FEEDING HABIT, FECUNDITY AND MOLECULAR CHARACTERIZATION OF SCHIZOTHORAX PLAGIOSTOMUS IN KHYBER PAKHTUNKHWA,

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 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 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. 4.4 Phylogenetic Tree using NJ method showing the Relationship 104 of 28-Sequences with 16 Schizothorax spp. COI sequences from NCBI GenBank. 4.4.1 Nucleotide sequence comparison (GeneDoc) of fish CO1 111

13 sequenced in the present study from different streams adjoining river Indus in Indus Kohistan, Pakistan. Black and grey indicate 100 and 80–90 % sequence identity, respectively.

14 LIST OF PLATES

Plate. No. Page 1 Snow Barbell, Kerai in Pashto, Khont in Kashmiri, 3 Schizothoraxplagiostomus

3.1 Fish Sampling along river Indus in Indus Kohistan KP, 37 Pakistan during 2016.

3.2 Gut Extraction from S. plagiostomus for Gut Content Analysis 39 3.3 Extraction of gonads from S. plagiostomus, for the assessment 40 breeding behavior.

3.4 Extraction of DNA from the muscle tissues of S. plagiostomus. 41 3.4.1 Bands visualization of PCR products (COI gene) in a GelDOC 42 showing COI amplification with 1kb gel ladder.

4.1 Different food items collected from the gut of S. plagiostomus. 75

15 LIST OF ABBREVATIONS A Adenine ANOVA Analysis of Variance APHA American Public Health Association b0, b1, b2 Beta Coefficients BWT Body Weight C Cytosine Cm Centimeter COI Cytochrome C Oxidase Gene I Sub-Unit CPUE Catch per Unit Effort DO Dissolved Oxygen G Guanine Gm Grams GSI Gonado-Somatic Index IUCN International Union for the Conservation of Nature Kg Kilo Gram KP Khyber Pakhtunkhwa mg Milligrams MT Metric Tons pH Hydrogen Ion Concentration ppm Parts per million r2 Coefficient of Determination S Schizothorax SD Standard Deviation Spp. Species T Thymine TDS Total Dissolved Solids TL Total Body Length

16 ACKNOWLEDGEMENTS

All glory be to God, the most gracious, compassionate and merciful, who bestowed on me the potential and ability to complete my research work and whose countless blessings enabled me to perceive and pursue higher ideal of life.

First of all, I am heartily thankful to Prof. Dr. Shamim Akhtar, Chairperson Department of Zoology/Biology, PMAS-AAUR for his motivation and affectionate assistance during the completion of my research work.

I owe my deep and sincere thanks and gratitude to my research supervisor Dr. Muhammad ZubairAnjum, Assistant Professor, Department of Zoology, PMAS- Arid Agriculture University, Rawalpindi whose dynamic supervision, constant guidance, illustrative advice, keen interest, encouragement, every possible cooperation and provision of relevant literature made possible the completion of my research work.

I am thankful to Prof. Dr. MazharQayyum and all facultyDepartment of Zoology, PMAS- Arid Agriculture University, Rawalpindi for their guidance, high concern, affectionate assistance and cooperation particularly in statistical approaches.

Many thanks to Dr. Abid Ali and Dr. Muhammad Khursheed, Assistant Professors, Department of Zoology AWKUM, for their support and high concern particularly in molecular analysis.

I am greatly thankful to my mother (late) and brothers, wife and children who have been a constant source of encouragement and enlightenment in the pursuit of knowledge and whose prayers and good wishes have always been a great strength to me.

Muhammad Qayash Khan

17 CHAPTER 1

INTRODUCTION

Pakistan is, basically, is a land of geological and geographical diversity.The country is blessed with large natural inland aquatic resources in the form of rivers streams and its tributaries, world largest irrigation system, natural and man-made lakes. In Pakistan the freshwater capture is dominated by river and its tributaries. The fish fauna of river Indus system in its northern part is cold-water type while the large middle and southern portion of the system is dominated by zones of warm- waterfisheries. The fisheries production from these resources and reservoirs accounts for more than 80 % of total inland fish production.Fisheries play an important rolein the national economy. In Pakistan the fisheries sector contributesabout 0.4% in the gross domestic product (GDP). During the year 2014-15, a totalof 99,203 MT fish and fish product were exported which has earned US $ 253,497 million(Azam & Shafique, 2017).

1.1 INDUS RIVER AND ITS ICHTHYOFAUNA

Pakistan is Hydro geographically divided in to three river systems, i.e., the Indus river system, Baluchistan drainage and land lock river system(Rafique, 2000). The Indus River originates from Kailas range in western Tibet, is the largest river system of the country covering over a length of 3058km and draining an area of 963,480 km²before discharging into the Arabian sea(Sehgal, 1988).

The ichthyofauna of sub-continent has been explored by a number of studies conducted at different localities and different times(Hamilton, 1822; McConnell & Lowe-McConnell, 1987); (Rafique & Qureshi, 1997); (Talwar & Jhingran, 1991). During the last few years the knowledge on ichthyofauna of Pakistan has substantially been increased (Rafique, 2000);(Rafique, 2001); (Rafique et al., 2003);( Mirza M.R, 2003).

Though these studies provide some base line information regarding species distribution and its diversity in different localities yet is deficient in providing information based on their ecology, behavior, genetics and reproductive biology.

18 Some information on the fresh water Ichthyofauna of river Indus has been documented previously. The Freshwater fish fauna of Pakistan is constituted by 193 fish species. All these species belong to class , sub-class Teleostei, 3 cohorts, 6 super orders, 13 orders 30 families and 86 genera (Rafique & Khan, 2012). On the basis of IUCN status, endemism, rarity and economic importance 86-fresh water fish species have been identified as species of special importance among which the S. plagiostomushas been identified as Vulnerable (Rafique & Khan, 2012). The present work will explore some aspects of the ecology of S. plagiostomus, inhabiting river Indus and its small tributaries in Indus Kohistan, Khyber Pakhtunkhwa, Pakistan.

Species of Snow Trout belongs to family and subfamily Schizothoracinae. A total of 68-species belonging to the same subfamily have been recorded worldwide (He & Chen, 2006).A total of 28 species of snow trout have been recorded from Himalayan and Sub-Himalayan regions. The short growth period and slow growth to gain maturity, are the inherent biological features of these species which hinder their growth and population on increase(Mir, et al.,, 2012). Schizothorax is represented by a number of species i.e. Schizothoraxesocinus, Schizothoraxcurvifrons, Schizothoraxniger, Schizothoraxplageostomus, Schizothoraxlabiatus, Schizothoraxrichardsoniietc. Variety of names have been referred to genus Schizothorax in English literature, such as Snow Carp, Snow Trout, Snow minnows and Mountain barbells( Mirza, 1991) or even Indian Trout (Tilak & Sinha, 1975). In this context the name Snow barbell is the most appropriate name as the names Snow Trout and Indian Trout are taxonomically misleading (Kullander, Fang, Delling, & Ahlander, 1999).

Majority of this snow barbells are restricted to the Trans-Himalayan Region of the Indus system where the temperature of the river does not exceed than 20°C. It is widely distributed in Gilgit Baltistan and Khyber Pakhtunkhwa while a rare population was recorded from Punjab and Balochistan.S. plagiostomus is a ray finned fish which belong to genus Schizothorax. It is locally known as khont in Kashmir and Punjab, Kerai in Khyber Pakhtunkhwa and its common name is snow trout.

19 S. plagiostomus colorsdark grey on dorsal side and lighter grey on lateral side. The ventral side is whitish. The species is typically lotic inhabiting fast flowing streams of Kashmir (Kullander et al., 1999). The body is elongate, fusiform with projecting snout. The mouth is inferior with very deep, sharp lower jaw having sharp keratinized antero-ventral cutting edge. The inter-orbital space is wide and flat. The pharyngeal teeth arranged in three rows. Rostral and maxillary barbless are present. has un-branched last thick fin-ray which is serrated posteriorly. Anal fin rays are smooth and flexible. The dorsal fin-rays are branched 7(4), 8(21); anal fin-rays branched 5(25), and gill rakers 15(1), 17(5), 18(4), 19(3), 20(6), 21(4) and 22(1). Scales are very small and elliptical. The scales along the base of caudal fin are distinctly enlarged. Caudal fin deeply emarginated. The number of scales ranges from 89 to 99(Kullander et al., 1999).

Plate 1: Snow Barbell, Kerai in Pashto, Khont in Kashmiri, Schizothoraxplagiostomus

S. plagiostomus is very similar to S. labiatus, both species are syntopic and their distinction sometimes leads to more confusion.The genus Schizothorax possess a group of species with a remarkable similarity in their general morphology which are often difficult to identify on their external morphological characters across Indian Himalaya (Chandra, Barat, Singh, Singh, & Matura, 2012) and (F. A. Mir, Mir, & Chandra, 2013).

SchizothoraxplagiostomusHeckel, 1838 is an indigenous freshwater fish of commercial importance. The species is phytophagous and inhabiting the reservoirs of Central and Eastern Persia in the West to the far reaches of the Mekong and Yangtzekiang in the East (Ganai, Yousuf, & Tripathi, 2011)

20 Knowledge about spatial and temporal distribution of is a central tenet in ecology. The special aim of ecologists focus on identification of spatial-temporal dynamics of habitat heterogeneity, which influences the abundance and distribution of organism, shaping biotic patterns and processes in natural landscapes (Hanski & Gaggiotti, 2004); (Lovett, Jones, Turner, & Weathers, 2005); (Molle, Mollinga, & Meinzen-Dick, 2008).

In monitoring studies, the catch per unit efforts (CPUE) is a standardize method used by fishermen and fisheries agencies. Catch per unit effort is commonly used method for estimating population size where counting method is difficult to apply(Hinton & Maunder, 2004). In fact analyzing and reporting data as actual numbers is not possible, the CPUE is commonly used method in fisheries sector. It is used to standardize the data based on catching efforts i.e. number of fish caught per number of traps used and total event time spend. It is assumed that CPUE has an impact of constant catchability which determine the same probability of all animals that being caught (Schwarz & Seber, 1999). Keeping in view these assumptions, CPUE can be used to estimate species abundance (Schaefer, 1954); (Harley, Myers, & Dunn, 2001); (Bigelow, Maunder, & Hinton, 2003); (Laloë, 2007) and(Zimmerman & Palo, 2011). A number of variables such as fishing techniques, fisherman behavior, water temperature, and substrate size and predator presence affect CPUE (Goodyear et al., 2003); (Hoyle & Maunder, 2006); (Laloë, 2007);(Abrahamsson, 1983); (Somers & Stechey, 1986) and(Dorn, Urgelles, & Trexler, 2005).

Schizothorax Spp.arewidely distributed in Indus river system of Northern Pakistan, KP and its range of distribution extend throughout Himalaya and Trans- Himalaya. The species aregeographically spread in different rivers, lakes and tributaries throughout Himalaya and its distribution reaches to the confines of , eastern , Pakistan, Turkistan, , Ladakh, Tibet, BhutanandNorth east (Bahuguna, Thapliyal, & Bahuguna, 2008). Although S. plagiostomus is widely distributed in IndusRiver system and previous studies have shown that they were abundant and common, but recent observations indicate the drastic decline in the populations of this indigenous fish due to introduction of exotic species, climate

21 change,damming and over-fishing.Deterioration of water quality is a major cause of stress in fishes which increases their susceptibility to diseases(Girijakumari Nisha, Rajathi, Manikandan, & Marimuthu Prabhu, 2014). The culturing of this species of economic importance in captivity has not been established which might be due to the problem of developing an appropriate feed for itsalevin.

1.2 FEEDING HABIT IN FISHES

All organisms depend on energy for their growth, reproduction and all other metabolic processes. Living organisms utilize food resources to obtain energy. Hence, food is considered as the most essential component for all metabolic processes of organisms. Fishes like all other organisms depend on the energy obtained from theirfood for their survival and growth. Food consumption is an essential factor in controlling fish reproduction. Information regarding natural food of fish play an important role to understand the fish nutritional requirements, its interactions with other organisms and its potential use for aquaculture (Royce, 1989).

The diet of most fish’s changes with age and growth. The food and feeding habit of fishes varies with time, space and as well as with respect to changes in different stages of their growth (Hardy, 1924). Assessment of the feeding habit of fish is an important factor before its stocking into a new ecosystem, so as to leave the native fish fauna with least disturbance in their natural habitat. Dietary changes in fish feeding habit in response to extrinsic and intrinsic factors provide information on basic functioning of fish assemblage which are very important for developing Ecosystem Based Fisheries Management models(Hanson & Chouinard, 2002). For better hatchery operation and management, understanding of fish feeding habit at different ages is very essential. In aquaculture sector, the nutritional information of brood stock plays a key role in the reproductive performance of many fish species (Bromage, 1995); (Brooks, Tyler, & Sumpter, 1997); (Izquierdo, Fernandez-Palacios, & Tacon, 2001).

The feeding habit of freshwater fishes has been extensively studied worldwide. Some outstanding work in this field has been contributed by(Al-Hussaini, 1949; Hynes, 1950); (Hyslop, 1980); (Melo, Machado, & Pinto-Silva, 2004); (Mamun, Tareq, & Azadi, 2004);(Beltrano, Cannizzaro, Vitale, & Milazzo, 2006);(Bhuiyan, Afroz,

22 &Zaman, 2006); (M. S. Islam, Hibino, & Tanaka, 2006), (Başçınar & Sağlam, 2009)and (Shalloof & Khalifa, 2009). (Jellyman, 1989);(Amezaga, Santamaría, & Green, 2002) and(Lima-Junior& Goitein, 2004).

Bony fishes being the most diverse group among have a wide range of food preference. They may be herbivore, carnivore, omnivore or detritivore. The feeding habit is directly related to the size of fish, its metabolic rate and environmental temperature. Smaller fishes have higher metabolic rate than larger ones, similarly the warm water fishes generally have higher metabolic rate as compared to cold water fishes. Different types of habitat conditions can potentially influence the fish feeding, reproduction and survival by effecting their physiology, behavior and genetics (Schoener, 1974)and (Southwood, 1977). The accurate description of fish diet and feeding habit is a very important aspect in fisheries management for the purpose of species conservation, breeding and culture. Diet of fishes reveal the integration of many ecological components i.e. energy intake, behavior, habitat use and inter/intra specific interactions. The practices of stomach contents analysis provide a strong insight to assess the fish feeding habit both quantitatively and qualitatively. The analysis of stomach contents has become a standard practice to reveal the feeding habit of fish and all other animals (Hyslop, 1980). S. plagiostomusis a phytophagous fish; its mouth is adapted to scrap attached from the surfaces of stones and pebbles. Its feeding habit have been reported by many authors (T. Shrestha, 1979);(J. Shrestha, 1999);(Terashima, 1984) and(Sharma, 1989).

Snow carp (S. plagiostomus)is an indigenous fish species of economic importance with a wide ecological range from Gilgit Baltistan to Punjab Province of Pakistan. This is the dominant fish species found in the River Indus and its tributaries throughout Khyber Pakhtunkhwa Province and northern parts of Pakistan. Information on the feeding habit of this species is scarce. Knowledge on food and feeding habit of this species of economic importance is a prerequisite for the purpose of its breeding and culture in captivity. The present study was undertaken to examine the food and feeding habit of S. plagiostomus.

1.3 REPRODUCTIVE BEHAVIOR IN FISHES

23 Reproduction is the fundamental characteristic of all living organisms. In fishes at least three types of reproduction are found i.e. bisexual, hermaphroditism and parthenogenesis. The bisexual reproduction is most common among fishes where sperms and eggs are produced in separate male and female individuals. According to the mode of egg production the fishes are categorized into the following three categories:

Oviparous:Majority offishes lay their eggs and are known as oviparous or egg layers. About 97% of all known fish species are oviparous.

Viviparous: Some fishes give birth to their young which are known as viviparous or live bearer. In such type of reproduction, the male and female fish mate in pairs and copulate, the fertilization of egg take place inside the body of mother fish. The developing embryo takes nutrition from the wall of mother’s reproductive tract.

Ovoviviparous: Such fishes are also known as egg retainers. The male and female fish copulate, the fertilization is internal and the fertilized eggs are retained inside the female body until they are mature enough to hatch.

The reproductive system in fishes is composed of male gonad as testes and female gonad as ovaries. The testes in most fishes are paired organs of similar size, which are partially or totally fused. Testes are found suspended by lengthwise mesenteries in the upper half of the body cavity. The color of testes is creamy white and when matured they produced milt. Majority of bony fishes lacks seminal vesicle, sperm sac and sperm duct. The sperm are shed into the body cavity from where the milt exudes through abdominal pore behind anus.

The female reproductive system of fishes is consisting of a pair of ovaries where the oocyte or ova are produced. The ovaries are partially fused where they are suspended in the body cavity by a fold of mesentery. As the oviduct is absent in most bony fishes and the ripe ova are shed directly to the body cavity and find exit through abdominal pore.

Reproduction in fishes is a complex process which depends on synchronized gametogenesis, development of accessory organs and secondary sexual characters, migration to breeding ground and mating behavior etc. The ultimate success of fish is

24 determined by the ability of fish species to reproduce successfully in varying environment and thereby to maintain its viable population (P. Moyle & Cech, 1988). Environmental factors have a great influence on the gonadal development of fishes and their fecundity. The environmental conditions in nature such as temperature, day length and food supply are closely correlated with the reproduction process in fishes(Sundararaj & Vasal, 1976).

For the purpose of better management and conservation in the area of fisheries sciences, the knowledge on various intricacies of reproduction is very important. Fecundity studies are very useful for tracing the different stock or populations of the same species in different areas (Gupta, 1968).Information on breeding biology, migration and breeding habitats of fishes is very useful in identifying those areas that require conservation in natural water resources. Aquaculture industry is based on the knowledge of reproductive biology, which is essential to breed the fish species of special importance in captivity.

The ratio of fish gonad weight to the fish body weight is known as gonado- somatic index (GSI). GSI is very helpful to identify the days and spawning season in fishes as the gonads in gravid female increase in size prior to spawning. The number of eggs being readied for the next spawning by a female among all egg-laying animals is called fecundity (P. K. Roy & Hossain, 2006). Fecundity has a many variations among different races of the same species. Estimation of fecundity in fishes is a basic determinant of productivity and hence contributes to its culture in captivity and population dynamics. Knowledge about fish fecundity is essential for the evaluation of commercial potentialities of its stock, captivity culture, life cycle and actual fisheries management (M. Islam, Sultana, Hossain, & Mondal, 2012).

The importance of Schizothorax Spp. and other genera inhabiting Himalayan and sub-Himalayan regions of the Indian sub-continent is understoodfor fisheries production and management. Most species of Schizothorax genera has a great economic importance from the sport and food point of view. S. plagiostomusis one of the most dominant fish species in the mountain water of Khyber Pakhtunkhwa and Gilgit Baltistan. It grows and breeds in cold, fast flowing torrential rivers like river Indus and

25 Swat. Introduction of exotic species, overfishing and degradation of spawning areas are the major constrains on its population to increase. Schizothorax Spp. are characterized by slow growth, low fecundity and late sexual maturity in their rigorous environment (Mohammad & Pathak, 2010). Such biological characteristicsare the major constrains which impede their intensive exploitation and breeding in captivity. For the purpose of their aquaculture exploitation and breeding in captivity, important studies on its maturation and spawning habit have been made by(Yamada, Aoki, & Mitani, 1998); (N. Roy & Gurung, 2007)and (Agarwal, Thapliyal, & Rawat, 2001).

Systematic information on GSI, fecundity and seasonal cyclicity of gonads through histological analysis in S. plagiostomus to establish its reproduction protocol in captivity is meager. Keeping in view the economic importance of this species, a systematic investigation on the GSI, fecundity and hatching period in relation to temperature variations was undertaken in the present study.

1.4 MOLECULAR CHARACTERIZATION OF FISHES

Fishes are the most diverse group of animals among chordates, constituting nearly half of all vertebrate species. According to Fish-Base Data (www.fishbase.org), the class Pisces includes approximately 15700 marine species and 13700 freshwater species. To ease species identification where traditional method are not applicable and to flag previously unidentified species, the Fish Barcode of life initiative (FISH-BOL; www.fishbol.org)has been established as a collaborative international research effort, which pursue to create a reference library for DNA barcodes for all fish species derived from voucher specimens with authoritative taxonomic identifications(Ward, Hanner, & Hebert, 2009)

FISH-BOL initiative gather, analyze and store COI barcode data from mitochondrial DNA. FISH-BOL enterprise is determined to obtain high quality sequence records for COI barcode region with the goal of analyzing 10-specimen per species. The effort will likely to sequence more than 0.5million specimens (Ivanova, Zemlak, Hanner, & Hebert, 2007).

Today the huge diversity of organisms is understood and it is fact that there are

26 too few taxonomists to discover, describe and identify the entire specimen collected by applied biologists. This taxonomic impediment is the main problem for those who are interested in the area of bio protection ((Ball & Armstrong, 2008);(Bleeker, Klausmeyer, Peintinger, & Dienst, 2008);(Chown, Sinclair, & van Vuuren, 2008) and conservation biologists (Guiliano & Blaxter, 2006),(Holmes, Steinke, & Ward, 2009), (Logan, Alter, Haupt, Tomalty, & Palumbi, 2008) and scientists identifying food items (Yancy et al., 2008). To remediate and resolve such taxonomic impediment, the DNA bar-coding has been proposed (P. D. Hebert, Cywinska, & Ball, 2003)(P. D. Hebert, Ratnasingham, & de Waard, 2003).

DNA barcoding is a modern taxonomic technique which involves the use of small genetic markers in organism mitochondrial DNA for the purpose of species identification and classification.DNA bar coding can be used to identify fish species from whole fish, fillets, fins, fragments, juveniles, larvae, eggs, or any properly region of the cytochrome c oxidase ׳preserved tissue available. In most animals, the 5 subunit I (COI) in the mitochondrial genome has been used as the target sequence, which encodes part of the terminal enzyme of the respiratory chain in mitochondria (Flomer, 1994). The ability of bar coding to provide species assignments also has important implications for the discovery of cryptic species (P. D. N. Hebert, Penton, Burns, Janzen, & Hallwachs, 2004), (Sriwattanarothai, Steinke, Ruenwongsa, Hanner, & Panijpan, 2010).

Species identification through DNA barcoding is based on sequencing a short standardize genomic region of the target specimen and the sequence information obtained, then compared to the sequence library from known species in a database. Cytochrome C oxidase I (COI) is a short fragment of 650-base pair on mitochondrial DNA which is used as standard barcode sequence in kingdom Animalia. Accurate identification of fish species from an egg to adult is very important in many areas especially in the area of management and conservation. Previously a number of protein and DNA based methods have been described for the genetic identification of fish species (Ward & Grewe, 1994)and (Tautz, Arctander, Minelli, Thomas, & Vogler, 2002). From the last 30-years DNA sequence analysis has been in practice for species

27 identification and different laboratories use different sequences for diverse taxonomic groups (Hubert et al., 2008).

Fishes are the most diverse group among chordates and DNA barcoding is a possible solution for their identification, description and discovery. However, this requires barcode databases with good nominal species coverage on databases such as Genbank and BOLD. The fish barcode campaign “FISH-BOL” estimates that there are DNA barcodes for about 10,267 fish species in their database (www.Fish-BOL.org) while the number of publically available COI sequences in Genbank is only 8,327 species. At present the sparse space for species coverage in the available databases such as BOLD and GenBank is a big challenge for DNA barcoding. Species cannot be identified without barcode and barcode for only ca. 60,000 of the 1.5 million described species are publically available via Genbank(Kwong, Srivathsan, & Meier, 2012).

On the basis of external morphological characters, the members of Schizothroax genus are difficult to distinguish and application of DNA barcoding may be useful for species identification. The wide distribution of S. plagiostomus in river Indus and its tributaries including river Swat, river Panjkora and river Chitral may have genetic variation due to habitat diversity. The purpose of this study was to examine COI diversity of S. plagiostomus distributed along river Indus and its tributaries.

The present study has been planned with the hypothesis that the assessment of the species abundance and distribution, analysis of feeding habit and fecundity estimation of this species of economic importance will assist its aquaculture practices in captivity. The molecular characterization of S. plagiostomus will resolve the issue of its identification and classification. The general objectives of the present study were:

• To observe the distribution and abundance of S. plagiostomus at different stream/tributaries flowing to river Indus in Indus Kohistan district of Khyber Pakhtunkhwa, Pakistan.

28 • To evaluate seasonal variation in the food and feeding habit of S. plagiostomus aiming to determine the dietary requirement as herbivorous, carnivorous, omnivorous or detritivorous.

• To study gonad histology during pre-spawning and spawning period of S. plagiostomus and to determine the seasonal cyclicity in its gonads. The estimation of absolute fecundity will be very advantageous to bring this species of economic importance in captivity.

• The application of DNA-barcoding, a modern taxonomic method using small genetic markers in mitochondrial DNA will resolve the issue of ambiguity inthe identification of S. plagiostomus with other Schizothoracine.

• To investigate the possible intra-specific sequence variationsin the COI gene of S. plagiostomusto be collected from different localities along river Indus and river Swat.

The specific objectives of the present study were:

• To investigate the feeding habit and proper feed of S. plagiostomus.

• Estimation of fecundity pattern to bring this fish species of economic importance under artificial culture.

• Molecular characterization through application of DNA-barcoding for species identification and conservation.

29 CHAPTER 2

REVIEW OF LITERATURE

2.1RIVER INDUS

Douglas Hill (2006) quoted river Indus as one of the most important water system in Asia which originate near Kailash range in Tibet, flows to the west and fall into Arabian Sea.According to Sehgal (1998)the origin of Indus River is Mansorawar Lake in Tibet, which is approximately 3058 km long draining an area of 963,480 km² before discharging into the Arabian Sea.

Mirza M. R, (1975) studied the physical geological, meteorological and hydro biological conditions along river Indus and concluded that these parameters vary substantially as do corresponding important human uses of the river and dependent economic conditions. He divided the river ecosystem into five categories: Mountain peak area,Foothill Mountains, Plain area, Semi-desert area and Delta region(Mirza M. R. 1975).

Karim &Veizer (2002)reported that Shingo, Shyok, Shigar, Hunza, Gilgit and Kabul Rivers form the headwater tributaries of river Indus in northern Pakistan. In Panjab the Jhelum, Chenab, Ravi and Sutlej rivers drain into the river Indus.

Inam et al.(2007) concluded that river Indus originates from Gangdise Mountains parallel to Himalayas in Tibet, now part of Chinese territory. The river flows through the arid plains of Pakistan and India covering a length of 1980 miles from its head waters and meets the Arabian Sea before depositing rich silt in the ecologically valuable delta.Coldwater fish species confined to the higher latitude of the northern half of Pakistan. The northern Pakistan has three ranges of mountain system extending from the west to east, the Hindukush, the Karakoram and the Himalayas.

2.2SPECIES DIVERSITY AND DISTRIBUTION

According to Chapin Lii et al. (2000) definedspecies diversity as the number of species in an area and their relative abundance which involve species richness, composition, evenness, interspecies interactions and theirspatio-temporal variations. I.

30 Schlosser (1987) and Tonn (1990)reported that both biotic and a-biotic factors on a spatial scale determine fish diversity and its distribution.

Conner & McCoy (1979) reported that larger land masses possess greater diversity of fish habitats supporting higher species diversity.De Silva et al. (2007) stated that freshwater fish diversity is directly related to land areas of different nations of East, South and . De Silva et al. (2007) worked ondifferent native fish species in relation to their endemism and land area in different Asian countries and reported a positive relationship between their endemism and species richness of native species.

Oberdoff et al. (1995)and Guégan et al. (1998)studied pattern of fish species richness on global scale and concluded that river size and energy availability are the most important factors influencing patterns of species richness.

Grossman (1982) and Ross et al. (1985) stated that patterns of species richness on local scale is related to biological factors such as inter-specific competition and by Moyle &Vondracek (1985). Similarly, physical factors such as habitat diversity were reported by Gorman & Karr (1979), Schlosser (1982) and Arunachalam (2000), andwater chemistry by Rahel (1986).

Horwitz (1978), Methews (1985) and Bhat (2004) reported thatchannel morphology, flow regime and water temperature effect patterns of species richness locally.Tonn (1990) concluded that patterns of species richness observed in local communities is determined both by local as well as large scale mechanisms.

2.2.1Fresh Water Fish Diversity

Berra (2011) reported that distribution pattern of freshwater fishes varies globally and each continent has its distinctive fish fauna. Levêque et al. (2008) concluded that most of the fishes belonged to 5-texa, the Siluriformes, the , the Charciformes, the Perciformesand the Cyprinodontiformes.

Kottelat& Whitten (1996) investigated that 35-40% of the world freshwater Ichthyo-fauna is found in East, South and Southeast Asian countries with arichnessrange of 7447 species. According to De Silva et al.(2007) individual Asian

31 countries have rich diversity of freshwater Ichthyo-faunaviz, China with 1287 species, Indonesia with 1037 species, India with 724 species, with 661 species and Malaysia with 527 species.

Bishtet al. (2009) and Soyinka et al. (2010) reported that changes in environmental factors such as water quality, depth, water current, food and substratum of the river effect the occurrence, abundance and distribution of fish fauna. Fitzpatrick et al. (2001)revealed that species distribution, abundance and assemblage composition of aquatic organisms is under the relative influence of environmental characteristics which are highly complex and interrelated.

Puertas&Bodmer (2004) hasreported that in the area of fisheries and conservation biology, catch per unit efforts (CPUE) is an indirect method of measuring species abundance of a target species therefore changes in CPUE inferring changes to the target species “the true abundance”.

Rafique (2000) reported that fish diversity of river Indus is fairly poor in comparison to other major rivers in the region, comprising of 177-fish species (dominated by carp family Cyprinidae and loach family Noemachcilidae) including two exotic species. He concluded that the major reasons for poor fish diversity are long torrential upper courses in Himalaya, glacier water and high sediment load.

Day (1888) has confirmed the geographical distribution of Schizothoraxspecies throughout Himalayan and sub Himalayan region (rivers, lakes, tributaries) extending to confines of China, eastern Afghanistan, Pakistan, eastern Turkistan, Nepal, Ladakh, Tibet, Bhutan, north-east India, including Kashmir, Himachal Pradesh, Uttaranchal (Garhwal and Kumaun region) and Assam.

Bahuguna (2002) hasfound Schizothoraxrichardsonii(Gray) and Schizothoraxplagiostomus as the most important food fish of Garhwal Himalaya among snow trout, occupying the status of highly economical indigenous food fish of Garhwal region.

According toRafiq (2007) and Rafique& Khan (2012) the area under Pakistan is occupied by a minimum of 193 freshwater fish species belonging to class

32 Actinopterygii, sub-class Teleostei, 3 cohorts, 6 super-orders, 13 orders, 30 families and 86 genera.Javedet al (2012) reported 12-species of sub-family Schizothorancinae from Pakistan.

Hussain & Mirza (2007) studied the fish fauna in the northern mountain areas of Pakistan and Kashmir. They reported snow-carps (Family Cyprinidae, subfamily Schizothoracinae), the loaches of the genus Triplophysa(Family Nemacheilidae), and the Glyptosternonreticulatum(Family ) which extend downstream along the main Indus River up to BeshamQila).

In these studies, the authors (Das &Subla, 1964; V. Jhingran, 1999) have presented that Schizothorax is believed to have migrated into Kashmir water from Central Asiatic watershed surrounded by Hindukush, Karakoram and inner ends of North-Western Himalayas and Sulaiman range. In these part of Himalayas, it evolved into a number of species in which some of these species became endemic.

2.2.2Fish Communities and their Ecological Pattern

In these studies, the authors (Horwitz, 1978; Angermeier& Karr, 1983; Pusey et al., 1995; Taylor et al., 1996; Magoulick, 2000; Silvano et al., 2000; Da Silva Abes&Agostinho, 2001; Bhat, 2004) have reported that both on spatial and temporal scales fish communities’ changes.

Lowe-McConnell (1975) has reported that habitat diversity such as upstream and downstream gradient effect changes in fish communities both in tropical and temperate ecosystems. In these studies, the authors (Kouamélanet al., 2003; da Silva Abes&Agostinho, 2001; Bhat, 2004) have observed the same pattern in the tropical ecosystemwhere they found increase tendency of species richness from upper stream to lower stream.

Authors like (Grossman et al., 1985; McConnell & Lowe-McConnell, 1987; Gido et al., 1997; da Silva Abes&Agostinho, 2001) have reported that on temporal scale, fish assemblage in streams may largely be determined by seasonal variations such as small-scale flood and drought.

33 2.2.3Habitat Association

Vannoteet al. (1980) noted that species distribution and organization in stream communities is effected by the physio-chemical features of the environment.Angermeier& Schlosser (1989) has investigated that size of habitat and complexity determine species richness and abundance in Panama streams. In these studies, authors (Hotwitz, 1978; I.J. Schlosser, 1982) presented that water chemistry, temperature and channel morphologyaffect fish composition and richness. Fish species composition and richness is determined by stream size and canopy openness (Angermeier & Karr, 1983)substrate complexity, stream current and depth.

2.2.4Threats to Freshwater Fish Fauna

López-Rojas & Bonilla-Rivero (2000) reported that in relation to habitat features and fauna, majority of freshwater ecosystem are unique which are extremely vulnerable to change. Bruton (1995) has reported that fresh water fishes are highly threatened taxa. In these studies the authors (Domínguez et al., 2006; Nguyen & De Silva, 2006) have confirmed that rapid decline of fishes were caused by factors identified as habitat alteration, pollution, overexploitation and introduction of non-native species.

2.2.5Introduction of Exotic Species

Authors like (Kaufman, 1992; Witte et al., 1992; P.B. Moyle et al., 2003) have reported that introduction of non-native species have cause decline of native fish fauna and in some instances its elimination or extinction. In these studies (Cambray, 2003; Casal, 2006) have presented that 568 fish species have been introduced in fresh water ecosystem around the world although many of the world worst invasive species were introduced for recreational fishing. In this connection authors (Kaufman, 1992; Witte et al., 1992; (Ogutu Ohwayo, 1993) have reported that introduction of Nile perch

(Latesnilotica) and‐ Oreochromisesculentus has caused extinction of many endemic cichlids in Lake Victoria east.

Scribner et al. (2000) reported in their review on hybridization in freshwater fishes that introduction of non-native fishes is a major contributing factor for hybridization. Gregg et al. (1998) found hybridization between native species

34 Oreochromismortimeriand non-native O. macrochirin Zimbabwe.Koppelman (1994)has successfully hybridized smallmouth bass (Micropterusdolomieu) with introduced spotted bass M. punctulatus.

2.2.6Effect of Alteration in Hydrology

In these studies the authors (Dudgeon, 1992; P. B. Moyle & Randall, 1998) has noted that hydrological alterations are the major threats to freshwater fish fauna and habitat. Similar observations were recorded by that fish communities are badly affected by hydrological alterations such as dam construction, diversion for irrigation and channelization.(Groom, Meffe, & Carroll, 2006) and (Dudgeon, 2000) reported that physical barrier like dams, embankments etc. affecting freshwater fish ecology by stopping fish movement and blockage of migratory routs and altering flood-pulse inundation cycles.

2.2.7Effect of Water Pollution

The authors (Richter et al., 1997; (Domínguez Domínguez et al., 2006) have worked on sources of water pollution. They found that ‐urban, industrial and non-point sources especially from agriculture sector constituting major sources of water pollution which leads to rapid deterioration of water quality.P. B. Moyle & Leidy (1992) havereported that water pollution can increase mortality rates and decrease the reproduction success, growth and behavior of fishes.

2.2.8Management and Conservation of Indigenous Fish Fauna

According to (Allan & Flecker, 1993; Richter et al., 1997) conservation of freshwater habitat and fauna has increasingly become a priority. (Johnston, 1999; Cambray, 2003) has presented that knowledge about ecology, behavior, life history, population dynamics, species habitat, species diversity and distribution are important in formulating plan for species conservation and management. In these studies the authors (Merrick, 1997; Saunders et al., 2002; Sundarabarathy et al., 2005; Sarkar et al., 2006)haveconcluded that conservation and management is a long term continuing activity which involve management of protective areas, breeding in captivity for reintroduction programs, restoration of freshwater ecosystems, involvement of local

35 communities and environmental education. Schindler & Parker (2002) havepresented that in conserving species and its habitat, the restoration of ecosystem is an important conservation effort. In these studies the authors (Poffet al., 1997; Saunders et al., 2002) have found that restoration of freshwater ecosystem needs provision of natural water flow, elimination of non-native fish species and maintenance of riparian vegetation.

In line with these studies the authors (Sundarabarathy et al., 2005; Sarkar et al., 2006) have found that breeding in captivity and their seed stocking into the natural water resources are important techniques for the conservation of rare, indigenous and endangered species.

Sarkar et al. (2006) has reported that due to overexploitation the wild population of endangered feather black Chitalachitala were declined drastically in last 10 years. The species were bred in captivity through synthetic fish hormone ova prim with 76-80 % success rate.

2.3 ANALYSIS OF GUT CONTENTS AND FEEDING HABIT

Staples (1975) has reported that food and feeding habit of fishes in different habitat varies in respect of time, space and at different stages of growth, therefore studying the feeding habit of fishes is important.Fagbenro et al. (2000)has presented that assessment of feeding habits in fishes based on stomach content analysis is a common practice in fish ecology which is an important mean to find out the tropic relationship in aquatic communities.

Hahn et al. (1997) has found that the tropic relationship in ecosystem is identified by studies on the natural feeding habit of fish which produce valuable information for the development of artificial foods used in psiciculture.Addition of vegetable crops to the food of rainbow trout has significantly enhanced their specific growth and weight gain during the term of experiment.

Bhuiyan et al. (2006) has reported that type of food depends to a great extent upon the nature of environment, which is interesting from specific to ecological point of view. Oso et al. (2006) has emphasized to study the food and feeding habit of fish as a

36 subject of continuous research for a purpose of developing successful fisheries management program on fish.

Gümüşet al. (2002) hasconcluded that identification of the stomach contents enables researchers to know about food consumption, feeding and assimilation rates, cannibalism and even habitat segregation.In these studies the authors (Spence et al., 2007; Shamsan, 2008) hasworked on fish feeding behavior and have mentioned fishes divided into the categories of herbivorous, omnivorous, carnivorous and herbivorous feeding habits.

Babare et al. (2013) hasanalyzed the gut contents of Mystussperata in comparison to Wallagoattufrom Godavari River in Maharashtra state and revealed no variations in their gut contents.

Imevbore&Bakare (1970) has revealed grasses, seeds and decaying plant matter in the gut of Cirrhinuscitharus.Fish fauna of Kashmir prefers to eat phytoplankton i.e. diatoms, green and blue green algae followed by detritus and sand.

Cabana et al. (1994) have revealed that the diet composition of fish is effected both by temporal and spatial conditions as well as by environmental factors. The diet of fish is influenced by fish size, maturity, condition, season (water level), bottom, depth, latitude, longitude and habitat types.

Shah et al. (2014) has analyzed the gut contents of Schizothorax Niger and Schizothoraxesocinus which revealed that the S. Niger contains more vegetable matter than animal matter, while gut content of S. esocinus contains slightly higher quantity of animal matter than plant matter.

2.4 FISH REPRODUCTIVE BIOLOGY

Murua&Saborido-Rey (2003) reported that prominent variations in fecundity among fish species often reflect different reproductive strategies.Khaironizam&Ismail (2013) studied the fecundity and spawning aspects of cyprinid fish Neolissochilussoroidesfrom the Gombak River ofPeninsular Malaysia.Bithy et al.

37 (2013) estimated the fecundity of JatPuti, Puntius sophore and concluded that the same was highly correlated with total length and body weight.

Mekkawy& Hassan (2011) has reported that the process of estimating fish population is based on fecundity or total egg production. According to Gómez-Márquez et al. (2003)fecundity is the reproductive potential which is an important biological parameter that playing a significant role in the evaluation of commercial potentials of fish stock.

In these studies, (Lagler, 1956; Zin et al., 2011) haverevealed that complete knowledge on the fecundity of fish is very important when evaluating the commercial potentialities, stock studies and life history especially in the culture and management fishery. Muhammad & Pathak (2010) has found that in general fecundity is species specific which varies from species to species.

Alikunhi (1956) has observed fecundity and larval development in Labeobata.Nagendran et al. (1981) have worked on the fecundity of Rasboradaniconius and observed relationship of the fecunditywith total body weight and gonad weight.Arthi et al. (2013) havestudied the reproductive biology of two freshwater fishes Ompokbimculatus and O. malabaricusand found that these two fishes breed throughout the year in river Amaravathy, Tamil Nadu, India.

(Webster & Lim, 2006) havedefined fecundity as the number of mature ova in the ovary, number of ovulated eggs or number of ova deposited in a spawning season.According to Kime (1995) spawning response in fishes relies both on internal as well as on external stimuli. Planas& Swanson (2008) have found that the gonadotrophic hormones stimulate the maturation of the gonads.

In these studies the authors (Shyam Sunder & Subla, 2013;Gandotra et al., 2009; (Bhat, 2004;M. Jan & Ahmed, 2016a) has made preliminary reports on the reproductive biology of Schizothoraxspp..

Y. Singh et al. (2016) has examined the relationship between fecundity (F) and total length of fish (L), total body weight (W), ovary length (OL) and ovary weight (OW) in S. plagiostomus.M. Jan & Ahmed (2016a) has carried out studies on the

38 assessment of fecundity (F), gonado-somatic index (GSI) and hepato-somatic index (HSI) of snow trout, Schizothoraxplagiostomusin river Lidder originating from Kashmir Himalaya of District Anantnag, India.

The authors like (Raizada, 1985;Kullander et al., 1999) have worked on reproductive behavior of S. plagiostomus. They found that S. plagiostomus attain maximum weight up to 2.5kg and total length up to 60cm. It gains sexual maturity at 18-24cm total length and twice in a year (September toOctober and March to April) both in natural and artificial environment.A. Jan et al. (2017) haverevealed that S. plagiostomus spawns twice in a year. However, according to V. Jhingran&Sehgal (1978)S. plagiostomus spawns only once a year in different month and at different elevation in a riverine stream.

2.5 DNA BARCODING AND ITS APPLICATION

DNA barcoding is a process of sequencing 650 base pair fragment of the mitochondrial gene known as COI, cytochrome C Oxidase I. Avise (2012) has reported that mitochondrial DNA has attracted a lot of attention from the last 15 years which has been proved to be very useful for several studies, especially in the area of population structure and evolution.Mitochondrial molecular markers have gained a rapid popularity and diverse application in fisheries sciences related to their phylogenetic and population structure.Billington (2003) has presented thatmitochondrial studies has been used for the identification stocks and mixed fishery, providing information on hybridization between fishes, serving as a genetic marker in forensic analysisand providing critical information for species conservation and rehabilitation.

Meusnier et al. (2008) has pointed out controversy over the application of DNA barcoding with a view that various researchers have proposed different purposes of DNA barcoding and reported that creation of the sequence library that can be used to identify previously described taxa, remains the most prevalent concept of barcoding. In these studies the authors (P. D. Hebertet al., 2013; Rubinoff, 2006b)havedelineated that mitochondrial genome is relatively conserved and is excellent for primer creation.

39 In comparison to nuclear DNA, mitochondrial DNA has no introns, with a rare recombination which is maternally inherited in haploid manner.Mitchell (2008) havereported that the use of barcode for routine species identification is the most widely accepted of the potential applications such as species description, phylogenetic analysis and conservation efforts although theseapplications are highly controversial.

P. D. Hebertet al. (2003)haveplanned to establish a data base for COI barcoding sequences which may be used to identify future specimens. The same would become part of a global bio identification system(GBS) to solve problems associated with morphological which will help to reduce misidentification. Mitchell (2008) havepointed out that barcoding can be used as a quality control system to ensure correct specimen identification. Will &Rubinoff (2004) has pin pointed that correct identification of the species through barcoding is possible only when its barcode sequence exactly matches the barcode of identified specimen already present in the database.

Rubinoff (2006b) has reported that genetic diversity within species is a problem when the database possess only a single representative of each species in it and in this way the researcher is unable to identify the specimen without the aid of taxonomist which nullify the purpose of barcoding.Ward et al. (2009)suggested that it is important for researcher to assume that intraspecific variation is significantly less than inter- specific variation in a barcoding sequence region.

Mitchell (2008) hassupported the use of DNA barcoding for species identification and discovery and agreed upon subsequent morphological and molecular analysis.Rubinoff (2006a) has discouraged the sole use of mitochondrial DNA for the identification and discovery of new species and for the understanding of global biodiversity.Kerr et al. (2009) have presented that the use of DNA barcodes to highlight areas of traditional taxonomy after its reevaluation has been gaining popularity in the recent years.

Langhoff et al. (2009) haveproposed the use of DNA barcoding for the purpose of cataloguing global biodiversity before it disappear and to focus on conservation effort where necessary.Rubinoff (2006b) was disagreed on the use of DNA barcoding as the

40 primary resource for biodiversity analysis because it provides insufficient information about the potential endangerment of species.

Pagès et al. (2009) has described that the process of species identification through DNA barcoding requires the assignment of taxa to clusters on a tree based on neighbor-joining phylogenetic analysis.Packer et al. (2009) havereported that the number of taxonomists is declining and there is already insufficient number of experts to handle the existing workload in the same area. Mitchell (2008) has accepted that due to shortage of taxonomists it is difficult to solve the problem because taxonomic techniques are time consuming and highly specialized.

Beamish & Rothschild (2009) havereported that mitochondrial genes proved as promising markers for fish species identification when compared to nuclear genes because of its several special features.Hubert et al. (2008) haveinvestigated that mitochondrial DNA produces high copy number in each cell which is a small circular DNA having a size range of 15-20 kb, which is easily recovered by applying various extraction methods.Sangthong&Jondeung (2003) havedescribed maternal inheritance pattern of mitochondrial DNA without recombinationwhile Moore (1995) has reported it as a decent tool for studying phylogeny and genealogy of taxa through matrilineage, because of its rapid mutation rate and small effective population size.

Meusnier et al. (2008) havepresented the success level of DNA barcoding as over 97% in birds, mammals, fishes, and arthropods. P. D. Hebert et al. (2003)has believed that barcoding studies are exceptionally successful in respect of species identification. However, Mitchell (2008)has reported over 5% failure to determine species assignment by barcoding alone. P. D. Hebert et al. (2003) has also agreed upon and admitted some misidentification during their studies.

Ward et al. (2009) has presented that these errors and misidentifications are due to hybridization, introgression, polyploidization, incomplete lineage sorting, Wolbachiainfections (in invertebrates) and “numts” (paralogous copies of mitochondrial genes that are inserted into nuclear DNA).

41 According to (DeSalle, 2006; Lakra et al., 2011) DNA barcoding is applicable to target a vast number of species which has been proved to be tremendous tool in description of the new species and their fast discovery.

Species in genus Schizothorax are remarkably similar in general morphology and are very difficult to distinguish from one another on the basis of morphological characters. Many authors have described the taxonomy of genus Schizothorax.M. Mirza (1991)have described that genus Schizothorax belongs to family cyprinidae, which are commonly known as Snow Trout having 15 genera and over 100 species throughout the world.Chandra et al. (2012) havereported the utility of (COI) DNA barcodes for the identification of two commercially important Coldwater fish species of Genus Schizothorax,Family Cyprinidae, from Uttarakhand Himalayas.

42 CHAPTER 3

MATERIALS AND METHODS

3.1 STUDY AREA

The Indus Kohistan is situated between 34° 54′ and 35° 52′ north latitudes and 72° 43′ and 73° 57′ east longitudes covering an area of 7492 square kilometers. The district is bounded on the north and northeast by Ghizer and Diamer districts of Gilgit Baltistan, on the south and southeast by Battragram and Mansehra District. From the west side the area touches district Shangla and district Swat. In 2014, the district has been bifurcated into upper Kohistan district and lower Kohistan. Locality sites for map were extracted from Google Earth Pro and were analyzed in ArcGIS version 10.2.2 (figure 3).

Figure 3: Sampling sites at different streams along River Indus in Indus Kohistan, KP, Pakistan.

43 3.1.2Description of the River Indus

River Indus is the largest river throughout the regions which originates from the northern slope of mount Kailash on the Gangdise range of Tibet and joins Arabian Sea covering a length of about 3000 kilometers. Many snow fed tributaries join the mighty river in Gilgit Baltistan and Indus Kohistan. The water flows with high speed in mountain until it reaches Tarbela Dam in KP and then enters the Panjab plain where five rivers join it. The flow is affected by monsoon season with minimum discharge during winter and maximum discharge during July and September.

3.2 PHYSICO-CHEMICAL PARAMETERS

The abiotic parameters such as water temperature, pH, DO, conductivity, hardness, alkalinity, nitrate and dissolved CO2 were recorded in all sampling stations on monthly basis. The neutral polythene bottles were used for water sampling. The water temperature was recorded at the site of collection by using centigrade thermometer. Conductivity and pH was measured by standard pH meter. Dissolved Oxygen, dissolved

CO2, total alkalinity, hardness and nitrate were measured by using standard protocol of APHA, 1995 (Dhoteet al, 2007). To study seasonal variations, the average of three month parameters were computed for autumn, winter, spring and summer and the data were analyzed statistically using one-way ANOVA (Tukey test) at 0.05% level of significance.

3.3 CATCHABILITY AND ABUNDANCE

The present study was conducted in the Indus River and its tributaries in the Indus Kohistan district of Khyber Pakhtunkhwa Province, northeastern Pakistan, located approximately at a latitude of 35°15′N73°30′E. Fish sampling were done from six stations along river Indus i.e. Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream.Specimens were caught by using 5-panels of gill nets settled in a stretch of 3-kilometers from the river confluence in each tributary. The overall sampling protocol was similar in all sections. Gill nets were set over night with a maximum soak time of 12-hours and month-wise sampling event lastedfor six nights (one night per sampling site). Five panels of gill net 6×25 feet overnight (12-hours)

44 were considered one unit of effort.

Catch per unit effort is defined as the total catch divided by the total fishing effort in a given time. The CPUE were calculated by using the following equation:

CPUE= C/F

Where

C= no of fish caught

F= unit effort

During this study the unit effort was calculated by multiplying the 5-numbers of gears (Gill nets& Cast net) used with maximum soak time of 12-hours. A total of 1799 S. plagiostomus were collected by 5-panels of gill net 6×25 feet (mesh size 1cm,1.5cm, 2cm, 2.5cm and 3cm) during first week of each month in 2016.

3.4 GUT CONTENT ANALYSIS

A total of 240 specimens (167 male & 73 females) were dissected for gut content analysis. The total body length (cm), standard length (cm) and body weight at each catch were noted; all the specimens were labeled and perforated at vent region for better preservation. After recording morphometricparameters, the specimen was immediately preserved in specimen jars containing 10% formalin solution and brought to Trout Hatchery, Alpuri district Shangla for further analysis. At Hatchery the samples were identified by using the key as prescribed by (Kullander et al., 1999). The specimens were dissected out to determine the sex and fullness of their guts. The gut length (GL) and gut weight (GW) were recorded to the nearest centimeter and gram, respectively. The removed guts were preserved in 4% formalin for further analysis. The gut was analyzed in the fisheries laboratory at Zoology department Abdul Wali Khan University, Mardan (Plate 3.2).

The guts were dissected and food contents were removed into petri dishes. In the first step the total wet weight ofeach stomach contents in the samples were taken. The gut contents were analyzed by the methods of (1) Frequency of occurrence as prescribed by (Hynes, 1950), (Hyslop, 1980) and (Bowen, 1996) (2) Volumetric Analysis Index

45 and (3) Importance Index as prescribed by (Lima-Junior & Goitein, 2001) and (Fritz, 1974) respectively.

3.4.1 Frequency of Occurrence Fi = 100ni / n

Where:

Fi: frequency of occurrence of the i food item in the sample;

ni: number of stomachs in which the i item is found;

n: total number of stomachs with food in the sample.

3.4.2 Volumetric Analysis Index

The Volumetric Analysis Index is used to indicate the relative abundance of a particular item found in the stomach samples. The method involves ascribing points to distinct food items by simple visual inspection of the gut contents. The calculations are performed by a constant reference known as Standard Weight (SW). SW is the arithmetic mean of weights of stomach contents caught in a previous collection.

3.4.3 Point Ascription After calculation of SW, the points were ascribed to each stomach contents for analysis. The points were given to each stomach contents according to its proportional weight in relation to SW. The ascribed points to each food items were transformed to arithmetic mean by using the following formula.

Mi = Σi / n

Where:

Mi: mean of the ascribed points for the i food item;

Σi: sum of the ascribed points for the i food item;

n: total number of stomachs with food in the sample.

The values of Mi calculated for each food item were transformed to percentages by using the following formula:

46 Vi = 25 Mi

Where:

Vi: Volumetric Analysis Index of the i food item in

the sample;

25: multiplication constant to obtain a percentage;

Mi: mean of ascribed points for the i food item.

3.4.4 Importance Index

Importance Index shows the relative importance of food item in the fish diet. Importance Index was obtained for each food item by using the following formula.

AIi = Fi ×Vi

Where:

AIi: Importance Index of the i food item in the sample;

Fi: Occurrence Frequency of the item;

Vi: Volumetric Analysis Index of the item.

For comparison between distinct samples based on values of Importance Index, the ranking method as proposed by (Fritz, 1974) was applied and the samples were compared with Spearman’s rank correlation coefficients. The values of Importance Index obtainedin different seasons (Kruskal-Wallis test SPSS)(Zar, 1999), were compared through Dunnett Tpost-hoc..

3.5 REPRODUCTIVE BEHAVIOR, GSI AND FECUNDITY

Gill nets of 1, 1.5, 2, 2.5 and 3cm stretched bar mesh having a length of 25 feet and depth of 6 feet were used for fish sampling. A total of 45-specimens per month were selected for breeding assessment, caught from each locality i.e. Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream along river Indus. The ratio of female to male was found as 263-female and 277 males. Fish

47 samples were identified by using the keys as described by (Kullander et al., 1999) and (Jayaram, 1981).

The morphometric measurement such as total length (0.1cm) and total body weight (0.1g) of each specimen were recorded. After dissection the gonad weight (0.01g) and length (.0.01cm) were taken and the gonad maturity of each specimen was identified by using a seven point maturity scale as described by (Nagelkerke, 1997) (Table 3.1). At the same time sex sorting were proceeded and the gonads were preserved in 5% formalin solution.

3.5.1 Length Weight Relationship

The relationship between total length and total body weight of S. plagiostomus caught from different tributaries of river Indus was calculated by the equation of W=aTLb as described by (Bagenal & Tesch, 1978)where the TW (g) is the total body weight and TL (cm) is the total length while a and b are the intercept and slope of the regression line, respectively. The following formula was used to determine the length weight relationship.

W=aTLb

Where W= body weight of fish (g)

L= total body length (cm) a=constant b= exponent between TL and weight

Quadratic model was used to estimate the regression curve by applying the following equation.

2 Y= b0+b1+b1x1+b2x1 where Y= weight of fish (g) b0=constant b1=total length

48 3.5.2 Condition Factor

Condition Factor is defined as the wellbeing of fish species under study. The Condition Factor of S. plagiostomus in river Indus was studied by using Fulton’s Condition Factor (Bagenal & Tesch, 1978). The following equation was used to calculate Fulton’s Condition Factor.

FCF= TW/TL3×100

TW= Total body weight (g), TL= Total body length (cm)

The multiple comparison of K-values across categories of all month were made by applying Tukey post hoc test in SPSS version 21.

3.5.3 Calculation of Female to Male Ratio

The proportion of female to male is known as sex ratio which was determined by the following formula during this study.

Sex ratio= no of females/no of males

To test the significant difference between female and male ratios the Chi square (X2) statistic was applied.

3.5.4 Determination of Gonado-Somatic Index (GSI)

The ratio of fish gonad weight to body weight is called GSI. During this study the monthly GSI of S. plagiostomus was calculated to determine its period and frequency of spawning throughout the year. The GSI was calculated by using the following formula as described by (Bariche, Harmelin Vivien, & Quignard, 2003).

GSI (%) = Gonad Weight (g)/Body Weight (g) ‐

3.5.5 Estimation of Fecundity

A total of 263 female S. plagiostomus out of 540 fish samples were selected for the assessment of fecundity caught from different locality along river Indus during 2016. Gravid females were easily recognized from their enlarge abdomen. The samples were immediately shifted to the Department of Zoology Abdul WaliKhan University, Mardan

49 for fecundity estimation. The fish samples were thoroughly washed with tap water and the total length was measured in centimeters. Excess water was removed with the help of blotting paper and theirweight was recorded in grams. Then the specimens were dissected and their gonadswere removed (Plate 3.3).

The matured ovaries were preserved in Gilson’s fluid in order to loosen ova from the ovarian wall. The gravimetric method were applied to determine the total fecundity of gravid fish as described by (Yeldan & Avşar, 2000). According to this method the two lobes of ovaries were dried up with blotting paper and weighed. Then 0.01g of each ovary was taken separately from the anterior, middle and posterior part of each lobe. Only the number of ripe oocytes in 0.01 g was counted with the help of magnifying lens and then multiplied by the total weight of the ovary by applying the following formula.

F1= Gonad weight (g) ×number of oocyte in sub-samples/sub-sample weight

FT= F1+F2+F3/3

3.6 MOLECULAR CHARACTERIZATION

3.6.1 DNA Extraction

DNA extraction procedure involves the separation of whole DNA from proteins, lipids, carbohydrates and other cellular materials. Recent development of automated DNA isolation kits has replaced the laborious, toxic and time consuming manual steps in DNA isolation protocols. In the last twenty years a variety of commercial kits has been developed for faster and safer PCR extraction.

In the present study the isolation of DNA from twenty-eight specimens of S. plagiostomus caught from different localities along river Indus in Indus Kohistan were done by using Thermo Scientific GeneJET Genomic DNA Purification Kit #K0721, #K0722 and its described protocol.

In the first step the morphometric measurement were taken from each specimen and the samples were identified morphologically by using the standard keys of

50 (Kullander et al., 1999) and (Jayaram, 1981). Muscle tissue equivalent to 20mg were taken and grinded by using mortar and pestle.

In the second step, grinded materials were collected in 1.5ml micro centrifuge tubes and suspended in 180µl of digestion solution. Then 20 µl of proteinase K solution were added to the suspended materials and mixed thoroughly by vortexing.

In the third step the suspension was subjected to incubation for three hours at 56 C by using thermo mixer in order to obtain a uniform suspension. After this 20 µl of

RNase⁰ -A solutions were added to the suspension, mixed by vortexing and then incubated for 10minutes on room temperature.

In step five, 200 µl of lysis solution were added and vortexed for 15minutes until a homogenous mixture was obtained. In the following step 400 µl of 50% ethanol was added to the lysate and mixed by vortexing.

In seventh step, the lysate was transferred to a GeneJET Genomic DNA Purification Column inserted in a collection tube which was centrifuged for 1 min at 6000 × g. The flow through solution obtained in the collection tube was discarded and the DNA purification column was kept in to a new 2ml collection tube.

In step eighth of the provided protocol, 500μL of wash Buffer-I (ethanol added) were added to GeneJET Genomic DNA Purification Column placed in a collection tube and centrifuged for one minute at 8000× g. After centrifugation the flow through solution were discarded and the purification column were placed back in to the collection tube.

In the following step, 500 μL of wash buffer-II (with ethanol added) were poured to the GeneJET Genomic DNA Purification Column placed in a collection tube and centrifuged for 03-minute at maximum speed of 13000× g. After this the collection Tubes were discarded and the GeneJET Genomic DNA Purification Column were shifted to a sterile 1.5ml micro centrifuge tubes.

The genomic DNA was eluted from GeneJET Genomic DNA Purification Column by adding 200 μL of Elution buffer. The eluted materials were incubated for

51 2minutes at room temperature and then centrifuged for 01minute at 8000× g. In the last step, the purification column was discarded and the resulted DNA was stored at -20ºC.

3.6.2 PCR Amplification

A pair of primer (FP1 and FR1) was design by using Primer3 and Bio-Edit version 7.0.5 program in order to identify by obtaining the COI sequences from the collected fishes, available for Schizothorax spp. in GeneBank. Prior to PCR reaction, twenty-eight PCR tubes were properly labeled (1-28) and racked in sequence. The DreamTaq Green PCR Master Mix (2X) Cat# K1081 M/s Thermo-Scientific was used for PCR reaction during this study. The following protocol was adopted as provided in the kit’s user manual. The kit reagents were kept on ice.

The composition of master mix was 25µL of 30X Ex Taq buffer, 0.1 uMdNTPs, and 2.5 µM of forward primer, 2.5 µM of reverse primer and 8.5 µL of 30 X sterile PCR water and 0.5 µL of Ex Taq for each reaction. The master mix was thoroughly mixed and 375 µL from it were added to each labeled tube along with 4 µL of DNA sample.

The reaction was started at initial denaturation of 95 C for 5-minutes followed by 35cycles of denaturation at 95 C for 30-seconds. The annealing⁰ temperature were set as 58 C for 45 seconds and the extension⁰ temperature of 72 C for 45 seconds. The final extension⁰ took place at 72 C for 4 minutes. The gel used ⁰during this study was 1.5% agarose gel with ethidium bromide⁰ to stain DNA. 4µL of PCR product from each tube were thoroughly mixed with 2 µL blue dye.

6 µL mixtures from each sample were loaded into twenty-eight wells on gel. Similarly, 6 µL of GeneRuler 1kb ladder (ready to use) Thermo-Scientific were loaded into a single well. The clear and bright bands of twenty six out of twenty eight amplified DNA were obtained and visualized by using GelDoc(Plate 3.4.1).

The GelDoc has confirmed the amplification of DNA in twenty-eight PCR products. The amplified DNA was then shifted from PCR tubes to 1.5ml appendrop tubes labeled as 1-28. The tip of each labeled tubes were carefully sealed by using parapylene film. The unpurified PCR products were sent to Macrogene Korea for sequencing.

52 3.6.3 In-Silico Analysis

The raw sequences were trimmed to remove noise and contamination of primer sequences by using BioEdit version 7.0.5.2 (Hall, 1999). The obtainedsequences were scanned in NCBI GenBank through BLAST algorithms against NCBI non-redundant nucleotide database for homologous sequences deposited for same gene from different regions (Altschul, Gish, Miller, Myers, & Lipman, 1990).

The homologous sequences were downloaded and used in various In-silico analysis. The obtained sequences were aligned with homologous sequences for various fish species already downloaded from NCBI GenBank through Basic Local Alignment and Search Tool (BLAST). Sequences were aligned and edited using GeneDoc version 5.10 (Nicholas, Nicholas Jr, & Deerfield II, 1997) and BioEdit version 7.0.5.2 (Hall, 1999).

The downloaded sequences were: S. labiatus (KT833092.1), S. esocinus (KT210882.1), S. richardsoni (KC790369.1), S. progastus (KF739399.1), S. esocinus (KU317702.1), S. richardsoni (KU595220.1), S. richardsoni (KF429953.1), S. nepalensis (AP011207.1), S. curvilabiatus (MF804977.1), S. plagiostomus (KU317682.1), S. plagiostomus (KT184924.1), S. plagiostomus (KU317688.1), S. plagiostomus (KU317687.1), S. plagiostomus (KU317690.1), S. plagiostomus (KU317693.1) and S. plagiostomus (KT833100.1).

Sequences were manually shaded after alignment in GeneDoc. Aligned sequences were used to perform phylogenetic relationship by using the MEGA v.7. software. The bootstrap confidence intervals for each branching pattern were calculated from 1,000 replicates by re-sampling.

53 Plate 3.1.: Fish sampling along river Indus in Indus Kohistan KP, Pakistan during 2016.

54 Table 3.1: Stages of gonad maturity for cyprinids as described by (Nagelkerke, 1997).

Gonad Male Female Stages

I Immature, indistinguishable male Immature, indistinguishable male from female, gonads are a pair of from female, gonads are a pair of elongated transparent strings elongated transparent strings running running along the body cavity. along the body cavity.

II Unambiguously male. Testes very Unambiguously female. Ovaries small, tube shaped white-reddish very small, tube shaped white- and non-lobed. reddish and invisible eggs.

III Testes larger and white reddish in Ovaries larger and white reddish in color. Lobed up to some extent and color. Starting flattened to starting flattened to sideways. sideways. Eggs very small.

IV Large testes white in color, lobed Large Ovaries, flattened to sideways and flattened to sideways and covering body cavity wall. Eggs yellowish.

V Large whitish testes, sperm run out Large and full ovary and completely when testes cutoff. covering the body cavity. Eggs yellowish and run out when ovaries cut.

VI Running, large white testes Running, eggs yellow and extruded out through stripping.

VII Spent, testes became empty, Spent, ovaries wrinkled reddish in wrinkled and reddish in color. color which contain few yellow eggs.

55 Plate 3.2: Gut extraction from S. plagiostomus for gut content analysis.

56 Testes

Ovaries

Plate 3.3: Extraction of gonadsfromS. plagiostomus, for the assessment breeding behavior.

57 Plate3.4: Extraction of DNA from the muscle tissues of S. plagiostomus.

58 Plate 3.4.1:Bands visualization of PCR products (COI gene) in a GelDOC showing COI amplification with 1kb gel ladder.

59 CHAPTER 4

RESULTS

A total of 1799 Snow Barbel (Schizothoraxplagiostomus) having an average size of 5 to 46cm (TL) were caught from river Indus and its tributaries in Indus Kohistan Khyber Pakhtunkhwa, Pakistan during the year 2015-16. For the assessment of feeding habit, a total of 240 specimens out of these were selected for gut content analysis. The gut contents of 99 males and 141 females were analyzed to investigate the diet composition and seasonal fluctuations in the feeding habit of Snow Barbel (S. plagiostomus). Similarly, 540 specimens out of 1799 were selected for the assessment of breeding biology and spawning behavior.

4.1PHYSICO-CHEMICAL PARAMETERS OF THE WATER

Table 4, shows the season-wise and table 4.2 shows localities-wise record of physico-chemical parameters (Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream) along river Indus. The sampling data was pooled by seasons i.e. autumn (September, October & November), winter (December, January & February), spring (March, April & May) and summer (May, June, July & August) on the basis of water temperature. The average water temperature was recorded as 10.93 C, 9.79 C, 15.28 C and 20.91 C in autumn, winter, spring and summer respectively.⁰ The ⁰ difference⁰ in mean water⁰ temperature during the four seasons was statistically significant (p < 0.05) as shown in table 4. The mean water temperature across categories of all localities was similar (P>0.05). Similarly DO, conductivity, hardness and alkalinity from different localities of the river Indus showed significant differences during the four seasons. However, table 4.2, showsstatistically insignificant variations for the above parameters across different localities except alkalinity that showed significant variations (p< 0.05). Seasonal variations in the concentration of

NO3, water pH and dissolved CO2 throughout the year were found non-significant across categories of seasons and sampling sites as well.

Table 4.1, has shown the post hoc multiple comparison (Dunnett T) of physico- chemical parameters across categories of different seasons. The mean differences of

60 water temperature recorded throughout all seasons were highly significant. The mean differences of pH for each season were found non-significant. The season wise mean differences in dissolved oxygen (DO) were found significant between autumn and summer. The mean differences in the water conductivity recorded in summer season in comparison to autumn, spring and winter were highly significant.

The mean differences of water hardness were non-significant among all seasons except winter when compared to summer season. The mean differences in alkalinity were found highly significant across categories of all seasons. The mean differences for nitrate and dissolved CO2 across categories of all seasons were found non-significant.

61 Table 4: Mean values of physico-chemical parameters with standard error in parenthesis of water from river Indus and its tributariesrecorded in differentseasons (autumn, winter, spring and summer).

Physico- Seasons P- chemical Value properties Autumn Winter Spring Summer

Water 10.930C(±0. 9.790C(±0.4 15.280C(±0. 20.910C(±0.8 0.000 Temperature 86) 4) 79) 3) pH 7.32(±0.12) 7.60(±0.16) 7.67(±0.15) 7.80(±0.15) 0.135

DO (mg/L) 8.36(±0.31) 9.08(±0.43) 9.75(±0.52) 10.11(±0.42) 0.030

Conductivity 47.54(±5.51) 69.58(±3.61) 82.33(±2.83) 107.50(±3.16) 0.000 (uS/cm)

Hardness 86.75(±3.23) 81.33(±5.36) 87.17(±3.71) 98.41(±3.56) 0.035 (mg/L)

Alkalinity 63.36(±4.31) 64.00(±3.21) 63.83(±2.53) 78.25(±2.8) 0.05 (mg/L)

Nitrate 0.83(±0.12) 0.95(±0.06) 0.69(±0.08) 0.88(±0.09) 0.232 (mg/L)

Dissolved 14.71(±0.66) 15.83(±0.74) 15.50(±0.7) 15.67(±0.56) 0.625

CO2 (mg/L)

*The mean difference is significant at 0.05 levels.

62 Table 4.1: Post hoc (Dunnett T) test of physico-chemical parameters of water from river Indus and its tributariesto reveal seasonal variations (autumn, winter, spring and summer).

Dependent Season Season Mean Std. Sig. Variable Difference Error Water autumn summer -9.98 1.06 P<0.05 Temperature winter summer -11.13 1.06 P<0.05 spring summer -5.63 1.06 P<0.05 pH autumn summer -0.48 0.21 0.06 winter summer -0.21 0.21 0.62 spring summer -0.14 0.21 0.83 DO (mg/l) autumn summer -1.76 0.61 P<0.05 winter summer -1.03 0.61 0.22 spring summer -0.37 0.61 0.88 Conductivity autumn summer -59.96 5.54 P<0.05 (uS/cm) winter summer -37.92 5.54 P<0.05 spring summer -25.17 5.54 P<0.05 Hardness (mg/l) autumn summer -11.67 5.73 0.12 winter summer -17.08 5.73 P<0.05 spring summer -11.25 5.73 0.14 Alkalinity (mg/l) autumn summer -14.88 4.64 P<0.05 winter summer -14.25 4.64 P<0.05 spring summer -14.42 4.64 P<0.05 nitrate (mg/l) autumn summer -0.04 0.13 0.98 winter summer 0.08 0.13 0.88 spring summer -0.18 0.13 0.34 Dissolved CO2 autumn summer -0.95 0.94 0.62 (mg/l) winter summer 0.17 0.94 1 spring summer -0.17 0.94 1 *The mean difference is significant at 0.05 levels.

63 Table 4.2: Mean values of physico-chemical parameters of water with standard error in parenthesis from various localities of Indus River and its tributaries.

Parameters Localities/Sampling sites P-Value

Jalkot Palas stream Keyal stream Barseen stream Kandia river Darel stream stream Water Temperature 14.560C 14.330C 14.430C 13.730C (±1.96) 13.280C 15.020C 0.99 (0C) (±0.19) (±1.67) (±1.81) (±1.56) (±2.68) pH 7.43(±0.19) 7.56(±0.17) 7.62(±0.19) 7.27±0.18 7.85(±0.19) 7.85(±0.13) 0.17

DO (mg/L) 8.75(±0.67) 9.21(±0.55) 9.76(±0.57) 10.03±0.55 9.12(±0.48) 9.07(±0.6) 0.62

Conductivity (uS/cm) 76.06(±6.87) 91.75(±4.69) 72.25(±11.22) 75.37±8.77 71.75(±10.79) 73.25(±10.9) 0.65

Hardness (mg/L) 83.63(±3.73) 92.63(±3.71) 85.00(±5.09) 81.75±7.73 87.63(±5.45) 99.88(±3.49) 0.14

Alkalinity (mg/L) 59.78(±2.65) 67.50(±3.26) 68.87(±3.42) 59.38±6.52 74.62(±1.94) 74.12(±5.28) 0.04

Nitrate (mg/L) 0.80(±0.16) 0.82(±0.11) 0.98(±0.12) 0.80±0.07 0.91(±0.09) 0.80(±0.11) 0.97

Dissolved CO2 14.33(±0.61) 14.75(±0.62) 16.36(±0.58) 17.00±0.78 15.50(±1.21) 14.62(±0.63) 0.11 (mg/L) *The mean difference is significant at 0.05 levels.

64 4.2FISH DISTRIBUTION AND ABUNDANCE

Table 4.3 and figure 4.1, shows significant variations in species abundance at the confluence of different tributaries along river Indus. The maximum sampling was done at Darel stream with a mean catch of 33.17, mean CPUE of 0.55 and a mean Kruskal- Wallis rank value of 47.74 while a minimum sampling was made at Jalkot stream with a mean catch of 15.83, mean CPUE of 0.26, mean Kruskal-Wallis rank value of 22.96. Kandia stream were recorded with a mean catch of 28.08, mean CPUE of 0.46 and mean Kruskal-Wallis rank value of 41.13.

The mean number of fish caught from Keyal stream were 25.00 with mean CPUE of 0.41 and mean Kruskal-Wallis rank value of 37.21. The mean catch of 19.50 samples were made from Palas stream with mean CPUE of 0.32 and mean Kruskal- Wallis rank value of 28.33. The mean catch recorded from Barseen stream was 28.33 specimens with mean CPUE of 0.47 and mean Kruskal-Wallis rank value of 41.63.The number of fish collected from these streams during this study were found significant with a p-value of 0.04.

Tables 4.3.1, 4.3.2, 4.3.3 and figure 4.1.1, has shownthe post hoc multiple comparison (Dunnett T) and significant variations in the number of this species across different seasons and collection sites. The lowest number of S. plagiostomus recorded in August with a mean catch of 7.17, mean CPUE of 0.12 and a mean Kruskal-Wallis rank value of 8.42. A peak abundance of fish was recorded in the month of November with a mean catch of 44.50, mean CPUE of 0.74, mean Kruskal-Wallis rank value of 63.25 and a p-value of 0.000.

4.3GUT CONTENT ANALYSIS AND FEEDING HABIT OF S. PLAGIOSTOMUS

Table 4.4, 4.4.1, 4.4.2, 4.4.3 and figure 4.2, shows the mean importance index of different food items consumed by S. plagiostomus in four seasons. Spirogyra and Ulothrix occurred as maximum food items in the gut of S. plagiostomus during summer while their minimum amount occurred during autumn. Both these items constitute the major food of S. plagiostomus found almost throughout the four seasons. Considerable amount of Mayflies, Caddis flies and Sand and mud were collected during winterand

65 autumn. In addition, a considerable amount of sand and mud were found in the gut during summer and spring.

The gut of S. plagiostomus also contained a considerable amount of detritus throughout the year. These finding revealed that aquatic algae Spirogyra and Ulothrix constitute the main food of S. plagiostomus, collected from river Indus and its tributaries independently of seasonal effects. However, other organisms such as Mayflies, Caddis flies and detritus were also found in the stomachs of sampled individual’s indicative of Herbi-omnivore type of feeding habit.

According to the ranking index spirogyra and ulothrix ranked higher with significant difference in comparison to other food items (p < 0.05, Kruskal-Wallis test). The concentration of Spirogyra and Ulothrix was significantly higher among other diet contents which revealed that S. plagiostomus is a phytophagous fish, however the presence of other food items shows omnivorous and opportunist feeding habit.

Regarding the seasonal dietary shift, it was observed that the food items consumed by the species, shows higher mean rank of importance index by food items during winter, which revealed the occurrence of all six food items in the gut of S. palgiotomus. The lowest mean rank of importance index by food items were recorded during spring. These findings revealed that S. plagiostomus feed on varieties of different food items during winter in comparison to all other seasons.

The increased intake of other food items such as and detritus as a food during cold winter might be due decreased growth of aquatic algae when temperature fall down. The increase in water temperature during spring, summer and autumn the growth of spirogyra and ulothrix increases and S. plagiostomus consumed algae as major food.

Table 4.4 showing the values of occurrence frequency, volumetric analysis index and importance index for Spirogyra and Ulothrix during spring season which were found higher in the gut of S. plagiostomus. The low values of Detritus, Sand & mud, Caddis flies and May flies found in the gut of S. plagiostomus during the same season indicate its herbivore mode of feeding due to the dominance of algal growth attached to

66 stones and pebbles at the streams bed.Multiple comparison/ post hoc test was not performed as the overall test did not showed significant differences among samples.

Table 4.4.1, showing the higher values of occurrence frequency, volumetric analysis index and importance index for Spirogyra and Ulothrix in the guts of S. plagiostomus during summer season. The hot summer facilitate the growth of algae at streams bed and the fish tends to eat more algae. The other food items were recorded in minor amount which has shown its herbivore feeding habit. The post hoc test was not performed due to non-significant difference across samples.

Table 4.4.2, showing the maximum values of occurrence frequency, volumetric analysis index and importance Index for Spirogyra and Ulothrix during autumn season. S. plagiostomus consumes algae as a major food item attached to stones and pebbles at the streams bed during autumn. Due to the non-significant difference among samples, the post hoc test was not performed.

Table 4.4.3, showing the values of occurrence frequency, volumetric analysis and importance index recorded for Spirogyra, Ulothrix, Mayflies, Caddies flies, Detritus and Sand & mud. Detritus, Sand & mud, Mayflies and Caddies flies constituted the major food item found in the gut contents of S. plagiostomus during winter. The difference among all samples were not significant due to which the post hoc test was not performed.

Table 4.3.4, seasonal variations in the mean Importance index with standard deviation in parenthesis, mean rank of Importance Index and mean Kruskal-Wallis rank values of different food items found in the guts of S. plagiostomus, caught at the confluence of different streams along river Indus in 2016. The variations were non- significant across categories of seasons p>0.05 due to which the post hoc test were not performed.

Table 4.4.5, showing the Mean Importance index of different food items with standard deviation in parenthesis, mean rank values and percentage of different food items consumed by S. plagiostomus, caught at the confluence of different streams along

67 river Indus in 2016. The variations were highly significant across categories of different food items p>0.05.

4.4REPRODUCTIVE BEHAVIOR AND SPAWNING PERIOD OF S. PLAGIOSTOMUS

Figure 4.3.4 and 4.3.5 indicate the month wise maturity stages (in percentages) of male and female S. plagiostomus, respectively. Stage I (immature) was observed in both thesexes during the month of June. Stage-II was observed during the month of July followed by Stage-III later during the same month in males. All the females were observed in stage-II during July. Both the testes and ovaries were observed to be indistinguishable in stage I and later distinguishable in stage-II. The gonads were translucent, minute and thread like localized in a small part of the body cavity.

Stage III was started at the end of July in males while in case of females it was started during August. It was observed till late August in both sexes where the gonads were developing and reddish in color. Both the gonads were found in Stage IV during late August, where it was extended to the early September in males. The gonads in stage IV were found lobed and white in color. The females got their maturity early at the start of September while the males matured later where they were observed in stage IV at the start during the same month.

The gonads of both sexes grown in size and occupied almost the entire body cavity with observation of a large number of visible and spherical ova in the ovaries. The matured stage of testes (Stage-V) was extended to early October in males where in female it was restricted only to September. The gonads of both sexes were found in running (stage VI) for the rest of October and November. The gonads of both sexes were observed in stage IV in the following December and January.

The immature (Stage-I), stage II and stage III were found missing in both the sexes where all the gonads were observed in stage IV during these months. The matured gonads (stage=V) were found in both sexes throughout the months of February and March. The running stage VI was recorded in both gonads during April. During May the gonads of both sexes were found shrunken, reduced in volume and became dull in

68 color. The shrunken gonads in spent condition (stage=VII) indicated the end of spring reproductive cycle.

It is note-worthy that the gonads of all the specimens collected during December and January were found in stage IV and almost no specimen were recorded with stage I, stage II and stage III of the maturity stages.

The results of the present study clearly show that S. plagiostomus spawn twice in a year, in March-April and September to November. The present study also revealed the fact that S. plagiostomus breeds for a short period of time during spring while for a long period of time during autumn. The running stage VI in both sexes persisted longer during autumn (October, November) in comparison to spring cycle (April). It was also observed that spent (Stage-VII), immature ambiguous stage (Stage-I), unambiguous (stage=II) and maturing (stage=III) were recorded only once throughout the year in May, June and July, August respectively.

4.4.1 Length vs Body Weight Male

The total body length (TL) ranges of male S. plagiostomus, body weight ranges, regression constant (b0), regression coefficient (b1, b2) and coefficient of determination (R2) are given in table4.5 and figure 4.3. In the present study the length-weight relationship of male S. plagiostomus showed some variation during the different months of the year.

According to table 4.5, the mean total length of male S. plagiostomus for the month of January was31.49 cm (range= 20.4cm to 44cm) with mean body weight of 463.4g (range=210g to 930g). The values of quadratic slope coefficients for the quadratic model fit calculated were (b1 =-60.45) and (b2 =1.378). The overall model fit for the month of January was highly significant as the P-value=0.00 with R2 =0.97. The body weight was increased when the total body length increased (Figure 4.3).

The same pattern of correlation was found during February where (b1 = -46.304) and (b2 =1.154) of male S. plagiostomus having mean total length of33.02cm (range=20.5cm to 41cm) and mean body weight of 499g (range=230g to 732g). The P value of 0.00 with R2 =0.97 has shown that the overall model fit was highly significant

69 which showed increase of body weight when the total length was increased. In March the mean total length of male S. plagiostomuswas26.58cm (rang=20cm to 43.8cm) and mean body weight of 354.1g (range=225g to 994g). The value of non-linear quadratic coefficient for quadratic model fit obtained were (b1 = -39.657) and (b2 =1.123). The overall model fit for the month of March was highly significant with P value of 0.00 and R2 value of 0.99 showed that the model was a good fit. The total body weight increased when the total length was increased (Figure 4.3).

During April the mean total length of 31.19cm (range=20cm to 45cm) and mean body weight of 502.6g (range=230g to 1234g) were recorded for male S. plagiostomus.

The quadratic coefficient values for model fit obtained are (b1 = -77.126) and (b2 =1.717).

The P value of 0.00 and R2 value of 0.98 has shown that the quadratic model for the month of April is best fitted. For the month of May, the same trend of curvilinear regression was found with quadratic coefficient values of (b1 = -114.013) and (b2 =2.451) to fit quadratic model in fishes having mean total length of 30.69cm (range=20cm to 42.5cm) and mean body weight of 552.6g (range=272g to 1197.5g). The highly significant P vale of 0.00 and R2 value of 0.97 indicate that the quadratic model is best fit for total length vs body weight (Figure 4.3).

For the month of June, the mean total length of 28.67cm (range=20cm to 42.7cm) and mean body weight of 453.4g (range=269g to 1211.5g) were recorded in male S. plagiostomus. Similarly, the quadratic slope coefficient for quadratic model fit is (b1 = -109.274) and (b2 =2.362). The overall model fit is significant as the P value=0.00 with R2 value of 0.93 that shows the model is a good fit (Figure 4.3).

During July the mean total length of male S. plagiostomus obtained is 31.18cm (range=20cm to 43cm) and mean body weight of 537.2g (range=289g to 1300g). The quadratic slope coefficient for model fit calculated as (b1 = -107.1) and (b2 =2.233). The P value obtained as 0.00 with R2 value of 0.95 indicates that the model fit is highly significant and is a good fit. During August the mean total length of male S. plagiostomus recorded is 28.84cm (range=20cm to 38.5cm) with mean body weight of 415.1g (range=265.5g to 662.5g). The values of slope coefficient to fit quadratic model

70 obtained as (b1 = -27.511) and (b2 =0.825). The overall model fit is shown by figure 4.3, which is significant as the P value= 0.00 with R2 value of 0.98 indicating model is a good fit.

The mean total length for the month of September is 29.96cm (range=20.5cm to 42.5cm) with mean body weight of 482.8g (range=246.7g to 1110g). The curvilinear coefficients to fit the quadratic model are (b1 = -78.022) and (b2 =1.787). The P value is highly significant at 0.00 with R2 value of 0.98showing model is a good fit. During October the mean total length of male S. palgiostomus is 27.76cm (range= 20.8cm to 44.6cm) with mean body weight of 371.9g (range=226.7g to 1172g). Similarly, the quadratic slope coefficients to fit quadratic model are (b1 = -80.343) and (b2 =1.793). The P value obtained is highlysignificant at 0.00 with R2 value of 0.97 which indicate that model is a good fit (Figure 4.3).

For the month of November, the mean total length of male fishes recorded as 29.11cm (range=20.4cm to 42cm) with mean body weight of 462.6g (range=256g to

1284g). The curvilinear coefficient to fit quadratic model are (b1 = -137.338) and (b2 =2.882). The overall model fit is significant as the P-value=0.00 with R2 value of 0.95 that shows the model is a good fit (Figure 4.3).

During December the mean total length of male S. plagiostomus30.18cm (range=21cm to 43cm) with mean body weight of 525.8g (range=278g to 1310g). The quadratic slope coefficients calculated to fit quadratic model are (b1 = -155.217) and (b2 =3.138). The model fit is highly significant as the P-value is 0.00 with R2 value of 0.96 showing model as good fit (Figure 4.3).

4.4.2 Length vs Body Weight Female

The data regarding total body length (TL) and body weight rangeswith regression constant (b0), regression coefficient (b1, b2) and coefficient of determination (r2) offemale S. plagiostomus, are given in table 4.5.1.

Table 4.5.1, has shown the mean total lengthfor the month of Januaryin female S. plagiostomusas 29.38 cm (range= 21.5cm to 42cm) with mean body weight of 399g (range=245.6g to 860g). The calculated values of quadratic slope coefficients to

71 fitmodel were (b1 = -72.15) and (b2 =1.53). The overall modelwas highly significant as the P-value was less than 0.05 with R2 =0.96, which has indicated that body weight was increasedwith the increase in total body length (Figure 4.3.1).

The increasing trend were also observedduring February where (b1 = -66.28) and

(b2 =1.44) of female S. plagiostomus having mean total length of 32.67cm (range=22.5cm to 34.8cm) and mean body weight of 484g (range=268g to 940g). The P value was highly significant with R2 =0.97 shows that the overall model was best fitted.

During March, the mean total length of female S. plagiostomuswas 32.22 cm (rang=22.5cm to 43.8cm) and mean body weight as 479g (range=235g to 999g). The curvilinear values obtained to fit model fit were (b1 = -44.95) and (b2 =1.15). The p- value obtained was highly significant in March and R2 value of 0.93 has shown that the model was best fitted. The unit increase in total body length has brought a unit increasethe total body weight (Figure 4.3.1).

In April, the mean total length and mean body weight of 29.96cm (range=20.5cm to 43.5cm) and 463g (range=238g to 1272g) were recorded infemale S. plagiostomus respectively. The quadratic coefficient values for model fit obtained 2 were(b1 = -108.51) and (b2 =2.29). The P value obtained was highly significant, R value was 0.93 has indicated that that the quadratic model for the month of April wasbest fitted.

During May, the same trend of curvilinear regression wasobserved as the quadratic coefficient values obtained were (b1 = -103.23) and (b2 =2.20). The model was best fitted with mean total length of 31.18cm (range=22cm to 45.5cm) and mean body weight of 547g (range=282g to 1250.5g). The highly significant P vale was obtained whereas R2 value of 0.96 has indicated that the quadratic model was best fitted. For the month of June, the mean total length of 32.05cm (range=20.5cm to 43.5cm) and mean body weight of 592g (range=290g to 1217.5g) were recorded in female S. plagiostomus.

The curvilinear coefficient values obtained to fit model were (b1 = -103.99) and (b2 =2.24). The overall model was highly significant with R2 value of 0.93 (Figure 4.3.1).

72 InJuly, the mean total length and mean body weight of female S. plagiostomuswere recorded as 29.06cm (range=21.5cm to 41.7cm) and 482g (range=283g to 1161.5g) respectively. The quadratic slope coefficient to fitmodel was calculated as (b1 = -118.46) and (b2 =2.53). The P value obtained was highly significant with R2 value of 0.98, which has indicated that the model was bestfitted.

During the month of August, the mean total length of female S. plagiostomus recorded is 29.58cm (range=21cm to 42cm) with mean body weight of 463g (range=279g to 1110g). The curvilinear regression values to fit quadratic model 2 obtained were (b1 = -93.05) and (b2 =2.00). The overall model fit wassignificant with R value of 0.95 s (Figure 4.3.1). Similarly, for the month of September, the mean total length of 30.53cm (range=21.6cm to 43.6cm) andmean body weight of 498g

(range=286g to 1213g) were recorded. The curvilinear coefficients to fit model were(b1 2 = -98.56) and (b2 =2.09). The P value washighly significant and R value of 0.98 has shown the model as a good fit.

InOctober, the mean total length of female S. palgiostomuswas29.58cm (range= 20.5cm to 43.5cm) and mean body weight was 475g (range=256.7g to 1250g). The quadratic slope coefficients to fit model were(b1 = -93.01) and (b2 =2.04). The P value obtained washighly significant whereas the R2 value of 0.96 hasindicated that model was of a good fit.

For the month of November, the mean total length of female fishes recorded was27.5cm (range=21.5cm to 39.5cm) andmean body weight was406g (range=266g to

995g). The curvilinear coefficient to fit quadratic model were(b1 = -94.62) and (b2 =2.11). The overall model fit wassignificant and the R2 value of 0.95 has indicated that the model was ofa good fit (Figure 4.3.1).

In the month ofDecember, the mean total length of female S. plagiostomuswas 31.41cm (range=21.4cm to 42.6cm) whereas themean body weight was 563g

(range=263g to 1264g). The quadratic slope coefficients calculated to fit model were(b1

= -128.09) and (b2 =2.68). The model was highly significant as the P-value was less than 0.05 with R2 value of 0.98 (Figure 4.3.1).

73 4.4.3 Condition Factor

The month wise condition factor for male and femaleS. plagiostomus observed during this study are shown in table 4.5.2, 4.5.3 and figures 4.3.2, 4.3.3. The month wise condition factor was compared by performing Tukey post hoc test in SPSS.The overall differences of K-valuesobserved month wise in male S. plagiostomus were found less significant p-vale=0.46. Comparatively to this the K-values observed month wise in female S. plagiostomus, were highly significant p-value= 0.004.

The month wise condition factor of female S. plagiostomus was calculated as shown in table 4.5.3 and figure 4.3.3. The mean condition factor recorded in female S. plagiostomus ranged from (0.92±0. 08) to (3.39±0.14). The K-value calculated was highest in the month of July and November (1.98) followed by August and October (1.9), June, September, May and December (1.8), January and April (1.7) and February (1.41). The lowest K-value was recorded during February.

74 Figure 4: Bar graph showing variation in mean CPUE of S. plagiostomus caught from different stream along river Indus in Indus Kohistan KP, Pakistan during 2016.

75 Table 4.3: Speciesabundance of Schizothoraxplagiostomucaught from different tributaries along river Indus.

Mean Catch Mean CPUE Mean Kruskal-Wallis ` Collection Sites rank Values Jalkot Stream 15.83 0.26 22.96

Palas Stream 19.50 0.32 28.33

Keyal Stream 25.00 0.41 37.21 P Value 0.04 Barseen Stream 28.33 0.47 41.13

Kandia Stream 28.08 0.46 41.63

Darel Stream 33.17 0.55 47.75

*The mean difference is significant at 0.05 levels.

76 Table 4.3.1: Multiple comparison of mean CPUE (post hoc Dunnett test) calculated for different collection sites along river Indus.

(I) Collection (J) Collection Sites Mean Std. Sig. Sites Difference (I-J) Error

Jalkot Stream Darel Stream -0.29 0.09 0.014

Palas Stream Darel Stream -0.23 0.09 0.074

Keyal Stream Darel Stream -0.14 0.09 0.461

Barseen Stream Darel Stream -0.08 0.09 0.862

Kandia Stream Darel Stream -0.09 0.09 0.832

*The mean difference is significant at 0.05 levels.

77 Table 4.3.2: Seasonal variations in CPUE of Schizothoraxplagiostomusrecorded in differenttributaries along river Indus in Indus Kohistan KP, Pakistan during 2016.

Collection Months Mean Catch Mean CPUE Mean Kruskal- Wallis rank Values January 20.17 0.33 31.67 February 20.67 0.34 31.67 March 29.00 0.48 41.92 April 41.67 0.69 59.83 May 24.33 0.40 36.92 June 14.17 0.23 21.25 P Value 0.000 July 7.67 0.12 8.83 August 7.17 0.12 8.42 September 29.00 0.48 43.50 October 42.17 0.70 59.83 November 44.50 0.74 63.25 December 19.33 0.32 30.92 *The mean difference is significant at 0.05 levels.

78 Figure4.1: Bar graph showing monthly variations in means CPUE of S. plagiostomuscaught from different stream along River Indus in Indus Kohistan KP, Pakistan during 2016.

79 Table 4.3.3: Month wise multiple comparison (post hoc Dunnett test) of mean CPUE calculated for different collection sites along river Indus.

(I) Collection (J) Collection Mean Difference Std. Sig. Months Months (I-J) Error January December 0.01 0.08 1 February December 0.02 0.08 1 March December 0.16 0.08 0.311 April December 0.37 0.08 0 May December 0.08 0.08 0.937 June December -0.09 0.08 0.882 July December -0.2 0.08 0.121 August December -0.21 0.08 0.096 September December 0.16 0.08 0.311 October December 0.38 0.08 0 November December 0.42 0.08 0 *The mean difference is significant at 0.05 levels.

80 Plate 4.1: Different food items collected from the gut of S. plagiostomus.

81 Table 4.4: Gut contents analysis of S. plagiostomus caught from river Indus and its tributaries during spring 2016.

Food items (i) occurrence Volumatric Imortance Index frequency (F) Analysis Index (AIi) (Vi) Spirogyra 100 59.5 5950

Ulothrix 100 52.25 5225

May flies 36.67 4.5 165.02

Caddis flies 33.33 4.25 141.65

Detritus 33.33 4.25 141.65

Sand & mud 81.67 10.25 837.12

82 Table 4.4.1: Gut contents analysis of S. plagiostomus caught fromriver Indus during summer, 2016.

Food items (i) Occurrence Volumetric Importance frequency (F) Analysis Index (Vi) Index (AIi)

Spirogyra 100 155 15500 Ulothrix 100 118.25 11825 May flies 35 4.25 148.75

Caddis flies 28.33 3.75 106.24

Detritus 36.67 5.25 192.52

Sand & mud 83.33 17.5 1458.28

83 Table 4.4.2: Gut Content Analysis of Schizothorax plagiostomus caught from river Indus and its Tributaries during autumn 2016.

Food items (i) Occurrence Volumetric Analysis Importance Index frequency (F) Index (Vi) (AIi)

Spirogyra 1 90 25.75 2317.5

Ulothrix2 88.33 23.75 2097.84

May flies 3 56.67 13.75 779.21

Caddis flies 4 51.67 11.5 594.21

Detritus 5 41.67 12 500.04

Sand & mud 6 68.33 13.25 905.37

84 Table 4.4.3: Gut Content Analysis of Schizothorax plagiostomus caught from river Indus and its Tributaries during winter 2016.

Food items (i) occurrence Volumetric Importance frequency (F) Analysis Index (Vi) Index (AIi)

Spirogyra 71.67 14.25 1021.3

Ulothrix 78.33 15.75 1233.7

May flies 96.67 26.25 2537.59

Caddis flies 96.67 25.5 2465.09

Detritus 95 36.5 3467.5

Sand & mud 95 33.5 3182.5

85 Figure 4.2: Bar graph showing seasonal variation in the mean importance index of different food itemsidentifiedin gut of S. plagiostomus collected from river Indus during 2016.

86 Table 4.4.4: Seasonal variations in the importance Index, mean rank of importance index and mean Kruskal-Wallis rank values recorded for the gut content analysis inS. plagiostomus caught from river Indus during 2016.

Seasons Mean Importance Mean rank of Mean Kruskal- Index of six food Importance Wallis rank Values items Index Autumn 1231.65 2.33 11.83 (±968.71)

Winter 2183.96 3.33 16.17 (±1405.38) P=0.525

Spring 2142.43 1.83 10.67 (±2972.32)

Summer 4914.87 2.50 11.33 (±7309.22)

*The mean difference is significant at 0.05 levels.

87 Table 4.4.5: Annual seasonal variations recorded in different food items in the Gut of S. plagiostomus caught from river Indus during 2016.

Food items Mean Mean ranks % Seasons p- importance of age value Index of food importance items Index Spirogyra 7520.35 21.00 Autumn, winter, (±6048.64) 47.87 spring and summer Ulothrix 5703.97 20.00 Autumn, winter, (±4451.21) 36.31 spring and summer Mayflies 502.29 7.00 Autumn and 0.006 (±673.25) 3.2 winter Caddis flies 512.63 7.25 Autumn and (±708.04) 3.26 winter Sand & mud 591.16 8.25 Autumn, winter, (±599.71) 3.76 spring and summer Detritus 878.97 11.50 Autumn, winter, (±48.05) 5.6 spring and summer *The mean difference is significant at 0.05 levels.

88 4.4.4 Sex Percentage

Table 4.5.4 and figure 4.3.6, has shown a total of 540 S. plagiostomus selected for reproductive assessment out of 1799 specimens caught during the year 2016. As the fishes migrate to shallow and upper part of the river during spawning period, most of the gravid samples were collected from the Indus river tributaries in the study area. The selected specimens were consisted of 277 males and 263 females. The overall female to male percentage was significant (P ≤ 0.05). The overall Chi square (χ2) value calculated was 19.65 with significant P-value (P ≤ 0.05).

4.4.5 Curvilinear Regression Analysis

4.4.5.1 Total Body Length vs GSI Male S. plagiostomus

The curvilinear regression analysis (quadratic model) were done in SPSS for total length versus GSI, and Body weight versus GSI, both for male and female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan, KP, Pakistan

The monthly mean total length TL and mean GSI ranges of male S. 2 plagiostomus, coefficient of determination (R ) and beta coefficients (b1, b2) are given in table 4.5.5. During January mean TL of male S. plagiostomus is recorded as 31.49cm with mean GSI value of 7.48, (b1) as 0.46 and (b2) as -0.01. The P-value obtained is 0.00 with R2 value of 0.92, which indicated that the model was of aa good fit.

The GSI remains constant initially and then decreases with increase in TL of male S. plagiostomus. Similarly, in February the mean total length of male S. plagiostomus calculated as 33.02cm with mean GSI of 17.5 and regression coefficients 2 (b1= 1.00), (b2 = -0.022) and (R =0.91). The P-value obtained has shown the significant correlation between TL and GSI.During May the testes of male S. plagiostomus shrunken and much reduced in size. The mean total length of fish was 30.69cm with 2 mean GSI of 0.16 and coefficients of regression as b1 =0.017, b2=0.00 and R =93. The P-value of 0.00 obtained has showna significant decrease in GSI when TL increases. In the following June the mean total length of 28.67cm with mean GSI value of 1.03 indicate the start of second reproductive cycle. The slope coefficients values for

89 2 quadratic model obtained are b1 =0.01, b2= -0.002 and R =0.85. Regarding the goodness of fit result, the quadratic regression model was found fit on the data.

During July the mean total length of male S. plagiostomus recorded as 31.18cm with mean GSI value of 1.76. The slope coefficients for quadratic model fit obtained as 2 b1 =0.26, b2= -0.005. The P-value (0.00) is significant. The R =0.40 has indicated that quadratic model was fairly fit. For the month of August, the mean total length of male S. plagiostomus is 28.84cm with mean GSI of 4.71.

The slope coefficients values for quadratic model fit calculated as b1 =0.127, b2= -0.002. The P-value (0.996) obtained is non-significant. The R2 value of 0.004 indicates that model does not fit the quadratic equation and the data is much deviated. The testes of male S. plagiostomus were found in stage III during early August and stage IV later during the same month with devastating effect on the data. The data deviated from quadratic law and model did not fit.

During September the mean total length of male S. plagiostomus recorded as

29.96cm with mean GSI of 11.76. The slope coefficients for model fit calculated as b1 2 =4.643 and b2= -0.072. The overall P-value (0.00) is significant and the R value of 0.40 indicated that the quadratic model was ofa good fit. Mean total length of male S. plagiostomus recorded during October is 27.76cm with mean GSI of 18.8. The curvilinear regression coefficients for model fit obtained are b1 =1.041 and b2= -0.023. The significant P-value (0.00) and R2 value of 0.68 has shown that model was best fitted.

In November the mean total length is 29.11cm with mean GSI of 15.67. The slope coefficients for the model fit calculated are b1 =1.38 and b2= -0.029. The overall significant P-value (0.00) with R2 value of 0.88 indicates that the model is a good fit. During December the mean total length recorded is 30.18cm with mean GSI of 6.52.

The slope coefficients for quadratic model fit obtained are b1 =0.919 and b2= -0.018. The overall highly significant P-value (0.00) indicates the decrease of GSI in response to TL of male S. plagiostomus. R2 value of 0.92 has indicated the modelas a good fit.

90 The testes entered into autumn cycle and the fish get started to spawn from September to November. The gonads were found in running condition during late September and throughout October and November. The present findings revealed the fact that maximum GSI in male S. plagiostomus was recorded during spring cycle (20.2) in March when compared to autumn cycle (18.8) in October.

4.4.5.2 Body Weight vs GSI (Male)

The month-wise mean body weight (BWT) and mean GSI ranges of male S. 2 plagiostomus, coefficient of determination (R ) and beta coefficients (b1, b2) are given in table 4.5.6. The negative b1 value (in this case BWT vs. GSI) explains decrease in mean GSI when the body weight increases by one unit. Similarly, the b2 value states the decrease in mean GSI (dependent variable) when the squared body weight (squared independent variable) increases by one unit. The R2has idicatedthe goodness of fit and when it is higher the model is better.

During January the mean body weight of 463.4g with mean GSI of 7.48 was recorded. The slope coefficients for quadratic model fit obtained were b1 = -0.008 while b2 value was less than zero (1.044E-006). The P-value is highly significant (0.00) with R2 value of 0.96 indicating that the model is a good fit. For the month of February, the mean body weight was 499g with mean GSI of 18.66. The slope coefficients to fit model were calculated as b1 = -0.007 while b2 value remained less than zero (-8.680E- 006). The increase in body weight cause significant decrease in the GSI of male S. plagiostomus as P-value is (0.00) with R2 value of 0.98 indicating that the model is a good fit.

In March the mean body weight of 354g with mean GSI value of 20.22 was recorded in male S. plagiostomus. The curvilinear regression coefficients to fit model obtained as b1 = -0.026 while b2 value remained less than zero (9.084E-006).

The increase in body weight has a significant effect on the decrease of GSI as P- value is less than 0.05 (0.00). The R2 value of 0.96 has shown that model was ofa good fit. During April the mean body weight of male S. plagiostomus recorded as 502.62g with mean GSI value of 16.23. The slope coefficients values recorded for b1 = -0.018

91 and b2 =4.507E-006 to fit the model for goodness. The overall P-value was highlysignificant at 0.00 level as P<0.05. The R2 value of 0.98 has shown that model was of a good fit.

In May the mean body weight of male S. plagiostomus recorded as 552.58g with mean GSI of 0.16. The curvilinear coefficients to fit the model for goodness were calculated as b1 = -0.00 and b2 =2.510E-009. The highly significant P-value obtained was at 0.00 level as P<0.05. The R2 value of 0.97 has indicated that model was ofa good fit. During the month of June, the mean body weight of 453.36g was recorded in male S. plagiostomus with mean GSI of 1.03. The slope coefficients to fit the model for goodness were b1 = -0.001 and b2 =-9.405E-008. The P-value of 0.00 was significant P<0.05. The R2 value of 0.93 has shown the model as a good fit.

In the following July mean body weight of 537.2g was recorded with mean GSI value of 1.76. The slope coefficients for model fit obtained were b1 = -0.002 and b2 =- 1.773E-006. The P-value obtained was significant at 0.00 P<0.05. The R2 value of 0.47 shownthemodel as a good fit. In the month of August, the mean body weight of male S. plagiostomus recorded as 415g with mean GSI of 4.71. The slope coefficients calculated to fit the model for goodness were -0.005 and b2 =-6.539E-006. The P-value of 0.964 was non-significant as P<0.05. The lowest R2 value of 0.004 has shown that model was not of a good fit.

During the following September the mean body weight of 483g was recorded with mean GSI value of 11.76. The curvilinear regression coefficients of -0.051 and b2 =-4.058E-005 were calculated to fit the model for goodness. The P-value was non- significant at 0.137 level as P<0.05. The R2 value of 0.22 has shown that model was notfitted. In the following month of October, the mean body weight of 372g was recorded with mean GSI of 18.8. The slope coefficients calculated to fit the model for goodness were -0.022 and b2 = 5.963E-006. The P-value was significant at 0.00 as P<0.05. The R2 value of 0.76 has indicatedthat model was a good fit.

For the month of November, the mean body weight of male S. plagiostomuswas 482.64g with means GSI of 15.94. The slope coefficients for model fit were -0.012 and 2 b2 = 1.135E-006. The P-value was significant at 0.00 level as P<0.05. The R value of

92 0.96 has shownthemodel as a good fit. In the following month of December,the mean body weight of male S. plagiostomus was 526g with mean GSI of 6.52. The slope coefficients for model fit were -0.006 and b2 = 7.281E-007. The P-value was significant at 0.00 level as P<0.05. The R2 value of 0.96 has indicated that the model was ofa good fit for the month of December.

4.4.5.3 GSI vs TL Female:

r The mean total length (TL) ,mean GSI coefficient of determination (R2) and beta coefficients (b1, b2) of female S. plagiostomusrecorded for each month were given in table 4.5.7. The b1 value has shown the increase in mean GSI when the body total lengthincreases by one unit. Similarly, the b2 value obtained has shownthe decrease in mean GSI when the squared totallength increases by one unit. The highest R2indicated that the model was best fitted.

During the month of January mean TL of female S. plagiostomus recorded as 29.4cm with mean GSI value of 5.93. The slope coefficients to fit model calculated as

(b1) as 0.47 and (b2) as -0.009. The unit change in GSI in response to TL wasvery low. The P-value of 0.00 andR2 value of 0.91 has indicated that the model was best fitted. In February the mean total length of 32.7cm and mean GSI of 18.4 was recorded in female 2 S. plagiostomus. The regression coefficients (b1= 1.42), (b2 = -0.028) and (R =0.91) were recorded. The P-value (0.00) calculated has shown that TL has a significant effect on theGSI.

Mean Total length of female S. plagiostomus calculated as 32.2cm with mean GSI value of 18.3 and regression coefficient of b1= 0.54, b2= -0.016) during March. The P-value obtained was highly significant. R2 value of (0.90) has shown that the model wasa good fit. The mean total length of S. plagostomuswas foundas 30.00cm, mean GSI of 16.7 2 and regression coefficients b1 =1.2 andb2 =-0.026 with R value of 0.92. The P-value calculated was statistically significant. The ovaries of female S. plagiostomuswere found shrunken and much reduced in size during the month of May. The mean total length of fish during May was calculated as 31.2cm , GSI =0.17 and coefficients of 2 regression as b1 =0.012, b2=0.00 and R =92. The P-value of 0.00 has shown significant effect between TL and GSI. The mean total length of the target species were calculated

93 as 32.1cm with mean GSI value of 0.93 during June indicating the start of second reproductive cycle. The values of slope coefficients for quadratic model were calculated 2 as b1 =0.09, b2= -0.002. The P-value obtained was statistically significant.The R value of 0.90 has shownmodel as a good fit.

In July the mean total length of female S. plagiostomuswas calculatedas 29.1cm andmean GSI value of 1.93. The values of slope coefficients obtained was b1 =0.31, b2= -0.006. The P-value calculated was statistically significant. The R2 of 0.40 has shown that quadratic model was of fairly fit. During August, t the mean total length of female S. plagiostomuswas calculated as29.6cm with mean GSI of 4.62. The slope coefficients values for quadratic model fit were calculated as b1 =0.36, b2= -0.007. The P-value (0.55) obtained wasstatistically non-significant. The R2 value of 0.06 has indicatedthatthe data was deviated from quadratic law and model did not fitted. . The ovaries of female S. plagiostomus were found in stage III during early August and stage IV later during the same month which causes devastating effect on the data.

The mean total length of female S. plagiostomusobtained during September were

30.5cm andmean GSI of 16.8. The slope coefficient values calculated wereb1 =1.023 2 and b2= -0.023. The overall P-value was statistically significant. R value was calculated as 0.94. In October, the mean total length of 29.6cm andmean GSI of 15.3, were found.

The slope coefficient values tofit model were b1 =0.82 and b2= -0.019. The significant P-value obtained was significant whereas the R2 value of 0.90 has shown that model was of good fit.

The mean TL and GSI data recorded during the month ofNovember were 27.5cm and 16.1 respectively. The slope coefficients for the model fit were calculated 2 asb1 =0.88 and b2= -0.022. The overall P-value wassignificant. R value of 0.90 has indicatedgoodness of model fit. In December the mean total length recorded was31.4cm with mean GSI of 4.77. The slope coefficients values were b1 =0.36 and b2= -0.008. The overall P-value was statistically significant. R2 value was calculated as 0.92.

The ovaries of female S. plagiostomus entered into autumn cycle and get started to spawn from September to November. The gonads were found in matured stage during September and in running stage throughout October and November. The present

94 findings revealed the fact that maximum GSI in female S. plagiostomus was recorded during spring cycle (18.4) in February when compared to autumn cycle (16.8) in the month of September.

4.4.5.4 Body Weight vs GSI (Female)

The mean body weight (BWT) and mean GSI of female S. plagiostomus, and 2 coefficient of determination (R ) and beta coefficients (b1, b2) for each month, are given in table 4.5.8. The negative regression coefficient values obtained in the present study indicate one-unit decrease in dependent variable (GSI) when independent variable increased by one unit (body weight).The value of R2shown in the present study, indicatethe goodness of model fit.The mean body weight and mean GSIrecorded during

January were 399.08g and5.93 respectively. The slope coefficients b1 = -0.002 andb2- 2.41E-06 were calculated. The P-value washighly significant. R2 value of 0.98 has shownthat the model was of good fit.In February the mean body weight was 484.19g andmean GSI was18.4. The slope coefficients to fit model were calculated as b1 = -0.02 b1 =3.07E-06. The results showed a significant decrease in the GSI of female S. plagiostomus when body weight gained. P-value was highly significant. The R2 value of 0.96 was calculated and the model was fitted good.

The mean body weight and mean GSI of 479.02g and18.3 were calculated during the month of March in female S. plagiostomus. The slope coefficients values to fit model were b1 = -0.025 b2= (-7.26E-06).It was observed that increase in body weight has a significant effect on the decrease of GSI during the same month as P-value wasless than 0.05. The R2 value calculated was0.97and modelwas of a good fit. In April, the mean body weight and mean GSIin femaleS. plagiostomuswere463.06g and16.7. The slope coefficients values wereb1 = -0.02 and b2 =-5.23E-06. The overall P- value was highly significant at 0.00 level as P<0.05. The R2 value was 0.97.

During May the values for mean body weight and mean GSI of female S. plagiostomuswere 547.19g and 0.17 respectively. The curvilinear coefficients to fit the model for goodness were calculated as b1 = 0.00 and b2 =-1.09E-06. The P-value obtained was highly significant. The R2 value was0.98 and the model was of a good fit. During June, the mean body weight was calculated as 591.74g and mean GSI as0.93.

95 The slope coefficients to fit the model for goodness were b1 = -0.001 and b2 =-7.96E-06. The P-value was significant P<0.05the R2 value was0. 96..

During July the values of mean body weight and mean GSI in female S. plagiostomus calculated were of 482.02g 1.93. The slope coefficients for model fit obtained were b1 = -0.001 and b2 =-3.68E-06. The P-value obtained was significant at 0.00 P<0.05. The R2 value was0.38 which has indicated that the model was of a good fit. In the month of August, the mean body weight of male S. plagiostomus recorded was 462.67g and themean GSI was 4.62. The slope coefficients calculated to fit the model for goodness wereb1=-0.005 and b2 =-6.09E-06. The non-significant P-value was0.39. The lowest R2 value of 0.09 has indicated that model were notfitted.

During the following September the mean body weight recorded in female S. plagiostomuswas 497.72g with mean GSI value of 16.8. The curvilinear regression coefficients of -0.018 and b2 =-4.50E-06 were calculated to fit the model for goodness. The P-value was significant at 0.00 level as P<0.05. The R2 value was 0. 97. In October the mean body weight of 474.9g with means GSI of 15.3 were recorded in female S. plagiostomus. The slope coefficients calculated to fit the model for goodness were - 2 0.018 and b2 = -4.74E-06. The P-value was significant at 0.00 as P<0.05. The R value of 0.95 has shown that model was good fitted.

For the month of November, the mean body weight of 406.11g with means GSI of 16.1 were recorded in female S. plagiostomus. The slope coefficients to fit model 2 were -0.02 and b2 = -5.71E-06. The P-value was significant. The R value of 0.95 has indicated that that model was good fitted.

In December, the mean body weight of female S. plagiostomuswere calculated as563.04g withmean GSI of 4.77. The slope coefficients obtained to fitmodel wereb1=- 2 0.006 and b2 = -1.73E-06. The P-value was significant as P<0.05. The R value of 0.98 has shown the model as a good fit.

4.4.5.5 Assessment of Fecundity in Female S. plagiostomus

Monthly variations of TL, BWT, mean fecundity and its ranges with standard error in female S. plagiostomus caught from river Indus and its tributaries in Indus

96 Kohistan are given in table 4.5.9 and figure 4.3.7.

The mean absolute fecundity of 2046.93 (±288.66) (range=659.89-5920) eggs with fish having mean total body length of 32.7cm and mean body weight of 484g were recorded during February. Similarly, in the following March, the absolute fecundity of 6209.94(±474.52) (range= 2350.23- 13484.9) eggs were recorded in female S. plagiostomushaving meantotal body length of 32.2 cm and mean body weight of 479.00g.

Mean absolute fecundity of 8504.59(±614.62) (range=2386.67-13610.19) eggs were recorded during the month of April in fishes having mean TL of 30.00 cm and body weight of 463g. The maximum absolute fecundity with higher spawning response in female S. plagiostomuswas observed during April. The spawning cycle in spring season was observed during March and April and the gonad immediately changed to spent condition in the following May. The ovaries during June get started to enter into immature Stage-I (ambiguous sex) followed by developing stage-II (unambiguous sex) during July. The ovaries gained weight rapidly during August and September and got entered into second spawning cycle during autumn. The second spawning cycle was observed to start in September and ended in November. The mean absolute fecundity of 5618.19(±298.28), (range= 3687.46- 9311.16) eggs in fishes having mean TL of 30.5cm and mean body weight of 498g were recorded during September.In October the mean absolute fecundity of 12534.95(±447.91), (range=10296.34-18188) eggs were recorded in fishes having mean TL of 29.6cm and mean body weight of 475g. Similarly, during November, the mean absolute fecundity of 14079.92(±648.93), range=9998.88- 20474.96 ova in female S. plagiostomus having mean TL of 27.5cm and body weight of 406g were recorded.The spawning response lasted for a period of three months (September, October and November) during autumn.

The ovaries immediately stopped spawning during December without entering to spent stage. All the ovaries sampled during December were found lobed and white in color and indicated stage-IV of the maturity stages. It is noteworthy that spent, immature and developing stages were found missing during December and January.

These findings revealed the fact that S. plagiostomus spawn twice in a year with

97 a low fecundity response during spring (February, March and April) and a higher fecundity response during Autumn (September, October and November) table 4.5.9 and figure 4.3.7.

4.5IN-SILICO ANALYSIS

Tables 4.6 and 4.6.1has shown the average percentage divergence of all 28- specimen sequences with species from NCBI-GeneBank. The average evolutionary divergence and average percent evolutionary divergence between specimen sequences and S. plagiostomus (KU317693.1) from GeneBank were estimated as 0.03 and 2.978%, for S. plagiostomus (KT833100.1) 0.031 and 3.117, for S. plagiostomus (KU317682.1) 0.031 and 3.105, for S. plagiostomus (KT184924.1) 0.031 and 3.133, respectively.Similarly, the average evolutionary divergence and average percent evolutionary divergence estimated for specimen sequences and S. plagiostomus (KU317690.1) from NCBI GeneBank was 0.033 and 3.271, for S. plagiostomus (KU317688.1) was 0.033 and 3.282, for S. plagiostomus (KU317687.1) was 0.033 and 3.272, respectively.

The average evolutionary divergence and average percent evolutionary divergence for specimen sequences and sequences from NCBI GeneBank was estimated 0.032 and 3.227 for S. esocinus (KT210882.1), 0.032 and 3.183 for S. progastus (KF739399.1), 0.032 and 3.238 for S. labiatus (KT833092.1), and for S. esocinus (KU317702.1) as 0.036 and 3.588, respectively.

The average evolutionary divergences and average percent evolutionary divergence for S. nepalensis (AP011207.1), S. richardsonii (KU695220.1), S. richardsonii (KF429953.1), S. curvilabiatus(MF804977.1) and for S. richardsonii (KC790369.1) were estimate as 0.039 and 3.88, 0.03 and 2.971, 0.029 and 2.934, 0.07 and 6.979, and 0.033 and 3.336, respectively.

The highest percent evolutionary divergence was estimate between specimen sequences (S 1 FWP- S28 FWP) and S. curvilabiatus (MF804977.1) from China (6.9795), while the lowest divergence (2.934%) were estimated between the specimens sequences (S 1 FWP- S28 FWP) and S. richardsonii (KF429953.1) from India.

98 4.5.1 Phylogenetic Tree

The complete COI sequences of the specimen sequences were blasted and searched in NCBI GeneBank database. All the closely related showing 99% similarity were downloaded. The NJ phylogenetic tree was constructed by using Kimura-2- parameter with 1000 bootstrap replication based on complete COI sequences figure 4.4. The aligned sequences were used to determine the evolutionary relationship among these sequences.

Homology analyses were performed taking the predicted nucleotide sequences into account, revealed that COI sequences were cluster into two majorclades and seven sub-clades belonging to genus Schizothorax. Each sequence was grouped with homologous COI that belong to same genus. On the other hand, sequence branched as an isolated clade, showing the divergence of CO1 gene among these fish species.

The first clade on phylogenetic tree has a division of three sub-clades. In a first sub-clade the specimen sequence (S_15_FWP), (S_9_FWP), (S_6_FWP) and (S_21_FWP) were cluster together with S. labiatus, S. esocinus, S. progastus and S. richardsoni. The second sub-clade of specimen sequences (S_23_FWP), (S_28_FWP), (S_16_FWP), (S_2_FWP), (S_20_FWP), (S_1_FWP), (S_22_FWP), and (S_27_FWP) were clustered closely to the first sub-clade on the phylogenetic tree. The sequences in first sub-clade were closely related to S. labiatus, S. esocinus, S. progastus and S. richardsoni and distantly related to 2nd sub-clade

In a 4th sub-clade sequence (S_13_FWP) and (S_17_FWP) clustered together with S. nepalensis and S. curvilabiatus which shared the same clade with S. plagiostomus.

The fifth sub-clade of specimen sequences (S_5_FWP), (S_8_FWP), (S_24_FWP), (S_3_FWP) and (S_19_FWP) and (S_11_FWP) clustered closely with the with sixth and seventh sub-clade of specimen sequence (S_7_FWP), (S_26_FWP), (S_10_FWP), (S_25_FWP) and (S_4_FWP) in a major clade of S. plagiostomus of the phylogenetic tree. The phylogenetic tree clearly indicated that 13-specimen sequences clustered closely in a clade of S. plagiostomus, while remaining 15- specimen sequences

99 clustered with other species from Schizothorax genus on a phylogenetic tree.

100 Table 4.5:Monthly descriptive statistics and regression parameters (quadratic) of length-weight relationship of S. plagiostomus (male) caught from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan.

Months N Total Length (cm) Total Weight (g) Regression parameters R2 p value

Mean Minimum Maximum Mean Minmum Maximum b0 b1 b2 January 32 31.49 20.4 44 463.4 210.6 930 928.038 -60.45 1.378 0.97 ˂0.05 February 20 33.02 20.5 41 499 230 732 720.666 -46.304 1.154 0.97 ˂0.05 March 19 26.58 20 43.8 354.1 225 994 575.797 -39.657 1.123 0.99 ˂0.05 April 22 31.19 20 45 502.6 230 1234 1133.077 -77.126 1.717 0.98 ˂0.05 May 29 30.69 20 42.5 552.6 272 1197.5 1614.355 -114.013 2.451 0.97 ˂0.05 June 27 28.67 20 42.7 453.4 269 1211.5 1559.408 -109.274 2.362 0.93 ˂0.05 July 27 31.18 20 43 537.2 289 1300 1599.945 -107.1 2.233 0.95 ˂0.05 August 21 28.84 20 38.5 415.1 265.5 662.5 490.676 -27.511 0.825 0.98 ˂0.05 September 19 29.96 20.5 42.5 482.8 246.7 1110 1139.602 -78.022 1.787 0.98 ˂0.05 October 19 27.76 20.8 44.6 371.9 226.7 1172 1161.45 -80.343 1.793 0.97 ˂0.05 November 22 29.11 20.4 42 462.6 256 1284 1918.5 -137.338 2.882 0.95 ˂0.05 December 20 30.18 21 43 525.8 278 1310 2235.937 -155.217 3.138 0.96 ˂0.05 *The mean difference is significant at 0.05 levels.

101 Figure 4.3: Scatter plot showing curve estimation (quadratic) between total 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.

102 Table 4.5.1: Monthly descriptive statistics and regression parameters of length-weight relationship of S. plagiostomus (female) caught from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan.

Months N Total Length (cm) Total Weight (g) Regression parameters 2 Mean Minimum Maximum Mean Minimum Maximum b0 b1 b2 R p value January 13 29.38 21.5 42 399 245.6 860 1120.36 -72.15 1.53 0.96 ˂0.05 February 25 32.67 22.5 43.8 484 268 940 1050.23 -66.28 1.44 0.97 ˂0.05 March 26 32.22 20.5 42.8 479 235 999 680.97 -44.95 1.15 0.93 ˂0.05 April 23 29.96 20.5 43.5 463 238 1272 1551.44 -108.51 2.29 0.93 ˂0.05 May 16 31.18 22 45.5 547 282 1250.5 1508.61 -103.23 2.2 0.96 ˂0.05 June 18 32.05 20.5 43.5 592 290 1217.5 1507.48 -103.99 2.24 0.93 ˂0.05 July 18 29.06 21.5 41.7 482 283 1161.5 1691.29 -118.46 2.53 0.98 ˂0.05 August 24 29.58 21 42 463 279 1110 1394.96 -93.05 2 0.95 ˂0.05 September 26 30.53 21.6 43.6 498 286 1213 1468.10 -98.56 2.09 0.98 ˂0.05 October 26 29.58 20.5 43.5 475 256.7 1250 1358.70 -93.01 2.04 0.96 ˂0.05 November 23 27.5 21.5 39.5 406 266 995 1360.27 -94.62 2.11 0.95 ˂0.05 December 25 31.41 21.4 42.6 563 263 1264 1835.78 -128.09 2.68 0.98 ˂0.05 *The mean difference is significant at 0.05 levels.

103 Figure 4.3.1: Scatter plot showing curve estimation (quadratic) between total body length (cm) and body weight (g) of female S. plagiostomus caught fromriver Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016.

104 Mean CF of males S. plagiostomus 2.5

2

1.5

Mean CF Mean Mean CF 1

0.5

0 JANUARYFEBRUARY MARCH APRIL MAY JUNE JULY AUGUSTSEPTEMBEROCTOBERNOVEMBERDECEMBER

Collection months

Figure 4.3.2: Monthly variation in the condition factor of S. plagiostomus (male) collected from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan.

105 Table 4.5.2: Month wisedescriptive statistics of condition factor (K-value) 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.

Collection N Mean Std. deviation Minimum Maximum P- months value

January 32 1.52 0.49 0.94 2.48 February 20 1.47 0.55 1.06 2.67 March 19 1.92 0.52 1.18 2.83 April 22 1.69 0.61 1.13 3 May 29 1.92 0.68 1.16 3.53 0.046 June 27 1.99 0.68 1.21 3.51 July 27 1.8 0.6 1.16 3.61 August 21 1.9 0.72 1.13 3.32 September 19 1.81 0.51 1.23 3.06 October 19 1.77 0.51 1.11 2.6 November 22 1.88 0.57 1.08 3.02 December 20 1.87 0.51 1.21 3 *The mean difference is significant at 0.05 levels.

106 Mean CF female S. plagiostomus 2.5

2

1.5

Mean CF Mean 1 Mean

0.5

0

Collection months

Figure 4.3.3: Monthly variation in the mean condition factor with std. error bar of S. plagiostomus (female) collected from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan.

107 Table 4.5.3: Month wise condition factor (K-value) with Std. deviation and overall p- value of female S. plagiostomus caught from river Indus and its tributaries during 2016, in Indus Kohistan KP, Pakistan.

Collection N Mean Std. deviation Minimum Maximum P- months Value

January 13 1.66 0.56 0.94 2.47 February 25 1.41 0.42 0.92 2.42 March 26 1.47 0.48 0.98 2.73 April 23 1.71 0.54 1.06 2.9 May 16 1.76 0.5 1.13 2.72 June 18 1.8 0.61 1.18 3.39 0.004 July 18 1.98 0.6 1.18 3.15 August 24 1.87 0.66 1.08 3.21 September 26 1.77 0.51 1.22 2.93 October 26 1.86 0.54 1.24 3.2 November 23 1.98 0.45 1.33 2.7 December 25 1.77 0.46 1.22 2.75 *The mean difference is significant at 0.05 levels.

108 Figure 4.3.4: Monthly variation in the percentage of gonad maturity stages recorded in male S. plagiostomus caught fromrive Indus during January to December 2016(Gonad stages= I-VII).

109 Figure 4.3.5: Monthly variation in the percentages ofgonad maturity stages infemale S. plagiostomuscaught from rive Indus during January to December 2016 (Gonad stages= I-VII).

110 Table 4.5.4: Month wise sex ratio of S. plagiostomuswith chi square and p-values caught from river Indus during 2016.

Collection Months Male Female Chi Square value P value

January 32 13

February 20 25

March 19 26

April 22 23

May 29 16 19.65 0.05

June 27 18

July 27 18

August 21 24

September 19 26

October 19 26

November 22 23

December 20 25

*The mean difference is significant at 0.05 levels.

111 Figure 4.3.6: Bar graph showing moth-wise count of male and female S. plagiostomus caught from river Indus during 2016.

112 Table 4.5.5: Monthly descriptive statistics and regression parameters between TL and GSI of male S. plagiostomus caught from river Indus and its Tributaries during 2016, in Indus Kohistan KP, Pakistan.

N Total body Length (cm) GSI Regression parameters Months Mean Std. error Mean Std. error b1 b2 R2 p value January 32 31.49 1.3 7.48 0.26 0.46 0 0.92 ˂0.05 February 20 33.02 1.5 17.5 0.65 1 0 0.91 ˂0.05 March 19 26.58 1.39 20.2 0.69 0.35 0 0.94 ˂0.05 April 22 31.19 1.7 15.8 0.73 0.81 0 0.96 ˂0.05 May 29 30.69 1.37 0.16 0.01 0.02 0 0.93 ˂0.05 June 27 28.67 1.18 1.03 0.03 0.1 0 0.85 ˂0.05 July 27 31.18 1.35 1.76 0.09 0.26 0 0.4 ˂0.05 August 21 28.84 1.38 4.71 0.45 0.13 0 0 0.97 September 19 29.96 1.54 11.8 1.11 4.64 -0 0.39 ˂0.05 October 19 27.76 1.36 18.8 0.73 1.04 0 0.68 ˂0.05 November 22 29.11 1.28 15.7 0.6 1.38 0 0.88 ˂0.05 December 20 30.18 1.39 6.52 0.32 0.92 0 0.92 ˂0.05 *The mean difference is significant at 0.05 levels.

Table 4.5.6: The monthly descriptive and curvilinear regression analysis (quadratic) of body weight vs GSI in male S. plagiostomuscaught from river Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016.

113 Months N Body Weight (g) GSI Regression parameters Mean Std. Mean Std. b1 b2 R2 p value error error January 32 463.4 39.31 7.48 0.26 -0.008 1.04E-06 0.96 ˂0.05 February 20 499 39.43 17.5 0.65 -0.007 -8.68E-06 0.98 ˂0.05 March 19 354 42.56 20.22 0.69 -0.026 9.08E-06 0.96 ˂0.05 April 22 502.62 60.76 15.79 0.73 -0.018 4.51E-06 0.98 ˂0.05 May 29 552.58 58.59 0.16 0.01 0 2.51E-09 0.97 ˂0.05 June 27 453.36 45.11 1.03 0.03 -0.001 -9.41E-08 0.93 ˂0.05 July 27 537.2 54 1.76 0.09 0.002 -1.77E-06 0.47 ˂0.05 August 21 415 28.89 4.71 0.45 -0.005 -6.54E-06 0.004 0.964 September 19 483 54.79 11.76 1.11 -0.051 -4.06E-05 0.22 0.137 October 19 372 47.16 18.8 0.73 -0.022 5.96E-06 0.76 ˂0.05 November 22 462.64 56.24 15.67 0.6 -0.012 1.14E-06 0.96 ˂0.05 December 20 526 68.25 6.52 0.32 -0.006 7.28E-07 0.96 ˂0.05 *The mean difference is significant at 0.05 levels.

Table 4.5.7: The monthly descriptive and curvilinear regression analysis (quadratic) of TL vs GSI in female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016.

Months N Total body Length (cm) GSI Regression Parameters

114 Mean Std. error Mean Std. error b1 b2 R2 p value January 13 29.4 2.07 5.93 0.26 0.47 -0.009 0.91 ˂0.05 February 25 32.7 1.4 18.4 0.71 1.42 -0.028 0.91 ˂0.05 March 26 32.2 1.36 18.3 0.72 0.54 -0.016 0.9 ˂0.05 April 23 30 1.49 16.7 0.75 1.2 -0.026 0.92 ˂0.05 May 16 31.2 1.92 0.17 0.01 0.012 0 0.92 ˂0.05 June 18 32.1 1.73 0.93 0.05 0.09 -0.002 0.9 ˂0.05 July 18 29.1 1.5 1.93 0.16 0.31 -0.006 0.4 ˂0.05 August 24 29.6 1.26 4.62 0.42 0.36 -0.007 0.06 0.55 September 26 30.5 1.3 16.8 0.61 1.023 -0.023 0.94 ˂0.05 October 26 29.6 1.24 15.3 0.53 0.82 -0.019 0.9 ˂0.05 November 23 27.5 1.08 16.1 0.47 0.88 -0.022 0.9 ˂0.05 December 25 31.4 1.32 4.77 0.23 0.36 -0.008 0.93 ˂0.05

*The mean difference is significant at 0.05 levels. Table 4.5.8: The monthly descriptive and curvilinear regression analysis (quadratic) of body weight vs GSI in female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan, KP, Pakistan during 2016.

Months N Body Weight (g) GSI Regression Parameters Mean Std. error Mean Std. error b1 b2 R2 p value

115 January 13 399.08 53.2 5.93 0.26 -0.002 -2.41E-06 0.98 ˂0.05 February 25 484.19 43 18.4 0.71 -0.02 -3.07E-06 0.96 ˂0.05 March 26 479.02 40.4 18.3 0.72 -0.025 -7.26E-06 0.97 ˂0.05 April 23 463.06 60.41 16.7 0.75 -0.02 -5.23E-06 0.97 ˂0.05 May 16 547.19 86.75 0.17 0.01 0 -1.09E-06 0.98 ˂0.05 June 18 591.74 76.27 0.93 0.05 -0.001 -7.96E-06 0.96 ˂0.05 July 18 482.02 62.36 1.93 0.16 -0.001 -3.68E-06 0.38 ˂0.05 August 24 462.67 41.74 4.62 0.42 -0.005 -6.09E-06 0.09 0.39 September 26 497.72 49.23 16.8 0.61 -0.018 -4.50E-06 0.97 ˂0.05 October 26 474.9 45.69 15.3 0.53 -0.018 -4.74E-06 0.95 ˂0.05 November 23 406.11 34.71 16.1 0.47 -0.02 -5.71E-06 0.95 ˂0.05 December 25 563.04 61.77 4.77 0.23 -0.006 -1.73E-06 0.98 ˂0.05 *The mean difference is significant at 0.05 levels.

116 Mean absolute fecundity of female S. plagiostomus 16000 14000 12000 10000 8000 fecundity - 6000 4000 Mean A Mean 2000 0 -2000

Collection Months 2016

Figure 4.3.7:Line graph showing monthly variations of mean absolute fecundity with standard error bar in female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan district, KP, Pakistan.

Table 4.5.9: Monthly variations of mean absolute fecundity and means of body parameters for female S. plagiostomus caught from river Indus and its tributaries in Indus Kohistan during 2016.

117 Collection No of Fish Mean TL Mean BWT Mean Absolute Fecundity Minimum Maximum months with Std. error January 13 29.4 399 0 0 0 February 25 32.7 484 2046.93 (±288.66) 659.89 5920 March 26 32.2 479 6209.94(±474.52) 2350.23 13484.9 April 23 30 463 8504.59(±614.62) 2386.67 13610.19 May 16 31.2 547 0 0 0 June 18 32.1 592 0 0 0 July 18 29.1 482 0 0 0 August 24 29.6 463 0 0 0 September 26 30.5 498 5618.19(±298.28) 3687.46 9311.16 October 26 29.6 475 12534.95(±447.91) 10296.34 18188 November 23 27.5 406 14079.92(±648.93) 9998.88 20474.96 December 25 31.4 563 0 0 0

118 Table 4.6: Average nucleotide frequencies (AT/GC composition) after COI amplification of specimen sequences.

COI sequences T C A G S 1 FWP 28.7 27.8 25.9 17.7 S 2 FWP 28.8 27.9 25.8 17.5 S 3 FWP 28.8 28.0 25.7 17.5 S 4 FWP 27.9 28.7 24.1 19.4 S 5 FWP 28.7 28.0 25.7 17.6 S 6 FWP 28.8 28.0 25.7 17.5 S 7 FWP 28.9 28.0 25.6 17.5 S 8 FWP 28.8 27.9 25.7 17.6 S 9 FWP 28.6 28.0 25.7 17.6 S 11 FWP 28.9 27.8 25.7 17.6 S 12 FWP 27.8 30.8 25.0 16.3 S 13 FWP 28.9 27.9 25.9 17.3 S 14 FWP 25.4 31.5 24.1 19.0 S 15 FWP 28.7 27.8 25.8 17.6 S 16 FWP 28.8 28.1 25.8 17.4 S 17 FWP 28.9 27.8 26.0 17.2 S 18 FWP 28.1 28.9 26.4 16.6 S 19 FWP 28.7 27.9 25.8 17.7 S 20 FWP 28.8 28.1 25.8 17.4 S 21 FWP 28.6 28.0 25.9 17.5 S 22 FWP 28.7 28.3 25.7 17.3 S 23 FWP 28.8 28.0 25.9 17.3 S 24 FWP 28.8 27.9 25.6 17.7 S 25 FWP 29.0 27.8 25.8 17.4 S 26 FWP 28.9 28.0 25.6 17.5 S 27 FWP 28.7 28.3 25.7 17.3 S 28 FWP 28.7 28.0 25.8 17.5 S10 FWP 28.8 27.9 25.9 17.5 Average 28.6 28.3 25.6 17.5

119 Table 4.6.1: Species names, AT/GC average composition identified during this study.

COI sequences T(U) C A G Total S 1 FWP 28.7 27.8 25.9 17.7 691.0 S 2 FWP 28.8 27.9 25.8 17.5 691.0 S 3 FWP 28.8 28.0 25.7 17.5 692.0 S 4 FWP 27.9 28.7 24.1 19.4 677.0 S 5 FWP 28.7 28.0 25.7 17.6 693.0 S 6 FWP 28.8 28.0 25.7 17.5 692.0 S 7 FWP 28.9 28.0 25.6 17.5 692.0 S 8 FWP 28.8 27.9 25.7 17.6 692.0 S 9 FWP 28.6 28.0 25.7 17.6 692.0 S 11 FWP 28.9 27.8 25.7 17.6 693.0 S 12 FWP 27.8 30.8 25.0 16.3 711.0 S 13 FWP 28.9 27.9 25.9 17.3 692.0 S 14 FWP 25.4 31.5 24.1 19.0 705.0 S 15 FWP 28.7 27.8 25.8 17.6 693.0 S 16 FWP 28.8 28.1 25.8 17.4 691.0 S 17 FWP 28.9 27.8 26.0 17.2 691.0 S 18 FWP 28.1 28.9 26.4 16.6 686.0 S 19 FWP 28.7 27.9 25.8 17.7 691.0 S 20 FWP 28.8 28.1 25.8 17.4 691.0 S 21 FWP 28.6 28.0 25.9 17.5 692.0 S 22 FWP 28.7 28.3 25.7 17.3 693.0 S 23 FWP 28.8 28.0 25.9 17.3 692.0 S 24 FWP 28.8 27.9 25.6 17.7 691.0 S 25 FWP 29.0 27.8 25.8 17.4 690.0 S 26 FWP 28.9 28.0 25.6 17.5 692.0 S 27 FWP 28.7 28.3 25.7 17.3 693.0 S 28 FWP 28.7 28.0 25.8 17.5 693.0 S10 FWP 28.8 27.9 25.9 17.5 692.0 Avg. 28.6 28.2 25.7 17.5 687.2

120 Table 4.6.2: Estimates of average evolutionary divergences between specimen sequences and NCBI–GenBank sequences.

Species NCBI-GeneBank Regions Specimen Sequences Evolutionary % Evolutionary accession No from River Indus Divergence Divergence S. plagiostomus KU317693.1 AJK Pakistan S 1 FWP- S28 FWP 0.03 2.978% S. plagiostomus KT833100.1 China S 1 FWP- S28 FWP 0.031 3.117% S. plagiostomus KU317682.1 AJK Pakistan S 1 FWP- S28 FWP 0.031 3.105% S. plagiostomus KT184924.1 Dir Pakistan S 1 FWP- S28 FWP 0.031 3.133% S. plagiostomus KU317690.1 AJK Pakistan S 1 FWP- S28 FWP 0.033 3.271% S. plagiostomus KU317688.1 AJK Pakistan S 1 FWP- S28 FWP 0.033 3.282% S. plagiostomus KU317687.1 AJK Pakistan S 1 FWP- S28 FWP 0.033 3.272% S. esocinus KT210882.1 Dir Pakistan S 1 FWP- S28 FWP 0.032 3.227% S. progastus KF739399.1 India S 1 FWP- S28 FWP 0.032 3.183% S. labiatus KT833092.1 China S 1 FWP- S28 FWP 0.032 3.238% S. esocinus KU317702.1 AJK Pakistan S 1 FWP- S28 FWP 0.036 3.588% S. nepalensis AP011207.1 Japan S 1 FWP- S28 FWP 0.039 3.88% S. richardsonii KU695220.1 India S 1 FWP- S28 FWP 0.03 2.971% S. richardsonii KF429953.1 India S 1 FWP- S28 FWP 0.029 2.934% S. curvilabiatus MF804977.1 China S 1 FWP- S28 FWP 0.07 6.979% S. richardsonii KC790369.1 India S 1 FWP- S28 FWP 0.033 3.336%

121 Figure 4.4: Phylogenetic tree using NJ method showing the relationship of 28-sequences with 16Schizothorax spp.COI sequences from NCBI GenBank.

122 123 124 125 126 127 128 Figure 4.1: Nucleotide sequence comparison (GeneDoc) of fish CO1 sequenced in the present study from different streams adjoining river Indus in Indus Kohistan, Pakistan. Black and grey indicate 100 and 80–90 % sequence identity, respectively.

129 CHAPTER 5

DISCUSSION

5.1 FISH DISTRIBUTION AND ABUNDANCE

The present work was designed to investigate species abundance, distribution and the food composition of S. plagiostomus and seasonal variations. Upper part of river Indus is highly torrential which passes through narrow gorges having high sediment load leading to poor Ichthyodiveristy. The present finding revealed that S. plagiostomus was the most dominant fish species both in fast flowing torrential and low velocity waters collected from different streams joining river Indus (Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream) in the study area. In line with this (Yousuf, 1996)investigated that the dominance of S. plagiostomusthroughout Liddar river in Kashmir seems to be related to its love for fast flowing water in torrential which is present both in low and high depth and velocity.

During this survey the low fish fauna was found in River Indus which tally the findings of (Rafique, 2000)that the fish fauna of river Indus is constituted by 177 fish species including 12 exotic species which is substantially lower than other major rivers in Asia like (350 species), Mekong (400) & Hwang Hu (320 species). Several authors worked on the feeding habit of S. plagiostomus(T. Shrestha, 1979) and(Rai, Pradhan, Basnet, & Sawr, 2002).

5.2 FEEDING HABIT AND ANALYSIS OF GUT CONTENTS

It is fact that during this study the gut contents of S. plagiostomus were analyzed by the method of Volumetric Analysis Index, which has been calculated on a constant referential of standard weight (SW) for all guts. The method of calculating Importance Index of distinct food items in a fish gut is a combination of Volumetric Analysis Index and Occurrence Frequency methods, which has been proved highly effective when applied. The Importance Index method used during this study yielded good results and the guts were analyzed effectively. In practice, the risk of inaccuracy in identifying different food items and diet determination of various fishes due to their variable age and sizes is a major issue in Point Method (Hynes, 1950). Such issues can be avoided when the method of Volumetric Analysis Index is applied. Similar results were found by (Lima-Junior & Goitein, 2001)who also revealed the efficiency of the same method independently of the degree of stomach fullness. (Lima-Junior & Goitein, 2001) also weighted the method of Volumetric Analysis Index over the method of Food Preference Degree described (Sipaúba-Tavares & de S Braga, 1999).

130 Analysis of gut contents in fisheries sciences is widely used for the purpose of conservation and aquaculture practices.In the present study the percentage of different food items in the gut of S. plagiostomushas been found for Spirogyra as (47.87%), Ulothrix (36.31%), Mayflies (3.2%), Caddies flies (3.26%), Sand and mud (3.76%) and detritus (5.6%). (Das & Moitra, 1963)classified the feeding habit of fishes as herbivores (phytophagous), if their food comprise of 75% vegetable matter, carnivore, if they feed on 75% animal matter, and omnivore if they consume mixed diet comprised of 50%animal food and50% plant materials. Two more categories Herbi-omnivore (consuming greater amount of plant materials) and Carni-omnivore (feeding on greater amount of animal matter) have further been added to the same scheme of classification (N. Singh, Bahuguna, & Bhatt, 1993).The results of the present study clearly indicate that S. plagiostomus is a Harbi-omnivorous fish consuming less than 75% of vegetative matter.

In the present study the mean Importance Index calculated for Spirogyra as (7520.35), Ulothrix (5703.97), Mayflies (502.29), Caddies flies (512.63), Sand and mud (591.16) and detritus (878.97). Variations in these food items were found significant between vegetative matter and animal matter etc. which revealed that S. plagiostomus feed on vegetative matter as well as on other non-vegetative food items. In contrast to the present finding the phytophagous feeding habit was reported in S. curvifrons by (S Sunder & Subla, 1985)and (Rasool, Jan, & Shah, 2012).

The Importance Index method applied during this study indicated the presence of Spirogyra, Ulothrix, Mayflies, Caddies flies, Sand and mud and detritus which proved that S. plagiostomus consume a greater amount of vegetative matter scraping spirogyra and ulothrix attached to stones and pebbles. The considerable amount of animal matter along with detritus has confirmed the Herbi- omnivore type of feeding habit in S. plagiostomus. In contrast to the present findings the phytophagous feeding habit of S. plagiostomus were reported by (T. Shrestha, 1979), (Rai et al., 2002), (Terashima, 1984)and (Sharma, 1989).

The mouth of S. plagiostomusis inferior, wide, with deep lower jaw having keratinized cutting edge. The lower lip is folded and expanded with numerous papillae making it best suited for scrapping algae attached to stones and pebbles. Similar adaptations in the mouth of S. plagiostomus were reported and described by (Kullander et al., 1999).

The highest feeding activity of S. plagiostomus was observed during summer and spring. The results showed that S. plagiostomus eats more during warmer months than what is observed in the cold months. S. plagiostomus spawn twice in a year in autumn and in spring. The highest feeding activity of S. plagiostomusseems to be link with a reflex of recovery strategy due to physiological process of

131 gonadal development. Similar results were found by (Lima junor & Goitein, 2004) working on the feeding activity of Pimelodusmaculatus.

The present findings revealed that S. plagiostomus consume highest amount of Spirogyra and Ulothrix as a major food item throughout the year with a considerable amount of animal matter, sand particles and detritus. The intake of animal matter, detritus and sand and mud slightly increased during autumn and winter while Spirogyra and Ulothrixstill constituted the major component of the dietduring the same seasons. Similar relationship was observed between the nature of diet and seasons in S. esocinus and S. nigerby (Raina & Petr, 1999).

Statistically no significant difference was observed in the ranking of different food items across all seasons when the seasonal data was compared. However, the clear drop in the importance index of the main food items (spirogyra and ulothrix) added to smaller stomach fullness indexes observed in autumn and winter, showed a decrease in feeding activity at a transition between the seasons. In agreement to this the findings of )(Wootton, 1991)and (Jobling, 1994) states that the fish feeding rate is directly proportional to environmental temperature.

The present findings will help to prepare a proper feed for S. plagiostomus in order to bring these fish species of economic importance in captivity for the purpose of their conservation and breeding.

5.3 REPRODUCTION AND STAGES OF MATURITY

Studies on the reproductive behavior of fishes involves the assessment of morphometric at maturity stages, estimation of fecundity, duration of reproductive season, spawning behavior and spawning fraction which contributes a greater value in quantifying the reproductive capacity of fish species. The ovaries of S. plagiostomus observed during this study are bi-lobed elongated structure lying on either side of the just below the vertebral column. A series of seasonal morphometric changes were observed in the gonads of both sexes during this study. Similar seasonal changes were observed in the gonads of S. richardsoniiby (S. Qadri, Sultana, & Anjum, 1982).

In the present study seven maturity stages in the gonads of S. plagiostomus were identified i.e. stage-I immature, stage-II unambiguous sex, stage-III developing, stage-IV maturing, stage- V mature, stage-VI running and stage-VII spent. Similar stages of gonad maturity were identified and described by (Nagelkerke, 1997) in cyprinids. (Raina, 1976) reported the Cystovarian type of ovary as a characteristic feature of all Schizothacids. The present study has confirmed the presence of cystovarian type of ovary in S. plagiostomus.

132 5.3.1 Spawning Studies

The present study revealed the fact that S. plagiostomus spawn twice in a year i.e. February to April and September to November. The spawning period of S. plagiostomus observed for almost equal period of time during spring and autumn in both male and female. Contrary to the present finding (A. Jan et al., 2017) reported a short breeding season of S. plagiostomus during March and April when compared to October and November. Disagreeing to the present study, (V. Jhingran & Sehgal, 1978) reported that S. plagiostomus spawn only once in different months of the year at different elevation in the specific river.

It is evident from the present findings that the spawning period of S. plagiostomus depends upon the provision of optimum external factors such as water temperature, food availability and duration of photoperiod. In agreement to the present finding (M. Qadri, Mir, & Yousuf, 2013) revealed that the spawning behavior of S. richardsonii depends upon the exteroceptive factors such as water temperature, food availability and duration of photoperiod.

5.3.2Length Weight Relationship and Condition Factor

It is fact that the growth of any animal increases with increase in body length. The growth of fishes has strong relation with body length and weight. Studies regarding length weight relationship of fishes constitute an important tool in the field of fisheries which help to understand whether variations from expected weight for certain groups are the indicator of fatness, wellbeing and gonadal development in respect to their environment (Le Cren, 1951),(Bagenal & Tesch, 1978)and (Yousuf, Bhat, Mehdi, Ali, & Ahangar, 2003).

The slope coefficient b2 for TL vs BWT estimated during this study are within the range of 0.825 to 3.138 in case of males while in case of females its range is from 1.15 to 2.68. In the present study the overall b2was found higher in males than females which is contradictory to the finding of(M.

Jan & Ahmed, 2016b). The highest b2value in males S. plagiostomusindicates that the males gain weight at a faster rate than females. The present results also contrast the findings of (Dar, Najar, Balkhi, Rather, & Sharma, 2012)who found females at a faster rate than males in Schizopygeesocinus.

During this study a significant departure of b2 value from the isometric value of 3 was noticed especially in case of female S. plagiostomus. The slope value of regression line less than 3 has also been reported in Tor tor by (Malhotra, 1982) in Labeodero by (Malhotra & Chauhan, 1984) in Labeodyocheilus by (Malhotra, 1985),Cyprinuscarpiocommunisand Ctenopharyngodonidella(Dhanze & Dhanse, 1997)and Rasboradaniconius(Sunil, 2000).

133 In the present study b2 value of females S. plagiostomusduring spring spawning cycle was higher (1.15 to 2.29) than males (1.123 to 1.717), during autumn it was 2.02 to 2.11 in female while in male it ranged from 1.787 to 2.882. The males S. plagiostomus gain less weight during spring than females while during autumn they slightly gain larger than females.(Le Cren, 1951)reported that due to difference in fatness and gonadal development the female fish are heavier than male fish of the same length. (Qadri & Mir, 1980)found the value of regression slope as 2.4487 in S. plagiostomus from the peripheral water bodies of Dal Lake.(M. Jan & Ahmed, 2016b)reported the regression slope values of 2.970 in case of male S. plagiostomus and 3.538 in case of female S. plagiostomus from Liddar river, Kashmir. The overall range of regression slope coefficients calculated for S. plagiostomus in the present study are near to the normal range of b coefficient values of 2.3- 3.5 for snow trout as suggested by (Froese, 2006).

The condition factor is used to detect the wellbeing of fishes. (Brown & Murphy, 1991)concluded that condition indices can be used as indicator of relative health. In the present study condition factor in both male and female S. plagiostomus shows variations in different month. The highest K-value of 1.99±0.13 for male S. plagiostomuswas noticed in the month of June while for females it was 1.98±0.12 in July and 1.98±0.09 in November. The K-value recorded for both sex of S. plagiostomus during the present study is slightly higher when compared to the K-values recorded for S. plagiostomus by (M. Jan & Ahmed, 2016b)from Lidder River Kashmir. The seasonal variations in condition factor seems to be attributed to high feeding activity, fat deposition and gonadal development as a preparation for coming breeding season. (Maguire & Mace, 1993)reported that increase in K- values indicates the accumulation of fat and sometime it reflect the gonadal development.

The slope coefficients values calculated to fit quadratic modelfor both sex shows that the body weight increases with the increase of total length. Considering the maximum length of S. plagiostomusduring breeding season, the males were observed with larger size (maximum TL-45cm) than females (maximum TL=43.8cm) which indicate the prior attainment of sexual maturity in female. The same finding is contrary to the finding of (Juras & Yamaguti, 1989)who worked on the spawning behavior of Macrodonancylodon.

5.3.3Sex Composition

The present study revealed the fact that male to female sex ratio was dominated by males during both spawning periods (spring and autumn). This is in agreement to the finding of (Nikolsky, 1969)who observed the surplus of males in the spawning period of some fishes. Higher occurrence of female S. plagiostomus in the breeding season during this study observed may be attributed to the feeding migration. 134 5.3.4Gonado-Somatic Index (GSI) and Spawning Season

During the present observations variations in GSI and gonads morphology were taken to ascertain the spawning season in S. plagiostomus. Monthly variations in GSI index provided a very good indication of gonads development round the year. In line to the same observations (Crossland, 1977) have reported that changes in gonads condition can be assess from the quantitative record of gonad weight. In the present study, the GSI in male fish showed two peaks in March (20.2) and in October (18.8) while in female fish the maximum GSI were recorded in February (18.4) and in September (16.8).

The peak GSI values recorded in both sexes during February to April, September to Novembercoincided with the highest incidence of ripe males and females, which indicated the full breeding responses in S. plagiostomus. Contrary to the present observations, (Bhatnagar, 1964), reported that S. plagiostomusspawn in July-August and again in December-January. Keeping in view the present observations, it can reasonably be inferred, that increase in water temperature, turbidity, water volume during summer and a subsequent flooding during monsoon, might be causatives to gonads suppression in summer. As a result, the breeding season in S. plagiostomus was observed to be moderately long during autumn as compared to spring.

Stage-wise calculation of GSI in the present study revealed that highest GSI were observed in ripe stages (stage-V and VI) while the lowest values were recorded in spent (stage-VII), immature (stage-I) and maturing (stage-II), respectively.

For the determination of maturity stages during this study, the gonads of S. plagiostomus were assessed for fecundity and GSI on monthly basis. In this study, seven maturity stages were identified in both the sexes of S. plagiostomus. In agreement to the present findings (Negi & Dobriyal, 1997)also reported seven maturity stages in Crosscheiluslatiuslatius. (Shyam Sunder & Bhagat, 1979) and (Raina, 1976)reported five maturity stages in Schizothoraxniger and Schizothoraxesocinus respectively. Similarly six stages of maturity in Schizothoraxrichardsonii were reported by (Agarwal, Rawat, Thapliyal, & Raghuvanshi, 2004)from Bhagirathi River in Garwal, India.

S. plagiostomus are remarkable regarding their spawning season and breeding behavior. Our observations revealed that S. plagiostomus spawns twice in a year i.e. The same spawning behavior in S. plagiostomus has been observed by (Bhatnagar, 1964) and (Qadri, Mir, & Yousuf, 2013). The present observations revealed that the first spawning cycle of S. plagiostomuswas started from February and ends in April (spring spawned) while the second spawning cycle (autumn spawned) was started from September and was continued till the end of November. The same breeding behavior of S.

135 plagiostomus were reported by (A. Jan et al., 2017)where they were of the view that the breeding season extend to some extent in December. Contrary to this all specimens in December were found in stage-VI. The stage-IV were observing throughoutDecember and January. Again this is contrary to the findings of (A. Jan et al., 2017)where they reported the presence of dormant and mature gonads in S. plagiostomus during these months.

In the present study the gonadal development was found to be initiated in February and it was observed that gradual increase in water temperature during February and March might cause to induce breeding when optimum temperature comes during March and April. The sudden increase in water temperature during May seems to inhibit the breeding response where gonads were observed totally in spent condition. (Munro, Scott, & Lam, 1990)reported that the reproductive behavior of fishes is controlled by endogenous biological rhythm as well as environmental factors.

5.3.5Relationship of GSI and Fecundity with TL, BWT, Ovary Length and Ovary Weight

In male S. plagiostomus the GSI was found to decrease curve-linearly with the increase in total body length (TL) and body weight while it was found to increase curve-linearly with the increase in testes length and testes weight initially and then decline(tables 4.5.5, 4.5.6, 4.5.7 and 4.5.8). Similarly, in female fish the GSI was found to decrease curve-linearly with the increase in total length, body weight, and initially increase with ovary length and ovary weight and then decreases. A linear relationship between these variables were observed by(Bahuguna & Khatri, 2009) and(Mohammad & Pathak, 2010).

The value of R2in the present study shows a close relation with the GSI and ovary weight (R2=0.835) followed by ovary length (R2=0.735) in female S. plagiostomus. Similar to the same results, a higher correlation were also reported by (Bahuguna & Khatri, 2009)and (A. Jan et al., 2017)in S. plagiostomus.

Fecundity has a significant role in fish biology. Fecundity is a major reproductive capacity of female fish and its analysis and relationship with morphological characteristics such as size, weight and age has provided a reliable index of density dependent factors in population studies. (Nikolsky 1969), describe fecundity as a specific feature which arises with adaptations in certain environments during species evolution due to which the continuation of species is maintained. Similar adaptations can easily be observed in Schizothoracids.

During this study the quadratic model did not fit between absolute fecundity and GSI of female S. plagiostomus .A linear relation of GSI and fecundity in S. plagiostomus were reported by (A.

136 Jhingran, 1968)and (Bahuguna & Khatri, 2009). All the relationship between the GSI and TL, body weight, ovary length and ovary weight were found highly significant (p<0.05).

In the present study the highest absolute fecundity of 8504.59±614.62 (2386.67-13610.19) was recorded in the month of April during spring cycle in gravid specimen of 29.24 (13.80-52) cm TL. During autumn the absolute fecundity of 14079.92±648.93 (9998.88-20474.96) was recorded in the gravid specimens of 27.5(21.5-39.5) cm TL in the month of November. Same relationship between absolute fecundity and TL in S. phulo were reported by (Pishka, Swamy, & Devi, 1991). It was also observed that gravid females were more fecund in November than April.

The curvilinearmodelwas best fitted between fish length and weight and between gonads weight and gonads length but the relationship of these parameters was non-significant with absolute fecundity. (Lehman, 1953) and(Bagenal, 1957)found a significant relationship of these variables with absolute fecundity. The length-weight relationship of S. plagiostomus observed in the present study is in accordance with the model of (Le Cren, 1951).

Fecundity of S. plagiostomus calculated in the present study, did not show allometric pattern of growthwith BWT and TL which is contradictory to the allometric relationship of fecundity with BWT and TL reported by (Goel, Barat, Pande, Ali, & Kumar, 2011)for Schizothoraxrichardsonii and by (S Sunder & Subla, 1985) for Schizothoraxcurvifrons. Generally all teleost fishes have linear relationship of fecundity with body weight and ovary weight, however (Bagenal, 1957)reported a non-linear relationship of these parameters.

5.4 MOLECULAR CHARACTERIZATION

The present study represents the first attempt to assess the species identification and phylogenetic of S. plagiostomus through application of DNA barcoding inhabiting river Indus in northern Pakistan. The ideal DNA barcoding procedure should be fast with highly conserved primers sequences and reliable amplification and accurate sequencing. Valentini et al. (2009) has presented that DNA barcoding should be robust, with highly conserved primers sequences, reliable amplification and sequencing, the sequenced DNA should be identical among individual of the same species and should have variations with other species (Valentini, Pompanon, & Taberlet, 2009).Both traditional methods and molecular methods applied during the present study for species delineation and phylogenetic relationship were mostly concordant which tally the finding of(Ward, Zemlak, Innes, Last, & Hebert, 2005;Bernardi, Robertson, Clifton, & Azzurro, 2000).

All the specimen sequences collected during this study (S 1 FWP- S28 FWP) were A+T rich which tally the report of (Johns & Avise, 1998). Average A+ T content of specimen sequences in the 137 present study was calculated as 54.2 % and G+C contents as 45.8%. Similar results were reported by (Ward, Zemlak, Innes, Last, & Hebert, 2005); (Lakra et al., 2011); (Vineesh et al., 2013)and (Bashir, Bisht, Mir, Patiyal, & Kumar, 2016). Similar strong correlation between the GC contents for COI gene and entire mitochondrial genome were reported by(Min & Hickey, 2007).

During the present study the average percentage sequence divergence between the specimen sequences and Schizothorax spp. estimated was as 2.934%-6.979%, which is in accordance to 4.4% for Canadian fishes as reported by (McCusker, Denti, Van Guelpen, Kenchington, & Bentzen, 2013) and 5.1% for fishes from Mexico and Guatemala reported by(Valdez Moreno, Ivanova, Elías Gutiérrez,

Contreras Balderas, & Hebert, 2009). However, Bashir et al.,‐ 2016 reported average‐ sequence divergence‐ of 2.8% in Schizothorax genus from India.(Rosso, Mabragaña, Gonzalez Castro, & Díaz de Astarloa, 2012) even reported lowest K2P genetic distance of 1.68 while analyzing the Neotropical freshwater fishes from Argentina.

5.4.1 Phylogenetic Analysis

NJ joining tree clearly indicate the highest COI sequence diversity among fish specimen which were identified morphologically as S. plagiostomus from river Indus and its tributaries in Indus Kohistan district KP, Pakistan. The molecular characterization of S. plagiostomus applied during this study proved authentic and reliable for species level identification. It is also noteworthy that the fish spp. previously identified morphologically as S. plagiostomus are closely related with S. nepalensis and S. curvilabiatus.

The COI barcoding and phylogenetic analysis of Schizothorx genus inhabiting Himalayan and sub-Himalayan region has been proved to be very useful for its taxonomy as well as for investigation of its evolutionary pattern. During this study the phylogenetic position of S. plagiostomus was constructed by using the cytochrome C oxidase gene I for the purpose of its molecular characterization and genetic variation. All the specimen collected during this study were preliminary characterized as S. plagiostomus on the basis of their morphological identification but their molecular characterization showed high sequences diversity.Contrary to the present findings (P. D. Hebert & Gregory, 2005)reported that DNA barcoding based on COI divergences has aimed to build an association between molecular and morphological taxonomists.

The present study indicate that S. plagiostomus is closely related with S. nepalensis from Nepal and S. curvilabiatus from China whereas (Khan, Khattak, He, & Chen, 2017), reported that S. plagiostomus is closely related with S. esocinus, S. progastus, S. niger, S. nepalensis, S. richardsonii

138 and S. labiatus. Bashir et al. (2016) reported that S. plagiostomus is closely related to S. labiatus and S. esocinus(Bashir et al., 2016).

The NJ analysis of COI gene during the present study has confirmed 13-specimen sequences as S. plagiostomus, whereas rest of the 15-specimens were clustered closely with other Schizothoraxspp, which proved the authenticity of COI barcoding for identification purpose. This is in agreement to the finding of Hebert et al. (2003) who reported that NJ analysis through COI barcoding is an effective tool for the identification purpose (P. D. Hebert, Cywinska, et al., 2003).

Ward et al., 2005 reported that DNA barcoding is not aimed to establish phylogenetic relationship even when there is a clear evidence of evolutionary relationship exist, it is mainly used to identify known species and to aid the discovery of new ones. Accordingly, the COI analysis during the present study revealed to delineate species boundaries however the presence of some clear phylogenetic signals have been evident from the COI sequence data, which clustered into distinct clades on phylogenetic tree.The 100% clustering of sequences on the phylogenetic tree during the present study indicate the diagnostic ability of COI barcoding for correct species identification.

This study has successfully proved and confirmed the authenticity of COI barcoding in identifying the Schizothorax Spp.

139 SUMMARY

The present study was aimed to investigate the feeding habit of economically important freshwater fish Schizothoraxplagiostomusin different seasons, its fecundity pattern and molecular characterization through application of DNA bar coding, collected from river Indus and its tributaries in Indus Kohistan, KP, Pakistan. Indus Kohistan district, KP, Pakistan, 34° 54′ and 35° 52′ North latitudes and 72° 43′ and 73° 57′ East longitudes covering an area of 7492 square kilometers. River Indus pass throughout the district inhabiting abundant population of Schizothorax spp. River Indus is the major and most important river system throughout Asia.

S. plagiostomus is a species of ray-fined, semi cold fresh water fish encountering severe population stress and pressure in the study area due to various hydel power generation projects, mining and introduction of exotic species in the study area. Specimens were collected from river Indus and its tributaries and were identified morphologically as S. plagiostomus by using the keys of Jayaram (1981) and Kullanderet al (1999).

Physicochemical parameters such as water temperature, pH, DO, conductivity, hardness, alkalinity, nitrate and dissolved CO2 were recorded month wise (Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream) along river Indus. Non-significant variations were recorded for these parameters across all categories of localities except alkalinity which shows statistically significant variations.

Species distribution and abundance were assessed across all categories of localities and different seasons. The mean CPUE and Kruskal- Wallis rank values indicated that maximum fish were caught from Darel Stream (mean catch=33.17, mean CPUE=0.55 and mean K-W value=47.74). The maximum fish were caught during November with a mean catch of 44.50, mean CPUE of 0.74, mean Kruskal- Wallis rank value of 63.25.

For feeding assessment the gut contents of 240 (167 males & 73 females) were analyzed by Frequency of Occurrence method (Hynes, 1950; Hyslop, 1980; Bowen, 1996), Volumetric Analysis Index (Lima-Junior &Goitein, 2001) and Importance Index (Fritz, 1974). The gut contents were identified by using the keys of (G.W. Prescott, 1964), (J. M. Elliott, U. H. Humpesch & T. T. Macan, 1988).

The analysis of gut contents revealed that S. plagiostomus consumed mainly spirogyra and ulothrix attached to stones and pebbles, however a considerable amount of other food items such as Mayflies, Caddis flies, detritus and Sand & mud were also identified. Seasonal variations in the mean rank of importance index and mean Kruskal- Wallis rank values from the gut contents of S. 140 plagiostomus showed the highest values of 3.33 and 16.17 respectively during winter. Similarly the mean importance index and mean Kruskal-Wallis values calculated for different food items clearly indicated that S. plagiostomus consumed Spirogyra and Ulothrix as major food items with mean importance index and mean Kruskal-Wallis values of (7520.35), (21) and (5703.97), (20) respectively, across all categories of seasons. The Mayflies and Caddisflies were found only during Autumn and Winter in the gut of S. plagiostomus while the detritus, sand and mud were found in cosiderable amount throughout the year.

The Kruskal-Wallis Chi-square value of 2.33 obatined for seasonal variation in the importance index of S. plagiostomus gut contents was non-significant (p=0.525). The Kruskal-Wallis Chi-square value of 16.43 obtained for different food items in the gut of S. plagiostomus was highly significant (p=0.006). A total of 540 fish specimens (263 females and 277 males) were selected for breeding assessment during the present study. Maximum of seven gonads maturity stages were determined in S. plagiostomus by using the standard method of (Nagelkerke, 1997). Length weight relationship in both male and females specimens were determined by the method of (Bagenal&Tesch, 1978). The curvilinear relationship for quadratic model fit showed significant relationship between total body weight and total body length in both sexes. The gonado-somatic index (GSI) was calculated by using the method of Baricheet al., 2003. The quadratic model (curvilinear) was fitted to sort out the relationship between GSI and Total body weight and total body length.

The length-weight relationship and condition factor was calculated for both male and female fish. The curvilinear relationship was calculated by fitting the quadratic model. The results showed the strong correlation of body length-weight in both sexes. The highest Condition Factor for were recorded during the month of July, while in female the highest mean CF was found during June.

The quadratic model showed significant relationship in both the sexes. Estimation of fecundity in female fish was determined by using the method of (Lagler, 1956, Yeldan&Avsar, 2000). Maximum mean absolute fecundity of 14079.92 was estimated in female fish having mean TL of 27.5cm and mean BWT of 406 g during autumn in November. The results showed that these fishes were more fecund in autumn than spring breeding season.

A total of 28 specimens previously identified morphologically as S. plagiostomuswere randomly selected for molecular characterization. The DNA extraction from muscle tissues were done by using Thermo scientific GeneJET Genomic DNA extraction Kit and its prescribed protocol. In-SilicoAnalysis were done through (NCBI, GeneBank, BLASTn). Sequences were aligned and edited using GeneDoc version 5.10 (Nicholas, Nicholas Jr et al. 1997) and BioEdit version 7.0.5.2 (Hall 1999). Phylogenetic tree was constructed by using MEGA7. Molecular and evolutionary analysis is a fast, standardize and 141 reliable molecular method used to sequence mitochondrial cytochrome oxidase I gene (COI) for species identification worldwide. Primers were designed to amplify a target sequence of S. plagiostomus available at GeneBank NCBI. Mitochondrial COI gene was amplified through PCR from 28 individuals collected from six different streams joining river Indus in Indus Kohistan and then sequenced.

The PCR results indicated that the amplified sequences has a similarity of (99.6 to 99.9%) sequences. Phylogenetic tree was constructed by using neighbor joining method showed that S. plagiostomus has distinct clades from 28 sequences. Collected fish specimen was identified morphologically as S. plagiostomus.However, thepresence of severalShizothorax species was confirmed from target study area on the bases of molecular analysis using COI sequence.

The phylogenetic analysis has confirmed 13-specimen sequences as S. plagiostomus, whereas rests of the 15-specimen sequences were clustered with other Schizothorax Spp. on a NJ tree. The present study has confirmed the authenticity of DNA barcoding application for identification purpose.

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166 Acta Scientiae Veterinariae, 2018. 46: 1550. RESEARCH ARTICLE Pub. 1550 ISSN 1679-9216 DOI: 10.22456/1679-9216.82439 Received: 18 December 2017 Accepted: 28 March 2018 Published: 29 April 2018 1Department of Zoology, Abdul Wali Khan University, Mardan, Khyber Pakhtunkhwa, Pakistan. 2Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University, Punjab, Pakistan. 3College of Veterinary Science and Animal Husbandry, Abdul Wali Khan University, Mardan. 4Department of Zoology, University of Peshawar. CORRESPONDENCE: A. Ali [[email protected] - Fax: +92 937-843357]. Department of Zoology, Abdul Wali Khan University, Mardan. ZC 23200 Khyber Pakhtunkhwa, Pakistan. 1

Diet Composition and Seasonal Fluctuations in the Feeding Habit of Snow Barbel (Schizothorax plagiostomus) in River Indus, Pakistan

Muhammad Qayash Khan1,2, Muhammad Zubair Anjum1, Shamim Akhter1, Irfan Khattak3, Muhammad Adnan4 & Abid Ali1

ABSTRACT Background: Schizothorax plagiostomus is widely distributed in river Indus and is most important food fish in Pakistan. The feeding habit of fish is directly related to the size of fish, its metabolic rate and environmental temperature. The accu- rate description of fish diet and feeding habit is a very important aspect in fisheries management for the purpose of species conservation, breeding and culture. The present work was aimed to investigate the specie abundance, the diet composition and seasonal variations in the feeding habit of Snow barbell Schizothorax plagiostomus. Materials, Methods & Results: A total of 1799 fish specimens were caught at the confluence of six tributaries along river Indus at Indus Kohistan, northeastern Pakistan. The fish were collected by 5-panels of gill net during first week of each month. The site specific Catch Per Unit Effort (CPUE) and season specific CPUE of fish fauna were assessed. For the gut content analysis 240 samples (99 male and 141 females) of S. plagiostomus were selected on monthly basis. Frequency of occurrence method and volumetric method were applied to record the different food items in the gut of S. plagiostomus. The physico-chemical parameters, NO3 concentration and dissolved Co2 of water from different localities of river Indus were recorded month wise by Hach sensION 156 meter, Horiba LAQUA Nitrate Meter and EA80 meter respectively. Significant difference was observed in water temperature during the four seasons. Except alkalinity no other water parameter showed significant variation across different localities. The results showed that highest Mean CPUE was observed for Darel Stream (0.55) and lowest for Jalkot stream (0.26). Peak abundance of fish was recorded in the month of November with a mean catch of 44.50, mean CPUE of 0.74 and mean Kruskal-Wallis rank value of 63.25. Spirogyra and Ulothrix occurred as maximum food items in the gut of S. plagiostomus during summer while their minimum amount occurred during autumn. According to the ranking index spirogyra and ulothrix ranked higher with significant difference in comparison to other food items. The results showed that S. plagiostomus is phytophagous in its feeding habit, which consumed mainly algae attached to stones and pebbles during the whole year. However, the presence of some secondary items such as animal matter, detritus, sand and mud might be due to the distinct availabilities of food along the seasons. The highest feeding activity of S. plagiostomus was recorded during summer while the lowest one occurred during autumn, spring and winter. Discussion: Catch per unit effort (CPUE) is an indirect measure of the abundance of a target species. It is used as an index of stock abundance in fisheries and conservation biology. During the study low fish fauna was found in River Indus as reported previously. Majority of the fish occurred in snow fed river tributaries in the study area as these tributaries are comparatively less turbulent. Previous studies have also recorded that Schizothoracine generally prefer clean waters. The present findings of gut contents analysis showed clearly that S. plagiostomus is a phytophagous fish which scrap and consumed spirogyra and ulothrix attached to stones and pebbles. Earlier it was reported that mouth of S. plagiostomusis is inferior, wide, with deep lower jaw having keratinized cutting edge and the lower lip is folded and expanded with numerous papillae making it best suited for scrapping algae attached to stones and pebbles. The highest feeding activity was observed during warmer months as compared to cold months. S. plagiostomus spawn twice in a year in autumn and in spring. The highest feeding activity of S. plagiostomus seems to be link with a reflex of recovery strategy due to physiological process of gonadal development. Keywords: Schizothorax plagiostomus, Snow Barbel, feeding habit, seasonal fluctuation, River Indus.

167 M . Q . K h a n , M . Z . A n j u n , S . A k h t e r , I . K h a t t a k & A . A l i . 2 0 18. Diet Composition and Seasonal Fluctuations in the Feeding Habit of Snow Barbel (Schizothorax plagiostomus) in River Indus, Pakistan. Acta Scientiae Veterinariae. 46: 1550. 2

INTRODUCTION Schizothorax plagiostomus is a famous and most important food fish of Pakistan, distributed widely in the mountain and sub-mountain tributaries of river Indus. Catch per unit effort is commonly used method for estimating population size where counting method is difficult to apply. [4,5,8,14,21]. The feeding habit is directly related to the size of fish, its metabolic rate and environmental temperature. Addition of vegetable crops to the food of rainbow trout has significantly enhanced their specific growth and weight gain during the term of experiment [22]. Low water quality may cause overfeeding in fishes due to which chances of pathogen ingestion increases. Deterioration of water quality is a major cause stress in fishes which increases their susceptibility diseases [23]. Diet investigation of a particular fish species is a valuable tool which interpret the trophic relationship in an aquatic ecosystem by providing information about fish life, its position in the food web, food resources and its possible competitors [2]. The accurate description of fish diet and feeding habit is a very important aspect in fisheries management for the purpose of species conservation, breeding and culture. Both extrinsic factors (biotope, region) and intrinsic factors (species, size, behavior) effect the diet of fishes and such information are important to understand the basic functioning of fish assemblage for the purpose of developing Ecosystem Based Fisheries Management Model (EBFM) models [3,7]. Assessment of fish feeding habits proved to be very useful in fisheries management and aquaculture. The practices of stomach contents analysis provide a strong insight to assess the fish feeding habit both quantitatively and qualitatively [9]. Keeping in view the importance of Schizothorax plagiostomus in natural fisheries and its role in food web in the ecosystem, the present work has been carried out to get a brief baseline information on its abundance & distribution, feeding habit and seasonal variations in its food preferences.

MATERIALS AND METHODS Experimental design The species abundance and feeding habit of snow barbell Schizothorax plagiostomus were studied in river Indus and its tributaries at Indus Kohistan, district of Khyber Pakhtunkhwa Province, northeastern Pakistan, located approximately at latitude of 35°15′N73°30′E. A total of 1799 S. plagiostomus were collected by 5-panels of gill net 5×15 feet (mesh size 1cm,1.5cm, 2cm, 2.5cm and 3cm) during first week of each month in 2016. The traps were settled in a stretch of 3 kilometer from its confluence in each tributary (Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream). The overall sampling protocol was similar in all sections. Month-wise sampling event lasted for five days and six nights with a maximum soak time of 12-hours. The physico-chemical parameters such as Water Temperature (WT), pH, Dissolved Oxygen (DO), Conductivity, Hardness, Alkalinity were recorded by Hach sensION 156 meter, NO3 concentration by Horiba LAQUA Nitrate Meter and dissolved Co2 were recorded by EA80 meter. Out of 1799 specimen 240 samples (5.00- 46.00 cm total length) were selected for gut content analysis to investigate the feeding habit. The selected specimens were preserved in 10% formalin prior to examination in the laboratory. Individual specimen was weighed to the nearest of 0.1g and the total length were taken to the nearest of 0.1cm. Fish were identified by using the key as prescribed by Nyman & Swedmar [11]. Gut content analysis Each fish specimen was dissected and gut was removed. The gut was weighed (in grams to the nearest of 0.01 g) and then preserved in 70% ethanol solution till analysis. In order to minimize the errors during gut content analysis the anterior most section of the gut was selected as the same was found with the most recent ingested food and was in the better state of gut contents identification. The gut contents were removed into petri dishes, diluted in water and shake well to obtain a homogenous mixture. The homogenous mixture was divided into sub- samples and inspected microscopically for recognition of each food item. Each recognized food item was assigned a value proportional to its abundance. The standard weight (SW) was used as a reference for these values, which is the approximate arithmetic mean of the weight of sample gut content [9]. It was assumed that SW of 5.04 g (during this study) is equivalent to 4-points and each gut content was assigned a value according to the proportion of their mass in relation to the SW. By visual inspection, the total value obtained were divided among food items according to their relative

168 M . Q . K h a n , M . Z . A n j u n , S . A k h t e r , I . K h a t t a k & A . A l i . 2 0 18. Diet Composition and Seasonal Fluctuations in the Feeding Habit of Snow Barbel (Schizothorax plagiostomus) in River Indus, Pakistan. Acta Scientiae Veterinariae. 46: 1550. 3 volume. The importance index was calculated according to the method described by Lima-Junior & Goitein [9]. The mean ascribed values of each food item were obtained by summing up of points given to each food item divided by total number of guts in the sample. The arithmetic mean thus obtained were then multiplied by a multiplication constant. From such calculation, volumetric analysis index (Vi) values were obtained (Vi = 25 Mi). The Importance Index of each food item was calculated through multiplication of Volumetric analysis Index (Vi) values with the values of Frequency of occurrence (Fi) as described by Lima-Junior and Goitein [9] (AIi = Fi .Vi). The results obtained were statistically analyzed by the method where the food items were ranked in each sample. [9] The values of Importance Index obtained in different seasons were compared through Kruskal-Wallis Test [20]. The ranking was compared by Spearman rank correlation coefficients, in which the correlation was considered statistically significant at P < 0.05.

RESULTS A total of 240 fish having an average size of 5 to 46 cm (total length) were selected for feeding as- sessment caught from the river Indus and its tributaries during the year 2015-16. The gut contents of 99 male and 141 females were analyzed to investigate the diet composition and seasonal fluctuations in the feeding habit of Snow Barbel (Schizothorax plagiostomus). Physico-chemical parameters of the water Physico-chemical parameters of water from different localities (Jalkot stream, Palas stream, Keyal stream, Barseen stream, Kandia stream and Darel stream) on river Indus were recorded month-wise. The sampling data was pooled by seasons i.e. autumn (September, October & November), winter (December, January & February), spring (March, April & May) and summer (May, June, July & August) on the basis of water temperature. The average water temperature was recorded as 10.93oC, 9.79oC, 15.28oC and 20.91oC in autumn, winter, spring and summer respectively. The difference in mean water temperature during the four seasons was statistically significant (P < 0.05) as shown in Table 1. The mean water temperature of all categories of localities was similar (P > 0.05). Similarly DO, conductivity, hardness and alkalinity from different localities of the river Indus showed significant differences during the four seasons. However, insignificant variations were recorded for the above parameters across different localities except alkalinity that showed significant variations (P < 0.05). Seasonal variations in the concentration of NO3, water pH and dissolved CO2 throughout the year were found non-significant across categories of seasons and sampling sites as well. Fish distribution and abundance Variations in S. plagiostomus abundance were found significant at the confluence of different tributaries along river Indus (Table 3). The maximum sampling were done at Darel stream with a mean catch of 33.17, mean CPUE of 0.55 and a mean Kruskal-Wallis rank value of 47.74 while a minimum sampling were made at Jalkot stream with a mean catch of 15.83, mean CPUE of 0.26, mean Kruskal-Wallis rank value of 22.96 and a P-value of 0.040. Similarly the variations in the number of this species was found significant across different seasons. The lowest number of S. plagiostomus recorded in August with a mean catch of 7.17, mean CPUE of 0.12 and a mean Kruskal-Wallis rank value of 8.42. A peak abundance of fish was recorded in the month of November with a mean catch of 44.50, mean CPUE of 0.74, mean Kruskal-Wallis rank value of 63.25 and a P-value of 0.000. Fish Diet The mean importance index of different food items consumed by S. plagiostomus in four season are shown in Figure 2. Spirogyra and Ulothrix occurred as maximum food items in the gut of S. plagiostomus during summer while their minimum amount occurred during autumn. Both these items constitute the major food of S. plagiostomus found almost throughout the four seasons. Considerable amount of Mayflies, Caddis flies and Sand and mud were collected during winter and autumn. In addition, a considerable amount of sand and mud were found in the gut during summer and spring. The gut of S. plagiostomus contained traces of detritus throughout the year. These finding revealed that aquatic algae Spirogyra and Ulothrix constitute the main food of S. plagiostomus, collected from river Indus and its tributaries independently of seasonal effects. However, other organisms such as Mayflies, Caddis flies and detritus were also found in the stomachs of sampled individuals. According to the ranking index Spirogyra and Ulothrix ranked higher with significant difference in comparison to other food items (P < 0.05, Kruskal-4 169 M . Q . K h a n , M . Z . A n j u n , S . A k h t e r , I . K h a t t a k & A . A l i . 2 0 18. Diet Composition and Seasonal Fluctuations in the Feeding Habit of Snow Barbel (Schizothorax plagiostomus) in River Indus, Pakistan. Acta Scientiae Veterinariae. 46: 1550. Wallis test). The concentration of Spirogyra and Ulothrix was significantly higher among other diet contents (Table 5) which revealed that S. plagiostomus is a phytophagous fish, however the presence of other food items shows omnivorous and opportunist feeding habit. Regarding the seasonal dietary shift, it was observed that the food items consumed by the species, shows higher importance index during summer followed by spring. The lowest importance index was recorded during autumn followed by winter (Table 4).

170 Table 1. Mean values of Physico-chemical parameters of water in all seasons from Indus River and its tributaries at Khyber Pakhtunkhwa Province, northeastern Pakistan. Physico- Seasons P-Value chemical Autumn Winter Spring Summer properties Water Temp. 10.93 9.79 15.28 20.91 0.000 (oC) pH 7.32 7.60 7.67 7.80 0.135 DO (mg/L) 8.36 9.08 9.75 10.11 0.030 Conductivity 47.54 69.58 82.33 107.50 0.000 (uS/cm) Hardness 86.75 81.33 87.17 98.41 0.035 (mg/L) Alkalinity 63.36 64.00 63.83 78.25 0.05 (mg/L) Nitrate (mg/L) 0.83 0.95 0.69 0.88 0.232 Dissolved CO2 14.71 15.83 15.50 15.67 0.625 (mg/L)

171