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, 79 (2), 525-34

Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing With Traditional Methods to Uncover Bacterial Diversity

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Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing With Traditional Methods to Uncover Bacterial Diversity

Stefanie Kautz et al. Appl Environ Microbiol.

Abstract

We are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle ant Cephalotes varians were Rhizobiales, Burkholderiales, Opitutales, Xanthomonadales, and Campylobacterales, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history of Cephalotes ants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with a Spiroplasma sp. (order Entomoplasmatales), a potential ant pathogen. Our work shows that the microbiome associated with Cephalotes varians is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity.

Figures

Fig 1
Fig 1
Phylogenetic tree of bacteria associated with Cephalotes varians turtle ants and their GenBank relatives. Shown is a maximum likelihood phylogeny of the most prevalent OTUs as generated by 454 sequencing and by cloning. For each method, we clustered reads (454 sequencing) or sequences (cloning) at 97% similarity and selected the top clusters that accounted for 90% of reads or sequences, respectively. The branch color and inner circle refer to the source from which the bacteria were isolated, the middle circle refers to the bacterial order, and the outer circle refers to the method used to acquire the respective sequence. Colony CSM1323 was excluded from these analyses due to an infection with Entomoplasmatales bacteria. Please note the misplacement of Campylobacterales, which should group with other Proteobacteria.
Fig 2
Fig 2
Comparison of bacterial communities in Cephalotes varians ants detected through 454 pyrosequencing (upper rows) and cloning (lower rows). Samples were prepared from dissected ant body parts or entire workers of Cephalotes varians. Sample names are given above pies and sample sizes below pies. The relative abundance of reads at the taxonomic level of bacterial orders is displayed. Orders that accounted for less than 1% in a sample are summarized in a category termed “other.” “Unclass. gamma” refers to an unclassified gammaproteobacterium that clustered with the Pseudomonadales bacteria.
Fig 3
Fig 3
PCoA analysis of bacterial communities from samples that were subjected to 454 sequencing and cloning. Positions of the bacterial communities for each species along the two first principal coordinate axes are illustrated, along with the percentage of variation explained by each axis. Results based on 454 sequencing are displayed with no lines, while the same symbols with black outlines illustrate results obtained through cloning and Sanger sequencing. Note the distinct clustering of samples prepared from colony CSM1323 (gray symbols), which was infected by bacteria of the order Entomoplasmatales (Spiroplasma sp.), and also the clustering of the samples prepared from the hindguts of colony CSM1280 (1280 hind-2 and 1280 hind-3), as well as of the midguts of colony CSM1280 (1280 mid-2 and 1280 mid-3). “Hind” refers to the hindgut, “mid” refers to the midgut, and “wk” refers to samples prepared from whole worker extractions. Results are based on weighted UniFrac distances.

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