chemical cues released by A. tonsa induce chain formation in C. polykrikoides. Ingestion rate of A. tonsa on four-cell chains of C. polykrikoides was lower than on single cells, suggesting that chain formation may be an effective anti-grazing defense. Finally, nutrient amendment experiments demonstrated that vitamins (B₁, B₇, and B₁₂) increased the chain length of C. polykrikoides both singly and collectively, while trace metals and inorganic nutrients did not, showing that vitamins may also influence chain formation in this species.     

Cochlodinium polykrikoides blooms and eco-physical conditions in the South Sea of Korea

Eco-physical conditions for the initiation and termination of Cochlodinium polykrikoides blooms in the South Sea of Korea are examined in this paper. The C. polykrikoides blooms generally occur in the sea near Naro-Do in late August every year. The submarine canyon near Naro-Do plays an important role in surface water intrusion from the open ocean driven by northeasterly winds. In late August, the monsoonal wind system in Korea changes from southwesterly to northeasterly winds, causing Ekman transport of warm, fresh Changjiang Diluted Water (CDW) into the sea near Naro-Do and creating a front between inland sea water and CDW. Along the front, aggregation of single C. polykrikoides cells in the CDW and downwelling yield favorable eco-physical conditions for development of C. polykrikoides blooms. When typhoons and strong northeasterly winds bring vertically well-mixed East China Sea water into the sea near Naro-Do again in September, the eco-physical conditions favor diatom growth and lead to the termination of C. polykrikoides blooms.     

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.     

The effects of temperature, growth medium and darkness on excystment and growth of the toxic dinoflagellate Gymnodinium catenatum from northwest Spain

The chain-forming dinoflagellate Gynmodinium catenatum Grahamcauses recurrent outbreaks of paralytic shellfish poisoning(PSP) in the Galician Rias Bajas (northwest Spain). A sedimentsurvey in Ria de Vigo in April 1986 indicated that the highestconcentrations of cysts of this species were located in themiddle sections of the ria, with maximum abundance of 310 cystscm^–3. The effects of temperature, growth medium compositionand irradiance on the germination of laboratory-produced restingcysts were investigated. Newly formed cysts required very littletime for maturation, as excystment was possible within 2 weeksof encystment. Growth media did not affect germination success.In contrast, the excystment rate was retarded signifiantly indarkness. Germination was also strongly affected by temperature,with {small tilde}75% excystment success at 22–28°Cand little or no germination below 11°C after 1 month ofincubation. In culture, the optimum growth rate of vegetativecells was between 22 and 28°C, the highest rate being 0.53divisions day^–1 at 24°C. Growth did not occur at temperatures< 11°C or >30°C. These results are important withrespect to the different hypotheses proposed to explain theinitiation of G.catenatum blooms in the Galician Rias Bajasand Northern Portugal. The pattern of G.catenatum bloom developmentalong this coast has been related to seasonal upwelling in thearea, with major blooms occurring during the autumn as warmeroffshore surface water is transported towards the coast whenupwelling relaxes. The landward transport of established offshorepopulations of G.catenatum with the warm surface layer remainsa viable explanation for the observed blooms within the rias,but alternatively, our data suggest that cysts within the riascan provide the inoculum population at times conducive to growthand bloom formation. Even though newly formed G.catenatum cystshave a very short maturation time and can germinate in darknessacross a wide temperature range, bloom development will be significantonly during the late summer and early autumn, since in othermonths light levels at the sediment surface and temperaturesthroughout the water column are too low for significant germinationor growth.     

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.     

Optimization of total RNA isolation from Cochlodinium polykrikoides

Dinoflagellates are eukaryotic microorganisms that represent a group of single-celled algae composed of a highly diversified phylum that is well known for displaying an amazing range of ecological adaptation. Cochlodinium polykrikoides is an unarmored dinoflagellate species belonging to the Family Gymnodiniaceae. It is one of the causative agents for harmful algal blooms occurring in the west coast of Sabah, which result in massive economic damages to the aquaculture and mariculture industries. The limited availability of information pertaining to the genetics of C. polykrikoides raises the need for an in-depth study into the expression mechanisms and regulation of the genes of this microorganism. However, isolation of good quality and intact RNA samples from C. polykrikoides seems to be quite a challenge for molecular biologists. Rapid degradation of freshly isolated RNA samples is a bottleneck for researchers to conduct studies on the gene expression and regulation patterns, which requires intact and high-quality RNA samples. In this study, an attempt to establish the best protocol for isolation of good quality and intact RNA from C. polykrikoides was carried out by testing four different total RNA isolation protocols. We conclude that the RNA isolation protocol using the TRIzol reagent produced the best results among the methods tried. The information from this study will allow further research pertaining to the molecular aspects of C. polykrikoides to be conducted with much ease in the future.     

Differential responses of the dinoflagellate Cochlodinium polykrikoides bloom to episodic typhoon events

To better understand the effect of typhoons on the harmful alga Cochlodinium polykrikoides, we investigated cell population dynamics in relation to hydrographic conditions in Korean coastal waters before and after the passage of typhoons. After typhoon Lingling passed through the Yellow Sea, significant accumulation of C. polykrikoides on the southern coast of Namhae Island was associated with southerly winds on September 8, 2019. Similar to field observations of red tide, a particle transport model simulation showed that the virtual particles were greatly influenced by wind-driven currents associated with typhoons, particularly when diel vertical migration was included in the model. However, a bloom of C. polykrikoides disappeared immediately after the passage of typhoon Tapah on September 23, 2019. Because of the different patterns of bloom behavior after the passage of these typhoons, characteristics of other typhoons that affected the Korean peninsula during previous C. polykrikoides blooms were investigated. Analysis of typhoon properties including wind direction, precipitation, and wave height and energy suggested that high wave energy during the passage of a typhoon plays a critical role in the termination of C. polykrikoides blooms, because of its generation of high turbulence relative to other factors. In our study, the wave energy associated with typhoon Tapah (753.6 kJ m^?2 over 48 h) was much higher than that associated with typhoon Lingling (441.7 kJ m^?2 over 48 h). The results indicate that typhoons have an important role in determining the accumulation and termination of C. polykrikoides blooms through the physical effects of wind direction and wave energy.

     

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.     

Molecular cloning reveals co-occurring species behind red tide blooms of the harmful dinoflagellate Cochlodinium polykrikoides

Red tides caused by the marine dinoflagellate Cochlodinium polykrikoides Margalef pose significant environmental problems worldwide. Recently, the existence of severe blooms attributable to a single Cochlodinium Schütt species has been questioned by many researchers. Herein we investigated the dinoflagellate composition of harmful algal blooms (HABs) attributed to C. polykrikoides in Korean coastal waters at nine different stations (St.). The component species of Cochlodinium blooms were examined by using microscopic and gene-cloning methods. In the nine study areas, C. polykrikoides was the predominant species of HABs in St. 2, 4, 7, and St. 9. Based on the morphological identification, the bloom was initially thought to be caused only by C. polykrikoides; however, we detected additional bloom-forming dinoflagellates (Polykrikos schwartzii Bütschli and Polykrikos kofoidii Chatton), and diatoms (Pseudo-nitzschia americana (Hasle) Fryxell) along with C. polykrikoides. The parasitic dinoflagellates Amoebophrya Koeppen and Euduboscquella Coats, Bachvaroff & Delwiche were found to be co-located with Cochlodinium in our study, and for the first time, Cochlodinium fulvescens Iwataki, Kawami & Matsuoka was detected in Korea (west coast). These results suggest co-existence of multiple dinoflagellates in bloom populations of Cochlodinium and describe the composition of other dinoflagellate blooms (e.g., Polykrikos spp.) in Korean coastal regions. This co-occurrence may be considered during efforts to monitor and control HABs.     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.     

Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea

In seasonal environments, strong gradients of environmental parameters can shape life cycles of phytoplankton. Depending on the rate of environmental fluctuation, specialist or generalist strategies may be favored, potentially affecting life cycle transitions. The present study examined life cycle transitions of the toxin producing Baltic dinoflagellate Alexandrium ostenfeldii and their regulation by environmental factors (temperature and nutrients). This investigation aimed to determine whether genetic recombination of different strains is required for resting cyst formation and whether newly formed cysts are dormant. Field data (temperature and salinity) and sediment surface samples were collected from a site with recurrent blooms and germination and encystment experiments were conducted under controlled laboratory conditions. Results indicate a lack of seasonal germination pattern, set by an endogenous rhythm, as commonly found with other dinoflagellates from the Baltic Sea. Germination of quiescent cysts was triggered by temperatures exceeding 10°C and combined nutrient limitation of nitrogen and phosphorus or a drop in temperature from 16 to 10°C triggered encystment most efficiently. Genetic recombination was not mandatory for the formation of resting cysts, but supported higher numbers of resistant cysts and enhanced germination capacity after a resting period. Findings from this study confirm that A. ostenfeldii follows a generalist germination and cyst formation strategy, driven by strong seasonality, which may support its persistence and possibly expansion in marginal environments in the future, if higher temperatures facilitate a longer growth season.     

Physical transport processes affect the origins of harmful algal blooms in estuaries

The effects of physical transport processes on the initiation of harmful algal blooms (HABs) in estuaries were investigated through both mathematical model analysis and numerical model experiments. This study highlights the influence of the flushing effect due to physical transport processes on the location of bloom initiation, which is comparable to or even more important than local processes. The theoretical analysis suggests that the differential flushing effect at different waterbodies due to complex geometry is one of the dominant factors causing inhomogeneous distribution of algal density during HAB initiation. The ratio of residence time to volume is one of the key variables that determine the differential timing of HAB occurrence in estuary-subestuary systems with multiple interconnected waterbodies. As a result, a HAB tends to be observed first in those locations with relatively long residence time and small waterbodies, such as tributaries or areas with large eddies. Multiple unconnected originating locations can co-exist within an estuary.Two three-dimensional model experiments with realistic forcings were conducted to demonstrate the flushing effect on annual Cochlodinium polykrikoides bloom in the lower James River. The results show that while the environmental conditions that affect local processes, such as salinity and temperature, are important in determining the originating locations of HABs, the differential flushing effect is the dominant factor driving the spatial difference in the density of C. polykrikoides in this region during the bloom initiation. This explains why the occurrence of the first bloom in this region is frequently observed in the Lafayette River, a relatively small waterbody with long residence time. Because of the relatively low growth rate of C. polykrikoides and because of the high water-exchange between the mainstem and tributaries of the James River, initial cyst distribution is suggested to have a relatively small impact on originating locations of the bloom compared to flushing effect and salinity, and the HAB originating locations do not have to be in the waterbody with abundant cysts.     

Wind and temperature controls on Alexandrium blooms (2000–2007) in Thau lagoon (Western Mediterranean)

Since 1998, blooms of Alexandrium catenella/tamarense in the lagoon of Thau developed regularly each autumn, reaching a maximum of several millions cells per liter in 2004. By contrast, spring blooms occurred only twice (in 2000 and 2007). During these periods, sea surface temperatures (SST) and the wind patterns appear to impact the bloom occurrences much more than the apparent limiting resources such as inorganic nutrients. The analysis of SST and wind from April to June and September to November (from 2000 to 2007) indicates first that there has to be an initial wind stress in order to resuspend the cysts buried in the sediment. Blooms then occur after a period of weak winds (<4 m s⁻¹) and of stable SST close to 20 °C (±2 °C). Those conditions appear to be most favorable for germination of Alexandrium cysts and its ensuing vegetative growth. This period of stability (a few days to a few weeks) allows the development of the inoculum from the cyst's germination, its cohesion because of reduced hydrodynamics, and development of vegetative cells that are sensitive to agitation. Strong winds during 1–2 day periods can interrupt the bloom dynamics by dispersing (advection due to southeasterly winds) and/or eliminating (turbulence due to northwesterly winds) the vegetative cells. In the spring, under the same conditions of optimal SST, strong wind episodes dominate and those, as well as biological factors very likely lead to a lower occurrence of blooms relative to the fall situation.     

Development of real-time RT-PCR for detecting viable Cochlodinium polykrikoides (Dinophyceae) cysts in sediment

Morphological observations have confirmed that cysts are produced by dinoflagellates. However, finding a seed bed or unknown cysts in field samples by microscopy is extremely time consuming. Real-time PCR has been used to facilitate the detection of dinoflagellate cysts in sediment. However, DNA from dead vegetative cells remaining on the surface sediment may persist for a long period of time, which can cause false positive DNA detection. In this study, a non-quantitative RNA targeted probe using real-time RT-PCR was developed for detection of viable cysts in sediment. Large-subunit rRNA was used to develop a species-specific RNA targeted probe for the ichthyotoxic dinoflagellate Cochlodinium polykrikoides. The sediment samples were sieved and incubated at 30 °C for 3 h prior to RNA extraction to remove RNA from dead cells remaining in the sediment. Nested-PCR was conducted to maximize assay sensitivity. A field survey to determine the distribution of cysts at 155 sampling stations in the western and southern part of the Korean peninsula showed that C. polykrikoides cysts were detected at five sampling stations.