DIFFERENCES IN MALE SCENT IN THE TWO HOST ASSOCIATED STRAINS OF SPODOPTERA FRUGIPERDA AND EVIDENCE OF MATE DISCRIMINATION BY FEMALES DISSERTATION Presented in Partial Fulfillment of the Requirements of the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Paulo Marques, M.S. The Ohio State University 2004 Approved by Dissertation Committee: Professor Larry. Phelan, advisor Professor David Horn Adviser Professor Woodbridge Foster Department of Entomology Professor John Wenzel ABSTRACT Spodoptera frugiperda (J. E. Smith), considered for a long time a generalist feeder, now is known to comprise two genetically differentiated host-associated strains, designated as corn strain (CS) and rice strain (RS). Previous studies have revealed that females attract males of both strains but mate almost exclusively with their own strain. Males are known to possess two sets of hairpencils (potential pheromone-producing structures), but their role has not been previously assessed. The species is a good model to test Phelan’s (1992) Asymmetric Tracking model, which predicts that the sex with the greater parental investment will be more discriminating in their response to sexual signals, and that signals produced by the other sex will evolve to track this preference. Courtship sequences of S. frugiperda were videorecorded in an arena under infrared light, and analyzed frame by frame. Individual behaviors were transcribed to three-dimension transitional matrices, which were submitted to Information Theory analysis for quantification of intra- and inter-individual communication. Information analysis confirmed the existence of an asymmetry in the response between the sexes in S. frugiperda. In both intra- and inter-strain courtships, female-to- male information flow was lower than for male-to-female. Moreover, both RS and CS females were more likely to show a receptive response to male behaviors associated with hairpencil displays when courted by males of their own strain. These findings of a male- ii signal-based female preference for assortative mating, particularly in the absence of mate discrimination by males are consistent with the predictions of the Asymmetric Tracking hypothesis (Phelan, 1992) and the more specific hypothesis of Phelan and Baker (1987) that many lepidopteran male pheromones may have evolved in the context of reproductive isolation through sexual selection. Chemical characterization of each set of hairpencils was also conducted by solvent extraction and analysis by gas chromatography/mass spectrometry. Among the ca. 150 chemicals identified in the extracts, two stood out as showing clear differences between males of the two strains, 2,4-diphenyl-4-methyl-1-(E)-pentene and 2,4-diphenyl- 4-methyl-2-(Z)-pentene. These compounds had not previously been described in any Lepidoptera. iii ACKNOWLEDGEMENTS I thank my advisor Professor Larry Phelan for welcoming me at his laboratory and for giving me the opportunity of doing research on such an interesting subject - the evolution of sex pheromones. It was a hard and stimulating work on several aspects very much as I had anticipated when I left Portugal to this new and exciting learning experience in a US University. I thank him also for letting me share his valuable experience, for all the support he provided me, and for all the discussions we had, that made me think sharper and express my thoughts clearer and overall mature as scientist. I am grateful to my committee (Professor David Horn, Professor Woodbridge Foster and Professor John Wenzel) for their valuable suggestions. I would also like to thank Professor Dorothy Prowell from the Louisiana State University for her help in gathering the collections of Spodoptera frugiperda specimens with which I started both colonies at the OARDC-OSU. I also thank her for kindly letting me use her laboratory resources and for introducing me the necessary tools to assess S. frugiperda strain status. My gratitude also goes to Dr. Margaret McMichael at LSU for the valuable time she took to teach me the techniques for assessing S. frugiperda strain status, and in solving all the problems associated with the method after I returned to OSU. I thank her also for helping me in the field collections of specimens. iv I am also grateful to Professor Sally Miller for letting me use her laboratory resources when assessing the status of the S. frugiperda specimens brought from Louisiana. I thank Emeritus Professor Roy Rings for letting me “explore” and “damage” some of the S. frugiperda specimens of his precious collections at the OARDC-OSU. I would like to thank LeAnn Beanland, Seppo Salminen, and the other graduate students and staff working at the OARDC. They all made me feel at home while I lived and worked in the USA and I was privileged for the time and experiences we shared. Here, I especially would like to thank my amigo Jim Mason for his friendship and his remarkably unselfish support. His long experience with lepidopteran colonies and his natural interest helped solve the small and big ordinary laboratory problems on a daily basis. And last but not least I would like to thank my wife Augusta for believing in me all the way, for her endless encouragement and support. Her extraordinary strength and contagious optimism were vital to my accomplishment of this task. Fundação para a Ciência e Tecnologia, of the Portuguese state office Minitério da Ciência e do Ensino Superior, supported this research (Grant:Praxis XXI BD/9402/96) v VITA November 08, 1963 Born, Lisbon, Portugal 1992 Finishing Undergraduate Coursework in Biology and obtaining the Degree Licenciatura, Faculdade de Ciências da Universidade de Lisboa (University of Lisbon's Faculty of Sciences, Portugal) 1992/1993 High School Biology Teacher, Escola Secundária das Olaias (Lisboa) 1993/1994 On a science research scholarship from Junta Nacional de Investigação Científica e Tecnológica) (National Board for Scientific Reasearch, Portugal). 1994/1995 High School Biology Teacher, Escola Secundária de Vendas Novas (Vendas Novas, Portugal) 1995/1996 High School Biology Teacher, Escola Secundária nº 1 de Loures (Loures, Portugal) vi 1996 M. S. in Ethology, Instituto Superior de Psicologia Aplicada (Sup. Institute of Applied Psychology, Portugal) 1996/1997 High School Biology Teacher, Escola C+S da Ericeira (Ericeira, Portugal) 2003 Invited TA at the Universidade de Évora (University of Evora, Portugal FIELD OF STUDY Major Field: Entomology vii TABLE OF CONTENTS Page Abstract………………………………………………………………………….. ii Acknowledgments……………………………………………………………….. iv Vita………………………………………………………………………………. vi List of Figures …………………………………………………………………... x List of Plates…………………………………………………………………….. xi List of Tables…………………………………………………………………….. xii Prologue...........................................................................................................…... 1 Chapters: Chapter 1………………………………………………………………………… 23 1. Introduction……………………………………………………. 23 2. Materials and Methods………………………………………… 33 3. Results…………………………………………………………... 38 3.1. Description of Spodoptera frugiperda Courtship………………. 38 3.2. Ethogram of Spodoptera frugiperda…………………………… 39 3.3. Courtship success within and between strains………………….. 51 3.4. Information Theory analysis…………………………………… 55 3.4.1. Intra-strain Courtship analysis……………….………………... 55 3.4.2. Inter-strain Courtship analysis……………….………………… 64 4. Discussion……………………………………………………... 68 viii Chapter 2………………………………………………………………………… 77 1. Introduction………………………………………………….…. 77 2. Materials and Methods……………………………………….… 86 3. Results………………………………………………………….. 91 3.1 Genital hairpencils…………………………………………….... 91 3.2 Femoral hairpencils……………………………………….…….. 97 4. Discussion…………………………………………….………... 104 Epilogue ………..……………..…………………………………………. 111 References ……………………………………………………………….…. 113 Appendix A Protocol for host strain status determination of S. frugiperda….. 125 Appendix B Chemicals identified in all extracts obtained from male and female S. frugiperda…………………………………………….. 130 ix LIST OF FIGURES FIGURE PAGE 1. Dendogram for the genital brushes’ dataset 96 2. Dendogram for the femoral hair-pencils dataset 103 x LIST OF PLATES PLATE PAGE 1. Female calling (CAL) 43 2. A mating sequence in the corn associated strain of Spodoptera 49 Frugiperda species. 3. Male displaying genital hairpencils and femoral hairpencils to 54 female’s head (AbH). xi LIST OF TABLES TABLE PAGE Table 1. Mating outcomes of pairings between female and males of the two 55 host-strain of Spodoptera frugiperda of Spodoptera frugiperda Table 2. Information Theory parameters for intra-strain courtships. 57 Table 3. Male-to-female information transmission 59 Table 4 Female-to-male information transmission 61 Table 5 Information theory parameters for inter-strain courtships 65 Table 6 Genital hairpencils chemicals 93 Table 7 Femoral hairpencils chemicals 100 Table 8. Chemicals identified in all extracts of male and female 131 xii PROLOGUE Species and species-isolating mechanisms Species of sexually reproducing animals remain distinct, primarily because they are genetically independent. Genes present within individuals of one species usually combine only, in future generations, with genes from other members of the same species. Thus members of a species are said to "share a common gene pool" and one species is separated from another by "barriers to gene exchange," according to the Biological Species Concept (Mayr, 1940; 1976). Since evolution is based on the accumulation of genetic modifications, and the boundaries