Rapid but narrow - Evolutionary adaptation and transcriptional response of Drosophila melanogaster to toxic mould.

Insects have adapted to a multitude of environmental conditions, including the presence of xenobiotic noxious substances. Environmental microorganisms, particularly rich on ephemeral resources, employ these noxious chemicals in a chemical warfare against predators and competitors, driving co-evolutionary adaptations. In order to analyse how environmental microbes may be driving such evolutionary adaptations, we experimentally evolved Drosophila melanogaster populations by exposing larvae to the toxin-producing mould Aspergillus nidulans that infests the flies' breeding substrate. To disentangle the effects of the mycotoxin Sterigmatocystin from other substrate modifications inflicted by the mould, we used the following four selection regimes: (i) control without fungus, (ii) A. nidulans wild type, (iii) a mutant of A. nidulans ΔlaeA with impaired toxin production, (iv) synthetic Sterigmatocystin. Experimental evolution was carried out in five independent D. melanogaster populations each, for a total of 11 generations. We further combined our evolution experiment with transcriptome analysis to identify evolutionary shifts in gene expression due to the selection regimes and mould confrontation. Populations that evolved in presence of the toxin-producing mould or the pure mycotoxin rapidly adapted to the respective conditions and showed higher viability in subsequent confrontations. Yet, mycotoxin-selected populations had no advantage in A. nidulans wild type confrontation. Moreover, distinctive changes in gene expression related to the selection-regime contrast were only associated with the toxin-producing-fungus regime and comprised a narrow set of genes. Thus, it needs the specific conditions of the selection agent to enable adaptation to the fungus.

Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

Immune Stimulation via Wounding Alters Chemical Profiles of Adult Tribolium castaneum.

Group-living individuals experience immense risk of disease transmission and parasite infection. In social and in some non-social insects, disease control with immunomodulation arises not only via individual immune defenses, but also via infochemicals such as contact cues and (defensive) volatiles to mount a group-level immunity. However, little is known about whether activation of the immune system elicits changes in chemical phenotypes, which may mediate these responses. We here asked whether individual immune experience resulting from wounding or injection of heat-killed Bacillus thuringiensis (priming) leads to changes in the chemical profiles of female and male adult red flour beetles, Tribolium castaneum, which are non-social but gregarious. We analyzed insect extracts using GC-FID to study the chemical composition of (1) cuticular hydrocarbons (CHCs) as candidates for the transfer of immunity-related information between individuals via contact, and (2) stink gland secretions, with analysis of benzoquinones as main active compounds regulating 'external immunity'. Despite a pronounced sexual dimorphism in CHC profiles, wounding stimulation led to similar profile changes in males and females with increases in the proportion of methyl-branched alkanes compared to naïve beetles. While changes in the overall secretion profiles were less pronounced, absolute amounts of benzoquinones were transiently elevated in wounded compared to naïve females. Responses to priming were insignificant in CHCs and secretions. We suggest that changes in different infochemicals after wounding may mediate immune status signaling in the context of both internal and external immune responses in groups of this non-social insect, thus showing parallels to social immunity.

Paternal knockdown of tRNA(cytosine-5-)-methyltransferase (Dnmt2) increases offspring susceptibility to infection in red flour beetles.

Intergenerational effects from fathers to offspring are increasingly reported from diverse organisms, but the underlying mechanisms remain speculative. Paternal trans-generational immune priming (TGIP) was demonstrated in the red flour beetle Tribolium castaneum: non-infectious bacterial exposure of fathers protects their offspring against an infectious challenge for at least two generations. Epigenetic processes, such as cytosine methylation of nucleic acids, have been proposed to enable transfer of information from fathers to offspring. Here we studied a potential role in TGIP of the Dnmt2 gene (renamed as Trdmt1 in humans), which encodes a highly conserved enzyme that methylates different RNAs, including specific cytosines of a set of tRNAs. Dnmt2 has previously been reported to be involved in intergenerational epigenetic inheritance in mice and protection against viruses in fruit flies. We first studied gene expression and found that Dnmt2 is expressed in various life history stages and tissues of T. castaneum, with high expression in the reproductive organs. RNAi-mediated knockdown of Dnmt2 in fathers was systemic, slowed down offspring larval development and increased mortality of the adult offspring upon bacterial infection. However, these effects were independent of bacterial exposure of the fathers. In conclusion, our results point towards a role of Dnmt2 for paternal effects, while elucidation of the mechanisms behind paternal TGIP needs further studies.

How Individualized Niches Arise: Defining Mechanisms of Niche Construction, Niche Choice, and Niche Conformance.

Organisms interact with their environments in various ways. We present a conceptual framework that distinguishes three mechanisms of organism-environment interaction. We call these NC3 mechanisms: niche construction, in which individuals make changes to the environment; niche choice, in which individuals select an environment; and niche conformance, in which individuals adjust their phenotypes in response to the environment. Each of these individual-level mechanisms affects an individual's phenotype-environment match, its fitness, and its individualized niche, defined in terms of the environmental conditions under which the individual can survive and reproduce. Our framework identifies how individuals alter the selective regimes that they and other organisms experience. It also places clear emphasis on individual differences and construes niche construction and other processes as evolved mechanisms. The NC3 mechanism framework therefore helps to integrate population-level and individual-level research.

Experimental evolution of immunological specificity.

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.

Climate change facilitates a parasite's host exploitation via temperature-mediated immunometabolic processes.

Global climate change can influence organismic interactions like those between hosts and parasites. Rising temperatures may exacerbate the exploitation of hosts by parasites, especially in ectothermic systems. The metabolic activity of ectotherms is strongly linked to temperature and generally increases when temperatures rise. We hypothesized that temperature change in combination with parasite infection interferes with the host's immunometabolism. We used a parasite, the avian cestode Schistocephalus solidus, which taps most of its resources from the metabolism of an ectothermic intermediate host, the three-spined stickleback. We experimentally exposed sticklebacks to this parasite, and studied liver transcriptomes 50 days after infection at 13°C and 24°C, to assess their immunometabolic responses. Furthermore, we monitored fitness parameters of the parasite and examined immunity and body condition of the sticklebacks at 13°C, 18°C and 24°C after 36, 50 and 64 days of infection. At low temperatures (13°C), S. solidus growth was constrained, presumably also by the more active stickleback's immune system, thus delaying its infectivity for the final host to 64 days. Warmer temperature (18°C and 24°C) enhanced S. solidus growth, and it became infective to the final host already after 36 days. Overall, S. solidus produced many more viable offspring after development at elevated temperatures. In contrast, stickleback hosts had lower body conditions, and their immune system was less active at warm temperature. The stickleback's liver transcriptome revealed that mainly metabolic processes were differentially regulated between temperatures, whereas immune genes were not strongly affected. Temperature effects on gene expression were strongly enhanced in infected sticklebacks, and even in exposed-but-not-infected hosts. These data suggest that the parasite exposure in concert with rising temperature, as to be expected with global climate change, shifted the host's immunometabolism, thus providing nutrients for the enormous growth of the parasite and, at the same time suppressing immune defence.

Parasite infection disrupts escape behaviours in fish shoals.

Many prey species have evolved collective responses to avoid predation. They rapidly transfer information about potential predators to trigger and coordinate escape waves. Predation avoidance behaviour is often manipulated by trophically transmitted parasites, to facilitate their transmission to the next host. We hypothesized that the presence of infected, behaviourally altered individuals might disturb the spread of escape waves. We used the tapeworm Schistocephalus solidus, which increases risk-taking behaviour and decreases social responsiveness of its host, the three-spined stickleback, to test this hypothesis. Three subgroups of sticklebacks were placed next to one another in separate compartments with shelter. The middle subgroup contained either uninfected or infected sticklebacks. We confronted an outer subgroup with an artificial bird strike and studied how the escape response spread through the subgroups. With uninfected sticklebacks in the middle, escape waves spread rapidly through the entire shoal and fish remained in shelter thereafter. With infected sticklebacks in the middle, the escape wave was disrupted and uninfected fish rarely used the shelter. Infected individuals can disrupt the transmission of flight responses, thereby not only increasing their own predation risk but also that of their uninfected shoal members. Our study uncovers a potentially far-reaching fitness consequence of grouping with infected individuals.

Immune memory in invertebrates.

Evidence for innate immune memory (or 'priming') in invertebrates has been accumulating over the last years. We here provide an in-depth review of the current state of evidence for immune memory in invertebrates, and in particular take a phylogenetic viewpoint. Invertebrates are a very heterogeneous group of animals and accordingly, evidence for the phenomenon of immune memory as well as the hypothesized molecular underpinnings differ largely for the diverse invertebrate taxa. The majority of research currently focuses on Arthropods, while evidence from many other groups of invertebrates is fragmentary or even lacking. We here concentrate on immune memory that is induced by pathogenic challenges, but also extent our view to a non-pathogenic context, i.e. allograft rejection, which can also show forms of memory and can inform us about general principles of specific self-nonself recognition. We discuss definitions of immune memory and a number of relevant aspects such as the type of antigens used, the route of exposure, and the kinetics of reactions following priming.

A multi-faceted approach testing the effects of previous bacterial exposure on resistance and tolerance.

Hosts can alter their strategy towards pathogens during their lifetime; that is, they can show phenotypic plasticity in immunity or life history. Immune priming is one such example, where a previous encounter with a pathogen confers enhanced protection upon secondary challenge, resulting in reduced pathogen load (i.e., resistance) and improved host survival. However, an initial encounter might also enhance tolerance, particularly to less virulent opportunistic pathogens that establish persistent infections. In this scenario, individuals are better able to reduce the negative fecundity consequences that result from a high pathogen burden. Finally, previous exposure may also lead to life-history adjustments, such as terminal investment into reproduction. Using different Drosophila melanogaster host genotypes and two bacterial pathogens, Lactococcus lactis and Pseudomonas entomophila, we tested whether previous exposure results in resistance or tolerance and whether it modifies immune gene expression during an acute-phase infection (one day post-challenge). We then asked whether previous pathogen exposure affects chronic-phase pathogen persistence and longer-term survival (28 days post-challenge). We predicted that previous exposure would increase host resistance to an early stage bacterial infection while it might come at a cost to host fecundity tolerance. We reasoned that resistance would be due in part to stronger immune gene expression after challenge. We expected that previous exposure would improve long-term survival, that it would reduce infection persistence, and we expected to find genetic variation in these responses. We found that previous exposure to P. entomophila weakened host resistance to a second infection independent of genotype and had no effect on immune gene expression. Fecundity tolerance showed genotypic variation but was not influenced by previous exposure. However, L. lactis persisted as a chronic infection, whereas survivors cleared the more pathogenic P. entomophila infection. To our knowledge, this is the first study that addresses host tolerance to bacteria in relation to previous exposure, taking a multi-faceted approach to address the topic. Our results suggest that previous exposure comes with transient costs to resistance during the early stage of infection in this host-pathogen system and that infection persistence may be bacterium-specific.

In vitro effects of the neuroactive substances serotonin and γ-aminobutyric acid on leucocytes from sticklebacks (Gasterosteus aculeatus).

The majority of parasites have evolved strategies to evade the immune responses of their hosts. Neuroactive substances produced by cestodes are possible candidate molecules for regulating host immune responses. The neurons of helminths can synthesize a wide range of molecules that are identical to the ones functioning in their host organisms, and host lymphocytes have receptors for these neuroactive substances. We hypothesized that in teleost fish, antihelminthic immune responses are regulated via 5-hydroxytryptamine (5-HT, or serotonin) and γ-aminobutyric acid (GABA). In the present study, we investigated the in vitro influence of serotonin, GABA and Schistocephalus solidus (helminth) antigens on basic characteristics of the three-spined stickleback Schistocephalus solidus cellular immune response. Head kidney leucocytes (HKLs) were analysed by flow cytometry for cell viability and the frequency of leucocyte subsets (the granulocyte-to-lymphocyte ratio) and by a chemiluminescence assay for the production of reactive oxygen species (ROS). In short-term (2-h) HKL cultures, 5-HT did not change the total numbers of live HKLs, but the production of ROS decreased significantly with all 5-HT concentrations. In long-term (96-h) cultures, high 5-HT concentrations induced a decrease in leucocyte viability. This coincided with elevated ROS production in cultures with all 5-HT concentrations. In short-term (2-h) HKL cultures, GABA did not change the total numbers of live HKLs, but the production of ROS decreased significantly with high (100 nmol L-1) GABA concentrations. In long-term (96-h) cultures, high and medium concentrations of GABA (100 nmol L-1 and 10 nmol L-1) elevated the numbers of live HKLs compared to controls. The granulocyte-to-lymphocyte ratios generally increased upon exposure to GABA at all concentrations. All concentrations of GABA alone elevated the ROS production of HKLs compared to controls. In the present work, we showed that the neuroactive substances serotonin and GABA regulate the teleost immune system. Our study supports the hypothesis that these substances might be immunomodulators in tapeworm-fish parasite-host interactions.

Parasite-infected sticklebacks increase the risk-taking behaviour of uninfected group members.

Trophically transmitted parasites frequently increase their hosts' risk-taking behaviour, to facilitate transmission to the next host. Whether such elevated risk-taking can spill over to uninfected group members is, however, unknown. To investigate this, we confronted groups of 6 three-spined sticklebacks, Gasterosteus aculeatus, containing 0, 2, 4 or 6 experimentally infected individuals with a simulated bird attack and studied their risk-taking behaviour. As a parasite, we used the tapeworm Schistocephalus solidus, which increases the risk-taking of infected sticklebacks, to facilitate transmission to its final host, most often piscivorous birds. Before the attack, infected and uninfected individuals did not differ in their risk-taking. However, after the attack, individuals in groups with only infected members showed lower escape responses and higher risk-taking than individuals from groups with only uninfected members. Importantly, uninfected individuals adjusted their risk-taking behaviour to the number of infected group members, taking more risk with an increasing number of infected group members. Infected individuals, however, did not adjust their risk-taking to the number of uninfected group members. Our results show that behavioural manipulation by parasites does not only affect the infected host, but also uninfected group members, shedding new light on the social dynamics involved in host-parasite interactions.

Host-Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes.

Reciprocal coevolution between host and pathogen is widely seen as a major driver of evolution and biological innovation. Yet, to date, the underlying genetic mechanisms and associated trait functions that are unique to rapid coevolutionary change are generally unknown. We here combined experimental evolution of the bacterial biocontrol agent Bacillus thuringiensis and its nematode host Caenorhabditis elegans with large-scale phenotyping, whole genome analysis, and functional genetics to demonstrate the selective benefit of pathogen virulence and the underlying toxin genes during the adaptation process. We show that: (i) high virulence was specifically favoured during pathogen-host coevolution rather than pathogen one-sided adaptation to a nonchanging host or to an environment without host; (ii) the pathogen genotype BT-679 with known nematocidal toxin genes and high virulence specifically swept to fixation in all of the independent replicate populations under coevolution but only some under one-sided adaptation; (iii) high virulence in the BT-679-dominated populations correlated with elevated copy numbers of the plasmid containing the nematocidal toxin genes; (iv) loss of virulence in a toxin-plasmid lacking BT-679 isolate was reconstituted by genetic reintroduction or external addition of the toxins. We conclude that sustained coevolution is distinct from unidirectional selection in shaping the pathogen's genome and life history characteristics. To our knowledge, this study is the first to characterize the pathogen genes involved in coevolutionary adaptation in an animal host-pathogen interaction system.

Early stages of infection of three-spined stickleback (Gasterosteus aculeatus) with the cestode Schistocephalus solidus.

Parasitic helminths have evolved strategies to evade their host's immune systems. Particularly, the early time of interactions between helminths and their hosts might be decisive for their infection success. We used the cestode Schistocephalus solidus, and its highly specific second intermediate host, the three-spined stickleback (Gasterosteus aculeatus) to investigate parasite infection and host cellular immune responses starting 1 day postexposure (dpe). We recovered live parasites from stickleback body cavities already 24 hr after exposure. Infection rates increased up to 50% and did not change from 4 dpe onwards. Thus, not all parasites had reached the body cavity at the early time points and clearance of the parasite at later time points did not occur. Stickleback head kidney leucocytes (HKLs) did not show distinct signs of activation and lymphocyte proliferation, granulocyte-to-lymphocyte ratios and respiratory burst activity of infected sticklebacks did not deviate from controls significantly. The immune system was activated only late, as indicated by an increase in the total count of HKL relative to stickleback weight (HKL per mg fish), which was significantly elevated in infected fish 32 dpe. S. solidus seems to evade leucocyte activity early during infection facilitating its establishment in the hosts' body cavity.

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.

Dissecting the dynamics of trans-generational immune priming.

The transfer of immunity from mother to offspring is a central way to endow the offspring with increased protection against pathogens. This phenomenon is not only found within the vertebrate domain: in some circumstances, invertebrate mothers can also give their offspring an immune kick-start, which is termed trans-generational immune priming (TGIP). TGIP has been uncovered for a number of invertebrate species, but it is not ubiquitously evident. The reasons for which are not known. In this issue of Molecular Ecology, Tate, Andolfatto, Demuth, and Graham () probe the molecular underpinnings of TGIP in concert with the temporal dynamics of the response in the red flour beetle, Tribolium castaneum, infected with the bacterium Bacillus thuringiensis (Figure ). They provide previously lacking evidence for the repeatability of TGIP, meaning that when averaged across several experiments, the offspring of mothers infected with heat-killed bacteria had better survival when they themselves were infected with live bacteria than offspring from mothers that had not encountered the bacterium. In a detailed temporal examination of the offspring's acute infection phase (zero to 24 hr after infection), Tate et al. () follow T. castaneum's gene regulation responses to infection while simultaneously documenting bacterial load. Such an approach gives considerable insight into the physiological processes that occur in primed offspring, and a first glance at a potential role for tolerance and effects on host metabolism that might even resemble trained immunity, which is a form of innate immune memory in vertebrates.

Specificity of oral immune priming in the red flour beetle Tribolium castaneum.

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, Tribolium castaneum For priming, we used pathogen-free supernatants derived from three different strains of the entomopathogen, Bacillus thuringiensis, 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.

An experimental approach to the immuno-modulatory basis of host-parasite local adaptation in tapeworm-infected sticklebacks.

The evolutionary arms race of hosts and parasites often results in adaptations, which may differ between populations. Investigation of such local adaptation becomes increasingly important to understand dynamics of host-parasite interactions and co-evolution. To this end we performed an infection experiment involving pairs of three-spined sticklebacks and their tapeworm parasite Schistocephalus solidus from three geographically separated origins (Germany, Spain and Iceland) in a fully-crossed design for sympatric and allopatric host/parasite combinations. We hypothesized that local adaptation of the hosts results in differences in parasite resistance with variation in parasite infection rates and leukocyte activation, whereas parasites from different origins might differ in virulence reflected in host exploitation rates (parasite indices) and S. solidus excretory-secretory products (SsESP) involved in immune manipulation. In our experimental infections, sticklebacks from Iceland were more resistant to S. solidus infection compared to Spanish and German sticklebacks. Higher resistance of Icelandic sticklebacks seemed to depend on adaptive immunity, whereas sticklebacks of German origin, which were more heavily afflicted by S. solidus, showed elevated activity of innate immune traits. German S. solidus were less successful in infecting and exploiting allopatric hosts compared to their Icelandic and Spanish conspecifics. Nevertheless, exclusively SsESP from German S. solidus triggered significant in vitro responses of leukocytes from naïve sticklebacks. Interestingly, parasite indices were almost identical across the sympatric combinations. Differences in host resistance and parasite virulence between the origins were most evident in allopatric combinations and were consistent within origin; i.e. Icelandic sticklebacks were more resistant and their S. solidus were more virulent in all allopatric combinations, whereas German sticklebacks were less resistant and their parasites less virulent. Despite such differences between origins, the degree of host exploitation was almost identical in the sympatric host-parasite combinations, suggesting that the local evolutionary arms race of hosts and parasites resulted in an optimal virulence, maximising parasite fitness while avoiding host overexploitation.

Downregulation of the evolutionary capacitor Hsp90 is mediated by social cues.

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.

Infection of Tribolium castaneum with Bacillus thuringiensis: quantification of bacterial replication within cadavers, transmission via cannibalism, and inhibition of spore germination.

Reproduction within a host and transmission to the next host are crucial for the virulence and fitness of pathogens. Nevertheless, basic knowledge about such parameters is often missing from the literature, even for well-studied bacteria, such as Bacillus thuringiensis, an endospore-forming insect pathogen, which infects its hosts via the oral route. To characterize bacterial replication success, we made use of an experimental oral infection system for the red flour beetle Tribolium castaneum and developed a flow cytometric assay for the quantification of both spore ingestion by the individual beetle larvae and the resulting spore load after bacterial replication and resporulation within cadavers. On average, spore numbers increased 460-fold, showing that Bacillus thuringiensis grows and replicates successfully in insect cadavers. By inoculating cadaver-derived spores and spores from bacterial stock cultures into nutrient medium, we next investigated outgrowth characteristics of vegetative cells and found that cadaver-derived bacteria showed reduced growth compared to bacteria from the stock cultures. Interestingly, this reduced growth was a consequence of inhibited spore germination, probably originating from the host and resulting in reduced host mortality in subsequent infections by cadaver-derived spores. Nevertheless, we further showed that Bacillus thuringiensis transmission was possible via larval cannibalism when no other food was offered. These results contribute to our understanding of the ecology of Bacillus thuringiensis as an insect pathogen.