Gene expression during bacterivorous growth of a widespread marine heterotrophic flagellate

Phagocytosis is a fundamental process in marine ecosystems by which prey organisms are consumed and their biomass incorporated in food webs or remineralized. However, studies searching for the genes underlying this key ecological process in free-living phagocytizing protists are still scarce, in part due to the lack of appropriate ecological models. Our reanalysis of recent molecular datasets revealed that the cultured heterotrophic flagellate Cafeteria burkhardae is widespread in the global oceans, which prompted us to design a transcriptomics study with this species, grown with the cultured flavobacterium Dokdonia sp. We compared the gene expression between exponential and stationary phases, which were complemented with three starvation by dilution phases that appeared as intermediate states. We found distinct expression profiles in each condition and identified 2056 differentially expressed genes between exponential and stationary samples. Upregulated genes at the exponential phase were related to DNA duplication, transcription and translational machinery, protein remodeling, respiration and phagocytosis, whereas upregulated genes in the stationary phase were involved in signal transduction, cell adhesion, and lipid metabolism. We identified a few highly expressed phagocytosis genes, like peptidases and proton pumps, which could be used to target this ecologically relevant process in marine ecosystems

Comparative genomics reveals new functional insights in uncultured MAST species

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles

Return of the marine heterotrophic flagellates: diversity, distribution and gene expression patterns

Memoria de tesis doctoral presentada por Aleix Obiol Plana para obtener el título de Doctor en Ciències del Mar por la Universitat Politècnica de Catalunya (UPC), realizada bajo la dirección del Dr. Ramon Massana i Molera del Institut de Ciències del Mar (ICM-CSIC).-- 204 pages[EN] Marine heterotrophic flagellates (HF) are very small (2-5 μm) unpigmented protists that are dominant bacterial grazers in the ocean, where they link the transfer of carbon from bacterial cells to higher trophic levels. Through their bacterivorous activity, they also act as nutrient recyclers that allow for regenerated primary production, and they are partially responsible of keeping bacterial abundances in the ocean fairly constant. HFs are widespread throughout the eukaryotic tree of life, ubiquitous in the plankton and display a high functional diversity. During the last decades of the twentieth century, a growing interest in this functional group occurred, and studies were performed to characterize their ecological role. However, this initial attention diminished due to the difficulty to study natural HF species, as they possess few morphological traits for identification and generally remain uncultured. Consequently, HFs have been often neglected in marine surveys, to the extent of becoming one of the most understudied components of the marine microbiome. With the advent of high-throughput sequencing and the reduction of sequencing costs, studying these protists at a high-resolution level became feasible. This thesis represents a return to the study of HFs using these newly developed tools. We first investigated the distribution patterns of eukaryotic diversity along the water column of the ocean by metagenomics and compared the results with metabarcoding approaches. This analysis revealed a clear separation of taxonomic groups between pico- (0.2-3 μm) and nanoplanktonic (3-20 μm) fractions, as well as between photic (0-200 m) and aphotic (>200 m) regions. While some groups were not well represented by metabarcoding approaches due to technical biases, HFs were generally not affected by them. We then studied the diversity and distribution of HFs in the ocean using global metabarcoding data sets. With this, we identified a few dozens of HF species, most of them uncultured, as the dominant in surface and deep ocean regions. Many of these dominant species were present at relatively constant abundances, while others were influenced by temperature or displayed patchy distributions. Finally, we jumped from global patterns to study the gene expression of HFs in natural assemblages growing by bacterivory in unamended incubations. The obtained results using metatranscriptomics sequencing showed similar functional dynamics between experiments done at different times of a seasonal cycle, with marked differences between incubation times. Genes related to cysteine peptidases as well as some glycoside hydrolases emerged as key components involved in the process of bacterivory. Overall, this thesis returns HFs back to the spotlight and creates a solid foundation on which to perform renewed research on the ecology and functional role of this group[CAT] Els flagel·lats heterotròfics marins (HF) són protists no pigmentats de mida molt petita (2-5 μm). Són els principals bacterívors a l’oceà i tenen un paper clau a la cadena tròfica com a enllaç en la transferència de carboni de les cèl·lules bacterianes cap a nivells tròfics superiors. A través de la seva activitat, també actuen com a remineralitzadors de nutrients, tot permetent una contínua producció primària, i són responsables parcialment de mantenir estable la concentració de bacteris a l’oceà. Els HFs es troben al llarg de tot l’arbre eucariota de la vida, tenen una gran presència en ambients planctònics i mostren una gran diversitat funcional. Durant les últimes dècades del segle XX, l’atenció cap aquest grup funcional va créixer de forma notable, i es van dur a terme estudis per caracteritzar millor el seu paper ecològic. Tanmateix, aquest interès va anar disminuint a causa de la dificultat per estudiar-ne les espècies naturals, atès que aquestes tenen pocs trets morfològics per a la seva identificació i generalment no han pogut ser cultivades. Com a conseqüència, els HFs han estat sovint oblidats en les campanyes oceanogràfiques, fins al punt de convertir-se en un dels components menys estudiats del microbioma marí. Amb el naixement de les tècniques de seqüenciació massiva i la reducció dels seus costos, l’estudi detallat d’aquests protists ha passat a ser possible. Aquesta tesi representa un retorn a l’estudi dels HF utilitzant aquestes noves eines. En primer lloc, hem investigat els patrons de distribució de la diversitat eucariota al llarg de la columna d’aigua de l’oceà per mitjà de dades de metagenòmica i els hem comparat amb els resultats obtinguts a partir de seqüències del gen ribosomal 18S (metabarcoding). Aquesta anàlisi mostra una clara separació taxonòmica entre les fraccions pico (0.2-3 μm) i nanoplanctòniques (3-20 μm), així com entre les regions fòtiques (0-200 m) i afòtiques (>200 m). Tot i que alguns dels grups no estan ben representats en l’anàlisi feta a través del gen 18S a causa de biaxos tècnics, la majoria dels HF no es veuen afectats per aquests. A continuació, hem estudiat la diversitat i distribució dels HFs a l’oceà utilitzant conjunts de dades globals de metabarcoding. Amb això, hem identificat unes poques espècies d’HFs, majoritàriament no cultivades, com les dominants en aigües superficials i profundes de l’oceà. Moltes d’aquestes espècies dominants es troben presents en abundàncies relativament constants, mentre que d’altres estan influenciades per la temperatura o mostren distribucions irregulars. Finalment, hem passat de l’anàlisi de patrons globals a l’estudi de l’expressió gènica de comunitats naturals d’HFs mitjançant una sèrie d’incubacions on la bacterivoria ha estat estimulada. Els resultats obtinguts fent servir dades de metatranscriptòmica mostren una dinàmica funcional similar entre experiments duts a terme en diferents estacions de l’any, amb diferències marcades entre els temps d’incubació. Els gens relacionats amb les peptidases de cisteïna, així com algunes hidrolases glicosídiques es presenten com a components clau implicats en el procés de bacterivoria. Amb tot, amb aquesta tesi hem tornat a posar els HFs al centre d’atenció i hem creat una base sòlida sobre la qual realitzar una investigació renovada de l’ecologia i el paper funcional d’aquest grupDuring the realization of this thesis, Aleix Obiol Plana held a PhD fellowship FPI (BES-2017-080765), funded by the Spanish Ministry of Science and Innovation and was a student of UPC’s PhD program in Marine Sciences. This thesis was also supported by ALLFLAGS (CTM2016-75083-R), SINGEK (H2020- MSCA-ITN-2015-675752), Malaspina-2010 (CSD2008–00077) and DIVAS (PID2019-108457RB-I00) projects and the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S)Peer reviewe

Pushing the limits of 18S rDNA variants: A temporal and spatial study on marine heterotrophic flagellates

5th Joint Meeting of the Phycological Society of America (PSA) & International Society of Protistologists Annual Meeting (ISOP), 29 July - 2 August 2018, Vancouver, BC, CanadaStudies on microbial diversity have greatly advanced by high throughput sequencing of phylogenetic gene markers retrieved from natural assemblages. Now, it is feasible to simultaneously obtain thousands of sequences from hundreds of samples, and one of the main challenges is to discern very-similar real variants from the numerous low-frequency generated errors. Our main aim here is to identify the relevant variability within marine heterotrophic flagellates, a functional group of small bacterivorous protists including many uncultured species, and survey these variants along extensive temporal and spatial datasets. We use metabarcoding data from the V4 region of the 18S rDNA targeting marine picoeukaryotes, which were sampled at the global Malaspina expedition and the Blanes Bay Microbial Observatory (monthly during 10 years). By comparing different clustering methodologies, we identify single-nucleotide variants with contrasted temporal and spatial distributions. The combination of extensive sampling efforts and powerful sequencing and analytic tools enables us, for the first time, to conduct a global study to identify the dominant heterotrophic flagellates and better define their prevalence and relevance in the oceans.Peer Reviewe

Oceanic heterotrophic flagellates are dominated by a few widespread taxa

14 pages, 5 figures, 1 table, supporting information https://doi.org/10.1002/lno.11956.-- Data availability statement: Raw data used in this study can be found at the European Nucleotide Archive (http://www.ebi.ac.uk/ena) with accession numbers PRJEB23913 and PRJEB25224 (surface data sets for 18S and 16S, respectively; Logares et al. 2020), and PRJEB23771 (vertical profiles; Giner et al. 2020). TARA Oceans (de Vargas et al. 2015; Callahan 2017) and Malaspina V9/mTags tables (Obiol et al. 2020) can be found at Zenodo with DOIs 10.5281/zenodo.581694 and 10.5281/zenodo.3629394, respectively. All tables and code used for data processing and analyses can be found at https://github.com/aleixop/Malaspina_HFMarine heterotrophic flagellates (HFs) form a diverse and ecologically relevant functional group of bacterial grazers and nutrient remineralizers in oceanic waters. Despite playing a crucial role in marine biogeochemical cycles, there is still a lack of information on which specific taxa dominate HF assemblages and what are their patterns of distribution in a global context. In the present work, we addressed this issue by analyzing amplicon sequencing data sets retrieved from samples taken in tropical and subtropical oceanic regions at depths from surface to 4000 m. Only a few dozens of widespread taxa, mostly affiliating to MAST clades, Picozoa, Bicosoecida and Chrysophyceae, seemed to dominate surface HF assemblages. The majority of these dominant HFs were present at relatively constant abundances, while others were influenced by temperature or displayed a patchy distribution. In the deep ocean, only a handful of taxa belonging to Bicosoecida and Chrysophyceae, together with Diplonemea and Kinetoplastida, explained most of the HF signal. Co-occurrence networks between HF and prokaryotic taxa at the surface ocean revealed two main clusters influenced by temperature that did not seem to show specific patterns of interaction. However, some correlations emerged outside these thermal groups that could represent new prey–predator interactions. Overall, we identified the putatively most ecologically relevant HF taxa in the ocean, which become promising targets for further experimental and genomic studiesThis research was supported by the Spanish Ministry of Science and Innovation projects Malaspina-2010 (CSD2008–00077), ALLFLAGS (CTM2016-75083-R) and the “Severo Ochoa Centre of Excellence” accreditation (CEX2019-000928-S), and the European Union project SINGEK (H2020-MSCA-ITN-2015-675752). AO was supported by a Spanish FPI granPeer reviewe

Ciliate diversity and distribution across horizontal and vertical scales in the open ocean

16 pages, 4 figures, 2 tables, supporting Information https://doi.org/10.1111/mec.15528.-- The data that support the findings of this study are openly available in the open‐access repository Zenodo at http://doi.org/10.5281/zenodo.3736633 (Canals, Obiol, Muhovic, Vaqué, & Massana, 2020)Ciliates are globally distributed eukaryotic organisms inhabiting virtually all environments on Earth. Although ciliates range from 10 µm to a few millimetres in cell size, they are repeatedly reported in the pico‐sized fraction (<2–3 µm) of molecular surveys. Here, we used existing data sets (BioMarKs and Tara Oceans) with different size fractions to demonstrate that the ciliate pico‐sized signal, probably derived from cell breakage during filtration, is informative and reliable to study marine ciliate biodiversity and biogeography. We then used sequences from the pico‐eukaryotic fraction of two circumnavigation expeditions, Malaspina‐2010 and Tara Oceans, to give insights into the taxonomic composition and horizontal and vertical distribution of ciliates in the global ocean. The results suggested a high homogeneity of ciliate communities along the ocean surface from temperate to tropical waters, with ciliate assemblages dominated by a few abundant and widely distributed taxa. Very few taxa were found in a single oceanic region, therefore suggesting a high level of ciliate cosmopolitanism in the global ocean. In vertical profiles, ciliates were detected up to 4,000 m depth, and a clear vertical community structuring was observed. Our results provided evidence supporting ciliates as deeply integrated organisms in the deep‐sea trophic web, where they may play a relevant role as symbionts of metazoans and grazers of prokaryotes and small eukaryotes in the water column and in aggregatesThis project was supported by the Spanish Ministry of Economy and Competitiveness through the projects Consolider Malaspina‐2010 (CSD2008–00077) and ALLFLAGS (CTM2016‐75083‐R)With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI

Unveiling the seasonality of Ciliphora: from the whole community to individual species

1st Iberian Ecological Society Meeting (2019); XIV Congreso Nacional de la Asociación Española de Ecología Terrestre (AEET), Ecology: an integrative science in the Anthropocene, 4-7 February 2019, Barcelona, SpaineCiliophora protists are key components of microbial communities in marine ecosystems, playing an essential role as a link betweenlower and higher trophic levels in the food web. In temperate areas, it has been demonstrated that the community structure ofciliates varies throughout the year, following a strong seasonal pattern, but much less is known about the dynamics at lower taxo-nomic scales, including well defined groups and individual species. Here, we studied the Ciliophora community of an oligotrophiccoastal site in the Mediterranean Sea (Blanes Bay) by using high-throughput sequencing of the 18S rDNA V4 region. The two small-size fractions analyzed (pico- and nanoplankton) were very similar and represented well the Ciliophora diversity in the system, whichwe divided in 11 taxonomic groups and about 950 species. During the 10-years long period investigated, the Ciliophora communityexhibited a marked seasonality, and a recurrent seasonal dynamic was observed for Hypotrichia, Nassophorea and Oligohy-menophorea groups. Hypotrichia was exclusively composed by seasonally recurrent species, while Nassophorea, Oligohy-menophorea, and the non-seasonal groups were composed by both seasonal and non-seasonal species. Unveiling the taxa drivingthe seasonal patterns of the Ciliophora community will improve our understanding about its ecology, and will lead to a deeper knowl-edge of the marine ecosystem dynamics, by determining, for example, the trophic role that these species play and the main factorsthat influence their population dynamicsPeer Reviewe

Expression of genes involved in phagocytosis in uncultured heterotrophic flagellates

Special issue Linking Metagenomics to Aquatic Microbial Ecology and Biogeochemical Cycles.-- 12 pages, 4 figures, 1 table, supporting information https://doi.org/10.1002/lno.11379Environmental molecular sequencing has revealed an abundance of microorganisms that were previously unknown, mainly because most had not been cultured in the laboratory. Within this novel diversity, there are the uncultured MAST clades (MArine STramenopiles), which are major components of marine heterotrophic flagellates (HFs) thought to be active bacterial grazers. In this study, we investigated the gene expression of natural HFs in a mixed community where bacterivory was promoted. Using fluorescence in situ hybridization and 18S rDNA derived from metatranscriptomics, we followed the taxonomic dynamics during the incubation, and confirmed the increase in relative abundance of different MAST lineages. We then used single cell genomes of several MAST species to gain an insight into their most expressed genes, with a particular focus on genes related to phagocytosis. The genomes of MAST‐4A and MAST‐4B were the most represented in the metatranscriptomes, and we identified highly expressed genes of these two species involved in motility and cytoskeleton remodeling, as well as many lysosomal enzymes. Particularly relevant were the cathepsins, which are characteristic digestive enzymes of the phagolysosome and the rhodopsins, perhaps used for vacuole acidification. The combination of single cell genomics and metatranscriptomics gives insights on the phagocytic capacity of uncultured and ecologically relevant HF speciesThis project was supported by the EU project SINGEK (H2020‐MSCA‐ITN‐2015‐675752) and the Spanish project ALLFLAGS (CTM2016‐75083‐R, MINECO). S.W. was supported by a Postdoctoral Fellowship from the European Union's Seventh Framework Program under Marie Sklodowska‐Currie grant agreement 6261882With the funding support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S), of the Spanish Research Agency (AEI

Assessment of the diversity and bacterivory-related genes of marine heterotrophic flagellates using multiomics

Association for the Sciences of Limnology and Oceanography (ASLO) Summer meeting, Water connects!, 10-15 June 2018, Victoria, CanadaThe smallest unpigmented eukaryotes in the marine plankton, known as heterotrophic flagellates (HFs), form a diverse collection of predominantly uncultured flagellated cells that are important grazers of bacteria, with crucial roles in nutrient remineralization. Here we explore the biodiversity of HF assemblages and their genetic repertoire, specifically those functions concerning bacterivory, using multiomics. First, we developed a pipeline to extract the taxonomic composition of HF cells in natural assemblages by retrieving and annotating V4 18S rDNA reads from metagenomes. Then, we promoted an enrichment of HFs by incubating natural communities in the dark, a setting where bacterivory was the main carbon metabolism process. Metatranscriptomic analyses of corresponding HF enriched samples pointed to the specific set of genes actively transcribed under this particular condition. Combining these bacterivory-related genes with a set of genomes from uncultured HFs obtained by single cell genomics allowed linking these functional genes to given taxa. Finally, we applied both the biodiversity and functional pipelines to a set of metagenomes obtained during the circumglobal Malaspina expedition and a seasonal study at the Blanes Bay Microbial Observatory. This study enabled the identification of the dominant heterotrophic flagellates in both systems together with their genetic repertoire associated to bacterivoryPeer Reviewe