This entry describes the protocol for measuring photosynthesis and respiration in early life history stages of Montipora capitata.

Overview

This protocol can be adapted for use in other species and locations. This protocol does not describe use of the SDR system, rather the application for this specific purpose. This protocol would also be improved with addition of measurement of Pi curves, this was not done in this study.

Equipment Required

• PreSens Oxy-10 Sensor Dish Reader (SDR) in combination with Loligo 24-well microplate
• Custom built acrylic chamber for alignment of plate on SDR reader
• Glass coverslips
• PreSens software downloaded with calibrations entered as provided by manufacturer
• Aquarium lighting
• Filtered seawater (1 um)
• Bubbler and aeration
• Microfiber towels
• PC computer
• 4 percent PFA and small (250 uL) sampling tubes
• Pipettes
• MilliQ filtered water

Steps

Prepare system

1. Connect all components of the SDR and complete steps to connect computer and enter oxygen calibration data from manufacturer. Here, calibration data was obtained from Loligo systems: PSt5-1624-01_25°C.dat
2. Clean and rinse microplate with filtered seawater. After rinsing, fill all wells with filtered seawater and allow to soak for 10-15 minutes prior to run. If running photosynthesis using an aquarium light, turn light on to allow temperature to equilibrate in the chamber. If using an incubator, put microplate in incubator to allow temperature of chamber to equilibrate.
3. Ensure that plate well numbers are aligned between the microplate and the SDR. Ensure that system is protected from water exposure and splashing.

Prepare biological samples

1. Prepare larvae, recruits, juveniles for measurement. Begin by rinsing them in filtered seawater to ensure they are clean and free of debris. It may be helpful to pre-count and allocate samples in droplets or well plates so that addition to the microplate is quick.
2. Preliminary tests may be required to determine the number of samples that are required to obtain a sufficient signal. For M. capitata in this protocol, 6 larvae, 6 recruits, and 1 juvenile per well gave detectable signals.

Loading samples

1. Load the samples in the wells. If using large cover slips, alternate wells so that coverslips will not overlap.
2. Add the samples to the wells with a pipette. Top each well with aerated filtered seawater such that there is a “bubble” of water rising above the well producing a magnifying effect into the well. This will ensure easy sealing once the coverslip is added.
3. It is helpful to make sure the surrounding areas of the microplate are dry to prevent water wicking away from the wells.
4. Fill at least 2 wells per run with filtered seawaters as blank controls. Randomize the location of blanks on each run.
5. Add glass coverslips to each well once they are all loaded such that the sealing process occurs quickly.
6. Drop the cover slip on by “rocking” it onto the well, this will encourage bubbles of air to be pushed out during sealing.
7. Make sure that wells do not have any bubbles inside. If so, remove the coverslip, add more filtered seawater, and attempt to reseal.

Measuring photosynthesis

1. Once the wells are sealed, start measurement on the SDR PreSens software. The light should be on from the setup steps above. This will start the photosynthesis run.
2. Observe the wells during the run. If bubbles form note this and exclude from analysis.
3. Observe the oxygen concentration during the run. The slope should increase, indicating increases in oxygen concentration as compared to blanks.
4. The SDR may be sensitive to light and oxygen readings under light may “jump”. Give sufficient time for sample wells to distinguish from blanks and for a slope to emerge for ~10 min.

Measuring respiration

1. After photosynthesis run is complete, turn off lights. This automatically starts the respiration portion of the run. Reduce all ambient lighting.
2. Allow to run until sufficient deviation is seen from blanks and slope is consistent for ~10 minutes.
3. Once this is complete, end the data collection on the computer.

Sampling biological samples

1. Samples can now be removed from the microplate. Remove with a pipette and transfer to tubes if sampling is desired.
2. In this protocol, sample to measure larval size/volume and store in 250uL of 4 percent PFA at 4°C.
3. Move all larvae from each well into one tube. Label the tube and write down the corresponding well and run number.

Data downloading

1. Export data files in .xlsx format.
2. Covert to .csv for use in analysis.

Shutting down the system

1. Turn off computer after data has been exported.
2. Disconnect all components.
3. Rinse microplate with milliQ water and dry gently with a microfiber towel. Use a pipette to rinse each well gently.
4. Gently wipe all equipment with a microfiber towel and store in case.