Elizabeth Velazquez
Fall Quarter 2011
Luisa Marcelino, Timothy Swain
Northwestern University, Field Museum, Shedd Aquarium
Abstract Coral samples were obtained from Shedd Aquarium to investigate the symbiotic relationship between the corals and their zooxanthellae under environmental stress. The zooxanthellae DNA were extracted, amplified, and sequenced. The sequences were then analyzed using Sequencher 5.0 and BioEdit where they were aligned individually and then against other sequences found in previous literature research. The aligned sequences were run in Genbank using the BLAST function to identify the zooxanthellae at the subclade level. Further research into current literature was done with the best matched subclades to our sequences to further investigate the thermal resilience of the zooxanthellae. It was found that thylakoid membrane lipid compositions as well as lipid energy reserves are correlated to bleaching susceptibility. Clade D symbionts have higher lipid energy reserves, allotting for more thermal resilience in comparison to clade C as well as increased abundances in D symbiont types among reefs after bleaching events. Bleaching susceptibility was also found to have variation within clades. More research is needed to fully understand the coral-zooxanthellae relationship and acclimatization under stress.
Introduction
Coral reefs harbor over a fourth of all marine species and therefore are one of the most biologically diverse systems of the oceans (Gills, 2010). The coral community is comprised of a collection of biological communities that all interact and depend on each other, thus creating a complex system. This great biodiversity is very important because it increases ecosystem productivity where each and every species plays a significant role such as predation, habitat, nutrition, and population balance, among many more. Greater biodiversity leads to new medical discoveries and creates opportunities for economic development (Baker, 2008). The dying of the coral reefs would affect the possibility of finding new medicines and eliminate a vital source of food for many countries that depend on the fish they harbor. Many countries also depend on the reefs for their effect on tourism and their decline will affect tourist economies that are worth billions (Gills, 2010). The significance of biodiversity, potential medical discoveries, and tourism are all reasons that imply how imperative it is to preserve these beautiful organisms.
Background Information Coral reefs are mostly confined to shallow waters where light intensity is highest; known as the photic zone (Baker, 2008). Being mostly near the shore, coral reefs protect coastal areas from storm damage by absorbing most of the energy and shock of incoming waves. The shallow waters consist of high light intensity because of the short distance between the surface and the sea floor. These waters are primarily oligotrophic and are therefore very clear, which also allows for more light to pass through; wherein these regions coral reef systems provide most of the nutrients (Enriquez, 2005). Corals have an ability to grow hard skeletons of calcium carbonate and interestingly, the actual organism is the thin layer of soft living material covering the calcium carbonate skeleton (Teran, 2010). The reef-building corals, including other reef dwelling cnidarians, host dinoflagellate symbionts. These symbionts are in the genus Symbiodinium and are marine algae commonly known as zooxanthellae. The zooxanthellae actually live inside of the living tissue of the animal, creating a symbiotic relationship upon which the corals are highly dependent. The algae that live inside of the coral reflect through the coral and give it the beautiful colors that are visible to the natural eye (Gills, 2010). Through the provision of photosynthates and acceleration of calcification of the skeleton, the zooxanthellae contribute to the coral’s energetic budgets (Baker, 2008). Corals receive most of their energy, sugar, and carbon requirements from the symbionts through the transfer of photosynthetic products from the algae. The coral in turn uses these products to create fats, carbohydrates, proteins, and produce calcium carbonate facilitating a recycling of nutrients between the two organisms. These energetic products provide for rapid calcification of the skeleton while in turn the coral provides safe housing and nutrients for the algae (Middlebrook, 2008). This is why it is vital that the coral live in shallow water, so that the algae can receive enough sunlight to properly undergo photosynthesis. The corals rely heavily on the efficiency of collecting solar energy because it is essential for the survival of the coral and maintenance of the reef structure.
Coral reefs have a very small temperature threshold but can adapt to their environment and create thermal tolerances over several years (Teran, 2010). The current progression of climate change has led to rapid increases of temperature of 1-2 ˚C, thereby instigating the breakdown of the symbiotic relationship between the coral and the zooxanthellae. In addition, the deterioration of ozone, also a cause of anthropogenic sources of climate change, increases solar UV radiation and thus UV absorption by the corals. Coral reefs respond to ultraviolet (UV) radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids [MAAs], and enzymes involved in both the protection of the symbionts and the host from oxidative stress. UV radiation, synergistically with elevated temperatures, lead to changes in DNA, lipid membranes, and protein structures in the algae. These factors increase the metabolic rate of photosynthesis in the algae and lead to damage of photosystem II which is a protein complex that provides all the electrons for photosynthesis to occur (Lesser, 2004). The D1 protein is the reaction center of photosystem II and is one of the components that are most susceptible to damage (Warner, 1999). Damage to the photosynthetic apparatus of algae leads to the overproduction of oxygen radicals and toxins, which further damage the photosynthetic machinery. In turn, the radicals start to cause harm to the zooxanthellae cells and eventually their relationship with the coral breaks down. Ultimately, the polyps of the coral recoil and the algae are expulsed from the host (shown in Figure 1). The loss of the symbionts leaves the coral colorless, such that they appear bleached; a phenomenon known as coral bleaching. If temperature returns to normal, the few remaining algae can begin reproducing and creating food for the polyps again, allowing them to recover. In their bleached state, the corals are vulnerable to disease and if heat stress continues and the corals remain without their symbionts for a long period of time, the corals eventually starve to death. Once they die, other algae organisms begin to grow on and cover the skeletons of the once living corals. Without the corals, the reef is no longer as productive at producing the necessary nutrients and food for it to thrive.
Figure 1: Diagram of a coral undergoing bleaching. http://www.cabrillo.edu/~jcarothers/lab/notes/radiata/index.html The Purpose of this Study
In collaboration with the Field Museum and Shedd Aquarium, Luisa Marcelino and her team have been working with corals analyzing the symbiotic relationship with the zooxanthellae that they host. For the past few months, I have been working closely with Luisa Marcelino and Timothy Swain, taking coral fragments from Shedd Aquarium and running DNA analysis in the Pritzker Laboratory at the Field Museum. We aim to understand the zooxanthellae at the subcladal level because it has not yet been identified as one of the most important risk factor of coral bleaching (Baker, 2008). This information will be vital for a future bleaching experiment where several coral species will be bleached to observe not only the resilience of the corals but of the most abundant zooxanthellae types.
Materials and Methods Corals were being held in holding tanks at Shedd Aquarium under controlled environmental conditions. The corals were fragmented into three pieces and placed into containers with the same water from the tank the coral came from. One piece, 2 inches in length, was used for zooxanthellae extraction while the other two smaller fragments were used for DNA extraction.
Zooxanthellae Extractions and Counts Zooxanthellae cell extractions were done using a WaterPik, which was first rinsed using the seawater from Shedd Aquarium. The large fragment was placed into a plastic Ziploc bag and the tissue was removed by running the WaterPik over the fragment’s surface at medium to high speed. The water containing the zooxanthellae tissue cells was placed into centrifuge tubes and then centrifuged at 700 RCF for 4 minutes. The supernatant was removed and if there was still water in the Ziploc bag, the process was repeated until only a pellet remained. The pellet was re-suspended in the seawater from Shedd Aquarium to a desired volume (5mL) and shaken to obtain the desired concentration. One 1.5mL tube was filled with the re-suspended extraction for same day counts. Another 1.5mL tube was filled with 1mL of extraction and centrifuged at 700 RCF for 4 minutes. The supernatant was removed and 108um of 37% Formalin and 892um of seawater for preservation, stored at the lab bench. A 1.8mL cryogenic vial also underwent the same process but was filled with 1mL of 15% Glycerol and stored in a -80̊C freezer for future use.
Figure 2: Zooxanthellae counts using the hemocytometer.
The zooxanthellae cells were counted using a hemocytometer and a bright field microscope. The hemocytometer was filled on both sides using a micropipette and 12µL of the extraction for each chamber. The cells were counted using the center of the 5 by 5 µL chamber and recorded onto a cell count template (see figure 2). This was done for both chambers. Cell counts were done for 6 replicates per zooxanthellae extraction and the average number of zooxanthellae cells for each grid was calculated.
Surface Area and Cell Density
The surface area of the coral fragment was done using either the foil wrapping method or the wax dipping method. The foil wrapping method was adapted from previous experiments that have used it successfully. With the foil wrapping, the fragment was wrapped in aluminum foil to cover the entire surface area without any excess or overlap. The foil was then weighed to obtain the mass; this was completed at least three times. The surface area was calculated by dividing the average mass of the foil wrap of the fragment by the average mass of the aluminum foil.
The wax dipping method was done by first gluing the broken edge of the cleaned coral fragment to a base. Paraplast was heated above its melting point in an aluminum beaker on top of a hotplate. If the coral fragment was broken in more than one location, the edge that was not affixed to the base was dipped for 2 seconds and removed for an initial mass measurement. All the fragments were then weighed and each fragment was dipped into the wax for 2 seconds, removed, and rotated quickly in the air to distribute the excess wax and then let sit for 15 seconds to dry. If the fragment had been broken in more than one location, the excess wax was scrapped off of the broken, double-dipped surface using a razor blade. Each fragment was weighed again and the difference was calculated. The surface area was determined using a calibration curve (see figure 3) (Veal, 2010). Cell density was then calculated using the average number of zooxanthellae cells and the surface area (see Table 1).
Figure 3: Calibration curve of the wax dipping method for a Single Dip Calibration.
Cell Density Calculations | * Hemacytometer: 5x5 grid = 1 mm2 = 0.1 L * * x Volume * Number of zoox. cells from coral fragment (cells) / Surface area of coral fragment (cm2) = Zoox. density (cells/cm2) |
Table 1: Cell density calculation of the coral fragments.
DNA Extraction
One of the small coral fragments was completely submerged in CHAOS buffer in a 1.5mL microfuge tube in case CTAB extraction did not yield good results. CTAB extraction was done using the last small fragment to extract the DNA from the coral tissue (see Table 2). CTAB Extraction Protocol | 1. Add 200L of 2x CTAB to a 1.5mL microfuge tube. | 2. Add 5L of Proteinase K (10mg/mL). Grind tissue and mix well. | 3. Incubate at 60°C for 60 minutes while being shaken (use rotating incubator) | 4. Add 400L of CIA (chloroform:isoamyl alcohol, 24:1). | 5. Centrifuge for 10 minutes at 13,000xg at room temperature. | 6. Remove upper aqueous phase (<200L) without disturbing the interface and transfer to fresh 1.5mL microfuge tube. | 7. Add 200L of ice cold isopropanol or 95% ethanol, mix by inverting and precipitate at -20°C for at least 1 hour. | 8. Centrifuge in refrigerated rotor for 10 minutes at 10,000xg with the tube hinges facing outwards. | 9. Remove the isopropanol or ethanol without disturbing pellet. | 10. Add 1mL of ice cold 70% ethanol to wash pellet. | 11. Centrifuge in refrigerated rotor for 10 minutes at 16,000xg with the tube hinges facing outward. | 12. Remove ethanol without disturbing pellet. | 13. Dry pellet in speed vacuum until ethanol is gone. | 14. Resuspend pelleted DNA in 50L of TE buffer. | 15. Nanodrop extractions using TE buffer as a blank. | 16. Dilute extractions to approximately 20ng/L. |
Table 2: CTAB extraction protocol for DNA extraction.
DNA Amplification, Sequencing, and Alignment
The focus of this research paper is the Internal Transcribed Spacer (ITS) region of the zooxanthellae from our DNA extraction. Polymerase Chain Reaction (PCR) was done on 15 different coral fragments using ITS primers newA/newB and SSU primers Ss5z/Ss3z (see Table 3). The coral species identified by Shedd Aquarium include Acropora yongei, Montipora capricornis, Seriatopora hystrix, Montipora digitata, Echinopora lamellosa, Turbinaria reniformis, Montipora foliosa, Stylophora pistillata, Pocillopora damnicornis, Acropora sp, and a Turbinaria sp. The PCR amplified the ITS region of the zooxanthellae 32 times. The amplicon was then separated by gel electrophoresis using a 1% low melt gel, loading every other lane with 5 minutes apart, run at 200 V for 35 minutes. The brightest bands were cut and Gelase was added to remove as much agarose as possible and the tubes were incubated overnight. After a day of incubation, the samples were then sequenced using a 96 well plate and a long run on a 3730 Sequencing machine.
Primer | Species | Sequence | Primer Location | Region | Ss5z | Symbiont | 5'- GCA GTT ATA RTT TAT TTG ATG GTY RCT GCT AC -3' | PCR, forward primer (symbiont-specific) | SSU rDNA, 18S | Ss3z | Symbiont | 5'- AGC ACT GCG TCA GTC CGA ATA ATT CAC CGG -3' | PCR, reverse primer (symbiont-specific) | SSU rDNA, 18S | newA | Symbiont | 5'- GTT TCC GTA GGT GAA CCT GCG -3' | External flanking forward primer | ITS | newB | Symbiont | 5'- TTA ART TCA GCG GGT TCA CTT -3' | External flanking reverse primer | ITS |
Table 3: Primers used in PCR.
The sequences from the reactions were aligned using Sequencher 5.0. The aligned sequences were then aligned against 20 sequences from Lajeunesse 2005 using BioEdit. Lajeunesse’s sequences were focused on the ITS2 region of zooxanthellae and the aligned ITS2 region for each sequence was run across the Genbank database using the BLAST function to find the best matched subclade identity.
Results and Conclusions
Calculated Cell Densities
Surface area measurements, along with counted zooxanthellae cells, were used to calculate the zooxanthellae cell densities of each coral (see Table 4). Our coral samples ranged from branching (thin and thick) to laminar species, with each being very different from the rest. Therefore, no correlation was able to be determined between increasing surface area and the total number of zooxanthellae cells on each sample. The calculated cell densities were consistent with those in previous experiments, ranging from 3.03E+04 to 1.95E+06.
ITS Zooxanthellae Sequences
The zooxanthellae subclades were identified using the data obtained from the sequencing reactions. The 3730 Sequencing machine identifies the most abundant nucleotides when it distinguishes the sequence of the particular gene being sequenced. Sequencher 5.0 program takes the data from the 3730 Sequencing machine and aligns the forward to the reverse sequence of each sample. The program allows you to go through the chromatographs and accurately identify the nucleotides. Once the sequences were accurately aligned, sequences obtained from Lajeunesse 2005 by obtaining the Genbank accession numbers and aligned to our zooxanthellae sequences. Lajenesse’s ITS sequences were primarily of the ITS2 region and therefore limited our focus to the second half of our ITS sequences, which began approximately after base pair 360 (see Figure 4). There were some differences in between the sequences when aligned and several were longer than others while some were shorter. This could have been due to errors in the sequencing reaction where several base pairs could not be identified and therefore were eliminated to obtain cleaner sequences. In Genbank, using the BLAST function, the longer sequences were run both at the original length and at the same base pair length as Lajeuness’s sequences in order to determine the proper clade and subclade identities (see Table 5).
Figure 4: ITS Zooxanthellae sequence aligned with Lajeunesse 2005 ITS2 sequences using BioEdit.
Subclade Identities, Similarities and Abundances It was determined that the most abundant symbiont types in our coral samples were C21, C15, D1a, C1, C8c, and C1d. The Query Coverage determines how close in length is the sequence to the subclade while the Max Identity finds the sublade that has the maximum percentage of nucleotide matches; also, the smaller the E Value the more confident the match is. The identified subclades are consistent in our coral species with all the Montipora species hosting C15 type zoxxanthellae, the Acropora species hosting C21, and the Turbinaria species hosting C1. The Seriatopora hystrix and the Echinopora lamellose both host D1a type; while the Stylophora pistillata hosts C8c and the Pocillopora damnicornis hosts C1d. Under clade C, subclades C1, C21 and C3 are widely distributed symbiont types and are considered as host generalists and are therefore descendants of many host specific and/or regionally endemic species (Lajeunesse, 2005). C15 is a “younger” species that is not as host specialized and is more geographically diversified. According to phylogenetic trees (see Figure 5), C1d is only 3 changes from C1 while C21 and C15 are much farther in phylogenetic similarity between each other. On the other hand, C21 and C15 are closer in similarity to each other than to C1. When looking at the geographical differences, clade C seems to be the most dominant in the Pacific and the Caribbean (see Figure 6). Clades A and B are very common in the Caribbean but are rare in the Pacific while the opposite is true for clade D (Lajeunesse 2003). Coral bleaching occurs more frequently but in lower severity in the Caribbean because it can be assumed that at least one of the 3 clades present will be affected. On the other hand, environmental stress will cause more severe bleaching events that will affect a higher proportion of coral taxa in the Pacific because those corals share more similar symbionts (Baker, 2008).
Figure 5: Phylogenetic Tree of Symbiont Clade C (Lajuenesse, 2005)
Figure 6: Symbiont Clade Abundances within the Pacific and Caribbean (Lajeunesse, 2003)
In the coral-zooxanthellae symbiotic relationship, zooxanthellae thrive in the cytoplasms’ of their hosts. Evidence has shown that most corals host different symbiont types but only one is most abundant and is selected for (Sampayo, 2008). It has been found that under particular environmental stress, corals tend to switch from one type to another that is fit to handle the environmental pressure which will be discussed later in further detail. Research has shown that although additional symbiont types were detected during a bleaching event, all colonies reverted back to their original symbiont post bleaching but that overall abundance in clade D increase after a bleaching event. Changes in symbiont type are not sustained during bleaching and permanent changes are instead more likely to occur over generations rather than within the life cycle of the host (Sampayo, 2008).
Thermal Tolerances and Mechanisms of Acclimation to Environmental Stress
Coral acclimatization to environmental stress, such as increased oceanic temperatures, includes changing the expression of heat shock proteins, increasing the production of fluorescent proteins to dissipate excess energy, and changing the heterotrophic plasticity. The zooxaxanthellae instead increase the production of oxidative enzymes to increase their ability to photoacclimatize (Baker, 2008). Each symbiont clade has particular mechanisms that aid in their survival under environmental stress but more research is needed to completely understand each mechanism. Research shows that thylakoid membrane lipid composition is one of the major factors that determine a symbiont’s survival under stress.
Lipid Composition
The physiological basis of coral bleaching is initiated when the thylakoid membrane integrity of the symbiont chloroplast cells are compromised due to elevated temperatures (Tchernov, 2004). The organized stacking pattern of the thylakoids is essential for efficient photochemical transduction and it is corrupted under thermal stress. Higher concentrations of saturated fatty acids in the thylakoid membrane increase the thermal stability of the chloroplasts. Most zooxanthellae are limited in the ability to acclimate their lipid content to thermal stress and are therefore restricted to low thermal regimes because the critical threshold temperatures are determined by the saturation of lipids. The thylakoid membranes of thermally sensitive clades are disrupted when exposed to bleaching temperatures because of the high amounts of unsaturated lipids to saturated lipids (Tchernov, 2004). The lower concentration of saturated fatty acids leads to the greater susceptibility of attack by reactive oxygen molecules and ultimately damaging the host’s cells (Berkelmans, ).
The leakage of protons from the membranes, along with the loss of ATP, restricts carbon assimilation and generates O2. O2 reacts with generated electrons which in turn oxidates membrane lipids; this creates a positive feedback loop that is accelerated by increased light intensity. This damages not only the symbionts’ cells but also the hosts’, leading to the expulsion of the algae. Evidence found that thermal resistant zooxanthellae showed lower amounts of polyunsaturated fatty acids while thermally sensitive zooxanthellae had a 40% decrease in photochemical energy conversion efficiency under thermal stress (Tchernov, 2004). The difference between thylakoid lipid composition with the heat sensitive and heat tolerant species demonstrates that susceptibility to rising temperatures results from changes in the lipid biosynthetic pathways that are regulated by the enzymes that control the desaturation of specific fatty acids. Evidence shows that heat resistance is not associated with only one clade but instead is found among different clades (Tchernov, 2004). The ability of zooxanthellae to acclimate to thermal stress by modifying their lipid content was acquired by a common ancestor but selectively lost; thought to be due to the ecological trade-off between growth and heat resistance.
Energy Storage, Resilience, and Ecological Trade-Off
Phylogenetic analysis demonstrates that the evolutionary history of symbionts in corals selected for reduced tolerances to elevated temperatures during cooling periods leading to the dominance of heat sensitive corals. The relationship between corals and specific symbiont types may be energetically advantageous in the form of an increase in the storage of lipids in comparison to structural lipids along with coping with climate change. Evidence suggests that shifts in symbiont types are regulated by the trade-off between adaptive strategies to cope with climate change. Metabolic costs are associated with bleaching-tolerant symbiont types which have greater energy reserves but grow much slower (Cooper, 2011).
Symbionts in clade D appear to be associated with corals that are exposed to stressful environmental conditions (Stat, 2011). Clade C symbionts assimilate more carbon than clade D and clade C juveniles have symbiont types that grow at much faster rates than those in clade D and were therefore selected for. Clade D types grow 1/3 slower than clade C types but have greater lipid storage reserves which aid in their heat resilience. Zooxanthellae in clade C have lower lipid fraction ratios where there is an equal amount of stored to structural lipids while clade D types have higher lipid fraction ratios, double the amount of stored lipids to structural lipids. This could possibly be a strategy to guard against lean times if the zooxanthellae are expulsed during a bleaching event (Cooper, 2011). This accounts for the higher abundance of clade D in reefs that are exposed to higher temperatures versus the rarity of this clade in reefs that do not undergo environmental stress often (Baker, 2008).
Thermal tolerance is not only among different clades but also within each particular clade. It has already been established that clade D symbionts are much more thermally robust than those in clade C with common shifts from clade C to D types under environmental stress (Jones, 2008). Zooxanthellae type D1a seems to be most thermally resistant in comparison to other clade C types. Researches within symbionts in clade C have established substantial results that are vital for the understanding of symbiotic relationship. Corals with clade C1 and C8 have lower symbiotic processes under heat stress in comparison to C15, which has been found to be more heat resistant (Fitt, 2009). On the other hand, C1 symbionts are more thermally robust than C2 types, which have been found to be bleaching sensitive and often switch to clade D under stress (Jones, 2008). Since subclade C1d is genetically similar to C1, it can be assumed to encompass similar thermal tolerances, with C1 being thermally robust (Abrego, 2008). Zooxanthellae types C78 and C8a have been found to be more thermally tolerant than types C79 and C35a. During a bleaching event, C79 and C35a starved due to low protein levels which affected post-bleaching survival. Other symbiont types were also found in C79 and C35a colonies during bleaching that were not found in C78 and C8a colonies (Sampayo, 2008). Although it is uncertain the similarity between C8a and our identified C8c, it can also be assumed that it is also similar in thermal resilience. Unfortunately, no evidence has been found to determine the thermal resilience and thus bleaching susceptibility of our identified C21 type.
Conclusions and Future Work
Understanding the lipid composition ecological trade-offs among different symbiont zooxanthellae types is just the first step to truly understanding the coral-algae relationship. More work must be done to distinguish between thermally resistant and sensitive types to gain more knowledge in corals’ bleaching susceptibility. This knowledge will aid in protection of the corals and ultimately the entire reef ecosystem. This is vital because the overall loss in biodiversity of both symbiotic partners will have a negative effect in the resilience and ecological function of the reef system (Baker, 2008). In the future, we plan to conduct a bleaching experiment where our coral samples will be induced to bleaching. The zooxanthellae abundances will be monitored and recorded to determine their thermal tolerances and to further understand the mechanisms of bleaching susceptibility.
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