Rudimentary Organs and Genes: Evolution of the Visual System in Caecilians By Samantha Mila Mohun Department of Molecular Genetics, UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V9EL, UK AND Department of Zoology, Natural History Museum, Cromwell Road, London SW7 5BD, UK Thesis submitted to the University of London in candidature for the degree of Doctor of Philosophy (2007) 1 UMI Number: U591554 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591554 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Declaration The work contained in this thesis is the author’s own, except where stated or referenced accordingly. The views expressed in this thesis are those of the author and do not represent those of the University of London. Signed Date 2 Abstract As Darwin (1859 ppl43-146) noted, each major group of animals has evolved a visual system exhibiting unique adaptations to visual tasks. Caecilians are elongate, snake-like amphibians (the order Gymnophiona) that, except for the secondarily aquatic Typhlonectidae, are to some extent fossorial as adults. Caecilian eye morphology shows various degrees of rudimentation but the literature contains many contradictory reports, and phylogenetic characters based on eye morphology are controversial. New physiological, molecular and morphological data are presented here in order to better characterise the caecilian visual system and to investigate the utility of visual gene sequences in phylogenetics. I provide the first description of the morphology of the eye of a member of the Rhinatrematidae, the sister group of all other caecilians and additional observations on a range of caecilian species. Morphological discoveries include the presence of a poorly developed iris in both the Rhinatrematidae and Ichthyophiidae. I elucidate the previously unknown complete sequence of the caecilian rod opsin gene and present new data on the physiology caecilian rhodopsin. The obvious reduction in visual system morphology in caecilians is associated with a change in the genes involved in visual function. The rod opsin is the only visual pigment detected in caecilians suggesting a loss of colour and diurnal vision, which are probably unnecessary in these predominately burrowing vertebrates. A change in the maximum absorbance of the caecilian rhodopsin compared to other amphibians is reported and experimental confirmation that this is caused by 3 amino-acid substitutions in the rod opsin gene is presented. The pattern of substitutions implies a previously unknown mechanism of spectral tuning. Phylogenetic analyses of caecilian rod opsin sequences give results that reflect current understanding of caecilian relationships and suggest both coding and non-coding regions are useful markers for inferring phylogenetic relationships within caecilians. 4 Acknowledgements I would like to give many thanks to the people I have met and learnt from whilst working on my PhD. 1 would like to thank my supervisors Mark Wilkinson and David Hunt for their encouragement and guidance. Members of the Herpetological research group past and present, Dave Gower, Hendrik Mueller, Simon Loader Julia Day, James Cotton, Claire Slater, Andrea Cobbett, and Davide Pisani. I would also like to thank members of the molecular labs, Jacqueline Mackenzie-Dodds, Pat Dyal, Andy Warlow, Julia Llewellyn-Hughes and Claire Griffin. I would like to thank members of the Molecular Genetics department at the Institute of Ophthalmology, UCL, Wayne Davies, Livia Carvalho, Susan Wilkie, Jill Cowing, Beverley Scott, Juliet Parry and Jim Bowmaker. I would like to thank Werner Himstedt for his help in understanding vision of caecilians. I would also like to thank my family and friends for their continued support throughout. This research was funded by BBSRC studentship number 10085 5 Table of contents Title 1 Declaration 2 Abstract 3 Acknowledgements 5 Table of contents 6 List of illustrations 11 List of tables 18 Chapter 1 Introduction 1.1 Overview of thesis 19 1.2 What are caecilians? 21 1.2.1 Caecilian Sensory Behaviour 22 13 Visual pigments 23 13.1 Spectral shift 24 1.4 The complexity and perfection of the vertebrate eye 26 13 Vertebrate Eye Structure 30 13.1 Morphology of Amphibian Eyes 31 13.2 Fibrous tunic 34 1 3 3 Uveal tract 35 6 1.5.4 Lens 39 1.5.5 Retina 40 1.5.6 Adnexa 44 1.6 Why study caecilian visual systems? 45 Chapter 2 Material and Methods 47 2.1 Materials 47 2.1.1 Specimens 47 2.1.2 Kits 47 2.1.3 General reagents and solutions 48 2.1.4 Primers 49 2.1.5 Electrophoresis reagents 49 2.1.6 Culture Media 49 2.1.7 Bacterial strains 50 2.1.8 Tissue culture cells, media and reagents 50 2.1.9 Additional reagents for reconstitution of opsin 50 with 11-cis retinal 2.1.10 Additional Southern blot reagents 51 2.1.11 Additional in-situ hybridization reagents 51 2.1.12 Additional cDNA library reagents 52 2.2 Methods 52 2.2.1 Microspectrophotometry (MSP) 52 2.2.2 Nucleic Acid Extraction 53 7 2.2.2 Nucleic Acid Extraction 53 2.2.2.1 mRNA isolation 53 2.2.22 Quantification of mRNA and DNA 54 2.2.2.3 Reverse Transcription Polymerase Chain 54 Reaction (PCR) to create cDNA 2.2.2.4 Extraction of gDNA from Liver 55 2.23 DNA amplification 56 2.23.1 Polymerase Chain Reaction 56 2.23.2 Gel electrophoresis of PCR fragments 57 2.233 DNA extraction from electrophoresis gel 57 2.23.4 Rapid Amplfication of cDNA Ends (RACE) 57 2.23.4.1 3’ RACE 58 2.23.4.2 5’ RACE 58 2.2.4 Cloning into vector and Transformation of bacterial cells 2.2.4.1 Ligation of pGEM T-Easy (Promega) vector and PCR product 60 2.2.4.2 Ligation into pMT4 vector 60 2.2.43 Transformation of cells 60 2.2.4.4 Plating and screening of colonies by blue-white detection 61 2.2.4.5 Isolation of plasmid DNA from 5ml overnight culture (miniprep) 61 8 2.2.4.6 Maxiprep of high quantities of plasmid 62 2.2.5 Southern blot 63 2.2.5.1 Preparation of gel for Southern blotting 64 2.25.2 Southern blotting Transfer of DNA to membrane 64 2.2.53 Construction of probes 65 2.23.4 Hybridization of probe to Southern blot membrane 66 2.233 Stringency Washing 67 2.23.6 Immunological Detection 68 2.2.6 In situ hybridization of Typhlonectes eye sections 68 2.2.6.1 Cryosectioning 68 2.2.6.2 Creation of riboprobes 68 2.2.63 Probing cryosectioning with labeled gene fragment 69 2.2.6.4 Hybridizating of probe to sections 70 2.2.63 Post antibody wash and chemiluminescent reaction 71 2.2.7 Reconstitution of opsin with 11-cis retinal 71 2.2.7.1 Amplification of full length opsin with modified primers 71 9 2.2.7.2 Transfection of HEK 293T cells with pMT4/opsin 72 2.2.73 Harvesting 73 2.2.7.4 Regeneration of pigment 75 2.2.7 £ Isolation of pigments 75 22.7.6 Measurement of absorption spectra and rhodopsin template fitting. 76 2.2.8 cDNA library construction 2.2.8.1 First strand synthesis 77 2.2.8.2 cDNA Amplification by PCR 77 2.2.8.3 Sfi 1 restriction enzyme digestion 78 2.2.8.4 cDNA size fractiontion by Chroma spin-400 78 2.2.8.5 Ligation into vector 79 2.2.8.6 Packaging intoX vector 79 2.2.8.7 Titering of packaging 79 2.2.8.8. Amplification 80 2.2.8.9 Screening of clones 81 2.2.8.9.1 Hybridization of DNA to membrane 81 2.2.8.9.2 Labelling of probe using ECL detection kit 82 10 2.2.8.9.3 Prehybridization and Hybridisation 82 2.2.8.9.4 Blot washing 83 2.2.8.9.5 Detection 83 2.2.8.9.6 Excision into plasmid 84 2.2.9 Cycle sequencing 85 Chapter 3 Morphology of Caecilian Eyes 86 3.1 Introduction 86 3.1.1 Overview 86 3.1.2 Previous research on caecilian eyes 87 3.2 Materials and Methods 94 3 3 Results 94 33.1 The eye of Rhinatrema bivittatum 94 33.1.1 Gross morphology 94 33.1.2 Fibrous tunic 97 33.13 Lens 97 33.1.4 Uveal tract 97 33.13 Extrinsic Muscles 99 33.1.6 Retina 101 3.3.2 Diversity of caecilian eyes 102 3.4 Discussion 3.4.1 Comparison with previous caecilian research 108 11 3.4.2 Comparison with other vertebrates with degenerate visual systems 111 3.4.3 Further Research 118 3.5 Conclusion 119 Chapter 4 Identification and Characterization of Visual Pigments of Caecilians 121 4.1 Introduction 121 4.1.1 Overview 121 4.1.2 Frog and salamander visual pigments 122 4.13 Caecilian visual pigments 125 4.2 Objectives 126 4 3 Results 126 43.1 Microspectrophotometry (MSP) 126 43.1.1 Microspectrophotometry (MSP) of photoreceptor cells in retina 126 43.1.2 MSP to measure transmission of light through skin covering the eye in Typhlonectes natans 129 43.2 Isolation of opsin sequences from cDNA 129 4 3 3 Expression of visual pigment gene 140 43.4 In situ hybridization 144 43.5 Detection of other visual opsin classes by construction of cDNA library, PCR and Southern blot techniques.