**7. Future prospects**

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.

survive first-generation and colonize second-generation first-instar progeny. Scale bars: 10–20 μm [14].

90 Metagenomics for Gut Microbes

**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*

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

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 enzymes allowing the microbes to adapt to the harsh conditions of the insect gut. The studies can be extended to the very special conditions in the pupae where fluorescent bacteria could be observed. Thus, 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 different developmental stages of the insect, as well as to study the impact of food-ingested plant toxins. 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. In conjunction with CRISPR/ CAS9-created specific knock downs of defined metabolic capacities of the insect, detailed questions concerning the molecular dialog between the insect host and the microbial consortium can be answered.

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