Description

What is Nosema ceranae?

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].

**Figure 1:** *Nosema ceranae* spores imaged using phase contrast microscopy. The threadlike structures are polar filaments which spores use to penetrate honeybee epithelial cells. Pictured by Gisder S, et. al [5]

**Figure 2:** A Western honeybee worker (*Apis mellifera*)—the new host of *N. ceranae*.

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 [6]. 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 [7].

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 [8][9]. 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.

**Figure 3:** The chemical structure of fumagillin [10].

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 contributes billions of dollars in value through the pollination of crops [8] and helps support the economy by providing employment directly through honey production, or by supporting other industries.

Figure 4: Percentage contribution of honey production of Canada’s provinces in 2016 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.

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 the bees’ diets were supplemented with porphyrin species, the spore count 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) [8].

Figure 5: (A) shows Nosema spores without porphyrin treatment and (B) shows the damage done on the spore walls after incubation with the PP(Asp)2 Figure 6: A comparison between the similar structures of (A) PP(Asp)2 and (B) PPIX where the hydrophilic end of each molecule is highlighted.

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 [9].

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
Testing the modified 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.