Protocol: Integrated Physiological Analysis (Protein, Chl a, and Symbiont Density)

Protocol: Physiological Quantification of S. pistillata Larvae and Spats

This protocol describes the multi-step processing of individual or pooled planulae and spats to quantify total protein content, chlorophyll a concentration, and algal symbiont density.

Version: May 7, 2026

1. Sample Preparation and Homogenization

To ensure all physiological metrics are captured from the same biological material, samples must be homogenized thoroughly.

  1. Thawing: Remove samples from the -80°C freezer and place on ice.
  2. Initial Buffer: Add 0.25 mL of filtered seawater (FSW) or PBS to each 1.5 mL Eppendorf tube.
  3. Mechanical Lyse: Homogenize the tissue using an electrical handheld homogenizer for 30 seconds or until no visible chunks remain.
  4. Final Volume: Add an additional 0.25 mL of buffer (Total volume = 0.5 mL) and vortex briefly.
  5. Aliquoting: * Transfer 0.1 mL to a new tube for the Protein Assay.
    • Reserve the remaining 0.4 mL for Cell Counting and Chl a.

2. Total Protein Quantification (BCA Assay)

Utilizing the Cyanagen µQPRO – BCA kit.

  1. Centrifugation: Spin the protein aliquot at 5,000 rpm for 5 minutes at 4°C.
  2. Standards: Prepare BSA calibration standards (range: 0–200 µg/mL).
  3. Working Reagent (WR): Mix Reagent A and Reagent B in a 25:24 ratio, then add 1 part Reagent C.
  4. Loading: Aliquot 100 µL of sample (diluted if necessary) and 100 µL of WR into a 96-well plate.
  5. Incubation: Incubate at 37°C for 120 minutes.
  6. Measurement: Read absorbance at 562 nm.

3. Algal Symbiont Enumeration

  1. Separation: Centrifuge the reserved 0.4 mL homogenate (5,000 rpm, 5 min, 4°C).
  2. Supernatant: Remove the supernatant thoroughly; this contains the host fraction. The pellet contains the symbionts.
  3. Resuspension: Resuspend the pellet in 0.1 mL of PBSX1.
  4. Counting: Load 20 µL onto a hemocytometer.
  5. Imaging: Use an inverted fluorescence microscope (Blue excitation, 440 nm emission) to capture chlorophyll autofluorescence.
  6. Automation: Process images using the custom Fiji/ImageJ Algae Counter Macro to determine cells/mL.

4. Chlorophyll a Extraction

  1. Separation: Centrifuge the reserved 0.4 mL homogenate (5,000 rpm, 5 min, 4°C).
  2. Supernatant: Remove the supernatant thoroughly; this contains the host fraction. The pellet contains the symbionts.
  3. Extraction: To the remaining algae pellet (after cell count aliquot), add 0.3 mL of pre-chilled 90% Acetone.
  4. Incubation: Vortex and incubate in the dark at 4°C for 8–24 hours.
  5. Clarification: Centrifuge at 5,000 rpm for 5 minutes.
    • Loading Sample Triplicate : For each individual planulae extract tube, load 3 separate wells with:
    • 80 µL of clear sample acetone supernatant.
    • 20 µL of your pure, unbuffered 90% acetone stock.
    • Total In-Well Volume = 100 µL per well. * Blank Wells: Load a minimum of 3 separate control reference wells with exactly 100 µL of pure 90% acetone.
  6. Spectrophotometry: Measure Optical Density (OD) at 630, 647, 664 and 750 nm.
  7. Calculation: Calculate Chl a concentration using the Jeffrey and Humphrey (1975) equations for dinoflagellates.

5. Chl a Operational Parameters Matrix

  • $N$ = Total count of individual planulae pooled inside that specific extraction tube (use $N=1$ for single-larva extractions).
  • $b = 0.52\text{ cm}$ (Fixed vertical pathlength correction constant for a 100 µL total volume footprint in a 96-well half-area well)
  • $\text{DF} = 1.25$ (In-well pipetting dilution factor derived from $\frac{100\ \mu\text{L Well Volume}}{80\ \mu\text{L Extract Added}}$)
  • $\text{RF} = 1.25$ (Biomass recovery factor derived from $\frac{100\ \mu\text{L Total Aqueous Volume}}{80\ \mu\text{L Saved for Acetone}}$ to mathematically re-integrate the 20% aliquot shifted to microscopy)

Step 5.1: Pathlength & Turbidity Adjustments

Clear out background light scattering artifacts by subtracting the zero-absorption value ($A_{750}$) and convert the vertical microplate optics into standardized horizontal 1-cm optical paths ($A_{1\text{cm}}$):

\[A_{1\text{cm at 664}} = \frac{A_{664} - A_{750}}{0.52}\] \[A_{1\text{cm at 647}} = \frac{A_{647} - A_{750}}{0.52}\] \[A_{1\text{cm at 630}} = \frac{A_{630} - A_{750}}{0.52}\]

Step 5.2: In-Well Cross-Correction Concentrations (Jeffrey & Humphrey, 1975)

Determine the active raw pigment densities ($\mu\text{g/mL}$) inside the well using the certified dinoflagellate linear equations:

\[\text{Chl } a_{\text{well}} = 11.43 \times (A_{1\text{cm at 664}}) - 0.64 \times (A_{1\text{cm at 647}}) - 0.12 \times (A_{1\text{cm at 630}})\] \[\text{Chl } c_{2\text{ well}} = -3.73 \times (A_{1\text{cm at 664}}) - 3.73 \times (A_{1\text{cm at 647}}) + 23.29 \times (A_{1\text{cm at 630}})\]

Step 5.3: Standardized Physiological Yields per Larva

Average Symbiodiniaceae Abundance

Scale your grid count from the 20 µL microscopy fraction back up to the parent 100 µL aqueous volume, and normalize by the number of pooled planulae ($N$):

\[\text{Symbiodiniaceae Cells / Planula} = \frac{\text{Cells counted inside } 20\ \mu\text{L fraction} \times 5}{N}\]

Standardized Absolute Chlorophyll Mass

Calculate absolute pigment weight ($\mu\text{g}$) per single larva by multiplying by your in-well dilution ($\text{DF}=1.25$), total extraction volume ($V_{\text{extract}}=0.3\text{ mL}$), and biological pellet recovery factor ($\text{RF}=1.25$), combined as a static scalar coefficient ($1.25 \times 0.3 \times 1.25 = 0.46875$):

\[\text{Total Chl } a\ (\mu\text{g/planula}) = \frac{\text{Chl } a_{\text{well}} \times 0.46875}{N}\] \[\text{Total Chl } c_2\ (\mu\text{g/planula}) = \frac{\text{Chl } c_{2\text{ well}} \times 0.46875}{N}\]

6. Integrated Spreadsheet Formulas (For Data Sheet Automation)

To process your raw microplate exports instantly in Excel, JMP, or Python scripts, use these fully expanded master expressions:

Direct Formula for Chlorophyll a Yield per Planula ($\mu\text{g}$):

\(\text{Total Chl } a\ (\mu\text{g/planula}) = \frac{\left[ 11.43 \left(\frac{A_{664}-A_{750}}{0.52}\right) - 0.64 \left(\frac{A_{647}-A_{750}}{0.52}\right) - 0.12 \left(\frac{A_{630}-A_{750}}{0.52}\right) \right] \times 0.46875}{N}\)

Direct Formula for Chlorophyll c₂ Yield per Planula ($\mu\text{g}$):

\(\text{Total Chl } c_2\ (\mu\text{g/planula}) = \frac{\left[ -3.73 \left(\frac{A_{664}-A_{750}}{0.52}\right) - 3.73 \left(\frac{A_{647}-A_{750}}{0.52}\right) + 23.29 \left(\frac{A_{630}-A_{750}}{0.52}\right) \right] \times 0.46875}{N}\)

Written on May 30, 2026