ODOR MASKING OF A VERTEBRATE CARCASS BY A BURYING BEETLE (Nicrophorus marginatus) by CHARMAINE WOODARD, B.S. A THESIS IN BIOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved Richard J. Deslippe Chairperson of the Committee Nancy E. McIntyre John C. Zak Accepted John Borrelli Dean of the Graduate School May, 2006 ACKNOWLEDGEMENTS I thank my advisor, Dr. Richard J. Deslippe, for accepting me into his lab and permitting me to work on research concerning burying beetles. His invaluable assistance in ideas for research and his excellence in his guidance in writing are most appreciated. I also thank my other committee members, Dr. Nancy E. McIntyre and Dr. John C. Zak. Both were always more than willing to assist me at any point of my research. Dr. Richard Strauss was kind enough to help me with the types of analyses appropriate for my research. Dr. George Cobb and Dr. Yu-Jie Guo were instrumental in the analyses of my data. Dr. Stephen J. Pressley was kind enough to assist me with the making of maps. I also appreciate the generosity of the Hurt family for permitting me to trap on the Ratliffe Ranch near Odessa, Texas. I extend my deep appreciation to them all. I give my undying love and gratitude to my family, especially my son, Winter J. Woodard, my father, Martin M. Woodard, my mother, Betty E. Woodard, and my sister, Nikki E. Sands, for all their encouragement all my life to follow my dreams. I greatly appreciate the friendship and support from my fellow lab mates, David Melvin, Jilla HaghiPour-Beasley, Leahann Borth, Issam Al Diri and Leticia Torres. We have all shared the struggle of graduate school together. Lastly, but most important of all, I would like to thank my husband, William D. Melvin, for his unwavering affection, support and faith in me. Without his extensive mentoring, I would never have been able to complete this project. Financial support was provided by the Department of Biological Sciences, Texas Tech University, Lubbock, Texas. ii TABLE OF CONTENTS AKNOWLEDGEMENTS………………………………………………………………...ii ABSTRACT…………………………………………………………………....…….......vi LIST OF FIGURES………………………………………………….……………....…..vii CHAPTER I. INTRODUCTION……….……………………………………………………...1 Part I – Carrion, Scavengers and Decomposers…………………………...1 Carrion Availability……………………………….………………1 Scavengers…………………….…………………………………..3 Facultative Scavengers………………………………..…………...3 Obligate Scavengers…………………………………..…………...4 Decomposers…………………………………..…………………..4 Part II –Nicrophorus………………………………………….……..….....7 Natural History……………………………………..……………...7 Reproductive Behavior……………………………….…………...9 Chemical Ecology………………………………………..……....12 II. MATERIALS AND METHODS……...……………….……………..………16 Part I – Preparation for Experiments………………………….….……...16 Collection of Burying Beetles………………..…………………..16 Rearing of N. marginatus…………………..…….………………18 Part II – Headspace Sampling………………………………..…….…….19 . Collection of Mouse Volatiles………………………..…….……19 iii Double-blind Human Smell Test………………………..….……21 Headspace Collection and Smell Test Apparatus..………….…...22 Determination of the Chemical Composition of Anal Secretions…………………………...………………...…24 Part III – Effect of Anal Secretions on Soil Bacteria…………..…...……25 Part IV – Statistical Analyses...….…………………….…..…………….25 Mouse Volatiles……………………………………….….….…..25 Human Assessment…………………………….…..…………….27 III. RESULTS……………………………….……………….………..…………28 Part I – Discriminant Function Analyses on Four Treatments…………. 28 Three Groupings of the Four Volatile………………….…..……28 Two Groupings With MPO Excluded………..…………………..29 Part II – Comparisons of Volatiles………………………..………....…...30 Unknown Volatile Compound at RT 8.1 Minutes……………….30 Dimethyl Disulfide………………………...............………..……31 . Dimethyl Trisulfide………………………………...………..…...32 Methoxy-Phenyl-Oxime……………………………...…....….…33 Part III – Associations Found with MPO and Mouse Volatiles………….34 Part IV – Compounds Detected in Anal Secretions…………...…………36 Part V – Double-blind Human Smell Test………………….……………38 Ratings of Treatments………….…………………..…….………38 Associations between Age and Treatments…………..………….39 Part VI – No Suppression of Bacteria with Anal Secretions…………….40 iv IV. DISCUSSION…………..…………………….………………..…………….41 Odorants of Carcasses………………………………….…………..…….41 Dimethyl Disulfide………………..……………………………………...42 Dimethyl Trisulfide……………………………………………..………..43 Decrease of Unknown Compound at RT 8.1 Minutes ...………………...43 MPO Detected in Treatments with Anal Secretions……………………..44 Increase in MPO as Mouse Volatiles Increase…………………..44 Patterns Among Treatments……………………………....….…..45 Compounds in Anal Secretions……………………...……….…..45 Double-Blind Human Smell Test……………...………………....47 No Suppression of Soil Bacteria………………………..………..48 V. SUMMARY..…………………………….……………………………….…..49 VI. CONCLUSION….………………….…...……………………….……….….50 Future Research………………………………………………………….50 LITERATURE CITED………………………………….……………………………….52 APPENDICES……...……………………………………………………………………57 A. PPM FOR SPE VOLATILES DATA…….………………..…………….57 B. PPM FOR SPE MOUSE WEIGHT-ADJUSTED VOLATILES DATA……..………..…………………..……….………..59 C. HUMAN SMELL TEST DATA………………………...………….……60 D. SPME OF ANAL SECRETIONS DATA…………………......….……..61 v ABSTRACT Burying beetles of the genus Nicrophorus (Coleoptera: Silphidae) use small vertebrate carcasses as food in raising their young with bi-parental care. These carcasses are stripped of hair and treated with oral and anal secretions by both males and females. Here, I report on whether Nicrophorus marginatus, a widely distributed species in North America, masks the odor of decomposing carcasses, and if so, how it accomplishes the masking. First, I compare the volatiles emitted by mouse carcasses from six different treatments, including mouse carcasses that are processed by male and female pairs of N. marginatus. The release of volatiles is assessed both by human subjects and by chemical analyses, employing solid phase extraction. Second, I examine the chemical composition of the anal secretions of both N. marginatus and N. carolinus by solid phase microextraction to gain insight into which compounds, if any, are involved in odor masking. The solid phase extraction and solid phase microextraction analyses were subsequently characterized by gas chromatography and mass spectrometry. The results of the solid phase microextraction analyses showed the commonalities and differences of several compounds between the two sexes and genders of burying beetles, but shed no light on whether any of them aids in odor masking. The solid phase extraction analyses showed an increase in an anal secretion of N. marginatus, methoxy-phenyl-oxime, with the increase in decomposing mouse carcass volatiles, suggesting that odor masking is taking place. vi LIST OF FIGURES 1. Sulfur and amine compounds are emitted from a carcass due to bacterial activity………………………………………..…………….…………6 2. Lower molecular weight compounds at low concentrations indicate to a scavenger an edible carcass………………………….………………7 3. Nicrophorus marginatus assessing a mouse carcass…………………………..….9 4. N. marginatus forms a brood ball from a mouse carcass………………………...11 5. The principle component in the anal discharge of Silpha beetles is 15β-hydroxyprogesterone…………………………………………………….14 6. The stars indicate the two locations of the trapping sites for N. marginatus………………………………………………………………...16 7. Beetle traps consisted of 2 L buckets, pieces of wood for flotation devices and mouse carcasses…………………………………………...17 8. The single star indicates the trapping site for N. carolinus…………...………….18 9. A mouse carcass is manually shaved of hair with a small electric shaver…………………………………………………………………....20 10. A solution of anal secretions collected from male and female N. marginatus beetles are applied to a manually shaved mouse carcass……………………………………………………………21 11. Testing apparatus for the collection of headspace of the treatments…………….23 12. A discriminant function analysis (DFA) on the mouse and beetle volatiles……..29 13. A DFA to determine what groupings were present based on the volatiles dimethyl disulfide and dimethyl trisulfide……………………………..30 14. ANOVA with pairwise comparisons for unknown volatile at RT 8.12 with the first five treatments that showed no identified compound omitted from treatments involving mice…………………………………………...……..31 15. ANOVA with pairwise comparisons for dimethyl disulfide in treatments involving mice……………………………………………………..32 vii 16. ANOVA with pairwise comparisons for dimethyl trisulfide in treatments involving mice………………………………………………………..33 17. ANOVA for MPO that was only detected in treatments involving anal secretions……………………………………………………………………34 18. Relationship between the amounts of methoxy-phenyl-oxime and dimethyl disulfide……………………………………………………………35 19. Relationship between the amounts of methoxy-phenyl-oxime and unknown mouse volatile…………………………………………………….35 20. Regression analysis of the relationship between amounts of methoxy-phenyl-oxime and dimethyl trisulfide………………………………….36 21. Identified compounds found in anal secretions across the two sexes of both species of burying beetles, N. marginatus and N. carolinus……………………..37 22. ANOVA for the human assessment of the six treatments……………………….39 23. Regression analysis correlating the odor of the beetles-mouse treatments with the women’s ages..……………………………………………...40 viii CHAPTER I INTRODUCTION The overall theme of this thesis is the masking of the volatiles of decomposing mouse carcasses by the application of anal secretions of burying beetles (Silphidae: