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).

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).

Pansteatitis: A Fat Inflammatory Disease in South African Fishes

Jack McAlhany, Wofford College

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I applied for College of Charleston REU uncertain of the specific research I would partake in this summer. The email finally arrived containing my area of focus and I immediately told my parents “ This is perfect and will unify the topics I studied in school this year. I am researching a disease of the pancreas. My project is titled ‘Pancreatitis: an environmentally-induced inflammatory disease of South African fishes. ’ ” I arrived at Grice Marine Laboratory well-read on the etiology and symptoms of pancreatitis, only to find that an exchange of two little letters could make all the difference in my summer plans.

My research this summer was actually on panSTeatitis, a non-transmissible,  inflammatory disease of the fat tissue in South African fishes, particularly the tilapia. The interest for this research arose in 2008 when, as seen in Figure 2, 170 of the 600 Nile Crocodiles in Kruger National Park in South Africa died and the cause of death was attributed to pansteatitis (Ashton, 2010). The crocodiles are considered a sentinel species (Botha et al. 2011) as well as one of the main tourist attractions of the park, so this mobilized considerable interest into determining the causes of pansteatitis. After considerable research from various laboratories, the general consensus is that pansteatitis is the oxidation and eventual death of fat tissue seen in Figure 1 (Huchzermeyer, 2012), although the cause of the oxidation is still questioned. The proposed roots of pansteatitis are consumption of rancid, already oxidized fats, consumption of a high polyunsaturated to saturated fatty acids, which are more readily oxidized, or ingestion of metal pollutants that exacerbate the oxidation process (Huchzermeyer et al. 2013).

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Figure 1: Early pansteatitis lesion in mesenteric fat of sharp tooth catfish (left). Adipose tissue necrosis after developed pansteatitis in the same fish (right) (Huchzermeyer, 2012).

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Figure 2: Nile Crocodiles found dead in Kruger National Park due to pansteatitis (Rickrideshorses, 2011).

We will be researching the fatty acid composition of tilapia, which act as a model organism, and attempting to find a relationship among the fatty acids that will be able to act as a biomarker for determining whether an organism is affected with pansteatitis. This biomarker will allow for field testing using a simple blood test rather than the current method, which requires euthanasia to observe the fat tissue. A success in our research will facilitate sampling of organisms for pansteatitis and hopefully hone in on a potential cause of pansteatitis.

Acknowledgements

Funding for analysis of data is through the National Institute of Standards and Technology (NIST). Funding for sample collection is from Dr. Bowden, MUSC and Dr. Guillette, as well as collaborative South African laboratories.

NIST        MUSC

Bibliography

Ashton, P. 2010. The demise of the Nile crocodile (Crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in South Africa: The case of the Olifants River. Aquatic Conservation: Marine and Freshwater Ecosystems. 20: 489-493.

Botha, H., Van Hoven, W., Guillette, L. 2011. The decline of the Nile crocodile population in Loskop Dam, Olifants River, South Africa. Water SA. 37(1): 103-108.

Huchzermeyer, D. 2012. Prevalence of pansteatitis in African sharptooth catfish, Clarias gariepinus (Burchell), in the Kruger National Park, South Africa. Journal of the South African Veterinary Association. 83(1).

Huchzermeyer, D., Osthoff, G., Hugo, A., Govender, D. 2013. Comparison of the lipid properties of healthy and pansteatitis-affected African sharptooth catfish, Clarias gariepinus (Burchell), and the role of diet in pansteatitis outbreaks in the Olifants River in the Kruger National Park, South Africa. Journal of Fish Diseases. 36(11): 897-909.

Rickrideshorses. 2011. The Pansteatitis Pollution that Turned Crocodiles to Rubber. HubPages.