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.     

Functional and evolutionary insights into the simple yet specific gut microbiota of the honey bee from metagenomic analysis

The honey bee, Apis mellifera, harbors a characteristic gut microbiota composed of only a few species which seem to be specific to social bees. The maintenance of this stable and distinct microbial community depends on the social lifestyle of these insects. As in other animals, the bacteria in the gut of honey bees probably govern important functions critical to host health. We recently sequenced a metagenome of the gut microbiota of A. mellifera, assigned gene contents to bins corresponding to the major species present in the honey bee gut, and compared functional gene categories between these species, and between the complete metagenome and those of other animals. Gene contents could be linked to different symbiotic functions with the host. Further, we found a high degree of genetic diversity within each of these species. In the case of the gammaproteobacterial species Gilliamella apicola, we experimentally showed a link between genetic variation of isolates and functional differences suggesting that niche partitioning within this species has emerged during evolution with its bee hosts. The consistent presence of only a few species, combined with strain variation within each of these species, makes the gut microbiota of social bees an ideal model for studying functional, structural, and evolutionary aspects of host-associated microbial communities: many characteristics resemble the gut microbiota of humans and other mammals, but the complexity is considerably reduced. In this addendum, we summarize and discuss our major findings and provide a detailed perspective on future research.     

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.     

Socially transmitted gut microbiota protect bumble bees against an intestinal parasite

Populations of important pollinators, such as bumble bees and honey bees, are declining at alarming rates worldwide. Parasites are likely contributing to this phenomenon. A distinct resident community of bacteria has recently been identified in bumble bees and honey bees that is not shared with related solitary bee species. We now show that the presence of these microbiota protects bee hosts against a widespread and highly virulent natural parasite (Crithidia bombi) in an experimental setting. We add further support to this antagonistic relationship from patterns found in field data. For the successful establishment of these microbiota and a protective effect, exposure to feces from nest mates was needed after pupal eclosion. Transmission of beneficial gut bacteria could therefore represent an important benefit of sociality. Our results stress the importance of considering the host microbiota as an "extended immune phenotype" in addition to the host immune system itself and provide a unique perspective to understanding bees in health and disease.     

Dynamics of persistent and acute deformed wing virus infections in honey bees, Apis mellifera

The dynamics of viruses are critical to our understanding of disease pathogenesis. Using honey bee Deformed wing virus (DWV) as a model, we conducted field and laboratory studies to investigate the roles of abiotic and biotic stress factors as well as host health conditions in dynamics of virus replication in honey bees. The results showed that temperature decline could lead to not only significant decrease in the rate for pupae to emerge as adult bees, but also an increased severity of the virus infection in emerged bees, partly explaining the high levels of winter losses of managed honey bees, Apis mellifera, around the world. By experimentally exposing adult bees with variable levels of parasitic mite Varroa destructor, we showed that the severity of DWV infection was positively correlated with the density and time period of Varroa mite infestation, confirming the role of Varroa mites in virus transmission and activation in honey bees. Further, we showed that host conditions have a significant impact on the outcome of DWV infection as bees that originate from strong colonies resist DWV infection and replication significantly better than bee originating from weak colonies. The information obtained from this study has important implications for enhancing our understanding of host‑pathogen interactions and can be used to develop effective disease control strategies for honey bees.     

A genome-wide signature of positive selection in ancient and recent invasive expansions of the honey bee Apis mellifera

Apis mellifera originated in Africa and extended its range into Eurasia in two or more ancient expansions. In 1956, honey bees of African origin were introduced into South America, their descendents admixing with previously introduced European bees, giving rise to the highly invasive and economically devastating "Africanized" honey bee. Here we ask whether the honey bee's out-of-Africa expansions, both ancient and recent (invasive), were associated with a genome-wide signature of positive selection, detected by contrasting genetic differentiation estimates (F(ST)) between coding and noncoding SNPs. In native populations, SNPs in protein-coding regions had significantly higher F(ST) estimates than those in noncoding regions, indicating adaptive evolution in the genome driven by positive selection. This signal of selection was associated with the expansion of honey bees from Africa into Western and Northern Europe, perhaps reflecting adaptation to temperate environments. We estimate that positive selection acted on a minimum of 852-1,371 genes or approximately 10% of the bee's coding genome. We also detected positive selection associated with the invasion of African-derived honey bees in the New World. We found that introgression of European-derived alleles into Africanized bees was significantly greater for coding than noncoding regions. Our findings demonstrate that Africanized bees exploited the genetic diversity present from preexisting introductions in an adaptive way. Finally, we found a significant negative correlation between F(ST) estimates and the local GC content surrounding coding SNPs, suggesting that AT-rich genes play an important role in adaptive evolution in the honey bee.     

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.     

Honey Bee Infecting Lake Sinai Viruses

Honey bees are critical pollinators of important agricultural crops. Recently, high annual losses of honey bee colonies have prompted further investigation of honey bee infecting viruses. To better characterize the recently discovered and very prevalent Lake Sinai virus (LSV) group, we sequenced currently circulating LSVs, performed phylogenetic analysis, and obtained images of LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, the first detection of LSV4 in the US, and the discovery of LSV6 and LSV7. We detected LSV1 and LSV2 in the Varroa destructor mite, and determined that a large proportion of LSV2 is found in the honey bee gut, suggesting that vector-mediated, food-associated, and/or fecal-oral routes may be important for LSV dissemination. Pathogen-specific quantitative PCR data, obtained from samples collected during a small-scale monitoring project, revealed that LSV2, LSV1, Black queen cell virus (BQCV), and Nosema ceranae were more abundant in weak colonies than strong colonies within this sample cohort. Together, these results enhance our current understanding of LSVs and illustrate the importance of future studies aimed at investigating the role of LSVs and other pathogens on honey bee health at both the individual and colony levels.     

Special Issue: Honey Bee Viruses

Pollination of flowering plants is an important ecosystem service provided by wild insect pollinators and managed honey bees. Hence, losses and declines of pollinating insect species threaten human food security and are of major concern not only for apiculture or agriculture but for human society in general. Honey bee colony losses and bumblebee declines have attracted intensive research interest over the last decade and although the problem is far from being solved we now know that viruses are among the key players of many of these bee losses and bumblebee declines. With this special issue on bee viruses we, therefore, aimed to collect high quality original papers reflecting the current state of bee virus research. To this end, we focused on newly discovered viruses (Lake Sinai viruses, bee macula-like virus), or a so far neglected virus species (Apis mellifera filamentous virus), and cutting edge technologies (mass spectrometry, RNAi approach) applied in the field.     

Regulation of behavioral maturation by a primer pheromone produced by adult worker honey bees

Previous research showed that the presence of older workers causes a delayed onset of foraging in younger individuals in honey bee colonies, but a specific worker inhibitory factor had not yet been identified. Here, we report on the identification of a substance produced by adult forager honey bees, ethyl oleate, that acts as a chemical inhibitory factor to delay age at onset of foraging. Ethyl oleate is synthesized de novo and is present in highest concentrations in the bee's crop. These results suggest that worker behavioral maturation is modulated via trophallaxis, a form of food exchange that also serves as a prominent communication channel in insect societies. Our findings provide critical validation for a model of self-organization explaining how bees are able to respond to fragmentary information with actions that are appropriate to the state of the whole colony.     

Multitrophic interaction facilitates parasite-host relationship between an invasive beetle and the honey bee

Colony defense by honey bees, Apis mellifera, is associated with stinging and mass attack, fueled by the release of alarm pheromones. Thus, alarm pheromones are critically important to survival of honey bee colonies. Here we report that in the parasitic relationship between the European honey bee and the small hive beetle, Aethina tumida, the honey bee's alarm pheromones serve a negative function because they are potent attractants for the beetle. Furthermore, we discovered that the beetles from both Africa and the United States vector a strain of Kodamaea ohmeri yeast, which produces these same honey bee alarm pheromones when grown on pollen in hives. The beetle is not a pest of African honey bees because African bees have evolved effective methods to mitigate beetle infestation. However, European honey bees, faced with disease and pest management stresses different from those experienced by African bees, are unable to effectively inhibit beetle infestation. Therefore, the environment of the European honey bee colony provides optimal conditions to promote the unique bee-beetle-yeast-pollen multitrophic interaction that facilitates effective infestation of hives at the expense of the European honey bee.     

Factors associated with the diversification of the gut microbial communities within chimpanzees from Gombe National Park

The gastrointestinal tract harbors large and diverse populations of bacteria that vary among individuals and within individuals over time. Numerous internal and external factors can influence the contents of these microbial communities, including diet, geography, physiology, and the extent of contact among hosts. To investigate the contributions of such factors to the variation and changes in gut microbial communities, we analyzed the distal gut microbiota of individual chimpanzees from two communities in Gombe National Park, Tanzania. These samples, which were derived from 35 chimpanzees, many of whom have been monitored for multiple years, provide an unusually comprehensive longitudinal depth for individuals of known genetic relationships. Although the composition of the great-ape microbiota has been shown to codiversify with host species, indicating that host genetics and phylogeny have played a major role in its differentiation over evolutionary timescales, the geneaological relationships of individual chimpanzees did not coincide with the similarity in their gut microbial communities. However, the inhabitants from adjacent chimpanzee communities could be distinguished based on the contents of their gut microbiota. Despite the broad similarity of community members, as would be expected from shared diet or interactions, long-term immigrants to a community often harbored the most distinctive gut microbiota, suggesting that individuals retain hallmarks of their previous gut microbial communities for extended periods. This pattern was reinforced in several chimpanzees sampled over long temporal scales, in which the major constituents of the gut microbiota were maintained for nearly a decade.     

The interplay between microbiome dynamics and pathogen dynamics in a murine model of Clostridium difficile Infection

Clostridium difficile infection (CDI) arises in the setting of antibiotic administration where disruption of the normal indigenous gut microbiota leads to susceptibility to C. difficile colonization and colitis. Using a murine model of CDI, we demonstrate that changes in the community structure of the indigenous gut microbiota are associated with the loss of colonization resistance against C. difficile. Several antibiotic regimens were tested in combination for the ability to overcome colonization resistance, including a five antibiotic cocktail consisting of kanamycin, gentamicin, colistin, metronidazole, and vancomycin administered in drinking water for three days, a single intraperitoneal dose of clindamycin or 10 days of cefoperazone in drinking water. Following antibiotic treatment animals were challenged with 105 colony forming units of C. difficile strain VPI 10463 via oral gavage. Animals that received the antibiotic cocktail and clindamycin prior to C. difficile challenge followed one of two clinical courses, either becoming clinically ill and moribund within 2-4 days post challenge, or remaining clinically well. Animals that became clinically ill developed histologically severe colitis. These histopathologic findings were significantly less severe in animals that remained clinically well. Analysis of 16S rRNA gene sequences retrieved from gut tissue at necropsy demonstrated that Proteobacteria dominated the gut microbiota in clinically ill animals. In contrast, the gut microbial community of clinically well animals more closely resembled untreated animals, which were dominated by members of the Firmicutes. All animals that received cefoperazone treatment prior to C. difficile challenge were clinically ill and moribund by 2-5 days post challenge in a dose dependent manner. The gut communities in these animals were dominated by C.difficile suggesting that cefoperazone treatment resulted in a greater loss in colonization resistance. Thus, the severity of colitis that arises in this system reflects the interplay between the expansion of C. difficile in the gut community and the ecologic dynamics of the indigenous microbial community as it recovers from antibiotic perturbation. We demonstrate that altering the balance of these two opposing processes alters clinical outcome and thus may lead to novel preventative and therapeutic approaches for CDI.     

Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors

In vertebrates, including humans, individuals harbor gut microbial communities whose species composition and relative proportions of dominant microbial groups are tremendously varied. Although external and stochastic factors clearly contribute to the individuality of the microbiota, the fundamental principles dictating how environmental factors and host genetic factors combine to shape this complex ecosystem are largely unknown and require systematic study. Here we examined factors that affect microbiota composition in a large (n = 645) mouse advanced intercross line originating from a cross between C57BL/6J and an ICR-derived outbred line (HR). Quantitative pyrosequencing of the microbiota defined a core measurable microbiota (CMM) of 64 conserved taxonomic groups that varied quantitatively across most animals in the population. Although some of this variation can be explained by litter and cohort effects, individual host genotype had a measurable contribution. Testing of the CMM abundances for cosegregation with 530 fully informative SNP markers identified 18 host quantitative trait loci (QTL) that show significant or suggestive genome-wide linkage with relative abundances of specific microbial taxa. These QTL affect microbiota composition in three ways; some loci control individual microbial species, some control groups of related taxa, and some have putative pleiotropic effects on groups of distantly related organisms. These data provide clear evidence for the importance of host genetic control in shaping individual microbiome diversity in mammals, a key step toward understanding the factors that govern the assemblages of gut microbiota associated with complex diseases.     

Impact of stressor exposure on the interplay between commensal microbiota and host inflammation

Exposure to stressful stimuli results in the activation of multiple physiological processes aimed at maintaining homeostasis within the body. These physiological processes also have the capacity to influence the composition of microbial communities, and research now indicates that exposure to stressful stimuli leads to gut microbiota dysbiosis. While the relative abundance of many different bacterial types can be altered during stressor exposure, findings in nonhuman primates and laboratory rodents, as well as humans, indicate that bacteria in the genus Lactobacillus are consistently reduced in the gut during stress. The gut microbiota, including the lactobacilli, have many functions that enhance the health of the host. This review presents studies involving germfree and antibiotic treated mice, as well as mice given Lactobacillus spp. to prevent stressor-induced reductions in lactobacilli, to provide evidence that the microbiota contribute to stressor-induced immunomodulation, both in gut mucosa as well as in systemic compartments. This review will also discuss the evidence that commensal gut microbes have bidirectional effects on gastrointestinal physiology during stressor exposure.     

Characterization of the human gut microbiome during travelers' diarrhea

Alterations in the gut microbiota are correlated with ailments such as obesity, inflammatory bowel disease, and diarrhea. Up to 60% of individuals traveling from industrialized to developing countries acquire a form of secretory diarrhea known as travelers'' diarrhea (TD), and enterotoxigenic Escherichia coli (ETEC) and norovirus (NoV) are the leading causative pathogens. Presumably, TD alters the gut microbiome, however the effect of TD on gut communities has not been studied. We report the first analysis of bacterial gut populations associated with TD. We examined and compared the gut microbiomes of individuals who developed TD associated with ETEC, NoV, or mixed pathogens, and TD with no pathogen identified, to healthy travelers. We observed a signature dysbiotic gut microbiome profile of high Firmicutes:Bacteroidetes ratios in the travelers who developed diarrhea, regardless of etiologic agent or presence of a pathogen. There was no significant difference in α-diversity among travelers. The bacterial composition of the microbiota of the healthy travelers was similar to the diarrheal groups, however the β-diversity of the healthy travelers was significantly different than any pathogen-associated TD group. Further comparison of the healthy traveler microbiota to those from healthy subjects who were part of the Human Microbiome Project also revealed a significantly higher Firmicutes:Bacteriodetes ratio in the healthy travelers and significantly different β-diversity. Thus, the composition of the gut microbiome in healthy, diarrhea-free travelers has characteristics of a dysbiotic gut, suggesting that these alterations could be associated with factors such as travel.     

Rapid changes in the gut microbiome during human evolution

Humans are ecosystems containing trillions of microorganisms, but the evolutionary history of this microbiome is obscured by a lack of knowledge about microbiomes of African apes. We sequenced the gut communities of hundreds of chimpanzees, bonobos, and gorillas and developed a phylogenetic approach to reconstruct how present-day human microbiomes have diverged from those of ancestral populations. Compositional change in the microbiome was slow and clock-like during African ape diversification, but human microbiomes have deviated from the ancestral state at an accelerated rate. Relative to the microbiomes of wild apes, human microbiomes have lost ancestral microbial diversity while becoming specialized for animal-based diets. Individual wild apes cultivate more phyla, classes, orders, families, genera, and species of bacteria than do individual humans across a range of societies. These results indicate that humanity has experienced a depletion of the gut flora since diverging from Pan.The human microbiome is shaped by host genetics, environment, and lifestyle (1–3); thus, humanity''s unique evolutionary and cultural histories must have altered our associations with microorganisms (4). Despite intensive investigation of the microbiomes of humans spanning a range of geographic locations and cultures (5–7), how the composition of the microbiome has changed since humans diverged from other species, and since human populations diverged from one another, remains unclear, owing to a lack of knowledge about the microbiomes of ancestral hominid populations.Understanding how the composition of the human microbiome has changed over evolutionary time requires the inclusion of the microbiomes of phylogenetic outgroups (i.e., the African apes) into analyses of human microbiomes. Previous comparisons of the gut microbiomes of humans and the African apes have been restricted to just a few individuals per host species (8), precluding detection of the precise compositional differences that distinguish the microbiomes of the host species. Comparing the microbiomes of populations of chimpanzees, bonobos, gorillas, and humans while considering the phylogenetic relatedness among the hosts can reveal how the composition of the microbiome has changed since the host species diversified.Here we used a phylogenetic approach to identify the shifts in the composition of the microbiome that occurred along the lineages leading to the extant species of Homo and Pan. This analysis shows that humans across a range of cultures and geographies harbor microbiomes that are disproportionately divergent from those within wild apes. In particular, among the living hominid species, humans harbor uncharacteristically low levels of microbial diversity within their gut microbiomes.     

Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies

It has been proposed that one route of behavioral evolution involves novel regulation of conserved genes. Age-related division of labor in honey bee colonies, a highly derived behavioral system, involves the performance of different feeding-related tasks by different groups of individuals. Older bees acquire the colony's food by foraging for nectar and pollen, and the younger "nurse" bees feed larvae processed foods. The transition from hive work to foraging has been shown to be socially regulated and associated both with decreases in abdominal lipid stores and with increases in brain expression of genes implicated in feeding behavior in Drosophila melanogaster. Here we show that division of labor is influenced by a canonical regulator of food intake and energy balance in solitary species, the insulin/insulin-like growth factor signaling (IIS) pathway. Foragers had higher levels of IIS gene expression in the brain and abdomen than did nurses, despite their low lipid stores. These differences are likely nutritionally mediated because manipulations that induced low lipid stores in young bees also up-regulated these genes. Changes in IIS also causally influenced the timing of behavioral maturation: inhibition of the insulin-related target of rapamycin pathway delayed the onset of foraging in a seasonally dependent manner. In addition, pathway analyses of microarray data revealed that nurses and foragers differ in brain energy metabolism gene expression, but the differences are opposite predictions based on their insulin-signaling status. These results suggest that changes in the regulation of the IIS pathway are associated with social behavior.     

Chronic Bee Paralysis Virus and Nosema ceranae Experimental Co-Infection of Winter Honey Bee Workers (Apis mellifera L.)

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.     

Crop pollination from native bees at risk from agricultural intensification

Ecosystem services are critical to human survival; in selected cases, maintaining these services provides a powerful argument for conserving biodiversity. Yet, the ecological and economic underpinnings of most services are poorly understood, impeding their conservation and management. For centuries, farmers have imported colonies of European honey bees (Apis mellifera) to fields and orchards for pollination services. These colonies are becoming increasingly scarce, however, because of diseases, pesticides, and other impacts. Native bee communities also provide pollination services, but the amount they provide and how this varies with land management practices are unknown. Here, we document the individual species and aggregate community contributions of native bees to crop pollination, on farms that varied both in their proximity to natural habitat and management type (organic versus conventional). On organic farms near natural habitat, we found that native bee communities could provide full pollination services even for a crop with heavy pollination requirements (e.g., watermelon, Citrullus lanatus), without the intervention of managed honey bees. All other farms, however, experienced greatly reduced diversity and abundance of native bees, resulting in insufficient pollination services from native bees alone. We found that diversity was essential for sustaining the service, because of year-to-year variation in community composition. Continued degradation of the agro-natural landscape will destroy this "free" service, but conservation and restoration of bee habitat are potentially viable economic alternatives for reducing dependence on managed honey bees.