GENETIC VARIATION IN TWO MORPHOLOGICALLY SIMILAR SOUTH AFRICAN MASTOMYS SPECIES (RODENTIA: MURIDAE) by ANDRE-KARL SMIT DISSERTATION Submitted in partial fulfilment of the requirements for the degree MASTER OF SCIENCE in ZOOLOGY in the FACULTY OF SCIENCE at the RAND AFRIKAANS UNIVERSITY SUPERVISOR: PROF F H VAN DER BANK NOVEMBER 2001 ABSTRACT Two species of multimammate mouse, Mastomys coucha and M. natalensis are common, and widely distributed in southern Africa, occurring sympatrically in some areas, and allopatrically in others. The limits of their distribution are only provisional so far. As they share a high degree of morphological similarity, they are, as yet, impossible to identify with certainty in the field. Each species of multimammate mouse carries important diseases: with M. coucha being a carrier for the bacterium causing plague, and M. natalensis carrying the virus causing Lassa fever. In many areas, multimammate mice, being highly adaptable and ecological generalists, have become co-habitants with humans. This fact, coupled to the medical significance of both species, lends importance to being able to identify each species where it occurs, especially in areas where they occur sympatrically. Thus, a total of 40 specimens of M. natalensis were trapped from Richards Bay and La Lucia ridge in KwaZulu-Natal, and 43 specimens of M. coucha from Montgomery Park in Johannesburg and from the shores of the Vaal Dam in the Free State, with the aim of comparing these two species via gel electrophoresis. These specimens were from allopatric populations from the centres of their provisional distributions. It was expected that there would be genetic differences between the two sibling species. Blood, liver, and muscle samples were taken, either in the field from dead specimens caught in snap-traps, or back in the laboratory from live-trapped specimens. Fifteen proteins or enzymes provided interpretable results at a total of 39 loci. Nineteen of these were polymorphic (AAT-1, -2, ADH, EST-1, -2, GAP, GPI-2, IDH-1, -2, LDH- 2, PGD-1, PGM-1 to —4, PT-2, -3, Hb-1 and Hb-2) in one or more of the four 1 populations analysed. Fixed allele differences between M. natalensis and M. coucha were detected at AAT-1, ADH, EST-1, PGD-1, Hb-1 and -2, whereas locus differences were found to be present at GPI-2, PT-2 and -3. The large separation distances between bands at the latter loci were the reason that these were considered to be isozyme rather than allozyme differences. Average heterozygosities for the species were 0.018 for M. coucha and 0.032 for M. natalensis, and a mean genetic distance between these two species was 0.26. Efforts were made to raise polyclonal antisera to the distinct erythrocytes of each species. Erythrocytes were extracted from each species, purified and injected into host rabbits. Serum was harvested from the rabbits after suitable intervals, and the plasma was then analysed to see if antibodies to the erythrocyte proteins were present using the Ouchterlony method. No precipitation reactions occurred. The discovery of these markers distinguishing each species, as well as being the first study quantifying the extent of genetic variation within and between these species, is an important contribution in terms of being able to identify multimammate mouse species, especially where they occur sympatrically, and thus being able to identify any health risk present to either animals or people. 2 OPSOMING Die twee Vaalveldmuisspesies, Mastomys coucha en M. natalensis, is volop en wyd versprei deur suidelike Afrika. Hul verspreiding is in sommige gebiede simpatries en allopatries in ander, en die grense hiervan is slegs tentatief tot dusver. Die twee spesies se morfologie is amper identies in alle sigbare aspekte, en dit is dus onmoontlik om hulle van mekaar te onderskei in die gebiede waar hulle saam voorkom. Elke spesie Vaalveldmuis is draers van belangrikte siektes; met M. coucha as gasheer vir die bacterium wat Builepes veroorsaak, en M. natalensis is 'n draer van die sogenaamde Lassa-koors virus. As gevolg van hiedie muise se algemene ekologiese vereistes, kom hulle in baie stedelike gebiede voor as bywoners met mense, en word beskou as die gewone huismuis. Dit is dus noodsaaklik dat tegnieke beskikbaar is om die twee spesies te identifiseer waar hulle simpatries voorkom, juis as gevolg van van hul mediese belangrikheid en algemene voorkoms. Vir hierdie studie is 'n totaal van 40 individue M. natalensis in Richardsbaai en die La Lucia rif (KwaZulu-Natal) versamel asook 43 M. coucha in die Montgomeriepark-area (Johannesburg) en die Vaaldam (Vrystaat) om die genetiese samestelling van hierdie twee spesies te vergelyk met behulp van jel-elektroforese. Bloed, !ewer- en spierweefsels is versamel in die veld van dooie muise (wat gevang is met gewone muisvalletjies) asook van muise wat in die laboratorium doodgemaak is nadat hulle met muisvalletjies gevang is. Vyftien proteene of ensieme by 39 lokusse het interpreteerbare resultate gelewer. Negentien hiervan is polimorfies (AAT-1, -2, ADH, EST-1, -2, GAP, GPI-2, IDH-1, -2, LDH-2, PGD-1, PGM-1 tot —4, PT-2, -3, Hb-1 en Hb-2) in een of meer van die vier bevolkings. Gefikseerde alleliese verskille tussen die 3 twee spesies is waargeneem by die volgende lokusse: AAT-1, ADH, EST-1, PGD-1, Hb-1 en —2, terwyl vaste lokusverskille by GPI-2, PT-2 en —3 gevind is. As gevolg van die groot afstand tussen bande, is laasgenoemde lokusse isosiem, in plaas van allosiemmerkers. Gemiddelde heterosigositeite vir die spesies was 0.018 vir M. coucha en 0.032 vir M. natalensis en die gemiddelde genetiese afstand tussen hulle was 0.26. Pogings was gemaak om polikloniese teenliggaampies teen die eritrosiete van albei spesies te produseer. Eritrosiete was van elke spesie geneem, gesuiwer en in konyne ingespuit. Bloed was dan weer geneem van konyne na gepasde tydsintervalle, en die plasma hiervan was geevalueer vir teenliggaampies teen die eritrosietproteTene met behulp van die Ouchterlony-metode. Geen neersiag reaksies was gekry nie. Hierdie is die eerste studie om geneties variasie tussen en binne hierdie twee spesies aan te toon. Die bevestiging en opsporing van die onderskeie merkers tussen hierdie twee spesies is veral belangrik waar die spesies simpatries voorkom en dit is dus nou moontlik om die gesondsheidsrisiko vir beide mens en dier te indentifiseer. 4 TABLE OF CONTENTS Section Page 1 ABSTRACT OPSOMMING (IN AFRIKAANS) 3 ACKNOWLEDGEMENTS 6 FOREWORD 7 CHAPTER ONE Introduction 8 CHAPTER TWO Biochemical Genetic Markers to Identify Two Morphologically Similar South African Mastomys Species (Rodentia: 30 Muridae) CHAPTER THREE Isozyme and Allozyme Markers Distinguishing Two Morphologically Similar, Medically Important Mastomys 50 species (Rodentia: Muridae) CHAPTER FOUR Immunological Study 73 CHAPTER FIVE Summary 81 APPENDIX A Localities and Apparatus 98 APPENDIX B BIOSYS Input 106 5 ACKNOWLEDGEMENTS I owe thanks to the following people, without whom this project would not have been possible: My supervisor, Prof. Van der Bank for his patience, advice and unfailing sense of humour. Shaun West, for the generous loan of traps on a regular basis. Tony de Castro, for his assistance in showing me where to trap my very first specimens of Mastomys in Montgomery Park. Jaco Delport, for his assistance in trapping in the Richards Bay area. The generous friends and family members who provided a place for me to stay while on field trips and their occasional assistance in the field. To the Rand Afrikaans University for laboratory facilities and financial assistance during this study. Wilma Crous of the RAU Graphics Department for all the pictures. To Sasol, for funding this project. Thanks must also go to colleagues for assistance in the laboratory, especially to Leticia and Monique Greyling, and to Dr Meyer and Dr Falk for their advice. And to my family, who never doubted me. 6 FOREWORD This dissertation is presented as a compilation of two peer-reviewed internationally published research articles, along with a general introduction to and summary of the research work done. The first manuscript (Smit, A., Van der Bank, F.H., Falk, T. and De Castro, A. (2001) Biochemical genetic markers to identify two morphologically similar South African Mastomys species (Rodentia: Muridae) Biochemical Systematics and Ecology 29: 21-30) was published as a preliminary study. The second manuscript (Smit, A. and Van der Bank, F.H. (2001) lsozyme and allozyme markers distinguishing two morphologically similar, medically important Mastomys species (Rodentia: Muridae) BioMed-Central Genetics 2:15) was published as the follow-up study, with additional populations included in the analyses to support the findings of the preliminary report, as well as additional markers detected between the species. This work was also presented at a national conference (Smit, A.K., Van der Bank F.H. and De Castro, A. lsosiem merkers om twee morfologiese eenderse Suid-Afrikaanse Mastomys-spesies (Rodentia: Muridae) te identifiseer. Conference: The South African Academy for Science and Arts, Section Biology. Date 29 September 1998. Venue: University of Pretoria) and published in the conference proceedings (Smit, A.K. and Van der Bank, F.H. (1999) lsosiem merkers om twee morfologiese eenderse Suid- Afrikaanse Mastomys-spesies (Rodentia: Muridae) te identifiseer. SA Tydskrif vir Natuurwetenskap en Tegnologie 18(2): 60-61). 7 CHAPTER 1 INTRODUCTION CHAPTER 1: INTRODUCTION 1.1 THE GENUS PRAOMYS/MASTOMYS The PraomyslMastomys species complex is a group of morphologically similar species that occurs in very diverse habitats throughout sub-Saharan Africa (Qumsiyeh et al., 1990). This complex, and especially the so-called sub-genus Mastomys, is certainly the most widespread and ubiquitous group of rodents of tropical and temperate Africa, extending from the southern and eastern edges of the Sahara right down the continent as far as Plettenberg Bay on the south coast of South Africa (De Graaff, 1981).