Abstract
The thermal tolerance–plasticity trade-off hypothesis states that acclimation to warmer environments increases basal thermal tolerance in ectotherms but reduces plasticity in coping with acute thermal stress characterized as heat hardening. We examined the potential trade-off between basal heat tolerance and hardening plasticity, measured as critical thermal maximum (CTmax) of a larval amphibian, Lithobates sylvaticus, in response to differing acclimation temperatures (15° and 25°C) and periods (3 or 7 days). A hardening treatment applied 2 hours before CTmax assays induced pronounced plastic hardening responses in the cool, 15°C treatment after 7 days of acclimation, compared to controls. Warm acclimated larvae at 25°C, by contrast, exhibited minor hardening responses, but significantly increased basal thermal tolerance. These results support the trade-off hypothesis and fill a knowledge gap in larval amphibian thermal plasticity. Elevated environmental temperatures induce acclimation in heat tolerance yet constrains ectotherm capacity to cope with further acute thermal stress.
Summary Statement A larval amphibian follows the trade-off hypothesis such that the group with the highest basal heat tolerance displays the lowest hardening response and vice-versa.
Introduction
Environmental temperature is one of the most important abiotic drivers of organismal physiology (Angilletta Jr, 2009). As temperatures increase due to climate change, ectotherms will be under greater risk of approaching their upper thermal limit that will lead to shifts in species distributions, altered biological interactions, and reduced activity periods, all of which can result in extinction (Bellard et al., 2012; Blois et al., 2013; Cox et al., 2022; Somero, 2010). Global declines in amphibians have been linked to climate change (e.g., Blaustein et al., 2010; Campbell Grant et al., 2020; Lowe et al., 2021; Rollins-Smith, 2017), highlighting the need for continued research on how they respond to warming and thermal extremes.
As thermal traits generally evolve slowly in herpetofauna (Bodensteiner et al., 2020), phenotypic plasticity is likely a primary response to climate change and increasing thermal stress. Thermal acclimation represents reversible plasticity in basal heat tolerance and develops over days to weeks of chronic exposure to altered environmental temperatures (e.g., Cupp Jr, 1980; Lapwong et al., 2021b; Li et al., 2009; Rohr et al., 2018; Sgro et al., 2010). However, acclimation does not necessarily protect organisms against acute exposure to short-term heat events such as heat waves which are projected to increase in frequency (Seneviratne et al., 2021). The related heat hardening response is another form of thermal plasticity that, by contrast, develops rapidly over minutes to hours of exposure to acute heat stress (Bowler, 2005). Heat hardening is generated by exposing organisms to temperatures near or at their upper thermal limit. While hardening rapidly increases heat tolerance, these increases are transient and disappear within 36 hours (Deery et al., 2021; Maness and Hutchison, 1980; Phillips et al., 2016; Rutledge et al., 1987; but see Moyen et al., 2020), highlighting its role as a short-term protection mechanism. Therefore, plasticity in heat tolerance occurs at two different levels: basal thermal tolerance, measured as the limits of thermal performance curves (Huey and Stevenson, 1979), following an acclimation period, and hardening, which temporarily increases basal thermal tolerance following an acute heat stress.
Under an ideal scenario, both high basal thermal tolerance and hardening would improve ectotherm persistence under climate change. However, there appears to be a physiological limitation such that elevated basal thermal tolerance constrains the capacity of an organism to further increase their heat tolerance. For example, Stillman (2003) found a negative relationship between basal thermal tolerance and acclimation capacity in Petrolisthes crab populations across a latitudinal gradient. Building upon this, van Heerwaarden and Kellermann (2020) identified that this negative link was widespread across ectothermic clades and named this pattern the tolerance–plasticity trade-off hypothesis. Heat shock proteins (HSPs) may underlie the trade-off hypothesis because of the central role they play in maintaining homeostasis during extreme temperatures (Feder and Hofmann, 1999; Sørensen et al., 2003) and improving basal thermal tolerance (Bahrndorff et al., 2009; Blair and Glover, 2019; Gao et al., 2014; Krebs and Feder, 1998; but see Easton et al., 1987; Jensen et al., 2010). Because HSPs are energetically expensive to produce and maintain (e.g., Hoekstra and Montooth, 2013), populations from warm environments may be ‘preadapted’ to favor relatively high constitutive HSP expression to elevate basal thermal tolerance but exhibit less flexibility in upregulation following an acute heat shock compared to cool environment populations (Gleason and Burton, 2015). Therefore, under the trade-off hypothesis, hardening may be more useful to species that are less likely to experience chronic heat stress but receive greater benefits in combating acute stress (van Heerwaarden and Kellermann, 2020; but see Sgro et al., 2010). Thus, acute upper thermal limits that are near or pushed near adapted thermal maxima restrict additional plasticity for further increased thermal tolerance through acclimation (Somero, 2010). While a meta-analysis on ectotherms by Barley et al. (2021) provided support for the trade-off hypothesis, there is mixed evidence in larval amphibians (e.g., Menke and Claussen, 1982; Simon et al., 2015; Turriago et al., 2022) suggesting a need for further exploration.
The role of heat hardening in adult (Maness and Hutchison, 1980) and larval amphibians (Sherman and Levitis, 2003; Sørensen et al., 2009) is understudied. We aimed to investigate the trade-off hypothesis by testing how acclimation temperatures (low or high) and duration (short or long acclimation periods) affect interactions between heat hardening and basal thermal tolerance - estimated via critical thermal maximum (CTmax). These tests were conducted on larval wood frogs, Lithobates sylvaticus (LeConte 1825). Because larval anurans display a positive relationship between acclimation temperature and CTmax (e.g., Cupp Jr, 1980; Ruthsatz et al., 2022), we predicted that longer acclimation to warmer temperatures would increase basal heat tolerance compared to those acclimated to cooler temperatures. In line with the trade-off hypothesis, we also expected the hardening effect would be most pronounced in larvae with the lowest CTmax suggesting greater acute thermal plasticity under these environments.
Materials and Methods
Field Collection and Husbandry
Freshly laid (< 36 hours old) wood frog egg masses were collected from wetlands in Jackson Co., IL under an Illinois Department of Natural Resources permit (HSCP 19-03). The egg masses were maintained in 60 L plastic containers with aerated, carbon-filtered water. After hatching, larvae were initially fed autoclaved algal flakes (Bug Bites Spirulina Flakes, Fluval Aquatics, Mansfield, MA, USA), followed by crushed alfalfa pellets at two weeks after hatching. Animals were fed twice weekly, and water was changed weekly. All experimental procedures were approved by the Southern Illinois University Institutional Animal Care and Use Committee (22–008).
Critical Thermal Maximum Assay
After larvae reached early pro-metamorphic stages, 64 individuals were randomly selected and staged, weighed, and transferred to individual 750 mL plastic containers filled with 600 mL of aged (>24 hours) aerated, carbon-filtered water. Larvae were split (N=32/treatment) into low (15°C ± 0.2) and high (25°C ± 0.3) acclimation temperatures. There were no differences in initial Gosner (1960) stage (range = 27 – 35) or mass (0.25 – 0.55 g) between these groups (P > 0.3). The larvae were further randomly split into four groups (n=8 per group) that differed in acclimation period and heat hardening treatment: 1) 3-day control, 2) 3-day hardened, 3) 7-day control, and 4) 7-day hardened. Larvae were acclimated to low or high temperatures for either three or seven days. On the last day of acclimation, the CTmax of control groups was measured. The hardened groups were heated for 10 minutes at 2–4°C below the CTmax of control groups, following the protocol of Sherman and Levitis (2003). After this heat hardening treatment, the animals were returned to their acclimation temperatures for 2 hours, after which their CTmax was measured. Sample sizes were reduced to seven for the 7-day hardened low and high temperatures groups, and the 7-day control low temperature group due to mortality.
CTmax was measured between 1000 – 1600 hrs to minimize potential diel effects on heat tolerance (Healy and Schulte, 2012; Maness and Hutchison, 1980). Larvae were staged, weighed, and then placed in individual 125 mL flasks filled with 75 mL of aged, aerated, carbon-filtered water and submerged in a hot water bath (Isotemp 220, Fischer Scientific) and given 5 minutes to acclimate prior to beginning the assay. Water temperatures increased 0.6 ± 0.01°C per minute from a starting temperature of 19.9 ± 0.2°C. Beginning at ~34°C, larvae were prodded with a spatula every 30 seconds until they did not respond to the stimulus. At this point, a thermocouple probe (Physitemp BAT-12) was placed in the flask, water temperature was recorded which represented the larval CTmax. Flasks were then placed in a water bath at room temperature to facilitate larval recovery, and all larvae recovered ≤ 5 minutes. Upon completion of CTmax measurements, all larvae were euthanized via snap-freezing in −80°C ethanol.
Statistical Analyses
We assessed how larval CTmax shifted in response to our various treatments using a general linear model. While Gosner stage recorded prior to the CTmax measurement was normally distributed, mass was log-transformed to achieve normality, and both were included as covariates in the model. Fixed effects included acclimation period (3 or 7 days), acclimation temperature (low or high), hardening treatment (control or hardened), and their interactions. Post-hoc analyses were conducted using Tukey tests. All analyses were conducted in R Studio v. 2022.02.3 (https://www.Rstudio.com/) and significance values were set as α = 0.05.
Results
Across all treatments, wood frog larvae displayed a moderate degree of variation in their CTmax (range = 35.8° – 39.6°C; Table 1). Two individuals were dropped from analyses due to abnormally low CTmax values (≤ 34.9°C) in relation to their group mean. Of the main effects, only acclimation temperature (F1,49 = 6.52, P = 0.014) had a significant effect (Table 2) with those in the high acclimation temperature treatment exhibiting greater heat tolerance (Fig. 1). While neither hardening (F1,49 = 0.088, P = 0.77) nor acclimation period (F1,49 = 2.55, P = 0.12) had significant effects on CTmax, there was significant hardening by acclimation period (F1,49 = 6.11, P = 0.017) and acclimation period by acclimation temperature (F1,49 = 18.71, P < 0.0001) interactions. The former was driven by a more pronounced hardening effect for day 7 individuals, while the latter was the outcome of a pronounced increase in CTmax among larvae in the high acclimation treatment on day 7 (Fig. 1). Lastly, a significant three-way interaction was found for acclimation period, acclimation temperature, and hardening treatment (F1,49 = 4.47, P = 0.040). Larvae in the low acclimation treatment on day 7 showed the largest hardening effect of 0.9°C, which was more than double the hardening effect of any other group (Fig. 1). Larval mass and Gosner stage were unrelated to CTmax (P ≥ 0.29).
Discussion
Phenotypic plasticity of heat tolerance provides ectotherms the ability to counter the threat of overheating due to temperature extremes associated with climate change. Heat hardening, a form of thermal plasticity, represents the “first line of defense” against heat stress (Deery et al., 2021) through rapid upregulation of HSPs and/or changes to cellular structure in response to an acute thermal shock that can increase short-term heat tolerance (Bowler, 2005). However, the tolerance–plasticity trade-off hypothesis (van Heerwaarden and Kellermann, 2020) proposes that basal heat tolerance and thermal plasticity are negatively correlated; such that individuals with high CTmax have limited hardening (Gilbert and Miles, 2019). While numerous studies have demonstrated that amphibians exhibit plastic basal heat tolerance (e.g., Cupp Jr, 1980; Ruthsatz et al., 2022), hardening remains understudied.
In our study, we found evidence in support of the trade-off hypothesis for larval wood frogs, although the effect was minor (Fig. 1), potentially due to low sample sizes. The group with the lowest mean CTmax (36.5°C) had the greatest hardening effect (0.9°C), while the group with the highest mean CTmax (39.0°C) had a minimal hardening effect (0.1°C). While the 0.9°C hardening effect was comparable to larval American toads (Anaxyrus americanus) and African clawed frogs (Xenopus laevis) (Sherman and Levitis, 2003), the remaining groups had a minor hardening response (≤ 0.4°C) that was similar with values for larval bullfrogs (L. catesbeianus)(Menke and Claussen, 1982). Additionally, the bullfrogs showed no evidence of the trade-off hypothesis as CTmax increased positively with acclimation temperatures while hardening effect was unchanged. Hardening effects in lizard, salamander, and fish species are variable ranging from –0.4°C (Anolis sagrei) to 2.1°C (A. carolinensis) (Deery et al., 2021; Lapwong et al., 2021a; Maness and Hutchison, 1980; Phillips et al., 2016; Rutledge et al., 1987). In relation to other species, larval wood frogs acclimated to cooler conditions have a relatively strong hardening effect indicating significant plasticity in heat tolerance to improve their tolerance of overheating. This may benefit wood frogs as ephemeral pond breeding species are threatened by climate change (Blaustein et al., 2010) during the larval stage (Enriquez-Urzelai et al., 2019).
We can only speculate on the mechanism that drove the observed results, but we propose that HSPs represent an intriguing answer. This is because they are intimately tied to environmental temperature (Dalvi et al., 2012; Gu et al., 2019; Jin et al., 2019) and basal thermotolerance (Bahrndorff et al., 2009; Blair and Glover, 2019). Warm-tolerant ectotherms often express higher constitutive levels of hsp70 relative to less-tolerant populations, but that an acute heat-stress results in greater hsp70 expression in those with lower basal thermal tolerance (Gleason and Burton, 2015; Zatsepina et al., 2000; Zatsepina et al., 2001). Zatsepina et al. (2000) proposed that this provided temperate populations the capability to rapidly and intensely synthesize HSPs after brief exposure to heat shock that was absent in low latitude populations. We propose a similar pattern in the wood frog larvae, such that higher constitutive HSP levels in warm-acclimated larvae provided increased basal heat tolerance compared to cold-acclimated larvae, yet hardened larvae from the latter group greatly upregulated HSP expression following a heat shock enhancing their hardening response. This is in line with Drosophila acclimated to cooler temperatures (Bettencourt et al., 1999), which exhibited pronounced hardening plasticity that was absent in the warm-acclimated group. Quantifying constitutive and heat-shocked hsp70 mRNA of larval liver and gill tissues would offer support to this conclusion. Additionally, many ectotherms appear to have hard upper-limits to thermal tolerance after which their pejus range constraints any further plastic responses (Denny and Dowd, 2012). Thus, the warm acclimated larvae in our study could have approached their physiologically and evolutionarily determined upper limit that constrained any further plastic responses. Future tests are required to understand 1) if there is a degree of plasticity to hard upper limits of thermal acclimation, 2) the cellular and physiological mechanisms underlying these limits, 3) how these mechanisms determine the trade-offs between hardening and acclimation to chronic heat stress, and 4) how these mechanistic interactions are shaped by evolution in comparative studies.
Wood frog larvae with low basal heat tolerance demonstrated a large hardening effect suggesting a trade-off between the two traits. There is an inherent link between CTmax and hardening which may bias the presence of the trade-off hypothesis (van Heerwaarden and Kellermann, 2020), and Deery et al. (2021) proposed that correlative evidence of the trade-off hypothesis is a statistical artifact. However, we believe our methodology of using different individuals for CTmax and hardening removed the risks of spurious correlation and strengthened our analyses. Based on our results, we propose that larval wood frogs support the trade-off hypothesis after a relatively short acclimation period. Hardening benefits cool-acclimated populations in response to acute heat stress but plasticity in basal heat tolerance in response to prolonged warming are likely to be more beneficial in reducing overheating risk.
Competing Interests
The authors declare no competing or financial interests.
Author contributions
Conceptualization: J.D.; Methodology: J.D.; Formal analysis: J.D.; Investigation: J.D.; Data curation: J.D.; Writing - original draft: J.D.; Writing - review & editing: J.D., R.W.; Supervision: R.W.; Project administration: J.D.; Funding acquisition: R.W.
Funding
This research was funded through a Southern Illinois University Carbondale (SIUC) startup grant to R. W. Warne.
Acknowledgements
We would like to thank Jared Bilak, Abigail Pratt, Matthew Walker, and Justin Remmers for feedback and comments on our manuscript. We would like to thank the two anonymous reviewers for helpful feedback on this manuscript.