ABSTRACT
This study aim to investigate the morphological and genetic characterization of strains of Clariid fish species in some river bodies in Kano Stateusing microsatellite markers.One hundred and seventy seven Clariid fish samples (Clariasgariepinus and Heterobranchuslongifilis)were collected from six rivers (Thomas, Ghari, Tiga dam, Duddurun Gaya, Karaye and Bagwai) in Kano state. Body weight, twenty-two morphometric characteristics and four meristic counts were measured on each fish sample to determine the influence of river location, strain of fish and sex.Body weight was measured in grams using sensory scale, the morphometric measurements were measured in centimetres using flexible tapewhile meristic counts were counted visually.The morphometric characteristics taken on the body were; Body weight (BW), Total length (TL), Standard length (SL), Pre-dorsal distance (PDD), Pre-anal distance (PAD), Pre-ventral distance (PVD), Pre-pectoral distance (PPD),Caudal peduncle depth (CPD), Body depth at anus (BDA); measurements taken on the fin were; Dorsal fin length (DFL), Anal fin length (AFL), Pectoral fin length (PFL), Pectoral spine length (PSL); measurements taken on the head region were;Dorso-caudal length (DDCF), Dorso-occipital length (DODF), Head length (HL), Head width (HW), Snout length (SNL), Inter-orbital distance (ID), Eye diameter (ED), Length of occipital fontanelle (OFL), Width of occipital fontanelle (OFW) and Snout-occipital length (DSO). The meristic counts were; Dorsal fin ray count (DFRC), Pectoral fin ray count (PFRC), Anal fin ray count (AFRC) and Caudal fin ray count (CFRC).The total length (TL) and body weight (BW) of each fish sample was used to compute Length-Weight relationships using the formula: W = log a + b log L and K = 100W/L3 was used to compute Condition Factor. Blood sample was taken from each fishsampleby severing the caudal peduncle and drained into FTA cards for DNA extraction, Polymerase Chain Reaction and electrophoresis to determine genetic variationbetween the Clariid fish populations. Data gotten from the morphometric measurements were analysed appropriately using GLM procedures of SAS 9.4 to show the influence of river location, strain, and sex, Duncan multiple range test was used for mean separation, Principal component analysis of SPSS was used for possible data reduction, and Genealex 6.4 software package was used to analyse the resolve bands from DNA extraction to determine their base pair and genetic variation. Body weight, morphometric measurements and meristic countswere significantly affected (P<0.01,0.05) by location
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and strain while sex had effect (P<0.01, 0.05) only on total length, standard length, dorsal fin length, dorso-caudal length, caudal peduncle depth, anal fin length, head length, inter-orbital distance, eye diameter and length of occipital fontanelle. The equation for the length-weight relationship for the three strains were: C. gariepinus = -329.86+17.56TL andH. longifilis= -241.49+14.28TL.The condition factors showed varying degree of wellbeing of fish samples in their habitat (K = 0.37 to 0.89). Tiga dam had the best condition factor (0.81-0.89) followed by fishes caught in River Ghari (0.74-0.88). Pearson correlation analysis for all the variables measured showed that relationship between Body Weight and all the morphometric measurements were positive and significant. The ‗r‘ values ranged from low (0.23) to high (0.80) for BW/PDD and BW/DDCF. The other measurements had positive and significant relationships with values ranging from 0.30 for ED/SL to 0.92 for TL/SL. Principal component analysis indicated that most of the variables could be used for discrimination with regard to the species with a total variance of 82.52% shared as 47.66%, 19.16%, 8.67% and 7.03% for PC1, PC2, PC3 and PC4, respectively. Among the populations sampled, the genetic similarity ranged from 0.018 to 0.079 whilethe genetic distance ranged from 0.112 to 0.998. The Fst values ranged from 0.000 to 0.663, Fit ranged from -0.041 to 0.115, Fis ranged from -0.350 to -0.262. The result indicated a large number of gene flow (exchange) among the populations with a range of 0.455 to 0.866. The populations were not genetically pure but heterogeneous with varying degrees of genetic similarity and distance. Since there was no inbreeding as shown in the study, none of the population exhibited genetic uniqueness. The populations had a high genetic differentiation between populations but moderate differentiation within populations. The populations were outbred populations; an indication that relatives avoided mating in the population. There was an established magnitude of genetic divergence (91.86%) among the populations as shown by the result of the percentage polymorphism which depends on the number of alleles detected per locus and their frequencies. The study indicated that river location and species of fish had a significant influence on Clariid fish morphometric measurements and meristic counts. The study also gave an indication that the growth pattern of Clariid species in Kano State Rivers was positive allometric growth pattern (b>3) and the Clariid fishes are in good condition of wellbeing as indicated in the condition factor.
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TABLE OF CONTENTS
Cover Page i Title Page ii Declaration iii Certification iv Acknowledgements v Dedication vi List of figures xi List of tables xii List of plates xvi Abstract xvii CHAPTER ONE 1 1.0 INTRODUCTION 1 1.1Animal variation 1 1.2Statement of research problem 3 1.3Justification of the study 4 1.4Objectives of the study 5 1.5Research hypothesis 6 CHAPTER TWO 7 2.0 LITERATURE REVIEW 7 2.1 The African catfish (Clariasgariepinus) 7 2.2Heterobranchuslongifilis 14
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2.3Fish farming in Nigeria 16 2.4Morphological and meristic characteristics 19 2.5Fish body shape and skin pigmentation 24 2.6 Length-Weight relationship and condition factor in aquatic organisms 25 2.7Identification of Clariid catfishes as important tools in breeding and genetics 30 2.7.1 Parameters necessary for fish identification 31 2.8 Genetic diversity or variability 32 2.9 Fish genetics 34 2.9.1 Emergence of fish genetics 34 2.9.2 Application of biotechnology in fish population genetics 37 2.9.3 Application of molecular markers 39 2.9.4 Marker-assisted selection (MAS) in fish 51 2.10 DNA extraction, Polymerase Chain Reaction and Elctrophoresis 52 2.11 Studies on Population genetics in aquaculture 57 CHAPTER THREE 62 3.0 MATERIALS AND METHODS 62 3.1 Study location 62 3.2 Morphometric characters, meristic count, length-weight relationship and condition factor in Clariasgariepinus andHeterobranchuslongifilisin Kano state 65 3.2.1 Morphometric measurements 66
3.2.2 Meristic counts 67
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3.2.3 Length-weight relationship and condition factor (Ponderal index) 68 3.3 Genetic variation within and among Clariasgariepinus and Heterobranchus longifilispopulations in Kano state 71 3.3.1 Sample collection and DNA extraction 71 3.3.2 Polymerase Chain Reaction 72 3.4 Data analysis 75 CHAPTER FOUR 77 4.0 RESULTS 77 4.1 Morphological and meristic characterization of Clariid species in Kano State 77 4.1.1 Condition factor of Clariid species in Kano state 98 4.1.2 Length-weight relationship in Clariid species in Kano state 98 4.1.3 Pearson correlation coefficient among morphometric masurements 101 4.1.4 Pearson correlation coefficient among morphometric masurements for Clariasgariepinus 105 4.1.5 Pearson correlation coefficient among morphometric masurements for Heterobranchuslongifilis 105
4.1.6 Principal component analysis for all the variables 112
4.2 Genetic characterization of Clariid (Clariasgariepinus and Heterobranchus
longifilis) species in Kano state. 124 CHAPTER FIVE 147 5.0 DISCUSSION 147 5.1 Effect of location, sex and strain on morphological and meristic counts in Clariid
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Species 147 5.2 Length-weight relationship and condition factor (ponderal index) for the clariid population sampled and their location 150 5.3 Relationships among the morphometric measurements 151 5.4 Principal component analysis for location and strains using the morphometric characteristics 151 5.5 Genetic variability and similarity of the strains of clariid species studied 152 Genetic similarity and relatedness 152 Genetic Distance 153 Population differentiation 153 Allelic variation 154 Genetic diversity and variability 155 CHAPTER SIX 156 6.0 SUMMARY CONCLUSION AND RECOMMENDATION 156 6.1 Summary 156 6.2 Conclusion 157 6.3 Recommendation 158 REFERENCES 161
CHAPTER ONE
1.0 INTRODUCTION 1.1 Animal Variation Variability is the fundamental and basic characteristics of life. Every level of organization of life displays variation in some parameters, in space or time, within and between cells, tissues, organisms, populations and communities. The existence of variations in natural populations of organisms is a necessary condition for evolution. While variability is both a product and foundation of the evolutionary process, biologists are still confronted with the basic problems of explaining the nature, extent and causes of this web of complexity (Reynaldo and Cesar, 2014). Genetic variation is one key factor in the survival of species. Natural populations are perhaps the best gene banks which are critical resources for genetic variation for current and future application in improvement of farmed species of fish (Dunham, 2004). Morphological differentiation is one of the several approaches which have proved useful in studying variability. Morphological data alone, however, is insufficient to explain variability. Molecular biology, biochemical analysis and other methods coupled with morphology are powerful means in understanding variability and evolutionary relationships among and within populations of organisms (Reynaldo and Cesar, 2014).
Among populations, genetic diversity can also be gained when populations that are not normally in contact with another hybridize that is when isolated population experienced migration, gene flow and genetic drift. This can occur when physical barriers are removed such as when fishes are introduced to an area or escape, or when migration patterns changes due to environmental condition. Populations of many species of organisms may
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respond differently, both morphologically and genetically, to a changed environment. Individuals tend to express different phenotypes (morphological, physiological or behavioural) when surviving in varied environments (Freeman and Herron, 1998). To this end, genetic studies of fish populations play an important role in the sustenance of genetic diversity (Seeb et al., 2007). Genetic markers can provide valuable information about geographic structuring, gene flow and demographic history of populations that can be highly relevant for conservation and management purposes (Maes and Volckaert, 2007).
Of all the animals and plants in the aquatic environment, fish is the most important source of human food (Yilmaz et al., 2000). Fish plays an important role in the development of a nation. Apart from being a cheap source of highly nutritive protein, it also contains other essential nutrients required by the body (Sikoki and Otobotekere, 1999). Fishes are highly important in the development of Nigeria both economically and health-wise as source of protein with low cholesterol level in the diets of many populace.Fish and fish products are economically significant as they provide jobs and investment opportunities and, for many countries, a means of improving the balance of international trade (Yilmaz et al., 2000). Fish is a high quality food and apart from its protein contents, it is also rich in vitamins and contains variable quantities of fat and minerals for human health (Adeniyiet al., 2010). Fish oil contains vitamins A, D, E and K which have been successfully used in controlling coronary heart diseases, arthritis, atherosclerosis, asthma, auto-immune deficiency diseases and cancer (Bhuiyan et al., 1993). Fish is often recommended for cardio-vascular disease patients because of its unique fat, which is composed mainly of Omega- 3 polyunsaturated fatty acid. In addition to its nutritious flesh, vitamins A and D present in fish oil are important especially for infants and children (Fasakin, 2006). Fish also supplies to the body, a range of inorganic minerals such as Phosphorus, Fluorine,
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Potassium, Iron, Zinc, Magnesium, and Copper and in marine species Iodine as well as vitamins A and B complex (Adeniyi et al., 2010). The proximate composition, nutritive values and mineral composition of fishes in Nigeria have been documented (Olatunde, 1980; Abdullahi and Abolude, 2006; Dankishiya and Kabir, 2006; Abdulkarim and Abdullahi, 2009). Most of the fish used for human consumption is obtained through exploitation of wild populations. Water quality tolerance of catfish is diverse due to environmental changes. The warmer the water, the less the dissolved oxygen likewise, the greater the altitude, the less the dissolves oxygen, causing severe cases and death aquatic organisms including catfish. According to F.A.O., (2003), water quality requirement for catfish are as follows; temperature – 26 to 32oC, dissolved oxygen – 3 to 10 mg/l or > 3ppm, pH – 6 to 8, Alkalinity – 50 to 250 mg/l, Ammonia – 0 to 0.03% and Nitrite – 0 to 0.6mg/l. It also reported that for advanced fry, the requirement are as follows; dissolved oxygen – 3-5ppm, temperature – 30oC, ammonia – 0.1 to 1.0ppm, nitrite – 0.5ppm, nitrate – 100ppm, pH – 6 to 9, carbon dioxide – 6 to 15ppm and salinity – 10 to 16ppt. 1.2 Statement of Research Problem
Reduction in the genetic resources of natural fish populations is an important management problem. Not only has the genetic diversity of many fish populations been altered, but many populations and species have been extirpated by pollution, overfishing, destruction of habitat, blockage of migration routes and other human developments (Ferguson, 1995). Loss of genetic diversity and locally adapted populations (and species) can compromise stability and recovery potential of marine ecosystems as well as impair their ability to adapt to changing environmental condition.There is generally limited information on
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genetic variation amongand within Clarias and Heterobranchus species and this greatly hampers an efficient and sustainable exploitation of these resources (Worm et al., 2009). 1.3Justification of the Study The quantification of specific characteristics of an organism or group of organisms is a demonstration of the degree of speciation induced by both biotic and abiotic conditions, thereby contributing to the definition of different stock of species (Bailey, 1997). The African catfish Clarias gariepinus and Heterobranchus longifilis are economically important species, but little is known about the genetic background of the natural populations of these species. Also, genetic study is needed for proper identification of the two species and determination of the genetic connection between them. Although, morphometric parameters have been used in the past to identify these too species but more specific tool is needed for a more concise differentiation.
Genetic improvement of aquaculture species offers a substantial opportunity for increasing production efficiency, health, product quality and ultimate profitability. It entails a lot of parameters including the description of their population and differences. When different populations arise, with little or no connection between them, they become genetically different from one another. Loss of such populations thus results in loss of genetic diversity within the species. The existence of multiple spawning units is an indicator that populations may be reproductively isolated, particularly if the species shows spawning site fidelity or homing ability. The organization of these populations in time and space, along with the ratio of within and among population variation are important to maintain in order to avoid negative genetic effects (Altukhov and Salmnekova, 1994). Scientifically, sound management of fish resources relies on the information on the biology of the species which include information on population structure.
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There is little information on the genetic characteristics of clariid species of fish in natural waters in Northern Nigeria, especially Kano State. The study is expected to made known some useful inferences about the magnitude of stock delimitation may be possible with the notion that genetics-cum-environment influences phenotypic expression. Microsatellite DNA marker has been the most widely used for genetic studies, due to its easy use by simple Polymerase Chain Reaction, followed by a denaturing gel electrophoresis for allele size determination, and to the high degree of information provided by its large number of alleles per locus(Vignal et al.,2002).
1.4 Objectives of the Study
The aim of the study is to characterize morphologically and genetically the clariid species in Kano State using microsatellite markers. The specific objectives are as follows:
i) To determine morphometric characters and meristic counts of Clariid species in Kano state
ii) To determine the influence of location, strain and sex on the morphometric and meristic attributes of Clariid species.
iii) To determine length-weight relationships and condition factor (ponderal index) in Clariid species in Kano State
iv) To evaluate genetic variation within and among Clariid species populations in Kano state
v) To determine relationships between morphometric measurements from the studied fish populations.
1.5 Research Hypotheses (Null and Alternative)
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i Ho: Location, strain and sex does not have any influence on the morphometric and meristic count in Clariid species. Ha: Location, strain and sex does have any influence on the morphometric and meristic count in Clariid species. ii. Ho: The length-weight relationship and condition factor of Clariid species do not differ. Ha: The length-weight relationship and condition factor between Clariid species differ. iii. Ho: There is no genetic variation within and among Clariid populations. Ha: There is genetic variation within and among Clariid populations. iv. Ho: There is no relationship between morphometric measurements in the studied clariid populations. Ha: There is relationship between morphometric measurements in the studied clariid populations.
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