This post details results from resazurin trials of PolyIC seed from 18 March 2025 run at control (20°C) and high (42°C) temperatures. This is a continuation of trials I ran earlier this week.

Overview

I have set up the MERLABs FLx800 plate reader for the resazurin assay and ran the first round of full resazurin trials today using our resazurin protocol.

All data and scripts are in our GitHub repository.

This run is repeating the protocol I used a few days ago and will be repeated 2x per week at different temperatures.

Proceedure

Plate 1: 20°C (counter top, control)
Plate 2: 42°C (incubator)

Each plate had a temperature logger with it.

I prepared oysters using the following plate map.

• Wells A1-A3 = blanks
• Wells A4-B12 = control tank 2
• Wells C1-C3 = blanks
• Wells C4-D12 = treated tank 5
• Wells E1-E3 = blanks
• Wells E4-F12 = control tank 1
• Wells G1-G3 = blanks
• Wells G4-H12 = treated tank 4

I set oysters on the lid of the plates and took an image with a scale bar for size. I used ImageJ to measure max length of each oyster for analysis.

Size for plate 1:

Size for plate 2:

I then measured resazurin fluorescence hourly from 10:00-2:00 for a 4 hour incubation.

I then performed survival measurements after the 4 hour incubation.

Data analysis

I analyzed data using scripts available on the GitHub repository and as described previously in my past notebook posts.

Resazurin fluoresence was standardized to initial values, blank corrected, and size normalized.

I then examined effects of time, temperature, and spat treatment on resazurin fluorescence as a proxy for metabolic rate.

Results

There was a significant effect of temperature on metabolic rates and metabolic rate was significantly different between spat treatments.

Metabolic rates were lower at 42°C than 20°C, indicative of metabolic depression under elevated moderate temperatures that we have seen before.

Metabolic rate tracks as expected over a thermal performance curve. Metabolic rates were elevated at 36°C (metabolic stimulation) and lower at 40 and even lower at 42°C (metabolic depression) compared to control (20°C).

I’ll run a trial at 38°C next.

Metabolic rates decreased at 42°C in both control and treated spat. There is more separation in rates between temperatures in treated spat.

Treated spat had higher metabolic rates at 36°C than control spat. Metabolic rates were depressed at 40°C and were lower in treated spat. Metabolic rates decreased at 42°C in both treated and control spat.

Metabolic rates were significantly different between treated and control spat at 36 and 40°C, but not at 20°C or 42°C.

Next steps

On Tuesday of this week, I will run another trial at control and 38°C to wrap up this resazurin experiment.