Team:MichiganState/Description

Project Description

Project Description
 Bees and CCD
 Snodgrasella alvi
 Biocontainment
 References

Bees and Colony Collapse Disorder

With over 450 species in Michigan, bees are the most essential animal pollinators of agricultural crops and wildflowers[1]. Bees are integral to maintaining biodiversity, with approximately 75% of the world’s flowering plants relying on animal pollinators[2]. In recent years, however, data has shown that honey and wild bee populations are plummeting. In 2006, U.S. beekeepers reported winter hive losses for honeybees spiked from 15% to 30-90%[2]. This has resulted in an increase in Colony Collapse Disorder (CCD), a phenomenon where the majority of worker bees in a colony disappear and the colony cannot rebuild. In addition, half of the wild bumblebee species in Michigan have declined by more than 50% over the past 20 years[3]. There are several possible causes for these population declines, including parasites, climate change, and loss of foraging habitat[4]. Another contributing factor is the use of agricultural pesticides that negatively impact bee species, which will be the focus for BeeTox.

The decline of bee populations has been linked to a class of pesticides known as neonicotinoids, which act on nicotinic acetylcholine receptors (nAChRs) to interfere with the nervous systems of insects. Bees can be particularly sensitive to neonicotinoids, possibly because of their deficit of detoxification enzyme genes associated with insecticide metabolism[5]. Even at sublethal doses, these pesticides can drastically alter bee behavior, resulting in reduced immunocompetence, pollen production, homing abilities, and nursing. Once picked up by one bee, neonicotinoids can be transmitted throughout the hive via pollen, nectar, and social contact[4].

Snodgrassella alvi

We plan to use gut microbiome engineering to aid bees in insecticide metabolism in order to reduce the detrimental effects of agricultural practices on bee populations. Because bees exhibit a deficit of genes involved in natural xenobiotic metabolism, we hypothesize that probiotic microbes engineered to express enzymes known to bind and conjugate insecticides will increase the rate of neonicotinoid metabolism in the bee gut, protecting their host from the detrimental effects of pesticide ingestion. We have chosen to focus on the cytochrome p450 and glutathione S-transferase families as they have been associated with increased pesticide resistance in many insects[7,8,9]. We will utilize bioinformatics techniques to evaluate proteins within these gene families to select optimal genes for the probiotic for effective metabolism and successful transformation. The team has selected Snodgrassella alvi as the primary microbial chassis for engineering and use as a probiotic due to its central role in the gut microbiome of both honey bees and wild bumblebees[11]. S. alvi has been successfully transformed and established in the gut of newly emerged worker bees previously[6]. We aim to adapt these protocols for gut microbiome engineering in bees with established microbial communities for more practical treatment. BeeTox also plans to investigate secretory mechanisms specific to S. alvi that will allow enzymes produced by the probiotic to be transported directly into the lumen of the gut.

Biocontainment

Biocontainment will be a central focus of this project as it is essential that non-target insects do not become resistant to pesticides. Several possible methods of biocontainment will be explored and integrated into a single solution. One biocontainment strategy involves taking advantage of the inherent characteristics of Snodgrassella alvi to prevent spread to agricultural pests. S. alvi is restricted to guts of corbiculate bees, so it is possible this bacterium would not establish in other insects that consumed the probiotic[11]. Furthermore, S. alvi does not reproduce in atmospheric conditions, which may limit its survivability and ability to spread once excreted from the bee gut[14]. In addition to preventing spread of the probiotic itself, it is imperative that we prevent lateral gene transfer of detoxification genes to other bacteria that could interfere with agricultural practices. To accomplish this, we plan to take a two-pronged approach to LGT prevention. The detoxification gene itself will be located on a plasmid and controlled by a T7 promoter, while we will integrate the T7 polymerase gene into the chromosomal DNA of S. alvi[15]. Because T7 polymerase is highly specific and unlikely to occur in any naturally occurring microbe[16], this approach will prevent successful transcription of detoxification genes should the vector be taken up by a non-target bacterium. We will also utilize the GhoST toxin-antitoxin system[17], with the toxin located on the same plasmid as the pesticide detoxification gene and the antitoxin integrated into chromosomal DNA at a separate location, ensuring recipient cell death in the event of plasmid transfer. With this combined strategy, two independent phage transfers and a plasmid transfer would have to occur for successful LGT, making expression of detoxification genes in a non-target microbe extremely unlikely. Another approach for biocontainment, specifically during the administration of the probiotic solution to wild bees, involves incorporation of a selective feeding device. BeeTox will explore the capabilities of image recognition technology to design a system that will selectively administer a probiotic feeding solution to bees. Various feeding devices will be modeled and tested to determine the optimal device for both attracting bees and separating the probiotic from potential pests.

With the use of an engineered gut symbiont, we hope to reduce the threat that neonicotinoids pose to bees and, subsequently, plant biodiversity, without genetically modifying insects or utilizing drone pollinators, which would both be costly and difficult to implement[12]. In contrast, our solution would be relatively inexpensive. Due to the social nature of some bee species, we believe the beneficial microbes will spread within a hive for widespread establishment, minimizing the time and resources required to inoculate a community[13]. There are no known commercial insecticides that are non-toxic to bees[10], and despite the use of best practices intended to minimize the impact of pesticide usage on non-target organisms, neonicotinoids are still widely used by commercial farms and consistently pose a threat to local pollinators[18]. Rather than necessitating a complete shift in global agricultural practices, our solution will protect biodiversity by increasing the fitness of valuable pollinators to their environment.



References

  1. “Bees of Michigan.” College of Natural Science Michigan Pollinator Initiative, Michigan State University, pollinators.msu.edu/bees-of-michigan/.
  2. “Insect Pollinators.” National Resources Conservation Services, United States Department of Agriculture, www.nrcs.usda.gov/wps/portal/nrcs/main/national/plantsanimals/pollinate/.
  3. Graham, Lester. “Half of Michigan Bumblebee Species Dropped by 50 Percent or More.” Michigan Radio, National Public Radio, 16 Jan. 2019, www.michiganradio.org.
  4. Raymann, Kasie, et al. “Imidacloprid Decreases Honey Bee Survival Rates but Does Not Affect the Gut Microbiome.” Applied and Environmental Microbiology, vol. 84, no. 13, 2018, doi:10.1128/aem.00545-18.
  5. Mary R. Berenbaum and Reed M. Johnson (2015) “Xenobiotic detoxification pathways in honey bees.” Insect Science, vol 10, August 2015, pp. 51-58, doi:10.1016/j.cois.2015.03.005
  6. Leonard, S.P.; Powell, J.E.; Perutka, J.; Geng, P.;,Heckmann, L.C.; Horak, R.D.; Davies, B.W.; Ellington, A.D.; Barrick, J.E.; Moran, N.A. Engineered symbionts activate honey bee immunity and limit pathogens. Science 2020, 367, 573–576.
  7. Chen, Xiaokun, et al. “The Cross-Resistance Patterns and Biochemical Characteristics of an Imidacloprid-Resistant Strain of the Cotton Aphid.” Journal of Pesticide Science, vol. 40, no. 2, 8 Jan. 2015, pp. 55–59., doi:10.1584/jpestics.d14-031.
  8. Reid, William R, et al. “Overexpression of a Glutathione S-Transferase (Mdgst) and a Galactosyltransferase-like Gene (Mdgt1) Is Responsible for Imidacloprid Resistance in House Flies.” Pest Management Science, vol. 75, no. 1, 2018, pp. 37–44., doi:10.1002/ps.5125.
  9. Højland DH, Kristensen M (2017) “Analysis of Differentially Expressed Genes Related to Resistance in Spinosad- and Neonicotinoid- Resistant Musca domestica L. (Diptera: Muscidae) Strains.” PLoS ONE 12(1): e0170935. doi:10.1371/ journal.pone.0170935
  10. J. Devillers, A. Decourtye, H. Budzinski, M.H. Pham-Delègue, S. Cluzeau & G. Maurin (2003) Comparative toxicity and hazards of pesticides to Apis and non-Apis bees. A chemometrical study, SAR and QSAR in Environmental Research, 14:5-6, 389-403, DOI: 10.1080/10629360310001623980
  11. Powell, E., Ratnayeke, N., & Moran, N. A. (2016). Strain diversity and host specificity in a specialized gut symbiont of honeybees and bumblebees. Molecular ecology, 25(18), 4461-4471.
  12. Potts, Simon G., et al. "Robotic bees for crop pollination: Why drones cannot replace biodiversity." Science of the total environment 642 (2018): 665-667.
  13. Kwong, W. K., & Moran, N. A. (2016). Gut microbial communities of social bees. Nature Reviews Microbiology, 14(6), 374.
  14. Kwong, W. K., & Moran, N. A. (2013). Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order ‘Enterobacteriales’ of the Gammaproteobacteria. International journal of systematic and evolutionary microbiology, 63(6), 2008-2018.
  15. Leonard, S. P., Perutka, J., Powell, J. E., Geng, P., Richhart, D. D., Byrom, M., ... & Barrick, J. E. (2018). Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids. ACS synthetic biology, 7(5), 1279-1290.
  16. Studier, F. W., & Moffatt, B. A. (1986). Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Journal of molecular biology, 189(1), 113-130.
  17. Wang, X., Lord, D. M., Cheng, H. Y., Osbourne, D. O., Hong, S. H., Sanchez-Torres, V., ... & Benedik, M. J. (2012). A new type V toxin-antitoxin system where mRNA for toxin GhoT is cleaved by antitoxin GhoS. Nature chemical biology, 8(10), 855.
  18. Douglas, M. R., & Tooker, J. F. (2015). Large-scale deployment of seed treatments has driven rapid increase in use of neonicotinoid insecticides and preemptive pest management in US field crops. Environmental science & technology, 49(8), 5088-5097.