Immunogenetics of lithium response and psychiatric phenotypes in patients with bipolar disorder.

The link between bipolar disorder (BP) and immune dysfunction remains controversial. While epidemiological studies have long suggested an association, recent research has found only limited evidence of such a relationship. To clarify this, we performed an exploratory study of the contributions of immune-relevant genetic factors to the response to lithium (Li) treatment and the clinical presentation of BP. First, we assessed the association of a large collection of immune-related genes (4925) with Li response, defined by the Retrospective Assessment of the Lithium Response Phenotype Scale (Alda scale), and clinical characteristics in patients with BP from the International Consortium on Lithium Genetics (ConLi+Gen, N = 2374). Second, we calculated here previously published polygenic scores (PGSs) for immune-related traits and evaluated their associations with Li response and clinical features. Overall, we observed relatively weak associations (p < 1 × 10-4) with BP phenotypes within immune-related genes. Network and functional enrichment analyses of the top findings from the association analyses of Li response variables showed an overrepresentation of pathways participating in cell adhesion and intercellular communication. These appeared to converge on the well-known Li-induced inhibition of GSK-3ß. Association analyses of age-at-onset, number of mood episodes, and presence of psychosis, substance abuse and/or suicidal ideation suggested modest contributions of genes such as RTN4, XKR4, NRXN1, NRG1/3 and GRK5 to disease characteristics. PGS analyses returned weak associations (p < 0.05) between inflammation markers and the studied BP phenotypes. Our results suggest a modest relationship between immunity and clinical features in BP. More research is needed to assess the potential therapeutic relevance.

Disentangling specific and unspecific components of innate immune memory in a copepod-tapeworm system.

Evidence that the innate immune system can respond with forms of memory upon reinfection has been accumulating over the past few years. These phenomena of "immune priming" in invertebrates, and "trained immunity" in vertebrates, are contrary to previous belief that immune memory and specificity are restricted to the adaptive immune system. However, while trained immunity is usually a response with rather low specificity, immune priming has shown highly specific responses in certain species. To date, it is largely unknown how specificity in innate immune memory can be achieved in response to different parasite types. Here, we revisited a system where an exceptionally high degree of innate immune specificity had been demonstrated for the first time, consisting of the copepod Macrocyclops albidus and its natural parasite, the tapeworm Schistocephalus solidus. Using homologous (same family) vs. heterologous (different family) priming-challenge experiments, we first confirm that copepods exposed to the same parasite family benefit from reduced secondary infections. We further focused on exposed-but-not-infected copepods in primary exposure to employ a transcriptomic approach, distinguishing between immunity that was either specific or unspecific regarding the discrimination between tapeworm types. A weighted gene co-expression network (WGCN) revealed differences between specific and unspecific immunity; while both involved histone modification regulation, specific immunity involved gene-splicing factors, whereas unspecific immunity was primarily involved in metabolic shift. We found a functional enrichment in spliceosome in specific immunity, whereas oxidative phosphorylation and carbon metabolism were enriched in unspecific immunity. Our findings allow discrimination of specific and unspecific components of an innate immune memory, based on gene expression networks, and deepen our understanding of basic aspects of immune systems.

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.

Herbicide exposure alters the effect of the enthomopathogen Beauveria bassiana on immune gene expression in mealworm beetles.

Concerns have grown worldwide about the potentially far-reaching effects of herbicides on functional biodiversity in agroecosystems. Repeated applications over time can lead to accumulation of residues in soil, water, and food and may have negative impacts on non-target organisms. However, the effects of herbicide residues on interspecific relationships, such as host-pathogen interactions, are poorly studied. In this study, we evaluated the effects of two different concentrations of a commercial pendimethalin-based formulation (PND), the residual contamination (S, 13 ppm) in treated soils and the maximum residue level allowed by the European Commission in cereals (EU, 0.05 ppm). We tested the effect of PND on the biological interaction between the mealworm beetle Tenebrio molitor Linnaeus, 1758 and the entomopathogenic fungus Beauveria bassiana Vuillemin, 1912 (Bb, strain KVL 03-144) at two concentrations (LC50 5 × 105 conidia mL-1 and LC100 1 × 107 conidia mL-1). We checked the survival of beetles exposed to PND or/and inoculated with B. bassiana, the expression of four antimicrobial peptides (AMPs), and finally how PND affects in vitro germination of fungus. The exposure to PND had no significant effects on the survival of either control or Bb-exposed beetles. In the mealworm beetle, upregulation of gene expression of the inducible AMPs Tenecin 1, 2, and 4 was observed in PND-treated beetles after inoculation with Bb, while the levels of the non-inducible AMP Tenecin 3 were similar between treatments. In conclusion, our findings demonstrate that admitted residual doses of currently used herbicides modify an important component of the inducible immune response of an insect. This did not translate into an effect on the survival to B. bassiana in our system. However, residual doses of the herbicide at 13 ppm may temporarily affect fungal germination. These results raise questions about the compatibility of bioinsecticides with synthetic pesticides and the effects of herbicide residues on host-pathogen interactions.

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.

A new technique to study nutrient flow in host-parasite systems by carbon stable isotope analysis of amino acids and glucose.

Stable isotope analysis of individual compounds is emerging as a powerful tool to study nutrient origin and conversion in host-parasite systems. We measured the carbon isotope composition of amino acids and glucose in the cestode Schistocephalus solidus and in liver and muscle tissues of its second intermediate host, the three-spined stickleback (Gasterosteus aculeatus), over the course of 90 days in a controlled infection experiment. Similar linear regressions of δ13C values over time and low trophic fractionation of essential amino acids indicate that the parasite assimilates nutrients from sources closely connected to the liver metabolism of its host. Biosynthesis of glucose in the parasite might occur from the glucogenic precursors alanine, asparagine and glutamine and with an isotope fractionation of - 2 to - 3 ‰ from enzymatic reactions, while trophic fractionation of glycine, serine and threonine could be interpreted as extensive nutrient conversion to fuel parasitic growth through one-carbon metabolism. Trophic fractionation of amino acids between sticklebacks and their diets was slightly increased in infected compared to uninfected individuals, which could be caused by increased (immune-) metabolic activities due to parasitic infection. Our results show that compound-specific stable isotope analysis has unique opportunities to study host and parasite physiology.

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.

Insights into amino acid fractionation and incorporation by compound-specific carbon isotope analysis of three-spined sticklebacks.

Interpretation of stable isotope data is of upmost importance in ecology to build sound models for the study of animal diets, migration patterns and physiology. However, our understanding of stable isotope fractionation and incorporation into consumer tissues is still limited. We therefore measured the δ13C values of individual amino acids over time from muscle and liver tissue of three-spined sticklebacks (Gasterosteus aculeatus) on a high protein diet. The δ13C values of amino acids in the liver quickly responded to small shifts of under ± 2.0‰ in dietary stable isotope compositions on 30-day intervals. We found on average no trophic fractionation in pooled essential (muscle, liver) and non-essential (muscle) amino acids. Negative Δδ13C values of - 0.7 ± 1.3‰ were observed for pooled non-essential (liver) amino acids and might indicate biosynthesis from small amounts of dietary lipids. Trophic fractionation of individual amino acids is reported and discussed, including unusual Δδ13C values of over + 4.9 ± 1.4‰ for histidine. Arginine and lysine showed the lowest trophic fractionation on individual sampling days and might be useful proxies for dietary sources on short time scales. We suggest further investigations using isotopically enriched materials to facilitate the correct interpretation of ecological field data.

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.

Oral Immune Priming Treatment Alters Microbiome Composition in the Red Flour Beetle Tribolium castaneum.

It is now well-established that the microbiome is relevant for many of an organism's properties and that its composition reacts dynamically to various conditions. The microbiome interacts with host immunity and can play important roles in the defenses against pathogens. In invertebrates, immune priming, that is, improved survival upon secondary exposure to a previously encountered pathogen, can be dependent upon the presence of the gut microbiome. However, it is currently unknown whether the microbiome changes upon priming treatment. We here addressed this question in a well-established model for immune priming, the red flour beetle Tribolium castaneum exposed to the entomopathogenic bacterium Bacillus thuringiensis (Bt). After priming treatments, the microbiota composition of beetle larvae was assessed by deep sequencing of the V1-V2 region of the bacterial 16S rRNA gene. We compared the effect of two established routes of priming treatments in this system: injection priming with heat-killed Bt and oral priming via ingestion of filtered sterilized bacterial spore culture supernatants. For oral priming, we used several strains of Bt known to vary in their ability to induce priming. Our study revealed changes in microbiome composition following the oral priming treatment with two different strains of Bt, only one of which (Bt tenebrionis, Btt) is known to lead to improved survival. In contrast, injection priming treatment with the same bacterial strain did not result in microbiome changes. Combined with the previous results indicating that oral priming with Btt depends on the larval microbiome, this suggests that certain members of the microbiome could be involved in forming an oral priming response in the red flour beetle.

Serial passage in an insect host indicates genetic stability of the human probiotic Escherichia coli Nissle 1917.

BACKGROUND AND OBJECTIVES: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to effectively prevent and alleviate intestinal diseases. Despite the widespread medical application of EcN, we still lack basic knowledge about persistence and evolution of EcN outside the human body. Such knowledge is important also for public health aspects, as in contrast to abiotic therapeutics, probiotics are living organisms that have the potential to evolve. This study made use of experimental evolution of EcN in an insect host, the red flour beetle Tribolium castaneum, and its flour environment. METHODOLOGY: Using a serial passage approach, we orally introduced EcN to larvae of T.castaneum as a new host, and also propagated it in the flour environment. After eight propagation cycles, we analyzed phenotypic attributes of the passaged replicate EcN lines, their effects on the host in the context of immunity and infection with the entomopathogen Bacillus thuringiensis, and potential genomic changes using WGS of three of the evolved lines. RESULTS: We observed weak phenotypic differences between the ancestral EcN and both, beetle and flour passaged EcN lines, in motility and growth at 30°C, but neither any genetic changes, nor the expected increased persistence of the beetle-passaged lines. One of these lines displayed distinct morphological and physiological characteristics. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that EcN remains rather stable during serial passage in an insect. Weak phenotypic changes in growth and motility combined with a lack of genetic changes indicate a certain degree of phenotypic plasticity of EcN. LAY SUMMARY: For studying adaptation of the human probiotic Escherichia coli strain Nissle 1917, we introduced it to a novel insect host system and its environment using a serial passage approach. After passage, we observed weak phenotypic changes in growth and motility but no mutations or changes in persistence inside the host.

Integrating evolutionary aspects into dual-use discussion: the cases of influenza virus and enterohemorrhagic Escherichia coli.

Research in infection biology aims to understand the complex nature of host-pathogen interactions. While this knowledge facilitates strategies for preventing and treating diseases, it can also be intentionally misused to cause harm. Such dual-use risk is potentially high for highly pathogenic microbes such as Risk Group-3 (RG3) bacteria and RG4 viruses, which could be used in bioterrorism attacks. However, other pathogens such as influenza virus (IV) and enterohemorrhagic Escherichia coli (EHEC), usually classified as RG2 pathogens, also demonstrate high dual-use risk. As the currently approved therapeutics against these pathogens are not satisfactorily effective, previous outbreaks of these pathogens caused enormous public fear, media attention and economic burden. In this interdisciplinary review, we summarize the current perspectives of dual-use research on IV and EHEC, and further highlight the dual-use risk associated with evolutionary experiments with these infectious pathogens. We support the need to carry out experiments pertaining to pathogen evolution, including to gain predictive insights on their evolutionary trajectories, which cannot be otherwise achieved with stand-alone theoretical models and epidemiological data. However, we also advocate for increased awareness and assessment strategies to better quantify the risks-versus-benefits associated with such evolutionary experiments. In addition to building public trust in dual-use research, we propose that these approaches can be extended to other pathogens currently classified as low risk, but bearing high dual-use potential, given the particular pressing nature of their rapid evolutionary potential.

Morphological Characterisation of Haemocytes in the Mealworm Beetle Tenebrio molitor (Coleoptera, Tenebrionidae).

The immunocompetence of the mealworm beetle Tenebrio molitor has been well investigated at molecular and physiological levels, but information on morphological and functional characteristics of its immune cells (haemocytes) is still scarce and fragmentary. This study provides an updated overview of the morphology of circulating immune cells from mealworm beetle adults, using light and transmission electron microscopy. Based on their affinities for May-Grünwald Giemsa stain, haemocytes were defined as either eosinophilic, basophilic or neutral. Ultrastructural descriptions allowed to detect four main cell types in the haemolymph: prohaemocytes, plasmatocytes, granular cells and oenocytoids. The morphological plasticity of haemocytes and the evidence of mitotic circulating cells, intermediate cell stages, as well as autophagic activities suggest haemocyte proliferation, turnover and transdifferentiation as constantly active processes in the haemolymph. Cytochemical tests revealed differences in the distribution of carbohydrates among cell types underling the great plasticity of the immune response and the direct involvement of circulating immune cells in the resource allocation. In addition, our results provide a detailed morphological description of vesicle trafficking, macro- and microautophagy, apoptotic and necrotic processes, confirming the suitability of T. molitor haemocytes as a model for studying evolutionarily conserved cellular mechanisms.

Survival of the Sawfly Athalia rosae Upon Infection by an Entomopathogenic Fungus and in Relation to Clerodanoid Uptake.

Larvae of the turnip sawfly Athalia rosae are a pest of Brassicacae plants, as their feeding can cause defoliation of various crops of economic importance. The larvae and the adults of this sawfly species are known to take up different classes of chemical compounds from their respective host plants, with potentially deterrent functions against predators. In addition, compounds taken up by the adults, the clerodanoids, are known for their antimicrobial activity. These features could be a challenge to biocontrol strategies. Several natural enemies of A. rosae have been identified, targeting larval and pupal stages of A. rosae, which could potentially be used as biocontrol agents. However, targeting the adult stage of a larval pest in addition to targeting the juvenile stages may improve population control. In this study, we ask whether a strain of the entomopathogenic fungus Beauveria bassiana shows biological activity against A. rosae adults. We also investigate whether the behavior of clerodanoid uptake by the adults, which is commonly found, affects their survival in response to a B. bassiana exposure. We found a clear dose-response relationship, i.e., with increasing fungal conidia concentrations survival of A. rosae decreased. However, there was only a low incidence of mycelial growth and sporulation from A. rosae cadavers, indicating that either the fungus is not successfully developing inside this host, or it is not able to re-emerge from it. Clerodanoid uptake decreased the survival of healthy adults; however, it did not increase their survival to B. bassiana. Our results revealed that this strain of B. bassiana if applied alone is probably not suitable for biocontrol of this sawfly species, because A. rosae showed a high baseline resistance against this fungus. The behavior of clerodanoid uptake is unlikely to have evolved as a defense against this entomopathogenic fungus.

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.

Comparative Mortality and Adaptation of a Smurf Assay in two Species of Tenebrionid Beetles Exposed to Bacillus thuringiensis.

Bacillus thuringiensis is a spore-forming bacterium which infects insect larvae naturally via the oral route. Its virulence factors interact with the epithelium of the digestive tract of insect larvae, disrupting its function and eventually leading to the death of susceptible hosts. The most cited B. thuringiensis killing mechanism is the extensive damage caused to the insect midgut, leading to its leakage. The mortality caused by B. thuringiensis has been shown to vary between serovars and isolates, as well as between host life stages. Moreover, whether susceptibility to B. thuringiensis-induced gut leakage is generalized to all host species and whether there is individual variation within species is unclear. In this study, we adapted a non-invasive "Smurf" assay from Drosophila melanogaster to two species of tenebrionid beetles: The mealworm beetle Tenebrio molitor and the red flour beetle Tribolium castaneum, during exposure to B. thuringiensis. We highlight a differential mortality between two age/size classes of T. molitor larvae, as well as different killing dynamics between B. thuringiensis var. tenebrionis and var. tolworthi in T. castaneum. The Smurf assay did not reveal a high occurrence of extensive gut disintegration in both host species upon ingestion during B. thuringiensis exposure.

Dscam in immunity: A question of diversity in insects and crustaceans.

In insects and crustaceans, thousands of Down syndrome cell adhesion molecules (Dscam) can be generated by alternative splicing of variable exons from a single-locus gene, Dscam-hv. This extraordinarily versatile gene (38,016 protein isoforms produced in Drosophila) was first proposed to be involved in exon guidance and subsequently implicated in immunity as a hypervariable immune molecule. Almost 20 y after discovery of Dscam-hv, there have been many studies in insects and crustaceans regarding roles of Dscam in immunity, with many similarities and concurrently, many differences. Here, we review the current status of Dscam-hv, presented as a comparison of similarities and differences in insects and crustaceans and discuss hypotheses of Dscam functions in immunity.

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.