Haloarchaea: Life at the Brink

Ben Farmer, University of Kentucky

IMG_20180705_150556The problem: What comes to mind when you think of extreme environments? The freezing tundra of Antarctica, or maybe the fiery lava flows of a Hawaiian volcanic zone? Those particularly interested in marine science may think of the deep ocean, perhaps the Marianas Trench. Whichever drastic environment you think of, one fascinating thing ties all of these extremes together: life finds a way to thrive in each of them.

Earth is home to as many as 1 trillion species, and the bulk of them are microbes (Locey and Lennon 2016). Microbes that are adapted to live in conditions that are inhospitable to most life on Earth are called extremophiles. Archaea and bacteria, the two domains of life aside from eukaryotes, represent the majority of extremophiles. While archaea were long thought to be a type of bacteria since the two appear very similar, archaea are more closely related to humans. Archaea are an important model organism because they have forged a niche in just about every habitat imaginable. Hot springs in Yellowstone National Park were among the first locations where archaea were discovered and owe their vibrant colors to these microbes (Oren and Rodriguez-Valera 2001).

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Man-made salt pans of Bonaire, Dutch Caribbean, tinged pink by archaea. Halophiles dominate these artificial habitats. Credit: Benjamin van de Water, Flickr, 2009.

Haloarchaea are what I am studying this summer at the College of Charleston. Halo– is a prefix meaning “salt,” and haloarchaea are halophilic, or salt-loving. Perhaps the most famous location that haloarchaea have been found is in the Dead Sea – evidently not so dead after all. Haloarchaea are commonly found in water 10 times as salty as the ocean, in conditions known as hypersaline. Our goal is to investigate what adaptations have made that possible.

We know that the amino acid composition of halophiles is unusually acidic (Martin et al. 1999). Proteins of halophiles are therefore also unusually acidic, which allows their proteins to properly fold in hypersaline conditions. What we do not know is whether the expression of proteins can change at different salinities. Better understanding how proteins are adapted in haloarchaea lends itself to understanding extremophiles on a broader scale.

Mechanisms that allowed microbes to function in seemingly inhospitable environments were likely responsible for evolution of life on Earth (Rampelotto 2010). There are many habitats today that mimic extreme environments from both ancient history and current conditions on other planets, such as Mars. Martian soil is incredibly salty, a result of surface water that evaporated long ago (https://dornsife.usc.edu/labs/laketyrrell/life-in-hypersaline-environments/). Halophiles may have once lived in those hypersaline Martian waters. Therefore, knowledge that we gain about haloarchaea adaptations is valuable to our understanding of life both on Earth and elsewhere.


Acknowledgements

Many thanks to my mentor, Dr. Matthew Rhodes, who has introduced me to everything from cell culturing to python. This project is funded through the National Science Foundation and supported by the Fort Johnson REU Program, NSF DBI- 1757899.


 



References

Locey KJ, Lennon JT (2016) Scaling laws predict global microbial diversity. Proc Natl Acad Sci 113:5970–5975

Martin DD, Ciulla R a, Roberts MF (1999) Osmoadaptation in Archaea. Appied Enviromental Microbiol 65:1815–1825

Oren A, Rodriguez-Valera F (2001) The contribution of halophilic Bacteria to the red coloration of saltern crystallizer ponds. FEMS Microbiol Ecol 36:123–130

Rampelotto PH (2010) Resistance of microorganisms to extreme environmental conditions and its contribution to astrobiology. Sustainability 2:1602–1623

 

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Bacteria in the Ocean? That Eat Iron??

Lauren Rodgers, Rutgers University

Version 2The problem: Have you ever asked yourself, what is iron? It is an element? A rock? Some weird orange-ish substance? Is it the tool that you use to get the wrinkles out of clothes? And what does iron even do? Does it just sit there? Does anything eat it? Can we make things out of it? Iron is one of the most abundant elements on earth, yet not many people know much about the important role it plays in our lives.

Iron is more than just an element, or something found within a rock. It’s a nutrient, something necessary for the growth and metabolism of almost every living organism on Earth (Hedrich & Johnson, 2011). In the ocean, iron is found in two different forms, ferrous iron or Fe(II), which is soluble in water, and ferric iron or Fe(III), which is insoluble in water (Hedrich & Johnson, 2011). Because ferrous iron is soluble it is the form of iron that can be used by most organisms in the water (Hedrich & Johnson, 2011). This ferrous iron, however, is limited in the ocean despite its abundance in the Earth’s crust. In fact, Fe(II) is present only in incredibly small concentrations, making it a major limiting factor of growth for all of the plants and algae in the ocean. This is important because these plants and algae serve as the base of many food chains, so if there is a limitation on the growth of these organisms, it affects every other organism throughout the food chain. Though iron is an extremely important nutrient for many living organisms, it is still not well understood. One of the least understood aspects is how iron specifically cycles through different marine environments. Does it ever change form? Does anything add iron to the ocean? Does anything take iron out of the ocean? These questions bring us to Zetaproteobacteria.

Zetaproteobacteria is a recently discovered class of iron-oxidizing microbes. This just means that the bacteria eat iron in the form of Fe(II) and produce Fe(III) as a waste product (Emerson et al., 2007; Chiu et al., 2017). In fact, these waste products can take on the form of hollow tubes, also called tubular sheaths, or twisted stalks that you can see under the microscope!

 

Zetaproteobacteria were initially described in 2007 near hydrothermal vents, utilizing the large concentrations of Fe(II) that were present in the fluid that spewed from the vents (Emerson et al., 2007).

Iron Mat

Iron mat composed of Zetaproteobacteria on a lava rock near the submarine Loihi volcano. (A. Malahoff, Hawaii, Loihi Volcano, July 1988)

How do Zetaproteobacteria relate to the cycling of iron? 

Zetaproteobacteria, with their role in eating iron and transforming it from its soluble Fe(II) state into its insoluble Fe(III) form may have an important role in the cycling of iron through the environment, functioning as an important source of iron removal.

Since their discovery, Zetaproteobacteria have also been observed in many other habitats, including coastal estuarine habitats with lower levels of iron, similar to that of Charleston, SC. (Laufer et al., 2017; Chiu et al., 2017). Our study will try to identify if these Zetaproteobacteria are present in the muddy soils around Charleston, as well as measure the levels of Fe(II) and Fe(III) in the rivers where these bacteria may be found.

 

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Hopefully, through the study of the distribution of Zetaproteobacteria across the globe, including the chemical characteristics of the different environments that they inhabit, we may get a clearer picture of how iron cycles in aquatic environments and the role that these Zetaproteobacteria play.


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research. I would also like to thank the National Science Foundation for funding this research as well as the College of Charleston and Grice Marine Lab for their support.


References 

Chiu, B. K., Kato, S., McAllister, S. M., Field, E. K., & Chan, C. S. (2017). Novel pelagic iron-oxidizing Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone. Frontiers in Microbiology, 8(JUL), 1–16. https://doi.org/10.3389/fmicb.2017.01280

Emerson, D., Rentz, J. A., Lilburn, T. G., Davis, R. E., Aldrich, H., Chan, C. S., & Moyer, C. L. (2007). A novel lineage of proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities. PLoS ONE, 2(8), e667. https://doi.org/10.1371/journal.pone.0000667

Hedrich, S., Schlömann, M., & Johnson, D. B. (2011). The iron-oxidizing proteobacteria. Microbiology,157(6), 1551–1564.

Laufer, K., Nordhoff, M., Halama, M., Martinez, R. ., Obst, M., Nowak, M., … Kappler, A. (2017). Microaerophilic Fe(II)-oxidizing Zetaproteobacteriaisolated from low-Fe marine coastal sediments – physiology and characterization of their twisted stalks. Applied and Environmental Microbiology, 83(February), AEM.03118-16. https://doi.org/10.1128/AEM.03118-16

Mori, J. F., Scott, J. J., Hager, K. W., Moyer, C. L., Küsel, K., & Emerson, D. (2017). Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. Nov. ISME Journal, 11(11), 2624–2636. https://doi.org/10.1038/ismej.2017.132

Is “Paraben-Free” the Way to Be?

Jaclyn Caruso, Salem State University

Me-WetLab Edit

In the wet lab, we wear a lei to remember to shut off the tank valve. Photo: Jaclyn Caruso, 2018.

The problem: Have you brushed your teeth today? Washed your hair? Put on deodorant, perfume, makeup, or lotion? If you (hopefully) have, you’ve used a cosmetic. According to the FDA, anything that is applied to your body with the intention of cleansing or beautifying it is a cosmetic (FDA, 2018). Because this category covers such a wide variety of products, it’s easy to imagine just how many are used worldwide on a daily basis.

Like anything people use, cosmetics are eventually washed off, and often end up in the ocean from sewage drains and wastewater treatment plants. The problem with this pollution is that cosmetics contain preservatives. Although these components prevent the growth of bacteria and mold, their actions when introduced to natural systems are not tested at great lengths when considering their frequent use. Until a few years ago, the most common preservatives were a group of chemicals called parabens.

But, you’ve probably heard of at least one product that claims to be “paraben-free.” This aversion to parabens followed a landmark study in 2004 which showed that parabens have the potential to accumulate in human breast tumors (Darbre et al., 2004). The authors explicitly stated that the source of the parabens (methylparaben, mainly) was unknown, but many people were shaken by the findings. Cosmetics manufacturers began changing their formulations by using newer, “safer” preservatives like 2-phenoxyethanol and chlorphenesin (Bressy et al., 2016). However, these alternative preservatives have not been extensively tested for their effects on marine animals, which may be at risk when these chemicals enter the ocean.

Me with Urchin

Collecting sea urchins at Breach Inlet! Photo: Dr. Podolsky, 2018.

My research this summer aims to explore the effects that these alternative preservatives have on marine animal development. We will use the local sea urchin Arbacia punctulata as a model, because it is easily collected in the wild and reared in the lab. Like many marine animals, A. punctulata is a broadcast spawner—males and females release their sperm and eggs into the water column. After fertilization, the embryos develop into free-floating larvae, which are highly sensitive to pollutants.

We will expose the sea urchin larvae to various concentrations of each chemical to test whether larval development is affected negatively by the chemicals. Such negative effects could inhibit the ability of sea urchins to develop properly, leading to death or inability to mature to adulthood. If we see effects in sea urchins, there is a possibility of similar effects in other species that may be more directly important to humans, like fish and crustaceans.

Our ultimate goal is to explore whether products that are safer for people are safer for the marine environment. If they are—great! If not, we need to think critically about the products we use that end up in the ocean, because human and ocean health are inextricably linked. Healthy oceans, for example, provide us with food, medications, recreation, and more (NOAA, 2018).

Blog 1 Photo

Left: A beautiful specimen of Arbacia punctulata. Scale bar = 1 cm. Right: Dr. Podolsky demonstrating how to induce spawning in sea urchins using a low voltage across the gonopores. Photos: Jaclyn Caruso, 2018.

 


Acknowledgements

Thank you to Dr. Bob Podolsky (CofC) for his mentorship and endless patience, Dr. Cheryl Woodley (NOAA) for graciously offering her procedures and resources, and Pete Meier (CofC) for teaching me the ropes of setting up aquaria. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.


References

Bressy, A. et al. (2016) ‘Cosmet’eau—Changes in the personal care product consumptionpractices: from whistle-blowers to impacts on aquatic environments’, Environmental Science and Pollution Research. Environmental Science and Pollution Research, 23(13), pp. 13581–13584. doi: 10.1007/s11356-016-6794-y.

Darbre, P. D. et al. (2004) ‘Concentrations of Parabens in human breast tumours’, Journal of Applied Toxicology, 24(1), pp. 5–13. doi: 10.1002/jat.958.

FDA (U.S. Food & Drug Administration) (2018) ‘Is It a Cosmetic, a Drug, or Both? (Or Is It Soap?)’ https://www.fda.gov/Cosmetics/GuidanceRegulation/LawsRegulations/ucm074201.htm (accessed Jul. 2, 2018).

NOAA (National Oceanic and Atmospheric Administration) (2018) ‘What does the ocean have to do with human health?’ https://oceanservice.noaa.gov/facts/ocean-human-health.html (accessed Jul. 2, 2018).

2018 Fort Johnson REU Program

Welcome to the 2018 cohort!  Jaclyn Caruso, Kaylie Anne Costa, Nicole Duran, Ben Farmer, Connor Graham, Cheldina Jean, Samera Mulatu, Nick Partington, Lauren Rodgers, and Kelly Townsend will be contributing three posts about their research: The Problem, Research Approach, and Findings.

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.

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.

Related image

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.

IMG_3882 (1)

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

shramp

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.