**The Microbiome of** *Spodoptera littoralis***: Development, Control and Adaptation to the Insect Host Control and Adaptation to the Insect Host**

**The Microbiome of** *Spodoptera littoralis***: Development,** 

DOI: 10.5772/intechopen.72180

Tilottama Mazumdar, Beng-Soon Teh and Wilhelm Boland Wilhelm Boland Additional information is available at the end of the chapter

Tilottama Mazumdar, Beng-Soon Teh and

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72180

#### **Abstract**

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pone.0020447

76 Metagenomics for Gut Microbes

The symbiotic microbial consortium in the gut of *Spodoptera littoralis* shows dramatic, but reproducible changes in line with the development of the insect from the egg via six larval instars to the pupa. Since the food is kept constant during development, factors from the insect host and certain microbial symbionts are assumed to control the composition of the microbiome. A GFP-tagged *Enterococcus mundtii*, one of the major players of the consortium, easily integrates into the microbiome and can be monitored in all gut segments at all developmental stages. The reporter organism can be recovered from the gut using a preparative flow cytometry allowing subsequent RNA extraction for transcriptomic analyses. The transcriptomic profile from the fluorescent *Enterococcus* cells provides information on the adaptation of the reporter organism to the local gut conditions. The concept of using a fluorescent reporter organism that can be recovered at any time from any area of the intestinal tract will allow a holistic analysis of adaptation strategies used by the microbes to adapt to the insect gut. In combination with the analysis of transcript patterns from the gut membranes, a first insight into the molecular interaction between the insect host and the microbiome can be expected.

**Keywords:** *Enterococcus mundtii*, *Spodoptera littoralis*, gut microbiome, transcriptomics, flow cytometry

## **1. Introduction**

The development of a gut in multicellular organisms is an evolutionary achievement of the highest order. The gut allows the host to exploit the metabolic and catabolic abilities of a multitude of microbial inhabitants to degrade and digest recalcitrant and complex organic matter.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

The symbionts may also be involved in the detoxification of poisonous metabolites in food [1, 2]. The membranes of the intestinum carefully separate the bacterial symbionts from the host organism and prevent infection by invasive and deleterious members of the microbiome. Accordingly, the gut membrane is a complex structure that allows the exchange of nutrients with both high- and low-molecular weight (signaling) compounds and, on the other, blocks the entry of microbes and many of their macromolecular components [3]. The flux of nutrients and even more complex metabolites across the membrane is controlled by transport proteins expressed in the gut membrane [4, 5]. The microbiome also defends against parasites or pathogens [6–8]. The diverse functions provided by the microbial partners are vital for the insect's survival, especially in adverse ecological niches.

**2. Fluorescent reporters and their applications for** *in vivo* **imaging in** 

The green fluorescent protein (GFP) isolated from the jellyfish, *Aequorea victoria*, is widely used as a reporter for studying gene expression [16], and the localization and structure of living cells [17]. The GFP has a major excitation peak at about 395 nm and an emission peak at about 508 nm. The GFP contains 238 amino acids with a molecular weight of 26.9 kDa. It emits green fluorescence when exposed to light in the blue to ultraviolet range [18]. The GFP requires only oxygen as a cofactor for chromophore formation, which gives it an advantage over other reporter proteins [19]. It is sensitive and non-toxic, and does not affect cell growth [20, 21]. In addition, the GFP is stable at temperatures below 65°C and pH 6–11 [22]. Since the GFP was discovered, many mutants have been developed with modification in spectral and folding properties, or enhanced fluorescence intensity [23–26]. The choice of a GFP variant depends on several factors, such as pH, environmental temperature, toxicity, multimerization and photostability [26]. The first *gfp* gene was cloned in 1992 [27], and 2 years later, the gene was successfully expressed in both eukaryotes and prokaryotes [20]. Apart from GFP, many variants of red fluorescent proteins, such as mCherry and tdTomato have been developed based on DsRed originally isolated from *Discosoma* sp. [28]. Since then, over 40 coral fluorescent proteins with different colors, from cyan to chromo-red, have been described [29].

The Microbiome of *Spodoptera littoralis*: Development, Control and Adaptation to the Insect Host

http://dx.doi.org/10.5772/intechopen.72180

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The reporter proteins provide important tools with which to monitor gene expression from within the cells in real time and in the *in vivo* environment, such as the gastrointestinal tract. For a gene to be selected as a reporter, it must be able to easily detect signals secreted by the expressed reporter gene in the cells [30]. The *lux* gene derived from bacteria, and luciferase from the firefly and click beetle (*luc*), are two other common reporter genes used in bioluminescence imaging. Fluorescence imaging is commonly associated with the use of green and red fluorescence proteins [31, 32]. Rats and mice are popular model organisms which study the proliferation and colonization of lactic acid bacteria (LAB) [33, 34]. LAB has been tagged with green fluorescent protein (GFP) and mCherry to study their colonization of the intestinal

LAB is widely used as probiotics due to the benefits they bring to human and animal health by balancing the gut microbiome and by eliminating pathogenic microorganisms through the production of antimicrobial peptides [40, 41]. Due to the importance of LAB in many applications, it is essential to study how they survive and colonize by monitoring their metabolic activities *in vivo* through the development of fluorescent reporter microorganisms. It is impor-

Plasmids are present in most of the members of LAB, including *Enterococcus*, *Lactobacillus*, *Lactococcus*, *Bifidobacterium*, *Streptococcus*, *Pediococcus, Leuconostoc*, etc. Plasmids found in LAB

tant that the reporter gene in the fluorescent bacteria is stably expressed [42].

tract of chickens, mice and zebrafish [35–39].

**2.1. Construction of a GFP fluorescent system for** *E. mundtii*

**microbiomes**

Although almost all organisms rely on core microbiomes [9], in many cases the gut community changes according to the insect's developmental stage. In early instars of *Spodoptera littoralis,* several *Enterococcus* spp. dominate, whereas in late instars, Clostridia contribute significantly (ca. 50%) to the microbial population [10]. A core community, consisting of Enterococci, Lactobacilli and Clostridia was revealed in the insect larvae. These bacteria were always present in the digestive tract at a relatively high frequency; although developmental stage and diet have a great impact on shaping bacterial communities, clearly the insect gut selects for particular bacterial phylotypes. Enterococci are also prominent in the gut of insects such as *Drosophila*, ground beetles and desert locusts [11, 12]. The strong dependence of the gut community on the developmental stage of the insect host suggests that unknown low- and high-molecular weight factors control symbiotic interactions among the partners. For example, in *Drosophila melanogaster*, the immune system not only plays a central role in preventing pathogen infection, but also controls the resident bacterial population. The intestinal homeobox gene *Caudal* regulates the resident gut microbial community by repressing the antimicrobial peptide genes that are dependent on the nuclear factor kappa B. Silencing the *Caudal* gene by RNAi resulted in the overexpression of antimicrobial peptides, which in turn reduced the microbial population in the gut [13].

To monitor such developmentally controlled changes in the microbiome of *S. littoralis,* a fluorescent member of the gut symbionts—in particular, the dominant *Enterococcus mundtii* appeared to be an ideal reporting organism. A GFP-labeled *E. mundtii* [14] would easily integrate into the gut community and survive adverse conditions embedded in the community of enterococci, bacteria which are largely resistant to environmental stresses, such as antibiotic exposure, disinfection, desiccation and starvation [15]. As the transgenic *E. mundtii* are fluorescent, their presence can be monitored in all gut areas of the larvae and at developmental stages such as the pupa and the adult. Moreover, the reporter organisms can be easily recovered from the gut and used for transcriptomic analyses. By comparing transcriptomes from adjacent gut tissue and from the microbes, a "dialog" between the insect host and the symbiotic bacteria could be unraveled. This concept is generally applicable and can be used to holistically analyze host microbial interactions. The protocol of the approach based on the use of a fluorescent reporter organism—for example, using GFP-tagged *E. mundtii*—is described in this chapter.
