An in-depth look at the recent paper on monarch caterpillar hearing (with images)
Image above from Wikimedia
Things have been hectic in the lab this month - lots of projects are ongoing - so I haven't had a lot of time to report on the monarch literature. But, one recent paper did catch my eye, and I bet it did for a lot of other folks too - the recent study that described how monarch caterpillars hear! Today, I'm going to dive into this paper, including giving a summary, describe some of the main findings, and most importantly, show you some of the pictures from the study. Hopefully this will help folks get a better understanding of the paper, especially since I don't believe the paper was freely-available.
So to begin, let's start with the link to the abstract, which most folks have probably read. The study is published in the Journal of Experimental Biology. As of this writing, the paper is not yet in its final form, but in an "accepted-and-online" form. The study came from the lab of the all-time guru of butterfly hearing - Dr. Jayne Yack, from Carelton University (in Ontario). It looks to me like this was a graduate student project in her lab (Chantel Taylor). Dr. Yack is perhaps one of the world-leaders in the niche field of insect sensory perception. Here is a link to her lab - https://www.yacklab.org/. Her lab has studied hearing and sound production (yes, sound production) a variety of butterfly species, both at the caterpillar and adult stage. I don't know her personally but I have corresponded with her in the past, and I always see work from her lab in the news. After all, how can "whistling caterpillars" not be news-worthy?
This study appeared to be one of their first where they examined monarch caterpillars. They describe in the introduction that monarch larvae have been known to be able to hear for a long time, but there has not ever been any detailed examination of exactly how they do this. This is where this lab, with all of its know-how and technical gear (which I describe below) comes in.
The authors started by purchasing some larvae from some Canadian breeders (I know, boo) and reared them in a greenhouse until they reached 4th and 5th instar. Then they started their experiments. I'm not sure why they focused just on these later stages. I might have just been that they needed some big, chunky caterpillars to work with.
From my read of this paper, it looks like they had two overall goals: first, to identify possible structures on the larvae that they use to hear (akin to the larval ears), and then they wanted to know what types of sounds do they hear. I'm going to focus mostly on the first one here. There were a lot of other results and details in this paper, so please try to find a copy if you want to know more.
The first of these goals was a little gross, so don't get queasy here. They examined larvae under a microscope and selected a number of hairs on the body that looked like they might be sensory apparatuses. Don't ask me how they selected these. To me they just look like ordinary hairs. But I guess they strongly suspected that some hairs might be important because of their work with other species, where hairs are indeed important. I know what your thinking - hairs on caterpillars? Yeah, I know, they do have hairs. If you look really, really, closely, especially with a magnifying glass, you can seen tiny hairs on the surface of the caterpillar. There are some running along the sides and importantly, there are two near the head on the side of the body. I'm going to include below an image from the paper that points to these two head hairs. They called these upper and lower "Filiform Sensilla".
So, they selected these hairs near the head to test, plus some of the hairs running along the caterpillar side, and they also chose to test the obvious structures, the big tubercles, or "antennae" of the larvae (they're not really antennae). Then there was a weird one - they chose to test whether the surface of the caterpillar "neck" was important for hearing. This is the upper surface above the letter U in the left image above. Again, this must have been chosen from experience.
Once they had these "candidate" structures to test, then they went about sequentially "removing" and/or blocking these structures from larvae (without killing them), then playing sounds to the caterpillars to see if they responded. For the tiny hairs, they simply clipped them off under a microscope. To remove the antennae, they anesthetized the larvae, then tied off the base of the antennae with a string, then cut them off. The string kept the wound closed. For the neck thing, they just painted it over! They used different caterpillars for each of these trials. Essentially, they placed each of the caterpillars into a sound chamber next to a speaker, then played a loud sound to see if they reacted, either by jerking or freezing, or curling into a ball. They tested different groups of caterpillars that had different structures missing, and then counted how many caterpillars responded in each group. They had a control group too, where nothing was removed.
Before getting to the results, let me tell you about their sound booth setup. As I mentioned, this lab has all the technical know-how for studying sounds. This was very apparent from reading this part of the study too. Their sound booth sounds like (see what I did there?) it's super-complicated, and extremely precise. I'm going to add a screenshot below from the paper that describes this setup.
Wow, right? I would have loved to see a picture of this setup in the paper. It sounds like a professional radio studio sound room. I did see that they had included some online supplemental files (which are free to view), and one of those is a video that shows a caterpillar being exposed to their sound recording. Here is a direct link to it - http://movie.biologists.com/video/10.1242/jeb.211862/video-1
OK, so let's get to their results. Of the 15 control larvae, 14 of them jerked or otherwise reacted when they heard the sound, which makes sense - they had all of their structures intact. Next, those with tubercles (antennae) removed, and those with their necks covered in paint also reacted "readily" to the sound. Therefore these structures were ruled out as sound receptors and not tested further. It looks like after this they zeroed in on these tiny hair thingys. It looks like most (80%) of the larvae with their body hairs cut off responded to the sound, so these appeared to make little difference.
Lastly, and most importantly, the larvae with hairs clipped from near their head (like in the picture above) did NOT hear the sound. When both of these upper and lower hairs were removed, zero caterpillars responded. Curiously, when only the lower ones were removed, 80% of the larvae responded, and when only the upper one was removed, 10% responded. So it looks like the upper hair is the most important, but both of them are needed for complete hearing. Who knew?
To me it seems like this was the main result of the study - finding the ears of the monarch caterpillar. I mentioned there was a heck of a lot of additional information here too. The authors also measured the behavioral responses of the larvae, in extremely fine detail too, like using high-speed video-recordings, and laser vibrometry! They measured how long it takes caterpillars to react, how high they jump when reacting, how many times they bob their heads, even the angle in which their body curves during a jump! They also measured the fine-scale details of those sensory hairs, like with a microscope. While I'm not sure all of this was needed, I think this information would be useful for comparison to other species.
Some other information that seems useful for monarch folks, is a test they had done using different levels of sound (loudness). They tested intact caterpillars in their sound booth and cranked up the sound in stages. The levels were 72,75,78,81,84,87,89, and 92 dB. To most folks, these numbers are meaningless. For context, let me point out that these levels are all pretty loud to begin with; 45db is a quiet library, and 75db is like standing next to a busy freeway. I only know this because of my recent study where I had tested if monarch larvae become stressed by road noise, which I blogged about here. The results of their loudness test showed that the caterpillars responded to increasing loudness by jumping or "flicking" more. You can see a caterpillar "flicking" in that online video. During a 1second playback of a sound at 72db, the larvae flicked once on average. At 92db, they flicked an average a 3 times per second! This result seems like it's very useful for those of us who work with monarch larvae. In essence, you can think of the caterpillar "flick-rate" as an indication of how pissed-off they are. If it's 3 times per second, they are hopping mad (see what I did there?).
There is a caveat to this "pissed-off" scale - it may not work if the larvae have been repeatedly pissed-off. The authors also tested if larvae "habituate" to noise, by exposing a number of caterpillars to sound once every ten seconds for a few minutes (the exact timescale was not stated). The larvae did in fact stop reacting after about 1 minute. To me, this is extremely interesting, since a lot of research on stress in animals (even insects) is beginning to focus on how animals react to repeated stressors. The fact that the larvae ceased reacting to repeated auditory stress makes me wonder - does this mean they no longer care about the noise, or does it mean they have run out of steam, and physically can no longer jump when they hear it?
While I'm on the subject of loudness, I should mention that I consulted with Jayne back when I did that road noise project, since she is the real expert on butterfly hearing. At the time, a student and I had plans to continue that work by studying how adult monarchs react to road noise. Jayne informed us that this may not be a good idea, because adult monarchs don't have any known auditory sensory structures! In other words, she thinks they can't hear! To my knowledge, this has not be tested officially, but we did stop with our plans at the time. If you think about it though, it's a little weird that this species appears to be capable of hearing at the larval stage, but not at the adult stage. Why?
The authors of this study proposed that the monarch larvae can sense sounds for detecting aerial predators like wasps. Being able to hear something coming perhaps would allow them to get ready, maybe. It's unclear to me, and the authors really just threw this idea out there, but it kind of makes sense.
I'm going to leave it here. Hopefully, we'll hear more (see what I did there?) from the Yack lab in the future.
So, now you know how monarch caterpillars hear - with their hairs, of course!
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