The second success: Why flea beetles and Heliconius have the same number of species.

Root feeding larva of Monomacra violacea

After discovering the larval stages of all four genera of flea beetle, I realized that the beetles could be divided into two groups of five based on their larval feeding behavior: root/stem feeders in the genera Monomacra and Parchicola, and leaf feeders in Ptocadica, Disonycha and Pedilia (Pedilia larvae also eat stem tissue but not roots).  Then, remembering which beetles shared which plants, I realized that most plants hosted one species from each group, but not more than one.  This suggested that perhaps there were five distinct sets of Passiflora host plant, each supporting a different pair of flea beetles.  This in turn raised the question: do these same sets of plants support unique sets of Heliconius butterflies?  other insects?  The answer turned out to be yes!

Leaf feeding larva of Red Ptocadica

Solitary feeding generalist larva of Heliconius cydno

To answer this I turned to my research done in the 1970's and 80's, in which I measured and analyzed the host plant relationships of Heliconius.  There, I treated the butterflies as belonging to two species groups (group I and group II), each of which laid eggs on two main groups of Passiflora vines.  The vines themselves fell into three subgenera now called Astrophaea, Decaloba and Passiflora (why did they have to name one subgenus Passiflora?  It makes it wordy to always have to distinguish the genus from the subgenus).  Group I uses Decaloba , with one species specializing solely on Astrophaea.  Group II uses mainly subgenus Passiflora, but some species occasionally lay eggs on Decaloba and Astrophaea.  A complicated picture, but if you step back there is a simple pattern: Astrophaea with 1 species of Heliconius, Decaloba with 4 species and subgenus Passiflora with 5 species.  Within Decaloba and subgenus Passiflora there were some "generalist" species that would lay eggs on more than one Passiflora species, and some monophagous species specialized on one species only.

Group feeding H. doris larvae only eat Passiflora ambigua

After remembering this relationship, I looked up references on the genus Josia, an orange and black moth that feeds on Passiflora costaricensis at La Selva.  I got lucky and found an incredible  opus by James. S. Miller, a complete modern revision of the tribe of moths including Josia, called the Dioptinae.   According to his work, the 150+ species of Josia relatives included several genera specializing on the same three subgroups on Passiflora.  Getta feeds on subgenus Astrophaea, Josia feeds on Decaloba, and Lyces feeds on subgenus Passiflora.  Miller suggests that these genera may have co-evolved with Passiflora as it diversified over the past 40 million years.

Josia frigida feeds on Passiflora costaricensis at La Selva

Like Heliconius and Josiini, the flea beetles also specialize on the three subgenera.  Only one species (the Blue Monomacra violacea) feeds on more than one subgenus, and that only in the adult stage.  The others are all specialized to feed on one subgenus or another.  Thus, although we don't yet understand the precise reasons, it is clear that most Passiflora-feeding herbivores diversify and specialize on the three principal Passiflora subgenera.  A second finding from my original research on Passiflora and Heliconius is that different species, plant and butterfly, are found in different habitats within and adjacent to the forest.  For example, P. pittieri, the only member of subgenus Astrophaea, is only found within the forest, but the other two subgenera have members adapted to either forest or second growth environments.  As a result there are five sets of Passiflora species, as can be seen in the chart below.  Just what the flea beetle larval feeding data suggested!

One more observation completes the picture.  The Heliconius species may be divided into two types that probably don't compete with each other, generalists that fed on more than one Passiflora species, and monophagous species that normally only use one host plant. They don't compete strongly because the generalist species usually lay eggs singly on small isolated plants of more than one species.  The monophages on the other hand lay clusters of eggs on larger plants, and only on one species.   With these groupings in the chart above it becomes obvious why there are the same number of flea beetles as Heliconius: there are two sets of five host plant groupings for each type of herbivore!  The number does not add up to 20, because two Heliconius and 2 flea beetles are very rare and I don't have enough information to fit them in the chart.  Perhaps they are stragglers from other habitats in which they are more common.  Even with this complication, the chart shows clearly why the number of species in each community is about 8-10 species.

Interestingly, the community structure is not a function of chemical diversification in cyanogenic glycosides, which was one of my first hypotheses when I began the study.  Chemistry is obviously important in defining the three subgenera, although we don't understand its role, and it is also almost certainly important in the life histories of the monophagous species, but the overall role suggests that herbivore communities "deal" effectively with whatever cyanogens the plants throw at them.  Of course you may see in the chart three Passiflora species not widely used by either flea beetles or Heliconius at La Selva - they may have some effective chemical defense.  But the take home message is that the communities are determined by a combination of host plant taxonomy, habitat specialization, and larval feeding syndromes.  The second message is that the two communities are basically "full" or "saturated" with species.  This may be seen by the fact that each "box" in the chart has only one species, meaning that there is no "room" for two species to share the same "box."  This may be the reason that the same species are found now as were found in 1975 when I began the study.  Apparently these are relatively stable communities!

Success! Twice Over!

Blue Flea Beetle on leaf.  Hard to photograph - too reflective!

This shorter stay at La Selva is coming to an end, but it has been a good four-month stay. The effort to propagate cuttings of Passiflora lobata resulted two months later in 6 small but healthy potted plants with about 10 leaves each.   I was able to put these in cages with several Blue flea beetles, and, after a few weeks, got some eggs and larvae.  This was crucial, since the Blue Flea Beetle, with scientific name Monomacra violacea, is one of the most common flea beetles at La Selva.  It is also the most tolerant and can be found feeding on nearly every species of Passifora except two.  In past field seasons I found the larvae of the other genera, but Monomacra eluded me.

Monomacra species are obviously related to the three species of yellow Parchicola.  Both genera have a similar trim, elongated shape and both have a flattened rectangular depression on the posterior end of the pronotum, the "shield" immediately behind the head.  In 2013-14 I found eggs and larvae of two of the Parchicola species, and I expected the Blue beetles to be similar.  What I found was indeed some amount of similarity, but also some differences.

Like Parchicola "yellow-legged" (there is no official species name in this case), M. violacea cylindrical eggs are laid near the base of the plant.  Unlike Parchicola, the eggs are attached at the end (see photo).  The eggs hatch after an unknown number of days, and the slender, tiny larva emerges.  The larva is quite mobile, looping tail to head like a tiny inchworm.  The six thoracic legs are sturdy, grabbing the substrate easily.  The anal clasper is also relatively large and is surrounded by a ring of bristles. Each segment of the thorax and abdomen has two rows of pigmented flaps, like deflated balloons.  This pattern also contrasts with Parchicola, which have only one row of flaps.  Also, the larva is white rather than yellowish.  Otherwise, the young larvae of the two genera are similar.

Newly hatched M. violacea larva.

Newly hatched larvae have a transparent, unpigmented head capsule, bearing two large bulbous antennae capable of retracting into tubes on the head.  The legs and anal ring are also initially transparent, but after one day they melanize and darken.  The head capsule in particular become shiny black. The larvae loop along the rootlets of the host plant, feeding occasionally by chewing on root hairs and root tissue.  Typical feeding damage includes small pits eaten out of the pithy part of the root.  The central fibrous core of the rootlet is not eaten.

Third instar larva, almost fully grown and ready to pupate.

Above I was describing the first instar M. violacea larvae.  I have not yet seen the second instars, but the third instars are, of course, much bigger and fatter in relation to their body length.  The biggest change is that the pigmented flaps are replaced by tiny transparent spheres in the larger larvae.  They look like droplets of liquid but they are in fact cuticular, dry not wet.  The root environment where the larvae live has many predators such as centipedes, roundworms and isopods, and I can easily imagine the spheres are filled with some chemical deterrent.  But this just a guess at this time.

I forgot to explain the title of the blog: Success Twice Over! "  The eggs and larvae described here are one success.  The other has been my effort to make sense of the parallel species diversity of the flea beetles and the Heliconius butterflies.  More on that in the next blog.

Larval feeding damage to rootlet of P. lobata

Back to La Selva - in the rainy season!

Cement path at La Selva

Finally, after a full year, I am back at La Selva, working on the Flea Beetle Project!  During the past year Kim and I fulfilled a promise we made to ourselves to live for a full year on our Palomar property.  This turned out to be very successful and comfortable for us, to the point where it was hard to leave!  In fact Kim stayed an extra month to complete her job at Mother's Kitchen restaurant; she will join me in early September.

During the year we installed a broadband internet uplink, enabling me to update the flea beetle web site  (as well as allowing us to shop  and watch utube and netflix).   Also during the year I created a draft project summary, which may be found on the web page:

Red Ptocadica flea beetle on Passiflora lobata leaf.

http://johnterahsmiley.com/heliconius-passiflora-flea%20beetle/FB%20summary%202014/summary%202014.html .  To me this summary is very exciting, portraying a wide-angle picture of this tiny, complex, colorful world.  Writing the summary has also prompted me to focus on some of project's shortcomings, two of which I hope to correct on the present trip.  One is my lack of natural history observations during the June through August rainy season.  Another is that I still have not found the juvenile stages of one of the most common flea beetles, in genus Monomacra.

The present trip has begun ideally for correcting the first   problem - I arrived to La Selva August 5 after an unusually intense rainy season.  Any observations I can make over the next few weeks will tell a great deal about rainy season effects on the beetles.  Thus far, after a few days of observing,  I can say that many of the flea beetles are actively reproducing, as indicated by aggregations of 5-15 beetles of the same species on a plant, including mating pairs.  These include two species of Parchicola, Monomacra violacea, red Pedilia, and red Ptocadica.  The only common species I haven't yet found is yellow-tibia Parchicola.  This suggests (with the possible exception of yellow-tibia Parchicola) that life goes on as usual for the beetles during an intense rainy season.  This is in contrast to the Heliconius butterflies, whose numbers and activity seem greatly suppressed.  Perhaps another important difference between the flea beetles and the butterflies?

Red Pedilia larva after moulting to 2nd instar

As for the second problem, I need to grow a set of potted Passiflora vines for caging with Monomacra violacea flea beetles.  To do this I need to make Passiflora cuttings and root them, which takes a few weeks.   Once ready, I will catch the flea beetles and put them in the cage.  Then, over time, I will check the cages for eggs and larvae.  I predict great similarity to Parchicola, which is thought to be closely related to Monomacra, but we will find out.  In any case, it's great to be back!

Eggs of Red Pedilia on Passiflora pittieri

John's web site has moved!

All, FYI my web site, including links to my projects such as the La Selva Flea Beetle Project, has moved to http://johnterahsmiley.com .

I had a productive visit with David Furth, flea beetle expert extraordinaire at the Smithsonian.  He encouraged me to put together my natural history and taxonomic observations for publication, and promised to help with the taxonomic aspects.

I made a gallery of graphs based on my HCN measurements (how exciting is that?).  It's on the new web site at http://johnterahsmiley.com/heliconius-passiflora-flea%20beetle/FB%20summary%202014/Appendix%202.%20%20HCN%20graphs/Appendix_2.html .

I am working on a project summary that brings it all up to date.  I will post a link as soon as it is ready.

Genetic Barcoding Reveals Another Species!

Parchicola "black tibia", feeding on Passiflora vitifolia

It's been a long time since my last posting - partly because I've been too busy!  Now I am home on Palomar Mountain and have time to reflect.  This posting will be wordy and not so photogenic (sorry!) but it's what I want to report.

The two groups shown here are "cryptic species"

I recently received printouts from "genetic barcoding" over 100 specimens of La Selva flea beetles.  Genetic barcoding is an analytical technique that compares a specific genetic region (in this case the Cytochrome Oxidase 1 gene) in different animals.  Members of the same species have a nearly identical sequence of base pairs (the "letters" of the genetic code), while members of different species differ substantially.  The analysis can be automated and streamlined to the point where large numbers of samples can be processed at low cost, and the technique is becoming widespread among field biologists interested in biological diversity. It is also used to identify insect larvae, which otherwise must be reared to the adult stage to determine their identity.

Carlos Garcia-Robledo of the Smithsonian Institution in Washington D.C. is carrying out a project at La Selva looking at beetle diversity along elevation gradients.  Genetic barcoding is an essential component of his study.  In his project and others, there are many instances of "cryptic species", where barcoding has revealed hidden species differences even though the animals in question are superficially the same.  Carlos kindly invited me to submit my Passiflora-feeding flea beetles for analysis using his high throughput system.  All I would have to do is to prepare the sample plates and create a spreadsheet of attribute data for the samples.  Barcoding would be useful for my project, since my species have not been formally described and there is much room for error in my assignment of temporary (morphospecies) names.  Also, I have not been successful in rearing flea beetle larvae to adulthood for many species, leaving larval identity in doubt. 

Black-legged (DF1) and yellow-legged (DF2) Parchicola

To carry out this analysis, I preserved 136 specimens in alcohol to represent the range of Passiflora feeding flea beetles at La Selva, including 10-20 individuals of most of the species.  Only the Red-black Monomacra chontalensis (0 specimens), the Ptocadica "yellow" (2 specimens) and Disonycha quinquelineata (0 specimens) were severely under-represented.  Adult beetles and larvae were collected in the field and recorded in my field notes.  Then back in the lab I froze the beetles to humanely kill them, and put them in vials of 95% ethanol.  The vials were carefully labelled, including a tiny label written in pencil placed in the vial with the specimens.  I kept these samples in the freezer.  Before leaving Costa Rica I obtained a legally required export permit to take the samples out of the country.  Then, back in California, I carefully removed a leg (using sharp forceps and tiny scissors) and placed it in a numbered sample well on a 96-well plate, keeping track of each well and the field origin of each leg.  I also kept the rest of each specimen in an individually labeled sample vial for later reevaluation or examination.  I sent the completed 96-well plates to Carlos and he ran them through his barcoding protocol.  He sent me the results a few days later.

Of the 136 samples representing 7 species, 109 gave successful, uncontaminated sequences (80%).  Six of the seven species fell into 6 clearly defined groups, indicating that they are unique, distinguishable species (as suggested by their morphology and behavior).  The seventh species, Parchicola DF1 ("black-legged yellow") included two totally distinct groups that differ at the same level as the other 6 species.  We had found a cryptic species! We also found that the 3 flea beetle larvae found eating Passiflora lobata belonged to Ptocadica "red", and the 4 similar-looking larvae found on P. auriculata were Ptocadica bifasciata.  This verified the identity of the larvae used in feeding trials in 2013, and leads us to believe that Ptocadica bifasciata is somewhat specialized to feed on P. auriculata as opposed to P. biflora.  The barcoding results also verified that Ptocadica "yellow" is a distinct species from Ptocadica "red", an important distinction since the two species appear identical in dried museum specimens.

Splitting Parchicola DF1, as required by the barcode finding, potentially creates many problems for my study.  If the difference is truly cryptic, such that the species can't be separated without genetic analysis, then it becomes necessary to collect every specimen and subject it to the barcoding process.  And what about all the previous data based on field observations and/or dried specimens?  How do you present and analyze the results?  I have faced this problem twice before, always with the yellow flea beetles.  In my original survey in 1975 I collected “yellow flea beetles” and pinned them in a collection.  When I showed my collection to taxonomists at the US National Museum they showed me that I had collected two species of yellow flea beetle that they called Strabala sp. and Monomacra sp.  Because I kept specimens I was then able to work backwards and rework my data with the new information.  Years later, taking up the flea beetle project again, I took my collection to David Furth, also of the US National Museum.  He showed me that the beetles I was calling yellow Monomacra actually belonged to two species which he had given provisional names of Parchicola  DF1 and DF2, one with yellow legs and other with black legs.  I again reworked my data based on the pinned specimens’ leg color.  However, some of the data did not have associated pinned specimens, and could not be reworked.  I handled this in my data set by setting those observations aside.  Now, with barcoding revealing yet another hidden species, I had the same problem again.

I decided to try to discover morphological or behavioral characters that could distinguish the two new species of black-legged yellow flea beetles in the field.  The barcode results divided Parchicola DF1 into two groups, a larger group of 14 specimens and a smaller group of 7.  Looking at the field notes I immediately noticed that 4 of the group of 7 were from P. vitifolia, a plant not commonly collected.  The rest were from either P. quadrangularis or P. oerstedii.  I wasn't sure which because my field notes failed to specify; I had collected several and had put them in the same container, thus mixing them up.  I then remembered from a previous field collection from P. vitifolia that  Parchicola DF1 seemed unusually large and robust, to the degree that, at first, I thought they might be Ptocadica "yellow".  That recalled another similar experience with beetles that Ron Vargas brought me from his garden P. quadrangularis.  They seemed unusually large. These field notes and recollections of mistakes suggested the possibility that the barcode "group of 7" species was larger, and fed upon P. vitifolia and P. quadrangularis.  It was clear from looking at the group of 14, that they were primarily found on P. oerstedii and P. ambigua.  This led me to go out and collect 5 Parchicola DF1 from P. vitifolia and 5 from P. oerstedii, and  measure them.  The P. vitifolia beetles averaged about 10% larger than the P. oerstedii beetles!  I felt I was on to a real difference, in behavior (host plant choice) and morphology (body length).

Note the yellow tibia (middle leg segment above)

I then examined the two groups of five newly collected beetles, looking for some other character that would separate them.  It took only a few minutes to discover that the hind tibia (the third leg segment of the hind leg) was black or dark brown ("melanized") in the larger "vitifolia" beetles, and was clear to light yellow in the smaller "oerstedii" beetles.  All the other tibia and tarsi in both species were melanized.  The hind-tibia color difference suggested new morphospecies names for the two species: Parchicola "black-tibia" and Pa. "yellow-tibia".  I then examined my small collection of dried "black-legged yellow" flea beetles, and was easily able to divide them according to hind tibia color.  Doing this I saw that both tibia colors could be found on P. oerstedii, P. ambigua, P. quadrangularis and P. auriculata.  Unfortunately I had no pinned specimens from P. vitifolia.  Even though I could not check the original barcode specimens because they were in storage in California, I felt the relationship was strong enough to begin separating Parchicola DF1 into the two species.  I also found I could see the hind tibia color if I could see the correct angle with my binoculars, and I began recording the new species in my field notes.  This far the evidence is that "black-tibia" is usually found on P. vitifolia at La Selva, and "yellow-tibia" is usually on P. ambigua and P. oerstedii.  After returning to my home in California I opened the bag of vials containing specimens used in the barcoding, and examined the color of the remaining hind tibia (usually the first one had been removed for the barcode analysis).  In 15/15 cases where the hind tibia color could be determined, the "group of 7" beetles had black hind tibia and the "group of 14" beetles had yellow hind tibia, confirming the validity of the hind tibia character as a way to distinguish the two species.

Note the black hind tibia for this flea beetle

The genetic barcoding analysis enabled me to characterize the Passiflora-feeding flea beetle community with greater precision and confidence than would otherwise have been possible.  It is doubtful anyone would have discovered the two hind-tibia Parchicola species until much later, perhaps during dissection of genitalia as part of the formal species description process.  The confirmation of species identity for the six remaining species is also a valuable result.  I had suspected that Ptocadica bifasciata might include two species groups, because the intensity of the brown color varies between individuals and possibly host plants.  However, the barcoding result for the specimens I analyzed clearly indicate only one species.  The analysis also enabled identification of flea beetle larvae, a very important tool for beetles that are difficult to rear from larvae to adult.  Interestingly, the "final" total species list for Passiflora feeding flea beetles at La Selva includes 10 species,  exactly the same as the total for Heliconius!

Does genetic barcoding replace classical taxonomy in studies of species diversity?  Not in most cases.  Perhaps it could replace the latin binomial system for labelling species, except that using it means reading and writing 300 letter words!  Unlike some other genetic techniques, barcoding is not very useful for looking at the classification and relationships between species, genera and higher groups.  Nor does it tell you anything about the natural history, ecology and biology of the living organisms.  Put simply, genetic barcoding is a tool for identifying and labeling species, without telling you anything else about them.

Missing Larvae Found!

Isolation cage in shade house

In the last few days I have begun to fill a major gap in our knowledge of the Passiflora-feeding flea beetles.  I found the missing larvae!  Or at least some of them.  And they are different!

After returning from our holiday trip to the USA, I saw that one of the isolation cages (cage C) had more flea beetle adults than expected:  I had put 9 Parchicola DF1 adults (Black-legged Yellow or YBL for short) in the cage but now had 10.  Other cages had good survival over the 6-8 week interval, but even 100% survival could not account for this result.  I reasoned that the beetles had successfully reproduced in that cage.  The cages are kept in a "shadehouse," the equivalent of a greenhouse here at La Selva.   Here, the only danger to potted plants outdoors is too much heat from the sun and herbivores such as leafcutter ants.  In a shade house, porous cloth is used to exclude herbivores and about 70% of sunlight.  This creates a good balance between sunlight for growth and reducing heat build up.  Rain goes right through the cloth so watering is usually not an issue unless there is a dry period.

Black-legged Yellow flea beetle egg, <1mm long

To see what might have happened I carefully opened a slit in the cage C door and shook the foliage, driving the 10 beetles to sit on the cage wall.  I then removed the entire potted plant (actually there were two potted plants in this cage, both Passiflora oerstedii "OER"), and started a close examination using magnifying glasses and a bright headlamp. The first thing I found were five loose clusters of 6-12 tiny spine-like eggs, on the underside of the oldest leaves of the plant.  Newer leaves had no eggs, and I could not find any larvae.  I took the leaves into the lab and photographed them using the stereo microscope.

Black-legged Yellow flea beetle larva. 

Next I looked at the soil near the base of the plant where the stem enters the ground.  There, near the surface, was a dead flea beetle larva, but one I had never seen before.  It looked like a smaller version of the Ptocadica larvae that I often find on the Passiflora (see previous blogs for photos) except that the dorsal and lateral protuberances (bumps on the top and sides of the body) had short spines tipped with tiny spheres!  I have not seen this form of larva before, and it is safe to say that the YBL larva does not feed upon Passiflora foliage!  I would have seen them in my thousands of hours of looking at plants in the field if they were there.  What is the function of the spheres?  They are composed of cuticle and are patterned.  Perhaps they have a defensive function, dispensing chemicals to potential predators such as ants and spiders that might be found at the base of the plants.  The tiny ants in the isolation cages I have not identified, but they seem to completely ignore the larvae, walking over and around them as if they were not there.

Larvae feeding on Passiflora stem tissue

I then looked closer at the base of the plant, and saw where the larvae were chewing on the stem epidermis.  This behavior is very much like that of Pedilia larvae, which also eat the stem-skin of the their host Passiflora pittieri.  However, looking lower down, about 10 cm into the soil, I saw where the larvae were chewing well into the woody part of the stem, perhaps 30% of the way through.  These stems are tough and woody, but also flexible, and it is not clear that there is a distinct layer of live "sapwood" as opposed to dead "heartwood."  Judging from the recruitment of new adults, and the amount of feeding required, it seems clear that the bulk of nutrition for these larvae comes from this woody tissue.  I also found a pupa in the loose soil near the base o the plant.  I suspect it pupated in an earthen cell, which I unknowingly cracked open in examining the plant.

  

Black-legged Yellow flea beetle pupa

These new findings are from only one species, but three other Passiflora-feeding flea beetles are closely related, Parchicola DF2, Monomacra violacea, and M. chontalensis.  I have not yet seen larvae of these species.  My guess is that they also have basal stem (or root) feeding larvae, and that with luck my other cages will revel their presence.  I set up cage C with a different Passiflora species (P. auriculata; AUR) to see if they will reproduce on that plant.  In nature, I find 5-10% of adult YBL on AUR, but who knows about the larvae?   I will try to find out.  Back to work!

Black-legged Yellow flea beetle adult

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.

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!

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...

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!

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!