Sep 18, 2025
Assessment of the Critical Thermal Maximum (CTmax) of Iberian chubs
- Joao Moreno1,2,
- Sofia L Mendes1,
- Vitor C Sousa1
- 1cE3c – Centre for Ecology, Evolution and Environmental Changes & CHANGE – Global Change and Sustainability Institute, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal;
- 2MARE – Centro de Ciências do Mar e do Ambiente (MARE) & ARNET—Aquatic Research Network, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal
Protocol Citation: Joao Moreno, Sofia L Mendes, Vitor C Sousa 2025. Assessment of the Critical Thermal Maximum (CTmax) of Iberian chubs. protocols.io https://dx.doi.org/10.17504/protocols.io.261gekndyg47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 30, 2025
Last Modified: September 18, 2025
Protocol Integer ID: 223654
Keywords: Critical thermal maximum, CTmax, Iberian chubs, freshwater fish, cyprinids, Thermal tolerance, iberian chubs the upper thermal tolerance, ctmax in the iberian freshwater fish, upper thermal physiological tolerance, upper thermal physiological tolerance of individual, iberian freshwater fish, upper thermal tolerance, other freshwater fish, assessment of the critical thermal maximum, critical thermal maximum, water temperature, progressive increase in water temperature, iberian chub, disorganized swimming, genus squalius
Funders Acknowledgements:
Fundação para a Ciência e a Tecnologia
Grant ID: SFRH/BD/143199/2019
Fundação para a Ciência e a Tecnologia
Grant ID: SFRH/BD/145153/2019
Fundação para a Ciência e a Tecnologia
Grant ID: PTDC/BIA-EVL/4345/2021
Human Frontier Science Program Young Research
Grant ID: RGY0081/2020
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Abstract
The upper thermal tolerance of organisms is usually reported as the Critical Thermal Maximum, or CTmax. Here, we describe an adapted protocol to assess the CTmax in the Iberian freshwater fish of the genus Squalius, commonly known as Iberian chubs. The protocol involves exposing individuals to a controlled and progressive increase in water temperature until the point of loss of equilibrium, i.e., uncontrolled and disorganized swimming followed by loss of dorsoventral equilibrium. This point is recognized as the upper thermal physiological tolerance of individuals (i.e., CTmax). Although we applied this protocol exclusively to the Iberian chubs, it can potentially be adapted to other freshwater fish.
Materials
5L water bath
thermometer
aeration water pump (or equivalent)
For fish measurement:
precision scale (accuracy of at least 0.1 g)
graduated ruler
Optional for fin collection:
scissors
ethanol 70% (v/v) for disinfection
1.5 mL tube with 100% (v/v) molecular biology grade ethanol
Troubleshooting
Prepare a 5L water bath with dechlorinated water and appropriate artificial aeration (e.g., water aeration pump).
Warm the water to approximately 19-20 °C .
Put 2 fish at a time in the water bath and wait 10 minutes.
Note: Record the initial time and water temperature.
Increase water temperature by 3 °C every 00:10:00
When fish reach the state of "Loss of equilibrium"*, remove them from the water bath, record the water temperature and time, and put them in an aquaria with cold water (around 20 °C ) and wait for recovery.
*Loss of equilibrium was defined as uncontrolled and disorganised swimming followed by loss of dorsoventral equilibrium for ten seconds (Becker and Genoay, 1979).
Note: the temperature recorded at the state of "Loss of equilibrium" corresponds to the CTmax of the individual.
Repeat the procedure for all the fish.
After fish recover from the experiment, measure and weigh the fish.
Optional: with properly disinfected scissors, obtain a small fin clip to a tube with 100 % (v/v) molecular biology grade ethanol. This sample can be used for further DNA extraction and genotyping.
Protocol references
Becker, C.D., Genoway, R.G. Evaluation of the critical thermal maximum for determining thermal tolerance of freshwater fish. Environ Biol Fish 4, 245–256 (1979). https://doi.org/10.1007/BF00005481