RNA viruses of Crithidia bombi, a parasite of bumblebees.

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi - a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

Impact of climate change on parasite infection of an important pollinator depends on host genotypes.

Climate change is predicted to affect host-parasite interactions, and for some hosts, parasite infection is expected to increase with rising temperatures. Global population declines of important pollinators already have been attributed to climate change and parasitism. However, the role of climate in driving parasite infection and the genetic basis for pollinator hosts to respond often remain obscure. Based on decade-long field data, we investigated the association between climate and Nosema bombi (Microsporidia) infection of buffed-tailed bumblebees (Bombus terrestris), and whether host genotypes play a role. For this, we genotyped 876 wild bumblebee queens and screened for N. bombi infection of those queens between 2000 and 2010. We recorded seven climate parameters during those 11 years and tested for correlations between climate and infection prevalence. Here we show that climatic factors drive N. bombi infection and that the impact of climate depends on mitochondrial DNA cytochrome oxidase I (COI) haplotypes of the host. Infection prevalence was correlated with climatic variables during the time when queens emerge from hibernation. Remarkably, COI haplotypes best predict this association between climatic factors and infection. In particular, two host haplotypes ("A" and "B") displayed phenotypic plasticity in response to climatic variation: Temperature was positively correlated with infection of host haplotype B, but not haplotype A. The likelihood of infection of haplotype A was associated with moisture, conferring greater resistance to parasite infection during wetter years. In contrast, infection of haplotype B was unrelated to moisture. To the best of our knowledge, this is the first study that identifies specific host genotypes that confer differential parasite resistance under variable climatic conditions. Our results underscore the importance of mitochondrial haplotypes to ward off parasites in a changing climate. More broadly, this also suggests that COI may play a pertinent role in climate change adaptations of insect pollinators.

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.

Function and mechanisms in defence strategies.

A useful discussion of defence strategies cannot do without linking defence mechanisms to their function, that is, their contributions to fitness. Whereas the former is the domain of immunology, the latter is the subject of evolutionary ecology. For this, the concepts of the defence chart and the disease space can be used to connect the two domains and to sharpen the focus. These use different approaches but converge to the same end, that is, to understand what fitness costs and benefits are associated with existing mechanisms and how to identify the best defence strategy in a given environment.

Sociality and parasite transmission.

Parasites and their social hosts form many different relationships. But what kind of selection regimes are important? A look at the parameters that determine fitness of the two parties suggests that social hosts differ from solitary ones primarily in the structure of transmission pathways. Because transmission is, both, the physical encounter of a new host and infecting it, several different elements determine parasite transmission success. These include spatial distance, genetic distance, or the temporal and ecological niche overlaps. Combing these elements into a 'generalized transmission distance' that determines parasite fitness aids in the identification of the critical steps. For example, short-distance transmission to genetically similar hosts within the social group is the most frequent process under sociality. Therefore, spatio-genetical distances are the main driver of parasite fitness. Vice versa, the generalized distance identifies the critical host defences. In this case, host defences should be primarily selected to defend against the within-group spread of an infection, especially among closely related group members.

Bee Parasites: Don't Lose Your Flagellum.

Bees can protect themselves from infection by accumulating specific nectars in the honey crop. A new study shows that as parasites pass through the crop, their flagellum is thereby removed, which prevents host infection.

Genomic Variation among Strains of Crithidia bombi and C. expoeki.

In this study, we sequenced and analyzed the genomes of 40 strains, in addition to the already-reported two type strains, of two Crithidia species infecting bumblebees in Alaska and Central Europe and demonstrated that different strains of Crithidia bombi and C. expoeki vary considerably in terms of single nucleotide polymorphisms and gene copy number. Based on the genomic structure, phylogenetic analyses, and the pattern of copy number variation, we confirmed the status of C. expoeki as a separate species. The Alaskan populations appear to be clearly separated from those of Central Europe. This pattern fits a scenario of rapid host-parasite coevolution, where the selective advantage of a given parasite strain is only temporary. This study provides helpful insights into possible scenarios of selection and diversification of trypanosomatid parasites.IMPORTANCE A group of trypanosomatid flagellates includes several well-studied medically and economically important parasites of vertebrates and plants. Nevertheless, the vast majority of trypanosomatids infect only insects (mostly flies and true bugs) and, because of that, has attracted little research attention in the past. Of several hundred trypanosomatid species, only four can infect bees (honeybees and bumblebees). Because of such scarcity, these parasites are severely understudied. We analyzed whole-genome information for a total of 42 representatives of bee-infecting trypanosomatids collected in Central Europe and Alaska from a population genetics point of view. Our data shed light on the evolution, selection, and diversification in this important group of trypanosomatid parasites.

High Gut Microbiota Diversity Provides Lower Resistance against Infection by an Intestinal Parasite in Bumblebees.

The microbiome, especially the gut flora, is known to affect the interaction between parasites and their hosts. In this context, a parasitic infection can be viewed as an invasion into the preexisting microbial ecological community. Hence, in addition to the intrinsic defense mechanisms of the host itself, infection success depends on the colonization resistance of the microbiota. In the bumblebee Bombus terrestris, the microbiota provides resistance to the intestinal parasite Crithidia bombi, yet which properties actually provide protection remains largely unknown. Here, we show that the community structure of the gut microbiota-in terms of bacterial operational taxonomic units (OTUs) of 16S ribosomal RNA gene sequences-before parasite exposure can be informative of the eventual infection outcome. Specifically, higher microbiota OTU diversity is associated with less resistance. However, the microbial community structure does not differ between infected and noninfected individuals or between infected individuals of varying susceptibility. This suggests that parasite infection success depends on the microbiota composition but that subsequent changes occur, although the exact alteration that occurs remains elusive. In fact, the bumblebee microbiota is surprisingly unaffected by parasite exposure and infection. Rather, the microbiota-host interaction before parasite exposure seems to be a key mechanism regulating resistance to infection.

The genomes of Crithidia bombi and C. expoeki, common parasites of bumblebees.

Trypanosomatids (Trypanosomatidae, Kinetoplastida) are flagellated protozoa containing many parasites of medical or agricultural importance. Among those, Crithidia bombi and C. expoeki, are common parasites in bumble bees around the world, and phylogenetically close to Leishmania and Leptomonas. They have a simple and direct life cycle with one host, and partially castrate the founding queens greatly reducing their fitness. Here, we report the nuclear genome sequences of one clone of each species, extracted from a field-collected infection. Using a combination of Roche 454 FLX Titanium, Pacific Biosciences PacBio RS, and Illumina GA2 instruments for C. bombi, and PacBio for C. expoeki, we could produce high-quality and well resolved sequences. We find that these genomes are around 32 and 34 MB, with 7,808 and 7,851 annotated genes for C. bombi and C. expoeki, respectively-which is somewhat less than reported from other trypanosomatids, with few introns, and organized in polycistronic units. A large fraction of genes received plausible functional support in comparison primarily with Leishmania and Trypanosoma. Comparing the annotated genes of the two species with those of six other trypanosomatids (C. fasciculata, L. pyrrhocoris, L. seymouri, B. ayalai, L. major, and T. brucei) shows similar gene repertoires and many orthologs. Similar to other trypanosomatids, we also find signs of concerted evolution in genes putatively involved in the interaction with the host, a high degree of synteny between C. bombi and C. expoeki, and considerable overlap with several other species in the set. A total of 86 orthologous gene groups show signatures of positive selection in the branch leading to the two Crithidia under study, mostly of unknown function. As an example, we examined the initiating glycosylation pathway of surface components in C. bombi, finding it deviates from most other eukaryotes and also from other kinetoplastids, which may indicate rapid evolution in the extracellular matrix that is involved in interactions with the host. Bumble bees are important pollinators and Crithidia-infections are suspected to cause substantial selection pressure on their host populations. These newly sequenced genomes provide tools that should help better understand host-parasite interactions in these pollinator pathogens.

Host effects on microbiota community assembly.

To what extent host-associated microbiota assembly is driven by host selection or simply by happenstance remains an open question in microbiome research. Here, we take a first step towards elucidating the relative importance of host selection on the establishing gut microbial community in an ecologically relevant organism. We presented germ-free bumblebee, Bombus terrestris, workers from 10 colonies with a "global" microbial species pool comprised of an equal mixture of the gut microbiota of all colonies. By means of 16S amplicon sequencing, we found that overall microbiota community composition was generally shifted between pool-exposed workers compared to workers that naturally acquired their gut microbiota, but that the specific composition of the established microbiota also depended on colony identity (e.g. genetic background). Because the microbiota is protective against parasite infection in this system, variation in the filtering of a beneficial microbial community can have important consequences for host resistance and eventual co-evolution with parasites.

Parasite infection of specific host genotypes relates to changes in prevalence in two natural populations of bumblebees.

The antagonistic relationship between parasites and their hosts is strongly influenced by genotype-by-genotype interactions. Defense against parasitism is commonly studied in the context of immune system-based mechanisms and, thus, the focus in the search for candidate genes in host-parasite interactions is often on immune genes. In this study, we investigated the association between prevalence of parasite infection and host mitochondrial DNA (mtDNA) haplotypes in two natural populations of bumblebees (Bombus terrestris). The two most common haplotypes of the host populations, termed A and B, differ by a single nonsynonymous nucleotide substitution within the coding region of cytochrome oxidase I, an important player in metabolic pathways. We screened infection by Nosema bombi, a common endoparasite of bumblebees, and the corresponding host mtDNA-haplotype frequencies in over 1400 bumblebees between 2000 and 2010. The island population of Gotland showed lower mtDNA diversity compared to the mainland population in Switzerland. Over time, we observed large fluctuations in infection prevalence, as well as variation in host haplotype frequencies in both populations. Our long-term observation revealed that N. bombi infection of specific host genotypes is transient: We found that with increasing infection prevalence, proportionally more individuals with haplotype B, but fewer individuals with haplotype A were infected. This suggests that the presence of N. bombi in specific host genotypes relates to infection prevalence. This may be a result of parasite competition, or differential resilience of host types to ward off infections. The findings highlight the important role of host mtDNA haplotypes in the interaction with parasites.

Parasites and Their Social Hosts.

The study of parasitism in socially living organisms shows that social group size correlates with the risk of infection, but group structure - and thus differences in contact networks - is generally more important. Also, genetic makeup or environmental conditions have effects. 'Social immunity' focuses on defence against parasites that are particular to social living. Recently, the role of socially transmitted microbiota for defence has become a focus, too. But whether and how parasites adapt to social organisms - beyond adaptation to solitary hosts - is poorly understood. Genomic and proteomic methods, as well as network analysis, will be tools that hold promise for many unsolved questions, but to expand our concepts in the first place is a much needed agenda.

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.

Perspectives on the evolutionary ecology of arthropod antimicrobial peptides.

Antimicrobial peptides (AMPs) are important elements of the innate immune defence in multicellular organisms that target and kill microbes. Here, we reflect on the various points that are raised by the authors of the 11 contributions to a special issue of Philosophical Transactions on the 'evolutionary ecology of arthropod antimicrobial peptides'. We see five interesting topics emerging. (i) AMP genes in insects, and perhaps in arthropods more generally, evolve much slower than most other immune genes. One explanation refers to the constraints set by AMPs being part of a finely tuned defence system. A new view argues that AMPs are under strong stabilizing selection. Regardless, this striking observation still invites many more questions than have been answered so far. (ii) AMPs almost always are expressed in combinations and sometimes show expression patterns that are dependent on the infectious agent. While it is often assumed that this can be explained by synergistic interactions, such interactions have rarely been demonstrated and need to be studied further. Moreover, how to define synergy in the first place remains difficult and needs to be addressed. (iii) AMPs play a very important role in mediating the interaction between a host and its mutualistic or commensal microbes. This has only been studied in a very small number of (insect) species. It has become clear that the very same AMPs play different roles in different situations and hence are under concurrent selection. (iv) Different environments shape the physiology of organisms; especially the host-associated microbial communities should impact on the evolution host AMPs. Studies in social insects and some organisms from extreme environments seem to support this notion, but, overall, the evidence for adaptation of AMPs to a given environment is scant. (v) AMPs are considered or already developed as new drugs in medicine. However, bacteria can evolve resistance to AMPs. Therefore, in the light of our limited understanding of AMP evolution in the natural context, and also the very limited understanding of the evolution of resistance against AMPs in bacteria in particular, caution is recommended. What is clear though is that study of the ecology and evolution of AMPs in natural systems could inform many of these outstanding questions, including those related to medical applications and pathogen control.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Insect antimicrobial peptides act synergistically to inhibit a trypanosome parasite.

The innate immune system provides protection from infection by producing essential effector molecules, such as antimicrobial peptides (AMPs) that possess broad-spectrum activity. This is also the case for bumblebees, Bombus terrestris, when infected by the trypanosome, Crithidia bombi Furthermore, the expressed mixture of AMPs varies with host genetic background and infecting parasite strain (genotype). Here, we used the fact that clones of C. bombi can be cultivated and kept as strains in medium to test the effect of various combinations of AMPs on the growth rate of the parasite. In particular, we used pairwise combinations and a range of physiological concentrations of three AMPs, namely Abaecin, Defensin and Hymenoptaecin, synthetized from the respective genomic sequences. We found that these AMPs indeed suppress the growth of eight different strains of C. bombi, and that combinations of AMPs were typically more effective than the use of a single AMP alone. Furthermore, the most effective combinations were rarely those consisting of maximum concentrations. In addition, the AMP combination treatments revealed parasite strain specificity, such that strains varied in their sensitivity towards the same mixtures. Hence, variable expression of AMPs could be an alternative strategy to combat highly variable infections.This article is part of the themed issue 'Evolutionary ecology of arthropod antimicrobial peptides'.

Immune response and gut microbial community structure in bumblebees after microbiota transplants.

Microbial communities are a key component of host health. As the microbiota is initially 'foreign' to a host, the host's immune system should respond to its acquisition. Such variation in the response should relate not only to host genetic background, but also to differences in the beneficial properties of the microbiota. However, little is known about such interactions. Here, we investigate the gut microbiota of the bumblebee, Bombus terrestris, which has a protective function against the bee's natural trypanosome gut parasite, Crithidia bombi We transplanted 'resistant' and 'susceptible' microbiota into 'resistant' and 'susceptible' host backgrounds, and studied the activity of the host immune system. We found that bees from different resistance backgrounds receiving a microbiota differed in aspects of their immune response. At the same time, the elicited immune response also depended on the received microbiota's resistance phenotype. Furthermore, the microbial community composition differed between microbiota resistance phenotypes (resistant versus susceptible). Our results underline the complex feedback between the host's ability to potentially exert selection on the establishment of a microbial community and the influence of the microbial community on the host immune response in turn.

The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions.

As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the microbiome. The bee microbiome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee microbiome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

Colony pace: a life-history trait affecting social insect epidemiology.

Among colonies of social insects, the worker turnover rate (colony 'pace') typically shows considerable variation. This has epidemiological consequences for parasites, because in 'fast-paced' colonies, with short-lived workers, the time of parasite residence in a given host will be reduced, and further transmission may thus get less likely. Here, we test this idea and ask whether pace is a life-history strategy against infectious parasites. We infected bumblebees (Bombus terrestris) with the infectious gut parasite Crithidia bombi, and experimentally manipulated birth and death rates to mimic slow and fast pace. We found that fewer workers and, importantly, fewer last-generation workers that are responsible for rearing sexuals were infected in colonies with faster pace. This translates into increased fitness in fast-paced colonies, as daughter queens exposed to fewer infected workers in the nest are less likely to become infected themselves, and have a higher chance of founding their own colonies in the next year. High worker turnover rate can thus act as a strategy of defence against a spreading infection in social insect colonies.

Large scale patterns of abundance and distribution of parasites in Mexican bumblebees.

Bumblebees are highly valued for their pollination services in natural ecosystems as well as for agricultural crops. These precious pollinators are known to be declining worldwide, and one major factor contributing to this decline are infections by parasites. Knowledge about parasites in wild bumblebee populations is thus of paramount importance for conservation purposes. We here report the geographical distribution of Crithidia and Nosema, two common parasites of bumblebees, in a yet poorly investigated country: Mexico. Based on sequence divergence of the Cytochrome b and Glycosomal glyceraldehyde phosphate deshydrogenase (gGPDAH) genes, we discovered the presence of a new Crithidia species, which is mainly distributed in the southern half of the country. It is placed by Bayesian inference as a sister species to C. bombi. We suggest the name Crithidia mexicana for this newly discovered organism. A population of C. expoeki was encountered concentrated on the flanks of the dormant volcanic mountain, Iztaccihuatl, and microsatellite data showed evidence of a bottleneck in this population. This study is the first to provide a large-scale insight into the health status of endemic bumblebees in Mexico, based on a large sample size (n=3,285 bees examined) over a variety of host species and habitats.