Hello, everyone! Welcome back to the last installment of silkmoth updates. Things are starting to wrap up here this summer, and I’ve begun to analyze the data I have been collecting.
Behavioral Data Analysis
Last post, I explained the behavior assay that I am using with the moths to demonstrate how the sex pheromone bombykol can alter behavior. I have run over 100 trials since then and have been working on visualizing my data set. The Google Sheets pivot table feature has been a serious life saver for quick filtering and data wrangling. If you haven’t used it before, do it! Or I can force you to sit through an overly-enthusiastic demo like the rest of my labmates (sorry guys).
The moths have three choices during the experiment: they can choose the stimulus side, the control side, or have no response. No response is defined as no movement after 30 seconds of being placed in the chamber. Overall, the moths tend not to move unless they have a really good reason to expend their limited energy. Thankfully, this makes my job as an observer fairly easy, although boring at times, and the behavior response to bombykol very obvious.
My first visualization was to show the spatial preferences of the moths in the arena for each stimulus. Below is a schematic of the arena paired with horizontal bar charts for males and females for each stimulus.
Schematic of behavior task. B. Choice results for all female trials. C. Choice results for all male trials
For the females, behavior does not appear to be influenced by stimulus type- overall the bars look about the same with the majority of the time ‘no choice’ being made. For the males, it can be seen that when a female or synthetic bombykol is present, there is a greater amount of response. I believe the bombykol response is more profound than the female response for two reasons. First, the concentration of synthetic bombykol I’m using is very high. Second, sometimes when a female is in the arena she does not protrude her gland that releases bombykol, which makes it impossible for the males to know she is there.
Although the plots above do a good job with spatial representation, they not account for what I truly care about- reproductive behavior. Sometimes a male can be doing reproductive behavior and will be so excited that he dances around the arena and ends up on the control side by the end of the trial. To account for this, I’ve made a second plot with average frequency of reproductive behavior for each stimulus type. It can be seen that bombykol and females induce far more reproductive behavior than linalool or mineral oil. Linalool and mineral oil should technically have no reproductive behavior, but sometimes things can get contaminated even while I’m using separate chambers. By the end of some days, I’m pretty sure I am covered in bombykol, so I have become more careful in what order I run experiments.
Average reproductive behavior frequency observed during behavior assay for male moths.
Now that the data has been visualized, I am working to run statistical analysis on it. I will need to run a model that accounts for testing cohorts of moths multiple times (versus each trial having a new sample). This will likely be a repeated measures analysis of variance (RM-ANOVA) with post hoc Tukey test. I’ve been communicating with one of my teachers back at Westminster to determine which model is most appropriate for my dataset and will have those results soon.
Electrophysiology Problems & Solutions
Now onto electrophysiology! Last time I checked in, I was having difficulties recording consistent electroantennogram (EAG) data from the moth antenna. I was able to determine that the issue I was having was due to a poor connection between the electrode and the antenna. I determined this through a variety of experiments using a resistor instead of the antenna and applying various types of conductive gels and pastes. When the connection was good, the resistor would flat line and have no response to any of the stimuli blown on it (as it should since it’s not alive). When the connection was poor, it would give inconsistent, biological-ish responses due to the connection moving around, gel interacting with different substances and additional unknown factors.
For a few days I thought the EAG portion of my project was done- how could I determine if the connection was ‘good’ if a poor connection gave something that could easily be mistaken as biological? After running many troubleshooting trials and reaching out to a variety of resources, it turns out that when I blow on the antenna and it gives a large, high frequency spike, the connection is poor. So, I sporadically blow on the prep throughout the trial to make sure things are going well. I also switched from using an electrode gel to a more expensive electrode paste that appears to last much longer and create a better connection.
With these new methods I have begun to collect data that is much more consistent. I am only testing 3 compounds now: mineral oil (solvent/negative control), linalool (positive control) and bombykol (sex pheromone). Last week I ran trials in males and females and observed different responses. The females have a larger response to linalool and no response to bombykol or mineral oil, while the males response to linalool and bombykol. Seen below is an overlay of raw data from a trial with males on the left and females on the right that displays the response. This is similar to what has been found in research and has given me full confidence that my new method is working.
As you can see, this data lacks alignment, which makes further analysis very difficult. To align the data, I created a DIY laser beam with an LED and photoresistor that indicates when the stimulus is present.
Image of set up. Cotton ball blocking light signal across LED and photoresistor. Stimulus is on cotton ball and pulled into fan by air current.
This setup allows me to align each trial to stimulus onset. I am currently working with Ben on a code in Python that will automatically sort through the data and the statistical values we need to run analysis. Below is an image of the raw data in spike recorder. The red line is the stimulus marker and the green line is the recording from the antenna. You can see that when the stimulus (bombykol) is presented there is a slow hyperpolarization in the antenna as seen in previous literature.
Raw EAG response to bombykol. Red line is stimulus onset and green line is antenna.
I am psyched with the progress I have made since the last post. While I’m collecting additional data this week I will also be building a poster and preparing the classroom manuals and visuals for my project. I believe communicating one’s work is often the most difficult, yet important part of the scientific process. I’m looking forward to sharing my work and receiving some feedback on how to make it better. I hope to have more updates as I continue to work on this project while finishing up my bachelors degree this Fall. Thanks for following along and stay tuned!
Hello, everyone! Jess here. Lots of exciting things have happened in the last two weeks. First, I have begun to raise a group of silkworms into moths (#mothmom). This involves feeding them Mulberry leaves from my backyard each day, keeping everything extremely clean and crossing my fingers in hopes that I know what I’m doing. If first grade classrooms can keep them alive, so can I, right? In addition to raising silkworms myself, I have been getting shipments of cocoons to ensure I have plenty of moths to work with this summer.
As I mentioned in my last post, I have been working with cockroaches to refine methods until the moths are ready. Rather than looking at pheromones, I have been investigating olfaction with natural attractants and repellents. Unlike silkmoths, cockroaches have multiple pheromones and they cannot be easily ordered. So, I chose something we all know cockroaches love and are able to locate through olfaction: food. Additionally, it is well known that getting rid of cockroaches without the help of a exterminator can be extremely difficult, so why not investigate a variety of repellents I found on a garden blog and see what happens?
The first (and most important) step was to design an experiment to observe the natural cockroach response to these odorants. After going through multiple iterations of a behavioral chamber that involved a lot of laser cutting (and a lot of recutting because measuring a box without parallel sides is hard), I’ve found a design that works well. It’s extremely simple: a large tupperware with a clear top, dixie cups, tape, filter paper and the odorants of your choice.
Experimental set up with shelters for the cockroaches
Initially, I had the idea of setting up a classic choice paradigm. Cockroaches would enter the arena and choose whichever chamber contained the odorant of their preference. I quickly ran into issues because they hate being out in the light, and had no interest in my contrived scent experiment. So, I used their hate for the light to my advantage. I placed shelters (dixie cups) around the arena with different odor filter papers taped inside. This way, they were making a choice of which shelter they preferred. So far this design has been working well, and I will continue to optimize it and run trails throughout the summer.
The second portion of my cockroach experiment has been focused on electrophysiology. I wanted to design a simple, DIY method for recording from cockroach antennas, also known as an electroantennogram (EAG). The type of electrical activity I am looking for is a low frequency summed potential sent from the end of the antenna towards the base. This electrical potential is then transmitted to higher order areas of the cockroach brain where the scent is perceived and a response is initiated.
To do this, I’ve made pad electrodes with sewing pins and solder to lay the antenna across. I then cut off the antenna, put it on the electrodes and apply plenty of electrode gel to prevent it from drying out. Using syringes and aquarium tubing, I blow odorants tested in the behavioral experiment on the antenna and examine the electrical response.
Antenna set up ready for odorant stimulation
Here is a snippet of my data below compared with results previously seen in literature:
EAG recording from cockroach antenna. Red line = control air, Yellow line = ethanol
The signal is similar to what has been published in literature, suggesting this DIY method works (yay!). Further, there are differential signals when ethanol is introduced and when the control is introduced. This suggests the cockroach is capable of sensing ethanol, and the response is not due to the puff of air itself. How this sensation may affect behavior will then be determined in the preference experiment I described earlier.
Now that I have some moths to work with, the cockroach portion of my project will be put on the backburner for a little bit. The silk moths only live for 5-10 days, so I’ve got to work with them while I can. Looking forward to sharing my moth data with you all next post!
For any questions or comments- feel free to contact me at jess.breda@backyardbrains.com. In the mean time, here’s a timelapse video I took of my silkworms spinning a cocoon:
Greetings, this is Trevor coming live from Ann Arbor in a basement…
We have one week left in the internship and things are finally starting to come together. Last time I made a post, I was without a doubt on the struggle bus in terms of getting data worthy of a poster, let alone a journal article. Since that point I have learned a hell of a lot of what not to do while recording data. First, always make sure everything is plugged in, second, make sure your specimen is alive, and last always make sure your equipment is hooked up everywhere it should be. You may think I’m weird for this, but I think my moths are pretty cute. A couple weeks ago I was having a really hard time getting clear recording from the BYB Spikerbox due to so much activity happening in the antennae, and as a result we switched to the SpikerShield to get an electroantennogram recordings. Electroantennogram recordings are low frequency, are the preferred way to record antennae stimulus and look like this:
Before I started testing the sex pheromone bombykol on the moths, I characterized how the male moths responded when introduced to their female counterparts, and it is best described as a circling motion in a zigzag path towards the female. It happens almost every time a female is brought within a couple feet of a male, but is not present when stimulated with just any odorant, like lemon oil or peppermint.
The male seems significantly less enthused about the lemon than about the female-I wonder why?
Once I had that complete I began trials with bombykol using a new delivery system that went through many revisions. At first we were using a valve system to deliver odors via an air stream, but we observed a change in airflow when opening valves, which meant we couldn’t be sure if the neuronal responses we got were from the change in airflow or the added odorant. To fix this problem, we inserted syringes into the piping with some T-junctions on the way to the moth, thus keeping a constant air stream.
Now I needed a way to consistently empty the syringes, so for that I added a linear actuator and a wood based housing to hold the actuator and syringes. The housing, entitled “Mr. Orange” due to its bright orange paint job and my love for Reservoir Dogs, has three ports for three syringes, to serve as an air control, other odorant positive control, and pheromone syringes all in one recording. Along the way, thanks to our fearless leader Greg I had to edit the housing from two to three syringe ports just minutes after completing the project…
Are you happy now, Greg?
Before “Mr. Orange” was built I was able to successfully record a bombykol stimulus on the last day of my second batch of moths. Displayed below is Bombykol compared to a negative control, aka clean air. Remember they only live 5-10 to days, and have to be ordered, so my time working with them is precious! I am currently awaiting new moths so that I can do my final trials which will involve mulberry leaves, which their caterpillars live on and eat, as a positive control for both male and female moths. This will allow me to show that both males and females react similarly to one stimulus (and ensure the prep is working), but also that they have evolved such that males are much more sensitive to bombykol than females.
Last but not least, the interns all presented last week at MID-SURE, a poster session on Michigan State University’s campus in East Lansing, and I have included my poster below for anyone curious.
Schematic of behavior task. B. Choice results for all female trials. C. Choice results for all male trials 
