Gut microbiota modulates numerous physiologic processes in insects, such as nutrition, metabolic homeostasis, and pathogen exclusion. Keeping the normal microbiota is an essential element of the gut homeostasis, requiring an extensive network regulatory immune. The molecular mechanisms driving these various effects and the events leading to the establishment of a normal microbiome in insects are still unknown. In this study, the NF-kB (Imd and Toll) signaling pathways in the gut of Galleria mellonella and their roles in the regulation of its gut microbes were assessed. By BLAST search we found Imd and Relish (involved in Imd pathway), and Spatzel and Dif (involved in Toll pathway), in the transcriptome dataset of this insect. To find whether these genes are expressed in gut of G. mellonella, total RNA was extracted from the gut of the fifth instar larvae and cDNA library was synthesized by using RT-PCR and oligodt primer. Transcript levels of the genes were analyzed by RT-qPCR utilizing RPL27 as the reference gene. The results showed that all the genes were expressed in the gut of G. mellonella. However, the expression level of Imd was higher than that of the other genes. To test if these pathways play any role in the regulation of gut bacteria of G. mellonella, the gut bacteria was removed from the fifth Instar larvae by orally administration of antibiotics for one week (germ-free: aposymbiotics) and their guts were pulled and subjected to DNA and RNA extractions.
Successful removal of gut bacteria was confirmed by qPCR using general bacterial 16s rRNA primers. The expression levels of Imd and Toll genes were decreased in the aposymbiotic larvae. Together, these data suggest that Imd and Toll pathways are active in the gut of G. mellonella and they (most particularly Imd pathway) might function in regulation of gut microbes in this insect model.