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Diversity and Functions of Protozoa in Soils EcoFINDERS Stefan Geisen University of Cologne ‐ AG Bonkowski 05 October 2011 Identification Intro Sequencing Interaction studies Outlook Diversity of Protozoa in Soils • • • • Single‐celled, heterotrophic eukaryots Up to 100,000 individuals/gram soil Reproduction asexually Produce cysts to resist adverse conditions • Classically split into three morphological distinct groups – Flagellates (one ore more flagella) tolweb.org starcentral.mbl.edu – Ciliates (many short, hair‐like cilia) www5.pbrc.hawaii.edu – Amoeba (one or more pseudopodia) Stefan Geisen designmatrix.wordpress.com bms.ed.ac.uk Protozoa in Ecofinders Identification Intro • Little known due to – Small size – Difficult isolation and cultivation – High diversity • Case study: amoebae Outlook Interaction studies Sequencing Diversity of Protozoa in Soils Modified from http://www.natur.cuni.cz/biologie/veda-a-vykum/vyzkumne-tymy/evolucni%20-%20protistologie/fylogenetika.jpg Stefan Geisen Protozoa in Ecofinders Identification Intro • Little known due to – Small size – Difficult isolation and cultivation – High diversity • Case study: amoebae – Absence of clear cell shape – ↑Variability → Hard to identify Hartmanella sp. Interaction studies Sequencing Diversity of Protozoa in Soils Outlook 10 µm Acanthamoeba sp. Stefan Geisen Cochliopodium sp. Vanella sp. Protozoa in Ecofinders Flamella sp. Identification Intro Sequencing Importance of Soil Protozoa • Respiration and biomass of soil organisms – A comparison of 14 ecosystem studies (Foissner 1996) 100% Respiration (%) Biomass (2.7 g dwt m‐²) animals arthropods Interaction studies 80% arthropods ‚worms‘ 60% 40% bacteria + fungi 20% Outlook Respiration (%) ‚worms‘ protozoa 69% protozoa 31% 0% Stefan Geisen Protozoa in Ecofinders • Grazers of bacteria – Control bacterial energy channel Outlook Interaction studies Sequencing Identification Intro Importance of Soil Protozoa eusoils.jrc.ec.europa.eu Stefan Geisen Protozoa in Ecofinders • Grazers of bacteria – Control bacterial energy channel – Feed selectively on bacteria → Positive feed‐back on plants Outlook Interaction studies Sequencing Identification Intro Importance of Soil Protozoa Modified from Bonkowski (2004) Stefan Geisen Protozoa in Ecofinders Identification Intro • Lab experiments confirm functional importance, BUT no knowledge on dominant taxa and diversity in soil – Cultivate and identify protozoan species from sampling sites – Find genetic barcodes for soil protozoa – Use those to compare different European sites via high‐throughput sequencing Outlook Interaction studies Sequencing Goals within EcoFINDERS leben.audena.de Stefan Geisen belecker-chronik.de maehdreschen.de8 Protozoa in Ecofinders Identification Intro Sequencing Interaction studies Outlook Identification of Soil Amoeba • Combination of morphology and phylogeny • First clonal cultures from Sardina (Italy) Acanthamoeba sp. Hartmanella sp. ??? sp. 10 µm Korotnevella sp. Stefan Geisen Cochliopodium sp. Vannella sp. Protozoa in Ecofinders Cochliopodium sp. Identification Intro • Barcoding – Specific sequence targeting a single taxon • Examples – Cytochrome C Oxidase I (CO1) – Small Subunit Ribosomal DNA (SSU rDNA) Outlook Interaction studies Sequencing Barcoding and Sequencing http://mic.sgmjournals.org/content/14 6/5/1045/F1.expansion.html Stefan Geisen Protozoa in Ecofinders Identification Intro Sequencing Interaction studies Barcoding and Sequencing • Barcoding – Specific sequence targeting a single taxon • Examples – Cytochrome C Oxidase I (CO1) – Small Subunit Ribosomal DNA (SSU rDNA) • BUT: No barcodes for protozoa! – Very diverse →Eukaryo c primers do not work Modified from http://www.natur.cuni.cz/biologie/veda-a-vykum/vyzkumnetymy/evolucni%20-%20protistologie/fylogenetika.jpg • Group specific primers needed Outlook – Used for comparison of European soils via high‐throughput sequencing Stefan Geisen Protozoa in Ecofinders Identification Intro • Evaluate interactions with other organisms → ecological and economical value of protozoa • Case study – Interaction study between protozoa and different bacteria Outlook Interaction studies Sequencing Functional Importance of Protozoa ? magicsoil.com Stefan Geisen Protozoa in Ecofinders Identification Intro Sequencing Interaction studies Outlook Interaction Study ‐ Setup • Organisms – Bacteria • Bacillus subtilis • Corynebacterium glutamicum (RFP labeled) • Pseudomonas fluorescence (GFP labeled) – Protozoa • Acanthamoeba castellanii • Tetrahymena pyriformis – Added in a full factorial design in a 96 well plate • Automatic plate reader constantly measuring for 3 days – Optical density (OD) – Relative fluorescence units (RFU) from GFP and RFP signals Stefan Geisen Protozoa in Ecofinders Identification Intro • Presence of Acanthamoeba and Tetrahymena → higher feeding pressure → increased reduction of bacteria • Grazing protection by – Bacillus through filaments and spores – Pseudomonas through production of toxic compounds Outlook Interaction studies Sequencing Interaction Study ‐ Hypotheses Stefan Geisen Protozoa in Ecofinders • Bacteria – Growth rate: Pseudomonas > Corynebacterium > Bacillus – Strong intra‐bacterial competition → Bacillus and Corynebacterium inhibited → Pseudomonas profits RFU (RFP)/OD Identification Intro Sequencing Interaction studies Outlook Interaction Study ‐ Results Measuring cycles Stefan Geisen Protozoa in Ecofinders • Protozoa – Food preference of protozoa • Bacillus > Corynebacterium > Pseudomonas – Strong competition between protozoa – Presence of Bacillus as prey for Tetrahymena Outlook → Corynebacterium and Pseudomonas benefit AxB 100 µm RFU (RFP)/OD Interaction studies Sequencing Identification Intro Interaction Study ‐ Results Measuring cycles Stefan Geisen AxTxB Protozoa in Ecofinders 100 µm Identification Intro Sequencing Interaction studies Outlook Interaction Study ‐ Discussion • Hypotheses – Presence of Acanthamoeba and Tetrahymena → higher feeding pressure → increased reduction of bacteria – Grazing protection by • Bacillus through filaments and spores • Pseudomonas through production of toxic compounds • Diversity of bacteria AND protozoan determines microbial community Stefan Geisen Protozoa in Ecofinders Identification Intro Sequencing Outlook • Further experiments evaluating – Detailed response of protozoan numbers → Labeling protozoa – Effects of protozoan diversity on bacteria → Add protozoan species • Determine influence of bacterial life stage on protozoa with spores of Bacillus Outlook Interaction studies → Do soil protozoa serve as trojan horses for (pathogenic) bacteria? • … Stefan Geisen Protozoa in Ecofinders Identification Intro Sequencing Interaction studies Outlook Acknowledgements • Philippe Lemanceau • Michael Bonkowski • Jan Weinert • Anne Winding AARHUS UNIVERSITY Thank you for your attention! Stefan Geisen Protozoa in Ecofinders • Bacteria – Growth rate: Pseudomonas > Corynebacterium > Bacillus Outlook OD Interaction studies Sequencing Identification Intro Interaction Study ‐ Results Measuring cycles Stefan Geisen Protozoa in Ecofinders • Bacteria – Growth rate: Pseudomonas > Corynebacterium > Bacillus RFU (RFP)/OD Interaction studies Outlook RFU (GFP)/OD – Presence of other bacteria leads to strong competition Pseudomonas profits Bacillus and Corynebacterium inhibited Sequencing Identification Intro Interaction Study ‐ Results Measuring cycles Stefan Geisen Protozoa in Ecofinders OD Sequencing Identification Intro Interaction studies ‐ Results Outlook Interaction studies 100 µm 100 µm Stefan Geisen 100 µm Protozoa in Ecofinders Identification Intro Interaction Study ‐ Results • Protozoa – Food preference of protozoa • Bacillus> Corynebacterium > Pseudomonas Outlook • Tetrahymena superior grazer – Presence of Bacillus as prey for Tetrahymena RFU (GFP)/OD Corynebacterium and Pseudomonas benefit RFU (RFP)/OD Interaction studies Sequencing – Strong competition between protozoa for food Measuring cycles Stefan Geisen Protozoa in Ecofinders Outlook Interaction studies Sequencing Identification Intro GFP/OD Stefan Geisen Protozoa in Ecofinders Outlook Interaction studies Sequencing Identification Intro RFP/OD Stefan Geisen Protozoa in Ecofinders Outlook Interaction studies Sequencing Identification Intro OD Stefan Geisen Protozoa in Ecofinders • Trophozoit and cyst of Acanthaamoeba castellanii Outlook Interaction studies Sequencing Identification Intro Life stage Acanthamoeba Stefan Geisen Protozoa in Ecofinders Identification Intro • Protozoa – Food preference • Bacillus > Corynebacterium > Pseudomonas – Strong competition between protozoa • Tetrahymena superior grazer AxTxB 100 µm Outlook Interaction studies Sequencing Interaction Study ‐ Results 100 µm AxB Stefan Geisen AxC Protozoa in Ecofinders 100 µm – Control bacterial energy channel – Also fungal feeding protozoa present – Feed selectively on bacteria → Positive feed‐back on plants Shoot biomass (mg dry wt‐1) Identification Intro • Grazers of bacteria Root biomass (mg dry wt‐1) Outlook Interaction studies Sequencing Importance of Soil Protozoa Modified from Bonkowski (2004) Stefan Geisen Protozoa in Ecofinders • Pyrosequencing /454 (Roche) Outlook Interaction studies Sequencing Identification Intro High‐Throughput Sequencing http://www.nature.com/nmeth/journal/v2/n10/images/nmeth800‐F1.gif Advantages Disadvantages Culture‐independent Only information: DNA sequence Multi‐parallel‐sequencing >1,000,000 sequences/run Complex bioinformatics Almost fully automated PCR bias Cheap price/base High total cost Stefan Geisen Protozoa in Ecofinders Identification Intro Sequencing Interaction studies Outlook Foraminifera Haplosporidae Cercozoa Apicomplexa Dinoflagellates Radiolaria Ciliates Green algae + plants Stramenopiles Red algae Cryptophytes Glaucophyta Telonema Breviata Haptophytes Lobose amoebae Heterolobosea Euglenozoa Slime moulds Jakobids Entamoebae Malawimonas Pelobionts Animals Oxymonads, Trimastix Choanoflagellates Trichomonads Ichthyosporea Nuclearid amoeba Centrohelids Diplomonads Retortamonads Carpediemonas Microsporida and Apusomonads Fungi Collodictyonids Modified from http://www.natur.cuni.cz/biologie/veda-a-vykum/vyzkumne-tymy/evolucni%20-%20protistologie/fylogenetika.jpg Stefan Geisen Protozoa in Ecofinders
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