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Supplementary Figure 1 Multicenter randomised control trial 2746 randomised 947 control 910 MNP without zinc 889 MNP with zinc 223 lost to follow up 219 lost to follow up 183 lost to follow up 34 refused 29 refused 37 refused 16 died 12 died 9 died 3 excluded 4 excluded 1 excluded 671 in follow-up 646 in follow-up 659 in follow-up at 24mo of age at 24mo of age at 24mo of age Selection for Microbiome sequencing 516 paired samples unavailable 469 paired samples unavailable 497 paired samples unavailable 69 antibiotic use 63 antibiotic use 67 antibiotic use 31 outside of WLZ criteria 37 outside of WLZ criteria 34 outside of WLZ criteria 6 diarrhea last 7 days 2 diarrhea last 7 days 7 diarrhea last 7 days 39 WLZ > -1 at 24 mo 10 WLZ < -2 at 24mo 58 WLZ > -1 at 24 mo 17 WLZ < -2 at 24mo 48 WLZ > -1 at 24 mo 8 WLZ < -2 at 24mo available for selection available for selection available for selection available for selection available for selection1 available for selection1 14 selected 10 selected 15 selected 14 selected 20 selected1 7 selected1 1 Two subjects (one in the reference WLZ group and one undernourished) had, at 12 months, no diarrhea within 1 day of stool collection but reported diarrhea within 7 days prior. Length, cm kg Weight, Supplementary Figure 2. Length (left) and weight (right) z-scores of children recruited into clinical trial NCT00705445 during the first 24 months of life. Median and quantile values are shown, with medians for participants profiled in current study indicated by red (undernourished) and black (reference WLZ) lines. Protozoa, rarefied to 1000 80 reference WLZ undernourished 60 hness c 40 Ri 20 0 CTL MNP MNP + Zn Supplementary Figure 3. Protozoan richness in stool samples, grouped by nutritional status and micronu- trient supplementation. Samples were rarefied to 1000 reads. a age (p<0.001) nutritional status b 12 month reference WLZ (rarefied to 25,000) 2.00 24 month undernourished 1.7 reference WLZ 1.75 undernourished 1.6 3 1.50 1.5 1.25 1.4 Shannon diversity (H’) Shannon diversity (H’) 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 2 Read depth Read depth supplementation site CTL rural MNP 1.7 urban MNP+Zn Shannon diversity (H') 1.8 1 1.6 1.6 1.5 1.4 1.4 Shannon diversity (H’) Shannon diversity (H’) 1.2 0 0 10000 20000 30000 40000 50000 0 10000 20000 30000 40000 50000 Urban Rural Read depth Read depth c (Bray-Curtis) ns ns ns ns ns 0.8 ns 0.7 0.6 0.5 Distance to centroid 0.4 CTL MNP CTL MNP MNP+Zn MNP+Zn 12 month 24 month Supplementary Figure 4. Bacterial evenness and beta diversity. a, Rarefaction curves comparing species evenness (H′) by age group, micronutrient supplementation arm, nuritional status and site. Shaded regions represent standard error. Significance was tested at 25,000 read depth using a generalized linear model with stewise AIC-based model selection to identify significant variables. b, Boxplots comparing bacterial evenness by nutritional status and site. c, Variance among Bray-Curtis dissimilarities, calculated as distance to the centroid, between samples (n=150) by treatment arm and age group. 12 month CTL MNP MNP + Zn Actinomucor Incertae Corynebacterium Cercozoa Lactobacillus Rhizopus Rothia Rhizomucor Lactobacillus ● ● Lactobacillus Rhizopus ● Candida−Lodderomyces clade ● ● ● ● ● Trichosporon Cercomonas ● ● Actinomyces Enterococcus Pichia ● ● ● Bifidobacterium ● ● ● Pediococcus Lactobacillus Wallemia Gemmiger Senegalimassilia ● Wallemia Blastobotrys Cryptosporida ● ● Toxoplasma ActinomucorTrichosporon ● ● Blautia ● ● Colpodella ● Blautia Clostridium_XlVb ● Terrisporobacter ● ● ● ● ● Dorea Pseudomonas ● ● ● Kodamaea Wallemia Eugregarinorida−distant−branch ● Rothia Romboutsia Eggerthella ● ● Conoidasida ● Enterococcus ● ● Romboutsia ● ● Flavonifractor● ● ●Clostridium_sensu_stricto Bifidobacterium● Blautia ● Kluyveromyces ● Trichomonascus ● Intestinibacter ● Candida Corynebacterium ● ● ● Senegalimassilia Saccharomyces Collinsella BlastobotrysAmoebozoa_unknown_group1 ● Pichia ● ● ● ● Lysinibacillus Acinetobacter ● ● ● ● Actinomyces ● ● ● ● Bacillus Collinsella ● ● StreptococcusLichtheimia Bifidobacterium CryptosporidiumSaccharomyces Lachnospiracea_incertae_sedis ● ● Clostridium_XlVb Bifidobacterium Streptococcus Bacillus StenotrophomonasLysinibacillus ● Colpodella ● ● ● ● ● ● ● ● ● ● ● Lactobacillus ● Lachnospiracea_incertae_sedis Ruminococcus2 ● Amylomyces Weissella Syndiniales● ● Trichomonascus ● Dorea Actinomyces Lactobacillus Dorea ● Streptococcus Candida ● ● ● Gregarina−distant−branch ● ● ● Paenibacillus Acinetobacter Eugregarinorida● Gregarinasina Clostridium_sensu_stricto Lachnospiracea_incertae_sedis ● Gregarinasina−distant−branch Gregarina ● Eggerthella ● ● Clostridium_XVIII Cercomonas ● Prevotella Dorea ● ● ● Olsenella Enterocytozoon ● ● Pseudomonas Rhizopus ● Actinomyces ● Bifidobacterium Streptococcus ● ● ● ● ● Anaerostipes ● Intestinibacter ● Bifidobacterium Bifidobacterium Clostridium_sensu_stricto ● ● Syndiniales ● Heteromita Actinomyces Anaerostipes ● ● ● ● ● FaecalibacteriumCandida−Lodderomyces clade ● ● Gemella ● Blastobotrys ● ● Neogregarinorida Actinomyces Collinsella ● ● Lachnospi● racea_incertae_sedis ● ● IncertaeKluyveromyces Lachnospiracea_incertae_sedis Actinomyces Bifidobacterium ● ● Conoidasida ● ● MegasphaeClostridium_XlVbra Faecalibacterium ● ● Lactobacillus Kodamaea ● Bacillus Penicillium Cryptomycota ● ● ● Streptococcus ● Clostridium_sensu_stricto ● Streptococcus ● ● Senegalimassilia Lichtheimiaceae Lichtheimia Eggerthella ● ● ● ● ● ● ● ● ● Pseudomonas ● Candida−Lodderomyces clade Lachnospiracea_incertae_sedisSlackia G●regarina−distant−branch Streptococcus Lachnospiracea_incertae_sedis Tritrichomonas● ● ● ● Urban Alveolata Colpodella Cryptosporidium ● Lachnospiracea_incertae_sedis● ● Lachnospiracea_incertae_sedis Gregarinasina Tritrichomonas ● ● Anaerococcus ● ● Streptococcus Gregarinasina Dorea ● Olsenella Holdemanella ● ● ● ● Lactobacillus ● ● Bromeliothrix ● ● ● ● ● Clostridium_XVIII Acinetobacter ● Bifidobacterium Pseudomonas Ruminococcus2 ● Lactobacillus Penicillium ● ● ● Streptococcus Clostridium_XI Collinsella ● Pediococcus ● Cryptosporidium Dorea Streptococcus ● ● ● ● ● ● Gregarinasina−distant−bCollinsellaranch Fusicatenibacter ● Streptococcus ● ● ● Enterococcus ● ● ● ● ● ● Streptococcus Varibaculum ● Faecalibacterium ● ● Lachnospiracea_incertae_sedis Alveolata ● Clostridium_XI uncultured ● Lactobacillus ● ● ● ● ● Prevotella● Bifidobacterium ● ● ● ● Lactobacillus Penicillium Lactobacillus ● Incertae Mucor Lactobacillus Lactobacillus ● ● ● Streptococcus Lachnospiracea_incertae_sedis Lichtheimia Streptococcus Anaerostipes Akkermansia Lactobacillus ● ● Rhizomucor ● Neogregarinorida● ● ● ● Collinsella Stenotrophomonas Bifidobacterium● Talaromyces ● Cryptosporida Cercozoa ● Lactobacillus Corynebacterium Pediococcus Gregarina Roseburia Lichtheimiaceae● ● ● ● Eubacterium ● ● Collinsella Pediococcus Alveolata Gemmiger ● ● ● Actinomyces ● Ruminococcus2 ● ● Clostridium_sensu_stricto Blautia Pediococcus ● ● Actinomucor ● Clostridium_XVIII ● ● ● Gregarina−distant−branch Blautia ● Pseudomonas ● ● ● ● Clostridium_sensu_stricto ● ● Roseburia ● Finegoldia Enterococcus uncultured Amylomyces ● ● ● ● ● Olsenella Flavonifractor Fusicatenibacter Prevotella ● ● ● Anaerococcus ● Streptococcus ● ● ● ● Paenibacillus ● ● ● ● ● Clostridium_sensu_stricto Saccharomyces Lichtheimiaceae ● Prevotella Flavonifractor Pichia Intestinibacter Finegoldia ● ● Mogibacterium Lactobacillus Blautia Lactobacillus Bifidobacterium ● Clostridium_XVIII Mucor ● ● ● ● ● ● AmylomycesAnaerostipes ● ● ● ● Hanseniaspora ● ● Lactobacillus ● ● ● ● Enterococcus ● ● Streptococcus Clostridium_XVIII Enterococcus ● Clostridium_XVIII Rhizomucor ● Lachnospiracea_incertae_sedis● ● ● ● ● (5) ● (10) ● (9) Saccharomyces AkkermansiaClostridium_XlVb BifidobacteriumRhizomucor Streptococcus Nakaseomyces−Candida clade Gemella Pseudomonas Lactobacillus ● ● Stenotrophomonas Pichia ● ● ● Dorea Bacillus ● ● Mucor Rhizopus ● ● Collinsella ● Penicillium ● CollinsellaAlveolata−distant−branch ● ● BifidobacteriumBifidobacterium ● Blautia Dorea Lactobacillus ● Clostridium_sensu_stricto● ● Pseudomonas Streptococcus ● ● ● ● Olsenella Cryptomycota ● Rothia ● ● Eugregarinorida−distant−branch ● PseudomonasEugregarinorida−distant−b● ranch ● ● ● Bifidobacterium CercomonasClostridium_XVIII ● Atopobium ● Geotrichum● Candida−Lodderomyces clade ● ● Blautia ● ● ● Neogregarinorida Syndiniales Stenotrophomonas Actinomyces Lactobacillus ● Cercozoa● PucciniaEugregarinorida−distant−branch Varibaculum ● ● ● Lactobacillus ● ● ● ● Lichtheimiaceae Lactobacillus Trichomonascus ● ● Lactobacillus Heteromita Geotrichum ● Lichtheimia ● ● Talaromyces Actinomyces● Lactobacillus● ● Streptococcus ● Actinomucor ● WallemiaSaccharomyces Cercomonas ● Lactobacillus● Euglyphida ● ● Atopobium ● ● Holdemanella Anaerococcus Lactobacillus Hanseniaspo● r●a ● ● ● Prevotella● ● Cercomonas ● ● ActinomycesPenicillium● ● ● ● LactobacillusCandida−Lodderomyces clade Gregarina Actinomyces ●LactobacillusPediococcus Lactobacillus ● Wicherhamiella−Candida clade Eugregarinorida ● ● ● ● Weissella● Streptococcus● Gregarinasina−distant−branch uncultured ● Incertae ● ● Amylomyces ● ● Lactobacillus Amoebozoa_unknown_group3 ● Romboutsia ● ● ● Prevotella Pediococcus ConoidasidaAnaerostipes ● Neogregarinorida ● Gemella Eugregarinorida Clostridium_XlVb Toxoplasma Gordonibacter Alveolata−distant−branch ● Mucor Collinsella● ● ● ● Enterococcus ● ● Eggerthella Gemmiger Lactobacillus ● ● ● ● Actinomyces Candida Weissella ● Hanseniaspora Alveolata−distant−branch
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  • Predatory Flagellates – the New Recently Discovered Deep Branches of the Eukaryotic Tree and Their Evolutionary and Ecological Significance

    Predatory Flagellates – the New Recently Discovered Deep Branches of the Eukaryotic Tree and Their Evolutionary and Ecological Significance

    Protistology 14 (1), 15–22 (2020) Protistology Predatory flagellates – the new recently discovered deep branches of the eukaryotic tree and their evolutionary and ecological significance Denis V. Tikhonenkov Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, 152742, Russia | Submitted March 20, 2020 | Accepted April 6, 2020 | Summary Predatory protists are poorly studied, although they are often representing important deep-branching evolutionary lineages and new eukaryotic supergroups. This short review/opinion paper is inspired by the recent discoveries of various predatory flagellates, which form sister groups of the giant eukaryotic clusters on phylogenetic trees, and illustrate an ancestral state of one or another supergroup of eukaryotes. Here we discuss their evolutionary and ecological relevance and show that the study of such protists may be essential in addressing previously puzzling evolutionary problems, such as the origin of multicellular animals, the plastid spread trajectory, origins of photosynthesis and parasitism, evolution of mitochondrial genomes. Key words: evolution of eukaryotes, heterotrophic flagellates, mitochondrial genome, origin of animals, photosynthesis, predatory protists, tree of life Predatory flagellates and diversity of eu- of the hidden diversity of protists (Moon-van der karyotes Staay et al., 2000; López-García et al., 2001; Edg- comb et al., 2002; Massana et al., 2004; Richards The well-studied multicellular animals, plants and Bass, 2005; Tarbe et al., 2011; de Vargas et al., and fungi immediately come to mind when we hear 2015). In particular, several prevailing and very abun- the term “eukaryotes”. However, these groups of dant ribogroups such as MALV, MAST, MAOP, organisms represent a minority in the real diversity MAFO (marine alveolates, stramenopiles, opistho- of evolutionary lineages of eukaryotes.
  • Why the –Omic Future of Apicomplexa Should Include Gregarines Julie Boisard, Isabelle Florent

    Why the –Omic Future of Apicomplexa Should Include Gregarines Julie Boisard, Isabelle Florent

    Why the –omic future of Apicomplexa should include Gregarines Julie Boisard, Isabelle Florent To cite this version: Julie Boisard, Isabelle Florent. Why the –omic future of Apicomplexa should include Gregarines. Biology of the Cell, Wiley, 2020, 10.1111/boc.202000006. hal-02553206 HAL Id: hal-02553206 https://hal.archives-ouvertes.fr/hal-02553206 Submitted on 24 Apr 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Article title: Why the –omic future of Apicomplexa should include Gregarines. Names of authors: Julie BOISARD1,2 and Isabelle FLORENT1 Authors affiliations: 1. Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245), Département Adaptations du Vivant (AVIV), Muséum National d’Histoire Naturelle, CNRS, CP52, 57 rue Cuvier 75231 Paris Cedex 05, France. 2. Structure et instabilité des génomes (STRING UMR 7196 CNRS / INSERM U1154), Département Adaptations du vivant (AVIV), Muséum National d'Histoire Naturelle, CP 26, 57 rue Cuvier 75231 Paris Cedex 05, France. Short Title: Gregarines –omics for Apicomplexa studies
  • Tracing the Origin of Planktonic Protists in an Ancient Lake

    Tracing the Origin of Planktonic Protists in an Ancient Lake

    microorganisms Article Tracing the Origin of Planktonic Protists in an Ancient Lake Nataliia V. Annenkova 1,* , Caterina R. Giner 2,3 and Ramiro Logares 2,* 1 Limnological Institute Siberian Branch of the Russian Academy of Sciences 3, Ulan-Batorskaya St., 664033 Irkutsk, Russia 2 Institute of Marine Sciences (ICM), CSIC, Passeig Marítim de la Barceloneta, 37-49, ES08003 Barcelona, Spain; [email protected] 3 Institute for the Oceans and Fisheries, University of British Columbia, 2202 Main Mall, Vancouver, BC V6T 1Z4, Canada * Correspondence: [email protected] (N.V.A.); [email protected] (R.L.) Received: 26 February 2020; Accepted: 7 April 2020; Published: 9 April 2020 Abstract: Ancient lakes are among the most interesting models for evolution studies because their biodiversity is the result of a complex combination of migration and speciation. Here, we investigate the origin of single celled planktonic eukaryotes from the oldest lake in the world—Lake Baikal (Russia). By using 18S rDNA metabarcoding, we recovered 1414 Operational Taxonomic Units (OTUs) belonging to protists populating surface waters (1–50 m) and representing pico/nano-sized cells. The recovered communities resembled other lacustrine freshwater assemblages found elsewhere, especially the taxonomically unclassified protists. However, our results suggest that a fraction of Baikal protists could belong to glacial relicts and have close relationships with marine/brackish species. Moreover, our results suggest that rapid radiation may have occurred among some protist taxa, partially mirroring what was already shown for multicellular organisms in Lake Baikal. We found 16% of the OTUs belonging to potential species flocks in Stramenopiles, Alveolata, Opisthokonta, Archaeplastida, Rhizaria, and Hacrobia.
  • Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut

    Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut

    Microorganisms 2018, 6, 119 1 of 20 Article Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut Chiara Nocciolini 1, Claudio Cucini 2, Chiara Leo 2, Valeria Francardi 3, Elena Dreassi 4 and Antonio Carapelli 2,* 1 Polo d’Innovazione di Genomica Genetica e Biologia, 53100 Siena, Italy; [email protected] 2 Department of Life Sciences, University of Siena, 53100 Siena, Italy; [email protected] (C.C.); [email protected] (C.L.) 3 Consiglio per la ricerca in agricoltura e analisi dell’economia agraria – Centro di Difesa e Certificazione CREA-DC) Research Centre for Plant Protection and Certification, via di Lanciola 12/A, 50125 Firenze, Italy; [email protected] 4 Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-0577-234-410 Received: 28 September 2018; Accepted: 23 November 2018; Published: 27 November 2018 Abstract: Protozoa, of the taxon Gregarinasina, are a heterogeneous group of Apicomplexa that includes ~1600 species. They are parasites of a large variety of both marine and terrestrial invertebrates, mainly annelids, arthropods and mollusks. Unlike coccidians and heamosporidians, gregarines have not proven to have a negative effect on human welfare; thus, they have been poorly investigated. This study focuses on the molecular identification and phylogeny of the gregarine species found in the midgut of two insect species that are considered as an alternative source of animal proteins for the human diet: the mealworm Tenebrio molitor, and the super-worm Zophobas atratus (Coleoptera: Tenebrionidae).