Measuring Photophysiology of Attached Stage of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer

This protocol measures the photosynthetic activity of attached algae, while they are still attaching to the plankton, just as in nature. The main advantage of this technique is that the measurement of photophysiology can be done in real-time without sample distraction. To examine the effects of zooplankton individuals on baseline fluorescence, prepare adult scapholeberis mucronata from the culture without any attached organisms.

Now starve the individuals in FLW at 20 degrees Celsius for at least 90 minutes to avoid fluorescence from the gut content. Pour 1.5-milliliters of FWL into a cuvette. Pick up the desired number of s.

mucronata individuals using a pipette under an optical microscope at 100-times magnification and transfer them into the cuvette. Add FLW to bring the sample volume up to 2-milliliters and allow it to acclimate under low light at 20 degrees Celsius is for 15 minutes before the fast repetition rate fluorometer measurement. Then click on act to run"to start the measurement and repeat the measurements more than three times per sample.

Read the F0 value from the resulting plot. Pick up colacium species with a molted carapace using a pipette under an optical microscope at 100-times magnification and wash them with FLW. Aseptically inoculate colacium species in the AF-6 medium in a 10-milliliter glass tube on a clean bench.

Maintain the culture at a NC2 temperature in a growth chamber, shaking the glass tube gently by hand at least once per day to prevent cell settlement. To examine the effects of zooplankton individuals on chlorophyll a fluorescence from colacium species, use adult s. mucronata without any attached organisms.

To avoid fluorescence from the gut contents, starve the individuals in FLW for at least 90 minutes. Set up a cuvette-type fast repetition rate fluorometer. Now pour a 1.5-milliliters of sub sample of pre-cultured colacium species into a cuvette.

Then transfer the desired number of s. mucronata individuals into these cuvettes and bring the sample volume up to 2-milliliters with the filtered medium. Acclimate the individuals under low light at 20 degrees Celsius for 15 minutes before taking the fast repetition rate fluorometer measurement.

Click on act to run"to start the measurement, repeating the measurements more than three times per sample. Read the F0 and FM values from the resulting plot. To correct the baseline fluorescence, filter the culture medium using a 0.2-micrometer pour-sized filter and measure the fluorescence.

Subtract F0 of the baseline sample from F0 and FM of colacium species or modify the blank correction value in the settings in the options tab. Isolate s. mucronata individuals with colacium species using a pipette under an optical microscope.

Then wash s. mucronata using FLW. Transfer s.

mucronata into 100-milliliters of FLW and keep them under dark conditions at NC2 temperature for 90 minutes for starvation. Pour 1.5-milliliters of FLW into a cuvette. Transfer around ten s.

mucronata individuals with colacium species into a cuvette and add FLW to bring the sample up to 2-milliliters. Acclimate the individuals under low light at N2C temperature for 15 minutes and measure chlorophyll a fluorescence, as mentioned in the text manuscript. To enumerate the number of attached cells, fix the sample with 2%glutaraldehyde after taking the fast repetition rate fluorometer measurement.

Then take pictures at several focal depths and positions of s. mucronata under a light microscope. There was no significant effect on baseline fluorescence or chlorophyll a fluorescence by s.

mucronata up to 5 individuals per milliliter. However, maximum photochemical efficiency and non-photochemical quenching were significantly affected when s. mucronata density was 7.5 individuals per milliliter.

Seasonal variation in the phytophysiology of colacium species during the attachment stage and the planktonic stage for the stationary phase in the AF-6 medium showed similar mean maximum photochemical efficiency and non-photochemical quenching. For the attached stage of colacium species under dark conditions, there was no significant difference in photophysiological parameters, except non-photochemical quenching being higher for manganese than calcium after three hours, and at a low light intensity at 21 hours. For the planktonic stage, PS2 absorption cross-section was significantly lower in the manganese than calcium treatment at three hours.

Effective photochemical efficiency was significantly higher, but non-photochemical quenching was lower in the manganese treatment than control at 21 hours. Under increasing light, manganese tended to decrease PS2 absorption cross-section at three hours, in non-photochemical quenching at 21 hours, while increasing the effective photochemical efficiency at three hours. Calcium slightly improved non-photochemical quenching under increasing light.

However, it decreased the effective photochemical efficiency at three hours. The most important thing is the effect of substrate organisms. If algae attached to different species or materials, the effect can be different.

In addition to this protocol, measurement of carbon fixation or oxygen production rates are useful to clarify the response of the calcium productivity to the experimental manipulations. This protocol enable us to clarify how the photosynthetic activity of attached algae responds to environmental changes.