The Biology of Plant-Mosquito Associations DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Babak Ebrahimi, M.S.P.H. Graduate Program in Entomology The Ohio State University 2013 Dissertation Committee: Professor Woodbridge A. Foster, Advisor Professor P. Larry Phelan Professor David L. Denlinger Professor Peter M. Piermarini Copyright by Babak Ebrahimi 2013 Abstract Mosquitoes need plant sugar to maximize their activities. While males cannot survive without sugar, females performance increases in the presence of sugar. Mosquitoes use phytochemicals that are emitted by plants to find the source of sugar. To study mosquitoes’ behavior toward the phytochemicals, olfactometers have been used by researchers. Chapter 2 gives a description of three types of olfactometers that were designed and developed in the vector behavior laboratory at The Ohio State University, to evaluate attractiveness of phytochemicals. Milkweed and goldenrod flowers are attractive sources of sugar for Aedes vexans, Culex pipiens, and Cx. restuans in the Midwestern United States. In Chapter 3, attraction of the flowers of these two plants, their extracts, and the synthetic blend of milkweed were compared to unbaited and honey-baited traps. Flower-baited traps were more attractive to Ae. vexans and Culex spp. than traps with other baits. In Chapter 4, the possibility that Anopheles gambiae, a major African malaria vector, can learn plant odors and single phytochemicals, was explored in a mesocosm and an olfactometer. Results showed that associative olfactory learning depends on release rates of compounds during testing and conditioning. They also showed that when mosquitoes associated a compound with a sugar reward during conditioning, they were still highly attracted to it, even in the presence of a novel compound. The effect of sugar availability on vectorial capacity, ii including survival, biting rate, and fecundity was assessed in Chapter 5. Vectorial capacity was increased in a sugar-rich environment, unlike an earlier study, probably because mosquitoes had access to human blood for long period of time, allowing a higher biting rate. Fecundity was slightly decreased in the sugar-poor environment, probably because of a lower insemination rate in females, owing to rapid male die-off, and the females’ ability to use blood to provide energy. In addition to sugar availability, reproductive fitness of An. gambiae can be affected by rain. The experiments described in Chapter 6 showed that water agitation stimulates egg hatching, a first for this genus of mosquito. Eggs stored as long as a month hatched upon agitation. This characteristic has led to a new method of handling and colony maintenance in this species. Mosquitoes and other animals rely on many aspects of natural light in their lives, but artificial light generated by incandescent and fluorescent lamps and used in laboratory experiments contains a flicker component, a byproduct of alternating current. Insects can detect this flicker, and it may affect their responses to light-based behavior and development. A newly developed flicker-free dimmer for LEDs is described in Chapter 7. A low-pass filter has been placed between an Arduino microcontroller and a strip of LEDs to remove flicker. The microcontroller controls dimming duration. iii Dedication To Daryoush, Maziyar, and Mehrnoosh Ebrahimi, Pourandokht Raeisi and to my best friend and love, Leila Farivar iv Acknowledgments Many people paved my path through my Ph.D. program, by their intellectual, emotional, and financial support. I thank my advisor, Woodbridge Foster, who patiently taught, inspired, trained, encouraged, and assisted me through all steps. My appreciation to him goes beyond words; he opened my eyes to another field of science, and to another philosophy. I appreciate Larry Phelan for teaching me the principles of chemical ecology and insect behavior, and for his valuable contribution and suggestions in Chapters 2, 3, and 4. I was fortunate to have David Denlinger in my committee for all of his support and suggestions, particularly for Chapters 5 and 6. I am also grateful to have had Glen Needham in my committee for his support for providing lab material for a part the study in Chapter 4. I also thank Peter Piermarini for his suggestions on all chapters of this dissertation. Many of my friends and colleagues contributed to my research and helped me since I have started my Ph.D. Program. I was fortunate to work alongside Bryan Jackson, whose constructive discussions and support helped me during the past 3 years. He also contributed to Chapter 5, for which I am grateful. I also thank Chris Stone particularly for his contribution in constructing the mesocosms and also for his suggestions in Chapter 5. It was a pleasure to work alongside Philip Otienoburu, who helped me in Chapters 2 and 3. I am also grateful to have collaborated with Reza Farivar v in Chapter 7, which would have been impossible to fulfill without his ideas and support. I am also thankful for the encouragement and support that I received from my parents, Daryoush Ebrahimi and Pourandokht Raeisi, my brother, Maziyar Ebrahimi, and my sister, Mehrnoush Ebrahimi. Finally, I thank my best friend and my wife, Leila Farivar, whom I have been fortunate to have for her support, inspiration, and for her comments on statistical analyses of this dissertation. vi Vita June 1993 ..............................................................................Adab High School February 1999 ......................................................................B.S. Plant Protection, Isfahan University of Technology February 2003 ....................................................................M.S.P.H. Medical Entomology and Vector Control, Tehran University of Medical Sciences 2004 – 2006 ......................................................................C.E.O., Isfahan Adrian Avin Co. 2007-2013 ...........................................................................Graduate Teaching / Research Associate, The Ohio State University Publications Otienoburu P.E., Ebrahimi B., Phelan P.L., Foster W.A. 2012. Analysis and Optimization of a Synthetic Milkweed Floral Attractant for Mosquitoes. Journal of Chemical Ecology. 38(7): 873-881. Nikbakhtzadeh M.R., Hemp C., Ebrahimi B. 2007. Further Evidence on the Role of Cantharidin in the Mating Behaviour of Blister Beetles (Coleoptera: Meloidae). Integrative Biosciences. 11: 141-146. vii Nikbakhtzadeh M.R., Ebrahimi B. 2007. Detection of Cantharidin Related Compounds in Mylabris impressa (Coleoptera: Meloidae). Journal of Venomous Animals & Toxins. 13(3): 686-693. Yaghoobi-Ershadi M.R., Akhavan A.A., Zahraie-Ramazani A.V., Abai M.R., Ebrahimi B., Vafaie-Nezhad R., Hanafi-Bojd A.A. and Jafari R. 2004. Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran. Letter to the editor, Eastern Mediterranean Health Journal. 10(4/5): 688. Yaghoobi-Ershadi M.R., Akhavan A.A., Zahraie-Ramazani A.V., Abai M.R., Ebrahimi B., Vafaie-Nezhad R., Hanafi-Bojd A.A. and Jafari R. 2003. Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran. Eastern Mediterranean Health Journal. 9(4): 816-826. Alempoor-Salemi J., Shayeghi M., Zeraati H., Akbarzadeh K., Basseri H., Ebrahimi B., Rafinejad J. 2003. Some aspects of head lice infestation in Iranshahr area (southeast of Iran). Iranian Journal of Public Health. 32(3): 60-63. Fields of Study Major Field: Entomology viii Table of Contents Abstract................................................................................................................................ii Dedication...........................................................................................................................iv Acknowledgments................................................................................................................v Vita.....................................................................................................................................vii List of Tables.......................................................................................................................xi List of Figures...................................................................................................................xiv Chapters 1. Introduction: …................................................................................................................1 Effect of Sugar on Mosquitoes.....................................................................2 Location of Host Plants by Volatiles............................................................5 Practical Use of Phytochemicals..................................................................7 Potential Influence of Learning on Attraction to Plants.............................11 How Plant Communities Can Alter Vectorial Capacity..............................12 Objectives...................................................................................................14 2. Olfactometer Design for Aedes vexans and Anopheles gambiae Abstract......................................................................................................16 Introduction................................................................................................17 Materials and Methods...............................................................................19 Results........................................................................................................31