Paternal knockdown of tRNA(cytosine-5-)-methyltransferase (Dnmt2) increases offspring susceptibility to infection in red flour beetles.

Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

Transgenerational Developmental Effects of Immune Priming in the Red Flour Beetle Tribolium castaneum.

Immune priming, the increased chance to survive a secondary encounter with a pathogen, has been described for many invertebrate species, which lack the classical adaptive immune system of vertebrates. Priming can be specific even for closely related bacterial strains, last up to the entire lifespan of an individual, and in some species, it can also be transferred to the offspring and is then called transgenerational immune priming (TGIP). In the red flour beetle Tribolium castaneum, a pest of stored grains, TGIP has even been shown to be transferred paternally after injection of adult beetles with heat-killed Bacillus thuringiensis. Here we studied whether TGIP in T. castaneum is also transferred to the second filial generation, whether it can also occur after oral and injection priming of larvae and whether it has effects on offspring development. We found that paternal priming with B. thuringiensis does not only protect the first but also the second offspring generation. Also, fitness costs of the immune priming became apparent, when the first filial generation produced fewer offspring. Furthermore, we used two different routes of exposure to prime larvae, either by injecting them with heat-killed bacteria or orally feeding them B. thuringiensis spore culture supernatant. Neither of the parental larval priming methods led to any direct benefits regarding offspring resistance. However, the injections slowed down development of the injected individuals, while oral priming with both a pathogenic and a non-pathogenic strain of B. thuringiensis delayed offspring development. The long-lasting transgenerational nature of immune priming and its impact on offspring development indicate that potentially underlying epigenetic modifications might be stable over several generations. Therefore, this form of phenotypic plasticity might impact pest control and should be considered when using products of bacterial origin against insects.

Dnmt1 has an essential function despite the absence of CpG DNA methylation in the red flour beetle Tribolium castaneum.

Epigenetic mechanisms, such as CpG DNA methylation enable phenotypic plasticity and rapid adaptation to changing environments. CpG DNA methylation is established by DNA methyltransferases (DNMTs), which are well conserved across vertebrates and invertebrates. There are insects with functional DNA methylation despite lacking a complete set of Dnmts. But at least one of the enzymes, DNMT1, appears to be required to maintain an active DNA methylation system. The red flour beetle, Tribolium castaneum, lacks Dnmt3 but possesses Dnmt1 and it has been controversial whether it has a functional DNA methylation system. Using whole genome bisulfite sequencing, we did not find any defined patterns of CpG DNA methylation in embryos. Nevertheless, we found Dnmt1 expressed throughout the entire life cycle of the beetle, with mRNA transcripts significantly more abundant in eggs and ovaries. A maternal knockdown of Dnmt1 caused a developmental arrest in offspring embryos. We show that Dnmt1 plays an essential role in T. castaneum embryos and that its downregulation leads to an early developmental arrest. This function appears to be unrelated to DNA methylation, since we did not find any evidence for this modification. This strongly suggests an alternative role of this protein.