Summary1. Parasitized females in mammals, fish and birds can enhance the immune defence of their offspring by transferring specific antibodies for the embryo. Likewise, social insect mothers transfer immunity despite the fact that invertebrates lack antibodies. 2. Female trans-generational immune priming is consistent with parental investment theory, because mothers invest more into rearing their offspring than fathers. However, when immune priming is not directly linked to parental care, as is often the case in insects that abandon their eggs after oviposition, both sexes might benefit from protecting their offspring. 3. Using the red flour beetle, Tribolium castaneum, we show that after parental exposure to heatkilled bacteria, trans-generational immune priming occurs through fathers as well as mothers. 4. This novel finding challenges the traditional view that males provide only genes to their offspring in species without paternal care, and raises the possibility of a division of tasks with respect to immune protection between parents.

BackgroundPathogens can infect their hosts through different routes. For studying the consequences for host resistance, we here used the entomopathogen Bacillus thuringiensis and the red flour beetle Tribolium castaneum for oral and systemic (i. e. pricking the cuticle) experimental infection. In order to characterize the molecular mechanisms underpinning the two different infection routes, the transcriptomes of beetles of two different T. castaneum populations – one recently collected population (Cro1) and a commonly used laboratory strain (SB) – were analyzed using a next generation RNA sequencing approach.ResultsThe genetically more diverse population Cro1 showed a significantly larger number of differentially expressed genes. While both populations exhibited similar reactions to pricking, their expression patterns in response to oral infection differed remarkably. In particular, the Cro1 population showed a strong response of cuticular proteins and developmental genes, which might indicate an adaptive developmental flexibility that was lost in the SB population presumably as a result of inbreeding. The immune response of SB was primarily based on antimicrobial peptides, while Cro1 relied on responses mediated by phenoloxidase and reactive oxygen species, which may explain the higher resistance of this strain against oral infection.ConclusionsOur data demonstrate that immunological and physiological processes underpinning the two different routes of infection are clearly distinct, and that host populations particularly differ in responses to oral infection. Furthermore, gene expression upon pricking infection entailed a strong signal of wounding, highlighting the importance of pricking controls in future infection studies.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-445) contains supplementary material, which is available to authorized users.

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.

The relationship between robustness and evolvability is a long-standing question in evolution. Heat shock protein 90 (HSP90), a molecular chaperone, has been identified as a potential capacitor for evolution, since it allows for the accumulation and release of cryptic genetic variation, and also for the regulation of novel genetic variation through transposon activity. However, to date, it is unknown whether Hsp90 expression is regulated upon demand (i.e. when the release of cryptic genetic variation is most needed). Here, we show that Hsp90 has reduced transcription under conditions where the mobilization of genetic variation could be advantageous. We designed a situation that indicates a stressful environment but avoids the direct effects of stress, by placing untreated (focal) red flour beetles, Tribolium castaneum, into groups together with wounded conspecifics, and found a consistent reduction in expression of two Hsp90 genes (Hsp83 and Hsp90) in focal beetles. We moreover observed a social transfer of immunity in this non-eusocial insect: there was increased activity of the phenoloxidase enzyme and downregulation of the immune regulator, imd. Our study poses the exciting question of whether evolvability might be regulated through the use of information derived from the social environment.

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.