Dataset Information

Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi.

ABSTRACT: 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.

SUBMITTER: Salathe R

PROVIDER: S-EPMC3498296 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi.

Salathé Rahel R Tognazzo Martina M Schmid-Hempel Regula R Schmid-Hempel Paul P

PloS one 20121114 11

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 me ...[more]

PMID: 23155449

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Project description: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.

Project description:Recent declines in bumble bee populations are of great concern and have prompted critical evaluations of the role of pathogen introductions and host resistance in bee health. One factor that may influence host resilience when facing infection is the gut microbiota. Previous experiments with Bombus terrestris, a European bumble bee, showed that the gut microbiota can protect against Crithidia bombi, a widespread trypanosomatid parasite of bumble bees. However, the particular characteristics of the microbiome responsible for this protective effect have thus far eluded identification. Using wild and commercially sourced Bombus impatiens, an important North American pollinator, we conducted cross-wise microbiota transplants to naive hosts of both backgrounds and challenged them with a Crithidia parasite. As with B. terrestris, we find that microbiota-dependent protection against Crithidia operates in B. impatiens Lower Crithidia infection loads were experimentally associated with high microbiome diversity, large gut bacterial populations, and the presence of Apibacter, Lactobacillus Firm-5, and Gilliamella spp. in the gut community. These results indicate that even subtle differences between gut community structures can have a significant impact on a microbiome's ability to defend against parasite infections.IMPORTANCE Many wild bumble bee populations are under threat due to human activity, including through the introduction of pathogens via commercially raised bees. Recently, it was found that the bumble bee gut microbiota can help defend against a common parasite, Crithidia bombi, but the particular factors contributing to this protection are unknown. Using both wild and commercially raised bees, we conducted microbiota transplants to show that microbiome diversity, total gut bacterial load, and the presence of certain core members of the microbiota may all impact bee susceptibility to Crithidia infection. Bee origin (genetic background) was also a factor. Finally, by examining this phenomenon in a previously uninvestigated bee species, our study demonstrates that microbiome-mediated resistance to Crithidia is conserved across multiple bumble bee species. These findings highlight how intricate interactions between hosts, microbiomes, and parasites can have wide-ranging consequences for the health of ecologically important species.

Dataset Information

Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi.

Publications

Probing mixed-genotype infections I: extraction and cloning of infections from hosts of the trypanosomatid Crithidia bombi.

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