Effects of hypomethylation on immune system of Galleria mellonella L. (Lepidoptera: Pyralidae)

Galleria mellonella is becoming increasingly used as a model organism for studying human pathogen infection disease. Therefore, all factors affecting the level of immune response of G. mellonella should be evaluated. One of these factors is DNA methylation, among the most important epigenetic modifications. Firstly, we investigated the presence of CpG dinucleotides and their methylation status of eggs, last instars, pupae, and adults using HpaII, MspI, and ScrFI methylation-sensitive restriction endonucleases. The gel analysis of the restriction pattern of HpaII, MspI, ScrFI, and MspI+ScrFI on G. mellonella genomic DNA showed that all enzymes produced a wide range of smears. The digestion of DNA samples of egg, pupa, and adult resulted in a smear at higher molecular weight for MspI+ScrFI, and these differences were statistically significant. These results confirm the presence of CpG dinucleotides and methylation at the internal and outer cytosines in different developmental stages of G. mellonella. Presence of global DNA methylation, which was by 3%-5% was investigated in various developmental stages of G. mellonella. Then, different levels of DNA methylation in the last instars of G. mellonella were created with the hypomethylation agent, zebularine (0.25-32 mg/mL), and immunity responses were examined. The most effective doses of zebularine, to create a global hypomethylation status in G. mellonella larvae, were 0.25 and 1 mg/mL. The humoral/cellular immune response of G. mellonella larvae became weak at high hypomethylation. These results present the level of DNA methylation during all developmental stages of G. mellonella and how zebularine treatment affects physiological processes.