Silk Moths, Enter The Arena: Binary Choice Paradigm

Hello, everyone! It’s Jess again. Since I last wrote, I have shifted all my work from cockroaches over to silk moths. I’ve had to make a few modifications to my protocols, but overall the transition has been smooth. Working with the silk moths has been far more enjoyable than the cockroaches (no offense roaches, but you freak me out), and I’ve really settled into my role as a moth mom. The moths will hang out anywhere you put them–sometimes I even walk around with them on my shoulder when I am getting ready for experiments and have to carry other things!

Left: Female silk moth perched on her cup.

Right: silk moth along for a ride

Behavior 

In my last post I mentioned how the experiment has two parts: behavioral observation and electrophysiology. Unlike the cockroach experiment, designing a behavioral paradigm for the moths was fairly simple because the male silk moth’s response to the sex pheromone bombykol is extremely profound. I suppose if you’re only alive for five days reproduction is kind of a huge deal? 

For my assay, I use a binary choice paradigm. The materials are simple, dixie cups (to raise the chemicals off the ground so moths can’t touch them), multiple tupperware containers, and the compounds of your choice.

Three females starting the behavior task

For the experiment, I place a stimulant and corresponding control dixie cups on opposite ends of the tupperware, place a group of same sex moths in the middle and record what half of the arena they are in after 5 minutes have passed. Sometimes, the moths will not move for the full 5 minute trial, so they receive a ‘no choice’ score. All trials are recorded to ensure correct scoring and for the potential to be used in Anastasiya’s awesome tracking program when it’s complete.

In addition to scoring how many moths end up on the stimulant or control side, I also record how many moths are performing reproductive behavior. When the females emit the pheromone from a gland in their posterior, the males begin to rapidly flap their wings and spin around in circles as they orient themselves to the location of the female. It looks like this:

Three males responding to a female emitting bombykol

Males also have this same response when I place synthetic bombykol in the arena. In addition to bombykol, I am testing linalool (plant terpene found in the Mulberry leaves silkworms eat), ethanol (accessible positive control for electrophysiology), de-ionized water (ethanol solvent), mineral oil (linalool and bombykol solvent), in male moths and female moths. I’ve run 40 trails, and it appears that bombykol and female moths are the only things that change behavior in the males, and the females have no response to any of the stimulants. By the next blog post, I will have a visualization of my results.

Electrophysiology

Observing consistent electrophysiology result has been (and still is) the challenge of this project. The silk moth antenna is significantly more sensitive to mechanosensation than the cockroach and can quickly become overstimulated. Additionally, many of the compounds are oil-based, so they coat the interior of the syringe and make deployment difficult and inconsistent. I’ve come up with a couple solutions to my problems, and now I just need to figure out how work them together.

First, have modified a fan from the BYB office using a milk jug container to direct light airflow on the prep. This reduces the noise from other wind artifacts (literally breathing on the prep gives signal).  Second, using a sponge soaked in DI water, I have humidified the airstream to make the prep last longer.

Modified fan and humidified air set-up

The last, most challenging step has been determining how to deliver the stimulus. Ideally, I would like to inject it into the airstream, but as I mentioned above, the deployment of the syringes does not work well. I have tried blowing air through the syringe onto the prep, and soaking sponges with stimulant and placing them in the airstream. This week I will be prototyping some new ideas. 

Some days my data is beautiful, and some days it looks like shit. A bit of the variation I am seeing is also due to the hardware. I am currently deciding which BYB board will work best to record at the low frequencies I need and this alone can distort the signal from preparation to preparation.

Example of two ethanol trials looking very different just a few minutes apart.

The good news is that I have plenty of time and moths to figure this out. Also, not getting consistent data is frustrating, but this is one of the best part of research- when nothing really makes sense, you kind of hate the procedure, and then one day after you’ve tried it enough times, it miraculously works and you want to do it all over again. I made some good progress today, I’m sure I’ll make good progress tomorrow, and hopefully by the next post I’ll have some consistent data to show you all!