The University of Alberta and Team UAlberta deeply value safe working practices and the safety of the public who is affected by our work. The policies and initiatives put forth by the University’s Environment, Health, and Safety (EHS) Unit make clear the safety requirements while also reinforcing a safety culture at our institution . The EHS guidelines are also in line with Canada’s and Alberta’s Occupational Health and Safety regulations . Team UAlberta has closely followed the guidelines of EHS in preparing our members and our workspace for our project.
Additionally, we have adhered to the policies of the University’s Research Ethics Office and have fulfilled the requirements of iGEM’s Safety and Security Committee for the special considerations of our project.
To work in our laboratories, which fall under the jurisdiction of the University's Department of Chemistry and the Department of Chemical and Materials Engineering, our team members had to complete safety training appropriate for both departments. Prior to working in the lab, all the members of Team UAlberta completed four online courses administered by EHS called Laboratory Safety, Chemical Safety, Concepts in Biosafety, and WHMIS 2018. We also attend the safety seminars and training sessions hosted by the department.
Laboratory-specific safety training was also administered by our mentors. This training was intended to show Team UAlberta the locations hazards, special disposal bins, and the laboratory safety features, such as the eyewash station, fire extinguishers, and fire exits. This training also provided specific instruction concerning safety precautions during routine lab activities. These training requirements definitely made Team UAlberta ready for a summer of safe, and secure work!
The laboratories where Team UAlberta worked are both classified as Containment Level 2, while our expression and cloning strain, E. coli BL21 and DH10B respectively, are both Risk Level 1 organisms. Despite this, our team still adhered to the safety requirements of a Level 2 lab:
Personal Protective Equipment: When in the laboratory, it was required that members wore long pants, closed toed shoes, a lab coat, safety goggles, and gloves.
Equipment and Protocol Training: Whenever new protocols were conducted or unfamiliar equipment used, and the lab-specific training did not cover these aspects, additional training from our mentors received.
Chemical and Biosafety: Chemicals were stored in appropriate containers and locations with other compatible reagents. The SDS of reagents used were consulted for proper disposal. Biohazards were stored in biological waste bags and were sterilized by EHS. Working surfaces were disinfected with 70% ethanol or isopropanol and decontaminated with 10% bleach.
Safety Equipment: Eyewash stations were checked weekly in accordance with federal regulations, while safety showers were by inspected by EHS personnel.
Experimenting with Honeybees
Team UAlberta's project revolves around developing a new antifungal treatment against Nosema ceranae, a microsporidian parasite of Western Honey Bees (Apis mellifera). Nosema ceranae is an obligate parasite of honeybees which infects a very specific section of the bee digestive tract. Our team hopes to demonstrate that protoporphyrin IX (PPIX), a biomolecule, is capable of inactivating Nosema spores much like porphyrin molecules have been shown to do. Our intended outcome from designing experiments that use live honey bees is to investigate the effects which PPIX has on both on honeybees and on Nosema infectivity.
Our team explored many options for characterizing the effects of PPIX, though, after reviewing the "Three R's" (Replace, Reduce, Refine) in the design of our project's experiments, it was determined that using live honeybees for experiments was the most justifiable course of action given technical restrictions and scientific significance which will allow us to adequately develop our project. Our justification for using live honey bees is as follows:
1. Replace: Before live honeybees were considered, our team made sure to exhaustively search for any non-animal models to replace honeybees which would also be compatible with the restrictions of our project. In our research, we found that there are no commercially available stable cell lines for honeybees, no researchers at our institution work with home-made bee cell lines and that the creation bee cell lines is difficult and does not provide permanent cell lines.
This lack of available bee cell lines is compounded by the issue that Nosema ceranae is an obligate parasite of Apis mellifera, meaning that Nosema can only infect and viably reproduce within actual honeybees. There have been some results showing that Sf9 cells (derived from moths) and related derivatives are capable of being infected with Nosema, though the information on the viability of spores propagated from Sf9 cells has not been documented. Therefore, using a substitute insect cell line rather than honeybees could alter the infectivity of the Nosema parasite which will provide further complications for analysis and possibly lead to incorrect interpretations of any collected data from these cells lines. In fact, our contacts in bee-related research advised against using non-bee cell lines for our work due to these reasons. This became the primary motivation behind our team's decision to pursue the use of live honeybees for our experiments.
2. Reduce: The publications underpinning the design of our experiments also used live honeybees for studying Nosema infections. Our group aims to emulate their methods in order to be consistent with the established work, ensure power in our analyses, and yet still reduce the total number of honeybees used per measurement by utilizing samples for multiple different types of analysis. For example, the same group of bees can be used to calculate survivor numbers of honeybees over time per treatment group, and the spore load for individual bees, rather than setting up separate bee experiments for each analysis.
A power analysis using G*Power has also shown that we can obtain statistically significant spore load measurements from samples of at least 30 bees. Thus, this result greatly reduces the number of bees that we will need to sample for each measurement and also reduces the redundancy needed to counteract expected attrition. Therefore, our team has put much effort into ensuring that no unnecessary honeybees are to be used, yet measurements can still yield statistical significance from which valid conclusions can be drawn from.
3. Refine: As the impact of using actual honeybees became more apparent, we consulted with bee researchers at the University of Alberta to understand the standards used in bee experimentation. By doing this we have collected protocols that outline the appropriate housing, feeding, termination, and measurement techniques that have been established in bee focussed research. Their input made clear the conditions needed to keep honeybees in the laboratory, and we have incorporated this knowledge into the design of our experiments. Thus, our methods account for and incorporate established methods such that the bees are appropriately taken care of.
From our consideration of the three principles of "Replace, Reduce, and Refine", it was apparent to our team that using live honeybees for our experiments would provide quality data needed to characterize our project and which would support work already completed in the field of Nosema research.
Animal Use Ethics
As our project aimed to used live honeybees (Apis mellifera) in our experiments, we first consulted our institution’s guidelines as well as other bee researchers for safety and ethics requirements. In studying the University’s policies, we found that our Animal Care and Use Committees do not cover the use of insects and the University’s Research Ethics Office does not require an ethics review for our use of honeybees as they are not included in the University’s definition of “animal use” . Only invertebrates in the class Cephalopoda, which honeybees do not belong, require ethics review. Thus, to use honeybees, Team UAlberta does not need animal use committee documentation or ethics review per the University of Alberta's policies.
After our team was sure that our proposed work with honeybees was in line with the University’s policies, we fulfilled the requirements of iGEM’s Saftey and Security Committee. As we planned on using both honeybees (A. mellifera) and Nosema ceranae, organisms that are not on iGEM’s white list, we filled out the Safety Check-in forms. Originally, we did not fill out the Animal Use Form as honeybees did not fit the description of “higher-order invertebrates” that iGEM provided. However, after special considerations, the Safety and Security Committee extended the definition of higher-order invertebrates to social insects like honeybees. Following that, we completed the Animal Use Form as required.
Special safety considerations were made for working with live honeybees. To outfit our workspaces for honeybee experimentation, we consulted one of our advisors, Courtney MacInnis, a bee researcher, about what engineering controls and safe operating procedures were needed. From our discussions with her, we decided to keep honeybee work in a dedicated laboratory. For our bee containment, we combined published designs with the the design of Ms. MacInnis’s cages and this led to building custom enclosures.
We had also set up a netted canopy over our work area to ensure that escaped bees can be recaptured. Ms. MacInnis also helped us practice standard protocols before our own experimentation began in order to familiarize our members with safe conduct and proper techniques when handling bees. Safety equipment like veils and suits were in fact not needed for the laboratory setting as the honeybees were very docile, though they were used during bee collections.
Our team also purchased an Epi-Pen, which is a device for self-administration of epinephrine in case of anaphylactic shock and closure of airways. This device was purchased as honeybee stings can cause allergic reactions in some individuals. We tried to be proactive against allergies by inquiring about workplace allergy testing to both EHS and our local healthcare service providers. However, allergy testing for the workplace is unprecedented and there was no framework in place that would allow our team to receive the service outside of individual medical examinations. Our experience here reveals an area for improvement for our occupational safety system.
 Government of Alberta, “Environment, Health & Safety,” Government of Alberta, 2018 [Online]. Available: https://www.alberta.ca/ohs-act-regulation-code.aspx [Accessed: Oct. 15, 2018]
 University of Alberta Research & Innovation, “Research Ethics Office: Ethics Review,” University of Alberta, 2018 [Online]. Available: https://www.ualberta.ca/research/support/ethics-office/research-ethics/ethics-review [Accessed: Oct. 15, 2018]