Formation and germination of temporary cysts of Cochlodinium polykrikoides Margalef (Dinophyceae) and their ecological role in dense blooms

While the initiation and development of dense bloom of Cochlodinium polykrikoides have been shown to be related to some environmental factors, little is known about the ecological role of the formation and germination of temporary cysts, nor of their significance for the rapid expansion of dense regional-scale blooms. This study examined the factors affecting the formation and germination of temporary cysts of C. polykrikoides, and provides details about the germination process. In the laboratory experiments, C. polykrikoides produced the chain-forming temporary cysts that are immobile and surrounded by a hyaline membrane. The encystment experiment indicated that darkness induces the formation of chain-forming temporary cysts, consistent with field observation of morphology and fluxes of temporary cysts. Germination occurred twice from a single four-celled temporary cysts within 24 h after exposure to light, and the germlings appeared as two-celled chain-forming vegetative cells. The germination behavior of temporary cysts of C. polykrikoides differs from that of other dinoflagellates, and this may be a survival strategy for the maintenance of population size during dense blooms.     

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.     

The influence of bloom intensity on the encystment rate and persistence of Alexandrium minutum in Cork Harbor,Ireland

Toxic Alexandrium minutum blooms recur annually in Cork Harbor, Ireland where they initiate in an inlet known as the North Channel. The dynamics of these blooms have been studied since 2003, and a high degree of inter-annual variability in the cell densities has been observed. Two intense blooms, with maximum cell densities >500,000 cells L⁻¹, were observed in the summers of 2004 and 2011. Annual cyst surveys during winter found that cyst densities decreased after the 2004 bloom, and by 2010 an average of ca. 40 cysts g dry wt sediment⁻¹ was recorded. The intensity of blooms was found to be independent of the cyst density measured the previous winter. The cyst input to the sediment during both intense and low density blooms was measured directly through the deployment of sediment traps in the North Channel. The data allowed an estimate of the proportion of the A. minutum vegetative cells that underwent successful encystment, which averaged at 2.5% across a range of cell densities spanning three orders of magnitude. Maturation times of fresh cysts were determined at 5, 10 and 15 °C. The maturation time at 15 °C was found to be approximately 5 months, a value which increased by two months for a 5° decrease in temperature. A cyst dynamics model was constructed based on the field data to simulate the temporal variation of A. minutum cysts in the oxic layer of sediment. It revealed that a degree of resuspension is required to prevent cyst stocks from becoming exhausted in the thin oxic layer at the surface of the sediment. The model also demonstrated that the cysts supplied by periodic intense blooms, which occur with a frequency of every 7–8 years, are not in themselves enough to allow the population to persist over long time scales (decades). The cyst input from interim blooms of lower density is however enough to ensure the annual inoculation of the water column with A. minutum cells.     

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.     

Germination fluctuation of toxic Alexandrium fundyense and A. pacificum cysts and the relationship with bloom occurrences in Kesennuma Bay,Japan

While cyst germination may be an important factor for the initiation of harmful/toxic blooms, assessments of the fluctuation in phytoplankton cyst germination, from bottom sediments to water columns, are rare in situ due to lack of technology that can detect germinated cells in natural bottom sediments. This study introduces a simple mesocosm method, modeled after previous in situ methods, to measure the germination of plankton resting stage cells. Using this method, seasonal changes in germination fluxes of toxic dinoflagellates resting cysts, specifically Alexandrium fundyense (A. tamarense species complex Group I) and A. pacificum (A. tamarense species complex Group IV), were investigated at a fixed station in Kesennuma Bay, northeast Japan, from April 2014 to April 2015. This investigation was conducted in addition to the typical samplings of seawater and bottom sediments to detect the dinoflagellates vegetative cells and resting cysts. Bloom occurrences of A. fundyense were observed June 2014 and February 2015 with maximum cell densities reaching 3.6 × 10⁶ cells m⁻² and 1.4 × 10⁷ cells m⁻², respectively. The maximum germination fluxes of A. fundyense cysts occurred in April 2014 and December 2014 and were 9.3 × 10³ cells m⁻² day⁻¹ and 1.4 × 10⁴ cells m⁻² day⁻¹, respectively. For A. pacificum, the highest cell density was 7.3 × 10⁷ cells m⁻² during the month of August, and the maximum germination fluxes occurred in July and August, reaching 5.8 × 10² cells m⁻² day⁻¹. Thus, this study revealed the seasonal dynamics of A. fundyense and A. pacificum cyst germination and their bloom occurrences in the water column. Blooms occurred one to two months after peak germination, which strongly suggests that both the formation of the initial population by cyst germination and its continuous growth in the water column most likely contributed to toxic bloom occurrences of A. fundyense and A. pacificum in the bay.     

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.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Alexandrium and Scrippsiella cyst viability and cytoplasmic fullness in a 60-cm sediment core from Sequim Bay,WA

Many marine protists produce a benthic resting stage during their life history. This non-motile cyst stage can either germinate near the sediment surface to provide the inoculum for subsequent blooms or, be buried by sediment deposits over time and entrained into the sedimentary record. Buried cysts can be resuspended into the water column by mixing events (e.g., storms) or other disturbances (e.g., dredging). It is not clear how long cysts can survive while buried in the sediments and still be capable of germinating given favorable conditions. Here, the germination success of cysts produced by the potentially toxic dinoflagellate genus Alexandrium and the non-toxic dinoflagellate genus Scrippsiella is reported from a 60-cm sediment core collected in Sequim Bay, WA, in December 2011. Cysts of Alexandrium spp. and Scrippsiella spp. were isolated from 2-cm sections of the core, placed in individual wells of a 96-well plate with growth medium, imaged, incubated at favorable conditions and monitored for germination. An image analysis program, DinoCyst, was used to quantitatively measure the amount of granular storage products, presumed energy stores, inside the cytoplasm to test the hypothesis that older cysts located deeper in the sediment core will have fewer energy stores available and will be less likely to germinate. An index of the area of the cytoplasm occupied with granular storage products relative to cyst size, termed ‘cytoplasmic fullness’, and age, based on ²¹⁰Pb dating of surrounding sediments, was compared with germination success or failure. This research indicates that cysts of Alexandrium spp. and Scrippsiella spp. can remain viable in sediments for 60 years or longer, show little visual evidence of cytoplasmic deterioration over this timescale (as measured by cytoplasmic fullness), and that germination success is statistically similar for cysts isolated from 0–60 cm deep in the sediment core. These results suggest that a cyst's cytoplasmic fullness is not indicative of viability and that cysts located as deep as 60 cm in the sediments are as likely to germinate as surface cysts given favorable conditions.     

Role of resting cysts in Chilean Alexandrium catenella dinoflagellate blooms revisited

The detection of sparse Alexandrium catenella-resting cysts in sediments of southern Chilean fjords has cast doubts on their importance in the recurrence of massive toxic dinoflagellate blooms in the region. The role of resting cysts and the existence of different regional Chilean populations was studied by culturing and genetic approaches to define: (1) cyst production; (2) dormancy period; (3) excystment success; (4) offspring viability and (5) strain mating compatibility. This study newly revealed a short cyst dormancy (minimum 69 days), the role of key abiotic factors (in decreasing order salinity, irradiance, temperature and nutrients) controlling cyst germination (max. 60%) and germling growth rates (up to 0.36–0.52 div. day⁻¹). Amplified fragment length polymorphism (AFLP) characterization showed significant differences in genetic distances (GD) among A. catenella populations that were primarily determined by the geographical origin of isolates and most likely driven by oceanographic dispersal barriers. A complex heterothallic mating system pointed to variable reproductive compatibility (RCs) among Chilean strains that was high among northern (Los Lagos/North Aysén) and southern populations (Magallanes), but limited among the genetically differentiated central (South Aysén) populations. Field cyst surveys after a massive 2009 bloom event revealed the existence of exceptional high cyst densities in particular areas of the fjords (max. 14.627 cysts cm⁻³), which contrast with low cyst concentrations (<221.3 cysts cm⁻³) detected by previous oceanographic campaigns. In conclusion, the present study suggests that A. catenella resting cysts play a more important role in the success of this species in Chilean fjords than previously thought. Results from in vitro experiments suggest that pelagic–benthic processes can maintain year-round low vegetative cell concentrations in the water column, but also can explain the detection of high cysts aggregations after the 2009-bloom event. Regional drivers that lead to massive outbreaks, however, are still unknown but potential scenarios are discussed.     

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.     

Influences of sedimentation and hydrodynamics on the spatial distribution of Alexandrium catenella/tamarense resting cysts in a shellfish farming lagoon impacted by toxic blooms

Since resting cysts are a potential seeding source for blooms, the presence of these cysts in sediments is a marker of an established population for a number of harmful algal species. The spatial patterns of cyst density in relation to sediment characteristics and hydrodynamics are still largely misunderstood. This study investigated the spatial distribution of resting cysts belonging to the Alexandrium tamarense species complex (Dinophyceae) in sediments of a Mediterranean coastal lagoon (Thau Lagoon, France). This lagoon, hosting shellfish farming, is regularly impacted by toxic Alexandrium catenella blooms. The average cyst density across the whole lagoon was rather low, <20 cysts g⁻¹ of dry sediment (DS). However, densities varied widely among sampled stations, with the highest density (∼440 cysts g⁻¹ DS) recorded in a shallow cove named Crique-de-l’Angle, which is the only area where dense blooms of A. catenella and A. tamarense have been recorded in the years preceding this survey. An analysis using spatial autoregressive models demonstrated that cyst densities were highly spatially autocorrelated (indicating that close stations tended to have more similar cyst densities) with accumulation sites. With respect to sediment characteristics (5 granulometric fractions <2 mm and biochemical components), the highest densities were found in silty sediments containing high proportions of water and organic matter. Nevertheless, the linkage between cyst density and sediment structure was not always verified; this reflected the influence of hydrodynamics on the sedimentation of cysts and sediment particles, and on the dispersal of cysts away from the bloom area by wind-induced currents, suggesting that hydrodynamics was responsible for the spatially autocorrelated distribution of cyst densities.     

Life cycle,physiology, ecology and red tide occurrences of the fish-killing raphidophyte Chattonella

The marine fish-killing raphidophytes of the genus Chattonella currently consist of five species, i.e. C. antiqua, C. marina, C. minima, C. ovata and C. subsalasa. The distribution of Chattonella species was confirmed in tropical, subtropical and temperate regions in the world accompanying mass mortalities of fishes in nature and in aquaculture. The fish-killing mechanisms are still unclear, but suffocation is the ultimate cause of fish death. Increasing evidence is pointing towards the generation of reactive oxygen species (ROS, e.g. superoxide), which are responsible for the gill tissue injury and mucus production that leads to death of fishes. A taxonomic revision was proposed based on morphology and genetic diversity that Chattonella antiqua and Chattonella ovata should be varieties of Chattonella marina possessing nomenclatural priority. Optimum temperatures for growth are 25 °C for C. antiqua and C. marina, 25–30 °C for C. ovata and 20–30 °C for Chattonella subsalsa. Adequate ranges of salinity for growth were about 20–30 for Chattonella species. Chattonella cells generally divide once a day. Laboratory culture experiments with artificial synthetic medium demonstrated that C. antiqua, C. marina and C. ovata used only Fe chelated with EDTA for growth, although tested diatoms and dinoflagellates used rather many kinds of chelated Fe. A suitable concentration of humic acid supplied with iron also had enhancing effects on the growth of C. antiqua. Diel vertical migration was observed in Chattonella, and the cells reached 7.5 m deep at night in the case of C. antiqua demonstrated by a mesocosm experiment in the Seto Inland Sea. Chattonella species have diplontic life history and have haploid cyst stage in their life cycle. Encystment was observed through formation of pre-encystment small cells after the depletion of nitrogen, and the small cells sink to the sea bottom to complete cyst formation by attachment to the solid surface such as diatom frustules and sand grains. Newly formed cysts are in the state of spontaneous dormancy and they need cold temperature period of four months or longer for maturation (acquisition of germination ability). Cysts germinate in early summer and resultant vegetative cells play an important role as seed populations in blooming in the summer season. However, relatively small part of cyst populations actually germinate from bottom sediments, and success of red tide formation is dependent on the growth in water columns. Since red tides of Chattonella were observed when diatoms were scarce in seawater, diatoms appear to have a key for the predominance of Chattonella in water columns. Diatom resting stages in sediments need light for germination/rejuvenation, whereas Chattonella cysts can germinate even in the dark, implying the selective germination of Chattonella cysts at the sea bottom under calm oceanographic conditions which contribute to bloom formation of Chattonella. As a mechanism of red tide occurrences of Chattonella in coastal sea, “diatom resting hypothesis” was presented. Biological control using diatoms is proposed through the germination/rejuvenation of resting stages suspending from bottom sediments to euphotic layer by sediment perturbation with submarine tractors or fishing trawling gears. Since diatoms have much higher growth rates, and newly joined diatom vegetative cells grow faster and prevent occurrence of Chattonella red tides as a result. As another prevention strategy for Chattonella red tides, algicidal bacteria inhabiting in seaweed beds and seagrass beds are presented. Co-culture of fish and seaweeds in aquaculture areas, and the developments of seaweed- and seagrass-beds would be practical and ultimately environment-friendly strategies for the prevention of harmful red tides of Chattonella by virtue of natural algicidal bacteria supplied from seaweeds and leaves of seagrass.     

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.     

Evidence for Production of Sexual Resting Cysts by the Toxic Dinoflagellate Karenia mikimotoi in Clonal Cultures and Marine Sediments

The toxic dinoflagellate Karenia mikimotoi has been well-known for causing large-scale and dense harmful algal blooms (HABs) in coastal waters worldwide and serious economic loss in aquaculture and fisheries and other adverse effects on marine ecosystems. Whether K. mikimotoi forms resting cysts has been a puzzling issue regarding to the mechanisms of bloom initiation and geographic expansion of this species. We provide morphological and molecular confirmation of sexually produced thin-walled resting cysts by K. mikimotoi based on observations of laboratory cultures and their direct detection in marine sediments. Light and scanning electron microscopy evidences for sexual reproduction include attraction and pairing of gametes, gamete fusion, formation of planozygote and thin-walled cyst, and the documentation of the thin-walled cyst germination processes. Evidence for cysts in marine sediments was in three aspects: positive PCR detection of cysts using species-specific primers in the DNA extracted from whole sediments; fluorescence in situ hybridization detection of cysts using FISH probes; and single-cell PCR sequencing for cysts positively labeled with FISH probes. The existence of sexually produced, thin-walled resting cysts by K. mikimotoi provides a possible mechanism accounting for the initiation of annually recurring blooms at certain regions and global expansion of the species during the past decades.     

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.     

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.     

Phylogenetic relationships of Cochlodinium polykrikoides Margalef (Gymnodiniales,Dinophyceae) from the Mediterranean Sea and the implications of its global biogeography

Although the diversity of dinoflagellates has been intensively studied in several locations in the Mediterranean Sea since the 1950s, it is only during the last two decades that the morphotype of the toxic unarmoured dinoflagellate Cochlodinium polykrikoides Margalef has been detected, coinciding with its apparent worldwide expansion in marine coastal waters. In this study, vegetative cells of C. polykrikoides morphotype from the Catalan coast (NW Mediterranean Sea) were detected and isolated, and the DNA from collected cells was sequenced. While in the Mediterranean Sea, detections are scarce and C. polykrikoides is consistently present at low concentrations, we reported exceptional blooms of this species, in which the maximum abundance reached 2 × 10⁴ cells L⁻¹. Partial LSU rDNA region sequences showed that most C. polykrikoides populations from the Catalan coast formed a new differentiated ribotype, but others were included within the ‘Philippines’ ribotype, demonstrating their coexistence in the Mediterranean Sea. Thus, the current biogeographic nomenclature of the ribotypes is likely to be invalid with respect to the available information from populations comprising the ‘Philippines’ ribotype. The phylogeny suggests the existence of cryptic species that should be evaluated for species-level status. Accordingly, the ribotype determination must be carefully evaluated for all detections and bloom events, since accurate characterization of the morphology, ecophysiology and distribution of the ribotypes are not well resolved.     

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.     

Sexual resting cyst production by the dinoflagellate Akashiwo sanguinea: a potential mechanism contributing to the ubiquitous distribution of a harmful alga

The dinoflagellate Akashiwo sanguinea is a well known, cosmopolitan harmful microalga that frequently forms harmful algal blooms (HABs) in marine estuaries from temperate to tropical waters, and has posed a severe threat to fish, shellfish, and sea birds. Therefore, it is important to understand the ecology of this species, particularly the mechanisms regulating its ubiquitous geographic distribution and frequent recurrence of. To date, the mechanisms regulating distribution and recurrence of HABs by this species have been poorly understood. While resting cyst production can play a central role in the geographic expansion and initiation of HABs, studies of the life cycle of this alga, including cyst production, have been lacking. Here, we demonstrate that A. sanguinea produces sexual resting cysts homothallically. We present evidence for cell pairs in sexual mating, biflagellated planozygote formation, and cysts of different morphologies, and we describe time series for germination of cysts to germlings with two longitudinal flagella, along with studies of possible factors affecting cyst production. Phylogenetic analysis of large sub‐unit rDNA sequences revealed a monophyly of this species and thus possibly a recent common ancestor for all global populations. The discovery of resting cyst production by A. sanguinea suggests its frequent recurrence of blooms and global distribution may have been facilitated by the natural and anthropogenic transport of resting cysts.     

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.