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A scientific note on the lactic acid bacterial flora in
honeybees in the USA - A comparison with bees from
Sweden
Alejandra Vásquez, Tobias C. Olofsson, Diana Sammataro
To cite this version:
Apidologie 40 (2009) 26–28 Available online at: c
INRA/DIB-AGIB/ EDP Sciences, 2008 www.apidologie.org
DOI:10.1051/apido:2008063
Scientific note
A scientific note on the lactic acid bacterial flora
in honeybees in the USA – A comparison with bees
from Sweden
*
Alejandra V
asquez
´
1, Tobias C. Olofsson
1, Diana Sammataro
21Microbiology Laboratory at Campus Helsingborg, Department of Cell and Organism Biology,
Lund University Campus Helsingborg, Rönnowsgatan 6, PO Box SE-25108 Helsingborg, Sweden
2USDA-ARS Carl Hayden Honey Bee Research Centre, Department of Entomology, University of Arizona, 2000 East
Allen Road, Tucson, AZ 85719, USA
Received 3 June 2008 – Revised 2 September 2008 – Accepted 2 September 2008
Lactobacillus/ Bifidobacterium / Apis mellifera / 16S rRNA genotyping / coevolution
Recently, it was discovered by Olofsson and Vásquez (2008) that a novel flora composed of lac-tic acid bacteria (LAB) of the genera
Lactobacil-lus and Bifidobacterium (Fig.1), exists in the honey stomach of the honeybee Apis mellifera. The ten different flora members varied numerically with the sources of nectar and the presence of other bacterial genera within the honeybee. Closely related phylo-types of some of the lactic acid bacteria have been encountered before in previous work (Scardovi and Trovatelli,1969; Jeyaprakash et al.,2003; Baben-dreier et al.,2007) but only in the intestines of hon-eybees. It appeared that honeybees and the novel LAB flora evolved in mutual dependence on one another, the LAB obtaining a niche in which nutri-ents were available, the honeybees and their honey in turn being protected by the LAB from harm-ful microorganisms. In addition, four novel bacte-rial phylotypes belonging to the family
Pasteurel-laceae, were discovered (Fig.1). In other studies, (Jeyaprakash et al.,2003; Babendreier et al.,2007) clones with high sequence similarities have been found (Fig.1), but again only in the intestines of honeybees.
By comparison, honey stomachs of honeybees (Apis mellifera and A. m. scutellata) collected in the USA were sampled. During spring, honeybees were foraging on flowers that were in bloom (see Tab.I) Corresponding author: T.C. Olofsson,
Tobias.Olofsson@cob.lu.se
* Manuscript editor: Klaus Hartfelder
around the Carl Hayden Honey Bee Lab (Tucson, AZ) which has over 50 colonies of bees on location. Thirty bees that were actively working flowers were collected from each flower type. Between ten and 20 honey stomachs filled with nectar were removed, placed in an Eppendorf tube with 100µL physiolog-ical saline kept on ice and refrigerated until express shipped with cold packs to Sweden for bacterial an-alyzes. In Sweden, a total of 45 cultivated isolates were identified using the 16S rRNA gene sequenc-ing technique (methods of Olofsson and Vásquez,
2008).
The results showed that the Swedish bacterial phylotypes, representing all the groups of
Lacto-bacillus and Bifidobacterium, were found in the
honey stomachs of American bees (Fig.1). In ad-dition, a novel Lactobacillus phylotype 3d was iso-lated. This phylotype is possibly a novel species be-longing to the Lactobacillus genus. The 16S rRNA gene sequence of 830 bp resembled the type strain of Lactobacillus helveticus DSM 20075Tby 92.3%, which is below the threshold level generally used to define a species (95–97%) (Ludwig et al.,1998).
Babendreier et al. (2007) isolated a clone with high sequence similarity to this phylotype but from the intestines of honeybees (Fig. 1). One of the American honey stomach isolates (B4) was iden-tical in sequence to the Swedish Bifidobacterium phylotype Trm9 (isolated only from the intestine), which suggests a honey stomach origin.
Lactic acid bacterial flora in honeybees 27 Bin2*S (EF187231) Hma3* (EF187236)S F2 (EU753693)*A Trm9 (EF187232)S B4 *A(EU753692) Bin7* (EF187234)S A1*A(EU753695)
Bifidobacterium asteroides CCUG 24607 (EF187235)T F8 *A(EU753694)
A2 *A(EU753696)
Bma6* (EF187237)S
Bifidobacterium coryneforme CCUG 34986 (EF187238)T Bifidobacterium indicum JCM 1302 (T )
D86188
Bifidobacterium thermacidophilum subsp. porcinum P3-14 (AY148470)T Lactobacillus buchneri JCM 1115 (AB205055)T
Lactobacillus ferintoshensis R7-84 (AF275311)T Lactobacillus plantarum JCM 1149 (D79210)T
Fhon2* S(EF187239)
Lactobacillus kunkeei YH-15 (Y11374)T MiHM4 *A(EU753691)
Lactobacillus versmoldensis KU-3 (AJ496791)T Hon2* S(EF187244) F7 *A(EU753690) Lactobacillus sp. (DQ837633) Bin4 S(EF187245) Bma5* (EF187242)S B3 *A(EU753697) Lactobacillus sp. (AY667699) Lactobacillus sp. (DQ837636) Hma8* (EF187243)S Lactobacillus sp. (DQ837637) Biut2* S(EF187241) Lactobacillus sp. (AY667698) Hma2* S(EF187240) 3b*A(EU753698) Lactobacillus sp. (AY370183) 3d *A(EU753689) Lactobacillus sp. (DQ837634)
Lactobacillus ultunensis LMG 22117 (AY253660)T Lactobacillus acidophilus DSM 20079 (M58802)T Lactobacillus helveticus DSM 20075 (AM113779)T
Lactobacillus kalixensis DSM 16043 (AY253657)T Lactobacillus gasseri ATCC 33323 (AF519171)T Lv2 S(EF187249)
C4 *A(EU753688)
Trm1 S(EF187248)
Uncultured gamma proteobacterium clone HBG_A2V5-3 (DQ837609) Uncultured gamma proteobacterium clone HBG_D2V2-2 (DQ837606)
Hma5* S(EF187247)
Uncultured Serratia sp. clone pAJ204 (AY370191) LvLi2 S(EF187250)
Uncultured gamma proteobacterium clone HBG_B1V3-1 (DQ837610)
Actinobacillus equuli subsp. haemolyticus F154 (AF247716)T Phocoenobacter uteri NCTC 12872 (X89379)T
Proteus mirabilis ATCC 29906 (AF008582)T Edwardsiella ictaluri JCM 1680 (AB050826)T
0.01 substitutions/site I II III 100 100 100 94 87 90 100 95 100 100 100 100 99 100 98 100 95 95 100 100 100 98 94 100 100 100 98 94
Figure 1. A phylogenetic tree based on a distance matrix analysis of 715 positions in the 16S rRNA gene.
Closely related type strains and reference strains are indicated in parenthesis, together with accession num-bers from GeneBank. Cluster I displays the Lactobacillus group, cluster II the Bifidobacterium group, and cluster III the Pasteurellaceae group, which served as the out-group. The phylotypes characterised in the study are in bold print and derived from isolates, the accession numbers being included. Honey stomach phylotypes are marked *, Swedish isolatesSand American isolatesA. Bar: 0. 01 substitutions per nucleotide position. The bacterial 16S rRNA gene sequences were deposited in GenBank using the accession numbers EU753688-EU753698.
dominate the LAB flora and in the American sam-ples it was again shown to dominate (phylotype MiHm4, Fig.1). The numbers and members of the LAB flora detectable in the Swedish study were low during early spring when the bees began collecting pollen and nectar after the winter. In the first sam-plings of the American bees, in the early spring, when the bees were foraging on rosemary flowers (Tab.I), LAB flora were again very low. In both studies the LAB increased when the foraging activ-ity increased.
Furthermore, one of the phylotypes (Lv2) be-longing to the Pasteurellaceae family, obtained from Swedish honeybee larvae, was discovered in
the honey stomach of American bees (C4) (Fig.1). Together with the Swedish phylotype Hma5 two different phylotypes belonging to the
Pasteurel-laceae family have now been obtained from the
honey stomach. Pasteurellaceae phylotypes have now been detected in honeybees from Sweden, Italy, USA and Switzerland (Fig.1) indicating that they could be bee specific.
28 A. Vásquez et al.
Table I. Bacterial phylotypes, closely related to the Swedish phylotypes, isolated from American honeybee
honey stomachs when bees were foraging on the flowers rosemary (Rosmarinus officinalis), wild orange rootstock (Citrus spp.), grapefruit (Citrus paradisi Red var.) and kumquat (Fortunella spp.). Numbers of isolates found, closely related to the Swedish phylotypes by the 16S rRNA gene, are given. Unique Ameri-can phylotype*.
Phylotype Rosemary Bitter orange Grapefruit Kumquat
Fhon2 2 2 7 6 Hon2 3 3 Bma5 1 Hma2 1 1 3d* 6 1 Bin7 1 2 Hma3 1 2 1 Trm9 1 Bma6 1 1 Lv2 2 ACKNOWLEDGEMENTS
This study was financed by Gyllenstiernska Krapperupstiftelsen, Ekhagastiftelsen and Spar-bankstiftelsen Skåne. We are grateful for the help of the beekeeper Tage Kimblad, late ass. Prof. Sten Ståhl and others for their knowledge and experi-ence, and for their comments and reflections on our work.
Note scientifique sur la flore des bactéries d’acide lactique chez les abeilles domestiques aux États-Unis – Comparaison avec les abeilles de Suède.
Eine wissenschaftliche Notiz zur Milchsäurebak-terienflora in Honigbienen aus den USA – Ein Vergleich mit Bienen aus Schweden.
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