Description

Nosema ceranae: Fungal Freeloader

Nosema ceranae is a microsporidian parasite that infects the midgut of honeybees. N. ceranae invades the epithelial cells of the bee midgut and as it’s dependent on its honeybee host for its energy source, it causes debilitating energetic stress [1]. For individual honeybees, Nosema infections have symptoms which include shortened lifespans and weakened immune function. On the scale of a colony, the symptoms of Nosema infections greatly decrease hive productivity and contribute to colony failure[2][3][4].

Unfortunately, N. ceranae has recently spread to the major commercial honeybee species—the Western honeybee, Apis mellifera—and is now regarded as the dominant Nosema species infecting honey bees globally. Through our conversations with Albertan beekeepers, we found that N. ceranae is a constant issue but when there is an outbreak it can lead to unsustainable rates of death. Some report over 80% hive loss due to Nosema. Nosema is also especially pervasive in cold climates, like the one found in Alberta, as colder temperatures contribute to increased hive losses in winter [6].

Previous Nosema Treatments

Previous methods of treating N. ceranae infections rely heavily on fumagillin, a potent antifungal agent that is not only expensive but is mutagenic and toxic to mammals. At low doses, fumagillin has actually been shown to increase N. ceranae spore count in honeybees [7][8]. Moreover, Medivet Pharmaceuticals Ltd., the only company that produced fumagillin for the whole of North America’s supply, went out of business earlier this year. This development led to the collapse of the fumagillin supply chain and means that there is no longer any protection available against Nosema. Thus, the issues with using fumagillin and its recent discontinuation motivate the development of alternative treatments for combating N. ceranae as a replacement is imperative to Alberta’s honey industry and the survival of honeybees. Interestingly, Medivet was based in Alberta and we were able to interview their former CEO, Ursula De Runga.

Why Nosema and Honeybees?

Team UAlberta was made cognizant of the Nosema problem through contact with an Albertan beekeeper, Elisabeth Goldie. She made it very clear that Nosema was a massive concern to her and her fellow beekeepers. Honeybees and beekeepers are an integral part of everyday life as their ecological and economic impacts are far-reaching. This is particularly true in Alberta as our province contributed to almost half of all honey production in Canada in 2016, which is a $157.8 million CAD industry. The beekeeping industry also contributed 4 to 5.5 billions of dollars (CAD) in value through the pollination of agricultural crops [9].

4 to 5.5 Billion CAD for Agroeconomy [9]

Having these different aspects of Alberta’s beekeeping industry come to light made it clear to our team just how important it was to tackle this issue. We were motivated by the hope that we could actually work towards a project that could positively impact our community.

Our Plan

Once we identified the issue we wanted to combat, we conducted preliminary research which led us to recent findings that porphyrins, a class of organic compounds, are capable of inactivating N. ceranae spores. When bees’ diets were supplemented with porphyrin species, spore counts in their midgut decreased significantly with no observed adverse effects on the bees. In particular, the porphyrin PP(Asp)2, a chemically synthesized derivative of protoporphyrin IX (PPIX) harbouring aspartic amide moieties, was used to inactivate N. ceranae. The antifungal action observed was attributed to porphyrin disrupting the spores’ cell wall (Figure 5) [10].

This advancement was extremely interesting since porphyrins are ubiquitous in nature, such as PPIX, which is an intermediate in the endogenous heme biosynthesis pathway of Escherichia coli [11][12]. Given the structural similarities of PPIX to PP(Asp)2 (Figure 6), we hypothesized that biosynthesized PPIX may have similar antifungal effects on N. ceranae spores. What makes PPIX an attractive substitute to PP(Asp)2 is that PPIX is found in nature and can be biosynthetically produced by microbes instead of the inefficient chemical methods used for PP(Asp)2. Motivated by these results, Team UAlberta decided to focus on using porphyrins for treating Nosema infections in honeybees.

To address the threat of Nosema, and provide an alternative to fumagillin, Team UAlberta presents our 2018 iGEM project:

APIS: an Antifungal Porphyrin-based Intervention System for treating Nosema infections in honey bees!

APIS aims to augment the endogenous heme synthesis pathway in E. coli to produce an excess of protoporphyrin IX, a heme synthesis intermediate, to be used for inactivating Nosema spores.

We planned to accomplish this by completing two objectives:

• Engineer genetic constructs exploiting the heme biosynthesis pathway to overproduce PPIX
• Test the engineered strains and PPIX for their ability to inactivate N. ceranae spores in honeybees.

With continued input from beekeepers, we designed for two routes of implementation:

• Honeybee probiotic by introducing PPIX-producing microbes into the bee microbiome
• Large-scale PPIX-production to generate PPIX for conventional treatment application methods

These project designs would allow us to develop a product that could address the issue of Nosema ceranae in a safer and potentially more effective way than fumagillin, while still keeping the feed form which beekeepers found preferable and were accustomed to as it was a common way to administer fumagillin.

References

[1] C. I. MacInnis, “Nosema ceranae: A sweet surprise? Investigating the viability and infectivity of the honey bee (Apis mellifera L.) parasite N. ceranae”, M.S. thesis, University of Alberta, Edmonton, 2017. [Online]. Available: https://era.library.ualberta.ca/items/7b26607f-08fb-4e85-9f7a-0fbac0afee68 [Accessed: Oct. 15, 2018]

[2] D. M. Eiri, G. Suwannapong and N. J. C. Endler, "Nosema ceranae can infect honey bee larvae and reduces subsequent adult longevity," PLoS ONE, 10(5) e0126330. [Online serial]. Available: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126330 [Accessed: Oct. 15, 2018]

[3] A. K., R. Hernandez-Martin and L. Prieto, "Immune suppression in the honey bee (Apis mellifera) following infection by Nosema ceranae (microsporidia)," Environmental Microbiology, vol. 11, no. 9, pp. 2284-2290, 2009. [Online serial]. Available: https://www.ncbi.nlm.nih.gov/pubmed/19737304 [Accessed: Oct. 15, 2018]

[4] S. L. Gage, C. Kramer, S. Calle, M. Carrol, M. Heien and G. DeGrandi-Hoffman, "Nosema ceranae parasitism impacts olfactory learning and memory and neurochemistry in honey bees (Apis mellifera)," Journal of Experimental Biology, vol. 221, no. 4, 2018. [Online serial]. Available: http://jeb.biologists.org/content/early/2017/12/18/jeb.161489 [Accessed: Oct. 15, 2018]

[5] S. Gisder, N. Mockle, A. Linde and E. Genersch, "A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia," Environmental Microbiology, vol. 13, no. 2, pp. 404-413, 2011.

[6] M. L. Smith, "The Honey Bee Parasite Nosema ceranae: Transmissible via Food Exchange?," PLoS ONE, vol. 8, no. 8 , pp. 1-6, 2012.[Online serial]. Available: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043319 [Accessed: Oct. 15, 2018]

[7] W. Huang, L.F. Solter, P.M. Yau, B.S. Imai, “Nosema ceranae Escapes Fumagillin Control in Honey Bees,” PLOS Pathogens, vol. 9, no. 3:e1003185, 2013. [Online serial]. Available: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003185 [Accessed: Oct. 15, 2018]

[8] v. d. Heever, "Fumagillin: An Overview of Recent Scientific Advances and their Significance for Apiculture," Journal of Agricultural and Food Chemistry, vol. 62, pp. 2728-2727, 2014. [Online serial]. Available: https://www.ncbi.nlm.nih.gov/pubmed/24621007 [Accessed: Oct. 15, 2018]

[9] Horticulture and Cross Sectoral Division Agriculture and Agri-Food Canada, "Statistical Overview of the Canadian Honey and Bee Industry and the Economic Contribution of Honey Bee Pollination 2013-2014," Government of Canada, 2016.

[10] A. A. Ptaszynska, M. Trytek, G. Borsuk, K. R.-J. K. Buczek and D. Gryko, "Porphyrins inactivate Nosema spp. microsporidia," Scientific Reports, vol. 8, no. 5523, pp. 1-11, 2018. [Online serial]. Available: https://www.nature.com/articles/s41598-018-23678-8 [Accessed: Oct. 15, 2018]

[11] J. Zhang, K. Zhen, J. Chen and G. Du, "Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli," Scientific Reports, vol. 5, no. 8584, pp. 1-7, 2015.

[12] S. J. Kwon, A. L. de Boer, R. Petri and C. Schmidt-Dannert, "High-Level Production of Porphyrins in Metabolically Engineered Escherichia coli: System Extension of a Pathway Assembled from Overexpressed Genes Involved in Heme Biosynthesis," Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4875-4883, 2003.