Environmental Microbiology (2006)

8

(2), 258–272 doi:10.1111/j.1462-2920.2005.00893.x

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2912Society for Applied Microbiology and Blackwell Publishing Ltd, 2005

8

2258272

Original Article

Bacterial diversity in the gut of beesK. I. Mohr and C. C. Tebbe

Received 4 April, 2005; accepted 23 June, 2005. *For correspon-dence. E-mail christoph.tebbe@fal.de; Tel. (

+

49) 531 596 2553; Fax(

+

49) 531 596 2599.

Diversity and phylotype consistency of bacteria inthe guts of three bee species (

Apoidea

) at anoilseed rape field

Kathrin I. Mohr and Christoph C. Tebbe*

Institut für Agrarökologie, Bundesforschungsanstalt für Landwirtschaft (FAL), 38116 Braunschweig, Germany.

Summary

The gut of insects may harbour one of the largestreservoirs of a yet unexplored microbial diversity. Tounderstand how specific insects select for their ownbacterial communities, the structural diversity andvariability of bacteria found in the gut of different beespecies was analysed. For three successive years,adults and larvae of

Apis mellifera

ssp.

carnica

(honeybee), and

Bombus terrestris

(bumble bee), as well aslarvae of

Osmia bicornis

(red mason bee) were col-lected at a flowering oilseed rape field. Total DNA wasextracted from gut material and the bacterial diversitywas analysed, independent of cultivation, by geneticprofiling with single-strand conformation polymor-phism (SSCP) of polymerase chain reaction (PCR)-amplified partial 16S rRNA genes. The SSCP profileswere specific for all bee species and for larvae andadults. Qualitative and quantitative differences werefound in the bacterial community structure of larvaeand adults of

A. mellifera

, but differences in

B. terres-tris

were mainly quantitative. Sequencing of the PCRproducts revealed a dominance of

Alpha

-,

Beta

-, and

Gammaproteobacteria

,

Bacteroidetes

, and

Firmicutes

in all bee species. Single-strand conformation poly-morphism profiles suggested a higher abundanceand diversity of lactobacilli in adults of

A. mellifera

than in larvae. Further phylogenetic analyses indi-cated common bacterial phylotypes for all three beespecies, e.g. those related to

Simonsiella

,

Serratia

,and

Lactobacillus

. Clades related to

Delftia aci-dovorans

,

Pseudomonas aeruginosa

or

Lactobacillusintestinalis

only contained sequences from larvae.Several of the bee-specific clusters also containedidentical or highly similar sequences from bacteriadetected in other

A. mellifera

subspecies from South

Africa, suggesting the existence of cosmopolitan gutbacteria in bees.

Introduction

Insects are hosts for a large diversity of microorganisms,and during the long period of coevolution, both groupshave developed many different interactions. One of themajor habitats for microorganisms in insects is the diges-tive system, in which nutrients are degraded by both hostenzymes and metabolic activities of the microbial commu-nities (Martin, 1991; Cazemier

et al.,

1997; Dillon andDillon, 2004). The diversity of microorganisms inhabitingthe gut of insects must be enormous, due to the largerange of types in different groups of insects, resulting indifferent gut structures and functions. Indications of thismicrobial diversity were already obtained in early studiesby Steinhaus, in which 83 different bacteria were isolatedby cultivation from the digestive tracts of 30 insect species(Steinhaus, 1941). Since then, many more bacteria havebeen characterized from the guts of insects (see, forexamples, Charpentier

et al.

, 1978; Ulrich

et al.

, 1981;Mead

et al.

, 1988; Gilliam, 1997; Sittenfeld

et al.

, 2002;Murrell

et al.

, 2003).The large diversity as indicated by cultivation tech-

niques seems to be only a minor part of the actual bac-terial diversity suggested by the recent application ofpolymerase chain reaction (PCR)-based cultivation inde-pendent techniques. Typically, these techniques arebased on sequencing of the 16S rRNA genes (16S rDNA),i.e. genes encoding for RNA of the small subunit of thebacterial ribosomes (Egert

et al.

, 2003; Hongoh

et al.

,2003; Jeyaprakash

et al.

, 2003; Reeson

et al.

, 2003).Genetic profiling techniques, i.e. denaturing gradient gelelectrophoresis (DGGE) (Reeson

et al.

, 2003; Schabere-iter-Gurtner

et al.

, 2003), terminal restriction fragmentlength polymorphism (TRFLP) (Broderick

et al.

, 2004) orsingle-strand conformation polymorphism (SSCP) (Czar-netzki and Tebbe, 2004) can be helpful techniques forstudying the insect gut microbiota based on 16S rDNA asthey allow a straightforward comparison of the bacterialcommunity structures from a relatively large number ofsamples. In addition, quantitatively important membersof such communities can be characterized by DNA-

Bacterial diversity in the gut of bees

259

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd,

Environmental Microbiology

,

8

, 258–272

sequencing and database comparisons (Muyzer andSmalla, 1998; Osborn

et al.

, 2000; Dohrmann and Tebbe,2004). One important objective of using genetic profilingof microbial communities is to indicate similarities anddifferences between community structures in differentinsects and thereby contribute to estimating the total bac-terial diversity (Curtis

et al.

, 2002; Schloss and Handels-man, 2004).

This study was conducted to detect, characterize, andcompare the dominant bacteria inhabiting the gut of bees.Three bee species were selected: the honey bee

Apismellifera

ssp.

carnica

(Hymenoptera, Apoidea, Apidae),the bumble bee

Bombus terrestris

(Hymenoptera,Apoidea, Apidae), and the red mason bee

Osmia bicornis

(Hymenoptera, Apoidea, Megachilidae). The species cancoexist in the same habitat, where they collect pollen fromthe same plants (Haydak, 1970; Gilliam

et al.

, 1988;Ribeiro, 1999; Babendreier

et al.

, 2004). Major differencesexist between the three selected bee species in regard totheir social behaviour:

A. mellifera

lives in perennial colo-nies (states) with one egg-laying queen and up to 80 000female workers.

Bombus terrestris

also builds states, buttheir state is annual – only the copulated queens survivethe winter period – and a state comprises a maximum ofonly 600 individuals. The third species of this study,

O.bicornis

, lives solitarily. The females lay their eggs intolongitudinal breeding cells, each receiving a single eggprovisioned with food (Seidelmann, 1999; Wilkaniec andGiejdasz, 2003).

In colonies of

A. mellifera

, workers have mouth-to-mouthcontact with larvae and with each other, the latter byreciprocal feeding (Beetsma, 1985). Adult forager beesthemselves use nectar as their food source. Young larvaeof

A. mellifera

are first fed with a secretion from thehypopharyngeal glands, but later on this secretion is mixedwith pollen, glandular material and nectar. In contrast,

B.terrestris

larvae immediately receive pollen mixed withnectar and glandular material (Ribeiro, 1999; Pereboom,2000). In colonies of

B. terrestris

, adults have only indirectcontact with their larvae and with each other by feedingon the same food (bee bread), which has been collectedand mixed with nectar by the forager bees (Ribeiro, 1999).In

O. bicornis

, there is no direct feeding contact betweenlarvae and adults at all (Seidelmann, 1999).

To reduce the influence of environmental factors causedby the climatic conditions, landscape or food source, pop-ulations of the

A. mellifera

,

B. terrestris

, and cocoons offemale

O. bicornis

were established at the same field site,in the direct vicinity of a flowering oilseed rape field, allow-ing the bees to utilize the same pollen for feeding. Inaddition, for

O. bicornis

, nesting aids were placed at thefield to attract egg-laying females and later collect larvae.The emphasis of this study was to compare larvae of thethree bee species with each other, but at several sam-

plings, adult bees of

A. mellifera

and

B. terrestris

werealso analysed. Samplings were conducted during theflowering seasons of three successive years. Genetic pro-files of the bacterial community were generated fromPCR-amplified partial 16S rDNA with the single-strandremoval SSCP method (Schwieger and Tebbe, 1998;Dohrmann and Tebbe, 2004). The profiles of differentsamples were compared with each other, and thecontributors to the profiles were characterized by DNA-sequencing and phylogenetic analyses.

Results

Bacterial community structure as seen by genetic profiles

Single-strand conformation polymorphism profiles weregenerated from specimens of all three bee species col-lected in three successive years, i.e. 2001–2003 (Fig. 1A–C). The differences in the profiles of the same species anddevelopmental stages from one year to the other wererelatively high. However, when the patterns of each par-ticular year were compared with each other, similar resultswere found: For

A. mellifera

and

B. terrestris

there wereclear differences between the profiles of larvae and adults.In

A. mellifera

these differences could be attributed todifferent bands in the profiles. Typical bands for larvaewere seen in the upper part of the profiles, between SSCPmarkers 1 and 3, whereas adult specific bands were moreprevalent in a lower region of the profiles, in the vicinity ofSSCP marker 3 (Fig. 1A–C).

In contrast to

A. mellifera

, many bands of the profilesfrom

B. terrestris

were found in both developmentalstages, but for each stage with a typical intensity (Fig. 1Aand C; in 2002, only larvae were analysed). This sug-gested mainly quantitative differences in the structure ofthe bacterial community of larvae and adults (not all faintbands are clearly visible in Fig. 1). Bands in the positionof SSCP marker 2 were much stronger in profiles ofadults, and, on the other hand, bands near SSCP marker3 were more prevalent in larvae.

The SSCP profiles of the gut bacteria from

O. bicornis

larvae were clearly distinguishable from those of the otherprofiles, except for profiles generated from two

A. mellifera

larvae, one from a sample collected in 2001 and anotherfrom 2003 (AmL3 in Fig. 1A and C) respectively. The

O.bicornis

profiles from samples collected in 2001 wererelatively similar to each other (Fig. 1A) but in 2002 andeven more in 2003 (Fig. 1B and C), the profiles were moreindividual. Digital image analysis confirmed the existenceof structurally different bacterial communities for eachspecies and each developmental stage (Fig. 1, rightpanel).

260

K. I. Mohr and C. C. Tebbe

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd,

Environmental Microbiology

,

8

, 258–272

Bacterial diversity in the gut of bees

261

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd,

Environmental Microbiology

,

8

, 258–272

Indications for bacterial species diversity and their distribution in the three bee species

An attempt was made to sequence all different bandsthat were detectable on the SSCP gels for each beespecies and developmental stage. In some instances,cloning and sequencing of selected bands resulted in thedetection of more than a single sequence from a band,indicating that these bands were heterogeneous. Comi-gration of DNA molecules with different sequences inelectrophoretic gels is a phenomenon that needs to beconsidered when bands of complex profiles are charac-terized by DNA-sequencing (Sekiguchi

et al.

, 2001;Schmalenberger and Tebbe, 2003). A total of 146 bandswere selected and 193 sequences were retrieved fromthe SSCP profiles in this study. Fourteen of thesesequences were identified as putative chimera andexcluded from further analyses (data not shown). Basedon

FASTA

searches, the degree of similarity between thesequences retrieved in this study and those found in thedatabases ranged from 88.5% to 100.0%, with a majorityof sequences (74%) in the range of 96.0% to 99.0%(data not shown).

To estimate the bacterial species diversity and consis-tency of their occurrence in the different bee species,developmental stages and years of sampling, allsequences retrieved from the SSCP profiles wereassigned to phylotypes. The criterion of a phylotype wasset to

>

97% DNA sequence similarity. It should be notedthat this similarity cannot be scaled to a genus or specieslevel as the partial sequences of this study only repre-sented approximately 24% of the complete 16S rDNA.Based on this criterion, a total of 68 different phylotypeswere detected (Table 1). The vast majority of phylotypescould be assigned to one of six major phylogeneticgroups, i.e.

Firmicutes

(23% of all phylotypes),

Betapro-teobacteria

(22%),

Gammaproteobacteria

(19%),

Alphaproteobacteria

(13%),

Bacteroidetes

(12%), and

Actinobacteria

(7%).The majority of phylotypes, i.e. 66%, was only detected

in single SSCP profiles (including single replicates). Thisinconsistent occurrence indicated a quantitatively lessimportant contribution to the total bacterial diversity in thebee guts. Another 10% was only detected twice. The fourquantitatively most important groups were FIR7, BET13,GAM8, and ALP 2 which represented 9%, 8%, and twice6%, of all cloned 16S rDNA sequences respectively.

These four groups were most closely related to unculturedbacteria from the genera

Lactobacillus

,

Simonsiella

,

Ser-ratia

, and

Gluconacetobacter

. Another

Serratia

phylotype,GAM7, was most closely related to the same 16S rDNAsequence than GAM8, increasing the number of cloneswhich could be attributed the same uncultured

Serratia

to10% of the total.

The consistency of the major phylotypes was evaluatedby their frequency of detection throughout the 3 years ofstudy in the different bee species. In interpreting Table 1,it should be noted that more sequences, i.e. 112, wereanalysed from samples collected in 2001 than from theother years, which were represented by a total of 35 and32 respectively. This explains the higher number of closestrelatives found in 2001. Most frequently,

Lactobacillus

related bacteria, represented by the phylotypes FIR7 andFIR9, were detected and they were found in adult bees of

A. mellifera

, in larvae and adults of

B. terrestris

and inlarvae of

O. bicornis

(no adults analysed). The

Serratia

phylotypes GAM7/GAM8 were detected several times inlarvae of all three bee species and also in adults of

A.mellifera

. Similar results were obtained with the uncul-tured

Simonsiella

phenotype (BET13). In contrast,

Glu-conacetobacter

sp. (ALP2) was exclusively detected inadults of

A. mellifera

collected in 2001.The

Delftia tsuruhatensis

phylotype BET10 was thelargest group that could be assigned to a cultivated spe-cies (9% of all clones). Interestingly this species wasfrequently detected in larvae of all three bee species, butnever in adult specimens. Frequent detection, which indi-cated a certain preference for a developmental stage, wasfound with

Pseudomonas

sp. (GAM2, 4% of all clones)and

Halomonas variabilis

(GAM11, 3%) in larvae of

B.terrestris

, and

Propionibacterium acnes

(ACT1, 3%) inlarvae of

A. mellifera

and

O. bicornis

respectively.The qualitative differences that were found between the

SSCP profiles could further be characterized by attributingthe sequences to specific bands. Sequences related to

Salmonella enterica

(phylotype GAM6), an uncultured

Fir-micutes

with no close relative (FIR16), uncultured

Simo-nsiella

(BET13) and

uncultured Serratia

(GAM7/GAM8)were predominant in the upper regions of the profiles,between Markers 1 and 2, from larvae of

A. mellifera ssp.carnica and O. bicornis. Typically, many bands thatoccurred in the lower part of the SSCP profiles, in thevicinity of Marker 3, were caused by 16S rDNA genesrelated to Lactobacillus (data not shown).

Fig. 1. Single-strand conformation polymorphism profiles of PCR-amplified partial 16S rDNA (~370 bp) from the gut bacterial communities of three bee species (left panels) and their similarities as analysed by digital image analysis, based on Pearson correlation and UPGMA (unweighted pair group method using arithmetic averages) (right panels). Profiles from bees collected in three subsequent years are shown in A, B and C, corresponding to the years 2001, 2002 and 2003 respectively. Profiles were obtained from Apis mellifera ssp. carnica (Am), Bombus terrestris (Bt) and Osmia bicornis (Ob), either from larvae (L) or from adults (A). The numbers behind L and A indicate different bee specimens. The bordering lanes (S) show SSCP markers.

262 K. I. Mohr and C. C. Tebbe

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 258–272

Tab

le 1

.D

iver

sity

and

phy

loty

pe c

onsi

sten

cy o

f bac

teria

isol

ated

from

gut

mat

eria

ls o

f thr

ee d

iffer

ent b

ee s

peci

es.

Phy

loty

pe

Cha

ract

eriz

atio

n of

clo

sest

rel

ativ

e (m

icro

orga

nism

or

16S

rR

NA

gen

e)B

ee s

peci

es a

nd y

ear

of s

ampl

inga

Bac

teria

l div

isio

n

Gen

us n

ame

in c

ase

of s

imila

rity

of p

artia

l16

S r

RN

A g

ene

≥95%

Nam

e of

spe

cies

, st

rain

or

sequ

ence

in c

ase

of a

sim

ilarit

y of

the

part

ial 1

6S r

RN

A g

ene

≥97%

(Gen

e ba

nk a

cces

sion

no.

)

Api

s m

ellif

era

Bom

bus

terr

estr

isO

smia

bic

orni

s

Nam

eN

umbe

rof

clo

nes

2001

2002

2003

2001

2002

2003

2001

2002

2003

ALP

12

Alp

hapr

oteo

bact

eria

Ace

toba

cter

LL

ALP

211

Alp

hapr

oteo

bact

eria

Glu

cona

ceto

bact

erU

ncul

ture

d G

luco

nace

toba

cter

(AY

3701

88)

AA

LP3

1A

lpha

prot

eoba

cter

iaG

luco

nace

toba

cter

AA

LP4

1A

lpha

prot

eoba

cter

iaB

arto

nella

Unc

ultu

red

Bar

tone

lla (

AY37

0187

)A

ALP

51

Alp

hapr

oteo

bact

eria

Met

hylo

bact

eriu

mU

ncul

ture

d M

ethy

loba

cter

ium

(AY

5692

83)

LA

LP6

1A

lpha

prot

eoba

cter

iaS

phin

gom

onas

Sph

ingo

mon

as s

p. (

Z23

157)

LA

LP7

1A

lpha

prot

eoba

cter

iaB

revu

ndim

onas

Bre

vund

imon

as a

lba

(AJ2

2778

5)L

ALP

81

Alp

hapr

oteo

bact

eria

Unc

ultu

red

rum

en b

acte

rium

(AY

2449

83)

LA

LP9

1A

lpha

prot

eoba

cter

iaU

ncul

ture

d ba

cter

ium

(A

B07

4652

)L

BE

T1

1B

etap

rote

obac

teria

Cur

viba

cter

Cur

viba

cter

gra

cilis

(A

B10

9889

)A

BE

T2

3B

etap

rote

obac

teria

Com

amon

asC

omam

onas

tes

tost

eron

i (A

B06

4318

)L

BE

T3

1B

etap

rote

obac

teria

Com

amon

asC

omam

onas

den

itrifi

cans

(A

F23

3879

)A

BE

T4

4B

etap

rote

obac

teria

Jant

hino

bact

eriu

mJa

nthi

noba

cter

ium

livi

dum

(A

F17

4648

)L

LB

ET

51

Bet

apro

teob

acte

riaJa

nthi

noba

cter

ium

Jant

hino

bact

eriu

m li

vidu

m (

AF

1746

48)

LB

ET

61

Bet

apro

teob

acte

riaJa

nthi

noba

cter

ium

LB

ET

72

Bet

apro

teob

acte

riaR

alst

onia

Ral

ston

ia d

etus

cula

nens

e (A

F28

0433

)A

LB

ET

81

Bet

apro

teob

acte

riaR

alst

onia

Ral

ston

ia d

etus

cula

nens

e (A

F28

0433

)A

BE

T9

1B

etap

rote

obac

teria

Wau

ters

iaW

aute

rsia

pau

cula

(A

F08

5226

)L

BE

T10

8B

etap

rote

obac

teria

Del

ftia

Del

ftia

tsur

uhat

ensi

s (A

Y30

2438

)L

LL

LL

BE

T11

1B

etap

rote

obac

teria

Del

ftia

Del

ftia

acid

ovor

ans

(AY

3670

28)

LB

ET

121

Bet

apro

teob

acte

riaB

urkh

olde

riaB

urkh

olde

ria c

epac

ia (

U96

927)

LB

ET

1314

Bet

apro

teob

acte

riaS

imon

siel

laU

ncul

ture

d S

imon

siel

la (

AY37

0189

)A

AL

AA

LA

LL

BE

T14

1B

etap

rote

obac

teria

AB

ET

151

Bet

apro

teob

acte

riaId

eone

llaL

GA

M1

1G

amm

apro

teob

acte

riaA

cine

toba

cter

Aci

neto

bact

er h

aem

olyt

icus

(A

F54

2962

)L

GA

M2

7G

amm

apro

teob

acte

riaP

seud

omon

asP

seud

omon

as s

p. (

AF

4597

97)

LG

AM

31

Gam

map

rote

obac

teria

Pse

udom

onas

Pse

udom

onas

sp.

(A

F45

6705

)L

GA

M4

1G

amm

apro

teob

acte

riaP

seud

omon

asL

GA

M5

2G

amm

apro

teob

acte

riaS

teno

trop

hom

onas

Ste

notr

opho

mon

as m

alto

phili

a (A

B18

0661

)L

LG

AM

64

Gam

map

rote

obac

teria

Sal

mon

ella

Sal

mon

ella

ent

eric

a (A

L627

280)

LA

LG

AM

76

Gam

map

rote

obac

teria

Ser

ratia

Unc

ultu

red

Ser

ratia

(AY

3701

92)

LL

LG

AM

811

Gam

map

rote

obac

teria

Ser

ratia

Unc

ultu

red

Ser

ratia

(AY

3701

92)

L A

L A

LL

GA

M9

1G

amm

apro

teob

acte

riaL

GA

M10

1G

amm

apro

teob

acte

riaA

GA

M11

5G

amm

apro

teob

acte

riaH

alom

onas

Hal

omon

as v

aria

bilis

(AY

6167

55)

L

a.co

llect

ed f

rom

larv

ae (

L) o

r ad

ults

(A

).

Bacterial diversity in the gut of bees 263

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 258–272

GA

M12

1G

amm

apro

teob

acte

riaU

ncul

ture

d so

il ba

cter

ium

(A

J252

686)

LG

AM

131

Gam

map

rote

obac

teria

Aqu

icel

laA

quic

ella

sip

honi

s (A

Y35

9283

)L

BA

C1

1B

acte

roid

etes

Em

pedo

bact

erL

BA

C2

1B

acte

roid

etes

AB

AC

31

Bac

tero

idet

esC

hrys

eoba

cter

ium

Chr

yseo

bact

eriu

m s

p. (

AY46

8453

)L

BA

C4

1B

acte

roid

etes

Ped

obac

ter

Ped

obac

ter

afric

anus

(A

J438

171)

AB

AC

52

Bac

tero

idet

esU

ncul

ture

d ru

men

bac

teriu

mL

BA

C6

1B

acte

roid

etes

Unc

ultu

red

bact

eriu

m (

AJ4

3712

4)A

BA

C7

1B

acte

roid

etes

AB

AC

81

Bac

tero

idet

esA

AC

T1

6A

ctin

obac

teria

Pro

pion

ibac

teriu

mP

ropi

onib

acte

rium

acn

es (

AB

0416

18)

LL

LA

CT

22

Act

inob

acte

riaR

hodo

cocc

usR

hodo

cocc

us e

ryth

ropo

lis (

AB

0463

62)

LL

AC

T3

1A

ctin

obac

teria

Cor

yneb

acte

rium

Unc

ultu

red

Cor

yneb

acte

rium

(A

F36

6275

)L

AC

T4

1A

ctin

obac

teria

AA

CT

51

Act

inob

acte

riaL

FIR

12

Fir

mic

utes

Pla

noco

ccus

Pla

noco

ccus

mar

itim

us (

AF

5000

07)

AF

IR2

1F

irm

icut

esB

roch

othr

ixB

roch

othr

ix t

herm

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Tab

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264 K. I. Mohr and C. C. Tebbe

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 258–272

Phylogenetic analyses of the partial 16S rDNA

These analyses were conducted to establish the relation-ship of the partial 16S rDNA sequences found in thisstudy to each other and to related sequences from thepublic databases. Separate maximum likelihood (ML)trees were calculated for each of the dominant phyloge-netic groups found in this study, i.e. Alpha-, Beta-,Gammaproteobacteria, Bacteroidetes, Firmicutes, andActinobacteria. To reduce the complexity of the phyloge-netic trees, redundant or very closely related sequences(> 97% sequence similarity) which had been retrievedfrom the same bee species, from the same developmen-tal stage, and from the same year, were not included inthe trees.

The phylogenetic trees for the Alphaproteobacteria,Bacteroidetes, Actinobacteria and Fibrobacter/Acidobac-teria showed sequences from the bacteria of the bee gut,scattered over different phylogenetic clades with no spe-cific grouping of the sequences retrieved in this study (nofigure). On the other hand, several bee-specific groupscould be seen in the other three ML trees. Four suchgroups were found in the class Betaproteobacteria(Fig. 2). One of these groups, with sequences exclusivelyretrieved from larvae, was identical or very similar to therRNA gene of Delftia acidovorans and Ralstonia detuscu-laneuse. Two other, more heterogeneous groups were inthe phylogenetic vicinity of the Janthinobacterium lividumand Ralstonia pickettii species respectively. The largestgroup of bee-specific sequences within the Betaproteo-bacteria tree clustered with a cultivated Delftia tsuruhat-ensis (AY302438) and an uncultured Simonsiella(AY370189) respectively. The latter sequence had beendetected in A. mellifera ssp. scutellata from South Africa(Jeyaprakash et al., 2003). The same phylogenetic clus-ters were found with Distance matrix as a treeing method,indicating the stability of the phylogenetic clades shownin the ML tree (Fig. 2).

In the tree of the Gammaproteobacteria, one largeclade with sequences from all three bee species wasdetectable, and the only identical sequence found in thedatabases originated from an uncultured Serratia(AY370192) that was detected in A. mellifera ssp. capen-sis from South Africa (Jeyaprakash et al., 2003) (Fig. 3).Within this clade there was another subcluster whichexclusively contained sequences from larvae of all threebee species analysed in this study. Another phylogeneticgroup, composed only of sequences from B. terrestrislarvae from the first year of sampling was closely relatedto H. variabilis (U85871; AY616755) and an unculturedbacterium (AF371860) from a pig gut (Leser et al., 2002).Other sequences from larvae of A. mellifera and O. bicor-nis were identical to Stenotrophomas maltophilia(AB180661), Pseudomonas fluorescens (AJ583090), or

closely related to a S. enterica (AF227869). One cladeconsisting of eight different sequences, with Pseudomo-nas aeruginosa as the closest relative, was exclusivelycomposed of sequences found in B. terrestris larvae in2001.

The Firmicutes group showed one major cluster withsequences from all species and developmental stagesanalysed in this study and no foreign database sequence(Fig. 4). The closest cultivated relatives were Bacillusmacroides and Lactobacillus crispatus respectively. Threeother clusters with similar properties (no foreign databasesequences) were found within the Firmicutes tree. One ofthese clusters, harbouring sequences from larvae but notfrom adults, was most closely related to a cultured Lacto-bacillus intestinalis (AJ306299), originally isolated fromthe gut of mammals (Fujisawa et al., 1990).

Discussion

The gut is a microbial habitat that is typically influencedby structural and physiological factors and in addition bythe quality of food, the latter being the major carbon andenergy source for most gut microorganisms. In this study,the structural diversity of gut bacteria from different beespecies and from larvae and adults was compared witheach other. The gut of adult bees is more structured thanthe gut of larvae. The bacterial cell densities have beenreported to be in the range of 108-109 cells per g of gutcontents, and the numbers are typically higher in the end-gut of adult bees than in their midgut (Rada et al., 1997;Kacaniova et al., 2004). Thus, the SSCP profiles detectedin this study for adult bees were probably more influencedby endgut than by midgut bacteria, whereas this structuraleffect was lacking for larvae. Interestingly, the majority of16S rDNA sequences indicated the abundance of aerobic,facultatively aerobic, or aerotolerant bacteria in the gut.This suggested that the gut of the bees in this study didnot provide strictly anaerobic conditions. For larvae, thein vivo redox potentials that have been measured in thegut of A. mellifera support this conclusion (Bignell andHeath, 1985).

In addition to structural differences, it should be notedthat the adult foraging bees collect pollen and nectar, butthey themselves only utilize the nectar, i.e. carbohydrates.In contrast, the major food source for bee larvae is theprotein rich pollen. In response to these differences, thestructural diversity of the bacterial communities, asdetected by the PCR-SSCP profiling technique, betweenlarvae and adults were clearly distinguishable. SeveralSSCP bands which typically occurred in profiles of A.mellifera adult specimens could be attributed to lactoba-cilli. On the other hand, certain bands were more typicalfor larvae, e.g. S. enterica serovar typhi (AL627280),uncultured Simonsiella (AY370189) or uncultured Serratia

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(AY370192). However, sequencing of all detectable bandsfrom the SSCP profiles indicated that sequences fromseveral of the bands which were assumed to be typical bySSCP profiles for one developmental stage could also be

detected in profiles from the other stage. Thus, differencesin the profiles may relate more to quantitative than toabsolute aspects. The suggested dominance of lactoba-cilli in forager bees compared with larvae of A. mellifera

Fig. 2. Maximum likelihood tree including the partial 16S rDNA, PCR-amplified from bee-gut inhabiting bacteria of the subclass Betaproteobac-teria, detected from A. mellifera ssp. carnica (A), Bombus terrestris (B) and Osmia bicornis (O), from larvae (L) and adults (A), collected in three subsequent years, i.e. 2001, 2002, 2003 (years indicated by the last two digits in the sequence names) and indication of their closest phylogenetic relatives and other representative members. The numbers of nucleotides of each partial sequence ranged from 366 to 372. The scale bar represents a 10% estimated difference in nucleotide sequences. The core tree was calculated with 98 sequences of Betaproteobacteria, each with a length of more than 1300 bp. LN, 21 106.32; sites, 1420. The asterisk indicates a sequence detected in A. mellifera ssp. scutellata (Jeyaprakash et al., 2003).

Delftia tsuruhatensis, AY302438

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is supported by the fact that the food source (nectar andhoney) already has a low pH of approximately 3.9 (Snow-don and Cliver, 1996), and lactobacilli are acidotolerantand capable of fermenting sugars. The fermentation pro-cess is likely to contribute to the low pH in the gut, whichprobably inhibits the growth of other bacteria. In contrast,the pH values of bee larvae are typically above 7 (Bignelland Heath, 1985) which is probably less favourable forlactobacilli and more suitable for other gut bacteria, suchas those mentioned above.

The gut of the larvae of the solitary bee O. bicornisopens already during the early development of the larvae,allowing defaecation, while that of the larvae of both socialbees of this investigation opens much later, just shortlybefore pupation (Raw, 1972; Rust et al., 1989; Velthuis,1992). These differences may lead to different physico-chemical conditions which could explain why the SSCPprofiles from O. bicornis were so different from the otherprofiles. Most bacterial sequences from O. bicornis gutbacteria clustered with the same phylogenetic clades in

Fig. 3. Maximum likelihood tree of the Gammaproteobacteria, including the partial 16S rDNA sequences retrieved in this study from the gut of bees. For further details see legend of Fig. 2. The core tree was calculated with 96 sequences from the Gammaproteobacteria, each one consisting of more than 1300 bp. LN, 25 110.00; sites, 1389. An asterisk (*) indicates a sequence detected in the pig gastrointestinal tract (Leser et al., 2002). Double asterisks (**) indicate a sequence detected in A. mellifera ssp. capensis (Jeyaprakash et al., 2003).

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Fig. 4. Maximum likelihood tree of Firmicutes including the PCR-amplified 16S rDNA sequences retrieved in this study from gut material of bees. For more details see legend of Fig. 2. The core tree was calculated with 99 sequences from the Firmicutes, each sequence with more than 1300 bp. LN, 29 981.16; sites, 1396. The asterisk indicates a sequence that was detected in A. mellifera ssp. scutellata (Jeyaprakash et al., 2003).

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which sequences from larvae of the other two bee specieswere found. But two phylogenteic clades were detectedwith no O. bicornis derived sequences, i.e. the Comamo-nas/Janthinobacterium lividum group in the Betaproteo-bacteria and the Halomonas/P. aeruginosa group in theGammaproteobacteria. In fact, the latter group only con-tained sequences found in B. terrestris larvae. The lack ofdetection, however, cannot be a proof of specificity in thisstudy, as the sampling size, in comparison to thesequence richness detected, was relatively small.

Despite the high annual variability that could beexpected due to the uncontrolled weather conditions inthis field study, the SSCP profiles of larvae and adultsfrom A. mellifera also showed bands that were consistentduring the years and in both developmental stages.Sequences that contributed to these bands were identifiedas 16S rDNA from an uncultured Simonsiella sp., anuncultured Serratia sp., and L. crispatus. It is probable thatsome or all of these gut bacteria survive the pupation andwould thus directly be transferred from the larvae to theadults. However, it may also be possible that such bacteriathat occur in larvae and adults, also colonize the gut byfood inoculation, and possibly by mouth-to-mouth contact.

Interestingly, all of the closest relatives found to beshared between larvae and adults of A. mellifera ssp.carnica were also detected in the other two bee speciesand in both developmental stages of B. terrestris. Phylo-genetic analyses confirmed that these sequences wereclosely related among each other. Recently, Jeyaprakashand colleagues (2003) analysed the gut bacterial commu-nity from two other A. mellifera subspecies in South Africa,and they found, based on 10 unique 16S rDNA sequences,bacteria from six genera to be shared between both sub-species. In this study, a total of 179 partial 16S rDNAsequences were retrieved from larvae and adults of threedifferent bee species, representing 68 different phylotypes.Despite the small sampling size of Jeyaprakash and col-leagues in comparison to this study, the overlap was veryhigh with five of six genera, and Bifidobacterium being theonly species that was not detected here. A more detailedphylogenetic analysis revealed that the five similarsequences of both studies fell into the same groups and,thus, it can be suspected that these genes were derivedfrom bacterial species, highly abundant and adapted tothe survival in the gut of bees.

For A. mellifera and some other bee species, a numberof different bacteria have been detected from faeces andgut material with classical cultivation techniques and itwas found that most of these bacteria could be attributedto the genus Bacillus and other Bacillales, including lac-tobacilli (Gilliam and Valentine, 1976; Gilliam, 1979; Radaet al., 1997). Interestingly, in this cultivation independentanalysis, members of Bacillus were only detected infre-quently, in fact only once in an adult bee and twice in

larvae, whereas the high abundance of lactobacilli wasconfirmed. Members of the genus Bacillus typically growat pH values above 6.0 (Gordon et al., 1973) and the pHin the gut of forager bees is normally below 5.0 (Snowdonand Cliver, 1996). It is therefore likely that most of theBacillus-related bacteria would be inactive in the gut andtheir frequent detection by cultivation could be explainedby spores germinating in the growth media.

In addition to the bacilli and lactobacilli, cultivation stud-ies have also detected Enterobacteriaceae, other bacteriafrom the class Gammaproteobacteria, and Actinobacteriafrom gut and faeces of bees (Drobnikova and Bacilek,1982; Snowdon and Cliver, 1996; Matras et al., 1998) andthese groups were also detected in this study. In accor-dance with many recent studies in which cultivationdependent and independent community analyses werecompared with each other, the diversity found in this studywas much higher than suggested by the cultivation. Fivephylogenetic groups contributed almost equally to the totaldiversity, i.e. the Alpha-, Beta- and Gammaproteobacteria,Firmicutes, and Bacteroidetes and in addition some Acti-nobacteria. The prevalence of bacteria from these phylo-genetic groups is not unusual for microbial communitiesfrom many different habitats which are associated withplants or soil invertebrates (Kaiser et al., 2001; Sessitschet al., 2002; Egert et al., 2003; Schmalenberger andTebbe, 2003; Broderick et al., 2004; Czarnetzki andTebbe, 2004). The common motif of these different habi-tats could be their, more or less, continuous supply ofvarious carbon sources to support the growth of het-erotrophic, mainly aerobic or aerotolerant bacteria.

In summary, our study demonstrates the importance ofthe gut of bees as a reservoir for a large diversity of asyet poorly characterized microorganisms. The comparisonof the bacterial communities from the gut of closelyrelated, coexisting hosts indicated that bees share thesame or very closely related bacteria in their gut and thus,each insect species should not be regarded as a host fora completely different microbial community. Some of thesebacteria are obviously associated with bees independentof their geographical location or local diet and may thusbe regarded as cosmopolitans.

Experimental procedures

Field and experimental design

The field studies and samplings were conducted in collabo-ration with the Institute for Integrated Plant Protection, Fed-eral Biological Research Centre (BBA, Kleinmachnow,Germany). The experimental field, located near Dahnsdorf,Brandenburg, Germany, contained field plots with the oilseedrape (Brassica napus) varieties Artus and its derivative Fal-con pat (GS40/90/pat/35S-CaMV) both kindly provided byAventis (Frankfurt, Germany) as well as those seeded with

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wheat, rye and maize. The total area of the field was 13 ha.The variety Falcon pat was transgenic by expressing therecombinant phosphinothricin acetyl transferase, whichmediates resistance against the herbicidal compound glufo-sinate (Wehrmann et al., 1996). This property, however, wasirrelevant for this study, as the gene is not expressed in pollenand bees do not differentiate between pollen of Artus andFalcon pat (Pierre et al., 2003).

Three bee species were investigated in this study, i.e. A.mellifera ssp. carnica, B. terrestris and O. bicornis. The stud-ies were conducted during the flowering period of the oilseedrape, while none of the other cultivated crops were flowering.Bees were released and sampled in three successive years(2001–2003) under the same experimental conditions. Ineach year, one hive of A. mellifera, nine hives of B. terrestris,and bamboo tubes acting as breeding cells to attract O.bicornis were placed next to the field plots. In addition to thenesting aids, approximately 1400 cocoons of O. bicornis werealso released by our collaborators (BBA Kleinmachnow) atthe beginning of the growing season to increase the densityof this species at the field site. Adult bees (foragers) werecollected at the entrance of their hives when returning fromtheir collection flights. Larvae, close to pupation, were takenout of their breeding cells. Larvae of all species from eachyear were analysed. Adults of A. mellifera were collected andanalysed each year, whereas adults of B. terrestris were onlycollected in 2001 and 2003. In 2001, a minimum of 20 indi-vidual specimens were included as replicates for each beespecies and each developmental stage (larvae or adult)respectively. From each specimen, separate SSCP profileswere generated (method see below) to evaluate the variabilityof their bacterial community structure. For the following years,2002 and 2003, the number of individual replicates wasreduced to a range of three to six specimens. After collection,each specimen was transferred into jars which were closedwith a cotton plug soaked with an acetic ether solution to killthe insects. The jars were then transferred to the laboratory.The identity of the bee larvae collected from breeding tubeswas confirmed in collaboration with entomologists from theabove-mentioned BBA Institute.

Isolation and extraction of bacterial DNA from the bee guts

The preparation of the bee guts was conducted in collabora-tion with the above-mentioned BBA Institute. The bee guts,including food bolus, were removed from the individual spec-imens under sterile conditions. From adult bee guts, thehoney stomach was dissected from the mid- and endgutregion and the latter was analysed in this study. From larvae,the whole tube-like gut was analysed. The guts with theircontents (‘gut material’) were placed one by one separatelyinto safe-lock microreaction tubes (total volume 2.0 ml) whichcontained acetone to preserve the bacterial DNA (Fukatsu,1999). The tubes were kept in the refrigerator at 4∞C.

Polymerase chain reaction amplification of partial16S rDNA

Each sample (gut with contents) was carefully rinsed threetimes with sterile water. Then, the material was transferred

into 0.5 ml sterile saline (0.85% NaCl, wt/vol) where it wascut with a sterile scalpel into smaller pieces, crushed with aspatula, and suspended for three hours using a thermostaticmixer (Thermomixer Comfort, Eppendorf) at 500 r.p.m. androom temperature. The tubes were briefly vortexed and thencentrifuged for 5 min at 30 g to separate the gut tissue andpollen from the suspended material. The supernatants weretransferred to fresh tubes and the pellet was washed twicewith saline. The supernatants of each washing step werepooled, transferred to fresh tubes and again centrifuged for1 min at 1700 g. The supernatants were transferred to freshtubes and the bacterial cells were then collected at 10 600 g.The supernatants were discarded and the cell pellets weresuspended in 170 ml of a lysozyme solution (100 mg ml-1).The suspensions were incubated in the thermostatic mixerfor 2 h at 37∞C and 500 r.p.m. After that, 25 ml Proteinase K(Promega) was directly added and the mixture was furtherincubated for 1 h at 56∞C. Then, total DNA was extractedusing the NucleoSpin Tissue Kit, following the protocol forhuman or animal tissue provided by the manufacturer (Mach-erey and Nagel, Düren, Germany). The DNA was eluted fromthe columns with 50 ml preheated BE buffer.

Genetic profiling with single-stranded conformation polymorphism (SSCP)

A nested PCR approach was chosen to generate PCR prod-ucts for SSCP analyses. The extracted DNA was directlyused as a template for the first PCR with primers 27f and1492r (Weisburg et al., 1991) to amplify the almost completeeubacterial 16S rRNA gene (16S rDNA). The second PCRwas then used to generate the PCR products for the SSCPprofiles with the universal Com1 and Com2-Ph primers(Schwieger and Tebbe, 1998). The reverse primer was phos-phorylated for further single-strand digestion (Schwieger andTebbe, 1998). This nested PCR was chosen to exclude theamplification of 18S rRNA genes from contaminating remain-ing pollen or gut tissue. Each PCR was conducted in a totalvolume of 100 ml and the following composition: 5 U of Taqpolymerase (Hot Star Taq DNA Polymerase, Qiagen), 1¥PCR buffer, 200 mM of each of the four deoxynucleotides, andeach primer at 0.5 mM and 2 ml of DNA. Polymerase chainreaction amplifications were conducted in a Primus 96P ther-mocycler (MWG-Biotech) using the following conditions forthe first PCR (27f, 1492r): initial denaturation for 15 min at95∞C followed by 30 cycles of denaturing at 94∞C for 60 s,annealing at 50∞C for 60 s and extension at 72∞C for 70 s.Conditions for the second PCR with primers Com1 andCom2-Ph were the same, but only 25 cycles of amplificationwere conducted. A final elongation for 5 min at 72∞C com-pleted both PCR amplifications.

The size and yield of the PCR products was analysed byelectrophoresis in a 1% agarose gel stained with ethidiumbromide (Sambrook and Russell, 2001). The PCR productswere then purified using Qiaquick PCR Purification kit(Qiagen) and eluted in 30 ml elution buffer. The double-stranded DNA-concentration of each sample was measuredwith the PicoGreen dsDNA Quantitation Reagent (MoBiTec)using a microtitre plate reader (Fluoroskan II, Labsystems)with the requested wavelengths. In order to later compareSSCP profiles of the same amounts of DNA, the concentra-

270 K. I. Mohr and C. C. Tebbe

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tion was adjusted to 800 ng before single-strand digestion.The phosphorylated strands of the double-stranded PCRproducts were removed by digestion with 2.5 U of theenzyme lambda exonuclease (Amersham Biosciences) inthe buffer supplied by the manufacturer and incubated at37∞C for 60 min (Dohrmann and Tebbe, 2004). The single-stranded DNA molecules were purified with the MinElutePCR Purification Kit (Qiagen) and eluted with 10 ml elutionbuffer. Pouring and staining the SSCP gel as well as loadingthe samples and electrophoretic conditions have beendescribed in detail elsewhere (Schwieger and Tebbe, 1998;Schmalenberger and Tebbe, 2003; Dohrmann and Tebbe,2004). Single-strand conformation polymorphism markerswere generated from bacterial pure cultures by PCR usingCom1 and Com2-Ph and subsequent single-strandremoval, as described above. Shortly, the single-strand PCRproducts were separated under non-denaturing conditions inpolyacrylamide gels (MDE, FMS Bioproducts) using a Mac-rophor apparatus (Amersham Biosciences) set at 400 V and8 mA and a running temperature of 20∞C for 6800 V h. DNAin the gels was visualized by silver-staining (Bassam et al.,1991).

Digital image analysis of SSCP profiles

Similarities of SSCP profiles of the gut bacterial communitiesfrom the three bee species were analysed with the GelCom-par program (version 4.1; Applied Maths). Similarity matrixcalculations were based on Pearson correlation coefficients.Dendrograms for clustering of similar groups were based onUPGMA (unweighted pair group method using arithmetic aver-ages).

Extraction and sequencing of bands from SSCP profiles

Bands of interest were cut out of the silver-stained polyacry-lamide gels with a sterile scalpel. The single-stranded DNAwas then eluted from the gel by the ‘crush and soak’ proce-dure (Sambrook and Russell, 2001) and resuspended in12 ml of 10 mM Tris-HCl (pH 8.0). The single-stranded DNAmolecules were amplified by PCR using primers Com1 andCom2-Ph under conditions described above. The PCR prod-ucts were then purified using the NucleoSpinExtract-Kit(Machery-Nagel). After purification, the PCR products wereligated in the pGEM-T-vector-system (Promega) and clonedin Escherichia coli JM 109, as supplied and described by themanufacturer. DNA of clones with vector inserts wasextracted by alkaline lysis (15 min at 95∞C in 0.05 M NaOH,0.025% SDS) and DNA was treated as a template in PCRwith primers puc18f (5¢-CAC GAC GTT GTA AAA CGA C-3¢)and puc18r (5¢-GGA TAA CAA TTT CAC ACA GG-3¢), hybrid-izing to the insert-flanking regions of the vector. A nestedPCR of the diluted PCR product obtained with primers Com1and Com2-Ph followed. These PCR products were run onSSCP gels to compare the band position of the selectedproduct with its designated position in the original communityprofile. Clones which carried the expected inserts were cul-tivated overnight in Luria–Bertania (LB) broth (Sambrook andRussell, 2001) containing ampicillin (final concentration(100 mg l-1) and plasmids were extracted with the Plasmid

DNA-Purification Kit of Macherey and Nagel, following theprotocol of the manufacturer. The sequencing reactions werecarried out with the Sequitherm Excel II DNA sequencing kit-LC (Epicenter Technologies) and sequences were run andread using a LI-COR DNA 4200 GeneRead IR apparatus (LI-COR) as described elsewhere (Schmalenberger et al.,2001). Each PCR product was sequenced in both directions.

DNA sequence and phylogenetic analyses

Consensus sequences were generated from forward andreverse sequences using the Consed program (Gordon et al.,1998) and then imported into the ARB database (version ‘6spring 2001’) for alignment (http://www.arb-home.de). Theattached primer sequences were removed and thesequences were compared with the public databases usingthe FASTA search tool (http://www.ebi.ac.uk/fasta33/nucleotide.html). Sequences from the most closely relatedorganisms were then imported into the ARB database andintegrated into the existing phylogenetic tree of ARB

(tree_1400_may01). Subsequently, core trees were calcu-lated from approximately 100 sequences selected from thephylogenetic framework provided by ARB, including the clos-est FASTA hits each with a minimum length of 1300 nucle-otides. For each phylogenetic group, individual 50%conservation filters were calculated and applied (Ludwiget al., 2004). The partial sequences of this study were addedusing the parsimony interactive tool. Distance matrix trees(not shown) were calculated using the method of Fitch andMargoliash, 1967) with the Jukes-Cantor model (Jukes andCantor, 1969) based on one category of substitution rates.Maximum likelihood trees were calculated with theAXML + FastDNAML tool provided by ARB. To simplify thegraphical display of the trees, only the most importantsequences were included in the figures (Figs 2–4).

Nucleotide sequence accession numbers

All new sequences described in this study have been sub-mitted to the EMBL database and can be found under theAccession numbers AJ880109 to AJ880274. Sequencesretrieved in this study from the same bee species, the samedevelopmental stage, and the same year, showing 100%identity to each other, were submitted only once.

Acknowledgements

We thank Martina Sick and Stefan Kühne (BBA, Kleinmach-now, Germany) for their highly valuable collaboration andAnja B. Dohrmann for many helpful suggestions and discus-sion. The work was financially supported by the GermanMinistry for Research and Education (Projektträger Jülich;Project no. 0312628E).

References

Babendreier, D., Kalberer, N., Romeis, J., Fluri, P., and Big-ler, F. (2004) Pollen consumption in honey bee larvae: astep forward in the risk assessment of transgenic plants.Apidologie 35: 293–300.

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Bassam, B.J., Caetano-Anolles, G., and Gresshoff, P.M.(1991) Fast and sensitive silver staining of DNA in poly-acrylamide gels. Anal Biochem 196: 80–83.

Beetsma, J. (1985) Feeding behavior of nurse bees, larvalfood composition and caste differentiation in the honey bee(Apis mellifera L). Forts Zool 31: 407–410.

Bignell, D.E., and Heath, L.A.F. (1985) Electropositive redoxstate of the 5th instar larval gut of Apis mellifera. J ApicultRes 24: 211–213.

Broderick, N.A., Raffa, K.F., Goodman, R.M., and Handels-man, J. (2004) Census of the bacterial community of thegypsy moth larval midgut by using culturing and culture-independent methods. Appl Environ Microbiol 70: 293–300.

Cazemier, A.E., Hackstein, H.P., Op den Camp, H.J.M.,Rosenberg, J., and van der Drift, C. (1997) Bacteria in theintestinal tract of different species of arthropods. MicrobialEcol 33: 189–197.

Charpentier, R., Charpentier, B., and Zethner, O. (1978)Bacterial flora of midgut of two Danish populations ofhealthy 50th instar larvae of turnip moth, Scotia segetum.J Invertebr Pathol 32: 59–63.

Curtis, T.P., Sloan, W.T., and Scannell, J.W. (2002) Estimat-ing prokaryotic diversity and its limits. Proc Nat Acad SciUSA 99: 10494–10499.

Czarnetzki, A.B., and Tebbe, C.C. (2004) Diversity of bacteriaassociated with Collembola – a cultivation independentsurvey based on PCR-amplified 16S rRNA genes. FEMSMicrobiol Ecol 49: 217–227.

Dillon, R.J., and Dillon, V.M. (2004) The gut bacteria ofinsects: nonpathogenic interactions. Ann Rev Entomol 49:71–92.

Dohrmann, A.B., and Tebbe, C.C. (2004) Microbial commu-nity analysis by PCR-single-strand conformation polymor-phism (PCR-SSCP). In Molecular Microbial EcologyManual. Dodrecht, the Netherlands: Kluwer AcademicPublisher, pp. 809–838.

Drobnikova, V., and Bacilek, J. (1982) Effect of some pesti-cides on microorganisms isolated from honeybees. B Envi-ron Contam Tox 29: 734–738.

Egert, M., Wagner, B., Lemke, T., Brune, A., and Friedrich,M.W. (2003) Microbial community structure in midgut andhindgut of the humus-feeding larva of Pachnoda ephippiata(Coleoptera: Scarabaeidae). Appl Environ Microbiol 69:6659–6668.

Fitch, W.M., and Margoliash, E. (1967) Construction of phy-logenetic trees. A method based on mutation distances asestimated from cytochrome C sequences is of generalapplicability. Science 155: 279–284.

Fujisawa, T., Itoh, K., Benno, Y., and Mitsuoka, T. (1990)Lactobacillus intestinalis (ex Hemme 1974) sp. nov., nom.rev., isolated from the interstines of mice and rats. Int J SysBacteriol 40: 302–304.

Fukatsu, T. (1999) Acetone preservation: a practical tech-nique for molecular analysis. Mol Ecol 8: 1935–1945.

Gilliam, M. (1979) Microbiology of pollen and bee bread –genus Bacillus. Apidologie 10: 269–274.

Gilliam, M. (1997) Identification and roles of non-pathogenicmicroflora associated with honey bees. FEMS MicrobiolLett 155: 1–10.

Gilliam, M., and Valentine, D.K. (1976) Bacteria isolated from

intestinal contents of foraging worker honey bees, Apismellifera: The genus Bacillus. J Invertebr Pathol 28: 275–276.

Gilliam, M., Lorenz, B.J., and Richardson, G.V. (1988) Diges-tive enzymes and microorganisms in honey bees, Apismellifera – Influence of Streptomycin, age, season andpollen. Microbios 55: 95–114.

Gordon, R.E., Haynes, W.C., and Pang, C.H.N. (1973) TheGenus Bacillus. Agriculture Handbook No. 427. Washing-ton, DC, USA: US Department of Agriculture.

Gordon, D., Abajian, C., and Green, P. (1998) Consed: agraphical tool for sequence finishing. Genome Res 8: 195–202.

Haydak, M.H. (1970) Honey bee nutrition. Ann Rev Entomol15: 143–156.

Hongoh, Y., Ohkuma, M., and Kudo, T. (2003) Molecularanalysis of bacterial microbiota in the gut of the termiteReticulitermes speratus (Isoptera; Rhinotermitidae). FEMSMicrobiol Ecol 44: 231–242.

Jeyaprakash, A., Hoy, M.A., and Allsopp, M.H. (2003) Bac-terial diversity in worker adults of Apis mellifera capensisand Apis mellifera scutellata (Insecta: Hymenoptera)assessed using 16S rRNA sequences. J Invertebr Pathol84: 96–103.

Jukes, T.H., and Cantor, C.R. (1969) Evolution of proteinmolecules. In Mammalian Protein Metabolism. Munro,H.N. (ed.). New York, NY, USA: Academic Press, pp. 21–132.

Kacaniova, M., Chlebo, R., Kopernicky, M., and Trakovicka,A. (2004) Microflora of the honeybee gastrointestinal tract.Folia Microbiol 49: 169–171.

Kaiser, O., Pühler, A., and Selbitschka, W. (2001) Phyloge-netic analysis of microbial diversity in the rhizoplane ofoilseed rape (Brassica napus cv. Westar) employingcultivation-dependent and cultivation-independentapproaches. Microbial Ecol 42: 136–149.

Leser, T.D., Amenuvor, J.Z., Jensen, T.K., Lindecrona, R.H.,Boye, M., and Moller, K. (2002) Culture-independent anal-ysis of gut bacteria: the pig gastrointestinal tract microbiotarevisited. Appl Environ Microbiol 68: 673–690.

Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H.,Yadhukumar, et al. (2004) ARB: a software environment forsequence data. Nucleic Acids Res 32: 1363–1371.

Martin, M.M. (1991) The evolution of cellulose digestion ininsects. Philos T Roy Soc B 333: 281–288.

Matras, J., Mizak, L., and Muszynska, J. (1998) Influence ofhoneydew containing food on intestinal microflora in bees.Acta Microbiol Pol 47: 195–202.

Mead, L.J., Khachatourians, G.G., and Jones, G.A. (1988)Microbial ecology of the gut in laboratory stocks of themigratory grasshopper, Melanoplus sanguinipes (Fab)(Orthoptera, Acrididae). Appl Environ Microbiol 54: 1174–1181.

Murrell, A., Dobson, S.J., Yang, X.Y., Lacey, E., and Barker,S.C. (2003) A survey of bacterial diversity in ticks, lice andfleas from Australia. Parasitol Res 89: 326–334.

Muyzer, G., and Smalla, K. (1998) Application of denaturinggradient gel electrophoresis (DGGE) and temperature gra-dient gel electrophoresis (TGGE) in microbial ecology. AVan Leeuw 73: 127–141.

Osborn, A.M., Moore, E.R.B., and Timmis, K.N. (2000) An

272 K. I. Mohr and C. C. Tebbe

© 2005 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 8, 258–272

evaluation of terminal-restriction fragment length poly-morphism (T-RFLP) analysis for the study of microbialcommunity structure and dynamics. Environ Microbiol 2:39–50.

Pereboom, J.J.M. (2000) The composition of larval food andthe significance of exocrine secretions in the bumblebeeBombus terrestris. Insect Soc 47: 11–20.

Pierre, J., Marsault, D., Genecque, E., Renard, M., Champo-livier, J., and Pham-Delègue, M.H. (2003) Effects of herbi-cide-tolerant transgenic oilseed rape genotypes on honeybees and other pollinating insects under field conditions.Entomol Exp Appl 108: 159–168.

Rada, V., Machova, M., Huk, J., Marounek, M., and Duskova,D. (1997) Microflora in the honeybee digestive tract:counts, characteristics and sensitivity to veterinary drugs.Apidologie 28: 357–365.

Raw, A. (1972) Biology of solitary bee Osmia rufa (L) (Mega-chilidae). T Ro Ent Soc London 124: 213–229.

Reeson, A.F., Jankovic, T., Kasper, M.L., Rogers, S., andAustin, A.D. (2003) Application of 16S rDNA-DGGE toexamine the microbial ecology associated with a socialwasp Vespula germanica. Insect Mol Biol 12: 85–91.

Ribeiro, M.F. (1999) Long-duration feedings and caste differ-entiation in Bombus terrestris larvae. Insect Soc 46: 315–322.

Rust, R., Torchio, P., and Trostle, G. (1989) Late embryogen-esis and immature development of Osmia rufa cornigera(Rossi) (Hymenoptera, Megachilidae). Apidologie 20: 359–367.

Sambrook, J., and Russell, D.W. (2001) Molecular Cloning:a Laboratory Manual. Cold Spring Harbor, NY, USA: ColdSpring Harbor Laboratory Press.

Schabereiter-Gurtner, C., Lubitz, W., and Rölleke, S. (2003)Application of broad-range 16S rRNA PCR amplificationand DGGE fingerprinting for detection of tick-infecting bac-teria. J Microbiol Meth 52: 251–260.

Schloss, P.D., and Handelsman, J. (2004) Status of themicrobial census. Microb Mol Biol Rev 68: 686–691.

Schmalenberger, A., and Tebbe, C.C. (2003) Bacterial diver-sity in maize rhizospheres: conclusions on the use ofgenetic profiles based on PCR-amplified partial small sub-unit rRNA genes in ecological studies. Mol Ecol 12: 251–261.

Schmalenberger, A., Schwieger, F., and Tebbe, C.C. (2001)

Effect of primers hybridizing to different evolutionarily con-served regions of the small-subunit rRNA gene in PCR-based microbial community analyses and genetic profiling.Appl Environ Microbiol 67: 3557–3563.

Schwieger, F., and Tebbe, C.C. (1998) A new approach toutilize PCR-single-strand-conformation polymorphism for16S rRNA gene-based microbial community analysis. ApplEnviron Microbiol 64: 4870–4876.

Seidelmann, K. (1999) The race for females: the mating sys-tem of the red mason bee, Osmia rufa (L.) (Hymenoptera:Megachilidae). J Insect Behav 12: 13–25.

Sekiguchi, H., Tomioka, N., Nakahara, T., and Uchiyama, H.(2001) A single band does not always represent singlebacterial strains in denaturing gradient gel electrophoresisanalysis. Biotechnol Lett 23: 1205–1208.

Sessitsch, A., Reiter, B., Pfeifer, U., and Wilhelm, E. (2002)Cultivation-independent population analysis of bacterialendophytes in three potato varieties based on eubacterialand Actinomycetes-specific PCR of 16S rRNA genes.FEMS Microbiol Ecol 39: 23–32.

Sittenfeld, A., Uribe-Lorio, L., Mora, M., Nielsen, V., Arrieta,G., and Janzen, D.H. (2002) Does a polyphagous caterpil-lar have the same gut microbiota when feeding on differentspecies of food plants? Rev Biol Trop 50: 547–560.

Snowdon, J.A., and Cliver, D.O. (1996) Microorganisms inhoney. Int J Food Microbiol 31: 1–26.

Steinhaus, E.A. (1941) A study of the bacteria associatedwith thirty species of insects. J Bacteriol 42: 757–790.

Ulrich, R.G., Buthala, D.A., and Klug, M.J. (1981) Microbiotaassociated with the gastrointestinal tract of the commonhouse cricket, Acheta domestica. Appl Environ Microbiol41: 246–254.

Velthuis, H.H. (1992) Pollen digestion and the evolution ofsociality in bees. Bee World 73: 77–89.

Wehrmann, A., van Vliet, A., Opsomer, C., Botterman, J., andSchulz, A. (1996) The similarities of bar and pat geneproducts make them equally applicable for plant engineers.Nature Biotechnol 14: 1274–1278.

Weisburg, W.G., Barns, S.M., Pelletier, D.A., and Lane, D.J.(1991) 16S ribosomal DNA amplification for phylogeneticstudy. J Bacteriol 173: 697–703.

Wilkaniec, Z., and Giejdasz, K. (2003) Suitability of nestingsubstrates for the cavity-nesting bee Osmia rufa. J ApicultRes 42: 29–31.