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.

Immune specificity is the degree to which a host's immune system discriminates among various pathogens or antigenic variants. Vertebrate immune memory is highly specific due to antibody responses. On the other hand, some invertebrates show immune priming, i.e. improved survival after secondary exposure to a previously encountered pathogen. Until now, specificity of priming has only been demonstrated via the septic infection route or when live pathogens were used for priming. Therefore, we tested for specificity in the oral priming route in the red flour beetle, For priming, we used pathogen-free supernatants derived from three different strains of the entomopathogen,, which express different Cry toxin variants known for their toxicity against this beetle. Subsequent exposure to the infective spores showed that oral priming was specific for two naturally occurring strains, while a third engineered strain did not induce any priming effect. Our data demonstrate that oral immune priming with a non-infectious bacterial agent can be specific, but the priming effect is not universal across all bacterial strains.

14Immune priming, the increased chance to survive a secondary encounter with a pathogen, has been 15 described for many invertebrate species, which lack the classical adaptive immune system of 16vertebrates. Priming can be specific even for closely related bacterial strains, last up to the entire 17 lifespan of an individual, and in some species, it can also be transferred to the offspring and is then 18 called transgenerational immune priming (TGIP). In the red flour beetle Tribolium castaneum, a pest 19 of stored grains, TGIP has even been shown to be transferred paternally after injection of adult 20 beetles with heat-killed Bacillus thuringiensis. 21

Naturally occurring withanolides are secondary metabolites with interesting biological activities in vivo and in vitro. The role of withanolide production in plants is still conjectural though numerous studies strongly indicate a potent feeding deterrent effect against insects and other herbivores. In contrast to the potential medical applications of withanolides, the ecological effects on herbivores have not been studied intensively. In this article, we collect and summarise present studies on herbivore–plant interactions in the light of potential insecticidal functions of withanolides. Moreover, taking the case of the specialist noctuid moth Chloridea ( Heliothis ) subflexa , we not only illustrate the known negative effects of these compounds seem to have on herbivorous insect species but also discuss how C. subflexa larvae might benefit from adaptation to their host plants in the genus Physalis . Further, we describe potential activation and detoxification mechanisms based on the current state of knowledge.