A novel method for measuring acute thermal tolerance in fish embryos

Abstract

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.

CTmax in brook trout (Salvelinus fontinalis) embryos shows an acclimation response to developmental temperatures but is more variable than in later life stages

“…That variability perhaps highlights a challenge with this approach. The high variation we observed was also noted in black gobies ( Gobius niger ), where embryos exhibit greater variation than larvae and adults (Cowan et al, 2023). Unexplained, additional variation in embryos would necessitate relatively larger sample sizes to tease apart treatment effects.…”

“…CT max is widely used as a standardized metric of upper thermal tolerance in ectotherms, but it typically involves assessing the temperature at which fish lose equilibrium (Lutterschmidt & Hutchison, 1997). Such an endpoint is obviously not possible with embryos, but Cowan et al (2023) have developed a CT max methodology that could be useful for comparing upper thermal limits of embryos against older fish.…”

“…In this study, we assessed the CT max response in brook trout ( Salvelinus fontinalis ) embryos acclimated to different thermal regimes using a methodology adapted from Cowan et al (2023). Brook trout are a freshwater fish native to eastern North America that require well‐oxygenated, clear flowing streams or stratified freshwater lakes, and occupy thermal habitat between 10°C and 19°C in summer (Morrison et al, 2020; Smith & Ridgway, 2019).…”

“…In juvenile and adult fish, CT max responds reliably to acclimation temperature (Chatterjee et al, 2004), but embryos are thought to be less physiologically flexible than later life stages (McCormick et al, 1972), so a reasonable alternative prediction might have been that their CT max would not significantly change with acclimation. To our knowledge, this study is the first of its kind to examine the effects of acclimation on upper thermal tolerance of the embryo life stage, though others have quantified CT max of embryos (acclimation effects in Del Rio et al, 2019; CT max in Cowan et al, 2023). To do so, we acclimated brook trout embryos to three controlled thermal regimes: (a) ambient, (b) chronically warmed, (c) chronic warming during the final ~5–20 days of incubation to stimulate an early spring.…”

“…LT 50 protocols are not ideal for determining upper thermal tolerance as the lethality makes it difficult to compare against the life stages at which CT max is typically used (i.e., post‐hatch stages). Additionally, using cessation of heartbeat as an embryonic endpoint is not ideal for comparison to other life stages because the heart continues to beat after loss of equilibrium in adult and juvenile fish (Cowan et al, 2023).…”