Temporary Cysts Research Articles

Viability assessed with the most probable number dilution culture method after chemical treatment of ballast water reveals the presence of false negatives from an approved vital stain method

The present study compares the CMFDA/FDA + motility- and the Most Probable Number (MPN) Dilution Culture + Motility methods for testing the viability of ≥10–<50 μm organisms in chlorine treated ballast water. The results of both methods were within the regulatory compliance criterion <10 organisms/mL, but the MPN-method revealed that growth-outs did occur. While the CMFDA/FDA method showed <0.5 organisms/mL, the MPN-method gave approx. 6 organisms/mL. This demonstrated that false negatives, i.e. living but not stained organisms, may occur when using the CMFDA/FDA-method for compliance testing of chemical treated ballast water. Organisms surviving the treatment were primarily the dinoflagellate Scrippsiella sp. and various coccoid chlorophytes present in a brackish- and freshwater test, respectively. It is suggested that their resilience to the chemical treatment is the ability to transform into a temporary cyst (Scrippsiella sp.) or the presence of a chemical resistant cell wall (certain chlorophytes).

Changes in Toxin Production, Morphology and Viability of Gymnodinium catenatum Associated with Allelopathy of Chattonella marina var. marina and Gymnodinium impudicum.

Allelopathy between phytoplankton organisms is promoted by substances released into the marine environment that limit the presence of the dominating species. We evaluated the allelopathic effects and response of cell-free media of Chattonella marina var. marina and Gymnodinium impudicum in the toxic dinoflagellate Gymnodinium catenatum. Additionally, single- and four-cell chains of G. catenatum isolated from media with allelochemicals were cultured to evaluate the effects of post exposure on growth and cell viability. Cell diagnosis showed growth limitation and an increase in cell volume, which reduced mobility and led to cell lysis. When G. catenatum was exposed to cell-free media of C. marina and G. impudicum, temporary cysts and an increased concentration of paralytic shellfish toxins were observed. After exposure to allelochemicals, the toxin profile of G. catenatum cells in the allelopathy experiments was composed of gonyautoxins 2/3 (GTX2/3), decarcarbamoyl (dcSTX, dcGTX2/3), and the sulfocarbamoyl toxins (B1 and C1/2). A difference in toxicity (pg STXeq cell−1) was observed between G. catenatum cells in the control and those exposed to the filtrates of C. marina var. marina and G. impudicum. Single cells of G. catenatum had a lower growth rate, whereas chain-forming cells had a higher growth rate. We suggest that a low number of G. catenatum cells can survive the allelopathic effect. We hypothesize that the survival strategy of G. catenatum is migration through the chemical cloud, encystment, and increased toxicity.

Liza ramada Juveniles after Exposure to the Toxic Dinoflagellate Vulcanodinium rugosum: Effects on Fish Viability, Tissue Contamination and Microalgae Survival after Gut Passage.

Pinnatoxins (PnTX) and Portimines (Prtn), two toxins produced by the benthic dinoflagellate Vulcanodinium rugosum, are known to be lethal to mice after intraperitoneal or oral administration. They are also known to accumulate in shellfish such as mussels and clams, but their effect on fish and the upper food chain remains unknown. In this work, juveniles of the fish Liza ramada (Mullet) were exposed to a strain of V. rugosum producing PnTX G and Prtn A. The fishes’ viability and contamination were recorded at times interval. Results showed that L. ramada juveniles were able to feed on V. rugosum and that their tissues could be contaminated by PnTX G and Prtn A without impact on fish viability. Furthermore, the microalgae temporary cysts survived and germinated after fish gut passage. This study showed the potential of L. ramada to transfer PnTX and Prtn toxins to the upper food chain and to disseminate V. rugosum in environment.

Revision of the life cycle of the harmful dinoflagellate Margalefidinium polykrikoides (Gymnodiniales, Dinophyceae) based on isolates from Korean coastal waters

Although the life cycle of Margalefidinium polykrikoides, formerly called Cochlodinium polykrikoides, has been described previously, the descriptions on asexual and sexual phases of this species collected from Korean coastal waters are strange and still unclear. We collected the resting cysts of M. polykrikoides from surface sediments and re-examined the germination process, and asexual and sexual life stages, and the mating behavior of the vegetative cells using a microscope equipped with high-resolution time-lapse digital camera. The resting cyst of M. polykrikoides had a distinct germination process; before germination, a mature cell in shape was visible within the resting cyst body, and the cell quickly emerged from the resting cyst. The asexual reproduction and cell chain formation of M. polykrikoides were generated in a slightly oblique direction at the epicone of cell and the chain-forming cells of M. polykrikoides could form the chain-forming temporary cysts without asexual or sexual reproduction. The planozygote produced by gamete fusion of M. polykrikoides may not necessarily develop to resting cysts but instead may divide into vegetative cells occasionally. In this phase, the armored cell, which was previously recorded by Kim et al. (2007), was not observed. The sexuality of M. polykrikoides had a homothallic mating behavior.

Studies on woloszynskioid dinoflagellates IX: ultrastructure, cyst formation and phylogeny of the ‘red-snow’ alga Borghiella pascheri (Suchlandt) Moestrup (= Glenodinium pascheri, Woloszynskia pascheri, Gyrodinium nivalis)

ABSTRACTRed snow caused by dinoflagellates is a phenomenon rarely reported, described from the European Alps from 1914 onwards, and subsequently observed outside Europe on several occasions in Ontario, Canada. Considerable taxonomic confusion exists regarding the identity of the organism(s) causing red snow, but the most recent occurrence in 2016 in Ontario has now allowed detailed studies, including LM, SEM, TEM and molecular sequencing of the causative species. We conclude that the two species originally described as the cause of red snow, Glenodinium pascheri and Gyrodinium nivale, are synonymous and that the appropriate name for the organism is Borghiella pascheri (syn. Woloszynskia pascheri) as suggested by Moestrup & Calado in the recent volume of the Süsswasserflora. The central part of Borghiella pascheri cells is tomato red and filled with numerous organelles, whose ultrastructure indicates modified chloroplasts. Lack of cultures has prevented chemical characterization of the red pigment. Formation of temporary cysts was common in the samples. Transformation of the motile cells into temporary cysts was followed in detail, and the cysts were shown to be surrounded by the fused inner membranes of the amphiesmal vesicles, which became the cell membrane of the cysts, covered by the fused pellicle precursors. The cell membrane from the motile cell was discarded together with the outer amphiesmal vesicle membrane and the thin thecal plates, and the temporary cysts were therefore not surrounded by any pattern of vesicles. Sexual reproduction resulted in the formation of hypnozygotes. Although the species possessed several unusual features, DNA sequencing showed it to belong to Borghiella. The culture established in 1965 from the Botanical Garden in Göttingen, Germany and generally identified as Woloszynskia pascheri belongs to a separate species of Borghiella, to be described separately.The occurrence of red snow caused by dinoflagellates is discussed.

Effects of modified clay used for the control of harmful algal blooms on Alexandrium pacificum cysts

Cyst formation plays an important role in the resistance of dinoflagellates to adverse environments, and cyst germination is considered one of the causes of harmful algal blooms (HABs). Among the methods for mitigating HABs, modified clay (MC) is considered a promising strategy because of its high efficiency and low environmental impacts. The typical HAB species Alexandrium pacificum was focused on in this study to clarify the effects of MC on cyst formation and germination. The results showed that more than 90% of the vegetative cells were removed under the 0.6 g/L MC treatment. The vegetative cell density was monitored over 90 d and increased slightly to the peak at 10 d after the cell removal experiment, but persistent growth was not observed. The amount of cysts was maximal at 20 d after removal, however, most of the cysts were temporary cysts that subsequently disappeared. After 80 d, all the remaining cysts were resting ones. The total density of resting cysts was higher under MC concentrations of 0.2 and 0.4 g/L and lower under concentrations of 0.6, 0.8 and 1.0 g/L compared with that in the control. The total formation rate of resting cysts was 29.6% in the control group, and the lowest formation rate in the experimental groups was 15.5% at 0.6 g/L MC. The total germination rate of resting cyst decreased as the MC concentration increased, and approximately 68.0% of the resting cysts in the control group germinated successfully, whereas the addition of MC reduced the germination rate to as low as 12.4%. Our results indicated that the application of appropriate MC concentrations may provide an effective mitigation strategy for A. pacificum blooms because it does not leave more residual cysts, which can act as “seeds” for the initiation of HABs.

Effects of copper and butyltin compounds on the growth, photosynthetic activity and toxin production of two HAB dinoflagellates: The planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata

Controlled laboratory experiments were conducted to test the effects of copper (Cu2+) and butyltins (BuT) on the growth, photosynthetic activity and toxin content of two HABs (Harmful Algal Blooms) dinoflagellates, the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata. Microalgae were exposed to increasing concentrations of Cu2+ (10−4 to 31 nM) or BuT (0.084 to 84 nM) for seven days. When considering the growth, EC50 values were 0.16 (±0.09) nM and 0.03 (±0.02) nM of Cu2+ for A. catenella and O. cf. ovata, respectively. Regarding BuT, EC50 was 14.2 (±6) nM for O. cf. ovata, while A. catenella growth inhibition appeared at BuT concentrations ≥27 nM. Photosynthetic activity of the studied dinoflagellates decreased with increasing Cu and BuT concentrations. For O. cf. ovata, the response of this physiological parameter to contamination was less sensitive than the biomass. Cu exposure induced the formation of temporary cysts in both organisms that could resist adverse conditions. The ovatoxin-a and -b concentrations in O. cf. ovata cells increased significantly in the presence of Cu. Altogether, the results suggest a better tolerance of the planktonic A. catenella to Cu and BuT. This could result in a differentiated selection pressure exerted by these metals on phytoplankton species in highly polluted waters. The over-production of toxins in response to Cu stress could pose supplementary health and socio-economic threats in the contaminated marine ecosystems where HABs develop.

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 24h 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.

Different life cycle strategies of the dinoflagellates Fragilidium duplocampanaeforme and its prey Dinophysis acuminata may explain their different susceptibilities to the infection by the parasite Parvilucifera infectans

Some marine dinoflagellates form ecdysal cyst (=temporary cysts) as part of their life cycle or under unfavorable growth conditions. Whether the dinoflagellates form ecdysal cysts or not may influence susceptibility to parasitism. In this study, parasite prevalence relative to inoculum size of the parasitoid Parvilucifera infectans zoospores for two dinoflagellate hosts (i.e., Fragilidium duplocampanaeforme and Dinophysis acuminata), which have different life cycle strategies, was examined. Further, susceptibility of cysts to parasitism, encystment signal, duration of encystments, and effects of induced encystment on diel periodicity, using ecdysal cyst-forming F. duplocampanaeforme were explored. The percent hosts infected by P. infectans plotted as a function of inoculum size showed a sharp increase to a maximum in D. acuminata, but a gradual linear rise in F. duplocampanaeforme: while the parasite prevalence in D. acuminata increased to a maximum of 78.8 (±2.4%) by a zoospore:host ratio of 20:1, it in F. duplocampanaeforme only reached 8.9 (±0.3%), even at a zoospore:host ratio of 120:1. In F. duplocampanaeforme, infections were observed only in the vegetative cells and not observed in ecdysal cysts. When exposed to live, frozen, and sonicated zoospores and zoospore filtrate, F. duplocampanaeforme formed ecdysal cysts only when exposed to live zoospores, suggesting that temporary cyst formation in the dinoflagellate resulted from direct contact with zoospores. When the Parvilucifera zoospores attacked and struggled to penetrate F. duplocampanaeforme through its flagellar pore, the Fragilidium cell shed all thecal plates, forming a ‘thecal cloud layer’, in which the zoospores were caught and immobilized and thus could not penetrate anymore. The duration (35±1.8h) of ecdysal cysts induced with addition of zoospores was significantly longer than that (15±0.8h) of normally formed cysts (i.e., without addition of zoospores), thereby resulting in delayed growth as well as influencing the pattern of diel periodicity. The results from this study suggest that in addition to the classical predator-prey interaction and allelopathic interaction, parasitism and its accompanying defense can make the food web dynamics much more complicated than previously thought.

Effect of various nitrogen conditions on population growth, temporary cysts and cellular biochemical compositions of Karenia mikimotoi.

The harmful algal bloom (HAB)-forming dinoflagellate Karenia mikimotoi was exposed to different nitrogen (N) conditions, in order to study the population growth, temporary cyst production and cellular biochemical compositions in laboratory. The results indicated the population growth of K. mikimotoi was inhibited by different levels of N starvation but showed similar fast recovery after the resupplement of N, and temporary cysts were induced in the period of N starvation. K. mikimotoi grew well in inorganic (NO3-, NO2- and NH4+) and organic (urea) nitrogen sources, but the growth parameters (K, Tp, r) showed differences when simulated by Logistic model regressions. When the cellular organic compounds were measured simultaneously, K. mikimotoi cultured in urea produced more short-chained fatty acids while K. mikimotoi cultured in NH4+ produced more non-fatty acids compounds, indicating the potential change of toxins production cultured by various N sources. We concluded that K. mikimotoi could adapt to fluctuating N environments typical of coastal environments including total N concentration (deficiency or recovery) and relative compositions (different N sources).

Disinfection of fertilized eggs of the edible ascidian Halocynthia roretzi for prevention of soft tunic syndrome.

Azumiobodo hoyamushi, the causative agent of soft tunic syndrome, was likely introduced to farming sites of the edible ascidian Halocynthia roretzi via ascidian spat. The source of infection is thought to be cysts of A. hoyamushi that reside in the substrates on which the ascidian spat are attached, but not the spat themselves. Thus, there is a need to develop methods to prevent contamination of the substrates with A. hoyamushi during seed production of the ascidian. We evaluated the protozoacidal effects of sodium hypochlorite and povidone-iodine against the flagellate and temporary cyst forms of A. hoyamushi. Additionally, we evaluated the effects of these disinfectants on the development of fertilized ascidian eggs. The flagellate form of A. hoyamushi was completely inactivated by povidone-iodine (5 ppm, 1 min) and sodium hypochlorite (1 ppm, 1 min). The temporary cysts of A. hoyamushi were completely inactivated by both disinfectants (5 ppm, 1 min). Disinfection with 50 ppm povidone-iodine for 15 min or 5 ppm sodium hypochlorite for 15 min had no effect on ascidian embryogenesis. Thus, horizontal transmission of A. hoyamushi via the substrates can be efficiently prevented by disinfecting ascidian eggs or tools used for spawning with povidone-iodine baths ranging from 5 ppm for 1 min to 50 ppm for 15 min without any side effects.

Allelopathic effect of Chattonella marina var. marina (Raphidophyceae) on Gymnodinium catenatum (Dinophycea)

The allelopathic effect of the raphidophyte Chattonella marina var. marina on the dinoflagellate Gymnodinium catenatum was determined. Both species are harmful algal bloom forming algae, produce toxic metabolites, and can co-exist in the environment. In general, raphidophytes tend to dominate over dinoflagellates, which may indicate an allelopathic effect of the former algae. Strains of C. marina var. marina and G. catenatum isolated from Bahía de La Paz were cultured in bi-algal cultures with and without cell contact. Additionally, cultures of G. catenatum were exposed to cell-free culture filtrates of the raphidophyte to test whether soluble allelopathic molecules are active. During late stationary phase, both species were cultivated in mixed cultures for 72h using the following cell abundance proportions: 20×103cellsL−1: 20×103cellsL−1 (1:1; G. catenatum: C. marina); 10×103cellsL−1: 20×103cellsL−1 (1:2), and 20×103cellsL−1: 10×103cellsL−1 (2:1). Cells of G. catenatum were also exposed to different volumes of cell filtrates of C. marina (10, 20, and 50mL) using the same cell abundance proportions for 24h. Samples were taken daily for cell counts and microscopic observations. Growth inhibition was higher when there was cell contact between both species, however mortality of G. catenatum was also observed without direct cell contact, indicating that toxic metabolites are liberated to the culture medium. Changes in cell morphology of G. catenatum occurred in the presence of cells and filtrates of C. marina, such as loss of flagella and motility, swelling, loss of girdle and sulci, prominent nucleus, rupture of cell membrane, and cell lysis. Induction of temporary cysts was also observed. These results suggest that toxic metabolites are liberated to the medium by C. marina, affecting G. catenatum by inhibiting its growth and causing changes in its life history, providing new insights of interactions between raphidophytes and dinoflagellates that could happen in the natural environment when both species are present.

Effect of Nitrate, Ammonium and Urea on Growth and Pinnatoxin G Production of Vulcanodinium rugosum.

Vulcanodinium rugosum, a recently described dinoflagellate species producing a potent neurotoxin (pinnatoxin G), has been identified in French Mediterranean lagoons and was responsible for recurrent episodes of shellfish toxicity detected by mouse bioassay. Until now, the biology and physiology of V. rugosum have not been fully investigated. We studied the growth characteristics and toxicity of a V. rugosum strain (IFR-VRU-01), isolated in the Ingril lagoon in June 2009 (North-Western French Mediterranean Sea). It was cultivated in Enriched Natural Sea Water (ENSW) with organic (urea) and inorganic (ammonium and nitrate) nitrogen, at a temperature of 25 °C and irradiance of 100 μmol/m2·s−1. Results showed that ammonium was assimilated by cells more rapidly than nitrate and urea. V. rugosum is thus an osmotrophic species using urea. Consequently, this nitrogen form could contribute to the growth of this dinoflagellate species in the natural environment. There was no significant difference (Anova, p = 0.856) between the growth rate of V. rugosum cultivated with ammonium (0.28 ± 0.11 day−1), urea (0.26 ± 0.08 day−1) and nitrate (0.24 ± 0.01 day−1). However, the production of chlorophyll a and pinnatoxin G was significantly lower with urea as a nitrogen source (Anova, p < 0.027), suggesting that nutritional conditions prevailing at the moment of the bloom could determine the cellular toxicity of V. rugosum and therefore the toxicity measured in contaminated mollusks. The relatively low growth rate (≤0.28 day−1) and the capacity of this species to continuously produce temporary cysts could explain why cell densities of this species in the water column are typically low (≤20,000 cells/L).

Development, morphological characteristics and viability of temporary cysts of Pyrodinium bahamense var. compressum (Dinophyceae) in vitro

Pellicle or temporary cysts of Pyrodinium bahamense var. compressum (Böhm) Steidinger, Tester & F.J.R. Taylor and their role in bloom dynamics have not yet been adequately characterized and understood. We investigated the role of temperature- and nutrient-mediated stress as factors that could induce pellicle formation in batch cultures. Cellular features and their implications for temporary cyst viability were examined using confocal laser scanning microscopy (CLSM). Our data suggest that temperature change is one of the key factors influencing pellicle formation, preserving viability at low temperature (i.e. 13°C). Hypnocysts (resting cysts) were not observed. During pellicle formation, motile cells generally undergo ecdysis, extrusion of cytoplasmic materials and bacteria, compaction of the nucleus and non-motility. The outermost covering of the temporary cysts shows red autofluorescence and it contains lower concentrations of chlorophyll (chl) a and no detectable chl c. The nuclear region is surrounded by transitional red bodies and other unidentified cellular structures. Temporary cysts can immediately revert back to the motile state upon exposure to optimum conditions. This is accompanied by the expansion of the nuclear region, regeneration of the chloroplasts and enlargement of the cell. Developmental changes during reversal of temporary cysts to motile forms were also observed to cause breaks in the cell covering that could serve as sites for bacterial entry. Though observed in vitro, such behaviour may also be occurring in nature especially as a response to drastic short-lived environmental changes. This is the first detailed report on the characteristics of temporary cysts of P. bahamense var. compressum.

The dinoflagellates Pfiesteria shumwayae and Luciella masanensis cause fish kills in recirculation fish farms in Denmark

Fish kills in two geographically separate fish farms in northern Denmark in 2012, one using marine, the other brackish water ‘Recirculation Aquaculture Systems’ (RAS), were found to be caused by Pfiesteria shumwayae and Luciella masanensis, two species of dinoflagellates belonging to the family Pfiesteriaceae. There were no other harmful algae present in either of the aquaculture plants. Serious fish kills in the US have been attributed to Pfiesteria during the past 20 years, but this type of mortality has not been documented elsewhere. L. masanensis, described recently from Korea and USA, has not been previously reported to be the source of fish kills. In the marine farm, the affected fish was rainbow trout, in the brackish water farm pikeperch. Light microscopy is presently insufficient to discriminate between the approx. 20 species of the family Pfiesteriaceae described. Identification of the two algal species was therefore based on molecular sequencing of nuclear-encoded LSU rDNA, confirmed by scanning electron microscopy and, eventually, also by examination of the very thin amphiesmal plates of the flagellates by calcofluor-stained cells in a fluorescence microscope.Although the two fish farms differed in light and salinity conditions, both farms used re-circulating water in closed circuit systems. The dinoflagellates were examined in detail and shown to feed on organic material such as live, damaged nematodes, as described for the single pfiesteriacean flagellate known from freshwater, Tyrannodinium edax. Algal cells were observed to attach to their prey by an attachment filament and subsequently used a peduncle to suck up the food. Fish farms utilizing water recirculation technology are gaining popularity due to their reduced effect on the environment. The two cases from Denmark are apparently the first RAS farms in which serious fish kills have been reported. In the marine farm (Luciella) fish mortality increased dramatically despite treatment of the water with peracetic acid and chloramine-T. The plant was temporarily closed down pending investigation into the cause of mortality and subsequently to determine a method of management to control the dinoflagellate and avoid future fish kills. In the brackish water farm (Pfiesteria), water was treated with chloramine-T, which caused the dinoflagellates to disappear temporarily from the water column, apparently forming temporary cysts. The treatment was repeated after a few days to a week, when the temporary cysts appeared to germinate and the dinoflagellates reappeared in the water column.

Cold-Induced Cysts of the Photosynthetic DinoflagellateLingulodinium polyedrumHave an Arrested Circadian Bioluminescence Rhythm and Lower Levels of Protein Phosphorylation

Dinoflagellates are microscopic, eukaryotic, and primarily marine plankton. Temporary cyst formation is a well-known physiological response of dinoflagellate cells to environmental stresses. However, the molecular underpinnings of cold-induced cyst physiology have never been described. Cultures of the photosynthetic dinoflagellate Lingulodinium polyedrum readily form temporary cysts when placed at low (8°C±1°C) temperature and excyst to form normal motile cells following a return to normal temperature (18°C±1°C). The normal circadian bioluminescence rhythm and the expected changes in Luciferin Binding Protein abundance were arrested in L. polyedrum cysts. Furthermore, after excystment, the bioluminescence rhythm initiates at a time corresponding to zeitgeber 12, independent of the time when the cells encysted. Phosphoprotein staining after two-dimensional polyacrylamide gel electrophoresis, as well as column-based phosphoprotein enrichment followed by liquid chromatography tandem mass spectrometry, showed cyst proteins are hypophosphorylated when compared with those from motile cells, with the most marked decreases found for predicted Casein Kinase2 target sites. In contrast to the phosphoproteome, the cyst proteome is not markedly different from motile cells, as assessed by two-dimensional polyacrylamide gel electrophoresis. In addition to changes in the phosphoproteome, RNA sequencing revealed that cysts show a significant decrease in the levels of 132 RNAs. Of the 42 RNAs that were identified by sequence analysis, 21 correspond to plastid-encoded gene products and 11 to nuclear-encoded cell wall/plasma membrane components. Our data are consistent with a model in which the highly reduced metabolism in cysts is achieved primarily by alterations in the phosphoproteome. The stalling of the circadian rhythm suggests temporary cysts may provide an interesting model to address the circadian system of dinoflagellates.

Stress‐related Responses in Alexandrium tamarense Cells Exposed to Environmental Changes

Organisms tend to be sensitive to drastic changes in environmental conditions. For unicellular microorganisms, variations in physico-chemical conditions are particularly challenging and may result in acclimation, entrance into quiescence, or death through necrotic or autocatalytic pathways. This study focuses on the thecate dinoflagellate Alexandrium tamarense. Cellular responses to oxidative, thermal, and nutrient stress were characterized using stress indicators, such as pigment content, efficiency of photosystem II or production of reactive oxygen species (ROS), as well as hallmarks of apoptosis including activity of caspase-like enzymes and expression of a metacaspase gene homolog. The formation of temporary cysts, a survival strategy of short-term quiescence, was also monitored. Cellular responses appeared to depend on multifactorial influences where type and intensity of stimulus as well as position in cell cycle may act in combination. Sequences of events observed implicate ROS production as a key determinant of stress-related pathways, playing potential roles in intracellular signaling, formation of temporary cysts, or cellular damage. Variations observed in caspase-like activities and metacaspase gene expression did not appear to be associated with programmed cell death pathways; our results suggest a wider range of functions for these proteases in phytoplankton cells, including roles in survival pathways and cell cycle progression.

Feeding, respiration, and excretion of the Black Sea Noctiluca scintillans MacCartney in summer

Studies were conducted at the end of June 2011 in the coastal region of the northeastern part of the Black Sea. The bulk of the Noctiluca scintillans population was observed in the thermocline and reached a density of 40000 ind./m3. Analysis of digestive vacuoles content showed that Noctiluca could consume cells of Neoceratium tripos and N. furca, which had been considered inedible for Black Sea zooplankton, as well as temporary cysts of dinoflagellates, presumably of the toxic genus Alexandrium. The Noctiluca population consumed in total 10–30% of the abundance of temporary cysts, 2–29% of primary production, and 2–9% of potential Calanus euxinus egg production. For the first time, the excretion rates of ammonium nitrogen and mineral phosphorus were measured for N. scintillans. Our calculations showed that in summer, excretion by Noctiluca contributed from 4 to 18% and from 15 to 53% of phytoplankton total nitrogen and phosphorus requirements, respectively. The specific growth rate of Noctiluca (0.17–0.35) in summer, estimated from data on the daily food intake and respiration rate, was close to the values obtained in spring.

Floating Desalination and Water Pumping Plants as Harmful Algal Bloom Control Technologies

Harmful Algal Blooms (HABs) have significant ecological and economic effects on the marine environment and use. In recent years, researchers have been increasingly developing and testing methods to treat and control HABs. General categories or strategies proposed as HAB control technologies include mechanical, biological, chemical, genetic, and environmental controls. The authors of this paper suggest using floating desalination plants to treat or control red tides. HAB producing dinoflagellates have been shown to be sensitive to physical and chemical changes in the environment, such as changes in temperature and salinity. The typical response of dinoflagellates is to form cysts that then settle out of the water column. The discharges from a floating desalination and water pumping plant can rapidly change the temperature and salinity in the water column. These changes could be expected to induce encystment in the dinoflagellate species that form cyst and could cause mortality in those species unable to form temporary cysts. Preventing population growth, inducing encystment, or causing mortality would effectively end a HAB. Discharges from a desalination plant are temporary in nature and include hypersaline water, freshwater (hyposaline water), and heated warmer. By discharging the heated hyposaline water at a low depth in the water column, due to its lower density than seawater, the discharge would move upward towards the surface. Since the hypersaline water would be denser than seawater, by discharging it at the surface, the hypersaline water would sink. In environments where a stratified water column exists, pumping water could disrupt the stratification without the need for additional desalination. The discharges from a floating desalination plant would stress the red tide with surfacing warmer fresh water and sinking hypersaline water. The stresses caused by these disturbances can disrupt a HAB. These temperature and salinity changes that could be created by a floating desalination plant would be achieved without the discharge of chemicals or other materials that could have other detrimental environmental impacts. A good aspect of this treatment is that, with continued mixing after discharge, the water would return to ambient temperature and salinity relatively quickly with minimal effect on the marine environment. Since the dinoflagellates have been shown to react quickly to environmental changes, the temperature and salinity of the discharges could be controlled to reduce adverse impacts on other marine organisms. B

PREY SPECIFICITY AND FEEDING OF THE THECATE MIXOTROPHIC DINOFLAGELLATE FRAGILIDIUM DUPLOCAMPANAEFORME1

In summer to autumn of 2008, a recently described thecate mixotrophic dinoflagellate, Fragilidium duplocampanaeforme Nézan et Chomérat, occurred in Masan Bay, Korea, where it frequently contained bright‐orange fluorescent inclusions. Using cultures of F. duplocampanaeforme isolated from Masan Bay, we investigated feeding, digestion, and prey specificity of this mixotroph. F. duplocampanaeforme fed exclusively on Dinophysis spp. when offered a variety of prey including dinoflagellates, a raphidophyte, a cryptophyte, a ciliate, and diatoms separately. In addition, F. duplocampanaeforme had allelopathic effects on other organisms, including cell immobilization/motility decrease (in Dinophysis acuminata, D. caudata, D. fortii, D. infundibulus, Gonyaulax polygramma, Heterocapsa triquetra, and Prorocentrum triestinum), breaking of cell chains (in Cochlodinium polykrikoides), cell death (in Prorocentrum minimum), and temporary cyst formation (in Scrippsiella trochoidea). F. duplocampanaeforme engulfed whole Dinophysis cells through the sulcus. About 1 h after ingestion, F. duplocampanaeforme became immobile and shed all thecal plates. The ecdysal cyst persisted for ∼7 h, during which the ingested prey was gradually digested. These observations suggest that F. duplocampanaeforme may play an important role in the Dinophysis population dynamics in the field.