[Annual Termite Issue] Now That's "Gutsy" Research

If you were to take a termite, open up its hindgut and spill out its gut contents onto a microscope slide, you would be dazzled by a diverse array of microscopic life. You would find protozoa swimming around under the force of pulsating flagella. Bacteria in the shape of tiny rods, coils and balls would dot the slide’s surface. One species after another of microorganisms would be available for your viewing.

These microorganisms perform a variety of important functions for the termite. Most importantly, the protozoa and bacteria help the termite digest the main component of wood — cellulose. In return, the host termite provides the microorganisms with a food supply and the appropriate environmental conditions for their survival and growth.

Since termites are dependent on their gut microorganisms, some researchers have begun to look at gut microorganisms as a possible target for managing termites. If the symbiotic bacteria and protozoa were removed, the termite would not be able to digest cellulose, leading to eventual starvation and death.

One of the problems, however, with developing measures to target the gut microorganisms has been a lack of practical tools for studying the bacteria and protozoa. The available tools (i.e., culturing, microscope and DNA techniques) are either ineffective or time consuming and expensive. Thus, scientists have been hampered in their ability to answer basic questions about the termite/microorganism relationship and even more in their ability to develop methods for targeting the gut microorganisms.

The following study describes preliminary work in describing what species of bacteria are present in the termite gut and the development of tools for their study. This research will open new doors, allowing researchers to more easily target the gut bacteria in an effort to control termites.

THE STUDY. The sequence (or basic pattern) of an individual’s DNA can be used as a personal "fingerprint." Just as different humans have different DNA sequences, so too do different species of bacteria. The DNA sequence of bacteria can be used to identify their species. Since I could not effectively culture the bacteria, I utilized DNA sequencing technology in order to characterize which species were present.

One of the things that must be understood about sequencing is the importance of non-contamination. If DNA from two different sources are sequenced together, the data would be contaminated and useless. I needed, therefore, to find some way to sort the "mixed soup" of bacteria into their respective species. Due to their small size and similar appearance, I could not simply pick out individual bacteria from the mixed group of bacteria. In order to accomplish this difficult task, I utilized DNA extraction, polymerase chain reaction (PCR) and cloning technologies. Without going into too much detail, these processes essentially separated the DNA from the bacterial cells, and I snipped out the gene that I wanted to sequence (the 16S rRNA gene) from the extracted DNA and then inserted the clipped gene into new bacterial cells that could be easily cultured (see figure 1 above). I was then able to utilize microbiological techniques to grow pure cultures of bacteria that represented — due to the gene insert — the individual species of bacteria with which I originally started (see figure 2 on page 52). Now that I had uncontaminated colonies representing individual bacterial species, I was able to sequence the DNA.

To analyze the DNA sequences, I input the sequences into a computer program that builds "genealogical trees" telling me how closely related each sequence is compared to all other sequences used in the analysis. I made sure to also include in the analysis several reference sequences that I obtained from the National Institute of Health’s online BLAST database. These reference sequences corresponded to known bacterial species and helped determine from which family or group of bacteria my sequences originated.

RESULTS. I have cloned and sequenced 24 16S rRNA gene sequences of bacteria from the midgut and hindgut region of Reticulitermes flavipes. Preliminary data indicate that I may have discovered several new types of bacteria. In addition, the data suggest that the termite gut of R. flavipes contains strikingly less bacterial diversity than is found in other termite species (Hongoh et al. 2003). All of the bacteria from which I obtained DNA sequences were in the bacterial group known as the gamma-proteobacteria.

SIGNIFICANCE. In addition to answering basic biological questions, the data acquired from this study provided information that can be used from a practical perspective. Because of the tight symbiotic relationship between termites and their gut bacteria, targeting the gut bacteria in an effort to indirectly manage termites has great potential. My research into identifying the gut bacteria is helping open up a new target area for the control of termites.

Dr. Claudia Husseneder (2004) at Louisiana State University has suggested one example of how the gut bacteria can be used as targets for controlling termites. In her studies, she has proposed that termites can be controlled using a method called "paratransgenesis." In paratransgenesis, bacteria are genetically modified to produce a toxin that kills protozoa. As the termites ingest the genetically modified bacteria — via some type of bait matrix — the protozoa die. And, since the protozoa play a crucial role in the digestion of wood for the termites, the termites eventually starve to death. My identification of the gut bacteria may result in the discovery of several native bacterial candidates for use in paratransgenesis termite control.

The sequence data I collected is also being used to develop simple and relatively inexpensive tools for studying the gut bacteria. These tools, known as oligonucleotide probes, are small pieces of DNA that act like miniature "search parties." Each probe is built to find a sequence of DNA — in this case the 16S rRNA gene — that only corresponds to a certain species of bacteria. After being added to a mixed population of bacteria, the probe finds its target sequence, latches on to the sequence and emits a fluorescent light, signaling its successful find. Thus cells of the bacterial species one wants to study are easily found and identified. I am currently utilizing the sequences I have obtained to make oligonucleotide probes that are specific to the gut bacteria of termites. This will give termite researchers an important tool for studying the gut microorganism community.

Studying the gut microorganisms in order to find ways to kill termites? Now that’s gutsy research.

Hongoh, Y., M. Ohkuma and T. Kudo. 2003. Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS Microbiol. Ecol. 44:231-242.

Husseneder, C. 2004. Paratransgenesis: a new approach to termite control. In Proceedings, 2004 International Congress of Entomology, 15—21 August 2004, Brisbane, Australia.