The Munching: Experimental procedures and data analysis

smiley punches

“How’s the Very Hungry Caterpillar?” My labmate Hannah asked, as she stepped inside Bay Three of the greenhouse. In this scenario, I was the Very Hungry Caterpillar; with my standard Office Depot holepunch, I have been simulating insect herbivory on my plants. My goal is to elicit increased chemical defenses called cardenolides in the stems I’m damaging, and to investigate whether these defenses will also increase in clones connected to damaged stems by the root system. Acting as an army of caterpillars, every two hours for two days I hole punched every other leaf of every stem in a pot except for one undamaged stem. In the lushest pots, this meant I was hole punching as many as twelve stems! Despite organizational concerns and long hours on my feet, the hardest part of this endeavor was actually a problem real insects face. In addition to chemical defenses, milkweed has physical defenses against herbivory too. When broken, the tissue of the plant secretes sticky latex which gums up the mandibles of whatever is eating it. This gives milkweed its name and me a lot of trouble as I worked! Every round of holepunching I had to stop and wash out my “mandibles” several times so they could smoothly remove tissue without tearing leaves. Fortunately, I only had two days of munching, and now I’m just harvesting leaves every morning to track how the cardenolide levels fluctuate over time.

This hasn’t left me with very much time for the microsatellite project, but it’s coming along beautifully. Half of our lab is at the conference Botany this week, including Angie, the grad student on our project. She spent the two weeks before the conference prepping a presentation of our current data, which we were scrambling to generate and summarize for the conference. I got a lot of practice in R working on my map (see below!) as well as cleaning our data for analyses. We finally have real numbers to work with, and they’re looking great! We have also begun running tests and calculating statistics, but because we don’t have our full data set ready yet, these are very preliminary results. As we get more numbers back, we fill in all the NAs in our datasets, and we have to re-run every test and re-make every figure. Now that the conference has passed, we’re back at running PCR and genotyping to fill in those holes. It’s finally starting to feel like this project could be finished someday!

Voila! Not particularly impressive, but it got done.

flagged plants

The damaged stem to be sampled was flagged with blue, the undamaged stem was flagged with green

latex on leaves

Latex seeps out through the hole punches


  1. omspencer says:

    This project sounds very interesting to me. It was creative of you to come up with the idea of using a simple office hole puncher to mimic insects feeding on your plants. I also really like the map you have created in R. I have tried working with R before and it definitely takes a bit to get used to. I hope the rest of your project goes well!

  2. egeichenberger says:

    Thank you very much! I had only ever used R for basic statistics before, so mapmaking was a pretty big leap for me. I’ll be using R for data analysis and figure making for the rest of my project, so this was a great way to get more comfortable with the language! I’m very glad I got to use a simple hole puncher and not actual caterpillars; someone else in my lab is working with live monarch larvae, and they’re such a hassle! Plus, I got to use pesticides on my plants to keep insects at bay, which wouldn’t have been an option if I’d used real caterpillars. Getting covered in latex was a small price to pay for that convenience!