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). Praomys was
originally proposed as a subgenus of Epimys (=Rattus) in 1915, with E.
tulbergi as the type species. It was subsequently raised to full generic rank in
1926. The taxonomy of these genera and subgenera has been clouded by a
diversity of opinions. Some researchers have given both Mastomys and
Praomys full generic rank, whereas others regard Mastomys, Hylomyscus,
and Myomys as subgenera of Praomys (De Graaff, 1981). This confusion has
been further compounded by many external and cranial similarities between
species, and thus the systematics of this group remains poorly understood,
with many species yet to be described (Qumsiyeh et al., 1990). Indeed, many
morphologically similar species of the genus Mastomys have been described
throughout sub-Saharan Africa (Britton-Davidian et al., 1995). The genus
Praomys, as here understood, ranges over tropical Africa from Sierra Leone
and Guinea to Ghana in the west, to southern and eastern Zaire, Uganda,
9 Kenya and Tanzania in the east and southwards to Zambia and Malawi, right through to the southern coast of South Africa (De Graaff, 1981). The genus is well represented in the southern African subregion, especially by the multimammate mice, M. coucha and M. natalensis.
1.2 THE MULTIMAMMATE MOUSE
The term multimammate mouse applies to two morphologically similar species of mouse, namely M. natalensis (Smith, 1834) and M. coucha (Smith, 1836).
While the known forms have been described under a great number of independent specific names, it was customary for many years to regard both species as Smith's (1836) M. coucha, until it was shown that M. natalensis was the prior name (Skinner and Smithers, 1990). Thus both species were considered as M. natalensis until karyological studies demonstrated a difference in chromosome number, with M. coucha having 2n=36 and M. natalensis being characterised by 2n=32 (Skinner and Smith, 1990). Both the common and generic names refer to the large number of mammae present in the female, whereas the specific name of natalensis refers to the province of
KwaZulu-Natal, where this species was initially collected (De Graaff, 1981).
Due to frequent and close association with humans, the multimammate mouse is fairly well known. It is commonly captured and the large number of mammae on females makes for easy identification by specialist and layman alike. Ten to 12 mammae on both sides of the belly, from breast to groin, gives the female a high suckling capacity, and pregnant females carrying up to 24 foetuses have been known to occur (De Graaff, 1981). Multimammate
10 mice are smaller than ordinary rats (Rattus spp.) and are thus not easily confused with this genus. Although multimammate mice are predominantly found in the veld, they have long since become commensal with people, and both M. coucha (Fig 1.1) and M. natalensis (Fig 1.2) are the common "House
Mouse" in many areas of southern Africa (De Graaff, 1981). The fur of the species is moderately long and relatively soft. The dorsal colour in adults is buffy, suffused with back hairs. However, this fur is variable in colour, even within populations from any one locality. Specimens from Botswana have been described as indifferently coloured, generally dark grey above, or with a distinct brownish tinge. Cohabitants have been found to vary in colour from dark grey to predominantly brown (De Graaff, 1981). Long, fine, and dense underfur is also present, as well dark tipped gutter hairs and a few slightly loner guard hairs, both of which taper to very fine bases and extend somewhat beyond the general contour of the coat. The underparts vary from white to darkish grey, with individual hairs being grey at the base, and often having a whitish tip. There is a distinct difference in colour between juvenile and older specimens. The former is dull smoky grey, becoming a paler drab and then more rusty coloured and grizzled in maturity (De Graaff, 1981).
The tail is relatively short, being rarely as long as or slightly longer than head-body length. It is sparsely covered with short, rigid hairs, brownish above and a dull white underneath. Rings are present on the tail, and are fairly close to each other. The length of the tail can also vary considerably in animals from the same area. Hands and feet are fairly narrow in both species.
In the hindfoot, the hallux (big toe) falls just short of the base of the second
11 digit. The D5 (digits on each hand or foot are numbered from the inside
outwards) reaches to more than the base of the D4.
Figure 1.1: Mastomys coucha (after Mills and Hes, 1997)
Figure 1.2: Mastomys natalensis (after Mills and Hes, 1997)
12 The ears are moderately proportioned and ovate, with a thin covering of sparse, short hair. Usually there are at least eight pairs of mammae, with the rows being clearly visible in adult females, as each teat is usually ringed with hairs that are lighter in colour than the surrounding fur. The teats do not always occur in pairs (De Graaff, 1981). There are a few species-specific characteristics in which M. coucha and M. natalensis diverge (discussed in
Section 1.5 below).
1.3 HABITAT AND DISTRIBUTION OF SOUTHERN AFRICAN
MULTIMAMMATE MICE
Until a detailed investigation of the ecology of each species is undertaken, it can be assumed that most of the information provided for M. natalensis is also valid for M. coucha (Skinner and Smithers, 1990). The multimammate mouse has a wide habitat tolerance, and is found from sea level to high lying ground.
However, it is absent from really dry or arid areas. The distribution limits of these species are only provisional at present, as it has only been possible to analyse the haemoglobin electromorphs, genetic composition and other identifying characteristics of a relatively small number of specimens. The results that are available reveal a provisional distribution, which shows that these two species are sympatric in some areas, and allopatric in others
(Skinner and Smithers, 1990). At present, M. natalensis has been recorded in the northern parts of Namibia, the Caprivi Strip, over wide parts of Zimbabwe, in the eastern Transvaal (Mpumalanga), KwaZulu-Natal and the eastern and southeastern parts of the Cape Province (Fig. 1.4).
13 Figure 1.3: The provisional distribution of M. coucha in southern Africa (after Mills and Hes, 1997)
Figure 1.4: The provisional distribution of M. natalensis in southern Africa (after Mills and Hes, 1997)
14 Mastomys coucha, on the other hand, has been recorded from Windhoek
(Namibia), from wide parts of Zimbabwe, from the old Transvaal area, Free
State, northern KwaZulu-Natal and from the eastern Cape (Fig 1.3.) (Skinner and Smithers, 1990).
These species are fond of grassland areas where there is some cover of low scrub. In Botswana they are often found in dry watercourses, on the edges of swamps in Ngamiland, in Acacia and Mopane scrub
(Colophospermum mopane) and woodland, as well as in Tenninalia scrub.
Smit et al. (2001) reported that M. natalensis appears to be a species of the warm moist parts of the Savanna biome, according to the biome boundaries determined by Rutherford and Westfall (1993). Vegetation based biomes have been shown to correspond with zoogeographical patterns, including mammals
(Rutherford and Westfall, 1993). In contrast, M. coucha seems to be a species which occurs predominantly in the Grassland biome, but also extends into cool, dry areas of the Savanna biome, and the moister parts of the Nama-
Karoo biome (Smit et al., 2001). The specimen localities within the Nama-
Karoo biome probably represent areas that were historically part of the
Grassland biome, but have since been invaded by vegetation of the Nama-
Karoo biome, and can thus be termed 'false' Karoo (Smit et al., 2001). It is known that the distribution ranges of both species overlap in the Mpumalanga region, with specimens of both having been recorded from a single locality in the Hectorspruit region, which is situated in close proximity to the boundary between the Grassland and Savanna biomes.
15 Both species are also well known to be commensals with humans, and therefore tend to be numerous where population concentrations are high.
They are thus often found in scrub fences put up around cultivated land (De
Graaff, 1981) and are familiar to householders, finding abundant supplies of food and water in houses and stores, as well as shelter in which to safely rear their young. In African kraals, they may occur in very large numbers, especially in the fabric and thatch of traditionally constructed pole and mud huts and also around grain storage bins. However, they are less common in modern towns and cities, due perhaps to the different type of construction used, which does not provide the nooks, crannies and wooden floors under which to nest (Skinner and Smithers, 1990). They are also found on the edges of pans, especially if there are calcareous outcrops nearby. Both species are fond of sandy ground, overgrown with scrub and grass, where they may excavate their own burrows. On the other hand, they may use any other type of cover available, e.g. rock crevices, under fallen logs, cavities within piles of stones and even holes in termite mounds. In Zimbabwe, they show a wide habitat range. They may be found in conditions ranging from dry grassland to
Terminalia scrub on Kalahari sand, to areas in close proximity to water on basalt soils with mopane woodland (De Graaff, 1981). Multimammate mice are common on the fringes of and within agricultural lands where crops such as peanuts (Arachis hypogaea), maize (Zea mays), sorghum (Sorghum vulgarae) or other grains are grown. To some extent they are dependent on water, but occur in areas where this is only available seasonally (Skinner and
Smithers, 1990). Both M. natalensis and M. coucha live under diverse climactic and geographic conditions. They are common in cultivated areas
16 where humans are present, and co-exist with various other kinds of small mammals from place to place. Multimammate mice can thus be considered as a tolerant species with very generalised ecological requirements. Fieldwork has demonstrated that where one or either species occurs, they are quite likely to be one of the first rodent species to establish itself in an area recovering from some kind of habitat destruction (De Graaff, 1981). These processes may include damage by fires as well as modification of the habitat by humans. While these conditions may be somewhat less than optimal for other small mammal species during the process of pioneer secondary succession, the increased plant species diversity due to high proportions of weeds and exotics present provides a wider variety of food and shelter for the multimammate mouse. As succession continues, either Mastomys species may be gradually replaced (sometimes within months) by more specialist grassland species such as the striped field mouse Rhabdomys pumilio, and at a later stage, by vlei rats (Otomys spp.). In the light of these factors, both M. natalensis and M. coucha may be regarded as pioneer species, with their presence indicating an area in an early stage of secondary succession after habitat damage of some sort (De Graaff, 1981). Nearly all Mastomys species are problematic in public health and agriculture. Population explosions of either species have been known to occur at least since colonial times, and rodent damage can be devastating during such outbreaks, and result in acute food shortages (Leirs et al., 1996).
17 1.4 THE MEDICAL IMPORTANCE OF MULTIMAMMATE MICE
Multimammate mice have been shown to be reservoir hosts for a number of organisms that cause diseases in humans. These include Yersina pestis (Fig.
1.6), which causes plague, Lassa fever virus (Fig. 1.5), Witwatersrand and
Banzi viruses, and pathogenic bacteria such as Salmonella typhimurium,
Pasturella pneumotropica and Escherischia coll. In addition, M. natalensis carries a Lassa-like virus called Mopeia. The effect of this virus on humans has yet to be established or researched to any extent (Murray et al., 1995).
Although multimammate mice are predominantly veld rodents, they have long since become commensal with people, and this resulting close contact with them makes the transmission of these diseases, of which they act as carriers of, much more likely (Skinner and Smithers, 1990).
Up to now, it has not been determined which of the two species has a greater tendency towards commensalism with humans, or whether there is indeed a difference in this respect. The medical implications of this fact are significant, as it has been discovered that M. natalensis is substantially more resistant to experimental infection with Y. pestis than M. coucha, and that the geographic distribution of the plague in southern Africa corresponds closely to that of M. coucha, the species more susceptible to plague (Skinner and
Smithers, 1990). In addition, only M. natalensis so far has been found to carry the arenavirus causing Lassa fever, an infection that has a high mortality rate, even in patients with hospital care (15-20%; Murray et al., 1995).
18 Figure 1.5: The arenavirus (spherical structures) that causes Lassa fever, as seen under an electron microscope (after Pollard, 1978)
Figure 1.6: Yersinia pestis, the bacterium causing plague in all three forms (after Boyd, 1988)
19 Mastomys rodents are also the carriers of encephalomyocarditis (EMC) virus, from the Picornavirus family (Grobler et al., 1995). This virus has a worldwide
distribution, and the presence of the EMC virus or antibodies to it have been
reported in wild rodents on all continents. The disease has been linked to
myocarditis, abortion, stillbirth and foetal death in pigs (Grobler et al., 1995).
A recent outbreak of encephalomyocarditis in the Kruger National Park may
have killed approximately 103 elephants in the mid-nineties, and possibly
many more before the symptoms were recognised (Grobler et al., 1995). The
highest prevalence of antibody to the EMC virus occurred in the ubiquitous
Mastomys rodents (Grobler et al., 1995), although as both M. coucha and M.
natalensis are known to occur within the borders of the park, it is uncertain at this point whether one or both species carries the virus.
While multimammate mice carry other diseases, it has still not been
determined whether only one or both of the biological species are involved in the transmission thereof. Banzi and Witwatersrand viruses have been isolated from multimammate mice, demonstrating that these wild rodents are among their maintenance hosts. Both species are also highly susceptible to experimental infection with Borrelia duttoni, the agent that causes relapsing fever (Skinner and Smithers, 1990).
The great pandemics of plague have almost certainly had a more
profound effect on human history than any other single infection. The fourteenth century pandemic alone took 25 million lives, or a quarter of the
population of Europe, and has been called the worst disaster that has ever
befallen humanity. It is also interesting to note that the plague may have been
used in one of the first recorded instances of biological warfare, with Roman
20 soldiers hurling infected corpses into cities under siege. Plague has played a significant role even in modern times; between 1900 and 1972 there were 992 cases of plague in the United States alone, of which 720 (73%) were fatal
(Roberts et al., 1996).
Plague occurs in three forms, namely bubonic (Fig. 1.7), primary
pneumonic and primary septicaemic (Fig. 1.8). Bubonic plague, which is generally the most common in epidemics, is fatal in about 25% to 50% of
untreated cases. Pneumonic plague, a highly contagious, airborne form, and
septicaemic plague, a generalised blood infection, are more rare forms and
usually fatal (Roberts et al., 1996).
Plague is now endemic in the southern African Subregion, with wild
rodents being the reservoir hosts of the plague bacillus, and multimammate
mice therefore play and important role in its transmission to humans. The
disease was originally introduced by ship rats (Rattus spp.) at our coastal
ports. It was then transmitted from these rats to the local, indigenous rodents
by way of infected fleas. The disease was transmitted in particular to
multimammate mice, which would have come into close contact with
shipboard rats in the warehouses and storage bins of ports. Thereafter, with
free movement from houses and stores to their natural homes in the veld,
infected multimammate mice passed this disease along to gerbils (Tatera
spp.), again through infected fleas, and gerbils are now one of the principal
hosts of plague in the Subregion (Skinner and Smithers, 1990).
21 Figure 1.7: The characteristic buboes that develop in the bubonic form of plague (after Roberts and Janovy (1996)
Figure 1.8: An example of the symptoms in the septicaemic form of plague. Such cases are often fatal (after Ingraham and Ingraham, 1995)
22 The occasional mice population explosions that occur in endemic
plague areas also imply a corresponding increase in the number of fleas that
parasitise multimammate mice (Xenopsylla, Dinopsyllus and Leptopsyllya spp.). Once the explosion peak has passed, and mice numbers begin to dwindle, a multitude of fleas, possibly infected with plague, is suddenly confronted with a shortage of food animals, and they thus abandon their dead
hosts to seek blood meals from other mammals, including humans (De Graaff,
1981). Plague has occurred in southern Africa since 1899, and by 1974, it had spread eastwards into Zimbabwe, in the areas of Kalahari sand, reaching as far east as St Paul's Mission near Lupane, with human deaths resulting. By
1987, it was widely distributed in Owambo, northern Namibia. This wide spread of the disease, northwards from the coastal ports, has resulted in plagues current endemic status in the Subregion (Skinner and Smithers,
1990). Of course, the different health threats that are potentially posed by both species are difficult to identify, due the high degree of morphological similarity. To date, only a few micro-morphological, ethological and physiological differences have been described.
1.5 DISTINGUISHING BETWEEN M. NATALENSIS AND M. COUCHA
As mentioned before, M. natalensis and M. coucha are indistinguishable to the naked eye. They were originally considered as one species until chromosomal examinations revealed differences. Gordon (1984) suggested that on this basis the name M. natalensis is valid for the genetic species
2n=32, and M. coucha for the genetic species 2n=36. He also demonstrated,
23 via horizontal starch-gel electrophoresis, that there were two distinct haemoglobin electromorphs for each of the karyotypes. With reference to a human blood marker standard, the individual components of the double- banded electromorphs were designated as either "slow" or "fast" migrating
(Fig. 1.9). The fast migrating component was only found in specimens with 36 chromosomes, while slow markers were present in those with 32. Gordon
(1984) could not detect any hybrid haemoglobin patterns from natural allopatric or sympatric populations, although they could be produced in captivity. He also showed differences in the urethral lappet shape of the phallus and spermatozoa tail length of the two species. However, more recent research into the morphology of the spermatozoa of these two species by
Breed (1995) revealed them to be extremely similar, if not identical. The reproductive behaviour and ultrasonic vocalisations of the two species are unique as well, and preliminary studies of their pheromones also indicate differences (Skinner and Smithers, 1990)
More recently, Dippenaar et al. (1993) made use of multivariate techniques to differentiate between the cranial characteristics of the two species. They were able to distinguish a high percentage of old museum samples previously identified via haemoglobin electromorphs. However, they had an unexpected finding in a series of six specimens from Hectorspruit in
Mpumalanga, which presented a bimodal a posteriori discriminant score profile. The six specimens had been identified as M. coucha based on their electromorphs, but the subsequent cranial analysis showed three of them to fall into the range of M. natalensis. Although this anomaly was ascribed to data transcription errors, the possibility exists that this technique is not 100%
24 accurate. The authors did not recommend their technique for use in localities far outside the limits of their study due to the unknown extent of geographic variation for each species.
Figure 1.9: Gordon (1984) used these haemoglobin markers to distinguish between the species. Both species shared an allele with the same mobility as the human standard (Hu), but M. coucha (C) had a "fast" and M. natalensis a "slow" migrating allele each. The hybrids (Hy) share all three.
25 1.6 AIMS FOR THIS STUDY
To summarise, it is impossible to positively identify these two sibling species
in the field at present. This presents problems in areas where they occur
sympatrically, especially when coupled to identifying the different health threats posed by each species. It also makes determining the current ranges
of either M. coucha or M. natalensis next to impossible without the need for
laborious analyses, which most mammalogists do not bother with at present.
Most field workers only identify these rodents down to the genus level, thus
negating any possible positive contribution to studies of these species' ecology and distribution. In the light of these problems, three aims were
identified:
To analyse the extent of genetic variation within, and differentiation
between, populations of M. coucha and M. natalensis by using starch and
polyacrylamide gel-electrophoresis. This would yield insight into the
relatedness of these two species.
To ascertain whether there are any reliable genetic markers that will
distinguish each species from the other across a large range of their
distribution, using the same methods listed above. Consultants
researching levels of biodiversity and their implications for large
companies such as Sasol and the purposes of future eco-tourism need to
be able to distinguish between the two species (De Castro, A. Pers.
comm., 1998).
26 For the purposes of aims 1 and 2, allozyme electrophoresis was considered the technique of choice, for several reasons. One such advantage is the relative inexpensiveness and rapidity of the technique, which starch gel electrophoresis, along with the histochemical visualisation of locus specific enzymes, offers (Qamaruz-Zaman et aL, 1998). It may also be applied to virtually any life form, and can be used with large sample sizes without the need of technologically advanced equipment in expensive laboratories
(Qamaruz-Zaman et al., 1998).
3. To research the possibility of raising polyclonal antisera to the distinct
haemoglobin chains of each species with the purpose of developing a kit
to distinguish between the two species in the field, thus further enabling
identification. This would be researched using simple gel immunodiffusion
procedures to test for antibody-antigen precipitation reactions.
1.7 REFERENCES
BOYD, R.F. (1988) General Microbiology, second edition. Times Mirror/Mosby
College Publishing, Toronto.
BREED, W.G. (1995) Spermatozoa of murid rodents from Africa:
morphological diversity and evolutionary trends. Journal of Zoology
London 237: 625 — 651.
BRITTON-DAVIDIAN, J., CATALAN, J., GRANJON, L. and DUPLANTIER, J.
(1995) Chromosomal phylogeny and evolution in the genus Mastomys
(Mammalia, Rodentia). Journal of Mammalogy 76(1): 248-262
27 DE GRAAFF, G. (1981) The Rodents of Southern Africa. Butterworths,
Pretoria.
DIPPENAAR, N.J., SWANEPOEL, P. and GORDON, D.H. (1993) Diagnostic
morphometrics of two medically important southern African rodents,
Mastomys natalensis and M. coucha (Rodentia: Muridae). South
African Journal of Science 89, 300-303.
GORDON, D.H. (1984) Evolutionary Genetics of the Praomys (Mastomys)
natalensis species complex (Rodentia: Muridae). PhD Thesis,
Witwatersrand University, Johannesburg.
GROBLER, D.G., RAATH, J.P., BRAACK, L.E.O., KEET, D.F., GERDES,
G.H., BARNARD, B.J.H., KRIEK, N.P.J., JARDINE, J. and
SWANEPOEL, R. (1995) An outbreak of encephalomyocarditis
infection in free-ranging African elephants in the Kruger National Park.
Onderstepoort Journal of Veterinary Research 62: 97-108.
INGRAHAM, J.L. and INGRAHAM, C.A. (1995) Introduction to Microbiology.
Wordsworth Publishing, California.
LEIRS, H., VERHAGEN, R., VERHEYEN, W., MWANJABE, P. and MBISE, T.
(1996) Forecasting rodent outbreaks in Africa: an ecological basis for
Mastomys control in Tanzania. Journal of Applied Ecology 33:937-943.
MILLS, G. and HES, L. (1997) The Complete Book of Southern African
Mammals. Struik Winchester, Cape Town.
MURRAY, P.R., BARON, E.J., PFALLER, M.A., TENOVER, F.C. and
YOLKEN, R.H. (1995) Manual of Clinical Microbiology, sixth edition.
ASM Press, Washington, D.C.
28 POLLARD, M. (1978) Perspectives in Virology Volume 10. Raven Press, New
York.
QUMSIYEH, M.B., KING, S.W., ARROYO-CABRALES, A., AGGUNDEY, I.R.,
SCHLITTER, D.A., BAKER, R.J. and MORROW, K.J. Jr. (1990)
Chromosomal and protein evolution in morphologically similar species
of Praomys sensu lato (Rodentia, Muridae). Journal of Heredity 81:58-
65.
QAMARUZ-ZAMAN, F., FAY, M.F., PARKER, J.S. and CHASE, M.W. (1998)
Molecular techniques employed in the assessment of genetic diversity:
a review focusing on orchid conservation. Lindleyana 13(4): 259-283.
ROBERTS, L.S. and JANOVY, J. (1996) Foundations of Parasitology, fifth
edition. WCB Publishers, Toronto.
RUTHERFORD, M.C. and WESTFALL, R.H. (1993) Biomes of Southern
Africa. Memoirs of the Botanical Survey of South Africa 63.
SKINNER, J.D. and SMITHERS, R.H.N. (1990) The Mammals of the
Southern African Subregion (second edition). University of Pretoria,
Pretoria.
SMIT, A., VAN DER BANK, 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.
29 CHAPTER 2
BIOCHEMICAL GENETIC
MARKERS TO IDENTIFY TWO
MORPHOLOGICALLY SIMILAR
SOUTH AFRICAN MASTOMYS
SPECIES (RODENTIA:
MURIDAE)
30 CHAPTER 2:
BIOCHEMICAL GENETIC MARKERS TO IDENTIFY TWO
MORPHOLOGICALLY SIMILAR SOUTH AFRICAN MASTOMYS SPECIES
(RODENTIA: MURIDAE)
21 INTRODUCTION
The Mastomys species complex of mice is widely distributed in South Africa, especially the so-called Multimammate mice, Mastomys coucha and M. natalensis. The limits of their distribution are only provisional at this stage
(Skinner and Smithers, 1990), but it is known that they are sympatric in some areas, and allopatric in others. Mastomys coucha acts as a reservoir for the bacterium Yersinia pestis, the organism causing Plague (Dippenaar et al.,
1993).
At present, three diagnostic forms of plague are known: bubonic, primary pneumonic and primary Septicaemic. Bubonic plague, which is the most common type in epidemics, is fatal in about 25% to 50% of untreated cases. Pneumonic plague, a highly contagious (airborne) form, and
Septicaemic plague, a generalised blood infection, are rarer forms, and usually fatal (Roberts et aL, 1996). Apart from Bubonic plague, this species complex is also a reservoir for the Banzi and Witwatersrand viruses
(Dippenaar et al., 1993) as well as a recently emerged disease in forested
West Africa.. Lassa fever is an infection caused by an arenavirus, and has a high mortality rate, even in patients with hospital care (15-20% fatal).
Mastomys natalensis, as well as being a documented carrier of Lassa fever
31 also carries a Lassa-like virus called Mopeia. Its effect on man has yet to be established or researched (Murray et al., 1995).
Morphologically, both species are almost identical in all visible characteristics, and were originally regarded as one (de Graaf, 1981) M. natalensis. However, Gordon (1984) has shown that, at the very least, both species are distinguishable by ethological and micro-morphological characteristics, by their chromosome number, and characteristic haemoglobin variations. More recently, Dippenaar et al. (1993) used multivariate analysis of cranial characteristics to distinguish between the two species. However, 50% of the specimens collected at a sympatric locality were identified differently according to the latter method.
In the present study, we examined new material of two allopatric populations of M. coucha and M. natalensis aimed at identifying species characteristic genetic markers. Allozyme and haemoglobin variations were analysed comparatively.
2.2 MATERIALS AND METHODS
Tissue extracts of 24 individuals of M. coucha caught at Montgomery Park,
Johannesburg (26° 09'22"S, 27°58'58"E) and 20 individuals of M. natalensis caught at La Lucia ridge in Durban North (29°44'44"S, 31°03'09"E) (see
Appendix A for sample sites), were analysed electrophoretically using standard horizontal starch gels and homogeneous polyacrylamide gels (Fig.
2.2). The localities were also studied to determine if there was a relationship between habitat types and the distribution of these two species (Fig. 2.1).
32 Specimens that were positively identified by Gordon (1984), either chromosomally or via characteristic haemoglobin variation were used as reference.
Specimens were caught using standard steel snap-traps or live
Sherman-type traps, and samples of blood, liver, muscle, heart and kidney tissue were taken. These were placed in vials, kept on ice and transported to the laboratory. Haemolysate samples were prepared from native blood samples according to Falk et al. (1996). Samples were stored at about -20°C.
Allozyme studies were carried out using 12% starch gels, as well as 7% standard polyacrylamide gels (Ferreira et al., 1984). Staining methods are as described by Shaw and Prasad (1970) and Harris and Hopkinson (1976), and the method of interpretation of gel-banding patterns and locus nomenclature as referred to by Van der Bank and Van der Bank (1995). A discontinuous lithium-borate tris-citric acid buffer (RW; electrode: pH 8; gel: pH 8.7; Ridgway et al., 1970) and two continuous buffers: a tris-citric acid buffer system (TC; pH 6.9; Whitt 1970) and a tris-borate-EDTA buffer (MF; pH 8.6; Market and
Faulhauber, 1965) were used to study variation at 34 protein-coding loci.
Locus abbreviations, enzyme commission numbers, buffers, tissues and monomorphic loci are listed in Table 2.1.
Haemoglobin variations were analysed by isoelectric focusing (IEF) according to Falk et al. (1998). IEF separations were conducted on Servalyte precotes (pH range 3-10, Serva, Heidelberg, Germany). Gels were prefocused at 6°C (200-500 V). Subsequently, haemolysate samples (10 pl) were applied to an applicator strip positioned 5.0 cm from the anode and the voltage was limited to 1700 V. Separations were finished when a constant
33 current of maximal 2 mA/gel was reached (after about 2.5 h). Prior to use, haemolysate samples were diluted in distilled water (final concentration: 20 mg Hb/ml) and treated with 2-mercaptoethanol (3%) for 1 h at 5°C.
IEF separated haemoglobins could be identified by their red colour. In addition, gels were incubated in 4-chloro-1-naphthoVH202 (Serva, Merck) mainly to intensify haemoglobin components by their pseudoperoxidase activity. The staining solution consisted of 60 ml methanol and 340 ml PBS
(pH 7.4) containing 120 mg 4-chloro-1-naphthol and 1 ml 30% H202 (Miribel and Arnoud, 1988). Gels were also counterstained with Coomassie Brilliant
Blue G-250 (Serva). The following pl marker proteins (Serva) were used: horse myoglobin: pl 6.90 and 7.40 and lectins of Lens culinaris: pl 7.80, 8.00, and 8.30.
2.3 RESULTS AND DISCUSSION
2.3.1 ECOLOGY AND DISTRIBUTION
It was not previously possible to accurately describe the geographic distribution and habitat requirements of either species, as a result of the extreme morphological similarity of M. natalensis and M. coucha, and the fact that the genetic composition of only a relatively small number of individuals has been studied. It does however seem likely that differences in the habitat requirements of the two species do exist. Figure 2.1 shows the localities of the
M. coucha and M. natalensis populations positively identified by means of morphometric analyses conducted by Dippenaar et al. (1993), the
34 chromosomal and haemoglobin analyses by Gordon (1984), as well as one population of each species identified during the genetic study presented here, in relation to the boundaries of the various biomes recognised within the southern African Subregion by Rutherford and Westfall (1994). These localities represent only a fraction of the known localities for Mastomys within
South Africa and do not include other southern African material.
The boundaries of the biomes as depicted in Figure 2.1, were determined by Rutherford and Westfall (1994) on the basis of dominant and co-dominant plant life forms in climax systems, at a scale of 1: 10 000 000.
Biomes determined on the basis of vegetation have been shown to correspond with zoogeographical patterns, though the correlation seems to depend strongly on the animal group (Rutherford and Westfall, 1993). Some correspondence has been found for mammals (Rautenbach, 1978), and this is to be expected as vegetation not only determines the structural nature of the habitat for animals (Odum, 1971), but also reflects the prevailing climatic conditions, such as rainfall and temperature, which also affect mammals both directly and indirectly.
The localities included in Figure 2.1 indicate that M. natalensis is clearly a species of the Savanna biome, and more particularly of the moist warm regions of this biome. M. coucha seems to be a species which occurs
predominantly in the Grassland biome, but also extends into cool, dry areas of the Savanna biome, and the moister parts of the Nama-Karoo biome. The
localities within the Nama-karoo biome probably represent areas that were
historically part of the Grassland biome, but have since been invaded by vegetation of the Nama-Karoo biome, and can thus be termed 'false' Karoo
35 (Rutherford and Westfall 1993). The distribution ranges of these two species are known to be sympatric in the Mpumalanga Lowveld, and both species have been recorded from a single locality at Hectorspruit in this region, which is situated in close proximity to the boundary between the Grassland and
Savanna biomes. An analysis of distribution patterns, based on a far larger number of localities would be required to establish whether there are indeed major habitat differences between these two species.
2.3.2 GENETIC VARIATION
Twenty-three (67%) of the thirty-four protein coding loci studied were monomorphic (Table 2.1), and products of the following loci migrated cathodally: ADH-4, GAP, GPD-4, GPI-1, -2 and LDH-3. Polymorphic loci together with allelic frequencies are listed in Table 2.2, and F (fixation index) statistic summaries are given in Table 2.3.
Fixed allele mobility differences between the two populations studied were obtained at three loci: GPI-2 in liver, PT-2 and PT-3 in muscle tissue.
The products of GPI-2 and PT-3 were absent in M. coucha, whereas the products of PT-2 were absent in M. natalensis (Fig. 2.2a, b). While it is possible that PT-2 and PT-3 are different alleles of the same locus, the large separation distance between the bands suggests that they represent individual loci. A study of more individuals from different populations may reveal polymorphism, and is required to verify this conclusion. Products of these three isozyme loci are useful to identify individuals from either of the populations studied. Moreover, significant allelic frequency differences
(P<0.05) were identified at EST-1. Allelic frequencies for M. coucha deviated
36 from Hardy-Weinberg equilibrium at EST-1 and IDH-1, while those for M. natalensis deviated at IDH-1 and 2. These are probably due to small sample sizes.
F values (Wright, 1978) can be used to estimate the amount of genetic differentiation between species. A mean FsT value of 0.68 suggests a large amount of genetic differentiation between the two species studied, confirming their present taxonomic status and the results of Gordon (1984). Moreover, our estimate for mean Fis and Frr values also suggests a high degree of positive assortative mating and inbreeding, and further demonstrates very little or no gene flow between both species in the wild. Gordon (1984) also found no evidence of hybrids in nature, but he was able to mate them in captivity. However, these hybrids were infertile when back-crossed. Nei's unbiased genetic distance (1978) was calculated to be 0.123. This measurement estimates the number of allelic substitutions per locus that have occurred since both species have diverged. This estimate falls outside Ayala's
(1982) estimate for genetic distances between local populations of the same species of mammals (D = 0.058), but well inside of his estimate for subspecies (D: 0.232). It is also low when compared with a D value of 0.46
between Peromyscus species (Rodentia: Cricetidae), but compares better with values for Thomomys species (Rodentia: Geomyidae) of 0.08 (Avise and
Aquadro, 1982). Thus our calculation may appear low, but it does fall into the
range of rodent variation, and could most probably be attributed to an on-
going process of speciation. Unbiased heterozygosity values (Nei, 1978) were
calculated to be 0.019 and 0.038 for M. coucha and M. natalensis,
respectively (Table 2.2). However, both of these values are less than that
37 calculated by Selander et al. (1971) for Peromyscus polionotus (H = 0.053), and Ayala (1982) for mammals (average = 0.051).
In addition, we also determined the degree of haemoglobin variation in both populations of M. coucha and M. natalensis. Characteristically, all samples investigated revealed heterogeneous haemoglobin patterns (Fig.
2.3). Two haemoglobin components of differing pl, ranging between pH 7.52 and pH 7.04, were detected by thin layer isoelectric focusing of individual samples. Characteristic variations in haemoglobin phenotypes were found to occur in samples of the two species studied. Mastomys coucha specimens were characterised each by a unique combination of two different haemoglobin components with pls of 7.04 and 7.31, whereas M. natalensis specimens displayed two haemoglobin components with pls of 7.25 and 7.52.
Haemoglobin phenotypes appeared to be consistent within both species.
Essentially, these findings are in accordance with previous studies on the haemoglobin types of both species of the genus Mastomys studied here
(Gordon, 1984). However, based on starch gel separations, only one distinct haemoglobin component could be identified for each of the species and definite species characteristic differences in haemoglobin profiles remained questionable. It should also be noted that isoelectric focusing of haemoglobins enables an identification of both mice species without the use of reference samples, only pl marker proteins are required.
In conclusion, this study provides evidence that the populations studied of these two species of the genus Mastomys are more genetically distinct than previous studies have shown. Gordon (1984) demonstrated the mobility differences between M. natalensis and M. coucha, but was unable to resolve
38 the bands at pl 7.25 and 7.31. With these results it can be seen that these species do not share any of the two major haemoglobin components, and can now be identified on the basis of mobility alone, without the need of comparative analyses. An extension of the current study that includes more populations of both species across the range is required to confirm the presence of these markers between both species. These results may be valuable for the routine identification of these two medically important species.
2.4 REFERENCES
AVISE, J.C. and AQUADRO, C.F. (1982) A comparative summary of genetic
distances in the Vertebrates: patterns and correlations. Evolutionary
Biology 15, 151-185.
AYALA, F.J. (1982) Population and Evolutionary Genetics.
Benjamin/Cummings Publishing Company, Inc, London.
DE GRAAFF, G. (1981) The Rodents of Southern Africa. Butterworths,
Pretoria.
DIPPENAAR, N.J., SWANEPOEL, P. and GORDON, D.H. (1993) Diagnostic
morphometrics of two medically important southern African rodents,
Mastomys natalensis and M. coucha (Rodentia: Muridae). South
African Journal of Science 89, 300-303.
FALK, T. M., ABBAN, E. K., OBERST, S., VILLWOCK, W., PULLIN, R. S. V.
and RENWRANTZ, L. (1996) A Biochemical Laboratory Manual for
Species Characterization of Some Tilapiine Fishes. ICLARM Education
Series 17, Manila, Philippines.
39 FALK, T.M., VILLWOCK, W. and RENWRANTZ, L. (1998) Heterogeneity and
subunit composition of the haemoglobins of 5 tilapiine species
(Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon.
Journal of Comparative Physiology B 168, 9-16.
FERREIRA, J.T., GRANT, W.S. and AVTALION, R.R. (1984) Workshop on
Fish Genetics. CSIR Special Publications, Council for Scientific and
Industrial Research, Pretoria, RSA.
GORDON, D.H. (1984) Evolutionary Genetics of the Praomys (Mastomys)
natalensis species complex (Rodentia: Muridae). PhD Thesis,
Witwatersrand University, Johannesburg.
HARRIS, H. and HOPKINSON, D.A. (1976) Handbook of Enzyme
Electrophoresis in Human Genetics. North-Holland Publishing
Company, Amsterdam.
MARKERT, C.L. and FAULHABER, I. (1965) Lactate dehydrogenase isozyme
patterns of fish. Journal of Experimental Zoology 159, 319-332.
MIRIBEL, L. and ARNOUD, P. (1988) Electrotransfer of proteins following
polyacrylamide gel electrophoresis - nitrocellulose versus nylon
membranes. Journal of Immunological Methods 107, 253-259.
MURRAY, P.R., BARON, E.J., PFALLER, M.A., TENOVER, F.C. and
YOLKEN, R.H. (1995) Manual of Clinical Microbiology, sixth edition.
ASM Press, Washington, D.C.
NEI, M. (1978) Estimation of average heterozygosity and genetic distance
from a small number of individuals. Genetics 89, 583-590.
ODUM, E.P. (1971) Fundamentals of Ecology. Saunders, Philadelphia.
40 RAUTENBACH, I.L. (1978) Ecological distribution of the mammals of the
Transvaal. Annual of the Transvaal Museum 31 10, 131-156.
RIDGWAY, G.J., SHERBOURNE, S.W. and LEWIS, R.D. (1970)
Polymorphism in the esterases of Atlantic herring. Transactions of the
American Fisheries Society 99, 147-151.
ROBERTS, L.S. and JANOVY, J. (1996) Foundations of Parasitology, fifth
edition. WCB Publishers, Toronto.
RUTHERFORD, M.C. and WESTFALL, R.H. (1993) Biomes of Southern
Africa. Memoirs of the Botanical Survey of South Africa 63.
SELANDER, R.K., SMITH, M.H., YANG, S.Y., JOHNSON, W.E. and
GENTRY, J.B. (1971) Biochemical polymorphism and systematics in
the genus Peromyscus. I. Variation in the old-field mouse (Peromyscus
polionotus). Studies in Genetics VI. University of Texas Publications
7103, 49-90.
SHAW, C.R. and PRASAD, R. (1970) Starch gel electrophoresis of enzymes
— a compilation of recipes. Biochemical Genetics 4, 297-320.
SKINNER, J.D. and SMITHERS, R.H.N. (1990) The Mammals of the
Southern African Subregion, second edition. University of Pretoria,
Pretoria.
VAN DER BANK, F.H. and VAN DER BANK, M. (1995) An estimation of the
amount of genetic variation in a population of the Bulldog Marcusenius
macrolepidotus (Mormyridae). Water South Africa 21, 265-268.
WHITT, G.S. (1970) Developmental genetics of the lactate dehydrogenase
enzyme of fish. Journal of Experimental Zoology 175, 1-35.
41 WRIGHT, S. (1978) Evolution and the Genetics of Populations, Vol. 4.
Variability Within and Among Natural Populations. University of
Chicago, Chicago.
42 TABLE 2.1: Locus abbreviations, buffer systenis and enzyme commission
numbers (E. C. no.) are listed after each protein.
Protein Locus E.C. No. Buffer Tissue
1 . Alcohol dehydrogenase ADH-1 a -4a 1.1.1.1 RW M, L
2. Creatine kinase CK-1 a —3a 2.7.3.2 RW M, L
3. Esterases EST-1, -2a' —3a 3.1.1.- MF M, L
b 4. General proteins PT-1 -5 M, BI
5. Glucose-6-phosphate isomerase GPI-1, -2 3.5.1.9 RW L
6. Glyceraldehyde-3-phosphate GAPa 1.2.1.12 RW M, L
dehydrogenase
7. Glycerol-3-phosphate dehydrogenase GPD-l a --4a 1.1.1.8 RW M, L
8. Isocitrate dehydrogenase IDH-1, -2, -r 1.1.1.42 TC M, L
9. L-Lactate dehydrogenase LDH-l a, -2, -3a 1.1.1.27 RW M, BI
10. Malate dehydrogenase MDHa 1.1.1.37 TC M, L
11 a Phosphoglucomutase PGM-1, —3 5.4.2.2 RW M
11 b. Phosphoglucomutase PGM-4 5.4.2.2 RW L
12 Superoxide dismutase SODa 1.15.1.1 RW M, L
a Monomorphic loci
b Polyacylamide Gel Electrophoresis
43 Table 2.2: Allele frequencies, average heterozygosity per locus (H) and mean
number of alleles per locus (A) with standard errors in parentheses. Also
listed is percentage of loci polymorphic (P) for the two populations.
Mastomys concha Mastomys natalensis Locus Allele
A 0.156 1 EST-1 B 0.844
a A 1 GPI-2 A 0.375 IDH-1 B 0.833 0.5
C 0.167 0.125
A 0.125 IDH-2 B 1 0.875
A 0.033 LDH-2 B 0.967 1
A 0.062 PGM-1 B 1 0.938
A 0.062 PGM-2 B 1 0.938
A 0.062 PGM-3 B 1 0.938
44 Table 2.2 continued
Mastomys coucha Mastomys natalensis Locus Allele
A 0.026 PGM-4 B 1 0.974
A a 1 PT-2 A 1 a PT-3
H 0.019 0.038 (lc 0.012) (± 0.020)
1.1 1.2 A (±0.00) (± 0.10)
8.80% 17.60% P a Locus absent.
45 Table 2.3: Summary of F-statistics at all polymorphic loci for both species.
Locus FIS FIT Fsr
0.763 0.936 0.730 EST-1 - 1.000 1.000 GPI-2 1.000 1.000 0.127 IDH-1 1.000 1.000 0.067 IDH-2 - 0.034 - 0.017 0.017 LDH-2 - 0.067 - 0.032 0.032 PGM-1 - 0.067 - 0.032 0.032 PGM-2 - 0.067 - 0.032 0.032 PGM-3 - 0.027 - 0.013 0.013 PGM-4 - 1.000 1.000 PT-2 - 1.000 1.000 PT-3 0.694 0.902 0.680 Mean
46 22° D Desert biome 24° Grassland biome
Succulent biome 26° 0 Forest biome
Nama-Karoo biome 28° 0 0 Savanna biome 300 Fynbos biome
32° M natalensis M. couches
34°
16° 18° 20° 22° 24* 26° 28° 30' 32° 34° 36 .
Figure 2.1: The distributions of Mastomys coucha and M. natalensis according to positively identified specimens. Biome types are shown and the sampling sites of this study are indicated with -V.
47 ORIGIN f- GPI-2
■••■•••••11 M. coucha M. natalensis
B F- ORIGIN PT-5 PT-4
PT-3
tr-- PT-2 PT-1
Ifri■•■••1
M. natalensis M. coucha
Figure 2.2: Isozyme differences between M. coucha and M. natalensis at (A) GPI (liver) and (B) PT (muscle) protein coding loci. The GPI-2 and PT-3 loci are absent in M. coucha, but the PT-2 locus is absent in M. natalensis.
48 Mn Mc
_ p17.52 p17.31 p17.25 p17.04 —
Figure 2.3: IEF separation of haemoglobin phenotypes of M. coucha (Mc) and M. natalensis (Mn) (pl = isoelectric point).
49 CHAPTER 3
ISOZYME AND ALLOZYME
MARKERS DISTINGUISHING
TWO MORPHOLOGICALLY
SIMILAR, MEDICALLY
IMPORTANT MASTOMYS
SPECIES (RODENTIA:
MURIDAE)
50 CHAPTER 3:
ISOZYME AND ALLOZYME MARKERS DISTINGUISHING TWO
MORPHOLOGICALLY SIMILAR, MEDICALLY IMPORTANT MASTOMYS
SPECIES (RODENTIA: MURIDAE)
3.1 INTRODUCTION
The Praomys/Mastomys species complex is a group of morphologically similar species that occur in very diverse habitats throughout sub-Saharan
Africa (Qumsiyeh et al., 1990). The taxonomy of this group has been clouded by a diversity of opinions and this confusion has been further compounded by many external and cranial similarities between species, and thus the systematics of this group remains poorly understood, with many species yet to be described (Qumsiyeh et al., 1990). The genus Mastomys is well represented in southern Africa, especially by the ubiquitous multimammate mice, Mastomys coucha (Smith, 1836) and M. natalensis (Smith, 1834). The distribution of both species is only provisional at this stage (Skinner and
Smithers, 1990), and the species are known to be sympatric in some areas, and allopatric in others. These common agricultural pests are also commensal with man, often sheltering in houses in order to safely rear their young. In
African kraals they occur in very large numbers, living in the fabric and thatch of pole and mud huts (Skinner and Smithers, 1990).
Because these mice carry important diseases, the medical implications of this cohabitation with man are obvious. Mastomys coucha acts as a reservoir for the bacterium Yersinia pestis, the organism causing plague
(Dippenaar et al., 1993). The three forms of plague are bubonic, primary
51 pneumonic and primary septicaemic. Bubonic plague, which is the most common type in epidemics, is fatal in about 25% to 50% of untreated cases.
Pneumonic plague, a highly contagious (airborne) form, and septicaemic plague, a generalised blood infection, are more rare forms, and usually fatal
(Roberts et al., 1996). Plague still exists as a threat in southern Africa, with much current concern over the possibility of outbreaks in rat infested inner cities.
Both species are also carriers of the Banzi and Witwatersrand viruses
(Dippenaar et al., 1993) and M. natalensis is a natural reservoir for the arenavirus causing Lassa fever. This infection, occurring predominantly in
West Africa up until now, has a high mortality rate, especially amongst pregnant women and even in patients with modern health care. The Mopeia virus is another similar virus carried by M. natalensis, but the effect thereof on man is as yet unknown (Murray et al., 1995). Mastomys rodents were also linked to an outbreak of encephalomyocarditis (EMC) among African elephants in the Kruger National Park in the last decade (Grobler et al., 1995).
However, due to the fact that both species occur within the boundaries of the park, it is uncertain as to whether only one or both of these species may carry the EMC virus.
The differences between the two species are thus neither ecologically
nor economically trivial. This illustrates the need for research into their
relatedness, especially of a genetic nature, as this could yield insights as to why only certain species carry the above mentioned diseases, especially as these two sibling species are impossible to distinguish on the basis of external
morphology alone. For this reason, both M. coucha and M. natalensis were
52 described as one species, that of M. natalensis (De Graaff, 1981). However, these species do differ in chromosome number, with M. coucha having 2n=36 and M. natalensis having 2n=32 (Gordon, 1984). Differences in behaviour,
urethral lappet and spermatozoal morphology and haemoglobin mobility in electrophoresis were also reported by Gordon (1984). In contrast, more recent
research into spermatozoa' morphology of these two species led Breed
(1995) to report that they were extremely similar, if not identical in this respect. Dippenaar et al. (1993) used multivariate analysis of the cranial characteristics of each species in order to identify them. However, there were discrepancies in identification of 50% of the specimens collected at a sympatric locality according to this method and that of haemoglobin mobility.
The preliminary report of Smit et al. (2001) showed isozyme differences at three loci, GPI-2 in liver, and PT-2 and —3 in muscle tissue. This study also revealed through iso-electric focusing that the haemoglobin components present in each species were unique from the other. In the present study, we have used electrophoretic techniques in an attempt to confirm the isozyme markers (locus differences) reported above in two additional allopatric populations of M. coucha and M. natalensis, as well as to identify additional genetic markers.
3.2 MATERIALS AND METHODS
The genetic analysis of an additional nine individuals of M. coucha caught on the shores of the Vaal Dam (26°51'49"S, 28°08'59"E), 16 individuals of M. natalensis from rehabilitated dunes in the Richards Bay area (28°38'48"S,
32°16'15"E) as well as a further ten individuals from Montgomery Park
(previous reference site for M. coucha) (26°09'22"S, 27°58'58"E) and four
53 reference samples of M. natalensis from La Lucia Ridge (Kwazulu-Natal)
(29°44'44"S, 31°03'09"E) was done using electrophoresis (see Appendix A for pictures of sample sites). The total number of specimens from all populations was 40 for M. natalensis and 43 for M. coucha. The additional specimens were caught using live Sherman-type traps and were then transported to the laboratory where samples of blood, liver, and muscle tissue were taken. All samples except blood were frozen at -20°C. Blood samples were prepared for polyacrylamide gel electrophoresis using the technique described by Falk et al. (1996). Whole blood samples are centrifuged at 750 x g (1800-2000 rpm) for 10 min, after which the plasma supernatant is removed with a pipette. Precooled (5°C) PBS buffer (1.815g KH2PO4; 9.496g
Na2HPO4; 5.072g NaCI; 0.094g MgCl2; 0.110g CaCl2; 0.125g MnCl2 per
1000m1 distilled water, pH 7.4) is added to the compacted erythrocyte pellet; about seven volumes of PBS to three volumes of pellet. The cells are then resuspended by gentle shaking, and the resulting suspension is centrifuged again at 750 x g for 10 min. Supernatants are again removed with a pipette.
Resuspension, centrifugation, and removal of supernatant are repeated three times in total. The entire supernatant and the thin, white cloudy layer of cells above the red erythrocyte pellet must be carefully removed and discarded after each centrifugation step. After the third washing, four volumes of ice cooled distilled water are added to each volume of pellet, and mixed well; centrifuged at 2 200 x g (4000-5000 rpm) for 30 min, and the haemolysate solution (supernatant) is removed, and transferred. For stabilisation of the haemoglobin samples, 1 ml of PBS buffered glycerol (same PBS as above, but without CaCl2, MgCl2 or MnCl2; 80m1 glycerol plus 20 ml PBS) is added to
54 each millilitre of haemolysate, and mixed well. The resulting suspension may be stored at —20°C for up to 1.5 years.
Analyses were done using 12% starch gels, as well as 7% standard polyacrylamide gels (130x135mm)(Ferreira et al., 1984). A discontinuous lithium-borate tris-citric acid buffer (RW; electrode: pH 8; gel: pH 8.7; Ridgway et aL, 1970) and four continuous buffers: a tris-citric acid buffer system (TC; pH 6.9; Whitt 1970), a tris-borate-EDTA buffer (MF; pH 8.6; Market and
Faulhauber, 1965), a tris-citrate system, (P; pH 8.6, Poulik, 1957), and a histedine-citrate system (HC; pH 6.5 Kephart, 1990) were used to study variation at a total of 32 protein coding loci on starch gels. Five loci (general protein and haemoglobin) were resolved by means of polyacrylamide gel electrophoresis (PAGE) (see Appendix A for figures). Staining methods are as described by Shaw and Prasad (1970) and Harris and Hopkinson (1976), and the method of interpretation of gel-banding patterns and locus nomenclature as referred to by Van der Bank and Van der Bank (1995) were used.
Statistical analysis of data (input files are given in Appendix B) was executed using BIOSYS-1 (Swofford and Selander, 1981).
3.3 RESULTS AND DISCUSSION
All 15 of the proteins stained for provided adequate resolution or activity to be interpreted. Locus abbreviations and enzyme commission numbers along with the buffers and tissues yielding the best results are given in Table 3.1. The following protein products migrated cathodally: AAT-2, ADH, GAP, GPI-1, -2,
MDH-2 and PGD-2.
55 Nineteen (49%) of the 39 loci studied were polymorphic in one or more of the four populations analysed (Table 3.2). These were: 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. This was somewhat higher than the previous 33% estimate in the preliminary study, and mainly due to the presence of the additional markers detected. Fixed allele differences between M. natalensis and M. coucha were detected at AAT-1 (blood; Fig. 3.2a), ADH (liver; Fig. 3.2b),
EST-1 (liver; Fig. 3.2e), PGD-1 (muscle; Fig. 3.2c), Hb-1 and –2 (blood; Fig.
3.2d). Locus differences were confirmed in the new material at GPI-2, PT-2 and -3. The differences at these last three loci were considered to be isozyme
rather than allozyme differences due to the large separation distance between the bands (Smit et al., 2001). Thus these three loci remain useful in identifying individuals from either species, along with the new allozyme markers.
PGD and Hb were not analysed in the La Lucia and Montgomery Park populations in the initial study. However, the results obtained via PAGE for the
Hb locus in the Vaal and Richards Bay populations, using the haemoglobin purification method described by Falk et al. (1996), are analogous to those by
Gordon (1984). Significant differences (FR =0.936, P<0.05) were obtained for
EST-1 in the earlier study, but the additional data from the new material and increased resolution at this locus allowed us to re-evaluate those results, and indicate this locus is useful as another marker (Fig. 3.5). Results for the ADH locus were also re-evaluated, as analysis of the new populations was performed on different buffer systems that provided better resolution than previously obtained (Fig. 3.2). The most cost and labour effective analysis for the routine identification of these two species would appear to be to stain for
56 the GPI-2 and EST-1 loci on an RW buffer system running muscle and liver samples. While PAGE may provide better resolution, it is more expensive, requires more time to run and only one protein can be stained for at a time.
The other markers at PGD-1 and ADH are on incompatible buffer systems, and the purified haemoglobin samples needed for the AAT-2 and Hb loci are rendered less economic by the additional time, materials and equipment needed for preparation.
The genotypes at the IDH-1 and LDH-2 loci in the Vaal (M. coucha) population and the PGM-1, -2 and —3 loci in the La Lucia (M. natalensis) population were close to the expected Hardy-Weinberg proportions for naturally occurring populations. Deficiencies of heterozygotes were observed at the following loci: AAT-2 (Richards Bay and Montgomery Park populations), ADH-1 (Richards Bay), EST-2 (Montgomery Park), GAP
(Richards Bay), IDH-1 (Montgomery Park and La Lucia), IDH-2 (La Lucia and
Richards Bay), LDH-2 (Montgomery Park) and PGM-4 (La Lucia). There were no deviations of allele classes from Hardy-Weinberg proportions in the Vaal population, which is surprising as this had the smallest sample size of all four sites.
Polymorphism (P) was 14.7 and 11.4% in the La Lucia and Richards
Bay populations of M. natalensis, whereas the values for Montgomery Park and Vaal populations of M. coucha were 8.8 and 5.7% respectively, with average values of 13.1% for M. natalensis and 7.3% for M. coucha (Table
3.2). These are comparable to values reported for other rodent species.
Johnson and Selander (1971) obtained 7.9% in the Heteromyid Dipdomys,
17.4% for Otomys irroratus (Taylor et al., 1992), 16.1% for Rhabdomys
57 pumilio (Mahida et aL, 1999), and 6.7% for Oryzomys palustris (Loxterman et al., 1998). The values for M. coucha are lower than those for M. natalensis, and although the lowest value of 5.7% for the Vaal population could be attributed to small sample size, one could expect that the much larger geographic range of M. natalensis (from southern Africa right through to west
Africa) and the greater diversity of biome type would give this species a somewhat larger degree of genetic variation.
Numbers of alleles per locus (A) values were similar, ranging from 1.06 for the Vaal population to 1.21 for the La Lucia population, whereas the
Richards Bay and Montgomery Park populations were calculated to be 1.11 and 1.12. Individual heterozygosity (h) values for the Montgomery Park population ranged from 0.064 (LDH-2) to 0.278 (IDH-1), for the Vaal population, 0.105 (LDH-2 was the only heterozygous locus within this population), La Lucia from 0.069 (PGM-4) to 0.594 (IDH-1) and from 0.124
(IDH-2) to 0.500 (AAT-2). The mean H value for M. coucha was 0.018, and
0.032 for M. natalensis. These values put these two species in the lower range of rodent variation (Fig. 3.1). A much higher mean heterozygosity
(0.073) was obtained by Mahida et al. (1999) in their genetic assay of the grassland specialist, Rhabdomys pumilio. This is interesting, as Rhabdomys is a species that prefers a more pristine habitat than the opportunistic and pioneering multimammate mice. Indeed, M. coucha and M. natalensis tend to be replaced over time by Rhabdomys and Otomys species in areas that are recovering from habitat damage of some sort (De Graaff, 1981). H values determined by Taylor et al. (1992) for 0. irroratus were also high
(mean=0.071). One would expect that generalist species like M. coucha and
58 M. natalensis might possess more variation in the genome to deal with more challenging types of habitats, especially human-disturbed, but it would appear that this is not the case. H for M. natalensis was calculated to be nearly double that of M. coucha, but the low value for the Vaal population was probably due to the small sample size, which resulted in that species' average being brought down. On the whole, M. natalensis does appear to possess more genetic variation than M. coucha, perhaps because of the much greater
range of biome types spanned by the former species, as mentioned above.
The F values of Wright (1978) are useful to estimate the amount of genetic differentiation between species (Table 3.3). High values of FsT are considered to reflect substantial differences at any given locus, and are expected when studying separate species or populations that have diverged to a certain degree. Wright (1978) used the following groupings for the evaluation of FsT values: the range 0 to 0.05 is considered to reflect little genetic differentiation; 0.05 to 0.15 is indicative of moderate differentiation;
0.15 to 0.25 indicates great genetic differentiation and values greater than
0.25 reflect very great genetic differentiation. All of the measures have increased slightly with the addition of the new material, with pair-wise FST values between the species of 0.882 (Vaal and Richards Bay populations) and 0.743 (La Lucia and Montgomery Park populations) indicating large amounts of differentiation between the two species by the abovementioned criteria (Table 3.3). As expected, this is higher than the FsT value of 0.375 for
populations of 0. irroratus (Taylor et al., 1992) and 0.459 for populations of the striped field mouse R. pumilio (Mahida et al., 1999). These are comparable to the FsT values of 0.558 between the two M. coucha
59 populations, and 0.228 between the two M. natalensis populations. Pair wise
x2 probability totals between both sets of populations (Vaal-Richards Bay and
Montgomery Park-La Lucia) were zero (Table 3.3), indicating significant (p=0)
differentiation as expected for the two species.
Genetic distance (D) is a measure of the amount of genetic divergence that has occurred between two species since a hypothetical separation point
sometime in their evolutionary past. The D value of Nei (1978) is specially
adapted for small sample sizes and the mean value, between M. coucha and
M. natalensis was 0.26, with a D of 0.31 between the Richards Bay and Vaal
populations and 0.21 between the La Lucia and Montgomery Park
populations. The discrepancy between these values is the result of the
additional allozyme markers detected in the new material. This value is
obviously slightly larger than those obtained from intraspecific calculations of
D between populations of R. pumilio (highest D = 0.189, Mahida et al., 1999)
and 0. irroratus (highest D = 0.117, Taylor et al., 1992).
3.4 CONCLUSIONS
Murid rodents are biologically interesting subjects for genetic analysis, due to
their rapid evolutionary radiation, and cytogenetic differences are common
between genera and species (Qumsiyeh et al., 1990). It would seem that the
sibling species of M. coucha and M. natalensis are more examples of this
phenomenon, with a chromosomal mutation within M. natalensis giving rise to
the possibly younger M. coucha, which has been subsequently isolated
reproductively. It is unknown as to why these two species, which have so
60 much in common biologically, do not act as reservoirs for the same pathogens, although it is possible that differing physiological biochemistry may be the reason. Still, multimammate mice remain the most important pest species of African rodents (Van Gulck et al., 1998) both medically and agriculturally, and the isozyme markers reported here, as well as the new allozyme markers are reliable methods of distinguishing between them. This is certainly an aid to identify various disease threats from either species in areas like the Kruger National Park, where the two species occur in sympatry, as well as assisting in determining the eventual distribution of both species.
For these reasons it would also be easier and more economical to develop a field kit that effects the identification of these two species without the need of sophisticated laboratory equipment, expensive chemicals and expertise in the field of genetic analyses. A study was thus undertaken to research the viability of such a field kit in the next chapter.
3.5 REFERENCES
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DIPPENAAR, N.J., SWANEPOEL, P. and GORDON, D.H. (1993) Diagnostic
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61 FALK, T. M., ABBAN, E. K., OBERST, S., VILLWOCK, W., PULLIN, R. S. V.
and RENWRANTZ, L. (1996) A Biochemical Laboratory Manual for
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FERREIRA, J.T., GRANT, W.S. and AVTALION, R.R. (1984). Workshop on
Fish Genetics. CSIR Special Publications,' Council for Scientific and
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GORDON, D.H. (1984) Evolutionary Genetics of the Praomys (Mastomys)
natalensis species complex (Rodentia: Muridae). PhD Thesis:
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GROBLER, D.G., RAATH, J.P., BRAACK, L.E.O., KEET, D.F., GERDES,
G.H., BARNARD, B.J.H., KRIEK, N.P.J., JARDINE, J. and
SWANEPOEL, R. (1995) An outbreak of encephalomyocarditis
infection in free-ranging African elephants in the Kruger National Park.
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HARRIS, H. and HOPKINSON, D.A. (1976) Handbook of Enzyme
Electrophoresis in Human Genetics. North-Holland Publishing
Company, Amsterdam.
HOGAN, K.M., HEDIN, M.C., SUN KOH, H., DAVIS, S.K. and GREENBAUM,
I.F. (1993) Systematic and taxonomic implications of karyotypic,
electrophoretic, and mitochondrial-DNA variation in Peromyscus from
the Pacific Northwest. Journal of Mammalogy 74(4), 819-831.
JOHNSON, W.E., and SELANDER, R.K. (1971) Protein variation and
systematics in kangaroo rats (Genus Dipodomys). Systematic Zoology
20, 377-405.
62 KEPHART, S.R. (1990). Starch gel electrophoresis of plant isozymes: a
comparative analysis of techniques. American Journal Botany 77, 693-
712.
LOXTERMAN, J.L., MONCRIEF, N.D., DUESER, R.D., CARLSON, C.R. and
PAGELS, J.F. (1998) Dispersal abilities and genetic population
structure of insular and mainland Oryzomys palustris and Peromyscus
leucopus. Journal of Mammalogy 79(1), 66-77.
MAHIDA, H., CAMPBELL, G.K. and TAYLOR, P.J. (1999) Genetic variation in
Rhabdomys pumilio (Sparrman 1784) — an allozyme study. South
African Journal of Zoology 34(3), 91-101.
MARKERT, C.L. and FAULHABER, I. (1965) Lactate dehydrogenase isozyme
patterns of fish. Journal of Experimental Zoology 159, 319-332.
MURRAY, P.R., BARON, E.J., PFALLER, M.A., TENOVER, F.C. and
YOLKEN, R.H. (1995) Manual of Clinical Microbiology, sixth edition.
ASM Press, Washington, D.C.
NEI, M. (1978) Estimation of average heterozygosity and genetic distance
from a small number of individuals. Genetics 89, 583-590.
PATTON, J.L., SELANDER, R.K. and SMITH, M.H. (1972) Genic variation in
hybridising populations of gophers (genus Thomomys). Systematic
Zoology 21, 263-270.
POULIK, M.D. (1957) Starch gel electrophoresis in a discontinuous system of
buffers. Nature 180, 1477-1479.
63 QUMSIYEH, M.B., KING, S.W., ARROYO-CABRALES, A., AGGUNDEY, I.R.,
SCHLITTER, D.A., BAKER, R.J. and MORROW, K.J. Jr. (1990)
Chromosomal and protein evolution in morphologically similar species
of Praomys sensu lato (Rodentia, Muridae). Journal of Heredity 81, 58-
65.
RIDGWAY, G.J., SHERBOURNE, S.W. and LEWIS, R.D. (1970)
Polymorphism in the esterases of Atlantic herring. Transactions of the
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ROBERTS, L.S. and JANOVY, J. (1996) Foundations of Parasitology, fifth
edition. WCB Publishers, Toronto.
SHAW, C.R. and PRASAD, R. (1970) Starch gel electrophoresis of enzymes
— a compilation of recipes. Biochemical Genetics 4, 297-320.
SKINNER, J.D. and SMITHERS, R.H.N. (1990) The Mammals of the
Southern African Subregion, second edition. University of Pretoria,
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SMIT, A., VAN DER BANK, H., FALK, T. and DE CASTRO, A. (2001)
Biochemical genetic markers to identify two morphologically similar
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K., 1992 Genetic variation in the African vlei . rat Otomys irroratus
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64 VAN DER BANK, F.H. and VAN DER BANK, M., (1995) An estimation of the
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P. and LEIRS, H. (1998) Short-term effects of avian predation on
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65 TABLE 3.1: Locus abbreviations, enzymes stained for, enzyme commission
numbers (E. C. no.) as well as the tissues and buffers giving optimal results
(buffer descriptions are given in Materials and Methods).
Protein Locus E.C. No. Buffer Tissue
11. Aspartate aminotransferase AAT-1, -2 2.6.1.1 P BI
12. Alcohol dehydrogenase ADH 1.1.1.1 TC L
13. Creatine kinase CK-1 to —3 2.7.3.2 TC M EST-1 to —3 14. Esterases 3.1.1.- RW, MF M, L PT-1 to -5 15. General proteins PAGE M GPI-1, -2 16. Glucose-6-phosphate isomerase 3.5.1.9 RW, P L GAP 17. Glyceraldehyde-3-phosphate 1.2.1.12 TC M, L
dehydrogenase • GPD-1 to —4 18. Glycerol-3-phosphate dehydrogenase 1.1.1.8 RW M, L Hb-1, -2 19. Haemoglobin PAGE BI IDH-1 to —3 20. Isocitrate dehydrogenase 1.1.1.42 TC M, L LDH-1, -2 21. L-Lactate dehydrogenase 1.1.1.27 RW BI, M MDH-1, -2 22. Malate dehydrogenase 1.1.1.37 TC M, L PGD-1 to —3 23. 6-Phosphogluconate dehydrogenase 1.1.1.44 HC M, L PGM-1 to —3 24. Phosphoglucomutase 5.4.2.2 P, RW M PGM-4 14b. Phosphoglucomutase 5.4.2.2 RW L SOD-1, -2 25. Superoxide dismutase 1.15.1.1 P, RW M, L
BI = blood, L = liver, M = Muscle
66 Table 3.2: Allele frequencies, average heterozygosity per locus (H), mean number of alleles per locus (A) and percentage of loci polymorphic (P) for all populations studied with standard errors in parentheses.
Montgomery Vaal La Lucia Richards
Locus Allele Park Ridge Bay
AAT-1 A 1.000 1.000
B 1.000 1.000
AAT-2 A 0.250
B 0.750 1.000 1.000 0.500
C 0.500
ADH A 0.062
B 1.000 0.938
C 1.000 1.000
EST-1 A 1.000 1.000
B 1.000 1.000
EST-2 A 0.156
B 0.844 1.000 1.000 1.000
GAP A 0.167
GPI-2 A 1.000 1.000
B 1.000 1.000
67 Table 3.2 continued
Montgomery Vaal La Lucia Richards
Locus Allele Park Ridge Bay
IDH-1 A 0.944 0.375 1.000
B 0.833 0.056 0.500
C 0.167 0.125
H 0.029 0.006 0.035 0.029
(±0.015) (±0.004) (±0.019) (±0.017)
A 1.12 1.06 1.21 1.11
(±0.06) (±0.04) (±0.08) (±0.05)
P 8.80 5.71 14.71 11.43
68 Table 3.3: Summary of F-statistics between the Richards Bay and Vaal populations, the Montgomery Park and La Lucia populations (bold) at all loci analysed, as well as contingency x2 analysis for the pooled data of the two species. 2 Locus FIS FIT FST X
AAT-1 1.000 (1.000) 1.000 (1.000) 0.000 (0.000)
AAT-2 1.000 (1.000) 1.000 (1.000) 0.333 (0.143) 0.040 (0.186)
ADH 1.000 1.000 (1.000) 0.889 (1.000) 0.000 (0.001)
EST-1 1.000 (1.000) 1.000 (1.000) 0.000 (0.000)
EST-2 (0.763) (0.783) (0.085) (0.063)
GAP 1.000 1.000 0.091 0.085
GPI-2 1.000 (1.000) 1.000 (1.000) 0.000 (0.000)
IDH-1 -0.059 (1.000) -0.029 (1.000) 0.029 (0.127) 0.555 (0.056)
IDH-2 1.000 (1.000) 1.000 (1.000) 0.034 (0.048) 0.263 (0.167)
LDH-2 -0.059 (-0.034) -0.029 (-0.017) 0.029 (0.017) 0.178 (0.410)
PGD-1 1.000 1.000 0.000
PGM-1 (-0.067) (-0.032) (0.032) (0.341)
PGM-2 (-0.067) (-0.032) (0.032) (0.341)
PGM-3 (-0.067) (-0.032) (0.032) (0.341)
PGM-4 (-0.037) (-0.018) (0.018) (0.240)
Hb-1 1.000 1.000 0.000
Hb-2 1.000 1.000 0.000
PT-2 1.000 (1.000) 1.000 (1.000) 0.000 (0.000)
PT-3 1.000 (1.000) 1.000 (1.000) 0.000 (0.000)
Mean 0.819 (0.734) 0.979 (0.931) 0.882 (0.743) Total 0.000 (0.000)
69 0.08 - Dipodomys spp. 0.07 Otonvs irrorat us ❑ Thomomys bottae -±*" 0.06 Y1 0 T. urnbrinus 0.06 0 Peromyscus ,app. o 0.04 o Olyzonys palustris 17)4 0.03 Rhabdomys pumilio 0.02 0 M. concha l4li. nataiensis 0.01 Mammalian mean 0 Species
Figure 3.1: Comparative heterozygosities (H) for various species of Rodentia, along with the mammalian mean.
70
a
PGD-1
Origin -4 PC;D-2
I h1, x~ruacha 11. natatensts
natalensis AAT-11
4- M. CMIC
Origin -+
Figure 3.2: Fixed allele differences between Mastomys coucha and M. natalensis at PGD-1 (a), AAT-1 (b).
71
M. natalensis M. concha
+—Origin
b-2
naralensis M. couch()
e T. 4— EST-
I I I Mcoucha M. natalensis
Figure 3.2 continued: Fixed allele differences between Mastomys coucha and M. natalensis at ADH (c), Hb (d) and at EST-1 (e).
72 CHAPTER 4
IMMUNOLOGICAL
STUDY
73 CHAPTER FOUR:
IMMUNOLOGICAL STUDY
4.1 INTRODUCTION
Antigen-antibody reactions are characterised by high levels of both specificity and sensitivity. For these reasons, this reaction is widely used in qualitative and quantitative determinations of organic and biological components
(Ferreira et al., 1984). In mammals, invading elements are distinguished from body cells by means of membrane-bound proteins encoded by the major histocompatibility complex (MHC) (Voet and Voet, 1995). The MHC complex genes are in fact the most polymorphic know in higher vertebrates, and for this reason even unrelated individuals of the same species are highly unlikely to have the same set of MHC genes (Voet and Voet, 1995) Thus, despite their morphological similarities, M. natalensis and M. coucha are biologically distinct species and it is possible that their physiology differs enough to be able to use a type of antigen-antibody reaction to qualitatively distinguish between them. Apart from having different chromosome numbers, it is already known that there are several allelic and locus differences between the two species, as well as distinct haemoglobin chains that are not shared by either species (see Chapters 2 and 3). One of the aims of this study was thus to exploit these differences in the context of developing a field kit that would enable one to distinguish between the two species. It was decided to try simple immunodiffusion tests (Ouchterlony method) to test the viability of this aim.
74 4.2 MATERIALS AND METHODS
4.2.1 PREPARATION OF POLYCLONAL ANTIBODIES
Two immunisation protocols were used in raising polyclonal antibodies against erythrocytes of both M. natalensis and M. coucha in rabbits. In the first
instance, haemoglobin samples prepared according to Falk et aL (see Section
3.2) were injected in 300p1 amounts into the thigh muscles of two male
rabbits; M. natalensis samples into the one, and M. coucha samples into the other. Male rabbits were preferred as females that have had young may give false results in immunological tests (Meyer, D. Pers. comm., 2000).
Immunisation was repeated two weeks after the first injection, in order to
heighten antibody response (Meyer, D. Pers. comm., 2000). Blood was
collected from both rabbits approximately two weeks after the first
immunisation, for maximal collection of primary immune response antibodies,
and then again five to six days after the second immunisation during the
projected peak secondary response. Blood was taken from the ear according to Campbell et al. (1970). The ear was both warmed with a lamp and treated with xylene in order to dilate the blood vessels as much as possible. The
samples were subsequently centrifuged at 2000rpm for approximately 10
minutes, then plasma was removed using a micropipette and stored at —20°C.
An additional immunisation procedure was carried out (Falk, T. Pers.
comm., 2001). The same purified haemoglobin samples were used as
antigens, however this time they were prepared (according to the methods in
Section 3.2) without PBS buffer. These were then mixed 1:1 with Freundz
75 Complete Adjuvant (an adjuvant is a substance which improves the level of immune response to a separate antigen; Chapel and Haeney, 1993), and
250p1 quantities were injected into the rabbits, again into the thigh muscles.
Immunisations were carried out three times, each three weeks apart. The blood was then collected 5-10 days after the last immunisation. Booster injections were used to raise antibody titres subsequently if necessary. Each rabbit was sacrificed at the end of the experiment, and blood samples taken.
4.2.2 IPJIMUNODIFFUSION
These tests were carried out by the Ouchterlony method, adapted from
Campbell et al., (1970). In this method, both antigen and antibody are present in solution in separate wells in an agar plate (see Fig. 4.1). The solutions diffuse toward each other, and are eventually precipitated by an antibody- antigen reaction in a medium that originally contained neither.
4.2.2.1 PREPARATION OF AGAR PLATES
The agar medium was made up as follows: 100m1 medium contains 1g of agar, 98m1 of borate-saline buffer, 1 ml of a 1% sodium azide in saline solution to inhibit bacterial growth, and 1m1 of a 1% aqueous trypan blue solution to aid the observation of precipitation lines. The agar is added to the borate- saline solution, and dissolved by heating in a flask of boiling water. Eight millilitres of hot agar solution is then pipetted quickly into the bottom of a
115mm Petri dish and then left for 30 minutes to solidify. Cylinders were then
76 placed on the agar layer according to the pattern in Fig. 4.1, and another 8m1 of hot agar is pipetted into the dish to form the wells in the top agar layer, and again left to solidify (adapted from Campbell et aL, 1970).
4.2.2.2 TEST PROCEDURE
Once the agar plates are prepared and have solidified enough, samples are added to the wells (about 50p1). The antigen (the same samples used to immunize the test subjects) is placed in the centre well; with antibody . solutions (the plasma removed from whole blood samples) are placed in the outer wells. Each outer well contains a different concentration of antibody due to serial dilutions of 1:10; 1:100 and 1:1000 are made of the original plasma in order to test for ideal antibody-antigen equilibrium, at which point maximum precipitation will occur. To avoid the accumulation of condensate on the agar surface, filter paper is cut and fit into the lid of each dish. The dishes are then placed into a humidified incubator at body temperature (37°C). Conversely, the dishes may be left at room temperature alongside a pan of distilled water in order to maintain humidity. At the lower temperature, the reactions develop slightly slower and thus a longer observation period. The plates are then observed daily for a period of about one week, which is a sufficient length of time to allow precipitin reactions to occur (Campbell et al., 1970).
77 4.3 RESULTS AND DISCUSSION
All attempts to obtain a positive antibody-antigen reaction within the agar medium were unsuccessful. A dark blue band of precipitation in the agar gel would have indicated a positive reaction. However, on no occasion was this found to be the case (see Fig. 4.1). The ideal result would have been a band of precipitation between a Mastomys spp. haemoglobin sample and the blood plasma (containing antibodies) sample taken from the rabbit inoculated with the same Mastomys spp. serum. It would have then been possible to test whether anti-natalensis plasma would react with coucha haemoglobin samples (and vice versa). If no reaction occurs, it would have been possible to discriminate between the species by this method.
It is uncertain why this experiment was not successful, despite numerous attempts. The techniques involved are well known, and were carried out according to those described above. It is known that it is necessary to test a range of dilutions for optimum results in immunodiffusion
(Meyer, D. Pers. comm., 2000), and this is why each plate had wells containing serial dilutions of plasma samples. However this was still ineffectual. It is possible that the rabbits for some reason (perhaps they were immune-compromised) did not generate antibodies to the injected haemoglobin, causing the lack of response.
78 Figure 4.1: An example of the agar gels used for testing antigen-antibody reactions. The centre well contains antigen (a dilution of the haemoglobin solution used to inoculate hosts) whereas the outer wells are filled with plasma containing antibody. A dark blue line between wells would indicate a positive reaction.
4.4 RECOMMENDATIONS
Promising techniques involving lectins were used by Falk et al. (1996), to differentiate between closely related species of tilapia. These techniques may well be applied in future to the same effect for M. coucha and M. natalensis.
Lectins are molecules that have very antibody-like properties and bind with high specificity to certain membrane bound proteins. Falk et al, (1996) tested
79 a wide range of lectins against tilapia erythrocytes and found that some lectins will bind to the erythrocyte membrane proteins of certain species but not to others. In this way it was possible to identify species of tilapia by simple agglutination tests that may be verified by visual inspection. While the cost of lectins did not permit this during this study, future studies between these two species could benefit from this line of research.
4.5 REFERENCES
CAMPBELL, D.H., GARVEY, J.S., CREMER, N.E. and SUSSDARK, D.H.
(1970) Methods in Immunology — A Laboratory Text for Instruction and
Research, second edition. WA Benjamin, New York.
CHAPEL, H. and HAENEY, M. (1993) Essentials of Clinical Immunology, third
edition. Blackwell Scientific Publications, London.
FALK, T. M., ABBAN, E. K., OBERST, S., VILLWOCK, W., PULLIN, R. S. V.
and RENWRANTZ, L. (1996). A Biochemical Laboratory Manual for
Species Characterization of Some Tilapiine Fishes. ICLARM Education
Series 17. Manila, Philippines.
FERREIRA, J.T., GRANT, W.S. and AVTALION, R.R. (1984). Workshop on
Fish Genetics. CSIR Special Publications, Council for Scientific and
Industrial Research, Pretoria, RSA.
VOET, D., and VOET, J. (1995) Biochemistry, second edition. John Wiley &
Sons, New York.
80 CHAPTER 5
SUMMARY: FACTORS
AFFECTING GENETIC
VARIATION IN M. COUCHA
AND M. NATALENS1S,
HYPOTHESES TESTED,
RECOMMENDATIONS FOR
FUTURE STUDIES AND
CONCLUSIONS
81 CHAPTER 5:
SUMMARY: FACTORS AFFECTING GENETIC VARIATION IN M.
COUCHA AND M. NATALENSIS, HYPOTHESES TESTED,
RECOMMENDATIONS FOR FUTURE STUDIES AND
CONCLUSICAS
In addition to the explanations outlined in Chapters 2 and 3 accounting for the levels of genetic variation in the two Mastomys species studied, the following might also apply.
5.1 FACTORS AFFECTING GENETIC VARIATION IN M. COUCHA AND M. NATALENSIS.
Significant deviations of allele classes from expected Hardy-Weinberg proportions may occur for many reasons, including factors such as sampling error, population bottlenecks and founder effects, inbreeding, crossing and linking, random genetic drift and directional selection (Ayala, 1982). Influences such as these may cause shifts in favour, or conversely to the detriment, of certain genotypes within populations, resulting in changes in the overall genetic profile of a population. This resulting profile may not conform to the expected Hardy-Weinberg equilibrium. Hardy-Weinberg proportions are a theoretical construct given ideal conditions such as random mating, infinite sample size, and the absence of mutation, migration and random selection.
Because these conditions are almost impossible for natural populations to meet, the more often than not do not conform to these criteria.
82 Factors such as bottlenecks and founder effects can occur when a new population is begun from a very small gene pool, or very few individuals. This can cause a large amount of genetic drift, and the consequent deviation of the new populations' genetic structure from the original stock. However, in the case of M. coucha and M. natalensis, it is unlikely that either of these effects occurred because multimammate mice are ubiquitous throughout their ranges, extremely hardy and adaptable, and there is no evidence that any of the populations studied have been subjected to such conditions. The fact that the ranges of these species overlap in so many areas is confirmation that the differences between them are not the result of any geographic separation in the past, but rather of a chromosomal mutation (probably either a centric fission or fusion that resulted in the discrepancies between the number of chromosomes occurring in the two species today). Research into the chromosomal phylogeny of Mastomys by Britton-Davidian et al. (1995) revealed that chromosomal evolution in this genus is characterised by seven different types of rearrangements, the most frequent being pericentric inversions. However, pericentric inversions, while modifying the fundamental chromosome number, do not alter the diploid number of an organism, so these modifications would have to be accompanied by other chromosomal rearrangements that do, most likely centric fissions or fusions. Reproductive isolation of the new species may have occurred as a direct consequence of the extra chromosomes, or subsequently as a result of fixation of different alleles and traits in later times. The latter scenario is more likely to be accurate, as it would have given mutated individuals the chance to interbreed with original stock in order to better establish the new trait. Gordon (1984)
83 reported that the two species could still interbreed in the laboratory, if not in the wild, although hybrids did become sterile after a few generations.
However, he did not find any evidence of hybrids in the wild in almost one thousand individuals studied. However, it is certain that both Mastomys species have been in southern Africa for a long time.
East African fossil lineages dating back to 3.6 x 10 6 years ago have
evidenced slight morphological divergence in multimammate rats in the
Mastomys genus (Britton-Davidian et al., 1995). Micromammalian evidence
(rodent skeletons) from Rose Cottage Cave in the eastern Free State has
shown that multimammate mice have been in that vicinity since at least the
Holocene period, from about 8500 years before present (BP) to 500 years BP
(Avery, 1997). It was not possible to distinguish between the sibling species
on the basis of the cranial material, but Lynch (1983) referred Free State
material to M. coucha which (based on current distribution patterns) is the
most likely. However, if founder effects or bottlenecks have probably not
occurred in the past, both species are certainly under considerable selection
pressure from artificial sources, such as human interference. Small mammals
make important contributions to the biodiversity of their ecosystems in sub-
Saharan Africa (Linzey and Kesner, 1997). Apart from being prey for avian,
mammalian and reptilian predators, they are important consumers of seed
and herbage (Linzey and Kesner, 1997). Thus, artificial selection pressures
are very interesting to consider, especially in the case of multimammate mice,
which seem to thrive in human impacted environments. All four populations of
the present study were affected in some way by human civilisation; the La
Lucia ridge and Montgomery Park sites are adjacent to suburban areas, and
84 the Richards Bay site was undergoing rehabilitation after heavy metal mining.
These populations are definitely subjected to artificial selection pressures
(e.g. the Montgomery Park site is close to roads that carry heavy traffic, vagrants use the area to sleep in, pollution is present to a moderate degree and the grass cover in the area is burned to the ground at least once a year).
Similar problems affected the La Lucia construction site. Small mammals are likely to be impacted by overuse of habitats because of their food and cover requirements (Linzey and Kesner, 1997), and regular burning as well. These factors may also change the prey-predator structure prevailing in the area, as people will also influence the natural predators of multimammate mice such as owls and snakes. Whether the cumulative effect of all these factors is positive or negative with regards to the multimammate mouse is difficult to say without comparison to populations living in pristine conditions. Indeed, many areas of sub-Saharan Africa are so severely impacted by humanity and domestic animals that it is no longer possible to determine baseline data on disturbed populations of native species (Linzey and Kesner, 1997). A study by
Caro (2001) in East Africa revealed that average species density and abundance of small mammals (including M. natalensis) were greater outside than inside a national park in Tanzania. Caro (2001) concluded that rodent abundance actually increased in mildly disturbed habitats. However, in contrast, Avenant (2000) associated changes in small mammal habitats in the
Willem Pretorius nature reserve (Free State) with changes in small mammal diversity, and that ecological disturbances of those habitats go generally hand in hand with decreases in small mammal species richness. If so, it would appear that decreases in small mammal species richness is to the advantage
85 of M. coucha and M. natalensis. Given the evidence so far, it certainly seems
as if the multimammate mice are thriving, perhaps due to reduced predatory
pressure, and the abundance of food introduced into the environment by
humans.
The Richards Bay site is subjected to different kinds of selection
pressures. After the coastal dunes have been stripped of their valuable heavy
metals, the sand is artificially reseeded with pioneer species of plants, and left for succession to gradually replace the invaded areas with original dune forest. The rodent population structures are highly unstable during these
periods of succession, with Mastomys species being among the first to move
into young stands, whereas older stands are dominated by either the red veld
rat (Aethomys chrysophilus) or the pouched mouse (Saccostomus
campestris) after succession (Ferreira and van Aarde, 2000). They take advantage of the lack of competition from other species, and quickly establish themselves. It was probably at this stage that specimens were taken from this site. The mining and the associated destruction of vegetation in these coastal dune forests can be considered a discrete disturbance event of high intensity
resulting in total destruction (Ferreira and van Aarde, 2000). After the initial discrete disturbance however, succession occurs similar to that of habitats recovering from non-human induced disturbances, and Ferreira and van
Aarde (2000) concluded that intermediate levels of disturbance probably maintained the rodent species richness in the coastal dune forests of this study area. The artificial selection pressures applicable in the situations above may very well cause deviations in allele classes, if not from the expected
Hardy-Weinberg equilibrium, then perhaps from naturally occurring
86 proportions. It is also worth noting that multimammate mice have the capacity to populate a disturbed area very quickly due to the large numbers of young they have. Inbreeding may thus occur on a large scale in early stages of rodent succession. The pressures mentioned above may have also caused the low variation levels in the two Mastomys species studied (compared to other southern African murid rodents — see Fig. 3.1). The Vaal site was probably the least affected, being a section that sees little human traffic, since access is restricted. This population also conformed closely to expected
Hardy-Weinberg proportions at all loci.
Deviations in allele classes from the expected proportions could also be due to restricted sampling (see Table 5.1 for sample sizes per locus) and the presence of rare alleles as well as the aforementioned factors. The deviation in the Richards Bay population at the ADH locus is probably due to the presence of the ADH*A allele that only occurred in one individual, and represents a rare allele. This locus may be in the process of fixation for the
ADH*B allele in M. natalensis, or the ADH*A allele itself may confer a selective disadvantage upon the individual, especially as this loci is present in the liver. The liver is an extremely important organ in all mammals, having a variety of vital functions, and different alleles at loci in this organ could impact on the eventual adaptability and survivability of an individual multimammate mouse. Thus, rare alleles could confer either an advantage or disadvantage to prevailing environmental conditions. This would cause future selection for or against that allele respectively. There were also deviations in the AAT-2 locus in both the Richards Bay and Montgomery Park populations. Although the
AAT-2*B allele was the most common allele present in both species (and the
87 only allele found in the La Lucia Ridge and Vaal populations), the presence of a single individual possessing the AAT-2*A allele in Montgomery Park, and the 1:1 ratio of AAT-2*B to AAT-2*C alleles in the Richards Bay population probably caused the difference. An increase in the number of individuals analysed might yield proportions nearer the ideal, given that neither the AAT-
2*A nor AAT-2*C alleles are rare. EST-2 also displayed deviations in the
Montgomery Park population, where there were EST-2*A alleles in three of the 16 individuals analysed. Similar deviations occurred at GAP in the
Richards Bay population, and at PGM-1, -2 and —3 in the La Lucia population, where there was a single heterozygote possessing an A-allele at all three loci.
Once again these differences may be due to the small sample size, or rare alleles (see Table 4.1 for sample sizes). However, it is equally possible that expanded sampling might not change these results significantly, and that this variation is indeed representative of M. coucha and M. natalensis. The IDH-1 locus was difficult to score in all populations, so although it did display variation, not all individuals were scored due to the lack of resolution of many bands, possibly resulting in deviations at this locus. Deviations at IDH-2 were caused by the exceptions of single individuals possessing IDH-2*A alleles (La
Lucia ridge) and IDH-2*C alleles (Richards Bay), whereas both remaining populations were fixed for the common IDH-2*B allele (see Table 5.1).
Deficiencies of heterozygotes (Table 5.1) were observed at the following loci: AAT-2 (Richards Bay and Montgomery Park populations), ADH
(Richards Bay), EST-2 (Montgomery Park), GAP (Richards Bay), IDH-1
(Montgomery Park and La Lucia), IDH-2 (La Lucia and Richards Bay), LDH-2
(Montgomery Park) and PGM-4 (La Lucia).
88 TABLE 5.1: Allele frequencies, coefficients of heterozygote deficiency
(negative values) or excess (d), degrees of freedom (DF), x2 values, individual heterozygosity values (h) and sample size (N) for each polymorphic locus in the four populations of Mastomys studied. Mastomys coucha populations include Montgomery Park (MP) and Vaal (V) localities, whereas M. natalensis were taken from La Lucia Ridge (LL) and Richards Bay (RB). Allege Frequency Locus Pop. N d x2 DF h A B C MP 1.000 V 9 AAT-1 1.000 LL 4 1.000
RB 16 1.000
MP 4 0.250 0.750 -1.000 7.200* 1 0.375
AAT-2 V 3 1.000
LL 3 1.000
RB 4 0.500 0.500 -1.000 5.333* 1 0.500
MP 2
ADH V 7 1.000
LL 4 1.000 1.000
RB 16 0.062 0.938 -1.000 31.034* 1 0.117
EST-1 MP 20 1.000
V 5 1.000
LL 10 1.000
RB 5 1.000
EST-2 MP 16 0.844
V 4 1.000
0.156 -0.770 11.556* 1 0.264 U_ 10 1.000
RB 5 1.000
MP 20 1.000
V 8 1.000 GAP U_ 1 1.000
RB 12 0.167 0.833 -1.000 15.439* 1 0.278
89 Table 5.1 continued. Allele Frequency Locus Pop. N d x2 DF h A B C MP 30
V 9 1.000 GPI-2 LL 23 1.000 1.000
RB 16 1.000
MP 6 0.833 0.167 -1.000 11.111* 1 0.278 V 9 0.944 IDH-9 0.056 0.000 0.000 1 0.105 LL 8 0.375 0.500 0.125 -1.000 24.571* 3 0.594 RB 3 1.000
MP 10 1.000
V 9 1.000 IDH-2 U_ 11 0.909 -1.000 21.053* 1 0.165 0.091 RB 15 0.933 0.067 -1.000 29.037* 1 0.124
MP 15 0.967
V 9 0.033 0.944 -0.034 0.000 1 0.064 LDH-2 LL 10 0.056 1.000 0.000 0.000 1 0.105
RB 16 1.000
MP -
V 9 PGD-1 1.000 LL - _ RB 16 1.000
MP 7 1.000
V 9 1.000 PGN1-1 U_ 8 0.062 0.938 0.000 0.000 1 0.117 RB 16 1.000
MP 7 1.000
V 9 1.000 PGM-2 U_ 8 0.938 0.062 0.000 0.000 1 0.117 RB 16 1.000
90 Table 5.1 continued. Allele Frequency Locus Pop. N d 7C2 DF h A B C MP 7 1.000
V 9 1.000 PGM-3 LL 8 0.938 0.062 0.000 0.000 1 0.117 RB 16 1.000
MP 1.000 19 V - - PGM-4 LL 0.036 0.964 14 -0.037 0.000 1 0.069 RB -
MP 9
V 9 1.000 PT-2 LL 9 1.000 1.000
RB 10 1.000
MP 9
V 9 1.000 PT-3 LL 9 1.000 1.000
RB 10 1.000
MP - _
V 9 Hb-1 1.000 LL - _
RB 10 1.000
MP -
V 9 1.000 Hb-2 U. -
RB 10 1.000
* indicates significant (p<0.05) deviation of allele classes from expected Hardy-Weinberg proportions. - indicates locus absent, or,not studied in that population.
91 From these results and discussion, and those mentioned previously in
Chapters 2 and 3, it would be appropriate to evaluate to what extent the original aims were satisfied and to summarise recommendations for future consideration in the following section.
5.2 HYPOTHESES TESTED AND RECOMMENDATIONS FOR FUTURE
STUDIES
1. The first aim was to analyse the extent of genetic variation within, and
differentiation between, populations of M. coucha and M. natalensis.
The results obtained in this study of two populations each of M. coucha and
M. natalensis indicated species differentiation that is consistent with
their taxonomic status (D = 0.26; Fs-r = 0.882). Intraspecific
differentiation (mean H = 0.018 for M. coucha, and 0.032 for M.
natalensis) was found to fall into the lower ranges of rodent variation as
compared to related southern African murids Otomys irroratus (Taylor
et at., 1992) and Rhabdomys pumilio (Mahida et al., 1999) (see Fig.
3.1). This low variation could be accounted for by a number of reasons
(artificial selection, inbreeding, rare alleles) dealt with in the preceding
section. Although the Mastomys species complex is one of the most
widely researched rodents in Africa, this research is the very first to
yield results for the genetic differentiation within and between these two
particular species.
92 This project was done in the preceding parts Chapters 1,2 and 3), as it was
necessary to confirm the results obtained in the preliminary study for other,
more geographically separated populations, as well as to increase the sample
size for statistical reliability. For this reason, additional populations were
sample to satisfy the second aim stated in Chapter 1.
2. The second aim was to ascertain whether there are additional genetic
markers that will distinguish each species from the other across a large
range of their distribution.
Fixed differences between the species were found at no less than nine loci.
These were: AAT-1, ADH, EST-1, PGD-1, Hb-1 and —2 (fixed allele
differences), and GPI-2, PT-2 and —3 (locus differences). The locus
differences were found to be reliable throughout the populations analysed, as
were the allozyme markers at AAT-1, ADH and EST-1. Although the markers
at Hb-1 and —2 were not analysed in the Montgomery Park and La Lucia
Ridge populations, it is not unreasonable to hypothesise that they would also
have been consistent, especially considering that these markers are
analogous to those obtained by Gordon (1984) in almost one thousand
individuals sampled. One might argue that the Hb markers are nothing new,
as Gordon reported fixed differences between the species in this protein in
1984. However, in the light of the results of this study contradicting his
assertion that both species share a haemoglobin component, and the fact that
his results could not be duplicated (without considerable refinement of other techniques) it is obvious that there has been a great deal of improvement in
93 this regard. These markers are an important contribution to the routine identification of these morphologically similar species, especially in the areas where their distributions overlap. One of the most important areas in which these species occur sympatrically is the Kruger National Park. Multimammate mice in this region need to be studied for a number of reasons. Firstly, to ascertain whether only one or both species acts as a carrier for the EMC virus that affects animals in the park. Secondly, to establish the ranges of both M. coucha and M. natalensis within the park so that the disease threats posed by each (to animals and people) can be identified. Lastly, to find out whether levels of genetic variation within the two species in a protected park (e.g. the
Kruger National Park) are consistent with those reported here or whether they follow trends mentioned in the previous section (4.1).
3. The third aim was to research the possibility of raising polyclonal
antisera to the distinct haemoglobin chains of each species with the
purpose of developing a kit to distinguish between the two species in
the field, thus further enabling identification.
Immunological tests were done (see Chapter 4) with this aim in mind.
However, these were unsuccessful as no visible results were obtained for any of the immunodiffusion tests done. It is recommended that future studies on immunological differences between these two species use the techniques of
Falk et al. (1996) outlined in Section 4.
If the aim to raise polyclonal antisera, or to find suitable lectins that would have effected identification had been successful, the next obvious step would
94 have been to ascertain whether that technique would be robust enough for fieldwork. This would be of great assistance to mammalogists, especially taking the difficulties of field identification of Mastomys species into account.
Lectins may be the technique of choice in this regard, and should be further researched if possible. Additionally, sympatric populations such as those occurring in the Kruger National Park area should be tested for the markers found in this study, and to determine whether hybrids occur in the wild.
5.3 CONCLUSIONS
It is remarkable that, even in this day and age of our modern 21 st century, the distributional boundaries and detailed ecology of a small species of rodent
(especially one as agriculturally and medically important as described) can remain provisional or even unknown as described in the relevant texts. Yet such is the case for the multimammate mouse, especially for M. coucha, seeing as most of the historical data that has been collected was only applicable to a single species that was then known as M. natalensis. With the advent of data distinguishing between these two species as described in this dissertation, there is a great deal of scientific ground to be covered in order to fully understand the numerous differences that exist, including geographical ranges and the ecological niches occupied by both species. The isozyme and allozyme markers identified here, as well as being the first thorough study into the genetic variation within and between these two species are steps forward in covering that scientific ground, and will hopefully be followed by others until our knowledge and understanding are complete. Furthermore, the results for
95 the multimammate mice where large sample sizes are involved are important for the future monitoring of gene flow in populations, to determine levels of inbreeding and crossbreeding, and to enhance the global information on natural animal diversity.
5.4 REFERENCES
AVENANT, N.L. (2000) Small mammal community characteristics as
indicators of ecological disturbance in the Willem Pretorius Nature
Reserve, Free State, South Africa. South African Journal of Wildlife
Research 30(1): 26-33.
AVERY, D.M. (1997) Micromammals and the Holocene environment of Rose
Cottage Cave. South African Journal of Science 93: 445-448.
AYALA, F.J. (1982) Population and Evolutionary Genetics.
Benjamin/Cummings Publishing Company, Inc, London.
BRITTON-DAVIDIAN, J., CATALAN, J., GRANJON, L. and DUPLANTIER, J.
(1995) Chromosomal phylogeny and evolution in the genus Mastomys
(Mammalia, Rodentia). Journal of Mammalogy 76(1): 248-262.
CARO, T.M. (2001) Species richness and abundance of small mammals
inside and outside an African national park. Biological Conservation 98:
251-257.
FALK, T.M., ABBAN, E.K., OBERST, S., VILLWOCK, W., PULLIN, R.S.V. and
RENWRANTZ, L. (1996) A Biochemical Laboratory Manual for Species
Characterization of Some Tilapiine Fishes. ICLARM Education Series
17, Manila, Philippines.
96 FERREIRA, S.M. and VAN AARDE, R.J. (2000) Maintaining diversity through
intermediate disturbances: evidence from rodents colonizing
rehabilitating coastal dunes. African Journal of Ecology 38: 286-294.
GORDON, D.H. (1984) Evolutionary Genetics of the Praomys (Mastomys)
natalensis species complex (Rodentia: Muridae). PhD Thesis,
Witwatersrand University, Johannesburg.
LINZEY, A.V. and KESNER, M.H. (1997) Small mammals of a woodland-
savannah ecosystem in Zimbabwe. I. Density and habitat occupancy
patterns. Journal of Zoology, London 243: 137-152.
LYNCH, C.D. (1983) The mammals of the Orange Free State. Memoirs of the
National Museum, Bloemfontein 18: 1-218.
MAHIDA, H., CAMPBELL, G.K. and TAYLOR, P.J. (1999). Genetic variation
in Rhabdomys pumilio (Sparrman 1784) — an allozyme study. South
African Journal of Zoology 34(3): 91-101.
TAYLOR, P.J., CAMPBELL, G.K., VAN DYK, D., MEESTER, J. and WILLAN,
K. (1992) Genetic variation in the African vlei rat Otomys irroratus
(Muridae: Otomyinae). Israel Journal of Zoology 38 293-305.
97 APPENDIX A
LOCALITIES AND
APPARATUS
98
APPENDIX A:
LOCALITIES AND APPARATUS
Montgomery Park (Johannesburg) Vaal Dam
Richards Bay
La Lucia Ridge (Durban)
Figure 1: Sample localities for M. coucha (squares) and M. natalensis (circles) in this study.
99 Figure 2: A total of thirty-four M. coucha individuals were trapped at this suburban park and vlei in Montgomery Park (26°09'22"S, 27°58'58"E) during June 1998 and February-April 1999. The ecosystem of this area is disturbed due to the amount of human traffic passing through, and proved to be home to a large population of the opportunistic and pioneering M. coucha. This site was also chosen, as it was more or less central to the distribution of the species concerned.
100 Figure 3: This locality at the Vaal Dam (26°51'49"S, 28°08'59"E) yielded nine specimens in May 2000. Fairly lush grassland on the banks of the dam proved habitable for a number of genera, including Rhabdomys, Otomys and Mastomys. Specimens of genera other than Mastomys were released. All coordinates were taken with a Garmin GPS12 (12 channel).
101 Figure 4: Twenty-four M. natalensis individuals were trapped at this construction site on an abandoned sugar cane plantation in La Lucia ridge (29°44'44"S, 31°03'09"E) during June 1998 and April 1999. Coastal Kwazulu-Natal is ostensibly in the centre of the distribution of this species, and this site was thus chosen to avoid any possible overlap with M. coucha. The site's disturbed ecosystem and close proximity to human habitation proved ideal for this species.
102 Figure 5: Sixteen mice were trapped on dunes being rehabilitated by Richards Bay Minerals near Richards Bay (28°38'48"S, 32°16'15"E) in June 2000. Once again, these rodents are among the first to move into an area recovering from habitat damage.
103
a
b
Figure 6: Live traps used were either (a) standard steel Sherman traps (used in Richards Bay) or (b) the plastic equivalent (Vaal Dam, La Lucia ridge and Montgomery Park).
104 a
b
Figure 7: Apparatus used for starch gel (a) and • polyacrylamide gel (b) electrophoresis.
105 APPENDIX B
BIOSYS INPUT
106 APPENDIX B: BIOSYS INPUT
The input files used in this study are included in this appendix so as to facilitate future comparisons with this study and between M. coucha and M. natalensis.
GENOTYPIC VARIATION IN FOUR POPULATIONS OF MASTOMYS
NOTU=4, NLOC=39, NALL=3,CRT; (9(1X,A5)/10(1X,A5)/10(1X,A5)/10(1X,A5) AAT-1 AAT-2 ADH CK-1 CK-2 CK-3 EST-1 EST-2 EST-3 GAP GPD-1 GPD-2 GPD-3 GPD-4 GPI-1 GPI-2 IDH-1 IDH-2 IDH-3 LDH-1 LDH-2 MDH-1 MDH-2 PGD-1 PGD-2 PGD-3 PGM-1 PGM-2 PGM-3 PGM-4 PT-1 PT-2 PT-3 PT-4 PT-5 HB-1 HB-2 SOD-1 SOD-2
STEP DATA: DATYP=2,NCOL=2,ALPHA; (6A,3(1X,2A1,1X,12))
1 VAAL AAT-1 BB:09 AAT-2 BB:03 ADH CC:07 CK-1 AA:09 CK-2 AA:09 CK-3 AA:09 EST-1 BB:05 EST-2 BB:04 EST-3 AA:03 GAP BB:08 GPD-1 AA:03 GPI-1 AA:09
107 GPI-2 BB:09 IDH-1 AA:08 AB:01 IDH-2 BB:09 IDH-3 AA:09 LDH-1 AA:09 LDH-2 AB:01 BB:08 MDH-1 AA:09 MDH-2 AA:09 PGD-1 AA:09 PGD-2 AA:02 PGD-3 BB:09 PGM-1 BB:09 PGM-2 BB:09 PGM-3 BB:09 PT-1 AA:09 PT-2 AA:09 PT-3 BB:09 PT-4 AA:09 HB-1 AA:09 HB-2 AA:09 SOD-1 AA:09 SOD-2 AA:09
2 RICH'S BAY AAT-1 AA:16 AAT-2 BB:02 CC:02 ADH AB:01 BB:15 CK-1 AA:16 CK-2 AA:16 CK-3 AA:16 EST-1 AA:05 EST-2 BB:05 EST-3 AA:03
108 GAP AA:02 BB:10 GPD-1 AA:10 GPI-1 AA:16 GPI-2 BB:16 IDH-1 AA:03 IDH-2 BB:14 CC:01 IDH-3 AA:15 LDH-1 AA:16 LDH-2 BB:16 MDH-1 AA:16 MDH-2 AA:16 PGD-1 BB:16 PGD-2 AA:04 PGD-3 AA:06 PGM-1 BB:16 PGM-2 BB:16 PGM-3 BB:16 PT-1 AA:10 PT-2 BB:10 PT-3 AA:10 PT-4 AA:10 HB-1 BB:10 HB-2 BB:10 SOD-1 AA:16 SOD-2 AA:16
3 MONT PARK AAT-1 BB:07 AAT-2 AA:01 BB:03 ADH CC:02 CK-1 AA:07 CK-2 AA:09 CK-3 AA:08
109 EST-1 BB:20 EST-2 AB:01 BB:13 EST-3 AA:14 GAP BB:20 GPD-1 AA:09 GPD-2 AA:08 GPD-3 AA:08 GPD-4 AA:08 GPI-1 AA:30 GPI-2 BB:30 IDH-1 BB:05 CC:01 IDH-2 BB:10 IDH-3 AA:10 LDH-1 AA:15 LDH-2 AB:01 BB:14 MDH-1 AA:05 MDH-2 AA:15 PGM-4 BB:19 PGM-1 BB:07 PGM-2 BB:07 PGM-3 BB:07 PT-1 AA:09 PT-2 AA:09 PT-3 BB:09 PT-4 AA:09 PT-5 AA:08 SOD-1 AA:10 SOD-2 AA:10
4 LA LUCIA AAT-1 AA:04 AAT-2 BB:03 ADH BB:04
110 CK-1 AA:11 CK-2 AA:12 CK-3 AA:12 EST-1 AA:10 EST-2 BB:10 EST-3 AA:10 GAP BB:01 GPD-1 AA:10 GPD-2 AA:08 GPD-3 AA:08 GPD-4 AA:08 GPI-1 AA:23 GPI-2 AA:23 IDH-1 AA:03 BB:04 CC:01 IDH-2 AA:01 BB:10 IDH-3 AA:11 LDH-1 AA:10 LDH-2 BB:10 MDH-1 AA:04 MDH-2 AA:13 PGM-4 AB:01 BB:13 PGM-1 AB:01 BB:07 PGM-2 AB:01 BB:07 PGM-3 AB:01 BB:07 PT-1 AA:09 PT-2 BB:09 PT-3 BB:09 PT-4 AA:09 PT-5 AA:09 SOD-1 AA:13 SOD-2 AA:13
111