Waldan K. Kwong

Gut microbial communities of social bees

The gut microbiota can have profound effects on hosts, but the study of these relationships in humans is challenging. The specialized gut microbial community of honey bees is similar to the mammalian microbiota, as both are mostly composed of host-adapted, facultatively anaerobic and microaerophilic bacteria. However, the microbial community of the bee gut is far simpler than the mammalian microbiota, being dominated by only nine bacterial species clusters that are specific to bees and that are transmitted through social interactions between individuals. Recent developments, which include the discovery of extensive strain-level variation, evidence of protective and nutritional functions, and reports of eco-physiological or disease-associated perturbations to the microbial community, have drawn attention to the role of the microbiota in bee health and its potential as a model for studying the ecology and evolution of gut symbionts.

Genomics and host specialization of honey bee and bumble bee gut symbionts.

Significance Gut microbes are increasingly recognized as influential components of animal biology. Genomic, mechanistic, and evolutionary aspects of gut symbiont specialization remain understudied, however, largely due to the complexity of gut communities, especially in vertebrate systems. We show that the simple microbiota of eusocial bees exhibits host specificity and that coresident species in the bee gut possess complementary capabilities for energy metabolism, implying their occupancy in distinct ecological niches. In addition, coresidence in the gut of a host species results in horizontal exchange of genes between unrelated symbionts. Strains in different hosts have diverged, and honey bee symbionts are evolutionarily and functionally distinct from their bumble bee counterparts, indicating that gut symbionts may be critical elements in biological differences among bee species. Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1T and S. alvi wkB2T (isolated from Apis), as well as draft genomes for four other strains from Bombus. G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell–cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi, demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host–microbe interactions, microbe–microbe interactions, and social transmission.

The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions

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.

Dynamic microbiome evolution in social bees

Honey bees, bumble bees, and stingless bees have related gut microbial communities that are shaped by host evolutionary history. The highly social (eusocial) corbiculate bees, comprising the honey bees, bumble bees, and stingless bees, are ubiquitous insect pollinators that fulfill critical roles in ecosystem services and human agriculture. Here, we conduct wide sampling across the phylogeny of these corbiculate bees and reveal a dynamic evolutionary history behind their microbiota, marked by multiple gains and losses of gut associates, the presence of generalist as well as host-specific strains, and patterns of diversification driven, in part, by host ecology (for example, colony size). Across four continents, we found that different host species have distinct gut communities, largely independent of geography or sympatry. Nonetheless, their microbiota has a shared heritage: The emergence of the eusocial corbiculate bees from solitary ancestors appears to coincide with the acquisition of five core gut bacterial lineages, supporting the hypothesis that host sociality facilitates the development and maintenance of specialized microbiomes.

Genome-wide screen identifies host colonization determinants in a bacterial gut symbiont

Significance Many bacteria are adapted to live in specific locations in animal hosts, even while these same locations exclude colonization by most microorganisms. However, the genetic underpinnings enabling successful colonization are largely unclear. We developed a system to genetically manipulate Snodgrassella alvi, a bacterium restricted to bees, and explored the factors permitting S. alvi to establish within its natural habitat in the bee digestive tract. Using high-throughput methods that screen the entire genome, we find that host colonization is dependent on genes mediating cell surface interactions (e.g., adhesion), metabolism under nutrient limitation, and responses to various stresses. This study demonstrates the genetic tractability of the bee gut microbiota, an emerging system with parallels to the human microbiome. Animal guts are often colonized by host-specialized bacterial species to the exclusion of other transient microorganisms, but the genetic basis of colonization ability is largely unknown. The bacterium Snodgrassella alvi is a dominant gut symbiont in honey bees, specialized in colonizing the hindgut epithelium. We developed methods for transposon-based mutagenesis in S. alvi and, using high-throughput DNA sequencing, screened genome-wide transposon insertion (Tn-seq) and transcriptome (RNA-seq) libraries to characterize both the essential genome and the genes facilitating host colonization. Comparison of Tn-seq results from laboratory cultures and from monoinoculated worker bees reveal that 519 of 2,226 protein-coding genes in S. alvi are essential in culture, whereas 399 are not essential but are beneficial for gut colonization. Genes facilitating colonization fall into three broad functional categories: extracellular interactions, metabolism, and stress responses. Extracellular components with strong fitness benefits in vivo include trimeric autotransporter adhesins, O antigens, and type IV pili (T4P). Experiments with T4P mutants establish that T4P in S. alvi likely function in attachment and biofilm formation, with knockouts experiencing a competitive disadvantage in vivo. Metabolic processes promoting colonization include essential amino acid biosynthesis and iron acquisition pathways, implying nutrient scarcity within the hindgut environment. Mechanisms to deal with various stressors, such as for the repair of double-stranded DNA breaks and protein quality control, are also critical in vivo. This genome-wide study identifies numerous genetic networks underlying colonization by a gut commensal in its native host environment, including some known from more targeted studies in other host–microbe symbioses.

Evolution of host specialization in gut microbes: the bee gut as a model.

Bacterial symbionts of eukaryotes often give up generalist lifestyles to specialize to particular hosts. The eusocial honey bees and bumble bees harbor two such specialized gut symbionts, Snodgrassella alvi and Gilliamella apicola. Not only are these microorganisms specific to bees, but different strains of these bacteria tend to assort according to host species. By using in-vivo microbial transplant experiments, we show that the observed specificity is, at least in part, due to evolved physiological barriers that limit compatibility between a host and a potential gut colonizer. How and why such specialization occurs is largely unstudied for gut microbes, despite strong evidence that it is a general feature in many gut communities. Here, we discuss the potential factors that favor the evolution of host specialization, and the parallels that can be drawn with parasites and other symbiont systems. We also address the potential of the bee gut as a model for exploring gut community evolution.

Immune system stimulation by the native gut microbiota of honey bees

Gut microbial communities can greatly affect host health by modulating the hosts immune system. For many important insects, however, the relationship between the gut microbiota and immune function remains poorly understood. Here, we test whether the gut microbial symbionts of the honey bee can induce expression of antimicrobial peptides (AMPs), a crucial component of insect innate immunity. We find that bees up-regulate gene expression of the AMPs apidaecin and hymenoptaecin in gut tissue when the microbiota is present. Using targeted proteomics, we detected apidaecin in both the gut lumen and the haemolymph; higher apidaecin concentrations were found in bees harbouring the normal gut microbiota than in bees lacking gut microbiota. In in vitro assays, cultured strains of the microbiota showed variable susceptibility to honey bee AMPs, although many seem to possess elevated resistance compared to Escherichia coli. In some trials, colonization by normal gut symbionts resulted in improved survivorship following injection with E. coli. Our results show that the native, non-pathogenic gut flora induces immune responses in the bee host. Such responses might be a host mechanism to regulate the microbiota, and could potentially benefit host health by priming the immune system against future pathogenic infections.

Metabolism of Toxic Sugars by Strains of the Bee Gut Symbiont Gilliamella apicola

ABSTRACT Social bees collect carbohydrate-rich food to support their colonies, and yet, certain carbohydrates present in their diet or produced through the breakdown of pollen are toxic to bees. The gut microbiota of social bees is dominated by a few core bacterial species, including the Gram-negative species Gilliamella apicola. We isolated 42 strains of G. apicola from guts of honey bees and bumble bees and sequenced their genomes. All of the G. apicola strains share high 16S rRNA gene similarity, but they vary extensively in gene repertoires related to carbohydrate metabolism. Predicted abilities to utilize different sugars were verified experimentally. Some strains can utilize mannose, arabinose, xylose, or rhamnose (monosaccharides that can cause toxicity in bees) as their sole carbon and energy source. All of the G. apicola strains possess a manO-associated mannose family phosphotransferase system; phylogenetic analyses suggest that this was acquired from Firmicutes through horizontal gene transfer. The metabolism of mannose is specifically dependent on the presence of mannose-6-phosphate isomerase (MPI). Neither growth rates nor the utilization of glucose and fructose are affected in the presence of mannose when the gene encoding MPI is absent from the genome, suggesting that mannose is not taken up by G. apicola strains which harbor the phosphotransferase system but do not encode the MPI. Given their ability to simultaneously utilize glucose, fructose, and mannose, as well as the ability of many strains to break down other potentially toxic carbohydrates, G. apicola bacteria may have key roles in improving dietary tolerances and maintaining the health of their bee hosts. IMPORTANCE Bees are important pollinators of agricultural plants. Our study documents the ability of Gilliamella apicola, a dominant gut bacterium in honey bees and bumble bees, to utilize several sugars that are harmful to bee hosts. Using genome sequencing and growth assays, we found that the ability to metabolize certain toxic carbohydrates is directly correlated with the presence of their respective degradation pathways, indicating that metabolic potential can be accurately predicted from genomic data in these gut symbionts. Strains vary considerably in their range of utilizable carbohydrates, which likely reflects historical horizontal gene transfer and gene deletion events. Unlike their bee hosts, G. apicola bacteria are not detrimentally affected by growth on mannose-containing medium, even in strains that cannot metabolize this sugar. These results suggest that G. apicola may be an important player in modulating nutrition in the bee gut, with ultimate effects on host health. Bees are important pollinators of agricultural plants. Our study documents the ability of Gilliamella apicola, a dominant gut bacterium in honey bees and bumble bees, to utilize several sugars that are harmful to bee hosts. Using genome sequencing and growth assays, we found that the ability to metabolize certain toxic carbohydrates is directly correlated with the presence of their respective degradation pathways, indicating that metabolic potential can be accurately predicted from genomic data in these gut symbionts. Strains vary considerably in their range of utilizable carbohydrates, which likely reflects historical horizontal gene transfer and gene deletion events. Unlike their bee hosts, G. apicola bacteria are not detrimentally affected by growth on mannose-containing medium, even in strains that cannot metabolize this sugar. These results suggest that G. apicola may be an important player in modulating nutrition in the bee gut, with ultimate effects on host health.

Genome Sequences of Lactobacillus sp. Strains wkB8 and wkB10, Members of the Firm-5 Clade, from Honey Bee Guts

ABSTRACT We sequenced two strains from the Lactobacillus Firm-5 clade, a dominant group of symbionts in the guts of honey bees and other social bees. The genome of strain wkB8, comprising a 1.93-Mb chromosome and a 6.4-kb plasmid, was fully closed, while strain wkB10 was assembled into 32 contigs. These genomes will provide insights into how gut symbionts evolve and interact with their host species.

Apibacter adventoris gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from honey bees.

Honey bees and bumble bees harbour a small, defined set of gut bacterial associates. Strains matching sequences from 16S rRNA gene surveys of bee gut microbiotas were isolated from two honey bee species from East Asia. These isolates were mesophlic, non-pigmented, catalase-positive and oxidase-negative. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and C16 : 0 3-OH. The DNA G+C content was 29–31 mol%. They had ∼87 % 16S rRNA gene sequence identity to the closest relatives described. Phylogenetic reconstruction using 20 protein-coding genes showed that these bee-derived strains formed a highly supported monophyletic clade, sister to the clade containing species of the genera Chryseobacterium and Elizabethkingia within the family Flavobacteriaceae of the phylum Bacteroidetes. On the basis of phenotypic and genotypic characteristics, we propose placing these strains in a novel genus and species: Apibacter adventoris gen. nov., sp. nov. The type strain of Apibacter adventoris is wkB301T ( = NRRL B-65307T = NCIMB 14986T).