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

[This corrects the article on p. 1970 in vol. 8, PMID: 29075241.].

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 (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N 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 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides 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, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N 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 M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

Identification of metabolically active bacteria in the gut of the generalist Spodoptera littoralis via DNA stable isotope probing using 13C-glucose.

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction(1), boosting the immune response(2), pheromone production(3), as well as nutrition, including the synthesis of essential amino acids(4,) among others.     Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms. To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing (13)C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA(5). The incorporation of (13)C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ((12)C) one. In the end, the (13)C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the (12)C-unlabeled similar one(6). Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species. Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, (13)C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.