I hope you are safe and well during these crazy times. Today, I'd like to tell you about a new project that was just published in the Journal of the Lepidopterists' Society, authored by yours truly! This is a project I have been working on for the past couple of years, in stages, and I'm excited to finally see it come to fruition. Warning, this post is long, and contains lots of info, so don't try to skim it! Also, if you don't want to hear my blathering about the paper and just want to see the real thing, here it is, completely free on my website.
OK, so let me first set the stage for this project. As you saw from the title, this project was another one from my lab that falls under the theme of physiological ecology, and it was aimed at figuring out how monarchs react, physiologically, to being handled. If you think about it, people handle monarchs a heck of a lot. Think of all of the outreach events, where caterpillars and butterflies are passed around. Think of all of the monarchs in classrooms. Think of all of the citizen science programs, like monarch tagging - do you know how many monarchs are captured and tagged each year? It's an astounding number, something like 200,000 per year. And then there's the rearing. Now, I know that everyone who rears will tell you "I never touch my monarchs", but it's not just the touching. As you'll see from this study, simply moving the containers of monarchs from one shelf to another can cause a physiological reaction in the monarchs. Handling is really just a catch-all term for physical agitation, jostling, or disturbance. And there are many scenarios out there where this happens, like transporting monarchs. Has anyone here put containers of monarchs in their car to take somewhere? What does the vibration (or noise) of the car do to the monarchs? And finally, here's a big one to think about - what about the commercial breeding industry? They send live adult monarchs in the mail! Those monarchs experience a lot of handling, jostling and disturbance that lasts for over 24hrs!
So you can see that there are lots and lots of ways that we humans (even us researchers) are directly handling or otherwise physically agitating monarchs. Contrast this to what happens in the wild, where a monarch larva grows on a milkweed plant in a meadow. Other than some breezes that may sway the milkweed stalk around, those monarchs never experience this kind of disturbance, UNLESS, they are attacked by a predator. Think about a scenario where a stinkbug grabs a monarch larva, for example, or an adult monarch gets caught by a spider. What does the monarch "feel" when this happens? And importantly, does the monarch see the act of us handling or disturbing them as the same thing? This here is essentially the question I was trying to answer with this study - do monarchs experience a "fight-or-flight" reaction when handled?
The fight-or-flight reaction is a physiological response that is universal in humans, animals, and even insects, that is triggered when the animal senses imminent danger. It involves a series of changes in the body that helps to defend against the attack, or the disturbance, or the stressor. These changes include heightened sensory perception, increases in immunity, faster muscle twitching and especially, enhanced blood flow from elevation in heart rate. All of these changes are beneficial to fighting off a predator, or escaping from it. I feel like I'm teaching now. Did you write that down? There will be a quiz later.
So the overall goal of this study was to look for evidence that monarchs experience a "fight-or-flight" reaction after handling, by monitoring their heart rate. Yes, insects have hearts. I use heart rate as a proxy for the stress reaction in my lab. Essentially, if the animal's heart rate goes up, then that's a fight-or-flight reaction. So in this case, while we can never really know how a monarch "feels" about being handled, if we do observe this reaction after the handling, then we can at least infer that they perceive an imminent danger.
Ok, so now that you have the gist of the study, let's get into the details.
I evaluated monarchs at three different life stages - larva, pupa and adult. I conducted the trials for each stage at different times over a couple of years, because of the logistics of doing this. So keep this in mind, that I did not track the same monarchs across stages.
To evaluate the cardiac reactions of larva, I used a collection of 50+ larvae that had been reared as part of unrelated projects in the lab. These larvae had experienced the same rearing protocols always used - that is, rearing in plastic containers, fed cuttings of milkweed, containers cleaned daily, gloves always worn, etc. While I would have liked to evaluate "wild" larvae that had never been in contact with humans before, that simply wasn't possible, because I needed to have 5th instars that were all exactly the same age. But the fact that these were reared in the lab actually factored into the results, as I show later.
In prior posts, I have explained how I measure heart rates of caterpillars in my lab. Basically, this involves placing the caterpillar under a low-power microscope and watching for movements of the insect "heart", which is essentially a long tube that runs along the back of the caterpillar. That tube is the heart and it pumps rhythmically. In some caterpillars you can see the heart pumping just below the skin. In others I have to peer through the skin. Below is a video of it that I had put on youtube a while back.
Now I know what you're thinking because everyone thinks this. Doesn't putting the caterpillar under the microscope cause them stress? The short answer to this is no - and believe me, I've checked on this. I've found, through trial and error, that by gently picking up the caterpillar by the leaf it is sitting on, then moving the leaf to the microscope, there is no immediate cardiac reaction.
So getting back to this experiment, I wanted to know if the caterpillars experienced a stress reaction after being handled. To know this, I first had to measure their "before" heart rate. Here I gently assessed heart rate of each larva, using the procedure above. Here I only moved the leaf the larva was sitting on. For most monarchs, their before rate was between 30 and 50 beats/min. Next, I picked the larva up from its leaf, holding it between thumb and forefinger, as shown in the initial photo above. I then gently "handled" them for three minutes. Here I gently passed them back and forth between hands once every 30 seconds. This whole procedure was intended to replicate a gentle bout of human handling.
I don't want people to get hung up on the fact that I handled them for three minutes. Recall that there are an enormous array of ways that people handle or disturb monarchs, and each of these scenarios has different timeframes, levels of disturbance, etc. There was no way I could have adequately replicated each of these in this one study. So I chose a single timeframe that seemed like a balance between all of them. And, this was also a timeframe that was logistically do-able for me, since I had to do this same thing for over 50 monarchs. And, I also don't want people thinking, well these results don't pertain to me, since I only handle monarchs for 1 min, or 30 seconds, or whatever. I'll explain later.
After the handling procedure I then re-checked their heart rates.
Below is a chart showing what I found for the larval response. The red boxes are the average heart rates of larvae before (left) and after (right) 3 minutes of human handling.
As you can see from this figure, the heart rates of larvae after the three minutes of handling increased, by an average of about 20%. In case you're wondering, this magnitude is the same that we humans experience when we become stressed - about 20%.
Recall that these monarchs had been reared in the lab. This seems like a very relevant point right now. The rearing involves very gentle bouts of physical disturbance daily, to clean their containers, move the containers, etc. Every day there is some gentle "jostling" of the larvae, no matter how hard you try to be gentle. So really, these larvae in the trials had already experienced repeated bouts of daily disturbance for about a week already leading up to the test. But, they still had a fight-or-flight reaction to being handled. This tells me that the reared monarchs did not "get used to" these daily bouts of disturbance. This is an important point, as I've heard some people claim that their monarchs "get used to" being handled - they do not.
Before wrapping up the results for larvae, let me say a few things about the 3 minute thing. I suspect there will be a lot of people who will claim that this does not apply to them because "they don't handle monarch larva that long". This is wrong, or at least, an unproven assumption. In this study I arbitrarily chose to use a 3 minute period because that's the length of time that I figured would be sufficient to see a cardiac response. These results DO NOT prove that handling for less time is not stressful to larvae. I simply did not measure the effect of shorter handling times. See the difference? It is still entirely possible that handling for 10 seconds is enough to trigger a response. Until someone does this experiment using these shorter time periods, then no one can legitimately claim that their handling protocol does not stress larvae.
OK, so now let me tell you about the trials I did with monarch pupae. Yes, that's right, I measured the heart rates of crysalids! Believe it or not, monarch pupae maintain a beating heart throughout their metamorphosis, although it's a little weird. A study conducted about 5 years ago (not by me) hooked up some pupae to an electronic gizmo that registered heartbeats on them! That study found that their hearts beat very irregularly throughout their development - they beat for about 15 minutes at a time, then stop beating (flatline) for 2 hours, then beat again, etc. I have no idea why they do this, but this did make this next part challenging for me.
I also know what you're thinking here too - handling of pupae? Who actually handles pupae? No one does, I know. But, there are lots of cases where pupae are jostled or bumped, such as during transport to outreach events, or simply moving their containers. Remember, handling is simply a catch-all term for physical disturbance.
Here's where it's a little technical. To monitor heart rates of monarch pupae, I have my own electronic gizmo in my lab, which I've pictured below. I've described this apparatus in prior posts, so in a nutshell, let me explain. It is a sensor that will send an infrared beam into living tissue (without harm) and read movement under the tissue, such as the heart beat. The beauty of this thing is the animal in question is never even touched. In the case of monarch pupae, I can assess them by simply placing the probe next to the pupae (if it is hanging), or by placing the pupa under the probe (if it is detached). If I position the sensor in just the right place on the pupa, it reads the beating heart and displays it on my computer screen, kind of like you would see at a hospital bed monitor! It's very cool.
This pupa in the picture is detached, as some of them were in this project. Others were still hanging. It turns out that it didn't matter which.
Another thing that made this part challenging was the fact that the pupal cardiac reaction is sooo fast! When I initially tried to read the heart rates, I found that even the slightest nudge of the pupa was enough to trigger an immediate reaction. So in the end I decided to forgo the "before" step altogether, and simply measure the heart rates after. In nearly all cases, the before heart rate was 0 beats/min anyway (flatline).
So for this test, I used about 50+ pupae that had each been reared for different projects in the lab. They had all been reared under the same conditions, only at different times of the year. But importantly, they had all been sitting as pupae (untouched) for at least 5 days prior to me messing with them. So I know they were not stressed to begin with. Then, I tried three different ways to gently disturb the pupae. These were all different forms of physical "jostling", if you'd like. They were, gently squeezing the pupae for 20 seconds, gently bumping the pupae for 20 seconds, and then (for detached pupae) gently rolling them in my hand for 20 seconds. These all sound weird, but I was attempting to replicate, as thoroughly as possible, any form of physical jostling that these pupae would incur if they were, say, transported in the back of a car, or if someone were to carry their container from one room to another, for example.
In the end, it didn't matter which procedure I used - they all resulted in dramatic heart rate reactions. In all cases, their hearts immediately began beating really, really fast, like going from flatline to nearly 100 beats/min! Below is a chart showing the (average) magnitude of the response in monarch pupae.
This was perhaps the most surprising finding of this study - that monarch pupae are extremely sensitive to disturbance. You may be asking why this is, and to be honest, I don't know. I did point out in the written study that it is possible this reaction is necessary to help monarchs defend against parasitoid wasps, that attack them in the pupal stage. These wasps poke through the pupal skin and lay eggs. The monarch pupa may have an aggressive stress reaction to help flood that area with immune cells quickly, and/or to repair the hole. Who knows, really.
OK, now let's move on to my trials with adult monarchs.
These trials involved measuring heart rates of adult monarchs. I can't recall if I've talked about this before, so I'll first describe how this can be done. The next time you catch an adult monarch, and if you have a low-power scope at home, you can try this. Recall that insects have that long tube that runs along their back, which acts as the heart - it pumps blood rhythmically. In adult insects, it runs along the upper surface of their abdomen. I found through trial and error, that if you position the monarch in such a way in your hand, and curl the abdomen over a bit, you can see the tube beating under the thin sections of the membrane between the abdominal segments. This position is pictured below. It looks weird, and no one ever holds monarchs this way, I know.
Next, here is a youtube video I recorded of the adult monarch heartbeat!
So now, you may be thinking, but doesn't holding the monarch in this crazy position cause them stress and affect their heartbeat? Not really either, as I'll show below.
To examine how handling affects adult monarch heart rates, I essentially replicated the scenario of a monarch being captured and tagged. this is by far the most common adult handling scenario anyway. So for this part I went out and collected migrating monarchs during the fall migration period through our area. In total I caught and measured over 70 adults. Most of these were caught here on the campus at the University of Georgia, and I actually brought a portable microscope with me in the field (a net in one hand, microscope in the other!). I went to a very nice flower garden where we tend to see lots of migrants. I looked for and captured monarchs with a net, and as soon as it was captured, I put it under my scope to get the first reading. With practice, I found that I could get an initial reading within 30 seconds of initially catching the monarch.
After the first reading, I then pretended to tag the monarch. I had a collection of stickers (not real monarch watch tags though), and I went through all of the steps to "tag" the monarch. This involved gently scraping away scales on one of the wings, placing a sticker on that section, and pressing firmly to make it stick. Next I measured the wing size of the monarch with a standard ruler, and then examined its wings for damage. These are all things that people do when tagging, and importantly, all of this involved gentle handling, as you would expect. This handling period was exactly 3 minutes. Then, I put the monarch under the scope and took another heart rate reading.
Below is a chart showing what I found with these adult monarch trials.
Wow, I was shocked to see this result too - there was really very little reaction in the adults to being handled! The heart rate in the adults was extremely variable to begin with, but overall, the average rate after handling was only about 6% greater than the pre-handling rate. And, this difference was not statistically significant. There are various explanations here for this. It is possible that my initial heart rate reading actually failed to catch the real "baseline" heart rate, even though I measured it within 30 seconds of capture, and, maybe the monarchs were "already stressed" in the pre-handling phase. One of the reviewers of the study raised this possibility. But, I doubt this. The heart rates in the chart above are actually not that high for a monarch to begin with - they're actually on the low side. In previous experiments, I have seen baseline readings around 60 beats/min in adult monarchs. So I don't think they were already stressed.
I think a more plausible explanation is the simplest - adult monarchs simply did not experience stress during the 3 minutes of handling. In fact, it's entirely possible too that adult monarchs are naturally tolerant of stressors. If you think about all that they have to go through during their migration - they spend over two months on a harrowing journey filled with dangers (avoiding cars, crossing water bodies, storms, etc.). Maybe they are designed to be very resistant to any type of stress? I don't know this answer, but I'm damn sure going to find out in the coming years!
So I know this post is getting long so let's wrap up here, and I'll just list the main findings from each life stage.
This study showed:
1. That when larval monarchs are directly handled, they experience a stress reaction, which indicates their perceive this action as a danger or a threat.
2. That monarch pupae are extremely sensitive to any form of physical jostling or disturbance, possibly as a mechanism for dealing with predators and parasitoids.
3. That adult monarchs seem to be unusually resistant to handling, possibly because of the rigors of the migratory journey.
I think I'll end this post in the same way I ended the published paper. How does it help us to know this information? These findings should not be taken as evidence for why we should stop doing important outreach activities or citizen science work. However, these results should at least change our own perception of how monarchs "feel" about being handled. While we know that we are not trying to hurt the monarchs, they do not. To them, the simple act of us taking a photo of them on our hand is perceived as a life-or-death situation.
That's all for now.
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