Summary
The European honey bee (Apis mellifera) provides an essential pollination service and is hence an integral part of food security. In recent decades, beekeepers in Europe and the United States have been reporting severe losses of beehives. The reasons behind the decline are manifold. Pesticides, such as glyphosate, are ingested by honey bees at sublethal doses but can still have various harmful effects. With Beecilli, we designed a probiotic that heterogeneously expresses glyphosate-degrading enzymes. By incorporating the foreign DNA into the bacterial chromosome, we provide a stable construct that does not require constant selection pressure and thus simplifies potential real-life applications.
The problem
Economic & Ecological Importance of Honeybees
The relationship between mankind and bees dates back millennia. The first evidence of humans using honey and wax from wild beehives originates from 7,000 BC while the first beekeeping techniques were developed in Egypt around 3,000 BC1. Today, one species in particular dominates the “bee world” – the European honey bee (Apis mellifera).
Honey bees are the single most relevant pollinator2 for the 70% of crop species that rely on pollination3. These include almonds, melons, lentils or rapeseed2. Beyond food security, pollinators also play an essential role in ecological networks and contribute to many areas of modern life e.g. fashion (through cotton pollination), medicine, and biofuel production4.
Honeybee Decline
Over the past decades, several regions of the world have reported severe losses of honey bee populations. In Europe, the number of beehives decreased by 26.5% since 1961 while estimates show a 49.5% loss in the US and Canada5.
The main drivers behind honeybee decline are climate change, habitat loss, malnutrition, infectious diseases, and pesticides3. Oftentimes it is not a single cause but multiple factors that act together – either simultaneously or over time. In the case of pesticides, most countries nowadays require data on acute toxicity towards honey bees, before a compound can be approved. However, even sublethal doses can already impact bee behaviour and health – especially when encountered over long periods or in combination with other stressors6.
Glyphosate Impact
Glyphosate is a herbicide which was first commercialised by Monsanto in the 1970s. Since its development, usage of glyphosate continuously grew and as of 2016, it is the most abundant pesticide worldwide7. Apart from agriculture, it is also used for weed control in gardens, parks, and train tracks.
The chemical acts as an allosteric inhibitor on the 5-enolpyruvoylshikimate 3-phosphate synthase (EPSPS, E.C. 2.5.1.19)8 which is part of the Shikimate pathway. The pathway plays a crucial role in the synthesis of aromatic compounds, including aromatic amino acids, across all kingdoms except animals9.
Although bees do not use the Shikimate pathway, they still suffer from a range of sublethal effects. Glyphosate is taken up by forager bees who come across glyphosate residues in the environment10 and then transfer the contaminated nectar or pollen to other bees in the hive. The chemical mainly affects bees by perturbing the gut microbiome and, as a result, increases their susceptibility to infections11. Furthermore, glyphosate can lead to a decrease in larvae weight and brood survival12. In open field experiments, foragers fed with glyphosate also showed difficulties finding their way back to the hive13 which can negatively affect food supply. In general, literature taking into account the diverse stressors the bees face under realistic conditions is scarce.
Our Solution
Honey Bee Probiotics
Probiotics are microorganisms that benefit gut health. While probiotics for human consumption are a well-established product, more recently, the potential use of probiotics in other species has been explored. For bees, commercial probiotics exist which are aimed at increasing general resilience. However, these strains often fail to successfully colonise the gut because they are not part of the native microbiome of bees14.
With Beecilli, we want to modify a native microorganism of the bee gut microbiome. Similar approaches have been taken by two previous iGEM teams in 201515 and 202016 for other pesticides. They originally focussed on the native species Gilliamella apicola and Snodgrassella alvi. However, due to issues at different steps of the project, neither team met the original goal and instead worked on e.g. development of a bee feeder system.
In our project, we focussed on the well-characterised lab strain Bacillus subtilis. Substrains of B.subtilis were isolated from the honey bee gut by a research team in Argentina17. Feeding an unmodified B. subtilis strain already showed benefits at the hive level including higher honey storage and decrease in Nosema sp. (fungus) and Varroa sp. (parasite)18. For Beecilli, we decided to introduce a non-native pathway for glyphosate degradation into B. subtilis.
The Ability to Degrade Glyphosate
The ability to degrade glyphosate is shared among several bacteria (and some fungi) from different taxonomic groups19. These organisms often originate from glyphosate-contaminated sites including soil and aquatic systems.
Glyphosate can be degraded by two different enzymes – glyphosate dehydrogenase or C-P lyase20. The former breaks down glyphosate into aminomethlyphosponic acid (AMPA) and glyoxylate in a FAD-dependent redox reaction20. The products are further degraded and enter different metabolic processes19. In our work, we focused on two specific glyphosate dehydrogenases (goxA & goxB) previously isolated from environmental soil samples21,22 .
In the C-P lyase pathway, the enzyme cleaves off the phosphate group which yields sarcosine and Pi20. Bacteria expressing these gene functions are shown to utilize glyphosate as a phosphorus source23. Introducing the operon for this multimeric enzyme complex originating from Sinorhizobium meliloti into our host offers an alternative approach to degrading glyphosate.
In our project, we prepared gene constructs for all three enzymes and combined them with different promoters (see Parts). The inserts were designed for chromosomal integration to circumvent the necessity for antibiotics as selection pressures. For one of the enzymes, we constructed a model to calculate the glyphosate degradation time required based on probiotic dosage. Another model estimates how pesticides affect the composition of the hive population (see Model).
The Implementation
The modified probiotic could be fed as a dietary supplement in combination with sugar syrup or water to the bees. Thereby, beekeepers could protect their hives from the chemical harm of glyphosate and increase their resilience to other stressors. More information can be found here. However, we limited the scope of our project to the modification of the microorganism itself.
Next Steps
Although B. subtilis has been previously isolated from honey bee guts, we believe that Lactobacillus Firm5 would be an even better host when it comes to compatibility with the native gut microbiome14. For now, we decided to still demonstrate our approach in B. subtilis due to the availability of the strain and its established cultivation and cloning procedures. However, after successfully demonstrating glyphosate degradation in the engineered B. subtilis, we would propose to use the constructs for the transformation of Lactobacillus.
When glyphosate degradation in the final host has been demonstrated, one can continue with testing the probiotic in honeybees. Here, the focus would lie in showing the successful colonisation of the engineered strain in the bee gut, analysing the effects of the probiotic on the microbiome composition, and testing glyphosate degradation. At the hive level, the transfer efficiency of the probiotic between foragers and other bees needs to be analysed. These experiments would also offer data to improve our probiotic dosage model which shall eventually serve as a tool for real-life applications.
References
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(2018). Glyphosate perturbs the gut microbiota of honey bees. Proceedings of the National Academy of Sciences, 115(41), 10305-10310.
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(2018). Glyphosate affects the larval development of honey bees depending on the susceptibility of colonies. PLOS ONE, 13(10), e0205074.
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(2015). Effects of sublethal doses of glyphosate on honeybee navigation. Journal of Experimental Biology, 218(17), 2799-2805.
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