Now that you know what reactive oxygen species (ROS) are, i.e. highly oxidizing and therefore toxic molecules derived from molecular oxygen, and that they are produced when oxygen is “partially reduced” by stealing an electron, I can tell you about what I’ve discovered about the production of ROS by Antarctica macroalgae and we can theorize about what roles they might play in Antarctica ecosystems.
First of all, how do I look for ROS in Antarctic seaweed? It would be nice to be able to just see ROS being produced, but unfortunately that is impossible. ROS are normally invisible to the naked eye. Luckily, I can make them glow in the dark!
To make ROS glow, I use what we call a probe. This probe is a liquid that I drop into the seawater containing the algae I am working with. The molecules in the probe then enter the cells of the algae. Once they are inside algal cells, there are cellular enzymes that break the probe molecules in two. One of these pieces fluoresces, or glows, bright green when it is oxidized by strong oxidants such as ROS. The more strong oxidants, the brighter green the algae will glow. By putting the algae under a special microscope (called an epifluorescence microscope) and looking for the areas that glow bright green, I can see where ROS are being produced.
There are many reasons that Antarctic macroalgae might produce ROS. For one, they may generate it when they are wounded to prevent infection. Another reason for production might be to kill an endophyte spore that is trying to settle and germinate inside its tissue. So far I have surveyed about 9 species of macroalgae for ROS production upon wounding.
I wound them by lightly scratching their surface with a needle. This is the best way to simulate the wound that would be made by a small grazer like an amphipod. Then I add the probe and look to see whether the wound glows and how brightly.
I’m finding that some species of algae release ROS all the time, or “constitutively”, even when they aren’t wounded, while others do not. The dominant brown algae here do not release ROS constitutively. If you were to look at a picture of the brown alga Desmarestia anceps, you would see that the glow is so pale that it hardly registers on the camera. It almost looks like it is a picture of nothing! (There is such a picture on our Flickr site but not linked directly to this blog because, well, it really looks like a picture of nothing!)
Now look at the picture of a red alga I am studying called Myriogramme smithii. Look how brightly the cells glow. They are producing strong oxidants all the time. Most of the red algae I have looked at produce ROS constitutively while all of the brown algae I have studied do not produce ROS constitutively.
The browns I have looked at produce ROS upon wounding, but not nearly as much as the reds do. Compare a picture of a wounded Desmarestia antarctica (brown) and a wounded Palmaria decipiens (red). This is a huge difference.
I want to know whether these differences in ROS production have ecological effects. That is, do ROS affect other organisms in the environment? Amphipods are very small. To them, algae are like huge trees or fields. Do ROS affect amphipods? If so, the fact that some algae produce a lot of ROS while others produce very little may be ecologically important.
One thing I know is that amphipods can sense micromolar concentrations of hydrogen peroxide in seawater. Remember that hydrogen peroxide is an ROS. A micromolar concentration of hydrogen peroxide is 34 molecules of hydrogen peroxide for every billion molecules of water, or less than 1/10 of a drop of pure hydrogen peroxide per million drops of water.
When you add small amounts of hydrogen peroxide to seawater in which amphipods are living, they don’t eat as much food. So there is some sort of direct physiological effect of hydrogen peroxide, an ROS, on the amphipods that live on macroalgae.
We have to remember, of course, that in the ocean water moves around, or circulates. The hydrogen peroxide being produced by seaweed is certainly close to 100% at the site where it is being produced, but it probably diffuses fairly quickly as water moves around the site of production. Maybe it diffuses so quickly that the amphipods aren’t normally bothered by it. Or maybe the benefits of living on the macroalgae far exceed the physiological cost of living in a highly oxidizing environment.
I’d like to see if amphipods eat less food when they are in the presence of an alga that constitutively produces lots of ROS. Maybe amphipods have evolutionarily adapted to certain levels or types of ROS. We know that some amphipods chow down on certain types of red algae that produce lots of ROS. Do these amphipods produce antioxidants in their guts? I wonder.
One big question I still want to investigate is whether algae use ROS to kill endophyte spores. Remember how Kate is studying whether endophytes, the small string-like algae that live inside bigger algae, are parasitic to the macroalgae they live inside. If she finds that endophytes are bad for the macroalgae they inhabit, it would make sense for macroalgae to have evolved ways of avoiding colonization by endophytes. Some algae in other parts of the world use ROS to kill endophyte spores. Maybe some Antarctic algae do as well.
This is how science works. You learn one thing and instead of finding a conclusion you get 100 more questions! I will keep working and if I am really lucky, maybe I will answer a few. At least I won’t get bored anytime soon…
So that ends the saga of ROS for now. Thanks for sticking with it. I hope you learned something new!