Health & Honey Bee Gut Bacteria

The Role of Microbial Communities in Honey Bee Health, Disease and Development

Much like our own microbiome, except far simpler, the honey bee gut microbiota is involved in metabolism and immune stimulation. When the gut microbiome is disrupted through antibiotic treatments, pesticide exposure or poor nutrition, it usually leads to increased colony adverse effects. In the healthy colony, there is a core group of 5 bacterial phylotypes (“group similarities”), each having their own role in disease resistance, immune function and larval development.

This article describes the honey bee gut microbiota, its composition and role in bees’ lives as well as variations between species and environments. It ends with suggestions on further research and integration into beekeeping.

Composition of the Honey Bee Bacterial Communities

In the post on Roundup and its effects on bees, I noted three main phylums and their main representative species:

• Phylum Firmicutes: Lactobacillus spp. firm-4 & firm-5.
• Phylum Actinobacteria: Bifidobacterium spp..
• Phylum Pseudomonadota: Prasaccharibacter apium, Snodgrassella alvi, Bartonella apis, Gilliamella apicola & Frischella perrara.

Many studies prefer to list five core bacterial phylotypes, those strains with >97% similarities in their ribosomal RNA sequences (“16s rRNA gene”) the adult bee gut microbiome: Gilliamella, Snodgrassella, Lactobacillus Firm-4 and Firm-5, and Bifidobacterium (Reviewed in Kwong et al., 2016). Other types exist, but they are not present in every bee, or only account for a small proportion of the gut bacteria composition.

Comparing Microbial Communities in Different Castes

Let’s compare the feeding habits of the various hive members:

• The male drones do not feed themselves and are dependent on the worker bees,
• The queen consumes a high protein diet,
• Forager bees consume nectar in the field, or honey inside the hives,
• Nurse bees consume nectar, pollen and honey (Crailsheim et al., 1992).

As social insects, bees share food and honey crop contents through oral trophallaxis, so it is reasonable to expect colony-wide transmission of gut microbiota. Kapheim and colleagues compared bee casts from three colonies and found that the largest microbial differences were between drones and workers, with small variations between queens and workers (Kapheim et al., 2015).

The greatest diversity being amongst worker bees was suggested to be a result of the regular exchange of various nectar types between the returning foragers and receiving nurses. Any new bacteria, including pathogens, are introduced into the colony by the foragers and distributed by the nurse bees¹.

In contrast, workers, particularly nurses and foragers, maintain high microbial diversity, playing a central role in sustaining colony microbiome stability. One possibility for such diversity is the aforementioned forager-mediated bacterial introduction. The gut microbiota shifts slightly as the nurse bee grows older and begins foraging (Anderson et al., 2016).

The Male Microbial Communities are Similar to Workers

Drone microbial variations are readily explained: their diet is controlled by the nurse bees. Whatever the nurse bees possess, so do the drones. In the review by Kwong and Moran, the male gut microbiome composition was found to be closer to that of workers, with minor variations and a larger population of Lactobacillus species (Kwong & Moran, 2016).

Queen Gut Bacteria Contain Species Unique to Royal Jelly

The queen’s microbiome is enriched with bacteria that can enhance energy conversion for egg production. Queens had a higher number of Parasaccharibacter apium compared to other hive members, a bacterial species abundant in royal jelly (Corby-Harris et al., 2014, Corby-Harris et al., 2016). Analysis of this species shows a high number of metabolic electron transfer enzymes (ferrodoxins and oxidoreductases), as well as enzymes for the development of the membrane – essential for egg development. Worker bees do not posses P. apium as they are not egg layers, however some solitary female species bees do possess it (Martinson et al., 2011).

Nurse Bees as the Controllers of Microbial Diversity

Considering that the entire colony’s feeding and nutrient transfer occurs through nurse bees, they can be considered as the central controllers of microbial diversity. One study showed that worker bees emerging from their cells in isolation lacked a gut microbiota, and will continue to do so if they are isolated from the rest of the colony (Powell et al., 2014).

The authors compared the gut microbiota of worker bees exposed to nurse bees, other workers’ fecal material, and hive components like comb, honey, and bee bread. They found that when newly emerged workers are exposed to nurse bees or their fecal material, they acquired the typical microbial communities resembling their nestmates. The presence of nurse bees or hindgut material was essential for colonisation by core species of Gram-negative bacteria like Snodgrassella alvi, Gilliamella apicola, and Frischella perrara.

Note that some bacterial species could only be transferred from contact with the hive surfaces, such as Lactobacillaceae Firm-5 or the non-core Firmicutes groups. However, when newly emergent workers were kept separate from the nurse bees and only exposed to hive material, they were colonised by a large diversity of non-core bacteria (i.e. not the main species listed above).

The authors suggest that nurse bees provide a form of social immunity, only allowing the core, essential bacteria to colonise the gut microbiota of a newly emerged bee. In worker bees, the normal gut microbiota structure emerges after 4-6 days of life as an adult through a combination of trophallaxis and exposure to hive material (Powell et al., 2014).

The Microbiota in Health and Disease

The honey bee gut microbiota is involved metabolism and immune function, while also acting as a physical protective barrier against disease and colonisation from pathogens. Disruption of the gut microbiota in early life impacts the expression of developmental and immunity genes, producing worker bees that are less able to respond to environmental stressors like poor nutrition or temperature extremes (Schwarz et al., 2016).

Perhaps diseases like Nosema, Chalkbrood or European foulbrood can be revisited through the lens of imbalances in the microbial community members? For example, probiotic supplementation with the core gut bacteria resulted in an improved stress tolerance and diseases response to chalkbrood (Iorizzo et al., 2022).

Under laboratory settings, the American and European Foulbrood diseases were managed by adding various members of the resident gut bacteria (Moharrami et al., 2022). Further field testing can confirm if microbiota enhancement via probiotic supplementation can prevent and treat these diseases. This would make for great advances in AFB management, as there are no current effective treatments… outside of a fiery death for the whole hive, of course.

Impact of Antibiotics on Bee Gut Microbiota and Disease Resistance

Antibiotic treatment can disrupt the gut microbiota, negatively impacting the honey bees’ immune system and making them more susceptible to diseases like Nosema (Li et al., 2017). Li and colleagues found that Nosema infection alone did not impact the gut microbial composition, however when bees were treated with antibiotics and later infected by Nosema, they had a higher disease burden, and the highest mortality rates of the four treatment groups studied.

The disruption in gut microbiota decreased the expression of genes encoding the antimicrobial peptides abaecin, defensin1, and hymenoptaecin. Many studies have shown that these peptides are downregulated following antibiotic exposure, indicating that the gut bacteria are closely linked to the proper functioning of the immune system in honey bees (some examples: Chaimanee et al., 2012 & Kwong et al., 2017).

Another study showed how tetracycline reduced the abundance of gut bacteria in honey bee workers (Jia et al., 2021). The study found that nurse bees in tetracycline-treated colonies would transfer an altered microbiome to the newly emerged bee, however there was no significant impact when compared to untreated bees. The disrupted gut community still came from the same phylotypes, however the composition and population counts were altered compared to untreated bees.

So the disruption of gut microbiota through antibiotic treatment impairs the bees’ ability to fend off infections. This makes alternative, microbiome-friendly approaches more feasible. If anything, the overuse of antibiotics in beekeeping not only leads to increased bacterial resistance, but also second-order infections.

Environmental Variations in Gut Bacteria

Diet is the major factor that makes up the honey bee gut bacteria, and this is dependent on the flowers in bloom. Pollen and nectar are not created equal; different plants will provide different nutrients to bees. Jones and colleagues tested two different bee groups: those fed on a monoculture diet of rapeseed (canola), and those kept on a distant farm with diverse forage (Jones et al., 2017). Colonies feeding on rapeseed had relatively lower populations of Bartonella apis, a bacterial species that encodes genes involved in the degradation of secondary plant metabolites (eg: carbohydrates, organic acids, amino acids and terpenes, amongst [many] others Segers et al. 2017).

Is this decrease due to the lack of secondary metabolites from rapeseed, or is it due to the inability of B. apis to metabolise rapeseed’s secondary metabolites? Fang and colleagues reported the two major secondary metabolites, glucosinolates and the compound sinapine are distributed thorough out the plant tissues (Fang et al., 2012). No doubt some are consumed in the nectar, so perhaps this serves as an area of future research for why B. apis is reduced under monoculture feeding, like rapeseed?

Could the pesticide exposure influence bacterial communities in rapeseed monocultures? Neonicotinoids are the most common insecticide class used in rapeseed farming. However, in this study, the authors note that winter-sowed rapeseed was used, where the “concentrations of neonicotinoid pesticides were on the low end of the scale compared to those reported in other studies” (Jones et al., 2017). When the gut microbiome of rapeseed-fed bees are compared with that of the farm field, we can see how the foraging environment impacts the gut bacteria. But what about the same environment in different seasons?

Geography & Gut Microbiota Composition

No surprise, geographical variations also influence gut microbial communities. For instance, honey bees from different regions of Saudi Arabia exhibited variations in gut microbial composition, with Proteobacteria, Firmicutes, and Actinobacteria being the most dominant phylum (Khan et al., 2017). The authors also reported bacteria that was not previously identified in honey bee studies: Citrobacter sp., Providencia vermicola, Exiguobacterium acetylicum, and Planomicrobium okeanokoites. Perhaps these specific gut members provide local adaptions to the Saudi Arabia-native bee Apis mellifera jemenitica?

Variations in gut microbiota from the same phylotypes are also shown when comparing the Japanese honey bee Apis cerana japonica and the giant honey bee Apis dorsata (Yoshiyama & Kimura, 2009; Tajabadi et al., 2011). I do not agree with making such comparisons, as these are different species, who are exposed to different environmental pressures and adaptions. For example A. dorsata can forage under moonlight, and nests in exposed comb (Young et al., 2021). Surely the genetic variations that lead to these abilities impact the gut microbiota level?

Gut Microbiota Variations Between European and Asian Honey Bees

Variation exists within the gut microbiota species between of Apis mellifera and Apis cerana (Ellegaard & Engel, 2019). Apis mellifera had the greater diversity, with more microbial communities involved in sugar breakdown. The authors attribute these variations to factors like:

• Bee management practices, such as the supplemental feeding of bees, using Langstroth/“non-natural” beehives & worldwide transportation,
• Ecological adaptation, due to the generalist (and long distance) foraging preferences of A. mellifera,
• Habitat size, in which a larger hive and individual bee size means greater chances for variation between hives and individual bees, based on probabilities.

The same five core phylotypes (Lactobacillus Firm 4 & Firm5, Gilliamella, Bifidobacterium and Snodgrassella) were evident, however the actual bacterial populations colonising bees differed between honey bee species. The authors conclude by stating the drawbacks of microbial diversity: while more metabolic flexibility enables foraging on more diverse pollen sources, the increased competition within the gut microbiota may lead to species diverting resource to “inter-bacterial warfare” as opposed to a beneficial host relationship (Ellegaard et al., 2020).

Seasonal Affects on Honey Bee Gut Communities

How does the gut bacteria differ between summer and winter bees, since the latter live far longer? Kesanervoa & colleagues found that winter bees had more Bartonella and Commensalibacter species compared to their summer populations in western Switzerland (Kesnervoa et al., 2020). One possible explanation for this is that winter bees feed on honey and pollen². The typical forager consumes nectar from the plants she gathers and honey when inside the hive – pollen is allocated to the brood and nurse bees.

Bee Bread is not the Fermentation of Nectar and Pollen

One of the more interesting studies explored whether bees make bee bread as a “fermentation of nectar and pollen to enhance its nutritional content”. Anderson & colleagues disproved the notion that bee bread is the fermentation of nectar and pollen, since the microbial count in the cells were not indicative of the “nutrient conversion” process that many beekeepers are told about (Anderson et al., 2014).

Analysis of the microbe counts per grain of pollen provided a ratio of 1 bacterium to 10,000 pollen grains (1:10,000). When converted into a biological relevant ratio, it implies 1 bacterium for every 1,000,000 (one million!) pollen grains in a wax cell. By contrast, a single pollen grain in the hindgut of a nurse bee is covered by hundreds or thousands of bacteria.

The addition of pollen and nectar was to preserve food for later consumption, rather than ferment it into a new product. Inside the hive, nurse bees prefer to consume freshly collected pollen under three days of age. The study suggests that this tendency to eat fresh vs. older pollen stores could be a result of the strength or type of chemical signals that are released from stored pollen, with more attractive signals emanating from freshly-stored forage (Anderson et al., 2014).

So how does this redefine bee bread? Instead of a product formed by the “fermentation of nectar and pollen”, it becomes “pollen preserved for storage via the addition of nectar”. I’m sure another study will come along and further reclassify it.

Future Research: Integrating Gut Microbiota into Beekeeping Operations

To maintain a healthy colony, and by extension a healthy honey bee gut microbiome, the general advice to beekeepers is to “provide plenty of natural forage to bees, keep strong colonies and avoid chemical use inside the hive”. My fellow beekeeper, do more of that.

Commercial or large scale operations may find greater benefit to supplement their sugar feeds with probiotics and other key honey bee nutrients, to ensure their colonies can withstand stressors like pesticide exposure. I am curious about the impacts of probiotic use for colonies fed on monocultural diets as in commercial pollination operations. Will it make them less prone to stressors, such as the long-distance transportation, agrichemical use or the an incomplete diet? A million dollar study, indeed.

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Footnotes

1. If the colony is healthy and the bacteria is pathogenic, then their immune system can manage it. If the colony is stressed or weakened, or the condition is more virulent like Deformed Wing Virus or American foulbrood, then infection is likely. ↩︎
2. Since foraging is not possible in cold winter, the stored bee bread becomes nutrition for the colony. This is when the gut microbiota shifts, as the colony is dependent on stored rather than fresh food. ↩︎