The Pennsylvania State University The Graduate School College of Agricultural Sciences INVESTIGATIONS OF SMALL HIVE BEETLE-YEAST ASSOCIATIONS A Dissertation in Entomology by Tracy M. Conklin 2012 Tracy M. Conklin Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2012 The dissertation of Tracy Conklin was reviewed and approved* by the following: James H. Tumlinson Ralph O. Mumma Professor of Entomology Dissertation Advisor Chair of Committee Christina Grozinger Associate Professor of Entomology John Tooker Assistant Professor of Entomology David Geiser Professor of Plant Pathology Gary W. Felton Professor of Entomology Head of the Department of Entomology *Signatures are on file in the Graduate School iii ABSTRACT The small hive beetle is an invasive pest of honey bees from Africa which has recently spread to the US and Australia. While not a serious pest of honeybees in its native range, the small hive beetle is a major problem for beekeepers its invasive range, frequently causing hives to abscond. Recently, a yeast (Kodamaea ohmeri) has been found associated with the small hive beetle. When growing on pollen, this yeast produces an odor blend that is very attractive to the beetle. It has been hypothesized that the presence of K. ohmeri may be a key factor in severity of small hive beetle infestations. This dissertation explores the connection between the small hive beetle and its yeast in three areas. The first area is the growth of K. ohmeri in bee hives. It has been hypothesized that the presence of K. ohmeri in the bee hive causes attraction and aggregation of the small hive beetle. However, no evidence has been provided that K. ohmeri is capable of multiplying in the hive environment when small hive beetle infestations are low. This dissertation, then, examines the factors that affect growth of K. ohmeri in the bee hive, including water activity, nutrient availability, the presence of beetles, and the presence of bees. This dissertation documents K. ohmeri is capable of growing on bee bread from bee hives without beetles present, and that water activity is important to the growth of K. ohmeri on bee bread and pollen. Beetle frass, which contains a high quantity of K. ohmeri, may be the source of yeast inoculum. The second area of research concerns the specificity of the interaction between K. ohmeri and the small hive beetle. Several species of yeast occur naturally in bee hives or in beekeeper-added pollen supplements. This dissertation reports that these yeasts, as well as other yeasts associated with beetles and other insects are capable of producing beetle-attractive volatiles and attracting small hive beetle adults, implying that the small hive beetle’s relationship with K. ohmeri is not an exclusive one, and the small hive beetle may be attracted to naturally occurring yeasts growing on pollen in the bee hive. Finally, the third area of research concerns the effect of K. ohmeri on the oviposition of the small hive beetle. Though K. ohmeri has been shown iv to increase reproduction of small hive beetles on pollen or oranges, the mechanism for this increased growth is unknown. This dissertation finds that pollen fermented by K. ohmeri is a preferred oviposition substrate for small hive beetle females, though fermentation by Saccharomyces cerevisiae or K. ohmeri did not increase the rate of ovary development over that of control pollen. Preference was most likely mediated by gustatory or olfactory cues in the pollen. Beetles switched ovipositional preference to control pollen after 24 hour hours of oviposition. This switch in preference appeared to be caused by changes in the K. ohmeri- fermented substrate, since adding fresh K. ohmeri-fermented pollen after 24 hours restored preference. The presence of yeast in the hive may influence small hive beetle host selection and beetle reproduction in bee hives. In addition, this dissertation provides initial data on the mating biology of the small hive beetle, including first descriptions of mating behavior and reproductive structures. Initial data gathered in this study indicates that small hive beetles may require an extended mating period in order to be fertile, providing an opportunity for control by mating disruption strategies. v TABLE OF CONTENTS ABSTRACT ............................................................................................................................. iii LIST OF FIGURES ................................................................................................................. viii LIST OF TABLES ................................................................................................................... x ACKNOWLEDGEMENTS ..................................................................................................... xi Chapter 1 Introduction and Literature Review ........................................................................ 1 Abstract ............................................................................................................................ 1 Introduction ...................................................................................................................... 2 The small hive beetle, not the large hive beetle: Identification and early description ..... 4 Pest status ......................................................................................................................... 5 Current control methods for small hive beetle ................................................................. 6 Sanitation .................................................................................................................. 6 Genetic resistance ..................................................................................................... 7 Chemical control methods ........................................................................................ 7 Attract-and-kill trapping ........................................................................................... 9 Alternative hive entrances ........................................................................................ 9 Life cycle of the small hive beetle ................................................................................... 10 Eggs and larvae ........................................................................................................ 10 Wandering stage and pupation ................................................................................. 11 Adults ....................................................................................................................... 12 Mating and oviposition............................................................................................. 12 Pest adaptations ................................................................................................................ 13 Anatomy of the small hive beetle ............................................................................. 13 Host manipulation .................................................................................................... 14 Diet flexibility .......................................................................................................... 15 Overwintering behavior ............................................................................................ 16 Invasive species ........................................................................................................ 17 European honey bees versus African honey bees .................................................... 18 Association with Kodamaea ohmeri as another possible pest adaptation ........................ 21 Small hive beetle and K. ohmeri .............................................................................. 21 Fungal associations among the Nitidulidae .............................................................. 24 Small hive beetle and K. ohmeri, directions for research ......................................... 29 Conclusions ...................................................................................................................... 32 Chapter 2 Factors affecting growth of Kodamaea ohmeri associated with the small hive beetle (Aethina tumida) in western honey bee (Apis mellifera) hives .............................. 33 Abstract ............................................................................................................................ 33 Introduction ...................................................................................................................... 34 Methods ............................................................................................................................ 37 Water activity tests ................................................................................................... 37 vi Growth of K. ohmeri on bee bread ........................................................................... 38 Yeast quantification on beetles ................................................................................ 40 Field experiments ..................................................................................................... 41 Pennsylvania bee hives and treatments .................................................................... 42 Pennsylvania data collected ..................................................................................... 42 Kenya bee hives and treatments ..............................................................................