**6. Discussion**

Adhesion to the host gut epithelial surface is another key to successful colonization. Endosymbionts employ certain proteins (motifs and domains) for this purpose. These are mostly surface proteins associated with the cell wall and employing certain motifs, which act as the signal peptide for attaching to the cell wall. For example, the motif called LPXTG is a sorting peptide. The endopeptidase sortase cleaves it at the site between threonine and glycine residues, and links the peptide covalently to the peptidoglycan of the cell wall [109]. There is up-regulation in the genes encoding this motif and also in the sortase enzymes, indicating attachment of *E. mundtii* to the insect gut wall and biofilm formation. The up-regulation of the WxL domain hints at the increased colonization of the bacteria by their adherence to the gut epithelium. The WxL domain proteins are also crucial for adapting to varying envi-

to overcome different kinds of stresses, such as oxygen starvation, heat or oxidative stress (unpublished).

**Figure 3.** The gut microbiome of *S. littoralis* was dominated by *E. mundtii* and *Clostridia* sp. (A) Overview of the gut structure of fifth-instar larva of *S. littoralis*. (B) Illustration from within the gut space, which harbors major symbionts *E. mundtii*, *Clostridia* sp. and other bacteria. Bacteria adhere to the mucus layer of insect gut epithelium. Unknown interactions occur between microbe-microbe and host-microbe. (C) Illustration of some major expressed pathways *E. mundtii* used for survival in the gut. (i) Secretion of mundticin, an antimicrobial peptide, keeps pathogens at bay and helps the *E. mundtii* dominate the colonization process. (ii) A two-component system involving the accessory gene regulator (*agr*) system, which directs a histidine kinase to phosphorylate the response regulator, leads to the activation of transcription factors required for adaption. (iii) The induction of superoxide dismutase and catalase to manage oxidative stress leads to the conversion of superoxide radicals to water and oxygen. (iv) General or universal stress proteins help

ronmental conditions [110].

88 Metagenomics for Gut Microbes

Lactic acid bacteria are important in the production of fermented foods, such as dairy products. LAB is potential probiotics that provide benefits to human health [116]. Modified LAB could also be used as live vaccines or vaccine delivery systems [117]. It has been shown that the genetically modified *L. lactis* can survive and colonize the digestive tract of humans [118] and gnotobiotic mice [119]. In this chapter, we report the use of GFP to tag *E. mundtii* to monitor the bacteria's survival and activities in the intestinal tract of cotton leafworm, *S. littoralis*.

It has been shown that spatial and temporal distribution of fluorescent *E. mundtii* was observed across all developmental stages (**Figure 4**), as well as in the foregut, midgut and hindgut of *S. littoralis*. Data from the colony forming units (CFUs) show that the midgut houses the most

lepidopteran larvae have an even higher pH (11–12) in their midguts [124, 125]. The hindgut harbors high bacterial diversity and density in several insects, such as cockroaches, crickets

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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Microorganisms that live in the hindgut benefit from the metabolites and ions transported from the malpighian tubules into the hindgut. The hindgut, which stores nitrogenous and food waste, may contain nutrients for insect gut bacteria [123]. The hindgut is involved in water resorption [129]. The microbiota in the ileum of the hindgut of scarab beetles metabolizes plant polysaccharides into components that can be used by the insect [130]. In contrast to the hindgut, the midgut is an unfavorable environment for microorganisms. Many antimicrobial peptides [131] and digestive enzymes (lysozymes) [132] are secreted by the midgut epithelium cells of *D. melanogaster*. The peritrophic matrix secreted by midgut epithelial cells tends to accumulate digestive enzymes and to serve as a barrier to separate food particles, toxins and microorganisms [133]. The high alkaline pH in the gut of lepidopteran insects could kill many microorganisms. However, alkaline conditions favor the dominance of Firmicutesrelated bacteria in the midgut of the beetle *Pachnoda ephippiata* [134]. Both culture-dependent and culture-independent methods have detected the presence of *Enterococcus* in the alkaline

The mechanisms of bacterial colonization in specific regions of the gut are not well understood. The gut of *S. littoralis* does not possess specialized structures called bacteriomes that contain endosymbionts, such as are found in aphids, whiteflies and other insects. How *S. littoralis* houses *E. mundtii* remains unknown, as no compartmentalized structures exist to protect the bacterium; for example, the gut of the pupae has been strongly reduced. Several mosquito species, especially newly emerged adults, that undergo metamorphosis eliminate their gut bacteria [136]. The host organism selects its own microorganisms as it depends on these for growth and development. As an example, see the case of the honeybee, whose bacterial symbionts were

Only a few of the important survival strategies of *E. mundtii* have been mentioned. There are several other pathways that are meant for their adaptation to the differential living conditions inside the gut. We anticipate that further RNA sequencing will help explain some of the other

*E. mundtii* is clearly a successful and a major symbiont in the gut of *S. littoralis.* The method that we have developed here can be used to investigate an indigenous bacterial species within the whole community. With further improvements and modifications, this kind of reporter

The survival strategies of *E. mundtii* in the gut of *S. littoralis* have been unveiled, yet the mechanisms employed by host insect to control the bacterium remain poorly understood. Transcriptomic analyses of the reporter organism indicated already a pattern of relevant

and termites [126–128].

midgut of the gypsy moth larva [135].

unable to survive in the gut of bumble bees [137].

**7. Future prospects**

mechanisms that help the bacteria to survive in the gut.

system may be useful in many other species-specific interaction studies.

**Figure 4.** Photo showing the localization of fluorescent *E. mundtii* in the intestinal tract of *S. littoralis* at different life stages. (A) Bacterial cells accumulate on the peritrophic matrix separated between gut lumen and epithelium of fourth-instar larvae. (B) Bacteria cluster in the gut of fifth-instar larvae. (C) Fluorescent bacteria are visibly colonizing the tissue of pupae, although no gut tissue has been formed. (D) Vertical transmission of symbiont is evident as fluorescent *E. mundtii* survive first-generation and colonize second-generation first-instar progeny. Scale bars: 10–20 μm [14].

abundant bacterial counts, followed by the hindgut and foregut. Interestingly, the fluorescent *E. mundtii* were also detected in the eggs of *S. littoralis* [14], supporting a direct symbiont transmission from one generation to another. Other studies have shown that fluorescent bacteria were transmitted from the gut to the eggs in *Tribolium castaneum* [120]. The symbiotic *E. mundtii* was transmitted to the second-generation progeny, suggesting that the bacteria co-evolve with the insect host (**Figure 4D**). In addition, the fluorescent bacteria were detected in fecal samples of the larvae, indicating they had traveled successfully along the intestinal tract of *S. littoralis* (data not shown). The details of how a bacterial symbiont is transmitted from one generation to the next remain to be clarified. The symbiont that co-evolves with a host has a great chance to secure vertical transmission, for example, a symbiotic relationship exists between the aphid and its endosymbiont *Buchnera aphidicola*. It has been shown that the GFP-tagged *Asaia* strain is vertically transmitted from the mother to the offspring in *Anopheles stephensi* [121]. Bacterial symbionts can be horizontally transferred via "egg smearing," a phenomenon that involves a female stinkbug covering the surface of its eggs with symbiotic bacteria during oviposition. The newly hatched juveniles acquire the symbionts by ingesting the egg case [122].

Several factors, including the pH, redox potential, oxygen availability, and the nutrient and immune systems, can shape the microbial composition of the gut of insects [123]. Furthermore, constant change in gut contents due to molting and metamorphosis can affect the colonization of microorganisms. Many insects have an intestinal pH in the range of 6–8, and some lepidopteran larvae have an even higher pH (11–12) in their midguts [124, 125]. The hindgut harbors high bacterial diversity and density in several insects, such as cockroaches, crickets and termites [126–128].

Microorganisms that live in the hindgut benefit from the metabolites and ions transported from the malpighian tubules into the hindgut. The hindgut, which stores nitrogenous and food waste, may contain nutrients for insect gut bacteria [123]. The hindgut is involved in water resorption [129]. The microbiota in the ileum of the hindgut of scarab beetles metabolizes plant polysaccharides into components that can be used by the insect [130]. In contrast to the hindgut, the midgut is an unfavorable environment for microorganisms. Many antimicrobial peptides [131] and digestive enzymes (lysozymes) [132] are secreted by the midgut epithelium cells of *D. melanogaster*. The peritrophic matrix secreted by midgut epithelial cells tends to accumulate digestive enzymes and to serve as a barrier to separate food particles, toxins and microorganisms [133]. The high alkaline pH in the gut of lepidopteran insects could kill many microorganisms. However, alkaline conditions favor the dominance of Firmicutesrelated bacteria in the midgut of the beetle *Pachnoda ephippiata* [134]. Both culture-dependent and culture-independent methods have detected the presence of *Enterococcus* in the alkaline midgut of the gypsy moth larva [135].

The mechanisms of bacterial colonization in specific regions of the gut are not well understood. The gut of *S. littoralis* does not possess specialized structures called bacteriomes that contain endosymbionts, such as are found in aphids, whiteflies and other insects. How *S. littoralis* houses *E. mundtii* remains unknown, as no compartmentalized structures exist to protect the bacterium; for example, the gut of the pupae has been strongly reduced. Several mosquito species, especially newly emerged adults, that undergo metamorphosis eliminate their gut bacteria [136]. The host organism selects its own microorganisms as it depends on these for growth and development. As an example, see the case of the honeybee, whose bacterial symbionts were unable to survive in the gut of bumble bees [137].

Only a few of the important survival strategies of *E. mundtii* have been mentioned. There are several other pathways that are meant for their adaptation to the differential living conditions inside the gut. We anticipate that further RNA sequencing will help explain some of the other mechanisms that help the bacteria to survive in the gut.

*E. mundtii* is clearly a successful and a major symbiont in the gut of *S. littoralis.* The method that we have developed here can be used to investigate an indigenous bacterial species within the whole community. With further improvements and modifications, this kind of reporter system may be useful in many other species-specific interaction studies.
