AN ABSTRACT OF THE THESIS OF Sarah A. Maxfield-Taylor for the degree of Master of Science in Entomology presented on March 26, 2014. Title: Natural Enemies of Native Bumble Bees (Hymenoptera: Apidae) in Western Oregon Abstract approved: _____________________________________________ Sujaya U. Rao Bumble bees (Hymenoptera: Apidae) are important native pollinators in wild and agricultural systems, and are one of the few groups of native bees commercially bred for use in the pollination of a range of crops. In recent years, declines in bumble bees have been reported globally. One factor implicated in these declines, believed to affect bumble bee colonies in the wild and during rearing, is natural enemies. A diversity of fungi, protozoa, nematodes, and parasitoids has been reported to affect bumble bees, to varying extents, in different parts of the world. In contrast to reports of decline elsewhere, bumble bees have been thriving in Oregon on the West Coast of the U.S.A.. In particular, the agriculturally rich Willamette Valley in the western part of the state appears to be fostering several species. Little is known, however, about the natural enemies of bumble bees in this region. The objectives of this thesis were to: (1) identify pathogens and parasites in (a) bumble bees from the wild, and (b) bumble bees reared in captivity and (2) examine the effects of disease on bee hosts. Bumble bee queens and workers were collected from diverse locations in the Willamette Valley, in spring and summer. Bombus mixtus, Bombus nevadensis, and Bombus vosnesenskii collected from the wild were dissected and examined for pathogens and parasites, and these organisms were identified using morphological and molecular characteristics. Queens of Bombus griseocollis, Bombus nevadensis, and Bombus vosnesenskii were reared in captivity, and those that died or did not initiate nests were also examined for pathogens and parasites. In addition, the immune responses of healthy and infected bees were compared, to examine the effects of a common bumble bee gut pathogen Crithidia bombi (Kinetoplastida: Trypanosomatidae) on its host. This thesis reports that wild bumble bees in western Oregon are infected with the protozoa Apicystis bombi (Neogregarinida: Ophrocystidae) and Crithidia bombi, the fungus/microsporidian Nosema bombi (Microsporidia: Nosematidae), the nematode Sphaerularia bombi (Nematoda: Tylenchida), and two dipteran parasitoids, one unknown and one belonging to the family Conopidae (Diptera: Conopidae). In addition, this research presents the first ever report of infection by the larval pathogen Ascosphaera apis (Ascomycota: Ascosphaerales), in reared bumble bee adults. New host and distribution records are also presented for C. bombi, N. bombi, and S. bombi. The study found that bumble bees infected with C. bombi had lower activated levels of immune responses than healthy bees. The thesis discusses the long-term implications of study findings, and addresses the threat that bumble bee diseases pose to bee pollinators through shared environments. © Copyright by Sarah A. Maxfield-Taylor March 26, 2014 All Rights Reserved Natural Enemies of Native Bumble Bees (Hymenoptera: Apidae) in Western Oregon by Sarah A. Maxfield-Taylor A THESIS Submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented March 26, 2014 Commencement June 2014 Master of Science thesis of Sarah A. Maxfield-Taylor presented on March 26, 2014. APPROVED: Major Professor, Representing Entomology Director of the Entomology Graduate Program Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Sarah A. Maxfield-Taylor, Author ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to Dr. Sujaya Rao. She has given my so many wonderful opportunities, and I credit her with being the person who first exposed me to the study of Entomology. I would also like to thank my other graduate committee members, Dr. Bill Stephen and Dr. Ramesh Sagili, for your time and involvement in my research thesis and for your many wonderful ideas. Thank you to Kim Skyrm, Jennifer Bergh, Melissa Broussard, Joe Kaser, and Julie Kirby for your lively discussions and support. I would also like to thank all the OSU faculty that have helped me with my research. You are too many to name and my education would not have been the same without you. Finally, I would like to thank my family. If not for them I never would have finished my studies. In particular I would like to thank my children, Felix and Agate Taylor, for giving me the strength to accomplish my goals, even during times of great stress. CONTRIBUTION OF THE AUTHORS Dr. Sujaya Rao assisted with study design, writing and manuscript preparation of Chapters 3 and 4. Alija Mujic assisted with the writing and manuscript preparation for Chapter 3. Kimberly Skyrm assisted with the writing and manuscript preparation of Chapter 4. TABLE OF CONTENTS Page Chapter 1: Introduction.................................................................................................. 1 Chapter 2: Diversity and Abundance of Pathogens and Parasites of Native Bumble Bees in Western Oregon..................................................................................... 9 Introduction........................................................................................................ 9 Methods............................................................................................................. 17 Bee Collection....................................................................................... 17 Bees from the Wild......................................................................... 17 Reared Bees.................................................................................... 17 Examination of Bees for Pathogens and Parasites................................ 18 Fungal Isolation.....................................…........................................... 18 Pathogen and Parasite Identification.................................................... 19 Morphological................................................................................ 19 Apicystis bombi….................................................................,.... 19 Crithidia bombi.......................................................................... 19 Nosema bombi…......................................................................... 20 Sphaerularia bombi.................................................................... 20 Parasitoids….............................................................................. 20 Filamentous Fungi….................................................................. 20 Molecular........................................................................................ 20 Apicystis bombi…...................................................................... 21 Crithidia bombi.......................................................................... 21 Nosema bombi…........................................................................ 22 Sphaerularia bombi.................................................................... 22 Parasitoids….............................................................................. 23 Filamentous Fungi….................................................................. 23 Immunocompetence Assay.............................................................. 25 Hemolymph Collection.............................................................. 25 TABLE OF CONTENTS (Continued) Page Estimation of Protein.................................................................. 25 Phenoloxidase Activity Assay.................................................... 25 Prophenoloxidase Activity Assay............................................... 26 Results............................................................................................................... 27 Molecular Confirmation........................................................................ 28 Immune Response to C. bombi............................................................. 29 Discussion......................................................................................................... 29 Immune Response to C. bombi............................................................. 37 Conclusion............................................................................................ 38 Acknowledgements......…................................................................................. 40 References......................................................................................................... 41 Chapter 3: Novel infection of the larval pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in bumble bee adults.................................. 59 Abstract............................................................................................................. 59 Introduction....................................................................................................... 60 Methods............................................................................................................. 62 Fungal Isolation..................................................................................... 63 Morphological Identification................................................................