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The Attraction of Bumble Bee (bombus Impatiens Cresson; Hymenoptera

Permanent Link: http://ufdc.ufl.edu/UFE0024736/00001

Material Information

Title: The Attraction of Bumble Bee (bombus Impatiens Cresson; Hymenoptera Apidae) Colonies to the Small Hive Beetle (aethina Tumida Murray; Coleoptera: Nitidulidae)
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Graham, Jason
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aethina, apis, bee, beetle, bombus, bumble, choice, hive, honey, impatiens, kodamaea, mellifera, ohmeri, parasite, small, social, symbiosis, tumida, yeast
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The small hive beetle (Aethina tumida Murray, Coleoptera: Nitidulidae; SHB) a pest of the Western honey bee (Apis mellifera L.; Hymenoptera: Apidae) has been found in commercial bumble bee (Bombus impatiens Cresson, Hymenoptera: Apidae) colonies. A genus level host shift may be devastating to commercial and wild bumble bee colonies, a significant problem due to the value of bumble bees as pollinators. Further, bumble bee colonies may serve as an unmonitored source of SHB reproduction. For these reasons, understanding what mediates the attraction of SHBs to bumble bee colonies is important. Previously, investigators discovered a multitrophic interaction in which Kodamaea ohmeri (Ascomycota: Saccharomycetaceae), yeast transmitted by SHBs, played an important role in host location. Presumably, SHB deposit K. ohmeri onto pollen while they feed. When exposed to pollen, K. ohmeri produces components of honey bee alarm pheromone found to attract SHBs. To better understand SHB attraction to bumble bee colonies, three studies were conducted to investigate bumble bee/SHB interactions. In the first study (Chapter 2), SHB attraction to components present in honey bee and bumble bee colonies was investigated under the hypothesis that SHB are as attracted to bumble bee produced volatiles as they are to honey bee produced volatiles. The bumble bee vs. control bioassay results suggest that SHB are attracted to bumble bee adults, stored pollen, brood, wax, and the whole bumble bee hive though not to honey. SHB did not show a preference for honey bee or bumble bee components in the honey bee vs. bumble bee assays. Collectively, the data suggest that SHBs are as attracted to bumble bee colonies as they are to honey bee colonies and this attraction is chemically mediated. In the second study (Chapter 3), airborne volatiles were collected from commercial bumble bee and honey bee colonies and from each component of both colony types (adult bees, brood, honey, pollen and wax) to determine the chemical profile of volatiles present. In general, the volatile profiles of bumble bee and honey bee colonies were dissimilar with only 7 of 148 total compounds detected common to both colonies. In the final study (Chapter 4), eight commercial bumble bee colonies were tested for the presence of K. ohmeri which was present on all of the colony swab samples (n = 7 colonies times 8 swabs/colony = 56 samples). Furthermore, yeast was found in 6 of 7 adult bee samples, 2 of 5 pollen samples, 4 of 8 wax samples but not in brood or honey samples (each sample was from a different colony). This demonstrates that bumble bee nests are suitable micro-environments for the growth of K. ohmeri. Collectively, these findings support the overall hypothesis that attraction of SHBs to bumble bee colonies is chemically mediated. The success of the SHB at expanding its host range may be due to both types of bee colonies harboring K. ohmeri. Every effort should be made to determine the susceptibility of wild bee colonies to SHBs and the role K. ohmeri plays in SHB persistence. Ultimately, such investigations should aid the conservation and restoration of wild bee populations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Graham.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ellis, James D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024736:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024736/00001

Material Information

Title: The Attraction of Bumble Bee (bombus Impatiens Cresson; Hymenoptera Apidae) Colonies to the Small Hive Beetle (aethina Tumida Murray; Coleoptera: Nitidulidae)
Physical Description: 1 online resource (123 p.)
Language: english
Creator: Graham, Jason
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: aethina, apis, bee, beetle, bombus, bumble, choice, hive, honey, impatiens, kodamaea, mellifera, ohmeri, parasite, small, social, symbiosis, tumida, yeast
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The small hive beetle (Aethina tumida Murray, Coleoptera: Nitidulidae; SHB) a pest of the Western honey bee (Apis mellifera L.; Hymenoptera: Apidae) has been found in commercial bumble bee (Bombus impatiens Cresson, Hymenoptera: Apidae) colonies. A genus level host shift may be devastating to commercial and wild bumble bee colonies, a significant problem due to the value of bumble bees as pollinators. Further, bumble bee colonies may serve as an unmonitored source of SHB reproduction. For these reasons, understanding what mediates the attraction of SHBs to bumble bee colonies is important. Previously, investigators discovered a multitrophic interaction in which Kodamaea ohmeri (Ascomycota: Saccharomycetaceae), yeast transmitted by SHBs, played an important role in host location. Presumably, SHB deposit K. ohmeri onto pollen while they feed. When exposed to pollen, K. ohmeri produces components of honey bee alarm pheromone found to attract SHBs. To better understand SHB attraction to bumble bee colonies, three studies were conducted to investigate bumble bee/SHB interactions. In the first study (Chapter 2), SHB attraction to components present in honey bee and bumble bee colonies was investigated under the hypothesis that SHB are as attracted to bumble bee produced volatiles as they are to honey bee produced volatiles. The bumble bee vs. control bioassay results suggest that SHB are attracted to bumble bee adults, stored pollen, brood, wax, and the whole bumble bee hive though not to honey. SHB did not show a preference for honey bee or bumble bee components in the honey bee vs. bumble bee assays. Collectively, the data suggest that SHBs are as attracted to bumble bee colonies as they are to honey bee colonies and this attraction is chemically mediated. In the second study (Chapter 3), airborne volatiles were collected from commercial bumble bee and honey bee colonies and from each component of both colony types (adult bees, brood, honey, pollen and wax) to determine the chemical profile of volatiles present. In general, the volatile profiles of bumble bee and honey bee colonies were dissimilar with only 7 of 148 total compounds detected common to both colonies. In the final study (Chapter 4), eight commercial bumble bee colonies were tested for the presence of K. ohmeri which was present on all of the colony swab samples (n = 7 colonies times 8 swabs/colony = 56 samples). Furthermore, yeast was found in 6 of 7 adult bee samples, 2 of 5 pollen samples, 4 of 8 wax samples but not in brood or honey samples (each sample was from a different colony). This demonstrates that bumble bee nests are suitable micro-environments for the growth of K. ohmeri. Collectively, these findings support the overall hypothesis that attraction of SHBs to bumble bee colonies is chemically mediated. The success of the SHB at expanding its host range may be due to both types of bee colonies harboring K. ohmeri. Every effort should be made to determine the susceptibility of wild bee colonies to SHBs and the role K. ohmeri plays in SHB persistence. Ultimately, such investigations should aid the conservation and restoration of wild bee populations.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Jason Graham.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Ellis, James D.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024736:00001


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THE AT TRACTION OF BUMBLE BEE (HYMENOPTERA: APIDAE, Bombus impatiens CRESSON) COLONIES TO SMALL HIVE BEETLES (COLEOPTERA: NITIDULIDAE, Aethina tumida MURRAY) By JASON R. GRAHAM A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORI DA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009 1

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2009 Jason R. Graham 2

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T o my family 3

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4 ACKNOWLEDGMENTS I thank Dr. Jamie Ellis, my advisor and Drs. Drion Boucias and Peter Teal for their guidance as my committee and for me ntoring me in the art and scie nce of research. I also thank members of the Honey Bee Research and Extens ion Laboratory, Department of Entomology and Nematology at the University of Florida and the University of Flor ida Insect Pathology Laboratory, the USDA-ARS, Center for Medical Veterinary Entomology (CMAVE), Chemical Ecology Unit, Gainesville, FL and all the staff a nd faculty who made this study possible. Thanks go to Drs. Mark Carroll, Nicole Benda and Panni pa Prompiboon for their help with bioassays and analyses. I also thank gra duate students Tricia Toth, Anthony Vaudo, Eddie Atkinson as well as lab technicians Hannah OMalley, Mike OMa lley, Sparky Vilsaint, Meredith Cenzer, Renee Cole, Jeanette Klopchin and Mark Dykes for thei r moral and technical support. I also thank my family for their encouragement and Marleen Re bisz, my fiance, for her patience and loving support.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAPTER 1 INTRODUCTION..................................................................................................................12 2 SMALL HIVE BEETLE, Aethina tumida MURRAY (COLEOPTERA: NITIDULIDAE) ATTRACTION TO VOLAT ILES PRODUCED BY HONEY BEE, Apis mellifera L. (HYMENOPTERA: APIDAE), AND BUMBLE BEE, Bombus impatiens CRESSON (HYMENOPTERA: APIDAE) COLONIES......................................27 Materials and Methods...........................................................................................................29 Bumble and Honey Bees..............................................................................................29 SHB..............................................................................................................................29 Olfactometer................................................................................................................30 Hive Components.........................................................................................................31 Bumble bee whole hive.......................................................................................31 Honey bee whole hive.........................................................................................31 Adult bees...........................................................................................................31 Brood...................................................................................................................31 Stored honey, pollen and wax.............................................................................31 Choice Bioassays.........................................................................................................31 Statistical Analysis.......................................................................................................31 Results.....................................................................................................................................34 Discussion...............................................................................................................................35 3 A COMPARISON OF THE VOLATI LES PRODUCED BY COMMERCIAL Apis mellifera L. (HYMENOPTERA: APIDAE) AND Bombus impatiens CRESSON (HYMENOPTERA: APIDAE) COLONIES..........................................................................49 Materials and Methods...........................................................................................................50 Bee Source and Establishment.....................................................................................50 Volatile Collections.....................................................................................................50 Whole colonies....................................................................................................50 Colony components............................................................................................51 Volatile Analysis..........................................................................................................52 Results.....................................................................................................................................52 Discussion...............................................................................................................................53

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6 Whole Colonies............................................................................................................53 Colony Components.....................................................................................................54 Adults..................................................................................................................54 Brood...................................................................................................................55 Honey, pollen and wax.......................................................................................56 4 THE PRESENCE OF Kodamaea ohmeri (ASCOMYCOTA: SACCHAROMYCETACEAE) IN COMMERCIAL Bombus impatiens CRESSON (HYMENOPTERA: APIDAE) COLONIES AND THE RESULTING ECOLOGICAL RAMIFICATIONS.................................................................................................................73 Materials and Methods...........................................................................................................75 Bombus impatiens Colonies.........................................................................................75 Yeast Collection...........................................................................................................75 Yeast Culture and Volatile Collection.........................................................................76 Yeast Replication Rate.................................................................................................77 Pollen Preparation and Inoculation..............................................................................77 DNA Isolation and Analysis........................................................................................78 Results.....................................................................................................................................79 Yeast Collection...........................................................................................................79 Morphological Comparisons and Growth Rate...........................................................79 Volatile Comparisons...................................................................................................80 PCR Reactions.............................................................................................................80 DNA Sequencing.........................................................................................................81 Discussion...............................................................................................................................81 Yeast Identification......................................................................................................82 K. ohmeri Presence In Bumble Bee Colonies..............................................................84 5 DISCUSSION................................................................................................................... ....103 LIST OF REFERENCES.............................................................................................................110 BIOGRAPHICAL SKETCH.......................................................................................................123

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7 LIST OF TABLES Table page 1-1 A brief comparison of bumble bee and honey bee natural history.......................................26 2-1 Attraction of SHBs to bumble b ee colony components (pooled controls)...........................40 2-2 Attraction of SHBs to bumble bee colony components (separate controls).........................41 2-3 Attraction of SHBs to honey bee co lony components (pooled controls)..............................42 2-4 Attraction of SHBs to honey bee co lony components (separate controls)...........................43 2-5 Attraction of SHBs to honey bee or bumble bee colony co mponents (pooled controls)......44 2-6 Attraction of SHBs to honey bee or bumble bee colony components (separate controls)...45 3-1 The amount of materials collected from bumble bees and honey bee colonies....................58 3-2 Chemical compounds corresponding to p eaks from total ion chromatographs of volatiles collected from bumble bee and honey bee whole colonies and individual colony components...............................................................................................................59 4-1 Yeast presence on commercial bumble bee colony constituents..........................................88 4-2 Chemical compounds corresponding to p eaks from total ion chromatograph of volatiles collected from sterilized bumble b ee stored pollen inoculated with yeast 1, 2, 3, L-27, 4, and dead L-27 isolates.........................................................................................89 4-3 The known function of volatile compounds collected from yeast isolates...........................92

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8 LIST OF FIGURES Figure page 2-1 Lateral view of the four-way olfactometer...........................................................................46 2-2 The four way olfactometer used for SHB choice tests.........................................................47 2-3 Lateral view of the four way ol factometer showing insect inlet...........................................48 3-1 Volatile collection from a whole bumble bee hives..............................................................66 3-2 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee adults....................................................................................................................67 3-3 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee brood....................................................................................................................68 3-4 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee whole hives..........................................................................................................69 3-5 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee honey...................................................................................................................70 3-6 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee pollen...................................................................................................................71 3-7 Representative total ion chromatograms of volatiles collected from bumble bee and honey bee wax.......................................................................................................................72 4-1 External photographs of the quad system.............................................................................93 4-2 Diagrams of the internal components of the quad system....................................................94 4-3 Photographs of cultured yeast isolates..................................................................................95 4-4 Replication chart of yeast isolates collected from commerc ial bumble bee colonies (1, 2, 3 and 4) and Kodamaea ohmeri (L-27).............................................................................96 4-5 Representative total ion chromatograms of volatiles collected from sterilized bumble bee stored pollen inoculated with yeast isolates...................................................................97 4-6 Ethidium bromide stained gel of the bumble bee yeast isolates amplified using primers NL-1/NL-4 and F17/R317....................................................................................................98 4-7 Ethidium bromide stained gel of the bumble bee yeast isolates amplified using primers AB28 and TW81...................................................................................................................99

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4-8 Clustal 2.0.10 multiple sequence alignm ent using primers NL1 and NL4, for the 5 divergent domain of the 28S rDNA of bu mble bee yeast isolates 1,2,3 and A-1, the known K. ohmeri isolate..................................................................................................100 4-9 Clustal 2.0.10 multiple sequence alignment using primers NL1 and NL4, for the 5 divergent domain of the 28S rDNA of bu mble bee yeast isolates 1,2,3,4 and A-1, a known K. ohmeri isolate..................................................................................................101 4-10 Clustal 2.0.10 multiple sequence alignment using primers AB28 and TW81 (Curran et al., 1994) for the ITS-5.8S region to dis tinguish the yeast is olates 1,2,3,4, and known K. ohmeri isolates A-1 and L-27......................................................................................102 9

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10 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science THE ATTRACTION OF BUMBLE BEE (HYMENOPTERA: APIDAE, Bombus impatiens CRESSON ) COLONIES TO SMALL HIVE BEETLES (COLEOPTERA: NITIDULIDAE, Aethina tumida MURRAY) By Jason R. Graham December 2009 Chair: James D. Ellis Major: Entomology and Nematology The small hive beetle ( Aethina tumida Murray, Coleoptera: Nitidulidae; SHB) a pest of the Western honey bee (Apis mellifera L.; Hymenoptera: Apidae) has been found in commercial bumble bee ( Bombus impatiens Cresson, Hymenoptera: Apidae) colonies. A genus level host shift may be devastating to commercial and wild bumble bee colonies, a significant problem due to the value of bumble bees as pollinators. Further, bumble bee colonies may serve as an unmonitored source of SHB reproduction. For thes e reasons, understanding what mediates the attraction of SHBs to bumble bee colonies is important. Previously, investigators discovered a multitrophic interaction in which Kodamaea ohmeri (Ascomycota: Saccharomycetaceae), yeast transmitted by SHBs, played an important role in host location. Presumably, SHB deposit K. ohmeri onto pollen while they feed. When exposed to pollen, K. ohmeri produces components of honey bee al arm pheromone found to attract SHBs. To better understand SHB attraction to bumble bee colonies, three studies were conducted to investigate bumble bee/SHB interactions. In the first study (Chapter 2), SHB attracti on to components presen t in honey bee and bumble bee colonies was investigated under the hypothesis that SHB are as attracted to bumble

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11 bee produced volatiles as they are to honey bee produced volatiles. The bumble bee vs. control bioassay results suggest that SH B are attracted to bumble bee adults, stored pollen, brood, wax, and the whole bumble bee hive though not to honey. SHB did not show a preference for honey bee or bumble bee components in the honey bee vs. bumble bee assays. Collectively, the data suggest that SHBs are as attracted to bumble bee colonies as they are to honey bee colonies and this attraction is chemically mediated. In the second study (Chapter 3) airborne volatiles were coll ected from commercial bumble bee and honey bee colonies and from each co mponent of both colony types (adult bees, brood, honey, pollen and wax) to determine the chemical profile of volatiles present. In general, the volatile profiles of bumble bee and honey bee colonies were dissimilar with only 7 of 148 total compounds detected common to both colonies. In the final study (Chapter 4), eight commercial bumb le bee colonies were tested for the presence of K. ohmeri which was present on all of the colony sw ab samples (n = 7 colonies 8 swabs/colony = 56 samples). Furthermore, yeast was found in 6 of 7 adult bee samples, 2 of 5 pollen samples, 4 of 8 wax samples but not in brood or honey samples (each sample was from a different colony). This demonstrates that bumbl e bee nests are suitable micro-environments for the growth of K. ohmeri Collectively, these findings support the overa ll hypothesis that attr action of SHBs to bumble bee colonies is chemically mediated. The success of the SHB at expanding its host range may be due to both types of bee colonies harboring K. ohmeri Every effort should be made to determine the susceptibility of wild bee colonies to SHBs and the role K. ohmeri plays in SHB persistence. Ultimately, such investigations shou ld aid the conservation and restoration of wild bee populations.

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CHAP TER 1 INTRODUCTION The social insects wasps, ants, bees and termites represent economic pests to homeowners, provide pollination se rvices to farmers, industries to beekeepers and exterminators as well as fascinating study subjects for many entomologists. Through c ooperative behaviors, individual altruism, reproductive division of labor, chemical communication, and defense, social insects have dominated many terrestrial ha bitats for over 50 million years (Wilson, 1990; Hlldobler & Wilson, 2008). In the lush Amazon rain forest for example, social insects are the true kings of the jungle, accounting for 80% of the total animal biomass (Fittkau & Klinge, 1973). Some social insect colonies, for example those of Dorylus wilverthi Emery, are comprised of millions of individuals acting in unison to procure the necessities of life (Hlldobler & Wilson, 1990). One of the costs of maintaining a social insect colony of such magnitude is in the energetic value of its resources. These resources attract predators, parasites, pa rasitoids, and scavengers (Michener & Michener, 1951; Wilson, 1971; Schmid-Hempel, 1998; Hlldobler & Wilson, 2008). Consequently, the social insects independently have developed arsenals of defensive mechanisms ranging from sticky excretions to barbed, venomous stings to protect their colony (Michener & Michener, 1951; Michener 1974; Prestwich, 1984; Hlldobler & Wilson, 1990). Chemical communication, for example, allo ws the social insects to discriminate a nestmate from other insects, even of the same species (Ribbands, 1954; Wilson, 1965; Free, 1987; Howard, 1993). These general defense mechanisms chemical communication coupled with the location and architecture of the nest can deter many unwanted intruders. Those intruders that manage to pe netrate these defenses use evasive techniques, communication mimicry, defensive morphology and other means in or der to coexist in the nest and benefit from 12

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the colony (W ilson, 1971; Kistner, 1982). They are called symbionts. Symbionts may be categorized according to how they affect their host. A commensalist is a symbiont that benefits while its host is unaffected. In mutualistic symb iosis, both the host and the symbiont benefit. Finally, a parasite is a type of symbiont that obtains some or a ll of its nourishment from its host (Wilson, 1971; Kistner, 1982). It is the parasite that will be consider ed further in this thesis, in particular, the social parasite whos e host is a soci al insect colony. Social parasites may be either generalists having many possible hosts or specialists depending upon a single species for its existence. A further distinction may be made regarding the necessity of the relationship. The obligate soci al parasite has evolved to the point where it can no longer exist in the absence of its spec ific host (Hlldobler & Wilson, 1990). Facultative social parasites are free-living and may choose from a wider variety of hosts (Paracer and Ahmadjian, 2000; DEttorre et. al, 2002). Many parasites, including social parasites, possess the ability to shift hosts, abandoning the old host completely in fa vor of a new host or expanding their host range and increasing th eir repertoire of hosts (DE ttorre et. al, 2002; Neumann & Moritz, 2002). To accomplish either possibility, the social parasite may use an existing host relationship or explore and infest a new host th rough horizontal transmission. For example, while searching for an existing host organism, the paras ite may encounter non-hosts potentially able to fulfill some or all of the parasi tes needs (Jaenike & Papaj, 1992; Foitzik et. al, 2003). These circumstances can lead to a ne w host-parasite relationship. When a host shift occurs, the new host often la cks the experience and adapted defenses that the original host uses to resist the parasite. Ther e are many of examples of this in agriculture, but none perhaps more notable than two parasites that have shifted hosts and now affect races of the Western honey bee (Apis mellifera L.) globally. The first of the parasites is the varroa mite 13

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( Varroa destructor Anderson & Truem an). Varroa mites are parasites of the Asian honey bee Apis cerana Fabr. and, through host shift expans ion, have become parasites of A. mellifera (Oldroyd, 1999). Varroa mites invade brood cells contai ning developing bee larvae. When the cell is sealed the female mites lay eggs and their offspring feed on the hemolymph of the developing bee (Schmid-Hempel, 1998; Caron, 1999). Varroa mite s are minor pests of Asian bees due to several defense behaviors the bees use. These behaviors include extensive grooming, hygienic behavior (removal of va rroa-infested worker brood and non-removal of varroa-infested drone brood), and the plugging of the central pore of infested cells with wax (Delfinado-Baker and Peng, 1995; Fries, 1996; Guzman-Novoa et al., 1996; Spivak, 1996; Schmid-Hempel, 1998; Oldroyd, 1999). The Western honey bee is less efficient at controlli ng varroa due to its relative inexperience with the mite (Oldroyd, 1999; Sanford, 2001; Mondragon et al., 2005). The varroa mite has been found in all major beekeeping coun tries with the exception of Australia and areas of Africa (Zhang, 2000). Using mathematical mode ling, Cook et al., (2007) concluded that keeping varroa out of Australia for the next 30 years would av oid an estimated annual loss of U.S. $16.4 38.8. The second example of a parasite host shift within honey bees is the small hive beetle (Coleoptera, Nitidulidae: Aethina tumida Murray, hereafter referred to as SHB), the principle subject of this thesis. The SHB is a faculta tive pest that has expa nded its host range from colonies of African subspecies of Western hon ey bees to include co lonies of European subspecies of Western honey bees. This host shif t is due to the recent introduction of the SHB into the United States and Australia from its na tive range in sub-Saharan Africa (Elzen et al., 1999; Hood, 2000, 2004; Neumann & Elzen, 2004; Elli s et al., 2004). The SHB is considered only a minor pest of African honey bees, due to bee behaviors such as confinement of adult 14

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beetles and the hygienic rem oval of beetle eggs and larvae (Neumann & Elzen, 2004; Ellis et al., 2004). The natural history of the SHB is reporte d in several reviews (Lundie, 1940; Schmolke, 1974; Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and Hepburn, 2006). In short, adult and larval SHBs feed on honey, pollen, and bee brood within the colony (Lundie, 1940; Schmolke, 1974). It is thought that SHB defecati on causes stored honey to ferment, which may promote nest abandonment by the bees (Lundi e, 1940; Elzen et al., 1999; Hood, 2000). SHBs are attracted to honey bee colonies as places to feed and reproduce. SHB attraction to honey bee colonies was investigated by Suazo et al. (2003) using wind tunnel choice and olfactometric bioassays. In these studies, the researchers found that both male and female SHBs were attracted to adult worker honey bees, freshly collected polle n and a mixture of honey/propolis/pollen/wax from honey bee colonies but not to brood, beeswax or commercially available pollen (Suazo et al., 2003). Once in colonies, SHBs s earch for food, avoid detection, and oviposit. The females lay eggs (1000-2000 ove r lifetime) on pollen and wax, in gaps around the periphery of the hive, and into sealed br ood cells (Lundie, 1940; Schm olke, 1974; Ellis et al., 2003; Hood, 2004; Neumann & Elzen, 2004). Although SHBs are attracted to honey bee coloni es, their existence in bee colonies is met with resistance. The honey bee employs defens ive tactics such as chasing, corralling and guarding SHBs, ejecting SHB larvae and eggs fr om the colony, and absconding (Lundie, 1940; Schmolke 1974; Neumann & Elzen, 2004). In contra st, the SHB has evasion tactics such as dropping from the comb, hiding in hard to reach areas of the hive and tucking its appendages beneath its tank-like body (Lundie, 1940; Neumann & Elzen, 2004). The honey bee/SHB relationship includes several newly documented be haviors such as SHBs ability to solicit food from guard bees (Ellis et al., 2003; Ellis et al. 2004; Ellis, 2005). 15

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Even though the host expansion of SHBs be tween subspecies of African and European A. mellifera has been detrim ental to European bees, a host expansion of the SHB from honey bees to another social bee may prove fatal to the ne w host. When considering this possibility, it is important to understand hypotheses surrounding host shift patterns. There are two opposing hypotheses that address hos t shift patterns, and these remain true for social parasites as well. The host relatedness hypothesis suggests that the parasite is more likely to select a new host that is related to the old host (S haw, 1998; Nikoh and Fukatsu, 2000). The host habitat hypothesis suggests that host shifts are due to th e overlap of ecological niches by unrelated hosts (Norton & Carpenter, 1998 ; Nikoh and Fukatsu, 2000). The bumble bee is both closely related taxonomically and shares a similar ecological niche to the honey bee making the bumble bee colony a likely target of a SHB host shift. To understand these similarities, it is important to understand the na tural history of both bees. Bumble bee natural history has been well documented (Sladen, 1912; Free and Butler, 1959; Wilson, 1971; Alford, 1975; Morse, 1982; Kearns and Thompson, 2001; Goulson, 2003; Heinrich, 2004). Approximately 250 known species of bumble bees exist throughout Europe, North America, Africa and Asia (Williams, 1985 ). Bumble bees, having dense hair, large body size, high body fat content, insulated nests and hibernating queens are adapted to colder climates (Sladen, 1912; Free and Butler, 1959). Bombus species generally have an annual life cycle in which the queens emerge from hibernation in th e spring, found and provision a nest, lay eggs and fulfill the duties of both queen and worker unt il the population of workers is strong enough to carry the responsibilities of the worker caste (Wilson, 1971; Alford, 1975). The nesting sites typically are subterranean in abandoned rodent burrows. The queen bumble bee lays 8-16 eggs in a pollen lump with a wax canopy. The larvae emerge from the eggs in ~5 days and feed on 16

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pollen and nectar for ~14 days. The larvae then spin a silken cocoon and pupate for an additional ~14 days. The developm ent time from egg to adult is approximately four to five weeks (Free and Butler, 1959; Alford, 1975). Like all bees, bumble bees are phytophagous, and feed primarily on nectar and pollen their entire lives. The bees stor e pollen and nectar in individual wax cells very similar to those in which brood develop. These cells are organized more haphazardly than the honey bees architectural comb (Free and Butler, 1959). Unlike bumble bees, honey bees are perennial, surviving winters in temperate regions by storing food and clustering for thermoregulation. Since the col onies may survive many years, they can grow quite large. The honey bee hive c ontains vertically hanging comb composed of horizontally oriented, hexagonal ce lls. These cells are used for pollen and honey storage, as well as worker and drone brood development chambers. The queens are reared in vertically oriented cells called queen cells or queen cups (Langstroth, 1878; Caron, 1999). When comparing honey bees and bumble bees (Table 1), bumble bee colonies are built horizontally and subterranean/at ground level wh ile honey bee colonies are built vertically and are arboreal. Bumble bee colonies have an annual life cycle and smaller populations (100-300 adults) in comparison to the pe rmanent life cycle and larger populations (30,000+ adults) of honey bee colonies (Free and Butler, 1959; W ilson, 1971). Bumble bees are long-proboscis pollinators which permits them access to nectaries in flowers of some plants that short-proboscis honey bees are unable to access. Bumble bees also pollinate by vibration, termed buzz pollination, which is more efficient for plants such as tomato, potato, eggplant, blueberry and cranberry to name a few (Morse, 1982). Bumble bees will forage on cold (below freezing) and rainy days, whereas honey bees are fair-weather po llinators. In fair weather, bumble bees tend to forage faster than honey bees thereby completi ng more trips per day and carrying more pollen 17

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per bee. Conversely, honey bees have m uch larger colonies with a greater number of foragers (Goulson, 2003). Despite these differences, bumble bees share many similarities with honey bees (Table 1), such as pollen and nectar collection and storag e habits, abdominal wax secretion glands that produce the wax used to build comb (their nesting infrastructure), and social colonies of a single queen, drones, and many female workers (W ilson, 1971; Michener, 1974; Alford, 1975; Caron, 1999). They also host the same or similar, pests and pathogens (A lford, 1975; Kistner, 1982, Bailey and Ball, 1991, Schmid-Hempel, 1998). Sim ilar colony compositions, nesting strategies, and natural histories are probable reasons that bumble bee and honey bee colonies host many similar pests and pathogens. It is important to cons ider a few of the shared pathogens in order to put the potential SHB host shift to bumble bees into greater context. First, bumble bees and honey bees can host th e same viruses. Acute paralysis virus (APV) of honey bees, vectored and activated by varroa, ha s been shown to infect bumble bees (Bailey and Ball, 1991; Schmid-Hempel, 1998). Similarl y, deformed wing virus, (DWV) vectored by varroa as well, can infect both bees (Genersch et al., 2005). While these experiments show the viruses could be transmitted to bumble bees, th e varroa vectoring the diseases have no known associations with bumble bees. Bumble bees and honey bees also serve as hosts for some of the same mollicutes. Mollicutes are mobile, bacteria-like prokaryotes that have been placed in the family Spiroplasmatacaea (Whitcomb, 1983; Whitcom b et al., 1987; Schmid-Hempel, 1998). Spiroplasma spp. often are associated with flowers a nd found in the gut of insects across several orders. Spiroplasma melliferum and Spiroplasma apis are lethal to honey bees. Twenty-eight different strains of Spiroplasma have been identified from the hemolymph of B. impatiens and B. 18

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pennsylvan icus Degeer (Clark et al., 1985). Aerobacter cloaca is a bacterium that causes Bmelanosis, a disease of honey bee queens that affects the ovaries. Aerobacter cloaca has also been isolated from bumble bee queens, wher e it may or may not cause the disease (SchmidHempel, 1998). Several fungal species have been discussed in both bumble bee and honey bee studies and including Aspergillus candidus, Aspergillus niger Beauveria bassiana Beauveria tenella Candida pulcherrima Cephalosporium and Paecilomyces farinosus (Schmid-Hempel, 1998). Kodamaea ohmeri is yeast that has been isolated from SHB infested European and African honey bee colonies (Torto et al., 2007); and its presence in bumble bee colonies is an important component of this thesis (Chapter 4). Nosema apis and Nosema bombi are microsporidia that inf ect honey bees and bumble bees, respectively. The honey bee pathogen Nosema ceranae (Fries et al., 1996; Higes et al., 2006, 2007, 2008) was also recently discov ered in three species of bumb le bees native to Argentina (Plischuk et al., 2009). Honey and bumble bees also host parasitic mites. Tracheal mites ( Acarapis woodi Renni) and varroa mites are notorious para sites of the honey bee. As thei r name suggests, tracheal mites crawl into the trachea of the th oracic spiracles on the adult honey bee and pi erce the cuticle in order to feed on hemolymph (Bailey & Ball, 1991, Schmid-Hempel, 1998). Neither of these mites have been found to parasitize the bumble bee. Despite this, bumble bees do host a mite that inhabits its tracheal system ( Bombacarus buchneri Stammer), as well as externally dwelling hemolymph feeders, Scutacarus acarorum Goeze and Locustacarus buchneri Stammer, which are not found in association with honey bees (Kearns and Thomson, 2001; Otterstatter and Whidden, 2004). There also are less specific mite s which have been found in both bumble bee 19

PAGE 20

and honey bee colonies. Mites from the order Astigmata of the genera Kuzinia, Imparipes, Leptus Pyemotes Pygmephorus Scutacarus Siteroptes Macrocheles, Melichares and Proctolaelaps have been discovered in association with both bees (Schmid-Hempel, 1998). Both bumble bees and honey bees also host multiple species of wax moths (Lepidoptera: Pyralidae). In general, wax moths burrow into be eswax as larvae, feeding upon impurities within the wax, pollen, honey and occasionally brood (La ngstroth, 1878; Schmid-Hempel, 1998; Caron, 1999). The European wax moth, Aphomia sociella L., and the American wax moth, Vitula edmandsii Packard, are harmful pests in the bum ble bee nest (Alford, 1975; Kearns and Thomson, 2001). Aphomia sociella also has been reported on rare occasion in honey bee colonies (Toumanoff, 1939). The greater wax moth, Galleria mellonella L. and lesser wax moth, Achroia grisella Fabr., are similar pests of the honey bee. G. mellonella has been reported in bumble bee colonies as well (Oertel, 1963; Spiewok and Neumann, 2006). Besides the SHB, other beetles are associated with social bees. Both the honey bee and bumble bee have been found to host beetles of the same family as SHB Nitidulidae. Epuraea corticina Erichson have been discovered in honey b ee colonies, and while apparently causing no damage, there is a concern that they could tr ansmit pathogens between colonies (Ellis et al., 2008). Epuraea depressa Illiger has been found in five species of bumble bee colonies as well as social wasp nests (Scott, 1920; Cumber, 1949; Kistner, 1982, Ellis and Hepburn, 2006). Collectively, the data leave open the possibility that the SHB could expand hosts from honey bees to include bumble bees since both bees host nitidulids in their colonies. Investigators have shown in recent studies that bumble bee (Hymenoptera; Apidae; Bombus impatiens Cresson.) colonies are potential alternat ive hosts for the SH B. There currently are three studies in which inve stigators reported th e potential host switch of SHBs from honey 20

PAGE 21

bee to bum ble bee colonies (S tanghellini et al., 2000; Ambros e et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008). These investigations provide the framework for the thesis research hypotheses. In the first study, Ambrose et al. (2000) and Stanghellini et al. (2000) found that commercial colonies of B. impatiens artificially infested with SHBs in vitro had fewer live bees, more dead bees and more comb damage than col onies without SHB. The destruction of infested bumble bee colonies was rapid, extensive a nd terminal (Stanghellini et al., 2000). Further, SHBs were able to complete a full lifecycle within three B. impatiens colonies infested with twenty SHBs under laboratory conditions. The aut hors concluded that if SHBs locate and invade commercial and wild bumble bee colonies, than bumble bees and the ecological communities they support may be at considerable risk (Amb rose et al., 2000; Stanghellini et al., 2000). In the second study, Spiewok and Neum ann (2006) found that commercial B. impatiens colonies maintained within close proximity (~ 100 or ~500 m) to infested honey bee colonies became naturally infested with SHBs and successful SHB reproduction occurred within these colonies. Spiewok & Neumann (2006) also found th at in four-square choice tests the SHBs were attracted to both adult bumble bee workers and pollen from bumble bee colonies. As a result of their work, Spiewok and Neumann concluded that SH Bs are expected to infest not only wild and commercial B. impatiens nests but also nests of other bum ble bee species (Spiewok & Neumann, 2006). Most recently, Hoffman et al. (2008) placed four commercial B. impatiens colonies and four honey bee colonies in a closed greenhouse and released 1,000 SHBs. The colonies were invaded by SHBs which oviposited readily in the colonies and showed no apparent preference to honey bee over bumble bee colonies. The authors al so investigated the defensive responses of 21

PAGE 22

bum ble bees toward the SHB, reporting that bumble bees removed SHB eggs, and stung and removed SHB larvae. As a result, Hoffman et al. (2008) concluded that SHBs do not prefer honey bee to bumble bee colonies and bumble bees are able to use general defense tactics against SHB invasions. In these studies, the SHB has demonstrated the ability to expand hosts from honey bees to bumble bees, a genus-level shift. The potential for a genus leve l host expansion is of immediate concern due to the possibility of magnified repercussions. Commercially managed honey bee populations are far larger than would be found naturally, and in the same proportion their parasite loads could decimate native bees if there were ecological spillovers. Additionally, bumble bee colonies typically are not monitored as closely as those of honey bees. Consequently, introduced honey bee pests could become bumble bee pests, possibly only noticed when bumble bee populations decline drastica lly. For these reasons, it is im portant to better understand the documented attraction of SHBs to bumble bee co lonies. An ignored SHB host shift could be devastating to bumble bee colonies while al so providing an unmonitored source for the reproduction of SHBs. The following ecological an alogy clarifies the risk of a SHB reproductive source. Tallamy (1982) explained how MacArthur a nd Wilsons (1967) equilibrium theory of island biogeography relates to parasites and their hosts. The number of species (parasites) that inhabit an island (host) can be predicted based on the distance the island (host) is from the mainland or other islands (hosts ) and the size of the island (host density or host population). Honey bee and bumble bee colonies can be cons idered islands from which the SHB may acquire all of the resources that they need to reproduce successfully. Th e parasite population most likely would expand beyond the carrying capacity of the host, driving the SHBs to disperse in search of 22

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a new host. Therefore, even if a treatm ent wh ich eliminated SHBs from the honey bee colony was developed and made available to beekeepers, hives could be re-inf ested regularly if SHB were reproducing in nearby bumble bee colo nies (MacArthur & Wilson, 1967; Tallamy, 1982; Price, 1997). Conversely, honey bee colonies are found commercially in mu ch larger population densities than would occur naturally. An overf low of SHBs into the smaller populations of bumble bees would be potentially disastrous to wild populations of bumble bees (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Ne wman, 2006; Hoffman et al., 2008). There is also the concern that the SHB could transmit pathogens from infected honey bee colonies to uninfected honey bee coloni es (Ellis & Hepburn, 2006). In addition to the symbiotic, social and biological commonalities that bumble bees and honey bees share, ecologically they are both im portant pollinators. The threat that the SHB signifies (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008) could have disastrous repercussions, not only on the bees but also on bee-pollinated plants and the communities supported by these pl ants (Allen-Wardell et al., 1998). Honey bee, bumble bee, and pollinator populations in general are declining (Buchman & Nabhan, 1996, Cane & Tepedino, 2001; Goulson, 2003; Goulson et al., 2008). Understanding the mechanisms that drive the host shif t of SHBs from honey bees to bumble bees may lead to practical applications for pollinator conservation, a gr eater understanding of host-parasite dynamics, possible pest-control approaches and a better understand ing of the host-finding cues that lead the SHB to bee colonies. While the SHB is attracted to B. impatiens colonies (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008), it is unclear what mediates this attraction. In general, the hypothesis is that SH B attraction to bumble bee colonies will be 23

PAGE 24

chem ically mediated. In this thes is, findings from three investiga tions conducted in an effort to better understand the mechanisms behind SHB attr action to bumble bee colonies are reported. First, to determine how attract ive airborne volatiles are to SHBs, a comparison was made of SHB responses to bumble bee and honey bee pr oduced volatiles using olfactometric bioassays (Chapter 2). The hypothesis is that SHBs will be as attracted to volatiles present in bumble bee colonies as they are to those present in honey b ee colonies. This is believed to be the case because both colonies release airborne volatiles that are products of similar colony components (pollen, wax, brood, adults and honey). Second, the chemical ecology of commercial B. impatiens colonies will be investigated. The volatile profiles of bumble bee colony components will be compared to those of honey bee colonies. This will be the first comprehensive an alysis of the volatile profile of a bumble bee colony and each of its components. Using standard chemical ecology procedures, airborne volatiles present in commercial bumble bee and honey bee nests as well as volatiles produced by bee adults, brood (eggs, larvae and pupae), wax, stored pollen and honey will be collected and analyzed (Chapter 3). The hypothesis is that bum ble bee-produced volatiles will be similar to those produced by honey bees because the inse cts and their societies are similar. Finally, knowing that K. ohmeri may be an important component of SHB attraction to honey bee colonies (Torto et al., 200 7; Benda et al., 2008), commercial B. impatiens nests will be screened for K. ohmeri by swabbing colonies for the yeast, comparing volatile profiles obtained from any discovered yeasts, and sequencing the yeast DNA (Chapter 4). The hypothesis is that K. ohmeri is an important component of the SHB/bumble bee paradigm and will be discovered in commercial bumble bee colonies. Kodamaea ohmeri is also predicted to produce an alarm 24

PAGE 25

pherom one signature similar to that produced by honey bees when placed on bumble bee collected pollen. Collectively, the data will help to addre ss the overall goal of understanding documented SHB invasion into bumble bee colonies (Stanghellin i et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008) while pl acing this invasion into a larger ecological context. 25

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26 Table 1-1. A brief compar ison of bumble bee and honey bee natural history. 1Sladen, 1912; Free and Butler, 1959; Wilson, 1971; Alford, 1975; Morse, 1982; Kearns and Thompson, 2001; Goulson, 2003; Heinrich, 2004 Bum ble bee1 Honey bee2 Development of worker bee from egg to adult ~35 days ~21 days Queen placement of worker eggs queen lays 8-16 eggs in a pollen lump queen lays eggs individually into wax cells Development of worker eggs ~5 days ~3 days Development of worker larvae ~14 days; larvae disperse through pollen, mass feeding on pollen and nectar ~9 days; larvae individually fed royal jelly days 1-2, afterwards, some pollen and nectar added Development of worker pupae ~ 14 days, in individual wax cell with spun silk ~10 days, in individual wax cell with silk and wax capping Adult bee diet nectar/pollen nectar/pollen Food storage nectar and pollen are stored in vertically oriented ovular wax cells nectar and pollen are stored in horizontally oriented hexagon wax cells Colony life cycle annual perennial Colony orientation horizontal vertical Feral nesting sites subterranean or ground level cavities arboreal or ground level cavities Commercial hives cardboard quads with plastic and cotton nesting material -unmonitored wooden Langstroth hives with wax or Plasticell foundation -monitored frequently Colony size 100-300 adults 30,000+ adults Pollination -long proboscis -buzz pollination -used commercially on variety of crops -forage in cold, wet, and fair conditions -short proboscis -used commercially on a variety of crops -forage only in fair conditions 2Langstroth, 1878; Wilson, 1971; Michener, 1974; Butler, 1975; Morse, 1975; Hooper, 1976; Caron, 1999

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CHAP TER 2 SMALL HIVE BEETLE, Aethina tumida MURRAY (COLEOPTERA: NITIDULIDAE), ATTRACTION TO VOLATILES PR ODUCED BY HONEY BEE, Apis mellifera L. (HYMENOPTERA: APIDAE), AND BUMBLE BEE, Bombus impatiens CRESSON (HYMENOPTERA: APIDAE) COLONIES The small hive beetle (Coleoptera, Nitidulidae: Aethina tumida Murray, hereafter referred to as SHB), is a facultative pest of Western honey bees (Hymenoptera: Apidae, Apis mellifera L.) that has shifted hosts from colonies of African subspecies of honey bee to those of European subspecies of honey bee. This host shift is due to the recent introduction of the SHB into the United States and Australia from its native ra nge of sub-Saharan Afri ca (Elzen et al., 1999; Hood, 2000, 2004; Neumann & Elzen, 2004; Ellis & Hepburn, 2006). Moreover, investigators have shown in recent studies that comme rcial bumble bee (Hymenoptera: Apidae, Bombus spp.) colonies can serve as alternative hosts for the SH B, a new relationship that may be detrimental to wild and managed populations of bumble bee coloni es (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Neumann, 2006; Hoffman et al., 2008). In the first study, Ambrose et al. (2000) and Stanghellini et al. (2000) found that commercial colonies of B. impatiens Cresson artificially infested with SHB in vitro had fewer live bees, more dead bees and more comb damage. Further, SHBs were able to complete a full lifecycle within three B. impatiens colonies infested with twenty SHB each in vitro The authors concluded that if SHBs are able to locate and in vade commercial and wild bumble bee colonies, then bumble bees and the ecological communities they support may be at considerable risk (Ambrose et al., 2000; Stanghellini et al., 2000). In the second study, Spiewok and Neum ann (2006) found that commercial B. impatiens colonies maintained within close proximity (~ 100 or ~500 m) to infested honey bee colonies became naturally infested with SHBs and successful SHB reproduction occurred within these colonies. Spiewok & Neumann (2006) also found th at in four-square choice tests the SHBs were 27

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attracted to both adult b umble bee workers a nd stored pollen (also ca lled bee bread) from bumble bee colonies. As a result of their wo rk, Spiewok and Neumann concluded that SHB are expected to infest not only wild and commercial B. impatiens nests but also nests of other bumble bee species (Spiewok & Neumann, 2006). Most recently, Hoffman et al. (2008) placed four commercial B. impatiens colonies and four honey bee colonies in a closed greenhouse and released 1,000 SHBs. The colonies of both bee types were invaded by SHBs which readily oviposited in the colonies and showed no apparent preference to honey b ee over bumble bee colonies. While the SHB is attracted to B. impatiens colonies (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008), it is unclear what mediates this attraction. The general hypothesis of th is thesis is that th is attraction is mediated chemically as is SHB attraction to honey bee colonies (Su azo et al., 2003, Torto et al., 2005, 2007). In this study, original research is reported where volatile s were collected from whole honey bee and bumble bee colonies as well as from individual hive com ponents (adult bees, brood, honey, stored pollen, and wax) and the attractivene ss of these compounds to SHBs were tested in four-way olfactometer choice tests. This was done in an attempt to understand the recent SHB host expansion from honey bees to bumble bees, hypothesizing that SHBs will potentially be as attracted to bumble bee produced volatiles as they are to honey bee produced volatiles. Kodamaea ohmeri a yeast that produces volatiles known a ttractant to SHBs (Arbogast et al., 2007; Torto et al. 2007ab, Benda et al., 2008, Arbogast et al., 2009) was found from washes of SHBs, adult honey bees and in swabs taken from honey bee colonies (Torto et al. 2007ab, Benda et al., 2008), and was found more recently on ad ult bumble bees, bumble bee stored pollen, wax and swabs from commercial bumble bee colonies (Chapter 4). Knowing that K. ohmeri is present 28

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in comm ercial bumble bee colonies, SHBs are exp ected to be as attracted to bumble bee colony components and whole hives as they are to hon ey bee colony components and whole hives. Materials and Methods Bumble and Honey Bees In January 2009, a quad containing four commercial B. impatiens colonies was purchased from Koppert Biological Systems, Inc. (Romulus, MI) and located at the Un iversity of Floridas Bee Biology Unit in Gainesville, FL (N 29 37.629" W 82 21.405"). Each colony contained a corn-syrup/water solution, provided by Koppert Biological, Inc., for periods of low nectar flow. All colonies had a reproductive queen, 200-250 workers, brood and nesting material (cotton and plastic). The quad was placed on a wooden pallet and the colony entrances opened, permitting the bees to forage. Four honey bee colonies were maintained at the University of Floridas Bee Biology Unit in Gainesville, FL. All colonies were housed in full-sized, Langstroth-style hives, headed by a single queen, and used as produc tion colonies during the previ ous year. The colonies were provided a corn-syrup/water solu tion during periods of low nectar flow. The bumble bee and honey bee colonies were maintained in the same location, exposing the bees to identical environmental conditions and forage availability. SHB The SHBs used in these bioassays were mass-produced at the H oney Bee Research and Extension Laboratory (HBREL, Univ ersity of Florida, Gainesvi lle) using modified rearing methods originally outlined by Mrrle & Ne umann (2004). All SHB used in the various bioassays were 1-2 w post-emergence. After emer gence, the SHBs were fed only sugar water for three days prior to all bioassays. The bioassays were conducted between 18:00 and 2:30 of the following morning when SHB seem most active (Ellis et al., 2003). 29

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Olfa ctometer A 4-way olfactometer designed by member s of the USDA-ARS, CMAVE (Center for Medical, Agricultural and Veterinary Entomology), Chemical Ecology Unit, Gainesville, FL was used to determine SHB attraction to honey b ee and bumble bee col ony components and whole colony volatiles. The olfactometer (3.66 m 3.66 m 30.5 cm) was a 4-port/4-choice arena olfactometer, with a removable lid (Figures 13). The main body and ports were composed of solid UHMW-poly ethylene with a 0.95 cm thick cl ear Plexiglas removable lid. At the 4 glass inlet ports, Internal Odor Source (IOS) Adapters/Insect Isolation Traps (IIT) were attached to collect insects responding to th e odor source and prevent them from returning to the arena (Figures 1 & 2). Corrugated FEP tubing (1.15 cm inside diameter (ID) 1.27 cm outside diameter (OD), Cole-Parmer, Vernon Hills, IL) wa s attached to the IOS/IIT leading to the Odor Source Container (OSC). The OSC held the hive component being tested and differed depending on the component being tested and the availability of materials. The sa me corrugated tubing led from these containers to carbol oy air flow regulators (Aalborg Instruments and Controls, Inc., Orangeburg, NY) set at 0.5 liter/min and connected to 2 portable filtered-air pumps developed by the USDA/ARS (CMAVE, Gainesville, FL) (Figures 1 & 2). The insect inlet port was modified to adap t to a SHB release port constructed of 0.32 cm industrial tubing (Cole-Parmer, Vernon Hills, IL), nylon mesh and 0.64 cm industrial tubing (Cole-Parmer, Vernon Hills, IL; see Figure 3). The release port was connected to Master flex Tygon tubing (Cole-Parmer, Vernon Hills, IL) wh ich led to a carboloy air flow regulator (Aalborg Instruments and Controls, Inc., Orangeburg, NY) set at 2 liter/min which was further connected via Master flex Tygon tubing (Col e-Parmer, Vernon Hills, IL) to the house vacuum at the USDA/ARS (CMAVE, Gain esville, FL) (Figures 1 & 3). 30

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The bioassay was conducted in a walk-i n incubator at the USDA/ARS (CMAVE, Gainesville, FL) m aintained at approximately 31 C and 65% RH with 4 banks of Ecolux fluorescent lighting (General Electric Company, Fairfield, CT). Hive Components Bumble bee whole hive Two bumble bee colonies were removed from a quad and the plastic housing in which they were shipped and placed into a chamber deve loped by members of the CMAVE. The chamber was a steel pan with 1.25 cm holes (for ports) drilled in to either end and enclosed with a glass lid. The two colonies were placed side by side within the container, and the lid was secured with duct tape to prohibit air from escaping. Honey bee whole hive To collect a whole honey bee colony, a 1.25 cm port hole was drilled into the front and back of a healthy, 5-frame nucleus colony. The colony contained 5 (23.2 cm x 47.9 cm) frames with 21.27 cm Plasticell foundation (Dadant a nd Sons, Inc., Hamilton, IL), a queen, brood, honey, pollen, wax and adult bees. A telescoping lid was placed on the top and bottom of the nuc body and any cracks on the periphery where air c ould escape were sealed with duct tape. Adult bees All adult bumble bees (~32 g per colony) were collected from four bumble bee colonies and placed by colony into separate glass OSCs (3.8 L each). The collection chambers were maintained at 33 C, 65% humidity and 12 h of daylight. The adults were provisioned with a cotton wick saturated with a 50% sugar water soluti on. The adult honey bees (~32 g per colony) also were collected from four colonies and handled identically. All bee OSCs were connected to the olfactometer within 0.5 h of bee capture and the bioassay began approximately 1 h after capture. 31

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Brood Honey bee brood of all ages (egg to pupae) we re collected by taking brood fra mes from two colonies and using tin snips to cut out a nd remove a square of brood weighing ~32 g each. The brood square was pushed back into the fram e immediately and return ed to the honey bee colony. This gave the bees time to remove the m acerated brood and repair the wax located on the edge of the newly-cut square. After 2 d, the br ood square was removed and placed into an OSC ~30 min prior to the bioassay. One square was used for the honey bee brood vs. blank bioassay and the other for the honey bee brood vs. bum ble bee brood bioassay (b oth described below). Two groups (~32 g each) of live larvae/pupae we re collected from the commercial bumble bee colonies and placed into separate OSCs. One group was used for the bumble bee brood vs. blank bioassay and the other for the bumble bee brood vs. honey bee brood bioassay. The majority of the bumble bee brood collected was en closed in wax cells which differ in color and texture from wax cells contai ning honey or pollen. To avoid disturbing or injuring the brood within, and to keep the bioassay as natural as possible, the brood cells were left unopened during the bioassays. Stored pollen, honey and wax Stored pollen (~ 32 g), honey (~32 g) and wax (~ 32 g) were collected separately from each of the honey bee and bumble bee colonies. One collection of stored pollen was made from two honey bee colonies using a small autoclaved spatula. The bumble bee pollen was collected from two bumble bee colonies by removing and opening pollen cells and extracting the stored pollen with an autoclaved spatula. All stored pollen portions were weighed on filter paper and then transferred directly into the OSC. Honey bee honey was collected from two fram es, one each from two separate colonies. The honey collection was made by scraping honey out of the wax cells using an autoclaved 32

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spoon. The honey then was transferred into a gl ass Petri dish and weighed. Bum ble bee honey was collected through squeezing the wax honey cells to force the honey from them and into a glass Petri dish. The components were transferred into their respective OSCs and used in studies within 2 d of collection. To collect honey bee wax, a frame containing honey, pollen and brood was placed close to managed bee colonies. The bees from these colo nies robbed the combs, leaving only the wax behind. Once the wax was empty, it was scraped from the frame using an autoclaved hive tool. The wax was put into an OSC approximately 0.5 h prior to the bioassay. Wa x cells containing no stored pollen, honey, or brood were collected from two bumble bee colonies, weighed, and then put into an OSC. Choice Bioassays The choice bioassays were designed to test SHB attraction to (1) bumble bee colony components in the absence of other attractants (bumble bee vs. blank, single odor source bioassay), (2) honey bee colony components in the absence of other attractants (honey bee vs. blank, single odor source bioassay), and (3) honey bee vs. bumble bee colony components (honey bee vs. bumble bee, dual odor source bioassay). The single odor bioassays were composed of the whole colony or individual component in an OSC connected to one of the four ports of the olfactometer. The other three ports we re connected to a supply of clean, filtered air. For the dual odor source bioassays, the four ports were connected to OSCs housing bumble bee whole colony or individual components at one port, the honey bee equivalent at a second port, and clean, filtered air in the ot her two ports. Each port delivered air at 0.5 L/min while the vacuum port pulled air through the sy stem at a rate of 2 L/min. The SHBs were held in a 1 L container with a cotton wick saturated with sugar water (50:50 solution) while the olf actometer was being set up. The sex of the SHBs was not 33

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determ ined and left to chance; therefore gender specific behaviors were not evaluated. The SHBs were never used twice. A hit (= choice) was determined after the SHB crossed the threshold from the olfactometer into a glass trap (see Figures 1 & 2). If a test SHB showed no response after 10 minutes, it was removed from the arena and repl aced with another SHB. The positions of the odor source and control ports were chosen randomly and changed af ter every 5 SHBs tested. For each trial, 40 responding SHBs were used and th e trials typically took ~5 h to complete. Each group of five individual SHBs re leased was considered a repli cate and the ports were randomly repositioned between replicates. Statistical analysis The data were analyzed two ways: with pooled and separate controls For pooled controls, the control data were pooled and considered one c hoice. For separate controls, control data were not pooled and each control stati on considered a single choice. Fo r all analyses, the data were compared between main effects (whole colonies and colony components) and controls (pooled and separate) using a 1-way ANOVA with means separated using Tukey tests (SAS Institute, 2008). Results SHBs were attracted to whole bumble b ee colonies, wax, stored pollen, and brood significantly more than to clean air (pooled and separate controls Tables 1 and 2). SHBs were not attracted to adult bumble bees more than to the pooled controls while they were more attracted to bumble bee adults than to two of the three separa te control air por ts (Table 2). Furthermore, SHB were more attracted to pooled control ports than to bumble bee honey but no differences existed when the controls were considered separate choices (Table 2) 34

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Honey bee wax, honey and brood were found to be m ore attractive to SHBs than clean air (pooled or separate controls, Ta bles 3 and 4). Honey bee adults and whole hives were not found to be significantly more attract ive to SHB than the pooled contro ls (Table 3) while they were more attractive to SHB than the separate contro ls (Table 4). SHB attraction to honey bee stored pollen was similar to that of pooled and separate controls (Tables 3, 4). The results from the bumble bee vs. honey b ee component bioassays are reported in tables 5 and 6. There was no significant difference in SH B attraction to the poo led controls or the honey bee and bumble bee adults, brood, stored po llen, wax or whole hive s (Table 5). Bumble bee honey was significantly less attr active to SHBs than the pooled controls, but the attraction of SHBs to honey bee honey was not significantly differe nt from either (Table 5). Even when the controls were not pooled, SHB attraction to honey bee and bumble bee components was never different than that to at least one of the controls; neither were they different from one another (Figure 6). Discussion In general, the choice bioassays provided an understanding of wh at olfactory stimuli attract SHBs to bumble and honey bee coloni es (Tables 1-6). The bumble bee vs. nothing bioassay results suggest that SH Bs are attracted to bumble bee wax, stored pollen, brood, and adults as well as the whole bumble bee hive alth ough not to honey (Table s 1 & 2). This supports findings by Spiewok & Neumann (2006) in which SHBs were attracted to bumble bee adults and stored pollen from bumble bee co lonies more than to the three control squares in four square choice tests. SHB attraction to wa x, which may absorb volatiles of components stored within it, is unsurprising as SHB were attracted to most of the components normally stored in wax (stored pollen and brood, Tables 1 & 2). 35

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Bum ble bee brood was also found to be attr active to SHBs (Tables 1 & 2). Boer and Duchateau (2006) recently found evidence that B. terrestris brood uses cuticular chemicals to indicate to worker bees a need for food. Us ing gas chromatography and mass spectrometry (GCMS) analyses, B. impatiens brood volatiles were found to be composed of octane, 2heptanone, limonene, (E)-beta-ocimene, nonanyl acetate, pentadecane, heptadecane (Chapter 3). Because the SHBs were attracted to bumble b ee brood (Tables 1 & 2) and the volatile analysis showed these chemical compounds present in the volatile profile (Chapter 3), further study of these compounds should provide a greater unde rstanding of SHB attraction to bumble bee colonies. It is clear that SHBs are at tracted to components collected from bumble bee hives (Tables 1 & 2), thus supporting data from previous i nvestigations (Spiewok & Neumann, 2006; Hoffman et al. 2008). While Spiewok & Neumann (2006) conducted their investig ations using field studies and with Hoffman et al. (2008) using a greenhouse, here data is presented from choice tests conducted with an olfactometer, allowi ng for the study of SHB decision making in the absence of visual cues. Collectively, the data sugg est that SHBs use airbor ne volatiles to locate host colonies. This seems intuitive as SHBs typica lly search for their host colonies in the evening and at night (Ellis et al., 2003) and, once they find them, live in host colonies where light is restricted. SHBs also were attracted to honey bee wa x, honey, brood, adults, and whole colonies but not to stored pollen (Tables 3 & 4). This is consis tent with some of the findings of Suazo et al (2003) who found in olfactometric and wind tunnel c hoice tests that SHB were attracted to adult worker honey bees, a mixture of honey/propolis/ pollen/wax from honey bee colonies, and to freshly collected pollen, but not to brood, beeswa x or commercially available pollen. The SHB 36

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attraction to a m ixture of honey/pr opolis/pollen/wax was not tested in this study but rather their attraction to honey, stored pollen and wax separa tely. All honey bee colony components except honey bee stored pollen were attractive to SHB, supporting the coll ective findings of Suazo et al (2003). In this study, honey bee stor ed pollen was not found to be attractive to SHB while bumble bee stored pollen was attractive to SHBs (Table 4). The reason for this is unclear, especially since the bumble and honey bees we re foraging in the same areas, presumably collecting the same/similar pollen sources. One po ssibility for the difference in attraction to stored pollen processed by both bees is that bumble bee and honey bee stored pollen have different volatile profiles (C hapter 3). Also possible is that the SHB-attracting yeast Kodamaea ohmeri played a role in differing SHB attraction to bumble and honey b ee stored pollen (Torto et al. 2007a). Kodamaea ohmeri was not tested for in the stored pollen used in this portion of the study. SHB are known carriers of K. ohmeri (Torto et al., 2007ab, Benda et al., 2008). While the test honey bee colonies did host SHBs, none we re found in the bumble bee colonies. However, K. ohmeri has been found in commercial bumble bee co lonies in the absen ce of detected SHB presence (Chapter 4). Other possible reasons exist for differing leve ls of attraction of SHBs to honey bee and bumble bee stored pollen. Suazo et al. (2003) found that SHBs are attracted to collected honey bee pollen while I found that they were not attracted to stored pollen. Pollen undergoes processing before it is stored. Gilliam (1979) studied yeast vi ability on honey bee collected pollen and stored pollen by isol ating 113 yeasts from almond ( Prunus communis Kom.) flowers, pollen from pollen traps, and stor ed pollen. Strikingly, most of the yeasts that were found on the flowers and in the bee collected pollen taken fr om pollen traps were not found in the stored 37

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pollen (Gilliam 1979). Consequently, there may be some other processing that pollen undergoes before worker bees place it in cells, making it una ttractive to SHBs. Furt her, bumble bee stored pollen and honey bee stored pollen emit different volatile profiles, possibly resulting in the differing level of attraction of SHB to stored hon ey bee and bumble bee pollens (Chapter 3). Suazo et al (2003) found that honey bee brood (pupae removed from the comb, 10 g) was not attractive to SHB, whereas my results suggest that honey bee brood (eggs, larvae, pre-pupae and pupae within the comb, 32 g) is There are three possible reasons for this difference. First, this study tested SHB attractiveness to all br ood stages simultaneously whereas Suazo et al (2003) tested attractiveness only to pupae. Secondly, SHB attraction to brood may be dose dependent and positively correlated. This study te sted SHB attraction to 3 times more brood per replicate than Suazo et al. (2003) did. Finall y, the chemical signals produced by the brood may have been different in both studies due to our respective methods of hand ling the brood prior to analysis. Immature bees removed from individual cells, as in Suazo et al. (2003), may have been stressed, thus altering their vo latile profile whereas brood within the comb (reported here) may have been less disturbed. In general, the data from the bumble bee vs. honey bee choice bioassays were not straightforward. The bioassays did not indi cate significant SHB attraction to honey bee components over bumble bee components (Table 5 & 6). Furthermore, the assays did not detect an overwhelming attraction of SHB to any partic ular bee component over that of the controls, pooled or otherwise. This was perhaps an artifact of the testing protocol rather than an indication of a biological phenomenon because of the clear attraction of SHB to colony components of both bees in the individual component bioassays. SHB may have been overwhelmed with stimuli from the two distinct odor sour ces (honey bee and bumble bee components), thus regularly going 38

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to the clean air controls in confusion. Furtherm ore, the choice test arena w as small, possibly making the direction of an odor source to a searching SHB unclear when two or more sources were used. As such, another choice test design should be used to determine the preference, or lack therefore, of SHB to either bumble bee or honey bee colonies. SHB attraction to bumble bee and honey bee colo nies was addressed in part by Hoffman et al. (2008) who placed four commercial B. impatiens colonies and four honey bee colonies in a closed greenhouse and released 1,000 SHBs. All colonies of both bee types were invaded by SHB which oviposited readily in the colonies and showed no apparent preference to honey bee over bumble bee colonies (Hoffman et al. 2008). Fu rther investigations ar e needed to determine whether SHB are more, less, or equally attr acted to honey bee and bumble bee colonies. Beekeepers have witnessed the devastation that SHB presence can exact on honey bee colonies. If bumble bee colonies face the same threat, the results could be disastrous to commercial and wild bumble bee populations as well as the ecol ogical communities they support. SHB spillover from commercial bumble bee colonies to wild colonies has not been documented, but wild colonies contain the same components as comm ercial ones and these components are known to attract SHB, making SHB attraction to wild colonies likely. Future investigations need to be conducted to determine if SHB have begun to infest wild bumble bee colonies. 39

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Table 2-1. Attraction of SHBs to bum ble bee colony components (pooled controls). Bumble bee Hive component Control ANOVA wax (32 g) 3.8 0.3a 1.2 0.6b (F = 0.5; df = 1,15; P < 0.01) honey (32 g) 1.6 0.4b 3.4 0.4a (F = 10.9; df = 1,15; P = 0.01) adults (32 g) 2.4 0.5a 2.6 0.5a (F = 0.1; df = 1,15; P = 0.73) stored pollen (10 g) 3 0.3a 2 0.3b (F = 7; df = 1,15; P = 0.02) brood (32 g) 3.9 0.4a 1.1 0.4b (F = 19.5; df = 1,15; P < 0.01) hive 3 0.2a 2 0.2b (F = 14; df = 1,15; P < 0.01) SHBs were released into a four-way olfactometer. Control represents th ree control ports that emitted filtered air; the control data were pooled and considered one choice. Hive component represents air passed through a ch amber containing one of the bu mble bee colony constituents and it was emitted through a single port. Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single beetle was re leased twice. Row data followed by different letters are different at 0.05. 40

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Table 2-2. Attraction of SHBs to bum ble bee colony comp onents (separate controls). Bumble bee Hive component Control 1 Control 2 Control 3 ANOVA wax (32 g) 3.8 0.3a 0.5 0.2b 0.1 0.1b 0.6 0.2b (F = 76.7; df = 3,31; P < 0.01) honey (32 g) 1.7 0.4a 1.1 0.2a 1.1 0.4a 1.1 0.3a (F = 0.6; df = 3,31; P = 0.61) adults (32 g) 2.3 0.5a 1.5 0.5ab 0.9 0.2b 0.3 0.2b (F = 6.1; df = 3,31; P < 0.01) stored pollen (10 g) 3 0.3a 0.6 0.3b 0.6 0.4b 0.8 0.3b (F = 15.9; df = 3,31; P < 0.01) brood (32 g) 3.8 0.4a 0.3 0.2b 0.5 0.3b 0.4 0.2b (F = 37.7; df = 3,31; P < 0.01) hive 3 0.2a 0.5 0.2b 0.6 0.2b 0.9 0.1b (F = 46; df = 3,31; P < 0.01) SHBs were released into a four-way olfactometer. Control represents th ree control ports that emitted filtered air; the control data were left separate and considered three choices. Hive component represents air passed through a ch amber containing one of the bumble bee colony constituents and it was emitted through a single port Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single b eetle was released twice. Row data followed by different letters are different at 0.05. 41

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Table 2-3. Attraction of SHBs to honey b ee colony com ponents (pooled controls). Honey bee Hive component Control ANOVA wax (32 g) 3.9 0.3a 1.1 0.3b (F = 43.4; df = 1,15; P < 0.01) honey (32 g) 3.1 0.4a 1.9 0.4b (F = 4.9; df = 1,15; P = 0.04) adults (32 g) 2.8 0.4a 2.2 0.4a (F = 0.9; df = 1,15; P = 0.35) stored pollen (10 g) 2 0.5a 3 0.5a (F = 2.3; df = 1,15; P = 0.15) brood (32 g) 2.9 0.2a 2.1 0.2b (F = 5.5; df = 1,15; P = 0.03) hive 2.6 0.3a 2.4 0.3a (F = 0.5; df = 1,15; P = 0.51) SHBs were released into a four-way olfactometer. Control represents th ree control ports that emitted filtered air; the control data were pooled and considered one choice. Hive component represents air passed through a ch amber containing one of the honey bee colony constituents and it was emitted through a single port. Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single beetle was released twice. Row data followe d by different letters are different at 0.05. 42

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Table 2-4. Attraction of SHBs to honey b ee colony com ponents (s eparate controls). Honey bee Hive component Control 1 Control 2 Control 3 ANOVA wax (32 g) 3.9 0.3a 0.4 0.2b 0.2 0.2b 0.5 0.2b (F = 67.2; df = 3,31; P < 0.01) honey (32 g) 3.1 0.4a 0.8 0.4b 0.8 0.3b 0.3 0.2b (F = 16.4; df = 3,31; P < 0.01) adults (32 g) 2.8 0.4a 0.9 0.4b 0.8 0.4b 0.5 0.3b (F = 9.2; df = 3,31; P < 0.01) stored pollen (10 g) 2 0.5a 1.3 0.4a 0.9 0.4a 0.8 0.4a (F = 2; df = 3,31; P = 0.14) brood (32 g) 2.9 0.2a 0.6 0.3b 0.6 0.2b 0.9 0.2b (F = 23.1; df = 3,31; P < 0.01) hive 2.6 0.3a 0.9 0.2b 0.6 0.3b 0.9 0.2b (F = 14.2; df = 3,31; P < 0.01) SHBs were released into a four-way olfactometer. Control represents th ree control ports that emitted filtered air; the control data were left separate and considered three choices. Hive component represents air passed through a chamber containing one of the honey bee colony constituents and it was emitted through a single port Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single beetle was released twice. Row data followed by different letters are different at 0.05. 43

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Table 2-5. Attraction of SHBs to honey b ee or bum ble bee colony components (pooled controls). Honey bee component Bumble bee component Control ANOVA wax (32 g) 1.6 0.3a 1.6 0.2a 1.8 0.2a (F = 0.1; df = 2,23; P = 0.89) honey (32 g) 1.7 0.3ab 1 0.2 b 2.3 0.3a (F = 6.1; df = 2,23; P = 0.01) adults (32 g) 1.5 0.2a 1.5 0.2a 2 0.2a (F = 2.3; df = 2,23; P = 0.12) stored pollen (10 g) 1.8 0.3a 1.6 0.3a 1.6 0.3a (F = 0.1; df = 2,23; P = 0.93) brood (32 g) 1.5 0.2a 1.6 0.2a 1.9 0.1a (F = 1.3; df = 2,23; P = 0.29) hive 1.4 0.3a 1.5 0.2a 2.1 0.2a (F = 2.5; df = 2,23; P = 0.1) SHBs were released into a four-way olfactomet er. Control represents two control ports that emitted filtered air; the control data were pooled and considered one choice. Honey bee and bumble bee component represents air passed through a chamber containing one of the honey bee or bumble bee colony constituents and ea ch was emitted through a single port. Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single beetle was released twice. Row data followed by diffe rent letters are different at 0.05. 44

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45 Table 2-6. Attraction of SHBs to honey bee or bumble bee colony components (separate controls). Honey bee component Bumble bee component Control 1 Control 2 ANOVA wax (32 g) 1.6 0.3a 1.6 0.2a 0.8 0.2b 1 0.2ab (F = 4.8; df = 3,31; P <0.01) honey (32 g) 1.7 0.3a 1 0.2ab 0.9 0.2b 1.4 0.2ab (F = 3.4; df = 3,31; P = 0.03) adults (32 g) 1.5 0.2a 1.5 0.2a 1.1 0.1a 0.9 0.2a (F = 2.7; df = 3,31; P = 0.06) stored pollen (10 g) 1.8 0.3a 1.6 0.3a 1.1 0.1ab 0.5 0.2b (F = 7.1; df = 3,31; P < 0.01) brood (32 g) 1.5 0.2ab 1.6 0.2a 1 0.2ab 0.9 0.1b (F = 4.5; df = 3,31; P = 0.01) hive 1.4 0.3a 1.5 0.2a 1 0.3a 1.1 0.3a (F = 0.7; df = 3,31; P = 0.56) SHBs were released into a four-way olfactomet er. Control represents two control ports that emitted filtered air; the control data were left separate and considered two choices. Honey bee/bumble bee component represents air pa ssed through a chamber containing one of the honey bee or bumble bee colony constituents and each was emitted through a single port. Data are mean s.e. N = 8 replicates of 5 individual SHB releases/replicate. No single beetle was released twice. Row data followed by different letters are different at 0.05.

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Figure 2-1. Lateral view of the four-way olfactometer (modified from Carroll et al., in prep). The olfactometer is connected to an insect inlet port attached to the vacuum, drawing air from the odor source over the insect as it is released into the olfactometer. The vacuum is attached to a flowmeter (2 L/min) drawing four times the air delivered by each of the four ports (0.5 L/min), each port delivering air through air flowmeters, odor sources and glass traps. 46

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Figure 2-2. The four way olfact ometer used for SHB choice tests at the Center for Medical, Agricultural and Veterinary Entomology (CMAVE, USDA-ARS, Gainesville, FL). Filtered air is passed over bee constituents being tested and released through ports connecting the glass insect traps to the choice arena. Photo: Jason R. Graham. 47

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Figure 2-3. Lateral view of the four way olfactometer showing in sect inlet (Carroll et al., in prep). The vacuum line connector tube is detached from the vacuum line and a SHB is loaded into the vacuum connector tube A mesh excluder prevents the SHB from entering the vacuum line while the SHB travels past the SHB entry point and up the modified insect inlet. 48

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49 CHAPTER 3 A COMPARISON OF THE VOLATILES PRODUCED BY COMMERCIAL Apis mellifera L. (HYMENOPTERA: APIDAE) AND Bombus impatiens Cresson (HYMENOPTERA: APIDAE) COLONIES The life histories of bumble bees ( Bombus spp., Hymenoptera: Apidae) and European honey bees ( Apis mellifera L., Hymentopera: Apidae) are well known (Langstroth, 1878; Sladen, 1912; Wheeler, 1928; Free and Butler, 1959; Wilson, 1971; Michener, 1974; Alford, 1975; Butler, 1975; Morse, 1975; Hooper, 1976; Mo rse, 1982; Caron, 1999; Kearns and Thompson, 2001; Goulson, 2003; Heinrich, 2004, among many others). Bumble bees share many similarities with honey bees. These similarities include pollen and nectar collection and storage habits, the use of wax from abdominal wax secr etion glands to build comb (their nesting infrastructure), and social colonies of a singl e queen, drones, and many female workers. Their colonies host similar, and in some cases th e same, pests and pathogens (Toumanoff, 1939; Oertel, 1963; Showers et al., 1967; Alford, 1975; Ki stner, 1982; Whitcomb et al., 1983; Clark et al., 1985; Whitcomb et al., 1987; Bailey and Ba ll, 1991; McIvor and Melone, 1995; SchmidHempel, 1998; Kearns and Thomson, 2001; Otters tatter and Whidden, 2004; Genersch et al., 2005; Plischuk et al., 2009). Studies have show n (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffma n et al., 2008) that commercial B. impatiens colonies are potential alternative hosts for the small hive beetle (SHB, Aethina tumida Murray, Coleoptera: Nitidulidae), a natural pest of African subspecies of honey b ees (Lundie, 1940; Schmolke, 1974; Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and Hepburn, 2006). In this thesis it was shown that SHB attract ion to bumble bee colonies is chemically mediated (Chapter 2), as is often the case fo r other insects (Wilson, 1965; Schneider, 1969; Free, 1987; Hansson, 2002; Meiners et al., 2003; Silva-Torres et al., 2005) Since others have shown an attraction of SHB to bumble bee colonies (Stanghellini et al., 2000; Ambrose et al., 2000;

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Spiewok and Newm an, 2006; Hoffman et al., 2008, Ch apter 2), the hypothesis for this study is that volatile profiles pro duced by bumble bee and honey bee colonies are similar. To test this hypothesis, airbor ne volatiles present in commercial bumble bee and honey bee nests as well as volatiles pr oduced by bumble bee and honey bee adults, brood (eggs, larvae and pupae), wax, stored pollen (= bee bread) and hone y were collected and analyzed. Ultimately, the comparison of bumble bee and honey bee nest chemical ecology may explain why some nest invaders, such at the SHB, are found within both bee colony types, while others are exclusive to only one. Materials and Methods Bee source and establishment In June 2007, three comm ercial bumble bee ( B. impatiens) quads (= 12 colonies; Koppert Biological Systems, Inc., Romulus, MI) were es tablished at the University of Floridas Bee Biology Unit in Gainesville, FL (N 29 37.629" W 82 21.405"). Each quad consisted of four bumble bee colonies containing a reproductive queen, 200-25 0 workers, brood and nesting material and was secured with nylon rope on top of two cinder blocks la ying parallel to one another. The cinder blocks were set inside trays filled with so apy water to limit ant invasion. For the honey bee source, 12 commercial honey bee colonies also maintained at the University of Floridas Bee Biology Unit in Gain esville, FL were used. These colonies were queenright and housed in typica l Langstroth style equipment, receiving no pest control treatments in order to av oid volatile contamination. Volatile collections Whole colo nies 50

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Beginning in June 2007, volatiles were collecte d from colonies using methods modified from Heath & Manukian (1994) and Suazo et al. (2003). To collect volatiles from whole bumble bee colonies, SuperQ filters (polymeric adsorbent; Alltech, Deerfield, IL) were placed at the end of Teflon tubing (Ozone Solutions, Inc., Hull, IA ) and inserted the tubing into the entrance hole of each of the 12 colonies (Figure 1). At the vacuum port of an air compressor (Gast Manufacturing, Inc., Benton Harbor, MI), Mast er flex Tygon tubing (Cole-Parmer, Vernon Hills, IL) was attached to a carboloy flowmete r (Aalborg Instruments and Controls, Inc., Orangeburg, NY) calibrated to 1222.0 ml/min. Fr om the flowmeter, Tygon tubing connected the vacuum to the Teflon tubing and SuperQ filt er. Volatile collections were made for 3 to 7 hours per colony over the course of ~30 days, with 171 h total volatiles collected from the 8 bumble bee colonies. Immediately after each collection, the volatiles trapped on the SuperQ filters were extracted by eluting the filter with 500 l of methylene chloride. The samples were pooled and stored in a -70 C freezer (Revco Scientific, Inc., Asheville, NC) at the USDA-ARS Center for Medical, Agricultural and Veteri nary Entomology (CMAVE, Gainesville, FL). Volatiles were collected from 12 honey bee colo nies for a total of 171 hours using the methods as described for bumble bee colonies. Colony components Each of the colony components were coll ected from 8 bumble bee and 8 honey bee colonies, collecting a standard amount of each component (Table 1). The components were then placed in individual glass volatil e collection chambers (3.8 L), by component and colony at the CMAVE. All volatile collections were conducte d in a controlled environmental chamber designed by members of the CMAVE. The chamber was maintained without light and at an ambient temperature of ~33 C. Charcoal filte red and humidified air, controlled by carboloy 51

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flowm eters, was passed through the volatile collecti on chambers and into Supe rQ filters at a rate of 0.5 liters per minute. All component volatiles were collected for 14 h with the exception of brood volatiles which were collected for 7 h to a void stressing the larvae. Adult bees also were provisioned with a cotton wick saturated with a 50% sugar water solution and an autoclaved steel mesh platform on which the adults could crawl to minimize stress. The volatiles trapped on the SuperQ filters were extracted by eluting the filter with 500 l of methylene chloride. These samples were pooled by component and bee type and stored in a -70 C freezer at the CMAVE until use. These same methods were used for each bumble bee and honey bee colony component. Volatile Analysis The pooled volatile samples were analyzed for both bee types by constituent (whole colony, adults, brood, honey, pollen, and wax) an d colony at the CMAVE using a HP-6890 gas chrom atograph (GC, Hewlett Packard, Palo Alto CA) equipped with a HP-1 column (30 m x 0.25 m, J&W Scientific, Folsom, CA). The colu mn was linked to a HP 5973 mass spectrometer using electron impact mode (70 eV, Agilent, Palo Alto, CA) with the gas carried by helium. The GCMS oven temperature began at 35 C for the fi rst min and then ramped up 10 C per min to 230 C and stabilized for 10 min. The ion source temperature was held at 230 C. The volatile compounds collected were compared to the USDAARS library of collected and commercially available standards based on the retention times and mass spectra. Results Chromatograms are provided for a graphic comparison of component by bee type in Figures 2-7. The chemical compounds present in the colonies and/or components, represented by peaks in the chromatographs, are provided by retention time in Table 2. Only chemical 52

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com pounds with a quality rating 70 were reported in this study. These are non-confirmed identifications but best approximati ons based on spectral comparisons. As shown graphically (Figures 2-7), there is a high level of va riability between the volatiles collected from bumble bee and honey bee colony components as well as volatiles collected from whole colonies. Only 7/148 dete cted volatiles produced by components collected from bumble bee and honey bee colonies were sh ared by colonies of both bee types (Table 2). Adult bumble bee and honey bee volatiles both contained 2-hepta none, sharing no other compounds. Bumble bee and honey bee brood volatil es both emitted limonene. Pentadecane was the only common compound emitted by both bumble bee and honey bee whole colonies. The compounds 1-phenoxy-2-propanol an d butylated hydroxytoluene we re found in bumble bee and honey bee honey volatiles. Butylated hydroxyltoluene is a preservative and is not considered a natural product. Nonanal was the only compound shared by bumble beeand honey beestored pollen. Finally, bumble bee and honey bee wax both contained 2-nonanone and nonanal (Table 2). Discussion Generally speaking, bumble bee and honey bee co lonies shared only a few like volatiles. This was true for both the individual colony co mponent volatiles as well as the whole hive volatiles (Table 2 & Figures 2-7). Collectively, the data do not support th e original hypothesis that states volatile profiles produced by bumble bee and honey bee colonies would be similar. Whole colonies Pentadecan e was the only compound present among honey bee and bumble bee whole colony volatiles (Table 2). Studying the chemical analysis of honey bee egg coatings, KatzavGozansky et al. (2003) found pentadecane from qu een-laid diploid eggs, worker-laid haploid 53

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eggs, as well as from queen bee Dufours gland secretion. Pentad ecane also was found in cuticular hydrocarbons and the volatiles from worker honey bees (Schmitt et al., 2007). Pentadecane has not been found previously from bumble bee colonies. More matches between bumble bee and honey bee colony vola tiles were expected, especially given the fact that most of the components shared at least one volatile. Pe rhaps minor volatile components were undetected due to the overwhelming abundance of other volatiles. Colony components Adults The com pound 2-heptanone was found in bumble bee and honey bee adult volatile profiles (Table 2). Originally identified by Shearer and Boch (1965), 2-heptanone, produced from mandibular glands of worker bees, was thought originally to act as the principal aggressionprovoking component of worker honey bees (Free & Simpson, 1968). More recently, Vallet et al. (1991) found that 2-heptanone does not act as an alarm pheromone but acts instead as a forage marking pheromone. Vallet et al. (1991) also di scovered that 2-heptanon e can attract and repel guard honey bees, depending on the time of year encountered. While 2-heptanone has not been identified previously in bumble bee col onies, it has been found in association with Kodamaea ohmeri grown on trap collected honey bee pollen (Torto et al., 2007ab; Benda et al., 2008), although not with stored bumble bee pollen (Table 2). Interest ingly, 2-heptanone was found not only in the volatile profile of bu mble bee adults but also from that of bumble bee brood, wax and whole hive volatiles indicati ng its wide-spread prevalence in bumble bee colonies. The presence of 2-heptanone in bumble b ee and honey bee colonies may imply that both bees use the chemical similarly. In a series of studies (Stout et al., 1998; Williams, 1998; Stout and Goulson, 2001), researchers found that honey bees mark flowers that they have visited with 54

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2-heptanone. Other foraging honey bees and bumble bees were able to detect the 2-heptanone m ark and avoid the marked flowers until the nectaries refilled. In choice tests conducted by Torto et al (2005), 2-heptanone was among the compounds found in volatiles collected from 150-200 adult worker honey bees Furthermore, Torto et al. (2005) included 2-heptanone as an ingredient in a volatile profile to which they tested SHB attraction in wind tunnel bioassa ys. The profile consisted of 7 compounds in addition to 2heptanone (isopentyl acetate, 2heptanone, octanal, hexyl acet ate, nonanal, 2-nonanone, methyl benzoate and decanal). The profile was modeled af ter the natural blend of volatiles released by 150-200 adult worker honey bees. SHBs were attr acted to this blend, although they were more attracted to the actual honey bees (Torto et al., 2005). Volatiles released by honeybees in my st udy contained 2-heptanone, methyl benzoate, citral, tetradecane, heptadecane, nonadecane, 2-methyl-pentadecane and 2-methyl-heptadecane (Table 2). The differences between volatile profile s of adult honey bees in this study and in the one of Torto et al (2005) may be due to several uncontrollable variables such as environmental stress, handling, collection timing, etc. It is likely that the volatil e profiles of adult honey bees and bumble bees are complex, varyi ng with circumstance, temperamen t, bee health, etc. It would be helpful to study volatiles across a spectrum of healthy and distressed col onies to see how adult bee volatile profile varies depending on circumstance. Brood Bumble bee brood volatiles were composed of octane, 2-he ptanone, limonene, (E)-betaocimene, nonanyl acetate, pentadecane, hept adecane, of which only limonene was found in honey bee brood volatiles (Table 2). Limonene along with 2-heptanone, heptanal, pinene, octanal, terpenine, methyl benzoate, nonanal, and decanal were found to be honey bee hive 55

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volatiles capable of be ing detected by SHB in Gas Chrom atography Electro-Antennographic Detection (GC-EAD) bioassays. While honey bees detected fewer volatiles than SHBs, they were also capable of detecting limonene (Torto et al. 2007a). Zimmerman et al. (2006) reporte d (E)-beta-ocimene as a com ponent of volatiles collected from the tibia of male orchid bees, Eulaema bombiformis Packard (Hymenoptera: Apidae). Ocimene was also found from volatiles co llected from lab-reared colonies of Bombus terrestris L. with 30 workers when foraging activity was hi gh. In contrast, ocimene was unquantifiable during times when no foraging occurred (Granero et al., 2005). Honey bee queens also have been shown to release (E)-beta-ocimen e after successful mating has occurred (Gilley et al., 2006). The source of ocimene in bumble bee brood is unknown although it is po ssible that it was deposited on the brood by adult bees, is released by brood, or has some other function altogether. Perhaps in studying the behavior of bumble bees, particularly B. impatiens in the presence of these semiochemicals we can learn the function that they serve. Honey, pollen and wax The compounds 1-phenoxy-2-propanol and butylated hydroxytoluene were found in bumble bee and honey bee honey volatiles. Prior information on either of these compounds has not been described from honey bees, bumble bees or their honey. Butylated hydroxytoluene has been used extensively in the food industry as a food antioxidant addi tive (Branen, 1975), and may have been present in the sugar/water so lutions fed to the honey bee and bumble bee colonies. Volatiles collected from bumble bee and hone y bee wax contained 2-nonanone and nonanal, while volatiles collected from bumble bee and honey bee pollen contained only nonanal (Table 2). Nonanal was found by Saleh et al. (2007) to be a chemosensory cue, more like a footprint 56

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than a pheromone, left behind by B. terrestris ,at food locations, nest lo cations, and neutral areas (areas neither associated with food locations or with nest locations). Nonanal and 2-nonanone were used in wind tunnel studies where SHB were found to be attracted to a blend of com pounds which were collected from adult honey bees; non anal also was detected by SHB and honey bees in GC-EAD (Torto et al. 2005 & 2007ab). The volatile compounds found in both bumble bee and honey bee colony components and whole colony volatile profiles shoul d be investigated further to de termine the attraction of SHB and other nest invaders to each compound. Comm ercial bumble bee colonies and honey bee colonies, while similar enough to host the same a nd similar pests and pathogens, remain different regarding their volatile profiles (Table 2 & Figures 2-7). The few volatile compounds that were found in both colonies may be very important to arthropod pests during ho st seeking endeavors. With an understanding of the chemical ecology asso ciated with both types of bee colonies, a tool may be found to help conserve these important pollinators and control the damage caused by SHBs and other nest invaders. 57

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58 Table 3-1. The amount of materials collected fr om bumble bees and honey bee colonies. On average, ~153 adult bumble bees and ~243 adult honey bees = 32.2 g bees. Component Amount collected # Colonies providing samples Adult 32.2 g 8 Brood 54.6 g 8 Honey 36.7 ml 8 Pollen 2.5 g 5 Wax 8 g 8

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Table 3-2. Che mical compounds corre sponding to peaks from total ion chromatographs (Figures 2-7) of vol atiles collected from b umble bee and honey bee whole colonies and individual colony components. Retention time refers to the x axis of the total ion chromatograph ( Figures 2-7). Quality refers to how closely the compound matches its result from the GCMS lib rary search (with 100 being an absolute ma tch and 0 being no match at all). Only ch emical compounds with a quality rating 70 are reported herein. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 3.711 3-hydroxy-2-butanone 80 88.052 000513-86-0 x x x 3.713 1-methoxy-2-methyl-propane 80 88.089 000625-44-5 x 4.628 2,3-butanediol 87 90.068 000513-85-9 x x x 5.045 ethyl butanoate 96 116.079 000105-54-4 x 5.212 octane 93 114.14 000111-65-9 x x x 5.264 furfural 91 96.019 000098-01-1 x x 5.625 2,4-dimethyl-heptane 78 128.157 002213-23-2 x 5.817 butanoic acid 72 88.052 000107-92-6 x 5.865 ethyl isovalerate 96 130.099 000108-64-5 x 6.182 butyrolactone 87 86.037 000096-48-0 x x 6.352 2-heptanone 81 114.104 000110-43-0 x x x x x 6.417 styrene 96 104.063 000100-42-5 x 6.426 1,3,5,7-cyclooctatetraene 94 104.063 000629-20-9 x 6.529 3-methyl-butanoic acid 78 102.068 000503-74-2 x 6.814 nonane 95 128.159 000111-84-2 x 7.327 alpha-pinene 96 136.128 000080-56-8 x x 7.37 benzaldehyde 92 106.042 000100-52-7 x 7.581 1-methyl-2-propylcyclohexane 87 140.157 004291-79-6 x 59

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Colony component Table 3-2. Continued. Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 7.845 1-heptanol 80 116.12 000111-70-6 x 7.911 6-methyl-5-hepten-2-one 94 126.099 000110-93-0 x 7.953 4-methylene-1-1-methylethylbicyclo-hexane 94 136.125 003387-41-5 x 7.991 beta-pinene 96 136.129 000127-91-3 x 8 hexanoic acid 86 116.084 000142-62-1 x x 8.181 octanal 76 128.12 000124-13-0 x x 8.223 ethyl hexanoate 98 144.12 000123-66-0 x 8.401 methyl ester 2,4-hexadienoic acid 97 126.068 001515-80-6 x 8.427 dihydro-3-hydroxy-4,4dimethyl-2(3H)-furanone, 76 130.063 052126-90-6 x 8.435 decane 93 142.169 000124-18-5 x 8.465 pantolactone 91 130.063 000599-04-2 x 8.498 octamethyl-cyclotetrasiloxane, 78 296.075 000556-67-2 x 8.542 delta-3-carene 97 136.128 013466-78-9 x x 8.547 tricyclene 91 136.128 000508-32-7 x 8.602 phenyl methanol (benzyl alcohol) 97 108.057 000100-51-6 x 8.656 benzeneacetaldehyde 93 120.058 000122-78-1 x x x 8.822 limonene 90 136.125 000138-86-3 x x x x 8.865 eucalyptol 96 154.136 000470-82-6 x 8.869 2,5-furandicarboxaldehyde 91 124.016 000823-82-5 x 9.102 trans-bocimene 94 136.129 027400-72-2 x 9.331 sorbic Acid 96 112.052 000110-44-1 x x 9.373 2,4-dimethyl-hexane 72 114.141 000589-43-5 x 9.425 linalool oxide (furanoid) 91 170.129 000000-00-0 x 9.478 di-tert-dodecyl disulfide 86 402.335 027458-90-8 x 60

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Table 3-2. Continued. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 9.62 methyl benzoate 95 136.052 000093-58-3 x x 9.622 2-nonanone 90 142.128 000821-55-6 x x 9.646 sorbic Acid 96 112.052 000110-44-1 x 9.69 ethyl sorbate 97 140.08 002396-84-1 x 9.793 ethyl heptanoate 94 158.129 000000-000 x 9.81 nonanal 98 142.136 000124-19-6 x x x x x x 9.852 3,7-dimethyl-1,5,7-octatrien-3ol 72 152.12 029957-43-5 x 9.896 phenyl ethyl alcohol 94 122.069 00006012-8 x 9.987 benzeneacetonitrile 96 117.06 000140-294 x 10.026 undecane 96 156.189 001120-21-4 x 10.187 methyl octanoate 91 158.129 000111-115 x 10.218 2,6,6-trimethyl-2-cyclohexen1,4-dione 97 152.082 001125-21-9 x 10.412 hexanoic acid, 2-ethyl72 144.115 000149-57-5 x 10.447 camphor 93 152.12 000076-22-2 x 10.488 2,2,6-trimethyl-1,4cyclohexanedione, 74 154.099 020547-99-3 x 10.521 lilac aldehyde 90 168.115 053447-464 x 10.867 6-ethenyltetrahydro-2,2,6trimethyl-2H-pyran-3-ol 91 170.129 014019-11-7 x 10.926 benzenecarboxylic acid 94 122.037 00006585-0 x 11.039 octanoic Acid 90 144.115 000124-07-2 x 11.197 alpha-terpineol 91 154.128 000098-555 x 61

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Table 3-2. Continued. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 11.222 4-carene 89 136.125 029050-33-7 x 11.288 ethyl octanoate 96 172.15 000106-321 x 11.319 6-methyl-2pyridinecarbaldehyde 94 121.053 053547-60-7 x 11.34 decanal 83 156.151 000112-31-2 x x x 11.532 dodecane 95 170.199 000112-40-3 x 11.623 benzenepropanol 95 136.089 000122-97-4 x 11.789 ethyl ester benzeneacetic acid 87 164.084 000101-97-3 x 11.815 1-phenoxy-2-propanol 96 152.084 000770-354 x x x 11.827 para-anis aldehyde 95 136.05 000123-115 x 11.843 3-phenoxy-1-propanol 95 152.084 006180-616 x x 11.848 citral 74 152.12 000106-26-3 x x 12.103 e-cinnamaldehyde 97 132.06 014371-10-9 x x x 12.237 geranial 94 152.12 000141-27-5 x 12.54 thymol 91 150.098 000089-83-8 x x 12.56 p-tert-butyl-phenol 94 150.104 00009854-4 x x 12.631 2-undecanone 91 170.169 000112-12-9 x 12.67 e-cinnamyl alcohol 96 134.068 00440736-7 x 12.695 3-phenyl-2-propen-1-ol 93 134.073 000104-54-1 x 12.696 ethyl ester nonanoic acid 94 186.162 000123-29-5 x 12.789 5-ethyl-2-methyl-octane 87 156.188 062016-18-6 x 12.796 8-methyl-heptadecane 86 254.297 01328723-5 x 12.861 nonanyl acetate 91 186.159 000143-13-5 x x x x x 12.944 tridecane 93 184.219 000629-50-5 x 13.5 dodecamethylcyclohexasiloxane 90 444.113 000540-97-6 x x x x 62

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Table 3-2. Continued. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 13.559 methyl para-anisate 90 166.06 000121-98-2 x 13.735 butyl ester butanoic acid 80 144.115 000109-21-7 x 13.921 1,2,3-trimethoxy-5-methylbenzene 96 182.094 006443-69-2 x 14.007 1,3,5-trimethoxy benzene 93 168.078 000621-23-8 x 14.039 ethyl decanoate 96 200.18 000110-38-3 x 14.156 dodecanal 90 184.18 000112-54-9 x 14.181 tetradecanal 90 212.21 000124-25-4 x 14.292 e-cinnamic acid 96 148.05 000140-10-3 x 14.674 6,10-dimethyl-(Z)-5,9undecadien-2-one 93 194.167 003879-26-3 x 14.698 geranyl acetone 93 194.169 003796-70-1 x 14.903 7,11-dimethyl-3-methylene,1,6,10-dodecatriene 97 204.188 018794-84-8 x 15.015 3-tert-butyl-4-hydroxyanisole 92 180.115 000121-00-6 x 15.117 cyclododecane 95 168.188 000294-62-2 x 15.291 2-tridecanone 95 198.199 000593-08-8 x 15.314 1-pentadecene 95 210.235 013360-61-7 x 15.334 3,7,11-trimethyl-1,3,6,10dodecatetraene 89 204.188 026560-14-5 x 15.41 aselinene 96 204.189 000473-13-2 x 15.441 2,4-bis-1,1-dimethylethylphenol 94 206.167 000096-76-4 x 15.485 longifolene 91 204.187 000475-20-7 x 15.494 butylated hydroxytoluene 94 220.183 000128-37-0 x x x x 15.534 tetradecane (C14) 91 198.229 000629-59-4 x 15.534 pentadecane 72 212.25 000629-62-9 x x x x x x 63

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Table 3-2. Continued. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 15.555 hexadecane (C16) 91 226.258 000544-76-3 x 15.573 heptadecane (C17) 91 240.28 000629-78-7 x 15.591 2-bromo dodecane 86 248.114 013187-99-0 x 15.616 heptadecane 72 240.282 000629-78-7 x x 15.616 heneicosane 90 296.344 000629-94-7 x 15.696 (+)-gcadinene 95 204.189 039029-41-9 x 15.787 7-epi-alpha-selinene 91 204.188 123123-37-5 x 15.982 aromadendrene 91 204.188 109119-91-7 x 16.12 pentacosane 83 352.407 000629-99-2 x 16.149 3,7,11-trimethyl-1,6,10dodecatrien-3-ol 83 222.198 040716-66-3 x 16.494 ethyl dodecanoate 98 228.21 000106-33-2 x 16.541 2-methyl-, 1-(1,1dimethylethyl)-2-methyl-1,3propanediyl ester propanoic acid, 78 286.214 074381-40-1 x 16.574 4-(1,1,3,3-tetramethylbutyl)phenol 91 206.167 000140-66-9 x x x 16.735 hexadecane 92 226.266 000544-76-3 x 17.216 carbamic acid, N-[1,1bis(trifluoromethyl)ethyl]-, 4(1,1,3,3tetramethylbutyl)phenyl ester 72 413.179 296242-69-8 x 17.255 azulene, 1,2,3,3a,4,5,6,7octahydro-1,4-dimethyl-7-(1methylethenyl) 90 204.188 022567-17-5 x 17.584 1-heptadecene 96 238.264 006765-39-5 x x x 64

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Table 3-2. Continued. Colony component Adults Brood Hive Honey Pollen Wax Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number B B H B B B H B B B H B B B H B B B H B B B H B 17.843 heptadecane (C17) 95 240.28 000629-78-7 x x x 17.843 octacosane 89 394.454 000630-02-4 x 17.844 hexadecane (C16) 91 226.258 000544-76-3 x 17.863 pentadecane 97 212.25 000629-62-9 x 17.87 docosane 91 310.36 000629-97-0 x 18.084 heneicosane 86 296.344 000629-94-7 x 18.117 7-(1-methylethyl)-, (E,E)-8methyl-3,5,7-Nonatrien-2-one 89 192.151 070372-94-0 x 18.712 ethyl tetradecanoate 98 256.24 000124-06-1 x 18.916 octadecane (C18) 91 254.288 000593-45-3 x 19.19 phenol, 2,4,6-tris(1,1dimethylethyl) 72 262.23 000732-26-3 x 19.931 nonadecane (C19) 96 268.31 000629-92-5 x x 19.932 hexadecane (C16) 97 226.258 000544-76-3 x 19.936 2-methyl-undecane 83 170.203 007045-71-8 x x 19.958 octadecane 91 254.295 000593-45-3 x 20.73 ethyl hexadecanoate 99 284.269 000628-97-7 x 20.896 oxybenzone 76 228.079 000131-57-7 x 22.035 2-methyl-pentadecane 90 226.266 001560-93-6 x 22.035 heneicosane 96 296.344 000629-94-7 x 24.267 octacosane 87 394.454 000630-02-4 x 24.589 1-docosene (C22) 91 308.338 001599-67-3 x 25.046 2-methyl-heptadecane 83 254.297 001560-89-0 x 25.099 eicosane 91 282.327 000112-95-8 x 25.435 9-octadecenamide 93 281.272 000301-02-0 x 65

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Figure 3-1. Volatile collection from whole bumble bee hives. The quad is shown to the left; SuperQ filters were inserted into the entrance holes and connected by tubing to the flowmeter and vacuum pump. 66

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4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Bumble bee adults 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000AbundanceHoney bee adults Retention time (minutes) 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Bumble bee adults 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000AbundanceHoney bee adults Retention time (minutes) Figure 3-2. Representative total ion chromatograms of volatiles collected from bu mble bee and honey bee adults. Abundance scale (0 500,000) is the same for both chromatograms. Peaks correspond to vol atiles listed in table 2 by retention time. Star indicates homologous compound. 67

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Retention time (minutes) 4.006.008.0010.0012.0014.00 16.0018.0020.0022.0024.00 Bumble bee brood 200000 400000 600000 800000 1000000 0 200000 400000 600000 800000 1000000 Honey bee brood4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00AbundanceRetention time (minutes) 4.006.008.0010.0012.0014.00 16.0018.0020.0022.0024.00 Bumble bee brood 200000 400000 600000 800000 1000000 0 200000 400000 600000 800000 1000000 Honey bee brood4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00Abundance Figure 3-3. Representative total ion chromatograms of volatiles collected from bumble bee and honey bee brood. Abundance scale (0 1,000,000) is the same for both chromatograms. Peaks correspond to volatiles listed in table 2 by retention time. Star indicates homologous compound. 68

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4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000 1000000Honey bee hive 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000 1000000Bumble bee hive Retention time (minutes)Abundance 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000 1000000Honey bee hive 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000 1000000Bumble bee hive Retention time (minutes)Abundance Figure 3-4. Representative total ion chromatograms of volatiles collected from bumble bee and honey bee whole hives. Abundance scale (0 1,000,000) is the same for both chro matograms. Peaks correspo nd to volatiles listed in table 2 by retention time. Star indicates homologous compound. 69

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4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000Honey bee honey 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000Bumble bee honey Retention time (minutes)Abundance 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000Honey bee honey 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 200000 400000 600000 800000Bumble bee honey Retention time (minutes)Abundance Figure 3-5. Representative total ion chromatograms of volatiles collected from bumble bee and honey bee honey. Abundance scale (0 800,000) is the same for both chromatograms. Peaks correspond to volatiles listed in table 2 by retention time. Star indicates homologous compound. 70

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4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 500000 1000000 1500000 2000000 2500000 3000000Bumble bee pollen 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 500000 1000000 1500000 2000000 2500000 3000000Honey bee pollen Retention time (minutes)Abundance 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 500000 1000000 1500000 2000000 2500000 3000000Bumble bee pollen 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 500000 1000000 1500000 2000000 2500000 3000000Honey bee pollen Retention time (minutes)Abundance Figure 3-6. Representative total ion chromatograms of volatiles collected from bumble bee and honey bee pollen. Abundance scale (0 3,000,000) is the same for both chromatograms. Peaks correspond to volatiles listed in table 2 by retention time. Star indicates homologous compound. 71

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72 Figure 3-7. Representative total ion chromatograms of volatiles collected from bumble bee and honey bee wax. Abundance scale (0 500,000) is the same for both chromatograms. Peaks correspond to vol atiles listed in table 2 by retention time. Star indicates homologous compound. 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Bumble bee wax 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Honey bee wax Retention time (minutes)Abundance 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Bumble bee wax 4.006.008.0010.0012.0014.0016.0018.0020.0022.0024.00 0 100000 200000 300000 400000 500000Honey bee wax Retention time (minutes)Abundance

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CHAP TER 4 THE PRESENCE OF Kodamaea ohmeri (ASCOMYCOTA: SACCHAROMYCETACEAE) IN COMMERCIAL Bombus impatiens (HYMENOPTERA: APIDAE) COLONIES AND THE RESULTING ECOLOGICAL RAMIFICATIONS Bumble bees (Hymenoptera; Apidae; Bombus spp.) share many similarities with Western honey bees (Hymenoptera; Apidae; Apis mellifera ). These similarities include pollen and nectar collection and storage habits, abdom inal wax secretion glands that produce the wax used to build their nest infrastructure, and social colonies consisting of drones, a single queen, and many female workers. They also host similar, and in some cases the same, pests and pathogens (Wilson, 1971; Michener, 1974; Alford, 1975; Ki stner, 1982; Schmid-Hempel, 1998; Caron, 1999; Kearns & Thompson, 2001; Goulson, 2003; Heinrich, 2004; Plisch uk et al., 2009; among others). The similarities in colony compositi on, nesting strategy, and natural history (Wilson, 1971; Michener, 1974, Alford, 1975; Caron, 1999; Goulson, 2003; among others) and the fact that both occupy similar ecological niches (h ost habitat hypothesis Norton & Carpenter, 1998; Nikoh and Fukatsu, 2000) are possible reasons th at bumble bee and honey bee colonies host many similar pests and pathogens, in cluding the small hive beetle ( Aethina tumida Murray, SHB). Investigators have shown that commercial bumble bee colonies are possible alternative hosts for SHBs (Stanghellini et al., 2000; Ambr ose et al., 2000; Spiewok and Neumann, 2006; Hoffman et al., 2008). SHBs are a natural pest of African subspecies of honey bees now also affecting European subspecies in the SHB s introduced range (Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and Hepburn, 2006; Neumann and Ellis, 2008). However, the investigators were unable to determine conclusively what attr acts SHBs to bumble bee colonies. Most early investigations into SHB host attraction concerned SHB attraction to honey bee colonies or fruit (Lundie, 1940; Schmolke, 1974; Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and 73

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Hepburn, 2006). More recently, res earchers have begun to identif y specific volatiles to which SHB are attracted (Suazo et al ., 2003; Torto et al., 2005, 2007ab). Torto et al. (2005, 2007a) found that SHBs can detect and are attr acted to isopentyl acetate (IPA) am ong other components of honey bee alarm pheromone. Torto et al. (2007b) later demonstrated that the yeast Kodamaea ohmeri ( previously known as Pichia ohmeri and Yamadazyma ohmeri, Ascomycota: Saccharomycetaceae), wh ich grows on pollen present in bee colonies, produces IPA and other components of bee alarm pheromones. Indeed, volatiles from K. ohmeri inoculated pollen dough were found to be attractive to SHB in wind tunnel paired choice tests (Torto et al., 2007ab) Futhermore, traps baited with K. ohmeri inoculated pollen dough captured significantly more SHB than unbaited traps (Torto et al 2007b, Arbogast et al., 2007, 2009). In this study, I attempted to address SHB a ttraction to commercial bumble bee colonies by determining if bumble bee colonies contain K. ohmeri Given the similarities between honey bee and bumble bee ecology (Wilson, 1971; Michener, 1974; Alford, 1975; Schmid-Hempel, 1998; Caron, 1999; Goulson, 2003; He inrich, 2004; among others), the evidence that commercial bumble bee colonies can host SHBs (Stanghe llini et al., 2000; Ambrose et al., 2000; Spiewok and Neumann, 2006; Hoffman et al., 2008), and the close relationship between K. ohmeri and the SHB (Torto et al. 2005, 2007a, 2007b; Arbogast et al., 2007, 2009), I hypothesized that K. ohmeri would be present in commercial B. impatiens colonies and is an important component of the SHB/bumble bee paradigm. Furthe rmore, I also predicted that K. ohmeri produces a pheromone signature similar to that produced by honey bees when placed on bumble bee collected pollen. I investigated these hypotheses using standard me thods of yeast isolation and comparison of cultured yeast vo latile composition, morphology, and gene sequences with those 74

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of L-27 (Florida) and A-1 (Kenya) strains of K. ohmeri which were isolated fro m SHB washes by Torto et al. (2007a). Materials and Methods Bombus impatiens Colonies In June 2007, three comm ercial bumble bee quads containing four B. impatiens colonies per quad were purchased from Koppert Biological Systems, Inc. (Romulus, MI). The quads (Figures 1 & 2) were located at the University of Floridas Bee Biology Un it in Gainesville, FL (N 29 37.629" W 82 21.405"). Each colony contained a corn -syrup/water solu tion in a plastic bag, part of Kopperts quad system, for peri ods of low nectar flow. All colonies had a reproductive queen, 200-250 workers, brood and nesti ng material (cotton and plastic). The quads were secured to the top of tw o cement blocks with nylon rope The blocks were then placed inside trays filled with soapy water to guard ag ainst ant invasion. The entrances to all colonies were open to permit the bumble bees to forage naturally. Yeast Collection To sam ple colonies for K. ohmeri eight bumble bee colonies were removed from their quad containers and took them to the Center for Medical and Veterinary Entomology (CMAVE, USDA-ARS, Gainesville, FL). Next, the colonies were opened and yeast samples were collected by rubbing two cotton swabs on the inside top and bottom of the colonies and one cotton swab on each of the four sides inside the colonies (n = 8 samples 7 colonies = 56 swabs). The samples were then plated on Candida Isolation Agar (CIA; Atlas, 1997). The plates were placed in a crisper with distil led water to provide humidity. These crispers were placed in a Napco 4200 water jacketed incubator (N ational Appliance Co., Portland Oregon) for 2-3 d at 27 C. Following this, The plates were analyzed for th e presence/absence of yeast under a microscope 75

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equipped with a digital cam era (Department of Entomology and Nematology, University of Florida, Gainesville, FL) and using Autom ontage software (Synchroscopy, Frederick, MD). While the colonies were open, samples were collected from each of the components of the bumble bee hives. The samples collected from each colony, when available, included one adult bee (n = 7 colonies), one larvae/pupae (n = 8 colonies), one gram of bee bread (bee stored pollen, n = 5 colonies), one gram of wax (n = 8 co lonies) and one ml of honey (n = 8 colonies). The samples were then homogenized separately a nd plated on CIA media. The inoculated plates were incubated at 28C for 24 48 h, following which the plates were analyzed under a compound microscope (Leitz Laborlux S, Lei ca Microsystems Inc., Bannockburn, IL) to determine the presence/absence of yeast. Yeast Culture and Volatile Collection To com pare the volatile compositions of yeasts collected from commercial B. impatiens colonies to those of L-27, the three most common yeast phenotypes found in the bumble bee colonies were cultured. These were represented by three isolates (1, 2 and 3) phenotypically similar to K. ohmeri and one filamentous phenotype isolate (4 ). These isolates originated from different quads and, for the most part, were collect ed from different substr ates: isolates 1 and 4 were cultured from colony interior swabs, isolate 2 was cultured from an adult bumble bee homogenate and isolate 3 was cultured from a wax homogenate. Afte r colony isolation, 5 colonies of each phenotype were added to separate flasks of YPD media (Atlas, 1997) and placed on a C24 incubator shaker (New Brunswick Scientific, Edison, NJ) set at 200 rpm, 30 C, for 14 h. The result was a suspension of 5 yeast colonies, each in its own flask, 10 L yeast broth, and 500 L MilliQ water. Slides were prepared of the yeast broth to evaluate the cells for morphological similarities to L-27 and A-1 usi ng a compound microscope (Leitz Laborlux S, 76

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Leica Microsystem s Inc., Bannockburn, IL) c onnected to a digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Yeast Replication Rate To determ ine the replication rate of the yeas t isolates (1, 2, 3 and 4) for comparison with that of K. ohmeri yeast suspensions were created for each as above. Two yeast suspensions were then created of L-27 (cultured from Torto et al., 2007a), one left viable as a positive control and the other boiled in a water bath over a Bunsen burner for 10 min as a negative control. After 0.5 h, yeast replication was measured at a 600 nm wavelength (Bausch & Lomb Spectronic 20, Rochester, NY). Measurements were recorded for all yeast suspensions using a 1/10 dilution (of yeast broth/MilliQ water) at 0.5, 3, 6, 8.5, 11.5, 14.5, 17.5, 29.5, 53.5, and 77.5 h. Pollen Preparation and Inoculation Stored pollen (= bee bread, 21 g) was collect ed f rom wax cells from five bumble bee colonies. Samples were combined and irradiated at the Florida Department of Agriculture & Consumer Services Division of Plant Industry (F DACS-DPI, Gainesville, FL) using a sterilization dose of 15 kilo-gray (15kGy) at 25 hours with a Cesium 137 source. The irradiated pollen was divided into 3g batches and these ba tches were saturated wi th 2 mL of one of 6 treatments. The treatments were represented by y east broths prepared as above and incubated for 14 h in a Napco 4200 water jacket incubator (T hermo Fischer Scientific, Waltham, MA) at 27 C with isolates 1-4 collected from commercial bumble bee colonies, L-27 as the positive control and L-27 boiled for 10 min as the negative contro l. The final products (yeast broth saturated pollen batches) were placed in autoclaved, alumin um weigh boats and held in an autoclaved Petri dish. The Petri dishes were then put into a crisper and were incuba ted in a Napco 4200 water jacketed incubator (Thermo Fischer Sc ientific, Waltham, MA) for 4-5 d at 27 C. 77

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After yeast growth, volatiles were co llected from these samples for 6 h each at the CMAVE. To do this, the yeast/pollen mixtures were placed separate ly into autoclaved aluminum weigh boats and then into glass volatile collectio n chambers (3.8 L) which were maintained at 33 C. Charcoal filtered and humidified air were passed through the volatile collection chambers and into SuperQ filters at a rate of 0.5 l/min. The volatiles trapped on th e SuperQ filters were extracted by eluting the filter with 500 l of met hylene chloride. The volatile samples were analyzed at th e CMAVE using a HP-6890 Gas Chromatograph (GC, Hewlett Packard, Palo Alto, CA) equipped with a HP-1 column (30 m x 0.25 m, J&W Scientific, Folsom, CA). The column was linked to a HP 5973 mass spectrometer using electron impact mode (70 eV, Agilent, Palo Alto, CA) with the gas carried by helium. The GCMS oven temperature began at 35 C for the first min and then ramped up 10 C per min to 230 C and stabilized for 10 min. The ion source temperat ure was held at 230 C. The volatile compounds collected from the yeast phenotypes were compar ed to the USDA-ARS library of collected and commercially available standards based on the retention times and mass spectra. DNA Isolation and Analysis The four yeast iso lates from commercial B. impatiens colonies and isolates A-1 & L-27 were plated in Petri dishes c ontaining SMY broth and then in cubated at 27 C overnight. The cells were pelleted and the DNA wa s extracted using a Masterpuretm Yeast DNA purification kit (Epicentre, Madison, WI). The DNA preparations were evaluate d for both quality and quantity and then were amplified on ethidium brom ide stained agarose gels. Taq DNA polymerase (Promega, Madison, WI) was used to amplify a liquots of DNA with primers NL1 and NL4 for the 5 divergent domain of the 28S rDNA (Kurtzman and Robnett, 1997). Primers F17 and R317 78

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(Benda et al., 2008) and prim ers AB28 and TW81 (Curran et al., 1994) were used for the ITS5.8S region to distinguish the yeast isol ates from other species of yeast. The PCR products were extracted with a QIAquick PCR extraction kit (Qiagen, Germantown, MD) and taken to the Interdisciplin ary Center for Biotechno logical Research Core Facility (ICBR) at University of Florida (Gaine sville, FL). The PCR-amplicons were sequenced bidirectionally with an ABI Prism DNA Sequencer. The sequences were proof read, trimmed and aligned using the Clustal 2.0.10 multiple sequence alignment tool (Larkin et al., 2007). The DNA sequences provided by ICBR were then compared to those in GenBank using BLAST (blastn). Results Yeast Collection As shown in Table 1, yeast colonies were dete cted in 6 of the 7 a dult bee hom ogenates, 2 of the 5 pollen homogenates and in 4 of 8 of the wax homogenates, but not in any of the honey or brood homogenates. The colony interior samples consistently grew yeast from the 56 swab samples. The sampling protocol was designed based on available materials from purchased bumble bee colonies. Morphological Comparisons and Growth Rate Of the 68 yeast iso lates discovered from commercial bumble bee colonies, four of the predominantly represented isolates were studied in depth. The first 3 isolates (1, 2 and 3) were selected due to their similar appearance to L-27 and A-1, milky-white in color, globular, butyroid colony formation, and smooth texture (isolates 1, 2 and 3, Figure 3). Isolate 1 was cultured from a colony swab, isolate 2 was cultured from an adult bumble bee homogenate while isolate 3 was cultured from a wax homogenate Isolate 4, also cultured fr om a colony swab, exhibited 79

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filam entous growth, a white powdery texture and was not similar to L-27 (isolate 4, Figure 3). Isolates 1, 2 and 3, matched the budding cell form ation of L-27 when grown in yeast broth and prepared on a slide. In contrast, isolate 4 displayed a branching growth (Figure 3). Yeast isolates 1, 2, 3 and L-27 replicated quickl y, absorbing 0.5 nm of light within the first 8 h and then reached a plateau wi th a slower rate absorbing anot her 0.3 nm of light over the next 2 d (Figure 5). The filamentous yeast 4 isolate on the other hand took 3 d to replicate sufficiently to reach a 0.5 nm absorbance (Figure 4). The negative control L-27, which had been boiled for 10 min, did not grow (Figure 4). Volatile comparisons The volatile compositions of isolates 1, 2 and 3 were similar to one another and to L-27 (Table 2, and Figure 5). The volatile composition of isolate L-27 shared 7 chemical compounds with isolates 1, 2 and 3 which were not exhi bited by dead L-27 or is olate 4. These compounds were: 2, 3, butanediol, ethyl sorbate, ethyl nonanoate*, ethyl decanoa te*, ethyl dodecanoate, ethyl hexanoate*, and methyl linoaleate (*co mpounds also found by Torto et al., 2007b from collected pollen, not bee bread, and attractive to SHBs). The volatile composition of isolate 4 was not similar to those produced by L-27, 1, 2 or 3 and produced several unique compounds such as isopentyl formate, 2-methyl-propanoi c acid, ethyl 3-methyl butanoate, a-pinene, 4methoxybenzhydrazide, a-copaene, 8,9-dehydro-neoisolongifolen e, 1,5,5-trimethyl-6-(3-methylbuta-1,3 dienyl)-cyclohexene and allo-aromadendrene (Table 2 and Figure 5). The only compounds shared by all samples, including the ne gative control, were 2-methyl-butanoic acid, ethyl hexanoate and 2phenylethanol (Table 2 and Figure 5). PCR Reactions PCR-a mplification using primers NL1 and NL4, for the 5 divergent domain of the 28S rDNA of the bumble bee yeast isolates 1,2 and 3 and known K. ohmeri isolates (A-1) and (L-27) 80

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all produced a 600 bp segm ent of DNA, while fo r isolate 4, using the same primers, a 700 bp segment was produced (Figure 6). Using primers F17 and R317 for PCR-amplification of the bumble bee and K. ohmeri isolates for the ITS-5.8S region produced a 300 bp segment while for isolate 4, a 700 bp segment was produced (Fi gure 6). PCR-amplification using primers AB28 and TW81 for the ITS-5.8S region of the yeast is olates produced a 300 bp segment for yeasts 1, 2 and 3 and K. ohmeri isolates while for isolate 4, a 200 bp segment was produced (Figure 7). DNA sequencing BLAST searches (Altschul et al., 1997) of the 5 divergent domain of the 28S rDNA and the ITS-5.8S region sequences produced matches between the bumble bee yeast isolates 1, 2, 3 and the A-1 isolate (accession number EU569326) as well as the L-27 isolate (accession numbers AY911384 and AY911385) submitted by Benda et al (2008). The bumble bee yeast isolates were 99-100% homologous to K. ohmeri (= Endomycopsis ohmeri GenBank U45702) and other strains of K. ohmeri (accession numbers AF335976, AY2678 21 and AY267824, among others). The ITSI region of L-27 differed from that of A1, as was found by Benda et al. (2008). The ITSI region of the Kenyan isolate, A-1, was identical to those of the bumble bee yeast isolates 1, 2 and 3. Discussion Yeast was discovered in all of the commercial bumble bee col ony interior swab samples (n = 56, Table 1) and three of the selected isolates were shown through genetic analysis, volatile profile, replication rate, and morphological evidence to be K. ohmeri This demonstrates that bumble bee nests are suitable micr o-environments for the growth of K. ohmeri Because K. ohmeri is a known attractant of SHB (Torto et al. 2005, 2007ab) it is logical to infer that bumble bee colonies harboring K. ohmeri are attractive to SHBs. 81

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Yeast Identification Through genetic analysis identification of th e commercial bumble bee yeasts, 1, 2 and 3 was determ ined using BLASTN 2.2.20 (Zhang et al., 2000), with primers NL-1 and NL-4, which produced 65 hits with 99-100% max identity and 0.0 E value as a match to the species K. ohmeri (Ascomycota: Saccharomycetaceae). Interestingly, the ITS1 region of A-1, the Kenyan isolate, was identical to those of the bumble bee isolates 1, 2, 3 but not to the L-27 isolate collected from Florida SHBs (Figure 9). Benda et al. (2008) noted that the A-1 ITS1 sequence produced 100% homology to the previously available K. ohmeri database sequences. This suggests that the A-1 isolate is more widespread than previously thoug ht and the differences between the ITS1 regions of A-1 and L-27 are not due to geographic sepa ration. There are several sources for different strains of K. ohmeri which has made use of many different habitats (Etchells et al., 1950; de Araujo et al., 1995; Potacharoe n et al., 2003; Rosa et al., 1999, Han, 2004; Otag et al., 2005; TajAldeen et al., 2005; Torto et al., 2005, 2007ab; Lee et al., 2007; Benda et al., 2008; Dek, 2008; Kutty & Philip, 2008). Yeast isolates 1, 2, 3, L-27 and A-1 did not ma tch yeast isolate 4. Yeast isolate 4 identification using BLASTN 2.2.20 (Zhang et al., 2000), with NL-1 and NL-4, produced 15 hits with 100% max identity and 0.0 E value as a match to the genera Geotrichum (Ascomycota: Saccharomycetaceae), and only one of these hits (Accession number: AB281297) named a species: G. silvicola. During a yeast survey, Pime nta et al. (2005) described this as a novel yeast found in Drosophila spp. (Diptera: Drosophilidae) from Brazilian rainforests and from an oak tasar silkworm larvae ( Antheraea proylei Jolly; Lepidoptera: Saturniidae) found in India. It is unclear what mediated the intr oduction of yeast 4 into th e commercial bumble bee colonies or what role, if any, it had therein. However, yeast isolate 4, if G. silvicola, was not 82

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hom ologous to yeasts 1, 2, 3, A-1 or L-27 (Figure 9 & 10) as expected. Through volatile analysis further differences were found between the K. ohmeri isolates found within commercial bumble bee colonies and isolate 4. Of the seven chemical compounds produced by yeast isolates 1, 2, 3, and L-27, three were discovered previously from volatiles of L-27 (Tab le 2 & 3) and were found to be attractive to SHBs (Torto et al., 2007b). Four compounds were identified from yeasts growing on pollen in this study (Table 2 & 3). Three of which are fe rmentation produced volatiles (Magee & Kosaric, 1987; Kinderlerer & Hatton, 1990; Siebert et al., 2005) while th e other has been found in a volatile composition collected from several yeas ts present on sweet corn and that attract Carpophilus humeralis F. (Coleoptera: Nitidulidae) (Table 2 & 3) (Nout & Bartelt, 1998). Many of the volatiles found are important to bumb le bees, associated with fermentation or used by other nitidulid beetles (Table 2 & 3). To begin we will discuss the compounds found consistently in all of the volat ile profiles of commercial bumble bee colony isolates 1, 2, 3 and live L-27, the known K. ohmeri yeast isolate, on bumble bee stored pollen; excluding any which were also found in volatile profiles of isolate 4 and dead L-27 (essentia lly negative controls). Ethyl dodecanoate is associated with marki ng and sex pheromones and has been found in secretions from male bumble b ees across several species: B. lucorum L. (Calam, 1969), B. patagiatus and B. sporadicus Nylander (Kullenberg et al., 1970), B. cryptarum F., and B. magnus Vogt., (Bertsch et al., 2005). Methyl linoleate: (1) is a component of honey bee and bumble bee semiochemical blends (Le Conte et al., 1990; Kreiger et al ., 2006), (2) part of a brood secretion that stimulates worker bees to cap brood cells, (3) is a kairomone indicating to varroa mites the opportunity to en ter brood cells (Le Conte et al., 1990), and (4) is a component of B. terrestris L queen pheromone (Kreiger et al., 2006). 83

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Another notable ferm entation product, 2-phenyle thanol (Zilkowski et al., 1999, Zhu et al., 2003), was found in all of the sample volatile compositions, including dead L-27 and yeast 4 isolates (Table 2). Torto et al. (2007b) found this compound in pollen dough conditioned by beetles feeding on it for 14 days. The heavy pres ence of 2-phenylethanol (~45 % of the total volatile composition at 14 d) was attributed to SHB frass accumulation (Torto et al., 2007b). In the current study however, there were no SHBs in any life stage found w ithin the bumble bee colonies so it is not clear what produced the volatile compound. K. ohmeri presence in bumble bee colonies Within the colony, K. ohmeri was not found in the honey or brood homogenates (Table 1). Honey is known for its antimicrobial properties (Jeddar et al., 1985; Zumla & Lulat, 1989; Efem & Iwara, 1992; Wahdan, 1998; Lee et al., 2008 ; among others). While honey is prone to fermentation, indicating that some yeast can su rvive and even flourish on honey, Boucias et al. (unpublished) found that due to osmotic pressure K. ohmeri is unable to grow on pure honey. The fact that brood did not harbor the yeast (T able 1) justifies further discussion. Little is known about the physiological response of bumb le bee brood to fungi, bacteria and other microbes. However, solitary and social insects are known to respond to foreign microbes in the nest with defensive behaviors and genetic, physiological and cellula r immune responses (Rothenbuhler, 1964; Rees et al., 1997; Kaltenpoth et al., 2005; Evans et al., 2006; Stow et al., 2007; Stow & Beattie, 2008; among ot hers). The European beewolf ( Philanthus triangulum Fabricius; Hymenoptera: Crabronidae) for example, has been shown to de posit antennal bacteria onto the ceiling of their brood chambers, a beha vior that protects the developing brood from fungal infestation (Kaltenpoth et al., 2005). This clearly benefits the beewolf, which is ground 84

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dwelling and therefore exposed to a variety of bacterial and funga l pathogens (Kaltenpoth et al., 2005). Sim ilar nesting conditions exist for the ground dwelling bumble bee, which may have developed a general antifungal symbiosis or physiological response to protect their brood. Antimicrobial defenses are present at higher ra tes in social bees, with correlations between colony size/relatedness and increas ing antimicrobial strength (S tow et al., 2007). Rothenbuhler (1964) described hygienic behavi or in honey bees as a geneti c trait whereby worker bees remove American foulbrood-infected pupae and larvae from the colony. The immune response of Bombus pascuorum Scopoli, was studied by Rees et al (1997) who identified defensive antimicrobial peptides in adult bumble bees. The bees synthesized th e peptides in their hemolymph in response to infection by both bacter ia and fungi. Further res earch into the immune response of the bumble bee colony is warranted. It would be interesting an d potentially valuable to find an existing mechanism through which bumble bee brood inhibits the growth of K. ohmeri The wax and stored pollen homogenates containe d yeast though not as consistently as the colony swabs or adult homogena tes. This also may be explained by general bee hygienic behavior or antimicrobial defens e. The stored pollen used in th is study was collected and stored by bumble bee foragers, often completely seal ed within wax cells. An important dietary requirement in larval development (Sladen, 1912; Michener, 1974; Pereboom, 2000), perhaps stored pollen is processed in some way by the bumble bee adults prio r to being stored in cell as it is processed by honey bee adults (Gilliam, 1979). Yeast was present in adult bumble bee homogenates and the colony interior swab samples. This suggests that bumble bees may transmit the yeast mechanically throughout the hive, thus explaining the yeasts presence on all swab samples. It currently is unknown how K. ohmeri 85

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enters bum ble bee colonies. One possibility is that SHBs transfer the yeast to bumble bee colonies as evidenced by the recently discovered association of SHB with K. ohmeri (Torto et. al, 2007a, Benda et al., 2008). However, SHB are not the likely pathway in this case since SHB were not found in the colonies. Another possibility is that K. ohmeri is introduced to the colonies on pollen brought back by foraging bees. Bumble b ees, like honey bees, are covered with hairs that facilitate the transfer of pollen (Sla den, 1912; Wilson, 1971; Michener, 1974, Alford, 1975; Caron, 1999; Goulson, 2003; among others). Kodamaea ohmeri and related species have been isolated from flowers (Rosa et al. 1999; Pot acharoen et al., 2003) maki ng it possible for foraging bees to acquire the yeast while visiting flowers. Kodamaea ohmeri is cosmopolitan in distribution and previously has been isolated from a variety of sources in vivo : in hospitalized neonates and immu nocompromised individuals (Han, 2004; Otag et al., 2005; Lee et al., 2007, Taj-Aldeen et al., 2 005), food (Etchells et al., 1950; Dek, 2008), in marine environments (de Araujo et al., 1995; Kutty & Philip, 2008), and from flowers (Potacharoen et al., 2003). Kodamaea ohmeri also was found in association with stingless bees (Rosa et al., 2003), honey bees (Torto et al., 2005, 2007a, 2007b; Benda et al., 2008) and now bumble bees, all of which are important pollinators and potential SHB hosts (Stanghellini et al., 2000; Ambrose et al., 2000; Mutsaers, 2006; Spiewok and Neumann, 2006; Greco et al., 2007; Hoffman et al., 2008). When K. ohmeri grows on fresh honey bee collected pollen, its volatiles contain many compounds fo und to attract SHB (Torto et al., 2005, 2007a; Benda et al., 2008). Since SHBs are attracted to K. ohmeri and may be responsible for its transmission, finding K. ohmeri in commercial bumble bee col onies should motivate continued research in this area. Commercial bumble bee colonies host both SHBs and K. ohmeri suggesting that wild colonies may as well (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and 86

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Neum ann, 2006; Hoffman et al., 2008). Wild bumble bee colonies should be sampled to determine if SHBs or K. ohmeri are present within the hive. Commercial bumble bee colonies acting as a source of unmonitored SHB reproduction could undermine any SHB eradication and control efforts of nearby beekeepers. Furtherm ore, SHB spillover into wild populations of bumble bees could lead to the further decline of native pollinators and negatively impact the surrounding ecosystems (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Neumann, 2006; Hoffman et al., 2008). 87

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88 Table 4-1. Yeast presence on commercial bumble bee colony constituents. Data are the number of samples positive for yeast/total number of samples tested. Row data correspond to the colony from which the samples were taken. Columnar data are the constituent sampled. Pollen from 3 of the 8 colonies and adult homogenates and interior swabs were unavailable for colony 1 (N/A). Colony Adult Brood Honey Pollen Wax Colony interior 1 N/A 0/1 0/1 N/A 0/1 N/A 2 1/1 0/1 0/1 0/1 0/1 8/8 3 1/1 0/1 0/1 0/1 0/1 8/8 4 1/1 0/1 0/1 1/1 0/1 8/8 5 1/1 0/1 0/1 1/1 1/1 8/8 6 1/1 0/1 0/1 N/A 1/1 8/8 7 1/1 0/1 0/1 N/A 1/1 8/8 8 0/1 0/1 0/1 0/1 1/1 8/8 Total 6/7 0/8 0/8 2/5 4/8 56/56

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Table 4-2. Che mical compounds corre sponding to peaks from total ion chromatograph (Figure 5) of volatiles collected from steri lized bumble bee stored pollen inoculated with yeast 1, 2, 3, L-27, 4, and dead L-27 isolates. Rete ntion time refers to the x axis of the total ion chromatograph (Figure 5). Quality refers to how closely the compound ma tches its result from the GC MS library search (with 100 being an absolute match and 0 being no match at all). Shaded rows indicate the compound was found in yeast isolates 1, 2, 3 and L-27 but not in 4 or dead L-27. yeast isolate Retention time (min) Chemical compound Quality Molecular weight (amu) CAS Number 1 2 3 L-27 4 Dead L-27 3.701 1-methoxy-2-methyl-propane 72 88.089 000625-44-5 x 3.71 3-hydroxy-2-butanone 59 88.052 000513-86-0 x x 4.209 isopentyl formate 83 116.078 000110-45-2 x 4.767 2,3-butanediol 90 90.068 024347-58-8 x x x x 5.034 2-methyl-propanoic acid 50 88.052 000079-31-2 x 5.795 ethyl 2-methylbutanoate 92 130.099 007452-79-1 x 5.798 3-methyl-pentanoic acid 53 116.084 000105-43-1 x 5.827 ethyl isovalerate 93 130.099 000108-64-5 x 5.83 3-methyl-ethyl ester butanoic acid 55 130.099 000108-64-5 x x x x 5.83 phospholane 59 88.044 003466-00-0 x 5.86 ethyl 3-methylbutanoate 86 130.099 000108-64-5 x 6.062 2-methyl-butanoic acid 72 102.068 000116-53-0 x x x x x x 6.995 1-methoxy-2-propyl acetate 43 132.079 000108-65-6 x 7.37 a-pinene 90 136.129 000080-56-8 x 7.904 6-methyl-5-hepten-2-one 50 126.104 000110-93-0 x 7.907 3-chloro-acetate-1-propanol 32 136.029 000628-09-1 x x 7.945 hexanoic acid 72 116.084 000142-62-1 x x 8.177 ethyl hexanoate 97 144.12 000123-66-0 x x x x x x 8.182 3-tetrazol-1-yl-propionic acid 43 142.049 1000304-09-3 x 8.274 ethyl ester 3-hexenoic acid 76 142.099 002396-83-0 x 8.614 phenyl methanol (benzyl alcohol) 97 108.057 000100-51-6 x x 89

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Table 4-2. Continued. RT ( min) Chemical Compound Quality Mol Weight (amu) CAS Number 1 2 3 L-27 4 Dead L-27 8.69 1-butoxy-2-ethylhexane 38 186.198 062625-25-6 x 8.692 2-ethyl-1-hexanol 53 130.136 000104-76-7 x x 8.7 benzeneacetaldehyde 38 120.058 000122-78-1 x 9.077 7,7-dimethyl-2-methyleneBicyclo[2.2.1]heptane 60 136.125 000471-84-1 x 9.34 sorbic acid 96 112.052 000110-44-1 x x 9.655 cis-linaloloxide 49 170.131 1000121-97-4 x 9.661 ethyl sorbate 97 140.08 002396-84-1 x x x x 9.811 nonanal 90 142.134 000124-19-6 x 9.887 2-phenylethanol 91 122.069 000060-12-8 x x x x x x 10.491 butyl-cyclopropane 49 98.11 000930-57-4 x 10.836 6-ethenyltetrahydro-2,2,6trimethyl-2h-pyran-3-ol 72 170.131 014049-11-7 x 10.896 ethyl octanoate 50 144.115 000124-07-2 x x x x x 11.007 decamethylcyclopentasiloxane 91 370.094 000541-02-6 x 11.258 ethyl octanoate 97 172.15 000106-32-1 x x x x x 11.355 decanal 55 156.151 000112-31-2 x x x x x 11.861 para-anis aldehyde 95 136.05 000123-11-5 x 12.544 thymol 90 150.104 000089-83-8 x x x 12.677 ethyl nonanoate 91 186.162 000123-29-5 x x x x 13.564 4-methoxybenzhydrazide 64 166.074 003290-99-1 x 13.583 methyl para-anisate 91 166.06 000121-98-2 x 13.588 4-methoxy-methyl ester benzoic acid 93 166.063 000121-98-2 x 14.015 ethyl decanoate 97 200.18 000110-38-3 x x x x 14.072 a-copaene 91 204.189 003856-25-5 x 14.163 8,9-dehydro-neoisolongifolene 56 202.172 067517-14-0 x 90

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91 Table 4-2. Continued. RT (min) Chemical Compound Quality Mol Weight (amu) CAS Number 1 2 3 L-27 4 Dead L-27 14.282 1,5,5-trimethyl-6-(3-methylbuta-1,3 dienyl)cyclohexene 32 190.172 056763-66-7 x 14.689 6,10-dimethyl-(E)-5,9undecadien-2-one 53 194.167 003796-70-1 x 14.692 geranyl acetone 91 194.169 003796-70-1 x 15.134 allo-aromadendrene 99 204.189 025246-27-9 x 15.528 pentadecane 95 212.25 000629-62-9 x x x x x 15.531 hexadecane (C16) 91 226.258 000544-76-3 x 16.475 ethyl dodecanoate 98 228.21 000106-33-2 x x x x 16.578 4-(1,1,3,3-tetramethylbutyl)phenol 91 206.167 000140-66-9 x x x 18.693 ethyl tetradecanoate 98 256.24 000124-06-1 x x x 20.257 oxacycloheptadec-8-en-2-one 98 252.209 000123-69-3 x 20.711 ethyl hexadecanoate 98 284.269 000628-97-7 x x x x 22.48 1,5-cyclododecadiene 95 164.157 031821-17-7 x 22.578 methyl linoleate 93 294.25 000112-63-0 x x x x 22.639 octadecatrienoic acid ethyl 90 306.256 001191-41-9 x x x 22.637 7-methyl-7H-purin-6-amine 60 149.07 000935-69-3 x 22.674 mono(2-ethylhexyl) ester 1,2benzenedicarboxylic acid 91 278.152 004376-20-9 x 24.541 1-docosene (C22) 90 308.338 001599-67-3 x 25.053 tricosane 94 324.37 000638-67-5 x

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92 Table 4-3. The known function of volatile com pounds collected from yeast isolates 1, 2, 3, and L-27. Chemical Compound Known Function Reference ethyl nonanoate SHB attraction Torto et al., 2007b ethyl decanoate SHB attraction Torto et al., 2007b ethyl hexanoate SHB attraction Torto et al., 2007b 2, 3, butanediol fermentation odor associated with yeast attractive to nitidulids on corn Magee & Kosaric, 1987 Nout & Bartelt, 1998 ethyl sorbate fermentation odor associated with yeast Kinderlerer & Hatton, 1990 ethyl dodecanoate fermentation odor associated with yeast male bumble bee semiochemical Siebert et al., 2005 Calam, 1969 Kullenberg et al., 1970 Bertsch et al., 2005 methyl linoleate honey bee brood semiochemical bumble bee queen semiochemical Le Conte et al., 1990 Kreiger et al., 2006

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Figure 4-1. External photographs of the quad system. The full quad with crosshair illustrates the division of colony nest boxe s (left) and an independent nest box (right) is shown outside the quad (K oppert Biological Systems, Inc.). 93

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94 Figure 4-2. Diagrams of the internal compone nts of the quad system From left to right: lateral view of nest-box hous ing and sugar feeder without packaging; lateral view of nest-box housing and sugar feeder with cross-sectioned packaging; and open nest-box housing with lid. (Koppe rt Biological Systems, Inc.).

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1 2 3 L-27 A-1 4 Figure 4-3. Photographs of cultured yeast isolates. Colum ns from left to right are images of isolates 1, 2, 3, L-27, A-1 and 4 Rows from top to bottom are the isolates A) in slide preparations of yeast broth scale bars with yeas t cells are set to 10 m, and photos were taken with a microscope (Lei tz Laborlux S, Leica Microsystems Inc ., Bannockburn, IL) connected to a digital camera (Diagnostic Instruments, Inc., Sterli ng Heights, MI) B) plated on CIA (photo taken with a microscope equipped with a digital camera using Automontage softwa re (Synchroscopy, Frederick, MD) C) plat ed on CIA, showing the entire Petri dish photos were taken with a handheld digital camera (Diagnostic In struments, Inc., Sterling He ights, MI) approximately 36 h after plate inoculation. 95

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96 Figure 4-4. Replication chart of yeast isolates collected from commercial bumble bee colonies (1, 2, 3 and 4) and Kodamaea ohmeri (L-27). The bumble bee colony yeast isolates 1, 2 and 3 follow a similar rate of replication to that of L-27, while 4 replicated at a slower rate and different pattern. Y east Growth in YPD Media0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.5 3 6 8.511.514.517.529.5 53.577.5Hours Inoculated Yeast 4 L-27 Yeast 3 Yeast 2 Yeast 1 L-27 (Dead)Abundance at 600 nm

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97 Figure 4-5. Representative total ion chromatogram s of volatiles collected from sterilized bumble bee stored pollen inoculated with yeast isolates Bumble bee yeast is olates (1, 2, 3 and 4) and K. ohmeri (L-27) from SHB larvae living in European honey bee colonies and control K. ohmeri (dead L-27) representative tota l ion chromatographs are shown. Abundance scale is the same for all chroma tograms; see scale on Yeast 1 for relative abundance scale. See tables 2-7 for compounds of each chromatogram by retention time.

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98 Figure 4-6. Ethidium bromide stained gel of th e bumble bee yeast isolates amplified using primers NL-1/NL-4 and F17/R317. Lanes 2 7 are, respectively, 1, 2, 3, 4, A-1 and L-27 amplified using primers NL-1 and NL-4 for the 5 divergent domain of the 28S rDNA. Lanes 8-14 are, respectively, 1, 2, 3, 4, A-1, a negative control, and L-27 amplified using primers F17 and R317 for the ITS-5.8S region to distinguish from other species of yeast. Lane 1 contains HyperLadder II with molecular weight markers (50-2000 bp).

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99 Figure 4-7. Ethidium bromide stained gel of th e bumble bee yeast isolates amplified using primers AB28 and TW81. Lanes 2-8 are, respectively, 1, 2, 3, 4, A-1, L-27, and a negative control amplified using primers (A B28 and TW81) for the ITS-5.8S region to distinguish from other species of yeast. La ne 1 contains HyperLa dder II with molecular weight markers (50-2000 bp).

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100 Figure 4-8. Clustal 2.0.10 multiple sequence alig nment using primers NL1 and NL4, for the 5 divergent domain of the 28S rDNA of bu mble bee yeast isolates 1,2,3 and A-1, the known K. ohmeri isolate. The dashes represent gaps used by Clustal 2.0.10 when aligning the sequences. The presence of a star i ndicates homogeneity between the above nucleotides, whereas the absence of a star indicates heterogeneity between these.

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101 Figure 4-9. Clustal 2.0.10 multiple sequence alig nment using primers NL1 and NL4, for the 5 divergent domain of the 28S rDNA of bu mble bee yeast isolates 1,2,3,4 and A-1, a known K. ohmeri isolate. The dashes represent gaps used by Clustal 2.0.10 when aligning the sequences. The presence of a star i ndicates homogeneity between the above nucleotides, whereas the absence of a star indicates heterogeneity between these.

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102 Figure 4-10. Clustal 2.0.10 multiple sequence alignment using primers AB28 and TW81 (Curran et al., 1994) for the ITS-5.8S region to distinguish the y east isolates 1,2,3,4, and known K. ohmeri isolates A-1 and L-27.from other spec ies of yeast. The boxed ITS2 region shows the homogeneity between isolates A-1, 1, 2, 3, and lack thereof with isolates 4, and L-27. The underlined portion represents the small subunit 5.8S rDNA. The dashes represent gaps used by Clusta l 2.0.10 when aligning the sequences. The presence of a star indicates homogeneity between the above nucleotides, wher eas the absence of a star indicates heterogeneity between these.

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103 CHAPTER 5 DISCUSSION Investigators have shown in recent studies that bumble bee (Hymenoptera; Apidae; Bombus impatiens Cresson) colonies are potential altern ative hosts for the small hive beetle ( Aethina tumida Murray, hereafter referred to as SHB) (Stanghellini et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008). To determine the attraction of SHBs to honey bee and bumble bee components and hives, it was hypothesized that SHBs would be as attracted to volatiles present in bumble bee coloni es as they are to those present in honey bee colonies. Using a four-way choice bioassay, it wa s found that SHBs are attracted to bumble bee and honey bee adults, brood, and wax, as well as to whole bumble bee and honey bee hives (Chapter 2). It was also hypothesized that bumble bee-pr oduced volatiles would be similar to those produced by honey bees because the insects and their societies are similar. By studying the volatile profiles, it was found, c ontrary to the hypothesis, that only 7 out of 148 chemical compounds detected in the volatile analysis matched those emitted by similar honey bee and bumble bee components (Chapter 3). Despite this those compounds that did match include some volatiles known to attract SHB and pres ent in honey bee colonies and/or Kodamaea ohmeri (Ascomycota: Saccharomycetaceae) volatile profiles (Suazo et al ., 2003; Torto et al., 2005; Teal et al., 2006; Arbogast et al., 2007; Torto et al. 2007, Benda et al., 2008, Arbogast et al., 2009). Researchers have demonstrated that K. ohmeri grows on pollen present in honey bee colonies and produces volatile components of bee alarm pheromones which are attractive to SHBs (Torto et al., 2005; Teal et al., 2006; Arbogast et al., 2007; Torto et al. 2007ab, Benda et

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al., 2008, Arbogast et al., 2009). In this study it was shown that K. ohmeri is found naturally in commercial bum ble bee co lonies (Chapter 4). Kodamaea ohmeri grows on bumble bee stored pollen and emits some of the same chemical compounds as it does while growing on honey bee stored pollen (Torto et al. 2007, Benda et al ., 2008). These findings support the original hypothesis which stated that K. ohmeri would be present in commer cial bumble bee colonies and, when grown on stored bumble bee pollen, would produce volatiles found in honey bee alarm pheromone and produced by K. ohmeri growing on honey bee collected pollen (Chapter 4). Collectively, these findings support the overa ll hypothesis that attr action of SHBs to bumble bee colonies is chemically mediated. This is not entirely surprising as many insects use chemicals to locate their hosts (S horey & McKelvey, 1977; Price, 1997). Insects respond to volatile chemicals bound to chemoreceptors often found within the antennae. This produces a nerve response that passes through the nervous system and expresses itself in a behavior (Kaissling, 1971; Shorey & McKelvey, 1977; Na tion, 2002). This can be compounded when two or more volatiles are present, resulting in the synergistic attraction of a parasite/pest to its host. Examples of the synergistic attraction of food odors and pheromones have been studied from an ecological and applied standpoint in recent years. Males of the dusky sap beetle ( Carpophilus lugubris Murray) release an aggregation phe romone that, when accompanied by yeast volatiles, attracts more beetles than eith er cue does alone (Bartelt et al., 1991; Lin et al., 1992). Phillips et al. (1993) found that phero mones and stored product volatiles interact synergistically to attract stored grain beetles a nd suggested the use of the synergisms in pest management of these beetles. Thus far, research ers have studied two aspe cts of SHB attraction host volatiles (Suazo et al., 2003; Torto et al., 2005) and K. ohmeri volatiles (Arbogast et al., 104

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2007; Torto et al. 2007ab, Benda et al., 2008, Arbogast et al., 2009). However, the tw o acting together may impact SHB host locati on more than either would alone. Investigators studying SHB attraction to bumble bee colonies in the past did not investigate the presence of K. ohmeri within these colonies (Stanghellin i et al., 2000; Ambrose et al., 2000; Spiewok and Newman, 2006; Hoffman et al., 2008). Therefore, it is unknown to what extent K. ohmeri facilitates the attract ion of bumble bee colonies to the SHB. With the understanding that (1) SHBs are attracted to bumble bee co lonies and components (Chapter 2) and K. ohmeri (Teal et al., 2006; Arbogast et al., 2007; Torto et al. 2007ab, Benda et al., 2008, Arbogast et al., 2009) and (2) K. ohmeri is present within bumble bee colonies (C hapter 4), it is logical to infer that SHBs benefit from the relationship with K. ohmeri due to host-location cues produced by K. ohmeri Kodamaea ohmeri also probably benefits from a relationship with SHB because the SHB may spread the yeast to new hosts. Ganther (2005 ) discussed the need of yeasts for animal vectors to assist in dispersal. In an experiment where beetles were excluded from flowers, beetle associated yeasts were not able to disperse and colonize the flower s (Lachance et al., 2001). More recently, it was discovered that long-range di spersal by nitidulid beetles is responsible for the introduction of at least 3 yeas t species to Hawaii from contin ental America (Lachance et al., 2006). The ability of yeast to mark the host of b eetle parasites is an interesting strategy of parasitic invasion. A similar strate gy has been found in bark beetle systems. Some bark beetles are associated with yeasts that the beetles use to mark thei r host. The yeasts convert the aggregation pheromone cisand trans-verbenol to an anti-aggregation pheromone verbonone. Once a certain threshold of beetle s and yeast is present at the decaying host tree, the negative 105

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feedback causes the beetles to disperse to ot her potential hosts (Hunt & B orden, 1990). It is possible that the same phenomenon may occur between K. ohmeri SHBs, and their host colonies. It is unclear if honey bees benefit from or are harmed by the presence of K. ohmeri in their colonies. Ganther (2005) recognized the importance of yeast mutualisms with variable, loosely interrelated members opposed to individual spec ies-specific relationships. Perhaps the SHB mechanically transmits K. ohmeri between bee colonies, while fo raging bees, robbing bees and swarming bees deliver K. ohmeri to and from flowers, other bee colonies and to new geographic locations. Honey bees have been found to harbor yeast internally and within colony components such as bee bread, stored pollen and nectar (Gilliam, 1979; Phaff and Starmer, 1987; Vega & Dowd, 2005; Benda et al., 2008). Bumble bees also have been found to harbor yeasts (Brysch-Herzbe rg, 2004; Ganter, 2005; Chapter 4). The source of K. ohmeri in bumble bee colonies is unclear, especially since no SHBs were found in the bumble bee colonies sampled within this study. Most likely, foraging bees bring K. ohmeri into the colony from external sources perhaps on pollen. Once in the colony, the yeast reproduces, emanating a suite of chemical cues that attracts SHBs. The SHBs reproduce in host colonies and may help spread K. ohmeri to adjacent hives. If K. ohmeri is introduced to the colony via bee pollen collection, it should be found in floral yeast surveys targeting beepollinated flowers within the vicinity of known K. ohmeri -infected colonies. Future investigators should consider any correlation between K. ohmeri presence and overall colony health. Furthermore, one should determine if K. ohmeri is important nutritionally to the SHB, honey bees and/or bumble bees. 106

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It is currently unknown if K. ohmeri plays an im portant role in addition to facilitating SHB host location. Other important insect-yeast symbioses exist within several insect orders such as Coleoptera, Dictyoptera, Dipt era, Homoptera, Hymenoptera, Isoptera, Lepidoptera, and Neuroptera (Vega & Dowd, 2005). Some of these asso ciations are so important that the yeast is passed down generationally via vertical transmission. For example, rice plant hoppers Nilaparvata lugens Stl (Hemiptera: Delphacidae), Laodelphax striatellus Falln (Hemiptera: Delphacidae) and Sogatella furcifera Horvath (Hemiptera: Delphacidae) have yeast symbionts within their fat bodies that mi grate to the primary oocyte in the ovariole. Thus the female inoculates the eggs through ve rtical transmission. For rice plan t hoppers, this y east association provides important proteins and other meta bolites for the developing embryo, post-embryo, nymph and adult. Kodamaea ohmeri may be important similarly to SHBs and/or their hosts. Within the bee colony K. ohmeri may be affecting the biod iversity of yeasts through competition. Benda, et al. (2008) noted that while healthy honey bee colonies contained many yeasts, SHB-infested colonies contained exclusively K. ohmeri In Chapter 4, it was shown that the primary yeast isolate discovere d from bumble bee colonies was K. ohmeri Only one yeast isolate was found that was not K. ohmeri This suggests that other ye asts within the hive are displaced by K. ohmeri What this means for bee colonies an d SHBs is unclear yet the potential effects of K. ohmeri on bee colonies should cause concern a nd stimulate research in this area. Further investigations are necessa ry, particularly given the complexity of the relationship, the current lack of knowledge regardi ng this relationship, and the succe ss of this yeast in infection, competition and spread from host to host. The success of the SHB at expa nding its host range from hone y bee colonies to include bumble bee colonies may be due to the fact that they both types of bee colonies harbor K. 107

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ohmeri It is not known if SHBs are capable of findi ng, infiltrating or utilizing new hosts if the new host does not harbor K. ohmeri If, however, K. ohmeri is im portant in host range expansion, it may aid expansion in three wa ys. First, it is possible that K. ohmeri provides a source of nutrition to SHB that would ot herwise be absent in a poten tial host. Second, SHB may be attracted to hosts that K. ohmeri also finds suitable (SHB/yea st niche overlap). Finally, K. ohmeri may serve as a cloak for invading SHBs wher eby the volatile profile of the would-be host contains volatiles of K. ohmeri that mask invading SHBs that also carry K. ohmeri That both SHBs and K. ohmeri have been found capable of existing within honey bee colonies as well as bumble bee colonies, seems mo re than coincidental. Associations also have been found recently between stingless bees and K. ohmeri (Rosa et al., 2003) as well as between stingless bees and SHBs (Greco et al., 2009). Other bees may harbor the SHB and/or K. ohmeri and may be at risk as well. Carpenter bees Xylocopa sp. (Hymenoptera: Apidae), for example, were found to have more yeasts than honey bees in a study of yeast diversity among flowers and various bee stomachs (Sandhu & Wa riach, 1985). In the same study, Apis dorsata Fabr. (Hymenoptera: Apidae), Apis florae Fabr. (Hymenoptera: Apidae), Apis indica Fabr. (Hymenoptera: Apidae), Apis cerana Fabr. (Hymenoptera: Apidae), and Halictus sp. (Hymenoptera: Apidae) also were found to harbor multiple species of yeast. Surveys of yeasts and beetles within these and othe r bee colonies nesting in clos e proximity to known SHB and/or K. ohmeri infected bumble bee or honey bee coloni es may provide a be tter understanding of K. ohmeri and SHBs host expansion. Honey bee, bumble bee, and pollinator popula tions in general are declining (Buchman & Nabhan, 1996, Cane & Tepedino, 2001; Goulson, 2003; Goulson et al., 2008; Grixti et al., 2009) and pest spillover from commerci al colonies can impact wild populat ions negatively (Colla et al. 108

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109 2006; Otterstatter & Thompson, 2008). While commercia l bee colonies are va lued in agricultural settings, the value of wild b ees to both agriculture and natural ecosystems should not be underestimated or forgotten. The diversity of be es provides ecological re dundancy in the event of an extinction of one or more pollinator species. Every effort should be made to determine the susceptibility of wild bee col onies to SHBs and the role K. ohmeri plays in SHB persistence. Ultimately, such investigations should also ai d the conservation and restoration of wild bee populations.

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BIOGRAPHICAL SKETCH Jason Graham is a masters student at th e Honey Bee Research and Extension Laboratory in University of Floridas Entomology and Ne matology Department. He received his bachelors degree at University of Delaware, with a major in entomology and a minor in wildlife conservation. As part of his undergraduate studies he conducted research on the use of the beetle, Galerucella calmariensis L. (Coleoptera: Chrysomelidae) a biological control agent of Lythrum salicaria L. (Lythraceae), an invasive plant in the Delaware wetlands. He plans to pursue a Ph.D. at University of Florida through the Entomology and Nematology Department in the Honey Bee Research and Extension Laboratory at the University of Florida.