POSSIBLE CAUSES OF SELECTIVE LERNAEA ATTACK ON DIFFERENT FISH SPECIES FARZANA ABBAS 2007-VA-532 A THESIS SUBMITTED IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF OF DOCTOR OF PHILOSOPHY IN FISHERIES & AQUACULTURE UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE- PAKISTAN 2014 i DEDICATED To MY PARENTS W hose hands are always raised for my well-being even at this moment of time ii To, The Controller of Examinations, University of Veterinary and Animal Sciences, Lahore. We, the supervisory committee, certify that the contents and form of the thesis submitted by Farzana Abbas, Reg. No. 2007-VA-532 have been found satisfactory and recommend that it be processed for evaluation, by External Examiner(s) for the award of degree. SUPERVISORY COMMITTEE: Chairman : ------------------------------------------------ (Prof. Dr. Muhammad Ashraf) Member : -------------------------------------------------- (Dr. Sumaira Abbas) Member : --------------------------------------------------- (Dr. Arshad Javid) iii ACKNOWLEDGEMENTS Praises are all attributed to ALMIGHTY ALLAH, the omnipotent and sovereign; blessed and bestowed me with the power of pen and knowledge, mightier than the sword, and raised my humble self to a status he choose for His loved ones, namely those who know. Sure, the teaching and preaching of HOLY PROPHET HAZRAT MUHAMMAD (SAW), the torch of guidance, source of knowledge and wisdom for mankind, have ever been an inspiration for getting on with unknown and to invade the land of erudition. How could I, all alone, be thrilled to achieve this task, the recognition certainly goes to my seniors, specially my kind supervisor, Prof. Dr. Muhammad Ashraf, Chairman Department of Fisheries and Aquaculture, University of Veterinary and Animal Sciences, Lahore, Pakistan. His intellectual support, expert advice and constant encouragement developed my confidence and pursued me for writing of this dissertation. I am indebted to Dr. Sumaira Abbas, Assistant Professor, Department of Fisheries and Aquaculture, Dr. Arshad Javid, Assistant Professor, Department of Wildlife and Ecology University of Veterinary and Animal Sciences, Lahore, Pakistan for their friendly advices and prompt assistance during the study. A special thanks, goes without saying, to my class fellows; Mr. Khalid Javed Iqbal, Mrs. Sydah Andleeb, Mr. Iftikhar Ahmed Qureshi, Mrs. Ghina Islam, Ms. Rida Younis and field workers for their intimate support and warm appreciations. Family members are true assets of love and prayers in the back. It gives me pleasure to extend my gratitude to my friendly husband, Dr. Muhammad Hafeez-ur-Rehman, who has been a constant and invariable source of moral as well as academic support, making me more and more proud of him. I am delighted to thank my father and mother whom hands are always raised for my successful career and my sisters and brothers for their untiring efforts to support me to my satisfaction. Their patience and deep prayers are always with me in every field of life. May ALLAH bless them all with long and peaceful lives. Amin! Farzana Abbas iv TABLE OF CONTENTS Page Chapter Contents No. Acknowledgement iv Table of Contents v List of Tables ix List of Figures xi Abstract xii Chapter 1 1 1 General Introduction Chapter 2 2 7 Review of Literature Lernaea, its prevalence, infestation and susceptibility 7 Body composition 11 Hematological studies 14 Immunity studies 16 Literature cited 21 Chapter 3 Experiment 1. 3 37 L. cyprinacea Susceptibility, Infestation and its Treatment in Indigenous Major and Exotic Chinese Carps under Polyculture System. Introduction 37 Materials and Methods 40 Experimental site 40 Experimental design 40 Parasite collection, preservation and identification 40 Growth parameters 41 Water quality parameters 41 Pond treatment 41 Statistical analysis 43 Results 43 v Parasite identification 43 Weight and length gain in control and treated group 44 Infestation of L. cyprinacea during the course of study in control and treated 44 groups Discussion 50 Literature Cited 54 Chapter 4 Experiment-2 4 60 Comparison of biochemical profile of healthy and infectious Chinese, Indian and Exotic carps. Introduction 60 Materials and Methods 64 Experimental site 64 Experimental design 64 Water quality parameters 66 Proximate Analysis 66 Moisture and Dry Matter Test 66 Ash contents 67 Crude protein 67 Crude fat (Ether extract) 69 Mineral analysis 69 Wet digestion of samples 69 Determination of Sodium (Na) and Potassium (K) by flame photometer 70 Determination of Phosphorus (P) by spectrophotometry 70 Determination of Iron (Fe), Zinc (Zn), Calcium (Ca), Copper (Cu) and 71 Magnesium (Mg) Statistical analysis 71 Results 72 Bio-chemical analysis 72 Mineral analysis 74 Water quality parameters 76 Discussion 78 vi Literature Cited 84 Chapter 5 Experiment-3 5 93 Comparison of Various Blood Indices among Chinese and Indian Major Carps From the Perspectives of Their Resistance Against Lernaeaosis Introduction 93 Materials and Methods 97 Experimental site 97 Experimental Design 97 Blood haematology 97 Blood chemistry 99 Ig M determination 99 Statistical analysis 99 Result 100 Blood hematology 100 Blood chemistry 107 Discussion 110 Literature Cited 115 Chapter 6 6 Experiment-4 126 Fish Mucus; A Physical Barrier to Pathogens Introduction 126 Materials and Methods 128 Experimental site 128 Experimental animal 128 Experimental procedure 128 Mucus collection 128 Total Protein Contents 129 Polyacrylamide Gel Electrophoresis (SDS-PAGE) 129 Lectin activity 129 Alkaline Phosphatase Test 130 vii Statistical analysis 130 Results 130 Protein concentration 130 Protein profile of mucus 131 Lectin activity 132 Alkaline Phosphatase Test 133 Discussion 134 Literature Cited 138 Chapter 7 Experiment 5 7 Essential and Non-essential Amino Acid Composition of Indian and 144 Chinese Major Carps from the Perspective of Fish Immunity against Lernaeosis Introduction 144 Materials and Methods 146 Experimental site 146 Experimental animal 146 Parasitic infestation 146 Amino acid analysis 146 Statistical analysis 147 Results 147 Parasitic prevalence 147 Amino acid profiling 149 Discussion 152 Literature Cited 155 Collective Summary 160 viii LIST OF TABLES Table Page Title No. No. Morphometric measurements of experimental fish species at the initiation of 1 42 trial. 2 Feed Formula; ingredient and nutrient composition 42 3 Weight and length gain in the control and treated group during the study period. 45 Average number of L. cyprinacea on different body parts during study period in 4 46 control group. (Mean ± SD) Average number of L. cyprinacea on different body parts during study period in 5 47 treated group. (Mean ± SD) Number of L. cyprinacea and its %age on different body parts of experimental 6 47 fish in control ponds (without treatment of DDVP) during the course of study. Number of L. cyprinacea and its %age on different body parts of experimental 7 48 fish in treated ponds (DDVP) during the course of study. 8 Mean values for water quality parameters during the course of study 48 Pearson correlation among various water quality parameters (weekly basis) 9 49 during the course of study Pearson correlation among various water quality parameters (fortnightly basis) 10 49 during the study period. Morphometric measurements of experimental fish species at the initiation of 1 65 trial. 2 Feed Formula used during the course of studies 65 3 Proximate Analysis of fishes from treated group 72 Proximate Analysis of fishes from control group with heavy L. cyprinacea 4 73 infestation 5 Comparison of macronutrients in fishes from treated and control group 73 6 Mineral contents of fishes from treated group 74 7 Mineral contents of fishes from control group infected with L. cyprinacea 75 8 Comparison of minerals (ppm) concentrations in treated and control group 76 9 Mean values for Water quality parameters during the course of study 77 Pearson correlation between the parameters (weekly basis) during the course of 10 77 study Pearson correlation between the parameters (fortnightly basis) during the study 11 78 period. Comparison of blood parameters related to red blood cells among experimental 1 100 fish species 2 Comparison of differential leukocyte count in experimental fish species 101 3 Comparison of less important blood parameters in experimental fish species 102 ix 4 Comparison of blood parameters of experimental fish species females 103 5 Comparison of blood parameters between males of experimental fish species. 104 6 Comparison of blood parameters of males and females of Indian major carps 105 7 Comparison of blood parameters of males and females of Chinese carps 106 8 Total protein, albumin and globulin in females of experimental fish species 108 9 Total protein, albumin and globulin in males of experimental fish species 108 Total protein, albumin and globulin between genders of experimental fish 10 108 species 11 Total protein, albumin and globulin between species of experimental fish species 109 Total protein, albumin and globulin between males and females of experimental 12 109 fish species Protein concentration, HA titer and alkaline phosphatase activity in mucus of 1 131 experimental fish species Molecular weight of protein found in the skin mucus of experimental fish 2 132 species (SDS-PAGE analysis) Number of L. cyprinacae and its %age on different body parts of experimental 1 148 fish in treated ponds (DDVP) during the course of study Essential amino acid composition of the whole body tissues of experimental fish 2 150 species Non-essential amino acid composition of the whole body tissues of experimental 3 151 fish species x