Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis.

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.

Small genome of the fungus Escovopsis weberi, a specialized disease agent of ant agriculture.

Many microorganisms with specialized lifestyles have reduced genomes. This is best understood in beneficial bacterial symbioses, where partner fidelity facilitates loss of genes necessary for living independently. Specialized microbial pathogens may also exhibit gene loss relative to generalists. Here, we demonstrate that Escovopsis weberi, a fungal parasite of the crops of fungus-growing ants, has a reduced genome in terms of both size and gene content relative to closely related but less specialized fungi. Although primary metabolism genes have been retained, the E. weberi genome is depleted in carbohydrate active enzymes, which is consistent with reliance on a host with these functions. E. weberi has also lost genes considered necessary for sexual reproduction. Contrasting these losses, the genome encodes unique secondary metabolite biosynthesis clusters, some of which include genes that exhibit up-regulated expression during host attack. Thus, the specialized nature of the interaction between Escovopsis and ant agriculture is reflected in the parasite's genome.

Recent advances in vertebrate and invertebrate transgenerational immunity in the light of ecology and evolution.

Parental experience with parasites and pathogens can lead to increased offspring resistance to infection, through a process known as transgenerational immune priming (TGIP). Broadly defined, TGIP occurs across a wide range of taxa, and can be viewed as a type of phenotypic plasticity, with hosts responding to the pressures of relevant local infection risk by altering their offspring's immune defenses. There are ever increasing examples of both invertebrate and vertebrate TGIP, which go beyond classical examples of maternal antibody transfer. Here we critically summarize the current evidence for TGIP in both invertebrates and vertebrates. Mechanisms underlying TGIP remain elusive in many systems, but while it is unlikely that they are conserved across the range of organisms with TGIP, recent insight into epigenetic modulation may challenge this view. We place TGIP into a framework of evolutionary ecology, discussing costs and relevant environmental variation. We highlight how the ecology of species or populations should affect if, where, when, and how TGIP is realized. We propose that the field can progress by incorporating evolutionary ecology focused designs to the study of the so far well chronicled, but mostly descriptive TGIP, and how rapidly developing -omic methods can be employed to further understand TGIP across taxa.

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens.

BACKGROUND: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. RESULTS: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. CONCLUSIONS: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.

Life-history strategy determines constraints on immune function.

Determining the factors governing investment in immunity is critical to understanding host-pathogen ecological and evolutionary dynamics. Studies often consider disease resistance in the context of life-history theory, with the expectation that investment in immunity will be optimized in anticipation of disease risk. Immunity, however, is constrained by context-dependent fitness costs. How the costs of immunity vary across life-history strategies has yet to be considered. Pea aphids are typically unwinged but produce winged offspring in response to high population densities and deteriorating conditions. This is an example of polyphenism, a strategy used by many organisms to adjust to environmental cues. The goal of this study was to examine the relationship between the fitness costs of immunity, pathogen resistance and the strength of an immune response across aphid morphs that differ in life-history strategy but are genetically identical. We measured fecundity of winged and unwinged aphids challenged with a heat-inactivated fungal pathogen, and found that immune costs are limited to winged aphids. We hypothesized that these costs reflect stronger investment in immunity in anticipation of higher disease risk, and that winged aphids would be more resistant due to a stronger immune response. However, producing wings is energetically expensive. This guided an alternative hypothesis - that investing resources into wings could lead to a reduced capacity to resist infection. We measured survival and pathogen load after live fungal infection, and we characterized the aphid immune response to fungi by measuring immune cell concentration and gene expression. We found that winged aphids are less resistant and mount a weaker immune response than unwinged aphids, demonstrating that winged aphids pay higher costs for a less effective immune response. Our results show that polyphenism is an understudied factor influencing the expression of immune costs. More generally, our work shows that in addition to disease resistance, the costs of immunity vary between individuals with different life-history strategies. We discuss the implications of these findings for understanding how organisms invest optimally in immunity in the light of context-dependent constraints.

Gene expression differences underlying genotype-by-genotype specificity in a host-parasite system.

In many systems, host-parasite evolutionary dynamics have led to the emergence and maintenance of diverse parasite and host genotypes within the same population. Genotypes vary in key attributes: Parasite genotypes vary in ability to infect, host genotypes vary in susceptibility, and infection outcome is frequently the result of both parties' genotypic identities. These host-parasite genotype-by-genotype (GH × GP) interactions influence evolutionary and ecological dynamics in important ways. Interactions can be produced through genetic variation; however, here, we assess the role of variable gene expression as an additional source of GH × GP interactions. The bumblebee Bombus terrestris and its trypanosome gut parasite Crithidia bombi are a model system for host-parasite matching. Full-transcriptome sequencing of the bumblebee host revealed that different parasite genotypes indeed induce fundamentally different host expression responses and host genotypes vary in their responses to the infecting parasite genotype. It appears that broadly and successfully infecting parasite genotypes lead to reduced host immune gene expression relative to unexposed bees but induce the expression of genes responsible for controlling gene expression. Contrastingly, a poorly infecting parasite genotype induced the expression of immunologically important genes, including antimicrobial peptides. A targeted expression assay confirmed the transcriptome results and also revealed strong host genotype effects. In all, the expression of a number of genes depends on the host genotype and the parasite genotype and the interaction between both host and parasite genotypes. These results suggest that alongside sequence variation in coding immunological genes, variation that controls immune gene expression can also produce patterns of host-parasite specificity.

Differential gene expression and alternative splicing in insect immune specificity.

BACKGROUND: Ecological studies routinely show genotype-genotype interactions between insects and their parasites. The mechanisms behind these interactions are not clearly understood. Using the bumblebee Bombus terrestris/trypanosome Crithidia bombi model system (two bumblebee colonies by two Crithidia strains), we have carried out a transcriptome-wide analysis of gene expression and alternative splicing in bees during C. bombi infection. We have performed four analyses, 1) comparing gene expression in infected and non-infected bees 24 hours after infection by Crithidia bombi, 2) comparing expression at 24 and 48 hours after C. bombi infection, 3) determining the differential gene expression associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours after infection and 4) determining the alternative splicing associated with the bumblebee-Crithidia genotype-genotype interaction at 24 hours post infection. RESULTS: We found a large number of genes differentially regulated related to numerous canonical immune pathways. These genes include receptors, signaling pathways and effectors. We discovered a possible interaction between the peritrophic membrane and the insect immune system in defense against Crithidia. Most interestingly, we found differential expression and alternative splicing of immunoglobulin related genes (Dscam and Twitchin) are associated with the genotype-genotype interactions of the given bumblebee colony and Crithidia strain. CONCLUSIONS: In this paper we have shown that the expression and alternative splicing of immune genes is associated with specific interactions between different host and parasite genotypes in this bumblebee/trypanosome model.

Delayed virulence and limited costs promote fecundity compensation upon infection.

Individuals invest limited resources across vital tasks such as reproduction and survival. Individuals can spread reproductive investment over their lifetime, but cues of death or reduced fitness can influence this investment. In some systems, cues of infection induce early but costly reproduction through fecundity compensation as future reproduction becomes uncertain. A key aspect of parasite biology is the delay between exposure to parasites and the onset of virulence. This creates an important window of opportunity for hosts to respond to infection. Existing models have not accounted for this delay or the costs borne by offspring. We combine a theoretical and experimental approach to assess the role of costs and the importance of delay in virulence on fecundity compensation. We find that a delay in virulence selects for plastic fecundity responses even with moderate offspring costs. We tested our model experimentally by exposing pea aphids, Acyrthosiphon pisum, to various ecologically relevant cues of infection and monitored lifetime reproduction and survival of these aphids and their offspring. Our challenges induced fecundity compensation, but we did not detect any costs in mothers or offspring. We predict that the relationship between the costs and the delay in onset of virulence, as found here, determines the success of fecundity compensation as an adaptation against parasitism.

Protein-poor diet reduces host-specific immune gene expression in Bombus terrestris.

Parasites infect hosts non-randomly as genotypes of hosts vary in susceptibility to the same genotypes of parasites, but this specificity may be modulated by environmental factors such as nutrition. Nutrition plays an important role for any physiological investment. As immune responses are costly, resource limitation should negatively affect immunity through trade-offs with other physiological requirements. Consequently, nutritional limitation should diminish immune capacity in general, but does it also dampen differences among hosts? We investigated the effect of short-term pollen deprivation on the immune responses of our model host Bombus terrestris when infected with the highly prevalent natural parasite Crithidia bombi. Bumblebees deprived of pollen, their protein source, show reduced immune responses to infection. They failed to upregulate a number of genes, including antimicrobial peptides, in response to infection. In particular, they also showed less specific immune expression patterns across individuals and colonies. These findings provide evidence for how immune responses on the individual-level vary with important elements of the environment and illustrate how nutrition can functionally alter not only general resistance, but also alter the pattern of specific host-parasite interactions.

Double trouble: trypanosomatids with two hosts have lower infection prevalence than single host trypanosomatids.

Trypanosomatids are a diverse family of protozoan parasites, some of which cause devastating human and livestock diseases. There are two distinct infection life cycles in trypanosomatids; some species complete their entire life cycle in a single host (monoxenous) while others infect two hosts (dixenous). Dixenous trypanosomatids are mostly vectored by insects, and the human trypanosomatid diseases are caused mainly by vectored parasites. While infection prevalence has been described for subsets of hosts and trypanosomatids, little is known about whether monoxenous and dixenous trypanosomatids differ in infection prevalence. Here, we use meta-analyses to synthesise all published evidence of trypanosomatid infection prevalence for the last two decades, encompassing 931 unique host-trypansomatid systems. In examining 584 studies that describe infection prevalence, we find, strikingly, that monoxenous species are two-fold more prevalent than dixenous species across all hosts. We also find that dixenous trypanosomatids have significantly lower infection prevalence in insects than their non-insect hosts. To our knowledge, these results reveal for the first time, a fundamental difference in infection prevalence according to host specificity where vectored species might have lower infection prevalence as a result of a potential 'jack of all trades, master of none' style trade-off between the vector and subsequent hosts.

Influence of social lifestyles on host-microbe symbioses in the bees.

Microbiomes are increasingly recognised as critical for the health of an organism. In eusocial insect societies, frequent social interactions allow for high-fidelity transmission of microbes across generations, leading to closer host-microbe coevolution. The microbial communities of bees with other social lifestyles are less studied, and few comparisons have been made between taxa that vary in social structure. To address this gap, we leveraged a cloud-computing resource and publicly available transcriptomic data to conduct a survey of microbial diversity in bee samples from a variety of social lifestyles and taxa. We consistently recover the core microbes of well-studied corbiculate bees, supporting this method's ability to accurately characterise microbial communities. We find that the bacterial communities of bees are influenced by host location, phylogeny and social lifestyle, although no clear effect was found for fungal or viral microbial communities. Bee genera with more complex societies tend to harbour more diverse microbes, with Wolbachia detected more commonly in solitary tribes. We present a description of the microbiota of Euglossine bees and find that they do not share the "corbiculate core" microbiome. Notably, we find that bacteria with known anti-pathogenic properties are present across social bee genera, suggesting that symbioses that enhance host immunity are important with higher sociality. Our approach provides an inexpensive means of exploring microbiomes of a given taxa and identifying avenues for further research. These findings contribute to our understanding of the relationships between bees and their associated microbial communities, highlighting the importance of considering microbiome dynamics in investigations of bee health.

Lack of genetic differentiation between monarch butterflies with divergent migration destinations.

Monarch butterflies are best known for their spectacular annual migration from eastern North America to Mexico. Monarchs also occur in the North American states west of the Rocky Mountains, from where they fly shorter distances to the California Coast. Whether eastern and western North American monarchs form one genetic population or are genetically differentiated remains hotly debated, and resolution of this debate is essential to understand monarch migration patterns and to protect this iconic insect species. We studied the genetic structure of North American migratory monarch populations, as well as nonmigratory populations in Hawaii and New Zealand. Our results show that eastern and western migratory monarchs form one admixed population and that monarchs from Hawaii and New Zealand have genetically diverged from North American butterflies. These findings suggest that eastern and western monarch butterflies maintain their divergent migrations despite genetic mixing. The finding that eastern and western monarchs form one genetic population also suggests that the conservation of overwintering sites in Mexico is crucial for the protection of monarchs in both eastern and western North America.

Ecological immunogenetics of life-history traits in a model amphibian.

Major histocompatibility complex (MHC) genes determine immune repertoires and social preferences of vertebrates. Immunological regulation of microbial assemblages associated with individuals influences their sociality, and should also affect their life-history traits. We exposed Xenopus laevis tadpoles to water conditioned by adult conspecifics. Then, we analysed tadpole growth, development and survivorship as a function of MHC class I and class II peptide-binding region amino acid sequence similarities between tadpoles and frogs that conditioned the water to which they were exposed. Tadpoles approached metamorphosis earlier and suffered greater mortality when exposed to immunogenetically dissimilar frogs. The results suggest that developmental regulatory cues, microbial assemblages or both are specific to MHC genotypes. Tadpoles may associate with conspecifics with which they share microbiota to which their genotypes are well adapted.

In vitro larval rearing method of eusocial bumblebee Bombus terrestris for toxicity test.

Bumblebees are important pollinators of wild and agricultural plants but recently have been declining due to various stressors, such as pesticides and diseases. Because of the haplo-diploid sex determination system in hymenopterans, experiments using micro-colonies (small sub colonies without a queen) to identify risks to bumblebee health are limited as they are only able to produce males. Therefore, an experimental protocol for rearing bumblebee larvae in vitro is needed to better understand effects on worker larvae. Here, we aimed to establish a rearing method for larvae of Bombus terrestris for use in risk assessment assays. To confirm the validity of our rearing method, we tested two insecticides used for tomato cultivation, chlorfenapyr and dinotefuran. Bombus terrestris larvae fed with a high nutrient quantity and quality diet increased growth per day. All chlorfenapyr-exposed individuals died within 10 days at 2000-fold dilution, an application dose used for tomatoes. There were significant differences in adult emergence rate among almost all chlorfenapyr treatments. On the other hand, sublethal dinotefuran-exposure did not affect rates of pupation and adult emergence, growth, or larval and pupal periods. Although larvae were smaller than in the natural colony, this rearing method for B. terrestris larvae proved to be effective at evaluating realistic sub-colonies to pesticide exposures.

Genetic variation and microbiota in bumble bees cross-infected by different strains of C. bombi.

The bumblebee Bombus terrestris is commonly infected by a trypanosomatid gut parasite Crithidia bombi. This system shows a striking degree of genetic specificity where host genotypes are susceptible to different genotypes of parasite. To a degree, variation in host gene expression underlies these differences, however, the effects of standing genetic variation has not yet been explored. Here we report on an extensive experiment where workers of twenty colonies of B. terrestris were each infected by one of twenty strains of C. bombi. To elucidate the host's genetic bases of susceptibility to infection (measured as infection intensity), we used a low-coverage (~2 x) genome-wide association study (GWAS), based on angsd, and a standard high-coverage (~15x) GWAS (with a reduced set from a 8 x 8 interaction matrix, selected from the full set of twenty). The results from the low-coverage approach remained ambiguous. The high-coverage approach suggested potentially relevant genetic variation in cell surface and adhesion processes. In particular, mucin, a surface mucoglycoprotein, potentially affecting parasite binding to the host gut epithelia, emerged as a candidate. Sequencing the gut microbial community of the same bees showed that the abundance of bacterial taxa, such as Gilliamella, Snodgrassella, or Lactobacillus, differed between 'susceptible' and 'resistant' microbiota, in line with earlier studies. Our study suggests that the constitutive microbiota and binding processes at the cell surface are candidates to affect infection intensity after the first response (captured by gene expression) has run its course. We also note that a low-coverage approach may not be powerful enough to analyse such complex traits. Furthermore, testing large interactions matrices (as with the full 20 x 20 combinations) for the effect of interaction terms on infection intensity seems to blur the specific host x parasite interaction effects, likely because the outcome of an infection is a highly non-linear process dominated by variation in individually different pathways of host defence (immune) responses.

Aphid reproductive investment in response to mortality risks.

BACKGROUND: Aphids are striking in their prodigious reproductive capacity and reliance on microbial endosymbionts, which provision their hosts with necessary amino acids and provide protection against parasites and heat stress. Perhaps as a result of this bacterial dependence, aphids have limited immune function that may leave them vulnerable to bacterial pathogens. An alternative, non-immunological response that may be available to infected aphids is to increase reproduction, thereby ameliorating fitness loss from infection. Such a response would reduce the need to mount a potentially energetically costly immune response, and would parallel that of other hosts that alter life-history traits when there is a risk of infection. Here we examined whether pea aphids (Acyrthosiphon pisum) respond to immunological challenges by increasing reproduction. As a comparison to the response to the internal cue of risk elicited by immunological challenge, we also exposed pea aphids to an external cue of risk--the aphid alarm pheromone (E)-ß-farnesene (EBF), which is released in the presence of predators. For each challenge, we also examined whether the presence of symbionts modified the host response, as maintaining host fitness in the face of challenge would benefit both the host and its dependent bacteria. RESULTS: We found that aphids stabbed abdominally with a sterile needle had reduced fecundity relative to control aphids but that aphids stabbed with a needle bearing heat-killed bacteria had reproduction intermediate, and statistically indistinguishable, to the aphids stabbed with a sterile needle and the controls. Aphids with different species of facultative symbiotic bacteria had different reproductive patterns overall, but symbionts in general did not alter aphid reproduction in response to bacterial exposure. However, in response to exposure to alarm pheromone, aphids with Hamiltonella defensa or Serratia symbiotica symbiotic infections increased reproduction but those without a facultative symbiont or with Regiella insecticola did not. CONCLUSIONS: Overall, our results suggest that pea aphids are able to increase their reproduction in response to specific cues and that symbiont presence sometimes moderates this response. Such increased reproduction in response to risk of death increases the fitness of both aphids and their vertically transmitted symbionts, and since these organisms have high reproductive capacity, slight increases in reproduction could lead to a very large numerical advantage later in the season. Thus both symbiotic partners can benefit by increasing host fecundity under dangerous conditions.

Royal Decree: Gene Expression in Trans-Generationally Immune Primed Bumblebee Workers Mimics a Primary Immune Response.

Invertebrates lack the cellular and physiological machinery of the adaptive immune system, but show specificity in their immune response and immune priming. Functionally, immune priming is comparable to immune memory in vertebrates. Individuals that have survived exposure to a given parasite are better protected against subsequent exposures. Protection may be cross-reactive, but demonstrations of persistent and specific protection in invertebrates are increasing. This immune priming can cross generations ("trans-generational" immune priming), preparing offspring for the prevailing parasite environment. While these phenomena gain increasing support, the mechanistic foundations underlying such immune priming, both within and across generations, remain largely unknown. Using a transcriptomic approach, we show that exposing bumblebee queens with an injection of heat-killed bacteria, known to induce trans-generational immune priming, alters daughter (worker) gene expression. Daughters, even when unexposed themselves, constitutively express a core set of the genes induced upon direct bacterial exposure, including high expression of antimicrobial peptides, a beta-glucan receptor protein implicated in bacterial recognition and the induction of the toll signaling pathway, and slit-3 which is important in honeybee immunity. Maternal exposure results in a distinct upregulation of their daughters' immune system, with a signature overlapping with the induced individual response to a direct exposure. This will mediate mother-offspring protection, but also associated costs related to reconfiguration of constitutive immune expression. Moreover, identification of conserved immune pathways in memory-like responses has important implications for our understanding of the innate immune system, including the innate components in vertebrates, which share many of these pathways.