Team:UAlberta/Description

PROJECT INSPIRATION & DESCRIPTION

The Big Picture

Alberta is the largest honey producer in Canada, providing 45% of Canada’s honey, forming a multimillion dollar industry. Products from the hive (honey and wax) are valued at $200 million CAD [1]. Honey bees also play an essential role in modern agriculture, as pollinators they pollinate ⅓ of the plant-based products we eat and pollinate commercial crops like canola and soybeans, which are feedstocks of numerous manufacturing processes. Such industries bring in over $10 billion in sales annually, while creating jobs, supporting families and our community [8]. Globally, as much as $577 billion USD in economic value is produced through crop pollination [6], with honey bees commonly being used as a cross-pollinator [7]. Ultimately, bees are an integral part of our economy, environment, and society.

Figure 1: Apis mellifera, commonly known as the European honey bee, is the most common species of bee used in the commercial apiculture industry.

Figure 2: Apis mellifera, 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].

Nosema ceranae

Nosema ceranae is the most widespread disease affecting Apis mellifera, the European honey bee, in Canada [2]. This microsporidian parasite lives in the midguts of honey bees, infecting midgut epithelial cells where it reproduces and bursts out of the cell as spores, ready to infect again [4]. There is a range of infection, as all honey bees have some level of N. ceranae spores, therefore, it is important to determine whether the spore level is above or below the pathogenic concentration. However, individually infected bees suffer from weakened immunity, shortened lifespan, and diarrhea [3]. Across an entire hive, this infection causes reduced productivity, increased susceptibility to other diseases, and ultimately more death and increased risk of hive death. This infection weakens both individual bees and entire hives, leaving them more susceptible to other infections [4].

Continued Consultation

In 2018, the UAlberta team developed the Antifungal Porphyrin-based Intervention System (APIS), a possible treatment to N. ceranae, a fungal infection in A. mellifera, the European honey bee [5]. After speaking with our stakeholders, particularly the Alberta Beekeeping Commission which represents all commercial beekeepers in Alberta, we realized that there is a lack of an accurate diagnostic method. As such, this year we have continued our work to help beekeepers combat this disease, developing the Beetector: an on-site detector for N. ceranae spore quantification.

Figure 3: Our further consultation with beekeepers alerted us that another root cause of the issue of Nosema is that diagnostic methods are lacking, which hinders the ability for our APIS system to be deployed effectively. Thus, as a response to this, we decided to tackle Nosema detection this year.

Current Methods

Currently, the gold standard for diagnosing N. ceranae requires collecting bees and shipping them to a central laboratory where they are homogenized and viewed under a microscope where spores are counted [2]. From speaking with beekeepers we learned that this process is inadequate and can potentially place hives in jeopardy as results can take weeks allowing the disease to spread, and it can also lead to practices of treating all hives regardless of infection which is both expensive and can harm the bees if done improperly. Moreover, these tests are expensive and time-consuming, which prevents hobbyist beekeepers from accessing these tests.

Figure 4: The main method used to diagnose Nosema infections in hives is a hemocytometer, which is labour-intensive and inaccessible to untrained personnel (i.e. beekeepers).

Due to the limitations presented by the gold standard, some beekeepers opt to use visual signals to detect N. ceranae, such as declining populations, poor honey production, dysentery in and around the entrance of the hive, and worker bees crawling around the hive with swollen or greasy looking abdomens [4]. The issue with this method is that it is difficult to detect accurately, and often, by the time these visual signals are detected, the infection has spread too far for treatment to be effective.

One of the main by-products of these ineffective detection methods is that the bees will be too sick for effective treatment, and can possibly fly off to other hives and infect other bees as well.

The Beetector: An on-site paper detection system for quantifying N. ceranae spore load in honey bees

The Beetector system consists of M13 phage particles and amajLime chromoprotein joined together enzymatically by sortase. Our approach to developing the detection system is to use an M13 phage and screening for N. ceranae spores through phage display for binding. Following this, the screened phagemid is cloned to express a sortase-tagged structural protein. A recombinant amajLime chromoprotein is expressed, carrying a separate sortase tag, which is then enzymatically ligated to the phagemid. Therefore, the phage particles and recombinant amajLime are integrated, with sortase fusing the tags together, producing N. ceranae spore-binding phage particles with a visual reporter.

These phage particles are stored in a solution and given to beekeepers. Beekeepers will be given instructions for extracting bee midgut and making a homogenized solution. Upon testing, the phage particle solution is applied to a paper strip that has been immersed in the homogenized bee solution. The strip is then washed, and the colour intensity is compared to a scale that correlates spore concentration with color, thereby determining the spore load per bee.

Figure 5: A schematic of the Beetector system. In the system, the beekeeper initially crushes a handful of bees and mixes them with the Beetector solution. A filter paper is then used to separate the different components of the mixture and displays colour at variable intensities, with the intensity directly correlating with the severity of infection.

How will the Beetector advance the diagnosis of Nosema ceranae?

In summary, this design is far more advanced than current detection methods as information can be collected quickly, allowing for treatment decisions to be made earlier, thereby saving hives. Moreover, this test is simple making it accessible to all beekeepers.

We hope that with the Beetector we are able to address apiculturists' concerns, providing them with access to a point of care test that is accurate and affordable. Ultimately, with the Beetector alongside APIS, we are able to democratize diagnosis and facilitate the effective treatment of Nosema ceranae, thus saving the bees.

References

• [1] Agriculture and Agri-Food Canada, Statistical Overview of the Canadian Honey and Bee Industry and the Economic Contribution of Honey Bee Pollination, Ottawa, ON: Horticulture and Cross Sectoral Division, 2017. Available: http://www.agr.gc.ca/resources/prod/doc/pdf/honey_2016-eng.pdf. [Accessed: Oct. 20, 2019]
• [2] Canadian Food Inspection Agency, Honey Bee Producer Guide to the National Bee Farm-level Biosecurity Standard, Ottawa, ON: Office Of Animal Biosecurity Canadian Food Inspection Agency, 2013. Available: https://www.inspection.gc.ca/animals/terrestrial-animals/biosecurity/standards-and-principles/honey-bee-producer-guide/eng/1378390483360/1378390541968?chap=9. [Accessed: Oct. 20, 2019]
• [3] A. J. Burnham, “Scientific Advances in Controlling Nosema ceranae (Microsporidia) Infections in Honey Bees (Apis mellifera)”, Frontiers in Veterinary Science, vol. 6, no. 79, 15 March, 2019. [Online serial]. Available: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428737/. [Accessed Sept. 27, 2019]
• [4] 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, AB, 2017. Available: https://era.library.ualberta.ca/items/7b26607f-08fb-4e85-9f7a-0fbac0afee68. [Accessed: Oct. 20, 2019]
• [5] Science and Policy for People and Nature, “Press Release: Pollinators Vital to Our Food Supply Under Threat” Science and Policy for People and Nature, 2007. [Online]. Available:https://www.ipbes.net/article/press-release-pollinators-vital-our-food-supply-under-threat. [Accessed: Sept. 12, 2019].
• [6] U.S. Forest Service, “What is Pollination” U.S. Forest Service, [Online]. Available: https://www.fs.fed.us/wildflowers/pollinators/What_is_Pollination/. [Accessed: Sept. 15, 2019].
• [7] “UAlberta iGEM Team 2018 Wiki” 2018.igem.org, Oct, 2018. [Online]. Available: https://2018.igem.org/Team:UAlberta. [Accessed Oct. 8, 2019]