Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements.

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent. In this study, using the cosmopolitan HABs-causing species Scrippsiella acuminata as a representative, we measured the transcriptional activity of the most efficient pathway of the energy catabolism tricarboxylic acid (TCA) cycle, cell viability (via neutral red staining), and the cellular ATP content of resting cysts under a set of mock conditions in marine sediments (e.g., 4 °C, darkness, and anoxia) for a maximum period of one year. Based on the correlation analyses among the expression levels of genes, cyst viability, and ATP content, we revealed that the TCA cycle was still a crucial pathway of energetic catabolism for resting cysts under aerobic conditions, and its expression was elevated at higher temperatures, light irradiation, and the early stage of dormancy. Under anaerobic conditions, however, the TCA cycle pathway ceased expression in resting cysts, as also supported by ATP measurements. Our results have laid a cornerstone for the comprehensive revelation of the energetic metabolism and biochemical processes of dormancy of resting cysts in marine sediments.

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the ß subunit of ATP synthase gene (ß-F1-ATPase), a key indicator for the status of cell's energetic metabolism. The full-length cDNA of ß-F1-ATPase gene from S.trochoidea (Stß-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stß-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stß-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stß-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

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.

Cloning and Partial Characterization of a Cold Shock Domain-Containing Protein Gene from the Dinoflagellate Scrippsiella trochoidea.

CSPs, cold shock domain (CSD) containing proteins, are demonstrated to be involved in low temperature responses and various cellular processes under normal growth conditions. Here, we used the cosmopolitan, toxic, and resting cyst-producing dinoflagellate Scrippsiella trochoidea as a representative harmful algal bloom-forming dinoflagellate to investigate the expression patterns of CSP in vegetative cells in response to temperature shocks and in resting cysts, with an objective to probe the possible function of CSP in dinoflagellates. The full-length cDNA of a CSP gene from S. trochoidea (StCSP) was obtained which has a solely N-terminal CSD with conserved nucleic acids binding motifs. The qPCR results together indicated StCSP expression was not modulated by temperature at the transcriptional level and implied this gene may not be associated with temperature stress responses in S. trochoidea as the gene's name implies. However, we observed significantly higher StCSP transcripts in resting cysts (newly formed and maintained in dormancy for different periods of time) than that observed in vegetative cells (at exponential and stationary stages), indicating StCSP is actively expressed during dormancy of S. trochoidea. Taking together our recent transcriptomic work on S. trochoidea into consideration, we postulate that StCSP may play roles during encystment and cyst dormancy of the species.

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.

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.

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 ß-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.

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.

First report of fossilized cysts produced by the benthic Bysmatrum subsalsum (Dinophyceae) from a shallow Mexican lagoon in the Gulf of Mexico.

Cysts belonging to the benthic dinoflagellate Bysmatrum subsalsum were recovered from palynologically treated sediments collected in the Alvarado Lagoon (southwestern Gulf of Mexico). The cysts are proximate, reflecting the features of the parent thecal stage, and their autofluorescence implies a dinosporin composition similar to the cyst walls of phototrophic species. This finding is important for our understanding of B. subsalsum life cycle transitions and ecology. Encystment may play an important role in the bloom dynamics of this species as it can enable the formation of a sediment cyst bank that allows reinoculation of the water column when conditions become favorable. This is the first report of a fossilized cyst produced by a benthic dinoflagellate recovered from sub-recent sediments.

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.

Spatiotemporal changes in the genetic diversity of harmful algal blooms caused by the toxic dinoflagellate Alexandrium minutum.

Organisms with sexual and asexual reproductive systems benefit from both types of reproduction. Sexual recombination generates new combinations of alleles, whereas clonality favours the spread of the fittest genotype through the entire population. Therefore, the rate of sexual vs. clonal reproduction has a major influence on the demography and genetic structure of natural populations. We addressed the effect of reproductive system on populations of the dinoflagellate Alexandrium minutum. More specifically, we monitored the spatiotemporal genetic diversity during and between bloom events in two estuaries separated by 150 km for two consecutive years. An analysis of population genetic patterns using microsatellite markers revealed surprisingly high genotypic and genetic diversity. Moreover, there was significant spatial and temporal genetic differentiation during and between bloom events. Our results demonstrate that (i) interannual genetic differentiation can be very high, (ii) estuaries are partially isolated during bloom events and (iii) genetic diversity can change rapidly during a bloom event. This rapid genetic change may reflect selective effects that are nevertheless not strong enough to reduce allelic diversity. Thus, sexual reproduction and/or migration may regularly erase any genetic structure produced within estuaries during a bloom event.

Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Evidence for the production of asexual resting cysts in a free-living species of Symbiodiniaceae (Dinophyceae).

Coral reef ecosystems are the most productive and biodiverse marine ecosystems, with their productivity levels highly dependent on the symbiotic dinoflagellates belonging to the family Symbiodiniaceae. As a unique life history strategy, resting cyst production is of great significance in the ecology of many dinoflagellate species, those HABs-causing species in particular, however, there has been no confirmative evidence for the resting cyst production in any species of the family Symbiodiniaceae. Based on morphological and life history observations of cultures in the laboratory and morpho-molecular detections of cysts from the marine sediments via fluorescence in situ hybridization (FISH), cyst photography, and subsequent singe-cyst PCR sequencing, here we provide evidences for the asexual production of resting cysts by Effrenium voratum, the free-living, red tide-forming, and the type species of the genus Effrenium in Symbiodiniaceae. The evidences from the marine sediments were obtained through a sequential detections: Firstly, E. voratum amplicon sequence variants (ASVs) were detected in the cyst assemblages that were concentrated with the sodium polytungstate (SPT) method from the sediments collected from different regions of China Seas by high-throughput next generation sequencing (NGS); Secondly, the presence of E. voratum in the sediments was detected by PCR using the species-specific primers for the DNA directly extracted from sediment; Thirdly, E. voratum cysts were confirmed by a combined approach of FISH using the species-specific probes, light microscopic (LM) photography of the FISH-positive cysts, and a subsequent single-cyst PCR sequencing for the FISH-positive and photographed cysts. The evidences from the laboratory-reared clonal cultures of E. voratum include that: 1) numerous cysts formed in the two clonal cultures and exhibited a spherical shape, a smooth surface, absence of ornaments, and a large red accumulation body; 2) cysts could maintain morphologically intact for a storage of two weeks to six months at 4 °C in darkness and of which 76-92 % successfully germinated through an internal development processes within a time period of 3-21 days after being transferred back to the normal culturing conditions; 3) two or four germlings were released from each cyst through the cryptopylic archeopyle in all cysts with continuous observations of germination processes; and 4) while neither sexual mating of gametes nor planozygote (cells with two longitudinal flagella) were observed, the haploidy of cysts was proven with flow cytometric measurements and direct LM measurements of fluorescence from cells stained with either propidium iodide (PI) or DAPI, which together suggest that the cysts were formed asexually. All evidences led to a conclusion that E. voratum is capable of producing asexual resting cysts, although its sexuality cannot be completely excluded, which guarantees a more intensive investigation. This work fills a gap in the knowledge about the life cycle, particularly the potential of resting cyst formation, of the species in Symbiodiniaceae, a group of dinoflagellates having unique life forms and vital significance in the ecology of coral reefs, and may provide novel insights into understanding the recovery mechanisms of coral reefs destructed by the global climate change and suggest various forms of resting cysts in the cyst assemblages of dinoflagellates observed in the field sediments, including HABs-causing species.

Drought Resistant Resting Cysts of Paraphysoderma sedebokerense Preserves the Species Viability and Its Virulence.

The blastocladialean fungus P. sedebokerense is a facultative parasite of economically important microalgae and for this reason it has gained a lot of interest. P. sedebokerense has a complex life cycle which includes vegetative and resting stages. The resting cysts were assumed to play an essential role in survival by resisting drought, but this ability was never tested and the factors that trigger their formation were not evaluated. This study was aimed to induce resting cyst formation and germination in P. sedebokerense. At first, we tested the survival of P. sedebokerense liquid cultures and found that infectivity is retained for less than two months when the cultures were stored on the bench at room temperature. We noticed that dry cultures retained the infectivity for a longer time. We, thus, developed a method, which is based on dehydration and rehydration of the biomass, to produce, maintain, and germinate resting cysts of P. sedebokerense in both saprophytic and parasitic modes of growth. When the dry cultures were rehydrated and incubated at 30 °C, resting cysts asynchronously germinated after 5 h and the "endosporangium" was protruding outside of the cyst. Our method can be used to preserve P. sedebokerense for research purposes with the advantage of no need for expensive equipment.

Description of a new oxytrichid ciliate, Oxytricha buxai n. sp. and redescription of O. quadricirrata Blatterer and Foissner, 1988 based on morphology and 18S rDNA analyses.

The morphology of a new oxytrichid ciliate, Oxytricha buxai n. sp., isolated from a soil sample collected from the Buxa Tiger Reserve, West Bengal, India, was studied based on live observation and protargol impregnation. The new species is characterised by a body size of 85×35 µm in vivo, two macronuclear nodules with one or two micronuclei attached at variable positions, a few colourless cortical granules scattered throughout cortex, adoral zone of membranelles about 35% of body length with 26 membranelles on average, about 18 cirri in left and 16 cirri in right marginal row, right marginal row starts at the level of buccal vertex, usually 18 frontoventral transverse cirri, five dorsal kineties including one dorsomarginal row, three caudal cirri. Further, a redescription based on live and protargol-impregnated specimens of Oxytricha quadricirrata Blatterer and Foissner, 1988, isolated from a moss sample collected from the Kangra district, Himachal Pradesh, India, is provided. The Indian population of O. quadricirrata is similar in morphology to the type population. However, the dorsal side shows some variation, i.e., the presence of a second dorsomarginal row with one or two bristles and incomplete fragmentation of dorsal kinety 3 (vs single dorsomarginal row and complete fragmentation). The resting cyst is spherical and about 20 µm across, with a wrinkled surface. Morphogenesis is in typical Oxytricha pattern. Based on 18S rDNA, phylogenetic analyses show Oxytricha to be a polyphyletic genus. Further, O. quadricirrata clusters away from O. granulifera, thereby supporting the validity of the former.

How Ciliated Protists Survive by Cysts: Some Key Points During Encystment and Excystment.

Forming cysts is a common and important bionomic strategy for microorganisms to persist in harsh environments. In ciliated protists, many species have been reported to form cysts when facing unfavorable conditions. Despite traditional studies on the morphological features of cysts and the chemical composition of cyst wall, recent research has focused more on the molecular mechanisms of encystment. The present work reviews studies on developmental features and molecular information of resting cysts in ciliates, and pays more attention to the following questions: what are the inducing factors of encystment and excystment? How does the cell change morphologically during these dynamic processes? And what molecular mechanisms underlie those changes? We also present and summarize the characteristics of cysts from diverse ciliate lineages in a phylogenetic framework, aiming to provide new perspectives for studies on adaptive evolution of unicellular eukaryotes.

Taxonomy and molecular phylogeny of two poorly known ciliate genera, Balantidion and Acropisthium (Protista: Ciliophora: Litostomatea), including a new species of Balantidion.

The class Litostomatea Small & Lynn, 1981 is a morphologically diverse ciliate group including hundreds of free-living and endocommensal species. The genera Acropisthium Perty, 1852 and Balantidion Eberhard, 1862 previously consisted of one free-living freshwater species each. Here, we not only highlight additional morphological features of the two type species, but also investigate a new species, Balantidion foissneri sp. nov., isolated from a river flowing through Lake Weishan, China, based on complementary methods, i.e., living morphology, stained preparations, and 18S rRNA gene sequence data. Balantidion foissneri sp. nov. can be distinguished from the type species, B. pellucidum Eberhard, 1862, by the body size (115-170 × 50-80 µm vs. 70-100 × 25-45 µm in B. pellucidum), oral bulge (distinct vs. indistinct), extrusome shape (filiform vs. rod-shaped), and the number of somatic kineties (46-60 vs. 25-40). In Balantidion species, pre-encystment trophonts show similarly-shaped polymorphic cytoplasmic lepidosomes destined to adorn the outer surface of the resting cyst. Based on the current knowledge, assignment of Balantidion to the family Acropisthiidae Foissner & Foissner, 1988 is proposed. In addition, phylogenetic analyses based on molecular data show that the two Balantidion species form a fully-supported clade to which Acropisthium mutabile has a sister relationship.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation.

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 µM; P∼ 0.0362 µM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.

Fine-structural characterization and phylogeny of Sphaerodinium (Suessiales, Dinophyceae), with the description of an unusual type of freshwater dinoflagellate cyst.

Two strains of Sphaerodinium were established from two mountain areas in Portugal and examined by light microscopy, scanning and transmission electron microscopy, and sequence analyses of nuclear-encoded SSU, ITS1-5.8S-ITS2 and LSU rDNA. Both strains were identified as S. polonicum var. tatricum on the basis of comparison with the original taxonomic descriptions within the genus. The two strains were nearly identical in morphology and ultrastructure, except for the presence of pseudograna-like thylakoid stacks within more rounded chloroplast lobes in one of them. Sexual reproduction occurred in culture batches and resting cysts with single or grouped processes with wide bases and distal platforms with slightly recurved margins were seen to develop by sudden retraction of planozygote cytoplasm. Morphological, fine-structural and molecular characters were compared with previously available information from S. cracoviense, allowing for a more robust characterization of the genus. Important characters include a type F eyespot, a pusule canal linking the transverse flagellar canal to a collecting chamber connected to regular pusular tubes, a ventral fibre extending from the proximal-right side of the longitudinal basal body, and a membranous, lamellar body with a honeycomb pattern near the flagellar base area. The latter two features are shared with Baldinia anauniensis.

Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus.

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca2+ to overpopulated vegetative cells. However, an increase in Ca2+ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca2+ concentrations is essential, but Ca2+ inflow does not necessarily occur during encystment. Ca2+ image analysis showed that Ca2+ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca2+ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca2+ from vesicles.