A novel DNA sequence that Long Beach High School seniors and International Baccalaureate Diploma candidates Savannah Kile and Leah Shokrian identified through their participation in the school’s Science Research program and via the Barcode Long Island program through Cold Spring Harbor Laboratory’s DNA Learning Center, was published to the National Center for Biotechnology Information’s international database, GenBank. The students were the first researchers to record a partial sequence for the Pleurogonius malaclemys isolate DNAS-104-145386 18S ribosomal RNA gene, and as an outcome of their explorations, scientists around the world now have access to their discoveries.
For three years of their high school careers, Savannah and Leah spent countless hours observing and analyzing Pleurogonius malaclemys, a parasite that often forms cysts on its hosts. The students learned about the parasites and turtles in consultation with two Hofstra University professors, Dr. Russell Burke and Dr. Jason Williams. The students investigated the infection rate of mud snails in the tidal wetland located on the campus of Long Beach High School. This trematode species relies on two hosts – turtles and snails, in this case – in order to complete its life cycle.
Specifically, the hosts for the Pleurogonius malaclemys are diamondback terrapin turtles and mud snails, formally known by the corresponding names of Malaclemys terrapin and Ilyanassa obsoleta. “Because the parasite is a trematode, its presence on one of the hosts indicates that the other is nearby,” said Leah.
Mud snails are abundant, often found in quantities of hundreds or even thousands in marshlands, ponds, and other wetlands. Diamondback terrapin turtle numbers are known to be declining and there is a need for more data regarding their population ecology. Therefore, as Leah and Savannah explained, presence of the parasitic cyst on snails likely means that the turtles are in the area as well, and measures can be taken to preserve their habitats and enforce efforts to protect their nests.
Leah and Savannah collected snails in sites local to western Long Island, including the Long Beach High School pond, and studied them for cysts in the building’s science lab under dissecting microscopes. While conducting their research, they realized a positive correlation between the size of the snails and presence of the parasite. This enabled the students to work more efficiently by narrowing their selection pool from hundreds of snails down to those that were most likely to be infected. “We knew that if larger snails were not infected with the parasite, it was most likely not going to be found at that location,” said Savannah.
Leah and Savannah took their research even further by conducting DNA barcoding of the cysts, which confirmed the identify that their samples were Pleurogonius malaclemys and now their sequences can used by others to identify the parasite species based on any life history stage. The in-depth barcoding technique allowed students to isolate the parasitic DNA and sequence a specific region of the 18s ribosomal RNA gene, which can be used to potentially identify a species when aligned to available sequence information on GenBank.
DNA barcoding is a relatively new innovation developed by Paul Hebert, a researcher at the University of Guelph in Ontario, Canada, in 2003. Recently, Cold Spring Harbor Laboratory’s DNA Learning Center developed a student driven DNA barcoding research program, Barcode Long Island, through funding provided by an NIH SEPA grant to work with Long Island schools and explore and map Long Island’s biodiversity. This project includes the potential for students to discover unpublished organismal DNA barcodes, or “novel sequences,” which students can in turn publish to GenBank after taxonomic identification of the organism by professionals in the field. This is the third year that the program has been underway, and Long Beach was one of the first high schools to become involved.
“Taxonomy, especially of smaller organisms, has traditionally been done by specialized experts,” said Long Beach High School science teacher Cody Onufrock. “DNA barcoding allows people to study biodiversity on a much more sophisticated level than ever before. Cold Spring Harbor Laboratory’s DNA Learning Center and local students are building a database of life.”
DNA sequences are stored in the GenBank database with the objective of providing scientists with a way to obtain genetic information about a species that they are studying, or to act as a “barcode” which can be matched by a researcher with a found sequence; this allows for ID without the need of a specialized taxonomist. Leah and Savannah found no record of a precise match for the Pleurogonius malaclemys in this system, but they did discover evidence that it was related to other parasites in the trematode class. The pair moved forward in uncovering their sequence and making it available to researchers worldwide.
Manipulating the obtained snail parasite to obtain desired DNA and determine the sequence involved many steps. First, the students identified which part of the snail would yield veritable findings. They recognized that the operculum, or foot of the snail, is a protein, therefore not comprised of DNA. “We removed the operculum that contained the cyst so none of the snail’s DNA would be included, only the trematode’s,” said Savannah. She and Leah then ground the cysts in a lysis solution to extract DNA from the cells and put their samples through a number of subsequent DNA isolation and purification phases. They made use of a trematode primer and PCR thermal cycler, which amplified the target DNA sequence and created millions of copies of the 18s rRNA gene.
Through a process known as gel electrophoresis, Leah and Savannah ran their samples through a gel that separates DNA molecules by size and allows for visualization of amplified DNA strands, and thus confirms that the preceding actions to obtain DNA were successful. The students replicated the DNA barcoding protocol three times to confirm their results. The amplified DNA was converted into base pairs at a sequencing facility and translated into text form. Dr. Diana Padilla from Stony Brook University conducted the taxonomic identification of the parasite. The students then used bioinformatics software tools to analyze their results and connect with GenBank.
After a year-and-a-half-long process of submitting their results and securing approval, Leah and Savannah’s work was published in GenBank this December. “The benefit is that this will come up as a match for anyone studying the Pleurogonius malaclemys Trematode,” said Savannah.
“This is a professional-level accomplishment for two high school students,” said Director of STEM 6-12 Deborah Lovrich.