Perkey/Rizk (A. Schnabel)
A Novel Method for the Identification of Hydra Species
We propose to investigate species diversity and genetic variation of hydra throughout the Great Lakes region using molecular and enzymatic markers. Hydra is a genus of animals that are freshwater relatives of corals and sea anemones and are widespread in lakes and ponds throughout the world. Because of their small size and morphological simplicity, hydra species have traditionally been difficult to identify. This has lead to confusion over the number and geographic distribution of species. We will reduce some of this ambiguity by developing a simple and accurate method of hydra species identification. This research will build upon a year of prior work with hydra during which time we have collected and maintained hydra cultures, isolated genomic DNA, and experimented with several genes. We have also studied molecular biology theory in L211 and laboratory techniques in molecular biology in L323. The research this summer will involve two major parts. First, we will sample and culture hydra from various lakes and ponds throughout the Great Lakes region. Second, we will employ two different molecular techniques to develop species specific genetic "fingerprints." By providing researchers with a fast and reliable method of hydra species identification, further study in the areas of hydra population genetics and species distribution will be possible.
Hydra is a genus of freshwater animals found in lakes and ponds throughout temperate regions of the world. Like jellyfish and sea anemones, hydra are members of the phylum Cnidaria. Their body structure is in the form of a tube, 5-20 mm long and 0.3-1.0 mm wide, bearing a whorl of tentacles at the oral end and a basal disk at the aboral end (Fig. 1) (Lenhoff 1983). The importance of hydra as a model system in developmental and molecular biological research lies in the simplicity of their body plan.
This simplicity allows researchers to view biological phenomena without taking into account many other factors present in more complicated systems. Since hydra are multicellular animals, data obtained from research using hydra can be applied to systems of greater complexity such as humans.
The small size and morphological simplicity of hydra species has traditionally made them difficult to identify. As a result, there has been much debate as to the number and geographic distribution of hydra species (Campbell 1989). The goal of our research is to reduce much of this ambiguity by providing an effective means of hydra species identification. The current procedure for the identification of hydra species is based solely on comparing differences between species in the structure of nematocysts, which are stinging cells specific to cnidarians (Lenhoff 1983). In addition to being laborious and time-consuming, this method relies on qualitative interpretation by the observer and results may vary depending on the observerís level of expertise. Needless to say, very few biologists posses such an intimate acquaintance with hydra cell structure.
Last summer we began a research project to investigate hydra species diversity and evolution. We found it necessary to first be able to identify hydra species collected, and we were surprised to learn that there was no reliable method available (Adams and Martin 1963). Without such a method our research could not continue. Therefore, we plan to develop a novel method of hydra species identification that will be simple, accurate, and easily repeatable by all hydra biologists. This technique will utilize differences in DNA sequences rather than differences in physical morphology. The method will then be used to map the distribution of hydra species throughout the Great Lakes region. This will enable us to gain a more complete understanding of hydra ecology and genetic diversity. This project will be conducted under the supervision of Dr. Andrew Schnabel, and we will also benefit from the expertise of Dr. Ann Grens, who uses hydra as a model organism to study developmental genetics.
Our plan is to develop a technique for the identification of hydra species using both genetic and enzymatic approaches. Hydra will be collected from lakes and ponds in Indiana, Michigan, Illinois, Ohio, and Wisconsin. In the laboratory, hydra will be kept at conditions that simulate their natural habitat. Genomic DNA will be extracted from the hydra collected and genetic analysis will be carried out. The data obtained will be applied to hydra species distribution around the Great Lakes region.
Hydra Collection and Culture
In summer 1999, we collected hydra from several locations in northern Indiana and Dr. Schnabel and Dr. Grens made collections in Michigan and Wisconsin. We still have several of the collections in culture, and have extracted DNA from several others that died during the project. New hydra samples will be collected and cultured in the laboratory using methods learned from Dr. Grens, who maintains stocks of several thousand hydra. To obtain hydra, substrates such as rocks, leaves, and twigs will be collected from lakes and ponds in the Great Lakes region. In the laboratory, the lake material will be examined under the microscope and each individual hydra will be removed and grown in a separate petri dish. Parameters such as ion concentrations and temperature will be kept at constant levels that reflect conditions favored by the organism to achieve a maximum growth rate (Lenhoff 1983). Hydraís ability to reproduce asexually through budding results in the formation of offspring that are genetically identical to the parent. This aspect of hydra reproduction is taken advantage of by allowing each animal to reproduce asexually, generating a clonal population in which all of the individuals are genetically identical (Haley and Forrest 1949). Because five to ten animals are needed to obtain sufficient amounts of DNA, clonal populations are essential to our research.
Our genetic work will begin with an analysis of the internal transcribed spacer (ITS) region of the ribosomal genes of hydra. This region has been used as a genetic marker in research with other cnidarians that share many of the difficulties associated with species identification of hydra due to ambiguity of morphological characteristics (Miller et al. 1996). In our past research, we have successfully generated data using the ITS region, by making use of Polymerase Chain Reaction (PCR), a method carried out to generate useful quantities of specific DNA fragments. Hydra genomic DNA will be extracted and the ITS region will be isolated using PCR. The ITS gene isolated from different species of hydra will be treated with restriction endonucleases, which are enzymes that break the DNA at specific sites, producing fragments of various sizes. The fragments of DNA generated from the endonuclease reaction will be separated based on their size through a gel matrix by the influence of an electric current, a technique known as gel electrophoresis. The DNA fragments in the gel are made visible using fluorescent dyes that bind to DNA, but not the gel matrix. Since the ITS region presumably varies from one species to another in the number and location of sites recognized by the restriction enzyme, the pattern produced from the restriction enzyme will be unique to each species (Fig 2). The resulting different banding patterns on the gel will act as a genetic fingerprint that is uniquely identifies each species.
Another approach to the development of a classification technique for hydra species is through the analysis of enzymes. Enzymes, which are proteins that are abundant in al living cells, will be used as an indirect measure of genetic differences between species. Because enzymes are coded for by DNA, each enzymes is a direct reflection of the DNA sequence from which it was synthesized. A method similar to that of restriction analysis will be employed to determine differences in enzymes, which in turn will determine differences in species. Enzymes will be extracted from hydra individuals. These proteins will be analyzed using gel electrophoresis in which proteins are separated according to size and charge. Like restriction analysis of the ITS gene, this technique will generate a pattern unique to each hydra species.
Due to the recent increase in the utilization of hydra as research models, it is apparent that a quick and accurate method for the identification of hydra species is essential for further hydra research in the fields of population genetics, ecology, developmental and molecular biology. Over the coming summer, the method outlined in this paper will be tested and refined. The development of this technique will enable further study of genetic diversity and species distribution of hydra around the Great Lakes and will allow researchers working with hydra to quickly and accurately identify the species at hand, as well as increase the effectiveness and importance of hydra as a model system.
We are asking for $3,000 each for full time work, but if funds are limiting we will accept one fellowship shared between the two of us.
The chemical reagents required for this project will be provided by Dr. Schnabelís research finds. Because we are planning to sample hydra from Indiana, Michigan, Illinois, and Ohio, the majority of funds will be required for traveling expences.
- Estimated mileage: 1300 miles at $0.315 per mile for a total cost of $403.00
- Lodging: $150.00
- Meal expenses: $50.00
- Total expenses estimated at $600.00
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- Grens, A., Gee, L., Fisher, D., Bode, H. (1996) CnNK-2, an NK-2 homeobox gene, has a role in patterning the basal end of the axis in hydra. Developmental Biology, 180: 473-488.
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Time Line Budget
Research will be performed full time for 10 weeks during the summer. It will build upon and continue research on hydra begun last summer and one year of experience in the field of molecular biology. The time budget for the research to be conducted is as follows:
Week 1-2 Local collections of hydra will be made. Enzyme analysis methods will be learned from Dr. Schnabel.
Week 3-5 Collection forays will be taken to more distant areas of the Great Lakes region. Restriction endonuclease analysis of PCR isolated DNA fragment will begin.
Week 5-7 DNA and enzyme analysis of hydra species will continue. Identification of hydra species will begin.
Week 8-10 Continued DNA and enzyme analysis. Data will be reviewed and conclusions will be drawn. We will begin mapping distribution of species around the Great Lakes.