2nd cohort of caterpillars hatched

My second set of Papilio canadensis eggs (laid by my second female) finally hatched today! Only 2 of the 9 eggs haven't hatched yet. Here is my current inventroy for those keeping track:

• 7 mid-late instar caterpillars
• 7 first instar caterpillars
• 2 eggs

And here are some photos I took today

Egg:

1st instar:

Late intar:

The caterpillar colour pattern shouldn't change much after it reaches the above instar, although some Papilio sp. caterpillars do turn brown just before they are about to pupate.

Proof of concept: caterpillars spend time on tree branches

Today as I was watching the caterpillars feeding and I had a 'proof of concept' moment. Last summer I did a research project where I pinned artificial caterpillars (made of flour and lard) to branches of trees in the field and tracked the rate that they were attacked by birds. Specifically I was examining the extent to which countershading and eyespots protect caterpillar-like prey from bird attacks. See the published article here.

Aside: Countershading is a coulour pattern observed on many animals where those parts of the body that are naturals illuminated have darker pigment, and lighter pigmentation on those body sections that are naturally shaded. One of the main hypotheses for the evolution of countershading is that it makes it harder for predators to find prey because they blend in to their background better (i.e., it’s a form of crypsis). Hannah Rowland has done some great work on the adaptive value of countershading check out her publications and website. Here is a publication that used a similar design to the one I used:

Rowland, H. M., Cuthill, I. C., Harvey, I. F., Speed, M. P., & Ruxton, G. D. (2008). Can't tell the caterpillars from the trees: countershading enhances survival in a woodland. Proceedings of the Royal Society of London - B, 275(1651), 2539–2545

I made 4 types of caterpillars: solid green-no eyespots, countershaded-no eyespots, solid green-eyespots, countershaded-eyespots. Here are some photos to give you an idea:

 

I'll post more details about results of this work later, but one possible criticism of this design is that caterpillars may not spend a substantial portion of their time on tree branches. Arguably, caterpillars spend most of their time on leaves.

*Update: This work is now published, and I you can find out more in this more recent post. Also, the full citation for this work is below:

Hossie, T. J., Sherratt, T. N., (2012) Eyespots interact with body colour to protect caterpillar-like prey from avian predators. Animal Behaviour 84(1): 167-173, doi:10.1016/j.anbehav.2012.04.027

Today I observed 2 different caterpillars on the branch of the tree they are currently feeding on. I found the first one apparently resting on the branch, I spent some time watching it then eventually I decided to get my camera. When I got back it had moved to a leaf, but a few moments later I saw another one travelling along a branch. I don't have an SLR camera yet so without a manual focus on my digital camera it was hard to get good photos, but here are some:

Before observing this for myself, I had argued that caterpillars must spend at least part of their time on branches. How else could they travel between leaves? Despite this it felt good to observe it first hand, particularly during the day when many cryptic caterpillars are relatively inactive.

Eyespots!

My first "cohort" of caterpillars now have eyespots! This cohort is now down to 7 individuals, down from about 9 eggs laid. I'm supprized that the eyespots showed up this early becasue the caterpillars are still very small (i.e., roughly 1-1.5 cm long) and I dont think they have reached the 5th larval instar. All the caterpillars have turned from brown to green now, but do still have the white "saddle". Here are 2 views of what they look like now:

Many Papilio caterpillars weave silk pads on the leaves that they rest on. Of course caterpillars are insects which means they only have 6 legs, but anyone who has looked closely at a caterpillar might have counted more. These addional legs (after the first 6 legs on the thorax) are called "prolegs". On the prolegs these caterpillars have what are called "crotchets" - little hooks that grasp these silk threads in their silk pads. This gives them security from being knocked off with heavy winds or rain.

There is no question that these guys are incredibly cryptic now. Can you see the 2 caterpillars in the photos below?

Flyways - Papilio canadensis

Here is an example of a "flyway". Basically it's just a open corridor lined with trees. The best ones for finding tiger swallowtails (at least Papilio canadensis) are old 4-wheeler trails, fire roads etc. that are surrounded by early to mid succession forest. Here is an example of one of my very productive flyways along a cottage road that is surrounded by forest:

In my experience good flyways are trails lined with:

1) The primary host plant (e.g. Trembling Aspen)

2) Good nectar sources - flowering bushes with broad leaves seem to be frequented most.

Here is a flowering bush I caught 2 females fluttering around this summer:

Please suggest IDs of the above species if you recognize it.

Perseverance

I went out looking for more female Papilio canadensis butterflies yesterday. Once again, Isa was helping me. We did not see many Swallowtails, but we did catch 2 females. Neither female was flying in what I characterized earlier as a flight pattern typical of a female. Both were flying rapidly along flyways. I brought the butterflies back to my field station and sleeved them over the live host plant – the same as I did for the first female. When I checked them today I had both good and bad news.

Bad news: One female had been swarmed and killed by ants.

Although I had been spot spraying Raid™ insecticide on all anthills in and around my set-up, it has proved largely ineffective. I have now taken more aggressive steps. I picked up some Diatomaceous earth and have spread it on anthills and under my host plants. Hopefully this does the trick.

Good news: The other female laid 7 eggs! This is great as I was starting to worry that I was doing something wrong even though my set-up seemed to work well with the first female.

Also the caterpillars are starting to get much larger now. Some - but not all – have also turned green even though they are still very small and otherwise have the bird-dropping colouration (i.e. a white saddle in the middle body segments). It looks like we may be seeing the beginnings of some eyespots as well on a few of them.

Field and Lab Notes

Yesterday was a beautiful day, sunny and reaching 31°C. I went out collecting and had some help from Isabellle Letourneau. We spent the morning out collecting. We caught a female at 10:30AM, and caught a few males too. Isa pointed it out as I was distracted by a large dragonfly that I thought might be some kind of darner species. The female was fluttering about 6 feet off the ground around some broad leaves on a tree at the edge of an early succession forest. I am becoming convinced that I can ID males and females based solely on their flight patterns. Here is what I have observed:

Males:  Fly quickly along flyways and tree lines, usually in more or less the same direction.  

Females: Flutter around broad leaves and nectar sources. Landing briefly from time to time.

In terms of the early instar caterpillars a few observations of note:

·         At least during the day they all seem to rest on the midrib of the leaf

·         Most, but not all, rest with their head pointing towards the stem of the leaf

·         Most seem to rest on leaves with little to no damage

·         Leaf damage seems to be mostly along the margins of the leaf stopping at the midrib

It will be interesting to see if these behaviours change as they progress through their instars. Here are some photos:

Including this new female I have 2 females now. The other female I’ve had for about a week now, but I have only seen 2 new eggs. Here is one:

Becasue of the limited number of eggs I am trying a new design to induce egg laying. The females also tend to damage their wings as they fluttering around the tree they are sleeved with. I cut some branches of Trembling Aspen and placed the cut ends in tied off bags of water. I laid these branches in a box and covered the box with a very fine mesh bag. Using cuttings isn’t ideal because the eggs take a few days to hatch and I suspect that they do better if they hatch on live leaves. I don’t know yet, but I can’t see the branches lasting more than 2-days before wilting, especially under a heat lamp (heat is supposed to help with egg laying too). Here is what it looks like:

I'm starting to wonder if I need to try hand-pairing the females. I might try this soon. Also, I have learned that the females won't always drink nectar even if placed directly on soaked cotton balls. I have literally been taking a fine metal wire and unfurling the female’s proboscis onto the nectar. Once you do this, they always drink their fill.

The basis for a some of the ideas in my set up (e.g., using cotton balls) came from this great website:

http://www.insectnet.com/photos/tech2/t_rut01.htm

They seem to have had great success with Papilio rutulus. Check out this website for some solid tips and ideas for rearing Swallowtails.

Papilio canadensis caterpillars (1st instar)

Today I was very happy to see that the 9 eggs that my first female had layed earlier have hatched! I now have my first batch of Papilio canadensis caterpillars - I'm very excited. You won't see any eyespots yet though - Papilio canadensis caterpillars only get their eyespots when they are much larger. As insects grow they progress through a series of "instars", moulting as they pass through each. For now, the caterpillars actually look more like bird-droppings. Almost all of the Papilio genus caterpillars have this bird-dropping colouration in their early instars. In fact, there is a group of Papilio species that retain this colouration throughout their entire caterpillar life stage. In these early instars the caterpillars spend a lot of time in the open on the center of leaves – by resembling a bird-dropping the caterpillars don’t need to hide. You could say that they are hiding in plain sight.

Aside: The adaptive resemblance of an inanimate object or feature of the environment to avoid predation is called masquerade. Check out the recent papers by John Skelhorn and colleagues for some really interesting work exploring the difference between masquerade and crypsis.

Skelhorn J, Rowland HM, Speed MP, Ruxton GD (2010) Masquerade: camouflage without crypsis. Science 327: 51

Masquerading as a bird-dropping enables the caterpillars to use microhabitats that would otherwise risk detection from a predator. If I had to guess I would say the primary advantage of sitting in the open is heat. Insects are ectotherms and their rate of growth depends critically on temperature. Most of their body is dark at this point too, which should help them obtain heat.

From my reading I believe that Papilio canadensis become green with eyespots when they reach the 5th instar. Why the delay? and if masquerading as a bird-dropping works so well why the change? It could be linked to their change in size. As the caterpillars grow eventually they get too big to be a convincing bird-dropping. In addition, the caterpillars wouldn’t make a very convincing or intimidating snake mimic at less than 1 cm long.

Here are some photos of the 1st instar Papilio canadensis caterpillars:

Note: In an earlier post I had originally posted photos of a caterpillar that hatched in my set-up. I thought it was a Papilio canadensis caterpillar becasue I had never seen one for myself, and this one resembled other photos I had seen. That caterpillar has gotten larger now and it clearly is another species. I apologize for the confusion. I have removed the reference in the earlier post and I post 3 pictures here instead for comparison. You can see how it becomes easier to distinguish this species from Papilio canadensis as it grows.

Sexing Tiger Swallowtail butterflies (Papilio canadensis)

As promised, I created some comparative photos that show how you can differentiate male and female Swallowtails. Photographs are of Papilio canadensis that I collected in Kemptville, Ontario, Canada.

Note the greater extent of blue along the postdiscal hindwing of the female:

Note the claspers on the end of the male's abdomen:

OPP

A few days ago I was out looking for Swallowtails and I decided that I would travel light. Basically I was only planning to be out for about an hour so no need to bring my backpack with all my gear. All I really needed was my wallet, cell phone, keys, net, a collapsible cage, and my collecting permit. 

Aside: Papilio canadensis is listed provincially as “Special Concern”. To collect them you need a special permit from the Ontartio Ministry of Natural Resources (MNR). There is an exception for 1 specimen “...for the purposes of personal education without a licence, provided that the species is not listed under the Ontario Endangered Species Act or the federal Species at Risk Act.” Note that the Bog Elfin and the Karner Blue are listed under the Ontario Endangered Species Act. The Toronto Entomologists’ Association (TEA) has a great section on the applicable laws for insects in Ontario. You can check it out here: http://www.ontarioinsects.org/Laws.htm

Although it’s probably unlikely that I would get accosted by a Conservation Officer (CO) while I was out netting butterflies, legally I am to have this permit on me when I am out collecting. So I stuffed the permit into my back pocket and I was off. The next day I realized it was missing and must have fallen out when I was collecting. I had an electronic copy so I just printed out a new one and retraced my path from the day before. No luck so I just kept my eyes open.

Today I got a call from Constable Stewart of the OPP (Ontario Provincial Police). Apparently he was on duty and came across it when doing rounds on back roads. I drove down to the Police station and picked it up this afternoon. Constable Stewart found what I was doing interesting, so we spoke briefly about my project. I signed a declaration that the paperwork was returned, then I was off.

Lesson: It’s not always bad news when the Police phone looking for you.

Field work struggles

When I first went out catching I just took everything I could catch. I ended up getting 11 adults on my first day – pretty good I thought. But when I got back to my tent I started sexing them, and was having difficulty. They all looked like what I thought were supposed to be males. After doing some more research I confirmed what I feared. They were indeed all males. Here are two good features you can use to sex Papilio canadensis:

1)      The blue spots on the top of the wings extend much further on females than it does on males

2)      The males have claspers that help them hold onto the female when pairing

When I get pictures of pinned specimens I’ll post them. I'm curious how extensive this extra amount of blue along the wing of females is in Papilio swallowtails.

I released almost all of these males a day after I confirmed their sex. Males are of little use to me since wild caught females are unlikely to hand-pair successfully and they are likely to lay eggs if you just sleeve them over their host plant anyway. So far I have been struggling to find females. I have caught over 30 Swallowtails, and only one was a female. To try and figure out what was going on here I turned to an amazing textbook that I picked up earlier this year:

Swallowtail Butterflies: Their Ecology and Evolutionary Biology by Mark Scriber, Yoshitaka Tsubaki and Robert Lederhouse (1992)

If you are studying Swallowtail biology buy this book. The massive male bias is explained by the breeding ecology of Papilio canadensis. In Ch. 13 of the text Lederhouse compares the mating behaviour and sexual selection of North American Swallowtail butterflies. In this chapter he explains that Papilio glaucus and P. canadensis have a very similar mating ecology; males patrol for females and may travel great distances searching. Apparently the operational sex ratio is strongly male-biased along flyways and in concentrations of host trees. Only at nectar sources will females be equally represented.

This is definitely consistent with what I have been seeing in the field. I will be shifting my search tactic to target habitats close to nectar sources. Fingers crossed.

Update: After changing my tactics I did end up catching 2 Females! There were both feeding at a yet to be ID'd nectar source.  I transplanted some new host plants to sleeve these females in, but they looked pretty wilted by the end of the day. It was hot today (30°C), but I did give them lots of water. We’ll see what they look like tomorrow. For now I have them sleeved on the 2 live host plants that I had transplanted successfully earlier.

Rearing set up

Here are a few details about my set up back at my home base.

I set up a large screen tent outdoors. I tried to set it up so it was halfway under tree cover and half in full sun. There are eyeholes along the bottom edges every 6 inches or so to peg it down. I was glad that I pegged down every hole because we had a huge windstorm (25-35 km/h) a few days after I got it set up. I think the fact that it was pegged down and that the whole thing is mesh (even the top) stopped it from becoming a kite! Here’s what it looks like:

Swallowtails are known to drink from puddles. Oddly enough this behaviour is called ‘puddling’. I have also seen male Swallowtails feeding on animal scat in this way. A friend of mine took a great photo of this out in Bobcaygeon, I’ll see if he lets me post it on here. I set up a puddle inside the tent for the butterflies to drink from. All I did was dig a hole, lay down a plastic garbage bag then cover the bag with some of the dirt from the hole. It evaporated fast and needed to be refilled with water almost every other day. I only rarely saw butterflies drinking from it though.

I set out sliced fruit for them to feed on too, but I never saw them fly down to it. Occasionally I would rub my finger on the fruit juice then try to get the swallowtails to crawl onto my finger. Sometimes they would uncurl their proboscis (feeding tube) and start dabbing. I would place them onto the fruit then try to get another.  In the end both the puddle and the fruit are good ideas, but they don’t really work well in practice. Other people feed swallowtails homemade nectar (e.g. 10%honey 90% water solution) by placing them near the a nectar-soaked cotton ball then unfurling the proboscis with an insect pin. I suspect this works better, and I am currently trying this method.

I also transplanted 2 trembling aspen saplings into the tent. Beside the saplings I placed 2 rods meant for hanging plants, then covered them with a mesh bag. The trees did surprisingly well after the transplant. I did give them lots of water and sun. Above this I hung a UV heating lamp. The goal here was to create a micro habitat where adult females would be likely to lay her eggs. The key is to provide live host plants with lots of leaves where the saplings touch the top of the mesh.  Live plants increases the chance that your eggs will hatch successfully too, because it prevents the leaves the eggs are on from wilting and going mouldy.  The added heat is supposed to induce egg laying, but might be unnecessary if it’s already hot outside. I wanted to maintain the temperature at about 25°C. I have seen temperatures 23-29°C recommended.

When I caught a female I placed her in this set up. On the first day I saw eggs, but I’m not sure they were from her. I also found another small caterpillar. Lesson: always check your host plants for unwanted company. These are the eggs I had originally thought were from the Swallowtail, but later I realized they are probably from somone else:

Later, I found a total of 9 eggs laid over the course of 3 days. Here is what they look like:

Note that I did not hand-pair this female, she must have mated before I caught her. I haven’t heard of anyone having success hand-pairing wild caught females, and sleeving them over their host plant worked fine for me so far.

Intro to Fieldwork 2011

So my “Live tweets” won’t be of much use for those people that have no idea what I am doing this summer. Currently, I am in the 2^nd phase of my PhD research. Again, I am examining caterpillar eyespots - their ecology and evolution. These caterpillars with eyespots are generally thought to be snake-mimics – caterpillars that resemble snakes and are therefore protected from their bird predators that fear and avoid snakes. At least that's the idea, it has yet to be demonstrated and fully understood.

Last year I tracked the survival of artificial caterpillars with and without eyespots in the field. This project indicated that eyespots alone may not be sufficient to deter birds from attacking; however our experimental design did not include caterpillar behaviour which may play a critical role in deterring bird attacks. The next logical step is to determine how well these live caterpillars which naturally possess eyespots can deter attacking birds. I’m currently finishing the manuscript from that work, and I’ll provide more details about it later.

This summer’s goal is to generate a better understanding of the behaviours expressed by caterpillars with eyespots. Specifically, I’m interested in behaviours that might help the caterpillars survive an attack from a bird. If you watch the YouTube videos in my last post you can see some examples. Many caterpillars that have eyespots will also contort or flick their body when they are threatened. These behaviours may ‘startle’ attacking birds, and I suspect that they augment the fear response associated with the eyespots. 

Overall, my project goals for this summer are:

1)             Describe and measure the anti-predator behaviours of Papilio canadensis caterpillars associated with real and simulated attacks.

2)            Determine the efficacy of ‘eyespot’ markings in deterring lethal bird attacks in conjunction with, and separated from, caterpillar behavioural responses.

As I mentioned, I am working primarily with Papilio genus (Swallowtail) caterpillars. I live in Ontario, Canada and the species here is Papilio canadensis. It is closely related to Papilio glaucus which distributed just to the south of us, and has a very similar ecology. I am currently out catching adult female Swallowtail caterpillars, bringing them into captivity, and then sleeving them over their host plant (Trembling Aspen) so lay eggs. These eggs will be reared to caterpillars that I can use to quantify behavioural responses to simulated and real attacks. I’ll post pictures and descriptions of this process in more detail later.

Twitter

I should mention that I will be live tweeting my progress through the summer. At times I include pictures (aka "tweetpics"). If you want to follow me on twitter my username is hossiet and I'll be marking my research related tweets with #eyespots.

If you don't have Twitter you can still view my tweets on the righthand side of my Blog. To view a picture or link directly from the Blog just hold ctrl when you click the link. You can also view my Twitter feed directly at: http://twitter.com/#!/hossiet

The specific group that most of my work focuses on are the Swallowtails, the Papilio genus in particular. Here are a few cool videos to show you what these guys look like:

http://www.youtube.com/watch?v=uQhWDBzlLCM
http://www.youtube.com/watch?v=i_i4-zxlkvo

The Origin of Mimicry Theory

Henry Walter Bates

“Every species in nature may be looked upon as maintaining its existence by virtue of some endowment enabling it to withstand the host of adverse circumstances by which it is surrounded. The means are of endless diversity. Some are provided with special organs of offence, others have passive means of holding their own in the battle of life...A great number have means of concealment from their enemies, of one sort or other. Many are enabled to escape extermination, or obtain subsistence, by disguises of various kinds; amongst these must be reckoned the adaptive resemblance of an otherwise defenceless species to one whose flourishing race shows that it enjoys peculiar advantages.”

- H.W. Bates, 1862

I start here with another great quote by Henry Water Bates. He was a naturalist who travelled with Alfred Russel Wallace to South America. You can read about his travels and experiences in his book “Naturalist on the river Amazons”. An amazing naturalist and great thinker he developed the theory of what is now called Batesian mimicry. In particular, he provided the theoretical foundation for how the mimics he observed might evolve. Although maybe not fully appreciated at the time his work captured the interest of Darwin who wrote:

"I have just finished after several reads your Paper. In my opinion it is one of the most remarkable & admirable papers I ever read in my life. The mimetic cases are truly marvellous & you connect excellently a host of analogous facts…I rejoice that I passed over whole subject in the Origin, for I should have made a precious mess of it. You have most clearly stated & solved a wonderful problem.”

Darwin goes on to say: 

“With respect to mimetic resemblance being so common with insects; do you not think it may be connected with their small size; they cannot defend themselves;— they cannot escape by flight at least from Birds; therefore they escape by trickery & deception?

I have one serious criticism to make & that is about title of paper; I cannot but think that you ought to have called prominent attention in it to the mimetic resemblances.—Your paper is too good to be largely appreciated by the mob of naturalists without souls; but rely on it, that it will have lasting value, & I cordially congratulate you on your first great work. You will find, I sh^d. think, that Wallace will fully appreciate it.”

See the full letter from Darwin here: http://www.darwinproject.ac.uk/entry-3816

Darwin was clearly impressed and saw the lasting value of Bates’ work. It’s a bit hard to find the manuscript but anyone seriously interested in mimicry should take a read:

• Bates HW (1862) Contributions to an insect fauna of the Amazon valley. Lepidoptera: Heliconidae.  Transactions of the Linnean Society of London 23:495-566

You can’t help but agree with Darwin about the title of Bates paper. Darwin's comments raise a few interesting and particularly relevant points to my project. Caterpillars, especially large ones, are under intense selection to avoid predation by birds. Caterpillars can’t outrun their attackers, and just dropping off their host plant might have serious consequences. Many caterpillars can only eat one plant species and there are a whole suite of different enemies on the ground like ants that could make short work of a caterpillar. So those caterpillars with a different solution - such as the mimetic resemblance to a dangerous snake - survive to breed as adult butterflies or moths.

Introduction

“The most extraordinary instance of imitation I ever met with was that of a very large Caterpillar, which ... startled me by its resemblance to a small Snake... [The caterpillar] had on each side a large black pupillated spot, which resembled the eye of the reptile”

- H.W. Bates, 1862

Many animals have conspicuous eye-like spots on their body. In most animals these ‘eyespots’ are thought to intimidate predators from attacking or deflect the predator strikes away from vulnerable body parts. That ‘eyespots’ could help prey by resembling the eyes of a predator’s own enemies is thought to be particularly true for butterfly and moth caterpillars. Caterpillars with eyespots are often cited to be snake mimics that startle attacking birds which mistake them for dangerous snakes. Despite widespread acceptance, this phenomena is surprisingly understudied. 

My name is Tom, and I recently completed my PhD at Carleton University in Ottawa, Canada where I examined this phenomenon in depth. I will continue posting updates about my ongoing research examining the ecology and evolution of caterpillar eyespots.

Read: Preface to my research on caterpillar eyespots