AN ABSTRACT OF THE THESIS OF

AN ABSTRACT OF THE THESIS OF M. Rockwell Parker for the degree of Doctor of Philosophy in Zoology presented on April 15, 2010. Title: Activation, Modification and Suppression of Sex Pheromone Production in Garter Snakes.

Abstract approved: _____________________________________________________________________ Robert T. Mason Vertebrates communicate with one another and coordinate intraspecific reproduction by using a variety of sexually dimorphic signals, such as plumage, ornaments, sounds, and/or scents. These sexual dimorphisms are maintained by physiological factors, typically sex-specific hormones (though see Chapter 3 for an exception). The purpose of the research in this dissertation was to explore the mechanisms regulating sexual dimorphism in the sex pheromone blends of red-sided garter snakes (Thamnophis sirtalis parietalis). The reliance on sex pheromones (both identified and unidentified) in the coordination of snake reproduction appears to be conserved in all groups of the ophidia (Chapter 2). The red-sided garter snake is a model reptile for studying the mechanisms regulating expression of chemical signals because both abiotic and biotic factors have been shown to shape the peculiar biology of this vertebrate. Temperature is known to regulate male garter snake reproductive behavior, and I found, in accordance with this, that females are maximally attractive upon emergence from

hibernation (Chapter 3). Thus, low temperature dormancy is critical for both sexes in this species for optimizing reproduction (behavior, pheromone production). The relationship between sex steroid hormones and pheromone production was incomplete prior to the work in this dissertation. I first discovered that estrogen implantation induced female pheromone production in males, suggesting that estrogen is the primary steroid hormone inducing female pheromone production (Chapter 4). Further, the effect of estrogen is purely activational since implant removal abolished attractivity (Chapter 5). Also in Chapter 4, I showed that castration induced female pheromone production in males. My last data chapter revealed that testosterone actively inhibited female pheromone production, which I saw after supplementing castrates with testosterone (Chapter 6). Further, aromatase inhibition in castrates changed at least one property of the pheromone blend that abolishes attractivity. My research into the control of pheromone expression in garter snakes has revealed a pattern of interaction between steroid hormones and sexually dimorphic signal production at the level of the skin that corroborates findings in their closest relatives (birds). The presence of estrogen promotes expression of the female trait (female plumage in birds; pheromone production in garter snakes). However, the absence of testosterone is also sufficient for expression of the female trait in male garter snakes, suggesting that, unlike any avian species studied thus far, the balance between testosterone and estrogen is critical for proper expression of sexual phenotype. Thus, garter snakes may be a model group for exploring the evolutionary origin of hormonal control of sexually dimorphic signals.

©Copyright by M. Rockwell Parker April 15, 2010 All Rights Reserved

Activation, Modification and Suppression of Sex Pheromone Production in Garter Snakes by M. Rockwell Parker

A THESIS submitted to Oregon State University

in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Presented April 15, 2010 Commencement June 2010

Doctor of Philosophy thesis of M. Rockwell Parker presented on April 15, 2010. APPROVED:

_____________________________________________________________________ Major Professor, representing Zoology

_____________________________________________________________________ Chair of the Department of Zoology

_____________________________________________________________________ Dean of the Graduate School

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

_____________________________________________________________________ M. Rockwell Parker, Author

ACKNOWLEDGMENTS The research presented in this dissertation would never have come to fruition without Bob Mason’s willingness to take me on as Ph.D. student. I had no idea how much creativity and motivation it would take to synthesize a series of questions on this model system. Bob has also taught me more than I realized was involved in academics. His directness and openness about academia as process have equipped me to be a successful researcher and colleague. I am indebted to him for the guidance and support he has given me throughout my pursuit of complex questions. The faith he has in his graduate students is his greatest contribution to our success. I thank the members of my graduate committee for taking the time to guide and question me throughout my Ph.D. I commend my graduate representative, Jean Hall, for her ability to deal with both Bob and Andy on the same committee. In the same vein, Virginia Weis has been incredibly supportive throughout my time at OSU, and she is a true inspiration. Craig Mosley never shied from imparting his wisdom and pure, unbridled sarcasm on the Mason lab, though he could no longer serve on my committee after moving from OSU. I have enjoyed true collegiality in having Andrew Blaustein on my committee, from his ability to quiz me at the drop of a hat to our discussions about (in)famous herpetologists both past and present. Andy has always made me enjoy being a member of this Department. Lastly, Patrick Chappell is, without a doubt, the most generous faculty member I have known at OSU, who graciously agreed to join my committee during my last year. Pat and I have interacted on every level during my Ph.D., from instructor to friend.

I also want to thank the people in Manitoba that have made my research possible and enjoyable. Bill Watkins and Dave Roberts provided us with the support we needed in the field, and Dave helped me accomplish many side projects during my summers in Manitoba. Al, Gerry, Susan, and Shirley enriched the environment we call home for 6 weeks every field season, and the kindness they showed me during my mom’s health problems touched me deeply. I also want to thank the kids that have either helped me with research (Connor, Evan) or were just a happy face to talk to after a long day in the field (Samantha, Michael). I especially want to thank Pat for being the sister I could talk to and the shoulder I needed. The Mason lab can be a challenging place, from snow in May to brown outs in July, but undergraduates and ASE apprentices manage to take the sting of it all away. We have had the fortune of having two impeccable work study students, Robert Cressman and Ben Burke, without whom we would be utterly lost. Kata Haeberlin, volunteer extraordinaire, made my life a lot easier, and I hope I can one day repay her. Paige (and her “awesomeness”), Miranda, Elliott, and Sean were fantastic apprentices to work with in the lab, and I know that they will become successful scientists in their own right. I would also like to thank numerous undergraduates who have also contributed either to my research or to my sanity: Anna Vigeland, Mattie Squire, David Chin, Dolly Do, Kristi Parks, and Adam Cole. You all motivate me to be a better scientist and to think a little bit harder before I speak, contrary to whatever may come out of my mouth.

Many faculty and graduate students have made my time here enjoyable and collegial. Barb Taylor has always been willing to let me darken her door, and Doug Warrick never ceases to impress me with his mastery of dry humor. The Zo-grads at OSU made this a fun experience, and they make me proud to be a product of Zoology. The Zoology staff go above and beyond the call of duty, especially Tara Bevandich, who has offered me great advice and support. Joe Beatty is a true ally to the Zo-grads, and we all owe him much for the support he gives us. The friendship I have enjoyed with Chris and MJ Stallings, Mike Bogan, Betsy Bancroft, Laura Petes, Karen Kiemnec-Tyburczy, Elisha Wood-Charlson, Angie and Santiago Perez, Kristin Latham, Christy Schnitzler, Kate Boersma, Sarah Eddy, and Cat Searle has made this a life experience, not just academic. My real nucleus of support in Corvallis throughout my doctorate has consisted of Doug DeGross, Amanda Sapp, Maria Kavanaugh, Jerod Sapp, Tiffany Garcia, Dave Paoletti, and Steph Gervasi. I, as well as they, know how hard it is to be my friend. They deserve something... epic. I have also shared my voyage as an individual with Luke Sugie, Shawn Anderson, Kate Scollan, Alex Davis, Tab Dansby, and Dau Ngyuen. More than anything, these people make me step back from the minutia and enjoy moments in life that would otherwise go unnoticed. My parents and family have always been supportive in my academic endeavors, and their ability to respect this career path astounds me. Mom and Dad, you have funded as much of my Ph.D. as the EPA or NSF, and I share all of my success with you. You both have inspired me to be the best instructor and mentor I

can be, and I only hope I can touch the lives of as many students as you both have in your careers as public school teachers. Being a graduate student in the Mason lab is a collective experience. I have enjoyed my field seasons with collaborators, such as Mike LeMaster (who showed me much about GC-MS) and Randy Krohmer (who always entertained my “thought processes” in the field). Don Nichols and Elaine Lamirande made the field a fun experience, especially in the wee hours regaling us with stories of Bob on Guam. Heather Waye and Arianne Cease did much to help me transition into Mason lab life, and I am now trying to pass on the same advice to our newest members, Emily Uhrig and Sarah Moore. I owe the completion of my doctoral work to two people. Deb Lutterschmidt had no idea what she was signing up for that day when we went to pizza. I have idolized her and the standards she set as a researcher and mentor in both the lab and the classroom. I am looking forward to a lifetime of friendship and collegiality with her. Chris Friesen is of critical importance to me. He is as much an inspiration to me as Deb, and he is as much my sibling as she is. Together, they have given me the drive and focus I needed to complete this journey. One person in particular has contributed greatly to my personal growth and professional success. Simon was my quiet in the cacophony, a steady voice of reason and reflection that made me stare as deeply into my own faults as I did into the errors and shortcomings of others. The joy he brought into my life is immeasurable, and Koi represents but a fraction of that. Though our future together is as friends, I owe you more than anyone for the love and compassion you showed me through it all.

CONTRIBUTION OF AUTHORS Robert T. Mason has served as my graduate advisor in the Department of Zoology. He contributed to the design, implementation, interpretation, and writing of all chapters of this dissertation.

TABLE OF CONTENTS Page CHAPTER 1 – INTRODUCTION .......................................................................................... 1 CHAPTER 2 – PHEROMONES IN SNAKES: HISTORY, PATTERNS AND FUTURE RESEARCH DIRECTIONS ........................................................................... 14 Introduction .......................................................................................................... 14 Methods for Assessing Pheromone Usage in Ophidian Reproduction ................ 15 History of Reproductive Chemical Ecology Studies in Snakes ........................... 20 Broad Themes in Snake Reproductive Behavior and the Role of Pheromones ... 32 Future Directions .................................................................................................. 35 References ............................................................................................................ 37 CHAPTER 3 – LOW TEMPERATURE DORMANCY AFFECTS THE QUANTITY AND QUALITY OF THE FEMALE SEXUAL ATTRACTIVENESS PHEROMONE IN RED-SIDED GARTER SNAKES.................................................................... 47 Introduction .......................................................................................................... 48 Methods and Materials ......................................................................................... 53 Results .................................................................................................................. 56 Discussion ............................................................................................................ 58 References ............................................................................................................ 63 CHAPTER 4 – FEMALE MIMICRY IN GARTER SNAKES: THE ROLE OF ESTROGEN .......... 73 Introduction .......................................................................................................... 75 Materials and Methods ......................................................................................... 79 Results .................................................................................................................. 84 Discussion ............................................................................................................ 87 References ............................................................................................................ 92

TABLE OF CONTENTS (Continued) Page CHAPTER 5 – ACTIVATION OF FEMALE SEX PHEROMONE PRODUCTION IN MALE RED-SIDED GARTER SNAKES.................................................................. 104 Introduction ........................................................................................................ 106 Methods and Materials ....................................................................................... 111 Results ................................................................................................................ 116 Discussion .......................................................................................................... 118 References .......................................................................................................... 121 CHAPTER 6 – A NOVEL MECHANISM REGULATING EXPRESSION OF A SECONDARY SEXUAL CHARACTERISTIC: TESTOSTERONE INHIBITION OF FEMALE PHEROMONE PRODUCTION IN GARTER SNAKES ..................................... 132 Introduction ........................................................................................................ 134 Methods and Materials ....................................................................................... 137 Results ................................................................................................................ 142 Discussion .......................................................................................................... 146 References .......................................................................................................... 153 CHAPTER 7 – CONCLUSION ......................................................................................... 170 Remaining Questions – Female Garter Snakes .................................................. 170 The Vertebrate Skin as a Source of Chemical Signals ....................................... 176 Discussion .......................................................................................................... 180 References .......................................................................................................... 182 BIBLIOGRAPHY ............................................................................................................ 184

LIST OF FIGURES Figure

Page

1.1

GC trace from a female pheromone blend of a red-sided garter snake. . ...... 12

2.1

Schematic of a typical Y-maze for assaying trailing behavior in snakes. ..... 44

2.2

Examples of outdoor arenas for observing snake mating behavior. ............. 45

2.3

Individual chromatogram for pheromone from a female Red-Sided Garter Snake (Thamnophis sirtalis parietalis). ............................................. 46

2.4

Individual chromatograms of pheromone blends from intact male (left), castrated male (middle), and intact female (right) Red-Sided Garter Snakes (Thamnophis sirtalis parietalis). ...................................................... 47

3.1

Change in mass (mg; mean + s.e.; N=8 for each bar) of both total skin lipids (black bars) and pooled fractions containing only the nonvolatile methyl ketones that comprise the pheromone (pheromone fraction mass; gray bars) of female red-sided garter snakes................................................. 69

3.2

Sex pheromone concentration (µg pheromone/cm2 skin; mean + s.e.; N=8 for each bar) extracted from female red-sided garter snakes in the field and during laboratory-simulated hibernation and emergence............... 70

3.3

Changes in the female sexual attractiveness pheromone blend of redsided garter snakes from three sampling periods (fall, winter, spring). (A) Representative total ion chromatograms of sex pheromone blends from individual females from the three sampling periods. (B) Non-metric multidimensional scaling plot showing individual pheromone profiles. (C) Masses (mg; mean + s.e.; N=8 for each bar) of prospective individual components (categorized by molecular mass; Da) of the female sex pheromone blend over the three sampling periods.. ..................................... 72

3.4

Mass of components (mg; mean + s.e.; N=8 for each bar) comprising the female sex pheromone blend of red-sided garter snakes from three sampling periods (fall, winter, spring) grouped by methyl ketone type (saturated, unsaturated). ................................................................................ 73

4.1

Proportion of males (n out of 10; mean + SEM) courting the stimulus male from each experimental group in arena trials. ...................................... 98

4.2

Average number of males (+SEM) attracted from a mating ball by the experimental male. ........................................................................................ 99

LIST OF FIGURES (Continued) Figure

Page

4.3

Results of Y-maze trailing experiments. ..................................................... 101

4.4

Non-metric multidimensional scaling plot for pheromone data collected from the experimental males. ...................................................................... 102

4.5

Ratio of unsaturated (U) to saturated (S) pheromone components for each group (mean+SEM). ........................................................................... 103

4.6

Within-group comparisons of the proportion of pheromone profiles comprised of low molecular weight (black bars) and high molecular weight (hatched bars) methyl ketones. Inset, Between-group differences in the ratio of proportions of high molecular weight methyl ketones (HMW) to low weight (LMW). .................................................................. 104

5.1

Number of males attracted from mating balls in the den (per min.; mean + SEM) in both 2008 (inset) and 2009. ....................................................... 127

5.2

Total pheromone mass (µg, mean +SEM) from control, estrogen implanted (E2) and implant-removed (REMOVAL) males........................ 128

5.3

NMS plot derived from relative proportions of the 17 unique methyl ketones comprising the pheromone blends from the three groups. ............. 129

5.4

Mass of methyl ketone (µg; mean+SEM) for each of the 17 unique methyl ketones (classified by molecular weight [Da]) between the three groups. ................................................................................................ 131

5.5

Ratios (mean+SEM) of unsaturated:saturated methyl ketone molecules in the pheromone blends of male red-sided garter snakes. ......................... 132

6.1

Bioassay results examining attractivity of experimental male red-sided garter snakes. ............................................................................................... 158

6.2

Total mass (µg) of methyl ketones (sex pheromone components; mean + SEM) produced by experimental males. .................................................. 159

LIST OF FIGURES (Continued) Figure

Page

6.3

Mass (µg; mean+SEM) of individual methyl ketones (grouped by molecular weight, Da) comprising the sex pheromone blend of intact (SHAM) and castrated (GX) male red-sided garter snakes. Inset: Nonmetric multidimensional scaling (NMS) plot for total pheromone composition based on relative proportions of methyl ketones comprising pheromone blends. ...................................................................................... 161

6.4

Mass (µg; mean+SEM) of individual methyl ketones (grouped by molecular weight, Da) comprising the sex pheromone blend of castrated (GX) and aromatase inhibitor-implanted males (GX+ATD). Inset: NMS plot for total pheromone composition based on relative proportions of methyl ketones comprising pheromone blends. .................. 163

6.5

Mass (µg; mean+SEM) of individual methyl ketones (grouped by molecular weight, Da) comprising the sex pheromone blend of castrated (GX) and castrated males implanted with tetosterone (GX+T). Inset: NMS plot for total pheromone composition based on relative proportions of methyl ketones comprising pheromone blends. ...................................... 165

6.6

Mass (µg; mean+SEM) of individual methyl ketones (grouped by molecular weight, Da) comprising the sex pheromone blend of intact males (SHAM), castrated males implanted with testosterone (GX+T), and intact males implanted with testosterone (T). Inset: NMS plot for total pheromone composition based on relative proportions of methyl ketones comprising pheromone blends. ...................................................... 167

6.7

Ratio (mean+SEM) of unsaturated (U) to saturated (S) components in the pheromone blends of experimental male garter snakes. ....................... 168

6.8

Ratio (mean+SEM) of high molecular weight (HMW, >463 Da) to low molecular weight (LMW,