Peptide Puzzle Pieces: Perceiving Peculiarly Produced Proteins

Jackson Eberwein, Sonoma State University

The Approach: In my previous post, I discussed some of the problems about Domoic Acid Toxicosis in California sea lions, closing with the potential benefit of finding a biomarker protein. The first step in finding anything is to take a good look! How does someone look at something as small as a protein, though? When trying to see which proteins are in a sample of blood plasma and count how many there are of each, it can get complicated. The way I am achieving this is by using an instrument called a mass spectrometer. With it, I can predict the identity and amount of each protein in a sample. The mass spectrometer is picky, however. To read my proteins, it wants them to be cut up first. 

Orbitrap mass spectrometer (right) with attached liquid chromatograph (left).

Proteins are similar in structure to a tangle of string. The tangle first has to be unraveled, then it is cut up into small bits called peptides. These peptides are put into the mass spectrometer to be read. The shape of each peptide is pretty unique, and that unique shape is used to detect and measure them. With the help of some very handy computer programs, peptide measurements can be compared to a California sea lion genetic database to predict the protein that each peptide came from and how many of those proteins there might have been in the sample. Once we have the names and amounts of the proteins in the sea lion samples, the protein differences between each sample can be looked at. This is where we look for our biomarker. If there are one or more proteins that appear at consistently different levels in sea lion samples with Domoic Acid Toxicosis than in samples without it, those proteins have potential as good biomarkers! 

Acknowledgements

I would like to thank Dr. Michael Janech, Dr. Benjamin Neely, Alison Bland, The Marine Mammal Center, & College of Charleston. Supported in part by the Fort Johnson REU Program, NSF DBI-1757899.

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One Fish, Two Fish…

Ana Silverio, The University of Texas at Austin

The Approach: In my previous post, I explained how important small fishes are to the food web and how their new found interaction with Gracilaria vermiculophylla came about. Now, measuring something such as diversity and abundance may sound confusing but it’s as simple as one, two, three!

Abundance is the number of individuals per species in an ecosystem and relative abundance is the overall evenness of those individuals. Diversity is more of a measurement of variation or how many different species are counted in a designated area/habitat.

Fine mesh seine net being dragged over the 15-meter transect to capture our fish.
Photo Credit: Norma Salcedo

Now that we understand what we are measuring… what’s next? As mentioned before, the Charleston harbor has been introduced with an invasive species of seaweed, but it has served as a home for the juvenile fish. To measure diversity and abundance we have to take samples from two different sites affected by this invasive species. Luckily, it’s a short stroll over to Grice Beach behind our marine lab to find a section of Gracilaria with 20% coverage for our sparse site and one with 80% coverage for our dense site. After establishing our sample sites, we take a 15-meter transect which we will pull our fine-mesh seine net through at about knee-deep water. We quickly but gently pull the net up to the beach and start sorting through our samples placing the fish in a half-gallon jar while discarding any invertebrates. We repeat this at our second site and voilà we have our samples!

Initial sorting process for our samples
Photo Credit: Norma Salcedo

Are we done yet? Of course not! Once we collect both of our samples from the different patches of Gracilaria, we take them back to the lab to set in preservatives for about a week and begin the sorting process. While we sort each jar, we try to identify each fish down to the lowest classification if possible (in a perfect world we would have all of our critters down to species). After identification is complete, we start our measurements of diversity and abundance by counting our fish. When we are finished counting, we organize our data and use statistical analyses to see if there is a significant difference in diversity and abundance in our two sample sites. We have followed procedures from the past two summers and each time we have sampled this summer to make sure we can compare our data at the end.

And now for the big reveal… Drumroll please! Will we find a difference in diversity? In abundance? In neither or both? Will we finally win a battle against the dreadful pluff mud? Although the last part seems unfortunately unlikely, join me next time to finally find out what secrets Gracilaria has tangled up in the Charleston Harbor!


Special thanks to my mentor, Dr. Harold for his support and guidance throughout this project. Also, to Dr. Podolsky and Grice Marine Lab for giving me the opportunity to conduct this research. This project is supported by the Fort Johnson REU program, NSF DBI-1757899.

Life in Plastic, It’s not Fantastic

Samuel Daughenbaugh, DePauw University

2DA71FE7-975A-4AA8-8A78-DF3D1E545F05The Problem: We live in a plastic world. Plastics have saturated all aspects of our daily lives and, as a consequence, have also entered the natural world.  About 8.3 billion metric tons have been produced in the past 60 years, playing a pivotal role in the advancement of modern society (Parker, 2018). Although they are used to create many things we enjoy and benefit from, there are serious consequences for the health of humans and the environment that are associated with their use.

We have found plastics in unexpected places, everywhere from human guts to the most remote locations on earth (Schwabl, 2018; Woodall, 2014). Plastics have a long list of negative effects on living organisms, but their impact in the ocean is of special concern. Pictures of turtles with straws up their noses, bottle caps spilling out of dead bird stomachs, and penguins strangled in plastic beverage rings are often posted on social media sites. Less widely known are the chemical additives that leach from plastics. Phthalates are one such group of additives that pose threats to the health of humans and marine life.

IMG_7571

Current Fort Johnson REU Interns (Julianna Duran not pictured) collecting plastic and sand dollars on Otter Island. (Photo credit: R. Podolsky)

Phthalates have been valuable to the plastic industry because they promote flexibility and durability in many plastics (EPA, 2017). An astounding 470 million pounds of phthalates are used in the United States every year (EPA, 2017). This presents a significant problem because phthalates interfere with the production of important hormones that regulate growth and metabolism in humans and other animals (Boas et al., 2012).

This summer I am exploring the effects of three different phthalates– dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP)–on the larval development of marine invertebrates, using the purple-spined sea urchin (Arbacia punctulata) as a model. Sea urchin larvae float freely in the water column for an extended period of time and, therefore, are vulnerable to many marine pollutants.

IMG_6347

Purple-spined sea urchin (Arbacia punctulata)

Sea urchins are an important model because they are closely related to humans. Both humans and sea urchins use a signaling hormone called thyroxine, which is especially important for growth in early developmental stages (Heyland et al., 2006). Exposure to phthalates can disrupt the production of thyroxine. Additionally, larvae are very important to study because they form the base of food webs. Being at the bottom of the food chain means they feed animals at higher levels, many of which humans rely on for protein. Therefore, understanding how phthalates affect sea urchin growth and metabolism can lead to new insights into how these pollutants directly and indirectly impact human health.

 Acknowledgements

I would like to thank my mentor, Dr. Robert Podolsky, for his continued support, guidance, and encouragement. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Boas, M., Feldt-Rasmussen, U., & Main, K. M. (2012). Thyroid effects of endocrine disrupting chemicals. Molecular and Cellular Endocrinology, 355(2), 240-248. 

Environmental Protection Agency (Ed.). (2017). Phthalates. America’s Children and the Environment, 3, 1-19.

Heyland, A., Price, D. A., Bodnarova-Buganova, M., & Moroz, L. L. (2006). Thyroid hormone metabolism and peroxidase function in two non-chordate animals. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, 306B(6), 551-566.

Parker, L. (2018, December 18). A whopping 91% of plastic isn’t recycled. Retrieved from  http://www.nationalgeographic.com

Schwabl, P. (2018, October). Assessment of Microplastic Concentrations in Human Stool. Conference on Nano and microplastics in technical and freshwater systems, Monte    Verità, Ascona, Switzerland.

Woodall, L. C., Sanchez-Vidal, A., Canals, M., Paterson, G. L., Coppock, R., Sleight, V., . . . Thompson, R. C. (2014). The deep sea is a major sink for microplastic debris. Royal      Society Open Science, 1(4), 140317-140317. doi:10.1098/rsos.140317

Pinniped Problems: Domoic Acid Diatom Denotes Death

Jackson Eberwein, Sonoma State University

The Problem: Imagine a new disease spreading through your community, and it is deadly. It injures the kidneys, affects heart muscles, and causes parts of the brain to wither away. Scientists and doctors know that it is caused by a toxin made by a microscopic organism that loves to suddenly appear with force in unpredictable restaurants across the country. Despite this knowledge, doctors have no good way of knowing that a person has the disease until it is too late.
This is the reality for California sea lions. Along the west coast, large blooms of algae have been producing a toxin called domoic acid, and sea lions have been getting stomachs full of it through their diet of alga-eating fish. According to California Marine Mammal Stranding Network records from 1998 to 2006, around one out of every four California sea lion beach strandings or deaths along most of the California coast were due to exposure to the biotoxin. Since 2006, blooms of the algae have increased, with stranding numbers rising along with them.

California Sea Lion (Zalophus californianus) spooked about eating bad fish.
Photograph by Pixiabay

No good blood test exists for Domoic Acid Toxicosis, as the biotoxin that causes it rapidly clears from the body of sea lions. This means veterinarians can’t see if a sea lion has it unless they use outdated or expensive tests, or guess based on how an animal acts. Since veterinarians don’t have a good way to measure how bad the disease is, they don’t really know for sure if what they do helps a sick sea lion. If they wait to use the behavior of the animal to judge, then it is already too late because the disease has done permanent damage.
So how do we get a viable blood test? Can something be measured in blood when it is simply not there? In this case, we think it can, though not directly. While the domoic acid is in the body, it will be doing what toxins do best: messing with a lot of things that should not be messed with. This will cause changes in the body, such as more or less of a protein being made than it usually is. An unusual change in production of a protein could be measured instead of the toxin that caused that change. In this situation, the protein is called a “biomarker”, or a proxy for measuring the real target. By finding a biomarker protein in sea lion blood, it will actually be possible to make a cheap and effective blood test for the impacts of domoic acid!

Acknowledgements

I would like to thank Dr. Michael Janech, Dr. Benjamin Neely, Alison Bland, The Marine Mammal Center, & College of Charleston. Supported in part by the Fort Johnson REU Program, NSF DBI-1757899.

Neely BA, Ferrante JA, Chaves JM, Soper JL, Almeida JS, Arthur JM, et al. (2015) Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis. PLoScONE 10(4): e0123295. doi:10.1371/ journal.pone.0123295

Bejarano, A.C., Gulland, F.M., Goldstein, T., Leger, J.S., Hunter, M.S., Schwacke, L.H., VanDolah, F.M., & Rowles, T.K. (2008). Demographics and Spatio-Temporal Signature of the Biotoxin Domoic Acid in California Sea Lion ( Zalophus californianus ) Stranding Records.

Laake JL, Lowry MS, DeLong RL, Melin SR, Carretta JV (2018) Populationgrowth and status of California sea lions.J Wildl Manage82: 583–595 .

Gracilaria: New Intruder Weeding Through Charleston

Ana Silverio, The University of Texas at Austin

The Problem: Invasive species are animals that enter a new habitat away from their own home and are known for usually bringing about negative effects on natives in the area. Invasive species thrive in new environments when they can adapt to local conditions, and cause troubles in the way it works. With their usual predators not around, chaos can erupt, as they take away from some resources from the animals who call this habitat home (Albins et al 2015). Gracilaria vermiculophylla is a type of seaweed but also an invasive species from Asia and first seen on the Virginia coast. Although it is an invasive species, this seaweed seems to be singing a different song than usual (Nyberg et al 2009). Since it was first seen on the beaches of North America, it has taken a different role by providing a new habitat to local fishes. Gracilaria vermiculophylla is a dark brownish red seaweed with tangled strands that brush up against anything wading through the shallow water. Perfect for smaller fish to hide in. Although this seaweed seems to be bringing good things to the fishes not much is understood about what life was like for them under the waters of Charleston before our new stranger came about so we can’t comment on that part of the story. On the other hand, an interaction is indeed unfolding before our eyes and the story behind our new visitor is a bit fishier than one may think.

Example of a sample site: sparse patch of Gracilaria vermiculophylla on Grice Beach.
Photo taken by: Norma Salcedo

Gracilaria vermiculophylla is hard to miss on the shorelines of Charleston, it can be found in patches when the tide dwindles or on the seafloor. Its branches provide an ideal habitat along with a hiding space for juvenile fish during their vital first years of life and increases their numbers (Munari et al 2015). The preservation of these fishes during their early life stages is important to maintaining a healthy food web that keeps marine life afloat. Food is energy and energy is moved up to some of the biggest fisheries in this country from the very bottom of the smallest animals. It is important to know how the bigger fish’s food source is interacting with its habitat to make sure it’s healthy. Understanding how the interaction is working is a key factor in creating conservation plans and maintaining the ecosystem in good health.

Dense patch of Gracilaria vermiculophylla.
Photo taken by: Norma Salcedo

This summer, my research focus is on untangling Gracilaria vermiculophylla’s ecological relationships with these small fishes for a better understanding how diverse life is underwater. Replicating a design from the past two summers, I am curious to see the differences in diversity and abundances based on different patches of seaweed and if body size plays a significant role. Will more seaweed correlate with more diversity? The past two summers revealed some common patterns between fish diversity and patterns of seaweed patches but also some surprising differences between the two field seasons. Will we have a tie breaker this summer? Stay tuned to find out!


Special thanks to my mentor, Dr. Harold for his support and guidance throughout this project. Also, to Dr. Podolsky and Grice Marine Lab for giving me the opportunity to conduct this research. This project is supported by the Fort Johnson REU program, NSF DBI-1757899.


References

 Albins MA (2015) Invasive Pacific lionfish Pterois volitans reduce abundance and species richness of native Bahamian coral-reef fishes. Mar Ecol Prog Ser 522:231-243. 

Munari, C., N. Bocchi, and M. Mistri. “Epifauna associated to the introducedGracilaria vermiculophylla (Rhodophyta; Florideophyceae: Gracilariales) and comparison with the nativeUlva rigida(Chlorophyta; Ulvophyceae: Ulvales) in an Adriatic lagoon.” Italian Journal of Zoology 82.3 (2015): 436-445.

Nyberg, C. D., M. S. Thomsen, and I. Wallentinus. “Flora and fauna associated with the introduced red algaGracilaria vermiculophylla.” European Journal of Phycology 44.3 (2009): 395-403.

Uncovering Seasonal Changes in the Algae Our Oceans Depend On

Emily Spiegel, Bryn Mawr College

As described in my previous posts, this study focused on a polar diatom, F. cylindrus.  Despite the harsh temperatures of its habitat, this diatom is awesomely productive. It can form blooms under sea ice so thick, it looks like grass! Marine organisms feed on these blooms, which contributes to productivity of the entire ecosystem.

Because the poles are situated at the ends of the Earth, they are subject to constant changes in light availability, from continuous light to continuous darkness. How are photosynthetic organisms like F. cylindrus able to adapt to this stressful change? Their ability to produce biomass is dependent on light levels: too much and these cells can be overwhelmed, too little and there may not be enough to balance against the costs of respiration.

I found that in the low light exposure of polar autumn (6h light: 18h darkness), F. cylindrus begins to reproduce sexually, instead of asexually. This was found through analysis of RNA expression, which is an indicator for how much a certain gene is being transcribed into proteins to do work within the cell. Sexual reproduction leaves behind a trace in the RNA, based on the particular genes involved. As opposed to the primary form of diatom reproduction (asexual), sexual reproduction conserves resources and produces fewer cells. So the population does not grow to the same extent as populations reproducing asexually, but it’s also able to survive in stressful and changing conditions better than asexual populations.

Interestingly, stress can also reduce the ability of F. cylindrus to remove carbon dioxide from the atmosphere, in a process known as carbon fixation. This shift could have major implications for how well the polar oceans remove CO2 from the atmosphere at different times of year. Could autumnal months in the poles show dramatically decreased carbon fixation rates? What would such a pattern mean for current global carbon models? Further research must be conducted at the poles themselves to determine whether this relationship exists in nature, and how it is affecting carbon flux within the polar oceans.

This research was conducted in the lab of Dr. Peter Lee from the College of Charleston at the Hollings Marine Laboratory in collaboration with the Medical University of South Carolina. Many thanks to all members of the lab, particularly Nicole Schanke, MSc.

The BMA of Today

Christine Hart, Clemson University

2017-06-22 10.29.36

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).

QualitativeLvM-MS

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.