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1. Development of duplex TaqMan-based real-time PCR assay for the simultaneous detection of Perkinsus olseni and P. chesapeaki in host Manila clam tissue samples

Author

Sarah Itoïz, Adeline Bidault, Aurélie Chambouvet, Isabelle Arzul, Nelly Le Goïc, Clara Mouronvalle, Philippe Soudant, Xavier de Montaudouin, Evelyne Derelle, Morgan Perennou, Laboratoire des Sciences de l'Environnement Marin (LEMAR) (LEMAR), Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université de Perpignan Via Domitia (UPVD), UMR 5805 Environnements et Paléoenvironnements Océaniques et Continentaux (EPOC), Observatoire aquitain des sciences de l'univers (OASU), Université Sciences et Technologies - Bordeaux 1-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Sciences et Technologies - Bordeaux 1-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Génétique et Pathologie des Mollusques Marins, 17390 La Tremblade, France. (LGPMM), Santé, Génétique et Microbiologie des Mollusques (IFREMER SG2M), Institut Français de Recherche pour l'Exploitation de la Mer - Atlantique (IFREMER Atlantique), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer - Atlantique (IFREMER Atlantique), and Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)

Subjects
0106 biological sciences, 0301 basic medicine, Gill, [SDV]Life Sciences [q-bio], Ruditapes, Biology, Real-Time Polymerase Chain Reaction, 01 natural sciences, Microbiology, 03 medical and health sciences, Species Specificity, Ruditapes philippinarum, TaqMan, Animals, 14. Life underwater, Ecology, Evolution, Behavior and Systematics, Shellfish, ComputingMilieux_MISCELLANEOUS, Inhibitory effect, Host (biology), Molluscs, biology.organism_classification, Co-infection, Bivalvia, qPCR, 010602 entomology, genomic DNA, 030104 developmental biology, Real-time polymerase chain reaction, Alveolata, Duplex (building), [SDE]Environmental Sciences
Abstract

The aetiological agent Perkinsus olseni is globally recognised as a major threat for shellfish production considering its wide geographical distribution across Asia, Europe, Australia and South America. Another species, Perkinsus chesapeaki, which has never been known to be associated with significant mortality events, was recently detected along French coasts infecting clam populations sporadically in association with P. olseni. Identifying potential cryptic infections affecting Ruditapes philippinarum is essential to develop appropriate host resource management strategies. Here, we developed a molecular method based on duplex real-time quantitative PCR for the simultaneous detection of these two parasites, P. olseni and P. chesapeaki, in the different clam tissues: gills, digestive gland, foot, mantle, adductor muscle and the rest of the soft body. We firstly checked the presence of possible PCR inhibitors in host tissue samples. The qPCR reactions were inhibited depending on the nature of the host organ. The mantle and the rest of the soft body have a high inhibitory effect from threshold of host gDNA concentration of 2 ng.µL−1, the adductor muscle and the foot have an intermediate inhibition of 5 ng.µL−1, and the gills and digestive gland do not show any inhibition of the qPCR reaction even at the highest host gDNA concentration of 20 ng.µL−1. Then, using the gills as a template, the suitability of the molecular technique was checked in comparison with the Ray’s Fluid Thioglycolate Medium methodology recommended by the World Organisation for Animal Health. The duplex qPCR method brought new insights and unveiled cryptic infections as the co-occurrence of P. olseni and P. chesapeaki from in situ tissue samples in contrast to the RFTM diagnosis. The development of this duplex qPCR method is a fundamental work to monitor in situ co-infections that will lead to optimised resource management and conservation strategies to deal with emerging diseases.

Published
2021
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2. Diverse alveolate infections of tadpoles, a new threat to frogs?

Author

Mark Wilkinson, Adam Monier, Aurélie Chambouvet, Julius Lukeš, Thomas A. Richards, Evelyne Derelle, Michael J. Yabsley, Sarah Itoïz, Miloslav Jirků, Vanessa Smilansky, Marcos Isidoro-Ayza, David J. Gower, Laboratoire des Sciences de l'Environnement Marin (LEMAR) (LEMAR), Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), College of Life and Environmental Sciences [Exeter], University of Exeter, Institute of Parasitology [České Budějovice] (BIOLOGY CENTRE CAS), Biology Centre of the Czech Academy of Sciences (BIOLOGY CENTRE CAS), Czech Academy of Sciences [Prague] (CAS)-Czech Academy of Sciences [Prague] (CAS), University of Wisconsin School of Medicine and Public Health, The Natural History Museum [London] (NHM), University of Georgia [USA], University of South Bohemia, University of Oxford [Oxford], AC was funded by the ANR project ACHN 2016 PARASED (ANR-16_ACHN_0003). This work was supported by the Czech Ministry of Education (ERD Funds 019/0000759) to JL. MJ and JL were supported by Institute of Parasitology CAS (RVO:60077344). AM and TAR are both funded by the Royal Society through University Research Fellowships. SI was funded by a French doctoral research grant from Ecole Doctorale des Sciences de la Mer (EDSM) and Region Bretagne. VS was supported by the EU Research and Innovation Programme Horizon 2020 (Grant No. H2020-MSCA-ITN-2015-675752). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., ANR-16-ACHN-0003,PARASED,Ecologie fonctionnelle des protistes parasites dans le compartiment benthique(2016), European Project: 675752,H2020,H2020-MSCA-ITN-2015,SINGEK(2016), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and University of Oxford

Subjects
0106 biological sciences, Life Cycles, Parasitic intestinal diseases, 01 natural sciences, Pearls, White Blood Cells, Animal Cells, Medicine and Health Sciences, Biology (General), Protozoan Infections, Animal, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Larva, biology, Eukaryota, Protists, Amphibian, Parasitic diseases, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Alveolata, Parasitic Intestinal Diseases, Vertebrates, Frogs, Anura, Cellular Types, QH301-705.5, Immune Cells, Immunology, Zoology, Infections, 010603 evolutionary biology, Microbiology, Alveolate, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, Amphibians, 03 medical and health sciences, Virology, biology.animal, Protozoan infection, Genetics, medicine, Animals, Molecular Biology, 030304 developmental biology, Parasitic life cycles, Blood Cells, Macrophages, Acl, Organisms, Biology and Life Sciences, Cell Biology, RC581-607, medicine.disease, biology.organism_classification, Parasitology, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, Immunologic diseases. Allergy, Tadpoles, Developmental Biology
Abstract

International audience

Published
2020
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3. Intracellular Infection of Diverse Diatoms by an Evolutionary Distinct Relative of the Fungi

Author

Finlay Maguire, Javier del Campo, Bente Edvardsen, Sarah Itoïz, Adam Monier, Aurélie Chambouvet, Wenche Eikreim, Philippe Elies, Thomas A. Richards, Laboratoire des Sciences de l'Environnement Marin (LEMAR) (LEMAR), Institut de Recherche pour le Développement (IRD)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Université de Brest (UBO)-Institut Universitaire Européen de la Mer (IUEM), Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), University of Exeter, Faculty of Computer Science, Dalhousie University [Halifax], Rosenstiel School of Marine and Atmospheric Science (RSMAS), University of Miami [Coral Gables], plateforme technologique RMN-RPE-SM, Université de Brest (UBO), University of Oslo (UiO), ANR-16-ACHN-0003,PARASED,Ecologie fonctionnelle des protistes parasites dans le compartiment benthique(2016), and Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Institut national des sciences de l'Univers (INSU - CNRS)-Université de Brest (UBO)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, BACTERIAL, PARASITES, Lineage (evolution), [SDV]Life Sciences [q-bio], [SDV.BID]Life Sciences [q-bio]/Biodiversity, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, General Biochemistry, Genetics and Molecular Biology, Host-Parasite Interactions, 03 medical and health sciences, 0302 clinical medicine, Holomycota, Phytoplankton, Ecosystem, 14. Life underwater, TREE, In Situ Hybridization, Fluorescence, Phylogeny, [SDV.EE]Life Sciences [q-bio]/Ecology, environment, Diatoms, biology, Primary producers, Ecology, Host (biology), RIBOSOMAL-RNA SEQUENCES, ACL, fungi, Fungi, 15. Life on land, Ribosomal RNA, biology.organism_classification, LIBRARIES, INTERFACE, ALIGNMENT, 030104 developmental biology, Diatom, 13. Climate action, DISCOVERY, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery, MARINE DIATOMS, [SDV.EE.IEO]Life Sciences [q-bio]/Ecology, environment/Symbiosis
Abstract

The Fungi are a diverse kingdom, dominating terrestrial environments and driving important ecologies. Although fungi, and the related Opisthosporidia, interact with photosynthetic organisms on land and in freshwater as parasites, symbionts, and/or saprotrophic degraders [1, 2], such interactions in the marine environment are poorly understood [3, 4, 5, 6, 7, 8]. One newly identified uncultured marine lineage has been named novel chytrid-like-clade-1 (NCLC1) [4] or basal-clone-group-I [5, 6]. We use ribosomal RNA (rRNA) encoding gene phylogenies to demonstrate that NCLC1 is a distinct branch within the Opisthosporidia (Holomycota) [7]. Opisthosporidia are a diverse and largely uncultured group that form a sister branch to the Fungi or, alternatively, the deepest branch within the Fungi, depending on how the boundary to this kingdom is inferred [9]. Using culture-free lineage-specific rRNA-targeted fluorescent in situ hybridization (FISH) microscopy, we demonstrate that NCLC1 cells form intracellular infection of key diatom species, establishing that intracellular colonization of a eukaryotic host is a consistent lifestyle across the Opisthosporidia [8, 9, 10, 11]. NCLC1 infection-associated loss and/or envelopment of the diatom nuclei infers a necrotrophic-pathogenic interaction. Diatoms are one of the most diverse and ecologically important phytoplankton groups, acting as dominant primary producers and driving carbon fixation and storage in many aquatic environments [12, 13, 14]. Our results provide insight into the diversity of microbial eukaryotes that interact with diatoms. We suggest that such interactions can play a key role in diatom associated ecosystem functions, such as the marine carbon pump through necrotrophic-parasitism, facilitating the export of diatoms to the sediment [15, 16].

Published
2019
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