Transmembrane carriers of cardenolide-adapted leaf beetles (Coleoptera, CHRYSOMELIDAE) Dissertation with the aim of achieving a doctoral degree at the Faculty of Mathematics, Informatics and Natural Sciences Department of Biology Universität Hamburg submitted by Michael Baum 2015 Day of oral defense: March 22nd, 2016 Admission of the dissertation is recommended by the evaluators 1. Prof. Dr. Susanne Dobler 2. Prof. Dr. Christian Lohr Geheimnisvoll am lichten Tag Läßt sich Natur des Schleiers nicht berauben, Und was sie deinem Geist nicht offenbaren mag, Das zwingst du ihr nicht ab mit Hebeln und mit Schrauben. J.W. Goethe – Faust (verses 672-675) The existence of a badly put-together watch proves the existence of a blind watchmaker. from T. Pratchett – Small Gods Transmembrane carriers in cardenolide-adapted leaf beetles Table of contents Abstract..……………………………………………………..............v Zusammenfassung…………………………………………………..vii Introduction ...................................................................... 1 1. Cardenolides .............................................................................. 4 1.1 Structure and distribution ................................................................ 4 1.2 The Na+/K+-ATPase ......................................................................... 9 2. Insects on cardenolide plants ...................................................... 12 2.1 Target site insensitivity ..................................................................12 2.2 Hide the target ............................................................................... 13 2.3 Sequestration of cardenolides ....................................................... 16 2.4 Investigated species ...................................................................... 18 2.4.1 Lilioceris (CRIOCERINAE) ......................................................... 18 2.4.2 Chrysochus (EUMOLPINAE) ......................................................21 3. Transport Proteins .................................................................... 24 3.1 Oatps ............................................................................................ 25 3.2 ABC transporters ........................................................................... 32 Methods and Materials ................................................... 39 1. Breeding animals ...................................................................... 40 1.1 Myrmica rubra ............................................................................... 40 1.2 Lilioceris ........................................................................................ 41 2. Feeding experiments ..................................................................43 2.1 Tracer feeding of Lilioceris ............................................................. 43 2.2 Feeding experiments with Myrmica ............................................... 44 i Transmembrane carriers in cardenolide-adapted leaf beetles Table of Contents 3. Molecular toolbox ..................................................................... 47 3.1 Isolation of RNA ............................................................................ 47 3.2 Synthesis of cDNA via reverse transcription (RT-) PCR ................... 48 3.3 PCR ............................................................................................... 49 3.4 Agarose gel electrophoresis with nucleic acids ............................... 59 3.5 Restriction endonucleases ............................................................. 60 3.6 Purification of linear dsDNA .......................................................... 61 3.7 Spectrophotometry of nucleic acid solutions.................................. 63 3.8 Molecular cloning .......................................................................... 63 3.9 Site-directed mutagenesis (SDM) ................................................... 73 4. Heterologous expression in Xenopus oocytes ............................... 75 4.1 Extraction of oocytes from X. laevis ............................................... 76 4.2 Production of cRNA ....................................................................... 78 4.3 Injection of cRNA into Xenopus oocytes ......................................... 80 4.4 IHC with oocytes ........................................................................... 80 4.5 Transport assays ........................................................................... 82 5. Heterologous expression in Sf9 insect cells .................................. 85 5.1 Cell culture .................................................................................... 85 5.2 Transient expression ..................................................................... 88 5.3 ICC ................................................................................................ 90 6. Protein analysis ......................................................................... 91 6.1 Beetle immunohistochemistry (IHC) .............................................. 91 6.2 Protein extraction.......................................................................... 93 6.3 Protein quantification (Bradford assay) .......................................... 95 6.4 Western Blot ................................................................................. 96 6.5 Dot Blot ...................................................................................... 100 6.6 Antibodies .................................................................................... 101 7. Computational tools .................................................................105 7.1 Structural predictions of protein conformation ............................. 105 7.2 Local transcriptome databases .................................................... 106 7.3 Sequence editing and analysis ..................................................... 106 7.4 Sequence alignments and phylogeny ............................................ 107 7.5 Statistic tools ............................................................................... 107 7.6 Illustration and image processing.................................................. 107 ii Transmembrane carriers in cardenolide-adapted leaf beetles Table of Contents Results ......................................................................... 109 1. Cardenolides in the fecal mask of Lilioceris merdigera .................. 110 1.1 Tracer feeding .............................................................................. 110 1.2 Myrmica assays ............................................................................ 111 1.3 L. merdigera ABCBs ...................................................................... 115 2. Transport proteins in Chrysochus ............................................... 119 2.1 Oatps ........................................................................................... 119 2.2 ABCB transporter ......................................................................... 144 Discussion ..................................................................... 153 1. Fecal defense and ABCB transporters of L. merdigera .................. 154 1.1 Defensive effects .......................................................................... 154 1.2 Cardenolides are retained in feces of L. merdigera ......................... 158 2. Oatps of Chrysochus species ...................................................... 162 2.1 Structure ...................................................................................... 162 2.2 Phylogeny .................................................................................... 163 2.3 Conserved motifs .........................................................................164 2.4 Tissue distribution ........................................................................ 171 2.5 Transport assays .......................................................................... 173 3. ABCB transporters of C. auratus ................................................ 178 3.1 Phylogeny .................................................................................... 178 3.2 BBB function ................................................................................ 180 References .................................................................... 183 Appendix ...................................................................... 211 1. Abbreviations ................................................................................... 212 2. Primer list ......................................................................................... 218 3. Amino acid identity tables ................................................................. 223 4. ABCB ORF sequences ...................................................................... 225 5. Oatp ORF sequences ........................................................................ 228 6. Oatp TMHMM .................................................................................. 237 7. Oatp alignment ................................................................................ 238 Acknowledgements…………………………………………….….253 iii Transmembrane carriers in cardenolide-adapted leaf beetles Abstract In the constant evolutionary arms race between plants developing new defense mechanisms and herbivorous animals struggling to overcome them, a general strategy of plants is the production of specialized metabolites with toxic or deterrent effects. Insects have evolved methods to circumvent these