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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.

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.

ABSTRACT 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.

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.

ABSTRACT 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.

We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis ("cloning"). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.

Mixed-genotype infections have major consequences for many essential elements of host-parasite interactions. With genetic exchange between co-infecting parasite genotypes increased diversity among parasite offspring and the emergence of novel genotypes from infected hosts is possible. We here investigated mixed- genotype infections using the host, Bombus spp. and its trypanosome parasite Crithidia bombi as our study case. The natural infections of C. bombi were genotyped with a novel method for a representative sample of workers and spring queens in Switzerland. We found that around 60% of all infected hosts showed mixed-genotype infections with an average of 2.47±0.22 (S.E.) and 3.65±1.02 genotypes per worker or queen, respectively. Queens, however, harboured up to 29 different genotypes. Based on the genotypes of co-infecting strains, these could be putatively assigned to either 'primary' and 'derived' genotypes - the latter resulting from genetic exchange among the primary genotypes. High genetic relatedness among co-infecting derived but not primary genotypes supported this scenario. Co-infection in queens seems to be a major driver for the diversity of genotypes circulating in host populations.