Green Autofluorescence in Dinoflagellates, Diatoms, and Other Microalgae and Its Implications for Vital Staining and Morphological Studies

Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.     

Green autofluorescence in dinoflagellates, diatoms, and other microalgae and its implications for vital staining and morphological studies

Green autofluorescence (GAF) has been described in the short flagellum of golden and brown algae, the stigma of Euglenophyceae, and cytoplasm of different life stages of dinoflagellates and is considered by some researchers a valuable taxonomic feature for dinoflagellates. In addition, green fluorescence staining has been widely proposed or adopted to measure cell viability (or physiological state) in areas such as apoptosis of phytoplankton, pollutant stresses on algae, metabolic activity of algae, and testing treatment technologies for ships' ballast water. This paper reports our epifluorescence microscopic observations and quantitative spectrometric measurements of GAF in a broad phylogenetic range of microalgae. Our results demonstrate GAF is a common feature of dinoflagellates, diatoms, green algae, cyanobacteria, and raphidophytes, occurs in the cytoplasm and particularly in eyespots, accumulation bodies, spines, and aerotopes, and is caused by molecules other than chlorophyll. GAF intensity increased with time after cell death or fixation and with excitation by blue or UV light and was affected by pH. GAF of microalgae may be only of limited value in taxonomy. It can be strong enough to interfere with the results of green fluorescence staining, particularly when stained samples are observed microscopically. GAF is useful, however, for microscopic study of algal morphology, especially to visualize cellular components such as eyespots, nucleus, aerotopes, spines, and chloroplasts. Furthermore, GAF can be used to visualize and enumerate dinoflagellate cysts in marine and estuarine sediments in the context of anticipating and monitoring harmful algal blooms and in tracking potentially harmful dinoflagellates transported in ships' ballast tanks.     

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.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

SEXUALITY AND CYST FORMATION IN PACIFIC STRAINS OF THE TOXIC D1NOFLAGELLATE GONYAULAX TAMARENSIS1,2

Sexuality has been established for a culture of Gonyaulax tarnarensis Lebour (strain NEPCC–71). The addition of a thick inoculum to a nitrogen–deprived medium results in the occurrence of anisogamous sexual fusion within the first three days in the new culture. Planozygotes, large “lumpy” cells recognizable by their four flagella, may persist up to 2 wk before forming a smooth–walled, oval hypnozygote. The latter resembles cysts released asexually by ecdysis but has a slightly thicker wall. Viable cysts resembling hypnozygotes (zygotic cysts), but with reduced photosynthetic pigmentation, have been isolated from natural murine sediments in Hidden Basin, British Columbia, and a culture (strain NEPCC–254) was initiated from excysted individuals. Zygotic cysts of NEPCC–71 remained encysted in the light at 17 C for 8 wk before excysting. The presence of a ventral pen with toxicity in the latter strain indicates that the taxonomy of G. tamarensis-like organisms is still in a stale of flux and the criteria for recognition of G. excavata (Braarud) Balech as a separate species are not satisfactory as presently formulated.     

CHARACTERIZATION OF OCEANIC PHOTOSYNTHETIC PICOEUKARYOTES BY FLOW CYTOMETRY1

To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right-angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell she was assessed by Coulter counter, and pigment composition was measured by reverse-phase high-performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from the fluorescence of DNA-specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not too complex. Using the relationships that we established between size and light-scattering properties of the cells, we estimated equivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 μm. Chlorophyll a content was between 6 and 16 fg·cel^?1 as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.     

Role of nitrogen fixation in the autecology of Polaromonas naphthalenivorans in contaminated sediments

Polaromonas naphthalenivorans strain CJ2 is a Gram‐negative betaproteobacterium that was identified, using stable isotope probing in 2003, as a dominant in situ degrader of naphthalene in coal tar‐contaminated sediments. The sequenced genome of strain CJ2 revealed several genes conferring nitrogen fixation within a 65.6 kb region of strain CJ2's chromosome that is absent in the genome of its closest sequenced relative Polaromonas sp. strain JS666. Laboratory growth and nitrogenase assays verified that these genes are functional, providing an alternative source of nitrogen in N‐free media when using naphthalene or pyruvate as carbon sources. Knowing this, we investigated if nitrogen‐fixation activity could be detected in microcosms containing sediments from the field site where strain CJ2 was isolated. Inducing nitrogen limitation with the addition of glucose or naphthalene stimulated nitrogenase activity in amended sediments, as detected using the acetylene reduction assay. With the use of fluorescence microscopy, we screened the microcosm sediments for the presence of active strain CJ2 cells using a dual‐labelling approach. When we examined the carbon‐amended microcosm sediments stained with both a strain CJ2‐specific fluorescent in situ hybridization probe and a polyclonal fluorescently tagged antibody, we were able to detect dual‐labelled active cells. In contrast, in sediments that received no carbon addition (showing no nitrogenase activity), no dual‐labelled cells were detected. Furthermore, the naphthalene amendment enhanced the proportion of active strain CJ2 cells in the sediment relative to a glucose amendment. Field experiments performed in sediments where strain CJ2 was isolated showed nitrogenase activity in response to dosing with naphthalene. Dual‐label fluorescence staining of these sediments showed a fivefold increase in active strain CJ2 in the sediments dosed with naphthalene over those dosed with deionized water. These experiments show that nitrogen fixation may play an important role in naphthalene biodegradation by strain CJ2 and contribute to its ecological success.     

POTENTIAL IMPORTANCE OF BENTHIC CYSTS OF GONYAULAX TAMARENSIS AND G. EXCAVATA IN INITIATING TOXIC DINOFLAGELLATE BLOOMS1,2,3

Thick-walled, nonmotile cysts (termed hypnocysts) of two dinoflagellates were isolated from estuarine sediments in Cape Cod, Massachusetts, and germinated to produce their respective motile, thecate stages. Hypnocysts from Orleans district were identified as Gonyaulax excuvata (Braarud) Balech sensu Loeblich & Loeblich. Visually identical hypnocysts from Falmouth district were provisionally identified as Gonyaulax tamarensis Lebour. Both species were toxic. A geographic survey in September detected hypnocysts in only the sediments of locations where toxic blooms developed the preceding and following Spring. Laboratory incubation (16 C) of hypnocysts from sediment samples stored in the dark (5 C) for 6 mo initiated excystment by the temperature increase, with no appreciable effect from light regime, nutrient, or chelator concentrations. Motility of excysted germlings was optimum in highly chelated medium and in the presence of light. We conclude that hypnocysts of both tasa are important in seeding recurrent annual blooms, synchronizing early bloom development with vernal warning of seawater and increasing the geographic range of the species. We suggest that many red tides in New England and eastern Canadian waters are initiated through the displacement of motile estuarine populations into nearshore area by tidal advection and surface runoff, although the potential existence and importance of offshore cyst reservoirs cannot be discounted. Evidence is presented that hypnocysts are probable sexual zygotes whereas the thin-walled cysts readily formed in laboratory cultures (pellicle cyst) are asexual. Pellicle cysts are of limited durability, do not overwinter in nature, and therefore do not play a significant role in initiating toxic blooms.     

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.     

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

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

Occurrence and phylogenetic significance of cytokinesis-related callose in green algae, bryophytes, ferns and seed plants

 In order to investigate the occurrence of callose in dividing cells, we cultivated a selection of 30 organisms (the prokaryotic cyanobacterium Anabaena and eukaryotic green algae, bryophytes, ferns and seed plants) under defined conditions in the laboratory. Samples from these photoautotrophs, which are members of the evolutionary 'green lineage' leading from freshwater algae to land plants, were analysed by fluorescence microscopy. The β-1,3-glucan callose was identified by its staining properties with aniline blue and sirofluor. With the exception of the prokaryotic cyanobacterium, all of the eukaryotic organisms studied were capable of producing wound-induced callose. No callose was detected during cytokinesis of dividing cells of unicellular green algae (and Anabaena). However, in all of the multicellular green algae and land plants (embryophytes) investigated, callose was identified in newly made septae by an intense yellow fluorescence. The formation of wound callose was never detected in cells with callose in the newly formed septae. Additional experiments verified that no fixation-induced artefacts occurred. Our results show that callose is a regular component of developing septae in juvenile cells during cytokinesis in multicellular green algae and embryophytes. The implications of our results with respect to the evolutionary relationships between extant charophytes and land plants are discussed. Received: 15 September 2000 / Revision received: 23 October 2000 / Accepted: 23 October 2000     

A NEW SPECIES OF ENSICULIFERA,E. IMARIENSE (DINOPHYCEAE), PRODUCING ORGANIC-WALLED CYSTS1

We describe a new organic-walled resting cyst from surface sediments of Imari Bay in western Japan. The cysts are spherical, 23–29 pm in diameter, and their surface is covered with spinous to membranous ornaments that are 5–7 μm long and 1.5–2.2 μm wide. The ornaments vary from slender and bifurcate to membranous and multifurcate distal extremities. No archeopyle was observed. The cyst shape is variable in both natural samples and clonal cultures. Vegetative cells are small and ovoid, 17–25 μm long and 14–21 μm wide, and are yellow-brown in color. The epitheca is conical with a conspicuous apical horn, and the hypotheca is hemispherical. The cingular transitional plate has a needle-like spine at its anterior right corner. The plate formula is Po, X, 4″3a, 7″, 5c, 5s 5″and 2″. Although vegetative cells of the present species correspond to Ensiculifera, it is distinct from other species in producing no calcareous cysts. No species of Ensiculifera has been reported to produce cysts composed of only an organic wall. The present species is provisionally placed in the genus Ensiculifera as E. imariense sp. nov.     

蓝藻红色荧光蛋白All1280 GAF2在E.coli BL21(DE3)中的表达及其突变体构建

采用PCR技术从鱼腥藻（Anabaena sp.）PCC 7120中扩增获得红色荧光蛋白基因all1280 gaf2,并利用BamHⅠ和SalⅠ酶切位点,将该基因插入到pET-30a（+）中,构建表达载体pET-all1280 gaf2。将该表达载体与藻胆色素生物合成质粒pACYC-ho1-pcyA同时转化到大肠杆菌E.coli BL21（DE3）,表达后获得大肠杆菌色素细胞。结果显示,该色素细胞在荧光显微镜下具有红色荧光,且在15E/15Z态之间具有可逆光效应。进一步以pET-all1280 gaf2为模板,通过定点突变技术在all1280 gaf2基因中引入C53A突变,获得了突变体All1280 GAF2（C53A）。将All1280 GAF2（C53A）与藻胆色素在E.coli BL21（DE3）中共表达,获得了比野生型红色荧光更强的大肠杆菌色素细胞。研究结果表明,与野生型相比,All1280 GAF2（C53A）具有较高的摩尔消光系数和荧光量子产率,红色荧光更强。     

CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS1

Two new fluorochromes, PicoGreen® and SYTOX Green? stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes)examined. The major advantages of these dyes over others are 1)suitability for direct use in seawater, 2)green fluorescence emission of the DNA-dye complex (wavelength 525 ± 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence > 100-fold compared to the unstained cell, and 4)dyes can be used to quantify double-stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phyto-plankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, preservation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence. Total DNA values measured in 90 algal species (ca. 121 strains) varied by a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinoflagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagic phytoplankton.     

Distribution, dynamics and in situ seeding potential of Scrippsiella hangoei (Dinophyceae) cyst populations from the Baltic Sea

The distribution and seasonal dynamics of cyst populations ofthe spring bloom dinoflagellate Scrippsiella hangoei were studiedin surface sediments on the southwest coast of Finland, BalticSea. In situ germination was assessed by monitoring the fractionof empty cysts and chlorophyll a fluorescence in cyst populationsat different coastal sites throughout the annual cycle. Scrippsiellahangoei resting cysts were widely distributed in the study areaand occurred in exceptionally large numbers (magnitudes of 10⁴–10⁶cysts cm^–3) at all sampling locations between the innermostparts of the coastal archipelago and the open Gulf of Finland.The decreases in cyst number in winter and the increases occurringin late spring reflected the dynamics of germination and encystmentof the species. Chlorophyll fluorescence appeared in mid-winterin ~40% of cysts from well-aerated basins and 6–15% ofcysts from temporarily anoxic sediments. A generally low increasein the proportion of empty cysts indicated that only a partof the potentially germinable cysts actually germinates. Giventhe high cyst concentrations in the sediments, the potentialfor germination is considerable, despite the environmentallyand physiologically determined losses. In contrast, the sizeof the vegetative inoculum is very low, indicating that thesurvival of germlings is problematic under harsh winter conditions.This is an unusual life cycle strategy; however, the early releaseof cells into the water column provides a high probability forsuccessful bloom initiation under the unpredictable meteorologicalconditions in winter and early spring, which often lead to thesudden onset of favourable growth conditions.     

九龙江西陂库区沉积物甲藻孢囊的分布

孢囊在甲藻的生活史中发挥重要作用,福建省九龙江从2009年起,暴发多次拟多甲藻水华事件。采用显微镜观察和单细胞PCR技术,对九龙江西陂库区2012—2013年不同月份沉积物中的甲藻孢囊进行种属判定,并对甲藻孢囊的分布及其影响因素进行分析。结果表明,西陂库区沉积物中的甲藻孢囊主要为拟多甲藻属,约占80%,其次为裸甲藻属,发现了2009年水华的优势种佩式拟多甲藻(Peridiniopsis penardii)孢囊。库区沉积物中甲藻孢囊的丰度在(13.7±1.2)—(105.2±8.3)个/g干重之间。多元相关分析结果显示甲藻孢囊的丰度与含水率呈现显著正相关性(P0.05),反映了甲藻孢囊沿水流方向逐渐积累。本研究结果填补了国内水库甲藻孢囊鉴定和萌发的研究空白,为九龙江甲藻水华的防治提供科学参考。     

Role of temporary cysts in the population dynamics of Alexandrium taylori (Dinophyceae)

Although temporary cyst stages are common in dinoflagellates,their role remains unclear. Every year Alexandrium taylori (Dinophyceae)forms dense patches (10⁶ cells l^-1) along La Fosca beach (Spain,northwest Mediterranean), which last for 2 months (July, August).One of the characteristics of the life history of A. tayloriis the shift from a vegetative motile stage to non-motile temporarycysts. Here we present the temporal changes in the abundanceof temporary cysts in sediments and their in situ encystmentand excystment rates. The in situ encystment rate of temporarycysts from the water column to the sediment ranged from 1.8x 10⁶ to 4.4 x 10⁶ cysts m^-2 day^-1, whereas the excystment ratewas between 0.9 x 10⁶ to 2.7 x 10⁶ cysts m^-2 day^-1 during thebloom period. Some of the temporary cysts in the sediment tookmore than 1 day to produce vegetative cells and remained viablefor at least 4 days. We propose that temporary cyst formationin this species is a tool for reducing population losses. Theproduction of temporary cysts can be an advantage since partof the population is stored in the sediments.     

DEVELOPMENT OF GENETICALLY ENCODED FLUORESCENT PROTEIN CONSTRUCTS OF HYPERTHERMOPHILIC MALTOSE-BINDING PROTEIN

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.     

Encystment of Peridinium gatunense: occurrence, favourable environmental conditions and its role in the dinoflagellate life cycle in a subtropical lake

1. The abundance of cysts of the bloom‐forming dinoflagellate Peridinium gatunense in the sediments of Lake Kinneret and the effects of environmental conditions on encystment were studied in relation to bloom dynamics. Peak cyst formation coincided with the highest growth rate of the population, prior to bloom peak. 2. Peridinium cysts were counted in water and sediment corer samples from 2000 to 2003 and in archived sediment trap samples collected during 1993–94. The cyst data were examined in relation to ambient temperature and nutrient records, and revealed no direct correlation. 3. In laboratory encystment experiments with Peridinium cells collected from the lake, 0.2–3% of the vegetative cells encysted. Temperature, light and cell density had no significant effect on the percentage of encystment. 4. Cysts were always present in the lake sediments but their abundance in ‘non Peridinium’ years was much lower than after a massive bloom. Vegetative cells were always present in the water column after the collapse of the annual dinoflagellate bloom, potentially serving as the inoculum for the next bloom. We propose that the hardy cysts serve as an emergency ‘gene bank’ to initiate population build up following catastrophic die outs.