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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    • "Nowadays, next generation sequencing (NGS) offers the opportunity to determine the compositions of bacterial communities in great detail. This has proven to be an effective tool to identify some rare groups that were not easily detected with older methods (Kautz et al. 2013). "
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    ABSTRACT: The springtail Folsomia candida is an important model organism for soil ecology, ecotoxicology and ecogenomics. The decomposer activities of soil invertebrates like Folsomia depend on their relationship with microbial communities including gut symbionts. In this paper, we apply high-throughput sequencing to provide a detailed characterization of the bacterial community associated with parthenogenetic F. candida. First, we evaluated a method to suppress the amplification of DNA from the endosymbiont Wolbachia, to prevent it from interfering with the identification of less abundant Operational Taxonomic Units (OTUs). The suppression treatment applied was effective against Wolbachia and did not interfere with the detection of the most abundant OTUs (59 OTUs, contributing over 87% of the reads). However, this method did affect the inferred community composition. Significant differences were subsequently observed in the composition of bacterial communities associated with two different strains of F. candida. A total of 832 OTUs were found, of which 45% were only present in one strain and 17% only in the other. Among the 20 most abundant OTUs 16 were shared between strains. Denaturing Gradient Gel Electrophoresis (DGGE) and clone libraries, although unable to capture the full diversity of the bacterial community, provided results that supported the NGS data.
    FEMS Microbiology Ecology 10/2015; DOI:10.1093/femsec/fiv128 · 3.57 Impact Factor
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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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