Gettysburg Meets Gamboa: How Tortoise Beetles Are Excelling in the Chemical Arms Race

In the mystical town of Gamboa, Panama, through the forest and over a rickety wooden bridge, lies a community full of bat people, frog people, bug people, and so much more. It is home to one of the globally leading research institutes, the Smithsonian Tropical Research Institute (STRI), which hosts numerous scientists and offers major contributions to the field of tropical biology.  Surrounded by beautiful creatures of the rainforest and the incredible Panama Canal, the research opportunities here are truly remarkable. Alongside Dr. Trillo and her collaborators, we are studying the effects of endophytic fungus on the chemical ecology and anti-predator strategies of the tortoise beetle, Acromis sparsa.

P8020758.JPGTiffany Lam ’19 standing in the lobby of the Smithsonian Tropical Research Institute’s Gamboa Lab

The Study System

Acromis sparsa (A. sparsa), a species of tortoise beetles, are dietary specialists. This means that, unlike some other species of beetles, the larvae feed only on one plant, Merremia umbellate (M. umbellate), a morning glory vine. A. sparsa have many anti-predator defense mechanisms: First, moms guard their progeny from eggs to adulthood. The mother even stays with her brood(s) until they reach adulthood – talk about an amazing mom!  Once hatched, the larvae ravenously consume the leaves on their vine. It is in this larval stage that the second type of defense, chemical compounds, kicks in. From a telescoping anus, these larvae create a fecal shield to protect themselves from predators. That’s right, a movable shield of poop! This shield contains nasty chemicals that the larvae acquire from consuming their host plant.  Finally, larvae form tight clusters with their fecal shield pointed outward and acquire increased defense from living in groups (Vencl, Trillo and Geeta, 2011, Vencl and Srygley 2013). Some of the known predators of A. sparsa are Azteca ants, wasps, and true bugs.

445A3138.jpgMother A. sparsa guarding her egg clutch.  Photo credits to Christian Ziegler.

IMG-20170630-WA0001.jpgTwo larvae with prominent fecal shields.

Endophytes are microfungi that live within the tissues of, and form a symbiotic relationship with, plants. They have been found in all species of plants, infecting above ground tissue. Endophytes use their hostplant as a source of carbon in return for plant protection, enhanced growth, and nutrient acquisition. Moreover, research conducted by Hammer and Van Bael (2015) showed that the A. sparsa larvae that fed on endophyte rich M. umbellate leaves had a greater risk of predation by Azteca ants than the larvae that fed on leaves low in endophytes. But what is the mechanism for this effect? Are endophytes modifying leaf chemicals and, in turn, affecting the chemical defenses of their specialist herbivores? Is this increase in larval predation only relevant to one predator or is this effect widespread? Why is this important, you ask? Because understanding how herbivores are affected by the fungal communities of their hostplants can give us important insights that can then be applicable to agriculture, biocontrol, and the management of ecological trophic interactions! So, our hope is to answer some of these exciting questions this summer.

 

Our Project

The research we are conducting is two-fold: First, we are testing the effects of an endophyte low versus and endophyte high plant diet on the chemical defense compounds of Acromis sparsa’s larvae. Then, assuming there is a difference in larval chemical defense compounds, we will conduct predator bioassays to test whether if these differences affect survival of larvae against a spectrum of predators.

How do we do all this?

Collecting and potting M. umbellate: We pulled local wild vines of M. umbellate out of the ground, transferred them into individual pots and assigned them to treatment groups of endophyte high (E+) or endophyte low (E-).  To keep our plants safely endophyte free during the day, we constructed a large plastic bubble within a greenhouse. This plastic keeps herbivory to a minimum and stops endophytic spores from flowing through the air onto our plants. Our E- plants are kept in this bubble for the entire experiment, but the E+ plants are removed every night and placed into the forest, where endophytic spores are abundant. For the E- plants, we constructed a field cage out of cut-open laundry bags, hot glue, and safety pins, all draped over a PVC frame. This cage’s holes are large enough to let endophytes through, but not big enough that large herbivores can pass through.  Every morning when these plants are returned to their bubble, they undergo an herbivore check to keep pesky insects like aphids and caterpillars off our plants. We verified our endophyte treatment by plating leaf fragments on agar and allowing the fungus within these fragments to grow over a period of four days and we just got back some relieving results that our E+ and E- treatments are significantly different! Our treatments are working!! Hooray!!!

IMG_0561.JPGIndoor Bubble

P8020760.JPGOutdoor Cage

Brian and Tiffany with plates.jpgBrian Ruether ’19 and Tiffany showing off newly assayed endophytic growth

Collecting and raising A. sparsa larvae: We walked and drove all over Soberanía National Park and Gamboa, looking for clutches of A. sparsa eggs. Once collected, we waited until they hatched and then divided each brood such that one half fed on E+ leaves and the other on E- leaves. We’re currently monitoring the larvae in each treatment/family each day and feeding them new leaves from their respective treatments as needed. Around day five after hatching (when the larvae reach 3rd instar), the larvae are frozen. Samples of the larvae themselves, as well as fecal shields, will be sent to a collaborator to assess the chemistry of the larvae.

download.jpg                                                     Surrounded by predators and larvae

Predator Bioassays: Within the next two weeks, we will conduct predator bioassays by using frozen larvae reared to day five on four different predators. We will use frozen larvae because we want to separate the chemical defenses from the behavioral defenses (larvae run away from their predators, of course!). We will present thawed larvae from E+ and E- treatments to each predator and we will measure the predator’s latency to attack, handling time, and rejection frequency of the prey. Collecting and utilizing these predators is Brian’s favorite part of the project because he gets to explore the wide array of different insects and arachnids found in Panama!  We will use Azteca ants and wasps, and we are doing trials for two more predators to use: right now our bets are on wolf spiders , reduiviids (assassin bugs), and mantids, like this dead leaf mantid.

20624627_1840904726223064_1600894663_n.jpg              Wandering spider with meal worm in mandibles

20614553_1840904729556397_695070123_n.jpg                      Wheel bug, a type of assassin bug trying to give us her version of a high 5

P8020750.JPGFemale Dead-Leaf Mantid

The Full Experience: Panama is a hot, humid country, forming the bridge between South and Central America.  With the humidity at or near 100% everyday, torrential downpours are frequent.  Panama City, a huge beachside metropolis, is the first jungle you lay eyes on.  However, after careening through unpredictable traffic for hours on end, you can see a different kind of jungle: the Panamanian rainforest.  It’s a beautiful place, housing astounding biodiversity. Parrots and parakeets chirp away every morning, with the buzz of cicadas filling the air as the sun heats up the day. In the afternoon, agoutis (large squirrel-like rodents) sprint through the grasses with mango pits in their mouths, and insectivorous bats swoop through the night air, catching their katydid dinners. Insects are everywhere it seems: in the trees, in the air, and unfortunately between our floorboards. Plus, we have frequent (and awesome!) sightings of sloths and monkeys.

IMG-20170701-WA0009.jpg2017 Panamanian Reboot of The Creation of Adam

In our downtime we are able to take trips to Taboga Island, a small Pacific island a little ways off the coast, and the Río Mendoza, a long hike up a river with the promising reward of a waterfall and swimming hole. Dr. Trillo and fellow Gettysburg professor/husband Dr. Caldwell truly incorporated us into their family this summer, and we both feel as if we’ve gained a tropical mom and dad.

IMG-20170707-WA0016.jpgTaboga Island beach and dock

GPTempDownload (1).jpgEl Río Mendoza

Aside from everything we have learned from researching our project itself, we have the unique experience of immersing ourselves in a culture of true field biology. We have the opportunity to converse with fellow undergraduate students, graduate students, postdoctoral researchers, and well-established scientists every day. Not only are we learning about the beetles we work with but we are exposed to the endless research by scientists here including studies on frogs, bats, birds, and plants. Especially in times where the importance of science has been continuously doubted, it is amazing to be surrounded by passionate scientists who have an undeniable respect for earth and all its stunning creatures.

 

Sources Used:

 

Hammer, T. J., & Van Bael, S. A. (2015). An endophyte‐rich diet increases ant predation on a specialist herbivorous insect. Ecological Entomology, 40(3), 316-321.

 

Vencl, F. V., Trillo, P. A., & Geeta, R. (2011). Functional interactions among tortoise beetle larval defenses reveal trait suites and escalation. Behavioral ecology and sociobiology, 65(2), 227-239.

 

Vencl, F. V., & Srygley, R. B. (2013). Enemy targeting, trade-offs, and the evolutionary assembly of a tortoise beetle defense arsenal. Evolutionary ecology, 27(2), 237-252.

 

 

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