Experimental evolution of immunological specificity

Innate immune memory (i.e., immune priming) is found in many invertebrates. In some cases, immune priming provides protection against infection only when the same bacteria are used for priming and challenge; that is, priming can be specific. However, we still know little about the conditions favoring the evolution of immunological specificity. We present evidence that immune priming and its specificity can rapidly evolve in an insect through experimental selection by repeated bacterial exposure. Our populations evolved treatment-specific differences in expression profiles of immune, metabolic, and transcription-regulatory genes, pointing to similar mechanisms acting in vertebrate trained immunity. Hence, immune memory combines deeply rooted resemblances across systems with enormous evolutionary plasticity.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

The Red Flour Beetle as a Model for Bacterial Oral Infections

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

Infection routes matter in population-specific responses of the red flour beetle to the entomopathogen Bacillus thuringiensis

Background: Pathogens 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. Results: The 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. Conclusions: Our 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

Oral immune priming with 'Bacillus thuringiensis' induces a shift in the gene expression of 'Tribolium castaneum' larvae

BACKGROUND: The 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.RESULTS: Whereas 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.CONCLUSIONS: Our 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.</p

Oral immune priming with 'Bacillus thuringiensis' induces a shift in the gene expression of 'Tribolium castaneum' larvae

BACKGROUND: The 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.RESULTS: Whereas 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.CONCLUSIONS: Our 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.</p

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species

Insulin resistance in cavefish as an adaptation to a nutrient-limited environment

Periodic food shortages are a major challenge faced by organisms in natural habitats. Cave-dwelling animals must withstand long periods of nutrient deprivation, as—in the absence of photosynthesis—caves depend on external energy sources such as seasonal floods1. Here we show that cave-adapted populations of the Mexican tetra, Astyanax mexicanus, have dysregulated blood glucose homeostasis and are insulin-resistant compared to river-adapted populations. We found that multiple cave populations carry a mutation in the insulin receptor that leads to decreased insulin binding in vitro and contributes to hyperglycaemia. Hybrid fish from surface–cave crosses carrying this mutation weigh more than non-carriers, and zebrafish genetically engineered to carry the mutation have increased body weight and insulin resistance. Higher body weight may be advantageous in caves as a strategy to cope with an infrequent food supply. In humans, the identical mutation in the insulin receptor leads to a severe form of insulin resistance and reduced lifespan. However, cavefish have a similar lifespan to surface fish and do not accumulate the advanced glycation end-products in the blood that are typically associated with the progression of diabetes-associated pathologies. Our findings suggest that diminished insulin signalling is beneficial in a nutrient-limited environment and that cavefish may have acquired compensatory mechanisms that enable them to circumvent the typical negative effects associated with failure to regulate blood glucose levels