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.

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Grac Attack!

Aaron Baumgardner, The University of Akron

The only thing more pervasive than the constant thoughts of, conversations about, and stress from Gracilaria vermiculophylla in Dr. Erik Sotka’s lab is the invasion of this red alga that is occurring along the coasts of North America and Europe (Sotka, et al. 2013). After only a few short weeks in Charleston, I have seen how prevalent and successful this seaweed is. The success of G. vermiculophylla along the southeastern coasts of the United States is due in part to the established mutualism between it and the decorator worm Diapatra cuprea. This mutualism provides a secure site for the seaweed to grow (Kollars, Byers, and Sotka unpublished manuscript), but it does not explain the success of G. vermiculophylla to thrive in environmental conditions that differ from its native Japanese range.

G. vermiculophylla colonizes a mudflat in Charleston Harbor by clinging to tube-building decorator worms. Credit, Erik Sotka

G. vermiculophylla colonizes a mudflat in Charleston Harbor by clinging to tube-building decorator worms. Credit, Erik Sotka.

Researching G. vermiculophylla can help us understand how aquatic invasions occur. Do introduced populations evolve novel characteristics or do they simply benefit from the phenotypic plasticity of their source populations? To answer this question, it is necessary to test plasticity in response to varying environmental conditions on native and non-native populations (Huang, et al. 2015). Since G. vermiculophylla has spread outside its latitudinal range and into high salinity environments (Kollars, et al. 2015), I will be testing the plasticity of native and non-native G. vermiculophylla populations to a range of temperatures and salinities. Photosynthetic efficiency will be measured using a PAM fluorometer to provide a more objective way to quantify stress (Rasher and Hay 2010).

I would like to thank the College of Charleston for this internship opportunity, Dr. Erik Sotka for mentoring me on my project, and the National Science Foundation for funding REU programs.

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References:

Huang, Q. Q., et al. (2015). Stress relief may promote the evolution of greater phenotypic plasticity in exotic invasive species: a hypothesis. Ecology and Evolution 5(6), 1169-1177.

Kollars, N. M., Byers, J. E.,  & Sotka, E. E. (unpublished manuscript). Invasive décor: a native decorator worms forms a novel mutualism with a non-native seaweed.

Kollars, N. M., et al. (2015, in review). Development and characterization of microsatellite loci for the haploid-diploid red seaweed Gracilaria vermiculophylla. PeerJ.

Rasher, D. B., & Hay, M. E. (2010). Chemically rich seaweeds poison corals when not controlled by herbivores. PNAS 107(21), 9683-9688.

Sotka, E. E., et al. (2013). Detecting genetic adaptation during marine invasions. Grant proposal to the National Science Foundation.

New Philadelphia to Charleston

Aaron Baumgardner, The University of Akron

Coming from the landlocked small town of New Philadelphia in the Midwest, I feel like I’m dreaming when I realize I’m spending my summer researching in Charleston, SC. I’m thankful for the opportunity that my mentor, the College of Charleston, and the National Science Foundation has given me to learn and grow in my scientific ability.

However, I do not believe I would be where I am today if it weren’t for my Aunt Jane. She is the only member of my family with a background in science, and even though she is hundreds of miles away at UPenn, she is always an email or phone call away. She has always shown an interest in my academics and will always be there for any advice I may ask. She has helped me develop my professionalism and offered insight on which graduate schools are worth going to.   Because of her, I can finally realize it’s not a dream. It’s reality that I’m spending my summer in Charleston, SC. It’s because I’ve worked hard in school and reached out for opportunities for me to mature as scientist. And I owe her so much for pushing me to succeed.

Thank you Aunt Jane!

Benthic Microalgae Research – A day at the beach

Jessica Lowry, Coker College

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I’m Jessie Lowry, a rising senior Biology major at Coker College, which is located in the tiny, homy town of Hartsville, SC. I am really excited to be here in my hometown this summer with opportunity to do research through the College of Charleston REU at Ft. Johnson. My research project this summer that I am working with Dr. Craig Plante on is investigating what factors influence the communities of benthic microalgae, or photosynthetic microorganisms in sediment.

Before we begin researching what species of diatoms make up the benthic microalgal communities, we need to do some preliminary sampling to measure things like pH, salinity, temperature, grain size, and moisture, at the three local beaches where I will be sampling at.

I’m not sure what I had envisioned for doing research this summer, but what I did not expect was for it to be like a day at the beach! Yesterday, Dr. Craig Plante and I went to Isle of Palms and Folly Beach, and today we are going to Kiawah.

My mentor, Dr. Craig Plante carrying research supplies out to the water at Folly Beach, SC.

We took several samples of seawater and sediment at each beach and we will do tests back at the lab. Unfortunately, I will not be spending every day researching at the beach. It was really great to get some sand, salt, and sun during a day of research.

Samples of seawater and sediment from Isle of Palms and Folly Beach that we collected to measure pH, salinity, grain size, and moisture.

Samples of seawater and sediment from Isle of Palms and Folly Beach that we collected to measure pH, salinity, grain size, and moisture.

Also, the temperature was in the 90s, the water was slightly warmer than the air, and the sand at one point at Folly Beach was a scorching 120° F! Dipping our feet in the water definitely felt great.

Funding

This research is funded by the National Science Foundation Reseach Experience for Undergraduates program at College of Charleston’s Grice Marine Lab.

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