AUSTRALIAN FRONTIERS OF SCIENCE, 2005
Walter and Eliza Hall Institute of Medical Research, Melbourne, 12-13 April
Photo-physiology of coral bleaching
Associate Professor Peter Ralph, Institute of Water and Environmental Resource Management, University of Technology, Sydney
When Madeleine van Oppen suggested
that I come and present here, I thought about the structure of the presentation
and how we could link our data together, and how I could possibly show the
nexus between the work in my lab and the work that Madeleine has been doing.
(And we co-share a couple of students.) We are actually getting to a much more
interesting point in understanding the problem of coral bleaching, which
Madeleine has very clearly explained.
We are a plant physiology lab. So, whereas Madeleine has been working at the scale of the entire reef distribution, we are looking at much finer-scale changes in the bleaching response within the corals, and trying to understand the photosynthetic mechanisms.
We are also very fortunate to be well linked with a German company that provides us with the infrastructure to do this work. Janice suggested that 10 or 15 years ago we wouldn’t have been asking these questions about the coral bleaching issue. Five years ago I could not have answered the questions, and two years ago we didn’t have the technology to obtain some of the data that I have got. So we are lucky to be well supported in asking and answering these questions with the work we are doing.
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The headline shown here is from the important literature that I read, the Age – I don’t quite read the ‘mags’ as Rob Brooks does, but this is also a nice cheap literature source. And it provides good evidence of some media responses that we are getting to the coral bleaching issues.
What I will be presenting today, as I suggested, is that we are looking for the physiological link between the clades, and how we are seeing the impact on the responses with the bleaching. I will show four data sets: combined micro-electrode data, a new imaging-PAM, a coral aspirator system, and some thermal ramping data. So that will be the structure of the data that I will be presenting.
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I won’t go through the explanations that Madeleine has already provided for you, but there are a couple of extra things that I need to get you up to speed with, for you to understand some of the data.
Basically, as Madeleine pointed out, we have got the zooxanthellae up in the tentacles and we have got tissue and structure, down the side, which is also called coenosarc tissue. So polyp and coenosarc are the two regions that I will be discussing, within a single colony. In these three photographs can see the structures: you have got shaded regions and you have got a whole range of polyp and structure giving changes to the light climate. As a physiologist we are looking at changes in light, and how the light affects the bleaching responses.
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Bleaching takes place with a couple of degrees of temperature change. It is interesting that on 15 February this year we just about had a bleaching event, but Cyclone Ingrid came through and moved all of the 2° elevated average mean temperature water off the reef. So, fortunately, 2005 won’t be a bad bleaching year. But 1998, 2004, 2002 were all bad years.
Madeleine suggested that we have got a highly variable bleaching response. It is different amongst all the different symbiotic combinations.
First, not all species bleach at the same temperature; even within the same reef they all change. Different species have different tolerances, as Madeleine’s data clearly showed.
Not all colonies of the same species bleach similarly – there is within-colony variation (this is some of the work that we are looking at) down to the point where not all regions of a single colony bleach similarly.
How does the coral survive? How does the coral bleach partially and then come back three months later? I go up in January, we look at the bleaching response, I come back in July with my undergraduate students and say, ‘Well, the reef was bleached but it is back again.’ It is understanding how the zooxanthellae are recolonising, getting back in to support the coral.
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This slide shows the initial paradigm that we were working with about 10 years ago, based on the technology that we had. Under normal temperatures of water, we would measure the zooxanthellae within the coral, and they were all nice and healthy. Elevate the temperature, under light as well, and you get damage. You get a change in the parameter PSII. So we are measuring a parameter of health.
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Here I present my one equation for today. Madeleine introduced the concept of what is quantum yield, and a measure of health. All of the data that I will be presenting today is fluorescence-based. So, with regard to spatial conservation of energy: a photon of energy comes into the photosystems. It can be used in photochemistry, heat or fluorescence. All three processes are complementary, so if we get an increase in photochemistry we get a decrease in fluorescence. If we get an increase in energy dissipation as heat – that is, protection – we will get a decrease in fluorescence. So we understand these two processes by measuring fluorescence.
Of the two parameters I will talk about, the first is Ft, which is basically standard, base fluorescence. As we put a tissue in front of the fluorometer it sends pulses of light to it and we measure how much chlorophyll is there. So that is Ft, which I will talk about later.
Effective quantum yield is measured in the difference between the maximum fluorescence, once we give it a flash of light, and the base fluorescence. So we have the variable fluorescence divided by the maximum fluorescence, as shown by the parameter here. And 0.6 is healthy.
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To get back to the paradigms, we originally have a nice healthy coral and a damaged coral. The question is: are all of these zooxanthellae dead, and from the two regions, have the sun zooxanthellae been expelled and damaged, have the shade zooxanthellae been expelled and damaged? So we will look at two questions, spatial complexity and the temporal response of the bleaching of the corals.
[SLIDE: Measurement at polyp scale]
The first data set I will present is a combined micro-electrode. Yesterday we were talking about optrodes. This isn’t quite at the scale of the optical fibres we were talking about yesterday, but this is still a 40-micron tip. So it is still half the thickness of human hair. Why are we bothering measuring coral oxygen production and photosynthesis at such a small scale? It is because we can’t measure photosynthesis when we are in bulk water.
All of these measurements are done in situ. So we have got a coral down the bottom here – this is Acropora valida. If we had an electrode up here in the bulk water we would just be measuring the oxygen in the water. We have got to be actually touching the surface of the coral to measure oxygen production. This is why we need a very, very fine-scale electrode and optrode. You see here a combined oxygen electrode and fluorescence microprobe. This was built for us by a Danish company and it is giving us fantastic new insight into understanding how different clades are affecting the photosynthetic activity of corals.
[SLIDE: Oxygen production]
This is some data by Karin Ulstrup, a student whom Madeleine and I co-supervise. This is a basic photosynthesis light curve that I am presenting here. We have got two different situations, a polyp and a coenosarc. So this first graph is for the area where the actual animal is encased inside the coral; the other is for the skeletal outside tissue. And what is shown is oxygen production over a range of different light intensities.
We have got sun and shade – the shaded region is the triangle, the round region is the sun – and within the polyp there doesn’t seem to be any difference in the rate of oxygen production. However, the coenosarc tissue in the shade has a very different response to the sun. And this is the same as normal terrestrial leaves, basically. You would look at a sun and a shade leaf and you would get this type of response. So within corals we are getting different responses, but do we have different clades affecting these different responses?
To start out we have got three different types of light responses. We have got a polyp response, and we have got a shade response and a sun response from the coenosarc tissues. And are these different regions affected by bleaching, and does a single colony have differential responses due to light climates?
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You see here the imaging-PAM. The top photo shows a submersible fluorometer. Instead of taking a single point measurement as we would have done 20 years ago when we could take fluorescence of the area of your fingertip, we are now imaging 96-well plates. This system could image about 2 cm to 3 cm square. So we are starting to move into larger imaging systems.
We have here the fluorometer and we have the coral in a flow chamber where we can put light of different intensities and change the temperature of the water to bleach the coral. And we have Acropora nobilis, which we actually did the measurements on.
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Ary Hoffmann presented a movie last night, and this is my first movie. But I can’t take credit for it; I stole it from one of my students – it is Ross Hill’s data set that he has presented as a movie of an Acropora during a bleach. Time is shown at the bottom of the panel, for example three hours at 32° and 500 microeinsteins of light. As time goes on, we are seeing a bleach response. The other parameters shown are Ft and PSII.
This is basically telling us how much chlorophyll is in the tissue. At one hour, as we look at the scale down here, it is about 30 units. It is getting down to about 15 units after four hours. You can see polyp and coenosarc differences here. And as it goes to about seven hours we are down to about 10 units; at eight hours, it has significantly declined. So we have lost tissue, we have lost photosynthetic substrate.
Here is its health. You should look at the tip and the base of the region. Once again we have the same scale. So after six hours, seven hours, it’s just about dead. At eight hours, it’s dead; there is no photosynthesis happening. Control: it’s starting out about 0.4; it goes down to about 0.3, et cetera.
So we have got spatial variability within a single branch. We have got variability between coenosarc and polyp tissues in a bleaching Acropora.
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We have here another species, Cyphastrea serailia. This is a more bleaching tolerant species, a very different story. We take the same parameters – loss of chlorophyll and loss of health. After six, seven, eight hours, it’s still healthy. So this is one of the robust, solid species.
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Is this linked to the cladal differences? We have got spatial heterogeneity. The branching Acropora nobilis supports a clade C3. It is a bleaching sensitive species; it did lose zooxanthellae as the Ft declined. It is damaged, because we have got a loss of photosynthetic health; the tip was more damaged than the base. We have got a wide range of spatial variations in this species. So it is a different clade to the massive Cyphastrea, which is C1. It basically survived our bleaching conditions. There are no problems; this is a robust species. And we do find that, generally, when there is a bleaching event on the reef, the Acropora go first, not the Cyphastrea. So it appears that bleaching at this scale is linked to cladal responses.
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The next question is whether the heterogeneity is linked to the exposure of these treatments.
Using this evil-looking contraption, we have set up a Cyphastrea with a range of different sampling devices and fluorescence probes at different regions on the coral – a shaded region, a sun-adapted region – and we have done the same thing with the branching corals as well. We put them in a water-jacketed bath, measuring their fluorescence and collecting the zooxanthellae as they bleach out. So, as the zooxanthellae come out, we are capturing them and working out whether they are healthy. This is working on single cells; we are measuring the photosynthesis of a single cell.
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We collected about 800 cells from Pocillopora damicornis, and we broke the data up into three different time periods: 0 to 4 hours, 4 to 8, and 8 to 12. Shown here is a frequency histogram of health the healthy guys are at the top here, 0.6 and the number of cells. You can see how the distribution changes. In the first four hours we have got a number of very healthy cells being expelled and not many damaged ones. By 12 hours the distribution has changed quite significantly. And this has statistically demonstrated these changes.
So we have got different types of cells, of different health, coming off at different times. That’s the result with Pocillopora damicornis, a bleaching sensitive species.
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We took also the robust Cyphastrea serailia. Inset here is the last diagram, so you can compare the two different histograms. There are vast numbers of healthy zooxanthellae coming off this robust species. These, potentially, could be the zooxanthellae that are going back into the environment and could be the ones that could support future reinfection of the bleached colonies. This is one of the things that we want to pursue. Now that Madeleine has the ability to start measuring the genetic clades of these single cells and we also have the ability to measure the photosynthesis, can we correlate these two data sets and understand whether or not Cyphastrea are the survivors and they are different clades?
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So what have we come to from this? Basically, not all expelled zooxanthellae are dead. We have got survivors. We have got healthy zooxanthellae. We have got quantum yields of 0.65 after 12 hours of bleaching. These are tough zooxanthellae. So probably the paradigm that I suggested earlier is partly incorrect, and the more cheerful paradigm is closer. We have got some dead zooxanthellae coming out; we have got some healthy guys; we have got some intermediate guys – we have got a wide range of health coming from the colonies. Now, is that linked to clades, is it linked to light climate, what parameters is it linked to? Okay, Pocillopora is C1c and is different from the Cyphastrea group of zooxanthellae. The overall health of Cyphastrea was 0.56 over 12 hours; it was 0.38 for P. dam, or Pocillopora damicornis. So you can see there is quite a difference there.
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This slide relates to where most of my lab’s work is directed: looking at the biochemistry of the photo-physiology. I am not going to present a lot of this data, which is the work of a couple of my PhD students in some of these areas. But I will present the data set for the thylakoid membrane. Potentially, we need to go through and say, ‘Are there cladal differences in the oxygen involving complex PSII, state transition, dark reactions, dysfunction and symbiosis?’ and demonstrate that it is linked to clades, not just to species. And there is a wide range of protective mechanisms as well that we need to link to clades. But so far I will present just one data set, the thylakoid membranes.
[SLIDE: Membrane integrity]
This is some work of Ross Hill, one of my PhD students. The thesis that he is working by is this: if thylakoid membrane fluidity is linked to bleaching capacity, we are going to see that the membranes will become liquid, fall apart, at about 32°. If they are robust and they can survive to higher temperatures, then it is not the membrane integrity that we are losing.
[SLIDE: Rapid thermal ramping]
The instrument that he is using is just a standard fluorometer. This slide shows a data set that he has collected from five species of tolerant corals. We haven’t correlated it yet to the clades, but what he is showing here is that as you increase the temperature this is increased at 1° per minute there is a rapid rise in fluorescence at about 37°. It is interesting that, with five different species, that is his data: 37.3 ± 0.1 °C. This is not changing.
If membrane integrity was breaking down, it would be breaking down at 32°. It is breaking down at 37°. So once again it doesn’t appear that there is a physiological response in some parts of the photosystem. There may be other causes, but it certainly isn’t the membrane integrity.
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So we are chipping away at different parts of it, and what I am hoping to do, with Madeleine’s collaboration, is to pursue a lot more of these questions and link them back to the clades.
On spatial complexity, we have got evidence that there are microclimates – that it is linked to the light environments, linked to flow, linked to the temperature effect. We need to validate that the actual clades are the factors that are causing these spatial differences.
On the temporal patterns, we need to collect the individual zooxanthellae and work out at what time and where they have been expelled, and whether or not they are different clades coming off at different rates.
On the bleaching trigger, we need to work with other species that are much more sensitive, and see whether or not their clades break down at 32°. That will be quite an interesting data set when we can find that, but so far everything is turning out to be robust.
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All of this work happens with an army of international collaborators as well as research students in my lab. I should acknowledge ARC for supporting some of this work, and a wide range of international collaborators, most importantly Rolf Gademann, the engineer who builds the fluorometers for us. Without him, and without his interest in developing tools specifically for our needs, we wouldn’t be able to do any of this work. And the workhorse of my entire lab is all of our students, who generate most of the work. I strongly acknowledge all of their work in this.
