Quinn Martin and Dr. Byron Adams, Biology

Abstract

Species-specific food web relationships involving free-living microbivorous nematodes are not well established, yet critical to our understanding of their involvement in nutrient cycling and ecosystem function. Antarctic soil ecosystems are the simplest on earth and serve as a model system for exploring the role of biodiversity in ecosystem function. To establish which bacteria are involved in trophic relationships with nematodes, we surface-sterilized nematodes and extracted their DNA, including bacterial DNA from the gut contents of the nematodes. From this bacterial DNA we PCR amplified the 16S gene and sequenced the product in order to determine the identity of the gut contents of the nematode. We successfully amplified and sequenced the 16s rRNA of Photorhabdus from the gut of the nematode genus Heterorhabditis. We are currently continuing these experiments with genus Scottnema and Plectus nematodes from soil samples collected in Antarctica. Given a broader sampling regimen, this approach can be used to establish food web relationships in more complex ecosystems.

Introduction

The study of food webs reveals how organisms of a certain area interact and can help determine the role or impact an individual species has in the community. For many years scientists have studied the interactions of organisms on the food web level. Two of the most common ways to study what an organism eats are to (1) observe the organism to see what it feeds on and (2) study what is in the gut or excreted from the organism.

Scientists have been able to analyze stomach contents of many animals. It is a process that is difficult because of the degradation of the food as soon as it enters the mouth. Scientists have been able to amplify DNA from the excrement of larger animals to and identified what they were eating. One example of this is a study with bears where they amplified and sequenced specific plant DNA from excrement (Höss et al., 1992).

PCR amplification of gut contents becomes increasingly difficult as the organisms get smaller. This is because there is less material to amplify and the DNA gets digested into even smaller fragments. A group of scientists PCR amplified zooplankton gut contents. This was a difficult process because of the size of the organisms. They amplified the DNA of algae and other prey found in their gut. They also stated that DNA of the prey is quickly degraded by the predator (Troedsson et al., 2008).

Thus far nothing has been published claiming the ability to amplify and sequence nematode gut contents. There are many scientists that have been able to amplify and sequence bacterial symbionts. Bacterial symbionts live on or in the nematode. Scientists have shown that this symbiotic relationship is a mutualistic relationship. They showed with Heterorhabitis and Photorhabdus that the nematode gives the bacteria protection and motility. The bacteria help the nematodes kill their host and create an environment suitable for nematode reproduction (Liu et al., 2001)

We collected from Antarctica and after picking them into tubes we spiked them with contaminant bacteria as a control for our surface sterilization techniques. We ground up the nematodes to access the gut contents and added the mixture to a PCR master mix with primers to amplify the 16s region of the nematode genome. We then sequenced our PCR product.

Methods

Soil samples were collected from various regions of Antarctica’s dry valleys. Nematodes were extracted from the soils using a sugar centrifugation method specialized for Antarctic soils (Freckman, 1994).

Gut bacteria were extracted after surface sterilizing the nematode coating by a series of bleach and water washes. These washes are to kill and remove any bacteria on the outside that could compromise the study. The solution was then treated with an enzyme that destroys all the DNA. The nematodes we then crushed up to free the gut bacteria.

Polymerase Chain Reaction was used to amplify the 16S region of bacterial DNA. Universal primers were selected to amplify a wide range of bacteria. Before running the PCR reaction the nematode mixture was put on a heat block for 10 min. at 95º. The PCR master mix was prepared and 3 μl of the crushed nematode mix was added to 23 μl of master mix. PCR product was electrophoresed on a 1% agarose gel to confirm amplification of bacterial DNA.

Results

Some positive results have been recovered but this is a continuing study. Bacterial DNA has been sequenced but much of the data is inconclusive. There might be multiple strains of bacteria in the gut, which would explain DNA sequence data.

I have presented my methods and some findings at the 2009 Long Term Ecological Research (LTER) All Scientists Meeting in Estes Park, Colorado. I am probably a couple months from having all the data necessary to writing and publishing a paper in a scientific journal.

References

• Höss, M., Kohn, M., Pääbo, S., Knauer, F., & Schröder, W. (1992). Excrement analysis by PCR. Nature, 359, 199.
• Lui, J., Berry, R. E., & Blouin, M. S. (2001). Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence. Journal of Invertebrate Pathology, 77, 87-91.
• Troedsson, C., Simonelli, P., Nägele, V., Nejstgarrd, J. C., & Frischer, M. E. (2009). Quantification of copepod gut content by differential length amplification quantitative PCR (dlaqPCR). Marine Biology, 156, 253-259.