This Is How We Do It ♫

Julianna Duran, Virginia Tech

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First and foremost, if you didn’t get the reference in the title please click here!

Now that I have educated you on the topic of music, let’s switch to science.

 The Approach: In my previous post I mentioned that I am studying the lipids of Nile Crocodile and Mozambique Tilapia. So the first thing I did is wrestle the reptile like Steve Irwin and hand catch my fish – just kidding, but imagine how cool that would be! My samples were collected from Lake Loskop, South Africa in 2014. Once they were in my possession, here is what I did.

  1. Sample Preparation
    • The muscle tissue samples I received looked like chicken breasts you buy from the grocery store – except the size of a fat bean. These solid chunks need to be turned into a fine powder for me to analyze them. This was done by freezing the sample in the cryomill machine – where the samples were shaken extremely fast and broken up

      Cryomill

      Cryomill

  2. Extraction
    • Think of what happens when you pour oil in water. They go to different ends and don’t mix, right? (Yes) That is exactly what I’m doing with my samples. We are adding lots of chemicals to break down fats into their building blocks: Fatty Acids! The muscle layer (organic layer) hates touching the chemicals, so I take that out and can use it for my next step!
    • Check out a video I made of one of my extractions
  3. Gas Chromatography
    • This instrument is how I will measure the amount of each fatty acid in my samples.
    • How does it work?
      • The sample is injected into the system and enters a narrow glass column. The sample separates in this column based on its weight and boiling point. The particle encounters a flame at the end of the glass, which detects what specific fatty acid it is. The computer then gets this signal and generates a graph showing a fatty acid profile. Each peak on the graph is a different fatty acid, and the height of the peak indicates how much of it there is in the sample.
      • For help envisioning this process, take a look at this video (I used it when I learned about this instrument!)

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        Chromatogram

Summary:

I will be physically and chemically breaking down my samples, then getting fatty acid profiles for each of my individual species. This is all to see if there is a difference between healthy and diseased species and what lipids are most affected by Pansteatitis!


Supported by the Fort Johnson REU Program (NSF DBI-1757899), Dr. Mike Napolitano, Dr. John Bowden, The College of Charleston, NOAA, and NIST. 


References:

CryoMill. https://www.retsch.com/products/milling/ball-mills/mixer-mill-cryomill/function-features/ (accessed Jun 18, 2019).

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Crikey! What’s in the Water?

Julianna Duran, Virginia Tech

1B7047D7-DD01-4D65-B081-9D809AC07271The Problem: South Africa is home to some of the most extraordinary wildlife and culture. This diverse ecotourism plays a major role in their economy and conservation efforts.

Crocodile

Nile Crocodile (Photo credit: Darren Poke)

The Olifants River System in the Mpumalanga Province is a large source of water that provides a habitat for several species. Over the last 30 years in this region, there have been dramatic declines of Nile Crocodile (Crocodylus niloticus), fish, and waterfowl.

The cause of this is a disease called Pansteatitis. It is hypothesized that contaminants from coal mining and agriculture contributed to the emergence of the disease. Invasive species and the stagnant water may also be enhancing the intensity of its effects.

Pansteatitis is an inflammatory disease that affects the lipids, or fats, of an animal. The fats become tough which cause pain and a reduction in mobility that can make the species easier prey or unable to hunt for food.

Mozambique Tilapia (Oreochromis mossambicus) have been frequently diagnosed with pansteatitis and maintain a large population size. These characteristics make them a perfect model organism to use for researching pansteatitis – which is why they were selected for my project. I will be analyzing muscle tissue samples of these fish to compare the fatty acid profiles between healthy and diseased specimen; infected Nile Crocodile muscle will also be key in understanding how pansteatitis affects different organisms.

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Mozambique Tilapia – Photo taken from John Snow

It is important that we study Mozambique Tilapia to influence management efforts for top predators like Nile Crocodile, whose presence and actions impact the food web. In addition, tilapia and other fish are harvested and I want to ensure that any diseased fish caught are safe to eat. Although there have been no studies that have found whether or not this disease can directly affect humans, I hope that my study can give us an indication of the indirect human health risks.

Research Questions

  1. What is the difference in Fatty Acid Profiles between healthy and diseased Mozambique Tilapia?
  2. What is the difference between diseased Mozambique Tilapia and Nile Crocodile?
  3. What lipids are most affected by Pansteatitis?

This Summer, I will be investigating these questions and reporting back my findings. To find more information on the topics check out these links:

Blood Chemistry of Pansteatitis-Affected Tilapia

Life History of Mozambique Tilapia

Life History of Nile Crocodile


Supported by the Fort Johnson REU Program (NSF DBI-1757899), Dr. Mike Napolitano, Dr. John Bowden, The College of Charleston, NOAA, and NIST. 


References:

Bowden, J., Cantu, T., Chapman, R., Somerville, S., Guillette, M., Botha, H., Hoffman, A., Luus-Powell, W., Smit, W., Lebepe, J., Myburgh, J., Govender, D., Tucker, J., Boggs, A. and Guillette, L. (2016). Predictive Blood Chemistry Parameters for Pansteatitis-Affected Mozambique Tilapia (Oreochromis mossambicus). PLOS ONE, 11(4), p.e0153874.

Poke, D. 5 Interesting Facts About Nile Crocodiles. https://haydensanimalfacts.com/2015/03/04/5-interesting-facts-about-nile-crocodiles/ (accessed Jun 27, 2019).

Snow, J. Mozambique Tilapia. https://www.mexican-fish.com/mozambique-tilapia/ (accessed Jun 17, 2019).

Getting warmer…

Kaylie Anne Costa, University of Miami

IMG_6879Findings: In my previous post, I outlined how lipidomics and metabolomics would be used with mass spectrometry to study changes in the lipids and metabolites in manatee plasma in response to cold stress syndrome. The purpose of this study to provide deeper understanding how cold stress syndrome impacts Florida manatees

Our original research question was: Can changes in the lipidome and metabolome of plasma samples of Florida manatees be seen in response to CSS? Although the metabolomics data is still being processed, lipidomics has already shown promising results. Through our research we have found an interesting correlation between an

individual having a plasma Serum Amyloid A (SAA) value outside the healthy range and changes seen in their plasma lipidome. SAA is an acute phase protein produced in response to inflammation. When comparing the healthy manatee plasma samples to the CSS plasma samples with a Serum Amyloid A value greater than 50 µg/mL, we have found 81 lipids that differ significantly between plasma samples from healthy manatees and manatees with cold stress syndrome (Figure 1).

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Figure 1: Percentages of each lipid category out of the 81 total significant lipids that differed between CSS and healthy manatees

Our results indicate that the plasma lipidome of Florida manatees can differ as a result of cold stress syndrome. Now the next question is: what does this difference mean in context of manatees’ physiological response to cold stress syndrome?

This question is harder to answer, but we hope to be able to trace these lipids back to specific biological pathways that are altered by CSS. When the analysis of the metabolomic data is complete, we will have more pieces to the puzzle that may allow us to hone in on specific biological pathways affected by CSS that produce a change in both the lipidome and metabolome.

This pilot study will hopefully pave the way for future studies that will help protect this threatened species and conserve them as a sentinel species for studying how environmental changes will impact human health for the future.

This summer I have gained crucial research experience by using advanced techniques of analytical chemistry to address a threat to health in the marine environment. Through this REU program, I have learned about the diverse ecosystems in the Charleston area as well as the history that makes Charleston such a unique place. I would recommend the Fort Johnson REU program to any student looking for an opportunity to further their marine science education through research.

I cannot say thank you enough to my mentors Dr. John Bowden and Dr. Mike Napolitano. Their knowledge and eagerness to guide me through this process made this project possible. I would also like to thank the College of Charleston’s Grice Marine Lab for hosting the Fort Johnson REU program, National Science Foundation (NSF DBI-1757899)for funding, and our collaborators with the USGS Sirenia project for supplying the samples used in this study.

References:

Harr, K., Harvey, J., Bonde, R., Murphy, D., Lowe, M., Menchaca, M., … & Francis-Floyd, R. (2006). Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris). Journal of Zoo and Wildlife Medicine37(2), 151-159.

 

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.

flow chart

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.

Brrrrr…..

Kaylie Anne Costa, University of Miami

The problem: Do you hate the cold? Well manatees can’t stand it either! Every year Florida manatees (e.g., West Indian Manatee, Trichechus manatus latirostris) migrate to warmer waters during the winter months. In the past, they have used locations such as springs, layers of warm water created by salinity anomalies, and even the effluents from coastal power plants to stay warm. Unfortunately, new developments and recreational activities are taking over the natural warm water sources and most of the power plants are shutting down, so manatees have no haven for warm water.

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Manatees utilize the warm effluent water of a coastal power plant in Riviera Beach, Florida (Source)

When water temperatures fall below 20 °C (68°F), Florida manatees become susceptible to cold stress syndrome (CSS), which is a breakdown of normal biological and immunological processes that often leads to death (Bossart 2001). Manatees experiencing CSS have characteristic lesions and other symptoms like emaciation, lethargy, fat atrophyand loss, epidermal hyperplasia, pneumonia, and myocardial degeneration (Bossart, 2001; Bossart et al., 2003). CSS plays a major role in major manatee die off events and the number of cases continues to increase as manatees lose more and more warm water refuges to development and recreation. I will be expanding the current scientific knowledge of CSS by analyzing the lipids (aka fats) and metabolites, which are the products remaining after biological processes such as digestion, respiration, maintaining homeostasis, etc. in manatee plasma samples using mass spectrometry in hopes of learning more about metabolism for therapeutic applications.

Protecting Florida manatees is important for so many reasons. First off, the US Endangered Species act listed the Florida manatee as endangered in 2001, but recently reduced their status to only threatened in 2017 (Public Affairs Office, 2018). Without intervention, this species could easily return to its endangered status. Secondly, marine mammals are great sentinels to model how environmental changes will impact human health due to their physiological similarities, long life spans, and thick blubber’s ability to store large amounts of contaminants (Bossart, 2011). Thirdly, manatees help control the growth of sea grass beds. The presence of healthy sea grass beds allows the ecosystems around them to thrive. Lastly, manatees support economies through ecotourism. This research is necessary to protect Florida manatees from this understudied condition.

A huge thank you to my mentor Dr. John Bowden and co-mentor Dr. Mike Napolitano as well as everyone at NIST 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:

Public Affairs Office. (2018, February 7). Florida Manatee -Issues and Information. Retrieved June 17, 2018, from https://www.fws.gov/northflorida/manatee/manatees.htm

Bossart, G. (2001) Manatees. In: L. Dierauf & F. Gulland (eds.) Marine Mammal Medicine, pp. 939–960. CRC Press, Boca Raton, FL.

Bossart, G. D., Meisner, R. A., Rommel, S. A., Ghim, S. J., & Jenson, A. B. (2003). Pathological features of the Florida manatee cold stress syndrome. Aquatic Mammals, 29(1), 9–17.

Bossart, G. D. (2011). Marine mammals as sentinel species for oceans and human health. Veterinary Pathology, 48(3), 676–690.