Oil Spills, Climate Change, and Grass Shrimp

Cheldina Jean, American University

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Getting down and dirty for egg carrying grass shrimp in Leadenwah Creek. Photo: Katy Chung

The Approach: In my previous post, I discussed how grass shrimp (Palaemonetes pugio) larvae can be used to test the effects of oil when paired with environmental conditions such as ultraviolet light (UV), temperature, and salinity. In the environment, salinity, temperature, and different levels of light can affect the health and survival of organisms. UV light is one of the three types of radiation the sun emits. Crude oil is made up of polycyclic aromatic hydrocarbons (PAHs), which are formed from the incomplete burning of fossil fuels. When oil spills happen, UV light can change the PAH chemistry, making oil up to 100 times more toxic to marine organisms (Alloy et al., 2017).

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Upclose image of grass shrimp eggs. If you look closely, you can see the black eye spots of the embryo. Photo: Cheldina Jean

In the environment, grass shrimp experience salinities ranging from 0-36 parts per thousand (ppt), temperatures ranging from 2 °C to 37 °C (DeLorenzo et al., 2009), and various levels of UV light, all depending on season, precipitation, and tides.  For this research project, we collected adult grass shrimp with eggs from Leadenwah Creek, which is located on Wadmalaw Island, Charleston, SC. Seawater from the Charleston Harbor estuary was filtered and used for all of the test conditions. The oil we use in our tests was obtained through NOAA from the DeepWater Horizon oil spill.

 

 

We are looking at two different types of oil exposures for this project:

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Undiluted HEWAF. Photo: Cheldina Jean

  1. High Energy Water Accommodated Fraction (HEWAF), which is dissolved oil in seawater. The HEWAF is diluted to concentrations of 0.25%, 1%, 4% for our different tests. 
  2. Thin oil sheen, which is a thin layer of fresh oil placed on the surface of the water.

Standard laboratory testing conditions for grass shrimp generally consist of a salinity of 20 ppt, temperature of 25 °C, and fluorescent lighting (DeLorenzo et al., 2016).

 

 

For both oil exposure scenarios (HEWAF and sheen) we set up larval shrimp under combinations of the different environmental conditions: UV or no UV (using UV light bulbs or cool-white fluorescent bulbs, respectively) temperatures of 32 °C (90 °F) and 25°C (77 °F), and salinities of 10 ppt, 20 ppt, and 30 ppt.

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Temperature HEWAF test under UV conditions. Photo: Cheldina Jean

Newly hatched larvae were acclimated in the different temperatures and salinities before each test. Every 24 hours, the amount of larvae that survived and the amount that died were recorded. Each test ran for 96 hours and on the 96th hour, water quality (temperature, dissolved oxygen, salinity and pH) was recorded.

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Field Collection! (Featuring Shelby, myself, and two Hollings Scholars). Photo: Katy Chung

Next, we will use statistical analysis to evaluate our results. Stay tuned!

I would like to thank my mentor Marie DeLorenzo and co-mentor Katy Chung for guiding me through this research. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

 

 

 

Citations:

  1. Alloy, M., Garner, T. R., Bridges, K., Mansfield, C., Carney, M., Forth, H., … & Bonnot, S. (2017). Coexposure to sunlight enhances the toxicity of naturally weathered Deepwater Horizon oil to early life stage red drum (Sciaenops ocellatus) and speckled seatrout (Cynoscion nebulosus). Environmental toxicology and chemistry, 36(3), 780-785.
  2. DeLorenzo ME, Wallace SC, Danese LE, Baird TD (2009) Temperature and salinity effects on the toxicity of common pesticides to the grass shrimp, Palaemonetes pugio. J Environ Sci Health B 44:455–460.
  3. DeLorenzo, M. E., Eckmann, C. A., Chung, K. W., Key, P. B., & Fulton, M. H. (2016). Effects of salinity on oil dispersant toxicity in the grass shrimp, Palaemonetes pugio. Ecotoxicology and environmental safety, 134, 256-263.

 

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Stirring up the sediment, are we opening Pandora’s box?

Samera Mulatu, Georgia Southern University

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The Approach: Would you believe if I told you that animals in the Charleston harbor are changing from female to male?! This process, known as imposex, occurs in marine snails when females develop male sex traits because they are exposed to harmful chemicals. One of my main goals in this project is to measure the rates of imposex in the Eastern mud snail (Tritia obsoleta, previously known as Ilyanassa obsoleta) within the Charleston Harbor to see if these rates increase over time due to the dredging of the harbor. There is a plan to begin dredging the Harbor later this fall, and the idea is that dredging will bring harmful chemicals in the sediment up into the water column. The data I am collecting now will be the imposex rates of the mud snail before the dredging brings up any harmful chemicals buried in the sediment of the harbor. However, we aren’t just collecting a bunch of snails and waiting for them to change sexes! No, there’s so much more to it than that!

As mentioned in my previous post, disruption of the Retinoid X Receptor (RXR) gene pathway is known to be central to inducing imposex in mud snails. By studying RXR we could learn a lot about what chemicals and how much of them are needed to induce imposex. However, the RXR gene for Tritia obsoleta has never been sequenced! So the first task in this project was to find the most closely related snails to the mud snail whose RXR sequences were already known. Primers were then designed based on these related RXR genes of known species. After this, mud snails were collected from the Charleston Harbor. 50 mud snails were collected that had a shell size of greater than 12 mM in height (to ensure that we were only using adults). The mud snails were dissected, and from different dissected parts RNA was then extracted to retrieve messenger RNA (mRNA). The mRNA was then reverse transcribed with reverse transcriptase enzyme into cDNA (‘reverse’ because DNA is usually transcribed into mRNA). The cDNA library generated represents all of the mRNAs in the mud snail tissue. The cDNA was then PCR amplified using the RXR-specific primers described above. Once the PCR products were obtained, they were column purified and sent off for sequencing!

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I was preparing the primers for purification. Picture taken by: Cheldina Jean

Once the mud snail RXR sequences are retrieved, we will distinguish them into the two types of RXR gene forms, isoforms a and b. Designing new primers specific to these RXR isoforms, we can determine the relative abundance of each isoform based on chemical (i.e. TBT, DOSS, or SPAN 80) exposure in the lab using adult females. Hopefully, my results will contribute to a better understanding of what effect the dredging of the harbor will have on imposex rates of the mud snail. Furthermore, if we see that dredging is harmful to mud snails, it is probably not healthy for consumable seafood and people, as well. Something that may be considered when making future plans of dredging not only in the Charleston Harbor but other waterways as well.

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Extracting the RNA of the mud snails. Picture taken by Samera Mulatu

I would like to give a big thank to Dr. Demetri Spyropoulos for guiding me in my research. Also to the Fort Johnson REU Program, NSF DBI- 1757899, for providing me with the funds to complete this project.

Related research

Hotchkiss, A.K, A.G.Leblanc, R.M. Sternberg. 2002. Synchronized expression of Retinoid X Receptor mRNA with Reproductive Tract Recrudescence in an Imposex- Susceptible Mollusc. Environ. Sci Technol. 42: 1345- 1351.

Ravitchandirane, V. S, M.Thangaraj. 2013. Phylogenetic Status of Babylonia Zeylanica (Family Babyloniidae) Based on 18S rRNA GENE FRAGMENT.Annals of West University of Timisoara, ser. Biology. 1(2): 135- 140.

Barron- Vivanco, B.S, D. Dominguez- Ojeda, I.M. Medina- Diaz, A.E. Rojas- Garcia, M.L. Robledo- Marenco. 2014. Exposure to tributyltin chloride induces penis and vas deferns development and increases RXR expression in females of the purple snail (Plicopurpura pansa). Invertebrate Survival Journal. 11: 204-2012.

Horiguchi, T., M. Morita, T. Nishikawa, Y. Ohta, H. Shiraishi. 2007. Retinoid X Receptor gene expression and protein content in tissues of the rock shell Thais clavigeraAquatic Toxicology. 84: 379-388.

Bat-Signal? Have you heard about Diatom-Signal?​​

Connor Graham, Francis Marion University

IMG_0079The approach: In my previous post I talked about using benthic microalgae (BMA) as bioindicators for South Carolina’s coastline. If they are truly the “superheroes” we need, we will be able to use BMA to test water quality that affects commercial and recreational fishing, tourism, and even human health. My job in all of this is to determine whether or not these diatoms are actually present and similar in the sediments of the saltmarshes. If they are similar in similar unimpacted habitats then they can be used as biological signals. The bat-signal illuminates the sky to alert the citizens of Gotham City that there is a problem and in the same way diatoms could potentially be our signal for the environment.

My team and I have traveled to five barrier islands on South Carolina’s coast to gather samples from the mudflat regions. On each island, I had three main sites, 0,10, and 100 meters. From these sites, each had a letter, A, B, C where our samples were collected. At 10 meters, each letter has three sub-sites.

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Field Sampling Map. Created by: Connor Graham

Using BMA as bioindicators will require the community structure to be similar among islands. Previously, I mentioned when concerning microbes we assume they are everywhere because of their incredible abundance, however that is not the case. I will look at the BMA community structure on the various islands and see if there is any correlation between them and geographical distance. If there is a correlation between community variation this relationship is called beta diversity and geographic distance, then is it possible that factors other than environmental one also affects the relationship between regional and local BMA communities (i.e. dispersal limitation).

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Edisto Beach Sampling Site. Photo: Connor Graham

Some of the environmental factors that were measured at each site at each island are sediment, air and water temperature, amount of light and PAR, humidity, wind speed, pressure and the amount of dissolved oxygen. Current, and water salinity were also measured. If the communities are dissimilar these measurements could be our contributing factors.

The collected samples from the salt marshes will also undergo an array of measurements that are also considered ecological factors. For example, each sample will be measured for the moisture content, organic matter, and chlorophyll. Moisture content data allows me to again compare the different mudflats to identify similarity. The same is for organic matter and chlorophyll. Chlorophyll a measurements, in particular, will allow my team and me to quantify the total mass of diatom species (biomass) of each island.

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Field supplies ready for the first day at Folly Beach. Photo: Connor Graham

The idea of microorganisms displaying geographical patterns is debatable. Some believe that patterns are based on ecological factors alone, while others believe that the community diversity geographical patterns are based on ecological factors plus historical factors such as dispersal limitation or competition (Soininen 2012). Either way, we will finally be closer to knowing whether diatoms can be the signals we are looking for. If they are will we be able to see the “Diatom Signal” warning us about the health of our coast and what will we do about it?

Acknowledgments

I would like to thank my mentors: Dr. Craig Plante and Kristina Hill-Spanik (CofC). Also, I would like to thank my lab partner Max Cook (CofC). This project is supported by the Fort Johnson REU Program, NSF DBI-1757899).

Literature Cited

Soininen J. (2012) Macroecology of unicellular organisms – patterns and processes. Environmental Microbiology Reports, 4(1): 10-22.

Gracilaria: What are you hiding?

Nick Partington, St. Olaf College

Screen Shot 2018-07-03 at 10.37.44 AMThe approach: In my previous post, I discussed how we will primarily be researching differences in abundance and diversity of fish and fish species that utilize Gracilaria vermiculophylla as habitat in the Charleston harbor. In order to do so, we have been collecting several samples of fish from the two habitat types this summer. We then sort and identify fish from each sample to determine the number of individuals per species found in each habitat type, and will later carry out statistical analyses to determine if any significant differences exist between the two habitat types. Each of these steps, from collecting to identifying to analyzing, consists of techniques that must be replicated for each sample in order to ensure consistency.

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Sampling in a “sparse” patch of G. vermiculophylla

The first step is to collect the samples. We do this at Grice Cove, just a few minutes’ walk from Grice Marine Lab. On site, we have identified a section where about 20% or less of the beach is covered by G. vermiculophylla. These are the “sparse” patches. The “dense” patches are further down the beach, where about 80% or more of the beach is covered by the algae. At each site, we pull a fifteen foot seine net through about 1-2 feet of water for a distance of 15 meters. We then sort through the net, saving all of the fish and discarding plant matter and invertebrates such as crabs and shrimp. The next step is to sort and identify the specimens that we collected.

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An early stage of sample sorting. This sample includes a flounder (upper right), pipefish (upper left), and several anchovies (middle).

 

 

After being fixed in preservatives for about a week, we sort through our samples, grouping identical fish and identifying specimens to the lowest classification possible (hopefully to the species level). After the sorting and identifications are complete, the numbers of fish of each species for each sample are recorded. Later, after we have collected all of our data, we will perform statistical analyses on the data to discern any significant differences in diversity and abundance of fish that might exist between dense and sparse patches of G. vermiculophylla. Stay tuned to hear about our findings!


Special thanks to Dr. Tony Harold for his guidance in this research project. This project is funded by the National Science Foundation and is supported by the Fort Johnson REU Program, NSF DBI-1757899.

Journey to the Center of the Pluff

Lauren Rodgers, Rutgers University

Version 2The Approach: In my previous blog post I discussed the importance of iron in ocean ecosystems. Because so many living things rely on iron to live and grow, it is important for us to understand how iron cycles, as it enters the ocean, exits the ocean, and changes from one form to another. Zetaproteobacteria are marine bacteria that rely on iron to create energy for themselves, but in this process, they also turn dissolved iron into solid iron. So these bacteria make rust as they grow. Unfortunately, rust isn’t very good for other organisms, and the Zetaproteobacteria effectually remove iron from the ocean. But still, these organisms are one half of the iron cycle and therefore play an prominent role. With our research, we aim to determine whether these bacteria are present in Charleston’s estuaries, and extrapolate how they might be impacting the local iron cycle.

Now, you most likely have one thing on your mind: How are they going to study all of this!? From our lofty research aims, we must simplify those down to into bite sized goals so we can have a successful summer of sampling.

Our Goals:

  1. Identify whether Zetaproteobacteria can be found in the sediments around Charleston.
  2. Measure the amount of Fe(II) and Fe(III) in the sediments

The first thing that we did in order to accomplish these goals is pick sampling sites. We wanted to sample the sediments for these Zetaproteobacteria, so we chose muddy regions close to tidal rivers that empty into the ocean. We wanted tidal rivers because Zetaproteobacteria live in salty waters, and these rivers mix with salt water from the ocean. We decided to look for these muddy regions along the Ashley River, Wando River, Stono River, and Cooper River, picking easily accessible sites far up the river where the water is fresher, midway down the river where the salt content is at a mid-range, and low down on the rivers, near the ocean, where the water is salty.

 

After identifying the sites that we wanted to sample at, we needed to figure out how to sample. We wanted to sample the mud at different depths, so we decided to use syringes to suck up the mud.

 

Once all of the samples were collected it was time to get back into the lab to analyze the data. In order to confirm the presence of Zetaproteobacteria we conducted PCR, which is a process that tells us if there was any DNA belonging to the Zetaproteobacteria in the samples.

 

 

 

To analyze the iron a ferrozine assay was conducted. In a ferrozine assay, different chemicals are added to the samples, which then turn different shades of purple depending on how much iron is present in them.

 

While we have already completed a lot of the data collection, we still have more to do. In the next few weeks we will focus on collecting the last few samples and analyzing them in the lab. Soon all of the results will be ready for interpretation!


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research. I would also like to thank the National Science Foundation for funding this research as well as the College of Charleston and Grice Marine Lab for their support.

Methods for the Manatees

Kaylie Anne Costa, University of Miami

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The Approach: In my previous post, I described cold stress syndrome (CSS) in Florida manatees and the major threat it poses to the survival of this integral species. To expand the current scientific knowledge of CSS, I will be analyzing the lipids (aka fats) and metabolites, which are the products remaining after biological processes such as digestion, respiration, and maintenance of homeostasis, in 12 healthy and 21 CSS-affected manatee plasma samples in hopes of learning more about the metabolism of this condition and potential avenues for therapeutic applications.

In order to study the lipids and metabolites in manatee blood, I will be using liquid chromatography and mass spectrometry (LC/MS) with an electrospray ionization source. Metabolomics and lipidomics will be separately analyzed. After a chemical extraction is performed to selectively separate either the lipids or metabolites in the plasma, each extract will be individually injected into the chromatographic column to separate the chemical compounds present so that only similar compounds are analyzed in any moment of time (methodology proposed by Bligh & Dyer, 1959 and Cambridge Isotope Laboratories, Inc.). Once the separated compounds reach the end of the column, they are

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Rescued manatee showing signs of cold stress syndrome. (Photo from: https://savethemanateenewtech.weebly.com/endangerment.html)

transferred to the electrospray ion source where a high temperature and voltage will be applied to evaporate the solvent and give the compounds a charge to form ions that are then directed into the mass spectrometer. Within the mass spectrometer, the ions will first be filtered by electric fields to remove anything other than either lipids or metabolites and then detected by mass to charge ratio. The most abundant ions will be fragmented and the mass to charge ration of the fragments will also be detected using an MS/MS scan. To see an animation of the flow of ions through the mass spectrometer, please click the following hyperlink: https://www.youtube.com/watch?v=_A6NBBBcdts

As a result of the above processes, retention times for each ion are displayed in a graphical form called a chromatogram and the mass spectrum is recorded. Since the masses and retention times will not change between scans, these parameters for each ion can be matched to known databases of known lipids and metabolites. By applying multivariate statistics, we can determine if there is a difference in the lipids and/or metabolites in the plasma of manatees with CSS compared to healthy manatees.

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The top left graph shows a chromatogram. The highlighted peak is then shown on the mass spectrum below with a mass to charge ratio (m/z) of 760.58607. By locating this m/z and the m/z of its fragments in the mass spectrum of a MS/MS scan and matching the values with a database, we know the original peak represents Phosphatidylcholine (16:0_18:1).                                        (Graphic by Dr. Mike Napolitano)

The goal of my project is to see if CSS alters the lipid and metabolite contents of manatee plasma. If differences exist, I will study them to learn more about the progression of cold stress syndrome in manatees and the particular systems and metabolic pathways that are affected. It is our hope that this information leads to developing both diagnostic and treatment options for these animals thereby reducing the impacts of this syndrome.


A huge thank you goes to my mentor Dr. John Bowden and co-mentor Dr. Mike Napolitano as well as everyone at NIST, HML, and Fort Johnson for all of their help and guidance. I would also like to thank the National Science Foundation for funding and the Fort Johnson REU program for making this research possible (NSF DBI-1757899).


References:

Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and

purification.Canadian journal of biochemistry and physiology, 37(8), 911-917.

Cambridge Isotope Laboratories, Inc. Metabolomics QC Kit For Untargeted/Targeted Mass

Spectrometry: User’s Manual. Tewksbury, MA: Author.

The Search for High Quality Data

Kelly Townsend, Elmhurst College

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Photo Cred: Ashley Shaw

The Approach: In my previous post, I mentioned the importance of sea turtles to ecosystems and ecotourism. While very important, the populations of these endangered animals are declining largely due to human impacts on our oceans. Headlines of sea turtles washing up on shore from such things as being strangled by plastic or boat strikes is no new occurrence. Since sea turtles are declining for reasons largely caused by us, so it is up to us to save these beloved animals. This study aims to investigate the stability of two important health indices, RNA and plasma protein in sea turtle blood, at different temperature treatments over time.  These indices are frequently used by researchers to answer health related questions. Therefore, my study will hopefully aid other researchers in determining if their samples are of the right quality to measure these indices.

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Collecting blood from a Loggerhead Sea Turtle (Caretta caretta) Photograph authorized by NMFS Section 10(A)(1)(a) permit 19621

For this project, whole blood was obtained from loggerhead sea turtles off the coast of South Carolin. The blood was collected in either Vacutainer blood collection tubes containing sodium heparin or PAXgene tubes. Sodium heparin tubes contain an anticoagulant and are typically used for blood collection. PAXgene tubes contain an RNA preservative; therefore, are best suited for RNA analysis. The sodium heparin tubes used for plasma were centrifuged on the boat to separate the blood components (i.e, plasma, white blood, and remaining cells) while the PAXgene tubes for RNA were left unspun. Once in the lab, the tubes were divided out into approximately 1.5ml aliquots in order to subject them to different treatments. Plasma was used for the plasma protein treatments while whole blood was used for the RNA treatments. Treatments in this study included 4⁰C for seven days, 20⁰C for three days, delayed freeze time, and never frozen. There were also treatments that lasted twenty-eight days consisting of storage in cryogenic conditions (< -150⁰C), -80⁰C, and -20⁰C (frost-free and non-frost-free freezers). Once the treatments end, the plasma will be analyzed for protein concentrations via plasma electrophoresis, and the whole blood will be analyzed for RNA quality via RNA isolation followed by a bioanalyser to obtain RNA integrity numbers (RINs).

 

As a result of the study, I hope to determine the conditions at which plasma proteins and RNA are most stable and begin to lose stability in order to aid scientists with their research. By knowing these conditions, this will hopefully guide others in deciding how to store samples along with which ones are best suited for a variety of analyses. In order to help the sick and endangered sea turtles, the highest quality of research will be necessary which means high quality data. I hope this study will guide researchers to make that possible.

I would like to thank Dr. Jennifer Lynch, Jennifer Trevillian, and Jennifer Ness with the National Institute of Standards and Technology for being my supportive and awesome mentors. This project was made possible by the samples collected by Dr. Michael Arendt and the funding from the National Science Foundation (NSF DBI-1757899) supported by the Fort Johnson REU program.