Experimental evolution of resistance against Bacillus thuringiensis in the insect model host Galleria mellonella results in epigenetic modifications

Abstract

Epigenetic mechanisms have been proposed to translate environmental stimuli into heritable transgenerational phenotypic variations that can significantly influence natural selection. An intriguing example is exposure to pathogens, which imposes selection for host resistance. To test this hypothesis, we used larvae of the greater wax moth Galleria mellonella as model host to experimentally select for resistance to Bacillus thuringiensis (Bt), the most widely used bacterial agent for the biological control of pest insects. To determine whether epigenetic mechanisms contribute to the evolution of resistance against pathogens, we exposed G. mellonella larvae over 30 generations to spores and crystals mix of Bt and compared epigenetic markers in this selected line, exhibiting almost 11-fold enhanced resistance against Bt, to those in a non-selected control population. We found that experimental selection influenced acetylation of specific histones and DNA methylation as well as transcription of genes encoding the enzymatic writers and erasers of these epigenetic mechanisms. Using microarray analysis, we also observed differences in the expression of conserved miRNAs in the resistant and susceptible larvae, resulting in the repression of candidate genes that confer susceptibility to Bt. By combining in silico minimum free energy hybridization with RT-PCR experiments, we identified the functions and biological processes associated with the mRNAs targeted by these miRNAs. Our results suggest that epigenetic mechanisms operating at the pre-transcriptional and post-transcriptional levels contribute to the transgenerational inherited transcriptional reprogramming of stress and immunity-related genes, ultimately providing a mechanism for the evolution of insect resistance to pathogen.

Galleria mellonella experimental model for bat fungal pathogen Pseudogymnoascus destructans and human fungal pathogen Pseudogymnoascus pannorum

“…Galleria mellonella models have become invaluable to study cellular and humoral immune responses against pathogens [31–33], epigenetic influences on the evolution of virulence [32,34,35], and for development and testing antimicrobials [36–39]. It is relevant to add that the complete genomes of P. destructans and G. mellonella have become available recently [40,41].…”

Seasonal phenotype‐specific expression of microRNAs during metamorphosis in the European map butterfly Araschnia levana

“…Here, we investigated whether miRNAs contribute to the regulation of transcriptional reprogramming associated with seasonal polyphenism in A. levana using microarrays containing more than 2,000 conserved insect miRNA sequences. These microarrays have previously been designed to study the differential expression of miRNAs in another lepidopteran species, the greater wax moth Galleria mellonella , during development as well as in response to infection (Mukherjee & Vilcinskas, 2014, Mukherjee et al, 2017). We analyzed RNA samples from last‐instar larvae and pupae originating from A. levana caterpillars exposed to either short‐day (short‐day larvae, short‐day pupae) or long‐day (long‐day larvae, long‐day pupae) conditions to identify differentially expressed miRNAs that may regulate seasonal polyphenism (Vilcinskas & Vogel, 2016).…”

Is there a transgenerational inheritance of host resistance against pathogens? Lessons from the Galleria mellonella-Bacillus thuringiensis interaction model

“…In the follow-up study published in this issue 9 Mukherjee et al extended the selection process up to 30 generations, leading to a selected line exhibiting about 11-fold enhanced resistance against Bt spores-Cry mixtures compared with the susceptible control. Using antibody-based approaches to quantify the global amount of methylated DNA and acetylated histones, they observed that the levels of 5-methylcytosines and acetylated histones were higher in the midgut and the fat body of resistant larvae than in susceptible larvae.…”

“…The understanding of the tolerance mechanisms of Galleria to Bt-Cry biocides has improved significantly thanks to the resistance evolution model in Galleria 20,9 There is now a need to explore in detail the relative importance on the selected resistance phenotype of Cry toxins and the other virulence factors expressed by Bt during infection. 15 Another important issue raised by this work is whether similar phenomena occur in insect pests, which are of high economical importance, such as Plutella xylostella.…”

“…8 It is therefore tempting to speculate that epigenetic mechanisms could play a role in the transmission of protective defenses against pathogens from parents to offspring. In this issue of Virulence, 9 Mukherjee et al investigates this fascinating hypothesis by using the larva of the greater wax moth Galleria mellonella as a model host to study the transmission of the resistance against the entomopathogen Bacillus thuringiensis.…”