Endogenous circannual cycles drive seasonal cold hardening in temperate ants

Endogenous circannual cycles drive seasonal cold hardening in temperate ants | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Endogenous circannual cycles drive seasonal cold hardening in temperate ants View ORCID Profile Quentin Willot , View ORCID Profile Vladimír Koštál , View ORCID Profile Johannes Overgaard doi: https://doi.org/10.1101/2025.07.09.663877 Quentin Willot 1 Research Unit of Environmental and Evolutionary Biology, Institute of Life, Earth & Environment, University of Namur , Namur, Belgium 2 Section for Zoophysiology, Department of Biology, Aarhus University , 8000 Aarhus C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Quentin Willot For correspondence: quentin.willot{at}unamur.be Vladimír Koštál 3 Institute of Entomology, Biology Centre, Czech Academy of Sciences , Branišovská, 1160/31, 37005 České Budějovice, Czech Republic Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Vladimír Koštál Johannes Overgaard 2 Section for Zoophysiology, Department of Biology, Aarhus University , 8000 Aarhus C, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Johannes Overgaard Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract The ability to acquire cold tolerance and cope with seasonality underlies the broad geographical distribution of ectotherms. Among insects, cold-adapted ants, which are successful terrestrial insects in temperate and boreal ecosystems, rely on complex circannual life cycles to survive multiple consecutive winters. Following winter reactivation, colonies enter a fixed developmental phase whose duration is controlled by “Kipyatkov’s sand-glass device”, an endogenous programmed timer that ultimately enforces a new dormancy period for colonies after a set period, even under permissive light or temperature cues. Here, using five temperate ant species, we leveraged this system to tease apart the contribution of intrinsic seasonal programming and extrinsic thermal cues on worker cold tolerance, metabolic rates, and metabolomic profiles. We show that obligate colony-level dormancy alone is sufficient to modulate both the critical thermal minimum (CT min ) and the temperature causing 50% mortality during acute cold exposure (LTe 50 ), and further interacts with cold acclimation to shape worker phenotypic cold tolerance. While colony-level dormancy was only associated with limited metabolomic reorganization, cold acclimation triggered the accumulation of metabolites potentially involved in osmotic balance and membrane reorganization. Our results highlight a circannual cycle of endogenous cold hardening that operates independently of environmental exposure in temperate ants, providing new insight into the physiological adaptations underpinning the evolutionary success of this insect family in highly seasonal climates. Introduction Seasonal temperate climates pose several challenges to ectotherms. Winter is typically associated with reduced energy availability and exposure to potentially lethal cold temperatures 1 . In insects facing such challenges, these environmental constraints have led to the emergence of complex metabolic dormancy phenotypes such as diapause and quiescence 2 – 5 . Diapause is a stage-specific and programmed developmental arrest characterized by metabolic suppression 2 – 7 . It is often triggered by environmental cues in advance of the actual challenging conditions (facultative diapause, e.g., signaled through shortening days), and in some species, can also be genetically encoded to occur at a specific life stage regardless of environmental conditions (obligate diapause) 2 – 5 . In contrast to diapause, quiescence is a form of dormancy that occurs only as an immediate response to limiting environmental factors (low temperature, dehydration, fasting, hypoxia, etc.) 3 – 5 . Numerous studies have associated both winter diapause and quiescence with energy saving and increased resilience towards environmental stress in cold-adapted invertebrates 3 , 5 – 9 . However, the direct relationship between acquisition of cold tolerance and programmed dormancy in insects remains poorly understood 3 , 10 In some species, diapause triggers the preemptive buildup of cryoprotective molecules 11 – 15 , indicating at least partially overlapping pathways 3 . In others, despite diapause, accumulation of cryoprotective molecules occurs only after exposure to low temperatures, implying cold hardening and diapause to be mainly coincidental 16 , 17 . Ants offer an opportune model system to study the interactions of seasonal biological cycles and cold tolerance. Ancestrally tropical, ants have successfully radiated and adapted to most seasonal climates on the planet 18 , 19 . Further, individuals are usually long-lived 20 , thus they have to endure several consecutive winters over their lifespan. For example, queens of the European black garden ants Lasius niger can reach up to 28 years in captivity and workers of the same species display an estimated lifespan of roughly 1 to 3 years 21 . At the colony-level, many temperate species are characterized by annual growth cycles that involve developmental arrest at collective level (colony dormancy), potentially coinciding with individuals’ diapause. As described by Kipyatkov and colleagues, three main broad categories of annual colony-level growth cycles have been identified in ants (Fig. S1) 22 – 25 . First, tropical ant species appear unable to slow or halt colony-level growth in response to external cues (homodynamic colony cycle); workers are chill-sensitive and only capable of limited quiescence 22 – 25 . In contrast, subtropical and temperate species are able to enter either facultative (exogenous-heterodynamic) or obligate (endogenous-heterodynamic) prolonged periods of colony-level dormancy linked to higher winter survival (Fig. S1) 22 . Furthermore, in species displaying obligate diapause (endogenous-heterodynamic), the duration of the yearly developmental phase of colonies (from dormancy to dormancy) appears largely governed by an endogenous timer with only limited sensitivity to external conditions (such as temperature and/or day length) 22 , 23 , 26 – 28 . Captive colonies invariably enter obligate dormancy after a finite time interval, with environmental factors able to delay but not prevent its onset ( Fig. 1A ) 22 . This phenomenon led Kipyatkov to propose the existence of a “Sand-glass device” in these species, a master switch governing the length of seasonal colonial development based on endogenous colony-cycles, with minimal integration of environmental cues ( Fig. 1A ) 22 – 25 . Despite their success in temperate ecosystems, how colony-level dormancy and environmental exposure integrate to modulate the cold tolerance of temperate ants remains unexplored. Download figure Open in new tab Figure 1. A. Yearly developmental cycle of endogenous-heterodynamic ants. A. Environmental cues, such as rising temperatures, trigger the end of winter dormancy for colonies in spring. B. Colonies then enter a fixed growth period, with queen egg-laying, worker activity, and brood development. C. Regulated by Kipyatkov’s “ Sand Glass Device ”, an internal clock determines the onset of the next dormancy with only limited integration of environmental cues (e.g. dormancy is initiated after a fixed period even when ants are exposed to warm temperatures or long days), typically coinciding with late summer or early fall locally. This phase is marked by the cessation of queen egg-laying, reduced worker activity, and depending on the species, the presence of diapausing larvae whose development will resume next spring. B. Experimental design. We leveraged the developmental cycle of endogenous-heterodynamic ants to establish a full-factorial design disentangling the relative effects of (i) acclimation and (ii) colony-level dormancy on the cold tolerance and metabolic reorganization of workers. Here, we disentangle the relative effects of colony-level dormancy and acclimation on worker cold tolerance. We used five common endogenous-heterodynamic European ant species spread across four distantly related genera, and leveraged a full-factorial design based on dormant vs non-dormant colonies and cold acclimated vs non-cold acclimated individuals. Through this system, we measured the (i) lower limits for neuromuscular activity (Critical Thermal Minimum, CT min ), (ii) acute limits of cold-stress mortality (LTe 50 ), (iii) metabolic rates putatively indicative of diapause, and finally (iv) the metabolomic reorganization hypothetically associated with increased cold tolerance in workers. Methods Species sampling, husbandry and acclimation Colonies of Lasius niger , Lasius flavus , Formica fusca , Myrmica rubra, and Leptothorax acervorum were collected near Aarhus, Denmark (56.16°N, 10.20°E) in summer 2021. These four genera encompass the majority of species diversity in subboreal and boreal ecosystems, accounting for around 80% of the native specific ant diversity found in Denmark 29 . Colonies were brought back to a climatized chamber and kept in plastic boxes with Fluon®-coated sides and provided with 16×150 mm plastic test tubes with a water-filled section in the bottom separated with a moist cotton plug for nesting. During the developmental phase of the colony, characterized by worker foraging activity, queen egg-laying, and developing brood 22 , colonies were kept under a 12:12 light:dark cycle at constant 26°C, and fed honey water and sliced mealworms twice a week. At the onset of colonies’ dormancy, characterized by the absence of worker foraging activity, of queen egg-laying, and diapausing larvae with halted development ( Lasius , Myrmica , and Leptothorax species) or no diapausing larvae at all ( Formica fusca ) 22 , colonies were moved into a cold room kept under a constant 12:12 light:dark cycle at 8°C for 4 months. Following this cycle, all colonies were simultaneously returned to developmental conditions (constant 12:12, light dark cycle at 26°C) where queen egg-laying, foraging activity and larval development usually resumed in colonies within 1 to 2 weeks. Experiments began in 2022 following colonies’ first artificial laboratory dormancy cycle. As a result, data are reported for workers exposed solely to the conditions defined in the husbandry methods for a full year (dormancy to dormancy). Experimental design We leveraged the endogenous-heterodynamic annual developmental cycle of the selected ant species to establish four experimental conditions ( Fig. 1B ). First, (1) control conditions, corresponding to workers taken from colonies in their active developmental phase, characterized by queen egg-laying and brood growth, maintained at a constant 12:12 light:dark cycle and 26°C. Second (2), acclimated conditions, corresponding to workers originating from the same control conditions but subsequently acclimated to 8°C for 10 days. Third, (3) dormancy conditions at warm temperature, corresponding to workers from the same colonies as control conditions but sampled later in their annual developmental cycle after the onset of colony-level dormancy (characterized by the absence of worker foraging activity, of queen egg-laying, and diapausing larvae with halted development or no diapausing larvae for Formica fusca 22 ). Finally (4), cold-acclimated and dormancy conditions, corresponding to workers from dormancy conditions that had been moved to 8°C for 10 days. These four conditions allowed us to exploit a full factorial design disentangling the relative effects of (i) acclimation and (ii) colony-level dormancy on the cold tolerance and metabolic reorganization of workers ( Fig. 1B ). Dynamic cold tolerance assays To investigate the lowest temperature limits for worker neuromuscular activity, Critical thermal minimum (CT min ) of workers were recorded as described previously 30 . Briefly, workers were placed individually into 5 mL closed glass vials mounted to a rack and submerged into a transparent tank filled with a 25% ethylene-glycol:water solution. Temperature in the bath was initially maintained at 20°C for 15 min before it was gradually decreased at a rate of 0.1°C/min using a programmable water bath (LAUDA-Brinkmann, NJ, USA). Chill-coma (CT min ) was then scored for each individual (N=10-20 workers per species) as the temperature resulting in a total absence of movement of workers (even after gentle vial shaking). Lower Lethal temperature To investigate mortality to acute cold stress in workers, Lower Lethal temperatures (LTe 50 ), defined as the temperature where 50% of individuals died following an acute cold exposure of 24h, were measured as previously described for other insects 31 . For each species and each condition, 35-mL vials containing 10-20 workers were placed for 24 h at a range of constant stressful temperatures. Each species was exposed to 6–12 different temperatures to obtain mortality data spanning from 100% to 0% (between all treatments and species this included temperatures ranging from +8 to −20 °C). After the 24h cold exposure, vials were returned to room temperature (ca. 21°C), and workers that were not able to move after an additional recovery period of 24h were scored dead. The temperature corresponding to 50% mortality (LTe 50 ) was calculated by fitting a sigmoidal curve to the mortality data for each species and condition (see data analysis). Metabolic rates measurements To investigate the relationship between colony-level dormancy and suppressed metabolic rates in workers, indicative of diapause, standard metabolic rates (SMRs) of our 5 species of ants were recorded across two conditions: (i) control, (workers taken from colonies in their developmental phase) and (ii) dormant (workers from the same colonies but sampled after the onset of colony-level dormancy). SMRs were estimated indirectly from the rate of CO 2 production (VCO 2 ) using repeated measurements of stop-flow respirometry as described previously for ants 30 . Measurements were made at 18°C as a conservative tradeoff between detectable metabolic signal and lower level of worker activity. On completion of measurements, the total fresh weight of workers was measured to the nearest 0.1 mg (Sartorius, Type 1712, Göttingen, Germany) to allow for calculation of SMRs. Metabolomics To link modulation of cold tolerance across our experimental design with putative accumulation of cryoprotective molecules in workers, we sampled ants corresponding to our 4 experimental conditions (see design) to analyze their metabolomic profiles. Workers were snap-frozen in liquid nitrogen and their gaster (the portion of the abdomen containing the crop) was removed to avoid potential contamination from food content. We sampled from 5 species x 4 treatments x 4 replicates, except for L. acervorum where too little biological material was available to analyze control conditions. This resulted in 76 analytical samples. Samples were transported on dry ice to Biology Center, České Budějovice for analysis, and the fresh mass of each analytical sample was measured to the nearest 0.1 mg using a balance MSE6.6S (Sartorius), resulting in 5 – 10 mg of worker tissues pooled for analysis for each analytical sample. To prepare analytical samples, they were melted on ice and homogenized in 500 µL of extraction buffer – methanol:acetonitrile:deionized water (2:2:1, v/v/v; Fisher Scientific, Pardubice, Czech Republic). Twelve additional blanks without ant tissues (4 per experimental condition) were also prepared for subsequent analysis of background noise to the signal. Internal standards, p-fluoro-DL-phenylalanine, and methyl-D-glucopyranoside (from Sigma-Aldrich, Saint Louis, MO, USA) were added to the extraction buffer to standardize measurements, both at a final concentration of 200 nmol mL −1 . Downstream metabolite extractions and analysis of 82 targeted metabolites were then performed as previously described 32 . Of the 82 initially targeted metabolites, 19 were not detected in our samples, leaving 63 metabolites for downstream analysis. A manual quality control (QC) check was then performed to curate the dataset: first, potential blank contamination values were subtracted from the analytical samples. Second, metabolites with a significant standard deviation (>33%) across replicates for any condition in one or more species were excluded. After these steps, 53 differentially expressed metabolites passed the QC check. These metabolites were quantified from their areas under their respective chromatographic peaks and then normalized to the amount of fresh mass of the respective analytical samples. Data analysis Data analysis was performed in R version 4.1.2 33 . The comparison of CT min values between our four experimental conditions for each species was performed using one-way ANOVAs followed by Tukey’s multiple comparison post-hoc tests. Assumptions of normality and homogeneity of variance were verified using the Shapiro–Wilk test and Bartlett’s test, respectively. LTe₅₀ values were computed by fitting sigmoidal curves to mortality proportions for all conditions and species, providing the temperature of 50% mortality after a 24h exposure (LTe₅₀). LTe₅₀ values are reported in the manuscript with their 95% confidence intervals (CIs), non-overlapping 95% CIs were considered indicative of statistically significant differences between conditions. As a redundant analytical step to assess the effects of (i) colony-level dormancy and (ii) acclimation on both CT min and LTe₅₀ across our entire dataset, a model-building approach was implemented using the lmer() function from the lme4 package 34 (after verifying the assumptions of a linear mixed-effects model). Model testing through Akaike Information Criterion (AIC) was performed using the aicw function from the package geiger 35 . The effect size of fixed variables included in the best-performing models (dormancy and acclimation) on CT min and LTe₅₀ values was calculated through a partial Omega Squared statistic (ωp 2 ) using the effsize package 36 . To test whether the onset of colony-level dormancy was associated with suppressed metabolism indicative of diapause in workers, we used a two-way ANOVA testing for the impact of species, dormancy, and their interaction on worker SMR, followed by a Tukey post-hoc test to assess pairwise differences between control and dormant conditions within each species. To provide a holistic examination of dormancy status and acclimation on the metabolomic profiles of workers, we first performed a visual investigation of metabolomic data based on heat-map profiles of area under the chromatographic peak of metabolites’ fold change (FC) across conditions. Second, we performed a Principal Component Analysis (PCA) of metabolites differentially expressed across conditions using the function prcomp from the R stats package 33 . This PCA investigated within a single analysis which relative fluctuation of metabolites (area under the chromatographic peak) were generally associated with fluctuations in cold tolerance (CT min and LTe 50 ) across all species and conditions within our dataset. Results Endogenous onset of colony dormancy influences the CT min of workers even in the absence of cold acclimation We recorded CT min values of workers across our 4 conditions to disentangle the relative effect of colony-level dormancy and cold acclimation on the cold tolerance of individuals. Workers from control conditions exhibited the highest CT min , with values ranging from 1.7°C ( L. niger ) to 0.2°C ( M. rubra – Fig. 2 A-E ). A cold acclimation period of 10 days at 8°C of workers from control, non-dormant conditions significantly lowered their CT min across all species, indicating that CT min values were reduced with cold acclimation. The onset of colony-level dormancy also lowered CT min values across all species ( Fig. 2 ), indicating phenotypic changes linked to cold tolerance in workers even in the absence of cold acclimation. Across the five species we found that the isolated effects of dormancy and acclimation were synergistic such that the lowest CT min values were observed for workers having been cold-acclimated at 8°C for 10 days after the onset of colony-level dormancy, with CT min values ranging from −3.2°C ( L. flavus ) to −4.3°C ( L. acervorum ) ( Fig. 2A–E ). The best fit model testing for the impact of both dormancy and acclimation on CT min values identified through AIC comparison (Table S1) included both acclimation and dormancy, as well as their interaction, as factors ( Table 1 ). Thus, the mixed-effect regression model confirmed that acclimation and colony-level dormancy were significant predictors of CT min across species, independently of each other (p < 0.001; Table 1 ) but also with a strong interactive effect (p < 0.01; Table 1 ). Download figure Open in new tab Figure 2. Effects of cold acclimation and endogenous colony-level dormancy on CT min values of individuals across studied species. CT min values (mean ± SD) were calculated for 10–20 individuals per condition and species. Both a cold-acclimation period and the onset of colony-level dormancy independently exerted a significant effect on CT min values as compared to workers from control conditions (growing colonies maintained at 26°C, 12:12 light/dark cycles), while further displaying a strong and significant interactive effect ( Table 1 ). Statistical significance between treatments within species is indicated (p < 0.05*, p < 0.01**, p < 0.001***; one-way ANOVA and Tukey post hoc test). View this table: View inline View popup Download powerpoint Table 1. Significance ( p ) and effect size (partial omega-squared values, ωp 2 ) of each variable included in the best fit linear mixed-effects regression models teasing apart the effect of acclimation and colony-level dormancy on CT min and LTe 50 values (See table S1 for best model selection). Overall, both acclimation and dormancy statistically affected CT min across species, yielding moderate to large effect sizes. In addition, these fixed effects displayed notable interaction, indicating that the effect of acclimation on CT min was more pronounced for species after the onset of colony-level dormancy. In contrast, LTe 50 values were significantly influenced across all species by acclimation only, with moderate effect size. Tolerance to acute cold stress is primarily driven by cold acclimation The proportion of dead ants following acute 24-hour cold stress was measured as a function of temperature across our five species for each of the four conditions ( Fig. 3 ). The temperature causing 50% worker mortality over 24 hours was extracted from the fitted sigmoidal relationships to provide LTe 50 values ( Fig. 3 , mean ± 95% CI). Workers from control conditions (26°C with a 12:12 light/dark cycle) exhibited the highest LTe 50 values and were thus the most sensitive to acute cold stress, displaying LTe 50 values ranging from −2.2°C ( L. niger ) to −9.5°C ( L. acervorum ). A 10-day cold acclimation at 8°C of workers from control conditions significantly lowered their LTe 50 across all species ( Fig. 3 ). The onset of colony-level dormancy also impacted LTe 50 values when investigating species-specific patterns ( Fig. 3A–E ), although this effect was less pronounced than acclimation and not significant for L. flavus ( Fig. 3B ). There was limited support for additive/synergistic effects of dormancy and acclimation on LTe 50 values when comparing dormant and non-dormant individuals, except for F. fusca ( Fig. 3C ). Overall, the lowest LTe 50 values and lowest mortality proportions to acute cold stress were observed in acclimated or acclimated + dormancy workers, with values ranging from −8.5°C ( M. rubra ) to −13.5°C ( L. acervorum ). The best fit model testing for the impact of both dormancy and acclimation on LTe 50 values identified through AIC comparison (Table S1) included both acclimation (p < 0.001) and dormancy (p = 0.07) as factors, without their interactive effect, and with dormancy approaching statistical significance only ( Table 1 ). Download figure Open in new tab Figure 3. Effects of cold acclimation and colony-level dormancy on survival to acute cold-stress (LTe 50, mean ± 95% CI). A. Lasius niger . B. Lasius flavus . C. Formica fusca . D. Myrmica rubra . E. Leptothorax acervorum . For each species, 10-20 workers were placed at a single temperature ranging from +8 to −20 °C for 24h, and survival was scored after an additional 24h recovery period to obtain survival data spanning from 100% to 0%. LTe 50 from workers as well as their 95% CI were extracted from fitted sigmoidal curves on survival data. Overall, cold-acclimation exerted a significant effect on LTe 50 values across all species. The onset of colony-level dormancy also exerted a significant effect on LTe 50 values, although to a lesser degree than acclimation, except for L. flavus . No differences in LTe 50 values were observed between acclimated and acclimated + dormancy workers, except for F. fusca . Statistical significance between treatments within species is indicated at p < 0.05* (non-overlapping 95% CI). Colony-level dormancy aligns with suppressed metabolic rates in most, but not all, species To confirm that the onset of colony-level dormancy coincided with suppressed metabolism indicative of diapause in individuals, we compared metabolic rates between workers from control and dormant conditions at 18°C (Fig. S2). The two-way ANOVA testing for the impact of species and dormancy on SMR values (Table S2) returned both factors and their interaction term as significant (p<0.001), indicating that across the global dataset and all species tested, dormancy had a significant effect on SMR values. A Tukey post-hoc test to assess pairwise differences specifically between dormant and active conditions within each species indicated, however, a statistically significant variation between SMR for three species only ( Lasius flavus , Formica fusca , and Myrmica rubra ), while no statistical differences in SMR were observed between control and dormancy for Lasius niger and Leptothorax acervorum (Fig. S2). Cold acclimation, but not colony-level dormancy, drives metabolomic shifts in workers A heatmap representation of metabolites’ fold-change (FC) value between species and conditions ( Fig. 4 ) supported that colony-level dormancy exerted only limited effect on workers’ metabolic profiles ( Fig. 4A, D ). In contrast, cold acclimation exerted a stronger effect on workers’ metabolomic profiles ( Fig. 4B, C ) with notable changes in the prevalence of some specific metabolites (see below). To analyze this metabolomic response more holistically, we performed a Principal Component Analysis (PCA). This PCA examined the patterns of relative metabolite abundance changes (area under the chromatographic peak) across our four experimental conditions and all species tested within a single analysis, incorporating CT min and LTe 50 values to assess potential alignment between increased cold hardiness and shift in metabolomic profiles of workers ( Fig. 5A ). The PCA analysis revealed that the first two principal components (PC) explained 24.5% and 15.5% of the total variance between conditions, respectively ( Fig. 5A ). CT min and LTe 50 strongly aligned with PC2, to which the four most contributing metabolites were glycerophosphocholine, glycerophosphoethanolamine, trehalose and inositol ( Fig. 5B , Fig. S3). Average FC for these metabolites across cold-acclimated conditions were 8.73, 4.91, 2.28 and 1.54 as compared to non-acclimated conditions, respectively ( Fig. 4 ). Other metabolites contributing to PC2 by decreasing relative contribution were D and L glutamic acid, Aspartic acid, Fumaric acid, Proline, Ribitol (Adonitol), Malic acid, Tryptophan, Lysine, Tyrosine. Together, these 13 metabolites accounted for 50% of the dataset contribution to PC2 ( Fig. 5B , Fig. S3). A focused graphical representation of metabolite changes due to cold acclimation in species, mapped on schematic pathways of intermediary metabolism, is available in Fig. S4. The 13 metabolites contributing to up to 50% of PC2 are mapped in red (increased relative abundance) and blue (decreased relative abundance), respectively. Download figure Open in new tab Figure 4. Heatmap visualization of metabolomic reorganization in workers. Data is structured to display the fold-change (FC) in metabolite area under the curve due to (A) the onset of colony-level dormancy on control workers, (B) the effect of cold acclimation on control workers, (C) the effect of cold acclimation on colony-level dormancy, and (D) the comparison between control-acclimated and diapausing-acclimated workers, singling out the effect of dormancy on acclimated states. Metabolomic data were deficient for L. acervorum in the control condition, resulting in metabolites’ FCs being fixed as 1 for this species as displayed in A and B. Overall, the onset of colony-level dormancy exerted a limited effect on metabolomic reorganization in species (A, D). In contrast, cold acclimation (B, C) was associated with more significant and consistent changes across profiles, with glycerophosphocholine, glycerophosphoethanolamine, and trehalose appearing to visually display the highest FC compared to control/dormancy conditions (also see PCA results in Fig. 4). A simplified comprehensive map of intermediate metabolism is provided in Fig. S5 Download figure Open in new tab Figure 5. A. Principal Component Analysis (PCA) of metabolomic data, showing that variations of CT min and LTe 50 across treatments strongly aligned with PC2, to which the 4 most contributing metabolites were trehalose, glycerophosphoethanolamine, glycerophosphocholine, and inositol. B. Scree plot depicting the relative contribution of each metabolite and cold-hardiness variable to PC2 up to 50% of cumulative variance (a complete plot detailing the relative contribution of our 53 metabolites to PC2 is presented in Fig. S4). A simplified overview of the schematic pathways of intermediary metabolism impacted by cold acclimation is available in Fig. S5. Discussion This work uses the endogenous developmental cycle of temperate ant colonies as a framework for understanding the relative contributions of colony-level dormancy and cold acclimation to cold tolerance and seasonal adaptation in ants ( Fig. 1A, B ). In endogenous-heterodynamic ants that display obligate colony-level dormancy, most individuals can experience multiple obligate diapause/colony-level dormancies over their lifespans 22 – 24 . Many species (including Lasius , Myrmica , and Leptothorax in this work) also exhibit brood development that can extend across more than one developmental season, leading to the presence of diapausing larvae within dormant colonies 22 , 24 , 37 . In contrast, other species (such as those from the genus Formica, e.g., Formica fusca in this work), complete their brood-rearing cycle within a brief annual period 22 , 24 , and colonies enter dormancy without brood 22 , 25 , 27 . Within our experimental system, we therefore used the combined cessation of worker foraging activity, the absence of queen egg laying, and the presence of arrested-development larvae (or complete brood absence in F. fusca ) to define the onset of “colony-level dormancy”. Suppressed SMRs are also usually indicative of diapause in insects 7 . Across our dataset, we found support for a trend indicative of preprogrammed diapause associated with reduced SMRs of individuals and colony-level dormancy (Table S2), though this was not statistically significant for all species (Fig. S2). Together, our design thus offered a strong conceptual approach teasing apart the relative contributions of colony-level dormancy and acclimation on species’ cold tolerance. Insects’ cold tolerance is typically quantified from one or more complementary traits 31 , 38 . Here, we integrated the use of CT min and LTe 50 values to characterize the cold tolerance of workers. CT min is defined as the lower temperature at which individuals lose neuromuscular function and enter chill coma 39 . In insects, the onset of chill coma appears primarily driven by spreading depolarization of the central nervous system due to the loss of ion homeostasis within the CNS 40 – 42 . By contrast, LTe₅₀ is a measurement of cold mortality caused by homeostatic failure of the whole body; captured here by the temperature at which 50% of individuals die after a 24-h period of cold exposure 31 . Temperate ants are categorized as freeze sensitive, chill-tolerant insects: they are able to tolerate relatively low subzero temperatures as long as internal ice formation is prevented, but will ultimately experience mortality due to the direct and indirect effects of cold on homeostatic function above the freezing points of their tissues 37 , 43 – 45 . In our work, CT min thus captured the lower functional temperature for neuromuscular activity (reversible, non-lethal chill coma), while LTe₅₀ captured mortality limits associated with whole body chill-injury. First, focusing on CT min , our results highlight a novel pattern for insect literature. The onset of colony-level dormancy alone was enough to significantly lower CT min values ( Fig. 2 , Table 1 ). Moreover, the onset of colony-level dormancy displayed a strong interactive effect with acclimation to impact CT min values further. This suggests two things. First, the onset of colony-level dormancy in endogenous-heterodynamic ants (to a degree associated with diapause, Fig. S2, Table S2) exerted significant phenotypic modification in workers modulating temperature activity thresholds. Second, that the onset of this “winter phenotype” potentiates workers to be more responsive to subsequent acclimation ( Fig. 2 , Table 1 ). CT min values in insects and ants are documented both highly plastic and locally adaptive across populations and species 30 , 46 – 49 . Colonies studied here were collected in Aarhus, Denmark (56.16°N, 10.20°E), a region with a temperate oceanic climate and long, mild, winters averaging minimum temperature of ca. –0.56°C in January (1991-2023) 50 . CT min values of dormant and cold-acclimated individuals (ranging from –3.2°C to –4.3°C; Fig. 2 ) closely matched the local winter air minima by a few degrees margin, resulting in a relatively low thermal safety margin when considering this trait. This suggests that workers may retain capacity for activity throughout much of the cold season, especially when relying on behavioral avoidance strategies via their thermally buffered subterranean nests. Contrasting to CT min , the effect of colony-level dormancy on LTe₅₀ values was less consistent ( Fig. 3 , Table 1 ). This indicates that endogenous colony-level cycles had a less pronounced (but still detectable) effect on acute cold tolerance in most species. As expected, cold acclimation, on the other hand, consistently increased workers’ acute cold tolerance ( Fig. 3 , Table 1 ). LTe₅₀ values in acclimated workers ranged from –8.5°C to –13.5°C, indicative of significant capacity for surviving temperatures well below local winter minima in Denmark 50 . Our results thus illustrates a dual thermal safety margin strategy depending on the cold-tolerance trait considered: a narrow margin for activity (CT min ), as previously documented in temperate insects 51 , but a much broader margin for lethal limits (LTe₅₀), maximizing survival capabilities. These findings underpin the importance of assessing multiple physiological traits when evaluating the ecological relevance of species’ cold tolerance, as different measures capture different aspects of thermal adaptation in ectotherms 31 . Furthermore, mortality from acute cold-stress exposure (LTe₅₀) in chill-tolerant insects like ants is partly related to their ability to supercool their body fluids through the accumulation of cryoprotective metabolites 52 . To explore whether such biochemical adjustments could underpin shifts in LTe 50 in heterodynamic ants, we conducted a targeted metabolomic analysis in workers following dormancy/acclimation. In this context, cold acclimation triggered a modest but consistent metabolomic shift in workers ( Fig. 4, B, C ), while colony-level dormancy had a more limited impact ( Fig. 4, A, D ). The metabolites most responsive to cold acclimation across our dataset were (i) two intermediates of phospholipid catabolism (glycerophosphoethanolamine and glycerophosphocholine) and (ii) two sugars (trehalose and inositol, Fig. 4 , 5 , Fig. S3). However, fold-changes in these metabolites remained relatively modest comparative to other studies investigating metabolomics reorganization to cold in insects 53 , 54 . This could reflect the milder acclimation treatment applied to colonies (8°C for 10 days), which may not have been sufficient to trigger stronger physiological responses. A tentative interpretation could link the observed modulation of phospholipid catabolism to biological membrane remodeling. Membrane fluidity is highly temperature sensitive, and its disruptions can contribute to osmotic and ionic imbalance leading to cold-induced neuromuscular paralysis 42 , 55 , 56 . Second, polyol accumulation can act through multiple protective pathways. At high concentration, they can limit ice formation and lower the supercooling points of tissues, but can also be involved in stabilizing membranes and support osmotic balance 57 – 63 . Their modest upregulation in our work is likely indicative of osmotic and ionic homeostasis roles, rather than direct colligative depression of supercooling points, as previously suggested in Drosophila 32 , 64 . Polyol accumulation (e.g., glycerol) was previously reported in wintering cold-tolerant species of ants as well 37 , 43 – 45 . Further work would be needed to fully elucidate their role in the cold-tolerance strategies of temperate species. In summary, our findings document the occurrence of endogenous cold hardening in seasonally adapted ants. These results provide the first evidence that thermal tolerance in social insects can be partly regulated by seasonal endogenous cycles, independently of environmental cues. They highlight the interplay between intrinsic seasonal programming and extrinsic thermal cues in shaping cold tolerance in temperate ants, offering insights into some of the key processes that have allowed this family of insects to thrive in highly seasonal climates. Authors’ contributions Q.W., VK and J.O. conceived and planned the study. Q.W. collected samples. Q.W. performed lab experiments. Q.W., and V. K analyzed the data. All authors contributed to drafting the article and approved the final published version. Competing interests The authors declare no competing or financial interests. Funding This project has received funding from the European Union’s Horizon Europe research and innovation program under the Marie Skłodowska-Curie grant agreement No. 101148613 (QW). Acknowledgements We thank L. Jørgensen for her help in taking care of captive ant colonies. Funder Information Declared Marie Skłodowska-Curie Actions , 101148613 References ↵ Danks , H. V. Seasonal adaptations in arctic insects . Integrative and Comparative Biology 44 , 85 – 94 ( 2004 ). OpenUrl CrossRef PubMed Web of Science ↵ Tougeron , K. Diapause research in insects: historical review and recent work perspectives . Entomologia Experimentalis et Applicata 167 , 27 – 36 ( 2019 ). OpenUrl ↵ Denlinger , D. L. Insect diapause . ( Cambridge University Press , 2022 ). Denlinger , D. L. Insect diapause: from a rich history to an exciting future . Journal of Experimental Biology 226 , jeb245329 ( 2023 ). OpenUrl PubMed ↵ Koštál , V. Eco-physiological phases of insect diapause . Journal of insect physiology 52 , 113 – 127 ( 2006 ). OpenUrl CrossRef PubMed Web of Science Lebenzon , J. E. et al. Reversible mitophagy drives metabolic suppression in diapausing beetles . Proceedings of the National Academy of Sciences 119 , e2201089119 ( 2022 ). OpenUrl CrossRef PubMed ↵ Hahn , D. A. & Denlinger , D. L. Energetics of insect diapause . Annual review of entomology 56 , 103 – 121 ( 2011 ). OpenUrl CrossRef PubMed Web of Science von Schmalensee , L. , Süess , P. , Roberts , K. T. , Gotthard , K. & Lehmann , P. A quantitative model of temperature-dependent diapause progression . Proceedings of the National Academy of Sciences 121 , e2407057121 ( 2024 ). OpenUrl CrossRef PubMed ↵ Roe , A. D. , Wardlaw , A. A. , Butterson , S. & Marshall , K. E. Diapause survival requires a temperature-sensitive preparatory period . Current Research in Insect Science 5 , 100073 ( 2024 ). OpenUrl CrossRef PubMed ↵ Denlinger , D. L. in Insects at low temperature 174 – 198 ( Springer , 1991 ). ↵ Lehmann , P. et al. Metabolome dynamics of diapause in the butterfly Pieris napi: distinguishing maintenance, termination and post-diapause phases . Journal of Experimental Biology 221 , jeb169508 ( 2018 ). OpenUrl Abstract / FREE Full Text Michaud , M. R. & Denlinger , D. L. Shifts in the carbohydrate, polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (Sarcophaga crassipalpis): a metabolomic comparison . Journal of Comparative Physiology B 177 , 753 – 763 ( 2007 ). OpenUrl CrossRef PubMed Web of Science Koštál , V. , Zahradníčková , H. & Šimek , P. Hyperprolinemic larvae of the drosophilid fly, Chymomyza costata, survive cryopreservation in liquid nitrogen . Proceedings of the National Academy of Sciences 108 , 13041 – 13046 ( 2011 ). OpenUrl Abstract / FREE Full Text Vesala , L. , Salminen , T. S. , Koštál , V. , Zahradníčková , H. & Hoikkala , A. Myo-inositol as a main metabolite in overwintering flies: seasonal metabolomic profiles and cold stress tolerance in a northern drosophilid fly . Journal of Experimental Biology 215 , 2891 – 2897 ( 2012 ). OpenUrl Abstract / FREE Full Text ↵ Colinet , H. , Renault , D. , Charoy-Guével , B. & Com , E. Metabolic and proteomic profiling of diapause in the aphid parasitoid Praon volucre . PLoS One 7 , e32606 ( 2012 ). OpenUrl CrossRef PubMed ↵ Koštál , V. , Tollarova , M. & Šula , J. Adjustments of the enzymatic complement for polyol biosynthesis and accumulation in diapausing cold-acclimated adults of Pyrrhocoris apterus . Journal of insect physiology 50 , 303 – 313 ( 2004 ). OpenUrl CrossRef PubMed Web of Science ↵ Uzelac , I. et al. Effect of cold acclimation on selected metabolic enzymes during diapause in the European corn borer Ostrinia nubilalis (Hbn .). Scientific Reports 10 , 9085 ( 2020 ). OpenUrl PubMed ↵ Economo , E. P. , Narula , N. , Friedman , N. R. , Weiser , M. D. & Guénard , B. Macroecology and macroevolution of the latitudinal diversity gradient in ants . Nature communications 9 , 1 – 8 ( 2018 ). OpenUrl CrossRef PubMed ↵ Schultheiss , P. et al. The abundance, biomass, and distribution of ants on Earth . Proceedings of the National Academy of Sciences 119 , e2201550119 ( 2022 ). OpenUrl PubMed ↵ Keller , L. & Genoud , M. Extraordinary lifespans in ants: a test of evolutionary theories of ageing . Nature 389 , 958 – 960 ( 1997 ). OpenUrl CrossRef Web of Science ↵ Kramer , B. H. , Schaible , R. & Scheuerlein , A. Worker lifespan is an adaptive trait during colony establishment in the long-lived ant Lasius niger . Experimental Gerontology 85 , 18 – 23 ( 2016 ). OpenUrl CrossRef PubMed ↵ Kipyatkov , V. E. Seasonal life cycles and the forms of dormancy in ants (Hymenoptera: Formicoidea) . Acta Soc. Zool. Bohem 65 , 211 – 238 ( 2001 ). OpenUrl ↵ Kipyatkov , V. Role of endogenous rhythms in regulation of annual cycles of development in ants (Hymenoptera, Formicidae) . ( 1994 ). ↵ Kipyatkov , V. E. The evolution of seasonal cycles in cold-temperate and boreal ants: patterns and constraints . Life cycles in social insects: behaviour, ecology and evolution , 63 – 84 ( 2006 ). ↵ Lopatina , E. B. Structure, diversity and adaptive traits of seasonal cycles and strategies in ants . The complex world of ants , 7 – 49 ( 2018 ). ↵ Lopatina , E. & Kipyatkov , V. The influence of daily thermoperiods on the duration of seasonal cycle of development in the ants Myrmica rubra L. and M. ruginodis Nyl . In Proceedings of the Colloquia on Social Insects . 3 , 4 ( Russian Language Section of the IUSSI St. Petersburg , 1997 Published). ↵ Kipyatkov , V. & Lopatina , E. The regulation of annual cycle of development in the ants of the subgenus Serviformica (Hymenoptera, Formicidae) . In Proceedings of the colloquia on social insects . 2 , 49 – 60 ( Russian-Speaking Section of the IUSSI St. Petersburg , 1993 Published). ↵ Kipyatkov , V. & Lopatina , E. Temperature and photoperiodic control of diapase induction in the ant Lepisiota semenovi (Hymenoptera, formicidae) from Turkmenistan . Journal of Evolutionary Biochemistry and Physiology 45 , 238 – 245 ( 2009 ). OpenUrl ↵ Collingwood , C. A. The Formicidae (Hymenoptera) of Fennoscandia and Denmark . ( Scandinavian Science Press ., 1979 ). ↵ Willot , Q. , Ørsted , M. , Malte , H. & Overgaard , J. Cold comfort: metabolic rate and tolerance to low temperatures predict latitudinal distribution in ants . Proceedings of the Royal Society B 290 , 20230985 ( 2023 ). OpenUrl PubMed ↵ Andersen , J. L. et al. How to assess Drosophila cold tolerance: chill coma temperature and lower lethal temperature are the best predictors of cold distribution limits . Functional Ecology 29 , 55 – 65 ( 2015 ). OpenUrl CrossRef ↵ Moos , M. et al. Metabolomic signatures associated with cold adaptation and seasonal acclimation of Drosophila: profiling of 43 species . Journal of Experimental Biology, jeb . 250076 ( 2025 ). ↵ Team , R. C. R: A language and environment for statistical computing . ( 2013 ). ↵ Bates , D. , Mächler , M. , Bolker , B. & Walker , S. Fitting linear mixed-effects models using lme4 . Journal of statistical software 67 , 1 – 48 ( 2015 ). OpenUrl CrossRef ↵ Harmon , L. J. , Weir , J. T. , Brock , C. D. , Glor , R. E. & Challenger , W. GEIGER: investigating evolutionary radiations . Bioinformatics 24 , 129 – 131 ( 2008 ). OpenUrl CrossRef PubMed Web of Science ↵ Torchiano , M. & Torchiano , M. M. Package ‘effsize’ . Package “Effsize ( 2020 ). ↵ Berman , D. , Alfimov , A. , Zhigulskaya , Z. & Leirikh , A. Overwintering & Cold-hardiness of Ants in the Northeast of Asia . ( Pensoft Publishers , 2010 ). ↵ Teets , N. M. , Marshall , K. E. & Reynolds , J. A. Molecular mechanisms of winter survival . Annual Review of Entomology 68 , 319 – 339 ( 2023 ). OpenUrl CrossRef PubMed ↵ Hazell , S. P. & Bale , J. S. Low temperature thresholds: are chill coma and CTmin synonymous? Journal of insect physiology 57 , 1085 – 1089 ( 2011 ). OpenUrl CrossRef PubMed ↵ Robertson , R. M. , MacMillan , H. A. & Andersen , M. K. A cold and quiet brain: mechanisms of insect CNS arrest at low temperatures . Current Opinion in Insect Science 58 , 101055 ( 2023 ). OpenUrl PubMed Andersen , M. K. , Willot , Q. & MacMillan , H. A. A neurophysiological limit and its biogeographic correlations: cold-induced spreading depolarization in tropical butterflies . Journal of Experimental Biology 226 ( 2023 ). ↵ Overgaard , J. , Gerber , L. & Andersen , M. K. Osmoregulatory capacity at low temperature is critical for insect cold tolerance . Current Opinion in Insect Science 47 , 38 – 45 ( 2021 ). OpenUrl PubMed ↵ Berman , D. I. , Zhigulskaya , Z. A. & Leirikh , A. N. Cold resistance of an ant Camponotus herculeanus (Hymenoptera, Formicidae) in various climates in North-East Asia . CryoLetters 38 , 17 – 28 ( 2017 ). OpenUrl PubMed Berman , D. , Leirikh , A. & Zhigulskaya , Z. A common strategy of cold hardiness in ants of the genus Myrmica (Formicidae, Hymenoptera) in Northeast Asia . Entomological review 92 , 247 – 261 ( 2012 ). OpenUrl ↵ Heinze , J. , Stahl , M. & Hölldobler , B. Ecophysiology of hibernation in boreal Leptothorax ants (Hymenoptera: Formicidae) . Ecoscience 3 , 429 – 435 ( 1996 ). OpenUrl ↵ Bujan , J. , Roeder , K. A. , de Beurs , K. , Weiser , M. D. & Kaspari , M. Thermal diversity of North American ant communities: Cold tolerance but not heat tolerance tracks ecosystem temperature . Global Ecology and Biogeography 29 , 1486 – 1494 ( 2020 ). OpenUrl Diamond , S. E. & Chick , L. D. The Janus of macrophysiology: stronger effects of evolutionary history, but weaker effects of climate on upper thermal limits are reversed for lower thermal limits in ants . Current zoology 64 , 223 – 230 ( 2018 ). OpenUrl CrossRef PubMed Heinze , J. , Foitzik , S. , Kipyatkov , V. & Lopatina , E. Latitudinal variation in cold hardiness and body size in the boreal ant Leptothorax acervorum . Entomol. Gener 22 , 305 – 312 ( 1998 ). OpenUrl ↵ Kirchner , M. , Sorenson , C. & Youngsteadt , E. Too cold to handle: Climatic constraints on arboreal ants in temperate forests . Journal of Animal Ecology ( 2025 ). ↵ World Bank, Climate Change Knowledge Portal (2025) , (1991–2023). ↵ Sunday , J. M. et al. Thermal-safety margins and the necessity of thermoregulatory behavior across latitude and elevation . Proceedings of the National Academy of Sciences 111 , 5610 – 5615 ( 2014 ). OpenUrl Abstract / FREE Full Text ↵ Overgaard , J. & MacMillan , H. A. The integrative physiology of insect chill tolerance . Annual review of physiology 79 , 187 – 208 ( 2017 ). OpenUrl CrossRef PubMed ↵ Lee , R. E. A primer on insect cold-tolerance . Low temperature biology of insects , 3 – 34 ( 2010 ). ↵ Storey , K. B. & Storey , J. M. in Insects at low temperature 64 – 93 ( Springer , 1991 ). ↵ Slotsbo , S. et al. Tropical to subpolar gradient in phospholipid composition suggests adaptive tuning of biological membrane function in drosophilids . Functional Ecology 30 , 759 – 768 ( 2016 ). OpenUrl CrossRef ↵ Overgaard , J. , Sørensen , J. G. , Petersen , S. O. , Loeschcke , V. & Holmstrup , M. Changes in membrane lipid composition following rapid cold hardening in Drosophila melanogaster . Journal of insect physiology 51 , 1173 – 1182 ( 2005 ). OpenUrl CrossRef PubMed Web of Science ↵ Salt , R. Role of glycerol in the cold-hardening of Bracon cephi (Gahan) . Canadian Journal of Zoology 37 , 59 – 69 ( 1959 ). OpenUrl Gehrken , U. Winter survival of an adult bark beetle Ips acuminatus Gyll . Journal of insect physiology 30 , 421 – 429 ( 1984 ). OpenUrl CrossRef Sformo , T. et al. Deep supercooling, vitrification and limited survival to–100 C in the Alaskan beetle Cucujus clavipes puniceus (Coleoptera: Cucujidae) larvae . Journal of Experimental Biology 213 , 502 – 509 ( 2010 ). OpenUrl Abstract / FREE Full Text Jain , N. K. & Roy , I. Effect of trehalose on protein structure . Protein Science 18 , 24 – 36 ( 2009 ). OpenUrl CrossRef PubMed Web of Science MacMillan , H. A. et al. Cold acclimation wholly reorganizes the Drosophila melanogaster transcriptome and metabolome . Scientific Reports 6 , 28999 ( 2016 ). OpenUrl PubMed Toxopeus , J. , Koštál , V. & Sinclair , B. J. Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect . Proceedings of the Royal Society B 286 , 20190050 ( 2019 ). OpenUrl CrossRef PubMed ↵ Toxopeus , J. & Sinclair , B. J. Mechanisms underlying insect freeze tolerance . Biological Reviews 93 , 1891 – 1914 ( 2018 ). OpenUrl CrossRef ↵ Olsson , T. et al. Hemolymph metabolites and osmolality are tightly linked to cold tolerance of Drosophila species: a comparative study . Journal of Experimental Biology 219 , 2504 – 2513 ( 2016 ). OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted July 14, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Endogenous circannual cycles drive seasonal cold hardening in temperate ants Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Endogenous circannual cycles drive seasonal cold hardening in temperate ants Quentin Willot , Vladimír Koštál , Johannes Overgaard bioRxiv 2025.07.09.663877; doi: https://doi.org/10.1101/2025.07.09.663877 Share This Article: Copy Citation Tools Endogenous circannual cycles drive seasonal cold hardening in temperate ants Quentin Willot , Vladimír Koštál , Johannes Overgaard bioRxiv 2025.07.09.663877; doi: https://doi.org/10.1101/2025.07.09.663877 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41909) Biophysics (21436) Cancer Biology (18576) Cell Biology (25479) Clinical Trials (138) Developmental Biology (13367) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15598) Genomics (22482) Immunology (17726) Microbiology (40359) Molecular Biology (17162) Neuroscience (88532) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7636) Plant Biology (15129) Scientific Communication and Education (2044) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)