CpG Methylation of polynucleotides is one of the most studied epigenetic mechanisms that enable organisms to change their phenotype without altering the genotype. More recently CpG methylation occurring on small noncoding RNAs, especially of certain transfer RNAs has come into focus. This modification is established by the most conserved member of the DNA methyltransferase family, Dnmt2. Dnmt2 has been indicated in transferring paternal phenotypes to offspring in mice and its absence leads to an increased sensitivity to a variety of stressors in Drosophila melanogaster. We therefore hypothesise that it also might play a role in paternal transgenerational immune priming, which can be observed in the red flour beetle Tribolium castaneum, where exposure to a non-lethal dose of bacteria in fathers protects their offspring against a potentially lethal dose of the same pathogen. We were able to show that Dnmt2 is expressed throughout the entire life cycle of the beetle and that expression is significantly higher in the testes. We then combined a knockdown of Dnmt2 via pupal RNAi with a bacterial priming treatment in the eclosed adults and monitored the effects on their offspring. We used the entomopathogenic bacterium Bacillus thuringiensis for priming and challenge injections in adult fathers and offspring respectively. In the paternal generation, neither viability nor fertility were affected by either RNAi or priming treatment compared to the respective controls. Dnmt2 RNAi treatment led to a significant downregulation and slowed down the development in the offspring larvae. Although, we could not observe a significant paternal priming effect independent of treatment, paternal knockdown led to increased mortality after bacterial injection with B. thuringiensis. This demonstrates again an increased stress sensitivity caused by a lack of Dnmt2. Furthermore, to the best of our knowledge this is the first instance where this effect was observed in the offspring generation. In conclusion, our results highlight the importance of Dnmt2 and show the need to further investigate this enzyme and its function in tRNA methylation and paternal non-genetic inheritance.

Paternal knockdown of Dnmt2 increases offspring susceptibility to bacterial infection