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Finishing the Silkmoth Story

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!


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!


Electrophysiology at the Cambridge Science Festival

This post comes to you from our friends at Biomakespace! They are biohackers and electrophysiology enthusiasts who work and hack with our kits along with inventions of their own! They recently presented and demoed their cool tech at the annual Cambridge Sci  ence Festival in Massachusetts. We asked them a couple questions about the event and their experience using and demoing the Backyard Brains SpikerShield, and they were kind enough to prepare this debrief for us which we’re sharing with you today!

The annual Cambridge Science Festival welcomes over 40,000 visitors of all ages to hundreds of events developed and run by staff and students from departments and organisations across the University and research institutions, charities and industry around Cambridge. Events include talks, interactive demonstrations, hands-on activities, film showings and debates.

Over 1200 visitors streamed through the Plant and Life Sciences Marquee where Biomakespace had an activity table, with many stopping to find out about Biomakespace and the types of equipment/activities we had on display and where people could participate. There were a lot of families with young children, but also groups of students and adults.

Roger, our resident electrophysiology expert was reading electrical signals from the gastrocnemius muscle in the lower leg and the brachioradialis muscle in the forearm using a battery powered bioamplifier and viewing them on an oscilloscope as well as creating noise from a speaker. Visitors were really interested to see how their signals varied in ‘intensity,’ both amplitude and frequency, depending on the degree of effort involved in muscle contraction and also how easy it is to observe the firing of individual ‘motor units.’

Roger also demonstrated ‘reflex arcs’ by tapping the Achilles tendon in the lower leg or the distal brachioradialis tendon in the forearm. This usually requires a ‘reflex hammer’ containing an accelerometer, but he rigged up a normal hammer with a rubber-cushioned micro switch and a 9V battery attached with heat shrink to record the timing and intensity of the strike and observe the response. Reflex arcs can be unconsciously enhanced by clenching your teeth and interlocking fingers (the classic Jendrassik Maneuver) and also by imagining something that makes you really angry (mental-imagery interference), so we tried that out as well with some great results.

Next along we had a demo with the Backyard Brains SpikerShield on an Arduino Uno. This has a series of green, yellow and red LEDs indicating the response amplitude. A long line of children excitedly tried to flex their arms to get the red LEDs to light up (which wasn’t too difficult with where we’d set the thresholds!) – we explained that although Roger’s full setup is large and can be expensive, it’s now possible to do some great experiments with low-cost hardware and a mobile phone – which is the message and ethos that Biomakespace and Backyard Brains value above all else.

Technical details:

We performed EMG recordings from the gastrocnemius muscle in the lower leg and the brachioradialis muscle in the forearm using Gold ECG Electrodes (Ag/AgCl/solid adhesive; pre-gelled: TIGA-MED, Deutschland GmbH). Differential signals were amplified 1000X or 5000X, and filtered (low pass: 30 Hz – 300 Hz, high-pass: 800 Hz – 15 kHz, depending on the experiment) using a battery powered bioamplifier and viewed on a Hameg HM407-2 Analog/Digital Oscilloscope and monitored by a conventional PC audio amplifier.

Volunteers were shown how these signals varied in ‘intensity’, both amplitude and frequency, depending on the degree of effort involved in muscle contraction and also how easy it is to observe the firing of individual ‘motor units.’

Practical demonstrations of ‘reflex arcs’ were made by localised tapping of: (i) the Achilles tendon in the lower leg, and (ii) the distal brachioradialis tendon in the forearm. In the absence of a ‘reflex hammer’ containing an accelerometer, an effective alternative was achieved with a rubber-cushioned micro push-switch (RS Components 336-74) and a 9V PP3 battery attached to a light (4 Oz/114 g) hammer using heat shrink. Activation of the switch upon tapping the tendons produced a signal that was made compatible with the ‘trigger input’ of the oscilloscope by a Grass Model SIU5B Stimulus Isolator Unit.

The principle of unconscious modulatory control of such reflex arcs was shown utilising the classic Jendrassik Maneuver and also by mental-imagery interference, both having the effect of markedly enhancing the reflex-based EMG activity.

Electrophysiology in the classroom

The Muscle SpikerShield is a great way to get people experimenting with neurobiology in an easy to understand way and the kit gives so many opportunities for other learning as well: basic electronics, soldering skills, how an amplifier works and coding with Arduino. The Science Makers monthly meetup at Cambridge Makespace, which was instrumental in getting the Biomakespace group together, has used the Muscle SpikerShield and other Backyard Brains boards at several meetups, from constructing the kits to experimenting with muscles, worms, plants and trying to control objects. Everyone has loved it and it’s fantastic for demos – we’ve also had several people asking where they can get one of their own.

Children were really attracted to the Muscle SpikerShield activity as it allowed them to learn something about themselves through the link between number of lights lighting up and muscle activity, which isn’t something many had experienced before. Adults were interested to see how devices like the Muscle SpikerShield can show similar scientific concepts to the more expensive lab equipment and also saw the value as a teaching resource. And it is a lot of fun too! Boys were very keen to show their strength and tried to light up all the lights on the SpikerShield as well as try and make most noise when their muscles were hooked up to the Analog/Digital Oscilloscope and amplifier — especially when they competed with their dads!

Teaching Electrophysiology is important because it helps people understand how the brain and the nervous system work, which is fundamental to understanding who we are as human beings. The great thing in terms of experimenting with electrophysiology is that even at a simple level, people can find out things new things for themselves and there are so many tricks that illuminate how our brains and bodies work on an subconscious level, like the reflex arc experiments Roger was carrying out in his demo. Although electrophysiology is not a new field of science, combined with new technologies, such as advanced genetic and optical techniques, it allows to gain understanding on a wide variety of scales- from single ion channel proteins to whole organs systems and whole organisms and that keeps it really exciting.

Electrophysiology is going to play a big role in our future. First off we’ll be building and collecting devices to set up the ‘electrophysiology corner’ in the workshop and after that the projects will be community-led. Some ideas that have already been bounced around include recording spontaneous activity in invertebrates, investigating neuroplasticity in snails and record currents associated with growth or movement in plants and slime moulds. Some people are very interested in combining 3D-printing with electrophysiology kit to look at customised fitting and modified control mechanisms. One group who are associated with the space and the Science Makers group have been working for over a year on a fork of the Backyard Brains plant electrophysiology board and have re recently rigged up custom controllers for electrode micromanipulators with Playstation 2 controllers — we’d love to see more of that!

Biomakespace

Biomakespace is a non-profit organisation creating a community laboratory on the Cambridge Biomedical Campus in Cambridge, UK. We aim to build a community of scientists, engineers, technologists, entrepreneurs, teachers, artists and members of the public interested in engineering with biology. Biomakespace provides members with affordable access to a well equipped lab and prototyping space as well as to training and social events.  By encouraging and supporting project based interdisciplinary collaborations, Biomakespace aims to contribute to awareness, knowledge and innovation in engineering with biology. As the community grows, we aim to open the space to the public for events.

Visit our website (http://biomake.space) to find out more. To become a member or support us, visit (https://biomake.space/support-us)