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PUTNAM LAB POLYP BAILOUT PROTOCOL
2020-11-13
Protocols
polyp bailout, hyperosmotic stress, Pocillora
Putnam Lab, Lewinski Lab, Synthetic Coral

Original: 20201113
Last Revised: 20210211

Protocol for inducing polyp bailout via hyperosmotic stress

older version of this document go here

Materials and Equipment

Before experiment prep

  1. Precondition settlement substrate (plastic petri dishes and/or glass cover slips) in aquaria with live rock. This can be started a few weeks prior to experimentation
  2. Make bailout/settlement chambers -take a small tupperware container and cut out most of the sides and bottom. Attach 153µm mesh around each hole with hot glue so the fragments can receive water flow. Secure mesh around the tupperware (with holes) using a hot glue gun. Soak the container in freshwater for 24h to remove any plastic or glue leechate. Precondition settlement chambers in the aquaria for a few weeks as well

experimental_setup

Bailout steps

  1. Rinse tank in freshwater to clean. Fill 4L of the tank with artificial seawater (salinity ~35 psu) leaving 2L of space for mixing high salinity water (~75psu) to get to desired ~60psu. -NOTE: take temperature, salinity, and pH measurements of control and treatment tanks -NOTE: use artificial seawater from the large mixing tank (this is because the water table water is recirculating and might have imbalanced nutrients)
  2. Add a clean pump to the tank.
  3. Attach (ziptie) tubing coming from the high salinity DOS head around the pump such that the solution will be delivered directly into the pump inflow and be mixed immediately.
  4. Program DOS to add 60 mL of high salinity seawater (75 psu) every 30 minutes. This increases salinity by ~0.85-1.1 psu/hour. This means you'll need to program two 14 hour doses (28-29 doses each)
  • NOTE: don't forget to program the dos to stop dosing after 14 hours so the tank will not overflow. -NOTE: take temperature, salinity, and pH measurements of control and treatment tanks
  1. Add a ~5mm Pocillopora acuta fragment into a mesh settlement chamber.
  • NOTE: the coral frag is in the mesh settlement chamber so the micropropagules (bailed out polyps) don't get caught in the pump.
  1. After the first 14 hour dos-ing period, check temperature, pH, and salinity. Siphon out 2 L of water and reprogram dos for next 14 hour dosing period
  2. After bailout occurs, move the settlement chambers with coral fragments into a seawater tank at ~35psu.
  • NOTE: include preconditioned plastic petri dishes or glass cover slips in mesh chambers as settlement substrate. -NOTE: FOR SETTLEMENT CHAMBERS - use artificial seawater from the large mixing tank (this is because the water table water is recirculating and might have imbalanced nutrients)
  • NOTE: take temperature, salinity, and pH measurements of control and treatment tanks before disassembling
  • NOTE: place coral fragment with remaining (attached) MPs in a separate settlement chamber than the one with the detached micropropagules counted earlier (this impacts settlement calculations)

Settlement tracking

NOTE in lieu of placing recently detached micropropagules in settlement chambers, use 6-well culture plates in incubator - examples here

Using the well plates can allow for a more controlled settlement tracking experiment because 1) micropropagules are easier to locate in well plates and 2) the mesh chambers sometimes attract flat worms.

incubator setup

  1. Open cover and add 2mL artificial seawater to each well in the 6-well plates -NOTE: use artificial seawater from the large mixing tank (this is because the water table water is recirculating and might have imbalanced nutrients)
  2. Place settlement substrate in each well. This can include
  • waterproof paper with and without red tape (do not pre-condition paper)
  • microscope slide cover slips with and without red tape (can pre-condition cover slips)
  • petri dishes with and without red tape (can pre-condition petri dishes)
  • bare coral skeleton
    • coral skeleton needs to be soaked in bleach overnight, dried overnight, and autoclaved
  1. After bailout, count up to 20 micropropagules and pipette into a well
  • do not combine micropropagules from different coral fragments
  • label the plastic near the well with the coral frag ID
  • write down the following information as a table in your lab notebook: date, time, date bailout trial started, date settlement trial started, frag ID, well plate ID, # micropropagules provided, settlement substrate provided
  1. Check for settlement weekly using the compound scope
  2. Add ~1.5 mL seawater to each well Monday/Weds/Friday

Additional notes

NOTE: photograph and video (~1min) control and treatment fragments before starting the experiment, T=24hr, T=48hr. Use a Snapzoom attachment for a dissecting scope for these pictures (see below). photo_station

More photos of the bailout tank setup can be found here

Water quality (ambient tank)

  • humidity (in room) 33-35
  • Temp (water): 23-27 C
  • pH: -59-63 mV
  • salinity: 33.5-36.5 psu

Checkpoints

  • 0hr ~35psu - photograph fragments.
  • 24hr ~48psu - photograph fragments and check for bailout
  • 48hr ~60psu - photograph and check for bailout
  • Phenotyping immediately after bailout and subsequent 24hr increments to track survivorship. Use a compound microscope at 4x magnification. Micropropagules can be transferred to a Sedgewick Rafter chambers via a bulb pipette for viewing.
  • Settlement - 4-5 days after bailout and weekly afterwards

Response variables

  • Lab notebook post on microporapgule Phenotyping here. Use a compound microscope at 4x magnification. Micropropagules can be transferred to a Sedgewick Rafter chambers via a bulb pipette for viewing.
  • Sizing of microprapagules with ImageJ
  • Settlement success

References

Chuang et al 2020

Chuang, P. S., & Mitarai, S. (2020). Signaling pathways in the coral polyp bail-out response. Coral Reefs, 1-14.manuscript link

"high-salinity artificial seawater (48‰) was pumped into the experimental tank at a constant rate of 4 mL min−1 using a peristaltic pump" "partial polyp detachment (an indicator of onset of polyp bail-out) was observed in the treatment group at 46.0‰ salinity (24 h) and bailed-out polyps could be easily removed from the skeleton by gently shaking the coral fragment in water, we collected both undetached coral tissues (whole fragment including undetached polyps, remaining coenosarc, and underlying skeleton; T46.0-U) and detached polyps (10–15 completely detached polyps, collected with a large-bore pipette; T46.0-D) separately."

Richmond 1985

Richmond, Robert H. (1985) Reversible metamorphosis in coral planula larvae." Mar Ecol Prog Ser 22, no. 1: 181-5.manuscript link
larvae in ambient conditions

Sammarco 1982

Sammarco, P. W. (1982). Polyp bail-out: an escape response to environmental stress and a new means of reproduction in corals. Marine ecology progress series, 10(1), 57-65. manuscript link

adults in ambient conditions
"Total dissociation of all polyps in a given colony, however, required 2 to 3 d. There was some sloughing of remaining coenosarc tissue at this time. Almost any water movement accelerated the dissociation of polyps and coenosarc. Transferring portions of colonies to clean seawater after polyp bail-out had begun did not appear to slow the phenomenon."

Shapiro et al 2016

Shapiro, Orr H., Esti Kramarsky-Winter, Assaf R. Gavish, Roman Stocker, and Assaf Vardi. (2016) A coral-on-a-chip microfluidic platform enabling live-imaging microscopy of reef-building corals. Nature communications 7, no. 1: 1-10. manuscript link

"a small (∼5 mm) branch tip is removed from the mother colony using a clean stainless-steel bone cutter. The branch tip is placed in an open glass Petri dish filled with filtered artificial seawater (FASW) just covering the coral fragment. A gradual increase in salinity due to water evaporation results in a polyp-bail-out response, with separate polyps released from the coral skeleton. Polyp release can be expedited by gently pipetting or stirring the water around the coral fragment. Precise conditions for the different coral species used here differed as detailed in the text. A 5-mm fragment from the corals P. damicornis or S pistillata typically yields 30–40 micropropagates. Expelled micropropagates are transferred to a small Petri dish containing FASW at ambient salinity for up to 10 min for initial recovery. Micropropagates are then screened for viability using a stereo-microscope, and scored by observing tissue integrity, the extension of mesenterial filaments and the presence of vortical ciliary flows"