# Rapid and reversible plasticity of upper thermal limit, but no effects of multigenerational warming in medaka

Juliette Vallin, Fabio Gerberon, Rémy Lassus, Mathieu Floury, Anthony

Maire, Martin Daufresne, Arnaud Sentis

► To cite this version:

Juliette Vallin, Fabio Gerberon, Rémy Lassus, Mathieu Floury, Anthony Maire, et al. Rapid and reversible plasticity of upper thermal limit, but no effects of multigenerational warming in medaka. Journal of Thermal Biology, 2025, 130, pp.104155. 10.1016/j.jtherbio.2025.104155. hal-05130431

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# Rapid and reversible plasticity of upper thermal limit, but no effects of multigenerational warming in medaka

Juliette Vallin<sup>a,*</sup>, Fabio Gerberon<sup>a</sup>, Rémy Lassus<sup>a</sup>, Mathieu Floury<sup>b</sup>, Anthony Maire<sup>c</sup>, Martin Daufresne<sup>a</sup>, Arnaud Sentis<sup>a</sup>

## ARTICLE INFO

### Keywords:

Climate change

Heatwave

Physiology

Mesocosms

Experimental biology

Freshwater

Fish

## ABSTRACT

The global rise of atmospheric temperature and the escalating occurrence of extreme climatic events urge for a better understanding of the factors influencing thermal tolerance to extreme temperatures. In particular, the critical thermal maximum (CTmax) is a proxy of the upper thermal tolerance that is key in assessing species vulnerability to warming. While several mechanisms, such as short-term acclimation and intergenerational effects, can influence the CTmax of ectotherm species, their relative contributions and interactions under natural conditions remain unclear. Using the medaka fish (*Oryzias latipes*), this study investigates whether multigenerational warming has shaped CTmax after multiple generations of laboratory rearing and explores the interplay between potential intergenerational effects and plastic responses during an artificial heatwave in mesocosms. Our findings reveal that seven years of multigenerational exposure to different temperatures (30 °C vs. 20 °C) did not cause any detectable durable change in CTmax. This suggests that evolution of CTmax is slow or requires strong selective pressures to evolve fast enough. In contrast, medaka fish exhibited rapid acclimation with a 1.5 °C increase in their CTmax following a two-week heatwave. Interestingly, two weeks after the heatwaves, we could not detect a significant effect of heatwave exposure on CTmax showing that this plastic response can reverse rapidly. These results, along with other studies on ectotherms thermal tolerance, emphasize the potential for short-term response of species to changing climate through acclimation. Our findings highlight the importance of integrating plasticity into vulnerability assessments to improve predictions of climate change impacts.

## 1. Introduction

Ongoing climate change is not only driving an increase in global mean temperatures but also intensifying the frequency and severity of extreme events such as heatwaves, droughts, and floods (Hosseinzadehtalaei et al., 2020; IPCC et al., 2023; Kharin et al., 2018; King and Karoly, 2017). Aquatic organisms have thermal boundaries characterized by lower and upper thermal limits, beyond which survival is impaired (Ern et al., 2023; Kingsolver and Woods, 2016; Kong et al., 2022; Ørsted et al., 2022). Climate change and, in particular, extreme warm temperatures, directly challenge these limits by increasing the frequency at which organisms encounter conditions near their thermal thresholds. During these stressful events, even slight increases in temperature can drastically reduce performance and survival rates, resulting in substantial physiological impacts (Bennett et al., 2021; Hoffmann

et al., 2013; Jørgensen et al., 2022; Morley et al., 2019; Ørsted et al., 2022). This can lead to widespread mortality, or even extirpation of whole populations, which in turn may destabilise entire communities and, ultimately, the trophic network (Buckley and Huey, 2016; Woodward, 2009). Consequently, accurately characterizing the upper thermal limits of organisms is essential for assessing their vulnerability to climate change and predicting their persistence over both short- and long-term timescales.

For ectotherms, vulnerability to warming is often assessed using the thermal safety margin, defined as the difference between environmental temperatures (which includes day-to-day and seasonal variability) experienced by an organism and its critical thermal maximum (CTmax) (Desforges et al., 2023; Morley et al., 2019). The CTmax corresponds to the temperature above which an organism loses its organized locomotor activity and the ability to escape conditions that compromise its survival

<footnote><sup>a</sup> INRAE, Aix-Marseille University, UMR RECOVER, Aix-en-Provence, France</footnote>

<footnote><sup>b</sup> INRAE, HYCAR, Antony, France</footnote>

<footnote><sup>c</sup> EDF R&D, LNHE (Laboratoire National d'Hydraulique et Environnement), Chatou, France</footnote>

<footnote>* Corresponding author. INRAE, Aix-Marseille University, UMR RECOVER, Aix-en-Provence, France.</footnote>

<footnote>E-mail address: juliette.vallin@gmail.com (J. Vallin).</footnote>
(Beitinger et al., 2000). It is thus a measure of thermal tolerance often used as a proxy for the upper thermal limit of organism above which survival is impaired. The CTmax is widely employed in studies of thermal ecology and physiology, particularly in fish, amphibians, reptiles, and arthropods (e.g. Carilo Filho et al., 2022; Desforges et al., 2023; Kellermann and Sgrò, 2018; Layne and Claussen, 1982; Ørsted et al., 2022).

CTmax can vary among individuals and populations due to, for instance, genetic differences and plastic responses to local environmental factors, such as temperature and resource availability (Comte and Olden, 2017; Hoffmann et al., 2013; Morley et al., 2019; Rohr et al., 2018). In particular, plastic responses to temperature changes occurring within the lifetime of an individual, often referred as thermal acclimation (hereafter acclimation), can influence the thermal tolerance (i.e. CTmax) of ectotherms (Morley et al., 2019). In addition, recent studies reported that these effects can be transferred across generations through transgenerational acclimation (Donelson et al., 2012). Conversely, heat stress can lead to the genetic selective evolution of CTmax (Geerts et al., 2015; Lewontin, 1970; Morgan et al., 2020). Additional mechanisms such as maternal effects and epigenetics can also lead to intergenerational responses to temperature and can modulate CTmax (Donelson et al., 2012; Hoffmann et al., 2013). Together, these mechanisms can explain variations in CTmax across individuals and populations, although they typically occur on different time scales and involve different biological processes. Yet, the relative importance of these different mechanisms for explaining variations in CTmax remains largely unexplored. While some studies have documented rapid evolution of CTmax in zooplankton, phytoplankton, and insects (Geerts et al., 2015; Schaum et al., 2017, 2022), others highlighted the slow pace of evolution and its limited abilities for further change, particularly in vertebrates with long generation times compared to invertebrates (Bennett et al., 2021; Hoffmann et al., 2013; Morgan et al., 2020). Although acclimation to warmer temperatures can increase CTmax (e.g. Dai et al., 2022; Earhart et al., 2023; Morley et al., 2019), we have little information about the rate of acclimation (i.e. how fast the phenotype of organisms changes). Recent studies showed high variation in plasticity rates of CTmax between species and taxa (Einum and Burton, 2022) and that fish from shallow habitats can increase their CTmax within 3h of exposure to warm temperatures (De Bonville et al., 2025). Yet, there is almost no information about the duration of these acclimation effects (i. e. how long they last once temperatures return to normal). Finally, there is an ongoing debate on the possible costs of CTmax modulation. Recent studies showed that genetic adaptation through selection to high constant temperatures may reduce CTmax acclimation capacity, because of hard physiological constraints often referred to as concrete ceilings (Comte and Olden, 2017; Kellermann and Sgrò, 2018; Morgan et al., 2020, 2022). For instance, Morgan et al. (2020) artificially selected offspring from wild-caught zebrafish to increase or decrease their CTmax over 6 generations. They found that CTmax can evolve under artificial selection but this leads to a decrease in acclimation capacity, raising concerns about the ability of fish to adapt to ongoing global warming. Additionally, increased heat tolerance may come at a cost to body size, growth, or reproductive performance (Donelson et al., 2011; Peralta-Maraver and Rezende, 2021; Tüzün and Stoks, 2022; Willi and Van Buskirk, 2022). Understanding how thermal tolerance changes after long and short-term exposure to warming, and whether this can increase the ability of organisms to cope with climate change and extreme events like heatwaves, is therefore important to improve our understanding of climate change impacts. In addition, most studies on CTmax plasticity were conducted under controlled conditions using organisms acclimated to constant temperatures (e.g. Morgan et al., 2020), which differs significantly from the fluctuating temperatures and abrupt thermal extremes, such as heatwaves, occurring in natural environments. As a result, it remains unclear whether laboratory findings on CTmax plasticity can be extrapolated to the complexity of natural conditions.

The aim of this study was to investigate the influence of

multigenerational warming on CTmax, and to test, under semi-natural conditions, whether these intergenerational effects could influence (1) the short-term acclimation response of CTmax to a heatwave and (2) the duration of this acclimation effect after the heatwave. In a previous study (Morla et al., 2025), we observed that medaka fish (Oryzias latipes) reared at 30 °C (hereinafter, warm fish) had a CTmax 4.9 °C higher than those reared at 20 °C (hereinafter, cold fish). However, the mechanisms explaining these differences have not yet been determined. To test if these effects were only due to acclimation, we reared the offspring of the fish tested in Morla et al. (2025) in a common garden experiment in outdoor mesocosms and measured their CTmax. Additionally, we investigated whether the two fish populations differed in their acclimation responses to an artificial heatwave or in the duration of this acclimation response after the heatwave.

Our first hypothesis (H1) was that a multigenerational warming would increase thermal tolerance in future offspring, therefore we predicted that warm fish would have higher CTmax than cold fish (Donelson et al., 2012; Morley et al., 2019; Munday et al., 2017). Our second hypothesis (H2) was that exposure to a moderate heatwave event would cause a transient and reversible increase in thermal tolerance, thus we predicted that exposure to heatwave conditions would increase CTmax in both cold and warm fish, and that CTmax would quickly return to baseline after the heatwave ended (Dai et al., 2022; Earhart et al., 2023; McDonnell and Chapman, 2015; Morley et al., 2019; Penny and Pavey, 2021). Finally, our last hypothesis (H3) was that there would be a trade-off between acclimation to past multigenerational warming and acclimation to heatwave conditions, thus we predicted that cold fish would exhibit a greater increase in CTmax following the heatwave than warm fish (Comte and Olden, 2017; Culumber and Monks, 2014; Morgan et al., 2018; Sandblom et al., 2016).

## 2. Methods

### 2.1. Study organism

The medaka fish (Oryzias latipes) is a small teleost species native from East Asia (Wittbrodt et al., 2002). It has a lifespan of around one year in the wild and an adult size up to 50 mm. It mainly lives in shallow ponds or rice paddies (Shima and Mitani, 2004; Wittbrodt et al., 2002) where temperature fluctuates rapidly compared to larger and deeper water bodies. This eurythermal species can tolerate a wide temperature range (0 °C–40 °C), with an optimum near 25 °C. The medaka fish exhibits a short life cycle, with a generation time of 2–3 months, reaching sexual maturity within 10–12 weeks and producing offspring year-round (Alberto-Payet et al., 2022; Leaf et al., 2011; Shima and Mitani, 2004). These characteristics make it a valuable model organism for rearing and multigenerational studies in thermal ecology, genetics, and ecotoxicology (Nor Azra et al., 2022; Wang and Cao, 2021; Wang and Bhandari, 2020).

### 2.2. Experimental design

Here we provide an overview of the experimental design before fully describing it in the following sections. This study involved two medaka lineages reared for multiple generations at constant temperatures of 20 °C and 30 °C, referred to as cold and warm fish, respectively. The last generation was raised under a common garden setting in outdoor mesocosms. After few months of common garden, fish were exposed to one of two treatments: (i) a control without heatwave or (ii) a two-week artificial heatwave. CTmax was measured immediately after the heatwave and again two weeks later. Fish growth was also monitored monthly by measuring body length and mass.

### 2.3. Multigenerational thermal exposure

The cold and warm fish lineages were kept in separated climatic
chambers at an average of 19.94 °C (±0.35 °C) and 30.04 °C (±0.44 °C), respectively, under controlled experimental conditions. The fish were kept in different replicate aquariums (25 × 40 × 20 cm, with 10 replicate tanks by treatment, see Morla et al., 2025), with a density of ~30 adult fish per aquarium. Aquaponics plants as well as rocks were placed in aquariums for enrichment. Furthermore, aquariums had three walls covered with an opaque blue film to limit fish stress. Photoperiod was set to 12L:12D all year, except during the four months of optimum physiological spawning when the photoperiod was switched to 16L:8D. The two lineages originated from the same starting F₀ population of 320 medakas (160 females and 160 males) belonging to the CAB strain from Carolina Biological Supply Company (USA) and provided by AMAGEN® (France). Fish were fed "ad libitum" with dry flakes food TétraMin® three times a day. For each generation, egg clutches were collected during optimal fecundity period and placed in hatcheries, which are separate aquariums with specific small nets to hold the eggs and allow visual verification of emergence, at a temperature identical to the parental one. When juveniles emerged, they were transferred back to the regular rearing aquariums. Over seven years (2016–2023), 7 generations of cold fish and 11 generations of warm fish were produced, reflecting faster generation turnover at 30 °C due to earlier sexual maturity (Blanck and Lamouroux, 2007; Niu et al., 2023; Verberk et al., 2021). Medaka fish from the 7th cold fish generation and the 11th warm fish generation were simultaneously tested for CTmax in 2023 (Morla et al., 2025). Morla et al. (2025) used the dynamic CTmax method, where the temperature is rapidly increased until the individual loses consciousness (average ramping rate of 0.15 °C/min). In this study we also used the dynamical method to test for differences in CTmax across our experimental treatments (see Section 2.5).

## 2.4. Common garden and heatwave

In October 2023, eggs from the 7th cold and 11th warm generations were hatched and reared in aquariums at an average water temperature of 25.08 °C (±0.33 °C) for 60 days. Subsequently, 176 fish (88 per lineage) were transferred to 16 outdoor 1000L mesocosms (8 mesocosms for each lineage with 11 fish per mesocosm). Mesocosms were equipped with oxygen and temperature probes (recording every 10 min) and inoculated in September 2023 with filtered water, sediments, and macrophytes (Ceratophyllum demersum) from nearby ponds to establish natural aquatic communities. Water from all mesocosms was homogenized by mixing 10L from each mesocosm into all others. Insect nets were placed over mesocosms to prevent insect colonization. The mesocosms were then left to their natural dynamics until medaka introduction in December. Medakas were left five months in the mesocosms. Mesocosms were left uncleaned to mimic natural conditions and minimize fish stress. A pierced brick was placed in each mesocosm to provide extra refuge, complementing the naturally present macrophytes and algae/biofilm that also offered shelter. Medaka fish fed on the living plankton present in the mesocosms, without any external feeding

supply. Mesocosms' water temperature was similar across mesocosms and followed natural day-night and seasonal air temperature fluctuations (Table 1, Fig. S1).

In the end of May, five months after the introduction of fish in the mesocosm, we artificially warmed half of the mesocosms by +6 °C during 14 days using heat resistors (spiral immersion heaters, Zoppas Industries, Vittorio Veneto, Italy). Two heating resistors were placed in each mesocosm, with the heating section near the bottom of the mesocosm. This setup allowed a water flux to be generated within the mesocosms during heating, as the heated water rose from the bottom, mixing the water and homogenising the temperature within a few tens of minutes (personal observations). The duration (14 days) and amplitude (+6 °C) of the artificial heatwave were chosen to match the mean duration and amplitude of the 2003 European summer heatwave (Charpentier, 2011). Overall, this resulted in a full factorial design with two fish treatments (cold or warm fish) crossed with two temperature treatments (heatwave or control without heatwave) with four randomly selected mesocosms for each combination of treatments (Fig. S2).

We increased water temperature by 6 °C while keeping track of the natural day-night and seasonal fluctuations of ambient temperature (Fig. 1). We initiated warming at 4:30 p.m. (Fig. 1), when ambient temperatures began to decrease. As a consequence, the water temperature in the ambient mesocosms decreased while the water temperature in the warmed mesocosms remained constant (or slightly decreasing) until the six degree difference was reached (Fig. 1). This ensured that fish would not be stressed by a rapid temperature increase due to artificial warming. During the heatwave, mean oxygen concentration was lower in heated mesocosms (average oxygen concentration of 8.99 mg/L ± 1.59) than in mesocosms that remained at ambient temperature (average oxygen concentration of 10.80 mg/L ± 1.22). As dissolved oxygen would likely be limited in the event of a natural heatwave, this added realism to the simulated mesocosm heatwave. To end the heatwave, the heat resistors were switched off and the temperature of all mesocosms returned quickly to similar levels (Fig. 1). Oxygen level in mesocosms also returned to similar levels (9.87 mg/L ± 1.48 and 9.89 mg/L ± 1.58 for previously heated and not heated mesocosms, respectively).

In addition to the initial measurements taken shortly before their introduction into the mesocosms, fish were counted, measured, and weighed every month from the end of February to the end of May. To this end, all medaka fish from a mesocosm were fished, individually weighed on a precision scale (STX123, OHAUS, Nänikon, Switzerland), photographed on a scale and immediately placed back in their mesocosm. Total body length was measured, from the tip of the heat to the tip of the caudal fin using the Piximètre software (5.10 version; www.piximetre.fr). A body condition factor was calculated for each fish at each sampling date using the Fulton condition factor (Froese, 2006; Nash et al., 2006):

$$ \text{Fulton index} = \frac{\text{weight}}{\text{length}^3} \times 100 $$

<caption>**Table 1**</caption>

Water temperature (mean, standard deviation, minimum and maximum values of each month) in the mesocosms during the 5 months medaka fish were present, before the simulated heatwave (from mid December to 21st of May), during the simulated heatwave (from the 22nd of May to the 4th of June) and after the simulated heatwave (from the 4th of June to the 19th of June).

<table><thead><tr><th>Month</th><th>Mean water temperature (°C)</th><th>Standard deviation (sd) of water temperature (°C)</th><th>Minimum water temperature (°C)</th><th>Maximum water temperature (°C)</th></tr></thead><tbody><tr><td>December</td><td>8.74</td><td>1.78</td><td>3.85</td><td>14.31</td></tr><tr><td>January</td><td>7.56</td><td>1.72</td><td>3.85</td><td>11.98</td></tr><tr><td>February</td><td>10.73</td><td>1.91</td><td>5.59</td><td>16.82</td></tr><tr><td>March</td><td>14.08</td><td>2.84</td><td>7.14</td><td>21.89</td></tr><tr><td>April</td><td>17.78</td><td>2.74</td><td>10.27</td><td>27.60</td></tr><tr><td>May (before the heatwave)</td><td>20.75</td><td>3.04</td><td>11.56</td><td>28.57</td></tr><tr><td>During the heatwave</td><td>25.15</td><td>3.68</td><td>16.75</td><td>33.86</td></tr><tr><td>After the heatwave</td><td>24.86</td><td>2.87</td><td>19.08</td><td>32.69</td></tr></tbody></table>
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Water temperature recorded in the mesocosms, distinguished by colours, during and after the two weeks artificial +6 °C heatwave. Half of the mesocosms were subjected to the artificial heatwave. The dashed red line marks the onset of the heatwave for these mesocosms, while the dashed blue line indicates its end.</figcaption></figure>

with weight in mg and length as total body length in mm.

## 2.5. Critical thermal maximum (CTmax) measurements

We measured the CTmax of the fish on the day we ended the heat-wave (session 1) and then two weeks later (session 2). For session 1, four medakas were randomly selected in each mesocosm (for a total of 32 cold fish and 32 warm fish) and placed overnight under controlled indoor aquarium conditions, to recover from handling stress and acclimate to lab conditions. Aquarium water temperature was at 25 °C for all fish to ensure they started the CTmax trial under the same conditions, and because it was the overall mean temperature observed in mesocosms during the two weeks preceding the first trial (Table 1). After this rest and fasting period, medakas were placed in pairs (to avoid stress of being alone, as medaka are gregarious) in an aquarium (40x25 × 20 cm) with water at 25 °C and air pump for oxygenation. Aquarium's water temperature was warmed using the water bath method (e.g. Cowan et al., 2023; Recsetar et al., 2012), where the aquarium containing the fish was placed in a larger aquarium (150x60 × 50 cm) containing water at 55 °C and equipped with a heat resistor. Water pumps were placed on both sides of the larger aquarium to ensure homogeneous water temperature. The water bath method increases the aquarium water temperature for the medaka without requiring water mixing, thereby preventing fish from expending energy swimming against a current. The warming rate of the aquarium with the medakas was monitored continuously using a thermal probe (Water Temp Pro v2, Onset HOBO, Bourne, USA). It recorded an average increase in water temperature of 0.48 °C (±0.10 °C) per minute although ramping rate was not strictly constant over the trial duration and varied across trials (see Fig. S3 for more details). When an individual remained without movement for at least 3 s, we initiated physical contact with the tip of a hand net, and if the fish remained unresponsive, the endpoint was declared. This protocol was clearly communicated to all persons (5 in total) taking part in the experiment so that this measure is homogeneous and repeatable. As soon as an individual reached its endpoint, we recorded time to determine the corresponding CTmax temperature from the thermal probe recordings. The fish was immediately removed from the aquarium and euthanized with Benzocaine© [250 mg/L] after anaesthesia. Euthanized fish were then individually measured and weighed. Two weeks after the heatwave (i.e. session 2), the same protocol was repeated with the individuals left in the mesocosms (for a total of 32 cold fish and 31 warm fish, since one mesocosm contained only three warm fish instead of four in all other mesocosms). One warm individual exhibiting abnormal behavior prior to CTmax assessment was excluded from the analyses, but results remained consistent when this fish was included. More details on the CTmax trial can be found in Table S1 (table based on Raby et al., 2025).

The above project was approved by the ethics committee for animal

experimentation n°071. The experimental protocol was performed in agreement with the ethical permits #49756-2024051417235067 v3.

## 2.6. Data analysis

Fish growth before the heatwave was analysed using linear mixed models LMM (*lmer* function from the *lme4* R package) as the growth curves did not show a saturating growth pattern. This model included thermal origin (cold fish and warm fish) and measurement dates as fixed effects and the mesocosm ID number and fish sex as random effects.

The effects of thermal origin, heatwave treatment, CTmax session (at the end of the heatwave vs. two weeks after the heatwave) and their interaction on fish CTmax were also analysed with LMM. We also added the Fulton body condition factor (K) as a fixed effect to account for the potential influence of body condition on CTmax. We included the mesocosm ID number as random effect to control for non-independent samples (i.e. mesocosms). A preliminary analysis using fish sex as fixed factors indicated CTmax differences between males and females (with females having a CTmax on average 0.4 °C higher than males) but these differences were not influenced by the experimental treatments or their interactions. We thus decided to keep sex as a random effect to control for male-female differences. Furthermore, the ramping rates of CTmax trials were not strictly constant (ramping rate tended to slow down with time) and showed variation between trials (Fig. S3). For each CTmax trial, we thus fitted a quadratic model with water temperature as a function of time to estimate initial ramping rate (linear coefficient of the model) and how it changed over time (quadratic coefficient of the model). We then included trial duration, as linear and quadratic coefficients, as random factors in our models to account for non-constant ramping rate and variations between trials.

For each complete model, Wald Chi-square statistic of each fixed effect was calculated in deviance analyses (*Anova* function from "car" R package; Fox et al., 2012) to determine the significance of the fixed effects. Residuals were inspected to verify the assumptions of each model. All statistical analyses were performed with the R software (version 4.3.1; R development Core Team).

# 3. Results

## 3.1. Fish growth

At introduction in the mesocosms in December 2023, when they were 60 days old, cold fish mean length was 18.8 (±2.4) mm and warm fish mean length was 15.8 (±3.4) mm. We do not have information on fish length at hatch or before 60 days old. We found that, before the heatwave, the medakas grew substantially in the mesocosm with an average of 0.12 mm per day ($\bar{\chi}^2 = 925.8, df = 1, p < 0.001$, Fig. 2). Model selection indicated that fish growth was not significantly influenced by
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Medaka fish (*Oryzias latipes*) length (mm) over the months spent in mesocosms before the heatwave, for each thermal origin (blue for cold fish: fish from the lineage raised at 20 °C for multiple generations; orange for warm fish: fish from the lineage raised at 30 °C for multiple generations). Lines represent the fitted linear regressions for each fish thermal origin. Error bars represent standard deviation for each treatment at each time point.</figcaption></figure>

their thermal origin ($p > 0.05$), but thermal origin significantly influenced mean fish length, with cold fish being on average 2.3 mm longer than warm fish, representing an increase in body length of 9.6 % ($\chi^2 = 54.4$, $df = 1$, $p < 0.001$, Fig. 2 and Table S2).

## 3.2. Fish CTmax

The CTmax of medaka fish was not significantly influenced by the

interactions between fish thermal origin, heatwave treatment and CTmax session ($p > 0.05$), nor by the interactions between fish thermal origin and heatwave treatment ($p > 0.05$) or between fish thermal origin and CTmax session ($p > 0.05$). Overall, the CTmax was not significantly influenced by the fish thermal origin ($p > 0.05$) but was significantly influenced by the interaction between heatwave treatment and CTmax session ($\chi^2 = 57.2$, $df = 1$, $p < 0.001$).

During the first CTmax measurement session, conducted the day after the end of the artificial heatwave, CTmax was significantly higher (on average + 1.67 °C) in fish that were exposed to the heatwave compared to those not exposed ($\chi^2 = 89.0$, $df = 1$, $p < 0.001$, Fig. 3 a and Table S3). Furthermore, fish with a better body condition (i.e. higher Fulton condition factor) had a significantly higher CTmax ($\chi^2 = 5.6$, $df = 1$, $p = 0.01$, Table S3 and Fig. S4).

For the second CTmax session, 14 days after the end of the artificial heatwave, fish CTmax did not significantly differ between treatments, with a mean CTmax value across all treatments of 42.2 °C (Fig. 3 b and Table S3). However, as for the first CTmax session, fish with a better body condition had a significantly higher CTmax ($\chi^2 = 23.1$, $df = 1$, $p = 0.005$, Table S3 and Fig. S4).

## 4. Discussion

This study represents a rare investigation of the influence of multi-generational warming combined with heatwave on CTmax in a semi-natural experiment. Our results indicate no evidence of intergenerational effect on the CTmax of medaka fish after seven years of laboratory rearing at two contrasting temperatures (20 °C and 30 °C). In contrast, rapid plastic adjustments in CTmax were observed following a two-weeks +6 °C heatwave. These effects were transient, as no differences were detected two weeks after the heatwave.

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Boxplots of the critical thermal maximum (CTmax) of medaka fish (*Oryzias latipes*) reared in mesocosms (a) one day after the end of the heatwave and (b) two weeks after the end of the heatwave. Thermal origin: cold fish (in blue), fish from the lineage raised at 20 °C for multiple generations; warm fish (in orange), fish from the lineage raised at 30 °C for multiple generations. Heatwave: N0 (cross-shaped points), mesocosms not heated; YES (dot-shaped points, with thermometer in front of the fish), mesocosms heated at +6 °C during 14 days.</figcaption></figure>
## 4.1. Impacts of multigenerational warming on CTmax

Morla et al. (2025) reported a nearly 5 °C higher CTmax in warm medaka fish compared to cold medaka fish after 7 years of rearing at 30 °C and 20 °C, respectively. However, our findings reveal that after only one generation in common garden, no significant differences in CTmax subsisted between cold and warm fish. This result indicates that laboratory rearing had no or limited intergenerational effects on CTmax, contrary to our first hypothesis. This suggests that seven years of experimental rearing under different temperatures were not sufficient to induce strong intergenerational effects on medaka fish CTmax, or that the temperature difference in our experiment was too low. Nevertheless, acclimation to moderately elevated temperatures and response to acute thermal stress (i.e. CTmax) probably involve different mechanistic pathways that may not evolve together. In other words, CTmax represents a hard thermal limit that may require stronger selective pressures than those imposed in our experiment. However, even under artificial selection for higher thermal tolerance, Morgan et al. (2020) found that changes in CTmax in zebrafish occurred only slowly, highlighting its limited potential for long-term, heritable shifts across generations.

Interestingly, significant differences in body size were observed under common garden conditions, with cold fish being consistently larger than warm fish. This pattern may result from the Temperature Size Rule (TSR), stating that individuals reared at warmer temperatures grow faster at the beginning of their life but ultimately reach a smaller mature body size than individuals reared at cold temperatures (Ahti et al., 2020; Kingsolver and Huey, 2008; Verberk et al., 2021). Furthermore, fish reared at warmer temperatures can hatch prematurely (Cowan et al., 2024), leading to potential smaller size at birth and thus adulthood. Given that warm and cold fish experienced identical temperatures in the final generation, the observed TSR effect may be attributed to transgenerational plasticity, or to genetic selection of traits related to body size. This could suggest that building a strong intergenerational impact on CTmax requires more generations than for traits related to body size, likely due to the lower heritability of physiological traits compared to morphological traits (Mousseau and Roff, 1987). Another explanation could be the existence of close "physiological boundaries" to the increase of heat resistance, called concrete ceiling (Bennett et al., 2021; Sandblom et al., 2016). Finally, our results could be explained by a difference in thermal sensitivity of CTmax and body size. Indeed, traits related to fitness, like growth for example, and ultimately body size, tend to have a critical temperature lower than the CTmax (Ørsted et al., 2022). Thus, the 30 °C rearing condition in our study may not have imposed sufficient pressure to drive intergenerational effects on CTmax, but may have caused heritable phenotypic changes on some fitness-related traits like growth. These findings highlight that heritable shifts of CTmax in medaka fish is likely slow, limited, or requires stronger selective pressures than those imposed in this study. Given these results and evidence from other species (Bennett et al., 2021; Hoffmann et al., 2013), the limited evolutionary potential of upper thermal limits raises concerns about the ability of ectotherms to durably modulate their upper thermal tolerance to cope with the current pace of climate change. However, all species are not equal facing rapid environmental changes. Some species, particularly those with small size and short generation times, demonstrate high ability for evolution of their CTmax, indicating a better potential for evolutionary rescue (Geerts et al., 2015; Munday et al., 2008; D. R. O'Donnell et al., 2018; Schaum et al., 2017, 2022).

## 4.2. Impacts of heatwave on CTmax

Medaka fish exposed to a two-week heatwave showed an average increase in their CTmax of 1.5 °C compared to unexposed individuals. This plastic response was transient, as CTmax differences disappeared within two weeks post-exposure. These results highlight that the medaka fish is able to rapidly modulate its CTmax with acclimation, as we

expected (H2). This may be related to the natural shallow environment of the medaka. Indeed, rapid temperature changes can occur in shallow environments, where little or no thermal refugia are available, thus exerting a selective pressure for enhanced phenotypic plasticity (De Bonville et al., 2025). Rapid plasticity of heat tolerance, after an hour to few weeks of heat exposure, has been reported for many ectotherm species (e.g. De Bonville et al., 2025; Earhart et al., 2023; Einum and Burton, 2022; Waterbury et al., 2024). Thus, acclimation appears to be a widespread mechanism used by ectotherms to deal with increasing temperatures, which could prove crucial in the face of climate change. Indeed, one predominant effect of climate change is the increase in the intensity and frequency of extreme events like heatwaves (Hosseinzadehtalaei et al., 2020; IPCC et al., 2023; Kharin et al., 2018; King and Karoly, 2017). Acclimation may thus be an adaptive mechanism for organisms to mitigate the effects of acute heat events by allowing them to rapidly adjust to abrupt temperature changes and revert to baseline physiological states after the stress subsides, thereby mitigating the long-term costs of thermal stress. However, plastic responses have inherent limits. In our study, a 1 °C increase in environmental temperature corresponded to less than a 1 °C increase in CTmax (indicating that the acclimation response ratio is lower than 1), consistent with results on other ectotherms (De Bonville et al., 2024; Ern et al., 2023; McKenzie et al., 2021; Morley et al., 2019). This phenomenon is likely tied to the "physiological boundaries" of increasing heat resistance, suggesting that the upper thermal tolerance is physiologically limited and cannot increase beyond a certain value, either through genetic evolution or phenotypic plasticity (Bennett et al., 2021; Sandblom et al., 2016). This causes a narrowing of the thermal safety margin with increasing temperatures, with extreme warm temperatures still leading to organisms' deaths, even for species with high plastic abilities. Overall, with plastic abilities of upper thermal tolerance likely to be widespread, projections of future species distribution and vulnerability could benefit from integrating thermal plasticity of species (Bush et al., 2016; Harishchandra et al., 2022; Morley et al., 2019). However, it is worth taking care not to overestimate plasticity ability across temperature ranges.

## 4.3. Costs of enhanced thermal tolerance

Enhanced thermal tolerance, regardless of the mechanism involved, likely incurs costs due to energy allocation constraints (Garland et al., 2022). For example, studies showed that increasing heat tolerance can cause reduced growth or decreased reproductive performance (Donelson et al., 2011; Tüzün and Stoks, 2022; Willi and Van Buskirk, 2022). These costs are ecological debts induced by heat-buffering mechanisms, which can impact the long term dynamics of populations and communities (Martínez-De León and Thakur, 2024). In addition, possible trade-offs between short-term and long-term modulation of the upper thermal tolerance have been reported (Barley et al., 2021; Comte and Olden, 2017; Kellermann and Sgro, 2018). This implies a trade-off between the ability to persist in consistently warmer environments and the capacity to survive abrupt and extreme heat. In this study, we could not test for a trade-off between intergenerational effects and short-term acclimation (H3), as we did not observe evidence for intergenerational effects of our experimental conditions on CTmax. Yet we found that females exhibited a higher CTmax than males. While some studies report no effect of sex on CTmax in fish (e.g., Bartlett et al., 2022; Morgan et al., 2018), others suggest that females have greater thermal tolerance than males (e.g., O'Donnell et al., 2020; Yanar et al., 2023). The underlying mechanisms driving this difference remain unclear but it would be interesting to test if these differences could result from reproductive trade-offs that would differ across sex. In general, studying costs and trade-offs associated with modification of thermal tolerance is crucial, because they can undermine the potentiality that species may buffer the effects of climate change through evolution or acclimation. Thus, a path forward for research on species adaptation to climate change would be to rigorously assess the costs and trade-offs of the
mechanisms underlying enhanced thermal tolerance to improve the accuracy of species climate tolerance estimates and future projections.

## 4.4. Implications for wild medaka populations in the context of climate change

The medaka is widely distributed across East Asia but faces multiple threats, including habitat destruction, reduced connectivity due to modern farming practices, and genetic disturbances from non-native medaka introductions (Iguchi and Kitano, 2008; Nakao et al., 2017; Takehana et al., 2003). A significant population decline in recent decades has led to its classification as vulnerable on the Red List of Threatened Animals of Japan since 1999 (Takehana et al., 2003). While this eurythermal species can be found in temperatures ranging from 0 °C to 40 °C, ongoing climate change poses new challenges to its survival. Heatwaves are becoming more frequent and prolonged and are projected to intensify across East Asia (Ha et al., 2022; Nakano et al., 2013; Seo and Ha, 2022). In particular, the projected increase in hot and dry events (Ha et al., 2022) is concerning for medaka, as its shallow-water habitat may experience exacerbated impacts. Males may be more at risk than females, as females in our study exhibited higher CTmax, which could bias populations' sex ratio on the long term. Moreover, if temperature increase is combined with limited food resources, this could have a synergistic effect further endangering medaka, as we found that individuals with poorer body condition tended to have lower CTmax. Finally, rising temperatures could intensify existing threats, notably by enhancing the spread and competition from invasive species like mosquitofish (Wang et al., 2014). Although medaka exhibits high plasticity in its upper thermal tolerance, allowing some resilience to climate change, extreme thermal events, alone or combined with other anthropogenic pressures, could still endanger wild medaka populations.

## 4.5. Limits and perspectives

A key limitation of this study is the lack of investigation into molecular mechanisms underlying CTmax variation. However, this is a common limit in studies investigating species thermal tolerance. Indeed, exact mechanisms linked to CTmax modulation are not well understood, because the physiological mechanisms causing heat failure are not entirely elucidated (Desforges et al., 2023; Lefevre et al., 2021; McKenzie et al., 2021; Ørsted et al., 2022). Several physiological mechanisms inducing heat failure have been proposed, such as oxygen limitation, changes in protein structure or function, mismatch in the thermal tolerance of crucial biological processes or membrane malfunction (Ern et al., 2023; Hofmann and Todgham, 2010; Ørsted et al., 2022). Many prior studies have investigated these different mechanisms, but none is entirely supported by the literature, with probably a complex interplay of several mechanisms occurring at the same time (Ern et al., 2023).

Additionally, while our design mimicked natural heatwave conditions, repeated CTmax measurements during the heatwave could provide crucial insights into the dynamics and limits of plastic responses, and allow better comparison between cold and warm fish. Indeed, it could allow to measure response induction time, phenotypic response rate and maximum capacity for acclimation (De Bonville et al., 2025; Einum and Burton, 2022), which are key values in the context of thermal fluctuations (Dupont et al., 2024).

Finally, one perspective for CTmax studies could be to examine more closely the different components that define the temperature range that organisms can withstand. The temperature range of organisms can be divided into two ranges: a permissive range where the individual is able to survive and complete its life cycle, and the stressful range where it cannot function properly and will accumulate heat stress until heat failure (Ern et al., 2023; Ørsted et al., 2022). The temperature denaturing these two range is called the critical Temperature (Ørsted et al., 2022). Thus, an increase in CTmax can be due to an increase of the

critical Temperature, or to an increase in the amount of heat stress that can be endured once the critical temperature is exceeded. An increase in the critical temperature may be more beneficial to organisms because it would enlarge the sustainable temperature zone of organisms allowing survival and completion of life cycle. It would thus be interesting for future studies to investigate both the plasticity of the critical temperature and of the CTmax of ectotherms (Ørsted et al., 2022).

## 4.6. Conclusion

Our study demonstrates that medaka fish can rapidly adjust CTmax via acclimation in response to acute heat exposure. However, we did not detect intergenerational effects of the 7 years of rearing at different temperatures on CTmax. This result, along with a consistent body of literature, emphasizes the weak potential for ectotherms to mitigate the impacts of climate change on their physiology through intergenerational modulation of their upper thermal tolerance.

Acclimation offers a more immediate, albeit constrained, mechanism for coping with heat stress, highlighting its critical role in species' short-term responses to climate change. Integrating these plastic responses into vulnerability models will enhance predictions of climate change impacts but requires careful consideration of their limits. Future research should prioritize uncovering the costs and trade-offs of thermal tolerance mechanisms and the molecular basis of CTmax modulation to refine our understanding of species' adaptive capacity.

## CRediT authorship contribution statement

**Juliette Vallin:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Fabio Gerberon:** Visualization, Investigation, Formal analysis, Data curation. **Rémy Lassus:** Project administration, Investigation, Data curation, Conceptualization. **Mathieu Floury:** Writing – review & editing, Supervision. **Anthony Maire:** Writing – review & editing, Supervision, Funding acquisition. **Martin Daufresne:** Writing – review & editing, Supervision. **Arnaud Sentis:** Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization.

## Data statement

All data are available in a public repository, at VALLIN, JULIETTE, 2025, "Medaka fish (Oryzias latipes) critical thermal maximum (CTmax)", https://doi.org/10.57745/JA0XII, Recherche Data Gouv, V1, UNF:6:IO3GcCB++d9MrHyHbWMYvQ== [fileUNF].

## Declaration of generative AI and AI-assisted technologies in the writing process

After drafting and editing the manuscript, the authors used ChatGPT to finalise the editing by rephrasing sentences that were difficult to read. The objective was to improve the language and the readability of the text without adding new information or affecting the meaning of the text. After using ChatGPT, the authors reviewed and edited the content as needed and take full responsibility for the content of the article.

## Funding sources

This work was supported by Électricité de France (EDF) as part of the project "Cascatroph".

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
## Acknowledgements

We thank Simon Bazin, Julie Morla, Ange Molina, Tiphaine Peroux and Virginie Diouloufet for their help with the experiments.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtherbio.2025.104155.

## Data availability

All data are available in a public repository, at VALLIN, JULIETTE, 2025, "Medaka fish (Oryzias latipes) critical thermal maximum (CTmax)", https://doi.org/10.57745/JA0XII, Recherche Data Gouv, V1, UNF:6:IO3GcCB++d9MrHyHbWMYvQ== [fileUNF]).

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