The BMA of Today

Christine Hart, Clemson University

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In previous blog posts I described the sand-dwelling microalgae, also known as benthic microalgae (BMA), which are essential to estuary ecosystems. Not only do they produce the air we breathe and food we eat, they also inform us about the subtle changes that are occurring in our environment. Changes that otherwise may go unnoticed.

How do BMA show these environmental changes? By forming the foundation of estuarine energy, they provide a snapshot of how the estuary is functioning as a whole. If changes occur in BMA patterns, this may indicate changes in the overall ecosystem. BMA are also easily characterized and compared using modern molecular approaches. These qualities make BMA living indicators, or bioindicators, that are important in monitoring future ecosystem health.

BMA become visible in the upper layers of sediment at low tide. Later, they decrease in density—or biomass—as the tide rises. Our project studied the mechanism for the increase of biomass during low tide. Previous studies suggested that the mechanism for biomass increase is vertical migration of BMA from lower layers to upper layers of sediment. We also tested whether BMA growth due to high light exposure contributes to the biomass increase.

Our results indicated that both vertical migration and growth due to sunlight exposure were important to the increase in biomass. This is the first contribution to literature that recognizes a multifaceted approach to BMA biomass changes.

Additionally, we studied in how the biomass increase was connected to patterns in the type of BMA in Charleston Harbor. Previous studies suggested that increasing biomass was connected to changes in the abundance of BMA species; therefore, we expected to see the amount of certain BMA species change based on their exposure to migration and sunlight.

We were surprised by our findings. In this study, we found that BMA did not vary over short time periods (by tidal stage or by exposure to migration and sunlight). Instead, we found that BMA varied spatially and over a period of 6 years. In fact, only one of the dominant species of BMA remained the same from 2011 to 2017 (Figure 1).  The long-term change in community coincides with geological changes in the sampling site (Figure 2).

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Figure 1. The relative abundance of each dominant BMA species from 2011 to 2017 is shown immediately after sediment exposure (T0) and 3 hours later (TF). Only one species—Halamphora coffeaeformis—remains dominant in 2017. This is evidence of a dramatic change in the dominant type of BMA in Grice Cove.

These are positive results for the use of BMA as bioindicators. If types of BMA are invariable over short periods of time, measurements of BMA will be more precise. Bioindicators must be capable of showing changes that are occurring on a larger environmental scale; therefore, it would be a good sign if the change in BMA community reflects the changing geological environment (Figure 2). Still, more studies on the temporal and spatial patterns of BMA communities should be conducted before BMA can be used as bioindicators.

Changes in Grice Cove

Figure 2. Aerial view of Grice Cove sampling site over time. The approximate location of the sampling site is shown by the white line. Sampling sandbar has changed over time, possibly contributing to community changes. Source: “Grice Cove” 32 degrees 44’58”N 79 degrees 53’45”W. Google Earth. January 2012 to March 2014. June 20, 2017.

This study contributed new information to the studies of BMA biomass during low tide, and showed that the BMA of today in Grice Cove are significantly different than in previous years.

 

Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance. This project is funded through the National Science Foundation and supported by College of Charleston’s Grice Marine Laboratory.

 

Literature Cited:

Holt, E. A. & Miller, S. W. (2010) Bioindicators: Using Organisms to Measure Environmental Impacts. Nature Education Knowledge 3(10):8.

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19:186-201.

Rivera-Garcia, L.G., Hill-Spanik, K.M., Berthrong, S.T., and Plante, C. J. Tidal Stage Changes in Structure and Diversity of Intertidal Benthic Diatom Assemblages: A Case Study from Two Contrasting Charleston Harbor Flats. Estuaries and Coasts. In review.

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Cells and Instruments, but no Folsom Prison Blues

Brian Wuertz, Warren Wilson College

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In my previous post, “Hiding in plain sight”, I introduced DOSS, a compound that has been recently identified as a probable obesogen. We are especially concerned about the potential of this compound to cause obesity symptoms in developing children through exposure from their mothers. While DOSS is in many products we use daily, such as homogenized milk and makeup products, it is commonly prescribed to pregnant women in the form of Colace stool softener. I am investigating both how much DOSS is in certain places in the body and how it may promote obesity.

One of the main concerns about obesity is that it elevates the risk of developing other diseases such as diabetes or cancer by causing a state of chronic inflammation (Bianchini 2002).  Chronic inflammation in  adipose tissue is regulated by immune cells, including macrophages. Macrophages are immune cells found throughout the body that help to fight against infection by recognizing invading bacteria and engulfing them in a process called phagocytosis, literally meaning to eat the other cells. In addition to phagocytosis macrophages are important regulators of the larger inflammatory response by secreting proteins that tell other cells to initiate or maintain a state of inflammation (Fujiwara 2005). This inflammatory reaction may be induced by DOSS. We have seen evidence of increased inflammation and obesity in mice treated with DOSS, so in order to figure out what causes that I am focusing on macrophages because of the way they regulate inflammation.

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I am isolating macrophages from breast milk samples under this hood in a sterile environment to make sure they are not contaminated with bacteria.

One way to study the inflammatory response of macrophages is to expose them to DOSS and then see if they produce the inflammatory proteins. Instead of trying to measure the secreted proteins, we can measure how much RNA is made in the cell. The RNA is the translator molecule that takes the plan for the protein from the DNA and makes it available for the cell to read and make the right protein. I identified genes for four different inflammatory proteins to measure the RNA so we can test if DOSS causes the macrophages to make more of any of them. I am testing macrophages that I am isolating from human placenta and breast milk tissue because the developing child is influenced by inflammation in the placenta and breast milk. Macrophages in these tissues could be the source of inflammation that influences how the child develops.

Okay so we have talked about cells, but what about the instruments? In my last post I introduced my instrument of choice, but did not call it that. It is not a guitar or a saxophone, but the HPLC, or high performance liquid chromatograph. This is simply a fancy instrument used to separate chemical compounds by forcing them through a tiny filter column filled with tiny beads. Some compounds stick more to the beads than others, so when you flow a liquid through the column the compounds come out of the column at different times. It is essential to separate the compounds in a sample because then you can measure the amounts of individual compounds.

We want to know where DOSS goes in the body, so we need to be able to measure how much of it is in a sample. I am working to get a system up and running to measure the amounts of DOSS in samples from different cells and tissues. We want to be able to measure DOSS in humans and in marine mammals such as dolphins. Dolphins are exposed to DOSS in the COREXIT oil spill dispersal agent that is applied to large and small scale oil spill issues along coastlines and in harbors. Dolphins are an important sentinel species, meaning that they can provide insight into human health issues.

I have to prepare a column and get the right mixture of solvents to make DOSS come off of the column in a timely fashion and in a way that we can measure it. The measurement is actually done with a mass spectrometer, which measures allows us to identify the compound based on how much it weighs. The number of atoms and types of atoms in the compound determine the mass of the compound. This mass is how the instrument measures the compound. The technique I am using is therefore called liquid chromatography mass spectrometry or LC-MS and the instrument is also referred to by LC-MS. Hopefully by the end of the summer I will be able to find beautiful data with this instrument that will make a coherent tune rather than a jumble of notes.

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This is the MS part. It measures the mass of the compound and then breaks it apart and measures the mass of the pieces of the compounds and the amount of the compounds.

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This is the LC or liquid chromatography part of the LC-MS instrument. Most of the work is figuring out the best solvent system to the sample through the small column with the red tag on it.

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

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References:

Bianchini, F., Kaaks, R., and Vainio, H. (2002). Overweight, obesity, and cancer risk. The Lancet Oncology 3, 565–574.
Fujiwara, N., and Kobayashi, K. (2005). Macrophages in Inflammation. Current Drug Target -Inflammation & Allergy 4, 281–286.

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.

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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!

 

 

 

The Problem with PFCs- Seeking Answers in Plasma

Kady Palmer, Eckerd College

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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

Some Dramatic Microorganisms and Targeted Genetic Analysis

Emily Spiegel, Bryn Mawr College

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Genetic analysis has become the name of the game in many fields of biological research. Genes encode proteins, and in biology, proteins are king. Proteins guide biological pathways throughout the entire organism, so if you can track the genes, you can understand how the animal functions. Advances in technology like CRISPR, RNA sequencing, and PCR have improved the accessibility and accuracy of high-level genetic analysis in laboratories across the world. Some scientists utilize this technology to study the entire genome of an organism, while others attempt to understand the response of specific genes to various environmental factors or other external influences. This summer, I’m conducting an experiment focused on the latter. I’ll be studying how the polar algae species, Fragilariopsis cylindrus (affectionately known as Frag) copes with environmental stress by reproducing sexually. To do so, I’ll use targeted RNA sequencing to track genes related to sexual reproduction.

In order to understand how a Frag, responds to environmental stresses, you need a lot of algae. I reared nearly 100 liters of this algae in different artificial conditions. These conditions varied by two factors: photoperiod (the length of day and night), and nutrient levels. If you missed my previous post, “Stressing Out My Algae,” you should check it out for more details on the background for this experiment. We suspect that in conditions of stressful light energy (24 hours of continuous light), Frag will respond by reproducing sexually as opposed to its normal asexual mode of reproduction. This could possibly be a mechanism to rid itself of excess energy in times of stress, since sexual reproduction is more energetically expensive than asexual reproduction. By reproducing sexually, Frag may improve its chances of survival against this stress. Compounded with this is our hypothesis on nutrient deprivation. Previous experiments have shown that when a major nutrient, nitrogen, is limited, the algae cannot grow at full capacity and sexual reproduction is inhibited. We predict that when the stress of nitrogen limitation is combined with the stress of high light energy, we’ll see a reduction in the algae’s ability to survive in the stressful conditions due to the inhibition of sexual reproduction. So if we stress out the Frag enough and take away their ability to have sex, they’ll probably die. They’re some very dramatic microorganisms.

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24 bottles of algae were grown in six different experimental conditions varied by length of light exposure and nutrient levels. Algae was reared in 4-liter bottles filled with seawater.

So we grew our Frag, four bottles per six experimental conditions. Every day for eight days we extracted biomass from the bottle. From this sample we could test chlorophyll levels and cell counts, both of which give us a good idea of how well the algae in that bottle are growing in their conditions. We also took samples to be used for RNA extraction. Remember how genes encode proteins and proteins are king? Well before you can have your protein product, you need RNA. You’ve probably heard of DNA, which is the double stranded genetic cookbook. RNA is its single stranded offspring, which is then used as a the direct template to make proteins. A lot of genetic analysis therefore looks at RNA instead of DNA in order to understand how genes are being transcribed for protein production. We’re currently working on extracting the RNA from the original biomass sample and then running that pure RNA through a specialized machine called Nanostring. This is extremely targeted analysis, as Nanostring focuses in on the specific RNA we’re most interested in. In this case, we’re interested in RNA which is encoded from genes related to sexual reproduction. Using Nanostring will tell us how active the genes for sexual reproduction are in each bottle, which we can analyze to derive any correlation between our environmental stress factors and sexual reproduction.

If our hypothesis is correct, then we’ll see the greatest expression of sexual reproduction genes in the conditions of high light energy (24 hours of continuous light). We’d expect to also see low growth performance in nitrogen limited populations, indicated by low cell counts and chlorophyll levels. In these populations we predict we’ll see little if any expression of genes related to sexual reproduction. By the end, we’ll hopefully have a clearer picture of how phytoplankton like Frag deal with environmental stress.

Funding for this project is provided by the National Science Foundation in collaboration with the College of Charleston Grice Marine Laboratory and the National Oceanic and Atmospheric Administration. Acknowledgements to the entire lab of Dr. Ditullio and Dr. Lee in the Hollings Marine Laboratory facility.

Searching in the Sand

Christine Hart, Clemson University

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In “Exploring the Secret Garden” I discussed our studies of the benthic microalgae (BMA) that inhabit the intertidal regions of beaches. The goal of our study is to identify the mechanisms involved in the visually noticeable increase of BMA during low tide. This mechanism will be linked to changes in the type of BMA dominating the sand flat. To accomplish these goals our study will incorporate field work, molecular techniques, and DNA analysis.

During field work we will collect and manipulate sediment to distinguish between an increase in BMA by either vertical migration or growth mechanisms. The sediment will be collected on a sand flat in Grice Cove (Figure 1). Sand will be sampled using corers, which pick up a layer of sand without disturbing the vertical organization. The collected sand will be split between measurements of biomass, or BMA density, and DNA analysis. Biomass is measured by finding the concentration of chlorophyll a in the sediment. BMA synthesize chlorophyll a; therefore, the concentration of chlorophyll a is proportional to the density of BMA.

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Figure 1. Aerial view of Grice Cove sampling site with the approximate location of the 50 m sand flat transect site. Sampling sand flat is open to the Charleston Harbor. Source: “Grice Cove” 3244’58”N 7953’45”W. Google Earth. March 20, 2017. June 20, 2017.

The methods for field work are represented in Figure 2. There are two vertical migration treatments: filter and mesh. Filter treatments prevent vertical migration between cored and surrounding sediment. Mesh treatments permit vertical migration. If migration is important to the biomass increase, biomass measurements in mesh will be greater than in filter treatments. Filter and mesh treatments will also be exposed to shade and light conditions to interpret the impact of growth on biomass. Sunlight provides the energy necessary for BMA growth. Without sunlight growth will be limited. If growth is the mechanism of biomass increase, the shaded samples will have a lower biomass than the light exposed samples.

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Figure 2. Field work methods visualization. Locations of replicates along the 50 m transect are chosen using a random number generator and marked with flags. Random coordinates and a quadrat of 50 cm by 50 cm are used to determine where sediment will be sampled and treatments will be placed. Three controls (T0, TM, and TF) are taken at time intervals 1.5 hours apart after sand exposure. During TM and TF time points, samples are taken from the 4 treatments shown above: filter, mesh, filter + shade, and mesh + shade. Filter treatments prevent vertical migration, while mesh treatments permit vertical migration. Shaded and non-shaded filter and mesh treatments will be important in determining the role of sun exposure in biomass increase.

To link the mechanism of biomass increase to the BMA composition, we will use molecular techniques and analyze the DNA found in the sediment. DNA will be extracted from the sediment and amplified using a polymerase chain reaction (PCR). The DNA will be sequenced using High Throughput Ion Torrent technology. The results from sequencing will identify the BMA present at each time point and within each treatment. This information will link the mechanism of biomass increase to the changes in BMA composition. Our understanding of BMA dynamics will establish a basis for the BMA ecology in the Charleston Harbor. In the future, BMA dynamics could be compared to our study to assess changes caused by human influences in Charleston estuaries.

 

Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance. This project is funded through the National Science Foundation and supported by College of Charleston’s Grice Marine Laboratory.

 

Literature Cited:

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19: 186-201.

Playing with Plutei

Hailey Conrad, Rutgers University

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Me! Photo Credit: Kady Palmer

Ocean acidification is known as climate change’s evil twin. When the pH of ocean water drops, carbonate ions in the water form carbonic acid instead of calcium carbonate. Calcium carbonate is the form of calcium that marine animals that have calcium-based skeletons (like us!) and shells use to build their bones and shells. Having smaller and weaker skeletons or shells impacts their ability to survive. However, some individuals within certain species or populations of species have genes that make them more resistant to ocean acidification. If these individuals are able to pass on these genes to their offspring, then the species has the ability to evolve in response to ocean acidification instead of going extinct. This summer I’m working with Dr. Bob Podolsky in College of Charleston’s Grice Marine Field Station to study the extent to which ocean acidification affects Atlantic purple sea urchins, Arbacia punctulata. We are specifically trying to see if any individuals within a population from Woods Hole, Massachusetts, have any heritable genetic resistance to the negative impacts of ocean acidification. We hypothesize that there will be genetic resistance given that the northern Atlantic coast naturally has lower levels of saturated calcium carbonate, so a population that has evolved to live in that type of environment should have some resistance to lower calcium carbonate levels already (Wang et al 2013). We’re using a basic cross breeding technique to rear Arbacia punctulata larvae to their plutei stage, when they have four main body rods. At this stage they look less like sea urchins than they do like Sputnik!

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A sea urchin pluteus larvae with four body rods

Then, we will look to see if any of the male parents consistently produce male offspring that are more resistant to ocean acidification.  If males like these exist within this population, then the species has the capacity to evolve in response to ocean acidification, instead of going extinct! This is a very big deal, and could potentially be very hopeful. Even if we don’t get the results that we are hoping for, the results of this research could inform policy and management decisions.

Literature Cited:

Wang, Z. A., Wanninkhof, R., Cai, W., Byrne, R. H., Hu, X., Peng, T., & Huang, W. (2013). The marine inorganic carbon system along the Gulf of Mexico and Atlantic coasts of the United States: Insights from a transregional coastal carbon study. Limnology and Oceanography, 58(1), 325-342. doi:10.4319/lo.2013.58.1.0325

Thank you to the National Science Foundation and College of Charleston’s Grice Marine Laboratory for funding my project. And, special thanks to Dr. Bob Podolsky for being a wonderful and supportive mentor!