Holdings: The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus

The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus: = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus

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Bibliographic Details
Main Author:	Kupper, Maria (Author)
Format:	Thesis Book
Language:	English
Published:	Würzburg September 2016
Subjects:	Symbiose Oogenese Camponotus floridanus Hochschulschrift
Online Access:	Volltext Inhaltsverzeichnis
Physical Description:	238 Seiten Illustrationen, Diagramme 1 CD-Rom: Digital Appendix

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adam_text	B AYERISCHE J ULIUS -M AXIMILIANS -U NIVERSITAET W UERZBURG F AKULTAET FUER B IOLOGIE L EHRSTUHL FUER M IKROBIOLOGIE THE IMMUNE TRANSCRIPTOME AND PROTEOME OF THE ANT CAMPONOTUS FLORIDANUS AND VERTICAL TRANSMISSION OF ITS BACTERIAL ENDOSYMBIONT BLOCHMANNIA FLORIDANUS D I S S E R T A T I O N ZUR ERLANGUNG DES NATURWISSENSCHAFTLICHEN DOKTORGRADES DER BAYERISCHEN JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG VORGELEGT VON MARIA KUEPPER AUS LUTHERSTADT EISLEBEN WUERZBURG, SEPTEMBER 2016 TABLE OF CONTENTS SUMMARY.......................................................................................................................................... 1 ZUSAMMENFASSUNG.......................................................................................................................... 5 1 INTRODUCTION........................................................................................................................ 9 1.1 THE NATURE O F BACTERIAL ENDOSYMBIOSES IN INSECTS..................................................................................... 9 1.2 STRATEGIES FO R THE VERTICAL TRANSMISSION O F INSECT ENDOSYMBIONTS...........................................................11 1.3 THE INSECT IMMUNE SYSTEM........................................................................................................................ 15 1.3.1 MICROBIAL RECOGNITION....................................................................................................................... 16 1.3.2 THE THREE MAIN IMMUNE SIGNALLING PATHWAYS ................................................................................ 17 1.3.3 PHAGOCYTOSIS.......................................................................................................................................19 1.3.4 MELANISATION...................................................................................................................................... 19 1.3.5 THE SYSTEMIC IMMUNE RESPONSE - FAT BODY AMP PRODUCTION.......................................................20 1.3.6 SOCIAL IMMUNITY................................................................................................................................ 22 1.4 ENDOSYMBIONT TOLERANCE AND REGULATION VIA THE INSECT HOST IMMUNE SYSTEM ......................................... 24 1.5 THE SYMBIOSIS O F CAMPONOTUS FLORIDANUS AND BLOCHMANNIA FLORIDANUS .............................................. 27 1.6 THE AIM O F THIS WORK................................................................................................................................. 30 2 MATERIAL..............................................................................................................................32 2.1 CAMPONOTUS FLORIDANUS COLONIES...............................................................................................................32 2.2 BACTERIAL STRAINS AND CELL LINES..................................................................................................................32 2.3 OLIGONUCLEOTIDES........................................................................................................................................ 33 2.3.1 OLIGONUCLEOTIDES FOR RECOMBINANT EXPRESSION OF HYMENOPTAECIN DERIVED PEPTIDES IN DIFFERENT EXPRESSION SYSTEMS............................................................................................................................ 33 2.3.2 OLIGONUCLEOTIDES FOR VALIDATION OF ILLUMINA SEQUENCING DATA VIA QRT-PCR ............................... 34 2.3.3 OLIGONUCLEOTIDES FOR GENE EXPRESSION ANALYSIS IN OVARIAN TISSUE VIA QRT-PCR .......................... 35 2.4 PLASMIDS......................................................................................................................................................36 2.5 ANTIBODIES.................................................................................................................................................. 36 2.6 CULTURE MEDIA, SOLUTIONS AND BUFFERS...................................................................................................... 37 2.6.1 BACTERIAL CULTURE MEDIA AND SUPPLEMENTARY SOLUTIONS.................................................................37 2.6.2 BUFFERS FOR AGAROSE GEL ELECTROPHORESIS, SDS-PAGE AND WESTERN BLOT ..................................... 37 2.6.3 BUFFERS FOR FLUORESCENCE IN SITU HYBRIDISATION (FISH )..................................................................38 2.6.4 BUFFERS FOR PROTEIN PURIFICATION...................................................................................................... 39 2.6.5 BUFFERS FOR VARIOUS APPLICATIONS..................................................................................................... 40 3 METHODS............................................................................................................................. 41 3.1 ANT REARING................................................................................................................................................ 41 3.2 IMMUNE CHALLENGE O F DIFFERENT C. FLORIDANUS STAGES...............................................................................42 3.2.1 PRODUCTION OF BACTERIAL SUSPENSIONS............................................................................................... 42 3.2.2 IMMUNE CHALLENGE OF C. FLORIDANUS .................... 42 3.3 RNA ISOLATION 43 3.4 HAEMOLYMPH COLLECTION.............................................................................................................................44 3.5 ANALYSIS O F THE IMMUNE DEFENCE INVENTORY O F C. FLORIDANUS BY TRANSCRIPTOME SEQUENCING ................ 44 3.5.1 RNA SAMPLE PREPARATION FOR ILLUMINA SEQUENCING.......................................................................44 3.5.2 BIOINFORMATICAL ANALYSES OF THE ILLUMINA SEQUENCING DATA SET .................................................... 45 3.5.3 VALIDATION OF ILLUMINA SEQUENCING DATA BY QR T-PCR .................................................................45 3.5.3.1 CDNA SYNTHESIS FOR QRT-PCR................................................................................................46 3.5 3.2 QRT-PCR USING THE DDCT METHOD .......................................................................................... 46 3.5.3 3 REFERENCE GENE SELECTION...........................................................................................................47 3.5.3 4 STATISTICAL ANALYSIS OF QRT-PCR DATA REGARDING IMMUNE CHALLENGE IN C. FLORIDANUS ..... 47 3.6 ANALYSIS O F THE C. FLORIDANUS HAEMOLYMPH PROTEOME............................................................................47 3.6.1 PREPARATION OF HAEMOLYMPH PROTEIN SAMPLES FOR MALDI-TOF ............................................... 47 3.6.2 MASS SPECTROMETRY ANALYSIS............................................................................................................. 48 3.6.3 BIOINFORMATICAL DATA ANALYSIS..........................................................................................................48 3.7 ANALYSIS O F C. FLORIDANUS HAEMOLYMPH PEPTIDES.................................................................................... 49 3.7.1 PREPARATION OF HAEMOLYMPH PEPTIDE SAMPLES FOR MALDI-TOF ................................................ 49 3.7.2 MASS SPECTROMETRY ANALYSIS............................................................................................................. 50 3.7.3 DATA ANALYSIS......................................................................................................................................50 3.8 GENERAL MOLECULAR CLONING METHODS......................................................................................................... 51 3.8.1 POLYMERASE CHAIN REACTION (PCR) AND AGAROSE GEL ELECTROPHORESIS ........................................... 51 3.8.2 PREPARATION OF CHEMICALLY COMPETENT E. COLI CELLS .............. 51 3.8.3 PURIFICATION OF PCR PRODUCTS.......................................................................................................... 52 3.8.4 ISOLATION AND PURIFICATION OF PLASMID DNA ................................................................................... 52 3.8.5 CLONING OF DNA FRAGMENTS INTO PLASMID VECTORS AND TRANSFORMATION INTO COMPETENT E. COLI CELLS..................................................................................................................................................... 52 3.8.6 DNA SEQUENCING OF PURIFIED PCR PRODUCTS AND PLASMID DNA...................................................52 3.9 PRODUCTION O F AN ANTISERUM FO R DETECTION O F C. FLORIDANUS HYMENOPTAECIN PEPTIDES..........................53 3.9.1 ISOLATION AND PURIFICATION OF INCLUSION BODIES FOR IMMUNISATION ................................................ 53 3.9.2 SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS FOR QUALITATIVE ANALYSIS OF PROTEIN SOLUTIONS................................................................................................................................53 3.9.3 MEASURING PROTEIN CONCENTRATIONS..................................................................................................54 3.9.4 IMMUNISATION WITH HYMENOPTAECIN PEPTIDES................................................................................ 55 3.9.5 QUALITATIVE ANALYSIS OF PRODUCED ANTIBODY SERA VIA WESTERN B LOT ............................................. 55 3.9.6 IDENTIFICATION OF PROTEINS VIA MASS SPECTROMETRY.......................................................................... 56 3.10 EXPRESSION AND PURIFICATION O F HYMENOPTAECIN PEPTIDES FO R FUNCTIONAL ANALYSES...............................56 3.10.1 EXPRESSION OF HYMENOPTAECIN PEPTIDES IN INSECT CELLS ............................................................ 56 3.10.1.1 INCORPORATION OF HYMENOPTAECIN GENES IN TRANSFER VECTORS AND MODIFICATION OF TEV CLEAVAGE SITES.............................................................................................................................59 3.10.1.2 TRANSFORMATION OF E. COLI DHLOMULTIBAC CELLS AND BLUE/WHITE SCREENING ........................ 60 3.10.1.3 ISOLATION OF RECOMBINANT BACMID DNA AND ANALYSIS VIA PC R ........................................... 60 3.10.1.4 PROPAGATION AND TRANSFECTION OF INSECT CELLS......................................................................... 61 3.10.1.5 AMPLIFICATION OF BACULOVIRUSES AND DETERMINATION OF THE NUMBER OF INFECTIOUS VIRUS PARTICLES BY END-POINT DILUTION................................................................................................62 3.10.1.6 PROTEIN EXPRESSION IN INSECT CELLS............................................................................................63 3.10.1.7 PURIFICATION OF GST-TAGGED HYMENOPTAECIN-LIKE PEPTIDE FUSION PROTEINS ......................... 63 3.10.1.8 PURIFICATION OF 6XHIS-TAGGED HYMENOPTAECIN PEPTIDE FUSION PROTEINS .............................. 63 3.10.1.9 CYTOBUSTER* PROTEIN EXTRACTION OF SOLUBLE PROTEINS IN INSECT CELLS ................................... 64 3.10.2 EXPRESSION OF HYMENOPTAECIN PEPTIDES IN E. CO LI....................................................................64 3.10.2.1 INCORPORATION OF HYMENOPTAECIN GENES IN THE PET32A PLASMID VECTOR .......................... 64 3.10.2.2 TRANSFORMATION OF COMPETENT E. COLI ROSETTA 2(DE3) CELLS.............................................64 3.10.2.3 BUGBUSTER PROTEIN EXTRACTION OF SOLUBLE PROTEINS IN E. COLI ROSETTA 2(DE3) ............. 65 3.10.2.4 LARGE-SCALE OVEREXPRESSION OF RECOMBINANT PROTEINS IN E. COLI ROSETTA 2(DE3) .......... 65 3.10.2.5 6XHIS-TAG PURIFICATION OF HYMENOPTAECIN FUSION PROTEINS WITH THE AEKTA PURE CHROMATOGRAPHY SYSTEM......................................................................................................66 3.10.2.6 PROTEOLYTIC REMOVAL OF THE FUSION TAG VIA ENTEROKINASE...................................................66 3.10.2.7 ION-EXCHANGE CHROMATOGRAPHY AND CONCENTRATION OF HYMENOPTAECIN PEPTIDE SAMPLES. 66 3.10.2.8 ANTIMICROBIAL ACTIVITY ASSAY..............................................................................................67 3.11 ANALYSING THE LOCALISATION O F B.FLORIDANUS WITHIN OVARIAN TISSUE O F C. FLORIDANUS.............................67 3.11.1 FLUORESCENCE IN SITU HYBRIDISATION...............................................................................................67 3.11.2 SAMPLE PREPARATION FOR TRANSMISSION ELECTRON MICROSCOPY AND TOMOGRAPHY ........................ 68 3.11.3 HIGH PRESSURE FREEZING OF C. FLORIDANUS OVARIES FOR TEM ....................................................... 69 3.12 ANALYSIS O F GENE EXPRESSION IN C. FLORIDANUS OVARIAN TISSUE ................................................................ 70 4 RESULTS ........................................................ 72 4.1 THE C. FLORIDANUS IMMUNE TRANSCRIPTOME................................................................................................72 4.1.1 FUNCTIONAL RE-ANNOTATION OF THE C. FLORIDANUS GENOME.................................................................72 4.1.2 THE IMMUNE SIGNALLING PATHWAYS OF C. FLORIDANUS........................................................................75 4.1.3 THE C. FLORIDANUS AMP REPERTOIRE IN COMPARISON TO OTHER INSECT SPECIES ................................. 81 4.1.4 DIFFERENTIAL EXPRESSION OF IMMUNE-RELATED GENES IN C. FLORIDANUS .............................................. 85 4.2 THE HAEMOLYMPH PROTEOME ANDPEPTIDOME O F C. FLORIDANUS..................................................................90 4.2.1 HAEMOLYMPH PROTEINS IN C. FLORIDANUS.......................................................................................... 90 4.2.2 REGULATION OF C. FLORIDANUS HAEMOLYMPH PROTEINS UPON INFECTION ............................................. 96 4.2.3 HAEMOLYMPH PEPTIDES IN C. FLORIDANUS........................................................................................ 104 4.2.3.1 PEPTIDES IN THE HAEMOLYMPH OF LARVAE................................................................................105 4.2.3 2 PEPTIDES IN HAEMOLYMPH OF WORKERS...................................................................................108 4.3 THE DETECTION O F HYMENOPTAECIN PEPTIDES IN THE HAEMOLYMPH FROM C. FLORIDANUS WITH A NEWLY GENERATED ANTISERUM................................................................................................................................. I L L 4.4 RECOMBINANT EXPRESSION, PURIFICATION AND FUNCTIONAL ANALYSIS O F HYMENOPTAECIN DERIVED PEPTIDES FROM C. FLORIDANUS................................................................................................................................... 113 4.4.1 EXPRESSION OF HYMENOPTAECIN DERIVED PEPTIDES IN INSECT CELLS...................................................113 4.4.2 PURIFICATION OF GSTHLD AND 6XHISHREP FROM INSECT CELLS...........................................................116 4.4.3 THE EXPRESSION OF THIOREDOXIN-S-HIS6 HYMENOPTAECIN FUSION PROTEINS IN E. COLI .................... 118 4.4.4 PURIFICATION OF THIOREDOXIN-S-HIS6 HYMENOPTAECIN FUSION PROTEINS VIA HPLC ....................... 121 4.4.5 FUNCTIONAL ANALYSIS OF HYMENOPTAECIN PEPTIDES.......................................................................... 124 4.5 THE DISTRIBUTION O F B.FLORIDANUS IN OVARIAN TISSUE O F C. FLORIDANUS DURING HOST OOGENESIS .............. 128 4.5.1 THE TISSUE LOCALISATION OF BLOCHMANNIA WITHIN AN OVARIOLE OF C. FLORIDANUS ............................ 128 4.5.2 A DETAILED ANALYSIS OF THE CELLULAR LOCALISATION OF BLOCHMANNIA IN OVARIAN TISSUE OF C. FLORIDANUS USING TRANSMISSION ELECTRON MICROSCOPY...............................................................134 4.5.3 THE INTRACELLULAR LOCALISATION OF BLOCHMANNIA FLORIDANUS..........................................................141 4.5.4 THE DISTRIBUTION OF BLOCHMANNIA FLORIDANUS IN MIDGUTS AND OVARIES OF RIFAMPICIN TREATED ANTS .......................................................................................................................................................... 145 4.6 THE EXPRESSION O F PUTATIVE IM M UNE GENES IN THE OVARIAN TISSUE OFC. FLO RID A N U S....................149 5 DISCUSSION........................................................................................................................ 155 5.1 THE IMMUNE GENE REPERTOIRE O F C. FLORIDANUS...................................................................... . 5.2 THE INFECTION-INDUCED DIFFERENTIAL EXPRESSION O F IMMUNE-RELATED GENES IN C. FLORIDANUS ................. 160 5.3 THE IMMUNE PROTEOME AND PEPTIDOME O F THE C. FLORIDANUS HAEMOLYMPH...........................................163 5.4 EXPRESSION AND FUNCTIONAL ANALYSIS O F HYMENOPTAECIN DERIVED PEPTIDES ........................................... 169 5.5 THE DOWN-MODULATION O F THE IMMUNE RESPONSE IN C. FLORIDANUS OVARIES MIGHT SUPPORT VERTICAL TRANSMISSION O F B. FLORIDANUS..................................................................................................................173 5.5.1 THE LOCALISATION OF B. FLORIDANUS IN OVARIAN TISSUE OF C. FLORIDANUS .......................................... 173 5.5.2 THE EXPRESSION OF IMMUNE-RELATED GENES IN C. FLORIDANUS OVARIES............................................177 5.6 CONCLUSIONS AND OUTLOOK..........................................................................................................................182 REFERENCES..................................................................................................................................... 184 APPENDIX .................................................. 213 I EVALUATION O F BACU/OVIRUS TITRES USING END-POINT DILUTION................................................................... 213 II C. FLORIDANUS PROTEIN ORTHOLOGS WITH IMMUNE-RELATED FUNCTION SHARED WITH D. MELANOGASTER, A. MELLIFERA AND N. VITRIPENNIS.................................................................................................................... 214 III STATISTICAL ANALYSIS O F GENE EXPRESSION DATA AFTER IMMUNE CHALLENGE..................................................217 IX IDENTIFICATION O F HYMENOPTAECIN PEPTIDES VIA MASS SPECTROMETRY........................................................226 X STATISTICAL ANALYSIS O F IMMUNE GENE EXPRESSION DATA IN C. FLORIDANUS OVARIES ................................... 227 X I ANNUAL REVIEWS, INC - LICENSE, TERMS AND CONDITIONS........................................................230 X II DIGITAL APPENDIX: LIST O F CONTENT ............................ 232 INDEX OF ABBREVIATIONS. 233
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owner_facet	DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174
physical	238 Seiten Illustrationen, Diagramme 1 CD-Rom: Digital Appendix
psigel	ebook
publishDate	2016
publishDateSearch	2016
publishDateSort	2016
record_format	marc
spelling	Kupper, Maria Verfasser (DE-588)1123654344 aut The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus vorgelegt von Maria Kuppper aus Lutherstadt Eisleben Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus Würzburg September 2016 238 Seiten Illustrationen, Diagramme 1 CD-Rom: Digital Appendix txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2016 Zusammenfassung in deutscher und englischer Sprache Symbiose (DE-588)4136029-1 gnd rswk-swf Oogenese (DE-588)4043568-4 gnd rswk-swf Camponotus floridanus (DE-588)4395029-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Camponotus floridanus (DE-588)4395029-2 s Symbiose (DE-588)4136029-1 s Oogenese (DE-588)4043568-4 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-142534 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-142534 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029410999&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Kupper, Maria The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus Symbiose (DE-588)4136029-1 gnd Oogenese (DE-588)4043568-4 gnd Camponotus floridanus (DE-588)4395029-2 gnd
subject_GND	(DE-588)4136029-1 (DE-588)4043568-4 (DE-588)4395029-2 (DE-588)4113937-9
title	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus
title_alt	Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus
title_auth	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus
title_exact_search	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus
title_full	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus vorgelegt von Maria Kuppper aus Lutherstadt Eisleben
title_fullStr	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus vorgelegt von Maria Kuppper aus Lutherstadt Eisleben
title_full_unstemmed	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus = Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus vorgelegt von Maria Kuppper aus Lutherstadt Eisleben
title_short	The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus
title_sort	the immune transcriptome and proteome of the ant camponotus floridanus and vertical transmission of its bacterial endosymbiont blochmannia floridanus das immuntranskriptom und proteom der ameise camponotus floridanus und die vertikale transmission ihres endosymbionten blochmannia floridanus
title_sub	= Das Immuntranskriptom und -proteom der Ameise Camponotus floridanus und die vertikale Transmission ihres Endosymbionten Blochmannia floridanus
topic	Symbiose (DE-588)4136029-1 gnd Oogenese (DE-588)4043568-4 gnd Camponotus floridanus (DE-588)4395029-2 gnd
topic_facet	Symbiose Oogenese Camponotus floridanus Hochschulschrift
url	https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-142534 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029410999&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
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