The Lizard Log

The Langkilde Lab in Action

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Washing Leaves and Spiking Feces: How to Trace Nutrients Through an Ecosystem

Continuing in our series of posts by Langkilde Lab undergraduates, we have a dispatch from Tyler Jacobs, a senior majoring in Immunology & Infectious Disease. Tyler has played the mellophone in the Blue Band as well as being in the Phi Mu Alpha Sinfonia Music Fraternity for all of his 4 years at PSU. After graduation he’ll be entering the Navy, getting a bachelors degree in nursing, and aiming to become a nurse anesthetist. Tyler’s post is below:

For the past year, my research has been focused on testing the accuracy and reliability of a new technique for field ecologists. The goal of my project is to determine whether nonradioactive (stable) isotopes can be used to trace the flow of nutrients through an ecosystem (specifically through the mangrove ecosystem).  Both macro- and micronutrients are important to the growth and development of plant tissues. This method can prove to be a useful new technique in future studies of nutrient cycles, such as the Nitrogen cycle, or, as in this case, and attempt to examine how an invasive species can affect nutrient flows within a specific ecosystem. In some mangrove ecosystems in Puerto Rico, invasive iguanas can cause severe damage by literally defoliating entire mangrove trees. These iguanas then do what all animals do after eating: they poop. This iguana gluttony on mangrove leaves can result in an accelerated delivery of nutrients, such as nitrogen, in the form of poop to other parts of the ecosystem. I attempted to describe this flow by using stable isotopes.

There were two components of this project: leaf assimilation of isotope and isotope uptake by animal tissues. The leaf assimilation involved the spraying of 15N isotope on three types of mangrove leaves in Jobos Bay, Puerto Rico. 15N, or “Nitrogen-15”, is an isotope of the naturally occurring element Nitrogen. However, while 15N is found naturally, it only makes up ~1% of the total nitrogen available to all living systems; most Nitrogen is 14N. The presence of an additional neutron (hence the number 15), can serve as a tag in various tests, allowing the nutrient to potentially be traced as it moves within an ecosystem. This type of research is often referred to as stable isotope research.

Red Mangrove Tree (Rhizophora mangle)

Red Mangrove Tree (Rhizophora mangle)

In our case, the sprayed leaves underwent a series of tests designed to strip away the surface of the leaf (via simulated rain, or by rubbing soap on the leaf’s surface). The theory behind this madness was simple: if the leaf did not absorb the sprayed-on isotope, it was just sitting on the surface of the leaf within all of the oils that make up the “waxy cuticle” outer layer (which is also responsible for the shiny appearance of certain leaves). To prove that this isotope wasn’t just sitting in this oil layer, the soap was used to completely remove it, leaving behind nothing but the organic leaf tissue. Therefore, if we did find any isotope, we could be sure that it had indeed been absorbed into the tissue of the leaf. All of the samples were processed in the lab and analyzed by GCMS for their isotope composition. GCMS stands for gas chromatography-mass spectrometry. It is a commonly used method for accurately determining the nutrient concentrations of small organic samples.

The uptake of isotope by animal tissues was the second portion of the project. This experiment allowed us to discover whether animals can absorb isotopes that have already been processed and used by another organism. To put it simply, the main question we are asking is: if tree leaves are enriched with isotope (15N) and eaten by animals, will the animals process and retain the original isotope in their digestive and muscular tissue? We tested this theory by setting up 5 plots, each containing feces of I. iguana. These invasive iguanas had been fed with the 15N enriched leaves, producing poop spiked with 15N. We allowed the local bottom-feeders, or detritivores (coffee been snails and fiddler crabs) to roam freely among the plots over a weeklong time period, during which they ate the iguana feces (delicious!) along with other decaying matter. To determine whether these detritivores were taking up the 15N, each day, a single crab and snail were collected from each plot. In the laboratory, I removed the digestive organ of the crabs, known as the hepatopancreas. Both digestive (digestive gland) and muscular tissue (muscular foot) were removed from the snail samples. All of the animal samples were analyzed by GCMS to determine their isotope composition, similar to the leaf samples.

Fiddler crab (Uca rapax) taken in Jobos Bay, Puerto Rico

Fiddler crab (Uca rapax) taken in Jobos Bay, Puerto Rico

The results from the isotope analysis of the leaf assimilation portion of the project showed promising results. All of the mangrove leaf samples retained a large amount of 15N in comparison to the control leaves. This result demonstrates that the method of spraying isotopes on leaves is an effective means of monitoring nutrient flow. The results of the animal tissue portion of the experiment proved generally inconclusive. We saw virtually no differences between the control concentration of 15N and the tissues collected over the weeklong period. This suggests that, while mangrove leaves took up the 15N, the isotope did not flow through the food web all the way to the detritivores (or at least was not at detectable levels).

To sum it all up, our main goal was to prove that this particular method of tracing nutrients through an ecosystem works. We discovered that although the animals did not take up the isotope as anticipated, the leaves were able to absorb 15N into biological tissues quite effectively. Although not as effective of a method as we had initially planned, this use of stable isotopes still proved useful in the tracing of nutrients through mangrove ecosystems.