ORIGINAL RESEARCH ARTICLE

Kodamaea ohmeri (Ascomycota: Saccharomycotina)presence in commercial Bombus impatiens Cresson and feralBombus pensylvanicus DeGeer (Hymenoptera: Apidae) coloniesJason R Graham1,2*, James D Ellis1, Nicole D Benda2, Cletus P Kurtzman3 and Drion G Boucias1

1University of Florida, Entomology and Nematology Department PO Box 110620, Bldg. 970 Natural Area Dr., Gainesville, FL32611, USA.2Center of Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, Chemistry Research Unit, U.S.Department of Agriculture, 1600 Southwest 23rd Drive, Gainesville, FL 32608, USA.3National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 N.University St., Peoria, IL 61604, USA.

Received 5 June 2010, accepted subject to revision 3 February 2011, accepted for publication 16 May 2011.

*Corresponding author: Email: jgraham@ufl.edu

SummaryIn this study, eight commercial and three feral bumble bee (Bombus impatiens Cresson and Bombus pensylvanicus DeGeer respectively,Hymenoptera: Apidae) colonies were tested for the presence of Kodamaea ohmeri (Ascomycota: Saccharomycotina), a yeast known to attractsmall hive beetles (SHB) (Aethina tumida Murray, Coleoptera: Nitidulidae) to honey bee (Apis mellifera L., Hymenoptera: Apidae) colonies.Swabs of commercial bumble bee colonies and homogenates of bumble bee colony components (adults, brood, honey, pollen and wax) wereplated on selective media. The resulting yeast isolates were compared to K. ohmeri previously isolated from SHB. Yeasts were detected in allof the commercial bumble bee colony swab samples (n = 56) and a selected subsample was shown through molecular, chemical, andmicrobiological evidence to be K. ohmeri. For the second part of the study, feral bumble bee colonies were excavated and evaluated for thepresence of any SHB life stage (none was found). Adult bees and swabs from the colonies were plated on selective media. Kodamaea ohmeriwas isolated in all samples collected from the feral bumble bee colonies. The presence of K. ohmeri in commercial and feral bumble beecolonies is of concern, as SHB, which harbour K. ohmeri, are attracted to the volatiles produced by K. ohmeri growing on bee collected pollen.

Kodamaea ohmeri (Ascomycota: Saccharomycotina) presente encolmenas comerciales de Bombus impatiens Cresson y silvestresde Bombus pensylvanicus DeGeer (Hymenoptera: Apidae)ResumenEn este estudio, ocho colmenas comerciales y tres silvestres de abejorros (Bombus impatiens Cresson y Bombus pensylvanicus DeGeerrespectivamente, Hymenoptera: Apidae) fueron analizadas para la presencia de Kodamaea ohmeri (Ascomycota: Saccharomycotina), unalevadura conocida por atraer a pequeos escarabajos de la colmena (Aethina tumida Murray, Coleoptera: Nitidulidae) hacia colmenas de laabeja melfera (Apis mellifera L., Hymenoptera: Apidae). Frotis de abejorros de colmenas comerciales y de homogeneizados de loscomponentes de las colmenas de abejorros (adultos, cra, miel, polen y cera) se sembraron en medios selectivos. Las cepas de levaduraresultantes fueron comparadas con las cepas de K. ohmeri aisladas previamente en pequeos escarabajos de la colmena. Se detectaronlevaduras en todas las muestras de frotis de colmenas comerciales de abejorros (n = 56), y una submuestra seleccionada por ser K. ohmeri secaracteriz a travs de pruebas moleculares, qumicas y microbiolgicas. Para la segunda parte del estudio, colmenas silvestres de abejorrosfueron excavadas y evaluadas para la presencia de cualquier estado de desarrollo del pequeo escarabajo de la colmena (el cual no fueencontrado). Las abejas adultas y los frotis de las colmenas se sembraron en medios selectivos. Kodamaea ohmeri fue aislada en todas las

Journal of Apicultural Research 50(3): 218-226 (2011) IBRA 2011DOI 10.3896/IBRA.1.50.3.06

IntroductionBumble bees (Hymenoptera; Apidae; Bombus spp.) share manysimilarities with western honey bees (Hymenoptera; Apidae; Apismellifera). These similarities include pollen and nectar collection andstorage habits, abdominal wax secretion glands that produce the waxused to build their nest infrastructure, and social colonies consisting ofdrones, a single laying queen, and many female workers (Wilson,1971; Alford, 1975;). Bumble bees and honey bees also occupysimilar ecological niches. Consequently, they often host many similarpests and pathogens (Alford, 1975; Kistner, 1982; Schmid-Hempel,1998; Plischuk et al., 2009; Singh et al., 2010), including small hivebeetles (SHB) (Aethina tumida Murray, Coleoptera: Nitidulidae, SHB).

SHB are a natural pest of the African subspecies of A. melliferanow affecting European subspecies in the SHBs introduced range(Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and Hepburn,2006; Neumann and Ellis, 2008). Investigators have reported thatcommercial bumble bee colonies (B. impatiens Cresson) are attractiveto and possible alternative hosts for SHB (Ambrose et al., 2000;Stanghellini et al., 2000; Spiewok and Neumann, 2006; Hoffmann etal., 2008). In an effort to determine potential sources of the attractionof SHB to bumble bee colonies, Graham et al. (2011), used four-wayolfactometer choice tests to show that SHB are attracted to volatilesproduced by adult bumble bees, stored pollen, brood, wax, and wholecolonies.

The yeast Kodamaea ohmeri (previously classified as Pichiaohmeri and Yamadazyma ohmeri, Ascomycota: Saccharomycotina)seems instrumental in the attraction of SHB to honey bee colonies,but its role in the attraction of SHB to bumble bee colonies isunknown. Kodamaea ohmeri, when associated with pollen in honeybee colonies, produces isopentyl acetate (IPA), a known SHBattractant (Torto et al., 2005, 2007a, 2007b). Volatiles from K. ohmeriinoculated pollen dough were shown to be attractive to SHB in windtunnel paired choice tests (Torto et al., 2007a, 2007b) and trapsbaited with K. ohmeri inoculated pollen dough captured significantlymore SHB than unbaited traps (Torto et al., 2007b; Arbogast et al.,2007, 2009).

Using this information, we developed an overall hypothesis that K.ohmeri may be an important component of SHB attraction to bumblebee colonies. In order to address this hypothesis adequately, threeseparate steps must be taken. Firstly, K. ohmeri must be isolated frombumble bee colonies and its volatile profile determined when growingon bumble bee collected pollen. Secondly, given that K. ohmeri isisolated from bumble bee colonies, it must determined how bumble

bee colonies acquire K. ohmeri. Finally, the overall effects of the yeaston bumble bee colonies, including its attraction to SHB, must bedetermined.

In this study, we took the first step toward determining the rolethat K. ohmeri may play in attracting SHB to bumble bee colonies byexamining eight commercial and three feral bumble bee (B. impatiensCresson and B. pensylvanicus Degeer respectively) colonies for thepresence of K. ohmeri. Given the similarities between honey bee andbumble bee ecology, the evidence that commercial bumble beecolonies can host SHB, the relationship between K. ohmeri and theSHB, and the cosmopolitan occurrence of K. ohmeri, we hypothesizedthat K. ohmeri would be present in the bumble bee colonies.Furthermore, we predicted that K. ohmeri growing on bumble beecollected pollen would produce volatiles similar to those producedwhen it grows on honey bee collected pollen. We isolated K. ohmerifrom bumble bee colonies and determined the composition of volatilesproduced by these strains. Further, we compared the strains isolatedfrom bumble bee colonies with those isolated from honey bee coloniesincluding K. ohmeri strains L-27 (Florida) and A-1 (Kenya)(Benda etal., 2008), that were isolated from SHB washes by Torto et al. (2007a).

Materials and methodsBombus impatiens coloniesIn June 2007, three commercial bumble bee quads containing four B.impatiens colonies per quad were purchased from Koppert BiologicalSystems, Inc. (Romulus, MI, USA). The quads were located at theUniversity of Floridas Bee Biology Unit in Gainesville, FL, USA (N 2937.629" W 82 21.405"). Each colony contained a corn-syrup / watersolution in a plastic bag as part of Kopperts quad system. This is usedby the bees in periods of low nectar flow. All colonies had areproductive queen, 200-250 workers, brood and nesting material(cotton and plastic). The quads were secured to the top of twocement blocks with nylon rope. The cement blocks were then placedinside trays filled with soapy water to guard against ant invasion. Theentrances to all colonies were open to permit the bumble bees toforage naturally.

Yeast isolationTo sample bumble bee colonies for K. ohmeri, eight commercial B.impatiens colonies were removed from their quad containers, openedand yeast samples were collected by rubbing two cotton swabs on theinside top and bottom of the colonies and one cotton swab on each of

Kodamaea ohmeri in bumble bee colonies 219

muestras recogidas de colmenas silvestres de abejorros. La presencia de K. ohmeri en colmenas comerciales y silvestres de abejorros es tanpreocupante como los pequeos escarabajos de las colmenas que albergan a K. ohmeri, ya que se sienten atrados por las sustancias voltilesproducidas por el crecimiento de K. ohmeri en el polen colectado por las abejas.

Keywords: Aethina tumida, Bombus impatiens, Bombus pensylvanicus, Kodamaea ohmeri, Apis mellifera

the four sides inside the colonies (n = 8 samples 7 colonies = 56swabs). The samples then were plated on Candida Isolation Agar(CIA; Atlas, 1997) for 2-3 d at 27C in a Napco 4200 water jacketincubator (Thermo Fischer Scientific, Waltham, MA). Plates wereexamined for the presence or absence of colonies displaying a yeastphenotype.

Additional samples, when available, were collected including oneadult bee (n = 7 colonies), one larvae / pupa (n = 8 colonies), 1 g ofstored pollen, (n = 5 colonies), 1 g of wax (n = 8 colonies) and 1.0 mlof honey (n = 8 colonies). Each of these samples was placed intoseparate micro centrifuge tubes containing MilliQ water andhomogenized for 1 min using a stand-mounted Tissuemiserhomogenizer (Thermo Fischer-Scientific, Waltham, MA, USA). Thehomogenates were then individually plated on CIA media. Theinoculated plates were incubated at 27C for 2-3 d, after which theplates were examined to determine the presence or absence of yeast.

Yeast culture and replication rateTo compare the volatile compositions of yeasts collected fromcommercial B. impatiens colonies to those of L-27, the three mostcommon yeast phenotypes found in the bumble bee colonies werecultured. These were represented by three isolates (1, 2 and 3)phenotypically similar to K. ohmeri, and one filamentous isolate (4).These isolates originated from different samples of different quads:isolates 1 and 4 were cultured from colony interior swabs, isolate 2was cultured from an adult bumble bee homogenate and isolate 3 wascultured from a wax homogenate. Isolates were inoculated intoseparate flasks of YPD broth (Atlas, 1997), incubated at 30C, withshaking at 200 rpm for 14 h in an Innova Biological ShakerIncubator (New Brunswick Scientific, Edison, NJ, USA). Yeast cellsfrom the broth cultures were examined with phase contrast optics andphotographed. The replication rates of these broth cultures, as well astwo additional cultures derived from L-27 (NRRL Y-27634 - one leftviable as a positive control and the other boiled in a water bath for 10min as a negative control Torto et al., 2007a) were estimated bymeasuring the increase in turbidity using a Spectronic 20spectrophotometer (Bausch and Lomb, Rochester, NY, USA) at 600nm after 0.5, 3.0, 6.0, 8.5, 11.5, 14.5, 17.5, 29.5, 53.5, and 77.5 hpost inoculation.

Pollen preparation, inoculation andvolatile collectionAdditional stored pollen (= bee bread, 21 g) was collected from waxcells from five bumble bee colonies. Samples were combined andirradiated at the Florida Department of Agriculture & ConsumerServices, Division of Plant Industry (FDACS-DPI, Gainesville, FL, USA)using a dose of 15 kGy for 25 h with a Cesium 137 source to sterilizethe samples. Aliquots of irradiated pollen (3 g) were saturated with 2 ml

220 Graham, Ellis, Benda, Kurtzman, Boucias

of yeast broth inoculated with either: isolates 1, 2, 3, or 4 collectedfrom commercial bumble bee colonies, L-27 as a positive control andL-27 boiled for 10 min as a negative control. Yeast broths wereprepared as previously described and incubated for 14 h in the waterjacket incubator at 27C prior to pollen treatment. Pollen batchessaturated with broth cultures were incubated in sterile aluminiumweigh boats at 27C. After 4-5 d, weigh boats were placed into glassvolatile collection chambers (3.8 L) maintained at 33C. Charcoalfiltered, humidified air was passed through the volatile collectionchamber for 6 h and collected onto SuperQ filters at a rate of 0.5 l/min. The volatiles trapped on the SuperQ filters were extracted byeluting the filter with 500 l of methylene chloride.

The volatile samples were analyzed using a HP-6890 GasChromatograph (GC, Hewlett Packard; Palo Alto, CA, USA) equippedwith a HP-1 column (30 m x 0.25 m, J&W Scientific; Folsom, CA,USA). The column was linked to a HP 5973 mass spectrometer usingelectron impact mode (70 eV, Agilent, Palo Alto, CA) with the gascarried by helium. The GCMS oven temperature began at 35C for thefirst min and then ramped up 10C per min to 230C and stabilizedfor 10 min. The ion source temperature was held at 230C. Thevolatile compounds collected from the yeast phenotypes werecompared to the USDA-ARS library of collected and commerciallyavailable standards based on retention times and mass spectra.

DNA sequencingThe four yeast isolates from commercial B. impatiens colonies andisolates Kenyan A-1 and L-27 isolates (Torto et al., 2007b; Benda etal., 2008) were plated in Petri dishes containing SMY agar and thenincubated at 27C for 12-16 h. DNA was extracted from each yeastisolate using a Masterpuretm Yeast DNA purification kit (Epicentre;Madison, WI, USA; Torto et al., 2007b). Taq DNA polymerase(Promega; Madison, WI, USA) was used to amplify aliquots of DNAwith primers NL-1 and NL-4 for the 5 divergent domain (D1/D2) ofthe nuclear large subunit ribosomal RNA gene (Kurtzman andRobnett, 1997). Primers F17 and R317 (Benda et al., 2008) andprimers AB28 and TW81 (Curran et al., 1994) were used for theinternal transcribed spacer (ITS) region (ITS1/5. 8S/ITS2). PCRamplicons were extracted with a QIAquick PCR extraction kit (Qiagen;Germantown, MD, USA) and sequenced bidirectionally using theApplied Biosystems Model 3130 Genetic Analyzer (Life TechnologiesCorporation; Carlsbad, CA, USA). The sequences were edited,trimmed, and aligned using the Clustal 2.0.10 multiple sequencealignment tool (Larkin et al., 2007). The DNA sequences werecompared to those in GenBank using BLAST (blastn).

Bombus pensylvanicus coloniesIn September 2009, three feral B. pensylvanicus colonies from theUniversity of Florida Natural Teaching Area, Gainesville, FL, USA (N

29 0.632245" W 82 0.372156") were excavated. Five adult beesfrom each nest were put into sterile vials, homogenized in MilliQwater, and plated separately on CIA media. Cotton swabs wererubbed across the wax cells and surrounding nest material and thenplated on CIA media. The inoculated plates were incubated at 27Cfor 2-3 d in a water jacket incubator, after which the plates wereexamined to determine the presence/absence of yeasts. The resultingyeast isolates were compared phenotypically to K. ohmeri isolates A-1and L-27, and then sent to the National Center for AgriculturalUtilization Research, Agricultural Research Service, United StatesDepartment of Agriculture, where they were identified on the basis oftheir D1/D2 nuclear large subunit ribosomal RNA gene sequence usingthe protocol of Kurtzman and Robnett (1997). The identity of earlierisolates was also verified at this time by resequencing.

ResultsYeast isolation and propertiesYeast colonies phenotypically similar to K. ohmeri were detected fromall of the swabs taken from the interior walls of the commercial B.impatiens colonies. Additionally, phenotypically similar yeasts weredetected in 6 of the 7 adult bee homogenates, 2 of the 5 pollenhomogenates and in 4 of 8 of the wax homogenates, but not in any ofthe honey or brood homogenates.

Of the 68 yeast isolates discovered in commercial B. impatienscolonies, four yeast colonies representative of the two major colonyphenotypes were selected. The first 3 isolates (1, 2 and 3) produced acolony phenotype and an ovoid to spherical cell (budding) phenotypesimilar to those observed with the L-27 isolate (Fig. 1). In contrast,

Kodamaea ohmeri in bumble bee colonies 221

isolate 4 displayed a different filamentous colony phenotype andproduced barrel-shaped cells (arthrospores) formed by fragmentationfrom hyphae (Fig. 1). Yeast isolates 1, 2, 3 and L-27 displayed similarreplication rates and shared similar lag phases, slopes, and onset ofstationary phases whereas the isolate 4 replicated more slowly undersimilar growth conditions and had a differing lag phase, slope andonset of stationary phase. The yeast isolates (1, 2, 3, 4 and Kenyan A-1) have been accessioned at the ARS Culture Collection (Table 1).

Yeast colonies phenotypically similar to K. ohmeri were detectedon all swab samples and adult bee homogenates taken from the nestmaterial of the feral B. pensylvanicus colonies. These yeast isolateswere accessioned at the ARS Culture Collection (Table 1).

Volatile comparisonsThe volatiles produced by isolates 1, 2 and 3 were similar to oneanother and to those of L-27.The volatile composition of L-27 shared7 chemical compounds with isolates 1, 2 and 3; these 7 were notreleased by isolate 4. The shared compounds were: 2, 3, butanediol,ethyl sorbate, ethyl nonanoate, ethyl decanoate, ethyl dodecanoate,ethyl hexanoate, and methyl linoaleate (Table 2). The volatilecomposition of isolate 4 was not similar to those produced by L-27, 1,2 or 3. The only compounds shared by all samples, including thenegative control, were 2-methyl-butanoic acid, ethyl hexanoate and 2-phenylethanol. A detailed table of all compounds detected throughvolatile analysis is available upon request by contacting the leadauthor.

DNA sequencingPCR-amplification using primers NL1 and NL4, for the gene sequenceof the D1/D2 domains of LSU rRNA for the bumble bee yeast isolates

Fig. 1. Photographs of cultured yeast isolates. Columns from left to right are images of isolates 1, 2, 3, L-27, A-1 and 4. Rows from top tobottom are the isolates: A. in slide preparations of yeast broth (scale bars with yeast cells are set to 10 m, and digital photos were taken witha phase contrast microscope microscope); B. plated on CIA (photo taken with a microscope equipped with a digital camera using Auto-Montagesoftware (Synchroscopy, Frederick, MD)); and C. plated on CIA, showing the entire Petri dish approximately 36 h after plate inoculation.

1, 2 and 3 and known K. ohmeri isolates (A-1) and (L-27) all produceda 450 bp segment of DNA, whereas a 700 bp 28S amplicon wasproduced using isolate 4 DNA. Amplification with primers F17 andR317 produced a 300 bp of ITS-5.8S fragment for the bumble bee 1,2, 3 and L-27 isolates and a 700 bp amplicon for isolate 4. PCR-amplification using primers AB28 and TW81 for the ITS-5.8S region ofthe yeast isolates produced a 300 bp segment for yeasts 1, 2 and 3and K. ohmeri isolates, while isolate 4 produced a 200 bp segment.

Graham, Ellis, Benda, Kurtzman, Boucias

BLAST searches (Altschul et al., 1997) of gene sequences for the5 divergent domain of the LSU rRNA gene and the ITS-5.8S regionsequences showed high homology between the bumble bee yeastisolates 1, 2, 3 and the A-1 (accession number EU569326) and L-27(accession numbers AY911384 and AY911385) isolates (Benda et al.,2008). D1/D2 gene sequences for the bumble bee yeast isolates wereidentical to the type strain of K. ohmeri (= Endomycopsis ohmeriGenBank U45702) and other strains of K. ohmeri (accession numbers

222

Table 1. Yeast isolates accessioned at the ARS Culture Collection for which the D1/D2 LSU rRNA gene sequence was determined.

Strainnumber

Source Species name Accession number

A-1 Aethina tumida(Torto et al., 2007b) Kodamaea ohmeri NRRL Y-48664

1 Bombus impatiens colony interior Kodamaea ohmeri NRRL Y-48665

2 Bombus impatiens adult homogenate Kodamaea ohmeri NRRL Y-48666

3 Bombus impatienswax homogenate Kodamaea ohmeri NRRL Y-48667

4 Bombus impatiens colony interior similar to Geotrichum silvicola(with 4 nucleotide differences) NRRL Y-48668

5 Bombus pensylvanicushive material (colony 1) Kodamaea ohmeri NRRL Y-48685

6 Bombus pensylvanicusadult bees (colony 1) Kodamaea ohmeri NRRL Y-48686

7 Bombus pensylvanicushive material (colony 2) Kodamaea ohmeri NRRL Y-48687

8 Bombus pensylvanicusadult bees (colony 2) Kodamaea ohmeri NRRL Y-48688

9 Bombus pensylvanicushive material (colony 3) Kodamaea ohmeri NRRL Y-48689

10 Bombus pensylvanicusadult bees (colony 3) Kodamaea ohmeri NRRL Y-48690

Table 2. Function of some volatile compounds collected from yeast isolates 1, 2, 3, and L-27. A detailed table of all compounds detectedthrough volatile analysis is available upon request by contacting the lead author.

Chemical compound Known function Referenceethyl nonanoate SHB attraction Torto et al., 2007bethyl decanoate SHB attraction Torto et al., 2007bethyl hexanoate SHB attraction Torto et al., 2007b

2, 3, butanediol fermentation odour associated with yeastattractive to nitidulids on cornMagee and Kosaric 1987Nout and Bartelt 1998

ethyl sorbate fermentation odour associated with yeast Kinderlerer and Hatton 1990

ethyl dodecanoate fermentation odour associated with yeastmale bumble bee semiochemical

Siebert et al., 2005Calam 1969Kullenberg et al., 1970Bertsch et al., 2005

methyl linoleate honey bee brood semiochemicalbumble bee queen semiochemicalLe Conte et al., 1990Krieger et al., 2006

AF335976, AY267821 and AY267824, among others). The ITS-1region of L-27 differed from that of A-1, as was found by Benda et al.(2008). The ITS-1 region of the Kenyan isolate, A-1, was identical tothose of the bumble bee yeast isolates 1, 2 and 3. Yeast isolate 4differed significantly from both L-27 and A-1 isolates and produced nomatches with these isolates. A BLAST search using isolate 4 produceda match with uncultured fungus clone FE17 (GenBank accessionnumber AY464893). The next closest match was the yeast Geotrichumsilvicola (AY158042), with 4 nucleotide differences. The yeast samplescollected from the feral B. pensylvanicus colonies were identified as K.ohmeri based on their D1/D2 sequences.

DiscussionIn this study, the dominant yeast samples representative of a muchlarger sample isolated from commercial and feral bumble bee colonieswere identified as K. ohmeri through genetic analysis, volatile profile,replication rate, and / or morphological data. This demonstrates thatcommercial and feral bumble bee nests are suitable micro-environments for K. ohmeri. Furthermore, this finding satisfies thefirst step in addressing our overall hypothesis that K. ohmeri may bean important component of SHB attraction to bumble bee colonies.The remaining steps that need to be completed before the hypothesiscan be addressed fully include determining how bumble bee coloniesacquire K. ohmeri and the overall effects of the yeast on bumble beecolonies, including its attraction to SHB.

Interestingly, the ITS-1 region of B. impatiens isolates 1, 2, 3were identical to the Kenyan A-1 isolate but not to the L-27 isolatecollected from Florida SHB. Benda et al. (2008) noted that the A-1 ITS-1 sequence produced 100% homology to the previously available K.ohmeri database sequences. This suggests that the A-1 isolate ismore widespread than previously thought and the differencesbetween the ITS1 regions of A-1 and L-27 are not due to geographicseparation. In addition, the B. impatiens hives harboured anotheryeast that produced a phenotype different from K. ohmeri. Isolate 4was identified as a new species closely related to G. silvicola(Ascomycota: Saccharomycotina) and represented a commonphenotype found in our samples of B. impatiens colonies.

Of the seven chemical compounds common to yeast isolates 1, 2,3, and L-27, three (ethyl nonanoate, ethyl decanoate and ethylhexanoate) were identified previously from volatiles of L-27 and foundto be attractive to SHB (Torto et al., 2007b). The other fourcompounds (2,3, butanediol, ethyl sorbate, ethyl dodecanoate andmethyl lineolate) were identified from yeasts growing on B. impatienspollen in this study (Table 2). Three of these (2,3, butanediol, ethylsorbate and ethyl dodecanoate) were fermentation-produced volatiles(Magee and Kosaric 1987, Kinderlerer and Hatton, 1990; Siebert etal., 2005) while methyl lineolate compound has been found in the

Kodamaea ohmeri in bumble bee colonies 223

volatile composition of several yeasts present on sweet corn andattractive to Carpophilus humeralis F. (Coleoptera: Nitidulidae; Noutand Bartelt, 1998) (Table 2).

Many of the volatiles found are important to bumble bees,associated with fermentation, or used by nitidulid beetles (Table 2).Ethyl dodecanoate is associated with bumble bee marking and sexpheromones found in secretions from male bumble bees of severalspecies: B. lucorum L. (Calam, 1969), B. patagiatus and B. sporadicusNylander (Kullenberg et al., 1970), B. cryptarum F., and B. magnusVogt., (Bertsch et al., 2005). Methyl linoleate is: 1. a component ofhoney bee and bumble bee semiochemical blends (Le Conte et al.,1990; Krieger et al., 2006); 2. part of a brood secretion thatstimulates worker bees to cap brood cells (Le Conte et al., 1990); 3. akairomone indicating to varroa mites the opportunity to enter broodcells (Le Conte et al., 1990); and 4. a component of B. terrestris Lqueen pheromone (Krieger et al. 2006). Another notable fermentationproduct, 2-phenylethanol (Zilkowski et al., 1999; Zhu et al., 2003),was found in all of the sample volatile compositions, including deadL-27 and yeast 4 isolates. Torto et al. (2007b) found this compound inpollen dough conditioned by allowing SHB to feed on it for 14 d. Inthe current study, however, SHB were not found in the B. impatienscolonies, so it is not clear what produced this volatile compound.

It currently is unknown how K. ohmeri enters bumble beecolonies. As a species, K. ohmeri is cosmopolitan in distribution andpreviously has been isolated from a variety of habitats includinghuman patients (Han et al., 2004; Otag et al., 2005; Lee et al., 2007;Taj-Aldeen et al., 2005), food (Etchells and Bell, 1950; Dek, 2008),marine environments (de Araujo et al., 1995; Kutty and Philip, 2008),and flowers (Potacharoen et al., 2003). Kodamaea ohmeri also hasbeen 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 andpotential SHB hosts (Ambrose et al., 2000; Stanghellini et al., 2000;Mutsaers, 2006; Spiewok and Neumann, 2006; Greco et al., 2008;Hoffmann et al., 2008). Kodamaea ohmeri may be introduced intobumble bee colonies on pollen or nectar brought back by foragingbees. Bumble bees, like honey bees, are covered with hairs thatfacilitate the transfer of pollen (Wilson, 1971; Alford, 1975; amongothers). Kodamaea ohmeri and related species have been isolatedfrom flowers (Rosa et al., 1999, Potacharoen et al., 2003) making itpossible for foraging bees to acquire the yeast while visiting flowers.The presence of K. ohmeri in both adult bumble bee homogenatesand the colony interior swab samples suggests that bumble bees maytransmit the yeast mechanically throughout the hive, thus explainingthe yeasts presence on all swab samples.

Since SHB are attracted to fermentation volatiles associated withK. ohmeri, they may be responsible for its transmission. As Benda etal. (2008) indicated, an assortment of yeasts were present in healthyhoney bee colonies but K. ohmeri was detected in colonies only at the

height of SHB invasion. Regardless, the data we present herein can beused in future studies to address ecological questions such as: 1. dobumble bee colonies serve as a source for SHB reproduction; 2. isK. ohmeri found in bumble bee colonies in areas not affected by SHB;and 3. is K. ohmeri crucial for SHB detection of bumble bee colonies?

By harbouring the SHB attractant K. ohmeri, both commercialbumble bee colonies and feral bumble bee colonies may serve as SHBreservoirs which would undermine any SHB eradication and controlefforts of nearby beekeepers. Additionally, SHB spillover could lead tothe further decline of native pollinators and negatively impactsurrounding ecosystems (Ambrose et al., 2000; Stanghellini et al., 2000;Spiewok and Neumann, 2006; Hoffmann et al., 2008). In conclusion,K. ohmeri may facilitate the movement of SHB into commercial andferal bumble bee colonies, a valid concern considering the importanceof these native pollinators, their vulnerability to SHB invasion, and thelack of bumble bee monitoring efforts.

AcknowledgementsWe thank Peter Teal (CMAVE) for technical input on experimentaldesign and with execution of the bioassays. We thank PannipaPrompiboon, Verena-Ulrike Blaeske and Kelly Sims of the University ofFlorida Insect Pathology Laboratory for their assistance with PCRwork, DNA preparation and analysis. We thank Tricia Toth, AnthonyVaudo, Eddie Atkinson, Hannah OMalley, and Meredith Cenzer(HBREL) for assisting with bioassay establishment and bee colonymanipulation.

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