Algae Microbiome: The Hidden World

Pressley Wilson, University of South Carolina Aiken

The Approach: In my previous post, I discussed (1) the importance of an organism’s microbiome in relation to its health and (2) the importance of algae in marine ecosystems due to their ability in producing oxygen, removing nitrogen and phosphorus from water, and exchanging inorganic carbon.

Considering the importance of these two variables, this summer I am researching the relationship between the algae microbiome and algae species in One’ula Beach, Honolulu, Hawai’i.  

DNA extraction in progress. Photo credit: Dr. Heather Fullerton.

The objectives of my research project are:

  • Identify relationship between algae microbiome and algae species
  • Identify relationship between the microbiome with algae’s morphology

Prediction

This study will predict there is a variation in the microbiome between algae species, due to the different species characteristics, such as calcification.

Sample Collection

The five algal species of different morphologies were hand-sampled by Dr. Heather Spalding at the intertidal region of One’ula Beach. The algae samples were rinsed with artificial saltwater to remove dirt and loosely associated bacteria. After cleaning, each species were placed into (1) a micro-centrifuge tube with 0.5 mL of RNA-later or (2) a 15 milliliter conical tube with 1.5 mL RNA-later. The RNA-later is a DNA preservation agent, was used to stabilize the algae DNA. The samples were stored at 4°C overnight. This overnight incubation allowed the RNA-later to penetrate the bacterial and algal cells to the DNA. After this incubation all tubes were frozen at -20°C and shipped overnight on dry ice to the College of Charleston, South Carolina, where they were stored in  -80°C freezer until DNA extraction.

Sample Analysis a

A completed gel electrophoresis.

A MoBio Fast DNA Spin Kit was used to extract the DNA from the algal samples. This DNA is then tested by PCR to determine if bacteria are present on the algae. To determine the number of bacteria present, qPCR is used. This molecular biology technique is used to quantify specific genes in a sample.

The PCR samples will be analyzed using gel electrophoresis, a molecular biology procedure that uses an electrical current to separate the components of the sample DNA by size. The qPCR data will be compared to a known DNA standard to determine the number of bacteria in our samples and calculations will be performed using excel.

Acknowledgements

I would like to thank Dr. Heather Fullerton for her guidance and support with this project and Dr. Heather Spalding for her sample collection. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899. 

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Meddling with Mysterious Macroalgae

Pressley Wilson, University of South Carolina Aiken

The problem: If you have ever been in the ocean, you have probably come across a piece of seaweed, which is a type of macroalgae. Macroalgae are simply algae that can be seen without a microscope. These organisms undergo photosynthesis, produce carbon, and can reduce the levels of phosphates and nitrates in water (Champagne et al. 2015).

Although algae are one of the most important parts of marine ecosystems, the algae microbiome (the bacteria that live in and around algae) is highly unknown and further research is needed to uncover this critical macroalgae information. Is there a relationship between bacteria and algal species? Is there a relationship between the algae’s physical features and the bacteria? Or is the microbiome the same throughout the algae, regardless of variation in species and physical features?

In order to answer these questions, I am conducting a research project this summer looking at the bacteria that are associated with intertidal macroalgae from One’ula Beach, Hawai’i.

Intertidal region of One’ula Beach, Hawaii

The five species below were chosen from the intertidal region of One’ula: Asparagopsis taxiformis (1), Avrainvillea sp (2), Halimeda discoidea (3), Padina sanctae-crucis (4), and Dictyota sandviscensis (5). These species were chosen because they range from red, brown, and green algae; have varying physical features; and all currently grow on the intertidal region of One’ula Beach.

Macroalgae from One’ula Beach, Hawaii (Photo credit: Dr. Heather Spalding)

Asparagopsis, Avrainvillea, and Dictyota are uncalcified, Halimeda is calcified, and Padina is lightly calcified. Asparagopsis has fluffy upright filaments, Avrainvillea and Padina have a fan-shaped thallus, Halimeda has flattened segments, and Dictyota has dichotomous branches. All species are native to One’ula Beach, Hawaii, except Avrainvillea which is an invasive species, meaning it is not native to the area. These species represent a diverse array of brown algae (Padina and Dictoyta), green algae (Avrainvillea and Halimeda) and one red alga (Asparagopsis).

This research will lead to a better understanding of algae, which could lead to a better understanding of all photosynthetic marine organisms. Furthermore, this research will be used as preliminary results for Dr. Heather Spalding’s work in the Northwestern Hawaiian Islands determining if there is a relationship between spatial patterns and the algae microbiome, beginning in August.

Acknowledgements

I would like to thank Dr. Heather Fullerton for her guidance and support with this project and Dr. Heather Spalding for her sample collection. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899. 

References

Champagne P, Hall G, Liu X, Wallace J, Yin Z. 2015. Determination of Algae and Macrophyte Species Distribution in Three Wastewater Stabilization Ponds Using Metagenomics Analysis. MDPI – Water. 7(7): 3225-3242.

Getting warmer…

Kaylie Anne Costa, University of Miami

IMG_6879Findings: In my previous post, I outlined how lipidomics and metabolomics would be used with mass spectrometry to study changes in the lipids and metabolites in manatee plasma in response to cold stress syndrome. The purpose of this study to provide deeper understanding how cold stress syndrome impacts Florida manatees

Our original research question was: Can changes in the lipidome and metabolome of plasma samples of Florida manatees be seen in response to CSS? Although the metabolomics data is still being processed, lipidomics has already shown promising results. Through our research we have found an interesting correlation between an

individual having a plasma Serum Amyloid A (SAA) value outside the healthy range and changes seen in their plasma lipidome. SAA is an acute phase protein produced in response to inflammation. When comparing the healthy manatee plasma samples to the CSS plasma samples with a Serum Amyloid A value greater than 50 µg/mL, we have found 81 lipids that differ significantly between plasma samples from healthy manatees and manatees with cold stress syndrome (Figure 1).

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Figure 1: Percentages of each lipid category out of the 81 total significant lipids that differed between CSS and healthy manatees

Our results indicate that the plasma lipidome of Florida manatees can differ as a result of cold stress syndrome. Now the next question is: what does this difference mean in context of manatees’ physiological response to cold stress syndrome?

This question is harder to answer, but we hope to be able to trace these lipids back to specific biological pathways that are altered by CSS. When the analysis of the metabolomic data is complete, we will have more pieces to the puzzle that may allow us to hone in on specific biological pathways affected by CSS that produce a change in both the lipidome and metabolome.

This pilot study will hopefully pave the way for future studies that will help protect this threatened species and conserve them as a sentinel species for studying how environmental changes will impact human health for the future.

This summer I have gained crucial research experience by using advanced techniques of analytical chemistry to address a threat to health in the marine environment. Through this REU program, I have learned about the diverse ecosystems in the Charleston area as well as the history that makes Charleston such a unique place. I would recommend the Fort Johnson REU program to any student looking for an opportunity to further their marine science education through research.

I cannot say thank you enough to my mentors Dr. John Bowden and Dr. Mike Napolitano. Their knowledge and eagerness to guide me through this process made this project possible. I would also like to thank the College of Charleston’s Grice Marine Lab for hosting the Fort Johnson REU program, National Science Foundation (NSF DBI-1757899)for funding, and our collaborators with the USGS Sirenia project for supplying the samples used in this study.

References:

Harr, K., Harvey, J., Bonde, R., Murphy, D., Lowe, M., Menchaca, M., … & Francis-Floyd, R. (2006). Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris). Journal of Zoo and Wildlife Medicine37(2), 151-159.

 

Living Life as a Sea Urchin Momma

Hailey Conrad, Rutgers University

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Me working hard to make my sea urchin babies

For my project I am using the same technique that the father of genetics, Gregor Mendel, used to establish his Laws of Heredity: cross breeding. So, I have to breed and raise a whole lot of sea urchins. For a refresher, I’m trying to determine if there is heritable genetic variation in how sea urchin (specifically an Arbacia punctulata population from Woods Hole, Massachusetts) larvae respond to ocean acidification. To do this, I’m rearing sea urchin larvae in low and high carbon dioxide conditions and measuring their skeletal growth. I’m breeding 3 sea urchin males with 3 sea urchin females at a time, for a total of 9 crosses. To tease apart the impact of genetic variation on just the larvae themselves, I will be fertilizing the sea urchin eggs in water aerated with either current atmospheric levels of carbon dioxide, about 410 parts per million, or 2.5 times current atmospheric carbon dioxide levels, about 1,023 parts per million. Then, I will be rearing the larvae in water aerated with either 409 ppm CO2 or 1,023 ppm CO2. This will give me four different treatments for each cross, giving me 36 samples in total. By fertilizing and rearing them in the same and different levels of carbon dioxide I will be able to see how much of an impact being fertilized in water with a higher carbon dioxide concentration has on larval growth versus just the larval growth itself. It’s important for me to make that distinction because I just want to identify genetic variation in larval skeletal growth, and separate out any extraneous “noise” clouding out the data. I’m rearing the larvae in a larval rearing apparatus. Each of the 36 samples will be placed in jar with water aerated with the correct CO2 treatment. Each jar will constantly have atmosphere with the correct CO2 concentration bubbled in. Each has a paddle in it that is hooked to a suspended frame that is swayed by a motor. This keeps the larvae suspended in the water column. The jars are chilled to 14 C by a water bath.

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My larval rearing apparatus

After a 6-day period the larvae are removed from the jars and their skeletal growth is measured. They are preserved with 23% methanol and seawater and frozen.

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An Arbacia punctulata pluteus

You’re probably curious how the heck I am able to measure the larva’s skeletons. They’re microscopic! Well, I use a microscope coupled to a rotary encoder with a digitizing pad and a camera lucida. Which, looks like this:

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A microscope coupled to a rotary encoder with a digitizing pad and a camera lucid hooked up to a computer

This complicated-sounding hodge-podge of different devices enables me to do something incredible. I can look through the microscope at the larva, and also see the digitizing pad next to the microscope, where I hold a stylus in my hand. When I tap the pad with the stylus and the coordinates of various points on the anatomy of the plutei that I am tapping at get instantly recorded on my computer! The rotary encoder is the piece attached to the left side of the microscope and it enables me to record coordinates in three dimensions. Then, I can use those coordinates to calculate the overall size of the skeleton. My favorite part of doing science is learning how scientists are able to do the seemingly impossible- like measuring something microscopic.

After I gather all of my data, I will do some statistical analysis to see the affect that the male parents have on the skeletal growth of their offspring. I will not be focusing on the impact that females have on the skeletal growth of their offspring. The quality of the egg itself could be an influencing factor on the size of the offspring, whereas sperm is purely genetic material. Like how I’m trying to isolate the influence of ocean acidification during larval rearing from during the act of fertilization, I am trying to isolate just genetic influences on larval skeletal growth from egg quality. Check back to see how it goes!

Special thanks to the National Science Foundation for funding this REU program, the College of Charleston and Grice Marine Laboratory for hosting me, and Dr. Bob Podolsky for mentoring me!

 

 

 

Playing with Plutei

Hailey Conrad, Rutgers University

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Me! Photo Credit: Kady Palmer

Ocean acidification is known as climate change’s evil twin. When the pH of ocean water drops, carbonate ions in the water form carbonic acid instead of calcium carbonate. Calcium carbonate is the form of calcium that marine animals that have calcium-based skeletons (like us!) and shells use to build their bones and shells. Having smaller and weaker skeletons or shells impacts their ability to survive. However, some individuals within certain species or populations of species have genes that make them more resistant to ocean acidification. If these individuals are able to pass on these genes to their offspring, then the species has the ability to evolve in response to ocean acidification instead of going extinct. This summer I’m working with Dr. Bob Podolsky in College of Charleston’s Grice Marine Field Station to study the extent to which ocean acidification affects Atlantic purple sea urchins, Arbacia punctulata. We are specifically trying to see if any individuals within a population from Woods Hole, Massachusetts, have any heritable genetic resistance to the negative impacts of ocean acidification. We hypothesize that there will be genetic resistance given that the northern Atlantic coast naturally has lower levels of saturated calcium carbonate, so a population that has evolved to live in that type of environment should have some resistance to lower calcium carbonate levels already (Wang et al 2013). We’re using a basic cross breeding technique to rear Arbacia punctulata larvae to their plutei stage, when they have four main body rods. At this stage they look less like sea urchins than they do like Sputnik!

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A sea urchin pluteus larvae with four body rods

Then, we will look to see if any of the male parents consistently produce male offspring that are more resistant to ocean acidification.  If males like these exist within this population, then the species has the capacity to evolve in response to ocean acidification, instead of going extinct! This is a very big deal, and could potentially be very hopeful. Even if we don’t get the results that we are hoping for, the results of this research could inform policy and management decisions.

Literature Cited:

Wang, Z. A., Wanninkhof, R., Cai, W., Byrne, R. H., Hu, X., Peng, T., & Huang, W. (2013). The marine inorganic carbon system along the Gulf of Mexico and Atlantic coasts of the United States: Insights from a transregional coastal carbon study. Limnology and Oceanography, 58(1), 325-342. doi:10.4319/lo.2013.58.1.0325

Thank you to the National Science Foundation and College of Charleston’s Grice Marine Laboratory for funding my project. And, special thanks to Dr. Bob Podolsky for being a wonderful and supportive mentor!

 

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.

 

Hook, Line and Sinker

Sierra Duca, Goucher College

Spotted seatrout, Cynoscion nebulosus, are important recreational fish that range from the Atlantic coast to the Gulf of Mexico. They are also good indicators of environmental changes in estuarine habitats since all of their life stages are found in estuaries1. To reiterate, I am studying muscle softness in spotted seatrout induced by the parasite Kudoa inornata. Several Kudoa species are notorious for causing this muscle softening, which makes the meat of the fish go bad faster than in uninfected fish2. This is an issue with fish that are consumed by people, such as the commercially important farmed Atlantic salmon2. seatrout for blogg

Fig 1. Spotted seatrout that were caught via trammel netting (PC: Sierra Duca).

First of all, to study this I need fish. While the mode of infection of Kudoa parasites is not well understood, it is presumed that wild spotted seatrout have a higher rate of infection of Kudoa inornata; therefore, I needed some wild spotted seatrout. In addition to the traditional hook and line approach of fishing for spotted seatrout, I was able to join a group at the South Carolina Department of Natural Resources (SCDNR) as they went trammel netting. In comparison to other nets, trammel nets have three layers of netting that vary in size in order to catch fish of various sizes. trammel net for blog

Fig 2. This illustration depicts the basic function and structure of a trammel net. A similar such device is used by the SCDNR to catalogue fish in specific sites over time in order to study the changing population dynamics of various fish species.

Once I have the fish I fillet, refrigerate, and take muscle biopsies at time points between 0-6 days, which is the most likely time that the fish would be consumed. I test the firmness of these muscle biopsies, as well as the parasite density. What I am trying to accomplish is to establish whether or not there is a link between parasite density and accelerated muscle softness (which causes the meat to go bad faster in infected fish), and if the rate of muscle softening changes over the course of 6 days. Ultimately the project will help increase our understanding of the effects of Kudoa inornata on the muscle of spotted seatrout. plasmodia for blog

Fig 3. This image (under 100x magnification) displays a plasmodium structure that contains a cluster of spores (known as myxospores) of Kudoa inornata in the muscle tissue of spotted seatrout. One way that I quantify parasite density is by looking at the average area of plasmodia. I can do this because generally larger plasmodia are found in the more infected fish  (PC: Sierra Duca).

Literature Cited 1Bortone SA (ed) 2003: Biology of the Spotted Seatrout. CRC Press. Boca Raton, FL, 328 pp 2Henning SS, Hoffman LC, Manley M (2013) A review of Kudoa-induced myoliquefaction of marine fish species in South Africa and other countries. S Afr J Sci. 109: 1-5

Photo Source (Fig 2): http://thewikibible.pbworks.com/w/page/22174694/Fishing%20in%20the %2Bible%20and%20the%20Ancient%20Near%20East

Acknowledgments The Fort Johnson REU Program is funded by the National Science Foundation. This research is made possible through the mentorship of Dr. Eric McElroy and Dr. Isaure de Buron.  In addition, I would like to thank the College of Charleston and the South Carolina Department of Natural Resources for providing the help and facilities necessary for my project.