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Feeding Habit, Fecun Characterization of Sc In FEEDING HABIT, FECUNDITY AND MOLECULAR CHARACTERIZATION OF SCHIZOTHORAX PLAGIOSTOMUS IN KHYBER PAKHTUNKHWA, PAKISTAN MUHAMMAD QAYASH KHAN 13-arid-3853 DEPARTMENT OF ZOOLOGY/BIOLOGY FACULTY OF SCIENCES PIR MEHR ALI SHAH ARID AGRICULTURE UNIVERSITY RAWALPINDI PAKISTAN 2019 FEEDING HABIT, FECUNDITY AND MOLECULAR CHARACTERIZATION OF SCHIZOTHORAX PLAGIOSTOMUS IN KHYBER PAKHTUNKHWA, PAKISTAN by MUHAMMAD QAYASH KHAN (13-arid-3853) A thesis submitted in partial fulfillment of the requirement for degree of Doctor of Philosophy in Zoology DEPARTMENT OF ZOOLOGY/BIOLOGY FACULTY OF SCIENCES PIR MEHR ALI SHAH ARID AGRICULTURE UNIVERSITY RAWALPINDI PAKISTAN 2019 ii iii iv v In The Name Of Allah, the Most Gracious, The Most Merciful. You (Alone) We Worship, And To You (Alone) We Turn For Help. Guide Us (O Lord) To the Path Is Straight. (Surah Al- Fatiha; Verse 4-5) vi In dedication to my late Mother and Father for making me be who I am, and my Wife and Sons for supporting me all the way! 7 CONTENTS Page List of Tables viii List of Figures xi List of Plates xiii List of Abbreviations xiv Acknowledgements xv Chapter 1 INTRODUCTION 1 Chapter 2 REVIEW OF LITERATURE 13 Chapter 3 MATERIAL AND METHODS 26 3.1 STUDY AREA 26 3.1.2 Description of River Indus 27 3.2 PHYSICO-CHEMICAL PARAMETERS 27 3.3 CATCHABILITY AND ABUNDANCE 27 3.4 GUT CONTENTS ANALYSIS 28 3.4.1 Frequency of Occurrence 29 3.4.2 Volumetric Analysis Index 29 3.4.3 Point Ascription 29 3.4.4 Importance Index 30 3.5 REPRODUCTIVE BEHAVIOUR, GSI AND FECUNDITY 30 3.5.1 Length Weight Relationship 31 3.5.2 Condition Factor 32 3.5.3 Calculation of Female to Male Ratio 32 3.5.4 Determination of Gonado-Somatic Index GSI 32 3.5.5 Estimation of Fecundity 32 3.6 MOLECULAR CHARACTERIZATION 33 3.6.1 DNA Extraction 33 3.6.2 PCR Amplification 35 3.6.3 In-Silico Analysis 36 Chapter 4 RESULTS 43 8 4.1 PHYSICO-CHEMICAL PARAMETERS OF WATER 43 4.2 FISH DISTRIBUTION AND ABUNDANCE 44 4.3 GUT CONTENT ANALYSIS AND FEEDING HABIT OF S. 45 PLAGIOSTOMUS 4.4 REPRODUCTIVE BEHAVIOUR AND SPAWNING PERIODS 47 OF S. PLAGIOSTOMUS 4.4.1 Length vs Body Weight male 48 4.4.2 Length vs Body Weight female 51 4.4.3 Condition Factor 53 4.4.4 Sex Percentage 53 4.4.5 Curvilinear Regression Analysis 54 4.4.5.1 Total body Length vs GSI Male S. plagiostomus 54 4.4.5.2 Body Weight vs GSI (Male) 56 4.4.5.3 GSI vs TL Female 58 4.4.5.4 Body Weight vs GSI (Female) 60 4.4.5.5 Assessment of Fecundity in Female S. plagiostomus 61 4.5 IN-SILICO ANALYSIS 63 4.5.1 Phylogenetic Tree 64 Chapter 5 DISCUSSION 112 5.1 FISH DISTRIBUTION AND ABUNDANCE 112 5.2 FEEDING HABIT AND ANALYSIS OF GUT CONTENTS 112 5.3 REPRODUCTION AND STAGES OF MATURITY 115 5.3.1 Spawning Studies 115 5.3.2 Length Weight Relationship & Condition Factor 116 5.3.3 Sex Composition 118 5.3.4 Gonad-somatic Index (GSI) and Spawning Season 118 5.3.5 Relationship of GSI and Fecundity with TL, BWT, Ovary 120 Length and Ovary Weight 5.4 MOLECULAR CHARACTERIZATION 121 5.4.1 Phylogenetic Analysis 122 SUMMARY 124 LITERATURE CITED 128 9 LIST OF TABLES Table. No. Page 3.1 Stages of gonad maturity for cyprinids as described by 38 (Nagelkerke, 1997). 4 Mean values of physico-chemical parameter with standard error 66 in parenthesis of water from river Indus and its tributaries recorded in different seasons (autumn, winter, spring and summer) during 2016. 4.1 Post hoc (Dunnett T) test of physico-chemical parameters of 67 water from river Indus and its tributaries to reveal seasonal variations (autumn, winter, spring and summer). 4.2 Mean values of physico-chemical parameters of water with 68 standard error in parenthesis from various localities of Indus River and its tributaries. 4.3 Speciesabundance of Schizothorax plagiostomu caught from 70 different tributaries along river Indus. 4.3.1 Multiple comparison of mean CPUE (post hoc Dunnett test) 71 calculated for different collection sites along river Indus. 4.3.2 Seasonal variations in CPUE of Schizothorax plagiostomus 72 recorded in different tributaries along river Indus in Indus Kohistan KP, Pakistan during 2016. 4.3.3 Month wise multiple comparison (post hoc Dunnett test) of 74 mean CPUE calculated for different collection sites along river Indus. 4.4 Gut Content Analysis of Schizothoraxplagiostomus caught from 76 river Indus and its tributaries during 2016. 4.4.1 Gut content analysis of Schizothoraxplagiostomus caught from 77 river Indus and its Tributaries during 2016. 4.4.2 Gut Content Analysis of Schizothoraxplagiostomus caught from 78 river Indus and its Tributaries during 2016. 4.4.3 Gut Content Analysis of Schizothoraxplagiostomus caught from 79 river Indus and its Tributaries during 2016. 10 4.4.4 Seasonal Variations in the Importance index, Mean Rank of 81 Importance Index and mean Kruskal-Wallis Rank Values recorded for the Gut Content Analysis in S. plagiostomus caught from river Indus during 2016. 4.4.5 Annual Seasonal Variations recorded in different Food Items in 82 the Gut of S. plagiostomus caught from river Indus during 2016. 4.5 Monthly Descriptive Statistics and Regression Parameters 83 (quadratic) of Length-Weight Relationship of S. plagiostomus (male) caught from river Indus and its Tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.5.1 Monthly Descriptive Statistics and Regression Parameters of 85 Length-Weight Relationship of S. plagiostomus (female) caught from river Indus and its Tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.5.2 Month wise descriptive statistics of condition factor (K-value) 88 with Std. deviation and overall P-value for male S. plagiostomus caught from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.5.3 Month wise condition factor (K-value) with Std. deviation and 90 overall p-value of female S. plagiostomus caught from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.5.4 Sex Ratio of S. plagiostomus (Month-wise) with Chi Square and 93 P-values caught from river Indus during 2016. 4.5.5 Monthly Descriptive Statistics and Regression Parameters 95 between TL and GSI of Male S. plagiostomus caught from river Indus and its Tributaries during 2016, in Indus Kohistan KP, Pakistan 4.5.6 The Monthly Descriptive and Curvilinear Regression Analysis 96 (quadratic) of Body Weight vs GSI in Male S. plagiostomus caught from river Indus and its Tributaries in Indus Kohistan, KP, Pakistan during 2016. 4.5.7 The monthly descriptive and curvilinear regression analysis 97 (quadratic) of TL vs GSI in Female S. plagiostomus caught from river Indus and its Tributaries in Indus Kohistan, KP, Pakistan during 2016 4.5.8 The Monthly Descriptive and Curvilinear Regression Analysis 98 (quadratic) of Body Weight vs GSI in Female S. plagiostomus caught from river Indus and its Tributaries in Indus Kohistan, KP, Pakistan during 2016. 4.5.9 Monthly Variations of Mean Absolute Fecundity and Means of 100 Body Parameters for Female S. plagiostomus caught from river Indus and its Tributaries in Indus Kohistan during 2016. 4.6 Average Nucleotide Frequencies (AT/GC composition) after 101 COI Amplification of Specimen Sequences. 4.6.1 Species Names, AT/GC Average Composition Identified during 102 11 this Study. 4.6.2 Estimates of Average Evolutionary Divergence between 103 specimen Sequences and NCBI–GenBank Sequences. 12 LIST OF FIGURES Figure. No. Page 3 Sampling sites at different streams along River Indus in Indus 26 Kohistan, KP, Pakistan. 4 Bar graph showing variation in mean CPUE of S. plagiostomus 69 caught from different stream along river Indus in Indus Kohistan KP, Pakistan during 2016. 4.1 Bar graph showing monthly variations in means CPUE of S. 73 plagiostomus caught from different stream along River Indus in Indus Kohistan KP, Pakistan during 2016. 4.2 Bar graph showing seasonal variation in the mean importance 80 index of different food items identifiedin gut of S. plagiostomus collected from river Indus during 2016. 4.3 Scatter plot showing curve estimation (quadratic) between total 84 body length (cm) and body weight (g) of male S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016. 4.3.1 Scatter plot showing curve estimation (quadratic) between total 86 body length (cm) and body weight (g) of female S. plagiostomus caught fromriver Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016. 4.3.2 Monthly variation in the condition factor of S. plagiostomus 87 (male) collected from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.3.3 Monthly variation in the mean condition factor with std. error 89 bar of S. plagiostomus (female) collected from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan. 4.3.4 Monthly variation in the percentage of gonad maturity stages 91 recorded in male S. plagiostomus caught from rive Indus during January to December 2016 (Gonad stages= I-VII). 4.3.5 Monthly variation in the percentages ofgonad maturity stages in 92 female S. plagiostomus caught from rive Indus during January to December 2016 (Gonad stages= I-VII). 4.3.6 Bar graph showing moth-wise count of male and female S. 94 plagiostomus caught from river Indus during 2016. 4.3.7 Line graph showing monthly variations of mean absolute 99 fecundity with standard error bar in female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan District, KP, Pakistan.
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