STUDIES on CHARACTERIZATION of MEMBERS of TWO FISH GENERA, Hydrocynus and Alestes, from the WHITE NILE and LAKE NUBIA

STUDIES ON CHARACTERIZATION OF MEMBERS OF TWO FISH GENERA, hydrocynus and Alestes, FROM THE WHITE NILE AND LAKE NUBIA

HAMID AHMAD MARGANI AWAD

B. Sc. (HONOURS), SUDAN UNIVERSITY, ANIMAL PRODUCTION, 1996

A THESIS SUBMITTED TO THE DEPARTMENT OF ZOOLOGY IN FULFILLMENT OF THE REQUIRMENTS FOR THE DEGREE OF M. Sc.

DEPARTMENT OF ZOOLOGY, FACULTY OF SCIENCE, UNIVERSITY OF KHARTOUM

October 2006

DEDICATION

TO MY LATE FATHER

TO MY LATE MOTHER

TO MY FAMILY

AND

TO MY FRIENDS

ACKNOWLEDGEMENTS

My thanks and grate appreciation are due to my supervisor Dr. Sumaia Mohamed Ahmed Abukashawa who encouraged me and whose keen guidance, valuable advice and patience throughout the period of this work made the essence for its accomplishment. In her person, I thank all the staff members of the Zoology Department, University of Khartoum, for their help and for offering the facility of the Genetics Lab. I would like to express special thanks to Professor Yousif Babikir Abu Gidieri, Zoology Department, for his interest in this work and his valuable advice. His willingness to share his experience and expertise was a tremendous help to me. Thanks are extended to Dr. Zuhair Nur Eldaim for his advice and support. I am grateful to my colleagues and friends who took the time to make suggestions for improvement of this work and for their encouragement and support. Thanks are extended to the Animal Resources and Research, Ministry of Science and Technology for sponsoring my study.

CONTENTS

Page DEDICATION I ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vii ABSTRACT x ARABIC ABSTRACT xii CHAPTER ONE 1 INTRODUCTION AND LITERATURE REVIEW 1.1 GENERAL INTRODUCTION 1 1.2 FISHES OF THE NILE 1 1.2.1 Lake Nubia 3 1.3 TAXONOMIC RELATIONSHIPS 3 1.3.1 The Genus Hydrocynus 6 1.3.1.1 Hydrocynus forskalii 6 1.3.1.2 Hydrocynus brevis 6 1.3.1.3 Hydrocynus lineatus 7 1.3.2.1 Alestes dentex 8 1.3.2.2 Alestes baremose 8 1.3.2.3 Alestes nurse 9 1.3.2.3 Alestes macrolepedidotus 9 1.4 CYTOGENETIC STUDIES 10 1.4.1 Chromosomes and karyotypes 10 1.4.2 Fish karyotypes 12 1.5 PROTEIN STUDIES 15 1.6 OBJECTIVES OF THE SUDY 17 CHAPTER TWO: MATERIALS AND METHODS 19 2.1. SAMPLE COLLECTION 19 2.2. MORPHOMETRIC MEASUREMENTS 19 2.3 STATISTICAL ANALYSIS 20 2.3 KARYOTYPING 22 2.4 PROTEIN ELECTROPHORESIS 23 2.4.1 Preparation of The Reagents Of SDS- Polyacrylamide Gel 23 2.4.2 Pouring SDS-Polyacrylamide Gels 26 2.4.3 Preparation Of The Samples 27 2.5 STATITICAL ANALYSIS 29 CHAPTER THREE: RESULTS 30

3.1 THE SPECIES 30 3.2 MORPHOMETRIC MEASUREMENTS 35 3.3 CYTOGENETIC DATA 68 3.4 PROTEIN POLYMORPHISM 84 CHAPTER FOUR: DISCUSSION AND CONCLUSIONS 90 4.1 DISCUUSION 90 4.2 CONCLUSION AND RECOMMENDATIONS 93 REFERENCES 95 APPENDICES: 101 APPENDIX 1: ABREVIATIONS APPENDIX 2: GLOSSARY

LIST OF TABLES

Table Page Table (I): Morphometric measurements and ratios of Alestes baremose from Lake Nubia 37 Table (II): Morphometric measurements and ratios of Alestes baremose from Jebel Aulia 38 Table (III): Morphometric measurements and ratios of Alestes dentex from Lake Nubia 39 Table (IV): Morphometric measurements and ratios of Alestes dentex from Jebel Aulia 40 Table (V): Morphometric measurements and ratios of Alestes nurse from Lake Nubia 41 Table (VI) Morphometric measurements and ratios of Alestes nurse from Jebel Aulia 42 Table (VII): Morphometric measurements and ratios of Alestes macrolepidotus 43 Table (VIII): Morphometric measurements and ratios of Hydrocynus lineatus from Lake Nubia 44 Table (IX): Morphometric measurements and ratios of Hydrocynus lineatus from Jebel Aulia 45 Table (X): 46 Morphometric measurements and ratios of Hydrocynus bevies from Lake Nubia Table (XI): Morphometric measurements and ratios of Hydrocynus brevis from Jebel Aulia 47 Table (XII): Morphometric measurements and ratios of Hydrocynus forskalii from Lake Nubia 48 Table (XIII): Morphometric measurements and ratios of Hydrocynus forskalii from Jebel Aulia Table(XIV): Comparisons of means and standard error of morphometric measurements for Alestes 51 Table (XV): Comparisons of means and standard error of morphometric measurements for Hydrocynus 52

LIST OF FIGURES

Figure Page Figure (1). Map of collection sites 21 Figure (2). Diagram of Hydrocynus and Alestes measurements 32 Figure (3). Photograph of Hydrocynus forskalii

Figure (4). Photograph of Hydrocynus lineatus 32 Figure (5). Photograph of Hydrocynus brevis 33 Figure (6). Photograph of Alestes baremose 33 Figure (7). Photographs of Alestes dentex 33 Figure (8). Photograph of Alestes nurse 34 Figure (9). Photograph of Alestes macrolpedotus 34 Figure (10). Morphometric relationships in Alestes nurse 55 a: Relationship between length & depth 55 b: Relationship between standard length & depth 55 c: Relationship between inter orbital width & eye diameter 56 d: Relationship between peduncle length & peduncle depth 56 57 Figure (11). Morphometric relationships in Alestes baremose:

a: Relationship between depth & length 57 b: Relationship between depth & standard length 57 c: Relationship between inter orbital width &eye diameter 58 d: Relationship between peduncle length & peduncle depth 58 59 Figure (12). Morphometric relationships in Alestes dentex

a: Relationship between length & depth 59 b: Relationship between standard length & depth 59 c: Relationship between eye diameter & inter orbital width 60 d: Relationship between peduncle length & peduncle width 60 Figure (13). Morphometric relationships Hydrocynus lineatus 61 a: Relationship between length & depth 61 b: Relationship between standard length & depth 61 c: Relationship between eye diameter & inter orbital width 62 d: Relationship between peduncle length & peduncle width 62 Figure (14). Morphometric relationship between in Hydrocynus forskalii 62 a: Relationship between length & depth 62 b: Relationship between standard length & depth 62 c: Relationship between eye diameter & inter orbital width 63 d: Relationship between peduncle length & peduncle width 63 Figure (15). Morphometric relationship in Hydrocynus brevis a: Relationship between length & depth 66 b: Relationship between standard length & depth 66 c: Relationship between eye diameter & inter orbital width 67 d: Relationship between peduncle length & peduncle width in 67 Figure (16): Prophase of Hydrocynus brevis in Jebel Aulia 70 Figure (17): Late prophase of Hydrocynus brevis in Lake Nubia 70 Figure (18): Metaphase of Hydrocynus brevis in Jebel Aulia 71 Figure (19): Metaphase of Hydrocynus lineatus in Lake Nubia 71 Figure (20): Interphase of Hydrocynus brevis in Lake Nubia 72 Figure (21): Telophase of Hydrocynus lineatus in Lake Nubia 72 Figure (22): Metaphase of Alestes baremose in Jebel Aulia 73 Figure (23): Metaphase of Alestes baremose in Jebel Aulia.73 73 Figure (24): Late Anaphase of Hydrocynus brevis in Jebel Aulia 74 Figure (25): Metaphase of Hydrocynus brevis in Jebel Aulia 74 Figure (26): Anaphase of Alestes baremose in Jebel Aulia 75 Figure (27) : Anaphase of Alestes dentex in Lake Nubia 75 Figure (28): Late prophase of Alestes dentex in Jebel Aulia 76 Figure (29): Metaphase of Alestes dentex in Lake Nubia 76 Figure (30): Early prophase of Hydrocynus brevis in Lake Nubia 77 Figure (31): Anaphase of Alestes dentex in Jebel Aulia 78 Figure (32): Anaphase of Alestes baremose in Lake Nubia 78 Figure (33): Metaphase of Hydrocynus brevis in Lake Nubia 79 Figure (34): Metaphase of Hydrocynus brevis in Jebel Aulia 79 Figure (35): Prophase of Hydrocynus lineatus in Jebel Aulia 80 Figure (36): Metphase of Hydrocynus forskalii 80 Figure (37): Late prophase of Hydrocynus 81 Figure (38): Late Anaphase of Hydrocynus 81 Figure (39): Metaphase of Hydrocynus lineatus 82 Figure (40): Ideogram of karyotype of Hydrocynus 83 Figure (41): Ideogram of karyotype of Alestes 84 Figure (42): Electrophoresis band of the soluble liver protein of Hydrocynus and Alestes collected from Lake Nubia 85 Figure (43): Electrophoresis band of the soluble liver protein of Hydrocynus and Alestes collected from Jebel Aulia 85 Figure (44): Electrophoresis band of the soluble muscle tissues protein of Hydrocynus and Alestes collected from Jebel Aulia 86 Figure (45): Electrophoresis band of the soluble liver protein of Hydrocynus and Alestes collected from Lake Nubia 86 Figure (46): Electrophoresis band of the soluble muscle tissues protein of Hydrocynus and Alestes collected from Lake Nubia 87 Figure (47): Comparison between two different populations of Alestes dentex in Lake Nubia and Jebel Aulia 87 Figure (48): Comparison between two different populations of Alestes baremose in Lake Nubia and Jebel Aulia 88 Figure (49): Comparison between two different populations of Hydrocynus brevis in Lake Nubia and Jebel Aulia 89

ABSTRACT

Members from species of two genera of the Nile fishes Hydrocynus and Alestes were collected from Lake Nubia at Wadi Halfa and from Jebel Aulia at the White Nile. Collection was done during the period from May 2001 to March 2005. Eight species were studied for morphometric measurements and ratios, cytogenetic patterns and protein polymorphism. Morphometric measurements taken included the total length (L), standard length (SL), depth (D), interorbital width (IOW), eye diameter (ED), peduncle length (PL) and peduncle depth (PD). Measurements and ratios revealed significant differences between members of the species collected at the two freshwater systems. The cytogenetic study revealed a chromosome complement of 2n= 56 for Hydrocynus species members and 2n= 42 for Alestes species members. Chromosomes were found to vary in structure within the complement where, metacentric, telocentric and acrocentric chromosomes were recorded. Chromosomal fusions and polyploidy were also recorded, and suggestion of recent chromosomal rearrangements in members from the two geographic locations is proposed. Profiles of total protein were done using electrophoresis; genetic variation is detected at this level between taxa of the two water systems. The electrophoretically separated soluble protein bands showed presence of polymorphic alleles in members of the same species caught from Lake Nubia and the White Nile.

ﺨﻼﺼﺔ ﺍﻟﺒﺤﺙ

ﺘﻡ ﺠﻤﻊ ﻋﻴﻨﺎﺕ ﻤﻥ ﺃﺴﻤﺎﻙ ﺍﻟﻨﻴل ﺘﺘﺒﻊ ﻷﺠﻨﺎﺱ ﺍﻟﻜﺎﺱ Hydrocynus ﻭ ﺍﻟﻜﻭﺍﺭﺓ

Alestes ﻤﻥ ﺒﺤﻴﺭﺓ ﺍﻟﻨﻭﺒﺔ ﻋﻨﺩ ﻭﺍﺩﻱ ﺤﻠﻔﺎ ﻭ ﻤﻥ ﺍﻟﻨﻴل ﺍﻷﺒﻴﺽ ﻋﻨﺩ ﺠﺒل ﺃﻭﻟﻴﺎﺀ

ﻟﻐﺭﺽ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ، ﺠﻤﻌﺕ ﺍﻟﻌﻴﻨﺎﺕ ﻓﻲ ﺍﻟﻔﺘﺭﺓ ﻤﻥ ﻤﺎﻴﻭ 2001 ﻭ ﺤﺘﻰ ﻤﺎﺭﺱ 2005

ﻭ ﺃﺠﺭﻴﺕ ﺩﺭﺍﺴﺔ ﻋﻠﻰ ﺜﻤﺎﻨﻴﺔ ﺃﻨﻭﺍﻉ ﺒﺘﺤﺩﻴﺩ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﻅﻬﺭﻴﺔ ﻭ ﻨﺴﺒﻬﺎ ﻭ ﻨﻤﻁ

ﺍﻟﻌﺒﻐﻴﺎﺕ ﻭ ﺘﻌﺩﺩ ﺍﻟﻨﻤﻁ ﺍﻟﻅﺎﻫﺭﻱ ﻟﻠﺒﺭﻭﺘﻴﻨﺎﺕ.

ﺍﺸﺘﻤﻠﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﺍﻟﻤﻅﻬﺭﻴﺔ ﻋﻠﻰ ﻗﻴﺎﺱ ﺍﻟﻁﻭل ﺍﻟﻜﻠﻲ ﻭ ﺍﻟﻁﻭل ﺍﻟﻤﻌﻴﺎﺭﻱ ﻭ ﺍﻟﻌﻤﻕ

ﻭ ﻋﺭﺽ ﺍﻟﻤﺤﺠﺭ ﻭ ﻗﻁﺭ ﺍﻟﻌﻴﻥ ﻭ ﻁﻭل ﺍﻟﺴﻭﻴﻘﺔ ﻭ ﻋﻤﻕ ﺍﻟﺴﻭﻴﻘﺔ. ﺃﻅﻬﺭﺕ ﺍﻟﻘﻴﺎﺴﺎﺕ ﻭ

ﺍﻟﻨﺴﺏ ﻭﺠﻭﺩ ﻓﺭﻭﻗﺎﺕ ﻤﻌﻨﻭﻴﺔ ﺒﻴﻥ ﺍﻷﻓﺭﺍﺩ ﻤﻥ ﺍﻷﺘﻭﺍﻉ ﺍﻟﺘﻲ ﺠﻤﻌﺕ ﻤﻥ ﺍﻟﻤﻴﺎﻩ ﺍﻟﻌﺫﺒﺔ ﻓﻲ

ﻜل ﻤﻥ ﺒﺤﻴﺭﺓ ﺍﻟﻨﻭﺒﺔ ﻭ ﺍﻟﻨﻴل ﺍﻷﺒﻴﺽ.

ﺃﻭﻀﺤﺕ ﺩﺭﺍﺴﺔ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻭﺠﻭﺩ56 ﺯ ﻭ ﺠ ﺎﹰ ﻤﻥ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻓﻲ ﺍﻷﻨﻭﺍﻉ ﺍﻟﺘﻲ ﺘﺘﺒﻊ

ﻟﻠﺠﻨﺱ Hydrocynus ﺒﻴﻨﻤﺎ ﺍﺤﺘﻭﺕ ﺍﻷﻨﻭﺍﻉ ﺍﻟﺘﻲ ﺘﻨﺩﺭﺝ ﺘﺤﺕ ﺍﻟﺠﻨﺱ Alestes ﻋﻠﻰ

42 ﺯ ﻭ ﺠ ﺎﹰ ﻤﻥ ﺍﻟﺼﺒﻐﻴﺎﺕ. ﺃﻭﻀﺤﺕ ﺍﻟﺩﺭﺍﺴﺔ ﻜﺫﻟﻙ ﺃﻥ ﻫﻨﺎﻟﻙ ﺘﺒﺎﻴﻥ ﻓﻲ ﺸﻜل ﺍﻟﺼﺒﻐﻴﺎﺕ

ﻓﻲ ﻜل ﻤﻥ ﺍﻟﺠﻨﺴﻴﻥ ﻓﻘﺩ ﺴﺠﻠﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺼﺒﻐﻴﺎﺕ ﺫﺍﺕ ﻨﻤﻁ ﻭﺴﻁﻲ ﻭ ﻗﻤﻲ ﻭ ﻁﺭﻓﻲ ﻜﻤﺎ ﻟﻭﺤﻅﺕ ﺤﺎﻻﺕ ﻟﻺﻟﺘﺤﺎﻡ ﺍﻟﺼﺒﻐﻲ ﻭ ﺘﻌﺩﺩ ﺃﻁﻘﻡ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻤﻤﺎ ﻴﺸﻴﺭ ﺇﻟﻰ ﺍﻓﺘﺭﺍﺽ

ﺇﻋﺎﺩﺓ ﺘﺭﺘﻴﺏ ﺍﻟﺼﺒﻐﻴﺎﺕ ﻟﻸﺴﻤﺎﻙ ﻤﻥ ﺍﻟﻤﻨﻁﻘﺘﻴﻥ.

ﺃﻅﻬﺭ ﺘﺤﻠﻴل ﺍﻟﺒﺭﻭﺘﻴﻥ ﺒﺎﻟﺭﺤﻼﻥ ﺍﻟﻜﻬﺭﺒﺎﺌﻲ ﻭﺠﻭﺩ ﺘﺒﺎﻴﻥ ﻓﻲ ﺍﻟﻤﺤﺘﻭﻯ ﺍﻟﺒﺭﻭﺘﻴﻨﻲ

ﻟﻸﻨﻭﺍﻉ ﻓﻲ ﺍﻟﻤﻨﻁﻘﺘﻴﻥ ﻜﻤﺎ ﺃﻭﻀﺤﺕ ﺤﺯﻡ ﺍﻟﺒﺭﻭﺘﻴﻨﺎﺕ ﺍﻟﺘﻲ ﻓﺼﻠﺕ ﻭﺠﻭﺩ ﺃﻨﻤﺎﻁ ﻤﺘﻌﺩﺩﺓ

ﻟﻠﺒﺩﻴل ﺍﻟﺠﻴﻨﻲ (ﻷﻟﻴل) ﺍﻟﻭﺍﺤﺩ ﻓﻲ ﺍﻷﻓﺭﺍﺩ ﻤﻥ ﻨﻔﺱ ﺍﻟﻨﻭﻉ ﻓﻲ ﺒﺤﻴﺭﺓ ﺍﻟﻨﻭﺒﺔ ﻭ ﺍﻟﻨﻴل

ﺍﻷﺒﻴﺽ. CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 GENERAL INTRODUCTION:

Fish present an important source of animal protein for a big sector of the world population (about 12% of the world animal protein) especially in the less developed countries (FAO, 1974). Studies on fish have started as early as the turn of the twentieth century. Investigations by then were mainly focused on systematics, general biology and distribution but very little biochemical or genetic studies were done. Recently, studies of the chromosomal, protein and DNA levels permitted better analysis of fish species and opened horizons to perform studies resulting in genetically improved fish material. Identification of the chromosome number and structure of fish helps to reduce or eliminate fish inherited diseases and enables scientists to make new hybrids with improved properties, and of course better economical value.

1.2. FISHES OF THE NILE The river Nile and its tributaries comprise the largest freshwater system in the globe. Diversified in its geography and habitat the Nile system was and still is the major water body that gained attention and deserved research. As early as the Nile voyages and adventures, the flora and fauna were recorded from inside the waters as well as from the surrounding shores. Being of economical, sentimental and worship values, fishes represented the centre of attention of researchers, tourists and natives. The Nile waters crossing the Sudan reflect the richness and variation of the system. Compared to the fast pace of man-made “developmental schemes”: dams, irrigation canals, agricultural schemes and human resettlements etc, studies are lagging behind. Fishes of the Nile in Sudan are represented by 17 native families: Alestiidae, Amphiliidae, Anabantidae, Aplocheliidae, Bagridae, Cichelidae, Citharinidae, Clariidae, Cyprinodontidae, Kneriidae, Mochokidae, Megalopidae, Mormyridae, Notopteridae, Scilbeidae and Sporidae. Four family members are introduced, they are Cprinidae of which the common carp was introduced, the family Ctenopharagodon represented by the grass carp, the family Poeciidae represented by the gambusia and the family Salmonidae represented by the rainbow trout. The families are represented by more than 200 fish species. Most of the work done to classify fish and to differentiate between the different genera was -almost- morphological and only little comparative biochemical or genetical studies have been done on those Nile fish (Babiker and Elhakeem, 1979). Boulenger 1907, Sandon (1950), Amirthalingam (1965) researched the Nile fishes and produced illustrated guides to their morphological identification. The comprehensive work on Nile fishes by Abu Gideiri (1984) presents the major reference for identification of fishes in Sudan until now.

1.2.1 Lake Nubia There is a lack of data on the important aspects of fishes and fisheries of Lake Nubia (Ali, 1980). Literature available deals with biology of fish communities (Fisheries Research Centre Annual Scientific Reports, 1967, 1968, 1979); George, 1971; Abu Gideiri and Ali, 1975), fish populations and fishing gears (Ali, 1977; Ali 1980) and fisheries management and development aspects (FAO,1974; Tanmiah,1977; Ali 1980). Hydrocynus and Alestes genera are very important fish in Lake Nubia with good processing qualities (salted fish or faseekh) that give these fish high value in the fish market. According to Ali (1999) the species members of the family Characinidae collected from Lake Nubia in the period 1967 to 1979 are Alestes dentex, Alestes baremose, Alestes nurse and Hydrocynus forskalii.

1.3. TAXONOMIC RELATIONSHIPS OF Alestes sp. AND Hydrocynus sp.:

The order Cypriniformes includes fishes that are chiefly freshwater representing two major groups which have common a Weberian apparatus, a bony structure which connects the ear with the swimbladder. The order is represented by three families: Characinidae, Citharinidae and Ichthyoboridae. Formerly the three families were grouped together as the Characinidae (Abu Gideiri 1984). All three families agree in having a scale-less head, no barbells and an adipose fin. The mouth is never protractile, but the three differ mainly in the position of the lateral line and in the nature of their teeth. The non- protractile mouth is usually bordered by the premaxillaries and maxillaries, rarely by the premaxillaries only. Pharyngeal bones normal, with small teeth. Branchiostegal rays 3-5, pectoral fin inserted very low down, folding like the ventrals, which are formed of 10-13 rays. An adipose dorsal fin is often present. The family is formerly represented by 3 genera: Alestes, Hydrocyon and Micralestes (Abu Gideiri 1984). The various taxonomic revisions applied to the family Characinidae and its relatives have provided neither a comprehensive treatment of the group nor a compelling phylogenetic relationship to account for the current variation between its members. Today members of the former Charicinidae are grouped under the family Alestiidae (formerly a sub family of Characinidae, Buckup, 1993). The family Alestiidae includes all African tetras with 18 genera and 109 species. Some members of this benthopelagic family are used in the aquarium trade. Reproductively, most members of this family are non-guarders may be found from 31° E to 26° S and 18° W to 46° E. Members of the family Alestiidae are a major component of freshwater fish fauna of the Nile system with respect to the number both of individuals and of species. The role of this family within the Nile ecosystems is therefore central. They have considerable morphological variability which is likely related to their highly diversified habitat. The relationship between this variability and the phylogeny of the group is an open, interesting question, relevant for the study of adaptive traits. A well supported phylogeny is required to address the question of hybridization: interspecific and even intergenic hybrids are thought to be common between Characids, and their taxonomic meaning is worth investigating.

1.3.1 The Genus Hydrocynus: Hydrocynus (Fish dogs or tiger fish) are strictly ichthyophagous known for their promptness and their voracity. All the fish belonging to this genus appreciably look the same and only an attentive examination makes it possible to differentiate them. They have a torpedo form, which they use to their advantage of nourishing themselves, since they continuously chase their prey. The fish are generally silver-plated and brilliant. The scales are marked with a dark spot, thus forming especially visible parallel stripes above the side line. According to certain species, these lines are more or less dark. The eye is almost entirely covered with a fatty eyelid. Members of this genus can further be distinguished by the teeth which are extremely strong. Each consisting of a single long, sharp flange arranged in a single row at the edge of each jaw. The mouth is larger and the snout rather longer than in other members of the family. The dorsal fin is inserted into the same level as the ventral fins or a little ahead. In all species the dorsal fin has 10-11 rays, the anal fin 13- 18, the pectoral fin 15-16 and the pelvic fin 10. The paired fins are more or less falcate (pointed and slightly curved), the lateral line nearer to the ventral than the dorsal outline. Gill membranes free from isthmus; a scaly process of the base of the ventral fin. Species representing the genus are: Hydrocynus brevis (Günther 1864), Hydrocynus forskahlii, (Cuvier 1819), Hydrocynus goliath (Boulenger 1898), Hydrocynus lineatus (Bleeker) Hydrocynus vittatus (Castelnau 1861) and Hydrocynus tanzaniae (Brewster 1986).

1.3.1.1 Hydrocynus forskalii: This fish inhabits the main Nile as well as both Blue and White Niles. It is recorded to grow up to one meter. Depth of body into length more than 4.5; length of head into length of body 4-5; caudal peduncle depth into length not less than 1.7; first ray of dorsal fin situated distinctly in advance of that of pelvic fin; scales on lateral line 48-54 with7.5-8.5 above and 4.5-5.5 below; scales between lateral line and scaly process at base of pelvic fin. Back grey, brown or olive-green, sides and belly silvery white; more or less distinct longitudinal rows of blackish spots, often confluent into streaks, run along the series of scales above the lateral line; these streaks and spots are entirely absent in very young specimens; dorsal fin and upper lobe of caudal yellowish or grey, often blackish towards the end; adipose fin grey. Or whitish, rarely with a black spot; ventral and anal fins, sometimes pectorals, tinted with pink or pale orange; lower lobe of caudal bright red (Abu Gideiri 1984).

1.3.1.2 Hydrocynus brevis (Gunther): Tiger-fish Kalb el bahr: Inhabits the main Nile and White Nile, grows up to 50 cm. Depth of body into length less than 4.5; length of head into length of body 3- 4; caudal peduncle depth into length not more than 1.7; first ray of dorsal fin situated directly above that of pelvic fin; scales on lateral line 47-54 with 8.5-9.5 above and 6.5 below; scales between lateral line and scaly process at the base of pelvic fin 3. Colouration very similar to that of H.forskalii; longitudinal dark streaks or series of spots, more or less distinct in adult specimens, usually confined to the region above the lateral line; a black spot sometimes present on adipose fin (Abu Gideiri 1984).

1.3.1.3 Hydrocynus lineatus (Bleeker): Inhabits the Blue and White Niles as well as Lake Nubia and grows up to 45 cm. Depth of body into length less than 4.5; length of head into length of body 3-4; caudal peduncle depth into length not more than 1.7; first ray of dorsal fin situated above or slightly in advance of pelvic fin; scales on lateral line 44-48 with 7.5-8.5 above and 4.5-5.5 below; scales between lateral line and scaly process at the base of pelvic fin 2. Black longitudinal streaks more strongly marked and extending to the series of scales below the lateral line; adipose dorsal fin with a deep black spot (Abu Gideiri 1984).

1.3.2 The Genus Alestes: The genus Alestes is a major representative of the family Alestiidae in the Nile system. Species worldwide in the genus Alestes can be summarized as follows: Alestes, A. affinis (Red fin Robber), A. ansorgii , A. baremose (Characin), A. bartoni, A. batesii, A. bimaculatu, A. bouboni, A. carmesinus, A. comptus, A. dentex (Characin), A. grandisquamis (Pink fin Alestes), A. humili, A. jacksonii (Victoria Robber), A. liebrechtsii, A. macrophthalmus (Torpedo Robber), A. peringuey, A. rhodopleura, A. schoutedeni, A. stuhlmannii , A. taeniuru, A. tessmann, and A. tholloni (Bailey, 1994). Members of the genus Alestes are smaller than those of Hydrocynus in general appearance. Although carnivorous they feed on insects rather than on other fish. Correlated with this is the structure and arrangement of the teeth which are much smaller than in Hydrocyon spp. and less pointed, mostly having more than one cusp and set in two rows along each jaw (Abu Gideiri 1984).

1.3.2.1 Alestes dentex (Characin /Kawara baladi): The species inhabits the main Nile and White Nile it grows up to 40 cm. First ray of dorsal fin situated just behind last ray of pelvic fin; anal fin rather long with 22 rays and body in this region tapers gradually; scales on lateral line 39-50 with 6.5-9.5 above and 3.5 below. The body colour is silvery, back dark grey or dark green; a more pr less distinct darker band may extend along each side of the back; lower lobe of caudal fin being bright red (Abu Gideiri 1984).

1.3.2.2 Alestes baremose (Joannis): Members of the species Alestes baremose or silversides is given the local name "kawara baladi". The fish inhabits the main Nile, Blue and White Niles, it grows up to 31 cm. First ray of dorsal fin situated equidistant from last ray of pelvic fin and first ray of anal fin; and fin with 25-30 rays; 30-38 gill rakers on lower part of first gill arch; scales on lateral line 44-51 with 8.5-9.5 above and 3.5 below. Colouration is the same as in Alestes dentex (Abu Gideiti, 1984).

1.3.2.3 Alestes nurse (Ruppell): This species inhabits the main Nile, Blue and White Niles. The smallest of the species, the fish has a local name ''Kawara, Hemaila". First ray of dorsal fin situated above root of pelvic fin or nearer to occipit than to caudal; anal fin rather short with 14-19 rays, and body in this region tapers rather abruptly; 16-20 gill rakers on lower part of first anterior gill arch; scales on lateral line 26-33 with 4.5-6.5above and 2.5-3.5 below. Silvery, dark grey or brown on back; a blackish spot above lateral line behind gill opening, and another on caudal peduncle (Sandon, 1950; Abu Gidieri, 1984).

1.3.2.4 Alestes macrolepidotus (Cuv. and Val.) Local name "Kawara safsaf" The fish inhabits the main Nile, the Blue and the White Nile and grows up to 42 cm. First ray of dorsal fin situated midway between pelvic and anal fins and nearer to caudal; anal rather short with 14-19 rays and body in this region tapers rather abruptly; 18-22 gill rakers on lower part of anterior gill arch; scales on lateral line 22-26 with 4.5-6.5 above and 2.5-3.5 below. Brownish above, the scales with a darker edge, silvery-white or pinkish beneath and on cheeks; a pinkish band often extends along the side, from the cheek to above the anal fin; fins pink or orange, the caudal fin is often edged with grey and blackish colours (Abu Gideiri 1984).

1.4. CYTOGENETIC STUDIES 1.4.1. 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 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 chromosome number is constant from individual to individual within a given species, with very rare exceptions; for 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, 1959). If it is difficult to identify the chromosomes according to the size alone, then, at least the chromosomes may be grouped according to their size similarity. The centromere is the point, on the chromosome, of attachment of the spindle fibers during cell division. According to the position of the centromere, the chromosomes may be classified into: (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: 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, 1986; Franthworth, 1988). Chromosome analysis is used to provide information about the genetical makeup of fishes and to identify any disorder which results from numerical abnormalities, chromosome polysomy, monosomy, mosaicism, polyploidy; and also from chromosomal changes such as structural abnormalities, translocations, deletions, duplications and inversions (Swanson, 1957; Barker, 1970; Avise et al, 1975; Al-Sabti, 1985; Suzuki, 1986).

1.4.2 Fish Karyotypes:- 1.4.2.1. The Techniques: Studies of the karyotype of fishes and their chromosome number were not as successful and common as those of other vertebrate groups; for only about 10% or even less of the more than 20,000 known species of fishes, have their karyotypes 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 usefulness of the resulting metaphase spreads and hence discourages karyotyping studies. Most of the methodologies used for preparation of fish chromosomes were, initially, borrowed from human and other vertebrate techniques. The commonly applied technique was that of the squash preparations from kidney cells or other organs (Ojima et al; 1963; Roberts, 1967; AL-Sabti et al; 1983). But this method has a great disadvantage: for the animal tested must be killed; but even so the mitotic index and the quality of preparations are generally inferior than what is expected (Blaxhall, 1975). Since 1970 onwards, many new improved techniques have been developed for fish chromosome preparations (Ojima et al; 1970; Barker, 1972; Summer, 1977; Amemiya et al, 1984; Rivilin at al; 1985; Reddy and John, 1986). These include blood leukocyte culture (Barker, 1972; Gold, 1974; Gold, 1979; Blaxhall, 1975, 1983; Hartley and Horne, 1983; AL-Sabti, 1985; Hartley and Horne, 1985; La Grande, 1989; Gold et al, 1990) and it is 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; Hartley, 1983; Gold et al, 1990). Although the techniques showed 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 are unclear (Hartley and Horne, 1983). Moreover, the technique required high quality serum. Most of the techniques are based on the use of colchicine to block the quickly proliferating organs at the 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; Steward and Levin, 1968; Klingerman and Bloom, 1977; Hartley and Horne, 1983; Rivilin et al., 1985). The best tissues for obtaining dividing cells include: the kidney, 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). Fish chromosomes vary greatly in their numbers. Generally, the chromosome number of fishes is found to be in the range of the low 2n=16 in Notabranchus (Post, 1965) to highest 2n=146 in Icthyomycon (Howell and Duckett, 1971).

1.4.2.2. Examples of Applications: Recently, some studies of Nile fish karyotypes have been carried out. Most studies are related to the Tilapia species. Of the 2400 Siluroidean species, which are distributed in all continents only about 81 species have their karyotypes been investigated, but none of which came from the Africa continent. Only when, in 1990, Agnese et al. carried out a study to investigate the karyotype of some African catfishes of the family Mochocidae that African representatives are used. The study was performed on a number of Synodontis spp; namely: Synodontis membranaceus, S. Bastiani, S.budgetti, S. courteti, S. filamentosus, S. ocellifer, S. schall, S. sorex and S. violaceus. The fish samples were collected from the River Niger at Bamako (Mali), the River Baoule at Dlaba (Mali) and the River Leraba (Ivory Coast).

1.5 PROTEIN STUDIES:- Genes located on the chromosome are expressed in the form of proteins. About 30-50% of the proteins coded by genes, among the majority of animal and plant populations proved to be polymorphic; whereas, the heterozygosity (relative number of loci in a given individual in the heterozygous state) was found to be in the range of 7- 15% (Lewontin and Hubby, 1966; Kirpichnikov, 1973; Kirpichnikov, 1981). One of the powerful tools for making informative comparisons between individuals and hence identification of taxa is the use of protein polymorphism. Protein polymorphism is the result of the slight structural differences of genetic material in the two homologous allelic genes which code for one protein. These differences in the gene action reduce different phenotypes of that certain protein (Levitan and Montagu, 1971). This can be used to detect differences between individuals of the same species. It is well documented that the main cause of protein polymorphism is the necessity to adapt to even altering environmental conditions. In the past, electrophoretically-detected enzyme polymorphism has been used extensively to study both the extent and patterns of genetic variation in the natural populations (reviewed by Lewontin, 1974). The development of modern biochemical techniques, particularly gel electrophoresis, has enabled the genetic diversity, population structuring and evolutionary relationship of many fishes to be directly appraised (Ferguson, 1980; Allendorf and Utter, 1987).

Species of fishes, like most other aquatic or terrestrial organisms, do not exist as one continuous or homogeneous population. 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; Allendorf and Phelps, 1981) 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; Mohammed, 1990; El-Fadil, 1999). So 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). As a matter of fact Russo et al (1996) found that nucleotide sequences (DNA) are less appropriate than amino acid (protein) sequences for constructing reliable trees for distantly related organisms. A population of highly heterozygous individuals would be more viable than a population of less heterozygous individuals, because individuals of the former population would have higher growth rates, fecundity, viability, developmental stability, resistance to environmental stresses and more survival rates than the individuals of the later population (Mitton and Grant, 1984; Allendrof and Leary, 1986; Zouros and Foltz, 1987; Quattro and Vrijenhoek, 1989; Leberg, 1990), and so polymorphism is believed to result in increased fitness. The most commonly used soluble proteins from the fish liver are the esterases (Kitamikado and Tachino, 1961 c), albumins and amylases. Most of the protein information, however, is from fish serum proteins. During a study to compare the morphological and biochemical features of two catfishes, from two different locations: Lake Nubia and the White Nile Mohammed (1990) found that the gene loci for the proteins: haemoglobin (Hb), glucose-6-phosphate dehydrogenase-1 (G6pd-1), 6-phosphogluconate dehydrogenase (6Pgd), malate dehydrogenase-1 (Mdh-1), phosphoglucomutase-1 (Pgm-1), glucose- phosphate isomerase-2 (Gpi-2), superoxide dismutase (Sod), catalase (Cat), glyceraldehydes-3-phosphate dehydrogenase (G3pd), erythrocytes esterase-II (EsII) and serum esterase-I (EsI), were polymorphic. The degree of polymorphism, in natural populations of fishes, was found to be 19% and its mean heterozygosity level is up to 65% (Altukhov et al; 1972; Avise et al; 1975).

1.6. OBJECTIVES OF THE STUDY:

This study is an attempt to determine the morphometric traits, the cytogenetic patterns and the genetical variation at the protein level of members from two fish genera Hydrocynus and Alestes from two different localities in the Nile system. The work aims to: 1. Study the morphological traits of species from the two genera 2. Provide information on the chromosome complements of species of Hydrocynus and Alestes 3. Investigate the karyotypic diversity within the family Alestiidae 4. Identify chromosomal peculiarities of the family such as polyploids and polymorphisms 5. Detect soluble protein polymorphism in the different species.

CHAPTER TWO

MATERIALS AND METHODS

2.1. SAMPLE COLLECTION The fish samples were collected from Lake Nubia (at Wadi Halfa) and from the White Nile (at the Fish Research Station, Jebel Aulia). Members of the genera from Lake Nubia are collected from Khor Musa Basha, Khor Grab and Khor Arab in Wadi Halfa. Figure (1) illustrates the collection sites. The collection was performed during the period from May 2001 to March 2005. Fifty samples from each species were collected at early morning using gill nets. The nets used in the study had a larger mesh size (3-4 cm) in Lake Nubia while those used in Jebel Aulia had a smaller mesh size (of 1-2 cm). The samples were kept in a tank, supplied with water and an aerator, in the laboratory till the time of use.

2.2. MORPHOMETRIC MEASUREMENTS. Measurements and ratios relating to various external features were performed for the identification of Hydracynus and Alestes fish species. These ratios are particularly characteristic to fish species. The measurements and ratios were taken according to the studies of Bishai and Abu Gideiri (1967), Boulenger (1905), Stubbs (1949) and Sandon (1950). The 50 fishes that were measured were put on a wooden board, and the measurements were performed using a vernier (of 0.01 cm accuracy). The measurements and ratios included were:- Total length (L): The distance from the tip of the snout to the tip of the dorsal lobe of the caudal fin. Depth (D): The greatest vertical measurement starting a few millimeters from the origin of the dorsal spine ventral ward. Standard length (SL): The distance from the tip of the snout to the origin of the caudal fin. Interorbital width (IOW): The distance between the two orbits. Diameter of eye (ED): The outward measurement from the inner rim. Peduncle length (PL): The distance from the origin of the anal fin to the point where the lateral line meets the caudal fin Peduncle depth (PD): The narrowest part posterior to the position of the anal fin and anterior to the caudal fin

2.3. STAFISTICAL ANALYSIS. The Statistical Package for Social Science (SPSS) was used to perform the one way analysis of variance (ANOVA) and the regression analysis for morphometric measurements. The ratios L/D, SL/D, I O W /E D, PL /PD were used.

Figure (1): Map of Lake Nubia showing stations of sampling sites. 2.3. KARYOTYPING:- A modification of the karyotyping techniques of Hitotsumachi et al. (1969), Barker (1970) and Cucchi and Baruffaldi (1990) was applied. The fish to be used was injected with 0.01ml of the 0.7% colchicine for every 1 gm of its body weight. After 3 hours the fish was dissected, and the kidney, liver and spleen were removed and each organ was squashed vigorously in a mortar with a glass rod to break the cell clumps. Then the tissues were transferred to labeled centrifuge tubes, and 5.0 ml of 0.5% KCl hypotonic solution was added to each tube. The contents of the tubes were mixed and the resultant cell suspension was set for 20 minutes. The tubes were then centrifuged at 2000 r.p.m. for 4 minutes, and the supernatant fluid was discarded carefully, leaving only the settled undisturbed cells at the bottom of the tubes. After that, 5.0 ml of freshly prepared Carnoy's fixative (3 parts absolute methanol: 1 part glacial acetic acid) was added and the settled cells were mixed gently until the cell suspension was homogeneous. The cells were allowed to fix for 10 minutes. The cells were then centrifuged for 4 minutes at 2000 r.p.m. and the supernatant fluid was decanted. The cells were re-suspended in 3.0 ml of fixative and centrifuged as before. The last step was repeated three times. After the removal of the supernatant fluid in the last round, the cells were suspended in 1.0 ml of the Carnoey’s fixative. During fixation of the cells, clean slides were washed by methanol and placed on a slide rack and transferred to the freezer (chilled). At the time of use, the slides were removed from the freezer; three to four drops of the cell suspension were dropped from a high distance onto the slides. The slides were stained using Giemsa stain (1 part Giemsa stock solution: 1 part distilled water) for 20 minutes. The slides were made permanent by passing them through a bath of Xylol for 3-4 minutes. The slides were then dried and mounted with cover slips; at that point, the slides were ready for examination under the microscope. Slides with well spread metaphase plates from the kidney were photographed using a Wild MP511 camera attached to a Leitz Dialux 20 phase-contrast microscope. Chromosome counts were made from slides directly and verified from photographs.

2.4. PROTEIN ELECTROPHORESIS:- Total protein was separated by Sodium Dodecyl Sulphate (SDS) using Polyacrylamide Gel Electrophoresis (PAGE) following a method modified after Sambrook et al. (1989). Detailed steps of protein electrophoresis used were summarized below.

2.4.1. Preparation of the Reagents of SDS-polyacrylamide Gel:

2.4.1.1. Preparation of 30% Acrylamide Stock Solution:- A stock solution containing 29% (w/v) acrylamide and 1% (w/v) N, N'-methylenebisacrylamide was prepared by dissolving 28.5gm acryl amide and 0.74gm bisacrylamide in 100 ml of distilled water. The pH of the solution was adjusted to 7.0 and the solution was stored in a dark bottle, coated by aluminum foil, in a refrigerator.

2.4.1.2. Preparation of Sodium Dodecyl Sulphate (SDS):- A 10% stock solution of SDS was prepared by dissolving 10 gm of SDS in 100 ml of distilled water. Then the stock solution was stored at room temperature.

2.4.1.3. Tris Buffers for the Preparation of Resolving and Stacking Gels:-

A- Resolving Gel Buffer:- A stock solution was prepared by dissolving 18.15 gm Tris OH (Tris base) in 80 ml distilled water. Then the volume was completed to 100 ml by adding 20 ml distilled water and pH was adjusted to 8.8. B -Stacking Gel Buffer: A stock solution was prepared by dissolving 3 gm Tris HCI (Tris acid) in 40 ml distilled water. Then the volume was completed to 50 ml and the pH was adjusted to 6.8. C- Running Buffer:- A stock solution was prepared by dissolving 3.02 gm OH, 14.4 gm glycine and 1 gm SDS in 500 ml of distilled water. Then the volume was completed to 800 ml pH was adjusted to 8.3. The stock solutions were kept in a refrigerator till usage.

3.4.1.4. Preparation of the Resolving Gel:- A 10% resolving gel was prepared by mixing: 3.1 acrylamide, 4.2ml distilled water, 2.5ml Resolving buffer, 100 µl of 10% TEMED (N-N-N-N tetra-methyl-ethylediamine).

2.4.1.5. Preparation of the Stacking Gel:- A four percent (4%) stacking gel was prepared by mixing:1.3 ml acrylamide, 6.0 ml Distilled water, 2.5 ml Stacking buffer, 100 ml of 10% SDS, 100 ml Ammonium persulphate solution and 10 ml TEMED.

2.4.1.6. Preparation of the 2x Loading Buffer:- A 2x loading buffer solution was prepared by mixing: 4 ml distilled water, 1 ml of 0.5 M Stacking buffer, 1.6 ml of 10% SDS, 0.8 ml Glycerol, 1.4 ml 2-mercaptoethanol and 0.2 ml of 0.05% bromo- phenol blue.

2.4.1.7. Preparation of the Staining Solution:- The staining solution was prepared by dissolving 0.2 g of coomassie brilliant stain in 62.5 ml distilled water and then adding 28.5 ml methanol and 5.75 ml glacial acetic. The staining solution was stirred and used instantly.

2.4.1.8. Preparation of the De-staining Solution:- The de-staining solution was prepared by mixing 10 ml of glacial acetic acid with 50 methanol and 100 ml distilled water.

2.4.2. Pouring SDS-Polyacrylamide Gels:- The glass plates of a mini-vertical electrophoresis apparatus were washed thoroughly with water and dried well before pouring the gel. The glass plates were assembled together, a short one and along one. Two spacers were inserted between the two plates, at each side, to leave space for pouring the gel and the two plates were held together by clips. The lower part of the assembled plates was dipped in a heated 2% agarose solution. The agarose the lower end of the gel was left to dry. The resolving gel was poured into the gap between the glass plates using an electric micropipette. Sufficient space was left to hold the stacking gel (the length of the teeth of the comb plus 1 cm). The resolving gel was overlaid with methyl alcohol to level the gel. The resolving gel was left for 30 minutes to polymerize, and then kept in the refrigerator overnight. After polymerization was complete, the overlay of methyl alcohol was poured off and the top of the gel was washed several times with distilled water to remove any un-polymerized acrylamide. The water was drained off completely and any remaining of it was removed by applying a paper towel to the top of the gel. Then, the stacking gel was poured directly on top of the polymerized resolving gel. Immediately, a clean Teflon comb was inserted carefully into the stacking gel solution, avoiding trapping air bubbles. More stacking gel was added to fill the spaces of the comb completely. The gel was then placed in a vertical position at room temperature to polymerize.

2.4.3. Preparation of the Samples:- Fish collected for protein assays were not treated with colchicine, but sacrificed freshly. Liver tissues were removed and squashed in a glass mortar with a small piece of ice, then the cell suspension was centrifuged and the supernatant containing the dissolved proteins was transferred to a labeled test tube and kept in the freezer at-20°C till the time of use. While the stacking gel was polymerizing, the sample were prepared for electrophoresis by mixing 20 µl of the sample with 10 µl of 2x SDS gel loading buffer in an eppendorf tube in an auto vortex mixer. Then the mixture was heated to 100 °C for 3 minutes to denature the proteins. After the polymerization of the stacking gel was complete (after about one hour), the Teflon comb was removed carefully. The gel was then mounted in the electrophoresis apparatus. The running buffer (Tris-glycine electrophoresis buffer) was added to the top and bottom reservoirs. A Gilson micropipette was used to load 20 µl of every prepared sample in a pre-determined order into the bottom of the gel wells. The Gilson micropipette was washed with running buffer after the loading of each sample. A protein marker of known molecular weight was added in a separate well. The electrophoresis apparatus was then attached to an electric power supply and a voltage of 80 V/cm was applied to the gel. After the dye front had moved into the resolving gel, the voltage was raised to 100 v/cm. The gel was run until the bromo-phenol blue reached the bottom of the resolving gel (about two and half-hours). Then the power supply was turned off. The glass plates were removed from the electrophoresis apparatus and were placed on a paper towel. The plates were pried apart carefully. The regions of the stacking gel (the wells at the top of the gel) and the agarose (at the bottom of the gel) were cut off by a blade. The orientation of the gel was marked by cutting the top corner of the gel near the most left well. The gel was then immersed in the coomassie brilliant blue stain solution and left to be stained, overnight, in a slowly rotating platform. The staining solution was removed and stored for future use. The gel was then immersed in the de-staining solution. The de-staining solution was changed 3-4 times until clear protein bands were seen on the gel. Desired gels with distinct protein bands were photographed. Molecular weights of the bands were estimated using the protein marker.

2.5. STATISTICAL ANALYSIS. The Statistical Package for Social Science (SPSS) was used to perform the one way analysis of variance (ANOVA) and the regression analysis for morphometric measurements and an excel program was used for analysis of protein electrophoresis.

CHAPTER THREE

RESULTS 3.1 THE SPECIES: The fish samples collected from Lake Nubia and The White Nile included four species of Hydrocynus and four species of Alestes. Following the keys of Sandon (1950) and Abu Gideiri (1984), specimens were identified as:

Scientific Name Common Name Reference Genus Alestes: Pebbly fish Alestes dentex Characin/ Kawwara Linnaeous baladi Alestes baremose Silversides/ Kawara Joannis 1835 baladi Alestes nurse Kawara Hemaila Ruppell Alestes macrolepidotus Kawara safsaf Cuvier and Val. Genus Hydrocynus: Tiger fish/fish dogs Hydrocynus brevis Tiger-fish/ Kalb el bahr Günther 1864 Hydrocynus forskahlii Tiger fish/kass Cuvier 1819 Hydrocycon lineatus Tiger fish/kass Bleeker Hydrocynus vittatus Tiger fish/Kass Castelnau 1861

Alestes macrolepidotus was not found among Lake Nubia catch, however, a single specimen was found among fishes collected from the White Nile at the Fisheries Research Station area in Jebel Aulia.

Figure (2): Photograph of Alestes spp. showing measurements taken: L= length; D= Depth; SL= PD= Peduncle Depth; PL= peduncle length

Figure (3): Hydrocynus forskalii

Figure (4): Hydrocynus lineatus Figure (5): Hydrocynus brevis

Figure (6): Alestes baremose

Figure (7): Alestes dentex

Figure (8): Alestes nurse

Figure (9): Alestes macrolpetouts

3.2 MORPHOMETRIC MEASUREMENTS:- Morphometric measurements and ratios are shown in Tables I through X for members of the two genera Alestes sp. and hydrocynus sp. from the White Nile (Jebel Aulia) and Lake Nubia; samples 1-25 are from Lake Nubia and samples 26-30 represent the White Nile (Jebel Aulia) catch in all tables. The range of total length for Alestes baremose was 20-29 cm in Lake Nubia and 19-35 in Jebel Aulia, while that for A. dentex was 20-37 cm in Lake Nubia and 19-25.5 in Jebel Aulia. Alestes nurse showed large fish sizes in Lake Nubia (20.5-49 cm) and small fishes from the White Nile (6.8-13.1) while the single specimen of Alestes macrolepidotus from Jebel Aulia recorded a total length of 38 cm. The range of the total length for Hydrocynus lineatus was 20.5-54 in Lake Nubia and 20-34 in the White Nile; that for Hydrocynus breviswas 21.5-41 in Lake Nubia and 16-49 cm in Jebel Aulia. Hydrocynus forskalii showed a range of 15-45 in Lake Nubia and 20-54 in Jebel Aulia. Of the 301 fishes measured, the largest specimens obtained were a specimen of Hydrocynus lineatus from Lake Nubia that measured 54cm long (Table VIII, sample 17) and a specimen of H. forskalii from Jebel Aulia (table XIII, sample 39). The smallest specimen of all was an Alestes nurse caught from Jebel Aulia. The ratios of the different measurements obtained were:-

Alestes baremose (Tables I and II): L/D = 5.6646 ± 1.202459; IOW/ED = 3.3384 ± 5.54531 and PL/PD = 1.0428 ± 0.412652. Alestes dentex (Tables III and IV): L/D = 6.0098 ± 3.076169; IOW/ED = 2.939 ± 0.638424 and PL/PD = 0.6838 ± 0.301884. Alestes nurse (Tables V and VI): L/D = 4.947 ± 1.79998; IOW/ED = 3.348 ± 1.614097 and PL/PD = 1.459 ± 0.438156. Hydrocynus lineatus (Tables VIII and IX): L/D = 6.6218 ± 1.583619; IOW/ED = 2.8666 ± 0.730517 and PL/PD = 1.0739 ± 0.178941. Hydrocynus brevis (Tables X and XI): L/D =6.2004 ± 1.073686; IOW/ED = 3.587 ± 0.518884; PL/PD = 1.356 ± 0.151929. Hydrocynus forskalii (TablesXII and XIII): L/D = 6.4474 ± 1.382907; IOW/ED = 3.7968 ± 1.028011 and PL/PD = 1.5384 ± 0.563507.

Measurements and ratios of the single sample of Alestes macrolepidotus is shown in Table VII.

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 22 5 17.3 4..43.9 1.5 1 1.5 2.1 2.5 .84 2 24 4.8 19 5 3.8 2.8 1.2 2.3 2.3 2.3 1.0 3 20 4.7 16.6 4.3 3.8 1.1 1 1.1 1.2 2.1 .57 4 20 5.3 15 3.8 3.9 1.1 1 1.1 2.2 2.3 .95 5 22 5.79 17 3.8 4.5 1.2 1 1.2 2.1 3 .7 6 22 4.9 19 4.5 4.2 2.1 1 2.1 2 2.5 .8 7 22 4.4 18 6 3.6 2.3 1 2.3 2.4 3.2 .75 8 24 5.3 16 4.5 3.6 2.4 1 2.4 2 2.4 .83 9 20 4 18 5 3.6 1.5 1 1.5 2.2 2.5 .88 10 23 6.1 18 3.8 4.7 2.3 1.9 1.2 2.5 3.2 .78 11 24 5 16 4.8 3.3 2.6 1 2.6 2.1 2.4 .87 12 21.5 6.8 16 3.2 4.7 1.5 1 1.5 2.2 2.5 .88 13 20 5 17 4 4.3 1.6 1 1.6 2.1 2.5 .84 14 22 5.7 15 8.8 3.8 1.3 1 1.3 3.1 3.1 1.0 15 25.5 6.5 16 3.9 4.1 1.3 1.9 .7 2.1 2.4 .87 16 21 4.4 18 4.8 3.8 1.3 1 1.3 2.3 2.1 1.09 17 23 5.8 16 4 4 1.2 .9 1.3 2 3.1 .64 18 21 6 15 3.5 4.3 1.5 1 1.5 2.1 2.2 .95 19 20 5.8 18 4.5 4 1.5 .9 1.7 2.4 2 1.2 20 23 6.1 15 3.8 3.9 2.1 1 2.1 2 2.3 .86 21 20 5.4 14.5 3.7 3.9 1.7 1.1 1.5 1.8 3.1 .58 22 19 4.8 17 4 4.3 2.2 1.2 1.8 2.1 3.3 .63 23 22 5.5 20 4 5 2.3 1 2.3 3.1 2.3 1.35 24 25 6.5 23.5 3.9 6 2.1 1 2.1 3.4 2 1.7 25 29 7.3 19 4 4.8 2.5 1 2.5 2.3 2.4 .55

Table (I) Morphometric measurements (cm) and different ratios of Alestes baremose from Lake Nubia

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 26 24 5.3 19.3 4.5 4.3 3.5 1.1 3.2 2.3 1 2.3 27 24 6 19 4 4.8 3.6 1 3.6 2.4 1.5 1.6 28 24 6.3 19.2 3.8 5 3.6 1 3.6 2.3 1.2 1.91 29 24.3 5.4 16.5 4.5 3.7 3.3 1 3.3 2.3 1.1 2.09 30 21 6 8 3.5 2.3 3.5 1 3.5 2.6 2.5 1.04 31 23 5.1 17 4.5 3.8 3.4 1 3.4 2.3 1.6 1.43 32 22 6.3 22 3.5 6.3 3.7 .9 4 2.3 3 .76 33 27 4.7 20 5.7 3.5 3.5 1.2 2.9 2.3 1.5 1.53 34 35 9.2 30 3.8 7.9 4.7 1.2 6.3 2.5 1.5 1.66 35 25 7.1 20 3.5 3.5 3.5 1.2 2.4 2.4 2.5 .96 36 23 5 19 4.6 4.13 4.7 1 4.7 2.5 2.5 1 37 24 6.32 29 3.8 7.63 3.5 1 3.5 2.4 2.6 .92 38 24 3.81 18 6.3 2.85 2.8 1 2.8 2.4 2.8 .85 39 26 5.77 21.5 4.5 4.77 3 1.1 2.72 2.4 1.5 1.6 40 26 7.43 22 3.5 6.28 4.3 1 4.3 2.6 1.8 1.44 41 27 5.09 22 3.5 6.28 3.1 1 3.1 2.4 4 .6 42 25.5 4.81 20 5.3 3.77 3.1 1 3.1 2.4 3.1 .77 43 21 4.37 17 4.8 3.54 4 1 4 2.5 2 1.25 44 19 5.42 14.5 3.5 4.14 4 1 41 2.5 2.5 1 45 20 5.26 15.5 3.8 4.07 3.1 1 3.1 2.3 2 1.25 46 23 4.79 18 4.8 3.73 3.2 1 3.2 2.4 3.1 .77 47 21 4.66 16 4.5 3.55 3.5 1 3.5 2.3 2.1 1.15 48 22 6.28 17 3.5 4.88 3 1 3 2.4 2.1 1.14 49 25.5 5.31 15 4.8 3.12 3.2 1 3.2 2.3 4.2 .54 50 34 10.62 21 3.2 6.56 3 1 3 2.4 5.1 .47

Table (II) Morphometric measurements (cm) and ratios of Alestes baremose from the White Nile (Jebel Aulia)

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 24 6.31 18 3.8 4.73 3 1.9 1.57 27 2.5 1.08 2 25 6.25 20 4 5 3.5 1.2 2.91 29 2.5 1.16 3 22 5.78 18 3.8 4.73 3.5 1 3.5 3 2.5 1.2 4 20 5.88 16 3.4 4.70 3.1 1 3.1 2.8 2 1.4 5 32 5.81 25 6.5 4.54 4.2 1.2 3.5 2 2.6 .76 6 27 4.21 22 6.4 3.43 4.4 1.2 3.66 2.8 2.5 1.12 7 28 4.66 23 6 3.83 4.4 1.2 3.66 2.9 2.8 1.03 8 31 3.75 25 8 3.12 4.2 1.4 3 3 3.5 .85 9 37 6.72 30 5.5 5.45 5 1.2 4.16 3.4 3.8 .89 10 29 5 22 5.8 3.79 4.5 1.3 3.46 3.8 2.5 1.52 11 33 5.5 25 6 4.16 4.3 1.3 3.30 3.2 3.9 .82 12 31 4.76 25 6.5 3.84 4.3 1.3 3.30 3.4 3.8 .89 13 30 5.17 24 5.8 4.13 3.2 1.3 2.46 3 3.6 .83 14 30 5.45 24 5.5 4.36 3 1.2 2.5 3 3.2 .93 15 32 7.27 25 4.4 5.68 2.8 1 2.8 26 3.3 .78 16 22 4.4 17 5 3.4 3.6 1.2 3 3 3.7 .81 17 24 5.58 19 4.3 4.41 3.5 1 3.5 3.2 3.9 .82 18 20.5 4.76 16.5 4.3 3.83 3.3 1 3.3 3 3.6 .83 19 21 5 16.5 4.2 3.92 3.2 1 3.2 3.2 3.5 .85 20 20 6.57 15 3.8 3.94 3.5 1 3.5 3 3.8 .89 21 22 6.11 17 3.6 3.72 3.3 1 3.3 3 3.5 .85 22 23.5 6.52 19 3.6 3.27 3.4 1 3.4 3.4 3.7 .91 23 24 6.66 18 3.6 5 3 1 3 3 3.5 1.03 24 23 3.76 18 3.4 5.29 3 1 3 3.5 3.4 .28 25 20 26.5 16 3.21 5 3 1 3 3 3.2 .29

Table (III) Morphometric measurements and ratios of Alestes dentex Lake Nubia

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 26 24 6.31 18 3.8 4.73 3 1.9 1.57 22 6.4 .34 27 21.5 4.17 16 4.8 3.33 3.5 1.9 1.84 2.2 5.3 .42 28 20 5 15 4 3.75 3.2 1 3.2 2.1 5 .42 29 22 6.87 17 3.2 5.31 3 1 3 2.3 4.2 .55 30 25.5 6.37 15.5 4 3.87 3.2 1.9 1.68 2.2 4 .55 31 21 6 16 3.5 4.57 3.4 1 3.4 2.3 5 .46 32 23 6.05 18 3.8 4.73 3.5 1.6 2.18 2.1 5.6 .38 33 21 6.17 16 3.4 4.70 3.3 1 3.3 2.4 5.3 .45 34 20 3.8 15 3.8 3.94 3.2 1.8 1.77 2.4 4 .6 35 23 5.26 18 3.7 4.86 3.4 1.5 2.56 2.3 5.5 .41 36 20 6.21 15 3.3 4.54 3.1 1 3.1 2.1 5 .42 37 19 6.06 14.5 3.73.91 3.2 1.6 2 2.3 4 .58 38 20.5 5.13 16.5 4.2 3.92 3.4 1.2 2.83 2.4 5.1 .47 39 25.5 4.88 16.5 4.5 3.66 3.8 1.2 3.16 2.6 5.3 .49 40 23 5.66 18 4 4.5 3.4 1 3.4 2.3 5.2 .43 41 22 5.75 18 2.8 4.73 3 1 3 2 5 .4 42 20.5 5.78 16.5 3.7 4.45 3.2 1 3.2 2.1 4.8 .44 43 25 5.54 20 4.1 4.87 4 1.9 2.10 2.3 5.3 .43 44 22 6.09 17 3.7 4.59 3.4 1 3.2 2 5 .4 45 24 5.94 19 4.2 4.52 3.2 1 4 2.4 5.4 .44 46 25 5.71 20 3.8 5.26 3.2 1.9 1.78 2.4 5 .48 47 20 6.57 15 3 5 3.2 1 3.2 2 5 .4 48 22 6.66 16 3 5.33 3.2 1 3.2 2.1 5.1 .41 49 19 5.13 14.5 3.73.91 3.4 1.6 2 2.3 4 .58 50 20 5 15 4 3.75 3.2 1 3.2 2.1 5 .42

Table (IV) Morphometric measurements and ratios of Alestes dentex from the White Nile (Jebel Aulia)

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 25.5 5.66 20.5 4.5 4.44 2.4 1.2 2 3.5 3.1 1.13 2 21.5 6.32 17 3.4 5 2.1 1.2 1.75 3.3 2.6 1.27 3 41 11.71 33.5 3.5 9.57 3 1.5 2 4 3.8 1.05 4 21.5 6.72 17 3.2 5.31 2.2 1.1 2 3 2.7 1.11 5 25 7.35 20 3.4 5.88 3.5 1.2 2.91 3.6 3.1 1.16 6 20.5 5.13 16.5 4 4.13 3.2 1.1 2.91 3.5 3.4 1.02 7 25 6.25 20 4 5 3.1 1.1 2.82 3.3 3.2 1.03 8 23 5.60 18 4.1 4.39 3.3 1.1 3 3.4 3.3 1.03 9 22 5.5 18 4 4.39 3.4 1.1 3.09 3.2 3 1.06 10 20.5 5.39 16.5 3.8 4.13 3.6 1 3.6 3.5 2.9 1.21 11 22 5.23 17 4.2 4.04 3.5 1 3.5 3.8 3 1.27 12 20.5 5.13 16.5 4 4.13 3.7 1 3.7 3.6 3.2 1.13 13 22 5.36 19.5 4.1 4.75 3.6 1 3.6 4.3 3.5 1.25 14 24 5.85 16.5 4.1 4.03 3.8 1 3.8 3.7 3.1 1.09 15 24 5.71 16.3 4.2 3.88 3.8 1 3.8 3.9 3 1.12 16 37 7.4 30 5 6 4.1 1.1 3.73 3.6 3.4 1.06 17 54 8.30 45 6.5 6.92 4.6 1.5 2.73 4 4 1 18 24 7.74 18 3.1 6.13 3.7 1 3.7 3.5 3.4 1.03 19 28 7 32.5 4 8.12 2.4 1.2 2 3.5 3.1 1.12 20 25 6 20 4 5.00 2.1 1.2 1.75 3.3 3.6 1.26 21 24 5 19 4.1 4.63 3 1.5 2 4 3.8 1.11 22 49 5.85 35 7 5.00 2.2 1.1 1 3 2.7 1.16 23 45 6.71 44 6.5 6.76 3.5 1.2 2.91 3.6 3.1 1.02 24 23 6.92 18 3.5 5.14 3.2 1.1 2.90 3.5 3.4 1.03 25 24 6.57 21 3.5 6 3.1 1.1 2.8 3.3 3.2 1.03

Table (V) Morphometric measurements and ratios of Alestes nurse from lake Nubia

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 26 10.5 3.11 9 3.1 2.8 2.5 .5 6 5 1.2 1.7 27 10.2 3.29 9 3.1 2.9 2.5 .5 5 2.1 1 2.1 28 12.1 3.17 9.5 3.5 2.7 2.8 6 4.7 3 1.3 2.3 29 10.3 3.27 9.3 4 2.3 3.2 1.2 2.7 3.4 1.5 2.3 30 12 3.15 10 3.8 2.6 3.6 1 3.6 2.5 2 1.3 31 8 3.66 9.8 3 3.3 2.7 .6 4.5 2 2.8 .7 32 12 3.42 10 3.5 2.9 3.5 1 3.5 2.5 1.5 1.7 33 8 3.33 5 2.4 2.1 2 .4 5 2.1 1.2 1.8 34 6.8 3.23 3.5 2.1 1.6 1.2 .5 2.4 2 1.2 1.7 35 8.5 3.4 5.1 2.5 2.4 1.5 .5 3 2 1.2 1.7 36 10.5 3.5 9.8 3 3.3 2.5 .5 5 3.2 2.3 1.4 37 13 3.33 13.7 4.5 3 4.1 .7 5.9 3.8 2.5 1.5 38 13.1 3.27 11.5 4 2.9 3.3 .5 6.6 3 2 1.5 39 9.3 3.72 8.1 2.5 3.2 .8 .5 1.6 2 1 2 40 10.8 3.6 6.5 3 3.2 2.7 .5 5.4 2.5 1.4 1.8 41 12.6 3.6 10.9 3.5 3.1 3.5 1 3.5 2.5 1.5 1.7 42 9 3.6 8 2.5 3.2 .5 .5 1 2 1 2 43 10.5 3 9.5 3.5 2.7 2.6 .6 4.3 2.5 1 2.4 44 7.3 3.7 4.9 2 2.5 .5 .5 1 2 1 2 45 8.5 4.0 5.2 2.1 2.5 .8 .5 1.6 2 1 2 46 11.4 3.6 10.1 3.2 3.2 3.7 .5 7.4 2.5 1.2 2.1 47 12.4 3.6 10.5 3.4 2.1 3.9 .5 7.8 2.4 1.3 1.8 48 6.8 3.4 4.9 2 2.5 .4 .5 .8 2 1 2 49 12.5 3.6 11.8 3.5 3.4 3.5 1 3.5 2.5 1.5 1.7 50 8.5 4.1 5.1 2.1 2.5 .8 .5 1.6 2 1.1 2

Table (VI) Morphometric measurements and ratios of Alestes nurse from the White Nile (Jebel Aulia).

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 38 5.06 25 7.5 3.3 8.2 1.3 6.3 2.8 1.3 2.15

Table (VII) Morphometric measurements and ratios of Alestes macrolepidotus caught at Jebel Aulia

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 25.5 5.66 20.5 4.5 4.44 2.4 1.2 2 3.5 3.1 1.13 2 21.5 6.32 17 3.4 5 2.1 1.2 1.75 3.3 2.6 1.27 3 41 11.71 33.5 3.5 9.57 3 1.5 2 4 3.8 1.05 4 21.5 6.72 17 3.2 5.31 2.2 1.1 2 3 2.7 1.11 5 25 7.35 20 3.4 5.88 3.5 1.2 2.91 3.6 3.1 1.16 6 20.5 5.13 16.5 4 4.13 3.2 1.1 2.91 3.5 3.4 1.02 7 25 6.25 20 4 5 3.1 1.1 2.82 3.3 3.2 1.03 8 23 5.60 18 4.1 4.39 3.3 1.1 3 3.4 3.3 1.03 9 22 5.5 18 4 4.39 3.4 1.1 3.09 3.2 3 1.06 10 20.5 5.39 16.5 3.8 4.13 3.6 1 3.6 3.5 2.9 1.21 11 22 5.23 17 4.2 4.04 3.5 1 3.5 3.8 3 1.27 12 20.5 5.13 16.5 4 4.13 3.7 1 3.7 3.6 3.2 1.13 13 22 5.36 19.5 4.1 4.75 3.6 1 3.6 4.3 3.5 1.25 14 24 5.85 16.5 4.1 4.03 3.8 1 3.8 3.7 3.1 1.09 15 24 5.71 16.3 4.2 3.88 3.8 1 3.8 3.9 3 1.12 16 37 7.4 30 5 6 4.1 1.1 3.73 3.6 3.4 1.06 17* 54 8.30 45 6.5 6.92 4.6 1.5 2.73 4 4 1 18 24 7.74 18 3.1 6.13 3.7 1 3.7 3.5 3.4 1.03 19 28 7 32.5 4 8.12 2.4 1.2 2 3.5 3.1 1.12 20 25 6 20 4 5.00 2.1 1.2 1.75 3.3 3.6 1.26 21 24 5 19 4.1 4.63 3 1.5 2 4 3.8 1.11 22 49 5.85 35 7 5.00 2.2 1.1 1 3 2.7 1.16 23 45 6.71 44 6.5 6.76 3.5 1.2 2.91 3.6 3.1 1.02 24 23 6.92 18 3.5 5.14 3.2 1.1 2.90 3.5 3.4 1.03 25 24 6.57 21 3.5 6 3.1 1.1 2.8 3.3 3.2 1.03

Table (VIII) Morphometric measurements and ratios of Hydrocynus lineatus from Lake Nubia

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 26 28 7 32.5 4 8.12 2.4 1.2 2 3.5 3.1 1.12 27 24 6.15 15 3.9 3.84 3.3 1.1 3 3.4 3.3 .103 28 24 6.85 19 3.5 5.42 3.4 1.1 3.09 3.2 3 1.06 29 24 8 15 3 5 3.6 1 3.6 3.5 2.9 1.20 30 20 5.71 19 3.5 5.42 3.5 1 3.5 3.8 3 1.26 31 24 6.31 20 3.8 5.26 3.7 1 3.7 3.6 3.5 1.028 32 25 8.33 19 3 6.33 3.6 1 3.6 3.8 3.5 1.085 33 24 5 20 4.8 4.166 3.8 1.1 3.45 3.6 3.4 1.058 34 34 9.71 19 3.5 5.42 4.1 1.5 2.73 4.3 3.9 1.102 35 24.5 5.56 20 4.4 4.54 3.5 1 3.5 3.7 3.4 1.088 36 45 12.85 18 3.5 5.14 3.6 1.1 3.27 3.9 3.5 1.11 37 23 5.75 21 4 5.25 3.7 1.1 3.36 3.6 3.2 1.12 38 24 6 19 4 4.75 3.6 1 3.6 3.5 3.1 1.13 39 20 4.87 15 4.1 3.65 4.3 1.5 2.86 4 3.4 1.18 40 25 6.75 20 3.7 5.40 4.5 1.5 3 4.6 4 1.15 41 25 7.35 19 3.4 5.58 3.2 1 3.2 3.5 3.1 1.12 42 24 6 20 4 5 3 1 3 3.2 3 1.06 43 22 5.36 19 4.1 4.63 2.6 1 2.6 3 2.3 1.304 44 21 6.17 20 3.4 5.88 3.1 1 3.1 3 3.1 .96 45 23 7.18 19 3.2 5.93 2.1 1 2.1 2.6 2.1 1.23 46 24 6.31 23 3.8 6.38 2.4 1.2 2 2.4 3.1 .774 47 23 5.60 20 4.1 4.87 2.1 1.3 1.61 2.8 3.2 .875 48 25 7.14 19 3.5 5.42 2 1.2 1.66 2.3 3.1 .741 49 28 9.03 24 3.1 7.74 2 1 2 3.5 2.6 1.029 50 24 5.71 16.3 4.2 3.88 3.8 1.1 3.8 3.6 3.4 1.058

Table (IX) Morphometric measurements and ratios of Hydrocynus lineatus from Jebel Aulia

Sampl L D L/D sL D SL/ IO E IOW/E pL pD PL/p e D W D D d 1 23 4 5.75 19 4 4.75 3.9 1.1 3.54 2.5 2.2 1.13 2 27 4.2 6.42 22 4.2 5.23 4.5 1.1 4.09 2.4 2.1 1.14 3 32 4.2 7.61 25 4.2 5.95 5.2 1.3 4 3 2.8 1.07 4 25 4.7 5.95 19 4.7 4.04 4.7 1.2 3.91 2.6 2.7 .96 5 27 4.3 6.27 22 4.3 5.11 4.5 1.2 3.75 2.5 2.6 .96 6 26 4.3 6.04 22 4.3 5.11 4.5 1.2 3.75 2 2.1 .95 7 31 5 6.2 26 5 5.20 5.3 1.2 4.41 2.9 2.5 1.16 8 25 4.2 5.95 20 4.2 4.76 4.2 1.1 3.81 2.5 2.2 1.14 9 21.5 3.45.65 17.53.4 5.14 3.8 1.1 3.45 2.4 2.1 1.14 10 25 4.5 5.5 20.5 4.5 4.55 4.1 1.3 3.15 3 2.8 1.07 11 25 4 6.25 19.5 4 4.87 4 1.2 3.33 2.6 2.7 .96 12 23 3.5 6.57 20 3.5 5.71 4.2 1.2 3.5 2.5 2.6 .96 13 27 4.2 6.42 20 4.2 4.76 4 1.2 3.33 2 2.1 1.16 14 25.5 4 6.37 19 4 4.75 3.9 1.2 3.25 2.9 2.5 .95 15 37 4 9.25 22 4 5.5 4.5 1.2 3.75 2 2.1 1.13 16 22 4.1 5.36 21.34.1 5.19 4.8 1.2 3.83 2.5 2.2 1.15 17 25 4.3 5.81 25 4.2 5.95 4 1.1 3.63 2.3 2 1.12 18 31 4 7.75 19 4 4.75 4.2 1.3 3.23 2.7 2.4 1.08 19 41 4 10.25 20 4 5 5 1.2 4.16 2.5 2.3 1.04 20 25.5 4.2 6.07 26 4.2 6.19 4.1 1.2 3.41 3.4 2.6 1.25 21 23 4.2 5.47 33 4.2 7.85 4 1.1 3.63 2.5 2 .92 22 22 4 5.5 20.5 4 5.12 4 1.1 3.63 2 2.6 1.25 23 25 3.8 6.57 18.53.8 4.86 4.2 1.2 3.5 2.4 2 1.05 24 27 4 6.75 16 4 4 4.1 1.2 3.41 2 1.9 1.09 25 23 3.5 6.57 20 3.5 5.71 4.3 1.2 3.58 2.4 2.2 1.09

Table (X) Morphometric measurements and ratios of Hydrocynus brevies from lake Nubia

Sampl L D L/D sL D SL/ IO ED IOW/E pL pD PL/p e D W D d 26 21 3.4 6.17 17.534 5.14 3.4 1.2 2.83 2.6 2 1.30 27 20 3.4 5.8 15.83.4 4.64 3.2 1.1 2.90 2.4 1.8 1.33 28 16 3 5.33 12 3 4.00 2.5 .1 2.50 2 1 2.00 29 22 3.5 6.28 19 3.5 5.42 3.9 1.2 3.25 2.8 2.3 1.21 30 28 4.1 6.82 20 4.1 4.87 4 1.3 3.07 2.9 2.5 1.16 31 20.5 4 5.12 16.5 4 4.12 4 1.2 3.33 2.7 2.3 1.17 32 22.5 4.1 5.48 18 4.1 4.39 4.3 1.2 3.58 2.8 2.4 1.16 33 24 4 6 19 4 4.75 4.2 1.1 3.81 2.7 2.5 1.08 34 37 4.5 8.04 30 4.6 6.52 4.8 1.2 4.00 2.9 2.5 1.16 35 28 4 7 23.5 4 5.87 4.2 1.1 3.81 2.5 2.2 1.13 36 25 4 6.25 20 4 5.00 4 1.1 3.63 2.4 2.1 1.14 37 49 6.2 7.9 35 5.2 5.64 4.4 1.2 2.00 2.9 2.5 1.16 38 20 4.2 4.76 16 4.2 3.80 4 1 4.00 2.6 2.2 1.18 39 25 4.1 6.09 20 4.1 4.87 4 1.2 3.33 2.7 2.4 1.12 40 23 4.1 5.60 18 4.1 4.39 4.3 1 4.30 2.4 2.1 1.14 41 22 3.5 6.28 18 3.5 5.14 3.5 1 3.60 2.2 2 1.10 42 20.5 3.8 5.39 16.5 3.8 4.34 4.2 1 4.20 2.5 2.2 1.13 43 22 4 5.5 17.5 4 3.12 4.2 1 4.20 2.5 2.2 1.13 44 24 4 6 19 4 4.75 4.2 1 4.20 2.5 2.2 1.13 45 24 6.5 3.69 19.56.5 3.00 4.9 1.2 4.08 2.9 2.5 1.16 46 27 4.8 5.62 20 4.8 4.16 3.5 1 3.50 2.5 2.2 1.16 47 24 4 6 20 4 5.00 4.1 1 4.10 2.9 2.4 1.12 48 24 4.1 5.85 19 4.1 4.63 4 1 4.00 2.5 2.2 1.16 49 24 4 6 21 4 5.25 4.4 1.2 2.00 2.7 2.4 1.18 50 20 4.2 4.76 16.34.2 3.88 4.1 1 4.10 2.5 2.2 1.15

Table (XI): Morphometric measurements and ratios of Hydrocynus brevies from the White Nile (Jebel Aulia).

Sample L L/D sL D SL/D IOW ED IOW/ED pL pD PL/pd 1 45 4.73 40 9.56.77 11 1.5 7.3 10 6 1.66 2 23 6.57 18 3.55.14 4 1 4 3.6 2.4 1.5 3 24 6.85 21 3.5 6 4.3 1 4.3 3 2.2 1.36 4 20 5.71 15 3.54.28 3.3 1 3.3 3.1 2.3 1.34 5 24 6 19 4 4.75 4 1 4 3 2.1 1.42 6 25 6.25 20 4 5 4 1.3 3.07 3.4 2.2 1.54 7 24 5.72 19 4.24.52 4 1.2 3.3 3.6 3.2 1.12 8 24 6.66 20 3.6 5.55 3.5 1.2 2.91 3.8 3.5 1.08 9 29 7.63 19 3.8 5 4 1.2 3.3 4.2 3.9 1.07 10 23.5 6.52 23 3.6 6.38 4 1.2 3.3 3.6 3.2 1.09 11 22 5.94 20 3.7 5.40 4 1.1 3.6 3.2 3.1 1.03 12 23 5.75 1 4 4.75 4.2 1 4.2 3.2 3 1.06 13 24 6 17 4 4.25 5 1 5 3.2 3 1.06 14 24 6 19 4.8 3.95 4 1.4 2.85 3.6 3.2 1.12 15 29 7.25 23 4 5.75 4.5 1.2 3.75 3.8 3.4 1.11 16 22 5.5 18 4 4.5 4.5 1 4.5 3.2 3.1 1.03 17 25 6.25 20 4 5 4 1 4 3.6 3.4 1.05 18 25 6.57 20 3.8 5.26 5 1 5 3.4 3.2 1.06 19 24 5.21 19 4.6 4.13 3 1 3 3.1 3 1.03 20 21 6.56 16 3.2 5 3.5 1.2 3.91 3.2 3 1.06 21 15 5 10 3 3.33 3.3 1.3 2.53 3 2.6 1.15 22 20 5.72 15 3.5 4.28 4 1.2 3.3 3.1 2.5 1.24 23 23 6.05 18 3.5 5.14 4 1 4 3.4 3 1.13 24 25 6.57 21 3.8 5.52 4 1.6 2.5 3.2 2.7 1.18 25 29 7.3 23 3.9 5.89 3.8 1 3.8 3.5 2.5 1.4

Table (X11) Morphometric measurements and ratios of Hydrocynus forskalii from Lake Nubia

Sampl L L/D sL D SL/D IO ED IOW/E pL pD PL/p e W D d 26 40 6.89 39.5 5.810.39 7.5 1.2 6.25 19.5 9 2.16 27 23 5.60 18.60 4.14.51 4 1 4 3.8 2.5 1.52 28 32 8 25.5 4 6.37 5 1 5 11.5 6.5 1.76 29 20.5 5.39 16.5 3.8 4.34 4 .9 4.4 3.6 2.2 1.63 30 45 12.85 44 3.5 12.57 4 1 4 18 10.5 1.71 31 23 6.57 18 3.5 5.14 4 1 4 10 5.8 1.72 32 22 6.28 21 3.9 5.52 4.3 1.2 3.58 8 4.3 1.86 33 20 5.72 15 3.5 3.94 3.3 1.3 2.53 4 2.1 1.90 34 24 6 19.3 4 4.82 4 1.2 3.3 9 2.1 4.28 35 25 6.25 20 4 5 4 .9 4.4 10 5.4 1.85 36 24 5.72 19.5 4.2 4.64 4 1.6 2.5 9 4.1 2.19 37 20 5.12 15.2 3.9 3.89 4.3 1 4.3 2.8 1.5 1.86 38 40 6.77 32 5.9 5.42 7.8 1.5 5.2 16.5 8.9 1.85 39 54 11.25 44 4 11 4.1 1.2 3.41 21 10.3 2.03 40 49 6.53 35 7.5 4.66 7.3 1.5 4.86 12.5 8.2 1.46 41 24 5.72 19.5 4.2 4.64 4.1 1 4.1 8 4.3 1.86 42 49 5.36 35 7.5 4.86 4.3 1 4.3 12 8.1 1.48 43 20 8.05 15 3.5 4.28 3.3 1.2 2.75 6 2.6 2.30 44 22 6.25 18.5 4.1 5.28 4.5 1.4 1.09 4 2.1 1.90 45 29 5.78 23.5 3.6 6.25 3.5 1 3.5 4.8 2.6 1.84 46 25 6.72 20 4 5.54 4.5 1.3 3.46 5.5 3 1.83 47 22 5.39 17.5 3.8 4.60 4.2 1 4.2 3.5 3.1 1.12 48 37 7.23 30.5 5.5 5.54 5 1.8 2.77 17 8.3 .84 49 20.5 5.39 16.5 3.8 4.34 4 1.6 2.5 6 2.4 2.5 50 47 7.23 35 6.5 5.38 8.5 1.8 4.72 20.2 12.6 1.58

Table (X111) Morphometric measurements and ratios of Hydrocynus forskalii from Jebel Aulia

Statistical analysis in Table XIV shows that A. baremose from Lake Nubia differs significantly from those caught at the White Nile in total length and interorbital width (p < 0.014 and p < 0.001). A. dentex from Lake Nubia differs significantly from the White Nile specimens in total length, standard length, depth, interorbital width and peduncle depth (p <0.002, p < 0.001, p < 0.003, p < 0.001). A. nurse from Lake Nubia differs significantly from those collected from the White Nile in all measurements except the eye diameter that showed no significant difference (p <0.1766). For Hydrocynus species studied, Table XV reveals that the total length and interorbital width differ significantly between species studied. The eye diameter, however, showed no significant difference between the species studied.

Comparison between the different ratios is shown in figures 10 to 15: Different ratios are shown for Alestes nurse in figure (10). Figure (10a) shows the relationship between total length and depth, figure (10b) shows the relationship between standard length and depth, figure (10c) shows the relationship between inter orbital width and eye diameter and figure (10d) shows the relationship between peduncle length and peduncle depth in Alestes nurse. Different ratios are shown for Alestes baremose in figure (11). Figure (11a) shows the relationship between depth and length, figure (11.b) shows the relationship between depth and standard length , figure (11c) shows the relationship between inter orbital width and eye diameter and figure (11d) shows the relationship between peduncle length and peduncle depth in Alestes baremose. Different ratios are shown for Alestes dentex in figure (12). Figure (12a) shows the relationship between length and depth in Alestes dentex, figure (12b) shows the relationship between standard length and depth figure (12c) shows the relationship between eye diameter and inter orbital with and figure (12d) shows the relationship between peduncle length and peduncle width in Alestes dentex. Different ratios are shown for Hydrocynus lineatus in figure (13). Figure (13a) shows the relationship between the length and depth, figure (13b) shows the relationship between the standard length and depth, figure (13c) shows the relationship between the eye diameter and inter orbital width while figure (13d) shows the relationship between the peduncle length and peduncle width in Hydrocynus lineatus. Ratios from statistical analysis are shown for Hydrocynus forskalii in figure (14). Figure (14a) shows the relationship between the length and depth in Hydrocynus forskalii, figure (14b) shows the relationship between the standard length and depth in Hydrocynus forskalii, figure (14c) shows the relationship between the eye diameter and inter orbital width and figure (14d) shows the relationship between the peduncle length and peduncle width in Hydrocynus forskalii. Different ratios are shown for Hydrocynus brevis in figure (15): Figure (15a) shows the relationship between the length, figure (15b) shows the relationship between the standard length and depth , figure (15c) shows the relationship between the eye diameter and inter orbital width and figure (15) shows the relationship between the peduncle length and peduncle width in Hydrocynus brevis. The results show that the fishes collected from Lake Nubia and those collected from the White Nile at Jebel Aulia are the same species. This is indicated by the straight lines in figures (10a to 10d), (11a-11d), (12a- 12d), (13a – 13d), (14a- 14d) and (15a-15d). The measurements and ratios showed only slight differences that are not significant between fishes collected from the two sites of the Nile system.

Linear Regression 50.00

40.00

length

30.00 length = 1.22 + 3.03 * d R-Square = 0.92

20.00

$ $ $$ $ $ $ $ $$ $ 10.00 $ $ $ $ $$

2.00 4.00 6.00 8.00 depth Figure (10a): Relationship between length and depth in Alestes nurse.

Linear Regression

40.00

gt 30.00

n standerd length = -1.73 + 3.39 * d e

l R-Square = 0.80

d d r

d 20.00

t s $ $ $ $ $ $ $$ $ 10.00 $$ $ $ $ $$ $ $ $

2.00 4.00 6.00 8.00 depth

Figure (10b): Relationship between standard length and depth in Alestes nurse

Linear Regression

10.00

7.50

5.00 $

inter orpital width orpital inter $ $ $ $ inter orpital width = 0.09 + 11.25 * led $ $ $ R-Square$ = 0.28 2.50 $ $ $ $ $ $ $ 0.00 0.15 0.20 0.25 0.30 0.35

log eye diameter+1

Figure (10c): Relationship between inter orbital width and eye diameter in Alestes nurse.

$ Linear Regression

$ 0.50 $ log pl = $0.33 + 0.37 * lpd

l R-Square = 0.37

o l

0.40 $ $$$ $ $ $

$ $

0.30 $ $ $ 0.00 0.10 0.20 0.30 0.40 log pd

Figure (10d): Relationship between peduncle length and peduncle depth in Alestes nurse

0.80 $ Linear Regression

0.70 $ $ $

2 $ $ $ $ $

th $

p $ $ $ $ $$ e

$ d

g o

l $$$ $ $ log depth2 = 0.61 + 0.01 * ll2 0.60 $$$ $$$ $ $ R-Square = 0.00 $ $ $ $ $ $ $$

$ $ 0.50 1.30 1.40 1.50 log length 2

Figure (11a): Relationship between depth and length in Alestes baremose

$ 0.80 Linear Regression $ $

0.70 $ $ $

2 $ $ $ $ $

th $

p $ $ $ $ $ $ $ $ e

$ d

o l $ $ $ $ $ 0.60 $ $ $ $ $ $ $$$ $ $ $ log depth2 = 0.72 + -0.06 * sqsl2 $$$ $ $ $ R-Square = 0.06

$ $ 0.50

1.00 1.50 2.00 sqrot standard length2

Figure (11b): Relationship between depth and standard length in Alestes baremose $ $ Linear Regression

0.20

r e

m a

e 0.10 y

e $ $ $$$

g o l $ $ $

$$$$ $$$ $$$ $$$ $ $ $$$$$$$$ $$ $ $ $ $ 0.00 log eye diameter = 0.02 + -0.01 * liow2 R-Square = 0.00 $ $ $

0.20 0.40 0.60 0.80 log iow 2

Figure (11c): Relationship between inter orbital width and eye diameter in Alestes baremose.

$ Linear Regression sqrot pendical depth2 = 0.00 + 1.00 * sqsl2 R-Square = 1.00

2.00 $ 2

t p

e $ $

d $

l $

a $ c i $ d $

n $ $ e 1.50 $ p $ $

t $ o

r $ q

s $ $

$ $ 1.00 $ 1.00 1.50 2.00 sqrot standard length2

Figure (11d): Relationship between peduncle length and peduncle depth in Alestes baremose

$ Linear Regression

1.40 $ lsl3 = 0.91$$ + 0.56$ * lld3 $ R-Square$ $ = 0.53 $

$ $ 3

l s l $ $ $ 1.30 $ $ $ $ $ $ $ $ $ $ $ $ $ $ $$$ $ $ $ $ $ $ 1.20 $ $ $ $ $ $ 0.50 0.60 0.70 0.80 0.90 log depth3

Figure (12a): Relationship between length and depth in Alestes dentex.

$ $ $ 1.30 $ $ $

$ $ $ $ $ $

$ $ $ $ $ $$$$ $ $ $ $ $ 1.20 $ $ $ $ $ $

0.50 0.60 0.70 0.80 0.90

log depth3

Figure (12b): Relationship between standard length and depth in Alestes dentex. $$$ $ $ Linear Regression $

$ $ $

1 0.20

+ e

t $

e m

a $

e $ $ $

y log eye diamete+1 = -0.06 + 0.26 * liow3 e

0.10 R-Square = 0.02 g $ $$$ $ $$$ $ o l

0.00 $ $$$$ $$$$$$$ $ 0.50 0.60 0.70 log inter orpital width

Figure (12c): Relationship between eye diameter and inter orbital width in Alestes dentex.

Linear Regression 10.00 $ $$$ $$$$$$ $$$ $ $$$$$ $ $$

$$$$ $ h

Peduncle t 0.00 p

depth e

c i

d n

e -10.00

pendical depth = 7.81 + -1.42 * pl -20.00 R-Square = 0.40

5.00 10.00 15.00 20.00 Pedunclependical length length

Figure (12d): Relationship between peduncle length and peduncle width in Alestes dentex.

$ Linear Regression

$ $ 0.80

log depth = -0.12 + 0.48 * logl4 h 0.70 $ t

$ R-Square = 0.30

p e

d $

$ g

$ $ o

l $ $$$ $ $ $$$ $ $$$ $ $ 0.60 $ $ $ $ $ $$$ $ $ $ $ $ $ $ $ $ 0.50 $ $ $ $

1.40 1.50 1.60 1.70 log length+5

Figure (13a): Relationship between length and depth in Hydrocynus lineatus

$ Linear Regression $$ 0.80

log depth = 0.06 + 0.41 * logsl4 $ h 0.70 R-Square = 0.36 $ pt

de $ $

$ $ og og

l $ $ $ $ $ $ $$$$ $ $ $ $ 0.60 $ $ $ $ $ $$$$ $ $ $ $ $ $ 0.50 $ $ $ $

1.20 1.30 1.40 1.50 1.60 log standerd length4

Figure (13b): Relationship between standard length and depth in Hydrocynus lineatus

Linear Regression 12.50 pendical depth = 1.17 + 0.58 * pl R-Square = 0.44

10.00 h

Peduncle p e

depth

l 7.50 a

n e

5.00 p

$ $ $$ $$$$$$$$ $$ $$$$$$ $$$$ $$$ 2.50 $ $

5.00 10.00 15.00 20.00 pendical length Peduncle length

Figure (13c): Relationship between eye diameter and inter orbital width in Hydrocynus lineatus

$ Linear Regression

0.90 $

0.80 5 $

h log depth 5 = -0.22 + 0.59$ * logl5 t $

p R-Square = 0.54 e

d 0.70

g $

o $

l $ $ $ $$$$$$$ $ $ $ 0.60 $ $ $ $ $ $ $ $ $$ $ $$$$$$ $

$ 0.50 $

Figure (13d): Relationship between peduncle length and peduncle width in Hydrocynus lineatus $ Linear Regression

0.90 $

0.80 5 $

h h log depth 5 = -0.22 + 0.59$ * logl5 $ pt R-Square = 0.54

de 0.70 $

og og $ l $ $ $ $$$$$$$ $ $ $ 0.60 $ $ $ $ $ $ $ $ $$ $ $$$ $$$ $

$ 0.50 $ 1.20 1.30 1.40 1.50 1.60 1.70 log length5 Figure (14a): Relationship between length and depth in Hydrocynus forskalii

$ Linear Regression

0.90 $ $

0.80 5

$ $ h

t log depth 5 = -0.05 +$ 0.51 * lsl5 p

e R-Square = 0.47

d 0.70 $

g $

o l $$ $ $ $$$$$$$$ $ $ $ 0.60 $ $ $ $ $ $ $ $ $ $ $$ $$$$$$ $

Figure (14b): Relationship between standard length and depth in

Hydrocynus forskalii$ Linear Regression

0.90 $ log depth 5 = 0.15$ + 0.73 * liow5 R-Square = 0.64 $ 0.80 5 $

h h $ $ pt

de 0.70 $

og og $ l $$ $ $ $$$$$ $ 0.60 $ $ $ $ $ $ $ $ $ $$$

$ 0.50 $ 0.50 0.60 0.70 0.80 0.90 1.00 liow5

Figure (14c): Relationship between eye diameter and inter orbital width in Hydrocynus forskalii

$ Linear Regression $ $ 1.00 $ $ log pd5 = 0.04 +$ 0.71$ * logpl5$ R-Square = 0.77 $ 0.80 $ $ $

pd5 $ $

0.60 $ og og

l $ $ $ $ $$$ $ $ $$$ $ $ $ $ $ $ $ $ 0.40 $ $ $ $ $ $ $ $$$

020 $

Figure (14d): Relationship between peduncle length and peduncle width in Hydrocynus forskalii

1.60 $ Linear Regression

$ $ $ 1.40 0 log total length-10 = 0.94 + 0.73 * ld2 $ 1 R-Square =$ 0.33 $

h- $ $ $ $ $ $ ngt $ e $ $

l 1.20 $ $ $ $ $ $ $ l $$$ a $ $ $ $ $ ot $ $ $ t $ $ $ $

og og $ $ l 1.00

0.80 $

0.00 0.20 0.40 0.60 log(depth-2)

Figure (15a): Relationship between length and depth in Hydrocynus brevis

$ Linear Regression 50.00

h t 40.00

e l

d r

a 30.00 $

d standard length = 11.01 + 31.14 * ld2 n

a R-Square = 0.33$ $

t $ $

s $ $ $ $ $ $ $$$$ $ $ $ $ 20.00 $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $

Figure (15b): Relationship between standard length and depth in Hydrocynus brevis

$ Linear Regression $

5.00 $ $ $ $

h $ $

t $ d

i $ etner orpital width = 1.70$ + 2.16 * ed

$ $ $ $ w

R-Square = 0.18

l $ $ $

a $$$ $ $ $

t 4.00

i $ $

p $

r $

e $

n t e $ 3.00

1.00 1.10 1.20 1.30 EYE DIAMETER

Figure (15c): Relationship between eye diameter and inter orbital width in Hydrocynus brevis

$ Linear Regression

3.00 $

h $ $ t pendical length = 1.73 + 0.16 * pdxpd g $ $

n R-Square = 0.43 e

l $ $ $

a $ $ $

c i

d 2.50 $ $ $ $ n

e $ $$$ p $

Figure (15d): Relationship between peduncle and peduncle width in Hydrocynus brevis

3.3 CYTOGENETIC DATA: Results of the cytogenetic study of species from the two genera are shown from different liver cells mitotic plates in figures (16) through (41). Figure (16): shows early prophase of Hydrocynus brevis from the White Nile, Jebel Aulia, figure (17) shows late prophase of Hydrocynus brevis from Lake Nubia (Wadi Halfa) while figure (18) shows late metaphase of Hydrocynus brevis from the White Nile. Figure (19) shows a well spread metaphase plate of Hydrocynus lineatus from Lake Nubia, figure (20) represents interphase from Hydrocynus brevis liver tissue and figure (21) shows telophase of Hydrocynus lineatus collected in Lake Nubia. Figure (22) shows a metaphase in Alestes baremose from the White Nile, Jebel Aulia and figure (23) shows metaphase of Alestes baremose from Jebel Aulia as well; both reveal the large chromosome number of Alestes species. Chromosomes of Hydrocynus brevis from the White Nile are shown in figure (24) revealing mitotic anaphase stage and figure (25) showing metaphase. The chromosome number appears less than that of Alestes species. Mitotic chromosomes of Alestes baremose show a phenomenon of chromosomal fusion clearly revealed in figure (26): small chromosomes can be seen surrounding “large” chromosomes. Alestes dentex karyotypes is represented by anaphase in figure (27) from a Lake Nubia specimen, a late prophase seen in figure (28) representing White Nile and a clear mitotic metaphase of a lake Nubia fish (figure 29). Figures (29) (30) and (31) show metaphase, prophase and anaphase stages from Alestes dentex. Comparison of karyotypes of the species from Lake Nubia and White Nile, Jebel Aulia reveals an obvious difference between the two geographic representatives as can be seen clearly in figures (29) and (31). Figure (31) suggests ploydy and figure (32) shows mitotic metaphase chromosomes arranged at the centre of a liver cell of Alestes baremose from Lake Nubia Figure (33) and (34) showing metaphase from Hydrocynus brevis from Lake Nubia and White Nile, Jebel Aulia can be examples of the small chromosome size of this species; figure (34) shows the differential staining of chromatin. A well stained prophase cell of Hydrocynus lineatus can be seen in figures (35) and (39). Hydrocynus forskalii karyotypes are shown by figures (36), (37) and (38). Ideograms of karyotypes representing Hydrocynus sp. and Alestes sp. are depicted in figures (40) and (41) respectively. Chromosome number was counted from several mitotic phases. The two figures confirm that Hydrocynus sp. has a karyotypes of a diploid chromosome number of 2n=56 and Alestes has a chromosomal complement of 2n=42 chromosomes as seen in figures (39) and (40) different chromosomes can be differenced by their centromere position; in Hydrocynus sp. six chromosomes are metacentric, fourteen are telocentric and eight are acrocntric while in Alestes sp. five chromosomes were found to be metacentric, three acrocentric and thirteen telocentric chromosomes.

Figure (16): Prophase from liver tissue of Hydrocynus brevis from the White Nile

Figure (17): Late prophase from liver tissue of Hydrocynus brevis collected from Lake Nubia

Figure (18): Metaphase from Hydrocynus brevis collected at the White Nile.

Figure (19): Metaphase plate from Hydrocynus lineatus collected at Lake Nubia

Figure (20): Interphase from Hydrocynus brevis caught at Lake Nubia

Figure (21): Telophase stage from Hydrocynus lineatus collected at Lake Nubia

Figure (22): Metaphase plate from Alestes baremose collected from the White Nile,Jebel Aulia

Figure (23): Metaphase plate from Alestes baremose specimens collected at Jebel Aulia in the White Nile.

Figure (24): Late Anaphase plate from Hydrocynus brevis caught from The White Nile (Jebel Aulia)

Figure (25): Metaphase of Hydrocynus brevis from the White Nile (Jebel Aulia)

Figure (26): Anaphase of Alestes baremose from the White Nile (note the “fusion” of chromosomes at the centre of the cell)

Figure (27): Anaphase from Alestes dentex from Lake Nubia

Figure (28): Late prophase in Alestes dentex from the White Nile.

Figure (29): Metaphase from Alestes dentex collected at Lake Nubia (metacentric chromosomes are seen as X-shaped)

Figure (30): Cells in early prophase stage from Hydrocynus brevis from Lake Nubia (note the differential chromatin staining)

Figure (31): Anaphase plate from Alestes dentex from Jebel Aulia (number of chromosomes suggests ploidy, note the differential staining)

Figure (32): Metaphase stage from Alestes baremose from Lake Nubia (chromosomes are lined at the middle of the liver cell)

Figure (33): Metaphase plate from Hydrocynus brevis caught from Lake Nubia (note the tendency of chromosomes to stick together)

Figure (34): Geimsa-stained liver cell showing metaphase from Hydrocynus brevis collected from the White Nile (note heterochromatic staining)

Figure (35): Prophase from Hydrocynus lineatus from the White Nile (note that chromosomes started to differentiate)

Figure (36): Metaphase plate from Hydrocynus forskalii

Figure (37): Late prophase from Hydrocynus forskalii.

Figure (38): Late Anaphase from Hydrocynus forsakalii.

Figure (39): Metaphase of Hydrocynus lineatus

Figu re (40): Ideo gram repre senti ng the kary otyp e of Hydrocynus sp. chromosomes arranged according to descending length.

Figure (41): Ideogram of karyotype of Alestes sp. chromosomes arranged according to length. 3.4 PROTEIN POLYMORPHISM: Figures (42) to (47) show the electrophoretically separated bands of liver soluble proteins of Hydrocynus and Alestes fish species from Lake Nubia and the White Nile sites. Results revealed the presence of polymorphic protein bands distinctive for populations of the same species in the two different localities. The 11 polymorphic bands designated have molecular weights ranging from 250 to 20 KDa. The results presented here are chosen from among 20 electrophoretic gels. Eight replicas are presented for the comparison of polymorphic bands. Figure (48) shows the very clear bands visible in the electrophoretic gel from Alestes dentex; band sizes ranged between 250 kDa (as band number one) to 20 kDa (as in band number 21). Polymorphism is shown between Alestes dentex and Alestes baremose in Lake Nubia, where Alestes baremose bands are slightly lighter. Figure (49) shows the comparison between two populations of Hydrocynus brevis and Hydrocynus lineatus from Lake Nubia and the White Nile.

Figure (42): Electrophoresis bands of the soluble liver protein of Hydrocynus and Alestes collected from Lake Nubia

Figure (43): Electrophoresis bands of the soluble liver protein of Hydrocynus and Alestes collected from the White Nile.

Figure (44): Electrophoresis bands of the soluble muscle tissue- proteins of Hydrocynus and Alestes collected from the White Nile.

Figure (45): Electrophoresis bands of the soluble liver proteins of Hydrocynus and Alestes collected from Lake Nubia

Figure (46): Electrophoresis bands of the soluble muscle tissue- proteins of Hydrocynus and Alestes collected from Lake Nubia

Figure (47): Electrophoresis bands of the soluble liver proteins of Hydrocynus and Alestes collected from Lake Nubia

0 21 19 17 15 13 11 9 7 5 3 1 A.B Wadi Halfa

A.B Jebal Aulia No.of Bands

1 Apperance of Precipitated Proten of Precipitated Apperance 0 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

A .D Wadi Half a No. of Bands A.D Jebal Aulia

Figure (48): Comparison between two different populations of Alestes dentex and Alestes baremose from Lake Nubia and the White Nile.

1 Apperance of Precipitated Proten

0 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 H.B Wadi Half a No. of Bands H.B Jebel Aulia

1 Apperance of PrecipitatedProten

0 H.L Wadi21 20Halfa19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

H.L Jebal Aulia No. of Bands

Figure (49): Comparison between two populations of Hydrocynus brevis and Hydrocynus lineatus from Lake Nubia and the White Nile. CHAPTER FOUR

DISCUSSION AND CONCLUSIONS

4.1. DISCUSSION: 4.1.1. Morphometric Traits Given the results obtained from the catch of the eight species of Alestes and Hydrocynus it is clear that there is variation in availability and number of species as well as individuals. This can be explained by variations in timing of collecting samples and variation in feeding and environmental factors depending on water content and the movement of fish groups along the Nile system. It is well established that the main Nile, the Blue Nile and the White Nile differ considerably in their general biological conditions and hence in their fish populations. As expected by Sandon (1950), great changes in the Nile system were brought in through dam and canal building activities which affected distribution of fish in the Nile. The presence of a single Alestes macrolepidotus specimen from the White Nile is quite interesting. Although it goes along with the previously recorded habitat for this species, it raises the question of whether Alestes macrolepidotus is becoming one of the endangered species. The magnificent work of Boulenger (1907), Stubbs (1949), Sandon (1950) and Abu Gideiri (1975, 1984) on identification of freshwater fish of the Nile system makes the basis for the study of fish and fisheries in Sudan. All these studies used morphometric traits to distinguish fish taxa of the Nile water system. Characters like body shape, fins, mouth, teeth, gills, operculum, gill rakers, nostrils, lateral line, scales, sexual differences, colour and markings, as well as measurements of body parts are the major distinguishing characters between fish. Although useful to differentiate fish to the generic level, keys based on morphological traits are sometimes limited in differentiating species and sub-species. For example the value of the length is limited by the well known fact that fish grow to different maximum sizes in different places. It is a fairly general rule that the fish of any species attain greater sizes in large lakes than in smaller ponds or rivers and in large rivers than in smaller ones (Sandon, 1950). The results obtained from morphometric measurements are comparable to those reported in the literature (Sandon, 1950; Abu Gedeiri, 1984). A suggestion of regrouping the species in both genera is well supported from this study for the fact that certain measurements and ratios revealed significant variation. Although morphometric measurements in this study are based on the same parameters of specific relationships accounting for L, L/D, SL, D, SL/D, IOW/ED, PL/PD, for the eight species studied from both localities (as presented in tables I to XV). The measurements and ratios differed from those reported in the literature by Abu Gideiri (1984). However differences from ratios obtained in the literature can be attributed to: a- Personal factors: Differences could be attributed to different tools used in this study (vernier) compared to the use of ruler in the literature. b- Differences in environmental conditions: Generally, morphological characters and measurements are often overlapping and affected by environmental factors that play an important part in the general growth of fishes and hence the variation in the ratio is expected (Bishai and Abu Gidieri 1987; Kirpichnikov, 1981; VanDer Bank and Ferreira 1986, 1987). c- Sex differentiation: Perhaps there are differences in the measurements and ratios between the two sexes of each species. There was a difficulty in differentiating males from females in this study because fishes were caught off the breeding season and the gonads appeared just as undifferentiated fatty bodies. The zigzag pattern and the deviation from the straight line seen in figures (10aÂ 15a) could also be due to differences in morphometric measurements and ratios between males and females. d- Insignificance in calculations: The measurements could have been more informative if the fishes were reared in the laboratory, for as stated by Bishai and Abu Gidieri (1967), the ratios recorded from fishes reared in the laboratory show slight differences from those found in natural waters. In addition, if the fishes were grouped into males and females, then the morphometric measurements could have been of better significance. Of course one could not rule out results of interbreeding and accumulation of mutations in both Hydrocynus and Alestes species especially from Lake Nubia.

4.1.1. Cytogenetic Patterns Fish taxonomy has witnessed a revolution in the last few decades by introduction of genetic data such as karyotyping, protein and DNA polymorphism as additional tools for accurate ranking of taxa. This study is meant to use some of these tools to review the taxonomical status of Hydrocynus and Alestes species. El-Fadil (1999) carried out a study to determine the karyotypes of two Nile fishes from the genus Clarias of the family Mochokidae, order Siluroidea, using the an enhanced squash method after the injection of the fishes with colchicine. The chromosome number wasfound to be (2n = 28) for Clarias spp. From the Blue Nile and (2n = 20) for Clarias spp. from the White Nile. The chromosome size was somehow large compared to other findings in the literature. This result suggests two genetically different fish and would not be of the same species because of the large difference in chromosome number between the two species. This is one example of the taxonomic issues encountered when more fine tools are used. The chromosome number and the size of the chromosomes found in this study fall within the range of fish chromosome number cited in the literature as Hydrocynus sp. has a diploid chromosome number of 2n=56 and Alestes sp. has a chromosomal complement of 2n=42. Hydrocynus sp. Members with the larger number of chromosomes showed small-sized chromosomes while the fewer-numbered chromosomes of Alestes species retained larger sizes. The chromosome number is constant for species within each of the two genera although there are differences in the structure of chromosomes between the species. For example the result of comparison of karyotypes of the Alestes dentex species from Lake Nubia and the White Nile reveals an obvious difference between the two geographic representatives as seen in figures (29) and (31). Again this result opens the door for re-grouping of the species in the two geographical localities. Chromosomal aberrations can be seen in the mitotic chromosomes of Alestes baremose which show a phenomenon of chromosomal fusions as revealed in figure (26) where small chromosomes can be seen surrounding “large” chromosomes. Fusion of chromosomes at certain mitotic stages is not odd to this species. This is of particular interest since chromosomal fusions are thought to be one way of change and hence divergence. More interestingly is the fact that the small chromosomes of Hydrocynus sp. are paralleled by the few, large chromosomes of Alestes sp.. This can be argued for explaining the very marked differences between members of the two genera (2n=56 and 2n- 42). It is tempting to speculate that the difference of 14 chromosomes between the two genera is encountered in chromosomal fusions especially when one considers the chromosome structures in figures (39) and (40) where in Hydrocynus sp. there are six metacentric chromosomes, fourteen telocentric and eight acrocntric, compared to Alestes sp. with five metacentric, three acrocentric and thirteen telocentric chromosomes. Another peculiarity in chromosomal pattern is the ploidy suggested in Alestes dentex.

4.1.3. Protein Polymorphism The enormous differences in the total protein profiles between the same species from Lake Nubia and the White Nile are expected since changes in the habitat are known to induce environmental adaptations in fish populations, which are reflected at the protein level. Observations made of the genetic variation between the populations in species of Hydrocynus and Alestes in the two water systems (Lake Nubia and White Nile) suggest that these species are not identical and there are polymorphisms existing between them. This supported by variation in the morphometric measurements of each species from the two water systems. Results of molecular and genetic variation within species from the water system may be attributed to the presence of different strains of the species which are not identified as yet.

4.2. Conclusions and Recommendations This study presented a preliminary investigation of Hydrocynus and Alestes species in Lake Nubia and the White Nile freshwater systems. The aspects touched by this study are very important in studies of the phylogeny of the family Alistidae. It suggests genetic variation within and between different species of two genera representative of the family that is supported by morphological data. Although the information gathered leads to assumptions of variable species in the two water systems (especially Alestes dentex) more studies are needed to assign the specific taxa accurate ranks. This could be done by more future studies. Three lines of studies are recommended: 1. A detailed genetic study of each species from both water systems where fluorescent techniques of chromosome banding are essential. 2. A thorough typing of enzymes from populations within each of the eight species. Serum and liver proteins should be studied extensively. 3. A parallel study using polymerase chain reaction (PCR) to amplify informative regions of the fish genome using universal fish primers (cytochrome b of the fish mitochondria being a conserved gene proved to be useful). Although this study might be expensive, it is essential as an accurate way of inferring fish phylyogenies.

The difficulties faced during this study were mainly of technical nature for example non availability of reagents, difficulties of sampling in the right season and relaying on the available facilities where the same efforts if used in advanced molecular phylogeny, results would have been more conclusive. However for the duration of the study with the limiting factors mentioned above, the results of this study shed light into one important area of fisheries. Overall the study makes important observations of genetic divergences among Hydrocynus and Alestes species in Lake Nubia and the White Nile freshwater systems. REFERENCES

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APPENDICES

APPENDIX 1 ABBREVIATIONS

ANOVA: One way analysis of variance APS: Amonium persulphate Cat: Catalase DNA: Deoxyribonucleic acid; the chemical material of which genes are made Es: Esterase FAO: Food and Agriculture Organization of the United Nations Agency G3pd: Glyceraldehyde-3-phosphate dehydrogenase G6pd: Glucose-6-phosphate dehydrogenase Gpi: Glucose phosphate isomerase Hb: Haemoglobin; the oxygen-transporting blob protein in most animals KDa: Kilo Dalton, a unit for measuring protein size Mdh:Malate dehydrogenase PCR: polymerase chain reaction (a method of replicating a DNA sequence once the sequence has been identified) Pgm: Phosphoglucomutase r.p.m.: Round per minute SDS: Sodium dodecyl sulphate Sod: Superoxidase dismutase SPSS: Statistical package for social science TEMED: (N-N-N-N tetra-methyl-ethylediamine) is a catalyst used to accelerate formation of free radicals from ammonium persulphate. WHO: World Health Organization

APPENDIX 2 GLOSSARY

Allele: One of a pair, or series, of alternative genes that occur at a single given locus in a chromosome Anaphase: An intermediate stage of nuclear division during which chromosomes are pulled to the poles of the cell. Autosome: A chromosome not involved in sex determination Centromere: Spindle-fiber attachment region of a chromosome Chromatin: The genetic term for any complex of DNA and protein found in a cell’s nucleus Deletion: Absence of a segment of a chromosome involving one or more genes Duplication: The occurrence of a segment more than once in the same chromosome or genome. Electrophoresis: The migration of suspended particles or components of a mixture of molecules (protein/DNA) in an electric field within a gel. Genome: A complete set of chromosomes (hence genes) in each cell of an organism inherited as a unit Genotype: The genetic constitution (gene makeup) of an organism Haploid: A cell having only one complete set (n) of chromosomes of one genome Heterochromatin: Densely-staining condensed chromosomal regions, believed to be genetically inert and functioning differently than the euhromatin that contains the coding region Heterozygote: An organism with unlike members of any given pair or series of alleles that consequently produce unlike gametes Heterozygsity: Relative number of loci in a given individual in the heterozygous state Homologous chromosomes: Chromosomes that pair with each other at meiosis Ideogram: Diagrammatic representation of karyotype in which chromosomes arranged according to descending size Inversion: A rearrangement of a linear array of genes in a chromosome in such a way that their order in the chromosome is reversed. Karyotype: The chromosome constitution of a cell or an individual arranged in order of size. Metaphase: A stage in mitosis or meiosis during which the chromosomes are aligned along the equatorial plane of the cell Monosomy: lack of one chromosome in a diploid organism (2n-1) Mosaic: A chimera; a tissue containing two or more genetically distinct cell types or an individual composed of such tissues. Phenotype: An observable characteristic Ploidy: The number of chromosome sets Polymorphism: The occurrence in a population (or among populations) of several phenotypic forms associated with alleles of one gene or homologs of one chromosome. Polyploidy: A condition where an organism has more than 2 sets of chromosomes Translocation: Change in position of a segment of a chromosome to another part of the same chromosome or to a different chromosome