Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae.

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.

Mixotrophic growth of the extremophile Galdieria sulphuraria reveals the flexibility of its carbon assimilation metabolism.

Galdieria sulphuraria is a cosmopolitan microalga found in volcanic hot springs and calderas. It grows at low pH in photoautotrophic (use of light as a source of energy) or heterotrophic (respiration as a source of energy) conditions, using an unusually broad range of organic carbon sources. Previous data suggested that G. sulphuraria cannot grow mixotrophically (simultaneously exploiting light and organic carbon as energy sources), its photosynthetic machinery being repressed by organic carbon. Here, we show that G. sulphuraria SAG21.92 thrives in photoautotrophy, heterotrophy and mixotrophy. By comparing growth, biomass production, photosynthetic and respiratory performances in these three trophic modes, we show that addition of organic carbon to cultures (mixotrophy) relieves inorganic carbon limitation of photosynthesis thanks to increased CO2 supply through respiration. This synergistic effect is lost when inorganic carbon limitation is artificially overcome by saturating photosynthesis with added external CO2 . Proteomic and metabolic profiling corroborates this conclusion suggesting that mixotrophy is an opportunistic mechanism to increase intracellular CO2 concentration under physiological conditions, boosting photosynthesis by enhancing the carboxylation activity of Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and decreasing photorespiration. We discuss possible implications of these findings for the ecological success of Galdieria in extreme environments and for biotechnological applications.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

An original mode of symbiosis in open ocean plankton.

Symbiotic relationships are widespread in nature and are fundamental for ecosystem functioning and the evolution of biodiversity. In marine environments, photosymbiosis with microalgae is best known for sustaining benthic coral reef ecosystems. Despite the importance of oceanic microbiota in global ecology and biogeochemical cycles, symbioses are poorly characterized in open ocean plankton. Here, we describe a widespread symbiotic association between Acantharia biomineralizing microorganisms that are abundant grazers in plankton communities, and members of the haptophyte genus Phaeocystis that are cosmopolitan bloom-forming microalgae. Cophylogenetic analyses demonstrate that symbiont biogeography, rather than host taxonomy, is the main determinant of the association. Molecular dating places the origin of this photosymbiosis in the Jurassic (ca. 175 Mya), a period of accentuated marine oligotrophy. Measurements of intracellular dimethylated sulfur indicate that the host likely profits from antioxidant protection provided by the symbionts as an adaptation to life in transparent oligotrophic surface waters. In contrast to terrestrial and marine symbioses characterized to date, the symbiont reported in this association is extremely abundant and ecologically active in its free-living phase. In the vast and barren open ocean, partnership with photosymbionts that have extensive free-living populations is likely an advantageous strategy for hosts that rely on such interactions. Discovery of the Acantharia-Phaeocystis association contrasts with the widely held view that symbionts are specialized organisms that are rare and ecologically passive outside the host.

Brandtodinium gen. nov. and B. nutricula comb. Nov. (Dinophyceae), a dinoflagellate commonly found in symbiosis with polycystine radiolarians.

Symbiotic interactions between pelagic hosts and microalgae have received little attention, although they are widespread in the photic layer of the world ocean, where they play a fundamental role in the ecology of the planktonic ecosystem. Polycystine radiolarians (including the orders Spumellaria, Collodaria and Nassellaria) are planktonic heterotrophic protists that are widely distributed and often abundant in the ocean. Many polycystines host symbiotic microalgae within their cytoplasm, mostly thought to be the dinoflagellate Scrippsiella nutricula, a species originally described by Karl Brandt in the late nineteenth century as Zooxanthella nutricula. The free-living stage of this dinoflagellate has never been characterized in terms of morphology and thecal plate tabulation. We examined morphological characters and sequenced conservative ribosomal markers of clonal cultures of the free-living stage of symbiotic dinoflagellates isolated from radiolarian hosts from the three polycystine orders. In addition, we sequenced symbiont genes directly from several polycystine-symbiont holobiont specimens from different oceanic regions. Thecal plate arrangement of the free-living stage does not match that of Scrippsiella or related genera, and LSU and SSU rDNA-based molecular phylogenies place these symbionts in a distinct clade within the Peridiniales. Both phylogenetic analyses and the comparison of morphological features of culture strains with those reported for other closely related species support the erection of a new genus that we name Brandtodinium gen. nov. and the recombination of S. nutricula as B. nutricula comb. nov.

Deep relationships of Rhizaria revealed by phylogenomics: a farewell to Haeckel's Radiolaria.

Rhizaria is one of the six supergroups of eukaryotes, which comprise the majority of amoeboid and skeleton-building protists living in freshwater and marine ecosystems. There is an overall lack of molecular data for the group and therefore the deep phylogeny of rhizarians is unresolved. Molecular data are particularly scarce for the clade of Retaria, which include two prominent groups of microfossils: foraminiferans and radiolarians. To fill this gap, we have produced and sequenced EST libraries for 14 rhizarian species including seven foraminiferans, Gromia and six taxa belonging to traditional Haeckel's Radiolaria: Acantharea, Polycystinea, and Phaeodarea. A matrix was constructed for phylogenetic analysis based on 109 genes and a total of 56 species, of which 22 are rhizarians. Our analyses provide the first multigene evidence for branching of Phaeodarea within Cercozoa, confirming the polyphyly of Haeckel's Radiolaria. It confirms the monophyly of Retaria, a clade grouping Foraminifera with other lineages of Radiolaria. However, contrary to what could be expected from morphological observations, Foraminifera do not form a sister group to radiolarians, but branch within them as sister to either Acantharea or Polycystinea depending on the multigene data set. While the monophyly of Foraminifera and Acantharea is well supported, that of Polycystinea, represented in our data by Spumellaria and Collodaria is questionable. In view of our study, Haeckel's Radiolaria appears as both, a polyphyletic and paraphyletic assemblage of independent groups that should be considered as separate lineages in protist classification.

Ancient DNA complements microfossil record in deep-sea subsurface sediments.

Deep-sea subsurface sediments are the most important archives of marine biodiversity. Until now, these archives were studied mainly using the microfossil record, disregarding large amounts of DNA accumulated on the deep-sea floor. Accessing ancient DNA (aDNA) molecules preserved down-core would offer unique insights into the history of marine biodiversity, including both fossilized and non-fossilized taxa. Here, we recover aDNA of eukaryotic origin across four cores collected at abyssal depths in the South Atlantic, in up to 32.5 thousand-year-old sediment layers. Our study focuses on Foraminifera and Radiolaria, two major groups of marine microfossils also comprising diverse non-fossilized taxa. We describe their assemblages in down-core sediment layers applying both micropalaeontological and environmental DNA sequencing approaches. Short fragments of the foraminiferal and radiolarian small subunit rRNA gene recovered from sedimentary DNA extracts provide evidence that eukaryotic aDNA is preserved in deep-sea sediments encompassing the last glacial maximum. Most aDNA were assigned to non-fossilized taxa that also dominate in molecular studies of modern environments. Our study reveals the potential of aDNA to better document the evolution of past marine ecosystems and opens new horizons for the development of deep-sea palaeogenomics.

The V-type ATPase enhances photosynthesis in marine phytoplankton and further links phagocytosis to symbiogenesis.

Diatoms, dinoflagellates, and coccolithophores are dominant groups of marine eukaryotic phytoplankton that are collectively responsible for the majority of primary production in the ocean.1 These phytoplankton contain additional intracellular membranes around their chloroplasts, which are derived from ancestral engulfment of red microalgae by unicellular heterotrophic eukaryotes that led to secondary and tertiary endosymbiosis.2 However, the selectable evolutionary advantage of these membranes and the physiological significance for extant phytoplankton remain poorly understood. Since intracellular digestive vacuoles are ubiquitously acidified by V-type H+-ATPase (VHA),3 proton pumps were proposed to acidify the microenvironment around secondary chloroplasts to promote the dehydration of dissolved inorganic carbon (DIC) into CO2, thus enhancing photosynthesis.4,5 We report that VHA is localized around the chloroplasts of centric diatoms and that VHA significantly contributes to their photosynthesis across a wide range of oceanic irradiances. Similar results in a pennate diatom, dinoflagellate, and coccolithophore, but not green or red microalgae, imply the co-option of phagocytic VHA activity into a carbon-concentrating mechanism (CCM) is common to secondary endosymbiotic phytoplankton. Furthermore, analogous mechanisms in extant photosymbiotic marine invertebrates6,7,8 provide functional evidence for an adaptive advantage throughout the transition from endosymbiosis to symbiogenesis. Based on the contribution of diatoms to ocean biogeochemical cycles, VHA-mediated enhancement of photosynthesis contributes at least 3.5 Gtons of fixed carbon per year (or 7% of primary production in the ocean), providing an example of a symbiosis-derived evolutionary innovation with global environmental implications.

Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution.

Photoautotrophs' environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.

Intracellular development and impact of a marine eukaryotic parasite on its zombified microalgal host.

Parasites are widespread and diverse in oceanic plankton and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and how the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the marine parasite Amoebophrya (Syndiniales) during intracellular infection, particularly following engulfment and digestion of nutrient-rich host chromosomes. Changes include a sequential acristate and cristate mitochondrion with a 200-fold increase in volume, a 13-fold increase in nucleus volume, development of Golgi apparatus and a metabolic switch from glycolysis (within the host) to TCA (free-living dinospore). Similar changes are seen in apicomplexan parasites, thus underlining convergent traits driven by metabolic constraints and the infection cycle. In the algal host, energy-producing organelles (plastid, mitochondria) remain relatively intact during most of the infection. We also observed that sugar reserves diminish while lipid droplets increase. Rapid infection of the host nucleus could be a "zombifying" strategy, allowing the parasite to digest nutrient-rich chromosomes and escape cytoplasmic defense, whilst benefiting from maintained carbon-energy production of the host cell.

Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging.

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.

A community perspective on the concept of marine holobionts: current status, challenges, and future directions.

Host-microbe interactions play crucial roles in marine ecosystems. However, we still have very little understanding of the mechanisms that govern these relationships, the evolutionary processes that shape them, and their ecological consequences. The holobiont concept is a renewed paradigm in biology that can help to describe and understand these complex systems. It posits that a host and its associated microbiota with which it interacts, form a holobiont, and have to be studied together as a coherent biological and functional unit to understand its biology, ecology, and evolution. Here we discuss critical concepts and opportunities in marine holobiont research and identify key challenges in the field. We highlight the potential economic, sociological, and environmental impacts of the holobiont concept in marine biological, evolutionary, and environmental sciences. Given the connectivity and the unexplored biodiversity specific to marine ecosystems, a deeper understanding of such complex systems requires further technological and conceptual advances, e.g., the development of controlled experimental model systems for holobionts from all major lineages and the modeling of (info)chemical-mediated interactions between organisms. Here we propose that one significant challenge is to bridge cross-disciplinary research on tractable model systems in order to address key ecological and evolutionary questions. This first step is crucial to decipher the main drivers of the dynamics and evolution of holobionts and to account for the holobiont concept in applied areas, such as the conservation, management, and exploitation of marine ecosystems and resources, where practical solutions to predict and mitigate the impact of human activities are more important than ever.

Subcellular Chemical Imaging: New Avenues in Cell Biology.

To better understand the physiology and acclimation capability of the cell, one of the great challenges of the future is to access the interior of a cell and unveil its chemical landscape (composition and distribution of elements and molecules). Chemical imaging has greatly improved in sensitivity and spatial resolution to visualize and quantify nutrients, metabolites, toxic elements, and drugs in single cells at the subcellular level. This review aims to present the current potential of these emerging imaging technologies and to guide biologists towards a strategy for interrogating biological processes at the nanoscale. We also describe various solutions to combine multiple imaging techniques in a correlative way and provide perspectives and future directions for integrative subcellular imaging across different disciplines.

Algal Remodeling in a Ubiquitous Planktonic Photosymbiosis.

Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.

Worldwide Occurrence and Activity of the Reef-Building Coral Symbiont Symbiodinium in the Open Ocean.

The dinoflagellate microalga Symbiodinium sustains coral reefs, one of the most diverse ecosystems of the biosphere, through mutualistic endosymbioses with a wide diversity of benthic hosts [1]. Despite its ecological and economic importance, the presence of Symbiodinium in open oceanic waters remains unknown, which represents a significant knowledge gap to fully understand the eco-evolutionary trajectory and resilience of endangered Symbiodinium-based symbioses. Here, we document the existence of Symbiodinium (i.e., now the family Symbiodiniaceae [2]) in tropical- and temperate-surface oceans using DNA and RNA metabarcoding of size-fractionated plankton samples collected at 109 stations across the globe. Symbiodinium from clades A and C were, by far, the most prevalent and widely distributed lineages (representing 0.1% of phytoplankton reads), while other lineages (clades B, D, E, F, and G) were present but rare. Concurrent metatranscriptomics analyses using the Tara Oceans gene catalog [3] revealed that Symbiodinium clades A and C were transcriptionally active in the open ocean and expressed core metabolic pathways (e.g., photosynthesis, carbon fixation, glycolysis, and ammonium uptake). Metabarcodes and expressed genes of clades A and C were detected in small and large plankton size fractions, suggesting the existence of a free-living population and a symbiotic lifestyle within planktonic hosts, respectively. However, high-resolution genetic markers and microscopy are required to confirm the life history of oceanic Symbiodinium. Overall, the previously unknown, metabolically active presence of Symbiodinium in oceanic waters opens up new avenues for investigating the potential of this oceanic reservoir to repopulate coral reefs following stress-induced bleaching.

A de novo approach to disentangle partner identity and function in holobiont systems.

BACKGROUND: Study of meta-transcriptomic datasets involving non-model organisms represents bioinformatic challenges. The production of chimeric sequences and our inability to distinguish the taxonomic origins of the sequences produced are inherent and recurrent difficulties in de novo assembly analyses. As the study of holobiont meta-transcriptomes is affected by challenges invoked above, we propose an innovative bioinformatic approach to tackle such difficulties and tested it on marine models as a proof of concept. RESULTS: We considered three holobiont models, of which two transcriptomes were previously published and a yet unpublished transcriptome, to analyze and sort their raw reads using Short Read Connector, a k-mer based similarity method. Before assembly, we thus defined four distinct categories for each holobiont meta-transcriptome: host reads, symbiont reads, shared reads, and unassigned reads. Afterwards, we observed that independent de novo assemblies for each category led to a diminution of the number of chimeras compared to classical assembly methods. Moreover, the separation of each partner's transcriptome offered the independent and comparative exploration of their functional diversity in the holobiont. Finally, our strategy allowed to propose new functional annotations for two well-studied holobionts (a Cnidaria-Dinophyta, a Porifera-Bacteria) and a first meta-transcriptome from a planktonic Radiolaria-Dinophyta system forming widespread symbiotic association for which our knowledge is considerably limited. CONCLUSIONS: In contrast to classical assembly approaches, our bioinformatic strategy generates less de novo assembled chimera and allows biologists to study separately host and symbiont data from a holobiont mixture. The pre-assembly separation of reads using an efficient tool as Short Read Connector is an effective way to tackle meta-transcriptomic challenges and offers bright perpectives to study holobiont systems composed of either well-studied or poorly characterized symbiotic lineages and ultimately expand our knowledge about these associations.