University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Spring 2016 Cuticular hydrocarbons of the small carpenter bee Ceratina calcarata Robertson (Hymenoptera: Apidae: Xylocopinae) Nicholas James Pizzi University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Pizzi, Nicholas James, "Cuticular hydrocarbons of the small carpenter bee Ceratina calcarata Robertson (Hymenoptera: Apidae: Xylocopinae)" (2016). Master's Theses and Capstones. 1080. https://scholars.unh.edu/thesis/1080 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. CUTICULAR HYDROCARBONS OF THE SMALL CARPENTER BEE Ceratina calcarata ROBERTSON (HYMENOPTERA: APIDAE: XYLOCOPINAE) BY NICHOLAS JAMES PIZZI BS Zoology, University of New Hampshire, 2014 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science in Zoology May, 2016 ii This thesis/dissertation has been examined and approved in partial fulfillment of the requirements for the degree of Masters of Science in Zoology by: Sandra M. Rehan, Assistant Professor of Biology Larry Harris, Professor of Zoology Winsor Watson, Professor of Zoology On April 19, 2016 Original approval signatures are on file with the University of New Hampshire Graduate School. iii ACKNOWLEDGEMENTS I would like to thank my adviser Sandra Rehan for her guidance throughout my graduate school career. It was a pleasure working under your mentorship and I am truly grateful for this experience. Thanks for always challenging me and teaching me more than I could have ever imagined. I would also like to thank all members of the UNH Bee Lab, past and present. Thank you Wyatt Shell, Jake Withee, Sarah Lawson, Erika Tucker, Michael Steffen, and Selena Helmreich. Special thanks to Sean Lombard, for without his assistance the completion of my second data chapter would not have been possible. Thank you Richard Kondrat and Ron New for assistance with GC/MS analyses, and the University of New Hampshire, New Hampshire Agricultural Experiment Station and the Tuttle Foundation provided support for this research. Thank you to my graduate committee members, Win Watson and Larry Harris. Your suggestions and feedback were most useful and greatly appreciated. Finally, I would like to thank my family, especially my parents Paul and Lisa Pizzi, for always being there for me night and day. Thank you for your unconditional emotional and financial support. iv ABSTRACT Cuticular hydrocarbons of the small carpenter bee Ceratina calcarata Robertson (Hymenoptera: Apidae: Xylocopinae) by Nicholas J. Pizzi University of New Hampshire, May, 2016 The formation and maintenance of eusocial insect groups, in which there are overlapping generations, cooperative brood care, and reproductive division of labor is a major evolutionary transition. To understand the origins of eusociality, simple societies must be studied. Subsociality is the simplest form of social behavior and is defined as prolonged maternal care for offspring. Studies with subsocial species can provide powerful insights into the transition from basic to advanced social behaviors. In this thesis I use the subsocial small carpenter bee Ceratina calcarata Roberton (Hymenoptera: Xylocopinae) as a model organism. Specifically I study cuticular hydrocarbons (CHCs) of this species to examine two important evolutionary questions. CHCs are long chain hydrocarbons present on the cuticle of insects and are used for signaling and communication purposes. In eusocial insects queen pheromones are CHCs that signal fertility and suppress worker reproduction. It is hypothesized that CHCs were first fertility signals in less social forms and subsequently coopted as queen pheromones in eusocial lineages. I test this hypothesis and show supportive evidence for it, as my results suggest that C. calcarata, a subsocial species, may use CHCs to signal fertility. Second, the use of CHCs to recognize non- nestmates is essential to the fitness of eusocial colonies. My results suggest that C. calcarata v may possibly use CHCs as recognition cues, indicating that the use of CHCs for recognition of conspecifics is not a derived trait unique to eusocial lineages, but was probably conserved and present in less social forms. Therefore, this thesis contributes to our understanding of the factors favoring the formation and evolution of eusociality. I show that fertility signals, and the use of CHCs for non-nestmate recognition are likely to be conserved traits. TABLE OF CONTENTS ACKNOWLEDGEMENTS .................................................................................................... iii ABSTRACT ............................................................................................................................ iv Chapter 1: General Introduction ............................................................................................... 1 Part 1: Social Evolution in Bees .......................................................................................... 1 Part 2: Behaviors of Bees .................................................................................................... 6 Part 3: Cuticular Hydrocarbons and Chemical Communication ........................................ 8 Part 4: Thesis Research Aims ............................................................................................ 11 Chapter 2: Characterization of cuticular hydrocarbons in a subsocial bee, Ceratina calcarata Robertson (HYMENOPTERA: APIDAE) ............................................................................. 13 INTRODUCTION .............................................................................................................. 13 METHODS ........................................................................................................................ 15 Nest Collections and Dissections ................................................................................... 15 Morphological Measurements ....................................................................................... 17 Cuticular Hydrocarbon Characterization ...................................................................... 17 Comparative Cuticular Hydrocarbon Analyses ............................................................. 18 Statistical Analyses ......................................................................................................... 18 RESULTS .......................................................................................................................... 19 Cuticular Hydrocarbon Profiles .................................................................................... 19 Morphological Measurements ....................................................................................... 20 Comparative Cuticular Hydrocarbon Profiles .............................................................. 20 DISCUSSION .................................................................................................................... 21 Reproductive Signaling and Ovarian Development ....................................................... 22 CHCs vary as a function of age ..................................................................................... 23 Comparative Analysis of Insect Cuticular Hydrocarbons ............................................. 25 Future Directions ........................................................................................................... 25 TABLES AND FIGURES ................................................................................................. 27 Chapter 3: Cuticular hydrocarbons serve as recognition signals in a subsocial bee, Ceratina calcarata Robertson (HYMENOPTERA: XYLOCOPINAE) ............................................... 33 INTRODUCTION .............................................................................................................. 33 METHODS ........................................................................................................................ 37 Bee Collections ............................................................................................................... 37 Nest Dissections and Behavioral Assay Preparations ................................................... 38 Preparation for Behavioral Assays ................................................................................ 38 Circle Tube Behavioral Assays ...................................................................................... 39 Morphological and Physiological Measurements ......................................................... 40 Statistical Analyses ......................................................................................................... 40 RESULTS .......................................................................................................................... 42 Circle Tube Assays ......................................................................................................... 42 Morphological and Physiological Measurements ......................................................... 43 DISCUSSION