Friday, November 29, 2013

Back at La Selva (it's nice to be back!)

Passiflora auriculata in cage.  Soil prepared for beetle pupae.
A few weeks ago Kim and I returned to La Selva for another extended stay (until the end of March).  Kim is already adding animals to her "accidental museum" and I hope to find the larvae of the flea beetles, and see which Passiflora they eat.  I also plan to continue testing the plants and insects for production of cyanide gas when crushed or damaged.

I brought down 6 cages, each large enough to hold a potted plant.  My plan is to put one species of adult beetles in each cage and see what the resulting larvae look like.  Right now I have 5 caged species: Red-brown-white (Ptocadica bifasciata that feeds on Passiflora subgenus Decaloba), Red-white (Ptocadica sp. RWh that feeds on P. lobata), Black-legged Yellow (Parchicola DF 1 that feeds on subgenus Passiflora), Yellow-legged Yellow (Parchicola DSF 2 that feeds on subgenus Decaloba) and Blue (Monomacra violacea that feeds on all species of Passiflora).  I have seen mating pairs in the cages, so I know there are females present in reproductive condition.

I don't need to isolate the red Pedilia flea beetle that eats Passiflora pittieri, since we have a large population already in residence in the lab clearing and I have tested its larvae.  Two other species are simply too rare to work with: Yellow Ptocadica that feeds on subgenus Passiflora, and Monomacra chontalensis  that feeds on subgenus Decaloba.  Perhaps these species are more common at higher altitudes or in other habitats than I can currently sample at La Selva. 

Passiflora garden in La Selva lab clearing.
Before leaving La Selva last April we established 12 species of Passiflora in an outdoor garden, with assistance from botanist Orlando Vargas and the La Selva staff.  The vines thrived in my absence and now, in spite of attacks by Atta leafcutter ants and wild peccaries, have grown into medium-sized vines.  Five species have flowered and set fruit: oerstedii, lobata, auriculata, biflora and megacoriacea.  Two species of flea beetle have been found on the garden plants: Parchicola DF 2 and Monomacra violacea.  I also have a Psiguria vine in the mix, in the hopes of attracting Heliconius butterflies seeking pollen.

Galerucine beetle on P. lobata
I have been using these vines to measure cyanogenesis in Passiflora foliage and for cuttings to use in the shade houses.  I also have found some unusual herbivores on the leaves including Juditha molpe, a metalmark caterpillar (Riodenidae) tended by the Ectatomma ants on P. auriculata.  I also have found a brown-striped species of galerucine Chrysomelidae eating the leaves of P. lobata.  Galerucines are the subfamily of chrysomelid leaf beetles that include the flea beetles (Alticini), and this brown striped beetle resembles a large flea beetle except that it lacks the jumping mechanism in the hind legs. The pattern of eating holes in the leaves also resembles the feeding damage made by a flea beetle. 

An example of looking at patterns of cyanogenesis in Passiflora ambigua can be seen in the graph below.  First, look at the red diamonds in the graph.  They show the amount of cyanide gas produced by the tips of leaves 1,3,5,7,9,and 11, (counting backwards from the tip of the branch).  The branch was fresh and undamaged prior to the leaf tips being removed for analysis.  The next day (day 1), I removed a strip from the cut end of the same six leaves, and measured that cyanide output (see the blue squares).   As you can see, the amount of cyanide increased in the newer leaves!  Also on day 1 I cut and measured HCN from the tips from the undamaged alternate leaves 2,4, and 6.  These amounts (green triangles) were comparable to the original amounts measured for non-damaged leaves.  Thus, the increase seen in leaves 1,3 and 5 (a phenomenon known as "induction") did not extend to the neighboring leaves.  Finally, still on day 1, I cut strips off the ends of the even-numbered leaves to see if there was somehow more cyanide in that penultimate part of the leaf as opposed to the tip.   There wasn't.  The next day (day 2) I sampled the twice-cut ends of the odd-numbered leaves (red circles). Here the HCN content seemed to even out between leaves, increasing in leaves with little HCN and decreasing in those with more.

Taken together, these results tell us that cyanide production roughly doubled 24 hours after a new leaf was damaged and that this response was localized to the leaf in question.  Adjacent leaves did not increase, and neither did older damaged leaves.  A more complex response seemed to occur in the next 24 hours.   One goal of this coming year at La Selva is to conduct studies like these in order to explore how Passiflora may be adapting to their complex herbivore community.  Ater all, they have simultanously to deal with herbivores that thrive on cyanogenic glycosides (Heliconius butterflies) and others that thrive when glycosides are absent (flea beetles).  These balancing agents of selection could seemingly lead to complex interactions within the whole community, including, perhaps, great variability in HCN production within and between species (something I have ample evidence for!).  More about this in my next blog posting.

Saturday, March 2, 2013

More similarity/differences and a sum-up statement!

Here's another similarity/difference between flea beetles and Heliconius:

Red-white Ptocadica on Passiflora lobata
I have been putting larvae of RWh Ptocadica on different species of Passiflora to see if they can survive and/or grow.  On their normal host plant the larvae grow and survive quite well, as summarized a few blog posts ago.  Although the results are sketchy, being based on only 2-3 larvae for each plant tested (alas, that was all I had!), there seems to be a correspondence between adult and larval food preference.    Adult RWh Ptocadica clearly prefer P. lobata as a host plant, with P. auriculata as a second choice (less than 10% occurrance).  No other Passiflora are acceptable.  RWh Ptocadica larvae clearly grow fastest and feed the most on P. lobata, but will eat and grow slowly only on P. auriculata of the other species tested.  Species which release high quantities of poisonous cyanide are not fed upon at all, while Passiflora with zero or low amounts may be eaten slightly but with no observable growth.

How does this result compare with Heliconius charithonia, the Heliconius that specializes on P. lobata at La Selva?  H. charithonia larvae feed and grow well on lobata along with many other species of subgenus Decaloba, including auriculata and biflora.  They grow slowly or not at all on members of subgenus Passiflora, including ambigua and oerstedii.  This is similar to RWh Ptocadica, with the exception that species of Decaloba other than the low-cyanide auriculata can't be eaten by the flea beetle.   All the other Decaloba are high in cyanide output.  As stated above, this flea beetle larvae cannot handle strongly cyanogenic food plants.
H. charithonia, only Heliconius that can eat P. lobata

It makes sense that the RWh Ptocadica might be very sensitive to cyanide poisoning, given that the normal, preferred host plant generates no measureable amounts of HCN when crushed.  In contrast, the Red Pedilia seem more tolerant of cyanide.  Although their feeding behavior seems designed to avoid cyanide release whenever possible, selecting thin leaf layers and stem epidermis, this species' host plant P. pittieri is highly cyanogenic.  The feeding trials on alternate Passiflora species indicate increased tolerance, with measureable feeding and growth on several Decaloba species.  This parallels the pittieri specialist butterflies H. sappho and sister species H. hewitsonii, which also show a limited feeding ability on Decaloba species, particularly arbelaezii and biflora.  Molecular evolution data tells us the connection between pittieri and these Decaloba is probably not phylogenetic, but the pattern of insect food choice suggests a similar chemistry or other nutritional factor.  The fact that flea beetles and butterflies share a similar response further suggests that both types of herbivore respond in the same way to these plant nutritional/chemical characteristics.

Flower of P. lobata.  Pretty!
To summarize, I have been able to work with two species of flea beetle larvae, the Red Pedilia and the RWh Ptocadica, both of which specialize on different species of Passiflora subgenus Decaloba.  Both species' larvae show feeding preferences and behaviors that reflect the preferences and behaviors of the adult flea beetles, with complete specialization of Red Pedilia on P. pittieri, and a weaker specialization of RWh Ptocadica on P. lobata and P. auriculata.  This suggests the hypothesis that flea beetle larval feeding preferences and tolerances may be accurately mirrored by those of the adult beetles, which are far easier to count and observe.  In addition, both species have a counterpart butterfly species, Heliconius sappho and charithonia, respectively, that have strong similarities in food tolerance and preference.

Taken together, these findings suggest that  unique chemical and physical defenses in Passiflora foliage create opportunities for herbivores to specialize if they can counteract the defenses.   The findings also suggest that one Passiflora species can host at least two specialist foliavores as long as their is substantial intraspecies variation in plant size, ant presence and/or foliage cyanogenesis.  If these results hold up as more Passiflora and herbivores are investigated, it will make a strong case that host plant foliage characteristics determine species diversity here at La Selva.  Obviously there is a lot more to be learned from these beautiful creatures.  Pura vida! as they say in Costa Rica.  Pure life!

Tuesday, February 26, 2013

Differences and Similarities!

I have found that flea beetle larvae are tricky and difficult to work with, at least as compared with butterfly caterpillars.  The biggest difference is that seem very willing to walk off the host plant; something butterflies such as Heliconius rarely do until they are ready to pupate.  That poses a mystery.  If they are willing to leave the host plant, then how do they relocate it later when they are ready to feed?  Do they walk at random until re-acquiring the plant?  Is there an odor trail or way to orient?  They also seem content to go for long periods without eating; again, very different from Heliconius larvae.  Perhaps their walking and waiting ability enables them to relocate the host plant before starving to death.  There is also the possibility that they may feed (sparingly?) on other plants besides their host Passiflora.  Something I need to test.

Red Pedilia eating vein of P. megacoriacea leaf
In the feeding trials I have conducted with flea beetle larvae, I often see signs of feeding from the epidermis of the stems and petioles, and from one layer of the leaf.  These larvae are experts at selectively taking tiny bites from very thin layers of plant tissue.  Even very thin membranous leaves such as those of Passiflora biflora may be selectively skeletonized, with one layer intact and the other removed.  As I mentioned in earlier blogs, this may allow the larvae to feed on cyanogenic Passiflora without getting poisoned.

Red Pedilia larvae turn pink-orange as does their frass.
Red Pedilia is related to Pedilia sirenae found on the Osa Peninsula in Costa Rica.  I have been told by colleagues that the La Selva Pedilia is very similar to P. sirenae except for being bright red rather than yellow-orange.  It is interesting to note that a few of the La Selva Pedilia are yellow-orange instead of the usual red.  Red Pedilia larvae range from pale white to orange or pink, getting pinker as they age.   Their diet changes from young leaves eaten by the tiny first and second instar larvae, to stem and petiole epidermis, eaten by the larger second and third instar larvae.  These latter plant tissues contain a lot of reddish pigment that is very apparent in the brightly colored frass left behind as the larvae feed.  For some unknown reason the larvae often chew leaf petioles, causing leaves to fall from the plant.

Red Pedilia larvae have a pronounced widening of the body over the front segments, effectively covering the head and front legs.  Since the host plant P. pittieri is often well-patrolled by ants visiting the nectar glands at the base of the leaves, this body shape probably protects the larvae from ant predation.  The P. pittieri plant in the La Selva lab garden is well tended by Ectatomma tuberculatum.  This large predatory ant seems to ignore the presence of Red Pedilia entirely.
3rd instar Ptocadica larva on leaf of P. lobata

Larvae of the Red-white Ptocadica ("RWh") and the Red-brown-white Ptocadica (Ptocadica bifasciata a.k.a. "RBrWh") are similar to Red Pedilia, but with recognizable differences.  These larvae have longer legs, and their body is somewhat more narrow, so that the head and legs are more visible from the side.  The lobes of the dorsal surface (the "back" of the larva) are rounded rather than pointed, and are very stiff and "armored" looking.  The legs are strong.  These larvae are more difficult to pick up than Pedilia larvae. 

I should note here that I am only 90% sure that these larvae belong to the genus Ptocadica.  It is possible though unlikely that they are Monomacra violacea, the Blue flea beetle.  For now, to keep things simple, I will assume that Ptocadica is correct.  I hope to make a definitive identification in the near future using genetic "bar-coding."

Tiny RWh Ptocadica larva feeding and growing on P. lobata
The is one very interesting difference between RWh Ptocadica and Red Pedilia.   RWh's preferred Passiflora is P. lobata, a species with hooked hairs covering the leaves and stems, like velcro.  These hooks are very sharp and are capable of killing caterpillars of most species of Heliconius butterflies.  The one exception: Heliconius charitonius.  Apparently charitonius has a thicker cuticle on legs and underside that is resistent to the hooks.  The RWh Ptocadica is also resistent to the hooks.  However, when larvae of Red Pedilia are placed on P. lobata they become hooked and die.  In future I hope to examine both species of flea beetle larvae with a scanning electron microscope to see if differences are visible than might explain these results.  I also would like to test larvae of the RBrWh Ptocadica (Pt. bifasciata) to see if they can survive on Passiflora lobata.  Since this species does not normally feed upon P. lobata I predict that, as in non-adapted Heliconius, the hooks will prove to be deadly.

Red Pedilia larvae hooked to death on P. lobata
I still have the goal of understanding the causes of species diversity and specialization in these two groups.  Comparisons like those above, between the flea beetle and the butterfly larvae, are all I have to go on so far.  In theory, there must be a balance between differences that allow the flea beetles and butterflies to coexist on the same plant species, and similarities that force both kinds of herbivore to split up the Passiflora species in the same way.

I have found major differences in sensitivity to ant predation (unlike butterflies, beetles are not very vulnerable to ants) and presence of cyanide (beetles avoid cyanide in their diet, butterflies do not).  These differences may be enough to prevent flea beetles and butterflies from competing face to face on the same host plant, at least most of the time.  Heliconius are generally more successful on the subset of plants which have few predatory ants, including the smaller, more isolated plants that can be searched out by the super-mobile highly visual females.  Flea beetles appear to be most successful on larger plants that can sustain "colonization" by less-mobile tiny-eyed beetles (are larger plants more likely to be colonized by ants?), also with reduced amounts of HCN-releasing chemicals.

The similarities have proven more difficult to figure out, but so far they are of the kind discussed above: specific adaptations to overcome specific plant defenses such as hooked trichomes, or, conceivably, special chemical defenses.  More about this topic later...

Friday, February 8, 2013

Visa trip to Panama

A funny quirk about visiting Costa Rica is that the travel permit (technically not a visa) to stay in the country last only 90 days.  In theory you can get a renewal in San Jose, but in practice everyone just leaves the country for three days and then comes back in.  It's a small country so travelling to the border is not difficult.  At least, not too difficult. 

Kim and I decided to go to the closest point in Panama, a beautiful set of islands named Bocas del Toro.  We took the bus to San Jose to do some shopping, and then the next morning we got on the bus named the "Bocatorenyo."  This took us first to Limon, on the Caribbean coast, and then down coast to Panama.  The border crossing was completely disorganized, only made slightly better by some taxi drivers who offered advice as to which line to stand in (but don't trip over the railroad tracks).  It took maybe an hour and a half to cross.  Then we zoomed through banana plantations and  couple of small cities in a very high speed mini-bus, to climb down into a water taxi (called a launch).  We then sped across 5-10 miles of open water and landed, just about dark, at a dock in "Bocastown".  Jose, a man sitting next to me in the taxi, showed us to a wonderful downtown hotel called "Cayo Zapatilla;" $20 per night, no air conditioning.  We gladly took the room.  We found the room to be very comfortable as long as we kept the hallway door open to allow air to circulate.  At night we blocked the door open 6" or so using one of the beds, so no one could get in while we were asleep.  We had no trouble.

Bocastown turned out to be a very entertaining and lively place to be and we stayed there for well over a week!  Our daughter Rose and her companion Eric joined us after a few days.  There were a variety of places to eat, and we rented bicycles and kayaks, and went around the island checking out beaches and places to snorkel and look at the marine life.  Bocas del Toro is home to huge and diverse mangrove forests and swamps, as well as fabulous beds of corals, sponges, seagrass and other marine life.  The Smithsonian Tropical Research Institute operates a small Marine field station there, which we visited.  We snorkeled in some shallow water just west of the marine station.  I found out that I need a special mask because I couldn't wear my eyeglasses with the mask we rented.  Still, what I saw was spectacular!

I spent a few days looking for Passiflora vines on the island.  The island, about 15 km across, has a fairly well-developed rainforest, with some large trees and typical fauna such as howler monkeys, parrots, toucans, and oropendolas.  I found five Passiflora: ambigua, menispermifolia, arbelaezii, biflora and auriculata, along with Heliconius hecale, sara, erato, maybe melpomene, and Dryas julia.  And only one flea beetle: Disonycha quinquilineata.  I have seen that flea beetle at La Selva (on P. biflora) many years ago, but never in the past few years.  The few plants I found had no sign of flea beetle feeding damage, so maybe they are rare or absent from the island.

 We cooked a few meals in the hotel kitchen, and spent a lot of time looking off the balcony at the street and central park below.  On the second day we were there, and for every night after, these constumed "devils" would appear around 5 pm and carry on down on the street below.  They carried whips and would try to whip any older boys or young men carrying sticks.  The boys would taunt the devils and run up to them, and they crack their whips in the sticks or on their legs if they could.  The number of devils escalated every night until there were about 7 or 8 the night before we left.  They would grab a bike from the boys and toss it in the street.  The boys would then try to get it back without getting whipped.  During the day we saw people were making the devil masks out of newspaper and plaster.  We were told that the devils were building up to Mardi Gras, on which day they have to run a gauntlet of citizens with whips of their own.

I was disappointed that there was so little music on the street.  One night there was a pretty awful band playing rock classics like Santana's "Oye como va", and another duo played andean flute music, but that was about it.  Oh well you can't have everything! 

We has a pretty good adventure for our required "visa trip"!  The only real downside was that we got a little bit sick with something "internal"; probably a virus going around.  We were extremely happy to get back to La Selva!

Sunday, January 27, 2013

Making real progress with larvae, but so many mysteries!

First instar larva, crawling up stem from lower leaf.
In my last posting I introduced the subject of flea beetle larvae, and how I hoped to observe them and measure how well they do on different Passiflora.  This past month I have been doing just that.  I have measured their growth rates, placed them on different Passiflora species, and taken hundreds of photographs.  I still don't know for sure which species of larva I am working with (but have narrowed it down to two).  Nor have I seen any of their eggs (although I think I should have since I was looking for them), and have not yet seen a pupa.  Even with all those unknowns, I now have a much better knowledge of what life is like as a flea beetle larva.

The first picture is of a first instar larva, crawling up a stem of P. lobata.  I believe it recently hatched from an egg down lower on the plant, and is now walking up to a leaf where it can feed.  Based on its size, I estimate this larva to weigh about 0.5 milligrams.  This is probably close to the weight of the egg that it hatched from.  Given that I can't find the empty egg shells, I suspect it eats the egg shell as its first meal.

Second instar larva, shown at the same scale as the one above.
After about 2 days the larva moults, shedding its first instar skin and head capsule.  The larva digs the hard parts of its legs and jaws into the leaf, and then moults by lifting its body away from the old parts.  These can usually be found later at the site of moulting.  Moulting seems to take at least a day, and maybe up to 2 days to complete.  The new hard parts are about 1.5 or 1.6 times as large (in linear dimension) as the shed parts.  The flea beetle moulting process takes substantially longer than moulting in Heliconius butterflies, which complete their moult in about 12 hours. 

Second instar larva actively walking across Passiflora vitifolia petiole.
The unusual shape of the flea beetle larva has probably evolved to protect the larva from predators such as ants.  The larva has large, strong legs and an enlarged anal clasper, both of which can grab the plant strongly.  The body is protected by heavy, thickened ridges on the back, and is flattened, covering the head and jaws from view.  The body shape is similar to larvae of lycaenid butterflies, many of which are adapted to live in and among ant colonies.  Ant protection is a very useful trait for a small, slow-growing insect feeding on Passiflora, plants well-protected by ants.

Third instar larva, same scale as the above two photos.
After a day or two in the second instar, the larva repeats the moulting process to the third instar.  This larva has even larger ridges and hard parts that are, again, about 1.6 times as large as the second instar parts.  After 1-2 days of feeding and growth, the larva reaches its full size of about 12-15mg.  Once the larva grows this much it begins crawling around the plant and neighboring plants.  Usually it disappears after a few days of moving and feeding, but what they are looking for and what cues are used to find a pupation site are still a mystery.  Larvae penned in containers usually die and are consumed by fungus.

Red-white Ptocadica, likely the same species as the larvae above.
The larvae shown in these pictures belong to either the Blue flea beetle Monomacra violacea, or, more likely, the Red-white species of Ptocadica.  Two weeks before the larvae appeared, I had placed adults of these two species on the leaves of Passiflora lobata., plant #1.  Most of the adults flew away and disappeared, but one of each beetle species remained on plant for several days.  They sat under leaves and moved around very little.  The Red-white beetle mainly stayed on the lower leaves, while the Blue beetle was more often on the upper leaves.  Two weeks later, when I found larvae on plant #1, they were mainly on the lower leaves, with a few up higher.  Also, after a few days the Red-white beetle moved across the shade house to the lower leaves of a second P. lobata, #2.  A similar number of larvae appeared 2 weeks later on this plant.  Based on the timing and location of the larvae I am fairly sure that most, if not all, of the larvae I found belong to the Red-white species.

The life-cycle drawing summarizes the findings to date.  Obviously there is a long way to go before being able to complete the drawing!

Comparing these larvae to the larvae of Heliconius, I find that the beetle larvae are slower-growing, with a relative growth rate of about 0.35 instead of 0.6.  The egg stage is longer, 14 days instead of 10 days.  The female beetle sits on the host plant for days in order to lay eggs, rather than the brief visit by the butterfly, and she eats the plant while waiting.    My previously published work shows that Heliconius larvae are extremely vulnerable to predation by ants whereas my observations here at La Selva suggest that most Passiflora-attending ants pay no attention to flea beetle larvae.  They seem effectively ant-proof, although experimentation is needed to determine this for sure.

I also began to test the shade house P. lobata larvae on a range of Passiflora species.   If Red-white Ptocadica is the correct species for these larvae, I predict best performance (survival and growth) on P lobata, since this species seems to specialize on P. lobata as an adult food plant.  More about his in the next blog posting!