An Assessment of Honeybee Foraging Activity and Pollination Efficacy in Australian Bt Cotton A Dissertation by MARWAN MOHAMED KESHLAF Presented in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY CENTRE FOR PLANT AND FOOD SCIENCES UNIVERSITY OF WESTERN SYDNEY AUSTRALIA July 2008 DEDICATION To my wife, my parents, my son Elyas and daughters Islam and Safeea, for their sacrifices, prayers, and constant support and encouragement. ii DECLARATION I declare that this thesis submitted is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution. Marwan M. Keshlaf 3 July 2008 iii ACKNOWLEDGMENTS I wish to express my sincere appreciation to my supervisors, Professor Robert Spooner- Hart and Dr. Robert Mensah, for their generous guidance, advice and support. I am especially grateful to Professor R. Spooner-Hart who clarified my ideas, and helped me to improve my writing skills. I am very grateful to Professor Andrew Beattie for his comments and valuable suggestion in the thesis. I would like to express my sincere gratitude to Dr. Albert Basta for supervising the bioassay experiments. Also, the assistance provided by UWS staff, Oleg Nicetic for his advice and statistical analysis, Liz Kabanoff for providing pollen SEM, Mr. Michael Duncan for bee management is greatly appreciated. Special thanks are given to Dr Michael Batley, from the Australian Museum of Natural History, Sydney, for insect identification. I am thankful to Australian Cotton Research Institute (ACRI) for providing their cotton fields, use of its research facilities, and processing yield measurements and ELISA test. Special thanks to Leah Austin and Raymond Morphew, employees at Narrabri (ACRI), for technical assistance. I thank Mr. Warren Jones, who provided all beehives and equipment during the research period; also his experience in crop pollination in Australia was useful for me. I also thank Al-Fateh University, Tripoli, Libya for the scholarship, and finally, I wish to thank my parents and family for their support and understanding. iv TABLE OF CONTENTS Page DEDICATION ………………………...……………………….………………… ii DECLARATION ………………………...……………………….……………… iii ACKNOWLEDGMENTS…………………...………………………..……….… iv TABLE OF CONTENTS………………………...……………………….……… v LIST OF TABLES xi LIST OF FIGURES xiii SUMMARY………………………...……………………….………..…………… xvi CHAPTER 1. GENERAL LITERATURE REVIEW 1.1 Introduction …………………………..……………………………………….. 1 1.2 Phenology of cotton ………………………...……………………….………… 4 1.2.1 The cotton plant ………………………………...……………...……….. 4 1.2.2 Flowering and bolls ……………………………………..………………. 4 1.2.3 The flowering pattern .……………………………………………...…… 6 1.2.4 Floral biology .…………………...……………………………...………. 7 1.2.5 Nectar and nectary glands ...………………….…………………………. 7 1.3 Cotton pollination …….………………………………………………...….… 9 1.3.1 Pollination ecology ……………………………………...…………....… 9 1.3.2 Pollinating agents …………………………………………………......… 13 1.3.3 The role of honeybees as pollinating agent in cotton ………………….... 15 1.4 Management of honeybee colonies during the pollination season ………… 18 1.4.1 The stocking rate ……………………………………………………….. 18 1.4.2 Scheduling the arrival and removal of honeybee colonies, in relation to the target crop .…………………….…………………………………… 20 1.4.3 The spatial distribution of colonies in relation to the target crop area ….. 21 1.5 Honeybee foraging behaviour in cotton flowers……………………………. 22 1.5.1 Pollen gathering activity ………………….…………………………...... 24 1.5.2 Nectar gathering activity …………………………..……………………. 27 1.5.3 Cotton as a honey plant ………………………………………..………… 28 1.5.4 Attractiveness of flowering cotton plants ………………………………. 30 1.6 Techniques used to increase activity of honeybees, and their pollination efficacy …………………………………………….…………………………. 31 1.6.1 The use of pollen trap to increase bee activity ...………….…...………... 31 1.6.2 Provision of supplementary food and its effect on bee activity ….……... 34 v 1.6.3 Use of pollination enhancement chemical on bee activity and pollination 35 1.7 The role of beetles in pollination ………………………..……….……...…... 38 1.8 Hazardous effects of insecticides on cotton pollination ……………….…… 40 1.9 Transgenic (Bt) cotton ..………………………………..……..…… 44 1.10 Thesis objectives …………………………..…………………………………. 46 CHAPTER 2. EFFECT OF TRAPPING POLLEN, AND FEEDING POLLEN SUPPLEMENTS, TO HONEYBEE COLONIES USED FOR COTTON POLLINATION 2.1 Introduction …………………………..…………………………....…………. 48 2.2 Materials and Methods .….……………………..……………...…………….. 50 2.2.1 Location …………………………..……………………..………..……... 50 2.2.2 Treatments ………………………..……………………………....……... 51 2.2.3 Flight activity …………..…………………………….…………..……… 53 2.2.4 Pollen gathering activity ………………………..…………………..…… 54 2.2.4.1 Trapping pollen ………………………..……………………….… 54 2.2.4.4 Area of stored pollen …………….……………..………………… 56 2.2.5 Brood rearing activity ……………………..………..…………………… 57 2.2.6 Nectar gathering activity ……………………..……..…………………... 58 2.2.7 Honeybee pollination efficacy ……………………..……..……………... 59 2.2.7.1 Foraging activity of honeybee visitors to cotton flowers.…….…... 59 2.2.7.2 Cotton yield measurements …………………………..…………... 60 2.2.8 Determination of the cotton flowering pattern …………………….……. 62 2.2.9 Determination of other species flowering within the experimental area ... 62 2.2.10 Recording of meteorological conditions during investigation period ….. 62 2.2.11 Statistical analysis …………………………..…………………………... 62 2.3 Results …………………………..………………………….…………………. 63 2.3.1 Influence of provision of supplementary pollen on honeybee activity ….. 63 2.3.1.3 Flight activity …………...…………………..………..………..…. 63 2.3.1.4 Pollen gathering activity …………...………………..……...……. 65 2.3.1.5 Brood rearing activity ………..………………..…………………. 67 2.3.1.6 Nectar gathering activity ………..……………………..……….… 68 2.3.2 Influence of restricting pollen entry to colonies (by using pollen trap) on honeybee activity …………………..…………..……………...………… 68 2.3.2.1 Flight activity …………..…………………...……………………. 68 2.3.2.2 Pollen gathering activity .…………………………………………. 69 2.3.2.3 Brood rearing activity …………………………..………………… 75 vi 2.3.2.4 Nectar gathering activity ………...……………..………………... 76 2.3.3 General flight activity …………………………………………………… 76 2.3.3.1 Diurnal flight activity …………………………………………….. 76 2.3.3.2 Weekly flight activity …………………………………………….. 77 2.3.4 Bee pollination efficacy ………………………..………………………... 77 2.3.4.1 Foraging activity of honeybee visitors to cotton flowers ………… 77 2.3.4.2 Non-Apis pollinators …………………………………..…………. 80 2.3.4.3 Cotton yield measurements ………………………..……………... 80 2.3.5 Determination of the cotton flowering pattern ………………………….. 81 2.3.6 Determination of other species flowering within the experimental area ... 82 2.3.7 Temperature and humidity during the experimental period ………..…… 82 2.4 Discussion ………………………..…………………….……………….……. 83 2.4.1 Influence of provision of supplementary pollen on honeybee activity ...... 83 2.4.2 Influence of restricting pollen entry to colonies (by use of pollen traps) on honeybee activity …………………………..……………..………….. 87 2.4.3 Bee pollination efficacy …………………………………………... 94 2.5 Conclusions ………………………..………………………………..………… 98 CHAPTER 3. THE EFFECT OF SYNTHETIC QUEEN MANDIBULAR PHERMONE ON HONEYBEE FORAGING ACTIVITY IN COTTON FLOWERS, AND SUBSEQUENT EFFECTS ON YIELD AND QUALITY 3.1 Introduction …………….……………………………….……………..……. 100 3.2 Materials and methods………….……………………………….…..…….. 103 3.2.1 Location ………….……………………………….…………………..… 103 3.2.2 Treatment ………….……………………………….………………....... 103 3.2.3 Assessment of bee activity …………….……………………………….. 104 3.2.4 Percentage boll set …………….……………………………….……….. 105 3.2.5 Assessment of cotton yield …………….……………………………….. 105 3.2.6 Recording meteorological conditions during investigation period …….. 106 3.2.7 Statistical analysis …………….……………………………….……….. 106 3.3 Results ………….……………………………….…………………………… 106 3.3.1 Assessment of bee activity …………….……………………………….. 106 3.3.2 Percentage boll set ……….………………………………..……………. 108 3.3.3 Assessment of cotton yield quality and quantity……………………….. 109 3.2.4 Temperature and humidity during the experimental period ..…………... 111 3.4 Discussion …..………….…………………………….………………………. 112 3.5 Conclusions …………………………………………….……………………. 116 vii CHAPTER 4. HONEYBEE FIELD ACTIVITY, POLLINATION EFFECTIVENESS AND INFLUENCE ON YIELD PARAMETERS IN TRANSGENIC BT COTTON 4.1 Introduction ………….……………………………….…..………………..... 118 4.2 Materials and methods …………….……………………………….…...…... 120 4.2.1 Location …………….……………………………..……………….….. 120 4.2.2 Honeybee pollination efficacy …………….……..….………………… 121 4.2.2.1 Treatments ..…….………………………………………………. 121 4.2.2.2 Assessment of honeybee foraging activity ………………........... 123 4.2.2.3 Cotton yield measurements …………….………………………. 125 4.3.2 Cotton gene flow …………….………………………………………… 125 4.2.3.1 Experimental design ……………………………………………. 125 4.2.3.2 Sampling and testing protocol ………………………………….. 126 4.2.4 Recording meteorological conditions during investigation period ……. 126 4.2.5 Statistical analysis ……….…………………………………………….. 126 4.3 Results ………….…………………………...…….…………………………. 127 4.3.1 Honeybee pollination efficacy …………….………………………..…. 127 4.3.2 Cotton yield measurements ……….………………………………….... 132 4.3.3 Cotton floral phenology ……………………………………………….. 134 4.3.4 Cotton gene flow ……….……………………………………………… 134 4.3.5 Temperature and humidity during experimental period ………………. 134 4.4 Discussion ………….……………………………….………………….……. 135 4.4.1 Honeybee pollination efficacy .…………….…………………….……. 135 4.4.2 Honeybee foraging activity …...…………………………………..…… 137 4.4.3 Cotton yield measurements ………….………..……………………….