Investigating the Integration of Small Hive Beetles (Aethina Tumida Murray, Coleoptera

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Investigating the Integration of Small Hive Beetles (Aethina Tumida Murray, Coleoptera Nitidulidae) into Western Honey Bee (Apis Mellifera L., Hymenoptera: Apidae) Colonies
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english
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Atkinson,Edward Blake
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University of Florida
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Gainesville, Fla.
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Entomology and Nematology
Committee Chair:
Ellis, James D.
Committee Members:
Teal, Peter E
McAuslane, Heather J
Brockmann, H. Jane Jane
Cline, Andrew

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Subjects / Keywords:
apis -- bee -- beetle -- behavior -- hive -- honey -- inquiline -- inquilinism -- insect -- melittophile -- mellifera -- nitidulidae -- olfactometer -- pest -- small -- social
Entomology and Nematology -- Dissertations, Academic -- UF
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Entomology and Nematology thesis, Ph.D.
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theses   ( marcgt )
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Abstract:
The small hive beetle (Aethina tumida Murray; Coleoptera: Nitidulidae) is native to sub-Saharan Africa, where it is considered an occasional nuisance in honey bee (Apis mellifera L.; Hymenoptera: Apidae) colonies. However, the species is considered a significant pest of honey bees in its introduced range of North America and Australia, where the beetle has been established since 1996 and 2002, respectively. The small hive beetle damages colonies through feeding and reproductive behaviors, and can cause absconding or complete colony collapse. Small hive beetles integrate into honey bee colonies via several adaptations, including: retraction of appendages beneath the body when encountering defensive honey bees, finding hiding areas (confinement sites) within the bee nest that are inaccessible to honey bees, and coercing host honey bees to feed them while confined. Other nitidulids have been found in honey bee colonies and they appear to have lower degrees of integration into honey bee nests than do small hive beetles. A series of experiments was conducted to investigate potential morphological (Chapter 3), behavioral (Chapters 2, 4-7), and chemical (Chapter 5) adaptations that enable small hive beetles to integrate successfully into honey bee nests. The results of the research suggest that small hive beetles are attracted to odors present in honey bee colonies (Chapter 2). Also, they possess leg modifications that allow them to retract their appendages beneath their bodies more fully (Chapter 3), thus resisting attack from honey bee hosts who treat them more defensively than they treat other beetles at the nest entrance (Chapter 4). Furthermore, small hive beetles have an altered chemical profile that is dependent upon their post-eclosion diet (Chapter 5), though the significance of the altered profile is unclear. Finally, small hive beetles are unique among other beetle species in their ability to find hiding places within the colony where they are confined by honey bee hosts (Chapter 6) until the ambient temperature decreases, whereafter the beetles enter the thermoregulatory cluster of honey bees (Chapter 7). The research presented herein contributes to a greater understanding of attributes of small hive beetles that enable them to integrate successfully into honey bee nests.
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In the series University of Florida Digital Collections.
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by Edward Blake Atkinson.
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Thesis (Ph.D.)--University of Florida, 2011.
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Adviser: Ellis, James D.
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1 INVESTIGATING THE INTEGRATION OF SMALL HIVE BEETLES ( AETHINA TUMIDA MURRAY, COLEOPTERA : NITIDULIDAE) INTO WESTERN HONEY BEE ( APIS MELLIFERA L., HYMENOPTERA: APIDAE) COLONIES By EDWARD BLAKE ATKINSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011

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2 2011 Edward Blake Atkinson

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3 To my wife and kids

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4 ACKNOWLEDGEMENTS I thank my advisor Dr. Jamie Ellis and other members of my committee, Drs. H. Jane Brockmann, Andy Cline, Heather McAuslane, and Peter Teal, for guidance and assistance with what was a wonderful introduction to my life of entomological research. Also, I th ank the University of Florida for funding me through my doctoral career. I acknowledge the following members of the University of Florida Honey Bee Research and Extension Laboratory who provided technical assistance with my projects: Katie Buckley, Meredit h Cenzer, Renee Cole, Jonnie Dietz, Mark Dykes, Katy Evans, Dr. Kamran Fakhimzadeh, Jason Graham, Dr. Alec Gregorc, Pablo Herrera, Michelle Kelley, Ben King, Jeanette Klopchin, Hannah OMalley, Mike OMalley, Catherine Zettel Nalen, Dr. Akers Pence, Andy S heffler, Marissa Streifel, Cindy Tannahill, Melissa Teems, Liana Teig en, Tricia Toth, Anthony Vaudo, Sparky Vilsaint, and Larry Wise. I am grateful to Jane Medley of the University of Florida Entomology and Nematology department for creating the wonderful diagrams used in this dissertation, Drs. Mark Carroll, Nicole Benda, Adrian Duehl and Richard Arbogast of the USDA ARS Center for Medical and Veterinary Entomology (CMAVE) for offering their scientific expertise during the experimental process, and Tredin a Davis, Dr. Matt Lehnert, and Curtis Murphy of CMAVE for providing beetles when I was in need. Also, I thank Alex Bolques of the Gad sden County Extension Office, Dr. Keith Delaplane and Amanda Tedrow of the University of Georgia, Jim McBride of Jims Farm, Jim Boyer and Buck Nelson of the University of Florida Plant Research and Education Unit, and Kyle Straughn of Straughn Farms for aiding me in my search for beetle species. Jerry Hayes of the Florida Department of Agriculture and Consumer Services, D ivision of Plant Industry (DPI) also was very accommodating when I needed more bee colonies, and Drs. Paul Skelley and Mike Thomas of DPI never hesitated to offer me help in identifying beetles and using the scanning electron microscope. Finally, I thank m y parents, sister, nephew,

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5 and my in laws for being excited for me, my wife for being so very kind to and supportive of me, our babies for making me the happiest daddy around, and my grandfather in law, Dr. Robert Woodruff, for introducing me to the field of Entomology.

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6 TABLE OF CONTENTS page ACKNOWLEDGEMENTS ......................................................................................................... 4 LIST OF TABLES ...................................................................................................................... 9 LIST OF FIGURES .................................................................................................................. 10 ABSTRACT ............................................................................................................................. 11 CHAPTER 1 INTRODUCTION ............................................................................................................. 13 Methods Used by Symbionts to Integrate into NonApis mellifera Social Bee Nests ........... 14 Morphological ............................................................................................................. 14 Passive defense .................................................................................................... 15 Phoresy ................................................................................................................ 16 Other .................................................................................................................... 16 Behavioral ................................................................................................................... 17 Phoresy ................................................................................................................ 17 Feeding/reproduction ............................................................................................ 18 Chemical ..................................................................................................................... 20 Methods Used by Symbionts to Integrate into Apis mellifera Nests .................................... 20 Morphological ............................................................................................................. 21 Passive defense .................................................................................................... 21 Phoresy ................................................................................................................ 21 Other .................................................................................................................... 22 Behavioral ................................................................................................................... 22 Phoresy ................................................................................................................ 22 Feeding/ reproduction ............................................................................................ 22 Other .................................................................................................................... 24 Chemical ..................................................................................................................... 25 Methods Used by SHBs to Integrate into Apis mellifera Nests ............................................ 25 Morphological ............................................................................................................. 26 Behavioral ................................................................................................................... 26 Chemical ..................................................................................................................... 27 Introduction to the Research Presented in this Dissertation ................................................. 28 2 ATTRACTION OF MULTIPLE BEETLE SPECIES TO HONEY BEE HIVE ODORS .... 47 Materials and Methods ....................................................................................................... 49 Beetles ........................................................................................................................ 49 Honey Bees ................................................................................................................. 50 Olfactometer ............................................................................................................... 50

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7 Choice Bioassay .......................................................................................................... 51 Statistical Analysis ...................................................................................................... 51 Results ............................................................................................................................... 52 Discussion .......................................................................................................................... 53 3 SHB ADAPTIVE LEG MORPHOLOGY .......................................................................... 58 Materials and Methods ....................................................................................................... 61 Results ............................................................................................................................... 61 Discussion .......................................................................................................................... 62 4 ADAPTIVE BEHAVIOR OF HONEY BEES TOWARD BEETLE INVADERS EXHIBITING VARIOUS LEVELS OF COLONY INTEGRATION ................................. 68 Materials and Methods ....................................................................................................... 70 Beetles ........................................................................................................................ 70 Observation Hives ....................................................................................................... 70 Behavioral Assays ....................................................................................................... 71 Statistical An alysis ...................................................................................................... 72 Results ............................................................................................................................... 72 Discussion .......................................................................................................................... 74 5 DIETARY EFFECTS ON THE CUTICULAR PROFILE OF SHBS AND ITS EFFECT ON BEHAVIORAL TREATMENT BY HONEY BEES .................................................... 82 Materials and Methods ....................................................................................................... 84 Beetles ........................................................................................................................ 84 Observation Hives ....................................................................................................... 84 Behavioral Assays ....................................................................................................... 85 Chemical Analysis ...................................................................................................... 86 Statistical An alysis ...................................................................................................... 86 Results ............................................................................................................................... 87 Discussion .......................................................................................................................... 88 6 DISTRIBUTION OF MULTIPLE BEETLE SPECIES INTRODUCED INTO HONEY BEE COLONIES ............................................................................................................... 97 Materials and Methods ....................................................................................................... 98 Beetles ........................................................................................................................ 98 Observation Hives ....................................................................................................... 99 Experiment 1: Hiding Behavior of Multiple Beetle Species ....................................... 100 Experiment 2: SHB Hiding Behavior ......................................................................... 100 Statistical Analysis .................................................................................................... 101 Results ............................................................................................................................. 101 Experiment 1: Hiding Behavior of Multiple Beetle Species ....................................... 101 Experiment 2: SHB Hiding Behavior ......................................................................... 102 Discussion ........................................................................................................................ 102

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8 7 TEMPERATUREDEPENDENT CLUSTERING BEHAVIOR OF SHBS IN HONEY BEE COLONIES ............................................................................................................. 116 Materials and Methods ..................................................................................................... 117 Beetles ...................................................................................................................... 117 Observation Hives ..................................................................................................... 117 Behavioral Assays ..................................................................................................... 118 Statistical An alysis .................................................................................................... 119 Results ............................................................................................................................. 119 Discussion ........................................................................................................................ 120 8 DISCUSSION .................................................................................................................. 125 Re examination of the Low Number of Honey Bee Nest Symbionts ................................. 126 The Future of SHB Research ............................................................................................ 129 LIST OF REFERENCES ........................................................................................................ 133 BIOGRAPHIAL SKETCH ..................................................................................................... 153

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9 LIST OF TABLES Table page 11 Arthropods found in nests of nonApis mellifera social bees and their means of integrating into the nests. ............................................................................................... 31 12 Arthropods found in nests of Apis mellifera and their means of integrating into the nests. ............................................................................................................................. 41 21 Beetle attraction to hive odors ........................................................................................ 57 31 Presence/absence of hypothesized inquilinous morphological characters in selected nitidulid species. ............................................................................................................ 65 41 Proportion of honey bee guard responses that were ignore and contact during the observation period ......................................................................................................... 79 42 Species colony interaction on the proportion of guard bee responses that were defend during the observation period .......................................................................... 80 51 Diet colony interaction on the proportion of guard bee responses that were ignore during the observation period .......................................................................... 90 52 Diet colony interaction on the proportion of guard bee responses that were contact during the observation period ......................................................................... 91 53 Diet colony interaction on the proportion of guard bee responses that were defend during the observation period .......................................................................... 92 54 Amounts of various hydrocarbons present on the cuticles of newlyeclosed beetles, beetles that have fed for 14 days on a diet of honey, pollen, and Brood Builder, and beetles that have fed for 14 days on sugar water ...................................................... 93 61 Proportions of different beetle species found in confinement sites, the interior walls of the colony, or missing altogether at different time periods following their introduction into observation honey bee colonies ......................................................... 108 62 Proportions of A. tumida found in confinement sites, on the combs, on the interior walls, or missing altogether at multiple time periods after their introduction into observation honey bee colonies .................................................................................... 110 71 Proportion of beetles confined at each temperature in experimental colonies ............... 122 72 Proportion of beetles confined at each temperature in control colonies ......................... 122

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10 LIST OF FIGURES Figure page 31 SEM images of entire legs ............................................................................................. 66 32 SEM images of tarsi ...................................................................................................... 67 41 Diagram of observation hive .......................................................................................... 81 51 Diagram of observation hive .......................................................................................... 94 52 Chromatograms from cuticle of newly eclosed, 14 day old beetles fed a diet of honey, pollen, and BroodBuilder, and 14 day old beetles fed a diet of sugar and water ............................................................................................................................. 95 53 Relative amounts of various hydrocarbons in newlyeclosed, 14 day old beetles fed a diet of honey, pollen, and BroodBuilder, and 14 day old beetles fed a diet of sugar and water ....................................................................................................................... 96 61 Diagram of experimental observation hive ................................................................... 112 62 Percentage distribution of each beetle species found in confinement sites, on the interior walls of the hive, or missing at each time period. ............................................. 114 63 Percentage distribution of A. tumida in colonies where the confinement sites were unwashed and washed .................................................................................................. 115 71 Diagram of experimental observation hive ................................................................... 123 72 The proportion of beetles in confinement sites as the ambient temperature decreased .. 124 73 The proportion of beetles in confinement sites as the ambient temperature increased ... 124

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11 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy I NVESTIGATING THE INTEGRATION OF SMALL HIVE BEETLES ( AETHINA TUMIDA MURRAY, COLEOPTERA : NITIDULIDAE) INTO WESTERN HONEY BEE ( APIS MELLIFERA L., HYMENOPTERA: APIDAE) COLONIES By Edward Blake Atkinson August 2011 Chair: James D. Ellis Major: Entomology and Nematology The small hive beetle ( Aethina tumida Murray; Coleoptera: Nitidulidae) is n ative to sub Saharan Africa, where it is considered an occasional nuisance in honey bee ( Apis mellifera L.; Hymenoptera: Apidae) colonies However, the species is consi dered a significant pest of honey bees in its introduced range of North America and Australia, where the beetle has been established since 1996 and 2002, respectively. The small hive beetle damages colonies through feeding and reproductive behaviors and c an cause absconding or complete colony collapse. Small hive beetles integrate into honey bee colonies via several adaptations, including: retraction of appendages beneath the body when encountering defensive honey bees, finding hiding areas (confinement si tes) within the bee nest that are inaccessible to honey bees, and coercing host honey bees to feed them while confined. Other nitidulid s have been found in honey bee colonies and they appear to have low er degrees of integration into honey bee nest s than do small hive beetle s A series of experiments was conducted to investigate potential morphological (Chapter 3), behavioral (Chapters 2, 47), and chemical (Chapter 5) adaptations that enable small hive be etles to integrate successfully into honey bee nests. The results of the research suggest that small hive

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12 beetles are attracted to odors present in honey bee colonies (Chapter 2). Also, they possess leg modifications that allow them to retract their appendages beneath their bodies more fully (Chapter 3), thu s resisting attack from honey bee hosts who treat them more defensively than they treat other beetles at the nest entrance (Chapter 4). Furthermore, small hive beetles have an altered chemical profile that is dependent upon their post eclosion diet (Chapter 5), though the significance of the altered profile is unclear. Finally, small hive beetles are unique among other beetle species in their ability to find hiding places within the colony where they are confined by honey bee hosts (Chapter 6) until the amb ient temperature decreases, whereafter the beetles enter the thermoregulatory cluster of honey bees (Chapter 7). The research presented herein contributes to a greater understanding of attributes of small hive beetle s that enable them to integrat e successfully into honey bee nests

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13 CHAPTER 1 INTRODUCTION Social insects typically host many colony co inhabitants, or symbionts, from a broad range of arthropod taxa. Using Wilsons definition (1971) a social insect symbiont is a species that has a clo se, dep endent relationship with a social insect species Social insect symbionts can be subdivided into three categories: parasites where one species benefits whi le the host suffers; commensals, in which one species benefits while the host is neither help ed nor harmed; and mutualists where both species benefit. The relationships between symbionts and their social insect hosts range from invaders merely using a hosts nest as temporary shelter to the invaders forming relationships with their hosts that may incur both immediate and evolutionary significance (Kistner 1979, 1982, Rosenheim 1990) A rthropod symbionts of termites and ants (termed termitophiles and myrmecophiles, respectively) are common wh ereas symbionts of bees (termed melittophiles) presumably are not (Wilson 1971, Kistner 1979) Several hypotheses have attempted to explain the relative scarcity of social bee symbionts two of which are discussed here. The first hypothesis relates to the qual ity of the hosts diet (Wilson 1971, Kistner 1979). Ma ny bees eat pollen and nectar derived foodstuffs that are concentrated, nutrient rich, and produce little debris. However ants and termites eat a variety of foodstuffs, such as insects and celluloserich plant material, which produce more refuse (Wilson 1 971) Therefore, bee colonies have less debris available in the colony, which min imizes potential food sources for symbionts while the opposite is true for colonies of termites and ants. Another explanation for the apparent lack of social bee symbionts is that social bee species tend to nest in trees, whereas most ants and termites nest in the ground. The increased abundance of grounddwelling arthropods compared to arboreal arthropods (see Andr et al. 1994, Osler and

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14 Beattie 2001) proportiona lly favor s symbiont development in ground nesting social insects. Consequently, arthropods presumably must be pre adapted to arboreal life to locate and thrive in social insect nests in trees. Thus, statistically one would expect fewer symbionts in social bee nests than ant or termite nests, which has been observed in nature (Wilson 1971). Despite these barriers, some symbionts have managed to establish themselves in social bee colonies. Sp ecific adaptations in morphology, behavior, and/or chemical use have been adopted by most or perhaps all, social insect symbionts (Wilson 1971, Kistner 1979). The purpose of this dissertation is to investigate the integration of the small hive beetle (SHB), Aethina tumida Murray (Coleoptera: Nitidulidae), into nests of th e western honey bee, Apis mellifera L. (Hymenoptera: Apidae). The following sections review arthropod integration into nonA. mellifera social bee nests (Table 1 1) and honey bee nests (Table 1 2) thereby providing the requisite background for understandi ng the relationship between SHBs and their honey bee hosts. This review is limited to arthropods which enter the nest of eusocial insect host s. It is not an exhaustive list of all melittophiles but, rather, is a representation of the various means of integ ration into social bee nests. Methods Used by Symbionts to Integrate into NonApis mellifera Social Bee Nests Morphological Symbiont morphology contributes significantly to successful integration into social bee nests. Morphological adaptations exhibit two main forms among social bee symbionts, which typically are linked to symbiont behavior. One form involves passive defense and includes adaptations that protect the symbiont from host attacks. The second form includes phoretic adaptations for using the host as a dispersal mechanism.

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15 Passive defense Several invader species utilize passive defense morphological adaptations to integrate into host bee nests. The springtail species Pseudocyphoderus melittophilous Mari Mutt (Collembola: Cyphoder idae) occurs in nests of the stingless bee Trigona testacea Klug (Hymenoptera: Apidae) (Mutt 1977) This species possesses a well sclerotized dorsal cuticle having heavy scales that are used for passive defense. Furthermore, they have an enlarged mesothora x with an anterior notch that protect s the back of the head from attack Also the meso and metathorax are expanded lateroventrally to protect the pleural body regions and legs, which can be pulled under the body due to their reduced size (Mutt 1977) Sco tocryptus species (Coleoptera: Leiodidae) are found frequently in nests of Melipona species (Salt 1929, Roubik and Wheeler 1982, Wheeler 1985, Davis and Gonzalez 2008) These beetles possess a strongly convex dorsum, which presumably makes them difficult t o grasp. Also, they are capable of fitting their head, antennae, and legs under their body (Salt 1929, Roubik and Wheeler 1982) The beetle Cleidostethus meliponae Arrow (Coleoptera: Corylophidae) is found with Melipona alinderi Alfken and has several mor phological adaptations for passive defense (Salt 1929) The pronotum and fused elytra, in dorsal view are flattened laterally thereby concealing the head and antennae. Also, the tibiae can be fold ed into the femora, which themselves can be situated into grooves on the venter T hese adaptations may allow the beetle to flatten to the substrate and avoid injury from its bee hosts (Salt 1929, Bowestead et al. 2001 ). The presence of these morphological adaptations among these melittophilic beetles led me to believe they are relevant in the SHB honey bee relationship.

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16 Phoresy Phoresy i.e. wherein one species is carried by another species (TorreBueno 1985), is a ubiquitous behavior used by many arthropods to gain entrance into bee nests. Mites (Acari) are among the most common phoretic invaders found in bee nests (Kistner 1982) In most mite species, the modified deut eronymph stage is adapted for phoresy. Adaptations include anal and ventral suckers for attachment, as in Kuzinia laevis (Dujardin) (Acari: A caridae) on Bombus species (Hymenoptera: Apidae) queens (Alford 1975, Schwarz and Huck 1997) and caudal suckers, as in Histiostoma halictonida Woodring (Acari: Anoetidae) on Halictus rubicundus (Christ) (Hymenoptera: Halictidae) pupae (Woodring 1973) Alte rnatively, t he leiodid genera Scotocryptus Scotocryptodes Parabystus and Synaristers have notched mandibles t o grasp the corbicular setae of their Melipona hosts, while retracting their appendages beneath the body leaving only the antennae expos ed (Roubik and Wheeler 1982, Bezerra et al. 2000) The triungulin first instar larvae of some ripiphorid beetles (Coleoptera: Ripiphoridae) are phoretic on sweat bees (Hymenoptera: Halictidae) visiting flowers (Falin 2002) These larvae attach to host wings via a terminal abdominal segment and tarsal pulvilli modified into suckers (Tomlin and Miller 1989 Cline and Huether 2011) Other Several other integrative morphological adaptations exist in melittophiles. For example, t he pseudoscorpion genera Dasychernes and Corosoma (Pseudoscorpiones: Chernetidae), which occur in Melipona nests, presumably disguise themselves with an abundance of vestitural setae (Salt 1929, Roubik 2006, Gonzalez et al. 2007) Similarly, larvae of Ecthrodape africana Burks (Hymenoptera: Perilampidae), which are parasites of Braunsapis pupae (H ymenoptera: Anthophoridae) closely resemble host larvae and are treated as host larvae as evidenced by

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17 adults frequently mov ing them about the nest with bee larvae. These larvae are covered with set ae which give them structural support, but also may provide tactile mimicry (Michener 1969) Behavioral The most prevalent adaptations for integrating into social bee nests are behavioral (Kistner 1982). Several behaviors enable symbionts to persist with s ocial bee hosts, in cluding adaptive feeding and reproduction The most common behavior, though, is phoresy, and this is largely performed by phoretic mites (Kistner 1982) Phoresy Successful phoresy involves morphological and behavioral traits working in t andem to enable the symbiont to use the host as a dispersal mechanism P horesy encompasses those behaviors the symbiont uses to rig the host for dispersal purposes, behaviors which are made possible by morphological adaptations. Scutacarus acarorum (Goeze) (Acari: Scutacaridae), which is phoretic on Bombus has been found attached to species of Parasistus (Acari: Parasitidae) that are subsequently attached to Bombus sp ecies (Richards and Richards 1976, Schwarz and Huck 1997) Similarly, Fuscuropoda marginata (Koch) (Acari: Uropodidae), which are phoretic on Bombus are also phoretic on Volucella bombylans (L.) (Diptera: S y rphidae), which inhabit Bombus colonies (Alford 1975) Cryptostigma (Hemiptera: Coccidae) are phoretic on stingless be es and secrete honeydew and wax within nests of Schwarzula in exchange for protection from natural enemies (Camargo and Pedro 2002, Roubik 2006) Antherophagus sp ecies (Coleoptera: Cryptophagidae) gain entrance to Bombus colonies by attaching to the legs a nd proboscis of foragers at flowers (Frison 1921, 1926, Wheeler 1928, Free and Butler 1959, Chavarria 1994, Gonzalez et al. 2004) Analogously Ripiphous smithi Linsey & McSwain (Coleoptera: Ripi p horidae) lay eggs in closed

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18 flowers. As the flowers open, th e eggs hatch, and triungulin 1st instar larvae attach to a visiting Lasioglossum and are taken to the nest where they parasitize the host larvae (Tomlin and Miller 1989, Majka et al. 2006) Feeding/reproduction Many species integrate into social bee coloni es via adaptations that allow them to feed and reproduce within nests. Some species are predatory/parasitic toward their host whi le others enjoy a commensalist or mutualist type relationship with a host. Predatory and parasitic taxa on host adults or brood are common For example, Locustacarus buchneri (Stammer) (Acari: Podapolipidae) reside within the tracheae of Bombus workers or queens and overwinte r in the tracheae of queens. This mite lays eggs about a week after emergence from hibernation. The resulti ng offspring ma te within the tracheae, and gravid females shift to other workers or larvae as the season progresses (Husband and Sinha 1970, Shykoff and SchmidHempel 1991, Otterstatter and Whidden 2004, Yoneda et al. 2008) Also, Varroa jacobsoni Oudemans V. underwoodi Delfinado Baker & Aggarwal (Acari: Varroidae), and Tropilaelaps clarae Delfinado Baker (Acari: Laelapidae) are parasitic on drone brood of Apis cerana F. (Eickwort 1997) Many mutillid wasp species are parasitic on halictid and apid bees ( Alford 1975). These wasps ei ther fight their way past guard bees at the entrance or dig a new nest entrance and lay eggs on the brood (Alford 1975, Roubik 1990, Brothers et al. 2000, Polidori et al. 2009) Cryptocerus elongata Mayr (Hymenoptera: Formicidae ) enter nests of Trigona mosquito Lutz (Hymenoptera: Apidae), kill the bees, consume the honey, a nd make a nest (Salt 1929) Brachycoma sarcophagina (Townsend), B. devia Falln (Diptera: Metopiidae), and Melittobia species (Hymenoptera: Eulophidae) quickly move into Bombus nests, oviposit (larviposit in the case of the Brachycoma species ) on or in larval cells, and the parasite subsequently consumes the

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19 developing larva (Frison 1926, Free and Butler 1959) Volucella sp ecies enter nests to oviposit in a similar fashion (Wheeler 1928) ; h owever, they are adapted to oviposit immediately if killed, and the resulting egg mass possesses a protective viscous cover that solidifies upon exposure to air (Free and Butler 1959) Achroia grisella (F.) and Galleria colonella Hbner (Lepidoptera: Pyralidae) feed on nest debris, but not brood, in Melipona spp. colonies (Hymenoptera: Apidae), where they can destroy weak nests (Salt 1929, Cepeda Aponte et al. 2002) Likewise, Ephestia khniella Zeller and Vitula edmandsii (Packard) (Lepidoptera: Pyralidae) deplete food stores of Bombus colonies (Frison 1926, Wheeler 1928, Alford 1975, Schmid Hempel 2001) Aphomia sociella (L.) (Lepidoptera: Pyralidae) consume food stores in addition to brood in Bombus spp. colonies (Frison 1926, Wheeler 1928) Many arthropod species that invade social bee nests are innocuous commensals being scavengers among the nest debris of their host species or using the nest as shelter. Tyrophagus m ites (Acari: Acaridae) feed on fungi in nests of stingless bees (Hymenoptera: Apidae) (Roubik 2006) Secondary invaders, which parasitize or predate upon primary invaders, include parasitic Apanteles species (Hymenoptera: Braconidae) and Stilpnus gagates (Gravenhorst) (Hymenoptera : Ichneumonidae), and the predacious mite genus Parasistus (Acari: Parasitidae) (Alford 1975) Springtails, such as Pseudosinella species (Collembola: Entomobryidae), feed on pollen and fecal material in nests of Lasioglossum zephyrum (Smith) and Augochlor a species (Hymenoptera: Halictidae) (Batra 1965, Eickwort and Eickwort 1972) Leafcutter ants, Acromyrmex octospinosus (Reich) (Hymenoptera: Formicidae), build fungus gardens within abandoned cells of active Bombus pullatus Franklin nests, do ing so without harming the hosts (Chavarria 1996)

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20 In some instances, inquilines activity benefit s the host creating a mutualistic relatio nship between the host and the invader For example, the feeding activity of mites (Acari) associated with Megalopta genalis MeadeWaldo and M. ecuadoria Friese (Hymenoptera: Halictidae) red uce harmful fungal loads on host brood (Biani et al. 2009) Also, the wasps Habrobacon juglandis (Ashmead) and Apanteles nephoptericis (Packard) (Hymenoptera: Braconidae) parasitize the potentially destructive moth Vitula edmandsii (Frison 1926) Chemical Chemical use is the most prevalent means of communication in insects and often is exploited by inquilines that integrate into social bee nests ( Wilson 1971). Nest or host semiochemicals may be produced by the in quiline de novo or are acquired from the colony itself. However, while chemical means of integration are common among myrmecophiles and termitophiles (e.g. Howard et al. 1980, Vauchot et al 1998, Lenoir et al. 2001 ), there are relatively few reported among melittophiles. Methods Used by Symbionts to Integrate into Apis mellifera N ests Apis mellifera is one of the most socially complex bee species as well as one of the most important pollin ators of agricultural crops, accounting for 8090% of all pollination, and having an estimated value of ~ $15 billion USD as of 2000 (Wilson 1971, Morse and Calderone 2000) Consequently, the study and control of its pests has been the focus of considerable research and attention (e.g Morse and Flottum 1997). Honey bees exhibit most of the traits hypothesized to be important for limiting symbiont invasion. Along with concentrated food sources and arboreal nests, honey bees exhibit a temporal division of lab or. At any given time, there are bees in the colony that perform different tasks for the colony, including defense (Breed et al. 2004) Thus, honey bee colonies are well defended at all times. Honey bee defense acts as an impediment to would be invaders, t h us

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21 minimizing invasion by opportunistic arthropods. De spite these adaptations, some arthropods succeed in infiltrating the nest. These invaders provide an opportunity to investigate how honey bees limit nest invasion, and the traits symbionts such as the SHB must possess to overcome honey bee defenses. Morphological behavior al and chemical adaptations all contribute to the success of arthropods at integrating into honey bee colonies (Table 1 2). Morphological Passive defense Passive defense mechanisms of nest invaders are achieved mainly through possession of a relatively thick cuticle For example, the cuticle of the deaths head hawkmoth Acherontia atropos enables them to deflect and subsequently survive po tentially lethal stings of honey bees (Moritz et al. 1991) Similarly, because of their thick cuicle, Euphoria sepulcralis (F.) (Coleoptera: Scarabaeidae) can enter honey bee hives unimpeded by their defensive stings (Woodruff 2006). Phoresy Afrocypholaelaps africana (Evans) (Acari: Ameroseidae) are phoretic mites that reside on flowers generally, but occasionally may be found in bee hives These mites possess sucker like ambulacral pads that lack claws and enable them to attach to honey bees at flowers (Seeman and Walter 1995) Another mite, Tropilaelaps clarae is devastating to honey bee colonies in southern and southeast Asia (Sammataro et al. 2000, Brown et al. 2002) This mite species has an elongate body that allows for movement between hairs of host bees, as well as large legs with claws for attaching to bees (Rath et al. 1991) Acarapis woodi (Rennie) (Acari: Tarsonemidae) is a mite that feeds and reproduces within the tracheae of honey bees. The females are phoretic for a brief time and have tarsal claws and specialized setae on the legs that enable attachment to bee hairs (Ochoa et al. 2005) Similarly,

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22 the destructive varroa mite, Varroa destructor Anderson & Trueman (Acari: Varroidae) has specialized structures on their legs called apoteles that adhere to hosts (Rose nkranz et al. 2010). The fly Braula coeca Nitzsch (Diptera: Braulidae) is phoretic on adult honey bees but rarely is problematic for host bees The adult is wingless and attaches to adult bees by means modified comb like pretarsal claws (Sammataro 1997) Other Acarapis woodi larvae possess enlarged pul villar pads, rendering their claws useless, but enabling them to move freely around the tracheae. Likewise both the body and leg setation are oriented distally which allows the mite to measure the radius of and navigate the tracheae (Ochoa et al. 2005) Furthermore, Acarapis mites collectively possess modified chelicerae for piercing the host integument and imbibing their fluids (Eickwort 1997) Behavioral Phoresy Many mite spe cies are phoretic on honey bees including Varroa destructor Tropilaelaps clarae and Acarapis woodi (Eickwort 1997) T he fly species Braula coeca also is phoretic on honey bee adults. Feeding/reproduction The phoretic mites Acarapis externus Morgenthaler A. dorsalis Morgenthaler and Varroa destructor all feed on the adult bees to which they are attached. Varroa destructor also feeds on brood once inside the colony (Eickwort 1997, Rosenkranz et al. 2010) Although T. clarae are phoretic on adult bees, they only feed on brood. The nonphoretic Pyemotes species (Acari: P yemotidae) feed on brood within the colony also (Eickwort 1997, Sammataro et al. 2000, Brown et al. 2002)

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23 Oplostoma fuligineus Olivier and Pachnoda sinuata flaviventri s Gory and Percheron (Coleoptera: Scarabaeidae) enter colonies and feed preferentially on open brood cells, but also will feed on capped brood, honey, and pollen stores secondarily (Donaldson 1989) Another scarab beetle Euphoria sepulcralis is found occasionally within nests feeding on food stores (Caron 1 997b, Woodruff 2006) Other predators of honey bee adults and brood include species of Meloe (Coleoptera: Meloidae), Paratemnus minor (Balzan) (Pseudoscorpiones: Atemnidae), and Salticus sp ecies (Aranae: Salticidae). The latter build silken cases on the inner cover of colonies and feed on passing bees (Wheeler 1928, Caron 1997a, b Sammataro 1997, Majka 2007) Highly effective predator ants in the genera Dorylus, Eciton Iridomyrmex Formica Crematogaster and Solenopsis (Hymenoptera: Formicidae) are swift in destroying honey bee colonies, and can be major honey bee pests in some regions of the world. Camponotus sp ecies can raid a colony of resources as w ell as destroy hive materials (Fell 1997) Similarly, Heterotermes tenuis ( Hagen) (Blattodea: Rhinotermitidae) and Dermestes lardarius L. (Coleoptera: Dermestidae) can feed on and destroy hive components, and subsequently produce sites that can harbor other pests (Caron 1997b) Mutilla europae a L. (Hymenoptera: Mutillidae) is a p arasite that lays eggs in pupal cells. The developing larva e then eat the bee larvae, and, after pupating within a host coc oon, the emerging adult feeds on the honey stores (Alford 1975) Several species of Blattodea, Coleoptera, and Lepidoptera feed on wa x, honey, and pollen within colonies (Nielsen and Brister 1979, Caron 1997b, Williams 1997) Acherontia atropos (L.) (Lepidoptera: S phingidae) and Vespula and Dolichovespula sp ecies (Hymenoptera: Vespidae) enter colonies occasionally to rob nectar (Moritz et al. 1991, Fell 1997) Eggs of

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24 Drosophila busckii Coquillett (Diptera: Drosophilidae) ar e laid on wax cappings, and developing larvae feed on honey stores, subsequently ferment ing them (Sammataro 1997) Similarly, Hermetia illucens L. (Diptera: Stratiomy idae) and Tenthredomyia australis Shannon (Diptera: Syrphidae) lay eggs in the colonies and the developing larvae feed on food stores (Sammataro 1997) Adults of the phoretic fly Braula coeca take food directly from honey bee mouthparts. Occas ionally, the fly will induce the host bee to regurgitate food by scratching the bees labrum (Sammataro 1997) Several scavengers can be found among the debris in honey bee nests, including mites, silverfish, earwigs, barklice, moths, and beetles (Lea 1910, 1912, Alfor d 1975, Caron 1997b, Williams 1997, Neumann and Ritter 2004, Ellis et al. 2008) Epuraea corticina Erichson (Coleoptera: Nitidulidae) has been found in supplemental protein patties placed within hives, and Carpophilus dimidiatus (F.) (Coleoptera: Nitidulid ae) has been found in pollen cells within active hives (Ellis et al. 2008) Species of Ellingsensis and Chelifer cancroides (L.) (Pseudoscorpiones: Cheliferidae) serve a beneficial purpose by feeding on destructive Varroa destructor and Galleria mellonella (L.) (Lepidoptera: Pyralidae) within colonies (Caron 1997a, Donovan and Paul 2005) Similarly, the parasitic wasps Bracon hebetor Say and Apanteles galleriae Wilkinson (Hymenoptera: Braconidae) parasitize pestiferous G. mellonella and A. grisella within colonies (Williams 1997, Dweck et al. 2010) Other Upon entering a colony, Galleria mellonella remains motionless if encountered by a honey bee, thereby evading detection. The moth can sustain this for up to five minutes, and is detected only if it is touched or approached closely by a bee (Nielsen and Brister 1977, Eischen et al. 1986) Acherontia atropos disorient s guard bees by producing a squeaking noise, which

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25 purportedly appeases the bees and helps the moth enter the colony to steal nectar and honey (Williams 1997) Chemical The most thoroughly studied chemical integration systems exist among melittophiles of honey bees For example, mass attacks on honey bee colonies by the predatory Vespa mandarinia japonica Smith (Hymenoptera: Vespidae) are l ikely assisted by pheromonal markings at the hive site (Fell 1997) Linepithema humilis Mayr (Hymenoptera: Formicidae) is a pest ant species that releases few volatiles, and Spangler and Taber (1970) suggest that a lack of identifying odors is one means of integr ating into bee colonies. Therefore, not only is a specific chemical profile helpful for integrating into a hive or marking its presence, but also so is a lack of any chemical profile at all Acherontia atropos enter s colonies by chemical camouflage, wherein the moth produces nest chemicals endogenously, resulting in virtual indifference to the moth by the bee hosts (Moritz et al. 1991) T he devastating pest mite V. destructor has a chemical profile nearly identical to that of honey bee s (Nation et al. 1992) Interestingly, a s the chemical profile of the developing immature bee changes, that of the mite changes correspondingly (Martin et al. 2001) Methods Used by SHBs to Integrate into Apis mellifera Nests The SHB gained significance in the mid 1990s being largely benign throughout its native range of sub Saharan Africa (Lundie 1940) I n its introduced range of North America and Australia, the SHB can cause devastation to even strong colonies, an d there is evidence that it may transmit bee pathogens mechanically (Neumann and Elzen 2004, Neumann and Ellis 2008, Eyer et al. 2009a, b Schfer et al. 2010a )

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26 Morphological There are few morphological adaptations reported for SHBs that would enable them to integrate into bee colonies. One adaptation occurs in the larval stage, wherein their bodies are covered with a series of dorsal and lateral protuberances. Lundie (1940) hypothesized that these protuberances prevent the larvae from being covered in honey while feeding. T he adult SHB s hard exoskeleton lik ely enables them to resist stings by bees as well (Neumann et al. 2001) However, neither of these hypotheses has been investigated quantitatively. Behavioral The majority of SHB integrative adaptations are behavioral and include passive defense, hiding, behavioral mimicry, and clustering. Upon entr ance into a honey bee colony, a SHB often is met with aggression (Schmolke 1974, Elzen et al. 2001) To avoid being stung and/or removed from the colony, the SHB retracts its appendages beneath its body, leaving nothing extended for bees to grasp, which is similar to what Salt (1929) proposed for the corylophid Cleidostethus meliponae in nests of Melipona alinderi (Schmolke 1974, Neumann et al. 2001) Some SHBs do not retract their appendages upon entering the hive, rather they use an alternative strategy of running quickly to an area of the hive inaccessible to host bees, such as a crack or crevice on the periphery (Schmolke 1974, Neumann et al. 2001) If the SHB is on a frame or wall of a colony and is pursued by a bee, it may drop to the bottom of the hive, which is a common defensive strategy employed by beetles and insects generally (Schmolke 1974, Dill et al. 1990, Leschen 2000, Neumann and Elzen 2004, Ohno and Miyatake 2007) Regard less, the SHB must find a place inaccessible to the bees to avoid removal. Once the SHB arrives in the space, the bees may station guards around the area (confinement sites), keeping the SHB s from escaping and reproducin g in the colony (Neumann et al. 2001, Ellis et al. 2003b, Ellis 2005)

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27 Within the confinement sites, the SHB s survive longer than would be expected without resources (Pettis and Shimanuki 2000, Ellis et al. 2002a) Survival is achieved through a form of be havioral mimicry, wherein the SHB s antennate the guarding bees mandibles thereby inducing the m to regurgitate a droplet of food, which is consumed by the confined SHB s (Ellis et al. 2002b) Finally, during the cold months following peak autumn infestation s (de Guzman et al. 2010) and once the SHB s are established within the colony, they regulate their body temperature by entering the thermoregulatory cluster of bees surrounding the brood (Pettis and Shimanuki 2000, Ellis et al. 2003a, Neumann and Elzen 2004) In this way, they are able to survive the cold weather experienced in temperate regions of the United States and Australia. Chemical Host location and recruitment are two interrelated chemical strategies used by SHBs to integrate into honey bee colo nies The SHB is attracted to components of honey bee colonies (Elzen et al. 1999, Suazo et al. 2003, Torto et al. 2005, 2007a, b, Graham et al. 2011 ) s pecifically components of honey bee alarm pheromone such as isopentyl acetate. The SHB can detect these compounds at lesser quantities than are detectable by the bees themselves (Torto et al. 2007a) This high degree of sensitivity explains the SHB s attraction to stressed colonies (Wenning 2001) as well as strong colonies under normal conditions (Neuman n and Elzen 2004) The second chemical method of integration involves recruitment of other SHB individuals to the hive Once SHBs have infested a colony, they cause a low level of stress to the colony. This stress leads to the release of alarm volatiles (e .g. isopentyl acetate) (Breed et al. 2004) This, in turn, attracts more SHB s to the colony. Furthermore, as the SHB s feed on bee collected pollen in the colony, a commensal yeast on their body, i.e. Kodamaea ohmeri (Etchells & T.A. Bell)

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28 invades the poll en and produces isopentyl acetate, thereby attracting more SHB s (Torto et al. 2007a) In both the beetle and yeast recruitment chemical cues there is a positive feedback mechanism that leads to the rapid collapse of the colony. Introduction to the Researc h Presented in this Dissertation Many arthropod species have been found in honey bee colonies and scavenge or prey on the inhabitants. However, only a few have a close, protracted relationship with honey bees. The SHB exhibit s a var iety of interactions wit h honey bee s including clustering among and being fed by the host bees (Pettis and Shimanuki 2000, Ellis et al. 2002b, Ellis et al. 2003a, Neumann and Elzen 2004). These interactions demonstrate SHBs high level of integration into honey bee colonies. A s eries of experiments was performed to investigate how t he SHB is able to integrate successfully into honey bee colonies. The majority of the experiments (Chapters 2, 3, 4, 6) were comparative, allowing me to gain insight into the origins of particular trai ts as well as their adaptive value through comparing traits among beetles exhibiting various levels of integration within honey bee colonies. Such levels of integration were assigned based on the species frequency of occurrence within honey bee nests, wit h higher frequencies of occurrence believed to reflect a higher level of integration by a beetle species The SHB is hypothesized to use morphological, behavioral, and chemical adaptations to integrate into honey bee colonies (Wilson 1971, Kistner 1979) C ollectively, the data will enable generalizations and predictions to be made concerning the attributes necessary to become a symbiont in social bee nests. Before invading a colony, symbionts first must find the nest. In Chapter 2, results are presented fro m an investigation involving the degree of attraction of various beetle species representing three levels of colony integration toward honey bee hive odors. A hypothesis was proposed that the most highly integrated beetle species (SHB) would be more attracted to hive odors than beetles marginally associated or not associated at all with honey bee colonies.

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29 M orphological adaptations are important to the success of symbionts in entering social insect colonies. An investigation of leg morph ology in SHB and other related nitidulid beetles was performed to determine physical features that may aid in colony integration (Chapter 3). M odifications in the femora and tibiae, as well as in tarsal setation were expected in the SHB, but not the other beetles M odifications exist in other beetle species known to inhabit social insect colonies, providing them with a way to survive host attacks (Attygalle et al. 2000, Eisner and Aneshansley 2000) The nest entrance is where beetles are presented with the first round of nest defense by the bees. Therefore a comparison of guard bee behavior toward SHBs and other beetles of varying levels of integration was performed. The other beetles included beetles fo und in honey bee colonies previously, and those never foun d in bee colonies (Chapter 4). A hypothesis was proposed that highly integrated SHB s would be treated more aggressively by bees than other beetles due to their status as a colony pest. The intruder s cuticular chemical profile also is important for successful entrance into the hive (Breed et al. 2004). T his profile c an be altered through diet. Therefore, a hypothesis was proposed that, although SHBs may be treated more aggressively than other beetle species (Chapter 4), SHBs provided a diet of honey bee products will have a chemical profile that enhances their acceptance by guard bees at the hive entrance (Chapter 5). Once beetle invaders successfully navigate bee defenses at the colony entrance, they enter the colony an d seek cracks/crevices to hide from attacking bees. In Chapter 6, an investigation was performed to test the hypothesis that a beetles ability to find confinement sites within a bee colony will vary according to the beetles level of i ntegration within bee colonies. Also tested

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30 was that previous SHB occupation of confinement sites predisposed those sites to hosting invading SHBs that were never before exposed to honey bee colonies. Despite the complicated interaction between SHBs and ho ney bees at confinement sites, the defensive system deteriorates during winter when bees cluster tightly in the nest to keep warm. In Chapter 7, the temperature at which the beetles leave confinement sites to enter the bee cluster, and the temperature at w hich they leave the cluster and return to their confinement sites was evaluated The research presented herein contributes to an understanding of the attributes that make the SHB successful at integrating into honey bee nests and provides several interesti ng lines of research for further study.

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31 Table 1 1. Arthropods found in nests of non Apis mellifera social bee s and their means of integrating into the nests. Symbiont Host Integrative characteristic Reference Morphology Acari Acaridae Acarus farris (Oudemans) Bombus terrestris (L.) Megabombus argillaceus Scopoli Megabombus zonatus (Smith) Pyrobombus niveatus Kriechbaumer reduced mouthparts in phoretic deutonymph Aytekin et al. 2002 Histiostoma halictonida Halictus rubicundus caudal suckers for attachment to pupae Woodring 1973 Kuzinia laevis Bombus spp. anal and ventral suckers for phoretic attachment Alford 1975, Schwarz and Huck 1997 Uropodidae Fuscuropoda marginata Bombus spp. pedicel secreted from glands for phoretic attachment Alford 1975 Pseudoscorpionida Chernetidae Corosoma spp., Dasychernes spp. Melipona spp. disguise themselves with vestitural setae Salt 1929, Roubik 2006, Gonzalez et al. 2007 Collembola Cyphoderidae Paracyphoderus spp., Cyphoderus spp., Partamora spp. Melipona spp. tough cuticle Roubik 2006 Pseudocyphoderus melittophilous Trigona testacea passive defense, well sclerotized dorsal cuticle, heavy scales, enlarged mesothorax, lateroventrally expanded meso and metathorax, shortened legs Mutt 1977 Coleoptera Leiodidae Parabystus spp., Melipona spp. toungue and groove Roubik and

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32 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference Scotocryptodes spp., Synaristers spp. mechanism to lock elytra together, notched mandibles to grasp setae on hosts corbiculae for phoresy, blind, lacking hindwings Wheeler 1982, Bezerra et al. 2000 Scotocryptus spp. Melipona spp. strongly convex dorsum, can fit head, antennae, and legs under body, larvae have flattened body and heavy dorsal setation, toungue and groove mechanism to lock elytra together, notched mandibles to grasp setae on hosts corbiculae for phoresy, blind, lacking hindwings Salt 1929, Roubik and Wheeler 1982, Wheeler 1985, Bezerra et al. 2000, Davis and Gonzalez 2008 Corylophidae Cleidostethus meliponae Melipona alinderi fused elytra and pronotum flattened at margins to conceal reduced head and antennae, shortened legs, tibiae fold within femora, which fit into grooves in the body, interlocking device to make body rigid Salt 1929 Bowestead et al. 2001 Monotomidae Cro wsonius spp. Trigona spp. reduced eyes, wingless Pakaluk and Ripiphoridae Ripiphoris spp. Halictus spp., Lasioglossum spp., Augochlora spp., Augochlorella spp. attach to host wings with terminal abdominal segment and tarsal pulvilli that have been modified into suckers, flattened, Falin 2002, Tomlin and Miller 1989

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33 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference heavily sclerotized bodies Diptera Phoridae Melittophora salti Brues Trigona amalthea Olivier head is close to thorax to protect junction between them, modified scutellum protects junction between thorax and abdomen, heavily sclerotized ovipositor, able to fit legs under body Salt 1929 Hymenoptera Perilampidae Ecthrodape africana Braunsapis spp. larvae have setae which give support tactile mimicry Michener 1969 Behavioral Acari Acaridae Acarus spp. Bombus spp., Megabombus spp., Pyrobombus spp. phoretic deutonymphs Frison 1926, Richards and Richards 1976, Schwarz et al. 1996, Huck et al. 1998, Aytekin et al. 2002 Glycyphagus spp., Tyroglyphus spp. Melipo na spp. feed on pollen Roubik 2006 Kutzinia laevis Bombus terrestris phoretic, predators of other nest invaders Allen et al. 2007, Alford 1975, Tyrophagus spp. Melipona spp. feed on fungi Roubik 2006 Ameroseiidae Garmamilla spp., Proctolaelaps spp. Bombus spp., Megabombus spp., Pyrobombus spp. phoretic deutonymphs, predators of other nest invaders Frison 1926, Alford 1975, Richards and Richards 1976, Schwarz et al. 1996, Huck et al. 1998, Aytekin et

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34 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference al. 2002 Neocypholaelaps spp. Melipona spp. feed on pollen Roubik 2006 Anoetidae Histiostoma halictonida Lasioglossum zephyrum, Megalopta spp. phoretic on wings of emerging adults Woodring 1973, Engel and Fain 2003 Chaetodactylidae Sennertia alfkeni (Oudemans) Xylocopa spp. phoretic in acarinaria Okabe and Makino 2002, Kawazoe et al. 2008 Gamasidae Urozercon melittophiles Silvestri Trigona cupira Smith Phoretic Salt 1929 Hemileiidae Hemileius spp. Melipona spp. feed on fungi Roubik 2006 Hypoaspididae Hypoaspis spp. Bombus spp., Megabombus spp., Pyrobombus spp. phoretic deutonymphs, predators of other nest invaders Frison 1926, Alford 1975, Richards and Richards 1976, Schwarz et al. 1996, Huck et al. 1998, Aytekin et al. 2002 Laelapidae Hypoaspis spp., Meliponaspis spp. Melipona spp., Meliponula spp., Trigona spp. parasites of host larvae Salt 1929, Roubik 2006 Laelapsoides spp. Megalopta spp. feeds on fungus on host brood Biani et al. 2009 Laelaspoides ordwayae Eickwort Augochlorella spp. phoretic on emerging in spring Eickwort 1966 Neohypoaspis ampliseta Delfinado Baker, Baker, & Roubik Melipona spp. predacious on mites in nests Roubik 2006 Pneumolaelaps spp. Bombus spp. phoretic on foragers, feed on honey and pollen Hunter and Husband 1973 Tropilaelaps clarae Apis cerana parasites of drone brood Eickwort 1997

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35 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference Tropilaelaps koenigi r um Delfinado Baker & Baker Apis dorsata (F.), A. florea (F.) parasites of host brood Eickwort 1997 Macrochelidae Macrocheles spp. Melipona spp. feed on fungi Roubik 2006 Trigonholaspis spp. Melipona spp., Meliponula spp., Trigona spp. parasites of host larvae, feed on fungi Salt 1929, Roubik 2006 Parasitidae Parasitellus fucorum de Geer Parasistus spp. Bombus spp., Megabombus spp., Pyrobombus spp. phoretic deutonymphs, predators of other nest invaders Frison 1926, Alford 1975, Richards and Richards 1976, Goldblatt and Fell 1984, Schwarz et al. 1996, Huck et al. 1998, Aytekin et al. 2002 Podapolipidae Locustacarus buchneri Bombus spp. feed and reproduce within tracheae, overwinter with queens Husband and Sinha 1970, Shykoff and Schmid Hempel 1991, Otterstatter and Whidden 2004, Yoneda et al. 2008 Podocinidae Lasiodeius spp. Melipona spp. feed on pollen Roubik 2006 Pyemotidae Parapygmephous spp. Agapostemon spp., Dialictus umbripennis (Ellis) Phoretic Eickwort and Eickwort 1969, 1971, Woodring 1973 Pyemotes spp. Melipona spp. feed on pollen Roubik 2006 Scutacaridae Imparipes eickworti Mahunka Dialictus umbripennis Phoretic Eickwort and Eickwort 1969, 1971, Woodring 1973 Scutacarus acarorum Bombus spp., Parasistus spp. phoretic deutonymphs, use Richards and Richards 1976,

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36 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference attached to Bombus spp. flowers to transfer between bee hosts Schwarz and Huck 1997 Tarsonemidae Acarapis woodi Apis cerana Apis dorsata feed and reproduce within tracheae Eickwort 1997 Tydeidae Neotydeolus spp. Melipona spp. feed on fungi Roubik 2006 Uropodidae Fuscuropoda marginata Bombus spp., Volucella bombylans within Bombus nests phoretic deutonymphs, scavenger Frison 1926, Alford 1975 Varroidae Euvarroa sinhai Delfinado & Baker Apis dorsata Apis florea parasites of host brood Eickwort 1997 Varroa jacobsoni Varroa underwoodi Apis cerana parasites of drone brood Eickwort 1997 Pseudoscorpionida Chernetidae Dasychernes inquilinus Chamberlin Melipona spp. phoretic Bezerra et al. 2000, Gonzalez et al. 2007, Davis and Gonzalez 2008 Collembola Entomobryidae Pseudosinella Lasioglossum feed on pollen and fecal Batra 1965, pettersoni Borner zehyrum, Augochlora spp. material Eickwort and Eickwort 1972 Dermaptera Forficulidae Forficula auricularia L. Bombus spp. feed on host brood Alford 1975 Hemiptera Coccidae Cryptostigma spp. Schwarzula spp. phoretic, secrete honeydew and wax Camargo and Pedro 2002, Roubik 2006 Coleoptera Cryptophagidae Antherophagus spp. Bombus spp. phoretic, attach to legs and proboscis at Frison 1921, 1926, Wheeler

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37 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference flowers, feed and reproduce within nest debris 1928, Free and Butler 1959 Chavarria 1994, Gonzalez et al. 2004 Silvan idae Silvan i s trivialis Grouvelle Melipona spp., Trigona spp. feed on nest debris Lea 1910, Salt 1929 Leiodidae Parabystus spp., Scotocryptodes spp., Scotocryptus melittophilus Reitter Synaristus spp. Cephalotrigona spp., Melipona spp., Partamona spp. phoretic, feed on pollen, fungi, and fecal debris Bezerra et al. 2000, Roubik 2006, Gonzalez et al. 2007, Davis and Gonzalez 2008 Nitidulidae Brachype p l us auritus Murray B. basalis Erichson Epuraea luteola Erichson Melipona spp., Trigona spp. feed on nest debris Lea 1910, Salt 1929 Glischrochilus fasciatus (Olivier) Bombus spp. scavenger Frison 1926, Alford 1975 Ripiphoridae Ripiphorous smithi Lasioglossum spp. phoretic 1 st instar larvae attach to host bees at flowers, feed on host larvae Tomlin and Miller 1989, Majka et al. 2006 Scarabaeidae Onthophagous hecate (Panzer) Bombus spp. scavenger Frison 1926, Alford 1975 Staphylinidae Belonuchus mordens Erichson Melipona spp. predacious Wheeler 1928 Tenebrionidae Tenebrio obscurus F. T. tenebrioides Beauv. Bombus spp. scavenger Frison 1926, Alford 1975 Diptera Metopiidae Brachycoma devia ( Falln ), B. sarcophagina (Townsend) Bombus spp. larviposit in or on larval cells, parasitic larvae consume host larvae Frison 1926, Free and Butler 1959

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38 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference Michiliidae Melipona capixaba Moure & Camargo feed on fecal debris Melo 1996, Roubik 2006 Muscidae Fannia canicularis L. Bombus spp. scavenger Frison 1926, Alford 1975 Phoridae Gymnoptera vitripennis (Meigen) Bombus spp. scavenger Frison 1926, Alford 1975 Phalacrotophora halictorum Melander & Brues Agapostemon spp. oviposit in brood cells, parasitic larvae consume host larvae Eickwort and Eickwort 1969 Pseudohypocera kerteszi Enderlein Melipona beecherii B. scavenger at low levels, larvae are predators of host brood at high levels Robroek et al. 2003, Roubik 2006 Pseudohypocera nigrofascipes Borgmeier & Schmitz Melipona spp., Trigona spp. feed on nest debris Lea 1910, Salt 1929 Syrphidae Volucella spp. Bombus spp. oviposit on or in host larval cells, parasitic larvae consume host larvae, oviposit immediately if killed Wheeler 1928, Free and Butler 1959 Lepidoptera Oecophoridae Endrosis sarcitrella L., Hofmannophila pseudospretella (Stainton) Bombus spp. scavenger Frison 1926, Alford 1975 Pyralidae Achroia grisella Galleria colonella Melipona spp. feed on nest debris Salt 1929, CepedaAponte et al. 2002 Aphomia sociella Bombus spp. feed on food stores and brood Frison 1926, Wheeler 1928 Ephestia khniella Bombus spp. feed on food stores Frison 1926, Wheeler 1928, Alford 1975, Schmid Hempel 2001 Galleria mellonella Apis cerana A. feed on food stores Williams 1997

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39 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference dorsata A. florea Vitula edmandsii Bombus spp. feed on food stores Frison 1926, Wheeler 1928, Alford 1975, Schmid Hempel 2001 Hymenoptera Braconidae Apanteles spp., Aspilota spp., Orthostigma pumilum (Nees) Bombus spp. parasites of other nest invaders Frison 1926, Alford 1975 Blacus paganus Haliday Hysteromerus mystacinus Wesmael Stenocyptus spp. Bombus spp. parasitize Antherphagus spp. Alford 1975 Habrobracon juglandis Bombus spp. parasitize Vitula edmandsii Frison 1926 Eulophidae Melittobia spp. Bombus spp. oviposit on or in host larval cells, parasitic larvae consume host larvae Frison 1926, Free and Butler 1959 Formicidae Acromyrmex octospinosus Bombus pullatus builds fungus gardens within abandoned cells Chavarria 1996 Cryptocerus elongata Trigona mosquito consume host colonys resources, establish colony within host nest Salt 1929 Lasius niger (L.) Solenopsis molesta (Say) Bombus spp. scavenger Frison 1926, Alford 1975 Oecophylla smaragdina F. Apis cerana A. florea inhabit host nest Fell 1997 Ichneumonidae Stilpnus gagates Bombus spp. parasite of other nest invaders Alford 1975 Mutillidae Mutilla europaea Bombus spp. parasites of host brood Brothers et al. 2000

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40 Table 1 1. Continued. Symbiont Host Integrative characteristic Reference Myrmilla capitata (Lucas) Lasioglossum malachurum (Kirby) parasites of host brood Polidori et al. 2009 Vespidae Vespa tropica L. Apis florea predate of host bees in nest Fell 1997

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41 Table 1 2. Arthropods found in nests of Apis mellifera and their means of integrating into the nests. Symbiont Integrative characteristic Reference Morphology Acari Ameroseidae Afrocypholaelaps africana sucker like ambulacral pads to attach to bees at flowers Seeman and Walter 1995 Laelapidae Tropilaelaps clarae elongated body for moving between hairs of bee and large legs with claws for attachment Rath et al. 1991, Sammataro et al. 2000 Tarsonemidae Acarapis woodi adults have tarsal claws and leg setae to accommodate attachment to bee hairs, larvae have reduced claws and enlarged pulvillar pads to allow for movement around tracheae, distally oriented leg setation to allow for measurement of tracheae, modified mouthparts for piercing i ntegument Eickwort 1997, Ochoa et al. 2005 Coleoptera Nitidulidae Aethina tumida larval protuberances to protect from drowning in honey, adults have thick cuticles to protect from sting Lundie 1940, Neumann et al. 2001 Diptera Braulidae Braula coeca wingless, pretarsal claws with modified combs for attaching to bee hairs Sammataro 1997 Lepidoptera Sphingidae Acherontia atropos thick cuticles to protect from sting Moritz et al. 1991 Behavior Acari Acaridae Acarus immobilis Griffiths Acarus siro L. Forcellinia galleriella (Womersley) Tyrophagus longior (Gervais) feed on comb, fungi, and debris Abrol et al. 1994, Eickwort 1997

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42 Table 1 2. Continued. Symbiont Integrative characterisitic Reference Tyrophagus palmarum Oudemans, Tyrophagus putrescentiae (Schrank) Ascidae Blattisocius spp., Lasoseius spp., Melichares spp., Proctolaelaps spp. feed on other arthropods Eickwort 1997 Carpoglyphidae Carpoglyphus lactis (L.) feed on comb, fungi, and debris Eickwort 1997 Cheletidae Cheyletus spp. feed on other arthropods Eickwort 1997 Glycyphagidae Glycyphagus domesticus de Geer feed on comb, fungi, and debris Eickwort 1997 Laelapidae Melittiphis spp. feed on other arthropods Eickwort 1997 Tropilaelaps clarae phoretic on adults, feed on brood Eickwort 1997, Brown et al. 2002 Macrochelidae Macrocheles spp. feed on other arthropods Eickwort 1997 Parasitidae Parasistus spp. feed on other arthropods Eickwort 1997 Pyemotidae Pyemotes spp. feed on brood Eickwort 1997 Scutacaridae Imparipes spp., Scutacarus spp. feed on comb, fungi, and debris Eickwort 1997 Tarsonemidae Acarapis dorsalis Acarapis externus feed on adults Eickwort 1997 Acarapis woodi phoretic Eickwort 1997 Tarsonemus spp. feed on comb, fungi, and debris Eickwort 1997 Varroidae Varroa destructor phoretic, feed on adults and brood Eickwort 1997, Rosenkranz et al. 2010 Pseudoscorpiones Cheliferidae Chelifer cancroides Ellingsensis spp. feed on Galleria mellonella and Varroa destructor Caron 1997b, Donovan and Paul 2005 Atemnidae Paratemnus minor feed on adults and brood Caron 1997b Aranae Salticidae Salticus spp. reside in silken cases on inner Caron 1997b

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43 Table 1 2. Continued. Symbiont Integrative characterisitic Reference cover, feed on passing bees Thysanura Lepismatidae Ctenolepisma lineate F. scavengers Caron 1997a Blattodea Blattidae Blatta orientalis L., Periplaneta americana (L.) feed on wax, honey, and pollen Caron 1997a Rhinotermitidae Heterotermes ten ui s feed on hive components Caron 1997a Termitidae Microcerotermes arbo re us (Emerson), Nasutitermes costalis (Holmgren) feed on hive components Caron 1997a Dermaptera Forficulidae Forficula auricularia scavengers Alford 1975, Caron 1997a Psocoptera Liposcelidae Liposcelis divinatorius ( M ller) scavengers Caron 1997a Trogiidae Trogium pulsatorium (L.) scavengers Caron 1997a Hemiptera Pyrrhocoridae Pyrrhocoris apterus (L.) feed on adults and brood Caron 1997a Coleoptera Cleridae Trichodes apiaries (L.) feed on adults and brood Caron 1997a Cryptophagidae Cryptophagous hexagonalis Tournier Cryptophagus scanius L. scavengers Caron 1997a, Haddad et al. 2008 Dermestidae Dermestes lardarius Dermestes vulpinus F. feed on hive components Caron 1997a Trogoderma glabrum (Herbst) feed on wax, honey, and pollen Caron 1997a Trogoderma ornatum (Say) feed on adults and brood, feed on hive components Caron 1997a Meloidae Meloe spp. feed on adults and brood Caron 1997a Nitidulidae Aethina tumida retract appendages when Schmolke 1974, Pettis and

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44 Table 1 2. Continued. Symbiont Integrative characterisitic Reference attacked, hide, drop when provoked, take food from bee mouthparts, induce regurgitation from host, enter thermoregulatory clusters during winter Shimanuki 2000, Neumann et al. 2001, Ellis et al. 2002b, Ellis 2003a, Neumann and Elzen 2004 Brachype p l us basalis scavengers Lea 1910, 1912, Brachype p l us blandus Murray Brachype pl us inquilinus Lea Cychramus luteus (F.) Epuraea luteola Glischrochilus fasciatus Lobiopa insularis ( Castelnau deLaPorte ) Caron 1997a, Neumann and Ritter 2004, Ellis et al. 2008 Carpophilus dimidiatus feed on pollen stores Ellis et al. 2008 Epuraea corticina feed on supplemental protein patties Ellis et al. 2008 Ptinidae Ptinus fur (L.) Ptinus raptor Sturm feed on wax, honey, and pollen Caron 1997a Pselaphidae Mesoplatus spp. scavengers Caron 1997a Scarabaeidae Anomola dimidiate Hope, Protaetia impavida (Janson), Torynorrhina opalina (Hope) feed on stored pollen Caron 1997a Copris lunaris L. Potosia opaca (F.) feed on honey Caron 1997a Euphoria lurida (F.), Euphoria sepulcralis feed on food stores Caron 1997a, Woodruff 2006 Macrocyphonistes kolbeanus Ohaus Potos ia hungarica Herbst, Rhyzoplatys auriculatus (Burmeister) feed on adults and brood Caron 1997a Oplostoma fulgeneu s Pachnoda sinuat a flaviventris (Gory & Percheron) feed on brood, honey, and pollen Donaldson 1989, Torto et al. 2010 Staphylinidae Polylobus quadratipennis Lea scavengers Caron 1997a Tenebrionidae Bradymerus sp., Platybolium alvearium Blair scavengers Caron 1997a Tenebrio madens Charpentier feed on stored pollen Caron 1997a Diptera

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45 Table 1 2. Continued. Symbiont Integrative characterisitic Reference Bombyliidae Anthrax spp. parasitize honey bee larvae Sammataro 1997 Braulidae Braula coeca phoretic on adult s, take food from bee mouth induce regurgitation from host Sammataro 1997 Drosophilidae Drosophila busckii larvae feed on honey stores Sammataro 1997 Phoridae Pseudohypocera kerteszi feed on adults and brood Sammataro 1997 Stratiomyidae Hermetia illucens larvae feed on food stores Sammataro 1997 Syrphidae Tenthredomyia australis larvae feed on food stores Sammataro 1997 Lepidoptera Oecophoridae Endrosis sarcitrella scavengers Williams 1997 Pyralidae Achroia grisella Vitula edmandsii feed on wax, honey, pollen Williams 1997 Galleria mellonella feed on wax, honey, pollen, evades detection by remaining very still Nielson and Brister 1977, Eischen et al. 1986 Plodia interpunctella ( Hbner ) scavengers Williams 1997 Aphomia sociella feed on brood, wax, honey, pollen Williams 1997 Sphingidae Acherontia atropos steal nectar, disorients guard bees by making squeaking noise Moritz et al. 1991, Williams 1997 Hymenoptera Braconidae Apanteles galleriae Bracon hebetor parasitize Achroia grisella and Galleria mellonella Williams 1997, Dweck et al. 2010 Crabronidae Bembex handlirschella Ferton parasitize Anthrax spp. Sammataro 1997 Eulophidae Melittobia acasta Walk parasitize honey bee larvae Fell 1997 Formicidae Camponotus spp. feed on food stores and destroys hive materials Fell 1997 Crematogaster spp., Dorylus spp., Eciton spp., Formica spp., feed on adults, brood, and food stores Fell 1997

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46 Table 1 2. Continued. Symbiont Integrative characterisitic Reference Mutillidae Iridomyrmex spp., Solenopsis spp. Mutilla europ ae a lays eggs in pupal cells, larvae feed on bee pupae, adults feed on honey stores Alford 1975 Vespidae Dolichovespula spp., Vespula spp. steal nectar Fell 1997 Vespa spp. feed on adults De Jong 1990, Fell 1997 Chemical Acari Varroidae Varroa destructor have chemical profiles that mimic honey bees Nation et al. 1992, Martin et al. 2001 Coleoptera Nitidulidae Aethina tumida detect pheromones at low concentrations to find host, recruit conspecifics through multitrophic interaction Elzen et al. 1999, Suazo et al. 2003, Torto et al. 2005, Torto et al. 2007a Lepidoptera Sphingidae Acherontia atropos have chemical profiles that mimic honey bees Moritz et al. 199 1 Hymenoptera Formicidae Linepithema humilis lack identifying odors to escape detection Spangler and Taber 1970 Vespidae Vespa mandarinia japonica use pheromonal markings to assist in mass attacks Fell 1997

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47 CHAPTER 2 ATTRACTION OF MULTIP LE BEETLE SPECIES TO HONEY BEE HIVE ODORS SHB s locate host honey bee colonies by detecting volatile chemicals including those emitted by honey bee workers and bee collected pollen (Suazo et al. 2003, Torto et al. 2005) Specifically, volatiles of honey bee alarm pheromone, including isopen tyl acetate (IPA), are highly attractive to the SHB which can detect IPA at lower quantities than the bees themselves (Torto et al. 2007a) Furthermore, SHBs carry a symbiotic yeast ( Kodamaea ohmeri ) which, when mixed with bee collected pollen, produces IPA and other components of the alarm pheromone that attracts SHB s (Torto et al. 2007a, b ) Therefore, the SHB exhibits a high degree of adaptation for finding host colonies. The SHB is not the only nitidulid beetle that occurs in honey bee colonies. Other nitidulids reported in honey bee colonies include Cychramus luteus (Neumann and Ritter 2004) Lobiopa insularis Carpophilus dimidiatus Glischrochilus fasciatu s Epuraea corticina (Ellis et al. 2008) and E. luteola ( personal observation). Although the SHB exhibit s a high degree of integration into honey bee colonies (Ellis and Hepburn 2006) it remains uncle ar if other nitidulid s share a similar relationship with honey bees. No damage has been reported from the presence of these nitidulids in honey bee colonies, and their ecological niche in honey bee colonies is likely that of a facultative scavenger Cychra mus luteus is a saproxylic species found in Europe (Kaila et al. 1994) In 2003, this species was found inhabiting two healthy honey bee colonies. However, this beetles inability to reproduce on hive components suggests that they are accidental inhabit ants (Neumann and Ritter 2004) Lobiopa insularis usually is found in agricultural settings feeding on fruits, e specially strawberries (Parsons 1938, Williams and Salles 1986). The species also has been found in colonies in Georgia, USA, bu t shows no evidence of integration beyond seeking shelter (Ellis et

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48 al. 2008) Carpophilus dimidiatus is found in tropical and temper ate climates worldwide (Parsons 1943) and often is found in agricultural settings feeding on corn (Connell 1975) over ripe fruits, stored products, and a variety of fermenting substrates (see Hinton 1945). Several individuals were collected from a single colony in Georgia, USA, and were assumed to be accidentals (Ellis et al. 2008) Glischrochilus fasciatus is found throughout North America, pre dominately in woodlots (Parsons 1943, Blackmer an d Phelan 1995, Majka and Cline 2006, Price and Young 2006). This species is a pest of raspberries, strawberries, tomatoes, and corn (Williams et al. 1981) and typically visits most fermenting substrates The species also was discovered in several honey bee colonies in Georgia, USA but upon further investigation, did not reproduce successfully on honey bee col ony components (Ellis et al. 2008) Epuraea corticina is found th roughout the United States and is associated commonly with sap f lows and oak wilt mats (Parsons 1969, Cease and Juzwik 2001). This species has been found in honey bee colonies in association with supplemental protein patties given to bees and is considered accidental (Ellis et al. 2008) Finally, a single specimen of E. luteola was found in a hone y bee colony in Florida ( personal observation) However, the presence of this species is assumed to be accidental. Herein I tested the hypothesis that beetle species highly integrated into honey bee colonies will be more attrac ted to hone y bee colony odors than beetles less integrated into honey bee colonies. To test this hypothesis, I recognized three distinct categories of beetle integration into colonies (Wheeler 1910). Synecthrans/symphiles (= highly integrated) are the most highly integrated beetles. This category includes species that are treated with hostility by hosts while they feed on host nest parts (synechthrans) and those species that are accepted to varying degree, and even fed by hosts oc casionally (symphiles) (Wheeler 1910, Ellis and Hepburn 2006). The SHB was the highly integrated species used in this study due to its high level of integration into

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49 honey bee colonies and its known ability to solicit food from worker honey bees (Ellis 2005, Ellis and Hepburn 2006). Accidental species occur in colonies, but more often are f ound outside of colonies (Smith 1886). Included in this category were L. insularis and E luteola which have been found in honey bee coloni es (Ellis et al. 2008, personal observation). Finally, nonintegrated beetles included Carpophilus hemipterus (L.), C. humeralis (F.) ( Coleoptera: Nitidulidae), and Oryzaephilus surinamensis (L.) ( Coleoptera: Silvanidae) that have never been captured in honey bee colonies. Beetle species were chosen based on their level of integration and availability in the study area. Furthermore, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) was the preferred non nitidulid (also used in Chapters 4 and 6), but O. surinamensis was substit uted in this experiment as T castaneum was unable to walk in the olfactometer Materials and Methods Beetles Adult O. surinamensis were obtained from in vitro rearing colonies at the USDA ARS facility in Gainesville, Florida (29.64 N, 82.35 W). Adult SHBs were captured from experi mental honey bee colonies maintained at the University of Florida Bee Biology and Research Unit (BBRU) in Gainesville, Florida ( 29.63 N, 82.36 W ). The SHBs were reared in an incubator (25C; 80% relative humidity; constant darkness) on a diet of honey, pollen, and Brood Builder (Dadant and Sons, Inc., Hamilton, IL) in a ratio of 1:1:2 respectively (Ellis et al. 2008, 2010) at the University of F lorida Department of Entomology and Nematology (29.64 N, 82.36 W). All other nitidulids were captured on rotting cantaloupe, Cucumis melo L., at the University of Florida Plant Science Research and Education Unit (PSREU) in Citra, Florida (29.41 N, 82.17 W) and reared in an incubator (24C; 40% relative humidity; constant darkness) on a tomato and prunebased diet developed by Peng and Williams (1990) for several

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50 generations prior to the experiment All nitidulids were lab reared and 2 4 weeks old at the time of the experiment. Oryzaephilus surinamensis were lab reared but were of unknown ages at the time of the experiment. All beetles were given water only for 24 hours prior to the experiment. Honey Bees Honey bees were of Russian honey bee descent an d were established in a 5 frame nucleus (nuc) hive of standard Langstroth dimensions at the PSREU before being taken to the BBRU one week prior to experiment ation The hive body of the nuc had a 1.25 cm hole drilled into the front and rear panels to accomm odate air flow. The colony contained honey, pollen, brood, worker bees, and a mature egg laying queen. During the experiment, cracks around the hive were sealed with duct tape so that airflow into and out of the colony was possible only through the drilled holes (see Graham et al. 2011) Olfactometer A 4 way olfactometer was used to determine beetle attractiveness to honey bee colony volatiles ( Vet et al. 1983, Graham et al. 2011). Glass traps designed to captur e beetles were attached at each of the four arms of the test arena (per Graham et al. 2011) The traps were attached to corrugated FEP tubing (1.15 cm inside diameter 1.27 cm outside diameter, Cole Parmer, Vernon Hills, IL). A tube from one of the traps connected to the hole at the front of the nuc while the tubes from the remaining three traps were connected to a carboloy air flow regulator (Aalborg Instruments and Controls, Inc., Orangeburg, NY) set at 0.5 liter/min. The flow regulators were attached vi a corrugated FEP tubing to a portable filteredair pump developed by the USDA ARS, Gainesville, Florida. The rear of the nuc also was attached to the air flow regulator (0.5 liters/min) and then to the pump. The bottom of the arena had an insect inlet port which was modified to accommodate the introduction of beetles. This consisted of a

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51 ~10 cm piece (diameter = 0.32 cm) and a ~4 cm piece (diameter = 0.64 cm) of industrial tubing (Cole Parmer, Vernon Hills, IL). The port was connected to corrugated FEP tubi ng, which connected to another air flow regulator set at 2 liter/min (Graham et al. 2011) The air flow regulator was connected to the house vacuum at USDA ARS by way of Master flex Tygon tubing (Cole Parmer, Vernon Hills, IL). Nylon mesh was attached at the inlet port and metal mesh was built into the glass trap ends distal to the arena to prevent beetle escape. Choice Bioassay The choice tests w ere conducted in November 2009 under red light conditions over two consecutive nights between 18:30 and 02:30 the next morning They were performed at the USDA ARS in Gainesville, Florida in a temperature controlled room held at approximately 31C and 65% relative humidity. Each replicate consisted of five consecutive, separate conspecific beetle introductions. Be etle sex was not determined. The bioassay was replicated eight times per beetle species (four replicates per beetle species per night) Species order and port assignments were randomized between replicates. Three ports delivered filtered air (control) and one delivered air pushed through the honey bee nuc. For each introduction, a beetle was placed in the ins ect inlet port and observed. A choice was made when a beetle completely entered one of the arms of the olfactometer. A beetle was given 10 minutes to m ake a choice, after which it was removed and a new beetle was introduced if no choice had been made ( per Graham et al. 2011) Beetles were used only once and non choices were not included in the analysis. Statistical Analysis Control means were averaged for each replicate (= number of beetles choosing control ports / 3 control ports = avg. # beetles going to a single control port). Beetle attraction to the olfactometer ports was tested recognizing treatment (filtered air or air from a honey bee colony) and beetle species (6 species) or level of integration (3 levels of integration) as main effects

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52 using a twoway factorial ANOVA. Within each treatment (filtered air and air f rom a honey bee colony), a one way ANOVA was used to te st beetle attraction to olfactometer ports by the main effects (1) beetle species and (2) beet le level of integration Where necessary, means were compared using Tukey Kramer tests and all analyses were conducted using JMP software (JMP 2008). Results Sinc e there was a significant beetle species treatment interaction ( F = 13.08; df = 5, 84; P < 0.01) and level of integration treatment interaction ( F = 21.68; df = 2, 90; P < 0.01), beetle attraction to the olfactometer ports was analyzed separately by be etle species a nd level of integration (Table 2 1). SHBs were sig nificantly more attracted to hive odors than to filtered air (Table 2 1). Carpophilus humeralis and O. surinamensis exhibited no preference to ports emitting either filtered air or air from a honey bee colony (Table 2 1). Carpophilus hemipterus and E. luteola were more attracted to the filtered air or were repelled by the hive odor whereas L. insularis consistently chose ports emitting filtered air in every assay (Table 2 1). When beetle speci es were grouped according to level of integration within honey bee colonies, the most highly integrated species (SHB) was attracted to hive odors more than to filtered air (Table 2 1). However, accidental species were more attracted to filtered air (or rep elled by hive odors) than to hive odors (Table 21) while the nonintegrated beetle species showed no preference to odors emanating from ports releasing either type of air (Table 2 1). SHBs, C. humeralis and O. surinamensis did not differ in their level o f attraction to air fr om a honey bee colony (Table 2 1). However SHBs were significantly more attracted to air from honey bee colonies than C. hemipterus E. luteola, and L. insularis whose level of attraction to honey bee colony odors did not differ sig nificantly from one another. Reciprocally, L. insularis E. luteola and C. hemipterus were attracted to the filtered air (or repelled by air

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53 from the honey bee colony) more often than C. humeralis and SHBs (Table 2 1). Concerning the levels of integration the highly integrated species (SHB) was more attracted to air from a honey bee colony than were the non integrated and accidental species, which decreased in their level of attraction to air from a honey bee colony respectively and significantly. That sa id, exactly the opposite relationship existed between beetles in the three levels of integration with respect to their attraction to filtered air with accidental species being most attractive to filtered air (or repelled by air from a honey bee colony) fol lowed by non integrated species and highly integrated species respectively (Ta ble 2 1). Discussion In general, there was a clear relationship between a beetles level of integration into honey bee colonies and its preference for hive odors though the rela tionship was different from that which I hypothesized originally. Although the highly integrated species (SHB) was more attracted to colony odors than beetles from the other two integration levels, I expected accidental species to be somewhat attracted to hive odors due to their known occurrence in honey bee colonies. This, however, was not the case. In fact, collectively, accidental species were less attracted to (more repelled by) hive odors than non integrated species that had never been reported from ho ney bee colonies. The SHB is well known to be highly integrated into honey bee colonies, and is attracted to honey bee nests and, more specifically, to components of honey be e alarm pheromone (Suazo et al. 2003, Torto et al. 2005, 2007a, b, Graham et al. 2011). Therefore, t he preference of SHBs for hive odors as demonstrated in this study was expected and is consistent with previous results (Suazo e t al. 2003, Torto et al. 2005, 2007a, b, Graham et al. 2011). Because the SHB represents the only beetle spe cies known to be highly integrated in honey bee colonies in this study, then

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54 this olfactory affinity for hive odors may be a key factor to its success at integrating into bee nests. Though SHBs were attracted to odors from honey bee colonies, no other beet le species in the study demonstrated a similar propensity Epuraea luteola and L. insularis the accidental associates were the species least attracted to (or most repelled by) odors emanating from the hive. Both species have been documented in honey bee coloni es (Ellis et al. 2008, personal observation) so a lack of attraction to the hive odors in this study is somewhat perplexing. T his result may be interpreted as an adaptation of the se beetles reflecting their level of integration into honey bee colonie s. Repulsion from the scent of alerted bees may be an adaptive strategy used by beetle species that do not possess other adaptive traits. Highly integrated species, such as the SHB, possess adapti ve traits that allow them to be attracted to honey bee colon ies while defending themselves against bee attacks. For example, the SHB possesses the ability to retract its appendages beneath its body (Neumann a nd Elzen 2004) thus lessening the likelihood that a bee can grab, sting, and/or remove it from a colony. In contrast, accidental species such as E. luteola lack such adaptations and defend themselves by fleeing from stressed or angered bees. Alternativ ely, accidental invaders may be attracted to odors from nest debris, pollen, or some other hive component that were not detectable in the odors emanating from the nuc used in this study. The suite of odors (e.g. brood stages and pollen reserves) may not be present in the nuc at the particular time of year the study was carried out (i.e. November). T heir observed repulsion may not be in response to pheromones produced by alerted bees, but rather to other odors associated with danger or unfavorable habitats. Overall, the nonintegrated beetle species were neither attracted to nor repelled by odors present in the nucleus colony. Oryzaephilus surinamensis, a nonintegrat ed beetle species, is a

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55 stored grain pest Despite a preference for stored grain s this spec ies may be attracted to honey bee colony odors for three reasons. First, the species has been found in pollen traps on bee colonies (Leonard 1983) Second, it is attracted to fungi produced volatiles, (Pierce et al. 1991) and fungi are present in honey bee colonies (Gilliam and Vandenberg 1997) Specifically, O. surinamensis is attracted to 3 methyl 1butanol (Pierce et al. 1991, Collins et al. 2008) which is a component of both fungi volatiles and honey bee alarm pheromone (Wager and Breed 2000) Third, it has been suggested that ancestral stored product beetles thrived on seeds that had accumulated in nests of rodents, birds, and social insects (Cox and Collins 2002) thus possibly predisposing some stored product beetles to attraction to honey bee nests. For example, Tribolium myrmecophilum a close relative of the pest species T. castaneum has been found in nests of ants and stingless bees (Lundie 1940, Angelini and Jockusch 2008) Despite these reasons to expect an attraction to hive odors by O. surinamensis, the results indicated no such preference. The other nonintegrated beetle species, C. hemipterus and C. humeralis also are attracted to 3methyl 1butanol (Phelan and Lin 1991, Nout and Bartelt 1998) just as with O. surinamensis. However, C. hemipterus was repelled by hive odors and C. humeralis wa s neither attracted nor repelled by hive odors. There are no records of these species occurring in honey bee colonies, but the related C. dimidiatus has bee n found in pollen cells within the hive (Ellis et al. 2008). Therefore, multiple Carpophilus species may forage occasionally inside honey bee colonies. Performing assay s of the behavioral responses of the accidental beetle species to d ifferent components of the hive, such as pollen, honey, brood, wax, and adult bees, would likely yield interesting far reaching results The presence of E. luteola and L. insularis in hives suggests that

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56 some colony component is attractive to the se species even though their overall response to odors in this study was one of repulsion or avoidance. In conclusion, the results from this study suggest that the most highly integrated beetle species, i.e. the SHB, was the species most highly attracted to the hive odors. Those beetle species exhibiting an intermediate level of integration into honey bee colo nies were less attracted to hive odors or even repelled by them.

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57 Table 2 1. Beetle attraction to hive odors Values are mean SE ( n ) number of beetles attracted to a port. The number of beetles attracted to filtered air was divided by three to account for multiple ports. Columnar means followed by the same capital letter are not different at Row means followed by the same lo wer case level of integration, beetle species were categorized together by level of integration into honey bee colonies using the terms highly integrated (HI) accidental (A) or non integrated (NI) (Smith 1 886, Wh eeler 1910). df = 1, 14 unless otherwise indicated. Treatment Beetle species (level of integration) H oney bee colony air Filtered air ANOVA A. tumida ( HI ) 2.250 0.453 (8)A a 0.875 0.125 (8)C b F = 8.56 P = 0.01 E. luteola ( A ) 0.125 0.125 (8)C D b 1.583 0.055 (8)A B a F = 114.33 P < 0.01 L. insularis ( A ) 0 (8)D b 1.667 (8)A a all beetles in every replication went only to the ports emitting filtered air C. hemipterus ( NI ) 0.375 0.263 (8)B C D b 1.542 0.088 (8)A B a F = 17.70 P < 0.01 C. humeralis ( NI ) 1.750 0.491 (8)A B a 1.083 0.164 (8)C a F = 1.66 P = 0.22 O. surinamensis ( NI ) 1.500 0.378 (8)A B C a 1.167 0.126 (8)B C a F = 0.70 P = 0.42 ANOVA F = 8.1; df = 5, 42; P < 0.01 F = 9.0; df = 5, 42; P < 0.01 Treatment Level of integration Air from a honey bee colony Filtered air ANOVA Highly integrated 2.250 0.453 (8)A a 0.875 0.125 (8)C b F = 8.56 P = 0.01 Accidental 0.063 0.063 (16)C b 1.625 0.028 (16)A a F = 517.64; df = 1,30; P < 0.01 Non integrated 1.208 0.248 (24)B a 1.264 0.083 (24)B a F = 0.05; df = 1,46; P = 0.83 ANOVA F = 13.4; df = 2, 45; P < 0.01 F = 14.6; df = 2, 45; P < 0.01

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58 CHAPTER 3 SHB ADAPTIVE LEG MORPHOL OGY Inquilinism is a ubiquitous lifestyle of many arthropods associated with social insect nests. Life with social insects provides inquilines a stable source of food that may be obtained either directly or indirectly from the host a s well as a means of prote ction from the environment ( Gullan and Cranston 2005). The most diverse social insect inquilines are beetles, which display a variety of integrative adaptations (Kistner 1982) Many of these adaptations are morphological, providing inquilines defensive postures or structures which effectively negate biting, stinging and/or removal tactics used by the social insect host to defend against the inquiline. There are many examples of beetles that possess adaptive morphology that permits them to integrate into social insect colonies. These include histerids, leiodids, chrysomelids, corylophids, cybocephalids, and nitidulids among others. Histerid beetles frequently are found in ant nests (Kistner 1982, Kovarik and Caterino 2001, Caterino and Vogler 2002, Kovarik and Tishech kin 2004, Caterino and T ishechkin 2008). Inquilinous histerids generally are considered to possess four morphological modifications associated with inquilinism, including: 1) a hard smooth exoskeleton; 2) a convex and often vaulted body; 3) a retractable h ead; and 4) short legs that can be retracted within body grooves (Reichensperger 1924, cited in Kistner 1982). Additionally, the myrmecophilous (ant inquilines) histerid Psiloscelis spp. have flattened and expanded tibiae (Kistner 1982) Myrmecophilous leiodids have proportionately shorter legs with reduced contiguous tarsal segments as well as more compact antennae than their freeliving counterparts (Kistner 1982) Melittophilous (bee inquilines) leiodids in the genus Scotocryptus are able to retract their head, antennae, and legs beneath a hemispheric body, making them difficult to grasp (Salt 1929, Peck 2003) Ventral grooves on the head accommodate the antennae and caniculate femora receive the

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59 tibiae (Roubik and Wheeler 1982) during retraction of the appendages. These beetles possess elytra that are held together tightly by a tongue and groove mechanism, thereby protecting the relatively soft, lightly sclerotized tergites from attack (Roubik and Wheeler 1982) The melittophilous corylophid Cleidostethus Arrow has similar adaptations to those of Scotocryptus (Bowstead et al. 2001, Salt 1929) T he head is small and can be retracted along with the antennae between the pronotum and prosternum, the margins of which are flattened and explanate The legs are small in proportion to the body, and the tibiae retractable within the femora, which subsequen tly fit into grooves on the body proper. The elytra are fused, and the only moveable region of the major body segments is between the prothorax and mesothorax. However, even this region can be made rigid, allowing the beetle to firmly grasp the substratum (Sa lt 1929) The chrysomelid Hemisphaerota cyanea (Say) can be found on palmetto plants in the southeastern United States (Woodruff 1965) and possesses a number of morphological features that allow it to survive ant attacks. The beetle has evolved highly modified tarsi that are covered with branched seta e and pores that secrete sticky oils. Upon attack by ants, the beetle presses its tarsi firmly to the plants surface, creating a higher area of contact between the hairs and sticky oil with the substratum. Additionally, H. cyanea has a hemispherical, glab rous, convex body that is difficult for ants to grasp with their mandbiles (Attygalle et al. 2000, Eisner and Aneshansley 2000) Ant removal of the beetle from the palmett o consequently becomes difficult or impossible to achieve. Nitidulidae contains many examples of inquilinism and the phenomenon purportedly has arisen independently at least four times within the subfamily Nitidulinae (Cline 2005). Cychramptodes murrayi Reitter is a predator of the waddle tick scale, Cryptes baccatus

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60 (Maskell) (Coccidae). These scales are tended by ants, and the nitidulid beetles possess morphological adaptations suited for passive defense. These inclu de a highly convex and glabrous body, ventrallyprojecting hypomera and epipleura that conceal the underside of the beetle including the legs, broad flattened tibiae, and caniculate femora. These leg structures allow the beetles to retract their legs and l ay flat against a surface (Kirejtshuk and Lawrence 1992, Leschen 2000) Species of Amphotis Erichson are associated commonly with ants (Parsons 1943, Hlldobler 1968, Audisio 1993) and have a broad, flat body which conceals the legs if attacked (Cline 2005). Amborotubus Leschen & Carlton is a recently described nitidulid genus that has been hypothesized to be a social insect inquiline based solely on its morphology, which includes flattened femora and tibiae and a shielded appearance. Cy lindroramus Kirejtshuk and Lawrence has a similar body shape as Amborotubus but its legs are hidden in lateral view, unlike Amborotubus (Leschen and Carlton 2004) Cylindroramus (Cline 2005) and Arhina Murray (A R C line pers. comm. ) are postulated as social insect inquilines based solely on their morphology. Herein, I tested the hypothesis that the SHB exhibits several inquilinous morphological adaptations specific to legs, and that other related species have a decreasing number of these structures based on their degree of integration into honey bee colonies. Specifically, sap beetles that have been found in colonies on occasion (accidentals) are expected to have fewer morphological leg adaptations than SHBs but more than those beetles which have not been found in colonies. To test this hypothesis the leg morphology of the SHB was compared to that of five bee colony accidentals ( Aethina villosa Reitter L. insularis G. fasciatus C. dimidiatus and E. luteola ) and four nitidulid species which have not been found in bee colonies including

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61 Stelido ta geminata (Say), Amphicrossus ciliatus (Olivier), Carpophilus hemipterus L., and C. humeralis (F.) Materials and Methods SHBs were collected from a honey bee colony at the University of Florida Bee Biology Research Unit in Gainesville, Florida ( 29.63 N 82.36 W ). Aethina villosa specimens were supplied from the Andrew R. Cline Collection (ARCC), which currently is housed at the California State Collection of Arthropods (CSCA) in Sacramento, California. Individuals from all other beetle species were col lected at the University of Florida Plant Science Research and Education Unit (29.41 N, 82.17 W) in Citra, Florida on rotting cantaloupes, Cucumis melo L. Beetle sex was not determined. Scanning electron micrographs were prepared according to standard pr otocols (Eisner and Aneshansley 2000) at the Division of Plant Industry in Gainesville, Florida using a JEOL JSM 5510LV SEM. Species were scored based on presence or absence of the following morphological traits: caniculate femora (i.e. Scotocryptus spp. i n Roubik and Wheeler 1982), flattened/expanded tibiae (= width (i.e. Psiloscelis spp. in Kistner 1982), undulate tarsal setation (i.e. Cantharis fusca in Beutel and Gorb 2001), and dense tarsal setation (i.e. Hemisphaerota cyanea in Eisner and Aneshansley 2000). These characters were chosen to represent adaptation to an inquilinous lifestyle (Kistner 1982). Scores were based on visual inspection of SEM images. Results SHBs exhibit all four leg morphological characters that were investigated in this study (Table 31, Figures 31, 3 2). All species except L. insularis G. fasciatus and E. luteola have dense setal pads on the ventral surface of the tarsomeres (Table 31, Figure 32). Also, all species except S. geminata A. ciliatus C. dimidiatus and E. luteola possess undulate tarsal setae (Table 31, Figure 32). The Carpophilus species have broad flattened tibiae (Table 31, Figure 31).

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62 Discussion The data may suggest that the presence of caniculate femora is correlated with inquili nous species since the SHB was the only species to posse ss this morphological characteristic (Table 31). That said, the SHB was the only beetle species to possess all of the studied leg characters. In sharp contrast, E. luteola exhibited none of the morph ological features investigated in this study though the beetle has been found in honey bee colonies before ( personal observation). Consequently, the data do not support the tested hypothesis that the number of leg morphological adaptations a beetle possess es is directly related to the degree of integratio n into honey bee colonies (i.e. E. luteola exhibits a moderate level of integration it is an accidental species though it possesses none of the studied leg characteristics). The caniculate femora pos sessed by SHBs which accommodate the broad flattened tibiae, allow the beetle to retract its legs underneath the body when encountering an aggressive bee (Neumann et al. 2001, Neumann and Elzen 2004) Furthermore, the SHBs dense, undulate tarsal setae presumably allow it to grasp a substrate, similar to the phenomenon found in t he chysomelid Hemisphaerota cyanea (Eisner and Aneshansley 2000) though this has not been tested nor has the presence of oil on the tarsi of SHBs A secure substrate attachment would prohibit bees from flipping the beetle over and stinging and/or removing them from the nest a feature which Schmolke (1974) detected in his behavioral observations. These leg adaptations accompany other defenseproviding morphological features th at the SHB possesses, including a hardened exoskeleton and overlapping body regions (Kistner 1979, Neumann and Elzen 2004), all of which allow the beetle to invade, colonize, and reproduce within honey bee colonies. The trophic habits of the beetles used in this study, and nitidulids generally, likely predispose them to possessing at least some of the morphological adaptations that would increase their ability to infest bee nests. Many nitidulids are associated with f ungal substrates in detritus,

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63 rotting fruits, subcortical spaces, and fungal fruiting bodies (Parsons 1943, Cline 2005) These conditions often favor flat to relatively flattened body forms. Aethina villosa L. insularis G. fasciatus A. ciliatus C arpophilus spp., and E. luteola not only are known from sap flows and fungal substrata but may also occur in flowers as well. Thus, their bodies should be somewhat compact and /or flattened so that they can enter small cavities such as a developing flower b ud (Parsons 1943, 1972, Vogt 1950, Nadel and Pena 1994, Majka and Cline 2006) Stelidota geminata occurs mainly in leaf litter but also at sap flows and overripe fruits (hence, the common name strawberry sap beetle) (Parsons 1943, Loughner et al. 2007) Therefore, it must be able to access small spaces as well. Furthermore, males of several beetle species exhibit dense tarsal setation used to grasp the female during copulation (A. R Cline, pers. comm.). However, the specimens used in this study were not sexed, so such a correlation between sex and setation density could not be surmised. Other species of Aethina are associated with decaying plant matter (including fruits) and flowers (Kirejtshuk 1997, Kirejtshuk and Lawrence 1999), as well as occur within leaf l itter (A R C line pers comm. ) Therefore, the general body form of Aethina should enable them to enter concealed places, including the small crevices of a bee nest. This hiding behavior is common in SHBs (Lundie 1940, Schmolke 1974, Neumann et al. 2001, Neumann and Elzen 2004, Ellis 2005) Also, some bee hive odors are identical to those associated with decay (Phelan and Lin 1991, Pierce et al. 1991, Nout and Bartelt 1998, Wager and Breed 2000, Collins et al. 2008, Graham 2009) so attraction to volatiles emanating from decomposition might predispose SHBs to attraction to honey bee nests. However the causative mechanism underlying bee nest see king behavior in SHB ancestors remains unclear.

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64 The striking differences between the leg characters of the two Aethina species investigated in this study provide further evidence that the SHB is welladapted for penetrating and persisting within honey bee nests. For example, the ability to retract appendages beneath the body is not crucial to the survival of the freeliving species A. villosa but it is for the inquilinous SHBs Thus, despite the two species close phylogenetic relationship, only the SHB displays caniculate femora and flattened tibiae. Also, the fact that the two species share dense, undulate tarsal setae suggests that this character is an exaptation which was adopted by SHBs for use within honey bee nests. These tarsal setation character s may have helped A villosa enter the honey bee colony in which it was found (A R C line pers. comm ); however the absence of other morphological and behavioral adaptations may have precluded this species from becoming fully integrated into the hive envi ronment. The degree of morphological adaptation in SHBs may suggest a long association with honey bee nests. This idea is supported when considered in conjunction with other characters the SHB displays. These include (1) behavioral mimicry, wherein a beetl e which is otherwise trapped within the colony is able to solicit food from bee prison guards trophallactically (Ellis et al. 2002b, Ellis 2005) (2) the beetles ability to detect and cue into honey bee volatiles (Suazo et al. 2003, Torto et al. 2005) and (3) the harboring of a yeast ( Kodamaea ohmeri ) which, when mixed with bee collect ed pollen, produces volatiles attractive to other conspecifics (Torto et al. 2007a ) These character s act synergistically to accommodate SHB entrance and integration within honey bee colonies.

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65 Table 3 1. Presence /absence of hypothesized inquilinous morphological characters in selected nitidulid species Leg character Femora Tibiae Tarsi Species caniculate flattened/expanded Undulate setae Dense setae Aethina tumida X X X X Aethina villosa X X Stelidota geminata X Lobiopa insularis X Glischrochilus fasciatus X Amphicrossus ciliatu s X Carpophilus dimidiatus X X Carpophilus hemipterus X X X Carpophilus humeralis X X X Epuraea luteola

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66 Figure 31. SEM images of entire legs. The species are as follows: A) Aethina tumida, B) A. villosa C) Stelidota geminata, D) Lobiopa insularis E) Glischrochilus fasciatus F) Amphicrossus ciliatus G) Carpophilus dimidiatus H) C. hemipterus I) C. humeralis J) Epuraea luteola. All images are the left proleg, except for J, which is the left m esoleg. C. humeralis (I) received a puncure in the femur during preparation.

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67 Figure 32. SEM images of tarsi. The species are as follows: A) Aethina tumida, B) A. villosa C) Stelidota geminata, D) Lobiopa insularis E) Glischrochilus fasciatus F) Amph icrossus ciliatus G) Carpophilus dimidiatus H) C. hemipterus I) C. humeralis J) Epuraea luteola. A, E, F, and H are images are the right mesotarsi; B is the right protarsi; C, D, G, and I are the left protarsi; and J is the right metatarsi.

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68 CHAPTER 4 ADAPTIVE BEHAVIOR OF HONEY BEES TOWARD BEETLE INVADE RS EXHIBITING VARIOUS LEVELS OF COLONY INTEGRATION Social insect colonies host many inquilinous species exhibiting various lev els of integration (see Kistner 1982, Chapter 1). This is true particularly of ant (Hymenoptera: Formicidae) and termite (Blattodea) nests. In contrast, bees seem to host fewer guests, and those that are present exhibit fewer adaptations (Wilson 1971, Kistner 1982). Some potential explanations for this have been offered, although fe w have been tested directly. One explanation offered by Wilson (1971) is the tendency of bees to nest in arboreal locations. This presents an obstacle that must be overcome by would be invaders as they must first locate and invade an arboreal colony Howev er, groundnesting bees are common, and researchers conducting a survey of parasites in wasp and bee nests found no significant difference in parasite loads between ground and tree nests (Wcislo 1996) Although discounted by Wilson (1971) as a potential explanation, Kistner (1982) suggests that the effectiveness of defense likely is part of the explanation for this apparent phenomenon. In ants, he points out, those with effective stings and other defen ses (e.g. Solenopsis sp.) host fewer guests than do species with lessprominent defenses (e.g. army ants). Western honey bees have particularly well defended nest entrances, so colony integration by wouldbe invaders begins at the colony entrance where hon ey bees station guard bees to keep out intruders. When a potential invader attempts to enter the nest, guard bees antennate the subject to determine whether it is a nestmate or an intruder (Breed et al. 2004) If the guard determines the invader is a threat, it either attack s the invader itself or, if the intruder is particularly large or aggressive, it recruit s more guards to the area (Breed et al. 2004) Thus, few arthropod species are successful at penetrating the nest entrance. Thus, intrusion into the hive by the SHB is the exception and not the rule for bee hive invasion

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69 The SHB is considered a highly integrated invader of honey bee colonies, an d possesses many adaptations that allow it to enter and thrive within host colonies (Ellis and Hepburn 2006). When approached by a honey bee, the SHB retract s its appendages beneath the body, thus inhibiting guard bees from grasping it to sting and/or remo ve it from the hive (Neumann and Elzen 2004) SHB leg morphology complements this appendage retraction behavior, i.e. the femora are grooved to a ccommodate broad flattened tibiae during leg retraction (Chapter 3). SHBs quickly find hiding places where honey bees cannot reach them upon host colony invasion. However, this inhibit s beetle escape nonetheless due to host bees confining them to these areas (Neumann et al. 2001, Ellis 2005) While honey bees can keep the SHB s confined in the hiding places indefinitely at low to moderate infestation levels, SHBs survive in these prisons through a form of behavioral mimicry, wherein they solicit food from honey bee guards (Ellis et al. 2002b Ellis 2005) To begin to address honey bee responses to beetles, I performed an experiment to ascertain if bees guarding colony entrances exhibit differential behav ioral responses to beetles that are integrated in honey bee colonies at various levels. Specifically, I looked at four different integration levels (C hapter 2). The first is synechthrans/symphiles (= highly integrated), which includes species that prey upo n host colonies while being treated with hostility by the hosts (synechthrans) as well as those which are accepted to some degree by their hosts, possibly being f ed by them (symphiles) (Wheeler 1910, Ellis and Hepburn 2006). The second group includes the a ccidentals (defined above). The third group includes non integrated species that have not been found in colonies previously. Five species of nitidulid beetles ( Coleoptera: Nitidulidae), one tenebrionid beetle ( Coleoptera: Tenebrionidae), and one glass bead (control) were used to test the hypothesis that guard honey bees will exhibit varying defensive responses toward invaders at the

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70 nest entrance in relation to the invaders degree of integration. The test subjects included the SHB (highly integrated), L. i nsularis (accidental), E. luteola (accidental), Carpophilus humeralis Fabr. (nonintegrated), C. hemipterus L. (nonintegrated), Tribolium castaneum (nonintegrated), and a small b lack jewelry bead as a control. Materials and Methods Beetles Adult SHBs and T. castanaeum were obtained from in vitro rearing colonies at the USDA ARS, Gainesville, Florida (29.64 N, 82.35 W) and were used to initiate rearing programs at the University of Florida Department of Entomology and Nematology (29.64 N, 82.36 W). Adults of the other nitidulid species were collected from rotting cantaloupe, Cucumis melo L., at the University of Florida Plant Science Research and Education Unit (PSREU) in Citra, Florida (29.41 N, 82.17 W) and used to initiate rearing pro grams. All species, with the exception of SHBs and T. castaneum were reared in an incubator (24C; 40% relative humidity; constant darkness) on a tomato and prunebased diet developed by Peng and Williams (1990). SHBs, E. luteola C. humeralis and C. hem ipterus were 2 4 weeks old at the time of the experiment. Due to limited availability, L. insularis used during the study were obtained from rearing programs or field collections. As such their age at the time of the experiment was unknown. Though T cas taneum were lab reared, their age also was unknown at the time of the experiment. Beetle sex was not determined. Observation Hives Four observation hives were created from previously established honey bee colonies of mixed European origin located at the PS REU. The observation colonies were given three frames containing pollen, capped honey, brood of all ages, worker bees, and a n egg laying queen. The observation hive structure (Figure 41) accommodated three, 23.0 42.6 cm (L W) wooden

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71 frames. The entran ce corridor to the hives contained a 10 20 cm (L W) test arena with delineated 1 cm squares, and could be closed at both ends with Plexiglas s doors containing holes to accommodate ambient airflow (Figure 4 1). The arena was used for the behavioral assa ys and included a side entrance where test beetles could be introduced directly into the arena. Behavioral Assays All trials were conducted in October 2009 and under red lights between 18:30 and 02:30 the following morning as the SHB has been found to be m ore active within the hive during the evening (Ellis et al. 2003a) For each trial, one beetle or a 60 mg black bead (Darice Inc., Strongsville, OH, USA) tethered to a 15 cm piece of monofilament fishing line (Zebco, Tulsa, OK, USA) was placed through the side entrance into the test arena. Beads and fishing line were auto claved before the trials. Prior to introducing the beet le or bead, the test arena was closed at both ends to trap guard bees in the arena. Once the beetle or bead was introduced, the guard bees responses to the beetle or control bead were recorded for 60 s. Three potenti al guard bee responses were recognized (described by Elzen et al 2001): ignore (a bee s head comes within 5 mm of the subject, but there is no contact); contact (the bee makes physical non defensive contact with the subject); and defend (the bee attempts to sting and/or remove the subject). Because some beetles, particularly SHBs, were able to escape from the arena despite efforts to limit this, only trials in which the beetles remained in the arena for were run simultaneously in the four observation hives with four different observers and one individual from a given beetle species or bead being inserted randomly into colonies (completely randomized design blocked on colony, N = 6 beetle species and 1 control (black bead) 15 individuals 4 observation colonies) ) All observers were trained prior to the experiment to reduce observer bias. The sliding Plexiglas doors on either side of the test arenas were opened briefly (

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72 reduce gua rd bee agitation. Also, the used bead was removed or the beetles were allowed to exit the test arenas at this time. Individual beetle and beads were used only once. Statistical Analysis Bee responses to beetles or beads were converted to proportion data due to the variable number of guard bees trapped in the test arena for each trial. This is not the proportion of bees performing a given response but rather the proportion of all responses that were either ignore, contact, or defend as a single bee may hav e demonstrated all of these behaviors multiple times during the 60 s observation period. All proportion response data ( i.e. dependent variable s ) were transformed with arcsine to stabilize the variance. For each type of response (ignore, contact, defend), the transformed proportions were analyzed using a two way ANOVA (JMP 2008) recognizing colony (A D) and either invader type (6 beetle species and 1 control bead) or level of integration (highly integra ted, accidental, non integrated, control bead) as main effects and colony invader type or level of integration as the interaction term. If t he interaction of colony invader and colony level of integration was significant then these variables were an alyzed within colony. When the ANOVA detected significant effects means were compared using Tukey Kramer tests, accepting differences at P Untransformed means are reported in this chapter Results The pro portion of bee responses to invaders t hat were ignore (invader type: F = 6.1; df = 3, 391; P < 0.01; level of integration: F = 4.6; df = 3, 403; P < 0.01) and contact (F = 5.4; df = 3, 391; P < 0.01; level of integration: F = 3.8; df = 3, 403; P = 0.01) varied significantly by colony. In gener al bees from colony C (0.52 0.02, 105) or D (0.48 0.03, 105) ignored invaders more so than bees from colony A (0.37 0.04, 105). Bees in colony B (0.42 0.02, 104) ignored invaders at a lev el equal to that of bees in all of the other colonies (data are mean SE

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73 proportion of responses that were ignore, n beetles ). Regarding contacting nest invaders, guard bees from colony B (0.48 0.02, 104) contacted nest invaders more than bees from colony D (0.35 0.02, 105). Bees in colonies A (0.41 0.03, 105) and C (0.40 0.02, 105) contacted invaders at a rate equal to bees in colonies B and D (data are mean SE proportion of responses that were contact, n ). In contrast to bee ignore and contact responses the proportion of responses that were defend d id not vary significantly by colony (invader type: F = 1.4; df = 3, 391; P = 0.23; level of integration: F = 1.7; df = 3, 403; P = 0.15). Colonies A (0.16 0.03, 105), B (0.10 0.01, 104), C (0.08 0.01, 105), and D (0.13 0.02, 105) exhibited defensiv e responses equally (data are mean SE proportion of responses that were defend, N). Overall, bees differed in their level of ignore and contact responses to the different nest invaders, as well as to the different levels of integration of the invaders (Table 4 1). Guard bees ignored T castaneum and E. luteola more than they ignored C. hemipterus L. insularis SHBs, and the control bead. Bees ignored C. humeralis more than they ignored the SHB and the control bead. When invaders were grouped by level o f integration, guard bees ignored accidental and nonintegrated species moreso than highly integrated species or the control bead (Table 4 1). In general, the control bead was contacted more by guard bees than were the six beetle species. Bees contacted L. insularis more than T. castaneum and E. luteola while their contact response to C. humeralis C. hemipterus and the SHB did not differ significantly from the other beetles. When considering invader level of integration, the control bead was contacted more by guard bees than any other invader group (Table 41). Guard bee ignore (invader type: F = 0.8; df = 18, 391; P = 0.65; level of integration: F = 0.8; df = 9, 403; P = 0.63) and contact (invader type: F = 0.68; df = 18, 391; P = 0.84; level of integr ation: F = 0.7; df = 9, 403; P = 0.72) responses were not affected by the interaction

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74 between colony and invad er type or level of integration. However, the defend response was affected by the interaction (invader type: F = 2.3; df = 18, 391; P = 0.01; leve l of integration: F = 2.2; df = 9, 403; P = 0.02). In colony A, the proportion of responses directed at SHBs that were defensive was higher than those toward any other species with the exception of C. hemipterus which itself was not treated more defensive ly than the other species. Also, the highly integrated species i.e. the SHB, was treated more defensively than groups at other levels of integration. In colony B, the SHB was treated defensively by bees more than any other species except for the two Carpophilus species. Bees in colony B responded to all non SHB beetles with equal defensive responses Regarding level of integration, bees in colony B treated the highly integrated species i.e. the SHB, more defensively than any other group. Also, the non int egrated species were treated more defensively than the control bead (Table 4 2). In colony C, the SHB elicited defensive responses from bees more than the control bead or any other beetle species. Also, the SHB was the sole representative o f the highly int egrated species and received more defensive responses from the bees (proportionally) than beetle groups at other levels of integration and the control bead. A similar pattern was seen in colony D except that bees responded to SHBs and the control bead with equal defensive responses (Table 4 2). All non SHB beetle species elicited similar defensive responses from guard honey bees, and all beetles except for T. castaneum were treated with the same level of defense as the bead. When grouping beetles by level o f integration in colony D, the highly integrated species was treated more defensively than the accidental and nonintegrated species. The control bead was treated similarly as compared to all other groups. Discussion Overall, the most integrated species, i .e. the SHB, was ignored the least and treated more defensively by guard bees than beetle groups representing all other levels of integration. T he se

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75 findings contrast with those of previous findings by Elzen et al. (2001) who demonstrated that European subspecies of honey bees did not treat SHBs and a control push pin differently (either ignore, contact, or defend). There are multiple possible reasons that these results differed from those of Elzen et al. ( 2001). First, Elzen et al. ( 2001) conducted their s tudy in wooden hoarding cages, which may affect bee/beetle behavior unpredictably (i.e. an artificia l bioassay). Secondly, the study herein was conducted nearly one decade later possibly suggesting evolving bee development of defensive adaptations toward SHBs over time. Finally, Elzen et al. (2001) collected bees randomly from colonies, whereas in this study, bees already guarding the colony entrance were sequestered. While one would expect bees guarding colony entrances ( e.g. this study) to be defensive (Breed et al. 2004) the same is not intuitive when randomly collecting bees from a colony ( e.g. Elzen et al. 2001) N urse bees, wax builders, etc. all would be sampled and are known to express reduced defensive behavior relative to guard bees (Breed et al. 1992a) Regardless, guard bees in this study were more defensive tow ard the SHB, the highly integrated species, than they were toward all other beetle groups at other levels of integration. The heightened defensive response by guard bees toward the SHB while exhibiting a consistently lower defensive response toward invaders at all other levels of integration may be an effort by bees to maximize energy efficiency. Like many other organisms, honey bees are known to engage in behaviors that are energetically conserved (see Dedej and Delaplane 2005) For example, this has been documented for honey bee foraging behavior, where honey bees are driven to nectar larceny to increase net energy profit (Dedej and Delaplane 2005) Though honey bee defensive behavior was not quantified energetically, one could see how expending energy to attack only those intruders known to threaten colony health (i.e. SHBs) while ignoring or minimizing defensive responses toward all other species is an effo rt to maximize energy

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76 conservation. If energy conservation is important in bee defensive responses to nest invaders at the colony entrance, then these data are consistent with the superorganism theory of social insect colony organization. Individual organi sms, including insects and mammals, are known to initiate immune responses to invading pathogens at an energy cost (e.g. Freitak et al 2003, Simmons and Roney 2009, Cutrera et al 2010). Consequently, one could argue that guard bee behavior at the nest en trance is initiated conservatively yet efficiently to limit invasion by organisms (i.e. pathogens) threatening the entire colony (or superorganism). The SHB is integrated more into honey bee colonies than the other beetle species tested. As such, one m ay hypothesize that the SHB has mechanism s to reduce defensive response s by guard honey bees at the colony entrance. Such mechanisms usual ly chemical or morphological (Wilson, 1971; Kistner, 1979), in highly integrated invaders of other social insects such as ants and termites are common (Dettner and Liepert 1994) The SHB employs an entire suite of morphological adaptations that allow it to penetrate honey bee colonies. These include a limuloid body form, the ability to retract its appendages beneath its body (Neumann and Elzen 2004, Ellis and Hepburn 2006), and flattened tibiae and grooved femora to accom modate retraction, (Chapter 3). In this study, guard bees displayed an interesting behavior toward nest invaders that may represent an i ntegral defensive response. This behavior began with a honey bee approach ing the invader, turning around so the abdomen faced the subject, and then kick ing the metathoracic legs backward s while fanning t he wings. Bees employed this behavior regularly (>50% of all introductions) against T. castaneum (less so with other invaders). This behavior has been documented in honey bees as a defense against ants (Spangler and Taber 1970) and other small

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77 insects (Yang et al. 2010) and may be advantageous to include an analysis of this behavior in future studies of arthropod invasion into honey bee colonies. B ee responses to nest invaders se emed to increase in intensity after initial bee contact with a ny potential invader. There are two possible explanations for increased intensity of responses during the observation period. First, a honey bee guard, when disturbed, may release pheromones fro m her sting gland that subsequently excite surrounding bees and heighten the overall defensive response toward the invader (Breed et al. 2004) Second, guard bees may m ark intruders with 2 heptanone, an alarm pheromone component that is pr oduced in the mandibular glands. This marking indicates an invader status to other guard bees (Breed et al. 2004) Thus heightened bee defensive responses toward an invader after initial contact is likely a dvantageous for colonies, and subsequently decreases the chance that a marked invader will infiltrate a colony successfully. In contrast to defensive responses toward beetle invaders, guard bee contact responses toward invaders were not significantly dif ferent among the three levels of colony integration. This constant guard bee response is consistent with the regular mode of intruder discrimination performed by guard bees at the nest entrance. Guard bees antennate all intruding arthropods to the hive and then subsequently decide to accept or attack the invader (Breed et al. 2004) B ee responses to the control bead were interesting. Guard b ees ignor ed the control bead less than any beetle species except SHBs, and they contacted it significantly more than any beetle species. Perhaps guard bees were curious toward the foreign object which did not release any biologically based volatiles Such a beha vior would be adaptive if the guard bee encountered invaders that emit low to undetectable (by bees) amounts of volatiles. For example, the argentine ant, Linepithema humile (Mayr), emits relatively low volatile titers compared to

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78 other ant invaders. Spang ler and Taber (1970) hypothesized that this enables the ant to enter honey bee colonies easily. Alternatively, bees may have recognized the bead as refuse and were assessing the size of the object to determine whether it could be removed effectively from t he colony. A difference in bead size assessment between colonies may explain why the bead was treated defensively in colony D (Table 4 2). T he se guards may have determined that the bead was an appropriate size to be expelled, and the expulsion attempts wer e recorded as defensive responses. Furthermore, the bead may have adsorbed odors as a result of observer handling, thereby eliciting curiosity from the bees, but not defense. The four colonies used in the study differed significantly with respect to the le vel they expressed ignore and contact responses toward invaders, thereby suggesting that some degree of genetic variation for these behaviors exists. Variation is important for bee populations to be able to adapt to environmental stresses, including wouldbe invaders. As such, variations in these traits (ignore and contact responses toward invaders) may be influenced by selection pressures, thus enabling colonies to adapt to invader pressures over times. In contrast to colony ignore and contact behavior, colonies did not differ in their pooled defensive res ponse toward invaders The SHB remains one of the most highly integrated insects in honey bee colonies and social bee colonies in general (Ellis and Hepbur n 2006) despite heightened defensive responses by honey bees toward SHBs These defensive responses include increased defensive activity at the nest entrance (this chapter ), overall aggression ( Elzen et al., 2001), and confinement behavior (Ellis 2005) The SHB accomplishes its high level of integration through morphological (Chapter 3), behavioral (Ellis e t al. 2002b) and potentially chemical adaptations. The data presented herein demonstrate that beetle invasion into honey bee colonies is met by bees in the form of a heightened defensive response.

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79 Table 4 1. Proportion of honey bee guard responses tha t were ignore and contact during the observation period. Data are mean SE ( n ) proportion of total responses that were either ignore or contact. Columnar means followed by the same letter are not ukey Kramer method. For Invader type, bee responses to each beetle species and the control bead were analyzed. For Level of integration, beetle species were grouped together by their level of integration into honey bee colonies. Invader type Species Ignore Contact A. tumida 0.314 0.028 (60)c d 0.368 0.018 (60) b c L. insularis 0.392 0.036 (59)b c 0.489 0.036 (60)b E. luteola 0.613 0.039 (60)a 0.279 0.032 (60) c C. humeralis 0.510 0.031 (60)a b 0.395 0.030 (60) b c C. hemipterus 0.436 0.041 (60)b c 0.362 0.033 (60) b c T. castaneum 0.643 0.031 (60)a 0.292 0.026 (60) c Control 0.220 0.022 (60)d 0.693 0.024 (60) a ANOVA F = 18.2; df = 6, 391; P < 0.01 F = 18.7; df = 6, 391; P < 0.01 Level of integration Level Ignore Contact Highly integrated 0.314 0.028 (60)b 0.368 0.018 (60)b Accidental 0.503 0.029 (119)a 0.383 0.026 (119)b Non integrated 0.529 0.021 (180)a 0.350 0.018 (180)b Control bead 0.220 0.022 (60)b 0.693 0.024 (60)a ANOVA F = 21.1; df = 3, 403; P < 0.01 F = 26.5; df = 3, 403; P < 0.01

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80 Table 4 2. Species colony interaction on the proportion of guard bee responses that were defend during the observation period. Data are mean SE ( n) proportion of total responses that were defend Columnar means followed by the same letter are not Kramer method. For Invader type, bee responses to each beetle species and the control bead were analyzed. For Level of integration, beetle species were grouped together by their level of integration into honey bee colonies. Invader type Species Colony A Colony B Colony C Colony D A. tumida 0.439 0.062 (15)a 0.216 0.031 (15)a 0.291 0.037 (15)a 0.329 0.054 (15)a L. insularis 0.144 0.069 (15)b 0.055 0.012 (14)b c 0.040 0.012 (15)b 0.032 0.019 (15)b c E. luteola 0.059 0.047 (15)b 0.069 0.015 (15)b c 0.045 0.016 (15)b 0.127 0.043 (15)b c C. humeralis 0.042 0.025 (15)b 0.109 0.022 (15)a b 0.049 0.016 (15)b 0.116 0.038 (15)b c C. hemipterus 0.217 0.094 (15)a b 0.135 0.026 (15)a b 0.081 0.028 (15)b 0.111 0.048 (15)b c T. castaneum 0.119 0.059 (15)b 0.079 0.019 (15)b c 0.036 0.012 (15)b 0.026 0.024 (15)c Control 0.084 0.034 (15)b 0.030 0.009 (15)c 0.053 0.023 (15)b 0.181 0.063 (15)a b ANOVA F = 5.7; df = 6, 98; P < 0.01 F = 7.8; df = 6, 97; P < 0.01 F = 11.6; df = 6, 98; P < 0.01 F = 6.6; df = 6, 98; P < 0.01 Level of integration Level Colony A Colony B Colony C Colony D Highly integrated 0.439 0.062 (15)a 0.216 0.031 (15)a 0.291 0.037 (15)a 0.329 0.054 (15)a Accidental 0.102 0.042 (30)b 0.062 0.009 (29)b c 0.042 0.010 (30)b 0.080 0.025 (30)b Non integrated 0.126 0.039 (45)b 0.108 0.013 (45)b 0.055 0.011 (45)b 0.084 0.022 (45)b Control bead 0.084 0.034 (15)b 0.030 0.009 (15)c 0.053 0.023 (15)b 0.181 0.063 (15)a b ANOVA F = 9.2; df = 3, 101; P < 0.01 F = 14.2; df = 3, 100; P < 0.01 F = 23.2; df = 3, 101; P < 0.01 F = 10.1; df = 3, 101; P < 0.01

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81 Figure 41. Diagram of observation hive. The white arrow indicates the location where beetle species or control beads were introduced into the test arena. The black arrows indicate the location of the Plexiglass doors that captured guard bees in the test arena.

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82 CHAPTER 5 DIETARY EFFECTS ON THE CUTICULAR PROFILE OF SHBS AND ITS EFFECT ON BEHAVIORAL TREATMENT BY HONEY BEES Nestmate recognition is a feature common to most, if not all, eusocial insect colonies (Breed 2003) This type of recognition is the ability of an insect to discriminate between conspecifics that are part of the colony and those that are not Nestmate recognition is often based on the specific blend of hydrocarbons found on the insects exoskeleton These hydrocarbons can be of a glandular source, as occurs in the ant Cataglyphis niger (Andre) (Hymenoptera: Formicidae) (Soroker et al. 1994) which is secreted on the exoskeleton surface. Hydrocarbons also can be cuticlederived, either resulting from endogenous chemicals from internal sources or diet based Diet based cues occur in the ant Linepithema humile which acquires its chemical profile from insect prey (Liang and Silverman 2000) Additionally these hydrocarbons can be externally derived, being acquired by contacting another source of the hydrocarbons which rub off onto the insect. This occurs in h oney bees and also in combination with other cues (e.g. genetic) (Breed 1983, Breed et al. 1995, Breed 1998, Downs and Ratnieks 1999) Though social insects can recognize nestmates, and by application non nestmates as well, other i nsects occasionally succeed at entering soci al insect colon ies There are three main methods by which arthropods can invade/integrate into social insect nests : (1) adaptations in body form, (2) behavior, and (3) chemical use (Wilson 1971, Kistner 1979) While not observable w ithout scientific equipment, chemical adaptations are well known among arthropods invading termite wasp, ant, and bee nests In honey bee colonies there are a few examples of invading arthropod use of chemical mimicry to invade/integrate into the colony (Chapter 1). Varroa destructor has a hydrocarbon profile similar to its host (Nation et al. 1992) Furthermore, this profile changes with the host as

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83 it under goes different developmental stages, thereby indicating a high degree of chemical adaptation (Martin et al. 2001) T he deaths head hawkmoth Acherontia atropos invades honey bee colonies and s ubsequently feed on honey within This is, in part, accommodated by its thick cuticle, which cann ot be penetrated easily by stings. Once inside the hive the moth can move about the colony rather unnoticed and with impunity This occurs because t he moth chemically mimick s the honey bee hydrocarbon profile and thus, the odor of the host colony (Moritz et al. 1991) The SHB is able to enter honey bee colonies due to several adaptive morphological fea tures (Chapter 3). The SHB is able to retract its appendages under neath its body and firmly gras p the substrate, thereby making it difficult for guard bees to eject the invader (Lundie 1940, Neumann et al. 2001) Once the guards cease aggression, the SHB s hide in cracks and crevices around the colony, often without further aggress ion from the bees. Bees detect the hiding SHB s and react by stationing guards around them, imprisoning them. T he SHB s do not starve in these prisons. Occasionally, they are able to solicit trophallaxis from their guards, thus allowing them to survive longe r and increasing their likelihood of successful reproduction within the colony (Neumann et al. 2001, Ellis et al. 2002b ) The mechanism that allows the SHB s to initiate trophallaxi s with bees i s not well understood. One possible method is via tactile stimulation of the bee However, this is unlikely, as honey bees have a highly developed nestmate recognition system based largely, if not entirely, on cuticular hydrocarbons (Breed et al. 1995, Breed 1998, Downs and Ratnieks 1999) Therefore, it is quite possible, especially given the wealth of recent literature on the subject, that SHB s use chemical means to aid in successful integrati on into honey bee colonies and subsequent ly solicit food from their hosts. T here is preliminary chemical evidence to support this

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84 assertion ( B. Torto, pers. comm ). Schmolke (1974) observe d that SHB s newly introduced into hives are treated more aggressively than SHB s that have been in the colony for some time. In this study, I hypothesized that the SHBs acquire cuticular hydrocarbons through diet on which they feed while in colonies, thus a fford ing them a higher probability of acceptance into bee colonies. Materials and Methods Beetles Adult SHBs were captured from experimental honey bee colonies maintained at the University of Florida Bee Biology and Research Unit (BBRU) in Gainesville, Florida ( 29.63 N, 82.36 W ). SHBs were reared in an incubator (25C; 80% relative humidity; constant darkness) on a diet of honey, pollen, and Brood Builder (Dadant and Sons, Inc., Hamilton, IL) in a ratio of 1:1:2 respectively (Ellis et al. 2008, 2010b ) at the University of Florida Department of Entomology and Nematology (29.64 N, 82.36 W). Wandering larvae were captured and put onto soil to pupate. From the resulting adult beetles, three groups (~150 beetles per group) wer e used for chemical and beha vioral analyses. 1) The first group was unfed and collected 12 hours after eclosion. 2) A second group was fed nothing but sugar water (1:1) for 14 days post eclosion 3) The third group was fed the honey pollen Brood Builder diet described above for 14 days post eclosion Beetles used for chemical analyses were frozen immediately at 80C until needed. For the behavioral trials, one additional group was created. This group consisted of beetles taken directly from managed honey bee colonies at the BBRU (no t one of the test colonies) ~3 hours prior to the behavioral assays. Observation H ives Four observation hives were created from previously established honey bee colonies of mixed European origin located at the University of Florida Plant Science Research and Education Unit in Citra, Florida (29.41 N, 82.17 W). The observation colonies were given

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85 three frames containing pollen, capped honey, brood of all ages, worker bees, and a laying queen. The observation hive structure (Figure 51) accommodated three, 23.0 42.6 cm (L W) wooden frames. The entrance corridor to the hives contained a 10 20 cm (L W) test arena with delineated 1 cm squares, and could be closed at both ends with Plexiglas doors containing holes to accommodate normal airflow (Figure 5 1). The arena was used for the behavioral assays and included a side entrance where test beetles could be introduced directly into the arena. Behavioral A ssays All trials were conducted per Chapter 4 under red lights between 18:30 a nd 02:30 the following m orning. Trials were run simultaneously in the four observation hives with four different observers who were trained in order to reduce observer bias. As a further measure to reduce bias, the observers were only given coded containers and the treatments wer e unknown to them. For each trial, one beetle or an auto claved 60 mg black bead (Darice, Inc., Strongsville, OH, USA) tethered to a n auto claved 15 cm piece of monofilament fishing line (Zebco, Tulsa, OK, USA) was placed through the side entrance into the test arena. T he t est arena was closed at both ends to trap guard bees in the arena prior to introducing the subjects Following their introduction, guard bees responses to the subject were recorded for 1 min The responses were determined to be either ignore (a bees head comes within 5 mm of the su bject, but there is no contact), contact (the bee makes physical nondefe nsive contact with the subject), or defend (the bee attempts to sting and/or remove the subject). Only trials in which the beetles rem ained in the arena for The design was a completely randomized design blocked on colony (4 colonies). The replicate schedule was beetles from 4 diet groups a nd 1 control (black bead) 15 individuals 4 observation colonies To allow ho ney bee movement through the entrance and to reduce guard bee agitation, t he sliding Plexiglas doors on either side of the test arenas were opened briefly ( trials. The used bead was removed or the beetles

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86 were allowed to exit the test arenas at this time. Individual beetle s and beads were used only once. Chemical Analysi s Ten frozen beetles (unsexed) from each of the three diet groups were used in chemical analyses. Each individual beetle was submerged in 1 ml of pentane for 10 minutes. Five g of tetracosane (C24) were added to the samples as internal standard. Samples were analyzed at the USDA ARS, Gainesville, Florida (29.64 N, 82.35 W) using a HP 6890 gas chromatograph (GC, Hewlett Packard, Palo Alto, CA) equipped with a HP 1 column (30 m 0.25 m, Agilent, Palo Alto, CA ). For chemical identification, the column was linked to a HP 5973 mass spectrometer operated in the electron impact mode (70 eV, Agilent, Palo Alto, CA) Helium was used as a carrier gas at a linear flow velocity of 18 cm/s. The GC oven temperature began at 35 C for the first min and then increased at 5 C per min to 200 C and held constant for 5 min. Then, the temperature was incr eased at 15 C per min up to 300 C and held constant for 10 min. The transfer line for t he mass spectrometer was held at 28 0 C Samples also were analyzed using a gas chromatograph flame ionization detector (HP 6890) using the same method as above to quantify amounts of compounds The flame ionization detector was held at 250 C. Statistica l Analysis B ee responses to beetles or beads were converted to proportion data. All proportion response data were transformed with arcsine T he transformed proportions were analyzed using a two way ANOVA (JMP 2008) reco gnizing colony and diet as main effects and colony diet type as the interaction term. Data for the chemical assays were transformed to adjust for normality, and the means were analyzed using a one way ANOVA (JMP 2008). Where appropriate, means were com pared using Tukey Kramer tests, accepting differences at P chapter

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87 Results Overall, bees at the colony entrance did not differ in their level of responses to the SHB groups (Tables 51, 52, 5 3). The i nteraction of colony diet type was significant for the all three responses (ignore, contact, and defend), so these variables were analyzed within colony. Concerning the ignore response, only in Colony C was there a significant effect, wherein SHBs from a ll of the diet groups, except for those fed sugar water, were ignored more than were the control beads (Table 5 1). Guard bees in Colonies B, C, and D contacted the control bead more than the SHB s (Table 5 2). I n Colony D, SHB s that were newly eclosed or collected from the honey bee colony were contacted more than the SHB s fed honey, polle n, and Brood Builder (Table 5 2). Finally, bees in Colonies B, C, and D treated all SHB s more defensively than they did the control bead B ees in Colony D treated SHB s fed honey, pollen, and Brood Builder more defensively than those from the honey bee colony (Table 5 3). The most prevalent hydrocarbons found on the adult SHBs were saturated and monounsaturated C23, C25C29 (Figures 52, 5 3). Saturated and monounsatura ted C26 and C28 are omitted from Figure 53 due to their relatively small abundance and insignificant differences between treatments (Table 5 4). Sugar water fed SHBs had all other detected hydrocarbons (saturated and monounsaturated C23, C25, C27, and C29) present on their cuticles in higher amounts than those of newly eclosed SHBs (Table 5 4). Furthermore, they had higher levels of all of these hydrocarbons except for saturated C25 and C27 than did the cuticles of SHBs fed honey, pollen, and Brood Builder (Table 5 4). Finally, SHBs fed honey, pollen, and Brood Builder had higher amounts of monounsaturated C23 and saturated C23, C25, and C27 than did newlyeclosed SHBs (Table 5 4).

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88 Discussion SHBs that were fed sugar water for 14 days differed in their c uticular profiles markedly and consistently from those fed honey, pollen, and Brood Builder for 14 days and from newlyemerged SHB s The former had a more pronounced profile However, based on the behavioral assays, this apparently did not alter their tre atment by guard bees at the nest entrance. Reducing or under producing, ones cuticular profile could be beneficial to a SHB trying to survive within a dark honey bee nest, where the majority of cues are chemical (Seeley 1998) SHB s that are in bee hives feed on pollen, honey, and brood (Lundie 1940) I f such a diet were to reduce a SHB s cuticular chemical signature compared to a sub optimal diet (like the sugar water in this study) as the results suggest, such a change can be considered adaptive, as in the ponerine ant Ectatomma ruidum (Roger) (Hymenoptera: Formicidae) (Jeral et al. 1997) To determine if this is the case in SHBs behavioral assays would be better done using younger bees withi n the nest where attack thresholds are higher than those of the guard bees at the nest entrance (Breed et al. 1992a) Furthermore, since confined beetles are fed by the bees troph allactically (Ellis et al. 2002b), it would be interesting to 1) determine the specific contents of the liquid food and 2) measure bee responses to SHBs fed this substance. All of the compounds found on the SHB cuticle have also been found associated with honey bees. Although the configurations were not determined directly in this experiment, most alkenes on the insect cuticle are in the (Z) configuration and have the double bond in the 9 position (Blomquist 2010). Breed (1998) found that (Z) 9tricosene (C23:1), which is a sex attractant in the housefly, Musca domestica L. (Diptera: Muscidae) (Carlson et al. 1971), is used in honey bee nestmate recognition while tricosane (C23), pentacosane (C25), and nonacosane (C29) yielded negative results (i.e. bees treated with the compounds were not treated differently by bees than untreated controls) suggesting they are not used in nestmate recognition (Breed and

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89 Stiller 1992). However, all of these (C25:1), 27:1), heptacosane (C2729:1) (Blomquist et al. 1980). Furthermore, Arnold et al. (1996, 2000) found several alkanes and alkenes between C21 and C33 to be associated with subfamilies (i.e. sister groups with the same drone father) within a honey bee colony, which may be used for kin recognition via contact cues. Schmitt et al. (2007) found tricosene, tricosane, pentacosene, pentacosane, heptacosane, and nonacosane in the headspace above foraging honey bees, suggesting that they may provide volatile cues. Similarly, Thom et al. (2007) found that returning foragers release (Z) 9tricosene, tricosane, (Z) 9pentacosene, and pentacosane while recruiting more foragers. In addition to cuticular chemical composition possibly being affected by diet, it would be interesting to determine if SHB s also can adsorb colony odors onto their cuticles from their host nests. This occurs in the myrmecophilous beetle Myrmecaphodius excavaticollis (Blanchard) (Coleoptera: Scarabaeidae) withi n nests of Solenopsis spp. ( Vander Meer and Wojcik 1982) as well as the thief ant species Ectatomma ruidum which, as discussed above, also reduces its cuticular profile (Breed et al. 1992b, Jeral et al. 1997) If a reduc ed cuticular profile aids SHB integration into honey bee colonies, this would be a novel case of adaptive diet mediated cuticular chemical reduction.

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90 Table 5 1 Diet colony interaction on the proportion of guard bee responses that were ignore during the observation period for newly eclosed beetles (new), beetles that have fed for 14 days on a diet of honey, pollen, and Brood Builder (1:1:2) (diet), beetles that have fed for 14 days on sugar water, beetles that are from a colony (colony), or a control bead Data are mean SE ( n beetles ) proportion of total responses that were ignore. Columnar means followed by the same letter are compared using the Tukey Kramer method. Diet Colony A Colony B Colony C Colony D new 0.314 0.064 (15) 0.422 0.064 (15) 0.369 0.030 (15)a 0.154 0.030 (13) diet 0.429 0.061 (15) 0.435 0.031 (15) 0.438 0.033 (15)a 0.233 0.031 (14) sugar water 0.496 0.061 (15) 0.374 0.042 (14) 0.319 0.036 (15)a b 0.196 0.027 (12) colony 0.346 0.044 (15) 0.419 0.046 (15) 0.378 0.039 (15)a 0.316 0.055 (13) control 0.503 0.077 (15) 0.363 0.045 (15) 0.228 0.037 (15)b 0.194 0.020 (15) ANOVA F = 2.2; df = 4, 70; P = 0.08 F = 0.4 ; df = 4, 70; P = 0.78 F = 5.3 ; df = 4, 70; P < 0.01 F = 2.1 ; df = 4, 62; P = 0.10

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91 Table 5 2 Diet colony interaction on the proportion of guard bee responses that were contact during the observation period for newly eclosed beetles (new), beetles that have fed for 14 days on a diet of honey, pollen, and Brood Builder (1:1:2) (diet), beetles that have fed for 14 days on sugar water, beetles that are from a colony (colony), or a control bead Data are mean SE ( n beetles ) proportion of total responses that were contact. Columnar means followed by the same letter are compared using the Tukey Kramer method. Diet Colony A Colony B Colony C Colony D new 0.398 0.064 (15) 0.321 0.030 (15)b 0.286 0.033 (15)b 0.309 0.036 (13)b diet 0.300 0.030 (15) 0.293 0.026 (15)b 0.345 0.029 (15)b 0.123 0.032 (14)c sugar water 0.311 0.043 (14) 0.367 0.034 (14)b 0.342 0.023 (15)b 0.243 0.045 (12)b c colony 0.343 0.037 (15) 0.282 0.029 (15)b 0.335 0.026 (15)b 0.272 0.032 (13)b control 0.373 0.054 (15) 0.632 0.042 (15)a 0.760 0.038 (15)a 0.710 0.021 (15)a ANOVA F = 0.7 ; df = 4, 70; P = 0.57 F = 16.7 ; df = 4, 70; P < 0.01 F = 35.6 ; df = 4, 70; P < 0.01 F = 34.8 ; df = 4, 62; P < 0.01

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92 Table 5 3 Diet colony interaction on the proportion of guard bee responses that were defend during the observation period for newly eclosed beetles (new), beetles that have fed for 14 days on a diet of honey, pollen, and Brood Builder (1:1:2) (diet), beetles that have fed for 14 days on sugar water, beetles that are from a colony (colony), or a control bead Data are mean SE ( n beetles ) proportion of total responses that were defend. Columnar means followed by the same letter are compared using the Tukey Kramer method. Diet Colony A Colony B Colony C Colony D new 0.288 0.061 (15) 0.257 0.046 (15)a 0.345 0.041 (15)a 0.537 0.049 (13)a b diet 0.271 0.062 (15) 0.272 0.036 (15)a 0.217 0.025 (15)a 0.643 0.055 (14)a sugar water 0.193 0.052 (15) 0.259 0.039 (14)a 0.339 0.031 (15)a 0.561 0.049 (12)a b colony 0.310 0.060 (15) 0.299 0.042 (15)a 0.287 0.036 (15)a 0.412 0.053 (13)b control 0.123 0.035 (15) 0.006 0.006 (15)b 0.016 0.006 (15)b 0.097 0.018 (15)c ANOVA F = 2.1 ; df = 4, 70; P = 0.08 F = 26.4 ; df = 4, 70; P < 0.01 F = 31.5 ; df = 4, 70; P < 0.01 F = 25.1 ; df = 4, 62; P < 0.01

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93 Table 5 4 Amounts (in g) of various hydrocarbons present on the cuticles of newly eclosed beetles (new), beetles that have fed for 14 days on a diet of honey, pollen, and Brood Builder (1:1:2) (diet), and beetles that have fed for 14 days on sugar water. Data are mean SE ( n beetles ) ng of the each hydrocarbon Columnar means followed by the same letter compared using the Tukey Kramer method. Diet C 23:1 C 23 C 25:1 C 25 C 26:1 C 26 C 27:1 C 27 C 28:1 C 28 C 29:1 C 29 new 5 3 (9)c 8 3 (9)c 7 4 (9)b 11 6 (9)b 1 1 (9) 2 2 (9) 2 2 (9)b 22 13 (9)b 2 2 (9) 9 5 (9) 0 0 (9)b 57 11 (9)b diet 21 4 (8)b 26 3 (8)b 44 11 (8)b 114 30 (8)a 5 3 (8) 0 0 (8) 13 6 (8)b 61 8 (8)a 9 4 (8) 60 28 (8) 0 0 (8)b 88 18 (8)b sugar water 152 26 (10)a 60 12 (10)a 477 91 (10)a 133 34 (10)a 2 2 (10) 4 4 (10) 116 23 (10)a 110 21 (10)a 18 7 (10) 13 6 (10) 56 13 (10)a 274 71 (10)a ANOVA F = 46.8 ; df = 2, 24; P < 0.01 F = 18.7 ; df = 2, 24; P < 0.01 F = 60.5 ; df = 2, 24; P < 0.01 F = 18.8 ; df = 2, 24; P < 0.01 F = 1.3 ; df = 2, 24; P = 0.29 F = 0.4 ; df = 2, 24; P = 0.65 F = 43.8 ; df = 2, 24; P < 0.01 F = 14.9 ; df = 2, 24; P < 0.01 F = 1.9 ; df = 2, 24; P = 0.17 F = 2.5 ; df = 2, 24; P = 0.10 F = 73.5 ; df = 2, 24; P < 0.01 F = 7.7 ; df = 2, 24; P < 0.01

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94 Figure 51. Diagram of observation hive The entrance to the test arena is denoted by the white arrow. The Plexiglass doors are denoted by the black arrows.

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95 Figure 52. Chro matograms from cuticle of newly eclosed (top), 14 day old beetles fed a diet of honey, pollen, and BroodBuilder (middle), and 14 day old beetles fed a diet of sugar and water (bottom). C23:1 C 23 C 25:1 C 25 C 26:1 C 26 C 27 C 27:1 C 28:1 C 28 C 29:1 C 29

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96 Figure 53. Relative amounts o f various hydrocarbons in newly eclosed (gray), 14 day old beetles fed a diet of honey, pollen, and BroodBuilder (white), and 14 day old beetles fed a diet of sugar and water (black) a a a a a a a a b b b b b a b b b a b b b b c c

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97 CHAPTER 6 DISTRIBUTION OF MULT IPLE BEETLE SPECIES INTRODUCED INTO HONEY BEE COLONIES Honey bees employ defen sive behaviors to prevent would be invaders from entering the colony. Many of these defensive tactics occur at the colonys entrance and are expressed by guard bees (Breed et al. 2004, Chapter 4). Honey bees also have colony level defensive behaviors for nest invaders once the y penetrate the nest. For example, honey bees use confinement behavior against SHBs (Neumann et al. 2001, Ellis 2005) This behavior involves confining the SHB s to peripheral cracks and crevices away from the colonys resources by stationing guard bees around the confinement sites in which SHB s hide naturally (Neumann and Elzen 2004, Ellis and Hepburn 2006) Such hiding behavior is important to the SHB s success, as it allows the beetle to avoid its honey bee hosts. Also, i t is crit ical to the hosts success, as it allows the bees to confine the SHB s away from valuable resources. Peculiarly, the behavior is present in SHB nave European races of honey bees even though they had not been exposed to the beetle prior to the mid 1990s. S eemingly, bees do not exhibit this behavior toward other nest intruders (Ellis et al. 2003a) As such, the purpose of the cu rrent study was to determine how honey bees react to nonSHB species once they enter colonies in an effort to better understand the origins of confinement behavior. I hypothesized that the presentation of confinement behavior by honey bees may be linked to the invaders level of colony integration. SHB s are considered highly integrated into honey bee colonies because they are able to endure defensive attacks from bees (Elzen et al. 2001, Chapter 4), exhibit a high degree of morphological specialization (Cha pter 3), and can induce their hosts to feed them trophallactically (Ellis et al. 2002b) Other Nitidulidae beetles have been found in honey bee colonies, but their respective degrees of integration remain unknown. However, they are

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98 assumed to be no more than innocuous shelter seeking visitors or facultative scavengers (Ellis et al. 2008) and their presence in bee colonies provides one the abilit y to test multiple aspects of confinement behavior. These beetle species include Cychramus luteus (Neumann and Ritter 2004) Lobiopa insularis Carpophilus dimidiatus Glischrochilus fasciatus Epuraea corticina (Ellis et al. 2008) and E. luteola (personal observation). These species are considered accidental within colonies, because they are not obligate inquilines and occur more frequently in other habitats (Smith 1886) I condu cted two experiments to address the hypothesis that the presentation of confinement behavior by honey bees may be linked to the invaders level of colony integration. In the first experiment, I compared intracolonial responses of honey bees toward beetles expressing varying degrees of colony integration to determine if 1) highly integrated species were more likely to find confinement sites than less integrated ones, 2) if there is a temporal pattern associated with confinement site location, and 3) whether confinement behavior is a general response toward all invaders (i.e. not specific to SHBs). Beetle species used in this experiment included : SHBs (highly integrated), L. insularis and E. luteola (accidental); and Carpophilus humeralis C. hemipterus and Tribolium castaneum (nonintegrated). In t he second experiment, I determined if previous SHB occupation of confinement sites predisposed those sites to hosting invading SHBs never before exposed to honey bee colonies. Materials and Methods Beetles Adult S HBs and T. castanaeum were obtained from in vitro rearing colonies at the USDA ARS, Gainesville, Florida (29.64 N, 82.35 W) and were used to initiate rearing programs at the University of Florida Department of Entomology and Nematology (29.64 N, 82.36 W). Adults from the other nitidulid species were collected from rotting cantaloupe, Cucumis melo L., in July

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99 2009 at the University of Florida Plant Science Research and Education Unit (PSREU) in Citra, Florida (29.41 N, 82.17 W) and used to initiate rea ring programs. All species, with the exception of SHBs and T. castaneum were reared in an incubator (24C; 40% relative humidity; constant darkness) on a tomato and prunebased diet developed by Peng and Williams (1990). SHBs were reared on a diet of hone y, pollen, and Brood Builder (Dadant and Sons, Inc., Hamilton, IL) in a ratio of 1:1:2 respectively (Ellis et al. 2008, Ellis et al. 2010) while T. castaneum were reared on flour. SHBs, E. luteola C. humeralis and C. hemipterus were 2 4 weeks old at the time of the experiment. Due to limited availability, the L. insularis used during the study were obtained from rearin g programs or field collections and, a s suc h their age at the time of the experiment was unknown. Though T castaneum were lab reared, their age also was unknown at the time of the experiment. No adult beetle used during the experiment had ever been in contact with honey bees or bee colonies prior to the study. Observation Hives In September 2009, four observation hives were created from previously established honey bee colonies of mixed European origin located at the PSREU. The observation colonies were given three frames collectively containing p ollen, capped honey, brood of all ages, worker bees, and a n egg laying queen. The observation hive structure (Figure 61) accommodated three, 23.0 42.6 cm (L W) wooden frames. I further modified each observation hive by cutting 8 semicircular grooves ( ~10 cm2 total area) on the periphery of both sides of the hives, totaling 16 grooves per hive (Figure 61). The grooves provided confinement sites for invading beetles. I drilled a small hole (1 cm diameter) into the side of the colony entrance to introduce beetles into the colony (Figure 6 1). The entrance corridor to the hives could be closed at both ends using Plexiglas doors.

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100 Experiment 1: Hiding Behavior of Multiple Beetle Species Because the study was conducted in an area where SHBs are present natur ally, I removed existing SHBs from each observation colony ~24 hours before each tr ia l. For each trial, conducted in October 2009, 25 beetles from a single species were introduced through the side of the entrance corridor of an observation hive at 21:00 ho urs. The Plexiglas door distal to the colony was closed to limit beetle escape from the colony. Once the beetles were introduced, I recorded the location of the beetles 5 min, 15 min, 30 min, 1 h, 2 h, 6 h, 12 h, and 24 h after their introduction into the colony. After conducting the 1 hour observation, I removed the Plexiglas s door from the colony to allow for natural bee movement (1 h was presumed adequate to limit any initial beetle escape from the hive) At each observation period, the beetles could be one of four places: (1) hiding where bees could not reach them (in the confinement sites), (2) in or on the comb, (3) roaming freely on the interior walls of the observation hive (interior walls), or (4) not present in the colony or hiding where they could not be seen by the observer (missing). The procedure was repeated until all beetle species were introduced into each of the four observation hives. Introductions into individual colonies were separated by 48 h. Experiment 2: SHB Hiding Behavior To account for the possibility that SHBs are attracted to confinement sites because other SHBs occupied the sites previously, I conducted a separate experiment in December 2009 using only SHBs and following the procedure outlined in experiment 1 with one modificatio n. In this experiment, the observation hives were opened before the trials and the surfaces of the grooved confinement sites were wiped with a cloth soaked with acetone to remove potential beetle pheromone or other marking residues. The outside surface of the glass side panels of the observation hives also were cleaned with acetone and rotated such that the cleaned outside surface was now the inside surface, having never been exposed to SHBs previously.

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101 Statistical Analysis For both experiments, I analyzed the data separately for each time period. For each beetle species in experiment one, the number of beetles at each location (confinement site, colony wall, missing) was converted into proportions of the total number of beetles observed in each colony. No beetles were found on the comb during the first experiment so this location was excluded from the analysis. Within each time period for the first experiment, I analyzed the proportion of each beetle species found at the three locations separately by locati on using a one way ANOVA. Where necessary, individual means were compared using the Students t method for multiple pairs For the second experiment, I analyzed the proportion of SHBs found at each location (confinement site, colony wall, comb, or missing) within washed colonies (with the confinement sites cleaned with acetone) or unwashed colonies (the main effect). Prior to all analysis, the proportion data were arcsine (2008). Results Experiment 1: Hi ding Behavior of Multiple Beetle Species At every recorded observation period, there were significantly more SHBs found in confinement sites than any other beetle species (Table 6 1, Figure 62). When not considering SHBs, few individuals of the other beet le species were present at confinement sites. That said, some beetle species were present at confinement sites during some time periods more so than were other beetle species, though no general trends in confinement site distribution of nonSHB species exi sted (Table 6 1, Figure 62). In general, the same proportions of all beetle species were found on the interior walls of the colony at all but one time period (1 hour Table 6 1). Finally, at all time periods issing than there were other beetles which could not be found.

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102 Experiment 2: SHB Hiding Behavior At every observation period through 1 h, there were significantly more SHBs found in unwashed confinement sites than in washed ones (Table 6 2, Figure 63). At 2 hours, 12 hours, and 24 hours, there were significantly more beetles found on the combs in washed colonies than in unwashed colonies. At 5 minutes, 6 hours, 12 hours, and 24 hours there were significantly more beetles found on the interior walls in wa shed colonies than in unwashed ones. At 15 minutes, 30 minutes, and 1 hour, there were significantly more beetles missing in washed than unwashed colonies (Table 6 2, Figure 63). Discussion The ability to navigate small spaces is a general characteristic of nitidulid beetles because many nitidulid species spend much of their adult lives under tree bark, in flowers, in fungus inhabited fruits, or in other similar decaying matter (Parsons 1943). Although not all nitidulid species tested in this study have be en found in honey bee colonies previously, I expected that individuals from all species would run from bee aggression and hide in the confinement sites around the colony perimeter. The generated data did not support this assertion. In the first study, more SHBs could be found in confinement sites than at other locations for all t ime periods. This behavior was unique to SHBs as only a few individuals from the other beetle species were observed hiding in confinement sites. Not only did the other beetle species not hide in the confinement sites, but >90% had left the colony within the first 1 hour of the observation period. Therefore, there was a strong pattern relating the level of beetle integration into honey bee colonies to beetle ability to find confinemen t sites when considering highly integrated species (SHB) versus all other levels of integration. However, I did not observe any temporal patterns related to beetle ability to find confinement sites.

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103 There are five possible reasons that SHBs were confined by honey bees while the other beetle species largely were not. First, it is possible that the honey bees were able to remove non SHB beetles from the colonies better than they were able to remove SHBs. There are mo rphological data that support this assertion as SHBs possess adaptations on their legs that allow them to avoid being flipped or lifted from a surface, making them more difficult to remove from a colony (Chapter 3). These morphological adaptations are abse nt in the other beetle species tested in this study. Despite this, other data (Chapter 4) suggest that the non SHB beetle species tested in this study usually are not treated defensively by bees, and often are ignored, at the nest entrance, and I did not o bserve any nonSHB removals This observation would lead one to believe that the nonSHB beetle species tested in this study can access a colony freely, thus predisposing them to being confined by the honey bees within as a second line of defense against c olony intrusion. In the current study, I show that this is not the case. The second possible reason that SHBs were confined by honey bees while the other beetle species largely were not may rely on other data which show that of the beetle species tested i n this study, only the SHB is attracted to bee colony odors (Chapter 2). This may suggest that the nonSHB beetles are more likely to leave the nest rather than willingly seek shelter within. This theory is strengthened by the observation that the beetles are not met with aggression at the colony entrance and could enter if they desired to do so. While in earlier work (Chapter 2) C humeralis did not show any attraction to or repulsion from the colony, the other beetles tested in the current study have been shown to be repelled by colony odors, possibly suggesting a reason they were not found in confinement sites. Interestingly, C. humeralis occurred at confinement sites at a greater rate than did the other non SHB species at all times except for 24 hours an d they were shown in Chapter 2 to not be repelled by bee colony odors. That the accidental species were

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104 not found in the colonies in nontrivial numbers is somewhat perplexing, as others have found L. insularis (Ellis et al. 2008) an d E. luteola (personal o bservation) within colonies on multiple occasions. However, they are not found in colonies nearly as often as SHBs, suggesting that they may enter colonies under specific conditions (weakened or stressed colonies, specific floral resources, etc.), which ma y not have been met in the four colonies used in this study. Third, confinement behavior in honey bees might be a specific defense against SHBs rather than against all colony intruders in general. However, this is unlikely given that the race of honey bee used in the study was first exposed to the beetle only in the mid 1990s (Hood 2000) and the b ehavior is identical to that exhibited by African races of honey bees toward SHBs. Recent evidence suggests that A. mellifera originated in Africa (Whitfield et al. 2006) If true, ancestors of European races may have been exposed to the SHB or its ancestors in their evolutionary history, thus expressing confinement behavior as a vestige. However, the SHB association with honey bees may have occurred after European honey bee races evolved thus erasing an ancestra l link between the two. Four th, confinement behavior may be an exaptation of drone corralling, which occurs when resources diminish in the fall when the drones become an expense to the colony (Free and Williams 1975). When this occurs, honey bees force the drones to the outside frames, then to the walls, then to the bottom board, where they are ultimately expelled from the colony, and no t allowed to re enter (Leventes 1956). Thus, SHBs may induce drone corralling behavior, which becomes expressed as confine ment. Drones are larger than workers and would not, therefore, be able to hide from them as the SHB does. The final possible reason I discuss for honey bee confinement of SHBs but not other beetle invaders could be that confinement behavior is an adaptat ion by the SHB producing favorable

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105 responses from their host. Upon entering a colony, beetles find hiding places quickly to escape aggressive bees (Neumann and Elzen 2004, Ellis and Hepburn 2006) However, they are unable to live much longer than a week without food resources (Pettis and Shimanuki 2000, Ellis et al. 2002a) and leaving the hiding areas may result in fatal encounters with honey bees As such, it is possible that SHBs elicit confinement behavior from adult bees (perhaps chemically) so that they can be tended while in cracks/crevices around the nest. Here the beetles are safe from attack and can induce the attracted bees to regurgitate liquid, which the beetles consume, thereby surviving for a longer period of time (Ellis et al. 2002b) Further support for beetle mediated confinement is offered in Chapter 4, wherein I observed that SHBs were treated defensively by honey bees more often than any of the other beetles observed. This may be due to the rapid movement s of SHBs when placed in the entrance, as no other beetle moved as rapidly as that species. Honey bees are attracted to objects that are moving rapidly and are dark (though this study was carried out under red light conditions) (Breed et al. 2004) Furthermore, the SHB vectors a yeast ( Kodamaea ohmeri ) which, when mixed with bee collected pollen, produces honey bee alarm pheromone, which is very attr active to honey bees (Breed et al. 2004, Torto et al. 2007a) However, this likely would not explain their treatment at the colony entrance since they have not yet accessed pollen at that point. Attracting defensive guard bees at the colony entrance is fatal for most potential nest invaders. However, SHBs have morphological and behavioral characters which allow them to survi ve attacks by honey bees For example, reportedly they have a hardened exoskeleton which allows them to resist honey bee stings and makes them difficult to grasp, although this has not been quantified (Lundie 1940, Schmolke 1974, Neumann et al. 2001) Also, they have grooved femora and flattened tibiae to accommodate their retracting behavior, wherein they pull all of

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106 their appendages beneath their body inaccessible to honey bees ( Neumann et al. 2001, Chapter 3). Furthermore, they have dense tarsal setation which may allow them to grasp to the substrate while their legs are beneath their body and resist being flipped by honey bees (Chapter 3), similar to Hemisphaerota cyanea, which uses its dense tarsal setae to avoid being moved by ants (Eisner and Aneshansley 2000) Such characters would enable the SHB to resist defensive bees There are several examples of parasites altering the behavior of their animal h osts to their own benefit (Moore 1995, SchmidHempel 1995, 1998) For example the trematode Diplostomum spathaceum (Rudolphi) causes diminished predator avoidance behavior in its intermediate fish host, thereby increasing the chance that it will be consumed by and infect its primary avian hosts (Seppl 2005) Similarly, the ant Leptothorax nylanderi ( Frs ter ) (Hymenoptera: Formicidae) becomes lethargic relative to unaffected workers when it becomes infected by the cestode Anomotaenia brevis (Clerc). The ant is the intermediate host for the cestode, which must be consumed by the primary host, which is a woo dpecker (Plateaux 1972, cited in Moore 1995). Also, the fungus Entomophthora myrmecophaga Turian & Wuest infects ants of the genus Formica When infected, ants leave their colonies in the evening and climb leaves of grass to which they become affixed with threads of fungus. From here, the fungal spores are able to disperse by air from the infected, dead ant (Balazy and Sokolowski 1977, S chmid Hempel 1998) If SHBs do initiate confinement behavior, as proposed here, they would be acting like a parasite within a superorganismal host (see Ellis and Hepburn 2006 ), altering the hosts behavior to increase its own fitness. Host finding behavior in SHBs is facilitated by their attraction to hive odors as well as attraction to volatiles produced by a yeast, Kodamaea ohmeri (Suazo et al. 2003, Torto et al. 2005, Torto et al. 2007a) When the yeast mixes with bee collected pollen, it produces

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107 components of honey bee alarm pheromone, a pheromone that is highly attractive to adult SHBs (Torto et al. 2007a) Though SHBs are attracted to components of honey bee alarm pheromone, this attraction likely does not explain their attraction to confinement sites within the colony, especially sites that have hosted SHBs previously as the sites typically do not contain pollen or associ ated debris. Other nitidulid species are known to produce aggregation pheromones (Bartelt et al. 1991, 1992 1994, 1995, 2004, Dowd and Bartelt 1993, Williams et al. 1993, Nardi et al. 1996, Cosse and Bartelt 2000) and the occurrence of such pheromones in the SHB has been suggested (Neumann and Elzen 2004) Though investigators in at least one study failed to sh ow that SHBs produce aggregation pheromones (Torto et al. 2007a) the data suggest that such pheromones may exist and be important in the hiding behavior of SHBs within honey bee colonies. Interestingly, over time the proportion of SHBs in confinement sites of both colony types (washed and unwashed confinement sites) converged, thus suggesting that free roaming SHBs were able to locate confinement sites easier once other SHBs found and occupied them. Confinement behavior is one of the most intriguing aspects of the honey bee SHB relationship. This study suggests that many beetles simply do not enter colonies, so there are no opportunities for confinement of those species to occur. However, there are no known examples of confinement behavior displayed by hone y bees toward other pests, or in other social insects in general. Therefore, it appears to be specific to SHBs, though what mediates this behavior remains unknown.

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108 Table 6 1. Proportions of different beetle species found in confinement sites, the interi or walls of the colony, or missing altogether at different time periods following their introduction into observation honey bee colonies. Within each time period and location, columnar means followed by different letters are significantly different at 0.05. Means were compared using the Students t method for multiple pairs Time Beetle species Location mean SE ( n ) proportion of beetles Confinement site Interior walls Missing 5 min A. tumida 0.570 0.148 (4)a 0.010 0.010 (4) 0.420 0.155 (4) C. humeralis 0.100 0.048 (4)b 0.170 0.081 (4) 0.730 0.060 (4) L. insularis 0.020 0.020 (4)b c 0.300 0.127 (4) 0.680 0.121 (4) T. castaneum 0.010 0.010 (4)b c 0.340 0.145 (4) 0.650 0.150 (4) C. hemipterus 0.010 0.010 (4)b c 0.120 0.054 (4) 0.870 0.053 (4) E. luteola 0.000 0.000 (4)c 0.100 0.038 (4) 0.900 0.038 (4) ANOVA F = 13.7; df = 5, 18; P <0.01 F = 2.4; df = 5, 18; P = 0.08 F = 2.4; df = 5, 18; P = 0.08 15 min A. tumida 0.600 0.121 (4)a 0.020 0.012 (4) 0.380 0.132 (4)b C. humeralis 0.060 0.038 (4)b 0.030 0.019 (4) 0.910 0.025 (4)a L. insularis 0.040 0.023 (4)b 0.210 0.117 (4) 0.750 0.131 (4)a T. castaneum 0.020 0.020 (4)b 0.120 0.107 (4) 0.860 0.101 (4)a C. hemipterus 0.020 0.020 (4)b 0.050 0.038 (4) 0.930 0.041 (4)a E. luteola 0.000 0.000 (4)b 0.110 0.044 (4) 0.890 0.044 (4)a ANOVA F = 15.0; df = 5, 18; P < 0.01 F = 1.3; df = 5, 18; P = 0.31 F = 4.7; df = 5, 18; P < 0.01 30 min A. tumida 0.590 0.125 (4)a 0.040 0.028 (4) 0.370 0.150 (4)b C. humeralis 0.100 0.050 (4)b 0.000 0.000 (4) 0.900 0.050 (4)a L. insularis 0.040 0.023 (4)b c 0.190 0.111 (4) 0.770 0.100 (4)a T. castaneum 0.000 0.000 (4)c 0.100 0.087 (4) 0.900 0.087 (4)a C. hemipterus 0.010 0.010 (4)b c 0.020 0.012 (4) 0.970 0.019 (4)a E. luteola 0.010 0.010 (4)b c 0.040 0.028 (4) 0.950 0.038 (4)a ANOVA F = 16.5; df = 5, 18; P < 0.01 F = 1.9; df = 5, 18; P = 0.14 F = 6.3; df = 5, 18; P < 0.01 1 hr A. tumida 0.580 0.141 (4)a 0.040 0.028 (4)a b 0.380 0.158 (4)b C. humeralis 0.170 0.104 (4)b 0.000 0.000 (4)b 0.830 0.104 (4)a L. insularis 0.030 0.019 (4)b c 0.120 0.080 (4)a 0.850 0.072 (4)a T. castaneum 0.010 0.020 (4)c 0.020 0.012 (4)a b 0.970 0.019 (4)a C. hemipterus 0.020 0.012 (4)c 0.000 0.000 (4)b 0.980 0.012 (4)a E. luteola 0.010 0.020 (4)c 0.100 0.053 (4)a 0.890 0.062 (4)a ANOVA F = 12.0; df = 5, 18; P < 0.01 F = 3.0; df = 5, 18; P = 0.04 F = 6.4; df = 5, 18; P < 0.01 2 hr A. tumida 0.630 0.127 (4)a 0.000 0.000 (4) 0.370 0.127 (4)b C. humeralis 0.070 0.030 (4)b 0.010 0.010 (4) 0.920 0.028 (4)a L. insularis 0.060 0.038 (4)b c 0.040 0.016 (4) 0.900 0.053 (4)a T. castaneum 0.000 0.000 (4)c 0.010 0.010 (4) 0.990 0.010 (4)a C. hemipterus 0.010 0.020 (4)b c 0.000 0.000 (4) 0.990 0.010 (4)a E. luteola 0.000 0.000 (4)c 0.010 0.010 (4) 0.990 0.010 (4)a

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109 Table 6 1. Continued. Time Beetle species Location mean SE ( n ) proportion of beetles Confinement site Interior walls Missing 2 hr ANOVA F = 20.9; df = 5, 18; P < 0.01 F = 2.1; df = 5, 18; P = 0.11 F = 16.6; df = 5, 18; P < 0.01 6 hr A. tumida 0.640 0.146 (4)a 0.010 0.010 (4) 0.350 0.143 (4)c C. humeralis 0.090 0.025 (4)b 0.000 0.000 (4) 0.910 0.025 (4)b L. insularis 0.030 0.030 (4)b c 0.010 0.010 (4) 0.960 0.028 (4)a b T. castaneum 0.010 0.010 (4)b c 0.010 0.010 (4) 0.980 0.012 (4)a b C. hemipterus 0.010 0.010 (4)b c 0.000 0.000 (4) 0.990 0.010 (4)a b E. luteola 0.000 0.000 (4)c 0.000 0.000 (4) 1.000 0.000 (4)a ANOVA F = 18.2; df = 5, 18; P < 0.01 F = 0.6; df = 5, 18; P = 0.70 F = 18.1; df = 5, 18; P < 0.01 12 hr A. tumida 0.710 0.172 (4)a 0.010 0.010 (4) 0.280 0.169 (4)b C. humeralis 0.040 0.016 (4)b 0.000 0.000 (4) 0.960 0.016 (4)a L. insularis 0.030 0.019 (4)b 0.010 0.010 (4) 0.960 0.016 (4)a T. castaneum 0.010 0.010 (4)b 0.010 0.010 (4) 0.980 0.012 (4)a C. hemipterus 0.010 0.010 (4)b 0.000 0.000 (4) 0.990 0.010 (4)a E. luteola 0.000 0.000 (4)b 0.000 0.000 (4) 1.000 0.000 (4)a ANOVA F = 14.5; df = 5, 18; P < 0.01 F = 0.6; df = 5, 18; P = 0.70 F = 14.9; df = 5, 18; P < 0.01 24 hr A. tumida 0.750 0.144 (4)a 0.000 0.000 (4) 0.250 0.144 (4)b C. humeralis 0.010 0.010 (4)b 0.000 0.000 (4) 0.990 0.010 (4)a L. insularis 0.010 0.010 (4)b 0.000 0.000 (4) 0.990 0.010 (4)a T. castaneum 0.000 0.000 (4)b 0.000 0.000 (4) 1.000 0.000 (4)a C. hemipterus 0.010 0.010 (4)b 0.000 0.000 (4) 0.990 0.010 (4)a E. luteola 0.000 0.000 (4)b 0.000 0.000 (4) 1.000 0.000 (4)a ANOVA F = 33.2; df = 5, 18; P < 0.01 No beetles found on interior walls F = 33.2; df = 5, 18; P < 0.01

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110 Table 6 2. Proportions of A. tumida found in confinement sites, on the combs, on the interior walls, or missing altogether at multiple time periods after their introduction into observation honey bee colonies. Significant effects were determined using a oneway ANOVA. Within each time perio d and location, columnar means followed by washed with acetone or unwashed prior to releasing A. tumida into the colonies. Time Condition of confinement site Location mean SE ( n ) proportion of beetles Confinement sites Combs Interior walls Missing 5 min Unwashed 0.570 0.148 (4) a 0.000 0.000 (4) 0.010 0.010 (4) b 0.420 0.155 (4) Washed 0.076 0.046 (4)b 0.089 0.053 (4) 0.093 0.044 (4)a 0.742 0.108 (4) ANOVA F = 11.0; df = 1, 6; P = 0.02 F = 5.5; df = 1, 6; P = 0.06 F = 7.4; df = 1, 6; P = 0.03 F = 3.0; df = 1, 6; P = 0.13 15 min Unwashed 0.600 0.121 (4) a 0.000 0.000 (4) 0.020 0.012 (4) 0.380 0.132 (4) b Washed 0.056 0.044 (4)b 0.000 0.000 (4) 0.057 0.035 (4) 0.887 0.047 (4)a ANOVA F = 19.4; df = 1, 6; P < 0.01 No beetles found on comb F = 0.8; df = 1, 6; P = 0.39 F = 13.6; df = 1, 6; P = 0.01 30 min Unwashed 0.590 0.125 (4) a 0.000 0.000 (4) 0.040 0.028 (4) 0.370 0.150 (4) b Washed 0.038 0.026 (4)b 0.000 0.000 (4) 0.062 0.014 (4) 0.900 0.016 (4)a ANOVA F = 23.0; df = 1, 6; P < 0.01 No beetles found on comb F = 1.4; df = 1, 6; P = 0.28 F = 12.6; df = 1, 6; P = 0.01 1 hr Unwashed 0.580 0.141 (4) a 0.000 0.000 (4) 0.040 0.028 (4) 0.380 0.158 (4) b Washed 0.029 0.018 (4)b 0.019 0.019 (4) 0.089 0.020 (4) 0.864 0.030 (4)a ANOVA F = 20.5; df = 1, 6; P < 0.01 F = 1.0; df = 1, 6; P = 0.36 F = 2.8; df = 1, 6; P = 0.14 F = 7.3; df = 1, 6; P = 0.04 2 hr Unwashed 0.630 0.127 (4) 0.000 0.000 (4)b 0.000 0.000 (4) 0.370 0.127 (4) Washed 0.220 0.111 (4) 0.070 0.025 (4)a 0.081 0.057 (4) 0.629 0.052 (4) ANOVA F = 5.5; df = 1, 6; P = 0.06 F = 8.6; df = 1, 6; P = 0.03 F = 2.6; df = 1, 6; P = 0.16 F = 3.4; df = 1, 6; P = 0.11 6 hr Unwashed 0.640 0.146 (4) 0.000 0.000 (4) 0.010 0.010 (4)b 0.350 0.143 (4) Washed 0.276 0.126 (4) 0.063 0.051 (4) 0.152 0.057 (4)a 0.510 0.060 (4) ANOVA F = 3.7; df = 1, 6; P = 0.10 F = 2.3; df = 1, 6; P = 0.18 F = 11.3; df = 1, 6; P = 0.02 F = 1.2; df = 1, 6; P = 0.32 12 hr Unwashed 0.710 0.172 (4) 0.000 0.000 (4) b 0.010 0.010 (4) b 0.280 0.169 (4) Washed 0.248 0.165 (4) 0.149 0.036 (4)a 0.074 0.029 (4)a 0.529 0.137 (4)

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111 Table 6 2. Continued. Time Condition of confinement site Location mean SE ( n ) proportion of beetles Confinement sites Combs Interior walls Missing 12 hr ANOVA F = 4.4; df = 1, 6; P = 0.08 F = 59.7; df = 1, 6; P < 0.01 F = 8.9; df = 1, 6; P = 0.02 F = 1.8; df = 1, 6; P = 0.23 24 hr Unwashed 0.750 0.144 (4) 0.000 0.000 (4)b 0.000 0.000 (4)b 0.250 0.144 (4) Washed 0.553 0.139 (4) 0.194 0.086 (4)a 0.123 0.042 (4)a 0.130 0.076 (4) ANOVA F = 1.2; df = 1, 6; P = 0.31 F = 17.0; df = 1, 6; P < 0.01 F = 24.1; df = 1, 6; P < 0.01 F = 1.0; df = 1, 6; P = 0.35

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112 Figure 61. Diagram of experimental observation hive. The white arrow indicates the location where beetles were introduced into the observation hive while the black arrows indicate the location of the eight grooves (confinement sites) located on the periphery of the observation hive. The confinement sites were present on both sides, totaling 16 sites.

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113 Figure 62. Percentage distribution of each beetle species found in confinement sites (black), on the interior walls of the hive (white), or missing (gray) at each time period.

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114 Figure 62. Continued

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115 Figure 63. Percentage distribution of A. tumida in colonies where the confinement sites were unwashed and washed. The four potential locations of A. tumida within a colony include confinement sites (black), comb (hashed), on the interior walls of the hive (white), or missing (gray).

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116 CHAPTER 7 TEMPERATUREDEPENDENT CLUSTERING BEHAVIOR OF SHBS IN HONEY BEE COLONIES Temperate races of honey bees are able to survive cold temperatures by forming thermoregulatory clusters when the ambient tempe rature falls below 18 C (Michener 1974, Stabentheiner et al. 2003) A honey bee cluster is a tight, contiguous mass of bees formed for the purpose of producing and conserving heat. They accomplish this by tightly grouping bees between frames and then having bees occupy empty cells in the combs to form a more solid cluster. The result is a sphereshaped mass of bees. Through this behavior, the bees are able to maintain a core temperature of 20 30 C. In contrast, African races do not form winter clusters well, as their native climates often do not necessitate clustering behavior. Consequently, African races of honey bees may perish when experiencing temperatures common in temperate climates (Hepburn and Radloff 1998) Honey bee clustering behavior also insulates and protects nest parasites present in the colony, such as Varroa destructor which survives by attaching to adults during the winter when few brood are reared (Bowen Walker et al. 1997, Schf er et al. 2010 b ) Si milarly, the SHB is able to endure temperate climates by entering the bee cluster when cold temperatures per sist (Pettis and Shimanuki 2000, Ellis et al. 2003b, N eumann and Elzen 2004, Schfer et al. 2010 b ). If fact, they have been found in individual comb cells with bees, thus illustrating how well they penetr ate the cluster (Ellis et al. 2003b). This is surprising since honey bees confine SHBs around the nest periphery most o f the year (Neumann et al. 2001, Ellis et al. 2003a, Ellis 2005) and beetles are met with aggression from bees when outside of confinemen t sites (Schmolke 1974, Elzen et al. 2001). As such, SHB/bee clustering behavior represents a behavioral change for both species during cold temperatures, resulting in an apparently less aggressive relationship between the two. Despite this, little is know n about the cluster entering behavior of SHBs,

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117 especially when the behavior is initiated, when it ceases, what happens during the behavior, and why the beetles, which have native hosts that do not cluster well (Hepburn and Radloff 1998) exhibit the behavior at all. The purpose of this study was to address the temporal aspects (initiation, cessation) of the cluster entering behavior in SHBs. To do this, I exposed honey bee observation colonies to different temperatures to determine the temperature at which SHBs (1) leave confinement sites on the nest periphery (N eumann et al. 2001, Ellis 2005) and enter the thermoregulatory cluster maintained by bees and (2) leave bee clusters and return to confinement sites. Materia ls and Methods Beetles Adult SHBs were captured from experimental honey bee colonies maintained at the University of Florida Bee Biology and Research Unit (BBRU) in Gainesville, Florida ( 29.63 N, 82.36 W ). SHBs were reared in an incubator (25C; 80% relative humidity; constant darkness) on a diet of honey, pollen, and Brood Builder (Dadant and Sons, Inc., Hamilton, IL) in a ratio of 1:1:2 respectively (Ellis et al. 2008, 2010 ) at the University of Florida Entomology and Nematology Department (29.64 N, 82.36 W). Observation Hives Seven observation hives (four for the experimental group, three for the control group) were created from previously established honey bee colonies of mixed European origin located at the University of Florida Plant Science Research and Education Unit in Citra, Florida (29.41 N, 82.17 W). The observation colonies were given three frames containing pollen, capped honey, brood of all ages, worker bees, and an egg laying queen and housed at the BBRU. The observation hive structure (Figure 71) accommodated three, 23.0 42.6 cm (L W) wooden frames. I further modified each observation hive by cutting 8 semicircular grooves (~10 cm2 total

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118 area) on the periphery of both sides of the hives, tot aling 16 grooves per hive (Figure 71). The groo ves provided locations where invading beetles could escape from bees, thus becoming confinement sites. I drilled a small hole (1 cm diameter) into the side of the colony entrance and used it to introduce beet les into the colony (Figure 7 1). The entrance corridor to the hives could be closed at both ends using Plexiglas doors. Behavioral Assays Experiments were performed in mid February 2010 to ensure winter bees inhabited the colonies (Fluri et al. 1982) Twenty SHBs were introduced into each colony 24 hours before the experiment to allow sufficient time for SHBs to find confinement sites ( Chapter 6 ). Two hours before the experiment, the observation hives were moved from the BBRU to a dark room (24.5 C) housing Florida Reach Ins (Walker et al. 1993) at th e Entomology and Nematology Department At 20:30, the number of SHBs in each of the 16 confinement sites was determined under red light conditions. Once the number of SHBs in confinement sites was determined at a given temperature, the four colonies were moved to another Florida Reach In offset at a different temperature. The colonies were left for one hour to allow bees and beetles to acclimate. This procedure was repeated eight more times (nine times total) at five different temperatures, exposing the colonies to thermostatic rooms that were descending in temperature successively, and then ascending in temperature (Table 7 1). The temperature of each room was determined using a digital thermometer (Acu Rite, Jamestown, NY) placed in each room for an hour prior to data collection. Control colonies were established and remained in a dark room at room temperature (~24.8 C, Table 7 2). The control colonies were moved out of the room and back into the room after each beetle count to replicate the movement of treated colonies between the rooms set at different temperatures. Beetle counts for these colonies were conducted at the same time period the treatment colonies were counted to control for time period effects on beetle

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119 presence in confinement sites. The control colonies permitted me to say that any discovered differences in the proportion of beetles in confinement sites were a function of descending/ascending temperature rather than the nightly time period at which the observations occurred. Statistical Analysis Because the total number of beetles found in confinement sites varied between colonies initially, the number of beetles in confinement sites in each colony was converted to proportion data relative to t he original number of beetles in confinement sites at room temperature. All proportion data (the dependent variable) were transformed with arcsine to analyses. For the experimental group data, the data were analyzed using a simple linear regression model (JMP 2008), recognizing descending or ascending temperature as main effects, and the transformed proportions of beetles in confinement sites as the response variables. For the control group data, the transformed proportions were ana lyzed using a one way ANOVA (JMP) recognizing observation time period (20:3004:30) as the main effect. Untransformed means are reported in this chapter Results The proportion of SHB s in confinement sites was positively correlated with temperature when ambient temperatures were descending (y = 0.08x 0.57; t = 6.67; P < 0.01; Figure 72) and ascending (y = 0.03x 0.18; t = 2.92; P < 0.01; Figure 73). Also, temperature accounted for a significant portion of the variance in SHB proportions in confinement sites when temperatures were descending (R2 = 0.72; F = 45.78; df = 1, 18; P < 0.01, Figure 72) or ascending (R2 = 0.32; F = 8.56; df = 1, 18; P < 0.01, Figure 73). Finally, the slo pe of the regression line for the proportion of SHB s exiting confinement sites when the temperature was descending was ~2.7 that for when the temperature was ascending (0.08 and 0.03, respectively). The slope describes

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120 the rate at which the SHB s exit the confinement sites and enter the cluster. For the control colonies, the proportion of SHB s in confinement sites at each time period did not differ significantly (Table 7 2). Discussion The data suggest that there is a regular pattern followed by SHBs when e ntering the thermoregulatory cluster of bees as the ambient temperature drops with the reciprocal being true when the ambient temperature rises. However, the difference in the slopes (0.08 and 0.03 for decreasing and increasing temperatures, respectively) suggests that the SHB s exit the confinement sites and cluster with the bees when the temperature decreases more quickly than they reoccupy the confinement sites when the temperature increases. Furthermore, the respective R2 values (0.72 and 0.32 for decrea sing and increasing temperatures, respectively) suggest that SHB s entered the cluster more regularly than they reoccupied the confinement sites One possible explanation for the slower return of SHB s to confinement sites is that the beetles were being conf ined within the cluster of bees. This may not have happened, however, given that age related polyethism (agebased division of labor) in bees does not follow the same pattern when bees cluster (Hepburn and Radloff 1998, Stabentheiner et al. 2010) and confinement behavior is governed by such a division of labor (Ellis et al. 2003c). That said, it is difficult to know exactly what happens within bee clusters since inner clusters cannot be observed directly. SHBs seem to cluster in cells with bees (Ellis et al. 2003b), but other possible interactions between beetles and bees in winter clusters remain unknown. Another potential explanation is that the cluster of bees may not heat up a s rapidly as it cools down. Thus, the bees themselves would remain in t he cluster and, likewise, the SHB s would exhibit a slower return to confinement sites as they wait for the cluster temperature to increase. Furthermore, the discrepancy between the R2 v alues may be the result of variation

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121 between colonies in the bees ability to warm up following exposure to low ambient temperatures, so the SHB s within the clusters did not leave and reoccupy confinement sites in a pattern that was consistent between the four colonies. The data from control colonies suggest that the proportion of SHB s confined does not vary throughout the night. This is consistent with previous results, which suggest that honey bees are capable of keeping a moderate number of SHB s in confinement sites indefinitely (Ellis et al. 2003a) The present study was performed at night, when SHB s are more act ive within the hive (Ellis et al. 2003a) Previous investigators have found that there are more bee guards keeping SHB s co nfined at night, which also may help explain why the number of SHB s in confinement sites remained constant throughout the study and why no SHB s were found on the combs (Ellis et al. 2003a, 2004a, b ) The findings suggest that SHBs have highly developed adaptation for coldweather tolerance facilitated through exploitation of their host bees thermoregulatory capabilities. The behavioral tug of war that likely occurs between SHB s and bees in bee clusters should be investigated to understand this adaptation in detail. These behavior s would be studied best through comparisons of the clustering behavior in colonies of European and African races of honey bees. Through such studies, I may be able to elucidate another adaptation that enables the SHB to integrate into honey bee colonies.

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122 Table 7 1. Proportion of beetles confined at each temperature in experimental colonies (colonies exposed to changing temperatures). Data are mean SE ( n) proportion of beetles in confinement sites of each colony. Time Temperature (C) Proportion Beetle s 20:30 24.5 0.829 0.075 (4) 21:30 21.6 0.938 0.063 (4) 22:30 16.1 0.231 0.149 (4) 23:30 9.1 0.094 0.094 (4) 00:30 6.6 0 0 (4) 01:30 9.4 0.108 0.079 (4) 02:30 15.9 0.088 0.030 (4) 03:30 21.7 0.219 0.129 (4) 04:30 24.3 0.538 0.197 (4) Table 7 2. Proportion of beetles confined at each temperature in control colonies (colonies remaining at room temperature throughout study). Data are mean SE ( n ) proportion of beetles in confinement sites of each colony. Time Temperature (C) Proportion Beetles 20:30 24.6 0.808 0.048 (3) 21:30 24.8 0.891 0.003 (3) 22:30 24.7 0.891 0.064 (3) 23:30 24.9 0.832 0.039 (3) 00:30 24.9 0.856 0.032 (3) 01:30 24.9 0.870 0.097 (3) 02:30 24.8 0.780 0.081 (3) 03:30 24.7 0.821 0.055 (3) 04:30 24.7 0.869 0.082 (3) ANOVA F = 0.39; df = 8, 18; P = 0.91

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123 Figure 71. Diagram of experimental observation hive. The white arrow indicates the location where beetles were introduced into the observation hive while the black arrows indicate the location of the eight grooves (confinement sites) located on the periphery of the observation hive. The confinement sites were present on both sides, totaling 16 sites.

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124 Figure 72. The proportion of beetles in confinement sites as the ambient temperature decreased. The line is the best fit line. The Y axis beetle proportions are arcsine For clarity, the X axi s has been reversed to show decreasing temperatures. Figure 73. The proportion of beetles in confinement sites as the ambient temperature increased. The line is the best fit line. The Y axis beetle proportions are arcsine

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125 CHAPTER 8 DI SCUSSION T h e panic that accompanied the discovery of the SHB in North America in the late 1990s gradually was followed by a reluctant acceptance of the pest as a way of life and a reason for beekeepers to alter their management techniques. Paranoia that fo llowed the SHB after its introduction largely has waned. In a survey of 571 beekeepers, less than 2% reported SHBs as a source of colony collapse (vanEngelsdorp et a l. 2010) Regardless the SHB remains a problem in many U.S. apiaries (including Hawaii) and recent evidence suggests that, similar to V. destructor it has the ability to carry and transmit honey bee pathogens (Eyer et al. 2009a, b, Schfer et al. 2010a ) Therefore, the study of SHBs as an applied problem is warranted. Basic research of this pest is fundamental to solving applied problems (see Ellis 2002). For example, the discovery of hiding and confinement behavior (Lundie 1940, Neumann et al. 2001, Ellis 2005) has guided control strategies including the design of adult SH B traps and their placement within colonies (Elzen et al. 1999, West 2004, Hood 2006, Cobey 2008, Levot 2008, Neumann and Hoffmann 2008) Also, studies of SHB feeding and attraction to colonies (Lundie 1940, Schmolke 1974, Ellis et al. 2002a, Kel ler 2002, Hood and Miller 2003, Suazo et al. 2003, Torto et al. 2005, 2007b, Arbogast et al. 2009, 2010) T he discovery of the SHB vectored yeast Kodamae a ohmeri (Torto et al. 2007a) ha s also led to the development of lures for attracting and capturing SHBs (Arbogast et al. 2007, Torto et al. 2007c, Nolan and Hood 2008) The results of the research presented herein indicate that the SHB is unique among nitidulids in its adaptive leg morphology (Chapter 3), treatment by honey bee hosts (Chapter 4), attraction to bee colony odors (Chapter 2), and hiding and subsequent confinement by honey bees (Chapter 6). Furthermore, the SHB exhibits adaptive behaviors which allow it to survive

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126 low t emperatures (Chapter 7) and, potentially, avoid recognition by hosts (Chapter 5), though the latter requires further investigation. Re examination of the Low Number of Honey Bee Nest Symbionts One of the motivations for this research was the perceived lack of symbionts in nests of social bees compared to that in ant and termite nests (Wilson 1971, Kistner 1982) However, an analysis of the diversity of arthropods found in bee nests (Chapter 1) provides evidence to the contrary, that social bee nests do in fact h ost many symbionts Furthermore, the number of symbiont species found in some bee nests may be comparable to th at found in ant and termite nests (Kistner 1979) However, the degree of specialization (morphological, behavioral, and chemi cal) is relatively low among bee nest symbionts (Wilson 1971) (Tables 1 1, 12) A discussion of why hone y bees have few nest invaders is warranted if one is to understand how SHB s and melittophiles in general, have been able to overcome obstacles to inha biting honey bee nest s One hypothesis offered to explain the scarcity of symbiont s in honey bee nests is that honey bees often nest in trees (Wilson 1971) though many nest in the ground as well (Seeley 1985) Therefore, a potential pest first would have to become adapted to arboreal life to become adapted to the nest environment. M any relatives of the SHB are associated with fungal mats and sap flows at trees (Cease and Juzwik 2001) so the association of SHB s with honey bee nests may have developed through regu lar contact Another explanation offered for the lack of honey bee symbionts is that the colonies are kept relatively free of debris through the cleaning behaviors of the nest inhabitants (Wilson 1971, Kistner 1982) Cavity nesting honey bees regularly remove debris and waste from their colonies (Seeley 1982) and these habits minimize the opportun ity for scavengers to invade This is done because the food stuffs (pollen and nectar) on which bees feed is concentrated, thus leaving l ittle

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127 refuse and r esources for scavengers that may become pests eventually. S cavenging arthropods compose the second largest group of symbionts found within ant and termite nests nests that often are full of debris (Kistner 1982) Finally, honey bee nests have limited entrances which reduce the number of potential nest invaders. Furthermore, t he nest entrances are guarded perpetual ly by a guild of guard bees that are physiologically specialized for the task (Breed et al. 2004) Ants and termites also excel at nest defense, but they have mul tiple entrances to their nest s which may represent multiple opportunities for nest invasion. Of the hypotheses offered to explain why honey bees have fewer symbionts, the efficient defense of limited entrances is, perhaps, the most probable. SHB s are able to overcome hive defenses in a variety of ways (Chapter 1). I hypothesized that SHBs are able to penetrate and persist within honey bee colonies as symbionts as a result of morphological, behavioral, and chemical adaptations. It is clear that they have morphological adaptations (Chap ter 3) that assist them when entering the nest. They can withstand attacks by defensive guard bees at the entrance by retracting their appendages beneath their body (Neumann et al. 2001, Neumann and Elzen 2004) a behavior that is accommodated by specialized morphological adaptations (Chapter 3). Specifically, SHBs have grooved femora that likely accommodate the flattened tibiae to allow for more complete retraction, though this has not been tested behaviorally. Also, SHBs have various behavioral adaptations (Chapters 2, 4, 6, 7) that are unique among related species that were used. Once within the colony, the SHBs find cracks and crevices around t he colony in which to hide from the bees with 60% of the beetles hiding within 15 minutes (Neumann and Elzen 2004, Chapter 6). Thus, although SHBs are treated defensively by guard honey bees (Chapter 4), the majority are able to enter the nest rather quickly (Chapter 6).

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128 What is not clear is whether they use chemical means to integrate into colonies In Chapter 6 I found that SHBs find the confinement sites more quickly if they have not been washed, suggesting that there is an attractive chemical or chemi cal blend present at these sites, potentially released by SHBs that previously occupied the sites. Furthermore, in Chapter 5 I found that SHBs reared on a presumably more nutriti ous diet of honey, pollen, and protein supplement produce a different cuticular chemical profile than that produced by SHBs reared on the less nutritious sugar water diet. That more lipids are present on the cuticle of SHBs reared on an essentially lipid free diet is intriguing, and may suggest an ability to blend in afte r feeding on hive products (particularly stored pollen). However, the differences in cuticular profiles do not appear relevant at the honey bee nest entrance. Thus, neither study (Chapters 5, 6) provides conclusive evidence for chemical use in SHBs. That s aid, were SHBs to lack either the morphological or behavioral traits that they possess, they likely would not be able to thrive within honey bee nests as they do. Similarly, the deaths head hawkmoth, Acherontia atropos has a suite of adaptations that all ows it to enter and feed on nectar and honey within honey bee colonies. However, this moth utilizes all three m ethods of integration. Morphologically they have a thickened cuticle that allows it to endure honey bee stings (Moritz et al. 1991). Behaviorally the moth emits a squeaking sound which is thought to reduce the defensive behavior of its hosts (Moritz et al. 1991). Finally, the moth endogenously produces a cuticular chemical profile that allows it to blend into the colony (Moritz et al. 1991). The m oth requires each method of integration, as the persistent odor allows it to blend in and, if noticed, it can emit the appeasing vibration and, if that fails, it is still protected by a thickened cuticle.

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129 The F uture of SHB Research A ggregation pheromone s, which are ubiquitous among Nitidulidae (Bartelt et al. 1991, 1992, 1994 1995 2004 Dowd and Bartelt 1993, Williams et al. 1993, Nardi et al. 1996, Cosse and Bartelt 2000) ha ve yet to be found in SHBs, al though results reported in Chapter 6 suggest that they may exist. Torto et al. (2007a ) report that no such pheromones have been detected after several bioassays Therefore, presenting the SHB s with an appropriate context to express such pheromones may be needed to discover the compound Aggregation pheromones have been utilized extensively t o trap various insect pests for monitoring and control (Phillips and Throne 2010) and elucidating such a pheromone for SHBs would be exceedingly useful in SHB management practices Regardless, the data presented in Chapter 6 suggest that such a pheromone may exist and this is further evidence that SHBs use chemical cues to integrate into honey bee colonies. Furthermore, testing the effects of the potential pheromone on heterospecific beetles would be worthwhile. The use of o ther chemical cues also may exist in SHBs. For example, although the implications of the data from Chapter 5 remain unclear, the cuticular profiles of SHB s may be beneficially altered as a consequence of their diet or surroundings. This chemical profile pl asticity might be relevant to survivability within the honey bee nest and may shed light on the confinement behavior of honey bees and the phenomenon of captive SHBs being fed by honey bees (Ellis et al. 2002b). Furthermore, the evolution of confinement be havior itself remains a mystery (Chapter 6). Future chemical studies will help answer many of these remaining questions. Recent evidence indicates that SHBs are capable of mechanically transmitting honey bee pathogens (Eyer et al. 2009a, b, Schfer et al. 2010a ) Following the consumption of infected honey bee brood, SHBs were found to be infected with sacbrood virus (Eyer et al. 2009b)

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130 deformed wing virus (Eyer et al. 2009a) and Paenibacillus larvae ( Schfer et al. 2010a ) The latter is a ba cterium that causes American foulbrood in honey bees. Furthermore, the study by Schfer et al. (2010a ) suggests that such infections can be transmitted to healthy honey bee colonies Nitidulids, including SHBs, are known to carry different fungi and some h ave the potential to transmit diseases between secondary organisms (Cease and Juzwik 2001, Torto et al. 2005, Benda et al. 2008) The potential for SHBs to carry fungal and viral pathogens is especially important in light of recent evidence which suggests that there is a correlation between the fungal microsporidian genus Nosema an invertebrate iridescent virus, and the recent phenomenon colony collapse disorder (CCD) (Bromenshenk et al. 2010) Thus, if SHBs are able to transmit these two pathogens, and these pathogens are involved in CCD, then the beetles may be contributing to these collapses. Relevant to all issues regarding SHB problems in apiaries is a means of measuring dispersal patterns and population dynamics. In Chapter 6 I investigated the intracolonial distributions of various be etle species, including SHBs. However, SHB infestations remain largely enigmatic in that they are very difficult to predict and can vary and change within an apiary almost daily. Mathematical modeling and, perhaps, epidemiological techniques may aid in unc overing intercolonial SHB distributions Also, the research presented in this dissertation, as well as that of others, focuses on adult morphology, behavior, and chemistry. It would be worthwhile to repeat many of the experiments using immature stages. Lit tle is known about interactions between honey bees and larvae beyond the fact that some honey bee races can remove them from colonies (Neumann and H rtel 2004, Spiewok and Neumann 2006a). Might the SHB larvae use similar techniques as the adults to get fed by the bees (Ellis et al. 2002b)? Do they possess any morphological adaptations which

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131 accommodate their persistence within the colony as the adults do (Chapter 3)? Lundie (1940) suggests that the protuberances act to prevent the larvaes drowning in the h oney. However, this is based entirely on anecdotal evidence and has yet to be demonstrated. Furthermore, though they can be removed effectively by African honey bee races, do the SHB larvae have any behavioral methods of avoiding honey bee aggression like the hiding behavior in the adults (Chapter 6)? Also, are there any other stages that can overwinter within or around honey bee colonies besides the adults (Chapter 7)? Finally, do SHB larvae produce chemicals to attract other larvae, as is suggested in adu lts in Chapter 6, and is evidenced in larvae by the common larval masses on foodstuffs; or does their cuticular profile change based on their diet, perhaps to their own advantage (Chapter 5)? Such investigations would provide further insight into the relat ionship between SHBs and their honey bee hosts. A final project on SHBs would compare the survivability, longevity, and reproductive capacity of several beetle species (as per Chapters 2, 3, 4, 6) on various diets of honey bee a nd nonhoney bee foodstuffs. Though I initiated this project multiple times, the developing larvae were overtaken repeatedly by the mold mite Tyrophagus putrescentiae (Acari: Acaridae), which has shown the ability to disrupt the SH B life cycle (Ellis et al. 2010 ). It would be interes ting to investigate whether these other (non SHB) beetle species, which include species that have been found in honey bee nests and beetles that have not, are able to survive and repro duce on the honey bee products. SHB s provide an excellent opportunity fo r studies on the development of inquilinism, as they are highly integrated into bee nests but do not complete their entire life cycle within the nest Furthermore, they are not obligate symbionts and can survive in nests of alternative hosts (Ambrose et al. 2000, Stanghellini et al. 2000, Spiewok and Neumann 2006b Hoffmann et al.

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132 2008, Greco et al. 2010) as well as on fruits (Ellis et al. 2002a, Keller 2002, Buchholz et al. 2008, Arbog ast et al. 2009, 2010) As such, SHBs can be considered a potential step toward obligate symbiosis within honey bee nests, and studies of a species with such plasticity can yield intere sting results with ecological and evolutionary ramifications.

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153 BIOGRAPHIAL SKETCH Edward Blake Atki nson was born in Ocala, Florida to a musician mother and a biologist father. He received a bachelors degree from the Florida State University in 2005 with a double major in physics and philosophy and a minor in mathematics Shortly after graduating, Eddie married Cryst al Taylor who also is an entomology graduate stude nt at the University of Florida. Soon after enrolling in graduate school, they had a daughter, Ella (now 3) and a son, Eli (now 1). Eddie plans on pursuing research on honey bee pests and inquiline integrat ion.