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.     

The life history of the toxic marine dinoflagellate Protoceratium reticulatum (Gonyaulacales) in culture

Asexual and sexual life cycle events were studied in cultures of the toxic marine dinoflagellate Protoceratium reticulatum. Asexual division by desmoschisis was characterized morphologically and changes in DNA content were analyzed by flow cytometry. The results indicated that haploid cells with a C DNA content occurred only during the light period whereas a shift from a C to a 2C DNA content (indicative of S phase) took place only during darkness. The sexual life cycle was documented by examining the mating type as well as the morphology of the sexual stages and nuclei. Gamete fusion resulted in a planozygote with two longitudinal flagella, but longitudinally biflagellated cells arising from planozygote division were also observed, so one of the daughter cells retained two longitudinal flagella while the other daughter cell lacked them. Presumed planozygotes (identified by their longitudinally biflagellated form) followed two life-cycle routes: division and encystment (resting cyst formation). Both the division of longitudinally biflagellated cells and resting cyst formation are morphologically described herein. Resting cyst formation through sexual reproduction was observed in 6.1% of crosses and followed a complex heterothallic pattern. Clonal strains underwent sexuality (homothallism for planozygote formation and division) but without the production of resting cysts. Ornamental processes of resting cysts formed from the cyst wall under an outer balloon-shaped membrane and were fully developed in <1 h. Obligatory dormancy period was of ∼4 months. Excystment resulted in a large, rounded, pigmented, longitudinally biflagellated but motionless, thecate germling that divided by desmoschisis. Like the planozygote, the first division of the germling yielded one longitudinally biflagellated daughter cell and another without longitudinal flagella. The longitudinal biflagellation state of both sexual stages and of the first division products of these cells is discussed.     

Factors regulating germination of resting cysts of the spring bloom dinoflagellate Scrippsiella hangoei from the northern Baltic Sea

The role of cyst germination as a factor in bloom initiationwas investigated for the dinoflagellate Scrippsiella hangoei(Schiller) Larsen from the northern Baltic Sea. This speciesblooms in very cold, often ice-covered waters, and is responsiblefor a significant fraction of the production in that region.Dormancy, temperature, oxygen and light were studied as factorspotentially controlling the germination of S.hangoei restingcysts. Laboratory-stored and field-collected cysts began togerminate in December following a mandatory dormancy periodlasting 6 months. Germination after this maturation intervalwas maximal when temperatures were within a 0–9°C‘window’. Mandatory dormancy is therefore the primaryfactor regulating the timing of germination in this species,as temperatures in the natural environment normally fall withinthis range at the time when S.hangoei cysts deposited the precedingyear have matured. Non-optimal temperatures, darkness and lowoxygen conditions all maintain a state of quiescence in thecysts. Cysts could germinate in darkness, but the rate of excystmentwas significantly higher in the light. Likewise, excystmentwas completely inhibited in anoxic conditions and was reducedunder severe hypoxia, with normal germination under moderatehypoxic concentrations. Temporary exposure to high sulfide concentrationspermanently reduced germination potential, indicating that S.hangoeicysts have low resistance to oxygen deficiency. Prolonged periodsof anoxia at the sediment surface, as frequently occurs in thestudy area, might reduce the size of the viable cyst pool andthus, alter the magnitude of the inoculum for S.hangoei bloominitiation. Together, these internal and external regulatoryfactors play important roles in the bloom dynamics of this importantdinoflagellate.     

LIFE CYLE AND SEXUALITY OF THE FRESHWATER RAPHIDOPHYTE GONYOSTOMUM SEMEN (RAPHIDOPHYCEAE)1

Previously unknown aspects in the life cycle of the freshwater flagellate Gonyostomum semen (Ehrenb.) (Raphidophyceae) are described here. This species forms intense blooms in many northern temperate lakes, and has increased in abundance and frequency in northern Europe during the past decades. The proposed life cycle is based on observations of life cycle stages and transitions in cultures. Viable stages of the life cycle were individually isolated and monitored by time‐lapse photography. The most common processes undertaken by the isolated cells were: division, fusion followed by division, asexual cyst formation, and sexual cyst formation. Motile cells divided by two different processes. One lasted between 6 and 24 h and formed two cells with vegetative cell size and with or without the same shape. The second division process lasted between 10 and 20 min and formed two identical cells, half the size of the mother cell. Planozygotes formed by the fusion of hologametes subsequently underwent division into two cells. Asexual cyst‐like stages were spherical, devoid of a thick wall and red spot, and germinated in 24–48 h. Heterogamete pairs were isogamous, and formed an angle of 0–90° between each other. Planozygote and sexual cyst formation were identified within strains established from one vegetative cell. The identity of these strains, which was studied by an amplified fragment length polymorphism analysis, was correlated with the viability of the planozygote. Resting cyst germination was described using cysts collected in the field. The size and morphology of these cysts were comparable with those formed sexually in culture. The excystment rate was higher at 24°C than at 19 or 16°C, although the cell liberated during germination (germling) was only viable at 16°C. The placement of G. semen within the Raphidophyceae family was confirmed by sequence analysis of a segment of the 18S ribosomal DNA.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.     

CHARACTERISTICS OF SEXUAL AND ASEXUAL RESTING CYST (STATOSPORE) FORMATION IN DINOBRYON CYLINDRICUM IMHOF (CHRYSOPHYTA)1

The reproductive significance of siliceous cysts (statospores) produced by the common vernal chrysophyte Dinobryon cylindricum Imhof has been investigated under defined culture conditions. Three types of statospores have thus far been induced in culture: 1) uninucleate, asexual; 2) binucleate, asexual (potentially autogamic); 3) binucleate, sexual (zygotic). The production of each type of cyst responds differently to an array of nutrient deficiencies (P, N, vitamins, micrometals). An individual clone may be capable of participating in the production of all or only a subset of these types of resting cysts. All D. cylindricum statospores are morphologically identical regardless of their reproductive significance. Sexual reproduction leading to zygotic statospore formation is anisogamous, heterothallic, and involves a gametogenic hormone (erogen) that is apparently continuously released from female clones. Only a single bipolar mating group is documented here and clonal compatibility varies considerably within the mating group. The dynamics of encystment for each type of statospore has been determined relative to the growth of the vegetative cell population. Statospores may be produced either during the exponential phase (intrinsic encystment) or stationary phase (extrinsic encystment) of culture growth depending on the clones involved. The effect of both asexual and sexual resting cyst production on the net growth rate and dynamics of natural chrysophyte populations is discussed. Statospores appear to represent a more flexible reproductive strategy than the resistant zygospores produced by the other common groups of planktonic microalgae.     

Gyrodinium undulans Hulburt, a marine dinoflagellate feeding on the bloom-forming diatomOdontella aurita, and on copepod and rotifer eggs

The marine dinoflagellateGyrodinium undulans was discovered as a feeder on the planktonic diatomOdontella aurita. Every year, during winter and early spring, a certain percentage of cells of this bloom-forming diatom, in the Wadden Sea along the North Sea coast, was regularly found affected by the flagellate. Supplied with the food diatomO. aurita the dinoflagellate could be maintained successfully in clonal culture. The vegetative life cylce was studied, mainly by light microscopy on live material, with special regard to the mode of food uptake. Food is taken up by a so-called phagopod, emerging from the antapex of the flagellate. Only fluid or tiny prey material could be transported through the phagopod. Larger organelles like the chloroplasts ofOdontella are not ingested and are left behind in the diatom cell. Thereafter, the detached dinoflagellate reproduces by cell division, occasionally followed by a second division. As yet, stages of sexual reproduction and possible formation of resting cysts could not be recognized, neither from wild material nor from laboratory cultures. Palmelloid stages (sometimes with a delicate wall) occurring in ageing cultures may at least partly function as temporary resting stages. The winter speciesG. undulans strongly resemblesSyltodinium listii, a summer species feeding on copepod and rotifer eggs. Surprisingly, in a few cases this prey material was accepted byG. undulans as well, at least under culture conditions. When fed with copepod eggs, the dinoflagellate developed into a large trophont, giving rise thereafter by repeated binary fission to 4, 8 or 16 flagellates, as a result of a single feeding act. A re-examination of both species under simultaneous culture conditions is planned.     

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.     

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.     

HIGH ENCYSTMENT SUCCESS OF THE DINOFLAGELLATE SCRIPPSIELLA CF. LACHRYMOSA IN CULTURE EXPERIMENTS 1

Close to 100% encystment efficiency and a yield above 10⁵ cysts·mL ^{? 1} were routinely achieved in full strength f/2 medium‐based batch cultures (883 μM NO₃ ^? and 36 μM PO₄ ^{? 3}) of the marine dinoflagellate Scrippsiella cf. lachrymosa Lewis. Increases in cell density led to nutrient depletion in this enriched medium, which was the most likely cause for initiation of cyst formation. Lowering the concentration of either nutrient to 1/10 the initial levels decreased the encystment efficiency, whereas use of ammonium as the N source resulted in both low cell yield and low encystment efficiency. The mandatory dormancy period was ca. 60 days and was not affected by cold dark storage of the cysts. Cysts produced in the initial phase of sexual reproduction were relatively large (length 47 μm, width 31 μm) with a heavy calcareous cover. Cysts produced thereafter lacked apparent calcareous cover and were smaller (length 29 μm, width 19 μm). The decrease of cyst volume (by a factor of 0.24–0.4) suggested strong resource limitation during the course of encystment. However, after the mandatory dormancy period, germination success of the smaller cysts was higher (80%), compared with the larger cysts that had been produced initially (50%). Germling survival (74%) was independent of cyst type but was enhanced by higher nutrient concentration during incubation. The ratio of initial nutrient concentration in the medium to the cyst yield was used as a proxy to estimate the cellular nutrient quota. The conservative estimates of 9 pmol N·cyst ^{? 1} and 0.4 pmol P·cyst ^{? 1} obtained in this manner are at the low end of the range of previous published estimates for other dinoflagellate cysts. Given the high encystment observed in laboratory experiments, we have no reason to assume an inherently lower encystment success in dinoflagellate field populations. Our results do not challenge the low nutrient paradigm for dinoflagellate sexuality. We believe that the high encystment success and cyst yield of this particular species is at least partly due to its ability to achieve very high cell densities in cultures, which evidently leads to nutrient depletion even in f/2 medium.     

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

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

EFFECTS OF PARENTAL FACTORS AND MEIOSIS ON SEXUAL OFFSPRING OF GYMNODINIUM NOLLERI (DINOPHYCEAE)1

Clonal strains of the dinoflagellate Gymnodinium nolleri Ellegaard and Moestrup were intercrossed to determine if cyst‐related traits are genetically regulated and to clarify unknown aspects in the sexuality of this species. The objectives were to determine whether the parental identity influenced the physiological and morphological aspects of the cyst offspring, and to describe and compare nuclear development and cell division of encysting and non‐encysting zygotes. Variables characteristic of each parental cross (difference in growth rates among parents, cyst production (CP), and genetic distance (GD) among parents assessed via an amplified fragment length analysis analysis) were studied to seek for possible relationships of the parental crosses with some characteristics of the cyst offspring (cyst size, length of dormancy period, germination success, and germling viability (V)). A principal component analysis using these variables showed three main results: (1) the dormancy period of cysts responded to a simple pattern of inheritance, (2) the larger the GD between parents, the smaller the CP, and progeny V, and (3) the size of cysts was influenced by both CP and the parental strain identity. A stable inheritance of the short dormancy period (14.6±5.5 days), dominant over medium (31.0±8.5 days) and long periods (52.7±9.2 days), was confirmed through two subsequent generations of cysts. The regulation of the sexual processes by a multiple loci system is discussed based on the pattern of inheritance of the dormancy period and the number of sexual recombination events recorded within cultures with self‐CP capability. Fusion of the gamete nuclei happened 0–48 h after the total cytoplasmic fusion. The nucleus of the zygote was bilobed and had thick and distinct chromosomes. Similar processes of nuclear and cell division occurred in the non‐encysting or encysting planozygote, and were characterized by the loss of the chromosomal structure, an apparent increase of the DNA content, and the formation of thinner chromosomes.     

CHANGES IN CELL CHEMICAL COMPOSITION DURING THE LIFE CYCLE OF SCRIPPSIELLA TROCHOIDEA (DINOPHYCEAE)1

The cellular content of carbon, nitrogen, amino acids, polysaccharides, phosphorus and adenosine trtphosphate (ATP) was determined at several stages during the life cycle of the dinoflagellate Scrippsiella trochoidea (Stein) Loeblich. Carbon per cell decreased slightly between exponential and stationary phase growth in vegetative cells whereas nitrogen per cell did not change. Both of these cellular components increased markedly on encystment and then decreased to vegetative cell levels during dormancy and germination. C/N ratios increased gradually during cyst dormancy and activation, reflecting a more rapid decrease in N than in C pools, even though both decreased through time. Amino acid composition was relatively constant during the vegetative cell stages; glutamic acid was the dominant component. Arginine was notably higher in cysts than in vegetative cells but decreased significantly during germination, suggesting a role in nitrogen storage. The ratio of neutral ammo acids to total ammo acids (NAA/TAA) decreased as cysts were formed and then gradually increased during storage and germination. The ratio of basic ammo acids to total ammo acids (BAA/TAA) changed in the opposite direction of NAA/TAA, whereas the ratio of acidic acids to total amino adds (AAA/TAA) was generally invariant. Ammo acid pools were not static during the resting slate in the cysts: there was degradation or biosynthesis of certain, but not all, classes of these compounds. The monosacchande composition of cold and hot water extracted polysaccharides was quite different between cells and cysts. A high percentage of glucose in cysts suggests that the storage carbohydrate is probably in the form of glucan. Total cellular phosphorus was higher in all cyst stages than in vegetative cells. However, ATP-cell^?1 decreased as vegetative cells entered stationary phase and encysted, and continued to decrease in cysts during dark cold storage. ATP increased only as the cysts were activated at warm temperatures in the light and began to germinate. The above data demonstrate that dormancy and quiescence are not periods of inactive metabolism but instead are times when numerous biochemical transformations are occurring that permit prolonged survival in a resting state.     

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.     

Interplay between the parasite Amoebophrya sp. (Alveolata) and the cyst formation of the red tide dinoflagellate Scrippsiella trochoidea

Syndiniales (Alveolata) are marine parasites of a wide range of hosts, from unicellular organisms to Metazoa. Many Syndiniales obligatorily kill their hosts to accomplish their life cycle. This is the case for Amoebophrya spp. infecting dinoflagellates. However, several dinoflagellate species known to be infected by these parasites produce diploid resting cysts as part of their life history. These resting cysts may survive several seasons in the sediment before germinating. How these parasites survive during the dormancy of their host remained an open question. We successfully established infections by Amoebophrya sp. in the red tide dinoflagellate Scrippsiella trochoidea. This host strain was homothallic and able to continuously produce typical calcified cysts covered by calcareous spines. Presence of the parasite significantly speeded up the host cyst production, and cysts produced were the only cells to resist infections. However, some of them were clearly infected, probably earlier in their formation. After 10 months, cysts produced in presence of the parasite were able to germinate and new infective cycles of the parasite were rapidly observed. Thus, a very novel relationship for protists is demonstrated, one in which parasite and host simultaneously enter dormancy, emerging months later to propagate both species.     

Comparative study of the life cycles of Alexandrium tamutum and Alexandrium minutum (Gonyaulacales,Dinophyceae) in culture1

The microalgal genus Alexandrium includes species known to produce paralytic shellfish poisoning (PSP). Due to the importance of discriminating between HAB‐forming species, we compared the undescribed life‐cycle pattern of Alexandrium tamutum Montresor, Beran et U. John and of its toxic relative Alexandrium minutum Halim. Sexual stages, asexual and sexual division, mating type, and nuclear morphology were studied in both species. Sexual cysts are known to be morphologically identical. However, the relative size of the U‐shaped nucleus may be used to differentiate between the cysts of these species since DNA packaging in the resting cysts was lower in A. tamutum than in A. minutum, species in which the planozygote nucleus was reduced to half its volume prior to encystment. The dormancy period of the cysts was <20 d for A. tamutum, but longer than 1 month for A. minutum. In both species, cyst appearance needed to be explained by the existence of more than two sexual types (+/–), which indicates a complex heterothallic mating type. However, planozygotes of both species may divide instead of encysting. This characteristic was used for nutritional and heritage studies. Isolated planozygotes of both species encysted in larger percentages in medium deficient in both nitrates and phosphates (L/15) than in medium without phosphates added (L‐P), a medium in which most planozygotes neither divide nor encyst. Parental strains of A. minutum with and without the ventral pore formed planozygotes and, later, offspring with the ventral pore, although apparently smaller than usual. A synchronization–flow cytometry method for discriminating diploids formed by sexual fusion (planozygotes) from cells with 2C DNA content resulting from self‐duplication of DNA (dividing cells) was described. The results indicated that the maximum percentage of A. minutum planozygotes (20%) was achieved only 3 to 5 d after crossing the parental strains, and that light might not be needed for the sexual fusion and formation of planozygotes.     

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.     

CYST FORMATION IN THE FRESHWATER DINOFLAGELLATE CERATIUM HIRUNDINELLA (DINOPHYCEAE)1

Cyst formation in Ceratium hirundinella (O. F. Müll.) Bergh was studied by light and electron microscopy, using material from several lakes and reservoirs and also laboratory cultures. Cells preparing to encyst build up large quantities of starch and lipid and at the same time reduce their other cell components. The cyst is released from the theca as a naked cell bounded by a double membrane. The most commonly found cyst deposits a layer of electron-dense granules containing silicon on the outer membrane and lays down a cellulose-like material between the two membranes. Cysts without the electron-dense granules are commonly formed in cultures but rarely found in lakes. These cysts appear less resistant to decay and do not show the reorganization of cell contents for dormancy. It is suggested that C. hirundinella has both a resting cyst, forming part of the life cycle, and a temporary cyst stage.