Team:UAlberta/BeeLab
BeeLab
Overview
Team UAlberta’s Beelab focused on performing in vivo experimentation on the efficacy of protoporphyrin IX (PPIX) at inactivating Nosema ceranae spores and the function of our Antifungal Porphyrin-based Intervention System (APIS) prototypes. Our work for our Beelab included collecting and housing honeybees for experimentation under various treatment conditions.
The main motivation behind using live honeybees rather than other animal models, or cell lines, is that N. ceranae is an obligate parasite of honeybees. N. ceranae infections have only been found to infect the Asian honeybee, Apis ceranae, and only recently has it been documented in the Western honeybee, Apis mellifera [1]. Use of cell line studies were also considered but honeybee cell lines are notoriously difficult to acquire and maintain. Published alternatives, such as Lepidoptera-based cell lines have been infected by N. ceranae, but are cited as not suitable for propagating spores [2].
As propagating spores was necessary for our experiments and due to the of the physiology of N. ceranae and limitations in vitro testing, Team UAlberta opted to use live honeybees for our experiments as it would provide the best route for evaluating our designs. Find the full justification for live honeybee experimentation here.
In order to demonstrate the feasibility of our APIS design, we identified four critical outcomes that our technology must achieve:
- Excess intracellular PPIX must not have any toxic effects on our E. coli chassis as the function of the APIS construct hinges on producing PPIX above natural concentrations. This aspect was focussed on in our Wetlab section.
- Our E.coli chassis itself must not be harmful to the health of both Nosema-infected or uninfected honeybees treated with it.
- The PPIX therapy must function as intended so that ingestion of PPIX must decrease Nosema spore loads in vivo or prevent the spread of infection.
- PPIX must not have a negative effect on the health of both Nosema-infected and healthy honeybees health. We must ensure that bees, both health and unhealthy, do not experience negative health consequences by consuming PPIX.
Achieving these four outcomes individually would provide a set of proof-of-concepts that demonstrate the functionality of APIS, and that our novel strategy of using biosynthesized PPIX as an antifungal therapeutic is effective against N. ceranae infections in vivo.
Establishing a Honeybee Hive
In order to perform our in vivo experimentation, we needed a source for Western honeybees (Apis mellifera). Luckily, we were introduced to Jason McKinnon, a friend of one of our supervisors, who had been looking to start a beehive of his own. With his cooperation, we set up a new honeybee hive in south Edmonton, AB, from which we would collect honeybees!
Our team purchased three hive boxes, a few dozen frames, and the basic accessories for starting a hive. With the hive ready to go, Jason and our team travelled to Hove Apiaries, owned by Alvin and Judy Hove, where we got a five frame nuc of Italian honeybees (Apis mellifera ligustica) which is a subspecies of the Western honeybee [3]. The nuc included brood frames, honey frames, a mated queen honey bees, and thousands of worker bees. Judy and Alvin were also very generous as they donated nine drawn frames for us to use when our hive grows and expands. After transplanting the nuc into our brand new hive, we let the honeybees get settled for a couple of weeks while Jason helped our team tend to the hive in the meanwhile. Watch our hive building process here!
Defining Specimens
Before any experimentation took place, our team defined our sample specimens as honeybee hives have different castes that exhibit different physiology. Thus, the subjects of our experiments were to be worker honeybees, as they are the most abundant caste [4] and would be the major carrier of N. ceranae in hives
However, workers are found in a distribution of ages in hives. So, our team had two options of which workers to collect:
- Newly emerged bees which are adult bees that have just finished pupation and have just emerged from their wax-capped cells. These bees ages can be determined and should have a known pathogen load as they would have not been in contact with other bees. Or,
- Non-newly emerged bees which are worker bees found in and around the hive. The age of these bees cannot be determined and their pathogen load is unknown as they have been potentially exposed to unhealthy bees. From now on, non-newly emerged bees will be referred simply to as adult bees.
If our team ended up collecting drone bees, or even the queen bee, our protocol was to return them to the hive.
Bee Collection
After letting our hive establish its new home, we began collection procedures for our various Beelab experiments. First, we consulted with Paul Greidanus, a commercial honey producer, about the effects of collecting honeybees on hive health. He told us that hives regularly lose hundreds of workers daily without adverse effects as healthy queens are consistently laying new brood (eggs). With this information, our team was reassured that we could collect enough bees for our experiments without harming our hive.
Our team’s first choice was to use newly emerged bees as more variables can be accounted for. To collect newly emerged bees, we placed frame cages around capped brood. Our frame cages were enclosed nets which would trap any workers that had just emerged. However, we quickly realized that bees did not emerge consistently in large numbers for our experiments which reduced the number of bees we could collect for a given day.
Due to this limitation, our team changed our approach and collected adult bees instead. Our decision was further justified by the idea that collecting a distribution of workers would be a closer approximation of the age variation within actual hive, thus providing a more realistic dataset for demonstration. To collect adult bees, we removed frames from the hive and ran our container across it. This caused multiple bees to gently fall into our container, which was faster than trying to individually collect bees. Paul Gredanius showed us this method of collecting bees and it drastically streamlined the process.
Bee Containment
To house our bees in our laboratory, we created custom bee cages according to previous methods [5]. Our design is depicted below. These cages had the appropriate openings for both feed inlets and removal of honeybees. Our mesh walls and flooring were designed to provide our honeybees with necessary ventilation and drainage.
Our standard feed for the honeybees was filter-sterilized 2M sucrose solution. These were poured into our feeding tubes which were 15 mL disposable centrifuge tubes with one to three holes drilled at the ends. When filled and capped, a vacuum is created which prevents the feed from pouring out while still allowing the solution to drip out slowly so that the honeybees are able to feed.
Check-in Procedures
For all our experiments, our members performed daily check-ins on the bees. Each day, the number of dead bees was counted and then removed from the cage. If the feeding tubes had a volume of less than 5 mL, they were refilled with the appropriate feed. The number of dead bees per cage and the initial and final feeding tube volumes were recorded.
Honeybee Termination
Our termination protocols prioritized the welfare of the honeybees. Through our conversations with bee-researchers, we found out that honeybees are classified to have no experience of pain, only a reflex to pain stimuli. However, our team still decided to create termination procedures that both reduce any pain stimuli the bees might experience and are consistent with established methods [6]. Termination, by instantaneous decapitation/physical destruction, was conducted swiftly in order to eliminate the possibility that the bees might experience a pain stimulus. Our termination protocols were only used when the honey bees had fulfilled their intended purpose for our experiments, meaning that we plan on terminating no bees unless we can obtain quality measurements from them.
Nosema ceranae Spore Counts
After dead honeybees were collected, or live bee sacrificed, N. ceranae, spore counts were conducted when appropriate. Individual or multiple bees were homogenized in 1 mL of PBS per bee and depending on the spore load, the liquid fraction was diluted further with PBS. 10 uL of each sample were then loaded onto a hemocytometer and Nosema spore were counted using a phase contrast microscope at 400x magnification.
With our bees specimens collected and with our four ultimate goals in mind, we designed several experiments to answer the following questions: