Incorporating the Functional Response of Rock Crabs into Ecosystem Management in California

— Written by Gabbie Baillargeon

After several weeks of spending quality Friday nights with my crabs recording behavioral observations during feeding trials, I finally had enough data to begin to piece together the larger puzzle.  The goal of this study was to measure rock crab’s functional response, or the per capita foraging rate of a predator across varying mussel densities, in order to gain a deeper understanding of the predator-prey relationship between rock crabs and California mussels in the nearshore California ecosystem.  The premise of this research is grounded in the knowledge that rock crab density varies naturally and aggression is common between individuals, and thus harvesting this species could have consequences for species interactions and community structure.  The results of this study show that the functional response of rock crabs at all three predator densities all fit a Hollings Type II functional response curve when plotted.  This means that predation rate increases with prey density until the organism becomes full, then the predation rate plateaus.

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Predator density of 2 crabs, feeding on 10 mussels

Comparing the predation rate of rock crabs on mussels, the results show conclusively that the predation rate was lower at all prey densities for the three-predator density in comparison to the two-predator density treatment.  This indicates that predator interference, most likely in the form of rock crabs fighting with each other as more crabs are present, decreases the number of mussels consumed per predator. However, it should be noted that the number of mussels consumed per tank did increase in proportion to predator density.  This means that per capita mussel mortality, or the chance of a mussel being consumed, does increase with the addition of more predators.  Breaking mussel consumption down on a per predator basis results in lower number of mussels being consumed per predator at higher predator density treatments due to dividing the number of mussels consumed by larger numbers of predators.

Behavioral studies show that number of aggression events, quantified by fights or aggressive posturing, were highest at the three-predator treatment.  However, there was no significant difference between average number of aggression events between the two and three predator treatments.  The size selection of mussels across all treatments was also measured in this study.  It was found that the average mussel consumed was 67 mm and that there was no significant difference between the average mussel size selected across predator treatment groups.

The main conclusion about the functional response of rock crabs is that they fit a Type II functional response and the shape of the curve is controlled by predator density, making it a predator dependent response. The first implication of this research is that rock crabs are clearly able to exert pressure on mussel populations, influencing their population dynamics.  Another important implication of this research is its application to fisheries management.  Pacific Rock Crabs and California Mussels are both harvested species and have strong trophic linkages to other organisms in the food web.  As fishing activity alters rock crab abundance, the biological interactions between rock crabs changes and in turn the degree of mussel consumption and availability of mussels for other species and harvesting fluctuates correspondingly.  Ecosystem-based management and co-management of species calls for better data that aims to understand key drivers of predator-prey relationships, such as the functional response.  The functional response is an important component of predator-prey relationships, and in the case of the rock crab, it presents strong evidence that rock crab stock assessments should be conducted in order to better assess the health of the population.  My research on these feisty crabs clarified one small piece of the puzzle in terms of learning how to incorporate knowledge of the nature of predator-prey relationships through studying the functional response in order to successfully implement ecosystem based management plans for fisheries.

Beach Recovery

— Written by Emma Saas

In my previous blog post, I explored how beach species might move from one beach to another, especially as their habitat is being lost. Now, I’d like to focus more on the recovery of sandy beach habitats as a whole. A number of stressors affect these narrow areas, including recreation, armoring, sea level rise, and extreme weather conditions.

Recreational activities such as driving on the sand, and preparation for recreational activities such as beach grooming, can lower the biodiversity of a beach drastically. Human impacts do not end there, unfortunately. Coastal armoring is put in place to protect roads, buildings, and homes. All are worthy of protection, as people need places to live and roads to drive on, however the armoring cuts into upper beach habitats. This reduces the amount of room the animals have to move, and as sea levels rise, the beach is narrowed even further. Essentially, the beach habitat is being squeezed and narrowed. Rising sea levels in conjunction with armoring and other stressors contribute to the loss of the upper beach first, which is where we find beach hoppers.

El Niño winters are an example of an extreme weather condition that can wipe out all the sand, fauna, and flora on a beach. Recovery from this type of event can be slow, but it is possible. With restoration efforts, some beaches could potentially slowly recover from all of these stressors. However, an important step in beach conservation is to reduce the initial impact we have on these habitats as much as possible.

Is the Culprit still on the Loose?

— Written by Vivian Lee

Through observing the presence of the 16s ribosomal unit from the bacterial samples that were cultured on various agar plates, the next step of identification was to get a closer look into the fingerprint. By using RFLP, which stands for Restriction Fragment Length Polymorphism, more distinctive banding patterns from the samples were differentiated through various enzymes. In RFLP, enzymes play an important role, as they break the DNA samples into small pieces at various restriction sites, they make each fragment distinguishable from one another on an agarose gel through gel electrophoresis. The enzymes that were used include HaeIII, DdeI, HinfI, HhaI, and RSAI. As the identification continued, the samples that underwent both a PCR and the RFLP were sent off for sequencing.

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Figure 1: Through Gel electrophoresis, Polymerase Chain Reaction (PCR) results displayed the presence of the 16s ribosomal unit in multiple sea star bacterial samples. The presence of the 16s ribosomal unit is identifiable at around 540bp.

So what does sequencing my samples mean? Sequencing the bacterial DNA samples allowed me to identify which strain of bacteria matched the DNA of our sample. Through this recognition, results have shown that there is a presence of Vibrio, as well as other bacteria, including the Pseudoalteromonas. Through these results, what can be said about the role of Vibrio and its effect on Sea Stars is still being questioned.  The identification of Vibrio puts us one step closer to understanding that among the Sea Stars that were sampled, sick stars had Vibrio present. There are still numerous questions that need to be answered and various parameters that need to be taken into consideration. Are there are Sea Stars in intertidal that are currently alive and healthy, and if so, how are these survivors different amongst different species? Genetically, are these survivors going to advance to defend themselves in the future from another possible disease outbreak? The next approach to isolating other strains of bacteria, not only Vibrio may lead to another possible culprit that may be responsible for the deaths of these stars.

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Figure 2: Gel results from a RFLP. Gel displays four different bacterial samples that experinced RFLP using five different enzymes (HaeIII, DdeI, HhaI, RsaI, HinfI, Sau3AI)

Multiple Physiological Factors Underly Phytoplankton’s Response to Stress

— Written by Ean Eberhard

When looking at the cell abundance across three light intensities, Low (60µmol(photon)/m2/s), Medium (400µmol(photon)/m2/s), and High (800µmol(photon)/m2/s) and four different temperatures, I found that the light intensity does have an effect on the cell abundance. At the lowest light intensity, the highest temperature of 30°C showed to have very little growth while all other temperatures steadily grew. However, when the light intensity increased at the highest temperature the cell abundance began to increase. This alone suggest that there is a complex response of phytoplankton to multiple stressors. If one was to do an experiment where all temperatures were only tested across the same low light intensity, then he would conclude that when temperatures are high there is very little growth in cell abundance and thus high temperatures are detrimental to phytoplankton. This statement is not true however because as stated previously, as the intensity of light increased, the high temperature cultures were growing well. The common trend across all three light intensities was that the 25°C temperature had the highest cell abundance. Now what about the growth rate?

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When looking at the growth rates across the four different temperatures I found a common trend that the lowest light intensity had the slowest growth rate. This low growth rate can be compared to the cell abundance and show a very steady increase in number. The other common trend is that the growth rate of both medium and high light intensities seems to be pretty similar across all four temperatures. Both these higher light intensities grow fast quick then decline in growth rate not soon after especially at 25°C. At 20°C the higher light intensity cultures do not decline in growth rate nearly as fast but also have smaller cell abundance. Again, this proves that there is an interaction between both temperature and light intensity in relation to both cell abundance and cell growth. So, we know the relationship between cell abundance and growth rate but what about the cell size? When the abundance increases are the cells increasing in size or decreasing in size?

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Taking a look at the cell size compared to the cell abundance across the three light intensities and four temperatures I found a general trend of a lower cell size when there is a higher abundance. However, there is more to look at. In the acclimation phase at the lowest light intensity there is reason to note that at the highest temperature of 30°C the cells abundance increases the least, as stated before. Although the cell abundance is not increasing the cell size is significantly increasing. The exact reason for this is unknown and further analysis is needed however, I can assume that the cells are allocating its resources towards cell growth and not cell division. This increase in the individual cell size may mean a greater number of chlorophyll, increasing the cells chance to obtain more light (as we know they want at the low light intensity and high temperature treatment) and grow in abundance.  The cell abundance in medium light shows that 30°C and 25°C have similar abundances at experimental day two but the 25°C has a much larger cell size. This means there is higher biomass at 25c. This higher biomass means that at a higher temperature there is less biomass. However, at the high light, the highest temperature of 30°C has the highest biomass as they have the largest cells while similar to the 25°C abundance. At medium light intensity, the 25°C has a higher biomass until we increase the light intensity and find that the 30°C has higher biomass. This again shows a more complex interaction between stressors; the higher light intensity counteracts the original assumption that the higher the temperature the lower the biomass. It seems that with a high light intensity and high temperature the cells seem to be doing fairly well in regard to biomass. The question is, is this biomass good biomass or not (quality food or bad food, this could affect the trophic cascade). In some cases, such as in medium light, the high temperature means less carbon will go into the system which means less carbon for trophic levels and less absorbed from atmosphere. In high light we can see that the 15 and 20 have larger cells than the 25 who has the highest abundance. Yet the highest temperature at highest light intensity is doing best, this means that with different combinations of stressors we have different complex responses. Again, we can ask are those larger cells better and more efficient than a bunch of smaller cells?

Does that mean that its producing more of lower quality cells? A further chemical analysis of the cells material composition is needed to understand the quality in regard to trophic cascade. Finally, I can take a look at the QY Max values of each culture to get an idea of the cells efficiency within its environment.

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A look at the QY Max values across the four temperatures at the three light intensities shows a general trend of higher light intensities across all temperatures had the lowest efficiency while the low and medium intensities would trade places for most efficient dependent on the temperature. When I compare the 25°C high light intensity treatments efficiency and cell size I find that it has a very small cell size with a low efficiency however when I compare this to the cell abundance I find that this treatment has a very high cell abundance. If one was to simply use cell abundance as proxy for the health of the phytoplankton than he would not have the full picture and say that these cells are doing very well, however, they are not. Another finding is that in the high light intensity at 20°C I found that as the cell size increases the cell efficiency also increases. This means that an overall finding is that the lower the cell size the lower the efficiency and vice versa.

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In conclusion, it is clear that one cannot simply look at one factor of a phytoplankton’s cell response to stress when determining its health. There are multiple physiological changes to consider and that must be compared to one another to effectively interoperate what is happening to the cells under stress. It is also clear that there is a complex response induced by interactions between multiple stressors that are not simply addictive.  We know this because the combination of High light and high temperature was actually positive for cell abundance and because if one was to do this experiment at one light intensity they would find that the high temp is detrimental however that is not always the case, high temperature at high light intensities means high growth.

There is still plenty to be considered and analyzed in this experiment. A few things include POC (particulate organic carbon), where we can ask about C/N ratios and how much carbon is being taken up by the cells or is the biomass of the same quality? Secondly, we should consider biogenic silica and ask about the difference in cell structure. One other consideration is looking at the light curves where we can ask about photosynthesis rates and whether CO2 is being processed well by the cells. There are plenty of other questions to be answered and are underway. This was only the beginning.

How do humans impact shark assemblages on coral reefs in Curaçao?

— Written by Maria Rivera

For my project this summer, I compared the relative abundance of different shark dermal denticle (“skin teeth”) functional morphotypes preserved in reef sediments across two locations on Curaçao, an island located in the southern Caribbean. Denticle morphology can provide insight into the identity and ecology of the sharks that they belong to, allowing me to investigate whether the shark community composition differs between these two reefs. But why should we care? Visual surveys, such as underwater visual censuses performed by divers and baited remote underwater video stations, have recorded few sharks along Curaçao’s reefs. However, sharks are sporadically spotted by divers and anecdotally were quite abundant along Curaçao’s coast in the past. Curaçao also recently declared its waters to be a shark sanctuary. However, it is challenging to protect and manage shark populations when we cannot readily monitor them or determine how they vary across space, particularly given their high mobility.

Using the denticle record, I investigated how shark community composition and size differed between a high and low human impact site on Curaçao. The low impact site (Klein Curaçao) is a small island a few kilometers away from the main island. This island has, for the most part, healthier reefs with higher coral cover and fish biomass than Curaçao proper and has experienced less human interference. Given its low impact and distance from most of Curaçao’s population, Klein Curaçao has been thought of, as least anecdotally, as a window into Curaçao’s historical reefs. In contrast, the high impact site (CARMABI) is located near Willemstad, Curaçao’s capital where most of the island’s population resides. This reef has been exposed to pressures from fishing, pollution, diving and tourism, and boat traffic. These sites were selected to represent a gradient of human impacts on Curaçao. I hypothesized that there would be a higher abundance of sharks and henceforth a higher abundance of denticles on the reef on Klein Curaçao, which has much less human influence, as opposed to CARMABI. I also hypothesized that human activities could alter the composition of shark communities. In particular, I predicted that that there would be more pelagic shark denticles (i.e. drag reduction morphotype) in the sediments on Klein Curaçao given that it is more exposed to the open ocean and historically has experienced less fishing pressure, which tends to selectively remove requiem and hammerhead sharks. In contrast, I predicted to find a higher relative abundance of nurse shark denticles (i.e. abrasion strength morphotype) at CARMABI since this species is not typically targeted by fishermen.

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Figure 1. Shows the differences in shark communities with differences in denticle morphology.

The samples at both sites were full of denticles, which was very exciting. We found a higher number of denticles per amount sediment at the low impact site (Klein Curaçao) than the high impact site (CARMABI). This suggests that there are larger shark assemblages at the low impact site. These sites also had higher time-averaged denticle abundances, unstandardized by reef accretion rates, than other regions in the Caribbean (Panama and the Dominican Republic) that we have surveyed using the denticle record. We also saw a difference in shark communities between sites. Klein Curaçao had a higher relative proportion of abrasion strength denticles, which are found on bottom dwelling sharks such as nurse sharks. In comparison, Carmabi had a higher relative proportion of drag reduction denticles, which are characteristic of open ocean, fast swimming sharks such as a great white shark. This finding was surprising and unexpected. While we have found a pendulum-like shift in community composition from abrasion strength to drag reduction denticles across time in Panama and the Dominican Republic due to human impacts, something different appears to be occurring in this space-for-time comparison across Curaçao. First, there could be spatial differences in habitat quality and environmental features. For example, the site at CARMABI might also be easily accessed by pelagic sharks or be providing habitat for reef-associated requiem and hammerhead sharks. Alternatively, Klein Curaçao has recently been exposed to higher fishing pressures by fishermen who are now venturing farther to access fishing resources given that the inshore reefs have been historically exploited. While our time-averaged samples are likely capturing a signal of the shark assemblage prior to the start of these pressures, we could be seeing a shift in community composition progress – from a community dominated by pelagics to one dominated by demersal sharks.  This could have potentially been due to changes in how humans affect these shark communities, for example, these effects could be caused by how and what humans have fished over the years and how that has changed. These results show us that shark communities do change over time and across regions, and also that humans can have an effect on these changes. This is important knowledge when it comes to better conservation and management of shark communities near the reefs of Curaçao.

Was the heat too hot for L. pictus to handle?

— Written by Carlos Estrada

After a temperate and not-so-long summer, the Lytechinus pictus project has come to an end. In that time, I’ve unfortunately caused the deaths of a few of my beloved urchin, but in the process raised hundreds of thousands of larvae. All in the name of science! And have I got science to report! We’ll start with what was surprisingly the most difficult part of project, taking pictures.

Morphometrics

Some quick analysis of variance (ANOVA) showed that there was no significance between the lengths of the larvae and the temperatures in which they were reared at any stage. The gastrula stage held the strongest relationship between rearing temperature and length, but very little relationship was seen with the prism and pluteus stages. This conclusion corresponds with that of Hammond and Hoffman (2013), which found that increased temperatures had no significant effect on larval skeletal length. Developmental timelines, however, differed as predicted with larvae reared in the 20°C buckets reaching all three stages much quicker than those reared in 17°C buckets.

Thermal Tolerance

Preliminary data shows significance between rearing temperature and thermal tolerance at both gastrula and pluteus stages. The prism stage, however, shows no significant difference between the two rearing temperatures.  All life stages showed little to no mortality until they reached higher temperatures, in which mortality increased dramatically. Although the beginning in mortality can differ, 31°C seems to be the temperature at which mortality reaches 90-100%.

Heat Shock: hsp70 Expression

Unfortunately, my first attempt at attaining any data on the expression of the heat shock protein hsp70 through gel electrophoresis did not go well. No bands appeared on my gel, but don’t panic! After a few tweaks to our formula, we’re ready to make another stab at it first thing in the morning. My partner-in-crime, Erin DeLeon Sanchez, has run her own gel in the past with pluteus DNA that showed bands. She also went to the trouble of conducting qPCR and found that hsp70 expression was stable throughout all both rearing temperatures and heat shock temperatures.

Discussion

Very little ecological research has been done on Lytechinus pictus and I can’t express how proud I am to have done my part. But more studies are needed to better understand the relationship between this urchin and it’s changing environment. Such studies could help us predict not only this species current and potential future biogeography, but that of its prey and predators as well.

Feeding Rate Variation in Purple Urchins

— Written by William Dejesus

My results showed feeding rate variation between trials with and without a 24-hour starvation period. A decrease in consumption rate was consistently observed in trials without the prior starvation period in the warm treatment. The ambient treatment did not show a significant pattern, which could have resulted from a less intense environmental constraint than what the warm treatment experienced.

It appears that consistent food availability could lead to decreased feeding rates. This can be applied to the kelp forest community in that a healthy abundance of giant kelp will prevent large spikes in feeding rate over time. The kelp forest community, like all ecological communities, requires a stable balance of trophic level interaction in order to function properly.

My investigation of the purple urchin’s feeding behavior has lead to the conclusion that the kelp forest community will need more urchin predators, such as the California sheephead in order to maintain the high abundance of biodiversity that it is famous for. Without these key predators, it is likely that urchins will consume kelp at a faster rate, inevitably leading to monoculture urchin barrens. These barrens are unsustainable for almost all species that call the kelp forest home, as well as the purple urchins, who will have eaten themselves out of house and home. Although urchins can survive for long periods of time without abundant food, previous studies have found that urchin health in barrens is extremely low with high potential for widespread disease.

Wrapping Things Up

— Written by Ara Yazaryan

Following extensive data collection, my research has yielded some interesting results. After sorting through several sheephead stomach contents, I have discovered some interesting trends. Sheephead tend to eat a lot of urchins! Their stomachs were full of spines and tests. They also have a high affinity for bivalves and algae covered in bryozoans. It was also interesting to directly observe that smaller individuals consumed more bivalves, whereas larger sheephead ate many more and larger urchins. I am currently still processing all this data to derive conclusions and make it ready for our upcoming research symposium.

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An Anacapa Sheephead that I caught, dissected and analyzed. Its stomach was filled with mostly algae, as well as a salt-and-pepper urchin.

In the future, I think it would be interesting to continue studying the Sheephead of Anacapa. By increasing sample size and studying more individuals, the composition of this diverse kelp forest community can be assessed. Other fish species could also be studied, using their guts to further elaborate our understanding of kelp forest food webs.
Overall, this research is important for quantifying the many branches that make up the kelp forest community structure. By studying the interrelated dependence and predator-prey interactions, the fine intricacies of kelp forest community structure will be revealed. The vital role that sheephead play in this community will also be further quantified. By limiting urchins and helping control other populations of invertebrates, sheephead play a vital role in helping maintain the kelp forest. Dare I say that they may even be considered a keystone species…?

Data collection has been completed, the microscope has been shut off, and the stomach contents are finally analyzed. Unfortunately, my time here in the Caselle laboratory as a REU researcher is drawing to a close. However, this summer full of fishy adventures will be remembered forever. The interactions, experiences, and techniques I have attained here have helped to mature me both as an individual and as a scientist. Research has captured me in its grip, and tempted me with its allures. The pursuit of the wonders contained in the unknown, as bottomless as the sea is deep, motivates me to carry onwards.

 

Materials and Methods of Ara’s Fishy Project

— Written by Ara Yazaryan

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Me and my prized catch, a nice sized male Sheephead (Semicossyphus pulcher)

My experiment, now finalized, is very exciting: I get to study what the Sheephead of Anacapa are eating! My project is a quantitative assessment of Sheephead (Semicossyphus pulcher) stomach contents across several different sites and populations at Anacapa, part of the Channel Island chain. Anacapa has a unique conservation history, as the MPAs established here are of varying ages. One site is 40+ years old, and is one of the oldest Marine Protected Areas in the state of California. Another was established relatively recently, and the remaining areas of the island remain open for public use. Across these three study sites, I seek to determine the diets of Sheephead across different ages and areas.

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A fully dissected and processed Sheephead, complete with gut contents in tray

First, Sheephead were collected from these three sites (with appropriate research licensing of course) via SCUBA spear-fisherman or – much to my angling delight – with hook-and-line. Next, the fish were dissected in lab. Various parts of the fish were saved for future study: the organs, otoliths, eyes, muscle tissue, gonads, and gills. But for my efforts, the stomach intestine was squeezed to release the slurry of partially digested fish food. These stomach contents were then weighed, placed into a Falcon Tube, and preserved in ethanol to prevent decomposition (and the horrendous smell that accompanies it). Upon preservation, I place these stomach contents into an examining tray to view under a dissecting microscope, under a snorkel vent to alleviate the less-than-stellar odour. Using a low-power scope to help me view the small pieces, I correspondingly separate the contents into categories. Based on my findings thus far, the majority of the sheephead gut contents consist of urchin spines and shell fragments, kelp encrusted in bryozoan colonies, small crabs, gastropods, and bivalves. However, a few unique finds have been unveiled: such as unidentified fish spines and scales (Sheephead

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A sample of Sheephead stomach contents under a microscope: a hermit crab, urchin spines and tests, as well as mussel shell fragments can be seen.

are not normally known to be piscivorous), octopi, pieces of gravel and sand, and even a plastic nurdle (a small bead of resin plastic such as polyethylene used in the production of larger plastic objects). Such variable stomach contents, once separated, will correspondingly be assessed to determine the composition by percent volume. Data will then be pooled from many fish from all three sites at Anacapa to determine dietary trends.

Though identifying, separating, and sorting each shell fragment may seem tedious at times, I am nonetheless driven by curiosity and the pursuit of scientific knowledge. Much like the Californian gold miners of old descending into deep, dark mines to unearth unknown treasures; I plunge my tweezers with nervous anticipation into the mushy lumps of partially digested Sheephead guts, facing constant perils and danger. Okay, maybe I am being a tad hyperbolic, but hopefully my point comes across. My stomach content examinations are part of a larger effort to understand fish populations, and the effects MPAs have on fish conservation. So I carry on with my unwavering efforts, to explore the vast unknown wilderness that is the world of fish guts!

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In the midst of gory dissection, complete with gloves, photo courtesy of Dr. Jenn Caselle

Method to the Madness

— Written by Ean Eberhard

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The experiment takes place in a cold room where the two multicultivators are shown side by side. Both are connected to the CO2 tank to the right which supplies a constant amount of CO2 to the 16 tubes.

The experimental design begins with the creation of artificial sea water. To this I add three different nutrient solutions; main stock, trace metal stock, and vitamin stock. This combination of nutrients and artificial sea water makes up F/2 media used for growing the culture of Phytoplankton that is specifically studied in this project. The name of this species is Thalassiosira pseudonana. Once the culture is healthy and acclimated the experiment can proceed. The experiment is split into two weeks, each week being split into two phases. The first phase is a three day long acclimating phase for the cultures to be monitored in their tubes. Monitoring the culture at this time assures that each tube is growing healthy before entering the second phase of 4 days, the experimental phase. The first week consist of two multicultivators (seen below), one set to maintain 15°C and the other 25°C. Each multicultivator contains eight tubes that are each inoculated with the same culture of Thalassiosira pseudonana. In each multicultivator there is a set of three tubes at low light intensities of 100µmol(photon)/m2/s, 200µmol(photon)/m2/s, and 300µmol(photon)/m2/s, a set of two tubes at medium light intensities of 400µmol(photon)/m2/s and 500µmol(photon)/m2/s, and a final a set of

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When taking a closer look at an individual multicultivator, one can see the aerators placed into each of the eight tubes. Behind the tubes are a set of LED lights which will be set to different intensities when running.

three tubes with high light intensities of 600µmol(photon)/m2/s, 700µmol(photon)/m2/s, and 800µmol(photon)/m2/s. Each tube is supplied with a continues flow of CO2 at 1000ppm with a pressure of 2psi. The multicultivators are set to begin a light cycle at 5am and go into a dark cycle at 5pm. These two multicultivators are designed to give us a total of 16 different environments for the phytoplankton to grow in, varying initially by temperature and light.

 

With the experiment started, the sampling begins. Each day, from each tube, I sample for, Non-photochemical quenching, light curve, chlorophyll, particulate organic carbon, nutrients, and pH. For the beginning, middle, and end of each experiment I sample for dissolved inorganic carbon, biogenic silica, and flow cytometry.

Sampling for Non-photochemical quenching and Light Curve

When sampling for NPQ and LC3, I remove the tubes and invert three times then pipette 3ml of the cultures from each tube into a cuvette, giving me a total of 16 cuvettes for NPQ measurements and 16 cuvettes for LC3 measurements. The cuvettes are placed in complete darkness for 30 minutes for a dark adaptation. After the 30 minutes the cuvettes are individually placed into an AquaPen, which will give a value for NPQ and LC3.

Sampling for Chlorophyll, Particulate Organic Carbon, Biogenic Silica, and Nutrients

Using a filtering apparatus and filter papers, I collect samples for Chlorophyll, POC, and Bsi by filtering the NPQ and LC3 samples. Each tube gets its own filter for Chlorophyll, POC, and Bsi. The Chlorophyll and Bsi samples are stored in the freezer for a later analysis while the POC filters are incubated and left to dry. The filtrate from these tubes are pooled together by light intensity, the low light intensities (100, 200, 300µmol(photon)/m2/s) are combined for a nutrient sample while the medium light intensities are pooled together for another nutrient sample and finally a third nutrient sample is taken from the high light intensities of 600, 700, and 800µmol(photon)/m2/s. These nutrient samples are also stored in the freezer for later analysis.

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The filtering apparatus uses a pump to create suction. A filtering paper specific to the material being collected is placed on a surface above the vacuum. The samples of NPQ and LC3 are filtered through the papers. The filtrate is collected for nutrients below and the material collected rest on the filter paper.

Sampling for Flow Cytometry

For flow cytometry measurements I pipette 1ml of the culture from each of the 16 tubes. I then fix these samples with 50µl of CH2O to kill the cell while preserving its structure. These samples are then stored in the freezer until processed and counted with the flow cytometer.

Sampling for Dissolved Inorganic Carbon

To sample for dissolved inorganic carbon (DIC) I fill a serum bottle full of the culture from each tube and add 50µl of mercury chloride. This gives me a total of 16 samples. The samples are then crimped shut to exclude gas exchange and stored in freezer for later analysis.

Sampling for pH

Sampling for pH is done for every tube. I pipette 3ml of the growing cultures from each tube into their own glass cuvette. These cuvettes are then placed in a dry bath for 5 minutes or until they reach room temperature. An initial reading is then taken by placing the cuvettes into a spectrophotometer where each cuvette is given three values at three different wavelengths. The cuvettes are then removed and treated with 50µl of m-cresol and then left for 5 minutes longer. The cuvettes are placed back into the spectrophotometer for another set of readings. Both the initial and treated values for each tube are entered into an excel spreadsheet with a preexisting equation that gives the value of pH. I should end with a total of 16 pH readings.

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To the left is the spectrophotometer that gives the values that are then used to calculate the pH for each tube. Above are 5 cuvettes demonstrating the visual grade of variance amongst pH when treated with m-cresol.