EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

PHYSIOLOGICAL AND ENVIRONMENTAL CONTROL OF GERMINATION IN SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE) RESTING CYSTS1

The effects of aging, temperature, and growth medium on germination in culture-produced resting cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich ore examined. Cysts undergo a mandatory period of dormancy lasting approximately 25 days, during which germination does not occur. The duration of this period is not affected by temperature. Once the dormancy period is completed, germination is regulated by external factors. Cysts germinate optimally in nutrient replete medium at temperatures greater than approximately 14° C. At lower temperatures or in nutrient-depleted media germination rate is dramatically slowed, although the final germination frequency appears unchanged. The large Q₁₀ of this temperature effect (ca. 11) suggests that the reduction in germination rate at lower temperatures is not merely the reflection of generally reduced metabolic rates, but rather the result of a temperature response specific to germination. At the highest temperatures tested (22–25° C), germination rate remains maximal although vegetative growth is greatly reduced. A shift in temperature or nutrient conditions, per se, is not necessary for germination. The relatively short dormancy period combined with the absence of a requirement for a dramatic shift in environmental conditions could facilitate rapid cycling between resting and vegetative stages in natural S. trochoidea populations. At the same time, the dramatic reduction in germination rate at low temperatures would permit cysts of this species to serve as overwintering cells as well.     

Different excystment patterns in two calcareous cyst-producing species of the dinoflagellate genus Scrippsiella

Scrippsiella rotunda and Scrippsiella trochoidea var. aciculifera (order Peridiniales, subfamily Calciodinelloideae) are autotrophic orthoperidinioid dinoflagellates producing calcareous resting cysts which are at times abundant in coastal marine sediments. We have carried out laboratory experiments to investigate features of cyst germination in the two species, including dormancy length, germination pattern and germination success, over an annual cycle and under different light and temperature conditions. The maturation period for S. rotunda cysts was between 17 and 24 weeks, while that of S. trochoidea var. aciculifera was much shorter, ranging between 2 and 5 weeks. Both species required exposure to light for germination, while temperature shifts (from 14 to 20C) in the dark did not induce excystment of mature cysts. In both species, germination was not synchronous, but distributed over a variable time interval, suggesting a high physiological diversity within the cyst pool. Moreover, exposure to light of S. rotunda cysts that had not completed maturation impaired the germination of a great percentage of the cysts. Differences in dormancy length may partially explain the distinct cyst production patterns observed for the two species in the Gulf of Naples.      

Dinoflagellate resting cysts as factors in phytoplankton ecology of the North Sea

The occurrence and distribution of dinoflagellate resting cysts were investigated at 11 locations in the south-eastern part of the North Sea. Twenty-six known cyst species and 7 unknown cyst types, which may act as seed population for planktonic dinoflagellate blooms, have been recorded for the first time in the area. The most common cysts in recent sediments were those ofScrippsiella trochoidea, Zygabikodinium lenticulatum, Peridinium dalei, Scrippsiella lachrymosa, Protoceratium reticulatum, Protoperidinium denticulatum, andP. conicum. At all stations,S. trochoidea dominated the cyst assemblages with a maximal abundance of 1303 living cysts/cm³ in the uppermost half centimetre. Cysts of the potentially toxic dinoflagellatesAlexandrium cf.excavatum andA. cf.tamarense were scarce. In the upper 2-cm layer of sediment, dinoflagellate cysts were found in concentrations of 1.8 up to 682 living cysts/cm³. Empty cysts constituted 22–56% of total cyst abundance. The comparative distribution of the cysts showed a general increase in abundance from inshore sites to the offshore area, whereby sandy stations exhibited the lowest cyst abundance and diversity. The wide distribution of living and empty cysts ofScrippsiella lachrymosa suggests that its motile form, which has not been officially recorded in the area until now, is a common plankton organism in German coastal waters. The relatively high abundance of cysts in recent sediments demonstrates the potential importance of benthic resting stages for the initiation of dinoflagellate blooms in the study area.     

Temperature‐induced changes in lipid biomarkers and mycosporine‐like amino acids in the psychrophilic dinoflagellate Peridinium aciculiferum

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BIOCHEMICAL COMPOSITION AND METABOLIC ACTIVITY OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE) RESTING CYSTS1

The composition and metabolic activity of cysts of the marine dinoflagellate Scrippsiella trochoidea (Stein) Loeblich were examined during dormancy, quiescence, and germination. On a per cell basis, newly formed cysts contained an order of magnitude more carbohydrate but significantly less protein and chlorophyll a than did exponentially growing vegetative cells. Loss of lipid and carbohydrate from cysts during the initial dormancy period reflected a respiration rate estimated to be 10% of the respiratory activity in vegetative cells. Among older, quiescent cysts the calculated respiration rate decreased further to approximately 1.5% of the vegetative rate and appeared to proceed largely at the expense of carbohydrate reserves. These estimated rates of respiration were in good agreement with direct measurements of cyst oxygen consumption. The transfer of quiescent cysts to conditions permissive for germination resulted in a rapid increase in respiration rate, as evidenced by carbohydrate loss and O₂ consumption. The increased respiratory activity was followed by an increase in protein content and, later, by an increase in chlorophyll a content and photosynthetic capacity. Just prior to germination the P/R ratio became greater than 1, and the estimated chlorophyll-specific photosynthetic activity reached 75% of the rate in vegetative cells. Complete restoration of photosynthetic and respiratory capacity apparently was not achieved until after excystment. These data confirm the common assumption that dinoflagellate cysts represent true “resting” cells, containing extensive energy reserves and displaying greatly reduced metabolic activity.     

The potential for dispersal of microalgal resting cysts by migratory birds

Most microalgal species are geographically widespread, but little is known about how they are dispersed. One potential mechanism for long‐distance dispersal is through birds, which may transport cells internally (endozoochory) and deposit them during, or in‐between, their migratory stopovers. We hypothesize that dinoflagellates, in particular resting stages, can tolerate bird digestion; that bird temperature, acidity, and retention time negatively affect dinoflagellate viability; and that recovered cysts can germinate after passage through the birds’ gut, contributing to species‐specific dispersal of the dinoflagellates across scales. Tolerance of two dinoflagellate species (Peridiniopsis borgei, a warm‐water species and Apocalathium malmogiense, a cold‐water species) to Mallard gut passage was investigated using in vitro experiments simulating the gizzard and caeca conditions. The effect of in vitro digestion and retention time on cell integrity, cell viability, and germination capacity of the dinoflagellate species was examined targeting both their vegetative and resting stages. Resting stages (cysts) of both species were able to survive simulated bird gut passage, even if their survival rate and germination were negatively affected by exposure to acidic condition and bird internal temperature. Cysts of A. malmogiense were more sensitive than P. borgei to treatments and to the presence of digestive enzymes. Vegetative cells did not survive conditions of bird internal temperature and formed pellicle cysts when exposed to gizzard‐like acid conditions. We show that dinoflagellate resting cysts serve as dispersal propagules through migratory birds. Assuming a retention time of viable cysts of 2–12 h to duck stomach conditions, cysts could be dispersed 150–800 km and beyond.     

PHYLOGENETIC POSITION AND MORPHOLOGY OF THECAE AND CYSTS OF SCRIPPSIELLA (DINOPHYCEAE) SPECIES IN THE EAST CHINA SEA1

Resting cysts of the marine phytoplanktonic dinoflagellate Scrippsiella spp. are encountered in coastal habitats and shallow seas all over the world. Identification of Scrippsiella species requires information on cyst morphology because the plate pattern of the flagellated cell is conserved. Cysts from sediments of the East China Sea were identified based on traits from both the cysts and the thecal patterns of germinated cells. Calcareous cysts belonged predominantly to S. trochoidea (F. Stein) A. R. Loebl., S. rotunda J. Lewis, and S. precaria Montresor et Zingone. The former two species also produced smooth and noncalcified cysts in the field. A new species, S. donghaienis H. Gu sp. nov, was obtained from six noncalcified cysts with organic spines. These cysts are spherical, full of pale white and greenish granules with a mesoepicystal archeopyle. The vegetative cells consist of a conical epitheca and a round hypotheca with a plate formula of po, x, 4′, 3a, 7′′, 6c (5c + t), 6 s, 5′′′, 2′′′′ and are morphologically indistinguishable from S. trochoidea. Results of internal transcribed spacer (ITS) sequence comparisons revealed that S. donghaienis was distinct from the S. trochoidea complex and appeared nested within the Calciodinellum/Calcigonellum clade. Culture experiments showed that the presence of a red body in the cyst and the shape of the archeopyle were constant within cell lines from one generation to the next, while the morphological features of the cyst wall, such as calcification and spine shape, appeared to be phenotypically plastic.     

The significance of sexual versus asexual cyst formation in the life cycle of the noxious dinoflagellate Alexandrium peruvianum

Alexandrium peruvianum (Balech et Mendiola) is a noxious phototrophic marine dinoflagellate. During the life cycle of this species, two kinds of cysts are produced: resting cysts, which are long-lasting and double-walled, and temporary cysts, which are short-lasting and thin-walled. In addition, short-lasting, but resting-like cysts can also be formed. Although it is crucial to identify sexual events in a dinoflagellate population, sexual and asexual cysts are morphologically very similar in this species. Therefore, we studied the complete life cycle and the nature of the cyst-like stages formed after individual isolation of specimens and crossing of clonal cultures established from germination of wild resting cysts. Asexual division in A. peruvianum takes place either in the motile stage by sharing of the theca (desmoschisis), or inside a vegetative cyst (temporary cyst), from which two, or at times four or six naked daughter cells can originate. The daughter cells completely synthesize new cell walls (eleutheroschisis). Sexuality was confirmed by the presence of fusing gamete pairs and longitudinally biflagellated planozygotes after out-crossing of compatible clonal strains. However, the clonal cultures had low levels of self-compatibility, since a flow cytometry analysis showed that synchronized self-crosses produced few zygotes (<5%). After isolation of individual cells, it was proved that the fate of the planozygotes depended on the nutritional status of the isolation media. Most of the planozygotes isolated to replete medium (L1) divided, whereas in medium lacking nitrates (L-N) or phosphates (L-P) they formed temporary, thin-walled cysts. Temporary cysts formed in L1 were always uninucleated and gave rise to one cell, while those formed in L-N or L-P produced 1–6 small cells. In addition, resting cysts were formed in culture, but never after individual planozygote isolation. Resting cysts were uninucleated and needed maturation time before entering dormancy. The resting cysts were considered sexual products, since longitudinally biflagellate germlings were liberated after germination in all cases studied. Mature resting cysts (52.3 ± 3.0 μm) had a dormancy period of 1–3 months, whereas temporary asexual cysts (32.5 ± 5.4 μm) germinated in less than 7 days.     

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.     

Dinoflagellate cyst records in recent sediments from Daya Bay, South China Sea

Nine sediment cores of 8–26 cm in length were collected from two basins of Daya Bay, the South China Sea, by Tokyo University Fisheries Oceanography Laboratory core sampler in August 2001 to investigate the distribution of dinoflagellate resting cysts. In the present study, 51 different cyst morphotypes representing 22 genera were identified from 65 sediment samples. Among them, there were 21 autotrophic species and 30 heterotrophic ones. Cyst species richness in each sample varied from 12 to 29, while the values of Shannon‐Weaver diversity index (H′) were between 0.15 and 4.13. There were an obvious increase in both species richness and values of H′in 2–6 cm sediments. Cyst concentrations varied from 154 to 113 483 cysts per gram dry weight sediment, and were much higher in upper sediments. Scrippsiella trochoidea was the most dominant cyst type, which took up over 90% of cyst assemblages in the upper sediments. The abrupt increase of S. trochoidea cysts in the surface sediments reflected the bloom of this species in Daya Bay in 2000. The results from cyst assemblages showed some trend of changes in water quality in this area, and indicated a typical type of pollution caused by cultural eutrophication, which started in the 1980s and greatly accelerated in the middle of 1990s. Cysts of Alexandrium, mainly those of Alexandrium catenella and Alexandrium tamarense complex, occurred frequently and abundantly in this area, with the highest concentration and relative frequency of 503 cysts per gram dry weight sediment and 22.3%, respectively. The high abundance of Alexandrium cysts provided rich ‘seed bed’ for Alexandrium blooms and was also an important source of paralytic shellfish poisoning toxins, especially in winter.     

Viability of dinoflagellate cysts after the passage through the copepod gut

Several dinoflagellate species form nonmotile, thick-walled resting cysts in their life cycle. Cysts can be ingested by planktonic and benthic organisms, but there is scarce information concerning their survival after the passage through the digestive apparatus of the grazers. We tested the germination capability of cysts produced by two neritic dinoflagellates, Scrippsiella trochoidea (F. Stein) A.R. Loeblich and Scrippsiella ramonii Montresor, after their ingestion by four copepod species. Experiments have been carried out with four species: Acartia clausi Giesbrecht, 1889; Centropages typicus Kröyer, 1849; Temora stylifera Dana, 1849; and Clausocalanus lividus Frost and Fleminger, 1968. Copepods were fed either with motile cells or cysts, and feeding and clearance rates were estimated for A. clausi, C. lividus and T. stylifera. Grazing rates on both dinoflagellates was much higher for vegetative cells than for cysts. Resting cysts were isolated from the faecal pellets and incubated to test their germination capability. S. trochoidea cysts eaten by C. typicus and T. stylifera showed a high germination rate, while cysts of the same species were not viable after the passage through the gut of A. clausi and C. lividus. In contrast, S. ramonii cysts were never able to germinate after being ingested by copepods. The observed variation in viability among the two cyst types and the different survival rates observed for S. trochoidea cysts might be related to differences in cyst morphology and to differences in the digestive process among the tested copepod species.     

INTERACTIVE EFFECTS OF SALINITY AND TEMPERATURE ON PLANOZYGOTE AND CYST FORMATION OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) IN CULTURE1

The factors regulating dinoflagellate life‐cycle transitions are poorly understood. However, their identification is essential to unravel the causes promoting the outbreaks of harmful algal blooms (HABs) because these blooms are often associated with the formation and germination of sexual cysts. Nevertheless, there is a lack of knowledge on the factors regulating planozygote‐cyst transitions in dinoflagellates due to the difficulties of differentiating planozygotes from vegetative stages. In the present study, two different approaches were used to clarify the relevance of environmental factors on planozygote and cyst formation of the toxic dinoflagellate Alexandrium minutum Halim. First, the effects of changes in initial phosphate (P) and nitrate (N) concentrations in the medium on the percentage of planozygotes formed were examined using flow cytometry. Second, two factorial designs were used to determine how salinity (S), temperature (T), and the density of the initial cell inoculum (I) affect planozygote and resting‐cyst formation. These experiments led to the following conclusions: 1. Low P/N ratios seem to induce gamete expression because the percentage of planozygotes recorded in the absence of added phosphate (‐P) was significantly higher than that obtained in the absence of added nitrogen (‐N), or when the concentrations of both nitrogen and phosphate were 20 times lower (N/20 + P/20). 2. Salinity (S) and temperature (T) strongly affected both planozygote and cyst formation, as sexuality in the population increased significantly as salinity decreased and temperatures increased. S, T combinations that resulted in no significant cyst formation were, however, favorable for vegetative growth, ruling out the possibility of negative effects on cell physiology. 3. The initial cell density is thought to be important for sexual cyst formation by determining the chances of gamete contact. However, the inoculum concentrations tested did not explain either planozygote formation or the appearance of resting cysts.     

First record of resting cysts of the benthic dinoflagellate Prorocentrum leve in a natural reservoir in Gujan‐Mestras,Gironde, France

The resting cysts of the benthic dinoflagellate Prorocentrum leve from a natural reservoir in Gujan‐Mestras (Gironde, France) were described in this study. The incubated urn‐shaped cysts gave rise to cells of P. leve. Morphological observations through light microscopy and scanning electron microscopy, particularly of the periflagellar platelets, combined with large subunit ribosomal DNA sequences obtained through single‐cell analysis confirm their affinity to the species P. leve. The cysts were characterized by a specific shape and the presence of an anterior plug. This is the first conclusive evidence for fossilizable resting stages within the Prorocentrales, one of the major orders within the Dinophyceae. Palynological treatments show that the cysts and endospores withstand hydrochloric and hydrofluoric acids. Micro‐Fourier transform infrared analysis on single specimens suggests that the composition of the endospore is cellulosic and the cyst wall a more robust, noncellulosic β‐glucan. The spectra overall are similar to other published spectra of resting cysts from autotrophic, planktonic dinoflagellates.     

Differences in pigmentation between life cycle stages in Scrippsiella lachrymosa (dinophyceae)

Various life cycle stages of cyst‐producing dinoflagellates often appear differently colored under the microscope; gametes appear paler while zygotes are darker in comparison to vegetative cells. To compare physiological and photochemical competency, the pigment composition of discrete life cycle stages was determined for the common resting cyst‐producing dinoflagellate Scrippsiella lachrymosa. Vegetative cells had the highest cellular pigment content (25.2 ± 0.5 pg · cell^?1), whereas gamete pigment content was 22% lower. The pigment content of zygotes was 82% lower than vegetative cells, even though they appeared darker under the microscope. Zygotes of S. lachrymosa contained significantly higher cellular concentrations of β‐carotene (0.65 ± 0.15 pg · cell^?1) than all other life stages. Photoprotective pigments and the de‐epoxidation ratio of xanthophylls‐cycle pigments in S. lachrymosa were significantly elevated in zygotes and cysts compared to other stages. This suggests a role for accessory pigments in combating intracellular oxidative stress during sexual reproduction or encystment. Resting cysts contained some pigments even though chloroplasts were not visible, suggesting that the brightly colored accumulation body contained photosynthetic pigments. The differences in pigmentation between life stages have implications for interpretation of pigment data from field samples when sampled during dinoflagellate blooms.     

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.     

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.     

The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

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

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.     

GREEN AUTOFLUORESCENCE OF DINOFLAGELLATE CYSTS CAN BE USED INSTEAD OF PRIMULINE STAINING FOR CYST VISUALIZATION AND ENUMERATION IN SEDIMENTS1

Primuline staining is widely used to visualize and enumerate dinoflagellate cysts in marine sediments. In staining cysts of Gymnodinium catenatum H. W. Graham, Scrippsiella trochoidea (F. Stein) A. R. Loebl., and cysts from estuarine sediments, we found their green fluorescence after primuline treatment to be seemingly no different from the green autofluorescence (GAF) inherent in vegetative cells and cysts of dinoflagellates fixed in formaldehyde. Although primuline subsequently proved to enhance green fluorescence of both species quantitatively, we nonetheless recommend taking advantage of dinoflagellates' GAF to detect and count their cysts in sediments. Doing so will reduce the time, chemical consumption, and possible loss of cells involved in the primuline‐staining procedure.