# Effects of duration and type of cold acclimation on the subsequent cold tolerance of a tenebrionid beetle

Samira Khodayari, Stéphane Derocles, David Renault

► To cite this version:

Samira Khodayari, Stéphane Derocles, David Renault. Effects of duration and type of cold acclimation on the subsequent cold tolerance of a tenebrionid beetle. Journal of Thermal Biology, 2025, 129, pp.104100. 10.1016/j.jtherbio.2025.104100. hal-05053921

HAL Id: hal-05053921

https://hal.science/hal-05053921v1

Submitted on 2 May 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>
<figure><img src="image_2.png" /></figure>

# Effects of duration and type of cold acclimation on the subsequent cold tolerance of a tenebrionid beetle

Samira Khodayari<sup>a</sup>, Stéphane A.P. Derocles<sup>b</sup>, David Renault<sup>b,* ID</sup>

## ARTICLE INFO

**Keywords:**

Lesser mealworm

Cold tolerance

Polyols

Alanine

Proline

## ABSTRACT

The effects of cold acclimation on the subsequent survival and recovery of cold-exposed adults of *Alphitobius diaperinus* (Coleoptera: Tenebrionidae) was examined. Acclimation conditions were: 1, 2, 4 and 8 days at 5, 10, 15, 25 °C and fluctuating temperatures of 5–10-15 °C (8:8:8 h). Acute and chronic cold stresses were induced by keeping beetles at –8 °C for 8 h and 0 °C for 2, 3, 4, 5 and 6 days respectively. Survival was recorded after 5 days of recovery at 25 °C. The insects acclimated to 5, 10, 15 and 25 °C for 1 and 4 days were selected for metabolic response studies which was performed by gas chromatography-mass spectrophotometry (GC-MS). Beetles acclimated at 25 °C (control) and beetles acclimated at 5 °C for 1 or 8 days did not survive after acute cold stress. Survival increased to 100 % after acute cold stress in beetles acclimated to fluctuating temperatures for 2 days. The lethal times for 50 % of the population (Lt50) were calculated for beetles after chronic cold stress and range from 4.89 days in beetles acclimated to fluctuating temperatures for 4 days–0.018 days in beetles acclimated to 5 °C for 8 days. The metabolic profile analysis showed that proline exhibited the highest concentration in all experimental groups. Increases of alanine and glycine in cold acclimated beetles were observed. Glutamic acid had opposite pattern and decreased significantly in acclimated beetles at 5 °C for 4 days. Alanine accumulation and glutamic acid decrease suggest that alanine has derived from transamination of glutamic acid and pyruvate. Erythritol, sorbitol, arabitol, adonitol, xylitol, glucose and maltose were the polyols and sugars that have been accumulated in cold acclimated beetles especially at 5 °C. Mannose has only accumulated in 1d acclimated beetles.

## 1. Introduction

In ectothermic animals, prolonged cold or heat exposures, thermal variations and cold shocks can affect numerous biological and physiological processes (Teets and Denlinger, 2013; Daly et al., 2024). The extent of the thermal stress intensity, which is a combination of temperature intensity and duration of exposure, may temporarily paralyze insects (chill coma, Findsen et al., 2014), thus preventing nutrition and escape from predators. Prolonged exposure to thermal stress also increases the risk of temperature-derived injuries with cascading consequences on insect lifespan and population dynamics (Chown and Terblanche, 2006; Colinet et al., 2011).

As there are predictable cues that insects can rely on in order to anticipate the start of wintertime, they have developed remarkable abilities to initiate behavioral and physiological changes that make them ready for overwintering (West and Wood, 2018). The capacity to

acclimate to the cold and develop cold-hardiness can be rapid, i.e. rapid cold-hardening corresponding to enhanced cold-hardiness after relatively short exposure (some few minutes or hours) to stressful cold temperatures. When adjustments take longer periods, they are described as acclimation which corresponds to enhanced cold-hardiness after prolonged exposure (several days or weeks) to novel thermal environments that are within the viable limits of the species (Sinclair and Roberts, 2005; Rako and Hoffmann, 2006; Renault, 2011; Teets et al., 2012). Both responses were demonstrated to have beneficial effects for the survival of cold-exposed insects (Hemmati et al., 2014; Tarapacki et al., 2021; Malekera et al., 2022). However, there are fewer studies which focused on the importance of the duration of acclimation on the subsequent ability of insects to handle low temperature exposure (but see Waagner et al., 2013; Tarapacki et al., 2021).

Thermal fluctuations within permissive temperatures can also enhance insects' survival at low temperatures by (i) preventing the

<footnote><sup>a</sup> Department of Plant Protection, Faculty of Agriculture, University of Maragheh, P.O.Box: 55181-83111, Maragheh, Iran</footnote>

<footnote><sup>b</sup> UMR CNRS 6553 ECOBIO [(Ecosystèmes, biodiversité, évolution)], Université Rennes, avenue Général Leclerc, 35042, Rennes cedex, France</footnote>

<footnote>* Corresponding author. University of Rennes, UMR CNRS 6553 ECOBIO, 263 avenue Général Leclerc, 35042, Rennes cedex, France.</footnote>

<footnote>E-mail addresses: khodayari@maragheh.ac.ir (S. Khodayari), stephane.derocles@univ-rennes.fr (S.A.P. Derocles), david.renault@univ-rennes.fr (D. Renault).</footnote>
formation of chill injuries, (ii) allowing the repair of chill injuries when temperatures are more favorable, and (iii) permitting hardening (Colinet et al., 2015; Lalouette et al., 2011). In most species tested to date, the survival probability was considerably improved when insects were maintained under fluctuating thermal regimes in comparison to their relatives maintained at constant low temperatures (Renault et al., 2004; Kostál et al., 2007; Lalouette et al., 2007, 2011; Colinet et al., 2011).

Cold acclimation and cold hardening generally improve both ionic and metabolic homeostasis of cold-exposed insects (Colinet et al., 2011; Overgaard and MacMillan, 2017; Enriquez et al., 2018). Several metabolic changes, in particular accumulation of sugars, polyols and amino acids, were repeatedly measured in cold-acclimated insects and likely contribute to enhanced cold hardiness (Renault et al., 2004; MacMillan et al., 2016). For instance, longer duration of cold-acclimation improved cold tolerance of the fruit fly *Drosophila melanogaster*, and increased survival rates at low temperature were accompanied with changes in the metabolomic profiles (Kostál et al., 2011b). In particular, proline and trehalose, which act as membrane and protein protectants, were accumulated in the flies (Kostál et al., 2011b). In other studies, accumulation of several amino acids, like proline, glycine, alanine and leucine were identified as cold stress responses (Storey and Storey, 1992; MacMillan et al., 2016). Importantly, most of these metabolites are essential intermediates for several metabolic pathways (for instance gluconeogenesis, synthesis and degradation of proteins) and also play important roles in setting responses to oxidative stress induced by cold exposure (Graney and Giesy, 1986; Terblanche, 2014). The activity of several antioxidant enzymes was also found to be upregulated in diapausing (Teets and Denlinger, 2013), cold-stored (Mojib-Haghghadam et al., 2023) and cold-acclimated insects (Grubor-Lajsic et al., 1997), in turn contributing to enhanced total antioxidant capacity.

The number of metabolites having key roles in cold acclimation and energy supply in insects makes metabolomics a valuable tool for examining the physiological underpinnings accompanying the enhancement of cold hardiness in insects (MacMillan et al., 2016). Metabolomics corresponds to the identification and quantification of the numerous metabolites required for maintenance, growth and cellular normal function of living organisms (Snart et al., 2015). Both gene expression and environmental stresses regulate metabolic fluxes meaning that the metabolome can provide a snapshot of the response of an organism to environmental stimuli (Colinet et al., 2012; Jones et al., 2013).

Using the lesser mealworm *Alphitobius diaperinus* (Panzer) (Coleoptera: Tenebrionidae) as a model species, this study examined the effects of the duration of cold acclimation at both constant and fluctuating thermal regimes on the subsequent cold tolerance of the species. In adults of *A. diaperinus*, cold was assumed to become injurious when temperatures fall below 15 °C in non-acclimated insects (Renault et al., 2004); temperatures lower than 8–10 °C, or prolonged cold exposure, force the insects to enter into chill-coma (Renault, 2011). The main objective of the study was to compare the effects of different types of cold acclimation on the survival of adults after they were exposed to acute or chronic cold stress, and their biochemical responses. We hypothesized that longer duration of cold acclimation would improve cold tolerance more significantly, and would modify the metabolic fingerprints and the total antioxidant capacity of the insects. We were also expecting that cold acclimation under fluctuating thermal regimes would improve survival to broader range of cold stress conditions.

# 2. Materials and methods

## 2.1. Insect rearing conditions

Individuals of the tenebrionid beetle *A. diaperinus* were hand-collected from two poultry houses at Taupont (N 47° 57' 27", W 2° 26' 15", France). Several thousand adults were then grown under controlled

conditions (25 ± 1 °C, 75 ± 5 % Relative Humidity, Light/Darkness: 14 h/10 h) in large plastic boxes containing bran and dry dog food *ad libitum*. Insects were supplied with tap water in 1.5 mL microtubes closed by cotton. The first adult generation that emerged from this rearing was then used for the below-described experiments. All adult insects were approximately 3-month-old before they were used for the experiments.

## 2.2. Cold acclimation and low temperature survival

### 2.2.1. Cold acclimation procedure

To examine the effects of cold acclimation on the subsequent survival ability of adult *A. diaperinus*, groups of 10 beetles were transferred into Petri dishes whose bottom was covered by a piece of filter paper and containing a 1.5 mL microtube filled with tap water and closed with cotton. A total of 240 Petri dishes was prepared. Then, 48 Petri dishes were placed into incubators (Sanyo MIR 153) at one out of three constant acclimation temperatures of 5, 10 and 15 °C, or at the fluctuating thermal regime oscillating between 5 and 15 °C on a 24 h cycle (5 °C: from 12.00 a.m. to 6.00 a.m.; 10 °C: from 6 a.m. to 12.00 p.m.; 15 °C: from 12.00 p.m. to 6.00 p.m.; 10 °C: from 6 p.m. to 12.00 a.m.) (Supplementary material 1). The beetles were transferred from the rearing condition at each of the four acclimation conditions at 12.00 p. m., and acclimated for 1, 2, 4 or 8 days (N = 12 Petri dishes for each acclimation temperature x acclimation time combination). Control insects (N = 48 Petri dishes) were maintained at constant 25 °C during the whole period (8 days). The effects of changes in daylength on cold acclimation was not considered in the study and the photoperiod of the rearing condition (14 h/10 h, Light/Darkness) was used. The beetles were collected at 12 p.m. at the end of the cold acclimation period, i.e. after 1, 2, 4 or 8 days of cold acclimation, and were directly used for assessments of their responses to acute or chronic cold stress, as well as for the evaluation of their physiological responses. During acclimation, the beetles were deprived from food but supplied with drinkable water.

### 2.2.2. Survival after acute and chronic cold stress

The survival after acute cold stress was measured in acclimated and non-acclimated (control) adult *A. diaperinus*. Four Petri dishes from each acclimation treatment were transferred at -8 °C for 8 h. After this 8 h-period, the beetles were returned at 25 °C. The recovery (or absence of recovery and death) from cold exposure may take hours to days; survival was thus scored 1 day after the insects were removed from the acute cold stress, and re-assessed 2 and 5d after the end of the cold treatment, except for insects acclimated under fluctuating thermal regimes whose survival was scored daily until the fifth day (Supplementary material 1). This procedure allowed us to test for the significance of recovery time (from 1 to 5d) in the survival models. When examined for survival, the beetles that exhibited locomotor activity or limb movement only (i.e., insects which were in chill coma) were considered as alive.

The survival of adult *A. diaperinus* to chronic cold stress was determined in acclimated and non-acclimated beetles which were exposed at constant 0 °C. For each acclimation treatment, five Petri dishes were used, and one Petri dish was removed at daily intervals from the second day to the sixth day of exposure, and returned to 25 °C. The survival was scored 1, 2, 3, 5 and 7d after the end of the cold exposure (Supplementary material 1), and was performed as described for the assessment of survival after the exposure to acute cold stress.

## 2.3. Physiological responses

The effects of the acclimation procedure (acclimation temperature, acclimation time) were examined by measuring the changes in fresh mass, total body metabolites and total antioxidant activity in adult *A. diaperinus* from eight out of the 20 acclimation treatments (five acclimation temperatures, 4 acclimation times). We focused on the beetles acclimated for one and 4 days at the constant temperatures. The beetles acclimated for 8d were not considered, as insects acclimated at
5 °C were immobile and not responding to mechanical stimuli. It was thus not possible to determine if they were in chill coma or if some of them were dead at the 5 °C-acclimation temperature.

### 2.3.1. Preparation of the samples for metabolic fingerprinting

Pools of three beetles were directly snap-frozen at the end of the acclimation period (N = 5 replicates per acclimation condition). Each batch of three beetles was then weighed (fresh mass, Sartorius microbalance, d = 0.01 mg), and stored at -20 °C before being used for the physiological measures.

The samples were prepared as described by Khodayari et al. (2013) with the following minor modifications. A volume of 300 µL of ice-cold methanol-chloroform solution (ratio 2:1, v:v) was added to all samples which were further crushed at 25 Hz for 90 s with a tungsten-bead beating apparatus (RetschTM MM301, Retsch GmbH, Haan, Germany). Ice-cold ultrapure water (200 µL) was added to each sample which was then vortexed and centrifuged (4000 g, 5 min, 4 °C). Afterwards, the upper aqueous phase (300 µL) of each sample was pipetted, put into new chromatographic glass vials, and vacuum-dried (Speed Vac Concentrator, MiVac, Genevac Ltd., Ipswitch, England). Methoximation was conducted by adding 30 µL of 20 mg. L⁻¹ methoxyamine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in pyridine to each glass vial before sylillation was realized by the addition of 30 µL of N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA; Sigma, #394866). The chemical preparation of the samples was performed under orbital shaking at 40 °C and lasted 135 min (methoximation for 90 min followed by sylillation for 45 min). The preparation of the samples was realized with a CTC CombiPal autosampler (CTC Analytics AG, Zwingen, Switzerland). This autosampler standardized the sample preparation steps, and their duration, prior injection to the gas chromatography.

### 2.3.2. GC-MS analyses

The GC-MS system and settings described by Khodayari et al. (2013) were used. The running order of the samples was completely randomized. Annotation of the detected peaks was based on mass spectra (two specific ions) and retention time, and quantification was obtained by the construction of calibration curves for each detected metabolite (58 pure reference metabolites with concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 750, 1000, 1500 and 2000 µM). The analysis was performed with XCalibur v2.0.7 software (Thermo Fischer Scientific Inc, Waltham, MA, USA).

### 2.3.3. Total antioxidant capacity

The total antioxidant capacity was determined in beetles acclimated at constant temperatures for one and four days, and was assessed using the antioxidant assay kit (#709001, Cayman chemical, USA) following the manufacturer's instructions. Briefly, the pools of three beetles composing each replicate were crushed in an ice-cold phosphate buffer (20 % (w/v), 5 mM potassium phosphate, 0.9 % sodium chloride, 0.1 % glucose, pH 7.4) before being centrifuged (15,000 g, 15 min, 4 °C). The supernatant was collected and diluted in the phosphate buffer (1/100). This diluted supernatant was then used for the analysis of the total antioxidant activity. The antioxidant capacity was assessed using 10 µL of the solution to inhibit the oxidation of ABTS® (2,2'-Azino-di-[3-ethylbenzthiazoline sulfonate]) to ABTS+®. The absorbance of the samples was measured at 750 nm (measurement of the quantity of ABTS+®), and the total antioxidant capacity was determined (expressed in mM Trolox equivalents) by running trolox standards.

## 2.4. Statistical analysis

The effects of acclimation temperature (qualitative: control, fluctuating, constant 5 °C, 10 °C, and 15 °C), recovery time (quantitative: 1, 2, and 5 days), acclimation time (quantitative: 1, 2, 4 and 8 days) and the two following interactions 1) between the acclimation temperature and the recovery time and 2) between the acclimation temperature and the

acclimation time on the probability of survival to an acute stress were tested with a generalized linear model (quasibinomial family). The effects of acclimation temperature (qualitative: control, fluctuating, constant 5 °C, 10 °C, and 15 °C), recovery time (quantitative: 1, 2, 3, 5 and 7 days), acclimation time (quantitative: 1, 2, 4 and 8 days), exposure time (quantitative: 2, 3, 4, 5, 6 days) and the three following interactions 1) between the acclimation temperature and the recovery time, 2) between the acclimation temperature and the acclimation time, and 3) between acclimation temperature and the exposure time on the probability of survival to a chronic stress were tested with a generalized linear model (quasibinomial family) performed with R (R Core Team, 2022). When a significant effect of the temperatures of acclimation was detected on the probability of survival, a pairwise comparison was performed with the Estimated Marginal Means (package emmeans).

For metabolomics, compounds exceeding 80 % correlation with other compounds were discarded from the analysis to avoid redundancy and overestimation in differences among groups during the analyses. Nineteen metabolites were thus discarded, and the 29 remaining compounds significantly accounted for the separation of the eight experimental groups. Group separation by the 29 discriminant variables was subsequently investigated in the Linear Discriminant Analysis (LDA). The main objective of this statistical procedure was to separate the experimental groups as a function of the discriminant compounds, and rejected the non-discriminating metabolites. The statistical significance of the LDAs was checked by permutation tests (10,000 permutations). Finally, MANOVA analysis was also conducted with duration (day), temperature, and interaction of these two terms as factors. These analyses were conducted with R™ 2.11.0 statistical software (R Development Core Team, 2024).

# 3. Results

## 3.1. Survival after acute and chronic cold stress

The acclimation temperature significantly influenced the survival probability of adult *A. diaperinus* after they were exposed to acute (GLM, F(4,253) = 80.4, p < 0.001; Fig. 1A) or chronic (GLM, F(4,567) = 34.56, p < 0.001; Fig. 1B) cold stress.

Survival probability after acute stress was significantly affected by the recovery and the acclimation times (GLM, respectively F(1,253) = 33.55, p < 0.001; F(1,253) = 19.3, p < 0.001; Fig. 2A and B). The interactions 'acclimation temperature x recovery time' and 'acclimation temperature x acclimation time' significantly affected the survival probability to acute cold stress (GLM, respectively F(4,253) = 6.29, p < 0.001; F(4,253) = 14.52, p < 0.001).

The recovery time, acclimation time and duration of exposure significantly affected the survival probability of adult *A. diaperinus* after they were exposed to chronic cold stress (GLM, respectively F(1,567) = 30.48, <0.001; F(1,567) = 44.62, p < 0.001; F(1,567) = 75.69, p < 0.001; Fig. 3A, B, 3C). The interactions 'acclimation temperature x acclimation time' and 'acclimation temperature x duration of exposure' also affected significantly the survival probability after to chronic cold stress exposure (GLM, respectively F(4,567) = 4.7, p < 0.001; F(4,567) = 3.71, p = 0.005). Finally, the interactions between the acclimation temperature and the recovery time did not affect the probability of survival after exposure to chronic cold stress (GLM, F(4,567) = 1.33, p = 0.026).

## 3.2. Metabolic responses to cold acclimation

Fresh mass of acclimated and non-acclimated beetles was similar (F = 1.041, p > 0.05). A total of 48 metabolites was identified from the whole body of adults of *A. diaperinus*, of which 16 amino acids, 8 polyols, 10 sugars and phosphorylated sugars, 4 metabolic intermediates from glycolysis, and 10 other compounds (Supplementary Material 2).

Total metabolites and amino acids did not differ among treatments (total metabolites: F<sub>7</sub> = 0.44, p = 0.86; total amino acids: F<sub>7</sub> = 1.01, p =
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Boxplots (minimum, 1st quartile, median, 3rd quartile, and maximum) of the number of surviving adults, independently of the recovery, acclimation and exposure times after A) an acute cold stress; B) a chronic cold stress. Letters indicate a significant difference (p < 0.05; GLM, Estimated Marginal Means).</figcaption></figure>

0.43), but physiological differences were detected among the distinct acclimation treatments (Table 1). Alanine, phenyl-alanine and proline were the most abundant amino acids in beetles from all experimental treatments. Alanine and glycine levels were significantly increased in beetles that were acclimated for 4 days at 5 °C, whereas glutamic acid and proline amounts were decreased significantly in these beetles. Valine was the only amino acid that was found in significantly lower amounts in 1d acclimated beetles at 15 °C. The polyols adonitol, arabitol, erythritol, sorbitol and xylitol, and the sugars glucose, fructose, and maltose were accumulated in acclimated beetles, in particular at 5 °C. Mannose accumulated in 1d acclimated beetles at all temperatures.

Class separation in the LDA was achieved using the 29 discriminant variables (Fig. 4). The first (LD1) and second (LD2) discriminant axes accounted for 76.4 and 20 % of the total inertia, respectively. The between-group inertia was 198.9 and 40.52 times higher than the within-group inertia, respectively. A clear separation among the groups was observed on LD1, which was explained by the acclimation temperature. The metabolites that contributed the most to this separation were adonitol, arabitol, alanine, glycine, xylitol, cadaverine and fructose (Fig. 4), which were measured in higher amounts in the 5 °C-acclimated beetles. The groups were secondarily separated on LD2 (Fig. 4) according to acclimation time. LD2 was characterized by the variations of glutamic acid, fumaric acid, isoleucine and threonine. A heatmap diagram showed that the metabolic signatures of the non-acclimated beetles (control) highly differed from the one of acclimated insects, with some metabolites that were upregulated, including gluconolactone and proline, whereas others were downregulated such as adonitol, arabitol and sorbitol (Fig. 5).

### 3.3. Total antioxidant activity

Total antioxidant capacity was significantly affected by the acclimation temperature ($F_{(3,32)} = 9.53, p < 0.001$), but not by the acclimation time ($F_{(1,32)} = 0.524, p = 0.474$). In addition, a significant interaction effect was observed between temperature and acclimation time ($F_{(3,32)} = 4.73, p < 0.01$). Acclimation at 10 °C for 1 and 4 days resulted in the highest total antioxidant activity compared with 15 and 25 °C. A 4d-acclimation period at 5 °C resulted in a similar increase in antioxidant activity (Fig. 6).

## 4. Discussion

So far, the acclimation kinetics over moderately short periods under constant or fluctuating thermal regimes has received less attention than the effects of short and long-term (seasonal) acclimation periods (but see Turriago et al., 2023). In the present work, we aimed at completing this knowledge gap by investigating the importance of the type of acclimation procedure (constant, fluctuating temperatures) and acclimation time on the subsequent survival probability of cold exposed insects. Adults of *A. diaperinus*, whose thermal biology is well-known (Salin et al., 2000; Renault et al., 1999, 2002, 2003, 2004, 2006, 2012), were acclimated at different temperature regimes before they were exposed to acute or chronic cold stress. To get a deeper understanding of the physiological rearrangements accompanying changes in thermal tolerance, metabolite fingerprinting and assessment of total antioxidant capacity were also measured.

Cold-acclimation significantly increased the cold tolerance of adult *A. diaperinus* which were thus better able to recover from low
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Probability of survival to an acute cold stress A) according to the recovery time and to the acclimation temperature independently of the acclimation time; B) according to the acclimation time and to the acclimation temperature independently of the recovery time. All data points are represented. Lines indicate the models (GLM, quasibinomial family) with the confidence intervals.</figcaption></figure>

temperature exposures in comparison to non-acclimated insects. The beneficial effect provided by cold-acclimation was more evident for insects exposed to acute cold stress, with little mortality being measured in beetles acclimated at constant 10 or 15 °C, or under fluctuating temperatures. A two-day acclimation period at 10 and 15 °C highly significantly increased survival probability in comparison to control (non-acclimated) beetles. This result is consistent with a former study conducted on the species and showing that chill coma temperature was reduced by about 3 °C after a short-term acclimation period, and remained unchanged when the beetles were acclimated for more than 2 days at 10 and 15 °C (Renault et al., 2012). Similarly, the survival probability of adult *A. diaperinus* whose acclimation duration at 15 °C was prolonged over two days before being exposed at - 8 °C was not further improved. This finding suggests that the 15 °C acclimation temperature may have not been cold enough for eliciting additional mechanisms that would have allowed further development of cold tolerance. Conversely, increased acclimation time (from 0 to 8 d) at 10 °C slightly but continuously improved cold tolerance. The enhancement of cold tolerance relies on several physiological, biochemical and molecular changes, including synthesis of compatible solutes, modifications of membrane composition, changes in the proteome and transcriptome (Teets and Denlinger, 2013; MacMillan et al., 2016), and this internal reorganization likely requires few days. In the spotted wing drosophila *Drosophila suzukii* (Matsumura, 1931), Enriquez and Colinet, 2019 reported that maximum cold tolerance was acquired after six days of acclimation, and was associated with several transcriptional changes in the expression of genes coding for neurotransmitter receptors and genes important for ion transport mechanisms. In a springtail species,

Kuyucu and Chown (2021) found that the critical thermal minimum stabilized after an acclimation period of seven days. Altogether, this information confirmed that both acclimation time and acclimation temperature are key drivers of cold tolerance acquisition in insects.

With the exception of beetles acclimated at fluctuating temperatures, the beneficial effect of cold acclimation was more limited for insects exposed to chronic cold stress. Acclimation at mild and moderate (10 and 15 °C) temperatures may have been progressively detrimental to the beetles, thus explaining the decreased survival probability when acclimation time was increased. In this species, reproduction stops when temperature falls below 15 °C (Rueda and Axtell, 1996). Prolonged exposures of several days at 10 and 15 °C may have disrupted physiological functions (Overgaard and MacMillan, 2017) and may have caused an accumulation of indirect chilling injury causing metabolic disorder, disturbance of ion homeostasis and oxidative stress (Kostál et al., 2007). The increased antioxidant activity measured in adult *A. diaperinus* acclimated at 5, 10 or 15 °C for four days is consistent with this assumption. Damages resulting from oxidative stress may have occurred if the capacity of the antioxidant system to scavenge Reactive Oxygen Species (ROS) was surpassed (Joanisse and Storey, 1996; Lalouette et al., 2011). In addition, the aerobic scope of non-feeding beetles was highly reduced at 15 and 10 °C (Renault et al., 2003), thus limiting energy production that is required for the restoration of physiological functions. As a consequence, physiological damages may have accumulated and extended in the insects' body, placing the insects at higher death risks.

In both acute and chronic cold stress assays, thermal acclimation at 5 °C had the lowest beneficial effect on the subsequent survival ability of the beetles. Survival rapidly fell below 20 % when insects were
<figure><img src="image_5.png" /><figcaption>Fig. 3. Probability of survival to a chronic cold stress A) according to the recovery time independently of the acclimation temperature, the acclimation time and the exposure time; B) according to the acclimation time and to the acclimation temperature independently of the recovery and exposure times; C) according to the exposure time and to the acclimation temperature independently of the recovery and acclimation times. All data points are represented. Lines indicate the models (GLM, quasibinomial family with the confidence intervals.</figcaption></figure>

<table><caption>**Table 1**

Results of the MANOVA model testing for the effects of acclimation temperature, acclimation time and their interaction on the metabolic profiles of adult *Alphitobius diaperinus*.</caption><thead><tr><td>Effect</td><td>F<sub>df</sub></td><td>Pillai</td><td>P-value</td></tr></thead><tbody><tr><td>Acclimation time</td><td>59.64<sub>29</sub></td><td>0.99942</td><td>0.10</td></tr><tr><td>Temperature</td><td>4.44<sub>87</sub></td><td>2.9317</td><td>0.009**</td></tr><tr><td>Acclimation time:Temperature</td><td>1.26<sub>87</sub></td><td>2.7729</td><td>0.37</td></tr></tbody></table>

acclimated for more than 2 days at 5 °C before being subjected to chronic or acute cold stress. At 5 °C, the insects quickly became motionless and fell into chill coma (Renault et al., 1999). Prolonged acclimation periods at 5 °C may have induced lethal physiological damages to the insects, in line with former studies which reported that lethal times for 50 % of the population ranged from ca. 8–11 days at 5 °C (Renault et al., 2004, 2011; Colinet et al., 2011). Acute direct chilling injury (i.e. protein denaturation and membrane lipid phase transition, Kostál et al., 2011a) generated by the transfer of the insects to the cold temperature likely caused physiological disorders reaching an extent that acclimation mechanisms cannot counteract.

Among the different tested acclimation regimes, thermal fluctuations had the greatest beneficial effect on survival probability of insects after they were exposed to acute or chronic cold stress. This finding is consistent with the available literature, with several studies reporting the beneficial effect of fluctuating thermal regimes for insects' survival (Renault et al., 2004; Colinet et al., 2006, 2011, 2015, 2016; Lalouette

et al., 2011). In general, less chill injuries accumulate in insects exposed to fluctuating thermal regimes as the bouts at warmer temperatures allow the restoration of physiological homeostasis (Kostál et al., 2007), and physiological changes such as alteration of membrane lipid composition (van Dooremalen et al., 2011) or upregulation of molecular chaperones (Arias et al., 2011) that are not otherwise possible at constant low temperatures. In the chill susceptible insects *Pyrrhocoris apterus* (Linnaeus, 1758) and *A. diaperinus*, it has been demonstrated that ion balance could be restored during daily pulses at the warmer temperature, which likely contributed to improve the survival of the insects (Kostál et al., 2007).

Mortality of the beetles was higher when the duration of recovery was increased, thus revealing the progression of chill injuries. At low temperatures, insects may enter a state of complete, yet reversible, neuromuscular paralysis defined as chill coma (Bale, 1996; Sinclair, 1999; Nedvěd, 2000). Here, the survival of the beetles decreased with the increased recovery time, thus revealing that chill injuries were not repaired and that latent chill injuries have occurred after the cold stress. This result highlights the importance of monitoring post stress survival over several consecutive days, with chill coma recovery time being a valuable metric of cold survival ability (Knapp et al., 2018). For example, Moraiti et al. (2022) used chill coma recovery time for assessing the existence of evolutionary changes in chill tolerance in *Ceratitis capitata* (Wiedemann, 1824) populations from Middle East to Central Europe.

In insects, physiological changes, in particular accumulation of polylols and sugars, were often reported in individuals exposed to a range of
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Linear discriminant analysis (LDA) on metabolomic data: sample projection onto the first LDA plane of adult *Alphitobius diaperinus* acclimated for one or 4 days at 5, 10, 15 or 25 (control) °C. The correlation circle reports the metabolite contribution to the separation of the groups.</figcaption></figure>

environmental stress (Issartel et al., 2005; Watanabe, 2006; Yoder et al., 2006; Mamai et al., 2014; Colinet et al., 2015). Cold acclimation often correlates with changes in hemolymph composition, with many amino acids (Hanzal and Jegorov, 1991; Lalouette et al., 2007) polyols (Enriquez et al., 2018) and sugars (Jedal et al., 2024) being accumulated. In the present work, temperature significantly affected the metabolic profiles of adult *A. diaperinus* with several polyols (e.g., adonitol, arabitol, xylitol), sugars (e.g. fructose, galactose, glucose and maltose) and amino acids (e.g., alanine, glycine, leucine, isoleucine, valine) exhibiting increased amounts in the beetles acclimated for one and 4 days at 5 °C, and 1 day at 10 °C. The alteration of the concentration of a wide number of metabolites suggested that cold exposure provoked large-scale physiological disruptions, in line with the high mortality we observed.

Higher amounts of proline and glutamic acid contributed to the separation of control and acclimated insects; alanine was also characterized by lower concentrations in control insects. In a former work, Renault et al. (2006) found higher amounts of alanine in adults of *A. diaperinus* exposed for 4 weeks at 10 °C, and concomitant decrease of proline levels. Increased levels of alanine found for one and four days acclimated beetles at 5 °C, and one day acclimated insects at 10 °C may have resulted from the transamination of glutamic acid and pyruvate. Alanine accumulation could have contributed to inhibit oxidative damage, prevent glycation of proteins, and buffer intracellular pH (Williams et al., 2014). Glycine also accumulated in cold acclimated beetles. Interestingly, glycine and alanine increase has been reported in ground beetles exposed to hypersaline conditions by Hidalgo et al. (2013), and glycine was assumed to have a protective role for macromolecules and enzymes.

In a previous study, it has been found that short acclimation periods (≤3 days) did not alter cold survival of adults of the tenebrionid beetle (Renault, 2011), probably because acclimation was not long enough to elicit beneficial physiological and molecular responses. In line with this observation, we found that some metabolites, in particular the polyols adonitol, arabitol, glycerol, sorbitol, and the sugars glucose and maltose, were measured in higher amounts in insects acclimated for four days in comparison to those acclimated for one day. Even if metabolomic profiles only provided correlative evidence of cold-induced physiological changes, this result suggested that the series of physiological and biochemical changes elicited during cold acclimated required few days. During cold-exposure, down-regulation of metabolic activities and alteration of energy production can be visualized by reductions of the amounts of several tricarboxylic acid (TCA) cycle intermediates, and a

larger dependence on glycolytic and gluconeogenic pathways (Colinet et al., 2012; Michaud and Denlinger, 2007; Hayward et al., 2014). As a result, it is difficult to determine the changes in metabolite amounts which are corresponding to cold-acclimation processes from those resulting from temperature-induced modification of TCA cycle activity (Hayward et al., 2014), i.e. if changes are direct protective responses or indirect consequences of cold acclimation and cold exposure.

In this study, we confirmed that both acclimation temperature and acclimation time affected the acquisition of cold tolerance in the lesser mealworm. The mild acclimation temperature (10 °C) and fluctuating thermal regimes were the most beneficial acclimation treatments, allowing the highest survival probability after the insects were exposed to acute or chronic cold stress. In the present work, investigations were performed on a chill susceptible species, making it difficult to distinguish the effects of cold acclimation from those of chilling accumulation in the survival and physiological results. Future studies will have to examine the time course of acclimation in cold-tolerant insect species to improve our understanding of the kinetics of cold acclimation. Finally, acclimation at fluctuating thermal regimes greatly improved survival probability. Future works should examine the effects of different acclimation time at fluctuating temperatures having different amplitudes on the development of cold tolerance.

### CRediT authorship contribution statement

**Samira Khodayari:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Formal analysis, Conceptualization.
**Stéphane A.P. Derocles:** Writing – review & editing, Writing – original draft, Visualization, Formal analysis, Data curation.
**David Renault:** Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Conceptualization.

### Code availability

R scripts from the analyses in this article were developed by ED and are openly available in GitHub at https://github.com/davidrenault/Anatalanta_Heat_stress_and_reproduction.

### Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Heatmap of the entire metabolomics dataset. The color gradient of the heatmap represents the cube root changes of each metabolite relative to the mean control level. Individual samples (horizontal axis) and compounds (vertical axis) are separated using hierarchical clustering (Ward's algorithm), with the dendrogram being scaled to represent the distance between each branch (distance measure: Pearson's correlation). 1d and 4d of acclimation at 5, 10, 15 and 25 °C are presented as 0 (red), 1 (green), 2 (blue), 3 (light blue) (one day), 4 (pink), 5 (yellow), 6 (grey), 7 (black) (4 days) respectively.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 6.** Total antioxidant activity of the adults *Alphitobius diaperinus* acclimated at 5, 10, 15 °C and non-acclimated (25 °C) for one and four days.</figcaption></figure>

Declaration of competing interest

The authors declare no competing interests.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtherbio.2025.104100.

Data availability

The data presented in this study are openly available in GitHub at https://github.com/davidrenault/Anatalanta_Heat_stress_and_reproduction.

References

Arias, M.B., Poupin, M.J., Lardies, M.A., 2011. Plasticity of life-cycle, physiological thermal traits and Hsp70 gene expression in an insect along the ontogeny: effect of temperature variability. J. Therm. Biol. 36, 355–362. https://doi.org/10.1016/j.jtherbio.2011.06.011.
Bale, J.S., 1996. Insect cold hardiness: a matter of life and death. Eur. J. Entomol. 93, 369-382.

Chown, S.L., Terblanche, J.S., 2006. Physiological diversity in insects: ecological and evolutionary contexts. Adv. Insect Physiol. 33, 50-152. https://doi.org/10.1016/S0065-2806(06)33002-0.

Colinet, H., Renault, D., Hance, T., Vernon, P., 2006. The impact of fluctuating thermal regimes on the survival of a cold-exposed parasitic wasp, *Aphidius colemani*. Physiol. Entomol. 31 (3), 234-240. https://doi.org/10.1111/j.1365-3032.2006.00511.x.

Colinet, H., Lalouette, L., Renault, D., 2011. A model for the time-temperature-mortality relationship in the chill-susceptible beetle, *Alphitobius diaperinus*, exposed to fluctuating thermal regimes. J. Therm. Biol. 36 (7), 403-408. https://doi.org/10.1016/j.jtherbio.2011.07.004.

Colinet, H., Larvor, V., Laparie, M., Renault, D., 2012. Exploring the plastic response to cold acclimation through metabolomics. Funct. Ecol. 26, 711-722. https://doi.org/10.1111/j.1365-2435.2012.01985.x.

Colinet, H., Renault, D., Javal, M., Berková, P., Šínek, P., Kostál, V., 2016. Uncovering the benefits of fluctuating thermal regimes on cold tolerance of drosophila flies by combined metabolic and lipidomic approach. Biochim. Biophys. Acta 1861 (11), 1736-1745. https://doi.org/10.1016/j.bbalip.2016.08.008.

Colinet, H., Sinclair, B., Vernon, P., Renault, D., 2015. Insects in fluctuating thermal environments. Annu. Rev. Entomol. 60, 123-140. https://doi.org/10.1146/annurev-ent-010814-021017.

Daly, E., Defourneaux, M., Legrand, C., Renault, D., 2024. The consequences of heatwaves for the reproductive success and physiology of the wingless sub-antarctic fly *Anatalanta aptera*. J. Therm. Biol. 123, 103910. https://doi.org/10.1016/j.jtherbio.2024.103910.

Enriquez, T., Colinet, H., 2019. Cold acclimation triggers major transcriptional changes in *Drosophila suzukii*. BMC Genom. 20, 413. https://doi.org/10.1186/s12864-019-5745-7.

Enriquez, T., Renault, D., Charrier, M., Colinet, H., 2018. Cold acclimation favors metabolic stability in *Drosophila suzukii*. Front. Physiol. 9, 1506. https://doi.org/10.3389/fphys.2018.01506.

Findsen, A., Pedersen, T.H., Petersen, A.G., Nielsen, O.B., Overgaard, J., 2014. Why do insects enter and recover from chill coma? Low temperature and high extracellular potassium compromise muscle function in *Locusta migratoria*. J. Exp. Biol. 217 (8), 1297-1306. https://doi.org/10.1242/jeb.098442.

Graney, R.L., Giesy Jr., J.P., 1986. Effects of long-term exposure to pentachlorophenol on the free amino acid pool and energy reserves of the freshwater amphipod *Gammarus pseudolimnaeus* Bousfield (Crustacea, Amphipoda). Ecotoxicol. Environ. Saf. 12 (3), 233-251. https://doi.org/10.1016/0147-6513(86)90015-1.

Grubor-Lajsic, G., Block, W., Telesmanic, M., Jovanovic, A., Stevanovic, D., Baca, F., 1997. Effect of cold acclimation on the antioxidant defense system of two larval Lepidoptera (Noctuidae). Arch. Insect Biochem. Physiol.: Publ. Collab. Entomol. Society of Am. 36 (1), 1-10. https://doi.org/10.1002/(SICI)1520-6327(1997)36:1<1:1:AID-ARCH1>3.0.C0:2%623.

Hanzal, R., Jegorov, A., 1991. Changes in free amino acid composition in haemolymph of larvae of the wax moth, *Galleria mellonella* L., during cold acclimation. Comp. Biochem. Physiol. A: Physiologist 100 (4), 957-962. https://doi.org/10.1016/0300-9629(91)90322-4.

Hayward, S.A.L., Manso, B., Cossins, A.R., 2014. Molecular basis of chill resistance adaptations in poikilothermic animals. J. Exp. Biol. 217 (1), 6-15. https://doi.org/10.1142/jeb.096537.

Hemmati, C., Moharrampourip, S., Talebi, A.A., 2014. Effects of cold acclimation, cooling rate and heat stress on cold tolerance of the potato tuber moth *Phthorimaea operculella* (Lepidoptera: gelechiidae). Eur. J. Entomol. 111 (4), 487-494. https://doi.org/10.1441/eje.2014.063.

Hidalgo, K., Laparie, M., Bical, R., Larvor, V., Bouchereau, A., Siaussat, D., Renault, D., 2013. Metabolic fingerprinting of the responses to salinity in the invasive ground beetle *Merizodus soledadinus* at the Kerguelen Islands. J. Insect Physiol. 59 (1), 91-100. https://doi.org/10.1016/j.jinsphys.2012.10.017.

Issartel, J., Renault, D., Voituron, Y., Bouchereau, A., Vernon, P., Hervant, F., 2005. Metabolic responses to cold in subterranean crustaceans. J. Exp. Biol. 208 (15), 2923-2929. https://doi.org/10.1242/jeb.01737.

Jedal, J.Y., Malmendal, A., Ramløv, H., 2024. Metabolites, ions, and the mechanisms behind seasonal cold hardening of *Pyrochroa coccinea* (Pyrochroidae) larvae. J. Insect Physiol. 153, 104610. https://doi.org/10.1016/j.jinsphys.2023.104610.

Joanisse, D., Storey, K., 1996. Oxidative stress and antioxidants in overwintering larvae of cold-hardy goldenrod gall insects. J. Exp. Biol. 199 (7), 1483-1491. https://doi.org/10.1142/jeb.199.7.1483.

Jones, O.A.H., Maguire, M.L., Griffin, J.L., Dias, D.A., Spurgeon, D.J., Svendsen, C., 2013. Metabolomics and its use in ecology. Austral Ecol. 38 (6), 713-720. https://doi.org/10.1111/aec.12019.

Khodayari, S., Moharrampourip, S., Larvor, V., Hidalgo, K., Renault, D., 2013. Metabolic changes associated with cold-hardiness in the two-Spotted spider mite: effect of acclimation and diapause. PLoS One 8 (1), e54025.

Knapp, M., Vernon, P., Renault, D., 2018. Studies on chill coma recovery in the ladybird, *Harmonia axyridis* ontogenetic profile, effect of repeated cold exposures, and capacity to predict winter survival. J. Therm. Biol. 74, 275-280. https://doi.org/10.1016/j.jtherbio.2018.04.013.

Kostál, V., Renault, D., Mehrabianova, A., Bastl, J., 2007. Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes: role of ion homeostasis. Comp. Biochem. Physiol. 147, 231-238. https://doi.org/10.1016/j.cbpa.2006.12.033.

Kostál, V., Zahradnícková, H., Šínek, P., 2011a. Hyperprolinemic larvae of the drosophilid fly, *Chymomyza costata*, survive cryopreservation in liquid nitrogen. Proc. Natl. Acad. Sci. USA 108, 13041-13046. https://doi.org/10.1073/pnas.1107060108.

Kostál, V., Korbelova, J., Rozsypal, J., Zahradnícková, H., Cimlova, J., Tomkala, A., Šínek, P., 2011b. Long-term cold acclimation extends survival time at 0°C and modifies the metabolic profiles of the larvae of the fruit fly *Drosophila melanogaster*. PLoS One 6 (9), e25025. https://doi.org/10.1371/journal.pone.0025025.

Kuyucu, A.C., Chown, S.L., 2021. Time course of acclimation of critical thermal limits in two springtail species (Collembola). J. Insect Physiol. 130, 104209. https://doi.org/10.1016/j.jinsphys.2021.104209.

Lalouette, L., Kostál, V., Colinet, H., Gagneul, D., Renault, D., 2007. Cold exposure and associated metabolic changes in adult tropical beetles exposed to fluctuating thermal regimes. FEBS J. 274, 1759-1767. https://doi.org/10.1111/j.1742-4658.2007.01723.x.

Lalouette, L., Williams, C.M., Hervant, F., Sinclair, B.J., Renault, D., 2011. Metabolic rate and oxidative stress in insects exposed to low temperature thermal fluctuations. Comp. Biochem. Physiol. A 158, 229-234. https://doi.org/10.1016/j.cbpa.2010.11.007.

MacMillan, H.A., Knee, J.M., Dennis, A.B., Udaka, H., Marshall, K.E., Merritt, T.J., Sinclair, B.J., 2016. Cold acclimation wholly reorganizes the *Drosophila melanogaster* transcriptome and metabolome. Sci. Rep. 6 (1), 28999. https://doi.org/10.1038/srep28999.

Malekera, M.J., Acharya, R., Mostafiz, M.M., Hwang, H.S., Bhusal, N., Lee, K.Y., 2022. Temperature-dependent development models describing the effects of temperature on the development of the fall armyworm *Spodoptera frugiperda* (JE Smith) (Lepidoptera: noctuidae). Insects 13 (12), 1084. https://doi.org/10.3390/insects13121084.

Mamai, W., Mouline, K., Blais, C., Larvor, V., Dabiré, K.R., Ouedraogo, G.A., Simard, F., Renault, D., 2014. Metabolic and ecotoxicological variations in *Anopheles gambiae* s.l. mosquitoes exposed to the stressful conditions of the dry season in Burkina Faso, West Africa. Physiol. Biochem. Zool. 87 (3), 486-497.

Michaud, M.R., Denlinger, D.L., 2007. Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated coldhardening in flesh flies (Sarcophaga crassipalpis): a metabolic comparison. J. Comp. Physiol. B 177, 753-763. https://doi.org/10.1007/s00360-007-0172-5.

Mojib-Haghighadam, Z., Sendi, J.J., Zibaee, A., Mohaghegh, J., 2023. Effect of cold storage on some biological and physiological performance of *Adalia decempunctata* L. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 263, 110797. https://doi.org/10.1016/j.cbpb.2022.110797.

Moraiti, C.A., Verykouki, E., Papadopoulos, N.T., 2022. Chill coma recovery of *Ceratitis capitata* adults across the Northern hemisphere. Sci. Rep. 12, 17555. https://doi.org/10.1038/s41598-022-21340-y.

Nedvéd, O., 2000. Snow white and the seven dwarfs: a multivariate approach to classification of cold tolerance. Cryo Lett. 21, 339-348.

Overgaard, J., MacMillan, H.A., 2017. The integrative physiology of insect chill tolerance. Annu. Rev. Physiol. 79, 187-208. https://doi.org/10.1146/annurev-physiol-022516-034142.

R Core Team, 2024. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/.

Rako, L., Hoffmann, A.A., 2006. Complexity of the cold acclimation response in *Drosophila melanogaster*. J. Insect Physiol. 52, 94-104. https://doi.org/10.1016/j.jinsphys.2005.09.007.

Renault, D., 2011. Long-term after-effects of cold exposure in adult *Alphitobius diaperinus* (Tenebrionidae): the need to link survival ability with subsequent reproductive success. Ecol. Entomol. 36, 36-42. https://doi.org/10.1111/j.1365-2311.2010.01243.x.

Renault, D., Bijou, A., Hervant, F., 2012. Impact of different acclimation temperatures and duration on the chill coma temperature and oxygen consumption in the tenebrionid beetle *Alphitobius diaperinus*. Physiol. Entomol. 37 (4), 354-359. https://doi.org/10.1111/j.1365-3032.2012.00851.x.

Renault, D., Bouchereau, A., Delettre, Y.R., Hervant, F., Vernon, P., 2006. Changes in free amino acids in *Alphitobius diaperinus* (Coleoptera: Tenebrionidae) during thermal and food stress. Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 143 (3), 279-285. https://doi.org/10.1016/j.cbpa.2005.11.012.

Renault, D., Hance, T., Vannier, G., Vernon, P., 2003. Is body size an influential parameter in determining the duration of survival at low temperatures in *Alphitobius diaperinus* Panzer (Coleoptera: Tenebrionidae)? J. Zool. 259 (4), 381-388. https://doi.org/10.1017/S0952836902003382.

Renault, D., Hervant, F., Vernon, P., 2002. Comparative study of the metabolic responses during food shortage and subsequent recovery at different temperatures in the adult lesser mealworm, *Alphitobius diaperinus* (Coleoptera: Tenebrionidae). Physiol. Entomol. 27 (4), 291-301. https://doi.org/10.1046/j.1365-3032.2002.00299.x.

Renault, D., Nedved, O., Hervant, F., Vernon, P., 2004. The importance of fluctuating thermal regimes for repairing chill injuries in the tropical beetle *Alphitobius diaperinus* (Coleoptera: Tenebrionidae) during exposure to low temperature. Physiol. Entomol. 29 (2), 139-145. https://doi.org/10.1111/j.0307-6962.2004.00377.x.

Renault, D., Salin, C., Vannier, G., Vernon, P., 1999. Survival and chill-coma in the adult lesser mealworm, *Alphitobius diaperinus* (Coleoptera: Tenebrionidae), exposed to low temperatures. J. Therm. Biol. 24 (4), 229-236. https://doi.org/10.1016/S0306-4565(99)00015-7.

Rueda, L.M., Axtell, R.C., 1996. Temperature-dependent development and survival of the lesser mealworm, *Alphitobius diaperinus*. Med. Vet. Entomol. 10 (1), 80-86. https://doi.org/10.1111/j.1365-2915.1996.tb00085.x.

Salin, C., Renault, D., Vannier, G., Vernon, P., 2000. A sexually dimorphic response in supercooling temperature, enhanced by starvation, in the lesser mealworm *Alphitobius diaperinus* (Coleoptera: Tenebrionidae). J. Therm. Biol. 25 (6), 411-418. https://doi.org/10.1016/S0306-4565(00)00003-6.
Sinclair, B.J., 1999. Insect cold tolerance: how many kinds of frozen? Eur. J. Entomol. 96, 157-164.

Sinclair, B.J., Roberts, S.P., 2005. Acclimation, shock and hardening. J. Therm. Biol. 30, 557-562. https://doi.org/10.1016/j.jtherbio.2004.09.001.

Snart, C.J., Hardy, I.C., Barrett, D.A., 2015. Entometabolomics: applications of modern analytical techniques to insect studies. Entomol. Exp. Appl. 155 (1), 1-17. https://doi.org/10.1111/eea.12281.

Storey, K.B., Storey, J.M., 1992. Biochemical adaptations for winter survival in insects. In: Steponkus, P.L. (Ed.), Advances in Low Temperature Biology, vol. 1. JAI Press, U. K, pp. 101-140.

Tarapacki, P.N.I., Jorgensen, L.B., Sorensen, J.G., Andersen, M.K., Colinet, H., Overgaard, J., 2021. Acclimation, duration and intensity of cold exposure determine the rate of cold stress accumulation and mortality in *Drosophila suzukii*. *J. Insect Physiol.* 135, 104323. https://doi.org/10.1016/j.jinsphys.2021.104323.

Teets, N.M., Peyton, J.T., Ragland, G.J., Colinet, H., Renault, D., Hahn, D.A., Denlinger, D., 2012. Combined transcriptomic and metabolomic approach uncover molecular mechanisms of cold tolerance in a temperate flesh fly. Physiol. Genom. 44, 764-777. https://doi.org/10.1152/physiolgenomics.00042.2012.

Teets, N.M., Denlinger, D.L., 2013. Physiological mechanisms of seasonal and rapid cold-hardening in insects. Physiol. Entomol. 38 (2), 105-116. https://doi.org/10.1242/jeb.203448.

Terblanche, J.S., 2014. Physiological performance of field-released insects. Curr. Opin. Insect Sci. 4, 60-66. https://doi.org/10.1016/j.cois.2014.06.003.

Turriago, J.L., Tejedo, M., Hoyos, J.M., Camacho, A., Bernal, M.H., 2023. The time course of acclimation of critical thermal maxima is modulated by the magnitude of

temperature change and thermal daily fluctuations. J. Therm. Biol. 114, 103545. https://doi.org/10.1016/j.jtherbio.2023.103545.

van Dooremalen, C., Suring, W., Ellers, J., 2011. Fatty acid composition and extreme temperature tolerance following exposure to fluctuating temperatures in a soil arthropod. J. Insect Physiol. 57, 1267-1273. https://doi.org/10.1016/j.jinsphys.2011.05.017.

Waagner, D., Holmstrup, M., Bayley, M., Sørensen, J.G., 2013. Induced cold-tolerance mechanisms depend on duration of acclimation in the chill-sensitive *Folsomia candida* (Collembola). J. Exp. Biol. 216 (11), 1991-2000. https://doi.org/10.1242/jeb.079814.

Watanabe, M., 2006. Anhydrobiosis in invertebrates. Appl. Entomol. Zool. 41, 15-31. https://doi.org/10.1303/aez.2006.15.

West, A.C., Wood, S.H., 2018. Seasonal physiology: making the future a thing of the past. Curr. Opin. Physiol. 5, 1-8. https://doi.org/10.1016/j.cophys.2018.04.006.

Williams, C.M., Watanabe, M., Guarracino, M.R., Ferraro, M.B., Edison, A.S., Morgan, T. J., Boroujerdi, A.F., Hahn, D.A., 2014. Cold adaptation shapes the robustness of metabolic networks in *Drosophila melanogaster*. Evolution 68 (12), 3505-3523. https://doi.org/10.1111/evo.12541.

Yoder, J.A., Benoit, J.B., Denlinger, D.L., Rivers, D.B., 2006. Stress-induced accumulation of glycerol in the flesh fly, *Sarcophaga bullata*: evidence indicating anti-desiccant and cryoprotectant functions of this polyol and a role for the brain in coordinating the response. J. Insect Physiol. 52 (2), 202-214. https://doi.org/10.1016/j.jinsphys.2005.10.005.