Kautz S, Rubin BER, Russell JA, Moreaua CS.. Surveying the microbiome of ants: Comparing 454 pyrosequencing with traditional methods to uncover bacterial diversity. Appl Environ Microbiol 79: 525-534

Field Museum of Natural History, Department of Zoology, 1400 S. Lake Shore Dr., Chicago, IL 60605, USA.
Applied and Environmental Microbiology (Impact Factor: 3.67). 11/2012; 79(2). DOI: 10.1128/AEM.03107-12
Source: PubMed


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.

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Available from: Jacob A Russell, Oct 08, 2015
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    • "This is the first genus-wide survey with broad phylogenetic sampling for Cephalotes and is consistent with previous results from small numbers of species (C. atratus, C. rohweri and C. varians; see Russell et al. 2009; Anderson et al. 2012; Kautz et al. 2013). These communities appear to be relatively simple, averaging just 20 unique 97% OTUs per 1000 sequences (Fig. S2, Table S3, Supporting information). "
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    • "While many microbiome studies examine animal feces, similar materials are often difficult to obtain from insects. Thus, dissected gut tissues, abdominal segments containing the entire digestive tract, or whole insects have been used instead (Jones et al. 2013; Kautz et al. 2013b). Here, we use several ant species as focal organisms to compare the quantity of bacterial DNA obtained from standard methods of nucleic acid extraction. "
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    ABSTRACT: The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.
    MicrobiologyOpen 09/2014; 3(6). DOI:10.1002/mbo3.216 · 2.21 Impact Factor
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    • "But other herbivorous ants engage in symbioses with large masses of gut bacteria, exhibiting novel gut morphologies that have arguably evolved to harbour these microbes (van Borm et al. 2002; Cook & Davidson 2006; Stoll et al. 2007; Bution & Caetano 2008; Russell et al. 2009). These communities have not generally been studied in depth (but see Kautz et al. 2013), but broad explorations of ant-associated microbes hint at trophic level and host phylogeny as correlates of gut community composition. Furthermore, phylogenetic analyses indicate that ant gut associates often come from ant-specific lineages on 16S rRNA phylogenies (Russell et al. 2009; Anderson et al. 2012). "
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    ABSTRACT: Insect guts are often colonized by multi-species microbial communities that play integral roles in nutrition, digestion, and defense. Community composition can differ across host species with increasing dietary and genetic divergence, yet gut microbiota can also vary between conspecific hosts and across an individual's lifespan. Through exploration of such intraspecific variation and its correlates, molecular profiling of microbial communities can generate and test hypotheses on the causes and consequences of symbioses. In this study, we used 454 pyrosequencing and TRFLP to achieve these goals in an herbivorous ant, Cephalotes varians, exploring variation in bacterial communities across colonies, populations, and workers reared on different diets. C. varians bacterial communities were dominated by 16 core species present in over two-thirds of the sampled colonies. Core species comprised multiple genotypes, or strains, and hailed from ant-specific clades containing relatives from other Cephalotes species. Yet three were detected in environmental samples, suggesting the potential for environmental acquisition. In spite of their prevalence and long-standing relationships with Cephalotes ants, the relative abundance and genotypic composition of core species varied across colonies. Diet-induced plasticity is a likely cause, but only pollen-based diets had consistent effects, altering the abundance of two types of bacteria. Additional factors such as host age, genetics, chance, or natural selection must therefore shape natural variation. Future studies on these possibilities and on bacterial contributions to the use of pollen, a widespread food source across Cephalotes, will be important steps in developing C. varians as a model for studying widespread social insect-bacteria symbioses. This article is protected by copyright. All rights reserved.
    Molecular Ecology 11/2013; 23(6). DOI:10.1111/mec.12607 · 6.49 Impact Factor
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