Next-generation sequencing sheds light on intricate regulation of insect gut microbiota


Correspondence: Takema Fukatsu, Fax: +81-29-861-6080; E-mail:


Next-generation sequencing (NGS) technologies are getting cheaper and easier and hence becoming readily accessible for many researchers in biological disciplines including ecology. In this issue of Molecular Ecology, Sudakaran et al. (2012) show how the NGS revolution contributes to our better and more comprehensive understanding of ecological interactions between gut symbiotic microbiota and the host organism. Using the European red firebug Pyrrhocoris apterus as a model system, they demonstrated that the gut microbiota consists of a small number of major bacterial phylotypes plus other minor bacterial associates. The major bacteria are localized in a specific anoxic section of the midgut and quantitatively account for most of the gut microbiota irrespective of host's geographic populations. The specific gut microbiota is established through early nymphal development of the host insect. Interestingly, the host feeding on different food, namely linden seeds, sunflower seeds or wasp larvae, scarcely affected the symbiont composition, suggesting homoeostatic control over the major symbiotic microbiota in the anoxic section of the midgut. Some of the minor components of the gut microbiota, which conventional PCR/cloning/sequencing approaches would have failed to detect, were convincingly shown to be food-derived. These findings rest on the robust basis of high-throughput sequencing data, and some of them could not be practically obtained by conventional molecular techniques, highlighting the significant impact of NGS approaches on ecological aspects of host–symbiont interactions in a nonmodel organism.

Almost all animals, except for rare cases of gutless nonfeeding organisms (Dubilier et al. 2008), are associated with a diverse array of gut microorganisms. In most animals, including humans, the gut microbiota consists of hundreds to thousands of bacterial and other microbial phylotypes. In many cases, the complex microbial community plays substantial biological roles for the host organisms: for example, cellulose digestion and nitrogen fixation in termites (Hongoh 2011), synthesis of nutrients and interactions with immune system in humans (Clemente et al. 2012). However, the large number of microbial components, the myriad interactions between them and often labile composition of the microbial community among species, populations and individuals make ecological studies a great challenge.

Recent studies have revealed several insect groups associated with highly specific gut bacteria. In several stinkbug families, for example, a particular gut section is differentiated into a symbiotic organ and harbours a specific bacterial symbiont therein. The symbiont provides nutrients deficient in host's diet and is essential for its survival and reproduction. The symbiont is strictly transmitted vertically and has cospeciated with the host over evolutionary time (Hosokawa et al. 2006; Kikuchi et al. 2009). Hence, there must be an evolutionary continuum among gut symbiotic associations in animals, ranging from very complex ‘one-host to many-microbes’ associations through moderately complex ‘one-host to several-microbes’ associations to simple ‘one-host to one-microbe’ associations. In the last simple cases, although the associations are biologically interesting and analytically straightforward, some important ecological aspects such as microbe–microbe interactions are totally lacking.

Therefore, for general understanding of ecological interactions between host animals and their gut microbes, model animals with ‘moderately complex’ gut microbiota are necessary. In this context, researchers have investigated the gut bacterial communities of the zebrafish Danio rerio (Kanther & Rawls 2010), the fruit fly Drosophila melanogaster (Broderick & Lemaitre 2012) and the honeybee Apis mellifera (Martinson et al. 2012). In these model systems, (i) the animals are easily maintainable in the laboratory, (ii) the animals are experimentally and genetically tractable, (iii) accumulated genetic and genomic resources are available for the host organisms, and (iv) their gut microbiota consists of relatively a small number (several to tens) of microbial components.

In this issue of Molecular Ecology, Sudakaran et al. (2012) highlighted the European red firebug Pyrrhocoris apterus (Fig. 1 top) as a new system with ‘moderately complex’ gut microbiota. Using the next-generation sequencing (NGS) technique called bacterial tag-encoded 454 FLX amplicon pyrosequencing (bTEFAP) (Sun et al. 2011), large quantities of bacterial 16S rRNA gene sequences were obtained from different tissues (a total of 52 357 reads from structurally differentiated regions of the midgut, namely M1, M2, M3 and M4, dissected from adult insects) (Fig. 1 bottom), different developmental stages (a total of 90 899 reads from eggs, first-fifth instar nymphs and male and female adults), different geographic populations (a total of 83 174 reads from insects collected at five localities in central Europe) and insects fed with different diets (a total of 46 423 reads from insects reared on either linden seeds, sunflower seeds or wasp larvae). Note that obtaining such large quantities of bacterial sequence data for revealing the microbial community in detail would have been impossible solely with conventional approaches. On the basis of these NGS data and conventional PCR, cloning, sequencing and quantitative PCR results, Sudakaran et al. (2012) demonstrated that (i) the gut microbiota of P. apterus consists of six major bacterial phylotypes (Coriobacterium glomerans, Gordonibacter sp., Lactococcus lactis, Clostridium sp., Klebsiella sp. and Rickettsiales sp.) plus other relatively minor bacterial associates, (ii) the major bacteria are preferentially localized in the anoxic M3 section of the midgut, (iii) the major bacteria account for most of the gut microbiota quantitatively, (iv) the major bacteria are consistently dominant in the gut microbiota across five European host populations, (v) the gut microbiota is established during the second or third nymphal instar in the host development, (vi) the host feeding on different food (linden seeds, sunflower seeds or wasp larvae) scarcely affects the composition of the major symbionts, and (vii) some of the minor components of the gut microbiota are food-derived. These results strongly suggest that the major bacteria in the anoxic midgut section are under a homoeostatic control, which might have been shaped through substantial co-evolutionary history between the host insect and the gut microbiota.

Figure 1.

The European red firebug Pyrrhocoris apterus (top) and the dissected midgut from the insect (bottom). The midgut consists of structurally distinct four regions (M1, M2, M3 and M4), of which M3 region is anoxic and specialized for harbouring six major bacterial phylotypes (photos courtesy of Martin Kaltenpoth and Sailendharan Sudakaran, respectively).

While P. apterus is easily maintainable in the laboratory and was used for ecological, biochemical, physiological and endocrinological studies (Socha 1993), researchers have recently paid little attention to this insect. However, application of NGS technologies in combination with experimental approaches to its biological aspects by Sudakaran et al. (2012) revived the red firebug as a promising model system with ‘moderately complex’ gut microbiota. Distinct from zebrafish, fruit fly and honeybee, wherein neither specialized symbiotic organs for nor apparent beneficial roles of their gut microbiota are known, the red firebug has a specialized midgut section for symbiosis, and the gut microbiota significantly contributes to the host fitness, which provides a unique opportunity to gain insights into the gut microbial ecology in the context of mutualism and co-evolution. The advent of NGS technologies means that the relative paucity of genetic and genomic background for this nonmodel insect matters less than before. In addition to the gut microbial diversity promptly unveiled in this study, not only the draft genome of the insect for candidate gene fishing but also the comprehensive transcriptome of the symbiotic organ for identifying symbiosis-related host genes is to be obtained easily. RNA interference generally works efficiently in diverse heteropteran bugs (Futahashi et al. 2011), and some of the major gut bacteria of P. apterus are culturable and thus possibly manipulatable genetically (Haas & König 1987; Kaltenpoth et al. 2009). These advantages will enhance the utility of the red firebug as a new model system.

In nature, organisms are interacting with their surrounding physical environments as well as other organisms. Thus, each of the organisms is regarded as a component of the ecosystem. Considering the diverse microbial community ubiquitously associated with the organisms, each of the organisms can also be regarded as constituting an ecosystem. NGS technologies have revolutionized molecular ecology by blurring the boundaries between ecology, molecular biology, genetics and genomics (Tautz et al. 2010). The ecological interactions in the compact internal ecosystem will be one of the priority research areas greatly boosted by the NGS revolution in coming years.

T.F., group leader of National Institute of Advanced Industrial Science and Technology (AIST) and also professor of the University of Tsukuba, Japan, is working on symbiosis and evolution of diverse insects and associated microorganisms.