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.

 

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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

css manatee

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.

The Search for High Quality Data

Kelly Townsend, Elmhurst College

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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.

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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.

Bacteria in the Ocean? That Eat Iron??

Lauren Rodgers, Rutgers University

Version 2The problem: Have you ever asked yourself, what is iron? It is an element? A rock? Some weird orange-ish substance? Is it the tool that you use to get the wrinkles out of clothes? And what does iron even do? Does it just sit there? Does anything eat it? Can we make things out of it? Iron is one of the most abundant elements on earth, yet not many people know much about the important role it plays in our lives.

Iron is more than just an element, or something found within a rock. It’s a nutrient, something necessary for the growth and metabolism of almost every living organism on Earth (Hedrich & Johnson, 2011). In the ocean, iron is found in two different forms, ferrous iron or Fe(II), which is soluble in water, and ferric iron or Fe(III), which is insoluble in water (Hedrich & Johnson, 2011). Because ferrous iron is soluble it is the form of iron that can be used by most organisms in the water (Hedrich & Johnson, 2011). This ferrous iron, however, is limited in the ocean despite its abundance in the Earth’s crust. In fact, Fe(II) is present only in incredibly small concentrations, making it a major limiting factor of growth for all of the plants and algae in the ocean. This is important because these plants and algae serve as the base of many food chains, so if there is a limitation on the growth of these organisms, it affects every other organism throughout the food chain. Though iron is an extremely important nutrient for many living organisms, it is still not well understood. One of the least understood aspects is how iron specifically cycles through different marine environments. Does it ever change form? Does anything add iron to the ocean? Does anything take iron out of the ocean? These questions bring us to Zetaproteobacteria.

Zetaproteobacteria is a recently discovered class of iron-oxidizing microbes. This just means that the bacteria eat iron in the form of Fe(II) and produce Fe(III) as a waste product (Emerson et al., 2007; Chiu et al., 2017). In fact, these waste products can take on the form of hollow tubes, also called tubular sheaths, or twisted stalks that you can see under the microscope!

 

Zetaproteobacteria were initially described in 2007 near hydrothermal vents, utilizing the large concentrations of Fe(II) that were present in the fluid that spewed from the vents (Emerson et al., 2007).

Iron Mat

Iron mat composed of Zetaproteobacteria on a lava rock near the submarine Loihi volcano. (A. Malahoff, Hawaii, Loihi Volcano, July 1988)

How do Zetaproteobacteria relate to the cycling of iron? 

Zetaproteobacteria, with their role in eating iron and transforming it from its soluble Fe(II) state into its insoluble Fe(III) form may have an important role in the cycling of iron through the environment, functioning as an important source of iron removal.

Since their discovery, Zetaproteobacteria have also been observed in many other habitats, including coastal estuarine habitats with lower levels of iron, similar to that of Charleston, SC. (Laufer et al., 2017; Chiu et al., 2017). Our study will try to identify if these Zetaproteobacteria are present in the muddy soils around Charleston, as well as measure the levels of Fe(II) and Fe(III) in the rivers where these bacteria may be found.

 

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Hopefully, through the study of the distribution of Zetaproteobacteria across the globe, including the chemical characteristics of the different environments that they inhabit, we may get a clearer picture of how iron cycles in aquatic environments and the role that these Zetaproteobacteria play.


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research. I would also like to thank the National Science Foundation for funding this research as well as the College of Charleston and Grice Marine Lab for their support.


References 

Chiu, B. K., Kato, S., McAllister, S. M., Field, E. K., & Chan, C. S. (2017). Novel pelagic iron-oxidizing Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone. Frontiers in Microbiology, 8(JUL), 1–16. https://doi.org/10.3389/fmicb.2017.01280

Emerson, D., Rentz, J. A., Lilburn, T. G., Davis, R. E., Aldrich, H., Chan, C. S., & Moyer, C. L. (2007). A novel lineage of proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities. PLoS ONE, 2(8), e667. https://doi.org/10.1371/journal.pone.0000667

Hedrich, S., Schlömann, M., & Johnson, D. B. (2011). The iron-oxidizing proteobacteria. Microbiology,157(6), 1551–1564.

Laufer, K., Nordhoff, M., Halama, M., Martinez, R. ., Obst, M., Nowak, M., … Kappler, A. (2017). Microaerophilic Fe(II)-oxidizing Zetaproteobacteriaisolated from low-Fe marine coastal sediments – physiology and characterization of their twisted stalks. Applied and Environmental Microbiology, 83(February), AEM.03118-16. https://doi.org/10.1128/AEM.03118-16

Mori, J. F., Scott, J. J., Hager, K. W., Moyer, C. L., Küsel, K., & Emerson, D. (2017). Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. Nov. ISME Journal, 11(11), 2624–2636. https://doi.org/10.1038/ismej.2017.132

Living Life as a Sea Urchin Momma

Hailey Conrad, Rutgers University

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Me working hard to make my sea urchin babies

For my project I am using the same technique that the father of genetics, Gregor Mendel, used to establish his Laws of Heredity: cross breeding. So, I have to breed and raise a whole lot of sea urchins. For a refresher, I’m trying to determine if there is heritable genetic variation in how sea urchin (specifically an Arbacia punctulata population from Woods Hole, Massachusetts) larvae respond to ocean acidification. To do this, I’m rearing sea urchin larvae in low and high carbon dioxide conditions and measuring their skeletal growth. I’m breeding 3 sea urchin males with 3 sea urchin females at a time, for a total of 9 crosses. To tease apart the impact of genetic variation on just the larvae themselves, I will be fertilizing the sea urchin eggs in water aerated with either current atmospheric levels of carbon dioxide, about 410 parts per million, or 2.5 times current atmospheric carbon dioxide levels, about 1,023 parts per million. Then, I will be rearing the larvae in water aerated with either 409 ppm CO2 or 1,023 ppm CO2. This will give me four different treatments for each cross, giving me 36 samples in total. By fertilizing and rearing them in the same and different levels of carbon dioxide I will be able to see how much of an impact being fertilized in water with a higher carbon dioxide concentration has on larval growth versus just the larval growth itself. It’s important for me to make that distinction because I just want to identify genetic variation in larval skeletal growth, and separate out any extraneous “noise” clouding out the data. I’m rearing the larvae in a larval rearing apparatus. Each of the 36 samples will be placed in jar with water aerated with the correct CO2 treatment. Each jar will constantly have atmosphere with the correct CO2 concentration bubbled in. Each has a paddle in it that is hooked to a suspended frame that is swayed by a motor. This keeps the larvae suspended in the water column. The jars are chilled to 14 C by a water bath.

IMG_9756_Moment

My larval rearing apparatus

After a 6-day period the larvae are removed from the jars and their skeletal growth is measured. They are preserved with 23% methanol and seawater and frozen.

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An Arbacia punctulata pluteus

You’re probably curious how the heck I am able to measure the larva’s skeletons. They’re microscopic! Well, I use a microscope coupled to a rotary encoder with a digitizing pad and a camera lucida. Which, looks like this:

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A microscope coupled to a rotary encoder with a digitizing pad and a camera lucid hooked up to a computer

This complicated-sounding hodge-podge of different devices enables me to do something incredible. I can look through the microscope at the larva, and also see the digitizing pad next to the microscope, where I hold a stylus in my hand. When I tap the pad with the stylus and the coordinates of various points on the anatomy of the plutei that I am tapping at get instantly recorded on my computer! The rotary encoder is the piece attached to the left side of the microscope and it enables me to record coordinates in three dimensions. Then, I can use those coordinates to calculate the overall size of the skeleton. My favorite part of doing science is learning how scientists are able to do the seemingly impossible- like measuring something microscopic.

After I gather all of my data, I will do some statistical analysis to see the affect that the male parents have on the skeletal growth of their offspring. I will not be focusing on the impact that females have on the skeletal growth of their offspring. The quality of the egg itself could be an influencing factor on the size of the offspring, whereas sperm is purely genetic material. Like how I’m trying to isolate the influence of ocean acidification during larval rearing from during the act of fertilization, I am trying to isolate just genetic influences on larval skeletal growth from egg quality. Check back to see how it goes!

Special thanks to the National Science Foundation for funding this REU program, the College of Charleston and Grice Marine Laboratory for hosting me, and Dr. Bob Podolsky for mentoring me!

 

 

 

Catch of the Day(s)

Melanie Herrera, University of Maryland College Park

South Carolina is known for its iconic southern cuisine, including a staple of fresh seafood which fuels the buckets of shrimp & grits and “catch of the day”. In order to support this huge industry (and fill the bellies of every South Carolinian), I am conducting an experiment to figure out where this seafood is holing up prior to its demise. Dr. Harold, his graduate student, Mary Ann McBrayer, and I are out on Grice Beach collecting fishes, crabs, shrimps, and much more in order to figure what exactly is there… And what they are using to survive.

Using a seine net, we encircle marine animals in dense and sparse patches of an invasive sea grass, Gracilaria, for collection. We hypothesize that Gracilaria is helping the local economy (a surprising contribution from an invasive species) by creating refuge for young animals. On the beach, we submerge separate samples of animals (from dense versus sparse areas of Gracilaria) into a euthanizing solution to bring them up to the lab for preservation and analysis (Figure 1).

Dense v Sparse.png

Figure 1: An example of animals caught in separate habitats at Grice Cove. The left exhibits animals caught in a dense area of Gracilaria and the right exhibits animals caught from a sparse area of Gracialaria. Credit: Melanie Herrera

In the lab, separate samples (dense versus sparse) undergo a few transfers into different fixatives (10% seawater formalin, 25% isopropyl, and 50% isopropyl consecutively) to keep the fish from decaying. After this preserving process, fish and other animals are separated and categorized by family, genus, and species. This categorization enables us to identify and analyze what types of animals and how many of each are using different habitat. Our analysis will give us insight on what type of habitat, either patches dominated by Gracilaria or areas with more open water, benefits fish. Specifically, we will be able to identify if Gracilaria is more advantageous to young fish or if their survivorship is independent from their habitat.

So far, we have collected lots of pipefish, narrow skinny fish that resemble a hair strand-size snake, Atlantic Silversides, a fish that looks exactly like it sounds, and more shrimp than anyone needs (Figure 2). Although some of these animals do not directly contribute to the seafood industry, its presence in the Charleston Harbor can tell us a lot of things. For example, we have seen some fishes that usually stay in warmer waters in the Southern U.S. Their expanding habitat can lead us to some more hypotheses on climate change and warm weather moving northward. In addition, we can find out if Gracilaria has a stake in rearing economically important fish in the future.

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Figure 2: (From left to right) Pipefish, Atlantic Silversides, and Grass Shrimp caught for analysis.Credit: Melanie Herrera

Thank you so much to my mentor Dr. Tony Harold and his lab for his advice and guidance. Thank you to Mary Ann McBrayer for helping me facilitate this project. This research is funded through the National Science Foundation and College of Charleston’s Grice Marine Lab.

 

The Problem with PFCs- Seeking Answers in Plasma

Kady Palmer, Eckerd College

Manatee_CR

I previously outlined the problem of perfluorinated chemicals (PFCs) in the environment and their unknown health effects.  In order to gain this knowledge, it is essential to determine what types of PFCs are frequently used and the mechanisms by which an individual would be exposed to them. Here, we are measuring the presence or absence of 15 PFCs that are commonly associated with non-stick cookware, firefighting foam, and water-resistant materials.

This compiled list of PFCs is the basis of my research procedure. From here, I must learn how these compounds interact with biological components in organisms in order to understand their subsequent health effects. With that being said, the type of samples I am analyzing is a topic worth explaining. PFCs are known to be “proteinophilic” or, attracted to proteins in the bloodstream of organisms like humans and, in the case of my study, manatees. Therefore, I am using manatee plasma to test for the total individual burden of PFCs. 

PFAAs1       PFAAS2

Fig 1. 69 collection tubes containing manatee plasma samples (left). Aliquots of 22 samples of manatee plasma for future studies (right). Photos taken by me!

With 69 different plasma samples, I am performing a series of procedures that allow me to extract the PFCs. After completing multiple chemical processes (methodology proposed by Reiner et al., 2012), I am left with a liquid (containing the PFCs), measuring no more than 1 mL to be placed into a small vial. From here the vials are inserted into a liquid chromatography tandem mass spectrometer (LC-MS/MS), a machine that reads each of the 15 unique chemical structures of the outlined PFCs of interest and determines their abundance in each vial. This system isolates the concentration of each perfluorinated chemical for every one of the 69 manatee samples.

Mass Spec

Fig 2. The basic process a mass spectrometer performs in order to provide the concentration of chemicals being studied. Photo from: http://www.emdmillipore.com/US/en/water-purification/learning-centers/Anwendungen/organic-analysis/lc-ms/lWib.qB.vb4AAAFA5fIBvVBh,nav?ReferrerURL=https%3A%2F%2Fwww.google.com%2F&bd=1

The concentrations of these chemicals is the ultimate goal of my research study. This data will be compared to manatee location, morphometrics, body condition, sex, and more, in order to gain a better understanding of the overall PFC burden on these animals. These factors, or variables, may also provide insight into what may be influencing the burden intensity an individual may face. Once this knowledge is gathered, potential links to the health effects of PFC accumulation can be investigated in both manatees and humans.

I’d like to thank the National Science Foundation for funding this research opportunity and the College of Charleston’s Grice Marine Laboratory REU program for making this experience possible. A special thanks to the NIST team who has been teaching and supporting me throughout this process, specifically, Dr. Jessica Reiner, Jacqueline Bangma, and my mentor, Dr. John Bowden.

Note: These samples were collected as part of a health assessment of manatees by the USGS Sirenia Project. No manatees were harmed in the process of obtaining them.

References

Reiner, Jessica, Karen Phinney, and Jennifer Keller. “Determination of Perfluorinated Compounds in Human Plasma and Serum Standard Reference Materials Using Independent Analytical Methods.” Analytical & Bioanalytical Chemistry 401, no. 9 (January 15, 2012): 2899–2907. doi:10.1007/s00216-011-5380-x.z