Development of compound microsatellite markers in the harmful dinoflagellate Cochlodinium polykrikoides (Dinophyceae)

We isolated 15 polymorphic microsatellites from Cochlodinium polykrikoides. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from two to 15, and the estimate of gene diversity was from 0.083 to 0.880 across the 15 microsatellites. We consider that these loci have a potential to reveal the genetic structure and gene flow among C. polykrikoides populations.     

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.     

Identification of the resting cyst of Cochlodinium polykrikoides Margalef (Dinophyceae,Gymnodiniales) in Korean coastal sediments

This study provides the first morphological features of resting cysts of Cochlodinium polykrikoides collected from Korean coastal sediments. Evidence for the existence of resting cysts of C. polykrikoides is based on the morphological and molecular phylogenetic data of the germinated cells and a resting cyst. The morphology of the resting cysts differed from that reported previously in sediments and culture experiments. The distinct feature is that the cyst body was covered by the reticulate ornaments and spines.     

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.     

Effects of temperature, salinity and irradiance on the growth of the harmful red tide dinoflagellate Cochlodinium polykrikoides Margalef (Dinophyceae)

The effects of temperature, salinity and irradiance on the growthof the harmful red tide dinoflagellate Cochlodinium polykrikoideswere examined in the laboratory. From 60 different combinationsof temperature (10–30°C) and salinity (10–40)under saturated irradiance, C. polykrikoides exhibited its maximumspecific growth rate of 0.41 day^-1 at a combination of 25°Cand salinity of 34. Optimum growth rates of >0.3 day^-1 wereobserved at temperatures ranging from 21 to 26°C and atsalinities from 30 to 36. The organism did not grow at temperatures10°C and only grew at salinities >30 if the temperaturewas >15°C. It was able to grow in temperatures rangingfrom 15 to 30°C and at salinities from 20 to 36. These valuesclosely resembled those observed for this species in situ. Itappears as if C. polykrikoides is a stenohaline organism thatprefers high salinities, indicative of offshore waters. Temperaturehad the greatest influence on the growth rate, followed by salinity,and then the interaction between temperature and salinity. Theoptimum irradiance for growth was >90 µmol m^-2 s^-1.Photoinhibition did not occur at 230 µmol m^-2 s^-1, whichwas the maximum irradiance used in this study.     

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.     

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.     

Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides

To elucidate the ichthyotoxic mechanisms of a harmful dinoflagellate Cochlodinium polykrikoides, biochemical responses of fish exposed to blooms were investigated. Particularly, based on our finding that oxidative damages of gill were associated with fish mortality (J. Plankton Res. 21 (1999) 2105-2115), dysfunction of ion-transporting enzymes and secretion of gill mucus of fish exposed to this bloom species were examined. The susceptibilities of several fishes to C. polykrikoides were different; the active pelagic fishes such as black scraper Thamnaconus septentrionalis, red sea bream Pagrus major, beakperch Oplegnathus fasciatus and seaperch Malakichthys wakiyae, were more vulnerable than the benthic fishes, flounder Paralichthys olivaceus and rockfish Sebastes inermis. In addition, the higher the algal cell density, the higher the fish mortality. When the test fishes were exposed to C. polykrikoides of 5000 cells ml(-1), the transport-related enzymes, carbonic anhydrase and Na(+)/K(+)-ATPase activities were significantly decreased. The activity of carbonic anhydrase was decreased with increasing algal cell density and exposure time. The quantity of total polysaccharide in gill mucus is higher in the fish exposed to C. polykrikoides than in the control fish; the magnitudes were higher in the pelagic fishes than that of benthic fishes. Moreover, a drop of blood pH and oxygen partial pressure (pO(2)) was also observed in red sea bream and flounder subjected to C. polykrikoides. These results suggest that the inactivation of gill transport-related enzymes activities, the fall in blood pO(2) and abnormal secretion of gill mucus by the C. polykrikoides may be one of the principal causes of fish kill.     

Harmful dinoflagellate Cochlodinium polykrikoides impairs the feeding behavior of larval sheepshead minnows (Cyprinodon variegatus)

Research evaluating the toxicity of the harmful dinoflagellate Cochlodinium (a.k.a. Margalefidinium) polykrikoides has been dominated by acute bioassays while the sublethal effects remain less well understood. This study examined the sublethal effects of C. polykrikoides exposure on the feeding behavior of larval estuarine fish. Sheepshead minnow (Cyprinodon variegatus) larvae were used in feeding experiments which assessed the total consumption of zooplankton prey (i.e., Artemia nauplii) over defined time periods. Larvae exposed to intermediate concentrations (i.e., 10² cells ml⁻¹) of clonal cultures of C. polykrikoides saw statistically significant reductions (range = 10%–81%) in the Artemia consumed compared to controls (i.e., filtered seawater, culture media or nontoxin producing dinoflagellate). These reductions were found independent of whether the larvae were fed or starved prior to experimentation. As these concentrations are similar to those typically found during mild blooms or at the periphery of dense blooms, these findings have significant implications for the feeding behavior of ichthyoplankton.     

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.

     

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.     

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.     

Ultrastructure of the Harmful Unarmored Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with Reference to the Apical Groove and Flagellar Apparatus

ABSTRACT. The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three‐dimensional structure of the flagellar apparatus. The apical groove is U‐shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5–1.0 μm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

The role of macroalgae in epiphytism of the toxic dinoflagellate Gambierdiscus toxicus (Dinophyceae)

Populations of the toxic, epiphytic dinoflagellate Gambierdiscus toxicus Adachi et Fukuyo are asSociated closely with Jania sp. on Hitiaa and Papara fringing reefs in Tahiti. Small populations were also observed to be asSociated with Amphiroa sp. and Halimeda opuntia (L.) Lamouroux. The cells attached themselves to the thallus by means of a short thread. When the thalli were irradiated, the cells began to detach from them and swim around the branches. The swimming cells stopped and attached to substrata when a disturbance occurred. The attached cells began to swim within a short time under light conditions when the thallus of Jania sp. were placed near the attached cells. Amphiroa sp. and H. opuntia also induced this re-commencement of swimming of the attached cells. These observations suggest that G. toxicus usually swims around macroalgal thalli on coral reefs. When sudden disturbance or strong water motion occurs, they attach to the surface of macroalgae and are not dispersed. Soon after water motion becomes slow, the cells begin to swim into the water around the thalli. The epiphytism of G. toxicus is different from epiphytic pennate diatoms, most of which adhere to the thallus all the time. The population of G. toxicus is maintained as an asSociation to a limited number of species of macroalgae which support the re-commencement of swimming after disturbance.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium catenella (Dinophyceae)

The outbreak of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium catenella (Dinophyceae) is a worldwide concern from the economic and food hygiene point of view. To assess the dispersal mechanism, a population genetic study using highly polymorphic genetic markers is one of the crucial approaches. We isolated 12 polymorphic microsatellites from this species. These loci provide one class of highly variable genetic marker as the number of alleles ranged from three to 12 and the estimate of gene diversity was from 0.248 to 0.897 across the 12 microsatellites; we consider that these loci have a potential to detail the genetic structure and gene flow among A. catenella populations.     

Temperature mediates secondary dormancy in resting cysts of Pyrodinium bahamense (Dinophyceae)

High‐biomass blooms of the toxic dinoflagellate Pyrodinium bahamense occur most summers in Tampa Bay, Florida, USA, posing a recurring threat to ecosystem health. Like many dinoflagellates, P. bahamense forms immobile resting cysts that can be deposited on the seafloor—creating a seed bank that can retain the organism within the ecosystem and initiate future blooms when cysts germinate. In this study, we examined changes in the dormancy status of cysts collected from Tampa Bay and applied lessons from plant ecology to explore dormancy controls. Pyrodinium bahamense cysts incubated immediately after field collection displayed a seasonal pattern in dormancy and germination that matched the pattern of cell abundance in the water column. Newly deposited (surface) cysts and older (buried) cysts exhibited similar germination patterns, suggesting that a common mechanism regulates dormancy expression in new and mature cysts. Extended cool‐ and warm‐temperature conditioning of field‐collected cysts altered the cycle of dormancy compared with that of cysts in nature, with the duration of cool temperature exposure being the best predictor of when cysts emerged from dormancy. Extended warm conditioning, on the other hand, elicited a return to dormancy, or secondary dormancy, in nondormant cysts. These results directly demonstrate environmental induction of secondary dormancy in dinoflagellates—a mechanism common and thoroughly documented in higher plants with seasonal growth cycles. Our findings support the hypothesis that a seasonal cycle in cyst germination drives P. bahamense bloom periodicity in Tampa Bay and point to environmentally induced secondary dormancy as an important regulatory factor of that cycle.     

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.     

Behavior of Peridinium bipes (Dinophyceae) resting cysts in the Asahi Reservoir

The role of resting cysts includes short- and long-term survival under extreme conditions, bloom initiation, species dispersal, reproduction, and preservation of genetic variation. Accordingly, it is important to understand their behavior in a water environment, especially in lakes and reservoirs where dinoflagellate blooms are observed. In this study, we estimated the behavior of the Peridinium bipes cysts in the Asahi Reservoir using laboratory experiments and field surveys. It was observed that the amount of light strongly influenced excystment, and few cysts germinated under dark conditions in the laboratory experiment. The minimum temperature on excystment was inferred from the laboratory experiment and field surveys to be about 5°C. Although the frequency of excystment did not depend on the water temperature from 10° to 20°C, the average preparation period for excystment decreased with the increase of water temperature. In the Asahi Reservoir, the excystment was estimated to occur during the months of March and April. If excystment did not occur in spring, the dinoflagellate bloom was not be observed until about July, although the bloom often began to appear in about May in the Asahi Reservoir. Consequently, the blooming season in the Asahi Reservoir is affected by the biomass of the germinated cysts in spring. Received: August 31, 2000 / Accepted: March 1, 2001