This protocol provides an overview of sampling early life history stages for Montipora capitata in July-August 2020.

Overview

Notebook entries that describe samples collected will reference to this protocol and will add specific information on sample volume, time of collection, etc.

Sampling Procedures

Sampling is conducted using three methods below. Each method will allow for different sample processing and assays/analyses. Liquid nitrogen snap freezing will allow for metabolomic, biomass, and genomic sampling. Preservation in paraformaldehyde will allow for microscopy approaches. Fixation in RNA/DNA shield will allow for genomic/molecular sampling. At each life stage, samples will be taken with each approach.

Snap Freezing in Liquid Nitrogen

1) Add live samples (larvae, eggs, recruits) to screw-cap cryo-tubes. Using a pipette, remove as much seawater as possible.
2) Immediately submerge in liquid nitrogen in dewar and allow to freeze for 1-2 minutes.
3) Move into -80°C freezer for long term storage.

Preservation in Paraformaldehyde (PFA)

1) Prepare 4 percent paraformadehyde solution by diluting concentrated PFA with millepore filtered water. Store this solution in the dark in a flammable cabinet. Transfer fixative to tubes within a properly ventilated hood and with PPE. 2) Add live samples (larvae, eggs, recruits) to screw-cap cryo-tubes. Using a pipette, remove as much seawater as possible.
3) Add 500-800ul 4 percent PFA, depending on sample volume. Samples should be completely submerged.
4) Slowly invert tube several times to encourage mixing of fixative in sample.
5) Store at 4°C overnight.
6) The next morning (8-12h later), remove 4 percent PFA from tube using a pipette and discard appropriately. 7) Add 70 percent ethanol to sample (500-800uL) for long term storage.
8) Store at 4°C.

Fixation in RNA/DNA Shield

1) Add live samples (larvae, eggs, recruits) to screw-cap cryo-tubes. Using a pipette, remove as much seawater as possible.
2) Add RNA/DNA Shield to tube in at least 1 : 3 to 1 : 5 ratio of sample : shield. 3) Store at -20°C until processing. Can store at -80°C for long-term storage.