Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity

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.

Cephaloticoccus gen. nov., a new genus of ‘Verrucomicrobia’ containing two novel species isolated from Cephalotes ant guts

“…By using PCR amplification and DNA sequencing techniques, previous studies have shown that members of the 'Verrucomicrobia' constitute part of a stable, autochthonous community of symbionts maintained in the guts of Cephalotes 'turtle' ants (Anderson et al, 2012;Kautz et al, 2013;Russell et al, 2009). These symbionts are protected by a proventricular filter (Lanan et al, 2016), remain stable in laboratory-reared colonies and likely coevolved with their hosts (Sanders et al, 2014).…”

The Core Gut Microbiome of the American Cockroach, Periplaneta americana, Is Stable and Resilient to Dietary Shifts

“…Heteropteran stinkbugs have developed highly species-specific associations with individual gut symbionts that are either maternally transmitted or acquired early in development (44)(45)(46). Other insects have established stable relationships with simple gut communities, including honey and bumble bees (47,48) and ants (49). While the mechanisms by which bees regulate their gut microbiome have not been established, the Sonoran Desert turtle ant, Cephalotes rohweri, was recently found to have a mechanical filter that blocks any bacteria or particles larger than 0.2 m from entering into the midgut and hindgut after an initial gut microbiome is established (50).…”

Quantifying the relative roles of selective and neutral processes in defining eukaryotic microbial communities

“…Massively parallel next-generation sequencing technologies have allowed significant leaps forward in the power with which we may sample microbial communities. These methods do not have the constraint of having to culture species to analyse them, but instead evaluate DNA directly isolated from the substrate of interest (Su et al, 2012;Kautz et al, 2013;Bokulich et al, 2014;Taylor et al, 2014). So far, studies utilising these techniques are providing increasing evidence that many microbial communities are not homogenised through space, but display significant structure just like many plant and animal communities do (Martiny et al, 2006;Hanson et al, 2012;Nemergut et al, 2013).…”