In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer ( Melolontha hippocastani )

The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer ( ), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining stable isotope probing (SIP) with C cellulose and N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In N urea-fed insects, the genera and were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, C cellulose- and N urea labeled bacteria. The incorporation of N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of , this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.