It Takes Guts –The Honey Bee Microbiome

By Emily Olson, Alberta Tech Transfer Program Technician

Do you ever wonder, what makes honey so good? How do bees convert nectar from flowers into this liquid gold? The answer to these questions is simple – bacteria!

the crop, midgut, and hindgut. Each with specialized functions and different bacterial communities. Bacteria in the crop are solely derived from food sources, bacteria in the hindgut are solely derived from other bees (e.g., bee-bee interactions), and in the midgut, the bacteria are from both food and social transmission6

The journey of bee and bacteria begins in the first interactions a developing bee has with its nest mates. Young larvae ingest their first bacteria ‘cocktail’ via brood food, a mixture of special gland secretions and pollen, given to larvae by nurse bees 5. However, a study by Martinsen et al. (2012), found that larvae have few bacteria present in their gut. They found that larvae’s microbiota varies from season to season and year to year, suggesting that the diversity of this bacterial community is determined by the brood food7

Prior to emerging, the developing bee sheds its gut lining, and the bee emerges with little to no gut bacteria. The first part of a bees’ life is dedicated to in-hive activities such as cleaning, caring for brood, and tending to the queen. Through interactions with other bees, especially foragers, microbes are transmitted to the young bee. By day 9, the bee’s microbiota is fully developed, and this community of bacteria remains with them for life7. As the bee ages and transitions from in-hive activities to foraging, it collects bacteria, good and bad, and brings them back to the hive.

Bees fly, in general, within a 5 km radius of the colony, collecting pollen, nectar, and propolis from the surrounding environment. While foraging, the nectar collected is stored in the bee’s honey stomach (crop), where enzymes are

Honey bees have bacteria in their guts, just like us! However, the diversity of these bacteria is much lower in bees than in humans. Honey bees have 8 different species that make up the “core gut bacteria”, whereas humans have around 1,000 1. These core bacteria are present in all honey bees, regardless of where they are in the world 2. Bees and their bacteria have evolved together, and this specialized relationship is mutualistic; bacteria receive nutrients from the bee and the bee, in return, receives protection from harmful microorganisms

3,4. Bacteria also aid in digestion, food preservation, pheromone production, caste and nest mate recognition, task cues, and much more5!

The honey bee digestive system is comprised of 3 main compartments: added from the hypopharyngeal gland and the conversion of nectar into honey starts3. These enzymes inhibit the establishment and growth of bacteria, resulting in the few bacteria present in the crop7. Among this small community of bacteria are lactobacillus bacteria (LAB), which continues to convert nectar to honey. Although LAB are only found in fresh honey and do not survive in stored honey, they contribute to its lasting antimicrobial properties by producing lactic acid8. It is suggested that LAB may inhibit the production of yeast that causes alcoholic fermentation3, allowing honey to be stored indefinitely. The pollen bees collect, once back in the hive, is mixed with regurgitated nectar from the honey stomach. In this process, LAB are also added to the pollen, transforming it into bee bread. These bacteria give the bee bread antimicrobial properties, allowing it to be stored for a long period of time, and helping defend bees and brood against pathogens4.

The midgut is the main site of digestion and nutrient absorption in the honey bee digestive system9. It contains few bacteria, mostly concentrated in the posterior section, near the hindgut. The hindgut, composed of the ileum and rectum, is important for the absorption of water, salt, minerals, and other substances10. It contains the highest density of gut bacteria, which remains relatively stable throughout the bee’s life11. Bees rely on the bacterial communities within the midgut and hindgut to digest and metabolize food, as they are unable to breakdown the carbohydrates present in pollen and nectar on their own12. The polysaccharides in pollen and nectar are broken down by enzymes produced by the gut bacteria into short chain fatty acids, which are then available as energy to the bee12. Bees collect food resources from a variety of plants producing different types of sugars, proteins, and defensive compounds, some of which are toxic to bees6 The gut bacteria detoxify and metabolize these substances by producing specific enzymes to break them down6. For example, pectin in plant cell walls is toxic to honey bees, but the enzymes produced by bacteria in the midgut degrade the pectin found in pollen grains and allow the bee to access the nutrients within6

The gut bacteria also play an important role in a bees’ immune response, by initiating the synthesis of antimicrobial peptides (AMPs), a crucial component of bee’s immune system13. As bees consume resources from outside the hive, they are exposed to foreign materials and pathogens. During an infection, AMPs are produced and work to damage cells and inhibit protein formation in the foreign microbes14. This immune response also helps to prime the bee’s immune system against future infections by enabling the immune system to recognize pathogens more quickly and be ready to fight off a reinfection12. Additionally, several strains of bacteria in the hindgut form a biofilm layer on the ileum’s epithelium, providing a physical barrier, which further acts to protect the bee against parasites, pathogens, and other foreign material6.

The impact of antibiotic use on the honey bee gut microbiota

Antibiotics are commonly used in honey bee hives to treat bacterial diseases, such as American Foulbrood and European Foulbrood. These antibiotics are broad spectrum, meaning they are effective against a variety of bacteria, and can impact the beneficial bacterial community within honey bees15. Given the importance of the gut bacteria for bee health, it’s important to understand the implications of the use of antibiotics, tylosin and tetracycline for example, as a management tool to fight honey bee bacterial diseases.

The bacterial community within honey bees is highly specialized, each species filling a niche. Therefore, the alteration of this community can impact the necessary functions these bacteria carry out, which may result in the disruption of nutrient breakdown and uptake, detoxification, and the level of bee susceptibility to pathogens. Powell et al. (2021) assessed the effects of Tylosin on the diversity of the honey bee microbiome16. Their results demonstrate that exposure to antibiotics decreases the abundance and diversity of bacteria species and causes bees to become more susceptible to bacterial infection. Similarly, a study by Deng et al. (2022) found that long term use of the antibiotic Tetracycline reduced the abundance of certain bacteria, which increased bee’s susceptibility to Israeli acute paralysis virus, suggesting that the bees microbiome plays a crucial role in resisting viral infection17

Prophylactic use of antibiotics has been a common practice in honey bee colonies here in Alberta. By limiting our use of antibiotics and incorporating alternative management practices, such as increased monitoring, isolating sick colonies, and implementing biosecurity protocols, we can avoid the negative effects the long-term use of antibiotics has on our colonies.

The impact of pesticides on the honey bee gut microbiota

Bees are often exposed to environment pesticides used on the landscape, as they are out foraging, and in-hive pesticides, placed on colonies by beekeepers.

Although some pesticides, such as glyphosate, are considered innocuous to honey bees, there is evidence they harm beneficial bacteria found within the bee gut and many studies have linked their use to the decline in honey bee health18. A study by Motta et al. (2018) analyzed the size and composition of gut bacteria of bees exposed to glyphosate18 Researchers found a significant shifts in the size of the microbiome on day 3 of glyphosate exposure, and that bees exposed to the pesticide were more likely to become infected with bacterial pathogens. They also noted that glyphosate may have sublethal effects on bees, such as impacting their ability to return to the hive after foraging18

Pesticide residues have been found not only on bees, but also in wax, pollen, and honey, resulting in a constant exposure to these chemicals. Because wax often remains in a colony year after year, pesticide residues can build up within the comb over time and have negative effects on overwinter survival, queen quality, and the overall fitness of the colony19. Kakumanu et al. (2016) examined how in-hive pesticides (coumaphos, fluvalinate, and chlorothalonil) alters the microbiome of honey bees. Exposure to these pesticides, commonly used to treat colonies, resulted in a significant change in the bacterial communities within the studied bees20 Decreased sugar and peptide metabolism was also observed, suggesting an altered bee microbiome impacts these important functions carried out by the bacteria.

Communication with farmers and pesticide applicators is essential to limiting exposure and preventing pesticide poisoning in our colonies. Ensure pesticides (in-hive and on the landscape) are applied properly by following the label, used only when thresholds are met, and Integrated Pest Management strategies have been considered.

For more information on how to prevent pollinator poisoning, check out “Protecting Pollinators – Best Management Practices for Foliar Application” on our website! https://www. albertabeekeepers.ca/preventingpollinator-poisoning/

The honey bee microbiome plays an important role in individual bee and overall colony health. The beneficial bacteria within the bee gut are important for nutrition, immune response, communication with nest mates, and much more. Honey bees rely on their internal bacterial community for survival and any alteration can have lasting impacts on colony health. There is significant evidence that antibiotics, as well as pesticides used in-hive and on the landscape, alter these communities resulting in lethal and sublethal effects on honey bees. By reducing our dependence on antibiotics, reducing exposure to pesticides, and incorporating organic treatments and cultural management practices into our operations, we can ensure the health of these important bacteria and therefore the health of our bees!

References

1. Engel, P., Martinson, V.G., & Moran, N. A. (2012). Functional diversity within the simple gut microbiota of the honey bee. PNAS, 109(27), 11002-11007. https://doi.org/10.1073/pnas.1202970109

2. Raymann K. & Moran N.A. (2018). The role of the gut microbiome in health and disease of adult honey bee workers. Curr Opin Insect Sci. 26, 97-104. doi: 10.1016/j.cois.2018.02.012.

3. Olofsson, T. C., & Vásquez, A. (2008). Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Current microbiology, 57(4), 356-363. doi: 10.1007/s00284-008-9202-0

4. Vásquez, A., & Olofsson, T. C. (2009). The lactic acid bacteria involved in the production of bee pollen and bee bread. Journal of apicultural research, 48(3), 189-195. doi: 10.3896/IBRA.1.48.3.07

5. Paris, L., Peghaire, E., Moné, A., Diogon, M., Debroas, D., Delbac, F., & El Alaoui, H. (2020). Honeybee gut microbiota dysbiosis in pesticide/parasite co-exposures is mainly induced by Nosema ceranae. Journal of invertebrate pathology, 172,107348. https://doi.org/10.1016/j.jip.2020.107348

6. Moran, N. A. (2015). Genomics of the honey bee microbiome. Current opinion in insect science, 10, 22-28. doi: 10.1016/j.cois.2015.04.003

7. Martinson, V. G., Moy, J., & Moran, N. A. (2012). Establishment of characteristic gut bacteria during development of the honeybee worker. Applied and environmental microbiology, 78(8), 2830-2840. doi: 10.1128/AEM.07810-11

8. Bogdanov, S. (1997). Antibacterial substances in honey. Swiss Bee Res. Center, 17, 74-76.

9. Harwood, G., & Amdam, G. (2021). Vitellogenin in the honey bee midgut. Apidologie, 52(4), 837-847. doi: 10.1007/s13592-021-00869-3ff. ffhal-03688791f

10. Caron, D. M., & Connor, L. J. (2013). Honey bee biology and beekeeping (No. 638.1 C293h). Wicwas Press,.

11. Maes, P. W., Floyd, A. S., Mott, B. M., & Anderson, K. E. (2021). Overwintering honey bee colonies: Effect of worker age and climate on the hindgut microbiota. Insects, 12(3), 224. https://doi.org/10.3390/insects12030224

12. Zheng, H., Perreau, J., Powell, J. E., Han, B., Zhang, Z., Kwong, W. K., ... & Moran, N. A. (2019). Division of labor in honey bee gut microbiota for plant polysaccharide digestion. Proceedings of the National Academy of Sciences, 116(51), 25909-25916. https://doi.org/10.1073/pnas.1916224116

13. Kwong, W. K., Mancenido, A. L., & Moran, N. A. (2017). Immune system stimulation by the native gut microbiota of honey bees. Royal Society open science, 4(2), 170003. doi: 10.1098/rsos.170003

14. Danihlík, J., Aronstein, K., & Petřivalský, M. (2015). Antimicrobial peptides: a key component of honey bee innate immunity: Physiology, biochemistry, and chemical ecology. Journal of Apicultural Research, 54(2), 123-136. https://doi. org/10.1080/0021 8839.2015.1109919

15. Aljedani, D. M. (2022). Antibiotic treatment (Tetracycline) effect on bio-efficiency of the larvae honey bee (Apis mellifera jemenatica). Saudi Journal of Biological Sciences, 29(3), 1477-1486. https://doi.org/10.1016/j.sjbs.2021.11.024

16. Powell, J. E., Carver, Z., Leonard, S. P., & Moran, N. A. (2021). Field-realistic tylosin exposure impacts honey bee microbiota and pathogen susceptibility, which is ameliorated by native gut probiotics. Microbiology spectrum, 9(1), e00103-21. https://doi.org/10.1128/Spectrum.00103-21

17. Deng, Y., Yang, S., Zhao, H., Luo, J., Yang, W., & Hou, C. (2022). Antibiotics-induced changes in intestinal bacteria result in the sensitivity of honey bee to virus. Environmental Pollution, 314, 120278. https://doi.org/10.1016/j.envpol.2022.120278

18. Motta, E. V., Raymann, K., & Moran, N. A. (2018). Glyphosate perturbs the gut microbiota of honey bees. Proceedings of the National Academy of Sciences, 115(41), 10305-10310. https://doi.org/10.1073/pnas.1803880115

19. Mullin, C. A., Frazier, M., Frazier, J. L., Ashcraft, S., Simonds, R., VanEngelsdorp, D., & Pettis, J. S. (2010). High levels of miticides and agrochemicals in North American apiaries: implications for honey bee health. PloS one, 5(3), e9754. https://doi.org/10.1371/journal.pone.0009754

20. Kakumanu, M. L., Reeves, A. M., Anderson, T. D., Rodrigues, R. R., & Williams, M. A. (2016). Honey bee gut microbiome is altered by in-hive pesticide exposures. Frontiers in microbiology, 7, 1255. https://doi.org/10.3389/fmicb.2016.01255

The Tech Transfer Program is funded by the Government of Canada and the Government of Alberta through the Canadian Agricultural Partnership.