Expect the Unexpected in Science

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Alessandra Jimenez, Whitworth University

As this internship has recently come to an end, I now begin to reflect on the wonderful yet challenging experience I had conducting observational research on Atlantic brown shrimp (Farfantepenaeus aztecus). In the last few weeks of this 10-week summer program, there was a fascinating yet unexpected turn of events. In particular, results of the experiment pointed to conclusions that I initially found myself unprepared for!

In summary, the focus of this experiment was to test effects of immune response on the ability to escape predators in shrimp. The escape mechanism, called tail-flipping (see video below) is actually powered anaerobically. However, recovery from this energetic behavior absolutely requires oxygen (is aerobic). As further explained in previous blog posts (click here and here), a recently discovered consequence of mounting an immune response against bacterial infection involves depression of aerobic metabolism. So, my mentor and I decided to focus on the recovery aspect (aerobic) of the escape response and predicted that this aerobic process would be impaired in shrimp injected with bacteria. At the same time, we predicted that the anaerobic part of this mechanism would be significantly impacted.

A slow-motion video of an Atlantic brown shrimp juvenile tail-flipping in an experimental tank (c) Alessandra Jimenez

The last few weeks of the internship mainly consisted of analysis, arriving at conclusions, and publicly reporting results. After testing tail-flipping ability (click here for an explanation of how this was tested) in a total of 42 shrimp juveniles, 30 of these were chosen for final analysis. Using a statistics software called Sigmaplot (version 12.5), I conducted tests that basically compared experimental groups based on the two variables I investigated: treatment type (bacteria or saline) and time given after injection (4 or 24 hours). Afterwards, results were deemed important based on significance values assigned by these Sigmaplot tests.

Significant results were very surprising!  Overall, results suggested that metabolic depression (indirectly caused by the immune response) did not have an impact on recovery (aerobic). At the same time, the most unexpected finding of all suggested that bacterial exposure actually increased anaerobic tail-flipping activity in Atlantic brown shrimp juveniles! Thus, this result called for a complete change in focus from the aerobic part to the anaerobic part of this particular escape response.

So, how could I possibly explain the increase in anaerobic processes found through this experiment? After much pondering and going through scientific literature, I formulated a new hypothesis. An important enzyme in crustaceans called arginine kinase is involved in the storage and creation of anaerobic energy that can be used for tail-flipping. Recent studies involved injecting bacteria into live crustacean tissue and comparing arginine kinase expression levels with controls. Results indicated a significant increase in expression in bacteria-injected tissue, especially in abdominal muscle (important for tail-flipping!). Based on these investigations, I now think that there may be a link between immune response and levels of anaerobic metabolism. Further research is required to explore this.

The final stages of the internship included creating and presenting a Powerpoint presentation of our work, and submitting a manuscript of my summer investigation. Overall, this REU internship experience has been challenging yet exciting, and has confirmed my love for marine biological research. As I mentioned at the end of my presentation, “expect the unexpected in science”.

powerpoint presentation - REU 2015

Picture of me right before giving my Powerpoint presentation (c) Alessandra Jimenez

References:

Burnett, L. E., Holman, J. D., Jorgensen, D. D., Ikerd, J. L., & Burnett, K. G. (2006). Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab. Biological Bulletin, 211(1), 50-57.

Gruschczyk, B., Kamp, G., 1990. The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp, Crangon crangon. J Comp Physiol B, 753-760.

Scholnick, D. A., Burnett, K. G., & Burnett, L. E. (2006). Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biological Bulletin, 211(1), 44-49.

Yao, C., Ji, P., Kong, P., Wang, Z., Xiang, J., 2009. Arginine kinase from Litopenaeus vannemai: Cloning, expression, and catalytic properties. Fish Shellfish Immunol 26, 553-558.

Many thanks to College of Charleston for hosting my project, Dr. Karen Burnett and Hollings Marine Laboratory for guidance and work space, and NSF for funding the REU program.

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To B12 or not to B12, that is the question…

Bryce Penta, University of Notre Dame

As the summer draws to an end, so too does this segment of my research with phytoplankton and vitamin B12. After completing three separate experiments, my project has finally reached its end.

One experimental design encompassed the first two experiments and used a mixed community of phytoplankton straight from the ocean while aboard the R/V Savannah, while the other relied on a culture of Phaeodactylum tricornutum, a phytoplankton species, that had all bacteria removed in laboratory settings. The first pair of experiments were conducted with an addition of both vitamin B12 and nitrate and the final experiment implemented a limitation of the same two nutrients. I specifically looked at the effect of varying availability of these nutrients on the photosynthetic efficiency and growth of the cultures.

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Photo confirming the lack of bacteria in the phytoplankton cultures in lab. The picture also shows that the cultures contained two body forms of Phaeodactylum tricornutum. Photo credit: Lena Pound

This study proposed that an increase in the availability of these nutrients would lead to an increase in efficiency and growth, as well as a decrease leading to a lower efficiency and growth. While we expected an effect of vitamin B12 on the phytoplankton functioning, in all three experiments B12 lacked any significant effect; however, nitrate showed a strong effect on the photosynthetic efficiency and growth in all experiments except the deep sea boat experiment.

While only nitrate exhibited a significant effect on the phytoplankton, this could be due to an alternate metabolic pathway that can bypass the need for vitamin B12. Using methionine synthase E (MetE) rather than the more efficient methionine synthase H (MetH) that requires vitamin B12, the Phaeodactylum tricornutum cultures functioned properly in the absence of vitamin B12 (Helliwell et al. 2011). Unlike the laboratory experiment, the ocean experiments may have lacked a B12 response due to microbes in the water already producing more than enough of the nutrient. Vitamin B12 lacked significant response in our experiments, but other experiments with species that lack the MetE synthase that allows for proper functioning without vitamin B12. Possible B12 effects on phytoplankton could lead to better climate modeling as phytoplankton form the basis of one of the world’s largest ecosystems.

These past ten weeks have culminated in a project that I am proud to have worked on this summer. Though my time here has ended, the people I have met here and the relationships formed over the summer will continue on in the future.

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Lee lab on the R/V Savannah showing off the catch of the trip, a 55 inch wahoo. Photo credit: Bryce Penta

Acknowledgements

This project is possible due to funding from the NSF College of Charleston Summer REU program and the Grice Marine Laboratory. Project ideation and collaboration with Dr. Peter Lee and the Di Tullio lab from the College of Charleston. Lab space and facilities provided by the Hollings Marine Laboratory.

References

Helliwell, K.E., Wheeler, G.L., Leptos, K.C., Goldstein, R.E., Smith, A.G. (2011) Insights into the Evolution of Vitamin B12 Auxotrophy from Sequenced Algal Communities. Molecular Biology and Evolution 28 (10): 2921-2933.

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What’s living in the sand?

Jessie Lowry, Coker College

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Visible microalgae seen on the surface of wet sand at Folly Beach.

Next time you go to the beach this summer, I want you to think about the sand that you are walking on. Did you know that there are tons of microscopic photosynthetic organisms, aka microalgae, that live on the surface of sand? Before this summer, I didn’t know about these organisms either. Here is a picture of visible microalgae on the surface of the sand. Look for this next time you’re at the beach!

Microalgae communities in sand are made up of single-celled eukaryotic algae and cyanobacteria living in the top several millimeters of the sand (Miller et al., 1996). These organisms play important roles in ecosystem productivity and food chain dynamics, as well as in sediment properties, such as erodibility (Miller et al., 1996).

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Dr. Craig Plante and Jessie Lowry collect samples of sediment from Folly Beach. Photo credit: Kristy Hill-Spanik.

I am studying these microalgal communities and what factors influence community structure. For example, does pH, salinity, nutrients, or grain size shape microalgal community structure? Or does geographic distance shape communities? To answer these questions, I am collecting samples from Kiawah Island, Folly Beach, Isle of Palms, and Pawley’s Island, SC. We are measuring environmental variables at each location, and using molecular tools to study microalgal community structure.

I am extracting the DNA from samples collected, amplifying specific regions from these samples using polymerase chain reaction (PCR), and then we will be getting these regions sequenced using Ion Torrent technology. We will then use QIIME to determine how similar these benthic microalgal communities are.

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Jessie Lowry preparing samples for PCR, or polymerase chain reaction, which is used to make millions of copies of a piece of DNA.

Diatoms, a group of microalgae, have been proposed as bioindicators of environmental health (Desrosiers et al., 2013). Bioindicators are really cool because instead of telling a snapshot of an environmental condition, such as pH, temperature, or amount of oxygen in an environment, biological indicators reflect those changes and can give an idea of how the ecosystem is being affected. This research will further our knowledge of what factors shape benthic microalgal communities, and give a better understanding of these organisms as a potential bioindicator. In addition, this research will add to knowledge about the distribution of microorganisms, which is also not fully understood.

Learn more:

http://web.vims.edu/bio/shallowwater/benthic_community/benthic_microalgae.html

http://www.aims.gov.au/docs/research/water-quality/runoff/bioindicators.html

References

Desrosiers, C., Leflaive, J., Eulin, A., Ten-Hage, L. (2013). Bioindicators in marine waters: benthic diatoms as a tool to assess water quality from eutrophic to oligotrophic coastal ecosystems. Ecological Indicators, 32, 25-34.

Miller, D.C., Geider, R.J., MacIntyre, H.L. Microphytobethos: The ecological role of the “Secret Garden” of unvegetated, shallow-water marine habitats. Estuaries, 19(2A): 186-212.

Acknowledgements

Thank you so much to my mentors Dr. Craig Plante, and Kristy Hill-Spanik. This research is funded through the National Science Foundation and College of Charleston’s Grice Marine Lab.

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Can extracellular enzymes functionalize electrodes?

Yoel Cortes-Pena, Georgia Institute of Technology

A microbial electrosynthesis cell (MEC) takes in electrical energy and converts it to chemical energy as fuels, but what supports that initial step of taking the electrons off the cathode so that they become available to the microbes? Are small redox molecules acting as intermediates between the electrode and the microbes? Or is there a more direct way?

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Photo of anaerobic cultures grown in a pressurized mixture of hydrogen and carbon dioxide. The tube on the far left is a sterile control of inoculated media that has no hydrogen inside. The other three tubes are biological triplicates.

Previous studies have shown that hydrogenases, enzymes that catalyze the conversion between hydrogen cations to hydrogen gas, are able to attach themselves to an electrode and remain active (1). Additionally, a study by Deutzmann el. al. showed that extracellular hydrogenases support the production of hydrogen on a cathode in an MEC that utilizes methanogenic bacteria (2). Therefore, It is possible that active hydrogenases are able to attach themselves onto an electrode, uptake the electrons directly off the cathode, and produce hydrogen which will later be consumed by the microbes!

Over this summer internship, I took the first step to test this hypothesis by asking the question “Are active hydrogenases released from the microbes?”. Active extracellular hydrogenases in the reactor suspension would support the hypothesis that hydrogenases are also active when attached to the cathode.

Hydrogenase_assayPhoto of hydrogenase activity as measured spectroscopically by methyl viologen. Oxidized methyl viologen is clear and reduced methyl viologen is blue. The test involves inserting the sample that may contain hydrogenases into a reaction mixture with oxidized methyl viologen and pressurized hydrogen. Hydrogenases would proceed to oxidize hydrogen gas and reduce methyl viologen, turning the mixture blue

The main experiment I am working on involves testing hydrogenase activity in anaerobic cultures that originated from the reactor suspension. These cultures are grown in a pressurized 80:20 mixture of hydrogen and carbon dioxide, which mimics the reactor environment.

By the end of the summer I hope to have hydrogenase activity data of spent cell-free culture medium that will answer our question  “Are active hydrogenases released from the microbes?”

References

(1) Baffert C, Sybirna K, Ezanno P, Lautier T, Hajj V, Meynial-Salles I, Soucaille P, Bottin H, Léger C(2012). Covalent attachment of FeFe hydrogenases to carbon electrodes for direct electron transfer. Anal Chem 84(18):7999-8005. doi: 10.1021/ac301812s.

(2) Deutzmann JS, Sahin M, Spormann AM. (2015) Extracellular enzymes facilitate electron uptake in biocorrosion and bioelectrosynthesis. mBio 6(2):e00496-15. doi:10.1128/mBio.00496-15.

Hook, Line and Sinker

Sierra Duca, Goucher College

Spotted seatrout, Cynoscion nebulosus, are important recreational fish that range from the Atlantic coast to the Gulf of Mexico. They are also good indicators of environmental changes in estuarine habitats since all of their life stages are found in estuaries1. To reiterate, I am studying muscle softness in spotted seatrout induced by the parasite Kudoa inornata. Several Kudoa species are notorious for causing this muscle softening, which makes the meat of the fish go bad faster than in uninfected fish2. This is an issue with fish that are consumed by people, such as the commercially important farmed Atlantic salmon2. seatrout for blogg

Fig 1. Spotted seatrout that were caught via trammel netting (PC: Sierra Duca).

First of all, to study this I need fish. While the mode of infection of Kudoa parasites is not well understood, it is presumed that wild spotted seatrout have a higher rate of infection of Kudoa inornata; therefore, I needed some wild spotted seatrout. In addition to the traditional hook and line approach of fishing for spotted seatrout, I was able to join a group at the South Carolina Department of Natural Resources (SCDNR) as they went trammel netting. In comparison to other nets, trammel nets have three layers of netting that vary in size in order to catch fish of various sizes. trammel net for blog

Fig 2. This illustration depicts the basic function and structure of a trammel net. A similar such device is used by the SCDNR to catalogue fish in specific sites over time in order to study the changing population dynamics of various fish species.

Once I have the fish I fillet, refrigerate, and take muscle biopsies at time points between 0-6 days, which is the most likely time that the fish would be consumed. I test the firmness of these muscle biopsies, as well as the parasite density. What I am trying to accomplish is to establish whether or not there is a link between parasite density and accelerated muscle softness (which causes the meat to go bad faster in infected fish), and if the rate of muscle softening changes over the course of 6 days. Ultimately the project will help increase our understanding of the effects of Kudoa inornata on the muscle of spotted seatrout. plasmodia for blog

Fig 3. This image (under 100x magnification) displays a plasmodium structure that contains a cluster of spores (known as myxospores) of Kudoa inornata in the muscle tissue of spotted seatrout. One way that I quantify parasite density is by looking at the average area of plasmodia. I can do this because generally larger plasmodia are found in the more infected fish  (PC: Sierra Duca).

Literature Cited 1Bortone SA (ed) 2003: Biology of the Spotted Seatrout. CRC Press. Boca Raton, FL, 328 pp 2Henning SS, Hoffman LC, Manley M (2013) A review of Kudoa-induced myoliquefaction of marine fish species in South Africa and other countries. S Afr J Sci. 109: 1-5

Photo Source (Fig 2): http://thewikibible.pbworks.com/w/page/22174694/Fishing%20in%20the %2Bible%20and%20the%20Ancient%20Near%20East

Acknowledgments The Fort Johnson REU Program is funded by the National Science Foundation. This research is made possible through the mentorship of Dr. Eric McElroy and Dr. Isaure de Buron.  In addition, I would like to thank the College of Charleston and the South Carolina Department of Natural Resources for providing the help and facilities necessary for my project.

Our complicated relationship with chemicals

Nina Sarmiento, Binghamton University

Chemicals found all around us that have been altered, mimicked, and synthesized to be added to our products, are behind the success of our modern society. They have made our plastics strong, our crops prosperous, and our medicines effective. But I have always wondered about the toxicity of these chemicals.  When you look at their biological activity, a chemical might possess the potential to do harm, like interfere with biological processes. The safety of a potentially harmful chemical is based on exposure and dose. It is important to know if we are touching it, eating it, or breathing it in, and for what period of time. The study of evaluating the harmful effects of substances on exposed organisms is what toxicology is all about. They have such an important job because their findings influence what we know is safe and unsafe, for us and organisms all around us.

I learned early on from pursuing biology that we are exposed to many things we are unaware of. Not only are we exposed to potentially harmful chemicals, but we facilitate exposure to other living organisms that may more sensitive. Take dogs for simple example. The toxic dose of something like chocolate for humans is very high, whereas leaving a small amount of chocolate out for a dog to eat could easily kill it.  Rachel Carson is someone I greatly admire, whose work on the pesticide DDT also exemplifies this reality. Food crops were the target for DDT, but birds were indirectly ingesting it, explaining the decline in the Bald Eagle population.  She is one of the people that sparked my interest in ecotoxicology, looking at the effects of harmful substances on ecology, not just humans.

unnamedThis is an example of some of the questions ecotoxicologists ask when there is a potentially harmful substance found in the environment. Photo credit: globe.setac.org.

Here is a chemical product you may not suspect as a threat, sunscreen. In sunscreens, UV filters protect you from getting burned, but also can act as endocrine disruptors, altering hormones and growth (1). Sunscreens are only meant for human skin, however they end up in our lakes, rivers and oceans through swimming or through waste water treatment effluent (2). Unintentionally, many more organisms become exposed.

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Photo credit: thesleuthjournal.com

In my project I will be using sea urchins as a model organism to study the effects sunscreens might be having on coral reefs.  I am learning how to preform toxicity tests on sea urchin sperm and embryos which involve an exposure period with sunscreen formulations and then evaluation of effects. I hope to investigate if the chemicals from sunscreens in the water can have negative impacts on coral reproduction.  My work can potentially help create understanding of how humans are contributing to coral reef decline, and influence others to take action to protect them.

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This is a picture of sunscreen water accomodated fractions (WAFs) I am making. They are a mix of sunscreen and seawater and I will be exposing the sea urchin embryos to each solution!

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This is me in the lab with a microscope I use to look at sea urchin sperm and embryos! Photo by Bob Podolsky

My research is funded by the National Science Foundation and College of Charleston partnered with National Oceanic and Atmospheric Administration

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Works cited:

1 Krause M.,, Klit A., Jensen M., Soeborg T., Fredrickson H., Schlumpf M., Litchensteiger W., Skakkebaek N E., Drzewieck K T. 2012. Sunscreens: are they beneficial for health? An overview of endocrine disrupting properties of UV-filters. International journal of andrology. 35 424-436.

2Kyungho C., Kim  S. 2014. Occurances, toxicities, and ecological risks of benzophenone-3, a common component of organic sunscreen products: a mini review. Environment International. 70 143-157.

Let’s talk plastic

Jimena B. Pérez-Viscasillas, University of Puerto Rico

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In a recent article published by The Conversation, researcher Richard Sharpe discusses his work on male reproductive efficiency and how it might be affected by phthalates. Phthalates are plasticisers, compounds added to plastic materials to make them more flexible.  “Plasticisers leach out of the plastic over time” Sharpe writes. “This is why if you use the same plastic water bottle over a long period it will eventually become brittle and break – indicating that you have drunk all of the plasticisers that leached out.”

Fig. 1  Plastics aren’t the only products with phthalates. These compounds can also be found in fragrances, personal care products, etc. (Taken from University of Michigan Formative Children’s Environmental Health and Disease Prevention Center

Yikes.

What could this mean for our health? Many different studies have been conducted to find out how these compounds might be affecting our bodies. Sharpe specifically focuses on how they might affect the male reproductive system. He argues that, because the available data on the subject is so variable, it is not yet completely conclusive whether phthalates are indeed the cause of lower male fertility.

What we do know, however, is that phthalates have been found to disrupt the production of some hormones in the body. Because hormones are the body’s chemical messengers, not having them able to deliver their messages to tissues properly could mean trouble for one’s health. One such case is that of prostaglandins, the hormone I’m looking at in this summer’s research. Prostaglandins are hormones that regulate inflammatory responses in the body. When you prick your finger and see it swell up, part of what you’re observing is due to prostaglandins doing their job, allowing more blood and immune cells into your tissue. Prostaglandins play major roles in the immune and reproductive systems. Therefore, it is hypothesized that if a pregnant woman suffers from hormonal inhibition because of exposure to phthalates, this could have long-term health effects on her baby too.

Fig 2. Human embryo. (Image taken from BBC)

Sharpe asks this research question in his article as well. However, part of the reason why effects of contaminants on human health are so difficult to research is precisely because exposure to them is so common among human populations. As he points out, “this means we don’t really have an unexposed group (“control”) against which to compare.” Luckily, we humans aren’t the only organisms around. Studying other species (such as, say, gators and chickens…) allows us to better understand these possible hazards.  Finding evolutionary relationships between us and sentinel species is an essential step towards understanding just what these phthalates and many other contaminants are up to in our bodies.

To learn more about phthalates, click here. For more about my research, stay tuned. Later, gators!

Acknowledgments

This research is made possible thanks to the Hollings Marine Laboratory, the Medical University of South Carolina, the College of Charleston and funding by NSF through the Fort Johnson REU program.

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References 

Kristensen, David M. (2011). Many Putative Endocrine Disruptors Inhibit Prostaglandin Synthesis.  ENVIRONMENTAL HEALTH PERSPECTIVES. Vol.119 No. 4: 534 – 541.

Milnes MR, Guillette LJ Jr. Alligator Tales: New Lessons about Environmental Contaminants from a Sentinel Species; BioScience 2008; Vol. 58 No.11 doi:10.1641/B581106

Sharpe, Richard. “Are plastics making men infertile?” The Conversation 25 Jun. 2015. Web 29 Jun. 2015.