The effect of light and temperature stress on toxin production
Pan et al. (1998) suggested that when a culture stops growing the energy that would have gone into cell division is rerouted into producing domoic acid (it has to go somewhere!). Pseudo-nitzschia cultures produce domoic acid when growth is limited by anything but nitrogen and light. So, if the cells must stop dividing because they don't have enough silica, but are still being exposed to the same light levels they must still process the same amount of photosynthetic energy. Lomas and Glibert (1999) hypothesized that diatoms in a cool, well-mixed environment take up surplus nitrate and store it internally. When the cell has extra energy to burn, this nitrate is reduced (the oxygen is removed) to nitrite and then again to ammonia, a very energy intensive process. Pseudo-nitzschia is a diatom. Could the Lomas and Glibert (1999) findings apply to Pseudo-nitzschia? How would Pseudo-nitzschia respond to high light, low temperature stress?
I designed some experiments based on the Lomas and Glibert (1999) and the Lomas et al. (2000) work on nitrate uptake by diatoms in cool water, high light environments to investigate domoic acid production in Pseudo-nitzschia. Below are some conceptual diagrams, first describing the findings in Lomas et al. (2000) and then showing my hypotheses for what might happen in Pseudo-nitzschia.
The diagram above shows a single diatom cell underwater. The temperature of the water is 10C and the cell has plenty of light. In cool, well-mixed water, enzymes that process photosynthetic energy are limited by temperature. Light energy, however, depends on depth in the water column, which near the surface (the mixed layer) can change relatively rapidly. There are times when the cell’s enzymes are temperature-limited and the cell is exposed to a rapid increase in irradiance, creating an energy imbalance similar to cessation of cell division due to limitation by Si or P. Lomas and Glibert (1999) found that diatoms exposed to a rapid change in light at a low temperature removed lots nitrate from the water and released lots of ammonia. Flagellates exposed to similar conditions removed the nitrate, but did not release ammonia. Their hypothesis was that the diatoms were taking up more nitrate than they needed in order to consume the energy that the carbon-fixing enzyme (RUBISCO) couldn't handle because of the low temperature. Instead nitrate reductase and nitrite reductase were using the energy to transform nitrate into ammonia. In order for this idea to work, nitrate reductase and nitrite reductase need to be able to function better than RUBISCO at lower temperatures. They do. But what happens at higher temperatures?
The cell above is still exposed to a rapid increase in light, but the water temperature is now 20C. That means RUBISCO is better able to handle the energy generated by photosynthesis. The cell doesn't need to take up large amounts of nitrate or expel lots of ammonia. The cell may even take up ammonia for amino acid and protein synthesis.
If Pseudo-nitzschia behaves like the diatom in the first panel by taking up lots of nitrate, would it still release ammonia or would it further process the nitrogen into DA? I designed a series of experiments to 1) determine if Pseudo-nitzschia behaves like a diatom or a flagellate under low temperature, high light conditions like in the Lomas and Glibert and the Lomas et al. studies and 2) see how these stressful conditions affect DA production, if at all. The results were interesting.
This graph shows nitrate concentrations in the growth media at the beginning of the experiment (0 min) and at the end of the experiment (180 min). The white bars represent nitrate in the flasks exposed to a rapid 100 fold increase in light. The grey bars represent nitrate in the flasks that did not experience any change in light. We can see that the nitrate was removed from the water in the high light flasks. The star indicates statistically significant difference between the white and grey bars.
This graph shows nitrite concentrations in the growth media at the beginning and end of the experiment (0 min, 30 min, 60 min and 180 min). We can see that nitrite is released into the water in the light exposed flasks while nitrite in the water does not change in the flasks with no change in light. Again, the star indicates a statistically significant difference in nitrite concentration between the white and grey bars. There is more nitrite in flasks exposed to high light.
This graph shows ammonium concentrations in the growth media at the beginning and end of the experiment. We can see that like nitrite, the ammonium concentrations are increasing in the high light flasks and not changing in the constant light flasks. This indicates a release of ammonium from the cells into the water.
So, this experiment has shown that Pseudo-nitzschia does take up nitrate and release nitrite and ammonium under low temperature/high light conditions similar to the diatoms in the Lomas and Glibert experiments. How does this affect DA production?
This graph shows total DA in the sample (DA in the cell and DA in the water in pg DA per cell). We can see that the flasks with constant light were producing DA while the flasks with high light were not! What is happening?
I suspect that the DA production in the constant light flasks represents normal production for the culture of Pseudo-nitzschia I used for this experiment instead of an increase or a stimulation of DA production caused by the experiment. The absence of this production in the high light flasks indicates DA production is being stopped or inhibited somehow. If our assumption that high nitrogen release in these flasks is due to an inability of the cell to consume energy by fixing carbon it is possible that the high light cells did not have the carbon skeletons available for the DA molecule. My results suggest that a rapid increase in light at low temperatures would reduce DA production.