Cloning our way to a perfect sequence

Kelsey Coates, Duquesne University

The Approach: In my first blog post, “FROM FEMALE TO MALE – MUD SNAILS TELL ALL!,” I described the goal of my research, to sequence isoforms of a hormone receptor called the Retinoid X Receptor (RXR) in the eastern mud snail.  

Mud snails all over a beach at Fort Johnson, SC.

These isoforms have yet to be sequenced in the mud snail! But what exactly is a DNA sequence? DNA is made of building blocks called nucleotides. A DNA sequence is the order of the nucleotides. A sequence like ACG could tell the organisms’ body to do one thing while a sequence like AGC could tell the organisms’ body to do another. A bit of the sequence has already been identified, but there is a gap in the sequence we are still trying to figure out.  

Theoretically, different chemicals or different concentrations of the same chemical can change the relative levels of the RXR isoforms. If this hypothesis is confirmed, mud snails can be used in the future to detect contaminants that affect marine organisms in the Charleston Harbor. Their patterns of isoform expression might suggest which seasonal contaminants are present in the environment where they live. For example, chemical one may trigger isoform A which has sequence ACG while chemical two may trigger isoform B which has sequence AGC.            

So how will we get these sequences? It starts with amplifying the known sequence of the mud snail that surrounds the isoform, including the mysterious gap. Amplification will be done by polymerase chain reaction (PCR) to ensure there are thousands of copies of the DNA to work with. After purification, the sequence is ready to be incorporated into a plasmid along with an antibiotic resistance component. Bacteria, like E. Coli, store their DNA in plasmid form compared to the double-helix form of humans.

Plates of E.Coli in the presence of Ampicillin set in the incubator.

Luckily for us, plasmids are easily manipulated and are reproduced rapidly in bacteria. E. Coli will be grown in the presence of the antibiotic ampicillin with the sequence we cloned into its DNA.  If the sequence is incorporated into the plasmid, the bacteria will have anti-biotic resistance and be able to grow on the ampicillin plates. The bacterial colonies with our plasmid will be PCR amplified. Then, after a final plasmid preparation, the samples from E. Coli can be sent to a lab that specializes in sequencing. Hopefully the lab will identify the gap and we will achieve our goal!

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Demetri Spyropoulos, Edwina Mathis, Dr. Bob Podolsky, The Fort Johnson REU Program, The Hollings Marine Lab, NOAA, and The Grice Marine Lab. This research was supported by the Fort Johnson REU Program, NSF DBI-1757899.

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From Female to Male – Mud Snails Tell All!

Kelsey Coates, Duquesne University

The Problem: Remember the big fuss over a chemical called tributyltin? Tributyltin (TBT) was used as an antifouling agent in paint on ships’ hulls (De Mora et al., 1997). Antifouling agents prevent marine organisms, such as barnacles, from growing on the bottom and sides of ships. TBT did that and more. In fact, it was banned in the United States in the 1980s when it was found to be a biocide – meaning it unintentionally killed marine plants and animals that were not on the ships’ hulls (De Mora et al., 1997). Long after being banned, TBT is still detectable in marine environments, categorizing it as a ‘legacy’ contaminant. It is also considered an endocrine disrupting chemical (EDC). EDCs are contaminants that mimic hormones in the bodies of people and other organisms, like the mud snail. EDCs can change the effects of hormones which can alter health and physical development.

Me at Grice Beach in Fort Johnson, SC collecting mud snails to examine their sexual organs and patterns of gene expression. Each bump in the mud is a snail! We verified that the snails were no longer in their reproductive season. Photo taken by Edwina Mathis.

The eastern mud snail, Tritia obsoleta, is abundant on the mud flats of the estuaries and rivers around Charleston, SC. They live in groups in the same intertidal zones for all 20 – 40 years of their lives. Mud snails use the winter season to reproduce and the summer season to feed and grow. Mud snails are detritivores, meaning they feed on decaying plant and animal matter bound to sediments and in the water column. This makes mud snails especially susceptible to chemical contaminants that associate with sediments, like TBT. One of the endocrine disrupting effects of TBT on mud snails is the induction of imposex, where female snails develop male sex organs to varying degrees (Sternberg et al., 2008). Because mud snails are so sensitive to TBT, elevated exposures lead to more extreme changes and infertility. This can happen to female snails of all ages over time! Mud snails are an ideal alert system for contamination because they stay in the same location all their long lives, spend months of the year solely focused on feeding, and show a spectrum of imposex based on exposure level. If there is any contaminant in the harbor water or sediment – the mud snail is sure to take it up.

TBT and other similarly acting EDCs may be of major concern due to the Charleston Harbor Dredging Project. The dredging project is going to make Charleston harbor the deepest harbor in the east coast (USACE, 2015). Dredging will likely resuspend sediments that had long past settled on the bottoms of waterways. Disturbing the sediment in this way could potentially release legacy contaminants, like TBT, into the water column and along the mud flats. This may increase imposex rates as well as other effects on a wide range of organisms and people. In the body, TBT acts like a hormone that binds to a receptor called the Retinoid X Receptor (RXR) (Iguchi et al., 2007). RXR in the mud snail is expected to come in three different forms called isoforms. My goal this summer is to sequence those isoforms to determine how different chemicals or different concentrations of the same chemical can change the relative levels of the RXR isoforms. By accomplishing this goal, mud snails can be used in the future to detect contaminants that affect marine organisms because their pattern of isoform expression might suggest which contaminants are present in the environment where they live.

Graduate student Edwina Mathis and I doing a NanoDrop to determine the purity of the mud snail DNA product we want to sequence. Photo taken by Katie Hiott.

Imposex is a concern for mud snails because interference with female snails’ sex organs can lead to infertility. Also, mud snails inhabit the same areas as crabs and juvenile fish. If crabs and small fish become contaminated, the larger fish and birds that prey on them would become contaminated in higher levels by the process of biomagnification. This could limit the amount and types of fish that humans can eat and sell which would disrupt the local marine economy. If the contaminants go undetected, it could lead to human reproductive and other health disorders. It is important to study imposex for the sake of all marine species and humans that use the harbor for food, shelter, and recreation.

Acknowledgements

I would like to acknowledge Dr. Demetri Spyropoulos, Edwina Mathis, Dr. Bob Podolsky, The Fort Johnson REU Program, The Hollings Marine Lab, NOAA, and The Grice Marine Lab. This research was supported by the Fort Johnson REU Program, NSF DBI-1757899.

Sources

  1. de Mora, S. J., and E. Pelletier. “Environmental Tributyltin Research: Past, Present, Future.” Environmental Technology 18, no. 12 (1997/12/01): 1169-77.
  2. Sternberg, Robin M., Andrew K. Hotchkiss, and Gerald A. LeBlanc. “Synchronized Expression of Retinoid X Receptor Mrna with Reproductive Tract Recrudescence in an Imposex-Susceptible Mollusc.” Environmental Science & Technology 42, no. 4 (2008/02/01): 1345-51.
  3. Iguchi, Taisen, Yoshinao Katsu, Toshihiro Horiguchi, Hajime Watanabe, Bruce Blumberg, and Yasuhiko Ohta. “Endocrine Disrupting Organotin Compounds Are Potent Inducers of Imposex in Gastropods and Adipogenesis in Vertebrates.” Molecular and Cellular Toxicology, Vol. 3, (2007): 1-10
  4. US Army Corps of Engineers. “Charleston Harbor Post 45 Final Integrated Feasibility Report/Environmental Impact Statement.” (2015/06)

Are Manatees the Key?

Kady Palmer, Eckerd College

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Contaminants. One word, countless different connotations. Therefore, the exposure to contaminants is a constant concern to both the public and the scientific community. The study I will be performing this summer focuses on perfluorinated chemicals, or PFCs. PFCs are a class of contaminants that are utilized in many commercially available products (ex: non-stick pans, stain resistant sprays, and water-resistant materials) and have been classified as highly abundant and persistent chemicals of concern, in relation to overall environmental and, subsequently, human health.

teflon

Photo from: “Should You Ban Your Teflon Pan? California.” Savvy California, January 1, 2015. https://savvycalifornia.com/teflon-pan-toxic-or-not/. 

Through various mechanisms, PFCs have been noted to integrate into the environment and end up in the air, soil, and water. As this is happening, the organisms living in these areas become exposed and are put into a precarious situation. Little research has been performed on examining exactly what the effect these compounds have on organisms in these types of environments. Although it would be just as interesting to scoop water samples from different places to determine a basis for this environmental change, my project will be delving a bit deeper. Because previous studies have shown data supporting PFC accumulation in the bloodstream of different marine animals and their subsequent health consequences, I will be expanding this research by analyzing the types and abundance of PFCs in the Florida manatee.

The Florida manatee (Trichechus manatus latirostris) inhabits areas of warm water, close to the shoreline. Unfortunately, manatees have a history of endangerment, as a result of human impacts (boat strikes, entanglements, drowning due to drainages) and environmental changes. Perfluorinated chemicals, as described above, could very well be impacting manatees in ways currently unknown. This study aims to isolate the types and abundance of PFCs in Florida manatees and potential health concerns associated with this exposure. While the health of manatees is undoubtedly important, the results of this research could provide insight as to the overall health of the ecosystems examined. Manatees could function as a model for other organisms, demonstrating the possible repurcussions of PFC exposure. If that is the case, the knowledge gained from this organism, living so close to the shoreline of human inhabited areas, may be applicable in aiding future human research.

manatee-cow-and-calf

Photo from: “West Indian Manatee.” Southeast Region of the U.S. Fish and Wildlife Service. Accessed June 23, 2017. https://www.fws.gov/southeast/wildlife/mammals/manatee/.

I’d like to sincerely thank everyone involved in the National Institute of Standards and Technology laboratories who have been a wealth of information and guidance, specifically Dr. Jessica Reiner, Jackie Bangma, and my mentor, Dr. John Bowden. This project would not be possible without samples and information provided by Robert Bonde with USGS, funding from the National Science Foundation, and the College of Charleston’s Grice Marine Laboratory.

References:

Bangma, Jacqueline T., John A. Bowden, Arnold M. Brunell, Ian Christie, Brendan Finnell, Matthew P. Guillette, Martin Jones, et al. “Perfluorinated Alkyl Acids in Plasma of American Alligators (Alligator Mississippiensis) from Florida and South Carolina.” Environmental Toxicology and Chemistry, no. 4 (2017): 917. doi:10.1002/etc.3600.

“CDC – NBP – Biomonitoring Summaries – PFCs.” Accessed June 19, 2017. https://www.cdc.gov/biomonitoring/pfcs_biomonitoringsummary.html.

West Indian Manatee”. Southeast Region of the U.S. Fish and Wildlife Service. Accessed June 23, 2017. https://www.fws.gov/southeast/wildlife/mammals/manatee/.