All Shrimp Go To Heaven

Carolina Rios, New York University

The Approach: In my previous post, I discussed the negative impact that polychlorinated biphenyls (PCBs), long-lived chemical contaminants, can have on marine ecosystems and human health. Currently, I am working to verify a proposed model to estimate the impact that PCB contamination has on benthic marine invertebrates.

One of the issues with this proposed model is that it is based on data generated from the 1970s; and the analytical methods now available to scientists are much more sensitive and precise. To verify this model, we will generate contemporary data by running a series of short (acute) toxicity tests.

Testing

Test setup for grass shrimp (P. pugio)

In these acute toxicity tests, we measure the response of three species of marine invertebrates to PCBs. The three organisms that we are testing are grass shrimp (Palaemonetes pugio), amphipods (Leptocheirus plumulosus), and mysids (Mysidopsis bahia). We will be measuring mortality from PCB contamination. The standard tests that we are running consists of 6 concentrations, ranging from 6.25 ppb (parts per billion) to 420 ppb. It is important that we also have a control, so that we can understand the response of the organisms unaffected by PCBs. For the grass shrimp and amphipods, the test will run for 96 hours and we will renew the PCB solutions every 24 hours. Samples will be taken for chemical analysis at 0 hours, 24 hours, and 72 hours, so as to measure both the loss of PCBs over the 24 hour period, as well as the consistency of dosing. Loss of PCBs can be attributed to PCBs binding to the glassware and differences in dosing can be attributed to user variability. For the mysids, the test will also run for 96 hours, but the dosing solutions will not be renewed after the initial dosing. Samples will be taken at 0 hours, 48 hours, and 96 hours, so as to measure the loss of PCBs over the 96 hour period. For all tests, mortality will be recorded every 24 hours until the end of the test.

Analysis

Solid Phase Extraction apparatus. Dosed samples are within the large reservoirs at the top of the apparatus. PCBs will be isolated on the nonpolar solid phase, which consists of the smaller columns below the reservoirs.

The PCBs from the collected samples will be isolated through solid phase extraction. Solid phase extraction consists of a nonpolar solid phase and a polar liquid phase; similar to how oil cannot be mixed into vinegar, PCBs are not very soluble in water. As PCBs are nonpolar and hydrophobic, they will bind to the solid phase. The PCBs can then be lifted off of the column by running a nonpolar solvent (ethyl acetate) through the nonpolar solid phase. The sample is then analyzed using gas chromatography-mass spectrometry (GC-MS). The essential concept of GC-MS is that molecules will separate based on differences in size. This is how the amount of each individual PCB is determined, which can then be used to calculate an actual concentration. Analysis of the chromatograph can give more accurate concentrations, allowing us to understand how concentrations vary over time. This will give us a better understanding of the relationship between dose concentrations and the mortality response.

Acknowledgements

I would like to thank Dr. Ed Wirth and Brian Shaddrix for their continued guidance and support, as well as my co-mentor Dr. Paul Pennington. Supported by the Fort Johnson REU Program, NSF DBI-1757899.

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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.

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)

Manatees and PFCs- The Future of Contaminant Studies

Kady Palmer, Eckerd College

IMG_1115

In my previous post, “The Problem with PFCs- Seeking Answers in Plasma”, the abundance of perfluorinated chemicals, or more specifically perfluoroalkyl acids (PFAAs), was analyzed in manatee plasma and correlated to variables such as site, water temperature, and blood chemistry. The purpose of this study was to develop a greater understanding of these chemical contaminants in regards to their routes of exposure and subsequent health effects.

Accumulation of PFAAs within organisms is proposed to be predominantly attributed through diet. Therefore, apex predators, like alligators, dolphins, and humans are found to be at a higher risk for increased concentrations of these chemicals in their body (Bangma et al., 2017, Fair et al., 2012). This is a result of biomagnification, or increasing levels of a compound as one continues up the food chain or trophic hierarchy. Manatees, however, are not predators, and are considered lower on the trophic hierarchy due to their herbivorous diet. With that knowledge, the amount of PFAAs within them, if any, was hypothesized to be very small.

After obtaining data from chemical extractions and liquid chromatography tandem mass spectrometry (LC-MS/MS), concentrations of at least two perfluoroalkyl acids were detected in all 69 manatee plasma samples. What that means is that PFAAs are integrating into the biological systems of manatees and accumulating within their bloodstream, presenting different results than our initial hypothesis.

pfos

One of the most common PFAAs found in manatee plasma, known as perfluorooctanesulfonic acid (PFOS). Photo from: http://pubs.sciepub.com/ces/2/1/3/

Data and statistical analyses determined location-based differences in PFAA concentrations. In addition, correlations were found between high PFAA burden, blood chemistry measurements, and water temperature at the time of sampling. With this information, a basis for further investigations is possible to begin determining potential health effects of PFAAs in not only manatees, but in humans as well.

manateesCR

Because manatees cannot tolerate cold water, they congregate in warm waters during the winter seasons. Interestingly, correlations between water temperatures and PFAA values were found in this study. Photo from: http://proof.nationalgeographic.com/2014/07/21/floridas-manatees-the-search-for-warmer-water/

In summary, the purpose of this experiment was to answer two questions: 1) Are PFAAs present in manatee plasma? 2) If so, can heavy burdens of PFAAs be statistically correlated to health variables?

The first question was answered within the first week of analysis, simply by identifying detectable levels of these chemicals in manatee plasma. The second question, however, is more complicated to answer. The statistics say that there are associations between PFAAs and differing health measurements, however, the significance and meaning of that data is something future research must focus on. The reasons behind the correlations are still unknown, even though some explanations may be proposed.

I would like to extend an enormous thank you to everyone who made this project possible, including Dr. Jacqueline Bangma, Dr. Jessica Reiner, and my extremely motivating mentor, Dr. John Bowden. I would also like to thank the National Science Foundation for their funding, the College of Charleston’s Grice Marine Lab for hosting this REU, and the USGS Sirenia project for supplying the samples I utilized in this project.

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 (2017a): 917. doi:10.1002/etc.3600.

Fair, Patricia A., Magali Houde, Thomas C. Hulsey, Gregory D. Bossart, Jeff Adams, Len Balthis, and Derek C.G. Muir. “Assessment of Perfluorinated Compounds (PFCs) in Plasma of Bottlenose Dolphins from Two Southeast US Estuarine Areas: Relationship with Age, Sex and Geographic Locations.” Marine Pollution Bulletin 64 (January 1, 2012): 66–74. doi:10.1016/j.marpolbul.2011.10.022.

 

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/.