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)

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.

 

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

Samera Mulatu, Georgia Southern University

IMG-0640

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!

IMG-54671

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.

IMG-5214

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.

Why Cleaning our Oceans Could Also Reduce Obesity

Samera Mulatu, Georgia Southern University

The problem: Charleston Harbor is undergoing massive dredging to make way for super tankers. There are concerns that banned legacy compounds buried in the sediments, such as tributyltin (TBT), will be brought up into the harbor waters as a result of the dredging. A related concern is that oil spills will become more common and involve the release of cleanup compounds into the water column, including Span 80 and dioctyl sodium sulfosuccinate (DOSS). These three compounds and others are known to act as endocrine disrupting compounds (EDCs), which disrupt the Retinoid X Receptor (RXR) pathway. In mollusks, RXR disruption induces imposex development (when female mollusks develop male sex traits). One goal of my project is to measure the rates of imposex in the Eastern mud snail (Tritia obsoleta) within different sites in Charleston Harbor and to see if these rates increase over time with dredging.

The same chemicals that cause imposex are used in medications, processed foods (e.g. homogenized milk), textiles, paints, and cosmetics.  Because the identification and study of many EDCs is fairly new and upcoming, their effects on the human body are still not fully understood. Those EDCs found to promote weight gain are called “obesogens.” Early exposure to obesogens can detrimentally affect a child’s health into adulthood! These obesogens can persistently alter hormonal signaling pathways in children, which can lead to permanent metabolic damage.

Obesity in the United States is at an all-time high. Approximately half of the population is predicted to be obese by 2020.  This is a serious health problem because obesity can drastically increase the likelihood of developing cancer, diabetes, and heart disease. Many people associate obesity with lack of exercise. However, it should be noted that obesogens can reduce energy, increase appetite and change behaviors associated with weight gain.

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The author (right) collecting mud snails (Tritia obsoleta).  Photo: Dr. Spyropoulos.

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 clavigera. Aquatic Toxicology. 84: 379-388.