Memory and specificity are hallmarks of the adaptive immune system. Contrary to prior belief, innate immune systems can also provide forms of immune memory, such as immune priming in invertebrates and trained immunity in vertebrates. Immune priming can even be specific but differs remarkably in cellular and molecular functionality from the well-studied adaptive immune system of vertebrates. To date, it is unknown whether and how the level of specificity in immune priming can adapt during evolution in response to natural selection. We tested the evolution of priming specificity in an invertebrate model, the beetle Tribolium castaneum. Using controlled evolution experiments, we selected beetles for either specific or unspecific immune priming toward the bacteria Pseudomonas fluorescens, Lactococcus lactis, and 4 strains of the entomopathogen Bacillus thuringiensis. After 14 generations of host selection, specificity of priming was not universally higher in the lines selected for specificity, but rather depended on the bacterium used for priming and challenge. The insect pathogen B. thuringiensis induced the strongest priming effect. Differences between the evolved populations were mirrored in the transcriptomic response, revealing involvement of immune, metabolic, and transcription-modifying genes. Finally, we demonstrate that the induction strength of a set of differentially expressed immune genes predicts the survival probability of the evolved lines upon infection. We conclude that high specificity of immune priming can evolve rapidly for certain bacteria, most likely due to changes in the regulation of immune genes.

Immune priming has now been demonstrated in a wide range of invertebrate species. Studies testing this phenomenon largely differ in terms of experimental design, host-parasite combinations, agents used for priming, and in particular the degree of demonstrated specificity of the primed response. This review provides an overview of known and putative mechanisms underlying broad-spectrum and specific immune priming in arthropods. We focus on insects and particularly the red flour beetle Tribolium castaneum, where priming has been demonstrated within and across generations. We will also draw attention to the relevance of routes of priming and infection, which can occur septically and orally, with largely differing physiology. For oral priming, an involvement of gut microbiota was demonstrated in mosquitoes and flour beetles. Generally, a primed state could result from long-lasting immune activation or a form of memory that does not entail lingering immune components. Moreover, the primed state could also be of a qualitatively different kind than the challenge response. Finally, we will consider that there should be natural variation in priming capability, and therefore a possibility to study this trait with experimental evolution approaches.

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.

The production of reactive oxygen species (ROS) is a normal consequence of the aerobic cell metabolism. Despite their high and potentially detrimental reactivity with various biomolecules, the endogenous production of ROS is a vital part of physiological, immunological, and molecular processes that contribute to fitness. The role of ROS in host–parasite interactions is frequently defined by their contribution to innate immunity as effectors, promoting parasite death during infections. In vertebrates, ROS and antioxidant system enzymes, such as superoxide dismutase (SOD) are also involved in acquired immune memory, where they are responsible for T-cell signalling, activation, proliferation, and viability. Based on recent findings, ROS are now also assumed to play a role in immune priming, i.e., a form of memory in invertebrates. In this study, the potential involvement of Cu,Zn SODs in immunity of the red flour beetle Tribolium castaneum is described for the first time, applying an approach that combines an in silico gene characterisation with an in vivo immune priming experiment using the Gram-positive entomopathogen Bacillus thuringiensis. We identified an unusually high number of three different transcripts for extracellular SOD and found that priming leads to a fine-tuned modulation of SOD expression, highlighting the potential of physiological co-adaptations for immune phenotypes.