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Background: Despite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey bee Apis mellifera and the eastern bumble bee Bombus impatiens. Results: We find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments. Conclusions: Overall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage. 6S3bFgh36a8k5RNed1PpF4 Video Abstract [ABSTRACT FROM AUTHOR]

The gut microbiota of bees affects nutrition, immunity and host fitness, yet the roles of diet, sociality and geographical variation in determining microbiome structure, including variant‐level diversity and relatedness, remain poorly understood. Here, we use full‐length 16S rRNA amplicon sequencing to compare the crop and gut microbiomes of two incipiently social carpenter bee species, Xylocopa sonorina and Xylocopa tabaniformis, from multiple geographical sites within each species' range. We found that Xylocopa species share a set of core taxa consisting of Bombilactobacillus, Bombiscardovia and Lactobacillus, found in >95% of all individual bees sampled, and Gilliamella and Apibacter were also detected in the gut of both species with high frequency. The crop bacterial community of X. sonorina comprised nearly entirely Apilactobacillus with occasionally abundant nectar bacteria. Despite sharing core taxa, Xylocopa species' microbiomes were distinguished by multiple bacterial lineages, including species‐specific variants of core taxa. The use of long‐read amplicons revealed otherwise cryptic species and population‐level differentiation in core microbiome members, which was masked when a shorter fragment of the 16S rRNA (V4) was considered. Of the core taxa, Bombilactobacillus and Bombiscardovia exhibited differentiation in amplicon sequence variants among bee populations, but this was lacking in Lactobacillus, suggesting that some bacterial genera in the gut may be structured by different processes. We conclude that these Xylocopa species host a distinctive microbiome, similar to that of previously characterized social corbiculate apids, which suggests that further investigation to understand the evolution of the bee microbiome and its drivers is warranted. [ABSTRACT FROM AUTHOR]

Bumble bees are important pollinators that face threats from multiple sources, including agrochemical application. Declining bumble bee populations have been linked to fungicide application, which could directly affect the fungi often found in the stored food and gastrointestinal (GI) tract of healthy bumble bees.We test the hypothesis that fungicides impact bee health by disrupting bee–fungi interactions. We examined the interactive effects of the fungicide propiconazole and fungal supplementation on the survival, reproduction and microbiome composition of microcolonies (queenless colonies) using two species, Bombus vosnesenskii and B. impatiens.We found that in B. vosnesenskii, fungicide exposure decreased survival, while fungal supplementation mitigated fungicide effects. For B. impatiens, fungicide application had no effect, but fungal supplementation improved survival and offspring production.Fungicides reduced fungal abundance in B. vosnesenskii microcolonies, but not in B. impatiens, where instead fungal addition decreased fungal abundance (ITS copy number). Fungal composition varied between treatments but differently between bee species. In B. impatiens, fungal addition increased microbiome diversity. In B. vosnesenskii, the abundance of the pathogen Ascosphaera was negatively associated with survival, while the yeast Zygosaccharomyces was positively associated with survival.Our results highlight that bumble bee species differ in response to fungicides and in the nature of bee‐fungi associations. Fungicides can alter bee–fungi interactions with consequences for bee survival and reproduction, and exploring the mechanisms of such interactions, including interactions among fungi in the bee GI tract, may offer insights into bumble bee biology and conservation strategies. [ABSTRACT FROM AUTHOR]