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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Some Dramatic Microorganisms and Targeted Genetic Analysis

Emily Spiegel, Bryn Mawr College

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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.

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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.

Stressing Out My Algae

Emily Spiegel, Bryn Mawr College

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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.

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

Bryce Penta, University of Notre Dame

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

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

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

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

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

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

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

Acknowledgements

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

References

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

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Even Phytoplankton Need Their Vitamins

Bryce Penta, University of Notre Dame

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Phytoplankton: unlike dolphins and other large marine organisms, these little creatures do not catch the attention of most people. Producing almost 50% of the world’s oxygen, phytoplankton provide a unique research opportunity to learn more about bottom-up controls on the environment. Phytoplankton have long been understood as key factors in ecosystem mechanisms, but the details of their functions still remain poorly understood. After spending the previous summer studying freshwater phytoplankton, I wanted to switch to marine environments to understand more about these small organisms.
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Figure 1. An example of the diversity of phytoplankton, all with various nutrient thresholds, especially in regard to vitamin B12. (Photo credit: Martin 2013)

My project aims to understand the effects of vitamin B12 limitation on the photosynthetic efficiency of the phytoplankton. Photosynthetic efficiency refers to the ability of the organism to funnel as much useable energy as possible into photosynthesis. Certain nutrients and trace elements in limited concentrations affect the ability of phytoplankton to photosynthesize by inhibiting key steps in the metabolic pathway. Vitamin B12, a possible limiting agent, can only be produced by microbes and recently the discovery of these organisms has exploded (New producer discovered, click here to find out more).  I will be altering the nutrient balance for my samples, subjecting them to higher or lower levels of B12 and nitrates. Until recently, most phytoplankton research has focused on inorganic compounds (nitrates, phosphates, etc.), disregarding the importance of biologically active compounds like B vitamins. Under stress of nutrient limitation, the phytoplankton no longer efficiently use the energy from photons and thus emit the energy as fluorescence. The hope of this project is to better understand the influence of vitamin B12 on both mixed phytoplankton samples and a single species culture.

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Figure 2. A fluorometer used to measure the photosynthetic efficiency of phytoplankton by taking the maximum fluorescence and the standard to get a ratio of efficiency.  (Photo credit: ACT Technologies Database)

Ultimately, the goal of this study is to elaborate on previous findings that implicate vitamin B12 in photosynthetic pathways. Few studies utilize B vitamins as a potential factor in phytoplankton systems. From this new understanding of the effect on photosynthetic efficiency, we can advise climate modelers to include or disregard vitamin B12 availability for their models as a potent limiting agent for phytoplankton.

Acknowledgements

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

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Resources

Panzeca, C., A.J., Tovar-Sanchez, Agusti, S., Reche, I., Duarte, C.M., Taylor, G.T., Sanudo-Wilhelmy, S.A. (2006) B Vitamins as Regulators of Phytoplankton Dynamics. 596-597.

Sanudo-Wilhelmy, S.A., Gomez-Consarnau, L., Suffridge, C., Webb, E.A. (2014) The Role of B Vitamins in Marine Biogeochemistry. Annual Review of Marine Science. 6: 339-367.

Bertrand, E.M., Allen, A.E. (2012) Influence of vitamin B auxotrophy on nitrogen metabolism in eurkaryotic phytoplankton Frontiers in Microbiology 3: 1-16.

Martin, Claire. (2013) Vanishing Marine Algae Can Be Monitored From a Boat With Your Smartphone. Smithsonian. http://www.smithsonianmag.com/science-nature/vanishing-marine-algae-can-be-monitored-from-a-boat-with-your-smartphone-2785190/?no-ist

“ACT Technologies Database -FLUOROMETER.” ACT Technologies Database -FLUOROMETER. N.p., n.d. Web. 16 June 2015.