Low-diversity bacterial community in the gut of the fruitfly Drosophila melanogaster

Abstract

The bacteria in the fruitfly Drosophila melanogaster of different life stages was quantified by 454 pyrosequencing of 16S rRNA gene amplicons. The sequence reads were dominated by 5 operational taxonomic units (OTUs) at ≤ 97% sequence identity that could be assigned to Acetobacter pomorum, A. tropicalis, Lactobacillus brevis, L. fructivorans and L. plantarum. The saturated rarefaction curves and species richness indices indicated that the sampling (85,000-159,000 reads per sample) was comprehensive. Parallel diagnostic PCR assays revealed only minor variation in the complement of the five bacterial species across individual insects and three D. melanogaster strains. Other gut-associated bacteria included 6 OTUs with low %ID to previously reported sequences, raising the possibility that they represent novel taxa within the genera Acetobacter and Lactobacillus. A developmental change in the most abundant species, from L. fructivorans in young adults to A. pomorum in aged adults was identified; changes in gut oxygen tension or immune system function might account for this effect. Host immune responses and disturbance may also contribute to the low bacterial diversity in the Drosophila gut habitat.

Fig. 1

Rarefaction curves of OTUs clustered at different %ID across life stages of D. melanogaster Canton-S. (A) 93%, (B) 95%, (C) 97%.

Fig. 2. Identification of candidate novel taxa from pairwise comparisons of %ID among 16S rRNA gene sequences

A and B. Minimum pairwise %ID of the V2 region of the 16S rRNA genes for publicly available sequences of each species of Acetobacter and Lactobacillus respectively. The values for the species detected in this study are highlighted as ◆ A. pomorum, △ A. tropicalis in (A), and + L. brevis, ◇ L. fructivorans and ▲ L. plantarum in (B). The equations and r² values for regression of minimum %ID (y) on number of valid sequences (x) are shown. [For (A), the asterisk refers to the regression equation and r² excluding outlier species (minimum %ID < 70%).] C and D. Relationship between minimum pairwise %ID of the V2 region of the 16S rRNA genes and corresponding (near-)full 16S rRNA gene sequences for the publicly available Acetobacter and Lactobacillus species used in (A) and (B) respectively. Regression equations for %ID V2 region of the 16S rRNA gene (y) on %ID near-full 16S rRNA gene (x) are shown. E and F. Comparison of the %ID of sequences in Table 3 of this study with known species of Acetobacter and Lactobacillus respectively. Species detected in this study are highlighted as in (A) and (B), and clusters are indicated by letter notations (a–r) used in Table 3. For clarity, only clusters with %ID < minimum %ID for the species represented by the top hit are shown.

Fig. 3. QRT-PCR of relative abundance of 16S rRNA gene sequence in technical replicates of the pyrosequencing experiment (November 2009) and independent biological samples (June 2010)

A. Lactobacillus fructivorans: A. pomorum in adults of different ages (*3–5 weeks for November 2009 samples; 4–5 weeks for June 2010 samples); 2- to 3-week-old flies were not obtained for the pyrosequencing experiment and its technical replicates in November 2009. B. Lactobacillus fructivorans: L. plantarum in third-instar larvae. C. Lactobacillus fructivorans: A. tropicalis in pupae. Ten replicates per sample.