The End of a Project, the Beginning of an Investigation

Meagan Currie, Swarthmore College

In the grand scheme of scientific research, 10 weeks is the bat of an eye. In the case of my research on the affects of the chemical nonylphenol on coral health, my work is just the first contribution to a hopefully comprehensive and informative investigation on this chemical. The more researchers understand nonylphenol and the ways in which it interacts with complex organisms such as coral, the better informed those developing water quality standards in  the future will be.

My work involved exposing the coral Acropora cervicornis to four different concentrations of nonylphenol (1, 10, 100 and 1000  μg/L) over 96 hours, and evaluating its effects on coral physiology, wound healing and the photosynthetic activity of symbiotic algae that live in the coral tissue. These results were compared to a control group that was not exposed to nonylphenol. The results of each of these tests were interesting in their own right. Put together, they will hopefully help direct future studies on nonylphenol.

In as little as 24 hours, there were visible changes in the overall appearance of coral fragments exposed to the highest two concentrations, which was monitored and quantified using a health index that evaluated tissue coverage, coloration and polyp retraction. This trend continued, and after 96 hours, the lowest three concentrations had significant reductions in physical health compared to controls. This indicates that nonylphenol does have a negative effect on coral health, and gives us a general sense of the concentration range at which nonylphenol exposure is toxic. 96-hours is a comparatively short exposure time, so longer experiments that monitor health of coral exposed to lower doses will better mimic natural exposure in the future.

Tissue regeneration was slightly different among the different treatments and the control, but most significant was the highest concentration of nonylphenol, which experienced a 12% tissue loss, compared to the 62% tissue gain of the controls. Below is an image comparing the initial (top row) and final (bottom row) images of one fragment from each concentration. Tissue has been stained purple to increase contrast, while exposed skeleton is white.

Coral tissue regeneration

Top row represents tissue coverage at time zero (before exposure). The image directly below shows the same fragment after 96 hours exposure to the given concentration of nonylphenol (or no chemical control). The highest concentration caused tissue loss and bleaching of the entire coral body, as well as a reduction in tissue coverage over the injury site.

We were surprised to see that the photosynthetic activity of the algal symbionts that live in coral tissue (known as zooxenthallae) did not have a reduction in activity during this exposure period. Fragments without an injury remained active at levels comparable to the control.

PAM complete fragmentsThis visual (left) shows the heat map produced by analyzing photosynthetic ability. The more blue the fragment, the more active the zooxenthallae in its tissue are. Next to each heat map is an image of the same fragment taken the same time point (96 hours of exposure). The top row shows a control fragment (no nonylphenol) while the bottom image shows a fragment exposed to 1000 μg/L nonylphenol (highest concentration). Given the visible loss in tissue and apparent bleaching, it is surprising that the differences between photosynthetic activity are very small.

It is very possible that there is a differential response to nonylphenol between species, and the zooxenthallae may respond positively to the presence of nonylphenol. There have been studies on nonylpheno that have shown another species of the same phylum as zooxenthallae (dinophyceae), to increase biomass when exposed to nonylphenol (Hense et al., 2003).While the coral and algae interact symbiotically, they are completely different organisms, and interact in different ways with the chemical.

This being said, there was a noticeable difference between the photosynthetic activity between the tissue regeneration fragments exposed to no nonylphenol (controls) and the highest concentration group. As I described earlier top centimeter of the tissue regeneration fragments was cut prior to chemical exposure, and then tissue coverage monitored. These fragments appeared to be more susceptible to the chemical because of their laceration than intact fragments.

Injured fragments PAM

Top Row: Heat map of control fragments that have been lacerated at the top. (Right) Images of the  corresponding fragments taken under the macroscope. No NP exposure.                            (blue/green = healthy).
Bottom Row: Heat map of injured fragments exposed to 1000 ug/L nonylphenol (highest test concentration). The injured fragments were more susceptible to nonylphenol, and experienced bleaching, tissue loss and complete polyp retraction.

Coral such as Acropora cervicornis are often injured by natural forces (waves, etc.) as well as boats hitting coral reefs and divers disrupting their natural environments. It is therefore important to acknowledge the greater sensitivity of injured fragments to the chemical. In the natural environment, this could mean that even lower concentrations of nonylphenol will negatively impact tissue growth and may encourage bleaching that will stop the coral from regaining health.

The findings of this study are an exciting first step, and the chemical will need to be studied further to better understand and interpret these results. This will be an ongoing investigation but my hope is that, in the future, any water quality standard for nonylphenol in the marine environment will be guided by an understanding of how the chemical interacts with stony corals. With strong information we will be able to sufficiently protect and prioritize these incredible, valuable and fragile organisms.

Many thanks to Dr. Cheryl Woodley of the Coral Health and Disease program at the Hollings Marine Laboratory for providing me the opportunity to work in her lab this summer. Thank you to NOAA and to the Fort Johnson REU program for this incredible experience, and to the NSF for the funding to make this project possible.

References

Hense, Ba., Jüttner, I, Welzl, G, Severin, GF, Pfister, G, Behechti, A, Schramm, KW. Effects of 4-nonylphenol on phytoplankton and periphyton in aquatic microcosms. Environ. Toxicol. Chem. 2003; 22(11): 2727- 2732.

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Sperm and egg variety show

Cecilia Bueno, Lewis & Clark College

This summer my research sought to look at the effects of low levels of salinity on the sperm activity and fertilization in squirrel treefrogs (Hyla squirella). In particular, we were interested in whether there was variation in how much sperm were slowed down by saltwater and whether this variation would correlate with the percentage of eggs fertilized by a particular male.

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Squirrel treefrogs have a wide variety of colors from brown to green. Preliminary results suggest they also vary in sperm response and fertilization success at elevated salinity. (photo credit)

In order to answer these questions, I performed two experiments- one looking at sperm activity in response to salinity, the other looking at fertilization at 6ppt salinity.

Our sperm experiment produced videos of sperm at different salinity levels (~0ppt, 4ppt, 5ppt, 6ppt, 7ppt, and 8ppt) from 30 different males. I then ran the videos through an updated Computer Assisted sperm Analysis (CASA) software on Imagej in order to find out how many sperm were moving in each video (percent motile) and how fast they were moving (average velocity). The percent motile and average sperm velocities were calculated for each male across trials.

Our preliminary results suggest that there are significantly lower levels of sperm activity at an increased level of salinity than the control- both with lower average velocity and lower percent motile. However, there does not appear to be a clear relationship between increasing salinity and sperm activity when comparing between 4 and 8ppt.

Since we used sperm from 30 males, we were able to look at variation in these males as a sample of their population. Our preliminary results suggest that there is significant variation in how different males’ sperm responds to increasing salinity. This is important because variation is needed if the population could to adapt, though future experiments need to be done to determine if this variation is heritable and can be passed down from parent to offspring.

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Male 2 (H. squirella) from my experiment where he was collected from at Dixie Plantation, Hollywood, SC.

The fertility experiment looked at fertilization success at 6ppt with 32 males and females. We found a large amount of variation in fertilization success overall within the population, from near 0% too near 100%, with many in-between. Preliminary results suggest that a significant amount of this variation can be attributed to both male and female influence.

When comparing sperm activity and fertilization success at 6ppt salintiy, our preliminary results do not find a significant relationship between the two. This proposes future questions to be studied about what could be causing variation among the two factors.

Overall, our preliminary results do suggest that salinity has a negative effect on sperm activity and fertilization. However, results also suggest that there is variation in the severity of these effects. There are many more questions to be answered about the effects of salinity on gametes (sperm and egg) and fertilization in frogs- from what could be causing variation, to effects on unfertilized eggs, to variation between different species and more. As freshwater systems continue to be threatened by increased salinity, answers to these questions will be increasingly important.

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.

Deanna Hausman, U. of Texas at Austin

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In my two previous posts, I discussed my research studying the effects of UV light on oil toxicity. Well, around two months and hundreds of larval shrimp later, my research has come to an end! And I’m discovered quite a lot of interesting things.

The first thing we discovered was just how much more toxic UV light makes oil. The average percent mortality for the various concentrations is shown in the following graph. This graph shows that UV light makes the three highest oil concentrations significantly toxic, while none of the concentrations tested were significantly toxic under non-UV light. After these data were analyzed, we determined that UV light essentially makes oil around 4.3 times more toxic than it would be under non-UV light.

initial mort

After 30 days, there was even more mortality, as shown in the following graph. This graph shows that after 30 days, all the shrimp exposed to the 3 highest concentrations of oil under UV light died, and only 1 of the shrimp exposed to the lowest concentration of oil under UV light hadn’t died. All the concentrations of oil were significantly more toxic under UV light. This study shows that even after shrimp are removed from oil, being exposed to oil can cause significant harm.

latent mort

Other studies showed that early oil exposure can harm shrimp development. Further studies looked at the dry weights of the shrimp at the end of 30 days and the concentration of ecdysteroid molting hormone at the end of 96 hours of oil exposure. This found that oil exposure increased the dry weight for some concentrations, and certain concentrations reduced the concentration of molting hormone. The reduced molting hormone is especially troublesome, because this hormone is very important in shrimp growth and development. Having reduced ecdysteroid could be inhibiting shrimps’ ability to properly grow.

Other experiments I conducted were on thin oil sheens. What I found was very mixed. There was higher average mortality in the shrimp exposed to the oil and to the oil and UV light, however, it wasn’t statistically significant. Therefore, more research is definitely needed.

In the future, some interesting research to conduct would be to look at the sublethal effects caused by lower concentrations of oil, and studying different species responses to the thin sheens. From the beginning, the goal of this work has been to better understand the harm caused by oil spills so that we can better respond to them, and I hope my work this summer has filled in some of the gaps!
A huge thank you to my mentors, Dr. Marie Delorenzo and Dr. Paul Pennington. I’d also like to thank the NSF, The National Centers for Coastal Ocean Science and the Grice Marine Lab for supporting this research.

Evolution is faster than you think

Killian Campbell, Eastern Washington University 

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My work here at Grice is concluding for the summer and I cannot believe how fast time has gone by!

In my last post, I revealed to you the primary tool (inhibitors) I would be using to understand the role heat shock proteins play in responding to stressors. I also mentioned that I would be subjecting samples of an invasive seaweed, Gracilaria vermiculophylla (or simply Gracilaria) to extreme heat, cold, and low salinity and observe how much they bleach in response.

After conducting the entirety of the experiment and reviewing our data, our hypotheses about heat shock proteins and Gracilaria, an invasive organism were largely supported!

We found that samples of Gracilaria when administered the heat shock protein inhibitor bleached at much higher rates than samples without the inhibitor. This relationship held true for all three stressors. This result is particularly interesting, because it demonstrates, at least experimentally, that heat shock proteins play a central role in tolerating stress events. However, when considering past research conducted on native populations these results become increasingly interesting. Research has shown that native populations bleach at much higher rates than the invasive populations they produced when subjected to the same stressor. Therefore, the amalgamation of this evidence has lead us to hypothesize that the Gracilaria population found here in Charleston rapidly evolved after it was introduced. Meaning that, Gracilaria in Charleston (and the greater SE USA) is thriving because it rapidly evolved to express a trait that allows it tolerate stressors. Given the experimental data produced so far, this type of response is not seen in the native populations.

Of course, we cannot claim this as absolute, but it definitely sets the stage for further research to pursue this idea.

Manatees and PFCs- The Future of Contaminant Studies

Kady Palmer, Eckerd College

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In my previous post, “The Problem with PFCs- Seeking Answers in Plasma”, the abundance of perfluorinated chemicals, or more specifically perfluoroalkyl acids (PFAAs), was analyzed in manatee plasma and correlated to variables such as site, water temperature, and blood chemistry. The purpose of this study was to develop a greater understanding of these chemical contaminants in regards to their routes of exposure and subsequent health effects.

Accumulation of PFAAs within organisms is proposed to be predominantly attributed through diet. Therefore, apex predators, like alligators, dolphins, and humans are found to be at a higher risk for increased concentrations of these chemicals in their body (Bangma et al., 2017, Fair et al., 2012). This is a result of biomagnification, or increasing levels of a compound as one continues up the food chain or trophic hierarchy. Manatees, however, are not predators, and are considered lower on the trophic hierarchy due to their herbivorous diet. With that knowledge, the amount of PFAAs within them, if any, was hypothesized to be very small.

After obtaining data from chemical extractions and liquid chromatography tandem mass spectrometry (LC-MS/MS), concentrations of at least two perfluoroalkyl acids were detected in all 69 manatee plasma samples. What that means is that PFAAs are integrating into the biological systems of manatees and accumulating within their bloodstream, presenting different results than our initial hypothesis.

pfos

One of the most common PFAAs found in manatee plasma, known as perfluorooctanesulfonic acid (PFOS). Photo from: http://pubs.sciepub.com/ces/2/1/3/

Data and statistical analyses determined location-based differences in PFAA concentrations. In addition, correlations were found between high PFAA burden, blood chemistry measurements, and water temperature at the time of sampling. With this information, a basis for further investigations is possible to begin determining potential health effects of PFAAs in not only manatees, but in humans as well.

manateesCR

Because manatees cannot tolerate cold water, they congregate in warm waters during the winter seasons. Interestingly, correlations between water temperatures and PFAA values were found in this study. Photo from: http://proof.nationalgeographic.com/2014/07/21/floridas-manatees-the-search-for-warmer-water/

In summary, the purpose of this experiment was to answer two questions: 1) Are PFAAs present in manatee plasma? 2) If so, can heavy burdens of PFAAs be statistically correlated to health variables?

The first question was answered within the first week of analysis, simply by identifying detectable levels of these chemicals in manatee plasma. The second question, however, is more complicated to answer. The statistics say that there are associations between PFAAs and differing health measurements, however, the significance and meaning of that data is something future research must focus on. The reasons behind the correlations are still unknown, even though some explanations may be proposed.

I would like to extend an enormous thank you to everyone who made this project possible, including Dr. Jacqueline Bangma, Dr. Jessica Reiner, and my extremely motivating mentor, Dr. John Bowden. I would also like to thank the National Science Foundation for their funding, the College of Charleston’s Grice Marine Lab for hosting this REU, and the USGS Sirenia project for supplying the samples I utilized in this project.

References:

Bangma, Jacqueline T., John A. Bowden, Arnold M. Brunell, Ian Christie, Brendan Finnell, Matthew P. Guillette, Martin Jones, et al. “Perfluorinated Alkyl Acids in Plasma of American Alligators (Alligator Mississippiensis) from Florida and South Carolina.” Environmental Toxicology and Chemistry, no. 4 (2017a): 917. doi:10.1002/etc.3600.

Fair, Patricia A., Magali Houde, Thomas C. Hulsey, Gregory D. Bossart, Jeff Adams, Len Balthis, and Derek C.G. Muir. “Assessment of Perfluorinated Compounds (PFCs) in Plasma of Bottlenose Dolphins from Two Southeast US Estuarine Areas: Relationship with Age, Sex and Geographic Locations.” Marine Pollution Bulletin 64 (January 1, 2012): 66–74. doi:10.1016/j.marpolbul.2011.10.022.

 

The Plutei: An Unexpected Journey

Hailey Conrad, Rutgers University

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Me, using a microscope with a camera lucida coupled to a rotary encoder and a digitizing pad to record landmarks for the plutei (Photo credit: Carly Lovas)

In previous blog posts I have documented my project studying the impact of ocean acidification on the larval skeletal growth of Atlantic purple sea urchin, Arbacia punctulata, from Woods Hole, Massachusetts. After breeding my sea urchin parents and rearing their larvae, I was able to record the coordinates of landmarks at different points on each larvae’s anatomy. Using those coordinates I calculated the distance between each point and was able to get the lengths of different skeletal segments (Fig. 1). During the actual experiment I had a lot of difficulty getting my larvae to survive- due to a variety of factors, some still unknown, I ended up collecting far less data overall than I wanted to. I wasn’t sure if I would get any meaningful results out of the data at all, and had mentally prepared myself for that to happen. However, at the very last minute, I got statistically significant results, that confirmed my hypotheses!

By comparing the average skeletal length of the larvae from specific parents reared in either current atmospheric CO2 levels (~400 ppm), or 2.5 times current atmospheric CO2 levels (1000 ppm) I was able to see the effect that being raised in higher CO2 had on the size and development of the larvae. I found that being reared in higher CO2 conditions had a negative impact on overall larval skeletal growth, as show in Fig. 2 below. In addition, I found that being reared in higher CO2 conditions caused larval to grow more bilaterally asymmetrical (Fig. 3). Interestingly, these results were only found in the post oral arms, not the anterolateral arms. After analyzing those results I also answered the main guiding question of my research- is there heritable genetic variation in these Arbacia‘s skeletal growth under higher CO2 conditions? And according to preliminary results, there is, at least when it comes to post oral arm length.

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Fig 1. A plutei with landmarks indicated and skeletal segments highlight.

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Fig. 2 Total skeletal length for larval reared in the two different carbon dioxide levels, as compared to the line y=x

 

 

 

 

 

 

 

 

 

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Fig. 3 Total skeletal evenness for larvae under two different CO2 conditions as compared to the line y=x. Large values indicate greater asymmetry.

But what do these results mean? The larval experience reduced skeletal size, and increased asymmetry under conditions of increased ocean acidification. The larvae use their cilia-coated arms to suspension feed and propel themselves. Reduced larval skeletal size directly impacts larval survival by reducing their ability to reach food. The larval development period may also increase to that larvae have time to grow to their full size, which means they spend more time vulnerable to predators who feed on larvae. Several NOAA climate change projections calculate that atmospheric CO2 will be 950 ppm by the year 2100, so any negative consequences of reduced skeletal growth and increased asymmetry we saw happening at 1000 ppm will occur in the near-future. However- heritable genetic variation in response to increased CO2 concentrations mean that Arbacia have the capacity to evolve in response to ocean acidification. Before you start celebrating- future studies are needed to determine the frequency of these resistive traits within the population overall to see if there are enough individuals with these traits for evolution to realistically happen. Now, my mentor can compare this data set to a previously collected data set from Arbacia in Charleston to see if there is variation between populations.

A special shout out to my incredibly supportive mentor Dr. Robert Podolsky! I’d also like to thank the National Science Foundation for providing funding for this REU program, and all of the staff at Grice Marine Lab who made this program possible.

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.