Paternal trans-generational immune priming, whereby fathers provide immune protection to offspring, has been demonstrated in the red flour beetle Tribolium castaneum exposed to the insect pathogen Bacillus thuringiensis. It is currently unclear how such protection is transferred, as in contrast to mothers, fathers do not directly provide offspring with a large amount of substances. In addition to sperm, male flour beetles transfer seminal fluids in a spermatophore to females during copulation. Depending on whether paternal trans-generational immune priming is mediated by sperm or seminal fluids, it is expected to either affect only the genetic offspring of a male, or also their step offspring that are sired by another male. We therefore conducted a double-mating experiment and found that only the genetic offspring of an immune primed male show enhanced survival upon bacterial challenge, while phenoloxidase activity, an important insect immune trait, and the expression of the immune receptor PGRP were increased in all offspring. This indicates that information leading to enhanced survival upon pathogen exposure is transferred via sperm, and thus potentially constitutes an epigenetic effect, whereas substances transferred with the seminal fluid could have an additional influence on offspring immune traits and immunological alertness.

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.

24Epigenetic mechanisms, such as CpG DNA methylation enable phenotypic plasticity and rapid 25 adaptation to changing environments. CpG DNA methylation is established by DNA methyltransferases 26 (DNMTs), which are well conserved across vertebrates and invertebrates. There are insects with 27 functional DNA methylation despite lacking a complete set of Dnmts. But at least one of the enzymes, 28 DNMT1, appears to be required to maintain an active DNA methylation system. The red flour beetle, 29Tribolium castaneum, lacks Dnmt3 but possesses Dnmt1 and it has been controversial whether it has 30 a functional DNA methylation system. 31Using whole genome bisulfite sequencing, we did not find any defined patterns of CpG DNA 32 methylation in embryos. Nevertheless, we found Dnmt1 expressed throughout the entire life cycle of 33 the beetle, with mRNA transcripts significantly more abundant in eggs and ovaries. A maternal 34 knockdown of Dnmt1 caused a developmental arrest in offspring embryos. 35We show that Dnmt1 plays an essential role in T. castaneum embryos and that its downregulation 36 leads to an early developmental arrest. This function appears to be unrelated to DNA methylation, 37 since we did not find any evidence for this modification. This strongly suggests an alternative role of 38 this protein. 39 40 10,11 . On the other hand, it also has been suggested that DNA methylation can have a more general, 55 basic role in insects and it might function in more ubiquitously essential processes, e.g. embryonic 56 development, genomic imprinting and alternative splicing 6,12-15 . 57 Insect methylation patterns can be highly diverse 15,16 . In cockroaches and some lepidopterans, DNA 58 methylation can reach levels similar to those observed in vertebrates and plants 15,17 . This is also true 59 for some regions of the genome of the stick insect, but here in addition to gene bodies also repetitive 60 elements are strongly methylated 18 . In contrast, holometabolous insects possess sparser DNA 61 methylation than vertebrates 15,16,19 . Most of these insects also differ from mammals in the location of 62 their methylation marks, as the majority of CpG methylation occurs within gene bodies 6,20,21 . The 63 placement of methyl groups at these mostly intragenic loci could hint at its possible role in alternative 64 splicing 22 . 65

Trans-generational immune priming (TGIP) describes the transfer of immune stimulation to the next generation. As stress and immunity are closely connected, we here address the question whether trans-generational effects on immunity and resistance can also be elicited by a nonpathogen stress treatment of parents. General stressors have been shown to induce immunity to pathogens within individuals. However, to our knowledge, it is as of yet unknown whether stress can also induce trans-generational effects on immunity and resistance. We exposed a parental generation (mothers, fathers, or both parents) of the red flour beetle Tribolium castaneum, a species where TGIP has been previously been demonstrated, to either a brief heat or cold shock and examined offspring survival after bacterial infection with the entomopathogen Bacillus thuringiensis. We also studied phenoloxidase activity, a key enzyme of the insect innate immune system that has previously been demonstrated to be up-regulated upon TGIP. We quantified parental fecundity and offspring developmental time to evaluate whether trans-generational priming might have costs. Offspring resistance was found to be significantly increased when both parents received a cold shock. Offspring phenoloxidase activity was also higher when mothers or both parents were cold-shocked. By contrast, parental heat shock reduced offspring phenoloxidase activity. Moreover, parental cold or heat shock delayed offspring development. In sum, we conclude that trans-generational priming for resistance could not only be elicited by pathogens or pathogen-derived components, but also by more general cues that are indicative of a stressful environment. The interaction between stress responses and the immune system might play an important role also for trans-generational effects.