Physiology Processing for Dec and July 2019 time points

KBay Bleaching Pairs Timeseries Project. Dec and July 2019: after clipping and extractions, the rest of the fragments will be airbrushed and processed for physiology metrics at URI. This is only for M. capitata. P. compressa clippings to stay at URI for future molecular analyses and fragment will not be airbrushed at this time. All fragments were clipped and clipping sent to UH for microbial analyses.

Molecular processing: DNA RNA extractions notebook post here.

Physiology excel spreadsheet with physiology processing information here.

Contents:

• Master List with airbrushing processing
• Cell counts
• Chlorophyll concentration

Master List with airbrushing processing

ColonyID	Species	Site	Pair	Bleach	Date	Airbrush Date	volume (mL)	Notes
3	Montipora capitata	13	1	Bleach	7/16/19
4	Montipora capitata	13	1	Non-bleach	7/16/19
11	Montipora capitata	13	2	Bleach	7/16/19			tiny
12	Montipora capitata	13	2	Non-bleach	7/16/19	20220411	8.5
19	Montipora capitata	13	3	Bleach	7/16/19	20220411	8
20	Montipora capitata	13	3	Non-bleach	7/16/19			tiny
201	Montipora capitata	13	9	Bleach	7/16/19	NA	NA	no more fragment
202	Montipora capitata	13	9	Non-bleach	7/16/19
203	Montipora capitata	13	10	Bleach	7/16/19	20220411	2	tiny; no extra homogenate; double the SA for 2 fragments b/c half of skeleton crumbled in bag
204	Montipora capitata	13	10	Non-bleach	7/16/19
209	Montipora capitata	13	11	Bleach	7/16/19	20220411	3
210	Montipora capitata	13	11	Non-bleach	7/16/19	20220411	7.5
211	Montipora capitata	13	12	Bleach	7/16/19	20220411	4.5
212	Montipora capitata	13	12	Non-bleach	7/16/19
217	Montipora capitata	13	13	Bleach	7/16/19
218	Montipora capitata	13	13	Non-bleach	7/16/19	20220411	7
219	Montipora capitata	13	14	Bleach	7/16/19	20220411	7
220	Montipora capitata	13	14	Non-bleach	7/16/19	20220411	12.5	11 + 1-2 mL leaked out
221	Montipora capitata	13	15	Bleach	7/16/19	20220411	11.5
222	Montipora capitata	13	15	Non-bleach	7/16/19
3	Montipora capitata	13	1	Bleach	12/4/19	20220317	9.5
4	Montipora capitata	13	1	Non-bleach	12/4/19	20220321	24	2 tubes
11	Montipora capitata	13	2	Bleach	12/4/19	20220321	13.5	10 + 3-4 mL spilled out
12	Montipora capitata	13	2	Non-bleach	12/4/19	20220411	9.5	fragment crumbled
19	Montipora capitata	13	3	Bleach	12/4/19	20220321	26.5	2 tubes
20	Montipora capitata	13	3	Non-bleach	12/4/19	20220321	28	2 tubes
201	Montipora capitata	13	9	Bleach	12/4/19	20220321	13
202	Montipora capitata	13	9	Non-bleach	12/4/19	20220321	11
203	Montipora capitata	13	10	Bleach	12/4/19	20220411	14
204	Montipora capitata	13	10	Non-bleach	12/4/19	20220321	17.5	2 tubes
209	Montipora capitata	13	11	Bleach	12/4/19	20220321	11.5	7 + Bag leaked ~4-5 mL spilled out
210	Montipora capitata	13	11	Non-bleach	12/4/19	20220321	4.5
211	Montipora capitata	13	12	Bleach	12/4/19	20220321	12
212	Montipora capitata	13	12	Non-bleach	12/4/19	20220321	8
217	Montipora capitata	13	13	Bleach	12/4/19
218	Montipora capitata	13	13	Non-bleach	12/4/19	20220411	16	2 tubes
219	Montipora capitata	13	14	Bleach	12/4/19	20220321	15
220	Montipora capitata	13	14	Non-bleach	12/4/19	20220321	7
221	Montipora capitata	13	15	Bleach	12/4/19	20220321	10
222	Montipora capitata	13	15	Non-bleach	12/4/19	20220321	10.5

Two of the skeletons don’t really look like M. capitata skeletons.. I don’t have any notes about these not looking right during extractions but circle back to this to double check these are actually Mcap..?

airbrushing

Cell counts

Chlorophyll concentration

Example Processing Workflow - not used as the final version for this project

After the URI and UH clipping, the fragment will be airbrushed with 1X PBS. The tissue slurry will be homogenized and 100 uL will be aliquoted out for holobiont assays (i.e. total protein, TAC) before being centrifuged for 3 min at 13,000 rcf. The host fraction will be aliquoted out and volume recorded, and this can be used for host assays (i.e. protein, TAC). The pellet will be resuspended in 1 mL of 1X PBS. From this aliquot, 100 uL will be separated for Sym assays (symbiont counts, protein, TAC) and the remaining 900 uL will be used in a chlorophyll content assay.

The holobiont, host, and symbiont fractions saved can be used for future assays we want to do like lipid content.

If the volume is >2 mL for a coral fragment, then only 1 mL will be spun down following the 100 uL holobiont assay aliquot.

Alternative protein kit:

https://benchling.com/s/prt-kaJmsboePaIXC1R9aATC/edit