STANDARD PROTOCOL FOR MEASURING PHOTOSYNTHETIC EFFICIENCY OF CORALS

This protocol is used to measure the flourescence of the coral polyps in order to understand the photosynthetic efficiency and health of the corals in our study system.

The equipment used in this protocol is called the Underwater Fluorometer Diving-PAM (Pluse Amplitude Modulated). The manual of this equipment could be accessed here.

General Overview of the steps for using PAM

1. Tagging the corals you want to measure the PAM data from
2. Assembling the Diving PAM and setup
3. Dark adapting the corals in the aquarium room
4. Measuring fluorescence using diving PAM after dark adaptation period ends
5. Equipment care and storage
6. Obtaining remaining PAM data from Memory
7. Turn back the lights in the aquarium room

Make sure you are timing the entire PAM process or schedule based on the availability of the lab members as well as how much time you have in the wetlab. Ideally, atleast two individuals should be present while following this protocol for a ideal and effective completion.

Please note: Before scheduling for any PAM measurements, make sure the Diving PAM has been charged for use. The process of charging it could be referred from the manual attached above on Page 31-32 under the section 8.1.3 Rechargeable Battery. A fully charged battery would display voltage between 12.5-12.9 V. Charge the battery whenever the voltage shows to be 11.2 V or below.

STEP 1: Tagging the corals

Tag a desired number of corals from both the sides of the blue tanks using zip ties avaliable with us. Make sure you select the right color of the zip tie as you should be able to see the zip ties in darkness while PAMing.

STEP 2: Assembling the Diving PAM and setup

This step could also be done while waiting for the dark adaptation wait period. However, if necessary you could also do this step before. In this step, we would be assembling two different parts of the equipment.

I. Assembling the equipment

In order to effectively PAM the corals, make sure you have this three components:

1. The DIVING-PAM
2. The Underwater Cable
3. Plastic connector piece/spacer

Follow the steps to assemble the Diving PAM equipment for measuring fluorescence:

1. Take out the DIVING-PAM from its protective case. The case also contains the underwater cable and the connector piece.

   Note: Place the DIVING-PAM gently on the table and make sure it is steady in order to prevent any damage to the equipment.

2. Now, take the underwater cable and insert on of its end onto the opening guarded by the fitted screw attachment on the side of the Diving-PAM. You are going to insert the cable in number 13 inlet shown in the diagram below. Make sure it is fitted nicely and not tightened all the way to prevent damage or breakage.

3. Attach the connector/spacer piece at the end of the other open tip.

Note: For measuring fluorescence of the corals in the blue tanks, this spacer is crucial to make sure the fibre optic tip does not directly touch or crush the coral tissue as well as cause damage to itself. The spacer also maintains safe amd consistent gap between the coral tissue and the fibre optic. In case of coral fragments, like that taken during a polyp bailout experiment for example., the spacer could be taken off as the coral colony is very small and the damage is very rare as the tip does not touch any branches in the coral colony. However, it is always advised to talk with your supervisor before doing PAM, as every experimental design might have different requirements.

1. Check if the DIVING-PAM is turning on. In order to check, click on the ON-switch (Number 1 in the diagram above). If everything you have done so far is proper, the display will illuminate and you would be able to see the menu. Now you can proceed with the next steps.

2. Choose the follow setting for PAMing different corals species in the tank present in Mode-Menu 50 (Measuring Light Intensity) & 49 (Gain). Using the decreasing arrow on the PAM from position 1, go to the respective menus mentioned above to select the settings shown below-

   i. For Pocillopora acuta and Montipora capitata,

   Select Measuring intensity: 11; Gain: 4

   ii. For Porites compressa,

   Select Measuring intensity: 12; Gain: 3

II. Other Required Setup

The other setup requirement is to have a datasheet present in the lab notebook. This datasheet could have columns like below:

|Date| Time|Initials| Crate Number/Tank ID/Coral ID | Mem # | F0 | FM | Y | Notes |——|—–|———|——————|————|————|————–|–|—|

This would be where you would enter the data after you have done PAMing of the corals.

STEP 3: Dark Adaptation of the corals

Before measuring the fluorescence, the corals are dark adapted. This step is CRUCIAL as dark adaptation helps the light reaction centers (Photosystems II) return to the baseline state and remain OPEN. This enables them to receive light and determines maximum quantum yeild that helps us to know if the corals are under any stress or experiencing damage from the light they are receiving in the aquarium system.

The corals could be dark adapted for a period of 30-60 minutes..

Follow the steps for dark adapting the corals:

1. Using the myAI app, turn off the ALL NINE AI Hydra LED Reef lights which are present on the roof of our blue tanks. Make sure the bluetooth is on on your device.

2. In the myAI app, Blue Tanks > Lighting > Click on the star (shows us saved presets) > Select the Dark apadt preset (you can identify this as it will be a blank graph mini icon with no lines).

   Note: Sometimes the myAI app if not properly updated or synced, does not turn all the lights off through the app. If this case arises, you can directly turn the switch for that respective light off from the electrical switchboard which is present on the side of the blue tank 3/4. You can find the light number on the light and the same number is helpful to turn off the switch from the electrical board.

3. Next step is to turn off the reef light of the display tank. In order to do this, unplug the switch at the electrical board or at the connector to successsfully prevent the light entering into the blue tanks. You might also need to pull the display tank outreach poster all the way down to attain complete darkness.
4. Turn off the main wetlab lights which are present on the roof from the light switch present in the wetlab. The light switch are present on the wall adjacent to the storing racks and above were our crates for the tanks are kept. You need to turn off the light switch on the right side.
5. Close off the big black curtains at the end of the incubators making sure that the tanks are entirely getting rid of any form of light entering into our aquarium system.

STEP 4: Measuring fluorescence using diving PAM after dark adaptation period ends

After the dark period is completed, the corals are now ready to get PAM. Before starting to PAM make sure:

• Individuals are assigned of taking notes and who would be handling the cable for PAMing. Discuss the plan of action and coordinate with your PAM partner.
• Turn off the pumps of the tank which you are first going to target.
• Now use the tall sitting chair to keep the DIVING-PAM on top of it, so you/your PAM partner do not have to hold the equipment in hand. Make sure you adjust the equipment in a way that isn’t prone to falling and the underwater cable is not tangled in any way.
• Remember: You are NOT going to turn on the lights until the NEXT STEP IS COMPLETED.

Follow these steps for obtaining PAM data:

1. Switch on the DIVING-PAM using the on switch.
2. Zeroing/Calibration check: Tap on the Mode option on the DIVING-PAM, select Menu 2: Auto Zero using the arrows. Now submerge the cable into the tank and do not hover/point to any specific coral colony for effective reset. Select a random area like base of the tank/tank walls for instance. Now, tap start so that the DIVING-PAM will measure the photosynthetic efficiency of that region (which would be zero) as it is pointed to a random surface lacking photosynthetic ability.
3. Now, proceed to tap on MEM, where you will now see that the DIVING-PAM display shows ZERO in the readings. You may now proceed to PAM the coral tissue. Note down the MEM # so you know where you’re PAM data starts to log in on that respective day.
4. Select your tagged coral fragment and point the tip onto the coral surface.

5. Now as your PAM buddy would be busy on making sure the fibre optic is not moving and we are getting a maxium yield, the other person would check the F* is depicting on the second row of the display.

   Ideally, if it is good targeted region, the values would be in the range F= 300-500 units. Coordinate with your cable holder partner and check with them the F-value you are seeing and let them know when you are ready to apply for a pulse. That is your measurement for that coral fragement.

   Note: A Pulse/A saturation pulse is a short but intense burst of light from the device. This pulse closes all the reaction centers in that targetted area forbidding the polyps to use this energy for photosynthesis. This causes the chlorophyll to emit the maximum fluorescence which we are interested in

6. The display screen would now highlight the values measured during that pulse like shown in the picture below.

The display shows various measurements:

i. F: Fluorescence yeild. It is measured before the last saturating light pulse triggered by START (your F value for the fragment).

ii. M: Maximal Fluorescence yield (M=Fm). Measured during the last sat

iii. m: Water depth measured with the built-in pressure sensor

iv. F* : Momentary fluorescence yield, which shows small fluctuations caused by electronic noise.

v. Y: Maximum Yeild

vi. E: relative rate of electron transport (ETR)

vii. L: Light intensity in units of PAR (Photosynthetic Active Radiation)

1. Note down the value in front of the Y (Yeild measurement) and M for example., 693 and 392 respectively from this picture above for that coral fragment entry in the datasheet you have created. You will go back to the memory to extract the other values later at the end.

2. Now proceed with repeating the steps 4-7 for all the other coral fragments you are wishing to obtain the PAM data off.

STEP 5: Equipment care and storage

Now that you have completed all the coral frags, turn on all the lights using the switch or the myAI app.

Now, carefully take out the underwater cable out of the water and dettach it from the DIVING-PAM by unscrewing it. Transport the DIVING-PAM onto the table and wash the underwater cable as well as the spacer with freshwater and keep it for drying on a cloth.

DO NOT wash the DIVING-PAM with water. Even though the equipment is safe to use underwater, it is ideal to have it in a case if using it in water. You can simply wipe any water droplets, if there are any on the DIVING-PAM.

Once you have obtained the data in the next step, you can turn off the DIVING-PAM by tapping on OFF and proceed to store the equipment and its parts back into the storage case.

STEP 6: Obtaining remaining PAM data from Memory

To obtain PAM data from the memory,

Tap MEM > Use arrow keys (∧ or ∨) > See previously recorded data > Look for your Y values and write down the corresponding required data we need to fill up the columns below.

|Date| Time|Initials| Crate Number/Tank ID/Coral ID | Mem # | F0 | FM | Y | Notes |——|—–|——-|——————–|————|————|————–|–|—|

Update the datasheet with the data and note down any observations from the day. Report to your supervisor about the day and any details.