Palo Verde, pt. 4: Independent projects

At the beginning of FLP week, I still had no solid idea to work with for the upcoming independent project. I knew that I was vaguely curious about trade-offs in anti-herbivore defenses in acacia trees, wondering if species with obligate ant interactions produced lower concentrations of secondary compounds than species with facultative ant interactions. However, I had talked to my professor Erika about this idea and she told me that because there are only about 4 species of acacia at Palo Verde, I wouldn't be able to gather enough data to make generalized conclusions about the potential trade-offs between biotic and chemical defenses in plants. I was also interested in the agroecosystem in and surrounding the rice fields that we had visited earlier for class, but didn't at first see any connection between these two ideas. However, at some point during the week it clicked for me, and I decided that I wanted to study anti-herbivore defenses in non-agricultural plants near the rice fields, predicting that they would have decreased defenses because of agrochemical use lowering the herbivore pressure in the region. Once I had my idea formulated, I talked to my professors about it and they gave it the OK. Rukhshana and Owen also decided to work with me, so by Thursday we had a team, a question, and a plan of attack.

Rice fields near Palo Verde where we did our IP

Wastewater flowing out from the flooded fields

The wastewater, contaminated with pesticides, flows out from the fields in exit canals like this one. The big trees on either side of the canal are guazumas.

 After our FLP presentations on Thursday morning, a bunch of students including me, Rukhshana, and Owen took a ride back out to the rice fields to make preliminary observations for the IPs. Most people were still looking for a question to work with, but we were out scouting for plants with quantifiable anti-herbivore defenses that grow abundently near the rice fields. We took about 20 samples and brought them back to Palo Verde, where the three of us met with our professor Susan. Our original plan was to compare plants near the rice fields to plants at Palo Verde, so Susan helped us sort through and identify our samples, pointing out the plants that plants that we might also find in the park. Unfortunately most of the abundant non-agricultural plants in the rice fields are weeds that don't  grow much in Palo Verde, so we had limited options to work with. However, we did notice some familiar faces growing along the edges of the irrigation canals in the fields: Guazuma ulmifolia, a large tree with trichomes on its leaves, and Vachellia collinsii, an acacia species that depends on agressive ants to protect itself against herbivores. Trichomes are like little tufts of hair on leaves that help prevent dessication and also discourage insect herbivory by making the leaf fuzzy and difficult to bite. Because they are discrete, they can easily be quantified just by counting their density using a dissecting microscope with a grid. The acacia trees use extrafloral nectaries (EFNs) to encourage ant inhabitation, so counting EFNs and measuring their size is a way to quantify the trees' defenses.

Guazuma trichomes, as seen through a dissecting microscope.

Vachellia extrafloral nectaries (EFNs)

To make our experimental design more robust, we wanted to find a third plant species to examine, and do some sort of additional analysis of chemical defenses on all three species. Unfortunately, one thing that OTS sorely lacks (in my opinion) is good lab space and equipment at the field sites, so most of the chemistry protocols we encountered for quantifying anti-herbivory chemicals in leaf tissue were way too advanced for our situation... The most serious equipment we had were mortars and pestles, and the most heavy-duty chemicals we could get our hands on were acetone, ethanol, vinegar, and  water (tap or bottled, but neither deionized or sterile). Needless to say, we were feeling pretty limited. When it came time to present our proposal on Friday morning, we still had not worked out the details of the third plant species or the second test we'd be using.

Regardless, we started to work. Data collection officially started on Saturday, but we spent the afternoon walking around Palo Verde and looking for forest edges facing the same direction as the irrigation canals in the rice fields. Because trichomes on leaves also help prevent water loss, it was important that our control site plants be getting approximately the same exposure as our plants at the fields. While we were flagging usable trees with fluorescent tape, something amazing happened: it rained! In the dry season! And when I say rained, I mean it POURED. After having been at Palo Verde for over a week already and sweating almost constantly, everyone was ecstatic. We all ran back to the building, dropped what we were doing, and sat in the rain, ecstatic. It felt amazing, and was a welcome last bit of freedom before the true work of IPs began the next morning.

Enjoying the rain on a hot and muggy day

Like I said, it was pouring!!! Miguel said that this is what the rainy season is like every day. I can't even imagine!

At dinner, we talked again with Susan, who told us that we should find a new control site closer to the rice fields to use in our experiment. Because Palo Verde is about 30 minutes away by car and is also partially wetlands, the soil types between the rice fields and the park are very different, and our professors thought it would be better to try to eliminate that as a potential confounding factor. So, the next morning when we began collecting samples, we went to a new site northeast (upstream of wastewater exit canals) of the rice fields and got 21 samples of vachellia. We needed 30 samples at each site with three individual leaves per sample, and getting just 21 took all morning because of the ants. As it turns out, vachellia's biotic defenses are pretty effective against herbivory, and all other sorts of threats. As soon as you touch a leaf to snip it off and take a sample, the ants come running down the stem and attack, biting all over your hands (or wherever else they land). We had to be really careful, and even so we still got eaten alive, literally. We came back for lunch and went back into the fields at 1:30, getting our 30 samples from the rice fields and our 9 remaining control samples. After dinner Rukhshana, Owen and I spent lots of hours up in the lab building counting and measuring the extrafloral nectaries on all 180 vachellia leaves, and then enetering our data into a spreadsheet. We also had decided on an herbivore preference test for chemical defenses, which our professors recommended because of the relative ease of the protocol. The general idea is to make a crude leaf extract and present it to ants, and see how much the ants eat compared to a sugar-water control. So, on night 1, we also made an ethanol-based extract from vachellia leaves from both sites. By the end of the first day, we were already exhausted!

The next morning, Owen and I went back out into the field to collect our guazuma samples. Rukhshana stayed behind to do our ant bioassays for the vachellia leaf extracts, which we had to repeat on 20 different ant colonies of the same species. Because another student needed to be driven to a different off-campus site to gather data, the driver couldn't stay with us all morning like he had the day before, and dropped us off at the control site. We collected 30 samples from our control site, and then started to walk to the rice fields since we weren't getting picked up until later that afternoon.

Owen climbing a tree to get a guazuma leaf sample

Unfortunately there are a LOT of rice fields near Palo Verde, and they all look really similar, so we got lost and were wandering around the roads for THREE HOURS, all while carrying around a garbage bag full of leaf samples and an 8-foot long pole pruner for clipping high-up branches. We realized in retrospect that we had walked all the way around the entire region because we missed a turn down the road that took us to the fields we were working at, and by the time we found our way back to the right place by walking back to Palo Verde and going back out from there, the driver was already waiting to bring us back. He doesn't speak any English and we were too hot and exhausted to explain what had happened in Spanish, so since we weren't sure whether or not someone else needed to be driven somewhere in the afternoon, we went back to Palo Verde with zero of the 30 samples we needed from the fields. After a bit of rest and SHADE (which does not exist in rice fields), we went back out for two more hours and collected all 30 of the samples we needed. I took the most glorious shower of my life when we had finished our hot and exhausting field work for the day, and ate a very satisfying dinner. Then I spent several hours counting the trichome density on some of the guazuma leaves, since lack of microscopes and issues of inter-observer reliabilty prevented us from sharing the work among all three of us. While I was counting, Rukhshana and Owen made the leaf extracts for the guazuma samples. Because I was so exhausted from our crazy day, I only got through about 1/4 of the leaves that I needed to look at, but I decided to just go to bed and finish it the next morning.

After getting some rest, I spent the entire next day counting trichomes at the microscope. Rukhshana completed all of the ant bioassays that we needed for the leaf extracts, and Owen went back out into the field with Susan to try to find a third species that we could use. Miraculously they were able to find one that grew at both sites, a shrub with trichomes called Malvaviscus arboreus, and collect the 30 samples from each one all in one day. I finished counting trichomes around 8:30 pm (seriously!), and handed off the trichome-counting responsibility to Owen for our malvaviscus samples. While he was working, Rukhshana made leaf extracts and I assembled a datasheet for our grazuma trichome information. We were all tired again, and Owen and I had gotten sunburn while lost the day before, so we went to bed at 9 pm, which felt wonderful.

Rukhshana observing ant responses to extracts made from leaves near the rice fields, far from the rice fields, and a sugar control.

By counting the number of ants present on each petri dish after a certain amount of time and dividing by the number at the sugar control, we could determine the relative palatability of the leaf extracts. A leaf extract that is less palatable to herbivores presumably contains more chemical defenses against herbivory.

Tuesday was our last day of data collection for the IP! Owen counted trichomes all day, literally from about 8 in the morning until about 1 am the next morning. Rukhshana and I did the remaining ant assays, and I started analyzing whatever datasets were complete. We met with Susan, and realized that we had significant results! She was very excited for us (as were we!), and it was amazing to comprehend that we had actually accomplished something both interesting and kind of important in just four days. The next day was dedicated to finishing our analyses and putting together our results, whichw e had to present on Thursday. We met with Susan several times to create our Powerpoint and interpret our results. The next morning was spent rehearsing our presetation, which we gave that afternoon. As soon as the presentations were done (which were really neat to listen to, since everyone had done completely different projects), we had a little bit of time to break down everything from our experiments, i.e. washing some glassware and un-flagging the 20 ant colonies we had used for bioassays. After dinner, I started writing my lab report rough draft, which was due the next day! I stayed up all night and it took me until 6:15 the next morning, but I finally finished, just in time for the first break we had had in a long time.