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.

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.

Energy metabolism and genetic information processing mark major transitions in the life history of Scrippsiella acuminata (Dinophyceae).

Many dinoflagellates perform sexual reproduction and form cysts as a life history strategy to survive adverse environmental conditions and seed annual harmful algal blooms (HABs). The molecular mechanisms underpinning the life stage transitions can provide clues about how key environmental factors induce encystment and initiation of a HAB but are still poorly understood. Here, we conducted an integrated physiological and transcriptomic study to unravel the mechanisms in Scrippsiella acuminata. We established a culture from a bloom, induced cyst formation, and divided the process into four life stages. Transcriptomic analysis of these stages revealed 19,900 differentially expressed genes (DEGs). The expression of genes related to photosynthesis was significantly up-regulated from vegetative stage to immature cyst stage, consistent with the marked increase in cell contents of energy-storing macromolecules (carbohydrates and lipids). When proceeding to resting cysts, most photosynthesis genes were down-regulated while "genetic information processing" related genes were up-regulated. Comparing germinating cysts with resting cysts revealed 100 DEGs involved in energy metabolism, indicating a high energy requirement of germination. In addition, the transition from germinating cysts to vegetative cells featured up-regulation of photosynthesis. Our results demonstrate that energy storage and consumption play a pivotal role in cyst formation and germination respectively and genetic information processing is crucial in cyst dormancy.

The responses of Scrippsiella acuminata to the stresses of darkness: antioxidant activities and formation of pellicle cysts.

In order to understand the strategy of Scrippsiella acuminata to cold dark environment, the antioxidant responses and the formation of pellicle cysts of S. acuminata to darkness at 8°C and 20°C were investigated. Cell densities decreased significantly after 96 h dark treatment, and no live cells were observed after 9-days dark treatments. The darkness stress generally resulted in an increase of antioxidant defenses, including soluble protein, superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA). Cellular soluble protein and SOD activity increased sharply under 20°C darkness, which protected algal cells against the oxidative stress from darkness, and resulted in relatively lower MDA levels. Soluble protein and SOD activity were enhanced under 8°C darkness as well however not in a sharp rise, and higher levels of MDA and GSH were recorded. The results suggested high SOD and protein levels protected cells against harsh darkness stress, while high GSH not only helped algae cells resist dark stress, but also played an important role in low temperature stress. Darkness promoted the formation of pellicle cysts of S. acuminata, and the maximum formation rates were 16.06% to 21.74% at 8°C and 20°C, respectively. Germination of pellicle cysts occurred within 24 h after light exposure, however pellicle cysts could not withstand long-time darkness stress, and all pellicle cysts died after 9-days darkness exposure. The results of this study suggest that S. acuminata is able to overcome temporary cold darkness through forming pellicle cysts.

Expression Patterns of the Heat Shock Protein 90 (Hsp90) Gene Suggest Its Possible Involvement in Maintaining the Dormancy of Dinoflagellate Resting Cysts.

Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone functioning in cellular structural folding and conformational integrity maintenance and thus plays vital roles in a variety of biological processes. However, many aspects of these functions and processes remain to be fully elucidated, particularly for non-model organisms. Dinoflagellates are a group of eukaryotes that are exceedingly important in primary production and are responsible for the most harmful algal blooms (HABs) in aquatic ecosystems. The success of dinoflagellates in dominating the plankton community is undoubtedly pertinent to their remarkable adaptive strategies, characteristic of resting cyst production and broad tolerance to stresses of temperature and others. Therefore, this study was conducted to examine the putative roles of Hsp90 in the acclimation to temperature stress and life stage alterations of dinoflagellates. Firstly, we isolated the full-length cDNA of an Hsp90 gene (StHsp90) via RACE from the cosmopolitan HAB species Scrippsiella trochoidea and tracked its transcriptions in response to varied scenarios via real-time qPCR. The results indicated that StHsp90 displayed significant mRNA augment patterns, escalating during 180-min treatments, when the cells were exposed to elevated and lowered temperatures. Secondly, we observed prominently elevated StHsp90 transcriptions in the cysts that were stored at the cold and dark conditions compared to those in newly formed resting cysts and vegetative cells. Finally, and perhaps most importantly, we identified 29 entries of Hsp90-encoding genes with complete coding regions from a dinoflagellate-specific environmental cDNA library generated from marine sediment assemblages. The observed active transcription of these genes in sediment-buried resting cysts was fully supported by the qPCR results for the cold-stored resting cysts of S. trochoidea. Hsp90s expressions in both laboratory-raised and field-collected cysts collectively highlighted the possible involvement and engagement of Hsp90 chaperones in the resting stage persistence of dinoflagellates.

Transcriptome and metabolome analyses of cold and darkness-induced pellicle cysts of Scrippsiella trochoidea.

BACKGROUND: Dinoflagellates are a group of unicellular organisms that are a major component of aquatic eukaryotes and important contributors to marine primary production. Nevertheless, many dinoflagellates are considered harmful algal bloom (HAB) species due to their detrimental environmental and human health impacts. Cyst formation is widely perceived as an adaptive strategy of cyst-forming dinoflagellates in response to adverse environmental conditions. Dinoflagellate cysts play critical roles in bloom dynamics. However, our insight into the underlying molecular basis of encystment is still limited. To investigate the molecular processes regulating encystment in dinoflagellates, transcriptome and metabolome investigations were performed on cold and darkness-induced pellicle cysts of Scrippsiella trochoidea. RESULTS: No significant transcriptional response was observed at 2 h; however, massive transcriptome and metabolome reprogramming occurred at 5 h and in pellicle cysts. The gene-to-metabolite network demonstrated that the initial transformation from vegetative cells into pellicle cysts was highly energy demanding through the activation of catabolism, including glycolysis, ß-oxidation, TCA cycle and oxidative phosphorylation, to cope with cold-darkness-induced stress. However, after transformation into pellicle cysts, the metabolism was greatly reduced, and various sugars, polyunsaturated fatty acids and amino acids accumulated to prolong survival. The identification of 56 differentially expressed genes (DEGs) related to signal transduction indicated that S. trochoidea received a cold-darkness signal that activated multiple signal transduction pathways, leading to encystment. The elevated expression of genes encoding enzymes involved in ROS stress suggested that pellicle cysts respond to increased oxidative stress. Several cell cycle-related genes were repressed. Intriguingly, 11 DEGs associated with sexual reproduction suggested that pellicle cysts (or some portion thereof) may be a product of sexual reproduction. CONCLUSIONS: This study provides the first transcriptome and metabolome analyses conducted during the encystment of S. trochoidea, an event that requires complex regulatory mechanisms and impacts on population dynamics. The results reveal comprehensive molecular regulatory processes underlying life cycle regulation in dinoflagellates involving signal transduction, gene expression and metabolite profile, which will improve our ability to understand and monitor dinoflagellate blooms.

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.

Interspecies competition between Scrippsiella acuminata and three marine diatoms: Growth inhibition and allelopathic effects.

Interactions between Scrippsiella acuminata and three diatoms, Chaetoceros curvisetus, Phaeodactylum tricornutum, and Skeletonema dohrnii, were investigated using bi-algal co-cultures and cell-free and sonicated-cell filtrates in this study. Volatile aldehydes in sonicated filtrates of the three diatoms were analyzed by GC/MS. Furthermore, effects of 2E, 4E-decadienal (2,4-D) on the growth and the photosynthetic efficiency of the four microalgal species were studied. The growth of Sc. acuminata was significantly inhibited by the three diatoms in all co-cultures, and the inhibitory effects were higher under nutrient-rich conditions. Both cell-free and sonicated-cell filtrates of the three diatoms showed significant inhibitions on the growth of Sc. acuminata, which highlighted that diatoms produce allelopathic compounds not only to the surrounding environments but also inside the cells. Fifteen aldehydes were detected in the sonicated-cell filtrates of the three diatoms, and 5, 5, and 12 types of aldehydes were detected in C. curvisetus, P. tricornutum, and Sk. dohrnii, respectively. Polyunsaturated aldehydes (PUAs) composition differed among the three diatom species. Phenylglyoxal (C8H6O2) dominated in C. curvisetus, 2,4-D (C10H16O) predominated in P. tricornutum, and high proportions of 2-hexenal (C6H10O), 2E, 4E-heptadienal (C7H10O), and 2,4-D were detected in Sk. dohrnii. 2,4-D showed significantly inhibitory effects on the growth of algal cells including diatoms themselves in a dose-dependent manner, and photosynthetic efficiency was significantly decreased as well. Sc. acuminata was the most sensitive species. The 96 h EC50 values of 2,4-D on the growth of the four microalgae were 1.64 µmol/L for Sc. acuminata, 3.09 µmol/L for C. curvisetus, 4.93 µmol/L for P. tricornutum, and 8.54 µmol/L for Sk. dohrnii, respectively. The results suggest that PUAs produced by diatoms may help them to take the competitive advantages in phytoplankton community, and thus to sustain diatom-dominated community structure in nutrient rich coastal waters.

Differences in Specific Mass Density Between Dinoflagellate Life Stages and Relevance to Accumulation by Hydrodynamic Processes.

One previously unstudied aspect of differences between sexual and asexual life stages in large-scale transport and accumulation is density (mass per unit volume) of cells in each life stage. The specific density was determined for Scrippsiella lachrymosa cells in medium with and without nitrogen (N) enrichment through density-gradient centrifugation. Growth medium without N addition is often called "encystment medium" when used for the purpose of resting cyst formation in cyst-forming dinoflagellates; mating gametes are usually seen after 2-3 days. Significant differences in specific density were found after 2 days in encystment medium simultaneously with the observation of typical gamete swimming behavior and mating. The specific density of cells in encystment medium was 1.06 g · cm-3 ; whereas, the specific density of cells in growth medium was 1.11 g · cm-3 . Cells in encystment medium were found to have significantly increased lipid content, reduced chlorophyll content, and reduced internal complexity. The findings may explain differential transport of less dense and chemotactically aggregating gametes into surface blooms in contrast to denser vegetative cells that perform daily vertical migration and do not aggregate. Passive accumulation of non-migrating gametes into layers in stagnant water also can be explained, as well as sinking of zygotes when the storage of highly dense starch increases. Resting cysts had a density of over 1.14 g · cm-3 and would sink to become part of the silt fraction of the sediment. We suggest that differences in behavior and buoyancy between sexual and asexual life stages cause differences in cell accumulation, and therefore large-scale, environmental transport could be directly dependent upon life-cycle transitions.

Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga Scrippsiella trochoidea (Dinophyceae).

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.

The effect of protocatechuic acid on the phycosphere in harmful algal bloom species Scrippsiella trochoidea.

The effects of allelopathy and the potential harm of several isolated allelochemicals have been studied in detail. Microorganisms in the phycosphere play an important role in algal growth, decay and nutrient cycling. However, it is unknown and often neglected whether allelochemicals affect the phycosphere. The present study selected a phenolic acid protocatechuic acid (PA) - previously shown to be an allelochemical. We studied PA at a half maximal effective concentration of 0.20 mM (30 mg L-1) against Scrippsiella trochoidea to assess the effect of PA on its phycosphere in an acute time period (48 h). The results showed that: 1) OTUs (operational taxonomic units) in the treatment groups (31.4 ± 0.55) exceeded those of the control groups (28.2 ± 1.30) and the Shannon and Simpson indices were lower than the control groups (3.31 ± 0.08 and 0.84 ± 0.02, 3.45 ± 0.09 and 0.88 ± 0.01); 2) Gammaproteobacteria predominated in the treatment groups (44.71 ± 2.13 %) while Alphaproteobacteria dominated in the controls (67.17 ± 3.87 %); 3) Gammaproteobacteria and Alphaproteobacteria were important biomarkers in the treatment and control groups respectively (LDA > 4.0). PA improved the relative abundance of Alteromonas significantly and decreased the one of Rhodobacteraceae. PICRUSt analysis showed that the decrease of Rhodobacterceae was closely related with the decline of most functional genes in metabolism such as amino acid, carbohydrate, xenobiotics, cofactors and vitamins metabolism after PA-treated.

Revival of Ancient Marine Dinoflagellates Using Molecular Biostimulation.

The biological processes involved in the preservation, viability, and revival of long-term dormant dinoflagellate cysts buried in sediments remain unknown. Based on studies of plant seed physiology, we tested whether the revival of ancient cysts preserved in century-old sediments from the Bay of Brest (France) could be stimulated by melatonin and gibberellic acid, two molecules commonly used in seed priming. Dinoflagellates were revived from sediments dated to approximately 150 years ago (156 ± 27, 32 cm depth), extending the known record age of cyst viability previously established as around one century. A culture suspension of sediments mixed with melatonin and gibberellic acid solutions as biostimulants exhibited germination of 11 dinoflagellate taxa that could not be revived under controlled culture conditions. The biostimulants revived some dinoflagellates from century-old sediments, including the potentially toxic species Alexandrium minutum. The biostimulants showed positive effects on germination on even more ancient cysts, showing dose-dependent effects on the germination of Scrippsiella acuminata. Concentrations of 1, 10, and 100 µM melatonin and gibberellic acid promoted germination. In contrast, 1,000 µM solutions, particularly for melatonin, drastically decreased germination, suggesting a potential noxious effect of high doses of these molecules on dinoflagellate revival. Our findings suggest that melatonin and gibberellic acid are involved in the stimulation of germination of dinoflagellate cysts. These biostimulants can be used to germinate long-term stored dinoflagellate cysts, which may promote studies of ancient strains in the resurrection ecology research field.

Effects of increasing temperature and acidification on the growth and competitive success of Alexandrium catenella from the Gulf of Maine.

Climate driven increases in ocean temperature and pCO2 have the potential to alter the growth and prevalence of future Harmful Algal Blooms (HABs), but systematic studies on how climate drivers influence toxic algal species relative to non-toxic phytoplankton are lacking. In particular, little is known about how future climate scenarios will affect the growth of the toxic dinoflagellate Alexandrium catenella, which is responsible for the paralytic shellfish poisoning (PSP) events that threaten the health and economy of coastal communities in the Gulf of Maine and elsewhere. The growth responses of A. catenella and two other naturally co-occurring dinoflagellates in the Gulf of Maine-Scrippsiella sp., and Amphidinium carterae-were studied in mono and mixed species cultures. Experimental treatments tested the effects of elevated temperature (20 °C), lower pH (7.8), and the combination of elevated temperature and lower pH on growth rates relative to those in near-current conditions (15 °C; pH 8.1). Growth rates of A. catenella decreased under elevated temperature and lower pH conditions, a response that was largely attributable to the effect of temperature. In contrast, growth rates of Scrippsiella sp. and A. carterae increased under elevated temperature and lower pH conditions, with temperature also being the primary driver of the response. These trends did not change substantially when these species were grown in mixed cultures (A. catenella + Scrippsiella sp., and A. catenella + A. carterae), indicating that allelopathic or competitive interactions did not affect the experimental outcome under the conditions tested. These findings suggest that A. catenella blooms may become less prevalent in the southern regions of the Gulf of Maine, but potentially more prevalent in the northeastern regions of the Gulf of Maine with continued climate change.

Scrippsiella acuminata versus Scrippsiella ramonii: A Physiological Comparison.

Scrippsiella is a cosmopolitan dinoflagellate genus that is able to form Harmful Algal Blooms in coastal waters. The large physiological, morphological, and genetic variability that characterizes this genus suggest the existence of cryptic species. In this study, flow cytometric analyses were carried out to compare the cell cycle and life cycle of two Scrippsiella strains from two different species: Scrippsiella ramonii (VGO1053) and Scrippsiella acuminata (S3V). Both species were also investigated by internally transcribed spacer rDNA sequencing and high-performance liquid chromatography-based pigment analyses. The reddish-brown color of S. acuminata and yellowish-green hue of S. ramonii were consistent with the quantitative differences determined in their pigment profiles. Our results indicate that the cell cycle is light-controlled and that it differs in the two species. S-phase was detected during the light period in both, whereas the G2/M phase occurred during the light period in S. ramonii but under dark conditions in S. acuminata. The detection of 4C stages, mobile zygotes (planozygotes), and resting cysts in S. ramonii (nonclonal) provided convincing evidence of sexuality in this species. Sexual related processes were not found in the clonal S. acuminata strain, suggesting its heterothallic behavior (i.e., the need for outcrossing). The differences in the genome size of these species were examined as well. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Metabolomic Fingerprints of Individual Algal Cells Using the Single-Probe Mass Spectrometry Technique.

Traditional approaches for the assessment of physiological responses of microbes in the environment rely on bulk filtration techniques that obscure differences among populations as well as among individual cells. Here, were report on the development on a novel micro-scale sampling device, referred to as the "Single-probe," which allows direct extraction of metabolites from living, individual phytoplankton cells for mass spectrometry (MS) analysis. The Single-probe is composed of dual-bore quartz tubing which is pulled using a laser pipette puller and fused to a silica capillary and a nano-ESI. For this study, we applied Single-probe MS technology to the marine dinoflagellate Scrippsiella trochoidea, assaying cells grown under different illumination levels and under nitrogen (N) limiting conditions as a proof of concept for the technology. In both experiments, significant differences in the cellular metabolome of individual cells could readily be identified, though the vast majority of detected metabolites could not be assigned to KEGG pathways. Using the same approach, significant changes in cellular lipid complements were observed, with individual lipids being both up- and down-regulated under light vs. dark conditions. Conversely, lipid content increased across the board under N limitation, consistent with an adjustment of Redfield stoichiometry to reflect higher C:N and C:P ratios. Overall, these data suggest that the Single-probe MS technique has the potential to allow for near in situ metabolomic analysis of individual phytoplankton cells, opening the door to targeted analyses that minimize cell manipulation and sampling artifacts, while preserving metabolic variability at the cellular level.

Comparative proteomic studies of a Scrippsiella acuminata bloom with its laboratory-grown culture using a 15N-metabolic labeling approach.

Comparative proteomic analysis was carried out using cells isolated from a natural bloom of Scrippsiella acuminata (formerly Scrippsiella trochoidea) in the early bloom (EB) and late bloom (LB) stages as well as with laboratory-grown cultures of cells isolated from the bloom in early growth (EG) and late growth (LG) stages. For quantitative proteomics, LG cells were grown for 20 generations in the presence of 15N as a reference (i.e. common denominator) for all comparison. In comparisons with early growth laboratory grown cells (EG/LG), nearly 64% of proteins identified had similar abundance levels, with the remaining 36% mostly more abundant in EG cells. Calvin cycle, amino acid metabolism, chlorophyll biosynthesis and transcription/translation were among the up-regulated processes. Cells from the early bloom (EB/LG) had a greater abundance of transporters and enzymes related to light harvesting and oxidative phosphorylation, while the abundance of these proteins decreased in late bloom cells (LB/LG). All natural bloom samples showed either constant or lower abundance levels of enzymes involved in sugar synthesis and glycolytic pathways compared to laboratory grown cells. Our results represent the first examination of the proteomic changes in the development of a natural dinoflagellate bloom. Importantly, our results demonstrate that the proteome of cells grown in the laboratory is distinctively different from cells in a natural bloom.

Transcriptomic Analyses of Scrippsiella trochoidea Reveals Processes Regulating Encystment and Dormancy in the Life Cycle of a Dinoflagellate, with a Particular Attention to the Role of Abscisic Acid.

Due to the vital importance of resting cysts in the biology and ecology of many dinoflagellates, a transcriptomic investigation on Scrippsiella trochoidea was conducted with the aim to reveal the molecular processes and relevant functional genes regulating encystment and dormancy in dinoflagellates. We identified via RNA-seq 3,874 (out of 166,575) differentially expressed genes (DEGs) between resting cysts and vegetative cells; a pause of photosynthesis (confirmed via direct measurement of photosynthetic efficiency); an active catabolism including ß-oxidation, glycolysis, glyoxylate pathway, and TCA in resting cysts (tested via measurements of respiration rate); 12 DEGs encoding meiotic recombination proteins and members of MEI2-like family potentially involved in sexual reproduction and encystment; elevated expressions in genes encoding enzymes responding to pathogens (chitin deacetylase) and ROS stress in cysts; and 134 unigenes specifically expressed in cysts. We paid particular attention to genes pertaining to phytohormone signaling and identified 4 key genes regulating abscisic acid (ABA) biosynthesis and catabolism, with further characterization based on their full-length cDNA obtained via RACE-PCR. The qPCR results demonstrated elevated biosynthesis and repressed catabolism of ABA during the courses of encystment and cyst dormancy, which was significantly enhanced by lower temperature (4 ± 1°C) and darkness. Direct measurements of ABA using UHPLC-MS/MS and ELISA in vegetative cells and cysts both fully supported qPCR results. These results collectively suggest a vital role of ABA in regulating encystment and maintenance of dormancy, akin to its function in seed dormancy of higher plants. Our results provided a critical advancement in understanding molecular processes in resting cysts of dinoflagellates.

Transcriptome Analysis of Scrippsiella trochoidea CCMP 3099 Reveals Physiological Changes Related to Nitrate Depletion.

Dinoflagellates are a major component of marine phytoplankton and many species are recognized for their ability to produce harmful algal blooms (HABs). Scrippsiella trochoidea is a non-toxic, marine dinoflagellate that can be found in both cold and tropic waters where it is known to produce "red tide" events. Little is known about the genomic makeup of S. trochoidea and a transcriptome study was conducted to shed light on the biochemical and physiological adaptations related to nutrient depletion. Cultures were grown under N and P limiting conditions and transcriptomes were generated via RNAseq technology. De novo assembly reconstructed 107,415 putative transcripts of which only 41% could be annotated. No significant transcriptomic response was observed in response to initial P depletion, however, a strong transcriptional response to N depletion was detected. Among the down-regulated pathways were those for glutamine/glutamate metabolism as well as urea and nitrate/nitrite transporters. Transcripts for ammonia transporters displayed both up- and down-regulation, perhaps related to a shift to higher affinity transporters. Genes for the utilization of DON compounds were up-regulated. These included transcripts for amino acids transporters, polyamine oxidase, and extracellular proteinase and peptidases. N depletion also triggered down regulation of transcripts related to the production of Photosystems I & II and related proteins. These data are consistent with a metabolic strategy that conserves N while maximizing sustained metabolism by emphasizing the relative contribution of organic N sources. Surprisingly, the transcriptome also contained transcripts potentially related to secondary metabolite production, including a homolog to the Short Isoform Saxitoxin gene (sxtA) from Alexandrium fundyense, which was significantly up-regulated under N-depletion. A total of 113 unique hits to Sxt genes, covering 17 of the 34 genes found in C. raciborskii were detected, indicating that S. trochoidea has previously unrecognized potential for the production of secondary metabolites with potential toxicity.

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.

Fine-structural characterization and phylogeny of Peridinium polonicum, type species of the recently described genus Naiadinium (Dinophyceae).

Peridinium polonicum is a freshwater peridinioid with an unusual tabulation that includes one or two anterior intercalary plates in the mid-dorsal axis, and in such a low position that it seems inset in precingular Plate 4. Although the species has been classified in both Peridinium and Peridiniopsis, evidence from nucleotide sequences consistently shows that its closest relatives are within the Scrippsiella group. The genus Naiadinium Carty has been recently described with P. polonicum as its type species. However, Naiadinium was separated from other peridinioids only on the basis of shape and plate arrangements and these characters do not allow reliable determination of its closest phylogenetic relatives. Serial section fine-structural analysis revealed the presence of a small peduncle supported by a conspicuous microtubular basket that extended far into the cell; a complex pusular system that included a collecting chamber from which about 70 pusular tubes radiated; a flagellar apparatus with general peridinioid characters but with an unusually large distance of nearly 700 nm between basal bodies. An ITS1-5.8S-ITS2 rDNA-based phylogenetic analysis grouped, with high statistical support, Naiadinium polonicum with three species currently placed in Scrippsiella, viz. S. irregularis, S. precaria and S. ramonii.