Identification and classification of endogenous bacteria within mole-rats of the Family Bathyergidae By James Henry du Toit van Sandwyk Submitted in partial fulfillment of the requirements for the degree of Magister Scientiae (Zoology) in the Faculty of Natural and Agricultural Science University of Pretoria Pretoria March 2007 © University of Pretoria Identification and classification of endogenous bacteria within mole-rats of theFamily Bathyergidae By J.H.d.T. van Sandwyk J.H.d.T van Sandwyk: Mammal Research Institute Department of Zoology and Entomology University of Pretoria Pretoria 0002 South Africa Supervisors: ProfN.C. Bennett: Mammal Research Institute Department of Zoology and Entomology University of Pretoria Pretoria 0002 South Africa Dr A.D.S. Bastos: Mammal Research Institute Department of Zoology and Entomology University of Pretoria Pretoria 0002 South Africa Email: [email protected] i DECLARATION I, James Henry du Toit van Sandwyk, declare that this thesis which I hereby submit for the degree of Magister Scientiae at the University of Pretoria is my own work and has not previouslybeenby me for a degree at this or any other tertiary institution. Signature 2 March 2007 Date ii ACKNOWLEDGEMENTS I would like to express my thanks my supervisors Nigel Bennett and Amanda Bastos for their help with all the unexpected problems which arose during my thesis. Nigel for all his help with sample collection and funding for the project, and Amanda for all her help with the molecular work, both of you have provided me with a solid foundation for my future pursuits in the field of science. I would also like to thank Dr. Fanus Venter for providing access to his micro-biology laboratory for all my bacterial culturing and for his help with understanding and interpreting the results of the bacterial study. Glen Malherbe and Babsie Potgieter are thanked for all their help with the technical details of my project as well as all the people in the Zoology Molecular Laboratory and Dr. Venter’s Microbiology Laboratory. Special thanks go to all my friends at the university and those in the real world, especially Leanne Hart and Lia Rotherham for all their help in all the various aspects of my project. Leanne and Craig Jackson assisted with the mole-rat pictures. A special word of thanks is given to my best “bud” Brian R. Smith for his help in my project and for helping me get over some hard times when nothing seemed to be going right by always having a spare ear and helping me to take a fresh look at the problem and find a solution. Finally I express my deep gratitude to my family, and in particular my Mom for all her help. iii “Omnia possum in eo qui me confortat” iv TABLE OF CONTENTS Declaration Page ii Acknowledgements Page iii-iv Table of Contents Page v-viii List of Figures Page ix-xi List of Tables Page xii Disclaimer Page xiii Chapter 1: General Introduction Introductory Page Page 1 1. The Family Bathyergidae and distributional range Page 2-3 of the five extant genera 2. The subterranean environment and its effects on the Page 3-11 bathyergids and their levels of sociality. Models put forward to explain how sociality occurs 3. Main Aims of the Project, Thesis structure and Page 11-13 Content 4. Literature Cited Page 14-18 Chapter 2: Identification of associated bacteria in three species of South African Mole-rats (Bathyergidae) v Introductory Page Page 19 Abstract Page 20 1. Introduction Page 20-24 2. Methods and Materials Page 25-27 2.1. Sample collecting Page 25 2.2. Bacterial culture an DNA extraction Page 25-26 2.3. 16S rRNA amplification Page 26-27 3. Results Page 27-40 3.1. Actinobacteria Page 28-29 3.2. Firmicutes Page 30-37 3.3. Proteobacteria Page 37-40 4. Discussion Page 40-44 5. Acknowledgements Page 44 6. Literature Cited Page 45-51 Appendix I Page 52-54 Appendix II Page 55-57 Appendix III Page 58-61 Appendix IV Page 62-65 Appendix V Page 66-68 Appendix VI Page 69-73 Appendix VII Page 74-79 Chapter 3: The recorded incidence of Bartonella infection amongst solitary and social species of South African bathyergids: a multi-gene approach Introductory Page Page 80 vi Abstract Page 81 1. Introduction Page 81-83 2. Methods and Materials Page 83-86 2.1 Sampling Page 83-84 2.2. Extraction of Bartonella genetic material for Page 84 screening 2.3. Primer design and target amplification region Page 84-85 2.4. Amplification of the gltA and inter-generic Page 85 region 2.5. Infection rate and genetic characterization of Page 85-86 positive samples 3. Results Page 86-89 3.1. Nucleotide sequence analysis Page 88 3.2. 16S-23S inter-generic region results Page 88 3.3. Phylogenetic analysis and species Page 89-90 identification 4. Discussion Page 91-92 5. Acknowledgements Page 93 6. Literature Cited Page 94-97 Appendix I Page 98-99 Appendix II Page 100-107 Chapter 4: General Conclusion Introductory Page Page 108 General Conclusion Page 109-113 1. Identified bacteria associated with three species of Page 109-110 vii bathyergids 2. Bartonella prevalence in mole-rats species varying in Page 110-112 the degree of sociality and possible implications 3. Pilot studies and continuing research Page 112-113 4. Literature Cited Page 114-115 viii LIST OF FIGURES CHAPTER 1: General Introduction Figure 1. A map of the African continent illustrating the distributions of the four species of solitary mole-rats. Page 4 Figure 2. A map of the African continent illustrating the distributions of 9 of the recognized species of the genus Cryptomys. Page 5 Figure 3. A map of the African continent illustrating the distributions of the 4 sub species of Cryptomys hottentotus. Page 7 CHAPTER 2: Identification of associated bacteria in three species of South African Mole-rats (Bathyergidae) Figure 1. Neighbour-joining tree showing the relationship between the two genera; Microbacterium and Arthrobacter ix which were isolated from mole-rat samples as well as and their relatedness to bacterial sequences in the Genbank database. Page 29 Figure 2. Relative positions of the four Firmicutes dataset on the 16S rRNA gene. Page 32 Figure 3. Neighbour-joining tree showing the relationship between the first dataset of the Firmicutes which was isolated from mole-rat samples as well as its relatedness to bacterial sequences in the Genbank database. Page 33 Figure 4. Neighbour-joining tree showing the relationship between the second dataset of the Firmicutes which was isolated from mole-rat samples as well as its relatedness to bacterial sequences in the Genbank database. Page 34 Figure 5. Neighbour-joining tree showing the relationship between the third dataset of the Firmicutes which was isolated from mole-rat samples as well as its relatedness to bacterial sequences in the Genbank database. Page 35 x Figure 6. Neighbour-joining tree showing the relationship between the fourth dataset of the Firmicutes which was isolated from mole-rat samples as well as its relatedness to bacterial sequences in the Genbank database. Page 36 Figure 7. Neighbour-joining tree showing the relationship between the different families recorded from the γ subclass of the Proteobacteria isolated from mole-rat samples as well as and their relatedness to bacterial sequences in the Genbank database. Page 38 CHAPTER 3: The recorded incidence of Bartonella infection amongst solitary and social species of South African bathyergids: a multi-gene approach Figure 1. Neighbour Joining (NJ) tree based on partial gltA sequences inferred using uncorrected p-distances. Bootstrap values from Minimum Evolution (ME), Maximum Parsimony (MP) and Maximum likelihood (ML) analysis that were > 50 % are indicated next to the relevant nodes as follows (NJ/ MP/ ML / ME). Page 90 xi LIST OF TABLES Chapter 3: The recorded incidence of Bartonella infection amongst solitary and social species of South African bathyergids: a multi-gene approach TABLE 1. Summary of Bartonella positives identified with the gltA primer- set identified using a BLASTn search. Page 87 TABLE 2. List of the most closely related Bartonella sequences in the Genbank database to those isolated from mole-rats. Page 87 xii DISCLAIMER This thesis comprises a series of chapters, some of which have beenprepared as stand alone manuscripts that will subsequentlybe submitted forpublicationpurposes. As a consequence there maybe some unavoidable repetition arisingbetween the chapters. xiii CHAPTER 1 General Introduction Contributing Authors: J. H. d. T. van Sandwyk, N. C. Bennett & A. D. Bastos Mammal Research Institute, Department of Zoology and Entomology, University of Pretoria, Pretoria 0002, South Africa 1 1. The Family Bathyergidae and distributional range of the five genera The family Bathyergidae is a monophyletic group of Hystricomorph rodents which occurs south of the Sahara (Faulkes et al. 2004, Ingram et al. 2004). The family is divided into two sub families, the Bathyerginae consisting solely of the genus Bathyergus (Bennett & Faulkes 2000) and Georychinae (Bennett & Faulkes 2000). Bathyergus is one of two polytypic genera along with the genus Cryptomys. The Bathyerginae comprises two species which are distributed along the western coast of Namibia and South Africa (Jarvis & Bennett 1990, 1991). The first, Bathyergus janetta (the Namaqua Dune mole- rat) is endemic to Namaqualand and the coastal dunes south of Namibia (Herbst & Bennett 2006) and the second, Bathyergus suillus (the Cape Dune mole-rat) occurs in the southern, southwestern and western regions of the Cape Province of South Africa (Bennett & Faulkes 2000). The other four genera within the family Bathyergidae (Figure 1) belong to the subfamily Georychinae (Figure 1, 2 and 3) (Bennett & Faulkes 2000), three of which are monotypic; these include the genus Georychus, which is endemic to the Republic of South Africa, the only species within this genus is Georychus capensis (the Cape Mole-rat), secondly Heterocephalus has its distribution in eastern Africa, the only species being Heterocephalus glaber (the Naked mole-rat).