• Andy Davis

Do monarch larvae get stressed from traffic noise? A ground-breaking new study examines heartbeats


Greetings blog readers, old and new!

I hope you brought some coffee, because today's blog entry is a doozy. I'm going to tell you about a brand new study conducted by yours truly (with the help of some talented undergraduate students), that is going to forever change the way you think about roadside habitat for butterflies - I guarantee it.

Since you read the blog title, you know that the project involved looking at stress in monarchs, which is something that has never before been done, and the way it was done was also ground-breaking. The project was just published online in the journal, Biology Letters - link here. If you have access to this journal, I encourage you to read the actual paper, although for those that don't, I'll try to cover the main elements of the project here (plus a little bit more).

Before I get to that, let me set the stage for you on this topic, with a youtube video that I came across a few years ago. It shows a larva on a milkweed right next to a road. As you watch the video, notice what the larva does when a car goes by. By the way, I didn't shoot this video - the owner appears to be a monarch enthusiast and has lots of video like this one.

Did you see the larvae jump (or jerk)? Play it again just to be sure. It does it when the first car (but not the second) goes by. The larva was reacting to the car - most likely the noise of the car - which means it can perceive, or sense the sound. Believe it or not, monarch larvae have the ability to perceive sound, even though we're not sure how they do it. In 1997, a researcher published a paper that demonstrated this - she observed that when jet airplanes passed overhead, the monarchs she was rearing (outside) all jumped.

OK, so the larvae jumped when it heard the car, but now imagine how many cars go by this little guy in the span of a day. In that 11 seconds of video, I counted 3 cars passing, which translates to about 16 cars per minute, 960 cars per hour, and over 9000 cars in 10 hours. What happens to this little guy when each car goes by? Does he react each time? Or does he eventually acclimate to this environment? Notice that he (or she) is a large caterpillar, meaning that he has already lived for at least a week or more on this plant, so that must mean he should be used to this noise, doesn't it? But he still jerked...

What I'm getting at here is something that has rarely been discussed in all of the talk and conversations around "roadside habitat for butterflies" - these areas are really, really, LOUD. They may look pretty when we drive by, but have you ever stopped your car on the side of a busy road and listened? It can be deafening, especially along freeways. And, these are the exact places where people are turning to plant milkweeds for monarchs. Remember the plans for the monarch highway? The point is, that these areas are loud, and this is something that has never been considered when we determine areas for planting milkweed. This is where my study comes in.

In my scientific career, I have studied monarchs (and other critters) for many years, but for most of this time I have studied their magnificent migration, and/or the physical appearance of these insects. For the past two years, I have undertaken a new direction for my monarch work - to study their physiological stress reactions. A lot of folks may not be aware of this, but monarchs, like all insects, can get stressed, just like humans and other vertebrates can. And importantly, one of the hallmarks of stress in all critters in the animal kingdom is an increase in heart rate. So, in my new direction of research on stress in monarchs, I have developed a way to monitor the heart rates of monarch larvae, as well as adults, which I'll tell you about next (it's very cool).

This whole process took me the better part of a year or two to develop, in terms of figuring out the procedures, designing the gear, and many, many months of trial and error. But now I am at a point where this approach is so effective, that I can use it to study heart rates of a wide variety of critters, even ants!

Let's start with the main question everyone always asks - how do you monitor heart rate in a caterpillar? There are actually two ways to do it, and in our paper, we used both. Let me first give you some insect biology 101. Insect hearts are not the same as ours - they have a single long tube that runs the length of their body (even in caterpillars), that conveys their blood. The tube (called the dorsal vessel) is the insect equivalent to a heart, and it contracts rhythmically to pump the fluid. For comparison, imagine a garden hose full of water, and the sides of the hose move in and out to push the water through. That's essentially what an insect 'heart' does. Also important is that fact that in caterpillars, it is situated just under the skin, running down the caterpillar's back. There is a way to actually see this heart beating if you have a good compound microscope (not the kind you use to check for OE). For everyone who doesn't have one, I'll add a video here of a larva under my scope. If you try this at home, look closely at the very center of the larva's back, and watch for movement - the larva needs to be very still to see it.

Notice the rhythmic pumping of the dorsal vessel (heart) in the larva. For our study, we simply viewed each larva under our scope and counted the number of beats per minute. I should point out that this larva happened to be one which had a very pronounced beating pattern - they aren't all this easy to see. In most cases, I need to find a thin section of skin and try to focus my scope on that section. With practice, I found that I usually can get a heart rate reading in 80-90% of larvae. So this was one way in which we monitored heart rates of larvae. The next is even cooler...

As my research on insect hearts has progressed, I've developed a more advanced, electronic way to do this counting, and it's completely harmless for the insects. A few years ago, I became aware of some obscure research that has been conducted on marine bivalves - clams, mussels, etc., where researchers glue an electronic sensor to the shell, that transmits a signal to a computer every time the clam's heart contracts. The actual way it does this is with an infrared beam, but I'll spare the nitty-gritty details here. I contacted one of these researchers and figured out how to get one of these devices, and he helped me set it up (thanks Nick). I wasn't sure if it would work for insects, especially ones with without a shell to glue it to! But I eventually figured out how to incorporate this sensor into an apparatus (shown below), so that I can monitor monarch larvae hearts without even touching the caterpillar.

The sensor is pointed out in the image on the right. I figured out that it really just needs to be close to the caterpillar to work (within a half inch). As you can see, the sensor is incorporated into an older-style microscope body (with the lenses removed). The larva is placed on the microscope stage under the sensor (they usually remain still if they are sitting on a milkweed leaf or stem). This setup allows me to position the sensor just in the right place over the caterpillar, and I can use the microscope controls to make fine-scale adjustments of its position up or down, left or right. Once in place, the sensor detects the heart pumping and relays this to a computer screen (pictured), so that I can see the heart beats in real-time. It looks a lot like a heart monitor that you find next to a hospital bed (beep...beep...beep).

I know what you're thinking - but doesn't the caterpillar get stressed by being handled, and/or placed on this device? I have gotten this question a lot too, and we actually went to great lengths to address this in our paper. First, we looked to see just how fast the larval heart rate increases once you pick up these critters. From all of our testing it seems that their hearts are surprisingly slow to react to handling. It seems to take them at least a minute before their heart rate even starts to increase, and then it slowly increases over a period of 10 minutes. For our project, we collected the heart rate on each caterpillar within 30 seconds. Moreover, we did not actually handle any of the caterpillars directly - we only picked up the stem or the leaf they were sitting on. So we are very confident that our procedures did not influence their heart rates.

OK, so I think this covers the methodology for the heart rate assessment. Next, I'll discuss the noise experiments.

We were interested in knowing if road noise stresses monarch larvae (by elevating their heart rate), and we conducted a series of short- and long-term experiments to do so. All experiments were conducted using monarchs we reared in our lab. We used two separate rooms in our building for the experiments. Each was identical in terms of lighting and temperature. In one we set up two large stereo speakers connected to an ipod, which played an audio file of a busy road (on a loop). We cranked up the volume so that it matched what is typical for a busy highway (75-80 decibels). This was our noise room. The other had no speakers and was basically quiet (45 decibels), and this was our control room. Below is a basic schematic showing this setup.

For our short-term experiments, we placed larvae into plastic containers (filled with milkweed) 3 days prior to the trials, to give them time to acclimate. Then on the day of testing, I monitored the resting heart rate of each caterpillar, using one of the procedures above. Next, we placed half of the larvae in the noise room, and half in the control room and left them for exactly two hours. Then, I took each larvae and measured its heart rate again. Remember, these larvae were all reared in our lab and had no prior exposure to traffic noise.

Of course, we know this experiment is not at all ecologically relevant - we had taken late-stage larvae that had no experience with traffic noise and suddenly exposed them to (a lot of) traffic noise. In reality, this wouldn't happen to a larvae, because they simply don't move around that much - a larvae would never all of a sudden find itself next to a busy road. These short experiments were only designed to tell us, biologically speaking, if 1) monarch larvae can perceive this sound at all, and 2) if they perceive it as a stressor. In reality, monarch larvae would be living on a milkweed plant next to a road for the duration of its larval life. So, we also had some long-term experiments that mimicked this. We placed groups of larvae in our noise room, and some in our control for 7 days, or 12 days. In the 12-day experiment, we actually started from the egg stage, i.e. as soon as the eggs were laid we placed them in the rooms. Once the larvae reached 5th instar I assessed the resting heart rate of all caterpillars in the exposed, or unexposed rooms. so this experiment essentially was designed to assess if long-term exposure to loud traffic noise alters the resting heart rate of monarch larvae.

Here is what we found.

First, short-term exposure to traffic noise significantly elevated the monarch larval heart rate, indicating that they do perceive this as a stressor. In other words, traffic noise is indeed stressful for monarch larvae. Their heart rates increased by about 16% in one experiment and 17% in another. In case you're wondering, this increase is on par with what we see in a wide variety of animals (and even humans) when they become stressed.

Meanwhile, in the long-term exposure trials, by the time the larvae reached 5th instar, their resting heart rates were no longer elevated. The rates of exposed larvae were statistically similar (though a tiny bit higher) to that of the control larvae. This was surprising to us, especially given the first results. But what we think is going on here is that the larvae eventually habituate to the stressful noise (or become desensitized to it?). Either way, it seems they learn to live with it. Fascinating!

Now, before you start feeling relieved about the long-term results, let me warn you that this may not be good for monarchs after all. Habituating to stress can be bad in the long run for animals from a physiological perspective. If you think about it, animals need to become physiologically stressed when faced with imminent danger, to help them during the stress event. This is the whole reason their (and our) heart rates become elevated in the first place - to help circulate more blood to muscles, which makes the muscles work better/faster during the stress event. In other words, without a working stress reaction, animals may not be able to effectively escape their predators, or deal with a storm, or what-have-you. AND, what animal on the planet is in most need of a working stress reaction? The monarch! Their incredible long-distance migration to and from Mexico each year is a journey that is chock-full of stressors and dangers. The point here is that monarchs need to have a fully-functional, operational stress response system (fight-or-flight reaction) in place when they migrate. In fact, there is new research coming out now on other migratory animals that suggests that how well an animal deals with stressors is a better predictor of migration success than even their locomotor ability is. So stress-reactions and migration ability seem to go hand-in-hand.

The other scary thing to point out about the long-term trials is that not all of the data we collected eventually made it into the final paper. We had conducted a number of other tests on these larvae, including on their behavior. You recall the larva from the video above, and how he jumped when the car went by, well larvae also do this when they are angry and fussing with other larvae. We've seen this when rearing monarchs in groups in the lab. In this experiment, one of my students actually attempted to assess the level of aggression seen in the exposed and unexposed larvae. She quantified how much larva-larva aggression she saw in each container, and also how the larvae reacted when picked up by us - i.e. whether it curled into a defensive ball, or wiggled, or even tried to bite us (well, her, really).

These behavior results were quite striking. It turned out that the larvae from the noise room were more aggressive to each other, and were much less likely to curl into a defensive ball when picked up. And then there was this - during the times when we were changing out the milkweed for the long-term trials, some of the larvae in the noise group BIT US! It was just a pinch, and we were really just surprised more than anything, but this happened on at least 3 different occasions on two different people. From conversations I've had with many experts who collectively, have raised tens of thousands of monarchs over the years, they tell me that this has never, ever, ever, happened to them.

All of this led us to conclude that chronic exposure to road noise makes monarch larvae angry. Unfortunately, the reviewers of the paper did not like this element of the project (too observational, too anthropomorphic, too... etc.), so it was eliminated prior to publication. It's too bad too (see what I did there?), because this information was important to the whole story on stress. In fact, there is a body of literature in the scientific world on invertebrates that indicates long-term stress causes behavioral changes, especially increasing levels of aggression. So I always thought this information tied in nicely with the other parts of the paper, and it demonstrated that there was in fact an effect of the chronic noise on the larvae.

Anyway, these behavioral changes are one more piece of information that tells us that we really have no idea what we're doing to monarchs when we plant milkweed right next to a road. Just because monarch caterpillars can (sometimes) be found on these plants, does not mean that they are normal.

I hope to work on this topic more in the future, especially now that I have this fancy setup in my lab. What I'd like to do next is to see if the larvae that are chronically exposed to traffic are less able to fend off predators, or deal with stressors. Then, we need to figure out if the stress reactions as larvae carryover into the adult stage, which is what I was getting at above. There are actually a whole slew of questions that this study raises, which really highlights how little we know about the long-term consequences of exposure to traffic noise. This study was really only the first, of what will hopefully be more to come.

Thanks for reading about this study. If you have comments, you can make them in the Facebook version of this blog - https://www.facebook.com/monarchscience/.

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Direct link to this blog entry:

http://www.monarchscience.org/single-post/2018/05/09/Do-monarch-larvae-get-stressed-from-traffic-noise-A-ground-breaking-new-study-examines-their-heart-rates-to-find-out

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The science of monarch butterflies

A blog about monarchs, written by a monarch scientist, for people who love monarchs