Turnover of strain-level diversity modulates functional traits in the honeybee gut microbiome between nurses and foragers.

BACKGROUND: Strain-level diversity is widespread among bacterial species and can expand the functional potential of natural microbial communities. However, to what extent communities undergo consistent shifts in strain composition in response to environmental/host changes is less well understood. RESULTS: Here, we used shotgun metagenomics to compare the gut microbiota of two behavioral states of the Western honeybee (Apis mellifera), namely nurse and forager bees. While their gut microbiota is composed of the same bacterial species, we detect consistent changes in strain-level composition between nurses and foragers. Single nucleotide variant profiles of predominant bacterial species cluster by behavioral state. Moreover, we identify strain-specific gene content related to nutrient utilization, vitamin biosynthesis, and cell-cell interactions specifically associated with the two behavioral states. CONCLUSIONS: Our findings show that strain-level diversity in host-associated communities can undergo consistent changes in response to host behavioral changes modulating the functional potential of the community.

Vast Differences in Strain-Level Diversity in the Gut Microbiota of Two Closely Related Honey Bee Species.

Most bacterial species encompass strains with vastly different gene content. Strain diversity in microbial communities is therefore considered to be of functional importance. Yet little is known about the extent to which related microbial communities differ in diversity at this level and which underlying mechanisms may constrain and maintain strain-level diversity. Here, we used shotgun metagenomics to characterize and compare the gut microbiota of two honey bee species, Apis mellifera and Apis cerana, which diverged about 6 mya. Although the host species are colonized largely by the same bacterial 16S rRNA phylotypes, we find that their communities are host specific when analyzed with genomic resolution. Moreover, despite their similar ecology, A. mellifera displayed a much higher diversity of strains and functional gene content in the microbiota compared to A. cerana, both per colony and per individual bee. In particular, the gene repertoire for polysaccharide degradation was massively expanded in the microbiota of A. mellifera relative to A. cerana. Bee management practices, divergent ecological adaptation, or habitat size may have contributed to the observed differences in microbiota genomic diversity of these key pollinator species. Our results illustrate that the gut microbiota of closely related animal hosts can differ vastly in genomic diversity while displaying similar levels of diversity based on the 16S rRNA gene. Such differences are likely to have consequences for gut microbiota functioning and host-symbiont interactions, highlighting the need for metagenomic studies to understand the ecology and evolution of microbial communities.

Genomic changes underlying host specialization in the bee gut symbiont Lactobacillus Firm5.

Bacteria that engage in long-standing associations with particular hosts are expected to evolve host-specific adaptations that limit their capacity to thrive in other environments. Consistent with this, many gut symbionts seem to have a limited host range, based on community profiling and phylogenomics. However, few studies have experimentally investigated host specialization of gut symbionts and the underlying mechanisms have largely remained elusive. Here, we studied host specialization of a dominant gut symbiont of social bees, Lactobacillus Firm5. We show that Firm5 strains isolated from honey bees and bumble bees separate into deep-branching host-specific phylogenetic lineages. Despite their divergent evolution, colonization experiments show that bumble bee strains are capable of colonizing the honey bee gut. However, they were less successful than honey bee strains, and competition with honey bee strains completely abolished their colonization. In contrast, honey bee strains of divergent phylogenetic lineages were able to coexist within individual bees. This suggests that both host selection and interbacterial competition play important roles in host specialization. Using comparative genomics of 27 Firm5 isolates, we found that the genomes of honey bee strains harbour more carbohydrate-related functions than bumble bee strains, possibly providing a competitive advantage in the honey bee gut. Remarkably, most of the genes encoding carbohydrate-related functions were not conserved among the honey bee strains, which suggests that honey bees can support a metabolically more diverse community of Firm5 strains than bumble bees. These findings advance our understanding of the genomic changes underlying host specialization.

Genomic diversity landscape of the honey bee gut microbiota.

The structure and distribution of genomic diversity in natural microbial communities is largely unexplored. Here, we used shotgun metagenomics to assess the diversity of the honey bee gut microbiota, a community consisting of few bacterial phylotypes. Our results show that most phylotypes are composed of sequence-discrete populations, which co-exist in individual bees and show age-specific abundance profiles. In contrast, strains present within these sequence-discrete populations were found to segregate into individual bees. Consequently, despite a conserved phylotype composition, each honey bee harbors a distinct community at the functional level. While ecological differentiation seems to facilitate coexistence at higher taxonomic levels, our findings suggest that, at the level of strains, priority effects during community assembly result in individualized profiles, despite the social lifestyle of the host. Our study underscores the need to move beyond phylotype-level characterizations to understand the function of this community, and illustrates its potential for strain-level analysis.

New Reference Genome Sequences for 17 Bacterial Strains of the Honey Bee Gut Microbiota.

We sequenced the genomes of 17 strains isolated from the gut of honey bees, including strains representing the genera Lactobacillus, Bifidobacterium, Gilliamella, Snodgrassella, Frischella, and Commensalibacter. These genome sequences represent an important step forward in the development of a comprehensive reference database to aid future analysis of this emerging gut microbiota model.

Disentangling metabolic functions of bacteria in the honey bee gut.

It is presently unclear how much individual community members contribute to the overall metabolic output of a gut microbiota. To address this question, we used the honey bee, which harbors a relatively simple and remarkably conserved gut microbiota with striking parallels to the mammalian system and importance for bee health. Using untargeted metabolomics, we profiled metabolic changes in gnotobiotic bees that were colonized with the complete microbiota reconstituted from cultured strains. We then determined the contribution of individual community members in mono-colonized bees and recapitulated our findings using in vitro cultures. Our results show that the honey bee gut microbiota utilizes a wide range of pollen-derived substrates, including flavonoids and outer pollen wall components, suggesting a key role for degradation of recalcitrant secondary plant metabolites and pollen digestion. In turn, multiple species were responsible for the accumulation of organic acids and aromatic compound degradation intermediates. Moreover, a specific gut symbiont, Bifidobacterium asteroides, stimulated the production of host hormones known to impact bee development. While we found evidence for cross-feeding interactions, approximately 80% of the identified metabolic changes were also observed in mono-colonized bees, with Lactobacilli being responsible for the largest share of the metabolic output. These results show that, despite prolonged evolutionary associations, honey bee gut bacteria can independently establish and metabolize a wide range of compounds in the gut. Our study reveals diverse bacterial functions that are likely to contribute to bee health and provide fundamental insights into how metabolic activities are partitioned within gut communities.

Beyond 16S rRNA Community Profiling: Intra-Species Diversity in the Gut Microbiota.

Interactions with microbes affect many aspects of animal biology, including immune system development, nutrition and health. In vertebrates, the gut microbiota is dominated by a small subset of phyla, but the species composition within these phyla is typically not conserved. Moreover, several recent studies have shown that bacterial species in the gut are composed of a multitude of strains, which frequently co-exist in their host, and may be host-specific. However, since the study of intra-species diversity is challenging, particularly in the setting of complex, host-associated microbial communities, our current understanding of the distribution, evolution and functional relevance of intra-species diversity in the gut is scarce. In order to unravel how genomic diversity translates into phenotypic diversity, community analyses going beyond 16S rRNA profiling, in combination with experimental approaches, are needed. Recently, the honeybee has emerged as a promising model for studying gut bacterial communities, particularly in terms of strain-level diversity. Unlike most other invertebrates, the honeybee gut is colonized by a remarkably consistent and specific core microbiota, which is dominated by only eight bacterial species. As for the vertebrate gut microbiota, these species are composed of highly diverse strains suggesting that similar evolutionary forces shape gut community structures in vertebrates and social insects. In this review, we outline current knowledge on the evolution and functional relevance of strain diversity within the gut microbiota, including recent insights gained from mammals and other animals such as the honeybee. We discuss methodological approaches and propose possible future avenues for studying strain diversity in complex bacterial communities.

Functionally Structured Genomes in Lactobacillus kunkeei Colonizing the Honey Crop and Food Products of Honeybees and Stingless Bees.

Lactobacillus kunkeei is the most abundant bacterial species in the honey crop and food products of honeybees. The 16 S rRNA genes of strains isolated from different bee species are nearly identical in sequence and therefore inadequate as markers for studies of coevolutionary patterns. Here, we have compared the 1.5 Mb genomes of ten L. kunkeei strains isolated from all recognized Apis species and another two strains from Meliponini species. A gene flux analysis, including previously sequenced Lactobacillus species as outgroups, indicated the influence of reductive evolution. The genome architecture is unique in that vertically inherited core genes are located near the terminus of replication, whereas genes for secreted proteins and putative host-adaptive traits are located near the origin of replication. We suggest that these features have resulted from a genome-wide loss of genes, with integrations of novel genes mostly occurring in regions flanking the origin of replication. The phylogenetic analyses showed that the bacterial topology was incongruent with the host topology, and that strains of the same microcluster have recombined frequently across the host species barriers, arguing against codiversification. Multiple genotypes were recovered in the individual hosts and transfers of mobile elements could be demonstrated for strains isolated from the same host species. Unlike other bacteria with small genomes, short generation times and multiple rRNA operons suggest that L. kunkeei evolves under selection for rapid growth in its natural growth habitat. The results provide an extended framework for reductive genome evolution and functional genome organization in bacteria.

Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut.

BACKGROUND: In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee A. mellifera. RESULTS: Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations. CONCLUSIONS: The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.