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.

Hiding in plain sight

Brian Wuertz, Warren Wilson College

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How much do we really know about all the chemicals that we are exposed to every day? Do we even know when we come into contact with them? How much do we know about what is in homogenized milk, soda, stool softeners, baby formula, and personal care products such as eyeliner? The answer may be “not enough” for one compound found in all of those products, dioctyl sodium sulfosuccinate, or DOSS. DOSS has recently been identified by my mentor, Dr. Spyropoulos and his Ph.D. student, Alexis Temkin, as a probable obesogen. Obesogens are a class of compounds that promote obesity by interfering with the body’s hormone signaling pathways related to energy use, fat cell regulation, and inflammation. These pathways are especially important in the developing fetus, where hormone signals influence development and may have long lasting effects on the health of the child after birth (Holder 2016).

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I am working on a High Performance Liquid Chromatography  (HPLC) system, in the early stages of developing a method to measure the amount of DOSS in cell extracts. (More to come in future posts!)

We are especially concerned with regards to the developing fetus and child because stool softeners containing DOSS are are commonly taken by pregnant women. Approximately 35% of over 20,000 women who gave birth at MUSC in recent years reported taking a stool softener containing DOSS during their pregnancy. I am working to help understand the biochemical pathways DOSS may follow to affect changes in the  developing fetus through a mother’s exposure to DOSS. I am also working on a method to measure the amount of DOSS in cells so that we can learn where in the body DOSS goes and how much of it there actually is.

You might be wondering how this fits into the theme of marine organism health at this point since all I have talked about is human health and a compound found in products we put in our bodies, DOSS. A red flag was raised about DOSS through research on COREXIT, one of the agents used to clean up the Deepwater Horizon oil spill. Over 40 million gallons of COREXIT was dumped into the ocean as a part of the cleanup effort and DOSS is one of the major components (Temkin 2016).  DOSS was flagged as a potential human health hazard because of the research done on marine environmental degradation. It amazes me how a perhaps seemingly unrelated topic can end up having human health implications. I am excited to keep working on this puzzle to learn more about DOSS and how it interacts with the systems in our bodies!

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

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Sources:

Holder, B., Jones, T., Sancho Shimizu, V., Rice, T.F., Donaldson, B., Bouqueau, M., Forbes, K., and Kampmann, B. (2016). Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal–Placental Messaging. Traffic 17, 168–178.
Temkin, A.M., Bowers, R.R., Magaletta, M.E., Holshouser, S., Maggi, A., Ciana, P., Guillette, L.J., Bowden, J.A., Kucklick, J.R., Baatz, J.E., et al. (2016). Effects of Crude Oil/Dispersant Mixture and Dispersant Components on PPARγ Activity in Vitro and in Vivo: Identification of Dioctyl Sodium Sulfosuccinate (DOSS; CAS #577-11-7) as a Probable Obesogen. Environ Health Perspect 124, 112–119.

 

 

 

Humans and gators and chickens, oh my!

Jimena B. Pérez-Viscasillas, University of Puerto Rico at Mayaguez

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When I first applied to this Marine Biology REU, in a lab that works mostly with alligators, at a Marine Science Campus right by the Charleston shore, I never thought I’d end up working with chickens. Yes, you read correctly: chickens, of the Chick-Fill-A and Kentucky Fried sort. I was surprised too, naturally, but it turns out the reason behind it is actually pretty important.

A couple of years ago, a group of scientists noticed some alligator populations in Florida weren’t doing too well. Their fertility levels were decreasing and a lower percentage of the eggs laid were hatching. Upon further study, evidence pointed towards a likely culprit: anthropogenic chemical contaminants in the environment. These contaminants were negatively affecting the gators’ hormone production and, in turn, their reproductive systems.

What do these gators have to do with chickens, though? Perhaps more importantly, what do they have to do with us? Let’s review some basic bio…

Figure 1: Vertebrate phylogenetic tree. Amniotes are organisms which have adapted to terrestrial reproduction. This group includes birds, reptiles, and mammals. (Graphic taken from: UCL)

There are some terrestrial animals which lay eggs (like chickens and gators) and some that carry their young in the womb, inside the placenta (like us). Both types of organisms, collectively called amniotes, have much of the same tissues surrounding their embryos during development. This shared characteristic means that we may be able to study some egg-laying animals to better understand our own reproductive systems.

Figure 2: A chick embryo and membrane. The membrane I’m going to be studying is that which lines the inside of the shell. Its called the chorioallantoic membrane, and it allows gas and waste exchange between the embryo and the environment. (Taken from Angiogenesis Laboratory Amsterdam)

Before we can use these organisms’ tissues as models of our own, however, we have to make sure we understand how they function. This is where I (and the chickens) come in. This summer, I’m going to be measuring how (and if), at different stages of development, the egg membrane of chickens produces hormones called prostaglandins. Prostaglandins play a major role in the immune system, as well as the body’s general regulation and reproduction. This preliminary research would help us better understand these sentinel species and allow us to later assess how their endocrine, immune and reproductive systems are being compromised by environmental pollutants. If we know how chemical contaminants in the environment are having negative effects on their reproduction, what might it tell us about how they’re affecting our health and reproduction?

To learn more about my project, check back for further posts!

Acknowledgements

This research, conducted at Dr. Louis Guillete’s MUSC Laboratory, is made possible thanks to funding from NSF and the College of Charleston. Further equipment and facilities are provided by the Hollings Marine Laboratory.

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References:

Bellairs, Ruth & Osmond, Mark. The Atlas of Chick Development.  San Diego, California: Elsevier Academic Press, 2005. Print.

Guillette LJ Jr. “The evolution of viviparity in amniote vertebrates—new insights, new questions.” J Zool  223 (1991): 521–526. Web. 10 June 2015.

Guillette LJ Jr. “The evolution of viviparity in lizards.” Bioscience 43 (1993): 742–751. Print.

Kalinski P. “Regulation of Immune Responses by Prostaglandin E2.” J Immunol 188 (2012):21-28. Web. 10 June 2015.

Kluge AG. Chordate Structure and Function. New York: Macmillan Publishing Co., Inc.; 1977. Print.

Milnes MR, Guillette LJ Jr. “Alligator Tales: New Lessons about Environmental Contaminants from a Sentinel Species.” BioScience 58.11 (2008): 1027-1036. Web. 15 June 2015.  doi:10.1641/B581106