Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Nia Riggins - Bryn Mawr College
Co-Author(s): Benardette Ezeabikwa, University of Maryland Eastern Shores, Maryland; Jorge Rodriguez, University of Maryland Eastern Shores, Maryland; A. Kwame Nyame, University of Maryland Eastern Shores, Maryland
In 1948, J.G. Mackin and H.M. Owen discovered a spherical organism in diseased eastern oysters that was associated with massive oyster mortalities in Louisiana. Today, this unicellular protozoan parasite, Perkinsus marinus, is prevalent throughout the warm waters of Gulf of Mexico and the east coast of the U.S. P. marinus is highly infectious and has caused hundreds of millions of dollars in losses over the last 35-45 years in mid-Atlantic states. Infected oysters die from extensive tissue lysis and blockage of major blood vessels. Dead and disintegrating oysters release highly infective stages of the parasite into the water where they come into contact with and are ingested by healthy oysters. It is not fully understood how P. marinus enters the oyster’s tissue. The present study investigates the interaction between P. marinus and oysters at the cellular level. Our hypothesis is that P. marinus has carbohydrates on their cell membrane that interacts with the sugars on the oysters’ cells. Glycoconjugates are believed to enable the protozoan to infect its host through a cell to cell interaction. We believe that healthy oysters become infected when the carbohydrates on P. marinus cells interacts with the oysters’ cells. Our experiment used cytological staining of P. marinus with plant lectins conjugated with Alexa 488 fluorophore as a marker for imaging (this included cell harvesting and the conjugation between the lectins). The lectins used were: Aleuria aurantia lectin (AAL), Sambucus nigra lection (SNL), Phaseoulus vulgaris Leucoagglutinin (L-PHA) and Mackia amurensis lectin (MAL). These lectins are known to bind specific cell surface glycan structures. Next, a protein assay was performed to determine the concentration of lectin-fluorescent fluid to add to the parasite’s cells. Finally, the cells were counted and a viability test was performed using trypan blue staining. The cells were pipetted in 8 tubes containing Hanks/2% buffer and 4 different Alexa labeled lectins were added to the tubes. In four of the tubes Haptins was added to prove that lectin binding was specific. Phrase contrast were used to establish that the cells were present in the samples. Negative controls were used to show the image in the absence of binding. This preliminary experiment shows that P. marinus binds strongly with SNL, which binds to cell surface α = 2, 6 linked sialic acid residues. This suggest the SNL synthesizes complex glycoconjugates which mediate the interaction between the P. marinus and its host. Sialic acid is known to interact with the environment and can also act as a mask or a recognition site. It is possible that the sialic acid enables the parasite to mask recognition by oysters’ immune system. Future studies should replicate the experiment and substantiate the finding with statistical evidence. Experiments should be designed to test the involvement of the interaction of the cell surfaces between oysters and P. marinus.
Not SubmittedFunder Acknowledgement(s): University of Maryland Eastern Shores; National Science Foundation; National Oceanic and Atmospheric Administration
Faculty Advisor: A. Kwame Nyame, aknyame@umes.edu
Role: Literature research; laboratory preparations; conducted and monitored experiments; recorded daily observations; wrote abstract and paper; created Powerpoint and Poster presentation