This post details results from resazurin trials of PolyIC seed from 13 March 2025 run at control (20°C) and high (36°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.

These trials were run on spat brought back from the hatchery last week (“batch 2”).

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: 36°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 = treated tank 4
• Wells C1-C3 = blanks
• Wells C4-D12 = control tank 1
• Wells E1-E3 = blanks
• Wells E4-F12 = treated tank 5
• Wells G1-G3 = blanks
• Wells G4-H12 = control tank 2

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 10:30-2:30 for a 4 hour incubation.

Noah 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 higher at 36°C than 20°C, indicative of metabolic stimulation under elevated moderate temperatures. Metabolic rates were lower at 40°C.

The difference in metabolic rates between 20°C and 36°C was greater for treated spat than for control spat. This was also true for 40°C trials from a couple days ago.

Treated spat had higher metabolic rates at 36°C than control spat. Metabolic rates were depressed at 40°C.

Next steps

On Tuesday of next week, I will run another trial at control and 42°C and again Thursday at 38°C and 20°C.