Data I’ll never publish: Antirrhinum herbivory

Inspired by this post, I’m going to try to put the results of small (but interesting) experiments up here every once and awhile. In the summer of 2014, I spent a lot of time washing plants. I was – and still am – curious of the function(s) of plant exudates. I primarily did this with Trichostema laxum and Atriplex rosea (in 2013), but I also did it with Mimulus layneae and Antirrhinum cornutum (California snapdragon). The snapdragon gave me interesting results.

(this post should also be regarded as potential project for someone else: I started it in May – there is plenty of time to get up to McLaughlin and do it again this year).

One of the experimental A. cornutum, showing leaf damage.

This snapdragon, while not as heavily glandular as Trichostema or that Mimulus, is fairly glandular-sticky, even entrapping a small number of minute insects (see the table/supplementary material). Under the microscope, you can see the fairly dense short glandular trichomes (the longer trichomes are mostly nonglandular) on the stalk and flower bud.

Stem of A cornutum with an entrapped insect.
Flower bud showing short glandular and long nonglandular trichomes.

Wondering whether the glandular exudate is defensive, I did an experiment where I removed it with water. Most glandular exudates in CA summer annuals seem water soluble, so a spray bottle rainfall takes off much of the exudate (observationally verified in situ with a 20x loupe – plus whatever was in this exudate made suds on the plant!). This manipulation was my first treatment group. Of course, adding water to a plant has an effect of its own, so I also had a water control group, where I added the same amount of water below the plant’s leaves, as to not wash off any exudates. Finally, I had a true control group, which received no water whatsoever. I instituted these treatments on the 30th of May and reapplied them on the 17th of June. Each time, I recorded the number of leaves, flowers, fruit, and plant height, as well as any damage. I also checked the plants, but did not reapply treatments on the 2nd and 19th of July (the last check all were senescent).

During the experiment, plants suffered two main forms of herbivory. The first type, which was most common and most destructive, was that the stems were entirely clipped off. I’m nearly positive this was by jackrabbits (indicated by a single flat cut diagonally across the stem) and it usually killed the plant. The photos below shows what remained.

A killed experimental A. cornutum plant. See it?!? Its the little stem to the bottom left of the flag. Also notice a nice healthy Lessingia in the background. They, too, are extremely glandular and sticky.
A survivor of mammalian herbivory. If the meristem was not completely destroyed, they often came back and branched like this. Like the classic overcompensation “herbivore-plant mutualisms”, the resulting plants were often bigger than the others, with more reproductive structures, but unlike this “mutualism”, it was too late in the season and they had low fitness, as they could not mature these structures.

The mammalian herbivory was not random. Of the 25 plants per treatment, 11 in the control group, 13 in the rainfall simulation (exudate removal) and a whopping 20 in the water control group were eaten by mammals (this is nonlethal, lethally was 10, 12, 18). With a simple chi-squared test, we can demonstrate that this was likely nonrandom (X2 = 7.3688, df =2, p = 0.025) (for lethal, X2 = 5.5714, df=2, p = 0.062). Why were the mammals targetting the water control plants so heavily?

Were they bigger and thus easier to find or just more profitable to eat? They were not significantly different in height, fruit or flower numbers from the other two groups during any check. I don’t have data on plant quality (perhaps the less water-limited plants were more nutritious or something?).

The other type of damage was equally-interesting. Heliothis phloxiphaga is a generalist caterpillar on glandular plants. It was the primary herbivore on my columbines, as well as a common herbivore on Trichostema laxum and other sticky plants. Like most heliothiine noctuids, it feeds primarily (but not exclusively) on reproductive structures. I only observed it once on Antirrhinum (eating a fruit), but all the fruit damage I found was consistent with it (and that’s one more time than I saw a jackrabbit eat it!).

The other type of damage: caterpillar fruit predation.

I had hypothesized, that if the exudate were defensive, the washed plants would be most heavily eaten. This hypothesis was supported with the fruit damage. Rainfall plants received far more damage than the other groups. (note: I didn’t actually analyze this with zero-inflated binomial, as it should be. There is a problem, in that only 7/25 of the water control plants had any fruit at all because of the rabbits.)

A crumby excel graph of proportion fruits damaged.

What does this all mean? Obviously, it means that mammalian and insect herbivores are responding to different plant traits. What they are exactly, I’m not sure (especially for mammals). If anyone (nudge, nudge, wink, wink) were to repeat this experiment, with a larger sample size, and maybe some other mechanistic experiments (perhaps cage controls and lots more trait data to see what is different in the water control and rainfall manip groups), I think its a pretty good system that someone could get a paper – if not a few – out of.

A Caterpillar Mystery in the Bahamas

I’ve been in the Bahamas for the last two weeks, studying the effect of resource pulses (hurricane-wrecked seaweed) on island communities with this project. In doing so, we kept coming upon these strange shelters on wild guava (Psidium longipes), locally called Bahama stopper, since the hard wooded bush/tree would apparently stop any progress you try to make into the thick coppice.

What on earth is this? ~  4 cm tall (pretty damn big by insect standards).
Inside some of these shelters (2/13), there was an odd larva, apparently a beetle larva, or so I initially thought. Because I am mostly a caterpillar person, I didn’t really pay it much mind. 
A really terrible picture, but notice the “antennae”. About 2-3 cm long. 
Louie, while processing insect samples one night, noticed that some things were not right about the apparent beetle larva – namely it had prolegs, the fleshy appendages that give caterpillars the appearance of having more than the six legs all insects have. I then looked at the “antennae” and found that they were not segmented, a dead giveaway that this was, in fact, a caterpillar and the “antennae” were actually tentacles (yes, that is the technical term for the fleshy projections that many caterpillars have – monarchs for instance).
Several of the shelters were torn like this, suggesting predation (by a bird [?]). This was the
only shelter with lines affixing it at the top – many had lower lines. 
I got much more interested after that, and sent along these pictures to Charley Eisemann, a good friend and probably the person on earth with the most knowledge about insect shelters. His blog – linked above – is simply phenomenal and if anyone was going to know the answer, he would. Very quickly (within a few minutes), he had correctly found the family of the moth – Mimallonidae. The amazing part here is that Charley has never seen a member of this family! Mimallonidae is an extremely small family by Lepidoptera standards, ~200 spp. – only 3 of which occur regularly in the US, a fourth is described from the US in Brownsville, TX, but is probably a tropical stray. He even dug pretty deep and found a very likely species identity, Ciccinus packardii – known from Cuba and known to feed on other Psidium species. While I do not know this for sure, it seems that is the most likely candidate as the larva matches very well the few images of Ciccinus online, and less so the other mimallonid genera. 

After a bit more searching, we came upon a young larvae feeding in a leaf press on P. longipes, which was not what I expected. This family is known as the “sack-bearers” and I was expecting something more along the lines of a bagworm (Psychidae), instead of a leaf presser.

A young (2nd, 3rd instar?) larva of this Ciccinus sp. ~ 8mm

Which brings us to the strange, pitcher plant-like shelters. The larva is oriented vertically inside the shelter, with a strange butt plate plugging up the bottom hole and the head just below the upper hole. What function the little hood forms is mysterious – perhaps shading the larva from the hot Bahamian sun or fierce rains? The better-known Ciccinus species of the US, C. melshiemeri, feeds on old oak leaves (too tannic for most caterpillars) and constructs a shelter, sort of like the pictured ones of frass pellets, silk and oak leaves in which it spends the winter as a larva, prior to pupation in the spring. This seems to be the case for this species as well – in two cases, I found spent pupal skins.

Spent pupal skin (successful emergence!) inside one of the shelters. You can also see the construction of silk and what
appears to be finely ground frass (caterpillar poop – a common building material for cats). 

Interestingly, I did find one that fit the description of the C. melshiemeri shelters well.

This was the only shelter anchored into leaves (it was vacant, unfortunately). You can see well the frass pellets forming the top of the shelter here.
The same shelter, with a Psidium leaf forming one side. 

These guys kept me occupied for quite awhile (I even dreamt about them!) and seem like a worthy avenue for future rearing efforts… there are a great deal of questions that remain about the shelters: Why the strange shape with a hood? Why build a free standing shelter, as opposed to anchoring it to a stem like most moths? Why wait around in a shelter instead of pupating right away? Do the shelters protect inhabitants from predators and parasitoids?

perhaps the prettiest of all found. I like the subtle banding.

Many thanks to Charley, Julia Blyth, John De Benedictus, Louie Yang, Jonah Piova-Scott and Jenn Mckenzie (who was the only one that could find occupied shelters) for help with the identification and finding of these guys.

Shelter-building caterpillars

This post is a bit late in coming, but I wanted to share what I worked on for several summers with Doug Morse for my undergrad thesis. We did most of the work in 2008 and 2009, but finished up some during the summer of 2012 and it was published earlier this year.

Caterpillars, like many other animals, construct shelters for themselves, by some estimates over 60% of lepidoptera (butterfly and moth) species make some sort of shelter. Most often these are made by folding or rolling the leaves which they feed on into shelters. The shelters likely serve a variety of purposes: protection from dessication, protection from predators, reduced probability of dislodgement, etc. Interestingly, several studies had found that caterpillars in shelters suffered less predation but more parasitism and indeed, at least one author had speculated that since parasitism was often so high (90%+ sometimes!), that shelters must not be good protection.

an unidentified caterpillar folding up a Malvaceae leaf, Chiloe Island, Chile

That seemed odd to me; if parasitism was so prevalent, it must be a strong selective force and clearly some survive, so there must be some way to beat it. Working with a cool leaf-rolling moth, Herpetogramma thesausalis, on ferns in Maine, I

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ran experiments to test two hypotheses:

1) Shelters provide protection from parasitoids
2) Shelters are costly for the caterpillars to produce

an adult male H. thesausalis on sensitive fern, its primary host

To test the first hypothesis, I thinned shelters during the moth’s pupal stage by carefully removing the outer layer of fern (the shelters usually involve 2-4 layers, so this did not expose the pupae inside). I used the pupal stage, as caterpillars would quickly rebuild the shelter when I thinned it – pupae are unable to. I found that parasitism almost doubled without that outer layer, though overall size of the shelter was only slightly smaller. Therefore, I found support for the first hypothesis – the first experimental evidence for this function of caterpillar shelters!

A cross-section of a shelter containing a parasitoid (Alabagrus texanus) cocoon.

For the second hypothesis, I divided caterpillars on ferns into three groups: the first I left alone (as controls), the second I dismantled their shelter every few days and the thirds I touched but did not destroy the shelters at the same interval. In essence, I was forcing one group to remake their shelters. Given my hypothesis, I expected that these caterpillars would either pupate smaller or later. Interestingly, the caterpillars delayed their pupation in order to rebuild shelters – an average of ~2 days later than the other groups – and they did not eclose smaller (or build smaller shelters!). Delaying their pupation puts them at increased risk for larval parasitism and delays their reproduction (the population is fairly synchronous, so this could be a big cost for some individuals). Hypothesis 2 supported!

A female ichneumonid (Itoplectis?) oviposits into a caterpillar shelter.

There is more to the story, it is a longer paper. But perhaps the most interesting thing is that there are two broad guilds of pupal parasitoids – those with long ovipositors (as seen in the above picture), which oviposit from outside the shelter, and those with short ovipositors which gnaw their way through the seams between leaves into the shelters. I suspect that larger shelters, with a longer distance between the outside and the pupa, are better protection against the former guild. However, in a larger shelter, there are more seams, which may make it easier for the latter guild to parasitize, an interesting trade-off, which I have only observational data to support.

Phaeogenes hebrus, the most common short-ovipositor species in this system.

The paper can be found here: http://link.springer.com/article/10.1007/s11829-013-9261-4#page-1 . Arthropod-plant interactions publishes lots of great – if limited interest – research, I read a very high number of the papers it publishes.

This would make a cool tattoo, eh?