The circadian clock of the carpenter ant Camponotus floridanus Die circadiane Uhr der Rossameise Camponotus floridanus Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Integrative Biology submitted by Janina Kay from Ochsenfurt Würzburg, 13.12.2017 Submitted on: …………………………………………………………..…….. Office stamp Members of the Promotionskomitee: Chairperson: Prof. Dr. Jörg Schultz Primary Supervisor: Prof. Dr. Charlotte Förster Supervisor (Second): Prof. Dr. Flavio Roces Supervisor (Third): Prof. Dr. Monika Stengl Date of Public Defence: …………………………………………….………… Date of Receipt of Certificates: ………………………………………………. CONTENT: Summary: ................................................................................................................................................. 1 Zusammenfassung: .................................................................................................................................. 2 1. Introduction.......................................................................................................................................... 5 1.1 The endogenous clock .................................................................................................................... 5 1.2 Rhythmic Behavior ......................................................................................................................... 7 1.2.1 Rhythmic behavior in selected ants ........................................................................................ 7 1.2.2 Rhythmic behavior in C. floridanus ....................................................................................... 10 1.3 The molecular basis of the circadian clock ................................................................................... 11 1.3.1 The transcriptional-translational feedback-loop .................................................................. 11 1.3.2 Components of the feedback loop in selected insects ......................................................... 11 1.3.2.1 The molecular feedback loop of Drosophila melanogaster ........................................... 11 1.3.2.2 The molecular feedback loop of hymenopteran species ............................................... 15 1.4 The anatomy of the circadian clock ............................................................................................. 17 1.4.1 The anatomy of the central clock in Drosophila melanogaster ............................................ 17 1.4.2 The anatomy of the central clock in Apis mellifera ............................................................... 18 1.4.3 The PDF-network ................................................................................................................... 20 1.4.3.1 The Pigment-Dispersing Factor ...................................................................................... 20 1.4.3.2 The PDF-network in Drosophila melanogaster .............................................................. 21 1.4.3.3 The PDF-network in Leucophea maderae ...................................................................... 22 1.4.3.4 The PDF-network in Apis mellifera ................................................................................. 24 1.5 Study Aims .................................................................................................................................... 27 2. Material and Methods........................................................................................................................ 28 2.1 Experiments: ................................................................................................................................ 28 2.1.1 Locomotor behavior in response to light at different temperatures.................................... 29 2.1.2 The PDF-network in minor and major workers of Camponotus floridanus .......................... 29 2.1.3 The localization and cycling of the Period protein in C. floridanus ....................................... 30 2.2 Animal rearing: ............................................................................................................................. 30 2.3 Monitoring of Locomotor Behavior: ............................................................................................ 30 2.3.1 Pre-entrainment in sub-colonies ........................................................................................... 31 2.3.2 Entrainment in TriKinetics LAM (LD-phase) .......................................................................... 31 2.3.3 Feeding .................................................................................................................................. 31 2.3.4 Monitoring Protocols ............................................................................................................ 32 2.3.5 Activity Recording ................................................................................................................. 33 2.3.6 Data analysis .......................................................................................................................... 33 2.3.6.1 Actograms ...................................................................................................................... 33 2.3.6.2 Average Day ................................................................................................................... 34 2.3.6.3 Period length .................................................................................................................. 35 2.3.6.4 Additional data analysis ................................................................................................. 35 2.4 Pre-processing of brain-samples .................................................................................................. 36 2.4.1 Collection of animals ............................................................................................................. 36 2.4.1.1 Collection from monitors ............................................................................................... 36 2.4.1.2 Collection from colonies................................................................................................. 36 2.4.2 Preparation of brains: ........................................................................................................... 36 2.4.3 Fixation .................................................................................................................................. 37 2.4.4 Embedding and Vibrating Blade Microtome Sectioning ....................................................... 37 2.5 Immunohistochemistry ................................................................................................................ 37 2.5.1 Immunofluorescent labelling ................................................................................................ 38 2.5.2 PDH staining in wholemounts ............................................................................................... 39 2.5.3 PDH staining on sections ....................................................................................................... 40 2.5.4 PER/PDH double-labelling ..................................................................................................... 42 2.6 Confocal Laser-Scanning Microscopy: .......................................................................................... 44 2.7 Data Processing ............................................................................................................................ 46 2.7.1 Image processing in FiJi ......................................................................................................... 46 2.7.2 Identification of PER cell clusters .......................................................................................... 46 2.7.3 Quantification of PER staining intensity ................................................................................ 46 2.7.4 Reconstruction of the PDF network: ..................................................................................... 47 2.7.4.1 Neuropil Reconstruction ................................................................................................ 47 2.7.4.2 PDH cell body reconstruction ......................................................................................... 48 2.7.4.3 PDH network reconstruction .......................................................................................... 48 2.7.4.4 Generation of the 3D-model .......................................................................................... 49 2.8 Statistics ....................................................................................................................................... 50 2.9 Materials ...................................................................................................................................... 50 3. Results: ............................................................................................................................................... 54 3.1 The endogenous Locomotor Behavior of C. floridanus: ............................................................... 54 3.1.1 C. floridanus is able to entrain to different light phases ....................................................... 54 3.1.2 C. floridanus has an endogenous period that is temperature compensated ....................... 61 3.2 Minor and major ants of C. floridanus have