ABSTRACT
The studies of mammalian chromosomes have constituted an effective area of
investigation to explain their relationship. The entire chromosome set of a species is
known as a karyotype. Many rodent species show a tendency for extensive chromosomal
variability within species and species complexes. The use of molecular methods to
provide more insight into the taxonomy and phylogeny of Cricetomys was recommended.
Karyotypic studies were carried out on the African Giant rat, (Cricetomys gambianus,
Waterhouse-1840) with the aim of determining its chromosome number, length,
centromeric indices and nomenclature. The chromosomes were prepared from the
conventional bone marrow of 10 African Giant rats – five male and five female Giant rats,
treated intraperitoneally with 2 ml of 0.04% colchicines for 3 hours. Chromosomes in
well-spread cells were counted and measured using KaryoType computer software. Arm
lengths, centromeric indices and nomenclature were determined from these measurements
and were expressed in micrometre (µm). The chromosomes were classified based on the
centromeric indices obtained as metacentrics, submetacentrics, acrocentrics,
subacrocentrics and telocentrics. Ideograms were also constructed from the
measurements. Photomicrographs of well-spread mitotic metaphase chromosomes were
used to construct a standard karyotype for the species. A diploid chromosome number of
2n = 80 with an autosomal fundamental number (NFa) of 66 to 95 were obtained for the
species of C. gambianus used in this study. From the constructed idiogram, there was
gradual decrease in length from one chromosome pair to another. The mean chromosomal
arm lengths were siginificantly (P < 0.05) higher in the males compared to those of their
female counterparts. There was no significant difference in the centomeric indices. The
chromosomal nomenclatures were predominantly terminal. The chromosomal numbers,
lengths, autosomal fundamental numbers and nomenclatures were similar with those
xviii
found in Benin Republic, Senegal, Niger Republic, Cameroun and other countries. The
comparative species of the Texas banner-tailed Kangaroo rat, Dipodomys spectabilis has
a diploid chromosome number of 2n = 72, and an autosomal fundamental number (NFa)
of 70, which was closely related to that of the Cricetomys
TABLE OF CONTENTS
S
Cover page ……………………………………………………………………………………………………………i
Fly leaf…………………………………………………………………………………………………………………ii
Title page…………………………………………………………………………………………………………….iii
DECLARATION…………………………………………………………………………………………………iv
CERTIFICATION……………………………………………………………………………………………….v
ACKNOWLEDGEMENTS …………………………………………………………………………………vi
TABLE OF CONTENTS ……………………………………………………………………………………vii
LIST OF TABLES ……………………………………………………………………………………………….x
LIST OF FIGURES ……………………………………………………………………………………………xii
LIST OF PLATES …………………………………………………………………………………………….xiv
LIST OF ABBREVIATIONS ……………………………………………………………………………..xv
ABSTRACT……………………………………………………………………………………………………..xvii
1.0 INTRODUCTION…………………………………………………………………………………………..1
1.1 Background ……………………………………………………………………………………………………1
1.2 Statement of Research Problem ………………………………………………………………………9
1.3 Justification of the Study…………………………………………………………………………………9
1.4 Aim and Objectives of the Study ……………………………………………………………………10
1.4.1 Aim of the study…………………………………………………………………………………………..10
1.4.2 Objectives of the study………………………………………………………………………………….10
1.5 Research Hypothesis……………………………………………………………………………………..10
2.0 LITERATURE REVIEW ……………………………………………………………………………..11
2.1 Karyogenetics of the African Giant Rat …………………………………………………………11
3.0 MATERIALS AND METHODS ……………………………………………………………………25
3.1 Materials………………………………………………………………………………………………………25
viii
3.1.1 Acquisition of the experimental animals …………………………………………………………25
3.1.2 Equipment…………………………………………………………………………………………………..25
3.2 Methodology …………………………………………………………………………………………………26
3.2.1 Injection and sacrifice of the animals ……………………………………………………………..26
3.2.3 Place where karyotype studies were carried out……………………………………………….27
3.2.4 Extraction of cells………………………………………………………………………………………..27
3.2.5 Fixation of cells……………………………………………………………………………………………27
3.2.6 Spreading of cells and air drying ……………………………………………………………………30
3.2.7 Preparation of Giemsa Stain ………………………………………………………………………….30
3.2.8 Staining of cells……………………………………………………………………………………………30
3.2.9 Chromosome analysis…………………………………………………………………………………..33
3.3 Components of karyotypic patterns of Cricetomys gambianus……………………………..34
3.4. Statistical Analyses……………………………………………………………………………………….36
4.0 RESULTS …………………………………………………………………………………………………….37
4.1 Anthropometric measurements of Cricetomys gambianus……………………………….37
4.1.1 The mean weight and length of Cricetomys gambianus…………………………………….37
4.2 Chromosomal Numbers, Lengths and Morphology of Cricetomys gambianus….41
4.2.1 The mean chromosomal arm lengths of Cricetomys gambianus…………………………67
4.2.2 Sexual dimorphism of chromosomal arm lengths of Cricetomys gambianus ……….69
4.2.3 Sexual dimorphism of the total chromosomal lengths of Cricetomys gambianus….71
4.2.5 Sexual dimorphism of the centromeric indices of Cricetomys gambianus……………73
4.2.7 Chromosomal nomenclature of Cricetomys gambianus…………………………………….75
5.0 DISCUSSION……………………………………………………………………………………………….77
6.0 CONCLUSION AND RECOMMENDATION ……………………………………………….85
6.1 Conclusion ……………………………………………………………………………………………………85
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6.2 Recommendations…………………………………………………………………………………………85
6.3 Contributions to Knowledge ………………………………………………………………………….87
REFERENCES…………………………………………………………………………………………………..88
APPENDIX I ……………………………………………………………………………………………………102
APPENDIX II…………………………………………………………………………………………………..134
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background
The African Giant rat (AGR) also known as Gambian pouched rat belongs to the order
Rodentia, Suborder Myomorpha, family Cricetidae, subfamily Cricetomyiane and genus
Cricetomys (Delany and Happold, 1979). It is a wild rodent consumed by the rural
population in Nigeria. Two species have been recorded in Nigeria, Cricetomys emni and
Cricetomys gambianus, Waterhouse-1840 (Happold, 1987). Other Gambian species exist
in South Africa and they include Cricetomys gambianus adventor, C. gambianus
selindensis and C. gambianus cunator. Cricetomys emni is distributed naturally in the rain
forest zone and is not associated with human habitation. It is less common than the
Gambian Giant rat (Happold, 1987).
African giant pouched rat is found throughout tropical and subtropical Africa, South of
the Sahara desert down to about 27o South latitude (Novak, 2009). Their native range
stretches from the Atlantic Ocean coast of West Africa, east across the Congo Basin to
the Indian Ocean Coast of East Africa (Peterson et al., 2006) and southwards into the
Transvaal and KwaZulu-Natal Provinces of South Africa (Malekani et al., 2002). The
African Giant rat is generally considered to be the Gambian Giant rat (Cricetomys
gambianus Waterhouse-1840) which has been separated as a species from the Southern
Giant Pouched rat.
The African giant pouched rat, because of its exceptional size and other interesting
attributes, is an economically important rodent within Africa. It is one of the most
common mammals exploited as bush meat (Ajayi, 1977; Kingdon, 1997; Assogbadjo et
al., 2005) and has been trained to aid in the detection of landmines (Verhagen et al.,
2
2003) and also in the medical diagnosis of pulmonary tuberculosis (Weetjens et al.,
2009). The distribution of this rodent (genus Cricetomys) spans almost the whole of subSaharan Africa, stretching from the savannah zone of West Africa through the GuineoCongolian forest block to the savannahs of East and southern Africa (Musser and
Carleton, 2005). These rodents have been proven to be carriers of disease pathogens
(Machang’u et al., 2004; Durnez et al., 2008) and recent reports show that they are
potential pest species as invasive populations have been discovered in the Florida Keys in
the USA (Engeman et al., 2006, 2007; Perry et al., 2006; Peterson et al., 2006).
In what can be regarded as a most authoritative reference and checklist for mammals,
Musser and Carleton (2005) recognized four species of giant pouched rat: Cricetomys
gambianus, Cricetomys emini, Cricetomys ansorgei, and Cricetomys kivuensis. Before
this, most notable publications such as those of Genest-Villard (1967), Rosevear (1969)
and Kingdon (1997), although noting the presence of several forms across the
geographical range of Cricetomys, recognized only two species. The first of these is the
broad-snouted C. gambianus, which is spread across the savannahs of Africa and
possesses a whitish-grey belly that is rather indistinctly defined in relation to the flanks.
The second is the slim-snouted C. emini, occupying the Guineo-Congolian forest block
and possessing a distinct white belly.
Several publications, employing alternative techniques such as karyotyping of the African
Giant rat (Granjon et al., 1992; Codjia et al., 1994; Dobigny et al., 2002; Corti et al.,
2005); plasma biochemical properties of the African Giant rat (Nssien et al., 2002;
Onwuka et al., 2003); Stereological estimation of the cerebral layers of African Giant rats
(Musa et al., 2017); multivariate craniometry (Bellier, 1973); anatomical and histological
studies of the digestive system of the African Giant rat (Nzalak et al., 2010);
3
morphologic, morphometric and histologic studies of cerebellum and forebrain of the
African Giant rat (Nzalak et al., 2002); morphometric studies of the cerebellum and
forebrain of the African Giant rat (Nzalak et al., 2005); morphometric characterization of
the African Giant rat (Cricetomys Waterhouse 1840) in the forest zone of south western
Nigeria (Olayemi and Akinpelu, 2008); weight assessment of some accessory digestive
organs in the adult African pouched rat (Nzalak et al., 2010a); gross anatomical,
histological and histochemical studies of the oesophagus of the African Giant rat (Nzalak
et al., 2010b); histological and histochemical studies of the colon of the African rat
(Nzalak et al., 2011) and gross anatomical aspect of gastro-intestinal tract of the wild
African giant rat – Cricetomys gambianus (Ali et al., 2008), have attempted to provide
additional information useful for characterization of the various giant pouched rat species.
However, the taxonomic impact of these studies has been of restricted importance
because they were conducted on limited specimen collections, underscoring the need for
more investigations covering the entire range of these rodents.
The use of molecular methods to provide more insight into the taxonomy and phylogeny
of Cricetomys was recommended. Preliminary molecular studies involving this genus
have helped to clarify its position and relationships with regard to other groups within the
rodent superfamily Muroidea (Peterson et al., 2006).
Until recently Cricetomys, based on dental morphology, was grouped alternatively under
the family Muridae by authors, who viewed its cheek teeth as triserial (Thomas, 1904;
Ellerman, 1941; Simpson, 1945; Roberts, 1951), or under the family Cricetidae by those
who consider its cheek teeth to be biserial (Petter, 1966; Rosevear, 1969; Reig, 1980,
1981).
4
Molecular techniques, however, have established this genus and others within the
subfamily Cricetomyinae as close relatives of archaic African muroids such as the
Nesomyinae, Dendromurinae, and Mystromyinae (DuBois et al., 1996), mitochondrial
12S rRNA (Verheyen et al., 1996; Jansa et al., 1999), mitochondrial cytochrome b (cyt b)
(Michaux et al., 2001), nuclear Lecithin Cholesterol Acyl Transferase (LCAT) and von
Willebrand Factor (vWF), and hence belonging to the family Nesomyidae as earlier
proposed by Lavocat (1973, 1978) and Chaline et al. (1977).
The African Giant rat or the Gambian pouched rat, Cricetomys gambianus (Waterhouse1840) (order Rodentia; family Muridae) (Plate I), is the world’s largest nocturnal rat and
is native to tropical Africa, where it is recorded from 29 countries, many thriving in urban
settings (Cooper, 2006). Some of the native names of African Giant rat in Nigeria are:
Burgu (Hausa), Okete (Yoruba) and Ikpukpa (Igbo).
The African Giant Rat has a long tail, which is bare with a white tip. The body is covered
with buff-grey, relatively long fur whereas the under parts are slightly paler. Front hands
are white. Face is characterized by long dark whiskers. An adult measures 75.0 cm from
the nose to the tip of the tail, and the tail is about 41.0 cm long. An average male weighs
about 1.3 kg and the female 1.2 kg. Small eyes are surrounded by a black eye-patch
(Rosevear, 1969).
These animals live up to 14 years in captivity, reaching maximum body weights of
approximately 2.80 kg in bucks and 1.39 kg in does. Male Gambian rats are larger than
females, achieving weights as high as 2.8 kg (Rosevear, 1969). The weights of adult rats
were 1.0-1.4 kg (the adult male was the largest) and the juvenile male weighs 0.5 kg.
Total lengths of the animals were 67.2-79.0 cm for males and 69.9 -73.5 cm for females.
Tails measured 37.2-40.0 cm for males and 37.4-40.5 cm for females (Perry et al., 2006).
5
The Cricetidae is a vast rodent family found not only throughout Africa but over much
Europe, Asia and as well as America. The Cricetids are overwhelmingly the majority of
the New World rodents (Rosevear, 1969). The ecological range extends from Senegal and
the Gambia east across West Africa and the Congo Basin to the Indian ocean coast of
East Africa (Halcrow, 1958; Coryndon et al., 1972), where it is mainly used as a meat
source (Lacasse et al., 2005). It is agriculturally important and is biologically interesting
in terms of its matriarchial social structure and its value to humans, for example when
trained to detect landmines. They are trained effectively by the Belgian firm Apopo at
Sokoine University of Agriculture in Morogoro, Tanzania, to detect landmines and sniff
out tuberculosis. Unfortunately, it is still widely persecuted by slash-and-burn and other
destructive practices (Cooper, 2008).
African Giant rats are omnivorous and are reported to consume vegetables, insects, crabs,
snails, palm fruits, and palm kernels (Ajayi, 1975). Members of this genus have been
linked to several potentially pathogenic zoonoses (leptospirosis, bartonellosis, and
trypanasomiasis), including monkeypox, which was introduced into the United States in
2003 (Gretillat et al., 1981; Hutin, 2001; Herder et al., 2002; Centres for Disease Control
and Prevention, 2003; Machang’u et al., 2004). Gestation period for C. gambianus ranges
from 27 to 42 days and litters consist of 1–5 in number; thus, members of the genus
Cricetomys must be considered highly fecund (Rosevear, 1969; Ajayi, 1975 and Hayssen
et al., 1993). Given their large body size, high fecundity, and omnivorous diet, these rats
pose a serious and potentially long-term threat to the indigenous ecological communities
within the Florida Keys.
Each gene maps to the same chromosome in every cell. Linkage is determined by the
presence of two or more loci on the same chromosome. The entire chromosomal set of a
6
species is known as a karyotype. In recent times, there has been much interest in
cytological studies of different species of organisms, especially vertebrates. This has lead
to the completion of genome sequencing in most of these species. Comparative
chromosome studies in related species have been of great value for the establishment of
systematic relationships in many plants and animals. A seemingly logical consequence of
descent from common ancestors is that more closely related species should have more
similar chromosomes. However, it is now widely appreciated that species may have
phylogenetically similar karyotypes because they are genomically conservative.
Therefore in comparative cytogenetics, phylogenetic relationships should be determined
on the basis of the polarity of chromosome differences (Graphodatsky, 2007).
Using cladistic analysis rearrangements that have diversified the mammalian karyotype
are more precisely mapped and placed in a phylogenomic perspective. “Comparative
chromosomics” defines the field of cytogenetics dealing with molecular approaches
(Claussen, 2005).
Mammalian comparative cytogenetics, an indispensable part of phylogenomics, has
evolved in a series of steps from a purely descriptive science to a heuristic science of the
genomic era. Technical advances have marked the various developmental steps of
cytogenetics (Graphodatsky et al., 2011).
In comparative cytogenetics, chromosome homology between species was proposed on
the basis of similarities in banding patterns. Closely related species often had very similar
banding pattern and after 40 years of comparing bands, it seems safe to generalize that
karyotype divergence in most taxonomic groups follows their phylogenetic relationship,
despite notable exeptions (O’Brien et al., 2006; Graphodatsky, 2006).
7
The studies of mammalian chromosomes have constituted an effective area of
investigation to explain their relationship. Genes provide instructions to build living
organisms and each gene maps to the same chromosome in every cell. Linkage is
provided by the co-localization of two or more loci on the same chromosome and the
largest linkage group is an entire chromosome. The entire chromosome set of a species is
known as a karyotype, which can be thought of as a global map of the nuclear genome.
The first step of the Human Genome Project took place when Tjio and Levan, in 1956,
reported the accurate diploid number of human chromosomes as 2n = 46 (Tijo and Levan,
1956). During this phase, data on the karyotypes of hundreds of mammalian species
(including information on diploid numbers, relative length and morphology of
chromosomes, presence of B chromosomes) were described. Diploid numbers (2n) were
found to vary from 2n = 6 – 7 in the Indian muntjac (Wurster and Benirschke, 1970) to
over 100 in some rodents (Contreras et al., 1990).
The second step derived from the invention of C-, G-, R- and other banding techniques
and was marked by the Paris Conference (1971), which led to a standard nomenclature to
recognize and classify each human chromosome (Paris Conference, 1971). Chromosome
painting data are now available for members of nearly all mammalian orders. It was
found that in most orders, there are species with rates of chromosome evolution that can
be considered as ‘default’ rates (Paris Conference, 1971).
The most widely used banding methods are G-banding (Giemsa-banding) and Rbanding (reverse-banding). These techniques produce a characteristic pattern of
contrasting dark and light transverse bands on the chromosomes. Banding makes it
possible to identify homologous chromosomes and construct chromosomal nomenclatures
for many species. Banding of homologous chromosomes allows chromosome segments
8
and rearrangements to be identified. The banded karyotypes of 850 mammalian species
were summarized in the Atlas of Mammalian Chromosomes (O’Brien et al., 2006).
Mammalian species differ considerably in heterochromatin content and location.
Heterochromatin is most often detected using C-banding (Hsu and Arrighi, 1970). Early
studies using C-banding showed that differences in the fundamental number (that is, the
number of chromosome arms) could be entirely due to the addition of heterochromatic
chromosome arms. Heterochromatin consists of different types of repetitive DNA, not all
seen with C-banding that can vary greatly between karyotypes of even closely related
species. The differences of the amount of heterochromatin among congeneric rodent
species may reach 33% of nuclear DNA in Dipodomys species (Hatch et al., 1976), 36%
in Peromyscus species (Deaven et al., 1977), 42% in Ammospermophilus (Mascarello et
al., 1977), and 60% in Thomomys species where C-value (haploid DNA content) ranges
between 2.1 and 5.6 pg (Patton and Sherwood, 1982; Sherwood and Patton, 1982).
Banner-tailed kangaroo rat (Dipodymos spectabilis, Merriam-1890) belong to the family,
Heteromyidae: A large, four-toed, long-tailed kangaroo rat; tail about 1.5 times as long as
head and body, with a distinct white tuft at end; hind foot broad and usually 50 mm or
more in length; upper parts dark buff; black facial markings and stripes on tail
conspicuous. External measurements average: total length, 350 mm; tail, 210 mm; hind
foot, 53 mm and weight, 115 g (Plate II). These kangaroo rats are extremely sexually
dimorphic. Males are significantly larger in characteristics such as total length, length of
tail, greatest length, width, and depth of cranium, and maxillary arch spread. Male banner
tails also have the largest baculum in the genus (Best, 1972).
9
1.2 Statement of Research Problem
There is no published research works on the molecular or genetic property of an African
giant rat from different geographical regions of Nigeria to the best of our knowledge.
Throughout the past decades, there has been an extensive effort to describe the
chromosomal constitution and variation in mammalian taxa, particularly of those
distributed in Europe (Zima, 2000, 2004). However, the karyotypes of many African
mammalian taxa still remain largely unknown. Their study could contribute to the
clarification of their taxonomy and phylogenetic relationships both within and among
related taxonomy.
Very little is known on the chromosomal constitution of the Cricetomys species in Africa,
especially in Northern Nigeria, apart from a study performed by Akintoye and Awopetu
(2005) on the genus Cricetomys emni in the South Western part of the country. The
current may fill this gap, using the G-banding staining techniques. (The results are
discussed and compared with those from Dipodomys spectabilis (Texas Banner-Tailed
Kangaroo Rat).
1.3 Justification of the Study
The result of the study will be of value in identifying the karyotypic patterns of African
Giant rat, which will be beneficial in comparing with other mammalian progeny.
The data to be obtained will be used to establish a reference data base for the karyotypic
patterns of African Giant rat. Knowledge of particular karyotypic patterns of African
Giant rat may be useful the in domestication of the rat and research and development in
the field of molecular genetics.
10
1.4 Aim and Objectives of the Study
1.4.1 Aim of the study
The aim of the study is to analyse the karyotypic patterns of the African giant rat.
1.4.2 Objectives of the study
This study is expected to:
i. Determine the nature of the chromosomal pattern and variability of African giant
rat.
ii. Compare karyotypic pattern of African giant rat with that of known Texas bannertailed kangaroo rat.
iii. Establish phylogenetic linkages between African giant rat with known Texas
banner-tailed kangaroo rat.
iv. Determine sexual differences in the karyotypic patterns of African giant rat.
1.5 Research Hypothesis
There is a relationship between the chromosomal numbers and differences in the
chromosomal lengths, centomeric indices and morphology of the male and female
African Giant rats (Cricetomys gambianus, Waterhouse-1840).
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