The Effect of Bleaching on Distribution of Symbiodinium Clades Present within Acropora spp. Meghan Sleeper 26/10/2010

Project Summary Coral reefs provide ecosystem services and have become economically important to many communities. They are often referred to as the “rainforests of the sea” due to the level of diversity found within the reef ecosystems. Coral species are fragile and sensitive to slight environmental changes in temperature. They receive the bulk of their energy from symbiotic algae (symbiodinium spp.). If environmental temperature shifts even a few degrees the coral may expel the zooxanthellae causing a bleaching or whitening of the coral tissue. Without endosymbionts the coral cells will eventually die. Some corals repopulate with zooxanthellae post bleaching. Studies have shown that the clade of zooxanthellae often changes after a bleaching event. This is called symbiont shuffling and tends to favor clade D post bleaching. This is most likely because clade D has thermotolerant properties. Although there have been many studies to determine if symbiont shuffling occurs post bleaching there have not been many controlled experiments to reduce external environmental factors and focus on the effect of bleaching due to increased temperature on symbiont distribution. A controlled study is the next step in understanding the relationship between coral bleaching events and distribution of zooxanthellae clades. A better understanding of the mechanisms of coral recovery post bleaching will allow us to better protect reef ecosystems. A study will be carried out at Lizard Island located off the coast of Australia on the Great Barrier Reef. A preliminary survey of the area will allow us to determine the most dominant

species of Acropora in the area. The species of Acropora chosen will be sampled off the shore and bleaching will be induced in a controlled experiment. The clades of zooxanthellae present in the control and treatment groups will be compared and statistically analyzed to determine if clade D is found more often in coral that has been recently bleached. The overall experiment should take no longer than 6 months to complete and will lead to a better understanding of what happens to different clades of zooxanthellae within coral cells post bleaching.

Introduction Coral reefs form one of the most diverse ecosystems on earth which supports many marine species including fish, sponges, mollusks and echinoderms (Mulhall, 2007). Although coral reefs cover less than 1% of the earth they provide an ecosystem for 25% of all marine species (Bryant et al. 1998). These diverse and beautiful ecosystems have always supplied humans with many beneficial services including recreation, coastal protection, habitat for fisheries and biodiversity (Rosenberg et al. 2007). Due to the beneficial services of coral reefs, they have a strong economic value with an annual global economic value estimated at USD 30 billion (Conservation International. 2008). A breakdown of the worldwide economic value of reefs can be seen in figure 1. The Great Barrier Reef alone has an economic value that ranges

Figure 1 the breakdown of services provided by reefs and the predicted value in billion dollars per year (USD).

from USD 700 million to 1.6 billion per year and accommodates around 2 million visitors per year (Carr and Mendelsohn, 2003). Coral reefs have been identified as an endangered ecosystem. According to some studies approximately 20% of the world’s reefs have already been lost and 26% are being threatened (Riegl et al. 2009). The World Wildlife Fund projects that approximately 95% of the world’s coral will be lost by 2050 if the ocean temperature continues to increase at a steady rate. Coral reefs are sensitive to small environmental changes. Temperature changes of even 1-2 degrees Celsius over a period of 5-10 weeks will cause severe bleaching and can often lead to coral death (Gabriel D. et al. 2005). Symbiodinium (often referred to as zooxanthellae) are autotrophic microorganisms. They are intracellular symbionts found within coral and provide their host with 90 percent of their required energy via photosynthesis. Zooxanthellae are also responsible for the bright coloration of coral reefs (Loram et al. 2007). There are 8 distinguishable clades of zooxanthellae that are titled A-H. The primary clade found within corals in the Indo-Pacific is clade C (Sampayo et al. 2008). Riegl et al. (2009) expressed that clade D is generally more thermotolerant than clade C

Bleached

and that there may be a shift in the distribution of clades C and D after a mass coral bleaching.

Normal

Environmental change in temperature can cause corals to induce cell adhesion dysfunction which will detach zooxanthellae from the cnidarians endodermal cells (Loram et al. 2007). At this point the zooxanthellae are either expelled or digested by the coral. This response

Figure 2 Multiple Acropora spp. that have and have not been whitened by coral bleaching within the same vicinity off the coast of Australia. Acropora is a genus of coral that is often referred to as staghorn coral. Acropora is one of the most abundant corals found on the Great Barrier Reef.

leads to coral bleaching and can be observed as a literal whitening of coral due to the loss of zooxanthellae (figure 2). A brief overview of what occurs during and after coral bleaching can be seen in figure 3.

Australian Institute of Marine Science Figure 3 demonstrates the mechanisms of coral bleaching; the expulsion of endosymbionts from cnidarians cells when corals become stressed. When coral completely dies the skeletal remains will be populated by filamentous algae. Before the coral tissue dies completely it is possible for the coral to recover if zooxanthellae repopulate within the coral cells,

Project Objectives Riegl et al. (2009), Sampayo et al. (2008) and Chen et al. (2005) have all noted the greater presence of clade D as compared to clade C post bleaching, but still it is difficult to conclude the coral bleaching is solely responsible for the the shift in zooxanthellae clades. The ocean ecosystem is not static which means that the environmental conditions are constantly shifting. This makes it difficult to determine the effect of one factor during field experiments. Therefore more controlled studies must be done to eliminate other factors and determine if zooxanthellae clades redistribute or “shuffle” based on thermotolerance after a coral is bleached. I am proposing a controlled study of zooxanthellae present in Acropora spp. coral post bleaching

that would be carried out on the reef surrounding Lizard Island, just off the coast of Australia. This study would aim to draw a more concise correlation between bleaching and the distribution of thermotolerant clades of zooxanthellae.

Experimental Procedures This study will be conducted at a site that shares many characteristics with Lizard Island (14°40′S 145°28′E), which is located in the Northern section of Australia’s Great Barrier Reef (Figure 4). As mentioned by Littman et al. (2008) Lizard Island is a mid shelf reef approximately 30 km off the coast of Australia and 19 km from the outer edge of the Great Barrier Reef. It is a continental island surrounded Figure 4 Map of Australia showing the location of Lizard Island

by fringing reef with three smaller islands nearby (Palfrey, South and Bird Island). The island itself

Mermaid Cove

covers an area of 7 km2. Acropora spp. (staghorn corals) will be the coral tested because it is one of the most abundant coral found in the Coral Sea. The samples would be taken from Mermaid cove located on the northern end of Lizard Island (Figure 5). This location is ideal because it is a protected marine park and fishing is prohibited within the cove. This

Figure 5 Map of Lizard Island demonstrating the location of Mermaid Cove on the island. Mermaid Cove is located on the Northern tip of the island.

removes the possibility of our data being skewed by the anthropogenic effects of overfishing. Mermaid cove is also ideal because it is protected from ocean currents on both the North/East and South/West side of the cove. A preliminary observational survey of the sample site will be conducted to ensure a significant presence of Acropora spp. A water sampling station would be set up in the center of Mermaid Cove in an area that is approximately 3-6 m deep. The station would take periodic measurements of the ocean temperature, salinity and pH. This allows us to ensure that the coral in our controlled experiment is exposed to the same conditions found in the sampling location. The coral samples will be collected by setting up a base line that transects the sampling station and runs parallel to the shore. Eleven transects will be placed perpendicular to the baseline every 20 meters with one intersecting the sampling station (Figure 6). At each transect, three 1 m2 quadrats will be placed using a random number generation. When the quadrat is first laid out, the surveyors will make visual observations and note the percent coverage as well as the presence of crown of thorns starfish (invasive species that can cause damage to corals). Within each quadrat, random number generation will be used again to select two samples of Acropora spp. to be collected and brought back to the lab. This will give us 33 quadrats and 66 Mermaid Cove

samples. Each sample will be placed in a plastic bag and labeled with a number indicating where within the site the sample was taken from.

Figure 6 Baseline running horizontal through Center of mermaid cove (~300m) with transects running perpendicular to baseline every 20 m from sampling station. Green triangle represents automatic water sampling station.

The coral samples will be kept in two different aquariums that will pump water directly from the ocean nearest the sampling site as possible. This will reduce the error rate and possibility of random factors skewing the results. The conditions in the aquariums will also be compared to the data collected from the sampling site and regulated when necessary. One sample from each quadrat (n=33) will be placed in one of the aquariums and used as a control for that sample location. The second sample from each quadrat (n=33) will be placed in the second aquarium and undergo simulated bleaching (treatment group). Bleaching will be simulated by exposing the treatment group to an increase of 2 degrees Celsius for a period of 8 weeks. This will cause the corals to expel most if not all of the Zooxanthellae from their cells. After the treatment is complete, the color of each coral sample will be recorded using a coral health chart supplied by coral watch (Figure 7). Tissue samples will be taken from each coral specimen in the

Not Bleached

Bleached

Figure 7 Coral health chart supplied by Project Aware coral watch program will be used to identify the health of coral samples and the extent of bleaching if any in the treatment and control groups.

control and treatment group. The tissue samples will be labeled to identify sampling location and preserved in 95% ethanol for DNA extraction. The samples from both control and treatment groups will be analyzed by using the DNA isolation, PCR amplification, RFLP (Restriction Fragment Length Polymorphism) and electrophoresis methods described by Dong et al. (2009). DNA Isolation: Zooxanthellae will be isolated from the coral samples by first homogenizing coral tissue in zooxanthellae buffer (ZB). The product would then be filtered through 125um mesh to remove large pieces of coral. The solution would be centrifuged for 1 minute at 10,000 g. The resulting pellet would be washed three to five times with 10ml ZB. The pellet would be resuspended in 0.75mL DNA isolation buffer (DNAB; 0.4 mol/L NaCl and 50 mmol/L EDTA, pH 8.0) that contains 1% Sodium Dodecyl Sulfate (SDS). The samples will be heated to 65oC for 30 minutes. Proteinase K will then be added (to a final concentration of 0.5mg/mL). Samples will be incubated at 50oC for 6 hours. A phenol:chloroform extraction will then be carried out on the samples to separate DNA from unwanted proteins and lipids. The DNA will be precipitated out overnight at -20oC by using ethanol and sodium acetate. Precipitate will be resuspended in 100uL of water. PCR Amplification: 520bp fragments that correspond to

Table 1 PCR thermocycler conditions

1. 95oC for 3 minutes

the 5’ end of the 28S rDNA gene of Symbiodinium will selected 2. 94oC for 30 seconds

for in the PCR amplification using oligonucleotide primers 28SZooxR (5-CCTTGGTCCGTGTTTCAAGA-3) and 28SZooxF

3. 50o for 1 minute

(5’-CCTCAGTAATGGCGAATGAAC-3’). This will be done by

4. 72oC for 2 minutes

combining 0.05mmol/L 28SZooxR and 28SZooxF, 20ng DNA in

*Repeat steps 2-4 thirty times

50uL 1X PCR buffer, 3 mmol/L MgCl2, 0.1 mmol/L dNTPs, and 5. 72oC for 10 minutes.

0.5 U Taq DNA polymerase (Promega). This mixture will then be

placed in a thermocycler to run cycles shown in Table 1. The PCR product will be assessed through gel electrophoresis on a 1% agarose gel (1X TAE buffer). If PCR is successful a band should be seen at 520bp. RFLP Analysis: RFLP analysis allows us to determine which Symbiodinium Clade is present in the sample by using restriction enzymes and analyzing the size of the DNA fragments produced. Several digestions using different restriction enzymes may need to be done to determine which clades are dominant within the sample site. If the dominant clades are C and D (as expected) the products of PCR will be digested with RsaI for 12 hours. The fragments will be examined using electrophoresis on a 1% agarose gel (1X TAE buffer). As shown in figure 7 by Dong et al. (2009) bands will be seen at 320/200 bp if the sample contains Symbiodinium Clade C and at 220/200/100 bp if it contains Symbiodinium Clade D (table 2).

Table 2 size of fragments created by Clade D and C when partial 28S ribosomal rDNA is digested with RsaI and size of fragment if rDNA is not successfully digested

Symbiodinium Clade Clade C

320/200 bp

Clade D

220/200/100 bp

Uncut rDNA

Figure 7 1% agarose, 1X TAE buffer, gel electrophoresis of RsaI restriction digest (RFLP analyses) of partial 28S ribosomal rDNA genes. C, Symbiodinium Clade C; D, Symbiodinium Clade D; CD, Dc, and Dc, a mixture of Symbiodinium Clades C and D; M, DNA ladder (Dong et al. 2009).

Fragment lengths when rDNA is cut with RsaI

520 bp

The data collected at this point will include the color and clades of zooxanthellae detected in the control group and treatment group at each sampling location. This data will be compiled into a table with the rows and columns found in table 3. The difference in Clade abundance between the control and treatment group will be calculated and analyzed using a T-test. This will allow us to determine if there is a significant difference in Symbiodinium clades found in the control and treatment group. Table 3 Example of table headings that will be used to organize the recorded data

Control Group (n=33)

Treatment Group (n=33)

Sample Site 1-33

Coral Color

Clades (A-H)

Coral Color

Clades (A-H)

Project Timeline and Budget The time needed to complete this research project from start to finish would be 5-6 months. 4-6 weeks would be allocated to planning which involves acquiring the materials, space and manpower needed to make the collection and analysis of data possible. One of the weeks during the 4-6 weeks of planning a survey of Mermaid Cove would occur. This will allow us to determine if there is a significant presence of Acropora spp. within Mermaid Cove and ensure the availability of Acropora spp. for sampling. Planning will continue 1-2 weeks after the survey is completed and will focus on the specific logistics of sampling. All 66 samples will be collected in the week immediately following the completion of all planning. As samples are collected throughout the week they will be placed (one from each site) in both aquariums. The aquariums will have been set up and allowed time for equalization with the ocean environment being sampled from (during the planning phase). Once samples have been randomly collected

and placed in the appropriate aquarium, they will be given 2 weeks to recover from any possible stress caused by the sampling methods. The controlled experiment will then begin, taking 8-9 weeks to complete. The temperature of the water in the treatment group’s aquarium will be gradually increased 2 degrees Celsius over one week and remain at the increased temperature for 8 more weeks to induce bleaching. When the experiment is complete, the next 1-2 weeks will consist of laboratory work. This includes the collection of tissue samples from each coral specimen, the DNA isolation of the zooxanthellae within each coral sample, the amplification of the DNA and the visualization of the DNA. Once the laboratory work is completed, the data will be analyzed to determine if there is a correlation between bleaching due to temperature and presence of clad D zooxanthellae. The statistical analysis and generation of a report will take 2-3 weeks to complete. A summary of the timeline described above can be seen in figure 8.

Task 6. Analysis + Write-up 5. Lab Work 4. Controlled Experiment 3. Sampling 2. Survey 1. Planning 0

2

4

6

8

10

12

14

16

18

20

Week Figure 8 Weeks since start of project vs. Task or phase of project. This timeline demonstrates the length of individual phases of the study as well as the overall length of the study.

The materials needed to complete this research project will primarily include materials for sampling in the field, conducting the controlled experiment and carrying out the laboratory work. A compiled list of items needed and the approximate cost can be seen in table 4.

Table 4 a list of items needed to complete each step of the experiment with amount and approximate prices of each item needed. Pricing was found online at Amazon.com and www.fishersci.com. Task

Item

Cost/Unit

Surveying and Sampling

Ocean sampling station Boat Scuba gear Plastic containers Sharpie markers 150m rope on a reel Spiral ground anchor 20 m segments of rope

750/month $300/day $25/day $0.50 $1.50 $80 $30

Controlled experiment

Laboratory work

Total Cost

2 months 7 days 6 units 7 days 33 2 1 1

$1,500 $2,100 $1,050 $16.50 $3 $80 $30

$10

3

$30

Homemade quadrats (PVC pipe and string)

$10

3

$30

Facility 55 gallon aquarium

$1,000/month $150

4 2

$4,000 $300

Thermometer Heater

$2 $20.00

2 1

$4 $20

Centrifuge

$1,000

1

$1000

Gel Doc UV imaging station

$10,000

1

$10,000

Thermocycler

$10,000

1

$10,000

Freezer 125um mesh

$200

1

$200

$20

1

$20

$20

3

$60

Hot water bath Proteinase K

$500

1

$500

$100

1

$100

phenol:chloroform (1:1) Sodium acetate

$50

1

$50

$50

1

$50

Sterile water

$50

1

$50

Taq DNA Polymerase

$90

1

$90

dNTPs

$100

1

$100

95% ethanol

$50

1

$50

PCR tubes

$100

1

$100

Micropipets/pipet tips

$500

2

$1,000

Agarose

$50

1

$50

1X TAE buffer

$30

3

$90

RsaI restriction enzyme

$60

2

$120

10X enzyme buffer

$10

2

$20

Gel electrophoresis unit, casting tray and comb(s)

$800

2

$1,600

Ethidium bromide solution

$30

1

$30

Gel loading dye

$40

1

$40

1Kb ladder

$60

1

$60

DNA isolation buffer (DNAB) w/SDS

Total

Units needed

$34,544

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