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.

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The Search for High Quality Data

Kelly Townsend, Elmhurst College

shark

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.

turtle pic

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.

Small Steps to Save the Sea Turtles

Kelly Townsend, Elmhurst College

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Turtle trawl on the R/V Lady Lisa. Photograph authorized by NMFS Section 10(A)(1)(a) permit 19621.

The problem: Do you like sea turtles? As for me, I have fallen in love with these cute creatures who occupy parts of the ocean. Seeing them pop their heads up or glide through the water always amazes me, but many species are endangered. A lot of effort has gone into saving them since sea turtles play an important role in the marine ecosystem. The marine ecosystem makes up a part of our world that is deeply loved but also threatened. Sea turtles help marine ecosystems function by limiting the amount of seagrass beds and sponges through consumption (McClenachan et al., 2006). Therefore, sea turtles presence in the environmental community is key to ecosystem restoration where their numbers have dropped and seagrass disease has been able to spread and coral overgrowth has increased. In addition, sea turtles also play an important role in ecotourism. Places like Costa Rica, United States, and Australia use sea turtles as a source of income by promoting tourism in areas where they live or nest, offering turtle walks, and selling souvenirs (Campbell, 2003). Since sea turtles act as an important resource for humans, there has been much effort into rehabilitating injured sea turtles and researching them in order to determine better prognostic indicators and courses of treatment. Sea turtles are important to us environmentally and economically, so saving them from going extinct requires the most reliable research and data possible to make that happen

 

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Turtle nesting beach located in Tortuguero, Costa Rica.

RNA and plasma proteins are both potential indicators for overall organismal health, but they can degrade quickly if not properly stored. Plasma protein concentrations in sea turtles can help wildlife veterinarians diagnose a disease and create a proper treatment plan (Gicking et al., 2004). Therefore, measuring plasma proteins in archived samples can indicate when or if a . particular disease developed in sea turtles. In addition, RNA concentrations and quality are good indicators of general health. High ratios of RNA/DNA has shown indications of increased cellular protein synthesis along with increased growth potential which means the sea turtle is growing properly (Vieira et al., 2014). However, in order to use archived samples to accurately track health indicators such as plasma proteins and RNA, it is vital to know if storage conditions allowed degradation of these molecules.

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Whole blood tubes used for RNA analysis.

This study aims to investigate RNA and plasma protein stability at different temperature treatments over periods of time. Samples will be maintained in favorable conditions along with unfavorable conditions to analyze the difference between the qualities. By knowing what happens on a molecular level to blood when storage conditions go wrong, we hope to eliminate the use of low quality samples used in research. Freezers malfunction, people forget to put samples away, and blood may not be put in the proper place so the results of this study will become a reference to those researchers who experience these tragedies.

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.

References:

Campbell L. 2003. Contemporary culture, use, and conservation of sea turtles. In: Lutz PL, Musick JA, and Wyneken J (Eds). The biology of sea turtles, volume 2. Boca Raton, FL:   CRC Press.

Gicking JC, Foley AM, Harr KE, Raskin RE, Jacobson E. 2004. Plasma protein electrophoresis of the atlantic loggerhead sea turtle, Caretta caretta. Herpetological Medicine and Surgery 14:13-18.

McClenachan L, Jackson JBC, Newman MJH. 2006. Conservation implications of historic sea turtle nesting beach loss. Front Ecol Environ 4:290-296.

Vieira S, Martins S, Hawkes LA, Marco A, Teodosio MA. 2014. Biochemical indices and life traits of loggerhead turtles (Caretta caretta) from cape verde islands. PLoS ONE 9:e112181.