Time for larvae

A bluish-grey larva - maybe Monomacra violacea?
6.2mm long

Larva, larvae, funny words.  Now that I have a picture (at least an idea) of how the adult flea beetles spread themselves across the Passionflower plants, it's time to tackle a harder question: what are the flea beetles' larvae eating?  There is a possibility that they are more specialized than the adults, since they not only have to survive out there, but also need to grow from egg to pupa.  Heliconius larvae grow extremely rapidly, quadrupling in size in just a few hours and then moulting, five times in rapid succession.  They grow 1000 times as large in 5-6 days!  They are true "growing machines."  But what about the beetle larvae?  How fast do they grow?  Which plants can they survive on?  How do they behave when feeding?

A yellowish larva - maybe the Red-white Ptocadica?
5.9 mm long

I began a couple of months ago to bring a few larvae from the forest into the laboratory, putting them on leaves similar to the ones they were found on.  The ones raised in containers in the lab did poorly, not feeding well and many dying in a few days.  I then decided to try to raise them on live potted plants in the greenhouse.  Actually we call it a shadehouse here at La Selva, a frame covered with shady mesh allowing the interior to stay cool by allowing free entry of air while creating shade to partially block the rays of the sun.  Just last week some of the Passiflora lobata plants in the shadehouse became large enough to use, and I put two flea beetle larvae on a new leaf.  They soon began feeding and seem to be thriving.

The Red-white Ptocadica adult.
The adults may stay on the shadehouse plants
for a few days.

I want to measure how fast the larvae grow, but they are delicate and should not be handled any more than necessary.  I found out a way to use my camera as a measuring device.  I put the camera on a single, consistent manual focus, and take a sharply focused picture.  All such pictures should have the same field of view (measured in millimeters) from side to side.  By comparing the length of the larvae in the photo to the full width I can then calculate low long the larva is in millimeters.  In this way I can see how fast a larva is growing by taking its picture every day.   I don't need to touch the larva at all.

A shield bug just happened to walk by,
with the brightest orange spots!

This dragonfly has amazing eyes. 
Are the brown parts sunglsses?

Of course I have to take photos of other creatures, such as the cute Red-white Ptocadica beetle, perhaps the parent of one of the larvae shown above.  Not to mention this spectacular brown-eyed dragonfly, er, brown on the top 1/3 of the eye dragonfly.  and the orange-spotted shield bug that happened to walk by.  The riches of nature never cease to amaze!

Sum up first weeks

It's been nearly 2 months here at La Selva, working on the flea beetle project.  Seems like a good time to sum up what I have found out.  I hope the amount of detail isn't too boring!

Ptocadica sp. ("fat yellow") prefers to feed on Passiflora ambigua

The basic idea is that flea beetles parallel Heliconius butterflies in the interactions with Passiflora species.  I have found 9 species of Passiflora here that are common enough to work with, four in the subgenus Passiflora and five in the subgenus Decaloba/Astrophea.  I have seen five species of Heliconius feeding on these plants, with three more species that are hard to find but that I know are here (total = eight species).  I also have five species of adult  flea beetles I can work with, with a sixth species found only in the lab clearing (Pedilia sp "red".) and two more that are rare but findable (total = eight species).  Four of the Heliconius and three of the flea beetle species feed on subgenus Passiflora.  Of these, two species of Heliconius and two species of flea beetles also feed on subgenus Decaloba (I call them "generalist" species).  The other four Heliconius and the other five flea beetles are restricted to feeding on subgenus Decaloba.

The correspondences are as follows:  Monomacra violacea (Blue), Parchicola d.f.1 (Black-legged Yellow),  Heliconius cydno, and H. hecale are generalists, feeding on most or all Passiflora species.  Passiflora ambigua (in subgenus Passiflora) hosts the specialist butterfly Heliconius doris and is the preferred host for Ptocadica sp. "yellow".  P. oerstedii and P. menispemifolia host the butterfly H. melpomene, but don't host a correspondingly specialist flea beetle.   In Decaloba/Astrophea, P. pittieri hosts the specialist butterfly H. sappho and the specialist flea beetle Pedilia sp "red".  P. lobata hosts Heliconius charitonia (which I have not seen yet at La Selva this trip) and is the preferred host for Ptocadica sp. "red-white".  P. auriculata hosts the specialist butterfly H. sara and is the preferred host for Ptocadica bifasciata, the red-brown-white flea beetle, and Parchicola d.f. 2, the Yellow-legged Yellow flea beetle.  P. biflora hosts H. erato and is the preferred host for Monomacra chontalensis.

Crematogaster ants sharing Passiflora auriculata nectary with flea beetle larva

The above list shows a strong correspondence between butterfly and flea beetle use of Passiflora species, but with some exceptions.  P. oerstedii and P. menispermifolia host the specialist H. melpomene, with no corresponding specialist flea beetle.   My earlier work suggests that the specialization of H. melpomene on P. oerstedii and P. menispermifolia is a consequence of the unusual petiolar nectaries on these plants, attracting parasitic hymenoptera rather than ants.  H. melpomene responds by seeking out and specializing on these ant-free plants, laying their eggs in shoot tips where the parasitic wasps will have difficulty finding them.  Flea beetles, in contrast to Heliconius caterpillars, are seemingly not affected by the presence or absence of ants or parasitic wasps.  A second difference is that there are seemingly two species of flea beetles which prefer P. auriculata, but only one species of Heliconius (H. sara) specializes on that plant.   

Pedilia sp "red" larvae eating stem epidermis on P. pittieri

One of the biggest differences I have seen between between flea beetles and Heliconiine butterflies is that the beetles tend to avoid plants or plant parts which produce high amounts of cyanide gas when crushed.   Even Pedilia sp. that feeds on the highly cyanogenic Passiflora pittieri seems to avoid some of the cyanide (perhaps 90%) by feeding on the epidermis of the stems and leaves (see photos of feeding damage).  The butterflies seem completely unaffected by the presence or absence of HCN, with caterpillars feeding and growing rapidly on plants with the highest HCN content such as P. arbelaezii and P. costaricensis.  Also, preliminary measurements indicate that Pedilia sp. "red" are not themselves cyanogenic, in either the larvae or the adults.  This is a strong contrast to Heliconiine butterflies and larvae, which are strongly cyanogenic.   Another big difference I mentioned above: flea beetles seem unaffected by the presence or absence of ants (see photo of flea beetle larva with ants).  Heliconius, by contrast, seldom survive to pupation on plants tended by the "wrong" species of ants (such as Ectatomma tuberculatum). 

P. pittieri leaf vein that has had its epidermis removed by feeding Pedilia flea beetles.

The biggest mystery remaining may be what the larvae are doing.  Which plants do they feed on?  I'm starting to observe mating behavior as the weather dries out a bit, so maybe we'll get a pulse of larvae to observe.

Overall summary of project:  Progress!  We're having fun!  Remarkably stable communities of plants and insects over the 40 year interval (thank you, La Selva!).

A new idea to test

Ptocadica bifasciata on Passiflora auriculata

 As I began measuring cyanide gas in more and more Passiflora, I began to notice a correlation: flea beetles seem more abundant on Passiflora with a reduced amount of cyanide.  The two species of beetles shown here are among the most abundant, and you can see the holes in the leaves left by the feeding beetles below.  The beetle on the right feeds on Passiflora auriculata, a plant with variable, but usually reduced, amounts of cyanide, and the P. oerstedii shown below makes none (that I can measure).  Other heavily attacked Passiflora include P. lobata and P. vitifolia, two other species with reduced or no cyanide.  The species with a lot of cyanide (P. ambigua, P. menispermifolia, P. biflora, P. costaricensis, P. pittieri) usually have fewer flea beetles.  (Sorry to throw so many latin names out there, but these plants don't have common English names).

Parchicola D.F. 1 on P. oerstedii, ate holes in leaves, and now seems to be drinking from the extraflora nectar.

Most, if not all, of the flea beetles species have the ability to eat cyanogenic Passiflora, but it remains possible that they survive better (or prefer) food with reduced amounts of cyanide.  One way to test this idea in a more controlled way is to choose a single, variable species such as P. auriculata, and then compare cyanide production in plants with and without flea beetle feeding holes.  I would predict that plants with many holes have low levels of cyanide, and that plants with few holes average more.  To carry out this plan I found 20 auriculata plants in the successional plots at La Selva, a site where I can easily see the plants.  Of these, 7 plants were extensively holed by flea beetles and 13 had few holes or none.  I then measured HCN production by grinding one leaf plucked from each plant.  Result: all seven of the flea-beetle-infested plants had cyanide levels below 0.1 μM/g (micromoles per gram of leaf weight), while the plants without flea beetles ranged up to 0.6 μM/g, six times as much!  Although these results tend in the direction of my prediction, they are not conclusive because the results depend on only four plants with high levels of cyanide.  I therefore went to another site where I could sample auriculata, and did a similar test on 10 plants.  In this set I found one flea-beetle-infested plant with high levels of cyanide (0.6 μM/g), and an even more cyanogenic plant (1.1 μM/g)with no beetles.  These results tell me that things are more complicated than a simple correlation between cyanide and presence of flea beetles, and that I need to dig in a little deeper to understand what is going on.  For example, the beetle holes in the two sites may have been caused by different beetle species, with Parchicola being more common at the second site.

Cebus capucinus at La Selva

Fortunately all my work is being supervised by troops of monkeys, including the White-faced Capuchin seen here!

Distractions of field work

Polybia species?

Now that I have a working lab protocol to measure cyanide, and have at least a rough idea of which species produce cyanide and which don't, I want to turn more attention to observing the flea beetles.  To accomplish this I have been hiking over to the successional plots, five experimental areas that are deliberately chopped to the ground on a five-year rotation, and allowed to recover naturally.  This repeated treatment provides good habitat for passionflower vines near ground level, and is one area where I can usually find flea beetles.  But, it can take a while to get there.

Lirometopum coronatum katydid on Passiflora auriculata

Ptocadica bifasciata on Passiflora auriculata

 The first photo shows the kind of distractions that happen every time I step out the door.  It shows a wasp colony (genus Polybia?) with 1-200 members, I expect with the queen (queens?) in the huddles.  A few workers seem busy building  new nest carton.  When I checked back a couple of hours later the carton was much more enclosed.  You can click on the photo to enlarge and see the wasps in detail.

The second photo shows a very bizarre katydid-like insect (Orthoptera: Tettigoniidae), with a rounded green cricket-like body, long antennae, and a strangely flattened head.  It took me a while to figure out what I was looking at.  The flat face is covered with light colored nodules and the jaws are black, giving the appearance of a crinkled, folded leaf with holes in it!  Only when it moved did I see what it was.  Apparently this species is carnivorous.

Monomacra violacea on Passiflora auriculata

When I finally arrived at the successional plots I did find some flea beetles to observe.  In fact I found one leaf of P. auriculata with three genera on it!  To the right is Ptocadica bifasciata, one of the larger species about 3 mm long.  I also saw Monomacra violacea, the shiny blue flea beetle, and Parchicola d.f. 2, the yellow-legged yellow flea beetle (not shown here).

I also saw 2 tiny orange flea beetles with black legs sitting on Passiflora vitifolia.  At first I thought it might be another species to add to my study, but after looking I couldn't find any sign that the beetles were feeding.  I suspect they were just sitting on the plant.  Later another large flea beetle landed near a P. auriculata, but it too showed no signs of feeding on Passiflora.  So far, in all my work here at La Selva, I have consistently found the same set of flea beetles.  The only exception is I haven't recently seen the "fat yellow" Ptocadica species nor the Dysonycha decemlineata species.  But no new species have cropped up.

unknown flea beetle (Coleoptera: Chrysomelidae: Alticini)

Working at La Selva

Cabina #3, researcher housing

I chose to work at the La Selva Biological Station partly because of the great scientific facilities there.  The cement trail system (see photo) provides safe and easy access to the different habitats, even allowing travel by bicycle.  Dangerous snakes such as the fer-de-lance are easily seen and avoided.  Trail-side plants such as Passiflora vines are protected because traffic is restricted to the walkway and does not spread out into the forest (by hikers avoiding muddy spots).  The station also provides comfortable although modest accommodations, and an excellent meal service.  Perhaps the best feature of all is the excellent scientific and logistical support that the station staff provides.  If I need something for the lab (for example a macro-photography set-up), or some scientific help with a question (for example, identifying Passiflora arbelaezii), I get immediate assistance.  This makes my work much more effective and allows me to make progress more rapidly.  Many of the facilities at La Selva have been funded by a special program at the US National Science Foundation, designed to support Field Stations and Marine Laboratories.  This program evaluates field stations on their scientific value and potential, and awards improvement grants for upgrading buildings, labs, and equipment.  By any measure, La Selva has been one of the most successful field stations in the world.  My 1978 PhD dissertation in the La Selva library has #21 on it, but there are well over 400 others listed.

Beautiful caterpillar

I just had to share a photo of this beautiful caterpillar, which I found on Passiflora arbelaezii.  I collected a pair of them a few days ago and they are growing fast.  I think it is a Dryas julia larva but will confirm when it ecloses from the pupa in a couple of weeks.  Dryas is related to Heliconius.  These larvae release very little or no cyanide gas when feeding on this highly toxic plant.  My measurements indicate that the leaves contain 3-7 micromoles of HCN per gram of leaf tissue, making it one of the more toxic species of Passiflora.

I measure HCN gas using a meter (yellow device in rear of photo) designed for emergency responders entering buildings with hazardous materials.  To use the meter effectively I had to deprogram all the built-in alarms, including physically removing the vibration alarm.  The built-in alarms were programmed go off at 4.7 part per million (ppm) cyanide gas (low alarm indicating danger) and at 10 ppm indicating extreme danger (get out!).  A square centimeter of crushed P. arbelaezii leaf is enough to set off the high alarm right away, but this caterpillar, even when rapidly feeding, usually releases no measurable amount.   The apparatus includes a special pump (yellow device center photo) which moves 5 milliliters of gas each second.  The ppm reading, along with the known volume of the glass flask enclosing the sample enbles me to calculate the micromoles of HCN gas contained in the flask.  The flask in the photo contains a Heliconius hecale larva feeding on P. arbelaezii.  The larva consumed the toxic plant with no difficulty.

New plant; first flea beetle record

Since I worked here in the 1970', researchers have named a new species of Passionflower Vine, Passiflora arbelaezii. Although I remember seeing it then, I was not sure it was a Passionflower.  Most Passionflowers have long, unbranched tendrils, filaments that grab the surrounding vegetation and by coiling, pull the plants higher up above the ground.  P. arbelaezii has thin, short tendrils that are branched into three smaller filaments, and these may even be sub-branched into three sets of three.  These tiny tendrils cling to almost anything, enabling this plant to climb up tree trunks as long as the surface is rough.  In the photo is my first record of a flea beetle using this plant species, in this case Monomacra chontalensis.  Although this is one of the less common species of flea beetle at La Selva, it makes sense that it was found on P. arbelaezii.  It's preferred host species is Passiflora biflora, the species that is physically most similar (and probably most closely related) to P. arbelaezii.

Blame this guy? gal? for our coming to La Selva

On the right is a Black-legged Yellow Flea Beetle, also known as Parchicola D.F. 1. This is a provisional name, needing further work by taxonomists such as David Furth of the Smithsonian National Museum (that's where the "D.F. 1" comes from). If questions about relationships with other beetles are resolved, and someone makes the effort to give the species a formal name, then our beetle will gain an official scientific Latin binomial name. This is important because among the million or so insect species, it is hard to store knowledge and information about any one unless there is an official unique name that everyone can use. There is a lot to learn about this and most other species of flea beetles. This one is unusual (lucky?) in that we are studying the live beetle in its natural habitat. Notice its rich yellow-orange color that shines in the light, and the lovely green leaf where it is having its picture taken. We still don't know for sure what its larvae look like, and really know very little still about its life cycle and other habits. But most flea beetles are known from sweep samples collected more or less at random, preserved in collections, and then sorted and identified as dried specimens. They turn a dull brown and most definitely lose the "twinkle in the eye" that you can see in the picture. The main reason I want to come to La Selva and spend six months is to see what I can find out about these attractive little creatures. See which plants they eat. See what their eggs, larvae and pupae look like. Take lots of pictures so everyone else can see also. And, as I said in the introductory paragraph, try to figure out why there are the same number of flea beetles as butterflies using the same set of plants.

To start with...isn't science wonderful!

Kim and I got to La Selva a little over 2 weeks ago, and we are now definitely settled in. Time for me to start a blog! After the somewhat painful process of setting up the photos (I miss my old PC software, photoshop and 11-view), I am ready to start posting things about my research here. So here goes... To start with, I have a slightly dated web site (copy and paste into your web browser without the quotes): "http://www.wmrs.edu/people/BIOs/john%20smiley/default.htm" that has background information and a summary of what I have found out so far under the link that starts with "Passionflower Vines...". If you can find them (hint: look in the little leaf icons on the third web page), I also have several hypotheses about the production of cyanide gas that I have already proven wrong! In only two weeks! Isn't science wonderful! Now I need better hypotheses.