SOME ASPECTS OF ECOLOGY AND GENETICS OF CHIRONOMIDAE (DIPTERA) IN RICE FIELD AND THE EFFECT OF SELECTED HERBICIDES ON ITS POPULATION By SALMAN ABDO ALI AL-SHAMI Thesis submitted in fulfillment of the requirements for the degree of Master August 2006 ACKNOWLEDGEMENTS First of all, Allah will help me to finish this study. My sincere gratitude to my supervisor, Associate Professor Dr. Che Salmah Md. Rawi and my co- supervisor Associate Professor Dr. Siti Azizah Mohd. Nor for their support, encouragement, guidance, suggestions and patience in providing invaluable ideas. To them, I express my heartfelt thanks. I would like to thank Universiti Sains Malaysia, Penang, Malaysia, for giving me the opportunity and providing me with all the necessary facilities that made my study possible. Special thanks to Ms. Madiziatul, Ms. Ruzainah, Ms. Emi, Ms. Kamila, Mr. Adnan, Ms. Yeap Beng-keok and Ms. Manorenjitha for their valuable help. I am also grateful to our entomology laboratory assistants Mr. Hadzri, Ms. Khatjah and Mr. Shahabuddin for their help in sampling and laboratory work. All the staff of Electronic Microscopy Unit, drivers Mr. Kalimuthu, Mr. Nurdin for their invaluable helps. I would like to thank all the staff of School of Biological Sciences, Universiti Sains Malaysia, who has helped me in one way or another either directly or indirectly in contributing to the smooth progress of my research activities throughout my study. My genuine thanks also go to the specialists, Prof. Saether, Prof Anderson, Dr. Mendes (Bergen University, Norway) and Prof. Xinhua Wang (Nankai University, China) for kindly identifying and verifying Chironomidae larvae and adult specimens. My sincere thanks to my beloved country, Republic of Yemen, and Ibb University for the scholarship and assistance provided during the course of my ii study. My great thank to all of my friends especially Hesham Hasan for his kind help during preparation of this draft. Last but not the least, I would like to thank all my family members, especially my father, mother and brother Abdullwahab. Great thanks to my wife and my sons Ali, Mohammad and Abdullaziz for their encouragement and patience during the time of my study. iii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ... ... '" ...... '" ............................ ,. ... ... ..... ii TABLE OF CONTENTS ......... ... '" .................... , ........ , ... ............. iv LIST OF TABLES '" .............. , ... '" .. , ............ '" ., ........ ,. ... ... ... ... ... xi LIST OF FIGURES................................................ ... ...... ...... ..... xv LIST OF PLATES ........................................................ :............ xxi LIST OF SYMBOLS AND ABBREVIATION ................................... xxiii LIST OF APPENDiCES............................................................. xxv LIST OF PUBLICATIONS ......... ................................ , .... ,. ...... .... xxvi ABSTRAK ............................................................................... xxvii ABSTRACT ............................................................................ xxix CHAPTER 1: INTRODUCTION 1.1 Introduction ... ............................................. ..................... 1 1.2 Objectives .... , ... , ........ , '" ..... , ........ , '" ......... " ........... , ... , ... 5 CHAPTER 2: LITERATURE REVIEW 2.1 Introduction ................................. , ........ , ........ , ............ ..... 7 iv 2.2 Life Cycle and Morphology of Chironomidae ............... ....... 8 2.2.1 Egg .................................................................... 8 2.2.2 Larvae .............................................. , ...... , ......... 8 2.2.3 Pupae ................................................................. 10 2.2.4 Adult ..... , .......................................... '" ................ 11 2.3 Importance of Chironomidae ........ , ..... , ..... , ..... , ... ~..... ...... 11 2.4 Classification of Chironomidae ..... , ..... , ................. , ......... 14 2.4.1 Tanypodinae ...................................................... 15 2.4.2 Chironominae ............................... , ..... ,. ... .......... 15 2.4.3 Telmatogetoninae ................... , ........... ,. .............. 16 2.4.4 Chilenomyiinae .......................................... ......... 16 2.4.5 Aphroteniinae ..................................................... 16 2.4.6 Podonominae ...... .................. .................. ...... ...... 16 2.4.7 Buchonomyiinae ... ......... ... ... ...... ............... ........... 16 2.4.8 Diamesinae ............................................. ........... 17 2.4.9 Prodiamesinae ...... ............ ...... ...... ...... ...... ........... 17 2.4.10 Orthocladiinae...................................................... 17 2.5 Chironomidae in the Oriental Region ....................... : ...... .. 17 2.5.1 Chironomida'e in South East Asia .......................... .. 19 2.5.2 Chironomidae in Malaysia,..... ............... ........ ......... 20 v 2.6 Rice Fields as a Habitat for Chironomidae ......... '" ... '" ........ , 21 2.7 Factors Influencing the Abundance of Chironomidae Population .................................................................... 22 2.7.1 Temperature ...................................................... 24 2.7.1 Oxygen .............................................................. 25 2.7.3 pH and Conductivity ......... '" .............. , .. , ............... 26 2.7.4 Total Suspended Solids (TS·S) ................................ 28 2.7.5 Total Organic Matter (TOM) ................................... 29 2.7.6 Water Depth ....................................................... 30 2.7.7 Nitrate-Nitrogen ... '" '" ......................................... 30 2.7.8 Phosphate .................................................... , .... 31 2.7.9 Pesticides ........................................................... 31 2.7.10 Chemicals and Heavy Metals ................................ 35 2.8 Methods in Identification of Chironomids ........................... 36 2.8.1 Morphological Method ...... '" .... ,. ... .... ..... ... ... ... ... ..... 36 2.8.2 Karyological Methods ......................................... , .. , 37 2.8.3 Molecular Methods ... .............................. ......... ... .... 38 2.8.3 (a) Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) 40 2.8.3 (b) Random Amplification Polymorphic DNA-PCR (RAPD-peR) ....... , ... , ... ... ... ............ .......... 42 2.8.3 (c) DNA Sequencing ........... , ...... ...... ... ...... ..... 42 vi CHAPTER 3: MATERIALS AND METHODS 3.1 Description of Study Area ....................... , ... ...... ............... 45 3.2 Rice Cultivation Phases ................................................... 47 3.3 Larval Study .................................... '" .. , ... '" .. , ...... ........... 52 3.3.1 Sampling of Chironomidae Larvae from the Rice Field..... 52 3.3.2 Sorting of Chironomid larvae from the Sample ... ...... ...... 52 3.3.3 Preparation of Slides ................ , ........ ,. ... ......... ........ 53 3.3.4 Morphological Identification .............................. ......... 53 3.4 Environmental Study...... .................. ............ ..................... 54 3.5 Bioassay .............................................. , ............ , .......... 55 3.5.1 The Larvae .......... , ........ , ............ , ........ , ... ......... .... 55 3.5.2 The Bioassay Procedures ............. , ............ , ........ , .... 56 3.5.3 The Herbicide........................ ... ............... ... ...... ... 56 3.6 Molecular Study ..... , ....... , .......................... , ........ , ............ , 59 3.6.1 DNA Extraction .... , .............................. , '" .............. 59 3.6.2 Electrophoresis, Amplification, and Sequencing ...... ..... 60 vii 3.7 Data Analysis .................................... ......... ... ......... ... ....... 61 3.7.1 Ecological Data Analysis ............ ............ .................. 61 3.7.2 Bioassay Data Analysis ... '" ........ , ..... , ..... , ... ... ... ........ 62 3.7.3 DNA Data Analysis ................................. ................. 62 CHAPTER 4: RESULTS 4.1 Larval Study .............. , ... ... ... ... ... ...... ... ... ... ... ... ... ... ... ..... 63 4.1.1 Distribution of Chironomidae Larvae in Rice Field.... 63 4.1.2 Influence of Environmental Variables on Chironomid larval Abundance... ........................... ...... ... ........ 72 4.1.3 Composition and Abundance of Chironomid Larvae in the Rice Field Plots at Bukit Merah Agricultural Experimental Station, Seberang Perai (BMAES).......... 79 4.1.4 Abundance of Chironomid Species.......................... 86 4.1.5 Distribution of Chironomid Species during Rice Cultivation Seasons ....... , ..... , ..... , ..... , ..... , ...... , '" ..... 95 4.1.6 Composition of Chironomid Species during Different Rice Growing Phases............ ...... ...... ...... .............. 97 4.1.7 Influence of Physico-Chemical Variables on Diversity of Chironomidae larvae in the Rice Field....................... 103 viii 4.2 Bioassessment of the Effect of Two Herbicides (2,4-0 Dimethylamine and Bensulfuron methyl) on Chironomus kiiensis .................... , ..... , ................................... , ... ... ... 106 4.3 Molecular Study ............................................................ 109 4.3.1 DNA Extraction, Amplification and Sequencing........... 109 4.3.2 Intra and Interspecific Variation