Fitness for the Ark: Are zoo bred amphibians ready to go back to the wild? Luiza Figueiredo Passos This thesis is presented to the School of Environment & Life Sciences, University of Salford, in fulfilment of the requirements for the degree of Ph.D. Supervised by Professor Robert John Young September 2017 2 Table of Contents Chapter 1 – General Introduction ........................................................................................ 13 1.1 Do the vocalisations of captive frogs differ from wild ones? ............................... 20 1.2 Does antipredator behaviour of captive frogs differ from wild ones? ............... 23 1.3 How does the colour of captive frogs compare with wild ones? .......................... 24 1.4 Does the skin microbiota of captive frogs differ from wild ones? ...................... 25 1.5 Conclusion ................................................................................................................ 27 1.8 Study Species ........................................................................................................... 28 1.9 Study sites ................................................................................................................ 30 1.10 Ethical Approval ..................................................................................................... 36 Chapter 2 –Neglecting the call of the wild: Captive frogs like the sound of their own voice ......................................................................................................................................... 37 2.1 Abstract .................................................................................................................... 37 2.2 Introduction ............................................................................................................. 38 2.3 Methodology ............................................................................................................ 39 2.3.1 Study subject .................................................................................................... 39 2.3.2 Study sites ......................................................................................................... 39 2.3.3 Recording Calls ................................................................................................ 39 2.3.4 Phonotaxis experiments ................................................................................... 41 2.3.5 Movement Analyses ......................................................................................... 44 2.4 Results ...................................................................................................................... 46 2.5 Discussion ................................................................................................................. 52 Chapter 3 – Do captive golden mantella frogs recognise wild conspecifics calls? Responses to the playback of captive and wild calls ........................................................... 55 3.1 Abstract .................................................................................................................... 55 3.2 Introduction ............................................................................................................. 56 3.2 Methodology ............................................................................................................ 57 3.2.1 Study subject .................................................................................................... 57 3.2.2 Recording Calls ................................................................................................ 57 3.2.3 Study sites ......................................................................................................... 58 3.2.4 Playback experiments ...................................................................................... 58 3.2.5 Ethogram .......................................................................................................... 60 3.2.6 Behavioural analysis ........................................................................................... 61 3.2.7 Statistical analysis ............................................................................................ 61 3.4 Results ...................................................................................................................... 62 3.5 Discussion ................................................................................................................. 65 Chapter 4 –The tonic immobility test: Do wild and captive Golden Mantella frogs (Mantella aurantiaca) have the same response? .................................................................. 67 4.1 Abstract .................................................................................................................... 67 4.2 Introduction ............................................................................................................. 68 4.3 Methodology ............................................................................................................ 70 3 4.3.1 Study subjects ................................................................................................... 70 4.3.2 Study sites ......................................................................................................... 70 4.3.3 TI Test ............................................................................................................... 71 4.3.4 Body Condition Index ...................................................................................... 73 4.3.5 Data Analysis .................................................................................................... 74 4.4 Results ...................................................................................................................... 74 4.5 Discussion ................................................................................................................. 78 Chapter 5– How does captivity affect skin colour reflectance of golden mantella frogs? .................................................................................................................................................. 82 5.1 Abstract .................................................................................................................... 82 5.2 Introduction ............................................................................................................. 83 5.3 Methodology ............................................................................................................ 85 5.3.1 Study subject .................................................................................................... 85 5.3.2 Study sites ......................................................................................................... 85 5.3.3 Spectrophotometric measurements ................................................................ 85 5.3.4 Colour analyses ................................................................................................ 87 5.4 Results ...................................................................................................................... 90 5.5 Discussion ................................................................................................................. 95 6.1 Abstract .................................................................................................................. 100 6.2 Introduction ........................................................................................................... 101 6.3 Methods .................................................................................................................. 103 6.3.1 Study subjects ................................................................................................. 103 6.3.2 Study sites ....................................................................................................... 103 6.3.3 Skin Bacteria Sampling and DNA Extraction ............................................. 104 6.3.4 16S Metagenomic Sequencing ....................................................................... 105 6.3.5 Statistical Analysis ......................................................................................... 106 6. 4 Results .................................................................................................................... 107 6.5 Discussion ............................................................................................................... 114 Chapter 7 - General Discussion .......................................................................................... 118 7.1 Research limitation ............................................................................................... 124 7.2 Conclusion .............................................................................................................. 129 References: ............................................................................................................................ 130 Appendices ............................................................................................................................ 154 4 List of Figures Figure 1. A wild specimen of Golden Mantella frog (Mantella aurantiaca) observed in Madagascar (Photo by Luiza Passos). .............................................................................. 28 Figure 2. Distribution map of the Golden Mantella frog in the wild (Source: