Distinctive gut microbiota of honey bees assessed using deep sampling from individual worker bees

Abstract

Surveys of 16S rDNA sequences from the honey bee, Apis mellifera, have revealed the presence of eight distinctive bacterial phylotypes in intestinal tracts of adult worker bees. Because previous studies have been limited to relatively few sequences from samples pooled from multiple hosts, the extent of variation in this microbiota among individuals within and between colonies and locations has been unclear. We surveyed the gut microbiota of 40 individual workers from two sites, Arizona and Maryland USA, sampling four colonies per site. Universal primers were used to amplify regions of 16S ribosomal RNA genes, and amplicons were sequenced using 454 pyrotag methods, enabling analysis of about 330,000 bacterial reads. Over 99% of these sequences belonged to clusters for which the first blastn hits in GenBank were members of the known bee phylotypes. Four phylotypes, one within Gammaproteobacteria (corresponding to "Candidatus Gilliamella apicola") one within Betaproteobacteria ("Candidatus Snodgrassella alvi"), and two within Lactobacillus, were present in every bee, though their frequencies varied. The same typical bacterial phylotypes were present in all colonies and at both sites. Community profiles differed significantly among colonies and between sites, mostly due to the presence in some Arizona colonies of two species of Enterobacteriaceae not retrieved previously from bees. Analysis of Sanger sequences of rRNA of the Snodgrassella and Gilliamella phylotypes revealed that single bees contain numerous distinct strains of each phylotype. Strains showed some differentiation between localities, especially for the Snodgrassella phylotype.

Figure 1. Bar graph showing relative abundances of bacterial phylotypes within the guts of individual honey bees from colonies in Arizona and Maryland.

Figure 2. Nonmetric multidimensional scaling ordination of gut bacterial assemblages from individual honey bees (N = 39).

(2D solution, 500 iterations, final stress = 17.56, final instability = 0.0008, Monte Carlo P = 0.0099, 0.0396). Axis 1 explains 65.5% of the variation, and Axis 2 explains 23.3% of the variation in the gut communities. Individuals from the same colony are represented by the same shape and color.

Figure 3. A.

Phylogenetic tree based on amplified and cloned sequences of 16S rRNA genes for the Snodgrassella phylotype from individual bees collected at the Arizona and Maryland sites, and with several previously published sequences from bee-associated bacteria corresponding to the Snodgrassella phylotype. Singletons were removed before analysis so clusters at branch tips represent sequences that are identical or differ only in single sequences. B. Sets of identical sequences within the 1250 bp 16S rRNA sequences for Gilliamella and Snodgrassella. Each set of identical sequences is represented as a single column of symbols corresponding to colony and site of the sample. Identical sequences were only found for samples from the same site and were usually clustered within an individual bee.