This entry details the sampling and collection of embryos, larvae, and recruits of Montipora capitata during July-Aug 2020 spawning period.

Overview

This sampling follows the protocol in the Mcapitata Sampling Protocol. Specific sampling notes and deviations from the protocol are detailed here. Preservation (PFA), fixation (RNA/DNA shield), snap frozen (liquid nitrogen) sampling described here.

Metadata for these samples (including sample identification numbers) can be found here: Sample Metadata.

HPF indicates hours post fertilization

Volume of sample (uL) indicates volume of concentrated embryo/larvae/recruit added to tube. Material was concentrated by removing water from tubes until a concentrated volume was reached so that the maximum amount of material could be sampled. This volume was adjusted based on available material and biomass/size of individuals. If limited, lower volumes were sampled for microscopy preservation.

See Fertilization entryand Larval rearing entry for further information on larval fertilization and rearing.

Sampling Activities

Embryos

• Eggs (0.5 hpf): Sampled fertilized washed (rinsed with FSW to remove sperm) eggs that were fertilized. Sampled at 2300 on 20 July 2020. n=4 tubes each for snap frozen (250 uL per tube), fixation (250 uL per tube with 1mL DNA/RNA shield), preservation (100 uL per tube with 1mL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 22 July 2020 at 1730.

• Embryos (4-5 hpf): Sampled developing embryos on 21 July 2020 at 0200. Embryos were in 1, 2, and 4 cell stages. n=4 tubes each for snap frozen (100 uL per tube), fixation (100 uL per tube with 1mL DNA/RNA shield), preservation (100 uL per tube with 1mL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 22 July 2020 at 1730.

• Embryos (12-14 hpf): Sampled developing embryos on 22 July 2020 at 1100. Embryos were in prawn chip/gastrula cell stages. n=4 tubes each for snap frozen (50 uL per tube), fixation (50 uL per tube with 500 uL DNA/RNA shield), preservation (20 uL per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 23 July 2020 at 1130.

Larvae

• Larvae (42-44 hpf): Sampled swimming larvae (displayed swimming activity) on 23 July at 1400. n=4 tubes each for snap frozen (50 uL per tube), fixation (50 uL per tube with 500 uL DNA/RNA shield), preservation (50 uL per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 24 July 2020 at 1830.

• Larvae (72-75 hpf): Sampled swimming larvae on 24 July at 1800. n=4 tubes each for snap frozen (50 uL per tube), fixation (50 uL per tube with 500 uL DNA/RNA shield), preservation (50 uL per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 25 July 2020 at 0800.

• Larvae (144-148 hpf): Sampled swimming larvae on 27 July at 1600. n=4 tubes each for snap frozen (50 uL per tube), fixation (50 uL per tube with 500 uL DNA/RNA shield), preservation (50 uL per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 28 July 2020 at 1600.

• Larvae (164-168 hpf): Sampled swimming larvae on 28 July at 1200. n=4 tubes each for snap frozen (20-30 larvae per tube), fixation (20-30 larvae per tube with 300 uL DNA/RNA shield), preservation (20-30 larvae per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 30 July 2020 at 1600.

• Larvae (200-210 hpf): Sampled swimming larvae on 30 July at 1200. n=4 tubes each for snap frozen (20-30 larvae per tube), fixation (20-30 larvae per tube with 300 uL DNA/RNA shield), preservation (20-30 larvae per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 30 July 2020 at 1600.

Recruits

• Recruits (~48 hours post settlement): Sampled free floating recruits, see Mcapitata Settlement entry for information on how these were settled. Recruits had formed a primary polyp. Concentrated recruits in a beaker and sampled volume with pipette. Where possible, estimated the number of recruits rather than volume. First sampling point occurred at 48 hours (or less) after settlement. Due to a hurricane, I was not able to check for settlement until 48 hours after larvae were introduced to substrate. Therefore, sampling points are identified as the window of time possible for settlement. Sampled recruits (48 h or less post settlement) on 28 July at 1200. n=4 tubes each for snap frozen (15-20 individuals per tube), fixation (15-20 individuals per tube with 300 uL DNA/RNA shield), preservation (15-20 individuals per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 30 July 2020 at 0900.

• Recruits (~96 hours post settlement): Sampled recruits on 30 July at 1200. n=4 tubes each for snap frozen (15-20 individuals per tube), fixation (15-20 individuals per tube with 300 uL DNA/RNA shield), preservation (15-20 individuals per tube with 500 uL PFA). Methods detailed in protocol linked above. Samples preserved in PFA were moved to 70 percent ethanol on 30 July 2020 at 1800.