Gut microbial communities confer protection against natural pathogens in important pollinators from the genera Bombus and Apis. In commercial species B. terrestris and B. impatiens, the microbiota increases their resistance to the common and virulent trypanosomatid parasite Crithidia bombi. However, the mechanisms by which gut microorganisms protect the host are still unknown. Here, we test two hypotheses: microbiota protect the host 1) through stimulation of its immune response or protection of the gut epithelium and 2) by competing for resources with the parasite inside the gut. To test them, we reduced the microbiota of workers and fed part of them with microbiota supplements. We exposed them to an infectious dose of C. bombi and characterised gene expression and gut microbiota composition. We examined the expression of three antimicrobial peptide (AMP) genes and Mucin-5AC, a gene with a putative role in gut epithelium protection, using qPCR. Although a protective effect against C. bombi was observed in bumblebees with supplemented microbiota, we did not observe an effect of the microbiota on gene expression that could explain alone the protective effect observed. On the other hand, we found an increased relative abundance of Lactobacillus bacteria within the gut of infected workers and a negative correlation of this genus with Gilliamella and Snodgrassella genera. Therefore, our results point to a displacement of bumblebee endosymbionts by C. bombi that might be caused by competition for space and nutrients between the parasite and the microbiota within the gut.

There is ample evidence that macroscopic animals form geographic clusters termed as zoogeographic realms (zones), whereas distributions of species of microscopic animals are still poorly understood. The common view has been that micrometazoans, thanks to their putatively excellent dispersal abilities, are subject to the ‘Everything is Everywhere but environment selects’ hypothesis (EiE). One of such groups, <1 mm in length, are limnoterrestrial water bears (Tardigrada), which can additionally enter cryptobiosis that should further enhance their potential for long distance dispersion (e.g. by wind). However, an increasing number of studies, including the most recent phylogeny of a eutardigrade genus Milnesium, seem to question the general applicability of the EiE hypothesis to tardigrade species. Nevertheless, all the Milnesium phylogenies published to date were based on a limited number of populations, which are likely to falsely suggest limited geographic ranges. Thus, in order to comprehensively test the EiE hypothesis, here, we considerably enlarged the Milnesium dataset both taxonomically and geographically, and we analysed it in tandem with climate type and reproductive mode. Additionally, we time-calibrated our phylogeny to align it with major geological events. Our results show that, although cases of long distance dispersal are present, they seem to be rare and mostly ancient. Overall, Milnesium species are restricted to single zoogeographic realms, which suggests that these tardigrades have limited dispersal abilities. Finally, our results also suggest that the breakdown of Gondwana may influenced the evolutionary history of Milnesium. In conclusion, phylogenetic relationships within the genus seem to be determined mainly by paleogeography.