The Pennsylvania State University The Graduate School College of Agricultural Sciences ECOPHYSIOLOGY OF OVERWINTERING IN HONEY BEES (APIS MELLIFERA, L.) A Dissertation in Entomology by Mehmet Ali Döke 2017 Mehmet Ali Döke Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2017 The dissertation of Mehmet Ali Döke was reviewed and approved* by the following: Christina Grozinger Distinguished Professor of Entomology Dissertation Advisor Chair of Committee Diana Cox-Foster Professor of Entomology Tom Baker Distinguished Professor of Entomology and Chemical Ecology Rudolf Schilder Assistant Professor of Entomology and Biology Naomi Altman Professor of Statistics Gary Felton Professor of Entomology Head of the Department of Entomology *Signatures are on file in the Graduate School ii Abstract Winter is a great challenge for all animals living in the temperate climates. Among insects, honey bees are unique for their overwintering behavior. While virtually all other insect species go into diapause (a dormant state), honey bee colonies comes to a reproductive arrest but existing worker bees form a thermoregulatory cluster to generate and maintain heat against the ambient temperature (Döke, Frazier, & Grozinger, 2015). During this process, the colony solely depends on food they have collected through the previous spring and summer and stored in the form of honey for energy (Owens, 1971). Moreover, the workers in fall must survive until the end of winter since there simply cannot be worker turn-over during this broodless period (Mattila, Harris, & Otis, 2001). Thus, in fall, workers with a distinct physiological state and increased longevity are produced in the colony, replacing the short-lived summer bees (Fluri, Lüscher, Wille, & Gerig, 1982; Z. Y. Huang & Robinson, 1995). Populations of honey bee colonies maintained in the same area for several generations can adapt to their environment and moving colonies between climatic regions can lower their overwintering survival, suggesting existence of selectable, genetic components for overwintering success (Büchler et al., 2014; Hatjina et al., 2014; Parker et al., 2010). On the other hand, all of the individuals throughout the year are half-sisters – daughters of a multiply mated queen – making them genetically similar. Yet worker bees of summer and winter are radically different in their physiology and behavior suggesting existence of differentially expressed genes that modify the individual phenotype according to environmental cues. The research presented in this dissertation establishes a solid foundation for the genetic, transcriptomic, physiological, and social components of honey bee overwintering and its regulation. Chapter 1 reviews the current state of research on honey bee overwintering and best practices to maximize survival rate of managed honey bee colonies. Additionally, using the literature, a testable hypothetical model for the environmental cues acting on honey bee overwintering is offered. We believe this model will be a useful framework for researchers in testing the effect of iii individual environmental parameters on honey bee overwintering, and indeed we initiate testing of this model in Chapter 5. Chapter 2 examines the molecular mechanisms underlying the unique “winter bee” physiological state. We evaluate expression patterns of key metabolic and antioxidant genes in the abdominal fat body tissues and thoracic flight muscles of nurses, foragers, and winter bees collected from the same colonies throughout the year. When the three groups are compared using gene expression patterns in the abdominal fat body tissues, winter bees resemble nurses. However, when the gene expression in thoracic flight muscle is compared, winter bees resemble foragers. These findings suggest two distinct genetic toolkits working in tandem in different tissues to mix- and-match expression profiles of summer workers tending different tasks to generate a novel phenotype; the winter bee. Chapter 3 explores the interactions between host physiological state and parasites, including the unique phenotype of the winter bee. Nosema is a microsporidian parasite infecting honey bees, which has been shown to alter the physiology of their hosts and increase behavioral maturation rates from nursing the foraging. However, these studies have been largely conducted by infecting one day old bees, and thus the impacts of Nosema on nurses, foragers, and winter bees have not been evaluated. Findings from this chapter show the effect of infection on longevity is greatly affected by host physiology. Moreover, previously identified “Nosema responsive” genes in honey bees (whose expression was altered by Nosema infection of young, cage-reared bees) were here shown to be primarily affected by the physiological state of the host rather than the infection state, suggesting that previous transcriptional studies were capturing the impact of Nosema on host maturation rather than a direct host immune response. Overall, these findings demonstrate the need for contextual experiments for realistic assessment of the effects infectious diseases have on their hosts. Chapter 4 compares the overwintering success colonies from southern (TX & FL) and northern (WV & VT) United States when placed in the same apiaries in temperate central Pennsylvania to examine the effect of genotype on winter survival and investigate whether northern stocks are locally adapted to colder and longer winters. Findings from this chapter suggest that while there iv are small – but detectable – genetic differences between populations of honey bees reared in the northern and southern United State, these differences are not associated with variation in overwintering survival. Instead, overwintering survival is greatly dependent on the size of the colony and can vary dramatically with the specific location – and its associated floral resources and climatic conditions - in which the colony is placed. Chapter 5 investigates the theory that landscape floral nutritional resources and climatic conditions influence social interactions and demographic structure in the colony, which then in turn mediates the production of winter bees and prepares the colony for winter. As was proposed in Chapter 1, two worker-produced pheromones (brood pheromone and forager pheromone) can act antagonistically, creating a push and pull model for the initiation and maintenance of winter phenotype. Testing this hypothesis, we did not find evidence to support a direct effect of the pheromones on colony vigor or survival. However, this study was limited by the fact that we experimented solely in the field where multiple confounding factors likely acted on the colonies. Lastly, Chapter 6 summarizes the findings from this dissertation, integrates results with current scientific literature, and discusses the future of research on honey bee overwintering. v Table of Contents List of Figures .................................................................................................................................. ix List of Tables .................................................................................................................................... x Acknowledgements ......................................................................................................................... xi Chapter 1 Overwintering honey bees: biology and management ................................................. 1 Chapter 2 Adapting to winter conditions: testing a 'mix-and-match' model of gene regulation 2 Abstract ....................................................................................................................................... 2 Introduction................................................................................................................................. 3 Methods ...................................................................................................................................... 5 Sample Collection. ................................................................................................................... 5 Dissection and Homogenization. ............................................................................................. 6 Gene Expression Analysis. ....................................................................................................... 7 Results ......................................................................................................................................... 8 Discussion .................................................................................................................................. 11 Chapter 3 Examining the interplay between host physiological state and parasite infection in honey bees (Apis mellifera, L.) ...................................................................................................... 14 Abstract ..................................................................................................................................... 14 Introduction............................................................................................................................... 15 Methods .................................................................................................................................... 18 Maintenance and selection of colonies. ................................................................................ 18 Generating Nosema spores for inoculations. .......................................................................