Generalist Life Cycle Aids Persistence of Alexandrium ostenfeldii (Dinophyceae) in Seasonal Coastal Habitats of the Baltic Sea.
ABSTRACT: 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.
Project description:Environmental conditions regulate the germination of phytoplankton resting stages. While some factors lead to synchronous germination, others stimulate germination of only a small fraction of the resting stages. This suggests that habitat filters may act on the germination level and thus affect selection of blooming strains. Benthic "seed banks" of the toxic dinoflagellate Alexandrium ostenfeldii from the Baltic Sea are genetically and phenotypically diverse, indicating a high potential for adaptation by selection on standing genetic variation. Here, we experimentally tested the role of climate-related salinity and temperature as selection filters during germination and subsequent establishment of A. ostenfeldii strains. A representative resting cyst population was isolated from sediment samples, and germination and reciprocal transplantation experiments were carried out, including four treatments: Average present day germination conditions and three potential future conditions: high temperature, low salinity, and high temperature in combination with low salinity. We found that the final germination success of A. ostenfeldii resting cysts was unaffected by temperature and salinity in the range tested. A high germination success of more than 80% in all treatments indicates that strains are not selected by temperature and salinity during germination, but selection becomes more important shortly after germination, in the vegetative stage of the life cycle. Moreover, strains were not adapted to germination conditions. Instead, highly plastic responses occurred after transplantation and significantly higher growth rates were observed at higher temperature. High variability of strain-specific responses has probably masked the overall effect of the treatments, highlighting the importance of testing the effect of environmental factors on many strains. It is likely that A. ostenfeldii populations can persist in the future, because suitable strains, which are able to germinate and grow well at potential future climate conditions, are part of the highly diverse cyst population. OPEN RESEARCH BADGES:This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.5061/dryad.c8c83nr.
Project description: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 ?-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.
Project description:The high evolutionary potential of phytoplankton species allows them to rapidly adapt to global warming. Adaptations may occur in temperature-dependent traits, such as growth rate, cell size and life cycle processes. Using resurrection experiments with resting stages from living sediment archives, it is possible to investigate whether adaptation occurred. For this study, we revived resting cysts of the spring bloom dinoflagellate Apocalathium malmogiense from recent and 100-year-old sediment layers from the Gulf of Finland, and compared temperature-dependent traits of recent and historic strains along a temperature gradient. We detected no changes in growth rates and cell sizes but a significant difference between recent and historic strains regarding resting cyst formation. The encystment rate of recent strains was significantly lower compared with historic strains which we interpret as an indication of adaptation to higher and more rapidly increasing spring temperatures. Low encystment rates may allow for bloom formation even if the threshold temperature inducing a loss of actively growing cells through resting cyst formation is exceeded. Our findings reveal that phenotypic responses of phytoplankton to changing temperature conditions may include hidden traits such as life cycle processes and their regulation mechanisms. This study emphasizes the potential of living sediment archives to investigate plankton responses and adaptation to global warming.
Project description:Ciliated protists are a large group of single-cell eukaryotes, leading to the resting cysts in unfavorable environmental condition. However, the underlying molecular mechanism of encystment in the free-living ciliates is poorly understood. Here we show that the resting cysts are better than the vegetative cells of Euplotes encysticus in adverse survivor with respect to energy metabolism. Therefore scale identification of encystment-related proteins in Euplotes encysticus was investigated by iTRAQ analysis. We analyzed a total of 130 proteins, in which 19 proteins involving 12 upregulated and 7 downregulated proteins were associated with encystment in the resting cysts in comparison with the vegetative cells. Moreover, direct fluorescent labeling analysis showed that the vegetative cells treated with shRNA-?-tubulin recombinant E. coli accumulated a large number of granular materials, and dramatic cell morphology changes. Importantly, the cell membrane rupture phenomenon was observed after three weeks of shRNA-?-tubulin interference as compared to the control group. These results revealed that different proteins might play an important role in the process of the vegetative cells into the resting cysts. These results will help to reveal the morphological changes and molecular mechanism of resting cyst formation of ciliates.
Project description:In recent years, blooms of toxic Alexandrium ostenfeldii strains have been reported from around the world. In 2013, the species formed a red tide in a shallow lagoon in western Japan, which was the first report of the species in the area. To investigate the genetic relatedness of Japanese A. ostenfeldii and global isolates, the full-length SSU, ITS and LSU sequences were determined, and phylogenetic analyses were conducted for isolates from western and northern Japan and from the Baltic Sea. Genotyping and microsatellite sequence comparison were performed to estimate the divergence and connectivity between the populations from western Japan and the Baltic Sea. In all phylogenetic analyses, the isolates from western Japan grouped together with global isolates from shallow and low saline areas, such as the Baltic Sea, estuaries on the east coast of U.S.A. and from the Bohai Sea, China. In contrast, the isolates from northern Japan formed a well-supported separate group in the ITS and LSU phylogenies, indicating differentiation between the Japanese populations. This was further supported by the notable differentiation between the sequences of western and northern Japanese isolates, whereas the lowest differentiation was found between the western Japanese and Chinese isolates. Microsatellite genotyping revealed low genetic diversity in the western Japanese population, possibly explained by a recent introduction to the lagoon from where it was detected. The red tide recorded in the shallow lagoon followed notable changes in the salinity of the waterbody and phytoplankton composition, potentially facilitating the bloom of A. ostenfeldii.
Project description:The transformation of a ciliate into cyst is an advance strategy against an adverse situation. However, the molecular mechanism for the encystation of free-living ciliates is poorly understood. A large-scale identification of the encystment-related proteins and genes in ciliate would provide us with deeper insights into the molecular mechanisms for the encystations of ciliate. We identified the encystment-related proteins and genes in Pseudourostyla cristata with shotgun LC-MS/MS and scale qRT-PCR, respectively, in this report. A total of 668 proteins were detected in the resting cysts, 102 of these proteins were high credible proteins, whereas 88 high credible proteins of the 724 total proteins were found in the vegetative cells. Compared with the vegetative cell, 6 specific proteins were found in the resting cyst. However, the majority of high credible proteins in the resting cyst and the vegetative cell were co-expressed. We compared 47 genes of the co-expressed proteins with known functions in both the cyst and the vegetative cell using scale qRT-PCR. Twenty-seven of 47 genes were differentially expressed in the cyst compared with the vegetative cell. In our identifications, many uncharacterized proteins were also found. These results will help reveal the molecular mechanism for the formation of cyst in ciliates.
Project description:In order to identify and reveal the proteins related to encystment of the ciliate Euplotes encysticus, we analyzed variation in the abundance of the proteins isolated from the resting cyst comparing with proteins in the vegetative cell. 2-D electrophoresis, MALDI-TOF MS techniques and Bioinformatics were used for proteome separation, quantification and identification. The comparative proteomics studies revealed 26 proteins with changes on the expression in the resting cysts, including 12 specific proteins and 14 differential proteins. 12 specific proteins and 10 out of the 14 differential proteins were selected and identified by MALDI-TOF MS. The identified specific proteins with known functions included type II cytoskeletal 1, keratin, Nop16 domain containing protein, protein arginine n-methyltransferase, epsilon-trimethyllysine hydroxylase and calpain-like protein. The identified differential proteins with known functions included Lysozyme C, keratinocyte growth factor, lysozyme homolog AT-2, formate acetyltransferase, alpha S1 casein and cold-shock protein. We discussed the functions of these proteins as well as their contribution in the process of encystment. These identified proteins covered a wide range of molecular functions, including gene regulation, RNA regulation, proteins degradation and oxidation resistance, stress response, material transport and cytoskeleton organization. Therefore, differential expression of these proteins was essential for cell morphological and physiological changes during encystment. This suggested that the peculiar proteins and differential proteins might play important roles in the process of the vegetative cells transforming into the resting cysts. These observations may be novel findings that bring new insights into the detailed mechanisms of dormancy.
Project description: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 <i>Hsp20</i> (<i>StHsp20</i>) and a <i>Hsp40</i> (<i>StHsp40</i>) 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, <i>Scrippsiella trochoidea</i>. 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 <i>StHsp20</i> and <i>StHsp40</i> 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 <i>StHsp20</i> was probably related to the heat tolerance of the species, and <i>StHsp40</i> was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of <i>StHsp40</i> was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied <i>StHsp40</i> might be engaged in resting cyst formation of <i>S. trochoidea.</i> 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.
Project description:During encystment of Azotobacter vinelandii, a family of alkylresorcinols (ARs) and alkylpyrones (APs) are synthesized. In the mature cyst, these lipids replace the membrane phospholipids and are also components of the layers covering the cyst. In this study, A. vinelandii strains unable to synthesize ARs were isolated after mini-Tn5 mutagenesis. Cloning and nucleotide sequencing of the affected loci revealed the presence of the transposons within the arsA gene of the previously reported arsABCD gene cluster, which encodes a type I fatty acid synthase. A mutant strain (SW-A) carrying an arsA mutation allowing transcription of arsBCD was constructed and shown to be unable to produce ARs, indicating that the ArsA protein is essential for the synthesis of these phenolic lipids. Transcription of arsA was induced 200-fold in cells undergoing encystment, but only 14-fold in aged cultures of A. vinelandii, in accordance with AR synthesis and cyst formation percentages under the two conditions. Although it was previously reported that the inactivation of arsB abolishes AR synthesis and results in a failure in encystment, the arsA mutants were able to form cysts resistant to desiccation. These data indicate that ARs play a structural role in the exine layer of the cysts, but they are not essential for either cyst formation or for desiccation resistance.
Project description:The expression of spore-specific marker transcripts at different stages of the asexual life cycle of Saprolegnia parasitica was analyzed. One of the markers, designated puf1, was found to be expressed transiently upon each of several cycles of zoospore encystment and reemergence. The transcript is induced immediately upon zoospore encystment and is rapidly lost when a cyst is triggered to germinate. In nongerminating cysts, puf1 is maintained until a time point when the cysts can no longer be triggered to germinate and thus have become determined for zoospore reemergence. The results show that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct and that the transcriptional machinery of oomycetes is also active in nongerminating spores. puf1 encodes a putative mRNA binding protein belonging to a conserved class of proteins including the Drosophila melanogaster Pumilio protein, Caenorhabditis elegans FBF, and Saccharomyces cerevisiae Puf5, all of which are involved in regulation of gene expression by post-transcriptional mechanisms.