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.

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The BMA of Today

Christine Hart, Clemson University

2017-06-22 10.29.36

In previous blog posts I described the sand-dwelling microalgae, also known as benthic microalgae (BMA), which are essential to estuary ecosystems. Not only do they produce the air we breathe and food we eat, they also inform us about the subtle changes that are occurring in our environment. Changes that otherwise may go unnoticed.

How do BMA show these environmental changes? By forming the foundation of estuarine energy, they provide a snapshot of how the estuary is functioning as a whole. If changes occur in BMA patterns, this may indicate changes in the overall ecosystem. BMA are also easily characterized and compared using modern molecular approaches. These qualities make BMA living indicators, or bioindicators, that are important in monitoring future ecosystem health.

BMA become visible in the upper layers of sediment at low tide. Later, they decrease in density—or biomass—as the tide rises. Our project studied the mechanism for the increase of biomass during low tide. Previous studies suggested that the mechanism for biomass increase is vertical migration of BMA from lower layers to upper layers of sediment. We also tested whether BMA growth due to high light exposure contributes to the biomass increase.

Our results indicated that both vertical migration and growth due to sunlight exposure were important to the increase in biomass. This is the first contribution to literature that recognizes a multifaceted approach to BMA biomass changes.

Additionally, we studied in how the biomass increase was connected to patterns in the type of BMA in Charleston Harbor. Previous studies suggested that increasing biomass was connected to changes in the abundance of BMA species; therefore, we expected to see the amount of certain BMA species change based on their exposure to migration and sunlight.

We were surprised by our findings. In this study, we found that BMA did not vary over short time periods (by tidal stage or by exposure to migration and sunlight). Instead, we found that BMA varied spatially and over a period of 6 years. In fact, only one of the dominant species of BMA remained the same from 2011 to 2017 (Figure 1).  The long-term change in community coincides with geological changes in the sampling site (Figure 2).

QualitativeLvM-MS

Figure 1. The relative abundance of each dominant BMA species from 2011 to 2017 is shown immediately after sediment exposure (T0) and 3 hours later (TF). Only one species—Halamphora coffeaeformis—remains dominant in 2017. This is evidence of a dramatic change in the dominant type of BMA in Grice Cove.

These are positive results for the use of BMA as bioindicators. If types of BMA are invariable over short periods of time, measurements of BMA will be more precise. Bioindicators must be capable of showing changes that are occurring on a larger environmental scale; therefore, it would be a good sign if the change in BMA community reflects the changing geological environment (Figure 2). Still, more studies on the temporal and spatial patterns of BMA communities should be conducted before BMA can be used as bioindicators.

Changes in Grice Cove

Figure 2. Aerial view of Grice Cove sampling site over time. The approximate location of the sampling site is shown by the white line. Sampling sandbar has changed over time, possibly contributing to community changes. Source: “Grice Cove” 32 degrees 44’58”N 79 degrees 53’45”W. Google Earth. January 2012 to March 2014. June 20, 2017.

This study contributed new information to the studies of BMA biomass during low tide, and showed that the BMA of today in Grice Cove are significantly different than in previous years.

 

Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance. This project is funded through the National Science Foundation and supported by College of Charleston’s Grice Marine Laboratory.

 

Literature Cited:

Holt, E. A. & Miller, S. W. (2010) Bioindicators: Using Organisms to Measure Environmental Impacts. Nature Education Knowledge 3(10):8.

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19:186-201.

Rivera-Garcia, L.G., Hill-Spanik, K.M., Berthrong, S.T., and Plante, C. J. Tidal Stage Changes in Structure and Diversity of Intertidal Benthic Diatom Assemblages: A Case Study from Two Contrasting Charleston Harbor Flats. Estuaries and Coasts. In review.

Some Dramatic Microorganisms and Targeted Genetic Analysis

Emily Spiegel, Bryn Mawr College

IMG_4143

Genetic analysis has become the name of the game in many fields of biological research. Genes encode proteins, and in biology, proteins are king. Proteins guide biological pathways throughout the entire organism, so if you can track the genes, you can understand how the animal functions. Advances in technology like CRISPR, RNA sequencing, and PCR have improved the accessibility and accuracy of high-level genetic analysis in laboratories across the world. Some scientists utilize this technology to study the entire genome of an organism, while others attempt to understand the response of specific genes to various environmental factors or other external influences. This summer, I’m conducting an experiment focused on the latter. I’ll be studying how the polar algae species, Fragilariopsis cylindrus (affectionately known as Frag) copes with environmental stress by reproducing sexually. To do so, I’ll use targeted RNA sequencing to track genes related to sexual reproduction.

In order to understand how a Frag, responds to environmental stresses, you need a lot of algae. I reared nearly 100 liters of this algae in different artificial conditions. These conditions varied by two factors: photoperiod (the length of day and night), and nutrient levels. If you missed my previous post, “Stressing Out My Algae,” you should check it out for more details on the background for this experiment. We suspect that in conditions of stressful light energy (24 hours of continuous light), Frag will respond by reproducing sexually as opposed to its normal asexual mode of reproduction. This could possibly be a mechanism to rid itself of excess energy in times of stress, since sexual reproduction is more energetically expensive than asexual reproduction. By reproducing sexually, Frag may improve its chances of survival against this stress. Compounded with this is our hypothesis on nutrient deprivation. Previous experiments have shown that when a major nutrient, nitrogen, is limited, the algae cannot grow at full capacity and sexual reproduction is inhibited. We predict that when the stress of nitrogen limitation is combined with the stress of high light energy, we’ll see a reduction in the algae’s ability to survive in the stressful conditions due to the inhibition of sexual reproduction. So if we stress out the Frag enough and take away their ability to have sex, they’ll probably die. They’re some very dramatic microorganisms.

IMG_4131

24 bottles of algae were grown in six different experimental conditions varied by length of light exposure and nutrient levels. Algae was reared in 4-liter bottles filled with seawater.

So we grew our Frag, four bottles per six experimental conditions. Every day for eight days we extracted biomass from the bottle. From this sample we could test chlorophyll levels and cell counts, both of which give us a good idea of how well the algae in that bottle are growing in their conditions. We also took samples to be used for RNA extraction. Remember how genes encode proteins and proteins are king? Well before you can have your protein product, you need RNA. You’ve probably heard of DNA, which is the double stranded genetic cookbook. RNA is its single stranded offspring, which is then used as a the direct template to make proteins. A lot of genetic analysis therefore looks at RNA instead of DNA in order to understand how genes are being transcribed for protein production. We’re currently working on extracting the RNA from the original biomass sample and then running that pure RNA through a specialized machine called Nanostring. This is extremely targeted analysis, as Nanostring focuses in on the specific RNA we’re most interested in. In this case, we’re interested in RNA which is encoded from genes related to sexual reproduction. Using Nanostring will tell us how active the genes for sexual reproduction are in each bottle, which we can analyze to derive any correlation between our environmental stress factors and sexual reproduction.

If our hypothesis is correct, then we’ll see the greatest expression of sexual reproduction genes in the conditions of high light energy (24 hours of continuous light). We’d expect to also see low growth performance in nitrogen limited populations, indicated by low cell counts and chlorophyll levels. In these populations we predict we’ll see little if any expression of genes related to sexual reproduction. By the end, we’ll hopefully have a clearer picture of how phytoplankton like Frag deal with environmental stress.

Funding for this project is provided by the National Science Foundation in collaboration with the College of Charleston Grice Marine Laboratory and the National Oceanic and Atmospheric Administration. Acknowledgements to the entire lab of Dr. Ditullio and Dr. Lee in the Hollings Marine Laboratory facility.

Searching in the Sand

Christine Hart, Clemson University

Interim report picture

In “Exploring the Secret Garden” I discussed our studies of the benthic microalgae (BMA) that inhabit the intertidal regions of beaches. The goal of our study is to identify the mechanisms involved in the visually noticeable increase of BMA during low tide. This mechanism will be linked to changes in the type of BMA dominating the sand flat. To accomplish these goals our study will incorporate field work, molecular techniques, and DNA analysis.

During field work we will collect and manipulate sediment to distinguish between an increase in BMA by either vertical migration or growth mechanisms. The sediment will be collected on a sand flat in Grice Cove (Figure 1). Sand will be sampled using corers, which pick up a layer of sand without disturbing the vertical organization. The collected sand will be split between measurements of biomass, or BMA density, and DNA analysis. Biomass is measured by finding the concentration of chlorophyll a in the sediment. BMA synthesize chlorophyll a; therefore, the concentration of chlorophyll a is proportional to the density of BMA.

Sampling Site.png

Figure 1. Aerial view of Grice Cove sampling site with the approximate location of the 50 m sand flat transect site. Sampling sand flat is open to the Charleston Harbor. Source: “Grice Cove” 3244’58”N 7953’45”W. Google Earth. March 20, 2017. June 20, 2017.

The methods for field work are represented in Figure 2. There are two vertical migration treatments: filter and mesh. Filter treatments prevent vertical migration between cored and surrounding sediment. Mesh treatments permit vertical migration. If migration is important to the biomass increase, biomass measurements in mesh will be greater than in filter treatments. Filter and mesh treatments will also be exposed to shade and light conditions to interpret the impact of growth on biomass. Sunlight provides the energy necessary for BMA growth. Without sunlight growth will be limited. If growth is the mechanism of biomass increase, the shaded samples will have a lower biomass than the light exposed samples.

Field Work Diagram.png

Figure 2. Field work methods visualization. Locations of replicates along the 50 m transect are chosen using a random number generator and marked with flags. Random coordinates and a quadrat of 50 cm by 50 cm are used to determine where sediment will be sampled and treatments will be placed. Three controls (T0, TM, and TF) are taken at time intervals 1.5 hours apart after sand exposure. During TM and TF time points, samples are taken from the 4 treatments shown above: filter, mesh, filter + shade, and mesh + shade. Filter treatments prevent vertical migration, while mesh treatments permit vertical migration. Shaded and non-shaded filter and mesh treatments will be important in determining the role of sun exposure in biomass increase.

To link the mechanism of biomass increase to the BMA composition, we will use molecular techniques and analyze the DNA found in the sediment. DNA will be extracted from the sediment and amplified using a polymerase chain reaction (PCR). The DNA will be sequenced using High Throughput Ion Torrent technology. The results from sequencing will identify the BMA present at each time point and within each treatment. This information will link the mechanism of biomass increase to the changes in BMA composition. Our understanding of BMA dynamics will establish a basis for the BMA ecology in the Charleston Harbor. In the future, BMA dynamics could be compared to our study to assess changes caused by human influences in Charleston estuaries.

 

Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance. This project is funded through the National Science Foundation and supported by College of Charleston’s Grice Marine Laboratory.

 

Literature Cited:

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19: 186-201.

Are Manatees the Key?

Kady Palmer, Eckerd College

unnamed

Contaminants. One word, countless different connotations. Therefore, the exposure to contaminants is a constant concern to both the public and the scientific community. The study I will be performing this summer focuses on perfluorinated chemicals, or PFCs. PFCs are a class of contaminants that are utilized in many commercially available products (ex: non-stick pans, stain resistant sprays, and water-resistant materials) and have been classified as highly abundant and persistent chemicals of concern, in relation to overall environmental and, subsequently, human health.

teflon

Photo from: “Should You Ban Your Teflon Pan? California.” Savvy California, January 1, 2015. https://savvycalifornia.com/teflon-pan-toxic-or-not/. 

Through various mechanisms, PFCs have been noted to integrate into the environment and end up in the air, soil, and water. As this is happening, the organisms living in these areas become exposed and are put into a precarious situation. Little research has been performed on examining exactly what the effect these compounds have on organisms in these types of environments. Although it would be just as interesting to scoop water samples from different places to determine a basis for this environmental change, my project will be delving a bit deeper. Because previous studies have shown data supporting PFC accumulation in the bloodstream of different marine animals and their subsequent health consequences, I will be expanding this research by analyzing the types and abundance of PFCs in the Florida manatee.

The Florida manatee (Trichechus manatus latirostris) inhabits areas of warm water, close to the shoreline. Unfortunately, manatees have a history of endangerment, as a result of human impacts (boat strikes, entanglements, drowning due to drainages) and environmental changes. Perfluorinated chemicals, as described above, could very well be impacting manatees in ways currently unknown. This study aims to isolate the types and abundance of PFCs in Florida manatees and potential health concerns associated with this exposure. While the health of manatees is undoubtedly important, the results of this research could provide insight as to the overall health of the ecosystems examined. Manatees could function as a model for other organisms, demonstrating the possible repurcussions of PFC exposure. If that is the case, the knowledge gained from this organism, living so close to the shoreline of human inhabited areas, may be applicable in aiding future human research.

manatee-cow-and-calf

Photo from: “West Indian Manatee.” Southeast Region of the U.S. Fish and Wildlife Service. Accessed June 23, 2017. https://www.fws.gov/southeast/wildlife/mammals/manatee/.

I’d like to sincerely thank everyone involved in the National Institute of Standards and Technology laboratories who have been a wealth of information and guidance, specifically Dr. Jessica Reiner, Jackie Bangma, and my mentor, Dr. John Bowden. This project would not be possible without samples and information provided by Robert Bonde with USGS, funding from the National Science Foundation, and the College of Charleston’s Grice Marine Laboratory.

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 (2017): 917. doi:10.1002/etc.3600.

“CDC – NBP – Biomonitoring Summaries – PFCs.” Accessed June 19, 2017. https://www.cdc.gov/biomonitoring/pfcs_biomonitoringsummary.html.

West Indian Manatee”. Southeast Region of the U.S. Fish and Wildlife Service. Accessed June 23, 2017. https://www.fws.gov/southeast/wildlife/mammals/manatee/.

Exploring the “Secret Garden”

Christine Hart, Clemson University

Interim report picture

On a walk along the beach, have you ever noticed the garden growing at the water’s edge? During low tide patches of green and gold speckle the sand, growing what researchers have called a “Secret Garden.”

The “Secret Garden” is made up of a variety of microorganisms like cyanobacteria, flagellates, and diatoms. These small, sand-dwelling organisms are collectively known as benthic microalgae (BMA). BMA are responsible for 50% of primary production in estuary systems through photosynthesis and an extracellular polymeric secretion. Though small, these photosynthetic powerhouses form the basis of ocean food webs. BMA are also important indicators of ecosystem health. Scientists have documented the response of BMA to a variety of environmental conditions. As humans change natural estuary conditions, BMA will serve as a bioindicator for changes in ecosystem health.

The visible patches of green and gold at low tide indicate an increasing density—or biomass—of BMA. Currently, researchers do not know the mechanism for the visible change in BMA biomass. Our study will focus on two possible mechanisms of biomass change. One mechanism may be the vertical migration of BMA to the top of the sand.  The increase in biomass could also result from growth among BMA species due to sunlight exposure.

In addition to the unknown mechanism, the particular BMA species associated with the green and gold sheen have not been well studied. Like plants in a garden, BMA species are diverse and serve their own roles in maintaining a healthy environment. To better use BMA as a bioindicator, we will characterize the type of BMA contributing to the visible biomass changes.

Our study will focus on the mechanism of changes in biomass during low tide, while also identifying changes in the presence of BMA species. The results from the study will give us a greater understanding of the BMA that are essential to estuary systems. This information will establish a basis of BMA dynamics that can be used as an indicator of the health of estuaries.

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Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance.  This project is funded through the National Science Foundation, and supported by The College of Charleston’s Grice Marine Laboratory.

 

Literature Cited

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19: 186-201.

Plante, C.J., E. Frank, and P. Roth. 2011. Interacting effects of deposit feeding and tidal resuspension on benthic microalgal community structure and spatial patterns. Marine Ecology Progress Series 440: 53-65.

Rivera-Garcia, L.G., Hill-Spanik, K.M., Berthrong, S.T., and Plante, C. J. Tidal Stage Changes in Structure and Diversity of Intertidal Benthic Diatom Assemblages: A Case Study from Two Contrasting Charleston Harbor Flats. Estuaries and Coasts. In Review.

Underwood, G.J.C., and J. Kromkamp. 1999. Primary production by phytoplankton and 
microphytobenthos in estuaries. Advances in Ecological Research 29: 93-153.

 

Stressing Out My Algae

Emily Spiegel, Bryn Mawr College

Emily Carboy 170612

One intern’s perspective on lab work, South Carolina, and the coolest organisms in and out of water: phytoplankton.

 

The lab itself is large, packed to bursting with equipment, boxes, cabinets, monitors, and glassware. An antechamber acts as a sterile room for the most delicate of procedures, demanding precision and care. Many things reside in this room, but never quiet. The constant whirling of a machine’s fan, the hum of a freezer housing samples from a time beyond easy recollection, the typing of a research assistant hunched over innumerable data sheets…all these and more cut through the quiet throughout all hours of the day and night.

 

And at the heart of it all is the algae.

 

Small, marine microorganisms constituting a larger class known as phytoplankton, algae are the unsung heros of the environmental world. Energy, or the basic ability to do work, is the key to survival, growth, and reproduction. Without it you (and your genes) aren’t going anywhere. Algae harness the energy readily available from sunlight and convert it into a useable currency in a process known as primary production. This energy is then distributed to the many higher animals that eat them. They are the foundation of the marine food web and of the world’s energy supply, contributing to 45% of the planet’s primary production (Brierley 2017). In short, algae are cool.

So cool in fact, I’ve decided to spend my entire summer studying them. More specifically, I’ll be studying patterns of their reproduction and growth. A grad student running an experiment in this lab last year got unexpected results when she raised algae in 24 hours of continuous light instead of the normal 12 hours of light:12 hours of darkness she had followed previously.  Despite a limitation in the nitrogen added to these samples, which typically inhibits growth, the populations grown in 24 hours of light were able to grow successfully. So researchers went looking for answers.

One potential explanation is that the continuous light conditions caused the induction of sexual reproduction in the algae samples. Algae, like the rest of us, don’t like to be stressed. And being constantly exposed to light, which they automatically begin to utilize for primary production, is very stressful. It’s kind of like giving a kid a bunch of candy bars. A little is nice, a lot induces a sugar high and headaches for anyone within a 20m radius. The algae have too much energy and so they start to adjust their behavior to accommodate for the stressful conditions. One accommodation is sex. That’s right, stress out your algae and they might just turn on the Marvin Gaye and set the mood. Normally the species I’m studying (Fragilariopsis cylindrus, or just Frag for anyone without a PhD) reproduces asexually allowing high growth rates within the population. My lab is also curious as to whether low light conditions (a cycle of 6 hours of light and 18 hours of darkness) might be equally stressful to the algae and cause a similar response.

This is where I come in. This summer I’ll be exposing algae to conditions of varying light and nutrient stress in order to determine if stress actually does cause them to start reproducing sexually. Along the way, we’ll keep track of growth rates by measuring biomass, or the amount of live material within a sample. This can be measured by a variety of cool devices which tell me the number of cells in a particular volume of sample and the amount of chlorophyll being utilized in that sample. Chlorophyll is a component of the cycle of photosynthesis and is therefore a measure of the primary producers (i.e. the algae) in the sample. Eventually I’ll also run genetic analyses, tracking the utilization of genes involved in sexual reproduction as a way to determine if the algae are reproducing sexually instead of asexually.

All in all, it’s bound to be an interesting summer. Full of days at the beach, early mornings with a culture counter, and lots and lots of algae.

 

I’d like to acknowledge the entire DiTullio/Lee lab at the National Oceanographic and Atmospheric Administration as well as the National Science Foundation’s Research Experiences for Undergraduates program organized by the College of Charleston Grice Marine Laboratory. This project would not be possible without the support and guidance from these institutions and individuals. 

 

Works Cited

Brierley, Andrew. “Plankton.” Current Biology Magazine 27 (2017): 478-83.