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BACHELOR THESIS Surveillance of Phytoplankton Key Species BACHELOR THESIS Surveillance of Phytoplankton Key Species in the “AWI-HAUSGARTEN” (Fram Strait) 2010-2013 via Quantitative PCR by Sebastian Micheller Matriculation No.: 739820 in the Bachelor Degree Course Biotechnology – Dept. of Applied Natural Sciences – Hochschule Esslingen, Germany tendered at July 1st, 2014 First Examiner: Prof. Dr. Dirk Schwartz 1 Second Examiner: Dr. Katja Metfies 2 Spaced out till: August 1st, 2015 1 Hochschule Esslingen, 73728 Esslingen a. N., Germany 2 Alfred-Wegener-Institute for Polar & Marine Research, 27570 Bremerhaven, Germany STATEMENT OF AUTHORSHIP I declare that the thesis Surveillance of Phytoplankton Key Species in the „AWI-HAUSGARTEN“ (Fram Strait) 2010-2013 via Quantitative PCR has been composed by myself, and describes my own work, unless otherwise acknowledged in the text. It has not been accepted in any previous application for a degree. Esslingen a. N., July 1st, 2014 Place/date Sebastian Micheller I ACKNOWLEDGMENTS First and foremost, I would like to express my deep gratitude to Dr. Katja Metfies, my supervisor at the Alfred-Wegener-Institute, Bremerhaven. Her patient guidance, enthusiastic encouragement and useful critiques were the cornerstones, making this thesis possible. Beside her, my supervisor Prof. Dr. Dirk Schwartz (Hochschule Esslingen) deserves my special thanks. During my studies at the Hochschule Esslingen, he inspired me for the field of molecular biology, being always a great mentor and support - even across the great distance during this study (Bremerhaven – Esslingen). I feel very great appreciation for them, giving me chances to grow as a person and as a scientist. I would also like to thank Dr. Christian Wolf and Dr. Estelle Kilias for their valuable and constructive suggestions during the planning, development and execution of this work. The willingness to give their time and energy so generously has been very much appreciated and was vital during setbacks and dead ends, appearing within this study. Another person, always helpful in so many ways was Kerstin Oetjen – her advice and assistance increased my motivation, scientific comprehension and confidence at the AWI. In additon, the remaining members of my working group has to be mentioned positively here: Kristin Hardge and Johanna Hessel always had a sympathetic ear for my problems during laboratory work, bioinformatical anlayses and evaluation processes of this study. I want to thank them for their advice and support. I like to thank Dr. Steffi Gäbler-Schwarz (cultivation/isolation/DNA-extraction of Phaeocystis spp.), Dr. Eva-Maria Nöthig (microscopic observations) and Annegret Müller (sequencing approaches) for their contributions to my thesis. Summing up, I had a great time joining the AWI – Helmholtz-University Young Investigator Group PLANKTOSENS. The positive experiences and achievements I gained during this time – no matter how difficult it has been sometimes – defininetly overweigh and I am pleased to say that I found new friends: Thank you for making a Bavarian feel like home in Northern Germany! PS: Not forgetting Juliane Riedel and her/my friends of the Havenhostel Bremerhaven. II LIST OF ABBREVIATIONS (1 of 2) Abbreviation Explanation Abbreviation Explanation Denaturating Gradient Gel Ø Average DGGE Electrophoresis % Percent DMS Dimethylsulfide °C Degree Celsius DMSO Dimethyl sulfoxide µL Microliter DNAa Deoxyribonucleic Acid µm Micrometer DOI Digital Object Identifier A Adenosine dsDNA Double-strand DNAa For example AC Accession Number e.g. (lat.: exempli gratia ) Amplified Fragment Length AFLP eds Editors Polymorphism Tree (lat.: arbor) (Software ARB EGC East Greenland Current package) European Multidisciplinary ARK Arctic cruise of Polarstern EMSO Seafloor Observatory European Seas Observatory AW Atlantic Water ESONET Network AWI Alfred-Wegener-Institute et al. And others (lat.: et alii/et aliae ) Federation of European A Absorbtion at wavelength X FEMS XXXnm Microbiological Societies Basic Local Alignment Search BLAST Fig. Figure(s) Tool Fluorescence In Situ BMC BioMed Central FISH Hybridization bp/bps Basepair/s FR France Fluorescence Resonance Energy BSA Bovine Serum Albumin FRET Transfer C Cytosine G Guanine Culture Collection of Algae and CCAP GC% GC-content in percent Protozoa Carbon dioxide concentration CCM h Hour(s) mechanism Culture Collection of Marine CCMP H2O Water Phytoplankton CO2 Carbon dioxide HAB Harmful Algae Blooms High-Performance Liquid CT Threshold cycle HPLC Chromatography Conductivity, Temperature and CTD i.e. That is (lat.: id est) Pressure (probe) International Council for the d Day(s) ICES Exploration of the Sea International Society for D1 Applied Dilution ISME Microbial Ecology D2 Searched Dilution km Kilometer Dideoxy Nucleoside ddNTP/s km2 Square kilometer Triphosphate/s deion. deionized km3 Cubic kilometer III LIST OF ABBREVIATIONS (2 of 2) Abbreviation Explanation Abbreviation Explanation Quantitative Polymerase Chain L Liter qPCR Reaction LM Light Microscopy ® Registered Trade Mark LTER Long-Term Ecological Research r2 Correlation coefficient m Meter RAC Return Atlantic Current Random Amplification of m. a. Modified after RAPD Polymorphic DNA MAST Marine Stramenopila RCC Roscoff Culture Collection Molecular Evolutionary Genetics Ribosomal Desoxyribonucleic MEGA rDNA Analysis (Software) Acid Mg2+ Magnesium ion (divalent) resp. Respectively Restriction Fragment Length min. Minute(s) RFLP Polymorphism mL Milliliter Rn Normalized ratio mm Millimeter RT Room Temperature Ribulose-1.5-bisphosphate mmol Millimolar RuBisCO carboxylase/oxygenase MT Megatons s Second(s) Stramenopila, Alveolata, Rhizaria n. a. Not available SAR group n. d. Not determined SD Standard Deviation n. p. Not published SEM Scanning Electron Microscopy n. s. Not specified sp./spp. Species (singular/plural) Single-stranded Conformation NAO North Atlantic Oscilation SSCP Polymorphism National Aeronautics and Space NASA ssDNA Single-strand DNAa Administration Norwegian Atlantic Slobe NASC SSU Small subunit Current National Center for NCBI T Thymine Biotechnology Information National Center for Marine Algae NCMA Ta Annealing temerature and Microbiota nm Nanometer Tm Melting temperature nt/nts Nucleotide/s TM Unregistered Trade Mark p-value in statistical significance p UK United Kingdom testing p. Page USA United States of America PAL Photosynthetic Available Light V Volt Variable 18S rDNA region PCR Polymerade Chain Reaction V4 number 4 preps Preperations WSC West Spitzbergen Current PW Pacific Water λ Wavelength IV LIST OF CONTENTS 1. ABSTRACT ........................................................................................................................................... 1 2. GENERAL INTRODUCTION ............................................................................................................ 3 2.1 Arctic Environment ......................................................................................................................... 3 2.1.1 AbioticCharacteristics ....................................................................................................... 3 2.1.2 Fram Strait, Currents and Deep-Sea Observatory HAUSGARTEN .................................. 4 2.1.3 Arctic Climate Change ...................................................................................................... 6 2.2 Arctic Ecosystem .............................................................................................................................. 7 2.2.1 Marine Arctic Food Web ................................................................................................... 7 2.2.2 Protista Diversity and Ultraplankton ................................................................................ 8 2.2.3 Phytoplankton Key Species – E. huxleyi, M. pusilla & P. pouchetii ............................... 10 2.3 Methods for Taxonomical Classification of Marine Protista Communities .................................. 14 2.3.1 Traditional Methods ........................................................................................................ 14 2.3.2 Genetic Targets – 18S rDNA ........................................................................................... 15 2.3.3 Molecular Methods – Quantitative Polymerase Chain Reaction .................................... 15 3. MATERIAL ......................................................................................................................................... 19 3.1 Equipment and Consumables ........................................................................................................ 19 3.2 Commercial Kits ........................................................................................................................... 20 3.3 Chemicals ...................................................................................................................................... 21 3.4 Buffers and Stock Solutions .......................................................................................................... 22 3.5 Media ............................................................................................................................................ 23 3.6 Microorganisms ............................................................................................................................ 24 3.7 Samples of Nucleic Acid – Retrospective Treatment.................................................................... 25 3.8 Primer and Probes ........................................................................................................................
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