Burn the Worm: Advancing Manduca Sexta as a New Model for Skin Injury and Infection
Abstract:
Bibliography/Citations:
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Additional Project Information
Research Plan:
Tobacco hornworms (Manduca sexta) were bred from adult Carolina hawkmoths, and raised on a commercial feed that was purchased from Oregon Silkworms, LLC. Through the first four instars, caterpillars were kept at room temperature and fed a steady diet of prepared food. A total of 180 healthy hornworms were raised.
After their emergence as fifth instars, at which point tobacco hornworms are at their largest, three burns were administered on the left side of each larva with a 60-watt soldering iron (LDK brand), between the segments where the second and third abdominal prolegs are located, third and fourth abdominal prolegs, and fourth and fifth abdominal prolegs are located. These locations were chosen because they were not close to the head nor heart (which are located near the front and top, respectively); care was also taken to avoid the spiracles, which is how insects breathe.
Upon receiving the three burns, which were timed at one second each, a sterile swab was used to administer one of four treatments: coal tar (negative control, considered toxic), cortisone (anti-inflammatory, but not anti-bacterial), honey (suspected to have antimicrobial properties), and triple-antibiotic ointment (positive control). In each test group, a total of 45 hornworms were used.
For incubation at 37oC, individual hornworms were placed in a clear, plastic tube (repurposed fruit fly culture tubes) with approximately 10 grams of food, capped with a foam plug (see Figure 5), and placed in a Happybuy 25 L incubator for three days. Caterpillars were checked each day and supplied with fresh food when necessary, as the fifth larval stage is a time of optimal eating and growth. Dead hornworms were removed as soon as melanization (darkening of the body) was apparent. After the three days, hornworms were returned to room temperature and allowed to eat until reaching pupation, at which point they were buried in moistened aspen wood shavings and kept in a 6 L Sterlite bin until emerging as adults. Once hawkmoths shed their pupal husks and assumed their adult form, all were visually assessed for any abnormalities. This was done by comparing multiple control hawkmoths and some treated with coal tar to check for any vivid abnormalities (enlarged body parts, seemingly underdeveloped ones.)
After the three-day incubation, every hornworm in each survival group was assigned a number: 0 for dead, and 1 for alive. These data were then entered into a table for analysis with One-Way Analysis of Variance (ANOVA) and post-hoc Tukey Honestly Significant Difference (HSD) tests in order to determine statistical significance.
Questions and Answers
1. What was the major objective of your project and what was your plan to achieve it?
My major objective of this project were two things. First, to test if the Manduca sexta were viable invertebrate models for burn injury and healing in humans. Second, was to see if coal tar is harmful and could cause cancer.
a. Was that goal the result of any specific situation, experience, or problem you encountered?
No
b. Were you trying to solve a problem, answer a question, or test a hypothesis?
I was both trying to answer a question and test my hypothesis. The testable questions to be investigated are as follows: “Can treatments that are suspected to cause skin cancer in vertebrates be replicated in an invertebrate model?”, and “Do certain compounds help to reverse skin damage and promote healing?” The hypothesis that guides this experiment states that if tobacco hornworms are exposed to coal tar, then they will exhibit uncontrolled cell growth in the same manner as cancerous cells because these insects exemplify many of the same genetic regulatory mechanisms that are found in vertebrates.
2. What were the major tasks you had to perform in order to complete your project?
a. For teams, describe what each member worked on.
3. What is new or novel about your project?
a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?
Honey has been studied as a burn treatment, but it has not been widely studied on invertebrates.
b. Is your project's objective, or the way you implemented it, different from anything you have seen?
The Manduca sexta have not been tested as models for burn injury and healing in humans. Silkworms and waxworms have some, but never the tobacco hornworm. Other animals commonly used for testing are vertebrates, but they have many issues (ethical review boards, hard to maintain, and they can feel pain.)
c. If you believe your work to be unique in some way, what research have you done to confirm that it is?
4. What was the most challenging part of completing your project?
a. What problems did you encounter, and how did you overcome them?
I completed this project as part of my school's research program (sophomore through senior year). My first year (teaching us about how to conduct research) was during the online learning year, and my second, the second half of which I collected most of my data (as well as a bit at the start of my third year) was when there were still many restrictions on what students could do. Thus, the hardest part was planning a project during my online year that could accommodate being online and not being able to use the school resources as much as I would have liked to. This meant keeping the worms in my house sometimes, having to plan my experiment on when the school would approve the materials I needed, etc.
I overcame these problems by simply pushing through them. When we (my teacher and I) couldn't get food fast enough to feed the hornworms, we planted tomato leaves so that they could eat. I went into school during the summers to accommodate the school's tight scheduling during the year.
b. What did you learn from overcoming these problems?
There were many things I learned. First, I learned how much resilience research takes. In a good way, the best researchers, I have learned, are the ones that strive to overcome difficulties and setbacks and ones who have a lot of drive. Until freshman year, I thought that the only way to use my love of science and biology was by being a doctor, but having the opportunity to do research taught me how much I love it. These setbacks taught me what it truly means to be a researcher so that when I continue doing this throughout my life, I know how to push through.
Additionally, it taught me the value of teamwork. I did my project on my own, but I always had people helping me and supporting me along the way. My mentors were always there for me to bounce questions off of and to help me plan, and my friends were always down to keep me company as I did my experiment (even sometimes helping me put the worms in their tubes.)
5. If you were going to do this project again, are there any things you would you do differently the next time?
I would definitely do more trials. I think it would just make my data better than it already is. Additionally, I also would have liked to culture bacteria from the worms (which is talked about in my paper.) I went into my project hoping to genome sequence them, but my school, unfortunately, did not have the resources to do that.
6. Did working on this project give you any ideas for other projects?
7. How did COVID-19 affect the completion of your project?
Talked about in number 4. It decreased how much time I had to complete the project and forced me to lower the scope of it.