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Experimental infection systems are important for studying antagonistic interactions and coevolution between hosts and their pathogens. The red flour beetle Tribolium castaneum and the spore-forming bacterial insect pathogen Bacillus thuringiensis (Bt) are widely used and tractable model organisms. However, they have not been employed yet as an efficient experimental system to study host-pathogen interactions. We used a high throughput oral infection protocol to infect T. castaneum insects with coleopteran specific B. thuringiensis bv. tenebrionis (Btt) bacteria. We found that larval mortality depends on the dietary spore concentration and on the duration of exposure to the spores. Furthermore, differential susceptibility of larvae from different T. castaneum populations indicates that the host genetic background influences infection success. The recovery of high numbers of infectious spores from the cadavers indicates successful replication of bacteria in the host and suggests that Btt could establish infectious cycles in T. castaneum in nature. We were able to transfer plasmids from Btt to a non-pathogenic but genetically well-characterised Bt strain, which was thereafter able to successfully infect T. castaneum, suggesting that factors residing on the plasmids are important for the virulence of Btt. The availability of a genetically accessible strain will provide an ideal model for more in-depth analyses of pathogenicity factors during oral infections. Combined with the availability of the full genome sequence of T. castaneum, this system will enable analyses of host responses during infection, as well as addressing basic questions concerning host-parasite coevolution.

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.

Immune priming is defined as enhanced protection upon secondary exposure to a pathogen. Such enhanced resistance after prior exposure has been demonstrated for a number of insect species including the red flour beetle, Tribolium castaneum. In testing this phenomenon, the majority of studies have focused on introducing the pathogen into the insect's hemocoel via septic wounding through the cuticle. Although such septic injury can occur in nature, many pathogens enter their hosts via the oral route, i.e. by ingestion. Bacillus thuringiensis bacteria are well-known insect pathogens that infect their host orally. We found that T. castaneum larvae showed increased survival after oral exposure to B. thuringiensis, when they had been orally primed with filter-sterilized media in which spores of B. thuringiensis had been raised. Such priming was achieved only with a naturally pathogenic strain of B. thuringiensis and a strain that was made pathogenic by transfer of plasmids. Moreover, primed larvae were smaller in size 24 h after priming and had a longer developmental time, indicating that investment in such a response comes at a cost. However, the increased survival in primed larvae was not caused by larval size differences upon challenge.

BackgroundThe phenomenon of immune priming, i.e. enhanced protection following a secondary exposure to a pathogen, has now been demonstrated in a wide range of invertebrate species. Despite accumulating phenotypic evidence, knowledge of its mechanistic underpinnings is currently very limited. Here we used the system of the red flour beetle, Tribolium castaneum and the insect pathogen Bacillus thuringiensis (Bt) to further our molecular understanding of the oral immune priming phenomenon. We addressed how ingestion of bacterial cues (derived from spore supernatants) of an orally pathogenic and non-pathogenic Bt strain affects gene expression upon later challenge exposure, using a whole-transcriptome sequencing approach.ResultsWhereas gene expression of individuals primed with the orally non-pathogenic strain showed minor changes to controls, we found that priming with the pathogenic strain induced regulation of a large set of distinct genes, many of which are known immune candidates. Intriguingly, the immune repertoire activated upon priming and subsequent challenge qualitatively differed from the one mounted upon infection with Bt without previous priming. Moreover, a large subset of priming-specific genes showed an inverse regulation compared to their regulation upon challenge only.ConclusionsOur data demonstrate that gene expression upon infection is strongly affected by previous immune priming. We hypothesise that this shift in gene expression indicates activation of a more targeted and efficient response towards a previously encountered pathogen, in anticipation of potential secondary encounter.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3705-7) contains supplementary material, which is available to authorized users.