Journal of Apicultural Research 50(3): 218-226 (2011)

IBRA 2011

DOI 10.3896/IBRA.1.50.3.06

ORIGINAL RESEARCH ARTICLE

Kodamaea ohmeri (Ascomycota: Saccharomycotina)

presence in commercial Bombus impatiens Cresson and feral

Bombus pensylvanicus DeGeer (Hymenoptera: Apidae) colonies

Jason R Graham1,2*, James D Ellis1, Nicole D Benda2, Cletus P Kurtzman3 and Drion G Boucias1
1

University of Florida, Entomology and Nematology Department PO Box 110620, Bldg. 970 Natural Area Dr., Gainesville, FL
32611, USA.
2
Center 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.
3
National 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

Summary
In 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 attract
small 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) were
plated on selective media. The resulting yeast isolates were compared to K. ohmeri previously isolated from SHB. Yeasts were detected in all
of the commercial bumble bee colony swab samples (n = 56) and a selected subsample was shown through molecular, chemical, and
microbiological evidence to be K. ohmeri. For the second part of the study, feral bumble bee colonies were excavated and evaluated for the
presence of any SHB life stage (none was found). Adult bees and swabs from the colonies were plated on selective media. Kodamaea ohmeri
was isolated in all samples collected from the feral bumble bee colonies. The presence of K. ohmeri in commercial and feral bumble bee
colonies 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 en

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

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 tan
preocupante como los pequeos escarabajos de las colmenas que albergan a K. ohmeri, ya que se sienten atrados por las sustancias voltiles
producidas por el crecimiento de K. ohmeri en el polen colectado por las abejas.
Keywords: Aethina tumida, Bombus impatiens, Bombus pensylvanicus, Kodamaea ohmeri, Apis mellifera

Introduction

bee colonies acquire K. ohmeri. Finally, the overall effects of the yeast

Bumble bees (Hymenoptera; Apidae; Bombus spp.) share many

determined.

similarities with western honey bees (Hymenoptera; Apidae; Apis

on bumble bee colonies, including its attraction to SHB, must be

In this study, we took the first step toward determining the role

mellifera). These similarities include pollen and nectar collection and

that K. ohmeri may play in attracting SHB to bumble bee colonies by

storage habits, abdominal wax secretion glands that produce the wax

examining eight commercial and three feral bumble bee (B. impatiens

used to build their nest infrastructure, and social colonies consisting of Cresson and B. pensylvanicus Degeer respectively) colonies for the
drones, a single laying queen, and many female workers (Wilson,

presence of K. ohmeri. Given the similarities between honey bee and

1971; Alford, 1975;). Bumble bees and honey bees also occupy

bumble bee ecology, the evidence that commercial bumble bee

similar ecological niches. Consequently, they often host many similar

colonies can host SHB, the relationship between K. ohmeri and the

pests and pathogens (Alford, 1975; Kistner, 1982; Schmid-Hempel,

SHB, and the cosmopolitan occurrence of K. ohmeri, we hypothesized

1998; Plischuk et al., 2009; Singh et al., 2010), including small hive

that K. ohmeri would be present in the bumble bee colonies.

beetles (SHB) (Aethina tumida Murray, Coleoptera: Nitidulidae, SHB).

Furthermore, we predicted that K. ohmeri growing on bumble bee

SHB are a natural pest of the African subspecies of A. mellifera

collected pollen would produce volatiles similar to those produced

now affecting European subspecies in the SHBs introduced range

when it grows on honey bee collected pollen. We isolated K. ohmeri

(Hood, 2000, 2004; Neumann and Elzen, 2004; Ellis and Hepburn,

from bumble bee colonies and determined the composition of volatiles

2006; Neumann and Ellis, 2008). Investigators have reported that

produced by these strains. Further, we compared the strains isolated

commercial bumble bee colonies (B. impatiens Cresson) are attractive

from bumble bee colonies with those isolated from honey bee colonies

to and possible alternative hosts for SHB (Ambrose et al., 2000;

including K. ohmeri strains L-27 (Florida) and A-1 (Kenya)(Benda et

Stanghellini et al., 2000; Spiewok and Neumann, 2006; Hoffmann et

al., 2008), that were isolated from SHB washes by Torto et al. (2007a).

al., 2008). In an effort to determine potential sources of the attraction

of SHB to bumble bee colonies, Graham et al. (2011), used four-way
olfactometer choice tests to show that SHB are attracted to volatiles
produced by adult bumble bees, stored pollen, brood, wax, and whole
colonies.

Materials and methods

Bombus impatiens colonies

The yeast Kodamaea ohmeri (previously classified as Pichia

In June 2007, three commercial bumble bee quads containing four B.

ohmeri and Yamadazyma ohmeri, Ascomycota: Saccharomycotina)

impatiens colonies per quad were purchased from Koppert Biological

seems instrumental in the attraction of SHB to honey bee colonies,

Systems, Inc. (Romulus, MI, USA). The quads were located at the

but its role in the attraction of SHB to bumble bee colonies is

University of Floridas Bee Biology Unit in Gainesville, FL, USA (N 29

unknown. Kodamaea ohmeri, when associated with pollen in honey

37.629" W 82 21.405"). Each colony contained a corn-syrup / water

bee colonies, produces isopentyl acetate (IPA), a known SHB

solution in a plastic bag as part of Kopperts quad system. This is used

attractant (Torto et al., 2005, 2007a, 2007b). Volatiles from K. ohmeri by the bees in periods of low nectar flow. All colonies had a
reproductive queen, 200-250 workers, brood and nesting material
inoculated pollen dough were shown to be attractive to SHB in wind
tunnel paired choice tests (Torto et al., 2007a, 2007b) and traps

(cotton and plastic). The quads were secured to the top of two

baited with K. ohmeri inoculated pollen dough captured significantly

cement blocks with nylon rope. The cement blocks were then placed

more SHB than unbaited traps (Torto et al., 2007b; Arbogast et al.,

inside trays filled with soapy water to guard against ant invasion. The

2007, 2009).

entrances to all colonies were open to permit the bumble bees to

Using this information, we developed an overall hypothesis that K. forage naturally.

ohmeri may be an important component of SHB attraction to bumble

bee colonies. In order to address this hypothesis adequately, three

Yeast isolation

separate steps must be taken. Firstly, K. ohmeri must be isolated from To sample bumble bee colonies for K. ohmeri, eight commercial B.
bumble bee colonies and its volatile profile determined when growing

impatiens colonies were removed from their quad containers, opened

on bumble bee collected pollen. Secondly, given that K. ohmeri is

and yeast samples were collected by rubbing two cotton swabs on the

isolated from bumble bee colonies, it must determined how bumble

inside top and bottom of the colonies and one cotton swab on each of

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Graham, Ellis, Benda, Kurtzman, Boucias

the four sides inside the colonies (n = 8 samples 7 colonies = 56

of yeast broth inoculated with either: isolates 1, 2, 3, or 4 collected

swabs). The samples then were plated on Candida Isolation Agar

from commercial bumble bee colonies, L-27 as a positive control and

(CIA; Atlas, 1997) for 2-3 d at 27C in a Napco 4200 water jacket

L-27 boiled for 10 min as a negative control. Yeast broths were

incubator (Thermo Fischer Scientific, Waltham, MA). Plates were

prepared as previously described and incubated for 14 h in the water

examined for the presence or absence of colonies displaying a yeast

jacket incubator at 27C prior to pollen treatment. Pollen batches

phenotype.

saturated with broth cultures were incubated in sterile aluminium

Additional samples, when available, were collected including one

adult bee (n = 7 colonies), one larvae / pupa (n = 8 colonies), 1 g of

weigh boats at 27C. After 4-5 d, weigh boats were placed into glass
volatile collection chambers (3.8 L) maintained at 33C. Charcoal

stored pollen, (n = 5 colonies), 1 g of wax (n = 8 colonies) and 1.0 ml filtered, humidified air was passed through the volatile collection
of honey (n = 8 colonies). Each of these samples was placed into

chamber for 6 h and collected onto SuperQ filters at a rate of 0.5 l/

separate micro centrifuge tubes containing MilliQ water and

min. The volatiles trapped on the SuperQ filters were extracted by

homogenized for 1 min using a stand-mounted Tissuemiser

eluting the filter with 500 l of methylene chloride.

homogenizer (Thermo Fischer-Scientific, Waltham, MA, USA). The

The volatile samples were analyzed using a HP-6890 Gas

homogenates were then individually plated on CIA media. The

Chromatograph (GC, Hewlett Packard; Palo Alto, CA, USA) equipped

inoculated plates were incubated at 27C for 2-3 d, after which the

with a HP-1 column (30 m x 0.25 m, J&W Scientific; Folsom, CA,

plates were examined to determine the presence or absence of yeast.

USA). The column was linked to a HP 5973 mass spectrometer using

electron impact mode (70 eV, Agilent, Palo Alto, CA) with the gas

Yeast culture and replication rate

carried by helium. The GCMS oven temperature began at 35C for the

To compare the volatile compositions of yeasts collected from

first min and then ramped up 10C per min to 230C and stabilized

commercial B. impatiens colonies to those of L-27, the three most

for 10 min. The ion source temperature was held at 230C. The

common yeast phenotypes found in the bumble bee colonies were

volatile compounds collected from the yeast phenotypes were

cultured. These were represented by three isolates (1, 2 and 3)

compared to the USDA-ARS library of collected and commercially

phenotypically similar to K. ohmeri, and one filamentous isolate (4).

available standards based on retention times and mass spectra.

These isolates originated from different samples of different quads:

isolates 1 and 4 were cultured from colony interior swabs, isolate 2

DNA sequencing

was cultured from an adult bumble bee homogenate and isolate 3 was The four yeast isolates from commercial B. impatiens colonies and
cultured from a wax homogenate. Isolates were inoculated into

isolates Kenyan A-1 and L-27 isolates (Torto et al., 2007b; Benda et

separate flasks of YPD broth (Atlas, 1997), incubated at 30C, with

al., 2008) were plated in Petri dishes containing SMY agar and then

shaking at 200 rpm for 14 h in an Innova Biological Shaker

incubated at 27C for 12-16 h. DNA was extracted from each yeast

Incubator (New Brunswick Scientific, Edison, NJ, USA). Yeast cells

isolate using a Masterpuretm Yeast DNA purification kit (Epicentre;

from the broth cultures were examined with phase contrast optics and Madison, WI, USA; Torto et al., 2007b). Taq DNA polymerase
photographed. The replication rates of these broth cultures, as well as (Promega; Madison, WI, USA) was used to amplify aliquots of DNA
two additional cultures derived from L-27 (NRRL Y-27634 - one left

with primers NL-1 and NL-4 for the 5 divergent domain (D1/D2) of

viable as a positive control and the other boiled in a water bath for 10 the nuclear large subunit ribosomal RNA gene (Kurtzman and
min as a negative control Torto et al., 2007a) were estimated by

Robnett, 1997). Primers F17 and R317 (Benda et al., 2008) and

measuring the increase in turbidity using a Spectronic 20

primers AB28 and TW81 (Curran et al., 1994) were used for the

spectrophotometer (Bausch and Lomb, Rochester, NY, USA) at 600

internal transcribed spacer (ITS) region (ITS1/5. 8S/ITS2). PCR

nm after 0.5, 3.0, 6.0, 8.5, 11.5, 14.5, 17.5, 29.5, 53.5, and 77.5 h

amplicons were extracted with a QIAquick PCR extraction kit (Qiagen;

post inoculation.

Germantown, MD, USA) and sequenced bidirectionally using the

Applied Biosystems Model 3130 Genetic Analyzer (Life Technologies

Pollen preparation, inoculation and

Corporation; Carlsbad, CA, USA). The sequences were edited,

volatile collection

trimmed, and aligned using the Clustal 2.0.10 multiple sequence

Additional stored pollen (= bee bread, 21 g) was collected from wax

alignment tool (Larkin et al., 2007). The DNA sequences were

cells from five bumble bee colonies. Samples were combined and

compared to those in GenBank using BLAST (blastn).

irradiated at the Florida Department of Agriculture & Consumer

Services, Division of Plant Industry (FDACS-DPI, Gainesville, FL, USA)

Bombus pensylvanicus colonies

using a dose of 15 kGy for 25 h with a Cesium 137 source to sterilize

In September 2009, three feral B. pensylvanicus colonies from the

the samples. Aliquots of irradiated pollen (3 g) were saturated with 2 ml University of Florida Natural Teaching Area, Gainesville, FL, USA (N

Kodamaea ohmeri in bumble bee colonies

221

29 0.632245" W 82 0.372156") were excavated. Five adult bees

isolate 4 displayed a different filamentous colony phenotype and

from each nest were put into sterile vials, homogenized in MilliQ

produced barrel-shaped cells (arthrospores) formed by fragmentation

water, and plated separately on CIA media. Cotton swabs were

from hyphae (Fig. 1). Yeast isolates 1, 2, 3 and L-27 displayed similar

rubbed across the wax cells and surrounding nest material and then

replication rates and shared similar lag phases, slopes, and onset of

plated on CIA media. The inoculated plates were incubated at 27C

stationary phases whereas the isolate 4 replicated more slowly under

for 2-3 d in a water jacket incubator, after which the plates were

similar growth conditions and had a differing lag phase, slope and

examined to determine the presence/absence of yeasts. The resulting

onset of stationary phase. The yeast isolates (1, 2, 3, 4 and Kenyan A

yeast isolates were compared phenotypically to K. ohmeri isolates A-1

-1) have been accessioned at the ARS Culture Collection (Table 1).

and L-27, and then sent to the National Center for Agricultural

Yeast colonies phenotypically similar to K. ohmeri were detected

Utilization Research, Agricultural Research Service, United States

on all swab samples and adult bee homogenates taken from the nest

Department of Agriculture, where they were identified on the basis of

material of the feral B. pensylvanicus colonies. These yeast isolates

their D1/D2 nuclear large subunit ribosomal RNA gene sequence using were accessioned at the ARS Culture Collection (Table 1).
the protocol of Kurtzman and Robnett (1997). The identity of earlier
isolates was also verified at this time by resequencing.

Volatile comparisons
The volatiles produced by isolates 1, 2 and 3 were similar to one
another and to those of L-27.The volatile composition of L-27 shared

Results

7 chemical compounds with isolates 1, 2 and 3; these 7 were not

Yeast isolation and properties

ethyl sorbate, ethyl nonanoate, ethyl decanoate, ethyl dodecanoate,

Yeast colonies phenotypically similar to K. ohmeri were detected from

ethyl hexanoate, and methyl linoaleate (Table 2). The volatile

all of the swabs taken from the interior walls of the commercial B.

composition of isolate 4 was not similar to those produced by L-27, 1,

impatiens colonies. Additionally, phenotypically similar yeasts were

2 or 3. The only compounds shared by all samples, including the

detected in 6 of the 7 adult bee homogenates, 2 of the 5 pollen

negative control, were 2-methyl-butanoic acid, ethyl hexanoate and 2-

released by isolate 4. The shared compounds were: 2, 3, butanediol,

homogenates and in 4 of 8 of the wax homogenates, but not in any of phenylethanol. A detailed table of all compounds detected through
volatile analysis is available upon request by contacting the lead
the honey or brood homogenates.
Of the 68 yeast isolates discovered in commercial B. impatiens

author.

colonies, four yeast colonies representative of the two major colony

phenotypes were selected. The first 3 isolates (1, 2 and 3) produced a

DNA sequencing

colony phenotype and an ovoid to spherical cell (budding) phenotype

PCR-amplification using primers NL1 and NL4, for the gene sequence

similar to those observed with the L-27 isolate (Fig. 1). In contrast,

of 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 to
bottom 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 with
a phase contrast microscope microscope); B. plated on CIA (photo taken with a microscope equipped with a digital camera using Auto-Montage
software (Synchroscopy, Frederick, MD)); and C. plated on CIA, showing the entire Petri dish approximately 36 h after plate inoculation.

222

Graham, Ellis, Benda, Kurtzman, Boucias

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

Source

Species name

Accession number

A-1

Aethina tumida
(Torto et al., 2007b)

Kodamaea ohmeri

NRRL Y-48664

Bombus impatiens colony interior

Kodamaea ohmeri

NRRL Y-48665

Bombus impatiens adult homogenate

Kodamaea ohmeri

NRRL Y-48666

Kodamaea ohmeri

NRRL Y-48667

number

3
4
5
6
7
8
9
10

Bombus impatiens
wax homogenate

Bombus impatiens colony interior

Bombus pensylvanicus
hive material (colony 1)

Bombus pensylvanicus
adult bees (colony 1)

Bombus pensylvanicus
hive material (colony 2)

Bombus pensylvanicus
adult bees (colony 2)

Bombus pensylvanicus
hive material (colony 3)

Bombus pensylvanicus
adult bees (colony 3)

similar to Geotrichum silvicola

(with 4 nucleotide differences)

NRRL Y-48668

Kodamaea ohmeri

NRRL Y-48685

Kodamaea ohmeri

NRRL Y-48686

Kodamaea ohmeri

NRRL Y-48687

Kodamaea ohmeri

NRRL Y-48688

Kodamaea ohmeri

NRRL Y-48689

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 detected
through volatile analysis is available upon request by contacting the lead author.
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

fermentation odour associated with yeast

Magee and Kosaric 1987

attractive to nitidulids on corn

Nout and Bartelt 1998

ethyl sorbate

fermentation odour associated with yeast

Kinderlerer and Hatton 1990

ethyl dodecanoate

fermentation odour associated with yeast

male bumble bee semiochemical

2, 3, butanediol

Siebert et al., 2005

Calam 1969
Kullenberg et al., 1970
Bertsch et al., 2005
methyl linoleate

honey bee brood semiochemical

bumble bee queen semiochemical

1, 2 and 3 and known K. ohmeri isolates (A-1) and (L-27) all produced

Le Conte et al., 1990

Krieger et al., 2006

BLAST searches (Altschul et al., 1997) of gene sequences for the

a 450 bp segment of DNA, whereas a 700 bp 28S amplicon was

5 divergent domain of the LSU rRNA gene and the ITS-5.8S region

produced using isolate 4 DNA. Amplification with primers F17 and

sequences showed high homology between the bumble bee yeast

R317 produced a 300 bp of ITS-5.8S fragment for the bumble bee 1,

isolates 1, 2, 3 and the A-1 (accession number EU569326) and L-27

2, 3 and L-27 isolates and a 700 bp amplicon for isolate 4. PCR-

(accession numbers AY911384 and AY911385) isolates (Benda et al.,

amplification using primers AB28 and TW81 for the ITS-5.8S region of 2008). D1/D2 gene sequences for the bumble bee yeast isolates were
the yeast isolates produced a 300 bp segment for yeasts 1, 2 and 3

identical to the type strain of K. ohmeri (= Endomycopsis ohmeri

and K. ohmeri isolates, while isolate 4 produced a 200 bp segment.

GenBank U45702) and other strains of K. ohmeri (accession numbers

Kodamaea ohmeri in bumble bee colonies

223

AF335976, AY267821 and AY267824, among others). The ITS-1

volatile composition of several yeasts present on sweet corn and

region of L-27 differed from that of A-1, as was found by Benda et al.

attractive to Carpophilus humeralis F. (Coleoptera: Nitidulidae; Nout

(2008). The ITS-1 region of the Kenyan isolate, A-1, was identical to

and Bartelt, 1998) (Table 2).

those of the bumble bee yeast isolates 1, 2 and 3. Yeast isolate 4

Many of the volatiles found are important to bumble bees,

differed significantly from both L-27 and A-1 isolates and produced no associated with fermentation, or used by nitidulid beetles (Table 2).
matches with these isolates. A BLAST search using isolate 4 produced

Ethyl dodecanoate is associated with bumble bee marking and sex

a match with uncultured fungus clone FE17 (GenBank accession

pheromones found in secretions from male bumble bees of several

number AY464893). The next closest match was the yeast Geotrichum species: B. lucorum L. (Calam, 1969), B. patagiatus and B. sporadicus

silvicola (AY158042), with 4 nucleotide differences. The yeast samples Nylander (Kullenberg et al., 1970), B. cryptarum F., and B. magnus
collected from the feral B. pensylvanicus colonies were identified as K. Vogt., (Bertsch et al., 2005). Methyl linoleate is: 1. a component of

ohmeri based on their D1/D2 sequences.

honey bee and bumble bee semiochemical blends (Le Conte et al.,
1990; Krieger et al., 2006); 2. part of a brood secretion that
stimulates worker bees to cap brood cells (Le Conte et al., 1990); 3. a

Discussion

kairomone indicating to varroa mites the opportunity to enter brood

In this study, the dominant yeast samples representative of a much

queen pheromone (Krieger et al. 2006). Another notable fermentation

cells (Le Conte et al., 1990); and 4. a component of B. terrestris L

larger sample isolated from commercial and feral bumble bee colonies product, 2-phenylethanol (Zilkowski et al., 1999; Zhu et al., 2003),
were identified as K. ohmeri through genetic analysis, volatile profile,

was found in all of the sample volatile compositions, including dead

replication rate, and / or morphological data. This demonstrates that

L-27 and yeast 4 isolates. Torto et al. (2007b) found this compound in

commercial and feral bumble bee nests are suitable micro-

pollen dough conditioned by allowing SHB to feed on it for 14 d. In

environments for K. ohmeri. Furthermore, this finding satisfies the

the current study, however, SHB were not found in the B. impatiens

first step in addressing our overall hypothesis that K. ohmeri may be

colonies, so it is not clear what produced this volatile compound.

an important component of SHB attraction to bumble bee colonies.

It currently is unknown how K. ohmeri enters bumble bee

The remaining steps that need to be completed before the hypothesis

colonies. As a species, K. ohmeri is cosmopolitan in distribution and

can be addressed fully include determining how bumble bee colonies

previously has been isolated from a variety of habitats including

acquire K. ohmeri and the overall effects of the yeast on bumble bee

human patients (Han et al., 2004; Otag et al., 2005; Lee et al., 2007;

colonies, including its attraction to SHB.

Taj-Aldeen et al., 2005), food (Etchells and Bell, 1950; Dek, 2008),

Interestingly, the ITS-1 region of B. impatiens isolates 1, 2, 3

were identical to the Kenyan A-1 isolate but not to the L-27 isolate

marine environments (de Araujo et al., 1995; Kutty and Philip, 2008),
and flowers (Potacharoen et al., 2003). Kodamaea ohmeri also has

collected from Florida SHB. Benda et al. (2008) noted that the A-1 ITS been found in association with stingless bees (Rosa et al., 2003),
-1 sequence produced 100% homology to the previously available K.

honey bees (Torto et al., 2005, 2007a, 2007b; Benda et al., 2008)

ohmeri database sequences. This suggests that the A-1 isolate is

and now bumble bees, all of which are important pollinators and

more widespread than previously thought and the differences

potential SHB hosts (Ambrose et al., 2000; Stanghellini et al., 2000;

between the ITS1 regions of A-1 and L-27 are not due to geographic

Mutsaers, 2006; Spiewok and Neumann, 2006; Greco et al., 2008;

separation. In addition, the B. impatiens hives harboured another

Hoffmann et al., 2008). Kodamaea ohmeri may be introduced into

yeast that produced a phenotype different from K. ohmeri. Isolate 4

bumble bee colonies on pollen or nectar brought back by foraging

was identified as a new species closely related to G. silvicola

bees. Bumble bees, like honey bees, are covered with hairs that

(Ascomycota: Saccharomycotina) and represented a common

facilitate the transfer of pollen (Wilson, 1971; Alford, 1975; among

phenotype found in our samples of B. impatiens colonies.

others). Kodamaea ohmeri and related species have been isolated

Of the seven chemical compounds common to yeast isolates 1, 2,

3, and L-27, three (ethyl nonanoate, ethyl decanoate and ethyl

from flowers (Rosa et al., 1999, Potacharoen et al., 2003) making it

possible for foraging bees to acquire the yeast while visiting flowers.

hexanoate) were identified previously from volatiles of L-27 and found The presence of K. ohmeri in both adult bumble bee homogenates
to be attractive to SHB (Torto et al., 2007b). The other four

and the colony interior swab samples suggests that bumble bees may

compounds (2,3, butanediol, ethyl sorbate, ethyl dodecanoate and

transmit the yeast mechanically throughout the hive, thus explaining

methyl lineolate) were identified from yeasts growing on B. impatiens

the yeasts presence on all swab samples.

pollen in this study (Table 2). Three of these (2,3, butanediol, ethyl

Since SHB are attracted to fermentation volatiles associated with

sorbate and ethyl dodecanoate) were fermentation-produced volatiles

K. ohmeri, they may be responsible for its transmission. As Benda et

(Magee and Kosaric 1987, Kinderlerer and Hatton, 1990; Siebert et

al. (2008) indicated, an assortment of yeasts were present in healthy

al., 2005) while methyl lineolate compound has been found in the

honey bee colonies but K. ohmeri was detected in colonies only at the

224

Graham, Ellis, Benda, Kurtzman, Boucias

height of SHB invasion. Regardless, the data we present herein can be ATLAS, R M (1997) Handbook of microbiological media. CRC Press;
used in future studies to address ecological questions such as: 1. do
bumble bee colonies serve as a source for SHB reproduction; 2. is

Boca Raton, FL., USA. 2036 pp.

BENDA, N D; BOUCIAS, D G; TORTO, B; TEAL P E A (2008) Detection

K. ohmeri found in bumble bee colonies in areas not affected by SHB;

and characterization of Kodamaea ohmeri associated with small

and 3. is K. ohmeri crucial for SHB detection of bumble bee colonies?

hive beetle Aethina tumida infesting honey bee hives. Journal of

By harbouring the SHB attractant K. ohmeri, both commercial

bumble bee colonies and feral bumble bee colonies may serve as SHB

Apicultural Research 47(3): 194201. DOI: 10.3827/IBRA.1.47.3.07

BERTSCH, A; SCHWEER, H; TITZE, A; TANAKA, H (2005) Male labial

reservoirs which would undermine any SHB eradication and control

gland secretions and mitochondrial DNA markers support species

efforts of nearby beekeepers. Additionally, SHB spillover could lead to

status of Bombus cryptarum and B. magnus (Hymenoptera, Apidae).

the further decline of native pollinators and negatively impact

Insectes Sociaux 52: 45-54. DOI: 10.1007/s00040-004-0761-1

surrounding ecosystems (Ambrose et al., 2000; Stanghellini et al., 2000; CALAM, D H (1969) Species and sex-specific compounds from the
Spiewok and Neumann, 2006; Hoffmann et al., 2008). In conclusion,

heads of male bumble bees (Bombus spp.). Nature 221: 856-857.

K. ohmeri may facilitate the movement of SHB into commercial and

DOI: 10.1038/221856a0

feral bumble bee colonies, a valid concern considering the importance

CURRAN, J; DRIVER, F; BALLARD, J W O; MILNER, R J (1994)

of these native pollinators, their vulnerability to SHB invasion, and the

Phylogeny of Metarhizium: analysis of ribosomal DNA sequence

lack of bumble bee monitoring efforts.

data. Mycological Research 98: 547-552. DOI: 10.1016/S09537562(09)80478-4

DEK, T (2008) Handbook of food spoilage yeasts (2nd edition). CRC

Acknowledgements

Press; Boca Raton, FL., USA. 352 pp.

DE ARAUJO, F V; SOARES, C A G; HAGLER, A N; MENDONA-HAGLER,

We thank Peter Teal (CMAVE) for technical input on experimental

L C (1995) Ascomycetous yeast communities of marine

design and with execution of the bioassays. We thank Pannipa

invertebrates in a south east Brazilian mangrove ecosystem.

Prompiboon, Verena-Ulrike Blaeske and Kelly Sims of the University of

Antonie van Leeuwenhoek 68(2): 91-99. DOI: 10.1007/

Florida Insect Pathology Laboratory for their assistance with PCR

BF00873096

work, DNA preparation and analysis. We thank Tricia Toth, Anthony

ELLIS, J D; HEPBURN, H R (2006) An ecological digest of the small

Vaudo, Eddie Atkinson, Hannah OMalley, and Meredith Cenzer

hive beetle (Aethina tumida), a symbiont in honey bee colonies

(HBREL) for assisting with bioassay establishment and bee colony

(Apis mellifera). Insectes Sociaux 53: 8-19. DOI: 10.1007/s00040

manipulation.

-005-0851-8
ETCHELLS, J L; BELL, T A (1950) Film yeasts on commercial cucumber

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