RXR sequenced, now on to imposex

Samera Mulatu, Georgia Southern University

IMG-0640Findings: My experience at the Fort Johnson REU Program was phenomenal! Towards the end of the program, I was able to retrieve the RXR gene sequences for the Eastern mud snail. While working towards this goal, I was able to get a first hand glimpse of the long and hard steps and techniques taken to retrieve DNA sequences. From generating primers, doing dissections, extracting RNA, making cDNA, and even making PCR products, these listed skills are only just a short list of what I learned during this research experience. Retrieving the RXR gene sequences for the mud snail, was a trial and error process. Sequences were sent in at least five times, and four of those five times did not give good results. This was a big lesson for me, and reminded me that science is a trial and error process because all of it is a learning process.

Now that the RXR gene sequence for the Eastern mud snail was retrieved, the next steps in this project would be to use the sequences to place the mud snail in its proper spot on the phylogenetic tree. Also, now that the gene sequences are retrieved they will be used next fall by Edwina Mathis (a graduate at MUSC who’s doing her research in this topic) and Dr. Demetri Spyropoulos to induce imposex in the Eastern mud snail while exposing the snails to TBT, SPAN 80, and DOSS. Afterwards, they will measure changes in isoform expression.

The significance of the results from this study will hopefully show that mud snail imposex is a sensitive indicator of endocrine disrupting compounds in the environment which may impact human health and the health of other organisms in the ecosystem. This is because high imposex rates in mud snail species could possibly be linked to higher levels of contamination found in that site within the Charleston Harbor. Hopefully this study will further future research on EDCs and their effects on different species.

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.

 

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Saving Samples for the Sea Turtles

lil turt and me

Photo Cred: Kaylie Anne Costa

Kelly Townsend, Elmhurst College

Findings: What an amazing summer this has been! I have been working to discover the quality and stability of RNA and plasma proteins from loggerhead sea turtle blood in different storage conditions. The results have showed that plasma proteins are quite stable while RNA degrades at a much higher rate. Therefore, we were able to conclude that samples that have been stored for many years are still viable for plasma protein analysis but not RNA analysis.

Throughout the summer, I have participated in many amazing opportunities to explore different field work and sampling techniques. I was fortunate enough to go on a four day cruise to do a health assessment of juvenile and adult loggerheads, volunteer on a turtle nesting beach to survey the loggerhead nests, and have a behind the scenes tour of the turtle hospital located at the Charleston aquarium. Even though my research pertained to turtles, I was also able to go shark lil turttagging for a day. Each experience has taught me something new and I have loved every minute of it.

During this project, I have also acquired new lab techniques and life skills that will make me a better scientist. Working alongside my mentors who are a part of the National Institute of Standards and Technology (NIST), I learned meaningful organizational and professional skills that I will be able to apply in any lab I work in. I have also learned new techniques in the lab involving new instruments that I have never used before this summer. All this new knowledge will greatly help me throughout my career. Overall, I had an awesome experience conducting research this summer and I have acquired so much new knowledge to apply in my life.

A huge thank you to Dr. Jennifer Lynch, Jennifer Trevillian, and Jennifer Ness with the National Institute of Standards and Technology for being my supportive and fantastic mentors. I would not have been able to complete this project and have amazing opportunities without them. 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.

Zetaproteobacteria: The Journey Continues

Lauren Rodgers, Rutgers University

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Findings: This past summer, we have been working furiously with one goal in mind: measuring the concentrations of iron in the sediments around Charleston to identify if they would be a good habitat for Zetaproteobacteria. We scoured Charleston for the perfect muddy sampling sights and battled the pluff, almost losing a few boots in the process, while also gaining lots of bug bites. We then spent hours in the lab, making solutions, extracting iron, running the spectrophotometer, and collecting data.  And now, after 10 long weeks, it is coming to an end.

This summer of researching Zetaproteobacteria with Heather, Alejandra, and Sarg has taught me so many things. One of which is that science does not happen overnight. You may have an idea of how you are going to do something, but when it comes to actually carrying it out, odds are that it will not go as you think. Science is a dynamic process. You are trying things out, failing, brainstorming for other ways to do things, revising methods, and most of all learning. And this part of the process is what makes it exciting. I experienced this first hand when it came to conducting the ferrozine assay. Much of the research detailing methods for a ferrozine assay were only written for liquid samples, not for sediment samples, so we had to come up with methodology for extracting iron from the sediments. As you can imagine, this took a lot of trial and error. Trial and error such as figuring out the hard way that the water you are using to make up the solutions is actually contaminated with iron, or that glass cuvettes tend to contain their own concentrations of iron in them as well. It was frustrating at times,  but this process was so important for me because it was one of the first times that I was able to take in ideas from many different sources and develop something of my own. It was hard work, but it was all worth it in the end. We were able to collect samples, successfully extract the iron, and measure the iron concentrations, gaining some very exciting results in the end.

What’s next for Zetaproteobacteria?

Now that we have optimized the ferrozine assay for measuring the concentrations of iron in the sediments, we can continue our research on Zetaproteobacteria. The first objective that we will work towards is identifying if Zetaproteobacteria are actually present in the sediments. If they are found, we will then quantify how many Zetaproteobacteria are actually present.

If Zetaproteobacteria are found in the sediments around Charleston, it could have many implications. The first implication is that they could be affecting the local iron cycle around Charleston through their transformations of Fe(II). Zetaproteobacteria have also been shown to be able to live on solid metal and use the Fe(II) present in it, quickening the metal’s rate of rusting. If they are found in Charleston, they could be speeding up the rusting of ships or even metal pipes. Lastly, their presence in Charleston would add evidence to their potential worldwide distribution.

The project from this summer may be finished, but the Zetaproteobacteria journey has just begun!

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The Fullerton Lab Team stopping to pose while sampling at Kearns Park on the Wando River. (p.c. Heather Fullerton)


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research, and my Fullerton Lab members for assisting me in the field and in the lab throughout the summer. 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.


 

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.

 

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.