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Aquatic ectotherms are vulnerable to thermal stress, with embryos predicted to be more sensitive than juveniles and adults. When examining the vulnerability of species and life stages to warming, comparable methodology must be used to obtain robust conclusions. Critical thermal methodology is commonly used to characterize acute thermal tolerances in fishes, with critical thermal maximum (CTmax) referring to the acute upper thermal tolerance limit. At this temperature, fish exhibit loss of controlled locomotion due to a temperature-induced collapse of vital physiological functions. While it is relatively easy to monitor behavioural responses and measure CTmax in larval and adult fish, this is more challenging in embryos, leading to a lack of data on this life stage, or that studies rely on potentially incomparable metrics. Here, we present a novel method for measuring CTmax in fish embryos, defined by the temperature at which embryos stop moving. Additionally, we compare this measurement with the temperature of the embryos’ last heartbeat, which has previously been proposed as a method for measuring embryonic CTmax. We found that, like other life stages, late-stage embryos exhibited a period of increased activity, peaking approximately 2–3°C before CTmax. Measurements of CTmax based on last movement are more conservative and easier to record in later developmental stages than measurements based on last heartbeat, and they also work well with large and small embryos. Importantly, CTmax measurements based on last movement in embryos are similar to measurements from larvae and adults based on loss of locomotory control. Using last heartbeat as CTmax in embryos likely overestimates acute thermal tolerance, as the heart is still beating when loss of response/equilibrium is reached in larvae/adults. The last movement technique described here allows for comparisons of acute thermal tolerance of embryos between species and across life stages, and as a response variable to treatments.

1. Aquatic ectotherms are particularly vulnerable to thermal stress, with certain life stages (embryos) predicted to be more sensitive than others (juveniles and adults). When examining the vulnerability of species and life stages to warming, it is particularly important to use appropriate and comparable methodology so that robust conclusions can be obtained. Critical thermal methodology (CTM) is commonly used to characterise acute thermal tolerance in fishes, with critical thermal maximum (CTmax) referring to a measured endpoint defining the upper acute thermal tolerance limit. This is the temperature at which fish exhibit loss of locomotory movements (i.e., loss of equilibrium) due to a temperature-induced collapse of vital physiological functions. While it is relatively easy to monitor behavioural responses and measure CTmax in juvenile and adult fish, this can be much more challenging in embryos. This has led to a lack of data on this life stage, or that studies rely on other, potentially incomparable, metrics.2. Here, we present a novel method for measuring acute upper thermal tolerance limits in fish embryos, where CTmax is defined by the temperature at which embryos stop moving. Additionally, we compare this measurement to the temperature at which the embryos' heart stops beating, which has previously been proposed as a method for measuring CTmax in this life stage.3. We found that, similar to other life stages, embryos exhibited a period of increased activity, which peaked approximately 2-3ºC before CTmax. Measurements of CTmax based on last movement are more conservative than measurements based on last heartbeat, additionally they are easier to record and work well with both large and small embryos. Importantly, measurements of CTmax based on last movement in embryos are similar to measurements from larval and adult stages based on loss of locomotory control.4. Using cessation of heart beats as CTmax in embryos likely overestimates acute thermal tolerance as the heart is still beating when CTmax based on loss of response/equilibrium is reached in larvae/adults. The last movement technique described here allows for comparisons of acute thermal tolerance between species, across life stages within species, and as a response variable to treatments.

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