Spelunking for Coral Health

Meagan Currie, Swarthmore College

Sadly for the scientists involved, understanding coral health doesn’t always require a wetsuit, air tank or coral reef access. My work with the coral species Acropora cervicornis – or staghorn coral – usually requires a mundane terrestrial dress-code and a green headlamp as my only light source. Working with coral in the lab is more akin to spelunking than scuba diving. This environment, however, allows researchers to control the conditions surrounding the coral in order to more accurately understand the effects of chemical exposure on coral health.

To refresh, I am looking at the effects of the common chemical nonylphenol, which is used in laundry and dish detergents, is a stabilizer in plastic food packaging, and is one of the chemicals that makes up nonylphenol ethyl oxalates, which are found in pesticides, paints, and personal care products. After looking at the development of sea urchins exposed to nonylphenol, I found that the environmentally relevant values (0.1 ug/L – 50 ug/L) did not have a noticeable effect on embryo health. While this is a good sign in terms of the toxicity of the chemical to marine organisms, I was surprised, as most papers that have investigated the toxicity of nonylphenol have found it to be harmful at these levels to a variety of different organisms. Either my urchins were more robust than I had expected, or the chemical was not dissolving in water evenly when I made up my solutions. I decided, because of this, to expose my coral to a wider range of nonylphenol concentrations to get a better sense of whether or not my chemical was reacting. The coral were exposed to nonylphenol at levels ranging from 1 ug/L to 1000 ug/L, and over 96 hours I measured the effects. Let me outline the ways in which researchers monitor coral health in the lab.

During this experiment I consistently measured three things. The first was how well coral tissue regrows when exposed to a chemical, in this case nonylphenol. To do this, I cut the top of half of my coral fragments off at the beginning of the experiment, and took a picture after staining the tissue left on the top of the coral. Over time, a healthy coral fragment will regrow tissue over this wound. An unhealthy coral will take longer to regenerate the tissue, and so at the end of the 96 hour period I took a second picture of the stained tissue to see whether nonylphenol affected the speed of regeneration over this time.

Control fragment regeneration: time 0 (left) to 96 hours (right)

Another way to measure the health of coral is to measure Pulse Amplitude Fluorometery (PAM). Coral have symbiotic algae called zooenthallae, which provide food and oxygen through photosynthesis to the coral polyps. In turn, the coral release carbon dioxide, and provide shelter for the zooenthallae. PAM exposes the coral to a flash of ultraviolet light, which then causes the zooenthalae living in the coral tissue to emit fluorescence.

PAM Example

By measuring the intensity of this florescence, we can better understand the concentration of zooenthallae in the tissue as well as how well they are photosynthesizing. Each day during the exposure I run PAM when the coral are most sensitive to light exposure, right before the light-cycle of their day begins. If you want to see more of the amazing fluorescent world of corals, watch this video created by the reef conservation group Coral Guardians.

https://www.youtube.com/watch?v=gXODm9qVkhM

Finally, each day I run a basic physioscore to assess how healthy the coral looks and to document changes over time. This involves three different indicators: the polyps; the coloration; and the tissue of coral. I measure these features on a scale of five, with five being completely healthy and zero being nonexistent. Coral extend their anemone-like polyps when they are healthy. The A. cervocornis is a rich brown when its zooenthallae are still present in the tissue, and its tissue should cover the entire fragment of the coral. Below are two images from my study, the first a healthy control after 96 hours and the second a less healthy fragment exposed to nonylpyhenol.

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Having finished my 96-hour spelunking experiment with the coral, I will now gather my data and try to draw conclusions about the effects that nonylphenol has on photosynthesis, regeneration and overall health of the coral. With luck, these data will help solidify our understanding about nonylphenol in the marine environment and its effects on coral and similar invertebrates.

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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.

Gametes galore!

By Cecilia Bueno, Lewis & Clark College

I am conducting my experiment in two parts: the first looks at how increased salinity affects fertilization success, the other looks at how salinity affects sperm function.

The fertilization experiments start with watching the weather- squirrel treefrogs mate on nights when there has been a lot of rain. We collected frogs from dixie plantation on nights when there had been 0.5 inches of rain accumulation or more. Since squirrel treefrogs mate at night, we would go out around 10:30pm and stay often until 3:00am when we found pairs.

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Grassy wetland at Dixie Plantation where several pairs of squirrel treefrogs were found

The frog pairs were taken back to the lab where they were placed in individual tubs with water at 6ppt salinity. When the pair had laid around 200 eggs, we removed them from the tubs and placed them with a new partner. This repeated until the frogs no longer went into amplexus- running from 4:30am to 8:00am.

After the eggs had been laid and the frogs stopped going into amplexus, the eggs were transferred from the large tubs to smaller weigh dishes. These weigh dishes with eggs were placed under the microscope and photographed for counting. I then counted how many eggs in each dish were fertilized. These counts- total eggs and number fertilized- will be used to calculate fertilization success of each of the pairs.

The second part of the experiment focused on sperm function of the frogs. Males from the initial pairs were kept for this experiment, while females and fertilized eggs were released. We created a sperm concentrate using the testes of each male. This sperm concentrate was then added separately to different tubes containing water at different salinity levels. We tested sperm at salinity levels which had previously been shown to have an effect on sperm function- 4ppt, 5ppt, 6ppt, 7ppt and 8ppt- as well as a control of 0.39ppt.

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Screenshot of a video of sperm from a squirrel treefrog male

Once the sperm concentrate has been added to a test tube, the sperm is activated so I have to act quickly. The diluted sperm solution is placed under a microscope and video taped.

After the videos have all been taken, they will be analyzing them with the CASA (Computer Assisted Sperm Analysis) software developed by Wilson and Leedy. This software, run on ImageJ, tracks motile sperm and calculates percent motility and average velocity of the motile sperm.

When I have counts on percent motility and average velocity of sperm in different treatments, I will be able to compare the results I get from the sperm experiments to the results from the fertility experiments.

I would like to thank the National Science Foundation for funding this REU program, and the Grice Marine Lab of the College of Charleston for hosting us. In particular, I would like to thank my mentor Dr. Allison Welch for her help and support.

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!

 

 

 

My Days with the Shrimp

Deanna Hausman, The University of Texas at Austin

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In “What can baby shrimp teach us about oil spills,” I discussed the problem of UV enhanced toxicity of oil. In other words, the fact that UV rays can cause molecules in oil known as PAHs to become more harmful than they would be otherwise. I also discussed the fact that this summer, I will be studying the effects of oil toxicity on grass shrimp, or Palaemontes pugio, an important estuarine species that cycles nutrients through the food chain. Because oil spills are always complex, and organisms can be exposed to oil in many different ways, from the sediment they walk on to the water they swim through, a variety of experiments are needed to get a better understanding of this issue.

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A few of the many PAHs- the compounds in oil that harm marine life Photo from: http://www.crawfordscientific.com/newsletter-2008-12-dedicated-HPLC-GC-columns-PAH-analysis.htm

The first and simplest of the experiments I conducted was the developmental test. In this test, I basically mixed oil and seawater in a giant blender, then took out the water with the oil dissolved in it. Then, I made several dilutions, creating several concentrations of the oily water. Then, I took 6-well plates and filled them up, and placed a single, 24-hour old shrimp in each well. Then, I put these plates in an incubator under UV and non-UV light, and waited for 4 days. After that, I moved the shrimp into clean water, counted how many died, and am currently monitoring them to see how the oil exposure in early life impacts their ability to grow into healthy juveniles.

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Shrimp being monitored after initial oil exposure

Another experiment I conducted essentially followed the same procedure as above, but instead of watching them as they grew, I analyzed the shrimp after their 96-hour oil exposure to see whether the oil affected the concentration of a hormone, known as an ecdysteroid, that controls their molting. Essentially, if the concentrations of this steroid are off in a shrimp it can’t grow properly, so it’s very important!

I’m also conducting an oil sheen test. In this experiment, I place 40 larval shrimp in an aquarium, some caged on the bottom and some swimming freely, and then place an extremely thin oil sheen on top. One aquarium goes under UV light and the other goes under fluorescent light, and after exposure I analyze whether the sheens have had a harmful effect. Whether thin oil sheens are toxic is something that’s not very well understood in this species, so it will be very interesting to see the results.

Finally, I’m conducting an experiment to see what occurs when oil is mixed in with sediment. Essentially, this involves putting sediment from an estuary in a jar, adding oil, and tumbling it around so that the oil is completely mixed in. Then, the sediment is placed into beakers along with water and 24-hour old shrimp, and put under UV and non-UV light for 24 hours, in order to see what mortality occurs. This will perhaps be the most informative experiment, as grass shrimp spend most of their time on the seafloor, so if they’re going to be exposed to oil, it will likely be from the sediment they’re walking on.

In short, I have my hands full this summer! It will be very interesting to see the results. Hopefully, this will increase our knowledge of the harmful impacts oil spills can have to estuarine organisms, and allow NOAA and oil spill analysts to make better predictions of the long-range impacts of oil spills. Ultimately, this may help them make better clean-up decisions.

Thank you to my mentors, Dr. Marie Delorenzo and Dr. Paul Pennington, for their guidance. I’d also like to thank Katy Chung for all her help and expertise. This research is funded through the National Science Foundation.

Stressed out Seaweed

 

Killian Campbell, Eastern Washington University

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In my last post, I introduced you to Gracilaria vermiculophylla (the invasive seaweed that I’m studying this summer) and the certain qualities it possesses that make it an interesting organism to study. I also mentioned that I am investigating the role of heat shock proteins (Hsps) in Gracilaria and how they potentially play a role in allowing Gracilaria to survive in different types of environments. However, I did not speak about the ways that I am going to actually understand those processes.

To put it plainly, I am stressing out my seaweed. Big time. In a biological context, this means that I am subjecting the Gracilaria samples to different conditions that it does not typically experience in the wild. As a result, the conditions that it will experience in the lab will be uncomfortable for the organism, and will thus produce “stress” in the organism.

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Fig. 1: Gracialaria samples in the incubator for storage after receiving stressors

In my project, the stressors we are choosing to subject Gracilaria to are: extremely hot and cold water temperatures, and extremely low salinity levels. After subjecting Gracilaria to these stressors, we will be looking for the amount of bleaching that occurs in the samples. Bleaching is an indication that the conditions the sample is experiencing are too stressful. Bleaching is a loss of pigment in the seaweed (turning from dark red to light pink or white) and it is similar to when leaves on a tree dry out, change colors and fall off—In other words, it is an indication of death in photosynthetic organisms. The data generated from these tests will tell is the types of stressors Gracilaria can endure, and how long it will take to produce a bleaching response in the Gracilaria afterwards.

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Fig. 2: 12-well plate with each well containing Gracilaria tips plucked from different individuals. For each tip, a specific stressor was applied to it.

To assess the importance of heat shock proteins in enduring various stressors, we will repeat the parts of the study above, except that we will also administer inhibitors to the Gracilaria samples this time.  Inhibitors are chemicals that stop the function of heat shock proteins. In theory, giving inhibitors to Gracilaria should decrease their ability to endure stressors.

As a result of the studies, we are curious to see if the data demonstrates that heat shock proteins confer a higher amount of stress tolerance in Gracilaria. By further understanding the role of Hsps in Gracilaria, we can begin to understand their greater ecological role. This may encourage future studies to develop knowledge about the role of Heat shock proteins play in allowing invasive organisms (such as Gracilaria) to inhabit so many diverse environments!

 

Thank you to Dr. Erik Sotka, Benjamin Flanagan and everyone else in the Sotka lab. I would also like to thank Grice Marine Laboratory at the College of Charleston and the NSF for this wonderful opportunity

 

Literature Cited:

Feder, M. E., and Hofman, G. E. (1999). HEAT-SHOCK PROTEINS, MOLECULAR CHAPERONES, AND THE STRESS RESPONSE: Evolutionary and Ecological Physiology. Annual Review of Physiology. 61, 243-282.

Flanagan, B., Kreuger-Hadfield, S., Murren, J., Sotka, E. E., and Strand A., (Unpublished) Increased heat tolerance and heat-shock protein expression in non-native range of a widespread marine invader

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).

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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.