Morphometric Traits, Karyotypic Features and Protein
Polymorphism of the African Lungfish (Um Koro),
Protopterus annectens annectens (Owen, 1839) and
Protopterus aethiopicus aethiopicus (Heckel, 1851) from
Sudan.
BY
Amna Saad Omer Khidir
B.Sc. (Honours), Department of Zoology
Faculty of Science, University of Khartoum
A thesis submitted to the Department of Zoology, University of
Khartoum in Fulfilment of the Requirements for the Degree of M.Sc.in
Zoology.
.
Department of Zoology
Faculty of Science
University of Khartoum
September, 2006
© Amna saad , 2006.
DEDICATION
TO MY:
DEAR HUSBAND
DEAR SONS
DEAR BROTHERS
DEAR SISTERS
i
ACKNOWLEDGEMENTS
I am greatly thankful and grateful to my supervisor Dr.
Sumaia Abukashawa for her valuable advise, keen guidance and
continuous encouragement throughout the period of my study.
I would like also to express my thanks to all the staff members
of the Zoology Department, University of Khartoum, for their help.
Thanks are also to Mr. Braima Musa who helped in the
collection of the fish samples, and Mr. Sayed Yousif, for the fine
photography.
Thanks are extended to the staff at the Genetics laboratory at
the Zoology Department for their help.
ii
CONTENTS
Page
DEDICATION
I
ACKNOWLEDGEMENTS
ii
CONTENTS
iii
LIST OF TABLES
v
LIST OF FIGURES
vi
ABSTRACT
ix
ARABIC ABSTRACT
xi
CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW
1
INTRODUCTION
1
1.1-
THE LUNG FISHES (DIPNOANS)
4
1.1.1- Neoceratodus forsteri (Australian lungfish)
6
1.1.2- Lepidosiren paradoxa (South American lungfish)
7
1.1.3- African lungfishes
10
1.2- MORPHOLOGICAL CHARACTERS OF Protopterus Spp.
14
1.2.1- Morphology of Protopterus annectens annectens
14
1.2.2- Morphology of Protopterus aethiopicus aethiopicus
17
1.3-
CHROMOSOMES AND KARYOTYPES
17
1.4-
FISH KARYOTYPING
19
1.5-
PROTOPTERUS KARYOTYPE
22
1.5.1-
Protopterus annectens Karyotype
22
1.5.2-
Protopterus aethiopicus Karyotype
23
1.6-
PROTEIN POLYMORPHISM
23
iii
1.7-
LUNG FISHES PROTEINS
25
CHAPTER TWO: MATERIALS AND METHODS
30
2.1-
SAMPLE COLLECTION
30
2.2-
IDENTIFICATION
32
2.3-
MORPHOMETRIC MEASUREMENTS
33
2.4-
KARYOTYPING
34
2.5-
PROTEIN ELECTROPHORESIS
37
2.5.1-
Reagents used
37
2.5.2-
Preparation of samples
38
2.5.3-
Electrophoresis
38
CHAPTER THREE: RESULTS
41
3.1-
41
GENERAL OBSERVATIONS
3.2-
MORPHOMETRIC MEASUREMENTS
48
3.3-
CYTOGENETICS
53
3.3.1-
Cytogenetics of Protopterus annectens annectens
53
3.3.1.1- The Karyotype
53
3.3.2-
54
Cytogenetics of Protopterus aethiopicus aethiopicus
3.3.2.1- The Karyotype
54
3.4-
63
PROTEIN POLYMORPHISM
CHAPTER FOUR: DISCUSSION
66
CONCLUSION
72
REFERENCES
74
iv
LIST OF TABLES
Page
Table (І): Morphometric measurements and ratios
of Protopterus annectens annectes (Owen, 1839).
50
Table (ІІ): Morphometric measurements and ratios
of Protopterus aethiopicus aethiopicus (Heckel, 1851).
51
Table (ІІІ): Comparison between morphometric
measurements and ratios of Protopterus annectens
annectens and Protopterus aethiopicus aethiopicus.±
standard error.
52
v
LIST OF FIGURES
Page
Plate(1).
Photograph of the Australian Lungfish
Neoceratodus forsteri.
8
Plate(2).
Photograph of the South American Lungfish
Lepidosiren paradoxa.
9
Figure(1). Aestivation-skeleton of Protopterus spp.
11
Plate(3). Photograph of the East African Lungfish
Protopterus amphibius.
15
Plate(4a and 4b). Photographs of The Slender Lungfish
Protopterus dolloi
16
Figure(2). "Map": Sudan, Showing Nile System and
Kordofan State.
31
Plate(5). A specimen of Protopterus annectens annectens
aestivating in a mucus cocoon inside mud.
44
Plate(6). Photograph of the Protopterus annectens
annectens.
45
Plate(7a and 7b). Photographs of the Protopterus
aethiopicus aethiopicus.
46-47
Plate(8). Mitotic prophase from the liver cells of
Protopterus annectens annectens.
55
vi
Plate(9). Mitotic anaphase from the liver cells of
Protopterus annectens annectens.
56
Plate(10). Mitotic metaphase from the liver cells of
Protopterus annectens annectens.
56
Plate(11). Mitotic telophase from the liver cells of
Protopterus annectens annectens.
57
Plate(12). Ideogram of Karyotype from Protopterus
annectens annectens .
58
Plate(13). Mitotic prophase from the liver cells of
Protopterus aethiopicus aethiopicus
59
Plate(14). Mitotic metaphase from the liver cells of
Protopterus aethiopicus aethiopicus.
60
Plate(15). Mitotic anaphase from the liver cells of
Protopterus aethiopicus aethiopicus.
60
Plate(16). Mitotic telophase from the liver cells of
Protopterus aethiopicus aethiopicus.
61
Plate(17). Ideogram of Karyotype from Protopterus
aethiopicus aethiopicus .
62
Plate(18). Electrophoretic bands of the serum of human,
Molecular weight marker.
64
Plate(19). Electrophoretic bands of the serum of:A: Polypterus spp. (garmout).
65
vii
B: Protopterus annectens annectens.
C: Protopterus aethiopicus aethiopicus.
viii
ABSTRACT
This study is meant to review the prevalence of the African
lungfish (Um koro) in Sudan, to study the genetic variation and
protein polymorphism within and between different populations of
lungfishes.
Fourty cocoons containing adults of the African lungfish
Protopterus annectens annectens (Owen, 1839) were collected from
dry ponds of Khor Al- Jogan in Northern Kordofan State. Five adult
specimens of the African lungfish Protopterus aethiopicus
aethiopicus (Heckel, 1851) were caught alive from the River Nile.
Morphometric measurements, including the Standard Length, Depth,
Distance Above Lateral Line, Distance Below Lateral Line,
Peduncle Length and Peduncle Depth were taken. Measurements and
ratios performed revealed that Protopterus aethiopicus aethiopicus
is longer and bigger in size than Protopterus annectens annectens.
The karyotypes of the two species were investigated,
Protopterus annectens annectens was found to have a diploid
ix
number of chromosomes of 2n=34, while Protopterus aethiopicus
aethiopicus showed a diploid number of 2n =28 chromosomes.
The serum of the two lung fish species and of the garmout fish
(Polypterus) were subjected to electrophoresis utilizing cellulose
acetate paper and lipo-protein paper. Human serum was used as a
molecular weight marker. The α- globulin and γ- globulin of the
serum protein appeared in the four specimens. An albumin band was
clearly demonstrated for the molecular weight marker and for the
Polypterus while no albumin band appeared in the electrophoresis
result of the sera of the two lungfishes.
Results were discussed against the available literature of the
lungfishes. Conclusions and recommendations were drawn to help in
direction of future research on lungfishes in Sudan.
x
‫ﺨﻼﺼﺔ ﺍﻟﺒﺤﺙ‬
‫ﺘﻬﺩﻑ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺇﻟﻰ ﻤﺭﺍﺠﻌﺔ ﻭﺠﻭﺩ ﺍﻷﺴﻤﺎﻙ ﺍﻟﺭﺌﻭﻴﺔ )ﺃﻡ ﻜﻭﺭﻭ(‬
‫ﺒﺎﻟﺴﻭﺩﺍﻥ‪ ،‬ﻭ ﺩﺭﺍﺴﺔ ﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻟﻭﺭﺍﺜﻴﺔ ﻭ ﺍﻟﻨﻤﻁ ﺍﻟﻅﺎﻫﺭﻱ ﻟﻠﺒﺭﻭﺘﻴﻥ ﺒﻴﻥ ﺍﻷﻨﻭﺍﻉ ﻭ‬
‫ﺍﻟﻌﺸﺎﺌﺭ ﻓﻲ ﻫﺫﻩ ﺍﻷﺴﻤﺎﻙ‪.‬‬
‫ﺘﻡ ﺠﻤﻊ ‪ 40‬ﺴﻤﻜﺔ ﻤﻥ ﺍﻟﻨﻭﻉ ‪ Protopterus annectens annectens‬ﻤﻥ‬
‫ﺩﺍﺨل ﺍﻟﺒﺭﻙ ﺍﻟﺠﺎﻓﺔ ﻓﻲ ﺨﻭﺭ ﺍﻟﺠﻭﻗﺎﻥ ﺒﻭﻻﻴﺔ ﺸﻤﺎل ﻜﺭﺩﻓﺎﻥ ﻜﻤﺎ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻰ‬
‫‪ 5‬ﻋﻴﻨﺎﺕ ﺤﻴﺔ ﻤﻥ ﺍﻟﻨﻭﻉ ﺍﻷﺜﻴﻭﺒﻲ ‪ Protopterus aethiopicus aethiopicus‬ﻤﻥ ﻨﻬﺭ‬
‫ﺍﻟﻨﻴل‪ .‬ﺃﺠﺭﻴﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﻅﻬﺭﻴﺔ ﻤﺘﻀﻤﻨﺔ ﺍﻟﻁﻭل ﻭ ﺍﻟﻌﻤﻕ ﺍﻟﻤﻌﻴﺎﺭﻱ ﻭ ﺍﻟﻤﺴﺎﻓﺔ‬
‫ﻓﻭﻕ ﺍﻟﺨﻁ ﺍﻟﺠﺎﻨﺒﻲ ﻭ ﺍﻟﻤﺴﺎﻓﺔ ﺘﺤﺕ ﺍﻟﺨﻁ ﺍﻟﺠﺎﻨﺒﻲ ﻭ ﻁﻭل ﻭ ﻋﻤﻕ ﺍﻟﺴﻭﻴﻘﺔ‪.‬‬
‫ﺒﻴﻨﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﻭ ﺍﻟﻨﺴﺏ ﺃﻥ ‪ Protopterus aethiopicus aethiopicus‬ﺃﻁﻭل‬
‫ﻭ ﺃﻜﺒﺭ ﺤﺠﻤﹰﺎ ﻤﻥ ‪. Protopterus annectens annectens‬‬
‫ﺘﻤﺕ ﺩﺭﺍﺴﺔ ﻨﻤﻁ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻟﻠﻨﻭﻋﻴﻥ ﻭ ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ‪Protopterus‬‬
‫‪ annectens annectens‬ﻟﻪ ﻁﺎﻗﻡ ﺼﺒﻐﻴﺎﺕ ﻤﻜﻭﻥ ﻤﻥ ‪2‬ﻥ=‪ 34‬ﺼﺒﻐﻲ ﺒﻴﻨﻤﺎ ﺍﺤﺘﻭﻯ‬
‫‪xi‬‬
‫ﻁﺎﻗﻡ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻓﻲ ﻨﻭﻉ ‪ Protopterus aethiopicus aethiopicus‬ﻋﻠﻰ ‪2‬ﻥ=‪28‬‬
‫ﺼﺒﻐﻲ‪.‬‬
‫ﺘﻡ ﺘﻌﺭﻴﺽ ﻤﺼل ﺍﻟﺩﻡ ﻟﻠﻨﻭﻋﻴﻥ ﺒﺎﻻﻀﺎﻓﺔ ﻟﺴﻤﻙ ﺍﻟﻘﺭﻤﻭﻁ ﻟﻠﺭﺤﻼﻥ‬
‫ﺍﻟﻜﻬﺭﺒﺎﺌﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻏﺸﺎﺀ ﺨﻼﺕ ﺍﻟﺴﻠﻴﻠﻭﺯ ﻭ ﻭﺭﻕ ﺍﻟﻠﻴﺒﻭﺒﺭﻭﺘﻴﻥ ﺒﻴﻨﻤﺎ ﺍﺩﺭﺝ ﻤﺼل‬
‫ﺍﻟﺩﻡ ﺍﻟﺒﺸﺭﻱ ﻜﻤﺠﺱ‪ .‬ﻅﻬﺭﺕ ﺤﺯﻡ ﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻟﻘﻠﻭﺒﻴﻨﺎﺕ ﻤﻥ ﺍﻟﻨﻭﻉ ‪ α‬و اﻟﻨﻮع ‪γ‬‬
‫ﻓﻲ آﻞ اﻟﻌﻴﻨﺎت اﻷرﺑﻊ‪ .‬ﻇﻬﺮت ﺣﺰم ﻟﺒﺮوﺗﻴﻦ اﻷﻟﺒﻮﻣﻴﻦ ﻟﺴﻤﻚ اﻟﻘﺮﻣﻮط ﺑﻴﻨﻤﺎ ﻟﻢ‬
‫ﻳﻈﻬﺮ أي ﺗﻌﺒﻴﺮ ﻟﺒﺮوﺗﻴﻦ اﻷﻟﺒﻮﻣﻴﻦ ﻓﻲ أي ﻣﻦ اﻟﻨﻮﻋﻴﻦ ‪Protopterus aethiopicus‬‬
‫‪ aethiopicus‬ﻭ‪. Protopterus annectens annectens‬‬
‫ﺤﻠﻠﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻭ ﻨﻭﻗﺸﺕ ﻋﻠﻰ ﺨﻠﻔﻴﺔ ﺍﻷﺩﺒﻴﺎﺕ ﺍﻟﻤﺘﻭﻓﺭﺓ ﻋﻥ ﺍﻷﻨﻭﺍﻉ ﺍﻟﺘﻲ ﺘﻤﺕ‬
‫ﺩﺭﺍﺴﺘﻬﺎ ﻭ ﺃﺩﺭﺠﺕ ﺍﻟﺘﻭﺼﻴﺎﺕ ﺍﻟﺘﻲ ﺘﺴﺎﻫﻡ ﻓﻲ ﺘﺤﺩﻴﺩ ﺍﺘﺠﺎﻫﺎﺕ ﺍﻷﺒﺤﺎﺙ ﺍﻟﻤﺴﺘﻘﺒﻠﻴﺔ‬
‫ﻟﻠﻨﻭﻋﻴﻥ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ‪.‬‬
‫‪xii‬‬
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
INTRODUCTION:Fishes are a major component of aquatic habitats with respect to
the number both of individuals and of species. They have considerable
morphological variability, which is likely related to their highly
diversified habitat. The relationship between this variability and the
phylogeny of some groups raise interesting questions relevant for the
study of adaptive traits and for discriminating between convergences
and shared traits due to common ancestry.
There are more than 200 fish species found in the River Nile in
the Sudan, but only little comparative or genetic studies have been done
on those Nile fishes (Babiker and Elhakeem, 1979). Most of the work
done to classify fishes and to differentiate between the different genera,
was almost morphological (Boulenger, 1907; Abu Gideiri, 1984).
Recently, the advancement in the methods using genetical information
at the chromosomal, protein and DNA levels allowed better analysis of
1
fish species and opened horizons to perform studies resulting in
improved fish culture and production. The knowledge of the genetic
material and the chromosome number and structure of fishes will help
to reduce or eliminate fish’s inherited genetical diseases, and will
enable scientists to make new hybrids with improved properties, and
hybrids of better economical value (Mohammed, 2000).
Species of fishes, like most other aquatic or terrestrial organisms,
do not exist as one continuous or homogenous populations, rather, they
consist of a collection of natural populations (Spanakis et al., 1989).
When investigating genetic variation in such natural fish populations,
polymorphism will be a very useful means to provide estimates of the
genetic variability within those natural populations and the amount of
genetic differentiation between them (Avise and Slender, 1972). It is
possible to differentiate between distant populations of the same
species, along a geographic range, by minor differences in the protein
patterns (Avise and Smith, 1974; Kirpichnikov, 1981; El Fadel, 1999).
Lungfishes have been the focus of more evolutionary controversy
than any other fish. Ever since their discovery, their proper
classification and significance have been a matter of serious scientific
2
debate, which has continued, in one form or another, right up to the
present. Unlike nineteenth century zoologists, current researchers may
no longer wonder whether lungfishes are amphibians or fishes, but they
search for sister groups among the tetrapods, lungfishes and other kinds
of sarcopterygians.
Bearing this background about the disputed phylogeny and
relationships of lungfishes and their interesting biology, the present
study is an attempt to review the existence and distribution of the
lungfish species in the River Nile and in Western Sudan. The study will
examine, and compare lungfishes using data of the morphometric traits,
the karyotypic features, and the genetical variation at the protein level.
Two species of the lungfish, P. annectens annectens and P. aethiopicus
aethiopicus will be studied using such available criteria and comparison
will be made between them.
The literature review below gives a summary of the current
research pursued and the hypothesis presented as to the origin,
phylogeny, general biology and recent genetical studies of lungfishes
with emphasis on African lungfishes.
3
1.1- THE LUNG FISHES (DIPNOANS):Lungfishes (or dipnoans, as they are `dual breathers') are an
archaic group of fishes, characterized by the possession of a 'lung'
opening off the ventral side of the esophagus (Chew et al., 2004). The
African fresh water lungfish Protopterus spp. belonging to the order
Dipnoi, are a group of Osteichthyes fishes, the relationship of which
with tetrapods have been disputed since their discovery. In the past,
they were variously considered as being related to actinistans,
tetrapods, and lower actinopterygians, though nowadays they are
considered a monophyletic group, the sister group of crossopterygians
(Morescalchi, et al., 2002). They are considered as a bridging creature
which moved onto the land from water (Neo Kotobuki, 1998).
Dipnoans first appeared in the geologic record in the early Devonian
with 50 extinct genera, surviving up to date, with only three genera,
Lepidosiren, Neoceratodus and Protopterus, including only six
recognized species; four in Africa and one each in South America and
Australia (Morescalchi, et al., 2002). They have been classified in a
variety of ways, ranging from class Dipnoi, to infraclass Dipnomorpha
4
to order Dipteriformes. However, there is a general agreement that
there are two main subcategories (orders).
Kingdom:
Animalia
Phylum:
Chordata
Subphylum: Vertebrata :
Chondrichthyes (cartilaginous fishes)
Osteichthyes (bony fishes)
Class:
Subclass:
Order:
Sarcopterygii (lobe-finned fish)
Dipnoi
1-Ceratodontiformes
Family: Ceratodontidae
Genus: Neoceratodus
2- Lepidosireniformes
2.1- Family: Lepidosirenidae
Genus: Lepidosiren
Species: N. Forsteri
Species: L. paradoxa
(South American lungfish).
(Queensland lungfish).
(Australian lungfish).
2.2- Family: Protopteridae
Genus: Protopterus
Species: 1. P. aethiopicus
(Marbled lungfish).
Subspecies:
•
P. aethiopicus aethiopicus
(Heckel, 1851)
•
P. aethiopicus congicus
(Poll, 1961)
•
P. aethiopicus mesmaekersi
(Poll, 1961)
Species: 2. P. amphibious (Peters, 1844)
(East African lungfish).
(Gilled lungfish).
Species: 3. P. annectens
(African lungfish).
Subspecies:
5
•
P. annectens annectens
(Owen, 1839)
•
P. annectens brieni
(Poll, 1961)
Species: 4. P. dolloi (Boulenger, 1900)
(Slender lungfish).
(De Courcy and Dolan, 2004; Gosse, 1984).
1.1.1- Neoceratodus forsteri (Australian lungfish):This is the most primitive form of lungfish of all, only one
family, one genus, one species occurs in Australia (Plate 1). It lives in
clear rivers and reservoirs, and is deep water-body fish with paddle-like
paired fins. It is thought that it can reach 1.5 meters long and weigh
about 45 kgs. Neoceratodus forsteri only uses its lung when stressed,
using its gills for respiration the remainder of the time, it does not
aestivate in underground. It is thought to resemble tetrapod ancestors in
the way they move by walking across the bottom of the pond using
their pectoral and pelvic fins (Neo Kotobuki, 1998).
The Australian lungfish, Neoceradotus forsteri, is considered to
be most closely related to other freshwater fish than other lungfish
species because of the well-developed gills on all gill arches.
6
1.1.2- Lepidosiren paradoxa (South American lungfish):According to Neo Kotobuki 1998, there is one species of this
lungfish which can grow up to 100 cm. Lepidosiren paradoxa, only
lives in the Amazon in South America. They breathe almost entirely
with their lungs, having degenerating gills which do not function well.
It is an elongate, rather eel-like fish with significantly shorter pectoral
and pelvic fins. Males protect the eggs and young in burrows, and
develop fringes around their pelvic fins which transfer oxygen (like
reverse gills) to the back of the burrow (Plate 2).
7
Plate (1): Photograph of a rare variety of the Australian lungfish
Neoceratodus forsteri.
(After Neo Kotobuki, 1998).
8
Plate (2): Photograph of the South American lung fish, Lepidosiren
paradoxa.
(After Neo Kotobuki, 1998).
9
1.1.3-African lungfishes (P. aethiopicus, P. amphibius, P.
annectens, and P. dolloi):The African lungfish, Protopterus spp., is generally held to have
survived for over 300 million years without marked change in the
anatomical and physiological features that characterized the first
terrestrial animals (Thomson, 1969). For this reason, the lungfish has
attracted the attention of those concerned with the adaptive mechanisms
involved in the evolutionary transition from aquatic to terrestrial life. In
addition, this genus has retained the distinctive ability to aestivate, a
type of dormancy that enables it to survive the seasonal periods of
drought (3-9 months) that characterize life in the tropics.
African lungfishes are almost unique amongst fish species in that
they are able to aestivate for long periods of time when faced with
drought conditions. An essential aspect of this ability is the production
of protective cocoon. To initiate the process of aestivation, the lungfish
burrows into the mud as the ambient waters recede and forms an
aestivation burrow (Figure 1).
10
Figure (1):- Aestivation – skelton of Protopterus spp.
11
As the surrounding mud dries, the mucous secretions of the skin harden
to form a waterproof cocoon that surrounds the body completely except
for the small opening at the mouth (Delaney et al., 1977). In this
subterranean nest, which is connected to the surface by a narrow
breathing channel, the lungfish is obliged to rely entirely on air
breathing for its external gas exchange, and is deprived of access to
food or water. Inevitably, the lack of food and water intake and the
cessation of gas exchange through skin and gills lead to dramatic
changes in metabolic functions (Delaney et al., 1977; Smith, 1935).
Survival during aestivation also implies accommodation to major
changes in acid-base balance and in the electrolyte composition of the
blood.
P. aethiopicus and P. annectens can aestivate in subterranean mud
cocoons for long periods of time (Smith, 1935; Janssens, 1964;
Janssens and Cohen, 1968a, 1968b). On land, there is often a lack of
water to flush the branchial and cutaneous surfaces, impeding the
excretion of ammonia and consequently leading to the accumulation of
ammonia in the body. Since ammonia is toxic therefore African
lungfishes have to avoid ammonia intoxication when out of water
12
(Cooper and Plum, 1987; Hermenegildo et al., 1996; Brusilow, 2002;
Felipo and Butterworth, 2002; Rose, 2002).
Protopterus spp., locally known as Um Koro, is an aggressive
carnivorous predator, while P. aethiopicus is an omnivorous. Food
includes mollusks, frogs and small fishes (Neo Kotobuki, 1998). It
inhabits brackish fresh water of small rivers and swamps in Senegal,
Niger, Gambia, Volta, Chad and Sudan (Basaglia, 2002). The fish is
known by its ability to breathe air by its 'lungs'. It inhabits areas that
flood in the wet season and dry out in the dry season. When water
levels begin to fall in the dry season, P. aethiopicus, P. amphibius,
(Plate 3) and P. annectens are capable of digging a hole in the mud, in
which they lie, using their 'lungs' to breathe air. When the water has
completely evaporated the lungfish folds itself up and secretes a thin
slime around itself which dries into a fragile cocoon. P. dolloi found in
Central Africa in the lower and middle Congo River basin is the slender
lungfish (Plate 4a and 4b) which can aestivate on land within a layer of
dried mucus (Brien, 1959; Poll, 1961) instead of inside a cocoon in the
mud like P. aethiopicus and P. annectens. African lungfishes can exist
in this state for over a year. The metabolic rate slows and the energy
13
necessary for survival comes from the breakdown of the muscle tissue.
The fish loose huge mass of body weight and is extremely lethargic.
Upon the rainy season the lungfish eats incredible amounts to regain its
normal body weight and increase its metabolism. Very little is known
about the genetical, molecular or evolutionary relations in the four
Protopterus spp. and in the dipnoan clade in general.
1-2-:-Morphological Characters of Protopterus spp:1-2-1-Morphology of Protopterus annectens annectens:The body of P. annectens annectens is lateral, elongated with a
circular cross section, with more or less straight dorsal head profile,
with terminal mouth, a prominent snout, small eyes, striking paired
long and filamentous pectoral fins with a basal fringe about three times
the head length and pelvic fins are about two times the head length the
dorsal fin is continuous with the caudal fin, cycloid scales embedded in
the skin. Dorsal side is dark grey color, ventral side lighter; great
blackish spots on the body and fins (Leveque, 1990).
14
Plate (3): The East African Lungfish (Gilled-lungfish), Protopterus
amphibius (After Neo Kotobuki, 1998).
15
Plate (4a): The Slender Lungfish, Protopterus dolloi aestivating in a
dried mucus cocoon on land (After Chew et al., 2004).
Plate (4b): The Slender Lungfish Protopterus dolloi (After Neo Kotobuki,
1998).
16
P. annectens annectens has a dioecism mode of fertilization,
spawning frequency. It spawns in swamps during the wet season; they
build nests in which the eggs, (white in colour and about 4mm.
diameter) are laid; the young are cared by the males, the larvae hatch in
eight days, and leave the nest in twenty days (Leveque, 1990).
1-2-2-Morphology of Protopterus aethiopicus aethiopicus:The body of P. aethiopicus aethiopicus (Plate 7a and Plate 7b) is
laterally elongated, with more or less straight dorsal head profile, with
terminal mouth, a prominent snout, small eyes, striking paired fins long
and filamentous, the dorsal fin is continuous with the caudal fin, the
color of the body is marble and shiny with the ventral side lighter
(Agbayani, 1999); the body shape is similar to P. annectens annectens.
This information on the morphology is used as the basis for
identifying the specimens collected for this study.
1.3- CHROMOSOMES AND KARYOTYPES:Chromosomes are very important tools in the classification and
taxonomy of animals and plants. The karyotype, which comprises the
complete haploid set of chromosomes in the cell, characterizes the
17
species by specific number, shape and relative size of the chromosomes
(Swanson, 1957; Goodenough, 1984). The karyotype is also of interest
in establishing evolutionary relationships between different species
(Goodenough, 1984).
Usually, the chromosomal number is constant from individual to
individual within a given species, with very few exceptions; for
example in some species, the number varies between sexes-in a very
regular way, though. Contrary, the numbers of chromosomes vary
remarkably between different species (Swanson, 1957; Schjeide and De
Vellis, 1970; Suzuki et al., 1986).
Generally, the chromosomes are morphologically identified by
two features: the relative size and position of the centromere. There can
be a considerable variation in the chromosome size within a genome
(Swanson, 1957). If it is difficult to identify the chromosomes
according to the size alone, then, at least the chromosomes may be
grouped according to their similarity.
According to the position of the centromere, the chromosomes
may be classified into:
18
(1) A metacentric chromosome: having the centromere in the middle
and the two arms of the chromosome are of about equal length.
(2) An acrocentric chromosome: having the centromere being located
slightly nearer to one end of the chromosome than the other.
(3) A telocentric chromosome: having the centromere located at one
end.
(Swanson, 1957; Strickberger, 1976; Ayala and Kiger, 1984;
Goodenough, 1984; Suzuki et al., 1986; Franthworth, 1988;
Mohammed, 2000).
Chromosomal analysis is used to provide information about the
genetical make up of fishes and to identify any disorder which results
from numerical abnormalities, chromosomal polysomy, monosomy,
mosaicism, polyploidy; and also from chromosomal changes such as
structural abnormalities, translocations, deletions, duplications and
inversions (Swanson, 1957; Suzuki et al., 1986).
1.4- FISH KARYOTYPING:Studies of the karyotype of fishes and their chromosomal number
were not as successful and common as those of other vertebrate groups;
19
for only about 10% or even less of the more than 20,000 known species
of fishes, have their karyotype been studied (Gold, 1979; Hartley and
Horne, 1985).
The
major
problem
encountered
in
dealing
with
fish
chromosomes is that, most fishes have a relatively large number of
comparatively small chromosomes (Gold, 1979). This limits the
fullness of the resulting metaphase spread and hence discourages
karyotyping studies.
Most of the methodologies used for preparations of fish
chromosomes were initially, borrowed from human and other
vertebrate techniques. The commonly applied technique is that of the
squash preparation from kidney cells or other organs (Ojima et al.,
1963; Roberts, 1967), but this method has a great disadvantage: for the
animal tested must be killed; but even so the mitotic index and the
quality of the preparations were generally inferior than what is
expected (Blaxhall, 1975).
Since 1970, many new improved techniques have been developed
for fish chromosome preparations (Ojima et al., 1970; Barker, 1972;
Amemiya et al., 1984; Rivilin et al., 1985; Reddy and John, 1986).
20
These include blood leucocytes culture (Barker, 1972; Gold, 1979;
Blaxhall, 1983; Hartley and Horne, 1983; Hartley and Horne, 1985; AlSabati, 1985) regarded to be the most useful and successful technique.
Another technique is cell suspension from tissues such as gills, kidney
and intestine (McPhail and Jones, 1966; Gold, 1974; Klingerman and
Bloom, 1977). Although the techniques show some advantages, such as
obtaining blood samples (for culture) easily from living fish without
affecting the viability of the animal, and also obtaining higher mitotic
indices than those obtained from any other technique, still fish
cytogeneticists gained only limited success from this method. Multiple
difficulties arose which affected the mitotic index, and they were
unclear (Hartley and Horne, 1983). Moreover the technique required
high quality serum.
Most of the techniques were based on the use of colchicine to
block the quickly proliferating organs at metaphase (by dissolving the
spindle fibers). Then, after the fishes are killed, cell samples are taken
and treated for slide preparation (Ohono et al., 1965; Scheel, 1966,
Stewart and Levin, 1968; Klingerman and Bloom, 1977; Hartley and
Horne, 1983; Revilin et al., 1985).
21
The best tissues for obtaining dividing cells include: the kidney,
liver, spleen, epithelial cells from gills, fins scales, eye cornea and other
quick proliferating organs, such as testes, which can only be used
during active spermatogonial proliferation (Gold et al., 1990). The
younger the animal, the higher probability of obtaining good spreads of
chromosomes and a high number of metaphase will be scored (ElFadel, 1999).
Generally, the chromosome number of fishes is found to be in the
range of 2n=16 in Notabranchus to 2n=146 in Icthyomycon (Howell
and Duckett, 1971).
1.5- Protopterus Karyotype:A neontological approach to the problem of the origin of
tetrapods relies on the examination of the available cytological and
molecular data of the genome of these vertebrates. Very little is known
about the evolutionary karyology in the four Protopterus species and in
the dipnoan clade in general.
1.5.1-Protopterus annectens Karyotype:-
22
Morescalchi and others (2002), carried out a study to investigate
the karyotype of Protopterus annectens, the fish sample were collected
from Nigeria, ten male and female specimens of P. annectens have
their karyotypes been investigated, but non of which came from Sudan,
the chromosomal number was 2n=34. No karyotyping was done on
Protopterus spp. from Sudan.
1.5.2-Protopterus aethiopicus Karyotype:In the literature reviewed no work on the karyotype of P.
aethiopicus was found.
1.6- PROTEIN POLYMORPHISM:The advances in modern biochemical techniques have improved
genetic diversity, population structuring and evolutionary relationships
of many fishes to be directly appraised.
Protein as a major component of muscle, enzymes, hormones,
hemoglobin and other body tissues, are composed of elements that can
be separated from one another by several different techniques :Chemical methods, ultracentrifugation and electrophoresis are the most
used today.
23
Thousands of proteins present in the body perform numerous
functions; these include serving as carriers of vitamins, oxygen and
carbon dioxide plus structural, kinetic, catalytic and signaling roles
(Murray et al., 1999).
The protein fingerprints for each type of tissue should look
different, the more similar the types of cells (e.g. skeletal muscle and
cardiac muscle), the more similar their proteins will be. The more
different the types of cells (e.g. skeletal muscle and liver), the more
different the proteins in those cells will be (De Courcy and Dolan
2004). While no universally accepted classification system exists,
proteins may be classified on the basis of their solubility, shape,
biological function, or three-dimensional structure (Murray et al.,
1999).
With the development of the sophisticated instrumentation, many
of the basic methods for protein analysis have changed little over the
past three decades. Electrophoresis at pH 8.4-8.6 using a cellulose
acetate membrane as a substrate is simple, rapid and sensitive; it is
generally satisfactory in detecting most common hemoglobin variants.
However protein detection and analysis have become more sensitive.
24
Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDSPAGE), has become the method of choice for the analysis and isolation
of small amounts of proteins.
The analysis of electrophoretically detectable genetic variation
can be a very useful means for both, inferring the genetic structures of
natural populations and for delineating taxonomic relationships (Van
Der Bank et al., 1989).
1.7- LUNGFISH PROTEINS:In the literature reviewed no adequate information about the
proteins of P.annectens annectens and P. aethiopicus aethiopicus from
Sudan was found. However a study on the kallikrein-kinin and RenninAngiotensin systems in the kidneys of the African lungfish, P.
annectens,(Masini et al., 1996), delt with the physiology of the fish.
Another study on the nitrogen metabolism in the African lung fish
Protopterus dolloi was carried by Chew et al., (2004).
According to Hyodo and colleagues (1997), neurohypophysial
hormones are nonapeptide proteins regulating various physiological
events related especially to water and salt metabolism and
25
reproduction, twelve distinct nonapeptide principles have been
chemically characterized in a wide variety of vertebrates and are
classified into two groups: the vasopressin (VP) and the oxytocin (OT)
families, they are believed to have developed from a common ancestral
molecule by gene duplication (Acher et al., 1997). All vertebrate
species, except for the cyclostomes, contain at least one VP family
peptide and one OT family peptide (Acher et al., 1997).
Complementary DNA and genomic analyses have shown that
neurohypophysial nonapeptides are synthesized as large precursor
molecules (Hyodo et al., 1997). Using statistical comparison of gene
structures and the predicted amino acid sequences of precursors, Hyodo
and colleagues (1997) have proposed that teleost neurohypophysial
hormone genes have their own evolutionary history separate from that
of the tetrapod genes. Their hypothesis is further supported by the
structural characteristics of neurohypophysial hormone precursors,
including composition and the presence or absence of post translational
modification sites (Hyodo et al., 1997).
The appearance of tetrapods is one of the most dramatic events in
vertebrate
evolution.
Sarcopterygians
26
(extinct
rhipidistians,
coelacanths, and lungfishes) are almost universally considered to be
ancestral to tetrapods (Hyodo et al., 1997). Several hypothesis on the
relationship among extant vertebrate groups have been proposed based
on molecular and morphological data (Hyodo et al., 1997). Analyses of
mitochondrial 12S rRNA and cytochrome b gene sequences suggested
that the lungfish and tetrapods are included in the same clade (Hyodo et
al., 1997). On the other hand, mitochondrial cytochrome oxidase I and
28S rRNA gene sequences support the hypothesis that lungfishes and
coelacanths form a monophyletic group and are equally closely related
to land vertebrates (Hyodo et al., 1997). Amino acid sequences of
growth hormone and the glycoprotein hormone α-subunit of lungfishes
have high homology with those of tetrapods (Hyodo et al., 1997).
African lungfish prolactin, like tetrapod prolactins, contains three
disulfide bonds and differs from teleost prolactins that lack the aminoterminal disulfide bond (Hyodo et al., 1997). The lepidosirenid
lungfishes
(Lepidosiren
and
Protopterus)
have
a
distinct
neurohypophysis that is more similar to that of amphibians than to that
of any other fish. In Neoceratodus, the thin neurohypophysis is located
posterior to the adenohypophysis and not dorsal to it as is the case in
27
the
lepidosirenid
lungfishes
(Hyodo
et
al.,
1997).
Because
Neoceratodus is considered to be closer to the Devonian ancestral
dipnoans and Lepidosiren and Protopterus are considered to be of
much more recent origin, parallel evolution between the lungfishes and
modern amphibians has been proposed for the above characteristic
observed in the Lepidosirenid lungfishes (Hyodo et al., 1997). On the
other hand, the pituitary of another extant Sarcopterygian, the
coelacanth Latimeria, is unique and different from the pituitaries of
lungfishes and amphibians (Hyodo et al., 1997). It has been pointed out
that the neurohypophysis is one character that appeared in the lineage
prior to the splitting of lungfish and tetrapods (Hyodo et al., 1997). The
morphological data, together with the molecular data, suggest that the
lungfishes have a hypothalamo-neurohypophysial system homologous
to that of amphibians.
The molecular and morphological traits raise interest in the
neurohypophysial hormones in lungfish. Intravenous injection of
vasotocin (VT) into free-swimming lungfish elicits a dose-dependent
diuretic response, as occur in some freshwater teleosts (Hyodo et al.,
1997). Lepidosiren and Protopterus live in shallow waters that are
28
subject to seasonal evaporation. They are able to survive in the absence
of environmental water by burrowing beneath the substratum and
encysting in a water-impermeable cocoon. Lungfishes have a full
complement of ornithine-urea cycle enzymes in the liver and become
exclusively ureotelic during aestivation. The cessation of water intake
results in hemo-concentration and marked oliguria (Janssens et al.,
1966 ; DeLaney et al., 1977).
The present work represents an attempt to fill in the gap of
information regarding protein polymorphism of the African lungfishes
from Sudan.
29
CHAPTER TWO
MATERIALS AND METHODS
2.1- SAMPLE COLLECTION:There are no rivers in Northern Kordofan State, the site chosen
for collection figure (2), and the rains are seasonal. The major seasonal
water system (Khor Abuhabil) was chosen as a location for collection.
An identified site at Khor AL-Jogan, near Umrawaba town was chosen.
Specimens were collected during the dry seasons of 2004 and 2005.
Fourty cocoons containing adult P.annectens annectens were collected
from dry ponds at Khor Al-Jogan, identified by the track left by the fish
on the dry mud. A two-headed dagger was used to dig carefully around
each cocoon. In some cases one opening that could be seen on the
surface would lead to other concealed cocoons that were not seen from
the surface of the muddy ground. The removed cocoons with the fish
inside were wrapped carefully in sterile cotton, and then placed singly
inside boxes. Specimens were transferred to the Genetics laboratory,
30
Figure 2"Map": Sudan, Showing Nile System and Kordofan State.
31
Zoology Department, Faculty of Science, University of Khartoum for
further studies. P.annectens annectens specimens lived inside the
cocoons at room temperature for more than a year. Some specimens
were freed and left in a plastic water container till the time of use. Two
specimens were taken to a farm Southern to Khartoum and left to swim
in a pond of 2.5 × 1.5 meters.
Five adult specimens of P. aethiopicus aethiopicus were
caught alive. Three of these were caught using serine nets from among
fish collected from Jebal Aulia dam on the White Nile while two
specimens were collected alive at Almorada fish market in Omdurman.
The five specimens were brought alive to the laboratory and maintained
in a water tank till the time of use.
2.2-IDENTIFICATION:Protopterus spp. were identified using a standard key of
morphological characters as described in the literature (Leveque, 1990;
Agbayani, 1999). The morphological descriptions and measurements
32
were carried on this study to stress the specificity of the specimens
collected.
Measurements and ratios relating to various external features
were performed for the identification of Protopterus spp., these ratios
are particularly characteristic to fish species.
2.3- MORPHOMETRIC MEASUREMENTS:The
measurements and ratios were taken according to the
studies of Bishai and Abu Gideiri (1967). The specimens were put on a
wooden measuring board, and the measurements were performed using
a ruler and vernier (of 0.01 cm accuracy). Measurements included:
1- Standard length (SL) (from the tip of the snout to the
origin of the caudal fin) / Depth (D) (the greatest
vertical measurement starting a few millimeters from
the origin of the dorsal fin ventral ward) =SL/D.
2- Peduncle length (PL) (the line joining the posterior
point of the origin of the anal fin to the point where the
lateral line meets the caudal) / Peduncle depth (PD)
33
(the narrowest part posterior to the position of the anal
fin and anterior to the caudal fin) =PL/PD.
3- Distance Above Lateral Line (DALL) / Distance
Below Lateral Line (DBLL) =DALL/DBLL.
2.4- KARYOTYPING:Karyotyping (for both somatic and germ cells) was made
following two techniques:2.4-1- A modification of the karyotyping technique of Hitotsumachi et
al., (1969), Barker (1970) and Cucchi and Baruffaldi (1990) was
applied.
Each fish was dissected, the liver, and the gonads were removed
and each organ was squashed vigorously, in a mortar with a glass rod,
to break the cell clumps. One ml of sodium citrate solution [0.5 gram
sodium citrate +5ml distilled water] was added while grinding. After
grinding, the mixture was transferred to an appendorf tube using a
dropper; the tube was marked and labeled. The contents of the tube
were centrifuged for 15 minutes at 2000 rounds per-minute, and the
supernatant fluid was discarded carefully, leaving only the settled
34
undisturbed cells at the bottom of the tube. One ml of freshly prepared
Carnoy's fixative (3 parts of absolute methanol: 1 part of glacial acetic
acid) was added, then the cells were centrifuged for 10 minutes at 2000
rounds per-minute. The last step was repeated three times. The cells
were kept in 1.5 ml of the fixative in an appendorf tube.
During the fixation of the cells, clean slides were washed by
methanol and were put on a slide rack inside a freezer. At the time of
use, the slides were removed from the freezer and were put on the
bench. Three drops of the cell suspension were dropped from a high
distance onto the slides. The slides were placed on a hot plate, and the
excess fixative was allowed to evaporate. The slides were then left to
dry, the slides were stained using Geimsa stain (1 part Geimsa stock
solution: 1 part distilled water) for 30 minutes. The slides were then
made permanent by passing them through a bath of Xylol for 3-4
minutes. The slides were dried and mounted with cover slips, ready for
examination under the microscope.
Slides with well spread chromosomes were photographed using a
digital camera attached to the microscope. Chromosome counts were
made from slides directly and verified from photographs.
35
2.4.2- A karotyping technique using Feulgen and Orecin stain was
applied. The fish was dissected, the liver and the gonads were removed
and kept in carnoy`s in the freezer till the time of use. Tissues removed
from carnoy's were washed 3 times in distilled water and dried on filter
paper. The specimens were warmed in 500 ml 10% HCl at 60-65ْC
water bath for 7 minutes, then washed and dried. Then, the specimens
were kept in the dark in 600 µL Feulgen stain for two hours and then
washed 3 times in distilled water. A small piece of the tissue was
removed by a needle and transferred to a clean slide carefully. The
tissue was covered with a cover slip and then pressed gently with the
thump. A drop of Orecin stain was added at the edge of each cover slip,
the slides were left on the bench to dry, then, the slides were ready for
examination under the microscope. A total of 250 slides were examined
for verification
Slides with well spread chromosomes were photographed using a
digital camera attached to a Leitz Dialux 20 contrast microscope which
was adjusted to the highest magnification; the oil immersion lens was
used. Chromosomal counts were made from slides directly and verified
from photographs.
36
2.5- PROTEIN ELECTROPHORESIS:Serum proteins were detected using a cellulose acetate membrane
as a substrate for electrophoresis at pH 8.4-8.6. Albumin, α-globulin
and γ-globulin proteins were assayed. Human and garmout (polypterus)
serums were used as marker proteins.
At alkaline pH serum is a negatively charged protein and in an
electric field will migrate towards the anode (+).
2.5.1 – Reagents used:The following reagents were prepared:• Electrophoresis buffer. Tris /EDTA/ borate (TEB) pH 8.5. 10.2 g.Tris(hydroxymethyl) aminomethane (Tris), 0.6 g. EDTA and 3.2 g. boric
acid were mixed and the volume was brought to one liter by adding
distilled water. The buffer was stored at 4ْC and could be used
repeatedly without deterioration.
• Protein stain. 5 g. ponceau S and 7.5 g. trichloracetic acid , were
added, and brought to one liter by adding distilled Water.
37
• Destaining solution. 50 ml of 5% acetic acid were dropped in one liter
of distilled water.
• Clearing solution. 125 ml of glacial acetic acid, 375 ml of methanol
and 20 ml of polyethylene glycol were mixed.
2.5.2-Preparation of samples:Fish used for protein analysis were kept alive and then
anesthetized using chloroform. Blood samples were taken directly from
the posterior vena cava. Blood samples were kept in labeled anticoagulant tubes. Samples were centrifuged for about (10- 15) minutes.
The supernatant fluid was transferred carefully to a labeled appendorf
tube, leaving only the settled undisturbed cells at the bottom. The
labeled samples were kept in the freezer at -20ْC till the time of use.
2.5.3-Electrophoresis:With the power supply disconnected, the compartments of the
electrophoresis tank were filled with TEB buffer. The wicks were
soaked and positioned. One cellulose acetate paper was taken and with
a blunt pencil initial end was marked. A faint starting line (origin)
38
approximately 3 cm from the initial end was also marked (the cellulose
acetate paper was handled at the ends only using a plastic forceps). In a
separate dish the cellulose acetate membrane was soaked in TEB buffer
for at least 5 minutes. The membrane was immersed slowly, to avoid
trapping air bubbles and ensuring even saturation of the membrane. The
membrane was blotted between two pieces of absorbent paper, without
letting it to dry out before sample application. A small volume (10µ) of
each diluted sample was placed into a sample well. The applicator was
dipped into the sample wells, (the samples were applied to the cellulose
acetate approximately 3 cm from one end of the membrane). The
applicator tips were allowed to remain in contact with the membrane
for 3 seconds. The membrane was placed upside down across the
bridge of the tank so that the cellulose acetate surface was in contact
with the buffer, with the line of application at the cathode end. The
power supply was connected and ran at 250-350 V for 10 to 15 minutes
or until a visible separation was obtained. The power supply was
disconnected; the membrane was removed with forceps and carefully
floated on the surface of the ponceau staining solution, allowing the
stain to impregnate the paper from below. When totally wetted, the
39
paper was immersed completely in the stain for 5 minutes and agitated
occasionally. The membrane was removed, drained, and the excess
stain was eluted with three changes of destaining solution for 2 minutes
each. The membrane was dehydrated in absolute methanol for 2-3
minutes, after which it was immersed in the clearing solution for 6-8
minutes, and then dried at 65ْ C for 4-6 minutes. The membranes were
labeled and stored in a protective plastic envelope.
Lipo protein paper, which is more sensitive, was used as well as
cellulose acetate paper. Conventional analysis in a medical laboratory
utilizing dip stick for detection of albumin was done.
40
CHAPTER THREE
RESULTS
3.1- GENERAL OBSERVATIONS:Plate (5) shows a cocoon of P. annectens annectens collected
from Khor AL-Jogan. A curved adult specimen can be seen in the
cocoon embedded in the dry mud. The colour of the dorsal side of the
body was light grey while the ventral side was lighter with red spots.
All the body was covered with thick layers of mucus. When the fishes
were freed in water, they started breathing immediately. The colour of
the body started darkening and the red spots disappeared.
Plate (6) shows two specimens of P. annectens annectens freed
from the cocoons. The body was found to be elongated with a circular
cross section, with straight dorsal head profile as described by
Agbayani (1999). The mouth was terminal with a prominent snout and
small eyes. A pair of long and filamentous pectoral fins about three
times the head length was a common morphological feature of all the
specimens, other fins were also typical: the pelvic fins, about two times
41
the head length, and a dorsal fin continuous with the caudal fin. The
scales were cycloid and embedded in the skin.
It was possible to maintain P. annectens annectens specimens
inside the cocoons for more than a year in the laboratory; however, the
two specimens freed in the plastic water container refused to feed on
small fish, meat or vegetables. The two specimens freed in a pond
2.5×1.5 meters in a farm Southern to Khartoum were active feed on
insects and frogs, they are still alive, when the dry season of 2006
started and no water in the pond was available, P. annectens annectens
formed cocoons which were not seen from the surface of the muddy
ground.
P. aethiopicus aethiopicus specimens collected from Jebal
Aulia dam area were found to comply well with morphological
descriptions of Agbayani (1999). Morphological features of P.
aethiopicus aethiopicus are depicted in Plate (7a) and Plate (7b). The
five adult specimens collected showed a marble shiny body colour of
the dorsal side with the ventral side lighter. The body shape is similar
to that of P. annectens annectens. Great blackish spots covered the
body and fins. P. aethiopicus aethiopicus, was observed to be very
42
active (used to jump out of the water tank), aggressive and strong; care
was taken while dealing with it.
When the water level was very low both P.annectens annectens
and P. aethiopicus aethiopicus curved their bodies, stopped moving,
their colour became pale and started to secrete mucus to form cocoons.
43
Plate (5): A specimen of Protopterus annectens annectens aestivating in a
mucus cocoon inside mud, photograph taken at Genetics Laboratory,
University of Khartoum.
44
Plate (6): Photogaph of Protopterus annectens annectens in a water tank
at the Genetics Laboratory, University of Khartoum.
Top: P. annectens annectens stretched on a wooden measuring board.
Bottom: The basal fringe of the pectoral fin is indicated by the arrow.
45
Plate (7a): Protopterus aethiopicus aethiopicus (The specimens
photographed were brought alive from Jebal Aulia dam).
46
Plate (7b): Photgraph of Protopterus aethiopicus aethiopicus (The
specimens were brought alive from Jebal Aulia dam).
Top: P. aethiopicus aethiopicus creeping on smooth surface.
Bottom: P. aethiopicus aethiopicus anesthetized, chest opened and blood sample
harvested.
47
3.2-MORPHOMETRIC MEASUREMENTS:3.2.1- Morphometric measurements of Protopterus annectens
annectens:Of the 20 fishes measured and identified as P. annectens
annectens, the longest fish was about 60 cm. and weighed about 1200
gm. The morphometric ratios obtained were as follows:1- Standard length / Depth (SL/D)
= 9.377 ± 0.351
2- Distance above lateral line / Distance below lateral line
(DALL/DBLL) = 1.000 ± 0
3-Peduncle length / Peduncle depth (PL/PD)
= 8.018 ± 0.353
The distance above the lateral line is equal to the distance below the
lateral line. The results of morphometric measurements and ratios were
shown in table (I).
3.2.2- Morphometric measurements of Protopterus aethiopicus
aethiopicus:-
48
Of the 5 fishes measured, the longest fish measured 140 cm. long
and had a maximum body weight of 6000 gm.
The ratios obtained were as follows:1-SL/D
= 5.351 ± 0.310
2-DALL/DBLL = 1.000 ± 0
3-PL/PD
= 3.733 ± 0.417
The results of morphometric measurements and ratios of P. aethiopicus
aethiopicus were shown in table (II).
The measurements and ratios obtained for the two species of
Protopterus revealed that, P. aethiopicus aethiopicus is longer and
bigger than P.annectens annectens Table (III).
49
Table (1): Morphometric measurements and ratios of Protopterus annectens annectens.
Sample no. S.L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
35
37
40
43
47
50
51
55
60
26
30
17
22
10
45
36
19
27
14
31
D
3.5
4.0
4.5
4.7
5.2
5.3
5.7
6.1
6.7
2.9
3.2
1.7
2.3
1.1
4.7
3.6
1.8
3.0
1.5
3.3
SL/D
DALL
DBLL
10.000
9.250
8.889
9.149
9.038
9.434
8.947
9.016
8.955
8.966
9.375
10.000
9.565
9.091
9.574
10.000
10.556
9.000
9.333
9.394
2.0
2.3
2.4
2.5
2.7
3.0
3.1
3.3
4.2
1.5
1.8
1.0
1.3
0.5
2.7
2.2
1.0
1.4
0.7
1.8
2.0
2.3
2.4
2.5
2.7
3.0
3.1
3.3
4.2
1.5
1.8
1.0
1.3
0.5
2.7
2.2
1.0
1.4
0.7
1.8
DALL/DBLL
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
PL
PD
PL/PD
weight
13.0
13.5
14.0
14.4
15.0
16.0
16.0
16.7
17.4
8.0
9.0
6.5
7.2
3.0
14.8
13.1
6.8
8.4
4.3
9.1
1.5
1.7
1.8
1.9
2.0
2.0
2.2
2.2
2.1
1.0
1.1
0.8
0.9
0.4
1.7
1.6
0.8
1.0
0.5
1.2
8.700
7.941
7.700
7.579
7.500
8.000
7.273
7.591
8.286
8.000
8.182
8.125
8.000
7.500
8.706
8.188
8.500
8.400
8.600
7.583
0270
0300
0560
0500
0910
1040
1200
1000
1200
0200
0300
0070
0214
0050
0800
0280
0080
0300
0065
0350
SL= Standard Length
D= Depth
DALL= Distance Above Lateral Line
DBLL= Distance Below Lateral Line
PL= Peduncle Length
PD= Peduncle Depth
50
Table (II): Morphometric measurements and ratios of Protopterus aethiopicus aethiopicus.
Sample No.
PL
PD
PL/PD
BODY Wt.
(gm)
1
20
5.5
3.636
2650
11
1
35
9.0
3.889
5000
13
13
1
40
10
4.000
6000
5.000
9.5
9.5
1
27
7.3
3.699
3500
5.294
4.5
4.5
1
13
3.5
3.714
1450
S.L
D
SL./D
DALL
DBLL
1
69.0
13.5
5.148
07
07
2
130
22.0
5.909
11
3
140
25.9
5.405
4
90.0
18.0
5
45.0
08.5
DALL/DBLL
SL= Standard Length
PL = Peduncle Length
D= Depth
PD=Peduncle Depth
DALL= Distance Above Lateral Line
DBL=Distance Below Lateral Line.
51
Table (III). Comparison between morphometric measurements and ratios of P. annectens annectens and P.
aethiopicus aethiopicus, ± standard error.
Ratios
Measurements of
Measurements of
P.annectens annectens
P. aethiopicus aethiopicus
Standard length
Depth
9.377 ± 0.351
5.351 ± 0.310
Distance above lateral line
Distance below lateral line
1.000 ± 0
1.000 ± 0
Peduncle length
Peduncle depth
8.018 ± 0.353
52
3.733 ± 0.417
3.3-CYTOGENETICS:The chromosomal number was counted from several mitotic
phases from the liver cells of the two species, following the two
protocols for karyotyping mentioned in the literature.
3.3.1-Cytogenetics of Protopterus annectens annectens:Plate (8) shows a mitotic prophase from liver cells while plate
(9) shows a plate with mitotic anaphase. Plate (10) shows a cell at
metaphase while telophase is shown in plate (11). Results reveal that
the chromosomes are very small and vary in size within the set.
3.3.1.1- The Karyotype:
The structure and number of chromosomes are represented in
plate (12). The chromosomes of P. annectens annectens are arranged
in an ideogram according to their sizes. Plate (12) shows the
karyotype of P.annectens annectens with a diploid number of
chromosomes 2n=34. The first pair of chromosomes is clearly
metacenrtric, while the remaining 16 pairs are acrocentric.
53
The structure of the chromosomes was not easily determined
because of the small size of the chromosomes, and moreover the
chromosomes were often found accumulated in a compact form.
3.3.2-The cytogenetics of Protopterus aethiopicus aethiopicus:Plate (13a,b ,c) shows a mitotic prophase from different liver
cells while plate (14a, b) shows a mitotic metaphase from liver cells.
Plate (15a, b) shows a mitotic anaphase while cells in late telophase
are shown in plate (16).
3.3.2.1- The Karyotype:
The ideogram shown in plate (17) shows the karyotype of P.
aethiopicus aethiopicus .The karyotype is characterized by a diploid
chromosome number of 2n = 28. Like P.annectens annctens, the
chromosomes were not easily determined because of their small size
(plate 13 a,b,c). Chromosome pairs 1, 2, 3, are distinctly larger than
the rest of the chromosomes in the set. Chromosome pairs 2 and 3 are
clearly metacentric. There is a clear variation in the sizes of the
chromosomes within the set.
54
Plate (8): Mitotic prophase from the liver cells of Protopterus
annectens annectens. Chromosomes started to separate and dark
heterochromatic material appear.
55
Plate (9): Mitotic anaphase from the liver cells of Protopterus
annectens annectens. Arrows indicate the 2 daughter nuclei.
Plate (10): Mitotic metaphase from the liver of Protopterus annectens
annectens.
The
chromosomes
distinguished easily.
56
though
separated,
cannot
be
Plate (11): Mitotic telophase from the liver cells of Protopterus
annectens annectens. The two daughter nuclei are already
distinguishable, note the light-stained chromatin scattered around
the nuclei.
57
Plate (12): Ideogram of karyotype from Protopterus annectens
annectens showing a diploid set with 2n = 34
58
a
b
c
Plate (13): Liver cells from Protopterus aethiopicus aethiopicus at
mitotic prophase.
a= one cell
b= 2 cells
c= a group of cells clumped together (this common on squash
preparations)
59
a
b
Plate (14): Mitotic metaphase from the liver cells of Protopterus
aethiopicus aethiopicus: chromosomes can be distinguished
individually and their number counted; a and b are presented to
show methods for verification of number and structure routinely
used in this study.
a
b
Plate (15): Mitotic anaphase from the liver of Protopterus aethiopicus
aethiopicus: 2 different stages of anaphase, (a) shows one of the
acrocentric chromosomes lagging behind as indicated by the arrow.
60
Plate (16): Mitotic telophase from the liver cells of Protopterus
aethiopicus aethiopicus.
61
Plate (17): Ideogram of karyotype of Protopterus aethiopicus
aethiopicus showing a diploid chromosome set of 2n =28. Pairs 1,
2, 3 are larger than the rest and 2 and 3 are metacentric.
62
3.4- PROTEIN POLYMORPHISM:The electrophoretically separated proteins albumin, α-globulin and
γ- globulin bands of the human serum, used as a standard, are shown in
plate (18). The serum of human, P.annectens annectens, P.aethiopicus
aethiopicus and of the ‘garmout’ fish (Polypterus) were subjected to
electrophoresis utilizing cellulose acetate paper and lipo-protein paper as
described in chapter two, are shown in plate (19). The α-globulin of the
serum protein appeared in Polypterus (plate 19, lane A), P. annectens
annectens (plate 19, lane B) and P. aethiopicus aethiopicus (plate 19, lane
C) is comparable to the molecular weight marker (plate 19, Mw). The γglobulin from serum protein is shown in plate (19) as well.
Plate (19) shows serum albumin for Polypterus (lane A) as well as
for the molecular weight marker (Mw), however the serum albumin is not
detected for either P. annectens annectens (lane B) or P. aethiopicus
aethiopicus (lane C).
63
Albumin ( m.w 66.4 kdl )
α globulin
γ globulin
Plate (18) : Electrophoretic bands of the serum of human, used as
a molecular weight marker.
*kDa = Kilodaltons
64
Albumin ( m.w 66.4 kdl )
α globulin
γ globulin
A
B
C
MW
Plate (19): Electrophoretic bands of the serum of three fishes:A: Polypterus spp. ( garmout ).
B: Protopterus annectens annectens. (um koro ).
C: Protopterus aethiopicus aethiopicus. (um koro ).
MW: Molecular weight marker.
65
CHAPTER FIVE
DISCUSSION
The lungfishes are a truly ancient group of vertebrates, known
from fossils dating to at least the lower Devonian, some 408 million
years ago (Neo Kotobuki, 1998). Like other lungfishes, P. annectens
annectens does indeed possess a 'lung' as well as gills, enabling it to
surface and breathe air when it lives in stagnant water with little
oxygen. In the dry season, when the water evaporates and little but
wet mud is left, the fish burrows down into substrate where it forms a
cocoon of mucus and mud. Its metabolic rate greatly decreases; it
requires only an occasional breath of air via a small opening in the top
of its cocoon, and can survive in this state of aestivation despite the
fact that the mud around it is completely drying out. Months later,
when the rains return, the lungfish re-emerges into the water and
resumes living like a fish. This study demonstrated this type of
lifestyle where P. annectens annectens formed cocoons which are
not seen from the surface of the muddy ground, when the artificial
pond was dried up and no water in the artificial pond was available.
66
P.aethiopicus aethiopicus specimens that were brought alive from the
Nile were very aggressive and active when freed in a water tank.
However, the results showed that P.annectens annectens specimens
when freed in a tank full of water showed no inclination to feed.
Another difference between the two subspecies is revealed by
differences
of
morphological
characters.
The
morphometric
measurements of the two Protopterus spp., show that P. aethiopicus
aethiopicus is longer and bigger than P. annectens annectens in all
ratios: S.L/D of P.aethiopicus aethiopicus< S.L/D of P.annectens
annectens and PL/PD of P. aethiopicus aethiopicus< PL/PD of P.
annectens annectens.
The sizes of the chromosomes were found to be small,
typical to the sizes of fish chromosomes cited in the literature
(Blaxhall, 1975; Gold, 1979). The chromosomal number of P.
annectens annectens agreed with those obtained by Morescalchi et al.,
(2002), 2n = 34. The karyotype of P. aethiopicus aethiopicus showed
a chromosomal number of 2n=28 chromosomes, however, no referral
value for the chromosomal number for this species was found in the
literature. The difference of chromosome number brings to the surface
67
the suggestion of revision of the taxonomic relationships of the two
taxa. It is well established that chromosome number is uniform for
species and deviation from a “standard” set got to be explained
(sometimes there are apparent chromosomal fusions in one of the
species categories). Another deviation in the karyotype between the
two subspecies is also calling for restructuring the lungfishes in
Sudan. The sizes of the chromosomes of P. aethiopicus aethiopicus
were found to be distinctly larger than those of P. annectens
annectens: chromosome pairs 2 and 3 (plate 17) appear as large
metacentric chromosomes. Fusion of chromosomes is proposed here
to explain the discrepancy in the karyotypes.
The protein polymorphism underlying the karyotypic variation
is quite interesting. The α- globulin and γ- globulin of the serum
protein appeared in the two species and no variation was seen This
expected as it is well known that globulin genes are conserved in
animals .As seen from plate (19) there is no albumin band in the
electrophoresis results of the sera of the two lungfishes, P. annectens
annectens and P.aethiopicus aethiopicus. The electrophoresis of the
garmut (Polypterus sp) and the marker of the human serum revealed
68
expression of the albumin protein. The same results obtained when
using the more sensitive lipo-protein paper as well as cellulose acetate
paper points toward some interesting pattern of gene expression in the
two lungfishes. Although the karyotypes showed differences, the
expression of globulin and albumin appeared the same. Absence of
albumin expression in the two species would lead to the suggestion
that either albumin genes are completely absent, or more likely that
they are genetically not expressed. There is evidence to suggest that
albumin is not present in the plasma of elasmobranches as well
(Fellows et al., 1980; Fellows and Hird, 1981; Peters and Davidson,
1991) and this may be a consequence of high urea concentrations. It is
possible then, that coelacanths also lack plasma albumin. It is
suggested that unequal evolutionary rates among gene or protein
lineages can cause erroneous maximum parsimony (MP) and
neighbor joining (NJ) topologies (Felsenstein, 1978; Felsenstein,
1983; Li WH et al., 1987; Huelsenbeck and Hillis, 1993). An
interesting observation is that the different phylogenetic trees using
serum albumin contained some inconsistencies in comparison with
known vertebrate phylogenies: in particular, all made incorrect
69
predictions for cobra albumin with consistent placement outside
amphibian albumins (Fellows et al., 1980; Fellows and Hird, 1981).
This could be due to the unusual cysteine pattern in cobra albumin
necessary for its self anti-venom activity (Clark and Voris, 1969;
Shao et al., 1993), coupled with a faster mutation rate which makes
this reptilian albumin appear phylogenetically older than it is.
While albumin has traditionally been used as a molecular clock
(based on data from mammalian species), albumins of lower
vertebrates may have different mutation rates (Metcalf et al., 2003).
While the evidence presented by Metcalf and others (2003)
provides some support for a close relationship of lungfish and
tetrapods, there is great debate as to whether lungfish or coelacanths
are more closely related to tetrapods. Unfortunately, they did not
include any coelacanth albumin in their study. However, coelacanths,
like elasmobranches (sharks and rays), use urea as an important
osmolyte (Brown and Brown 1967), while lungfish and tetrapods do
not. Although most teleost (bony) fish species so far examined lack
albumin in their plasma, however, in a study to infer vertebrate
phylogenies albumin nucleotide sequence was obtained from the
70
Australian lung fish Neoceratodus forsteri. Like mammals, it is
reported to have a typical sized (~ 65 kDa) albumin protein. This is
quite expected since the Australian lung fish is distantly related to the
African lungfishes as mentioned in the literature review (Hyodo et
al.,1997; Metcalf et al., 2003). The most needed information now is
whether albumin in African lungfishes is represented by DNA
sequences in the lungfish genome or not, and if the DNA sequence is
found to be there what causes the repression of the expression. The
lack of expression in both P. annectens annectens and P.aethiopicus
aethiopicus with different lifestyles and its lack in the non-aestivating
fishes is interesting.
It is noteworthy that, all recent studies of phylogenetic
relationships between
lungfishes and tetrapods have found that
different phylogenetic methods were unable to resolve these
relationships, with either a lungfish+ tetrapod clade or a (lungfish
+coelacanth)+ tetrapod clade (Zardoya et al.,1998). It is clear that
more genetic and molecular studies are needed.
71
CONCLUSION
This study reveals useful information about lungfishes in Sudan. It
shows that there are two subspecies of lungfishes in Sudan, P.
aethiopicus aethiopicus which is found in the River Nile system and
P. annectens annectens found in the western and central Sudan. The
study shed light on the genetic variation within and between the two
sub species.
This study presents interesting results of genetic and molecular
nature; however, more genetic and molecular investigations are
needed especially for explaining the lack of expression of the albumin
protein in the two species. Population studies are also needed as well
as studies of the different developmental stages, questions need to be
answered are: do all developmental stages lack albumin? Is the
protein expressed during non-aestivating periods? Are there DNA
sequences of albumin in the form of pseudogenes? The application of
DNA
analysis
techniques
(DNA
typing,
DNA
sequencing,
polymerase chain reaction methods (PCR)) will be of great use to
72
detect and investigate the genetic make up and the variation between
the two fish species and between populations of either species. More
protein profiles are needed to construct the phylogenetic relationships
between the two species and other possible species inhabiting
different areas in Sudan. A survey of the dry ponds associated with
Bahr Alarab in western and southern Sudan is essential.
Considering the limited time of the study and the lack of proper
resources for molecular investigations, the study paves the way for
future research on such an important fish.
73
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