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![Hypothetical pathways for reactive oxygen species (ROS) detoxification. Pathways evidenced in both ciliates (white background) or only in Strombidium rassoulzadegani (yellow background) are shown. In red, ROS (O 2-= superoxide radical; H 2 O 2 = hydrogen peroxide); in dark blue, antioxidant enzymes (SOD = superoxide dismutase; Cat = catalase; APX = ascorbate peroxidase; GPX = glutathione peroxidase; TPX = thioredoxin peroxidase); in light blue, enzymes involved in metabolism of non-enzymatic antioxidants (Nec3 = bifunctional monodehydroascorbate reductase/carbonic anhydrase nectarin-3-like; TRed = thioredoxin reductase; GRed = glutathione reductase; GSyn = GSH synthetase; GCLig = Glutamate-cysteine ligase); in green, non-enzymatic antioxidants (AsA = ascorbic acid; GSH = glutathione; Trx = thioredoxin). MDAs = monodehydroascorbate; GSSG = glutathione disulfide; TrxS 2 = thioredoxin disulfide; NAD(P) + = nicotinamide adenine dinucleotide (phosphate). Based on literature [61,63,75]. doi:10.1371/journal.pone.0101418.g004](profile/Luciana-Santoferrara/publication/263585806/figure/fig8/AS:669587737698314@1536653565634/Hypothetical-pathways-for-reactive-oxygen-species-ROS-detoxification-Pathways.jpg)
Hypothetical pathways for reactive oxygen species (ROS) detoxification. Pathways evidenced in both ciliates (white background) or only in Strombidium rassoulzadegani (yellow background) are shown. In red, ROS (O 2-= superoxide radical; H 2 O 2 = hydrogen peroxide); in dark blue, antioxidant enzymes (SOD = superoxide dismutase; Cat = catalase; APX = ascorbate peroxidase; GPX = glutathione peroxidase; TPX = thioredoxin peroxidase); in light blue, enzymes involved in metabolism of non-enzymatic antioxidants (Nec3 = bifunctional monodehydroascorbate reductase/carbonic anhydrase nectarin-3-like; TRed = thioredoxin reductase; GRed = glutathione reductase; GSyn = GSH synthetase; GCLig = Glutamate-cysteine ligase); in green, non-enzymatic antioxidants (AsA = ascorbic acid; GSH = glutathione; Trx = thioredoxin). MDAs = monodehydroascorbate; GSSG = glutathione disulfide; TrxS 2 = thioredoxin disulfide; NAD(P) + = nicotinamide adenine dinucleotide (phosphate). Based on literature [61,63,75]. doi:10.1371/journal.pone.0101418.g004
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Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich St...
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... under normal physiological conditions and as a response to oxidative stress, autotrophic and heterotrophic cells have enzymatic and non-enzymatic mechanisms to control ROS concentrations [25,26,28]. We found evidence for these pathways in the transcriptomes of Strombidium rassoulzadegani and Strombidi- nopsis sp. (Fig. 4, Tables S3 and S4). In this case, transcripts were clearly linked to known antioxidant enzymes and most of them had highly significant BLASTP hits against ciliates or other non- photosynthetic organisms, thus minimizing the possibility that these genes belong to the food algae (Tables S3 and S4). Although we show simplified antioxidant ...
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... non-enzymatic antioxidants, GSH is a tri-peptide that can be synthetized and recycled in both ciliates, according to the transcriptome data (Fig. 4, Table S4). Similarly, transcripts for the protein Trx and its recycling enzyme were found in both species. In contrast, transcripts related to the synthesis of AsA or other antioxidants found in plants and algae, such as carotenoids and tocopherols [65], were not detected in the ...
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... we found evidence for a group of enzymes that can recycle AsA (nectarin-3-like enzymes, Nec3) in S. rassoulzade- gani but not in Strombidinopsis sp. (Fig. 4, Table S4). Nec3 has monodehydroascorbate reductase (MDAR) activity, i.e. it trans- forms the oxidized form of AsA back to its reduced form, thus providing the advantage of keeping constant levels of AsA without the necessity of a constant supply [66][67][68][69][70]. Although AsA can also be recycled spontaneously or through a cycle that ...
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... (Fig. 3). In autotrophs, enzymes such as SOD, APX and GPX scavenge ROS also in the chloroplast and they are key in order to keep this organelle active [29,63,72]. However, these enzymes are nuclear encoded [60,73] and, even if they initially exist in kleptochlor- oplasts of S. rassoulzadegani, their activity is very likely lost after some time (Fig. 4). Inactivation of chloroplast antioxidant enzymes is known to limit photosynthetic efficiency [74]. Thus, oxidative damage may contribute to the lack of kleptochloroplast function- ality and the fact that a continuous supply of fresh chloroplasts is needed for the growth of S. rassoulzadegani ...
Citations
... In the Mediterranean Sea, most of the studies on ciliate metabarcoding analysis focused mainly on the geographical differentiations of the benthic community (Santoferrara et al., 2014;Ganser et al., 2021) and the characterization of parasitic ciliate species (Scheifler et al., 2019). ...
... Despite the geographical distance between the two stations being small, we could assess differences in the ciliate community, as the dynamics were affected according to the depth at the coastal station, and according to season in the offshore one. Since coastal stations are easily accessible, and the maximum depth is recorded at around 200 m, a good body of literature exists on aloricate ciliates in coastal environments based on morphospecies (Cariou et al., 1999;Agatha, 2011;Ganser et al., 2021), while metabarcoding data are mostly available from benthic ciliate species or only on tintinnids (Santoferrara et al., 2014;Massana et al., 2015;Tucker et al., 2017). Little is known about ciliate dynamics in offshore environments, especially in the Mediterranean Sea, where the maximum depth can reach down to 1500 m, like in this study. ...
Ciliates have been recognized as one of the major components of the microbial food web, especially in ultra-oligotrophic waters, such as the Eastern Mediterranean Sea, where nutrients are scarce and the microbial community is dominated by pico- and nano-sized organisms. For this reason, ciliates play an important role in these ecosystems since they are the main planktonic grazers. Regardless the importance of these organisms, little is known about the community structure of heterotrophic and mixotrophic ciliates and how they are associated to their potential prey. In this study, we used 18S V4 rRNA gene metabarcoding to analyze ciliate community dynamics and how the relationship with potential prey changes according to different seasons and depths. Samples were collected seasonally at two stations of the Eastern Mediterranean Sea (HCB: coastal, M3A: offshore) from the surface and deep chlorophyll maximum (DCM) layers. The ciliate community structure varied across depths in HCB and across seasons in M3A, and the network analysis showed that in both stations, mixotrophic oligotrichs were positively associated with diatoms and showed few negative associations with ASVs annotated as marine Stramenopiles (MAST). On the other hand, heterotrophic tintinnids showed negative relationships in both HCB and M3A stations, mostly with Ochrophyta and Chlorophyta. These results showed, in first place that, although the two stations are close to each other, the ciliate dynamics differed between them. Moreover, mixotrophic and heterotrophic ciliates may have different ecological niches since mixotrophic ciliates may be more selective compared to heterotrophic species regarding their prey. These findings are the first glimpse into an understanding of the dynamics between heterotrophic and mixotrophic ciliates and their role in microbial assemblages and dynamics of ultra-oligotrophic environments.
... An interesting comparative study was published by Santoferrara et al. (2014) on transcriptomes from marine planktonic mixotrophic and heterotrophic ciliates (i.e., an oligotrich Strombidium rassoulzadegani and choreotrich Strombidinopsis sp., respectively). Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. ...
The application of molecular phylogenetics to research on protists has substantially transformed our understanding of their evolution and systematics. More recently, advances in molecular technology, including high throughput sequencing, has opened new avenues for genomic analyses that elucidate major aspects of protistan biology across all levels of biological organization from cellular to ecosystems. This is a review of recent advances (particularly in the last two decades) of transcriptomic research on heterotrophic and autotrophic protists within three major topics: (i) Physiology and metabolism, (ii) Development and life cycles, and (iii) Environmental and ecological studies. Emphasis is placed on selection of representative research that highlights findings across diverse taxonomic groups within each of the three topics. Examples are drawn from parasitic as well as free-living taxa to provide a broad overview of some of the research strategies, and major findings, that have emerged from application of transcriptomics and related techniques in advancing our understanding of protistan biology.
... The mixed prey sequences were removed from the G. jinhaense AP17 transcriptome bioinformatically by creating a mixed prey transcriptome BLASTN reference database and using QIIME's filter script to remove sequences that matched with an E-value of 10 − 6 or less with the G. jinhaense AP17 transcriptome (Altschul, 1990;Caporaso et al., 2010;Santoferrara et al., 2014). An E-value of 10 − 6 was chosen because this is a common significance threshold and was used to generate both ciliate and Oxyrrhis marina prey-free transcriptomes (Santoferrara et al., 2014: Rubin et al., 2019. ...
... The mixed prey sequences were removed from the G. jinhaense AP17 transcriptome bioinformatically by creating a mixed prey transcriptome BLASTN reference database and using QIIME's filter script to remove sequences that matched with an E-value of 10 − 6 or less with the G. jinhaense AP17 transcriptome (Altschul, 1990;Caporaso et al., 2010;Santoferrara et al., 2014). An E-value of 10 − 6 was chosen because this is a common significance threshold and was used to generate both ciliate and Oxyrrhis marina prey-free transcriptomes (Santoferrara et al., 2014: Rubin et al., 2019. The resulting "G. ...
... The process of bioinformatically removing prey was carried out in a similar manner to previously published transcriptomes for heterotrophic alveolates (Hehenberger et al., 2019;Rubin et al., 2019;Santoferrara et al., 2014). In the process, approximately 26.9% of the contigs were removed as prey contamination from the original assembled transcriptome, which is only slightly higher than in the previous published transcriptomes with ranges from 7%− 24% (Rubin et al., 2019;Santoferrara et al., 2014). ...
First found in Korean coastal water, the dinoflagellate Gyrodinium jinhaense is a recently established species with unclear global distribution and unexplored genomic characteristics. From a laboratory fish mortality event off Long Island Sound, USA, we isolated a dinoflagellate, and by microscopic and molecular (18S rRNA gene; >99% identical) analyses found that it resembles G. jinhaense, hence named G. jinhaense strain AP17. Towards developing a genetic database for this dinoflagellate, a transcriptome of this species was sequenced using RNA-seq, producing 6 Gbp of data that was assembled into over 70,000 unigenes. The assembled transcriptome GC content was approximately 56% and the total Benchmarking Universal Single-Copy Orthologs for Eukaryota and Alveolata databases were approximately 50% and 57%, respectfully. Genes involved in grazing, energy generation, genome architecture, and protein synthesis, processing, and degradation were highly represented in the transcriptome. Moreover, fragments of polyketide synthase and saxitoxin genes were found but saxitoxins were not detected in high performance liquid chromatography measurements. With the first reported transcriptome for the Gyrodinium genus, this study will serve as a baseline for future Gyrodinium genomics and toxicological studies.
... The function of the sequestered chloroplasts in these ciliates is thus currently thought to depend on their innate robustness and ability to survive inside the ciliate host. Based on studies on the kleptoplastidic Strombidium rassoulzadegani, kleptoplastidic ciliates in the genus Strombidium are thought to depend on more frequent reacquisition of prey plastids compared to M. rubrum because they do not express genes related to plastid maintenance and replication (Santoferrara et al., 2014;Mcmanus et al., 2018). ...
Many marine ciliate species retain functional chloroplasts from their photosynthetic prey. In some species the functionality of the acquired plastids is connected to the simultaneous retention of prey nuclei. To date, this has never been documented in plastidic Strombidium species. The functionality of the sequestered chloroplasts in Strombidium species is thought to be independent from any nuclear control and only maintained via frequent replacement of chloroplasts from newly ingested prey. Chloroplasts sequestered from the cryptophyte prey Teleaulax amphioxeia have been shown to keep their functionality for several days in the ciliate Strombidium cf. basimorphum. To investigate the potential retention of prey genetic material in this ciliate we applied molecular marker specific for this cryptophyte prey. Here, we demonstrate that genetic material from prey nuclei, nucleomorphs and ribosomes is detectable inside the ciliate for at least five days after prey ingestion. Moreover, single-cell transcriptomic revealed the presence of transcripts of prey nuclear origin in the ciliate after four days of prey starvation. These new findings might lead to the reconsideration of the mechanisms regulating chloroplasts retention in Strombidium ciliates. The development and application of molecular tools appear promising to improve our understanding on chloroplasts retention in planktonic protists.
... Unlike the red Mesodinium spp., the mixotrophic oligotrichs have been shown to retain and utilize chloroplasts from many different species of algal prey (Johnson and Beaudoin, 2019;Maselli et al., 2020), and thus, are generally considered GNCMs. Mixotrophic oligotrichs are believed to depend on frequent reacquisition of prey plastids, as they do not appear to express genes related to plastid maintenance and replication (Santoferrara et al., 2014;Mcmanus et al., 2018). These plastidic oligotrichs make up, on average, 30% of ciliate biomass (Dolan and Pérez, 2000;Stoecker et al., 2009), and during spring-and summer-time peaks, they can even comprise >90% of ciliate biomass (Bernard and Rassoulzadegan, 1994;Haraguchi et al., 2018). ...
Many species of the ciliate genus Strombidium can acquire functional chloroplasts from a wide range of algal prey and are thus classified as generalist non-constitutive mixotrophs. Little, however, is known about the influence of irradiance and prey availability on their ability to exploit the photosynthetic potential of the chloroplasts, and how this may explain their spatial and temporal distribution in nature. In this study, inorganic carbon uptake, growth, and ingestion rates were measured for S. cf. basimorphum under three different irradiances (10, 40, and 120 μmol photons m–2 s–1) when acclimated to three different prey densities (5 × 103, 1 × 104, and 4 × 104 cells mL–1), as well as when allowed to deplete the prey. After prey depletion, cultures survived without prey longest (∼6 days) at the medium irradiance treatment (40 μmol photons m–2 s–1), while ciliate density, inorganic carbon uptake rates, and cellular chl-a content declined fastest at the highest irradiance treatment. This indicates that the ciliates may be unable to maintain the chloroplasts functionally without replacement at high irradiances. Ingestion rates were not shown to be significantly influenced by irradiance. The maximum gross growth efficiency (GGE) in this study (1.1) was measured in cultures exposed to the medium test irradiance and lowest prey density treatment (5 × 103 cells mL–1). The relative contribution of inorganic carbon uptake to the ciliate carbon budget was also highest in this treatment (42%). A secondary GGE peak (0.99) occurred when cultures were exposed to the highest test irradiance and the medium prey density. These and other results suggest that S. cf. basimorphum, and other generalist non-constitutive mixotrophs, can flexibly exploit many different environmental conditions across the globe.
... The elevated actin and tubulin proteins in the mesopelagic zone could be driven by higher phagotrophic activity since cytoskeletal components are upregulated in mixotrophic and heterotrophic protists under grazing conditions 44,55-58 . However, the cytoskeletal machinery is not upregulated in all grazing mixotrophs 47,59 and the trait may therefore be taxon-specific and depend on feeding mechanism. A reprolysin-like zinc-binding metallopeptidase was the only peptidase detected to be elevated in mesopelagic waters (Fig. 2) and may be an important contributor to protein degradation. ...
Marine microeukaryotes play a fundamental role in biogeochemical cycling through the transfer of energy to higher trophic levels and vertical carbon transport. Despite their global importance, microeukaryote physiology, nutrient metabolism and contributions to carbon cycling across offshore ecosystems are poorly characterized. Here, we observed the prevalence of dinoflagellates along a 4,600-km meridional transect extending across the central Pacific Ocean, where oligotrophic gyres meet equatorial upwelling waters rich in macronutrients yet low in dissolved iron. A combined multi-omics and geochemical analysis provided a window into dinoflagellate metabolism across the transect, indicating a continuous taxonomic dinoflagellate community that shifted its functional transcriptome and proteome as it extended from the euphotic to the mesopelagic zone. In euphotic waters, multi-omics data suggested that a combination of trophic modes were utilized, while mesopelagic metabolism was marked by cytoskeletal investments and nutrient recycling. Rearrangement in nutrient metabolism was evident in response to variable nitrogen and iron regimes across the gradient, with no associated change in community assemblage. Total dinoflagellate proteins scaled with particulate carbon export, with both elevated in equatorial waters, suggesting a link between dinoflagellate abundance and total carbon flux. Dinoflagellates employ numerous metabolic strategies that enable broad occupation of central Pacific ecosystems and play a dual role in carbon transformation through both photosynthetic fixation in the euphotic zone and remineralization in the mesopelagic zone.
... Mixotrophic oligotrichs do not seem to retain prey nuclei (Laval-Peuto and Febvre, 1986;Stoecker et al., 1988a), or to express genes related to maintenance of plastids (Santoferrara et al., 2014). Thus, it is possible that the functionality of the sequestered plastids is affected by ageing upon sequestration. ...
Ciliates represent an important trophic link between nanoplankton and mesoplankton. Many species acquire functional chloroplasts from photosynthetic prey, being thus mixotrophs. Little is known about which algae they exploit, and of the relevance of inorganic carbon assimilation to their metabolism. To get insights into these aspects, laboratory cultures of three mixotrophic Strombidium spp. were established and 35 photosynthetic algal species were tested as prey. The relative contributions of ingestion and photosynthesis to total carbon uptake were determined, and responses to prey starvation were studied. Ciliate growth was supported by algal species in the 2-12 μm size range, with cryptophytes and chlorophytes being the best prey types. Inorganic carbon incorporation was only quantitatively important when prey concentration was low (3-100 μgCL −1), when it led to increased gross growth efficiencies. Chla specific inorganic carbon uptake rates were reduced by 60-90% compared to that of the photosynthetic prey. Inorganic carbon uptake alone could not sustain survival of cultures and ciliate populations declined by 25-30% during 5 days of starvation. The results suggest that mixotrophy in Strombidium spp. may substantially bolster the efficiency of trophic transfer when biomass of small primary producers is low.
... For example, membrane production and (auto)digestion processes occur in cells that are not phagocytic. So, to what extent molecular methods can provide help in establishing rate processes and relative contributions of photo-versus osmo-versus phagotrophy in mixoplankton is far from clear at present, though various studies have explored the topic (Santoferrara et al., 2014;McKie-Krisberg et al., 2018). ...
Many protist plankton are mixotrophs, combining phototrophy and phagotrophy. Their role in freshwater and marine ecology has emerged as a major developing feature of plankton research over recent decades. To better aid discussions, we suggest these organisms are termed “mixoplankton”, as “planktonic protist organisms that express, or have potential to express, phototrophy and phagotrophy”. The term “phytoplankton” then describes phototrophic organisms incapable of phagotrophy. “Protozooplankton” describes phagotrophic protists that do not engage in acquired phototrophy. The complexity of the changes to the conceptual base of the plankton trophic web caused by inclusion of mixoplanktonic activities are such that we suggest that the restructured description is termed the “mixoplankton paradigm”. Implications and opportunities for revision of survey and fieldwork, of laboratory experiments and of simulation modelling are considered. The main challenges are not only with taxonomic and functional identifications, and with measuring rates of potentially competing processes within single cells, but with decades of inertia built around the traditional paradigm that assumes a separation of trophic processes between different organisms. In keeping with the synergistic nature of cooperative photo- and phagotrophy in mixoplankton, a comprehensive multidisciplinary approach will be required to tackle the task ahead.
... Genes potentially involved in predation, such as phagotrophy, were not identified likely because the predators were maintained on dissolved organic matter instead of prey cells. Interestingly, a comparison of gene content in a mixotrophic alga and a heterotroph (both ciliates) did not reveal significant differences in gene content in relation to trophic mode (Santoferrara et al., 2014). One study that examined differentially expressed genes and thus potential metabolic differences in feeding status focused on a mixotrophic alga either feeding on prey or photosynthesizing (Liu et al., 2015). ...
Grazing by heterotrophic protists influences plankton population dynamics, community composition, and the flux of carbon through marine planktonic food webs. To gain insight into the molecular underpinnings of grazing in dinoflagellates, a group of important heterotrophic protists, we used a RNA-Seq approach to investigate the transcriptomic response of Oxyrrhis marina under fed and starved conditions with three different phytoplankton prey (Isochrysis galbana and two strains of Heterosigma akashiwo). In response to fed and starved conditions, 1,576 transcripts were significantly differentially expressed in O. marina. Fed O. marina cells upregulated transcripts involved in the synthesis of essential fatty acids and storage carbohydrates suggesting that the predator was food satiated and excess glucose was being stored as an energy reserve. Transcripts encoding voltage-gated ion channels were also upregulated during grazing, and they are known to be involved in the detection of mechanical stimuli and the regulation of swimming behavior in several eukaryotic protists. Fed O. marina cells upregulated kinases, which can dictate cell shape changes and may be associated with phagocytosis. During starvation, upregulated O. marina transcripts included those involved in the degradation of energy-storage molecules like glucan 1,4-alpha-glycosidase and those involved in antioxidant activities and autophagy, like acid ceramidase that are associated with the digestion of polar lipids present in cell membranes. Starved O. marina also upregulated transcripts with high similarity to proton pumping proteorhodopsins suggesting that this heterotrophic protist may supplement its energy requirement during starvation with a light harvesting mechanism. Although herbivorous grazing is a pivotal transformation in the C cycle, logistical constraints limit our investigations of environmental and biological drivers. The molecular signals identified here provide new insights into the metabolic regulation of feeding and starvation in marine heterotrophic protists and can fuel hypothesis-driven research into predators’ metabolic response to prey availability.
... It is common for de novo transcriptomes for marine protists to have low annotation rates (e.g. Kuo et al. 2013;Lauritano et al. 2017;Santoferrara et al. 2014). Additionally, haptophyte transcriptomes will have low annotation rates because haptophytes occupy a phylogenetically unique position on the eukaryotic tree of life and are poorly represented among reference genomes (Read et al. 2013). ...
Phaeocystis globosa forms dense, monospecific blooms in temperate, northern waters. Blooms are usually dominated by the colonial morphotype—non‐flagellated cells embedded in a secreted mucilaginous mass. Colonial Phaeocystis blooms significantly affect food‐web structure and function and negatively impact fisheries and aquaculture, but factors regulating colony formation remain enigmatic. Destructive P. globosa blooms have been reported in tropical and subtropical regions more recently and warm‐water blooms could become more common with continued climate change and coastal eutrophication. We therefore assessed genetic pathways associated with colony formation by investigating differential gene expression between colonial and solitary cells of a warm‐water Phaeocystis globosa strain. Our results illustrate a transcriptional shift in colonial cells with most of the differentially expressed genes downregulated, supporting a reallocation of resources associated with forming and maintaining colonies. Dimethylsulfide and acrylate production and pathogen interaction pathways were upregulated in colonial cells, suggesting a defensive role for producing colonies. We identify several protein kinase signaling pathways that may influence the transition between morphotypes, providing targets for future research into factors affecting colony formation. This study provides novel insights into genetic mechanisms involved in Phaeocystis colony formation and provides new evidence supporting a defensive role for Phaeocystis colonies.
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