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Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The “9+2” axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked if there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana, have an extensive RSP complement including two divergent RSP9 orthologs, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither ortholog is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 ortholog. deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked with the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/-) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

International audience; We previously showed that injection of recombinant human group IIA secreted phospholipase A2 (hGIIA sPLA2) to Plasmodium chabaudi-infected mice lowers parasitaemia by 20%. Here, we show that transgenic (TG) mice overexpressing hGIIA sPLA2 have a peak of parasitaemia about 30% lower than WT littermates. During infection, levels of circulating sPLA2, enzymatic activity and plasma lipid peroxidation were maximal at day-14, the peak of parasitaemia. Levels of hGIIA mRNA increased in liver but not in spleen and blood cells, suggesting that liver may contribute as a source of circulating hGIIA sPLA2. Before infection, baseline levels of leukocytes and pro-inflammatory cytokines were higher in TG mice than WT littermates. Upon infection, the number of neutrophils, lymphocytes and monocytes increased and were maximal at the peak of parasitaemia in both WT and TG mice, but were higher in TG mice. Similarly, levels of the Th1 cytokines IFN-γ and IL-2 increased in WT and TG mice, but were 7.7- and 1.7-fold higher in TG mice. The characteristic shift towards Th2 cytokines was observed during infection in both WT and TG mice, with increased levels of IL-10 and IL-4 at day-14. The current data are in accordance with our previous in vitro findings showing that hGIIA kills parasites by releasing toxic lipids from oxidized lipoproteins. They further show that hGIIA sPLA2 is induced during mouse experimental malaria and has a protective in vivo role, lowering parasitaemia by likely releasing toxic lipids from oxidized lipoproteins but also indirectly by promoting a more sustained innate immune response.

International audience; High-throughput screening assays have been designed to identify compounds capable of inhibiting phenotypes involved in cancer aggressiveness. However, most studies used commercially available chemical libraries. This prompted us to explore natural products isolated from marine-derived fungi as a new source of molecules. In this study, we established a chemical library from 99 strains corresponding to 45 molecular operational taxonomic units and evaluated their anticancer activity against the MCF7 epithelial cancer cell line and its invasive stem cell-like MCF7-Sh-WISP2 counterpart. We identified the marine fungal Paradendryphiella salina PC 362H strain, isolated from the brown alga Pelvetia caniculata (PC), as one of the most promising fungi which produce active compounds. Further chemical and biological characterizations of the culture of the Paradendryphiella salina PC 362H strain identified (-)-hyalodendrin as the active secondary metabolite responsible for the cytotoxic activity of the crude extract. The antitumor activity of (-)-hyalodendrin was not only limited to the MCF7 cell lines, but also prominent on cancer cells with invasive phenotypes including colorectal cancer cells resistant to chemotherapy. Further investigations showed that treatment of MCF7-Sh-WISP2 cells with (-)-hyalodendrin induced changes in the phosphorylation status of p53 and altered expression of HSP60, HSP70 and PRAS40 proteins. Altogether, our study reveals that this uninvestigated marine fungal crude extract possesses a strong therapeutic potential against tumor cells with aggressive phenotypes and confirms that members of the epidithiodioxopiperazines are interesting fungal toxins with anticancer activities.

Giardia intestinalis is a protist intestinal parasite responsible for giardiasis, a disease whose impact is recognized in public health. After ingestion of Giardia cysts from either contaminated food or water, the trophozoite proliferative form, responsible for pathogenic effects, develops in the proximal small intestine of the host where it coexists with gut microbiota. Several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. intestinalis infections through partially known mechanisms (Travers et al. Journal of Parasitology Research, 2011). In the last years, our team has shown, using biological and biochemical approaches, that some probiotic strains of Lactobacillus, in particular L. johnsonii La1 and L. gasseri CNCM-I 4884, display anti-Giardia effects both in vitro and in vivo (Travers et al. Frontiers in Microbiology 2016; Allain et al. Frontiers in Microbiology 8:2707, 2018a, Frontiers in Microbiology 9:98, b). Our investigations have demonstrated that the supernatant of these strains contains Bile-Salt-Hydrolase (BSH)-like activities mediating toxic effects on Giardia. This effect is not directly, but by converting non-toxic components of bile (conjugated bile salts) into bile salts deconjugates proved to be highly toxic to the parasite. These anti-Giardia effects could be mimicked in vitro by treating Giardia cultures with either commercially available BSH bacterial enzymes (Travers et al. 2016) or two L. johnsonii La1 BSH enzymes produced and purified from recombinant Escherichia coli strains (Allain et al. 2018a), in the presence of bile, or even directly with some deconjugated bile salts (Travers et al. 2016). Currently, we are focusing on understanding the mechanism of action (MoA) of toxic metabolites generated by these BSH activities on the parasite itself using imaging and RNA sequencing methods in order to explore the changes in gene expressions in Giardia. Altogether, these data pave the way for new approaches for the treatment of this widespread neglected infectious disease.

SummarySexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin-8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B-gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses is unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal “9+2” structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.

Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell-and gene-based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space.These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.

Giardia duodenalis (syn. G. lamblia, G. intestinalis) is the protozoan parasite responsible for giardiasis, the most common and widely spread intestinal parasitic disease worldwide, affecting both humans and animals. After cysts ingestion (through either contaminated food or water), Giardia excysts in the upper intestinal tract to release replicating trophozoites that are responsible for the production of symptoms. In the gut, Giardia cohabits with the host's microbiota, and several studies have revealed the importance of this gut ecosystem and/or some probiotic bacteria in providing protection against G. duodenalis infection through mechanisms that remain incompletely understood. Recent findings suggest that Bile-Salt-Hydrolase (BSH)-like activities from the probiotic strain of Lactobacillus johnsonii La1 may contribute to the anti-giardial activity displayed by this strain. Here, we cloned and expressed each of the three bsh genes present in the L. johnsonii La1 genome to study their enzymatic and biological properties. While BSH47 and BSH56 were expressed as recombinant active enzymes, no significant enzymatic activity was detected with BSH12. In vitro assays allowed determining the substrate specificities of both BSH47 and BSH56, which were different. Modeling of these BSHs indicated a strong conservation of their 3-D structures despite low conservation of their primary structures. Both recombinant enzymes were able to mediate anti-giardial biological activity against Giardia trophozoites in vitro. Moreover, BSH47 exerted significant anti-giardial effects when tested in a murine model of giardiasis. These results shed new light on the mechanism, whereby active BSH derived from the probiotic strain Lactobacillus johnsonii La1 may yield anti-giardial effects in vitro and in vivo. These findings pave the way toward novel approaches for the treatment of this widely spread but neglected infectious disease, both in human and in veterinary medicine.

International audience; Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 μg L−1 microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 μg L−1 of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100–400 genes are differentially expressed under 5 μg L−1 MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 μg L−1 MC-LR potentially induces a health risk for aquatic organisms.

Macrophages have been shown to be essential for muscle repair by delivering trophic cues to growing skeletal muscle precursors and young fibers. Here, we investigated whether human macrophages, either proinflammatory or anti-inflammatory, coinjected with human myoblasts into regenerating muscle of Rag2(-/-) γC(-/-) immunodeficient mice, could modify in vivo the kinetics of proliferation and differentiation of the transplanted human myogenic precursors. Our results clearly show that proinflammatory macrophages improve in vivo the participation of injected myoblasts to host muscle regeneration, extending the window of proliferation, increasing migration, and delaying differentiation. Interestingly, immunostaining of transplanted proinflammatory macrophages at different time points strongly suggests that these cells are able to switch to an anti-inflammatory phenotype in vivo, which then may stimulate differentiation during muscle regeneration. Conceptually, our data provide for the first time in vivo evidence strongly suggesting that proinflammatory macrophages play a supportive role in the regulation of myoblast behavior after transplantation into preinjured muscle, and could thus potentially optimize transplantation of myogenic progenitors in the context of cell therapy.

Numerous strategies are under development for the correction of deleterious effects of mutations in muscular dystrophies, and these strategies must be validated in compelling models. Cellular models seem straightforward to set up; however, the proliferative capacity of muscle cells isolated from dystrophic patients is limited, and in addition it is difficult to envisage the use of large muscle biopsies from patients to obtain enough cells for ex vivo assessments. To overcome these problems, we have devised a strategy to obtain, from a patient with Duchenne muscular dystrophy (DMD), an inexhaustible source of myogenic progenitor cells with a deletion of exons 49 and 50 in the dystrophin gene. Starting material consisted of dermal fibroblasts isolated from a skin biopsy taken in a noninvasive way. These fibroblasts were first immortalized by telomerase gene transfer. Subsequent cell lines were converted into myogenic cells by means of a lentiviral vector encoding an inducible MyoD construct. Before myogenic induction, engineered DMD fibroblasts were able to proliferate infinitely. Under induction conditions, they were converted into myogenic cells, which differentiated into large multinucleated myotubes. We used these DMD fibroblast cell lines to assess dystrophin rescue by using engineered U7 small nuclear RNAs harboring antisense sequences required to restore an in-frame dystrophin mRNA by skipping exon 51. Further molecular analyses showed dystrophin rescue ex vivo as well as in vivo after engrafting of treated cells into regenerating muscles in immunodeficient mice.

Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

International audience; ABSTRACT: INTRODUCTION: Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle. METHODS: As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation. RESULTS: After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group. CONCLUSIONS: In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.

Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

International audience; Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 213 and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSE. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathics. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.

In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle-derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2(-/-) gammaC(-/-) C5(-/-) mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle-derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.

International audience; Population-based association studies have identified several polymorphic variants in genes encoding ion channel subunits associated with the electrocardiographic heart-rate-corrected QT (QTc) length in healthy populations of Caucasian origin (KCNH2 rs1,805,123 (K897 T) and rs3,815,459, SCN5A rs1,805,126 (D1,819D), 1,141-3 C>A, rs1,805,124 (H558R), and IVS24+116 G>A, KCNQ1 rs757,092, KCNE1 IVS2-128 G>A and rs1,805,127 (G38S), and KCNE2 rs2,234,916 (T8A)). However, few of these results have been replicated in independent populations. We tested the association of SNPs KCNQ1 rs757,092, KCNH2 rs3,815,459, SCN5A IVS24+116 G>A, KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 with QTc length in two groups of 200 subjects presenting the shortest and the longest QTc from a cohort of 2,008 healthy subjects. All polymorphisms were in Hardy-Weinberg equilibrium in both groups. The minor allele SCN5A IVS24+116 A was more frequent in the group of subjects with the shortest QTc, whereas the minor alleles KCNQ1 rs757,092 G and KCNH2 rs3,815,459 A were more frequent in the group with the longest QTc. There was no significant difference for KCNE1 IVS2-128 G>A and KCNE2 rs2,234,916 between the two groups. Haplotype analysis showed a twofold increased risk of QTc lengthening for carriers of the haplotype, combining alleles C and A of the two common KCNE1 SNPs, IVS2-129 C>T (rs2,236,609) and rs1,805,127 (G38S), respectively. In conclusion, our study confirms the reported associations between QTc length and KCNQ1 rs757,092 and KCNH2 rs3,815,459.

International audience; The complete sequences of the two major RNAs of Chronic bee paralysis virus (CBPV) have been determined. RNA 1 (3674nt long) and RNA 2 (2305nt long) are positive single-stranded RNAs that are capped but not polyadenylated. The 3' ends of both RNAs are unreactive to polymerisation or ligation even in denaturing conditions, a feature already observed in alphanodavirus RNAs. The three previously described smaller RNAs [Overton, H.A., Buck, K.W., Bailey, L., et al., 1982. Relationships between the RNA components of Chronic bee-paralysis virus and those of chronic bee-paralysis virus associate. J. Gen. Virol. 63, 171-179], were not detected in this study, supporting the hypothesis that they would correspond to the three RNAs of the Chronic bee paralysis satellite virus (CBPSV). RNA 1 and RNA 2 encoded three and four overlapping open reading frames (ORFs), respectively. The amino acid sequences deduced from the ORF 3 on RNA 1 shared the conserved motifs of the RNA-dependent RNA polymerase (RdRp) sequence and presented similarities with members of the Nodaviridae and Tombusviridae families. However, no similarities were found between the other CBPV deduced amino acid sequences and sequences in the NCBI databases, suggesting that CBPV is the prototype of a new family of positive single-stranded RNA viruses.

The peer review history for this article is available at https://publons.com/publon/10.1111/1365-2745.13986.Raw data have been deposited in the Sequence Read Archive (SRA) under the BioProject accession no. PRJNA692353.; International audience; In soils, plants and fungi can form complex mycorrhizal networks allowing nutrient transfers between plant individuals and species. It is less clear, however, whether such networks exist on the bark of trees where epiphytic plant communities thrive in rainforests. Previous work showed that tropical epiphytic orchids especially, harbour symbiotic fungi in their roots, but the structure and determinants of the resulting networks remain unknown at the tree scale.We tested the hypothesis that epiphytic orchids rooted in the same area on the bark share mycorrhizal fungi, regardless of their species (i.e. spatial determinant). For this purpose, we selected the trunk of six trees of two common species in a rainforest and sampled orchid roots, protocorms and surrounding bark. We identified mycorrhizal fungi including Tulasnellaceae using high-throughput sequencing of the ITS2 marker, and reconstructed orchid-fungus bipartite networks for each tree to analyse their structure and the spatial turnover of this symbiosis.We found that epiphytic orchid communities form antinested and highly modular networks with mycorrhizal fungi spread on the bark. As expected, modules of interactions are explained by their spatial structure, with nearby roots sharing fungi, but also by the orchid species involved. These results reveal the presence of shared mycelial networks in epiphytic habitats, whose roles in the resilience and facilitation of epiphytic plant communities need to be assessed.Synthesis. Tropical tree barks are densely colonized by certain mycorrhizal fungi that can form symbioses in nearby adult and young orchids simultaneously. These mycorrhizal networks may allow water and nutrient transfers to alleviate the stressful conditions of the epiphytic habitats.