Adult Stress Research Articles

Effects of early life stress and subsequent re-exposure to stress on neuronal activity in the lateral habenula

Early life stress can result in depression in humans and depressive-like behaviour in rodents. In various animal models of depression, the lateral habenula (LHb) has been shown to become hyperactive immediately after early life stress. However, whether these pathological changes persist into adulthood is less well understood. Hence, we utilised the maternal separation (MS) model of depression to study how early life stress alters LHb physiology and depressive behaviour in adult mice. We find that only a weak depressive phenotype persists into adulthood which surprisingly is underpinned by LHb hypoactivity in acute slices, accompanied by alterations in both excitatory and inhibitory signalling. However, while we find the LHb to be less active at rest, we report that the neurons reside in a sensitised state where they are more responsive to re-exposure to stress in adulthood in the form of acute restraint, thus priming them to respond to aversive events with an increase in neuronal activity mediated by changes in glutamatergic transmission. These findings thus suggest that in addition to LHb hyperactivity, hypoactivity likely also promotes an adverse phenotype. Re-exposure to stress results in the reappearance of LHb hyperactivity offering a possible mechanism to explain how depression relapses occur following previous depressive episodes.

Parenting Stress and Attachment Insecurity in Young Adulthood: A Social Relations Model.

We used the Social Relations Model to inspect the individual- and dyad-specific components of attachment among young adults and their parents, and examined relations between these components and parenting stress. Young adulthood is a transitional period in which the whole family is concerned with "launching" the young adult and exploring new ways to interact with and attach to one another. However, research on young adulthood attachment has primarily focused on young adults' attachment style rather than reciprocal attachments among family members. When the young adults were age 23, mothers, fathers, and young adults from 156 families reported their mutual attachment security. At ages 18 and 23, parents of the adolescent/young adult reported their parenting stress in interparental and parent-child relationship domains. Attachment in the families of young adults can be separated into three components: 1) actor effects (each family member's internal working model of attachment), 2) partner effects (characteristics of each family member as an attachment figure), and 3) relationship effects (dyad-specific attachment between family members). Increase of parenting stress in a family subsystem (dyad of family members) predicted attachment insecurity within the subsystem. Additionally, compensatory effects across family subsystems were observed. Attachment in the family during young adulthood is explained by family members' own characteristics as well as dyad-specific interactions and is predicted by parenting stress in family subsystems.

Prenatal inflammation shapes microglial immune response into adulthood

Hayes and collaborators recently unraveled that maternal immune activation in mice led to a long-lasting decrease in microglial immune reactivity. Thus, microglia exhibited a reduced immune response to a second proinflammatory stressor in adulthood. This altered microglial response impacted both astrocytic reactivity and neuronal circuitry.

Social threat and safety learning in individuals with adverse childhood experiences: electrocortical evidence on face processing, recognition, and working memory

ABSTRACT Background: Adverse childhood experiences (ACEs) are often associated with stress and anxiety-related disorders in adulthood, and learning and memory deficits have been suggested as a potential link between ACEs and psychopathology. Objective: In this preregistered study, the impact of social threat learning on the processing, encoding, and recognition of unknown faces as well as their contextual settings was measured by recognition performance and event-related brain potentials. Method: Sixty-four individuals with ACEs encoded neutral faces within threatening or safe context conditions. During recognition, participants had to decide whether a face was new or had been previously presented in what context (item-source memory), looking at old and new faces. For visual working memory, participants had to detect changes in low and high load conditions during contextual threat or safety. Results: Results showed a successful induction of threat expectation in persons with ACEs. In terms of face and source recognition, overall recognition of safe and new faces was better compared to threatening face-compounds, with more socially anxious individuals having an advantage in remembering threatening faces. For working memory, an effect of task load was found on performance, irrespective of threat or safety context. Regarding electrocortical activity, an old/new recognition effect and threat-selective processing of face–context information was observed during both encoding and recognition. Moreover, neural activity associated with change detection was found for faces in a threatening context, but only at high task load, suggesting reduced capacity for faces in potentially harmful situations when cognitive resources are limited. Conclusion: While individuals with ACE showed intact social threat and safety learning overall, threat-selective face processing was observed for item/source memory, and a threatening context required more processing resources for visual working memory. Further research is needed to investigate the psychophysiological processes involved in functional and dysfunctional memory systems and their importance as vulnerability factors for stress-related disorders.

Transcriptome and morphological analysis on the heart in gestational protein-restricted aging male rat offspring.

Background: Adverse factors that influence embryo/fetal development are correlated with increased risk of cardiovascular disease (CVD), type-2 diabetes, arterial hypertension, obesity, insulin resistance, impaired kidney development, psychiatric disorders, and enhanced susceptibility to oxidative stress and inflammatory processes in adulthood. Human and experimental studies have demonstrated a reciprocal relationship between birthweight and cardiovascular diseases, implying intrauterine adverse events in the onset of these abnormalities. In this way, it is plausible that confirmed functional and morphological heart changes caused by gestational protein restriction could be related to epigenetic effects anticipating cardiovascular disorders and reducing the survival time of these animals. Methods: Wistar rats were divided into two groups according to the protein diet content offered during the pregnancy: a normal protein diet (NP, 17%) or a Low-protein diet (LP, 6%). The arterial pressure was measured, and the cardiac mass, cardiomyocytes area, gene expression, collagen content, and immunostaining of proteins were performed in the cardiac tissue of male 62-weeks old NP compared to LP offspring. Results: In the current study, we showed a low birthweight followed by catch-up growth phenomena associated with high blood pressure development, increased heart collagen content, and cardiomyocyte area in 62-week-old LP offspring. mRNA sequencing analysis identified changes in the expression level of 137 genes, considering genes with a p-value < 0.05. No gene was. Significantly changed according to the adj-p-value. After gene-to-gene biological evaluation and relevance, the study demonstrated significant differences in genes linked to inflammatory activity, oxidative stress, apoptosis process, autophagy, hypertrophy, and fibrosis pathways resulting in heart function disorders. Conclusion: The present study suggests that gestational protein restriction leads to early cardiac diseases in the LP progeny. It is hypothesized that heart dysfunction is associated with fibrosis, myocyte hypertrophy, and multiple abnormal gene expression. Considering the above findings, it may suppose a close link between maternal protein restriction, specific gene expression, and progressive heart failure.

A life course approach to understanding stress exposures and cognitive function among middle-aged and older adults

BackgroundMany studies have evaluated the stress-cognition association, but few have captured the cumulative nature of stress or distinguished the influences of stressors occurring in childhood versus adulthood. Using a lifecourse approach, we investigated whether cumulative stress exposures are associated with poorer cognitive function and faster cognitive decline. MethodsWe used data from the Midlife Development in the United States Study (N = 3,954, mean baseline age: 56 years). We fit marginal structural generalized estimating equations models to estimate the difference in baseline cognitive function per SD increment in the continuous stressor score, and, separately, between persons in each life course stressor profile and those who did not experience high stress in either childhood or adulthood. We also characterized differences in cognitive decline across levels of stress exposures. ResultsHigher cumulative stress exposure was associated with lower executive function (difference per SD in continuous stressor score = −0.12 SD units, 95% CI = −0.16, −0.08) and episodic memory (difference = −0.09 SD units, 95% CI = −0.13, −0.05). Baseline executive function and episodic memory were lower among those with high stress only in childhood, only in adulthood, and both, than among those without high stress in childhood or adulthood. There was little evidence that rate of change in executive function and episodic memory differed across levels of cumulative stress exposures. ConclusionsThese findings offer support to the hypothesis that stress exposures, accumulated over the life course, worsen cognitive performance, but limited support for the hypothesis that these exposures promote cognitive decline.

Early life maternal separation induces sex-specific antidepressant-like responses but has minimal effects on adult stress susceptibility in mice.

Early life stress is known to increase the risk of depression and anxiety disorders, which are highly prevalent conditions that disproportionately affect women. However, the results of preclinical studies have been mixed, with some work suggesting that early life stress promotes anxiety-like behavior and/or increases susceptibility to subsequent stressors, and other research suggesting that early life stress reduces anxiety-like behavior and/or confers resilience to subsequent stress exposure. It is likely that factors such as sex and the timing and severity of early life and adult stress exposure dictate whether a particular early life experience promotes adaptive vs. maladaptive behavior later in life. Most work in this area has focused exclusively on males, but several sex differences in the effects of early life stress on subsequent stress susceptibility have been reported. The current study examined the impact of early life maternal separation on susceptibility to behavioral alterations induced by 3 days of variable stress in adulthood in male and female c57BL6 mice. Our results indicate that 3 days of adult stress is sufficient to increase anxiety-like behavior in several paradigms and to increase immobility in the forced swim test. In contrast, a history of maternal separation reduces anxiety-like behavior in several tests, particularly in males. These findings could contribute to our understanding of sex differences in mental illness by demonstrating that males are more likely than females to display adaptive responses to mild early life stressors.

Differential impact of two paradigms of early-life adversity on behavioural responses to social defeat in young adult rats and morphology of CA3 pyramidal neurons

Early life stress (ELS) is an important factor in programing the brain for future response to stress, and resilience or vulnerability to stress-induced emotional disorders. The hippocampal formation, with essential roles in both regulating the stress circuitry and emotionality, contributes to this adaptive programing. Here, we examined the effects of early handling (EH) and maternal deprivation (MD) as mild and intense postnatal stressors, respectively, on the behavioural responses to social defeat stress in young adulthood. We also evaluated the interaction of mild and intense ELS with later social defeat (SD) stress on the morphology and dendritic spine density of Golgi-cox-stained CA3 hippocampal neurons. SD stress in adult rats, as expected, increased anxiety and depressive-like behaviours in the open field, elevated plus-maze and forced swimming test. These effects were associated with reduction of dendritic spines and soma size of CA3 neurons. Both behavioural and structural alterations were significantly ameliorated in socially defeated rats that experienced early handling (EH-SD). Basal dendrites of CA3 neurons in EH-SD rats also showed longer dendrites and more intersections with Sholl circles in the distal portion, compared to both control and SD rats. On the other hand, in socially defeated rats with maternal deprivation experience (MD-SD) the stress-induced behavioural and structural alterations were generally intensified compared to SD rats. In MD-SD rats, apical dendrites of CA3 neurons demonstrated remarkable retraction; an effect that was not detected in SD rats. The reduction of dendritic spines density on the apical dendrites of CA3 neurons was also more pronounced in MD-SD rats compared to SD rats. Dendritic arbors and spines comprise the major neuronal substrate for the circuit connectivity, and cell region-specific alterations of dendrites and spines in CA3 neurons reveal plausible mechanisms that can underlie the impact of different ELSs on risk for affective disorders in response to social stress in adulthood.

Investigating the role of environmental enrichment initiated in adolescence against the detrimental effects of chronic unpredictable stress in adulthood: Sex-specific differences in behavioral and neuroendocrinological findings

Environmental Enrichment (EE) improves cognitive function and enhances brain plasticity, while chronic stress increases emotionality, impairs learning and memory, and has adverse effects on brain anatomy and biochemistry. We explored the beneficial role of environmental enrichment initiated in adolescence against the negative outcomes of Chronic Unpredictable Stress (CUS) during adulthood on emotional behavior, cognitive function, as well as somatic and neuroendocrine markers in both sexes. Adolescent Wistar rats housed either in enriched or standard housing conditions for 10 weeks. On postnatal day 66, a subgroup from each housing condition was daily exposed to a 4-week stress protocol. Following stress, adult rats underwent behavioral testing to evaluate anxiety, exploration/locomotor activity, depressive-like behavior and spatial learning/memory. Upon completion of behavioral testing, animals were exposed to a 10-m stressful event to test the neuroendocrine response to acute stress. CUS decreased body weight gain and increased adrenal weight. Some stress-induced behavioral adverse effects were sex-specific since learning impairments were limited to males while depressive-like behavior to females. EE housing protected against CUS-related behavioral deficits and body weight loss. Exposure to CUS affected the neuroendocrine response of males to acute stress as revealed by the increased corticosterone levels. Our findings highlight the significant role of EE in adolescence as a protective factor against the negative effects of stress and underline the importance of inclusion of both sexes in animal studies.

Epigenetic Mechanism of Early Life Stress-Induced Depression: Focus on the Neurotransmitter Systems.

Depression has an alarmingly high prevalence worldwide. A growing body of evidence indicates that environmental factors significantly affect the neural development and function of the central nervous system and then induce psychiatric disorders. Early life stress (ELS) affects brain development and has been identified as a major cause of depression. It could promote susceptibility to stress in adulthood. Recent studies have found that ELS induces epigenetic changes that subsequently affect transcriptional rates of differentially expressed genes. The epigenetic modifications involved in ELS include histone modifications, DNA methylation, and non-coding RNA. Understanding of these genetic modifications may identify mechanisms that may lead to new interventions for the treatment of depression. Many reports indicate that different types of ELS induce epigenetic modifications of genes involved in the neurotransmitter systems, such as the dopaminergic system, the serotonergic system, the gamma-aminobutyric acid (GABA)-ergic system, and the glutamatergic system, which further regulate gene expression and ultimately induce depression-like behaviors. In this article, we review the effects of epigenetic modifications on the neurotransmitter systems in depression-like outcomes produced by different types of ELS in recent years, aiming to provide new therapeutic targets for patients who suffer from depression.

Early Life Stress-Induced Epigenetic Programming of Hippocampal NPY-Y2 Receptor Gene Expression Changes in Response to Adult Stress

Early Life Stress (ELS) can critically influence brain development and future stress responses and thus represents an important risk factor for mental health and disease. Neuropeptide Y (NPY) is discussed to be a key mediator of resilient vs. vulnerable adaptations and specifically, the NPY-Y2 receptor (Y2R) may be involved in the pathophysiology of depression due to its negative regulation of NPY-release. The present study addressed the hypotheses that ELS and adult stress (AS) affect the expression of hippocampal Y2R and that exposure to ELS induces an epigenetically mediated programming effect towards a consecutive stress exposure in adulthood. The specific aims were to investigate if (i) ELS or AS as single stressors induce changes in Y2 receptor gene expression in the hippocampus, (ii) the predicted Y2R changes are epigenetically mediated via promoter-specific DNA-methylation, (iii) the ELS-induced epigenetic changes exert a programming effect on Y2R gene expression changes in response to AS, and finally (iv) if the predicted alterations are sex-specific. Animals were assigned to the following experimental groups: (1) non-stressed controls (CON), (2) only ELS exposure (ELS), (3) only adult stress exposure (CON+AS), and (4) exposure to ELS followed by AS (ELS+AS). Using repeated maternal separation in mice as an ELS and swim stress as an AS we found that both stressors affected Y2R gene expression in the hippocampus of male mice but not in females. Specifically, upregulated expression was found in the CON+AS group. In addition, exposure to both stressors ELS+AS significantly reduced Y2R gene expression when compared to CON+AS. The changes in Y2R expression were paralleled by altered DNA-methylation patterns at the Y2R promoter, specifically, a decrease in mean DNA-methylation in the CON+AS males compared to the non-AS exposed groups and an increase in the ELS+AS males compared to the CON+AS males. Also, a strong negative correlation of mean DNA-methylation with Y2R expression was found. Detailed CpG-site-specific analysis of DNA-methylation revealed that ELS induced increased DNA-methylation only at specific CpG-sites within the Y2R promoter. It is tempting to speculate that these ELS-induced CpG-site-specific changes represent a “buffering” programming effect against elevations of Y2R expression induced by AS.

Stressful life events and trajectories of depression symptoms in a U.S. military cohort

Depression is a common mental disorder that may comprise distinct, underlying symptom patterns over time. Associations between stressful life events throughout the civilian lifecourse—including during childhood—and adult depression have been documented in many populations, but are less commonly assessed in military samples. We identified different trajectories of depression symptoms across four years in a military cohort using latent class growth analysis, and investigated the relationship between these trajectories and two domains of civilian life experiences: childhood adversity (e.g., being mistreated during childhood) and more proximal stressful experiences (e.g., divorce). A four-group depression model was identified, including a symptom-free group (62%), an increasing symptom group (13%), a decreasing symptom group (16%), and a “chronic” symptom group (9%). Compared to the symptom-free group, soldiers with childhood adversity were more likely to be in the chronic depression, decreasing, and increasing symptom groups. Time-varying adult stressors had the largest effect on depression symptoms for the increasing symptom group compared to other groups, particularly in the last two years of follow-up. This study indicates the importance of considering events from throughout the lifecourse—not only those from deployment—when studying the mental health of servicemembers.

Early-life exposure to economic stress and metabolic risks in young adulthood: the children of the reunification in East Germany

BackgroundResearch on the long-term health consequences of early-life exposure to economic crises is scarce. We examine for the first time the long-term effects of early-life exposure to an economic crisis...

Adverse childhood experiences and perceived stress in early adulthood in the context of disadvantage

BackgroundAdverse childhood experiences (ACEs) may sensitize individuals to view situations in adulthood as more stressful, which may contribute to poor health outcomes. In populations facing disadvantage, ACEs may lead to the accumulation of stressors (stress proliferation or mediation hypothesis) throughout the life course. ACEs could also heighten perceived stress later in life due to its enduring impact (stress sensitization or effect modification hypothesis). ObjectiveWe examine the associations between ACEs and perceived stress in early adulthood, considering concurrent life stressors, in a longitudinal cohort of Puerto Rican youth exposed to a high degree of disadvantage. Participants and settingA community-based sample of 1626 Puerto Rican children living in disadvantaged contexts was followed longitudinally in the Boricua Youth Study from 2000 to 2017. MethodsACEs were measured prospectively during childhood (<18 yrs), and life stressors and past year perceived stress were measured in early adulthood (EA; mean age = 23.4, sd 2.22). Causal mediation analysis tested ACEs' effects on EA perceived stress indirectly through life stressors including potential effect modification. ResultsACEs influenced perceived stress in EA (standardized total effect = 0.13, p < .001) with 35% mediated by increased exposure to life stressors in EA due to ACEs. There was no evidence of increased sensitization to EA life stressors among those with higher ACEs exposure. ConclusionsACEs contribute to perceived stress in EA, albeit with small effect, partially through accumulating effects of ongoing stressors, supporting the stress proliferation hypothesis. Policies aimed at reducing exposure to adversity from childhood to EA are needed to reduce the experience of ACEs and negative sequelae.

Effect of social instability stress in adolescence or adulthood on sensitivity to sucrose concentration in a social context in male and female Long-Evans rats.

Although there is evidence of sex differences in responding to social stress, and that age when stressed matters, females are understudied and adult-stress comparisons are few. Here, we investigated stress effects on reward sensitivity by examining rats' choice of social versus sucrose reward in a continuous spatial allocation design. We predicted social instability stress (SS) in adolescence would result in greater social discounting (spend less time near a novel peer when provided access to sucrose) relative to nonstressed controls (CTLs) and relative to SS in adulthood. All increased sucrose intake as the concentration increased, with no evidence of social discounting. SS males tested soon after the stress had a decrease in intake, whereas those tested long after had an increase in both time near the peer and in intake. CTL and SS females did not differ in intake, although their dose-response curves differed when tested soon after the SS. We also tested whether SS changed the stimulus value of the rat as a social peer; when tested in triads, CTL rats spent similar time in interaction with SS versus CTL rats. In sum, effects of SS on reward sensitivity were greater for males irrespective of administered in adolescence versus adulthood.

Does Overparenting Hurt Working Turkish Mother’s Well-being? The Influence of Family–Work Conflict and Perceived Stress in Established Adulthood

Does Overparenting Hurt Working Turkish Mother’s Well-being? The Influence of Family–Work Conflict and Perceived Stress in Established Adulthood

Distinct behavior in early life stress dams predicts heterogeneity in future stress response in offspring over lifespan

IntroductionExposure to early life stress (ELS) strongly predicts prevalent, impairing, and costly psychiatric illness throughout life including mental disorders. The reason, some individuals are more vulnerable to ELS whereas others remain resilient, is poorly understood. There is a need for better understanding of early biological changes triggered by ELS with responsibility to negative outcomes in health.ObjectivesWe stratify animals after ELS according to corticosterone levels. [1] Re-challenging the animals to a second stressor, chronic social defeat (CSD) [2], in adulthood was performed to understand individual trajectories depending on corticosterone exposure during early adverse conditions.MethodsWe performed ELS as previously reported [1]. Behavior of mothers was observed during ELS. Correlation between level of corticosterone and behavior observed in dams. ELS animals were exposed to a second stress in adulthood. A battery of tests for different behavioral domains was performed. Behavioral analyses was combined with assessment of litter HPA system reactivity and observed behavior in dams.ResultsStress dams where significantly higher in number of sorties over whole observation period, time dams spent outside the nest differed. We could correlate the number of sorties on p3 with corticosterone plasma level at p9. Control dams spent significantly more time outside in 9pm recordings than stress animals. We could show reduced interaction with social juvenile targets in sociability test for CSD mice. Light dark transition was significantly higher for control mice compared to CSD but lower for control vs ELS animals.ConclusionsBehavior in dams during ELS correlates with chronic stress coping mechanisms in offspring’s adulthood.DisclosureNo significant relationships.

Racial Differences in Cumulative Disadvantage Among Women and Its Relation to Health: Development and Preliminary Validation of the Cumulative Stress Inventory of Women's Experiences.

Background:Cumulative disadvantage (CD) is a measure of accumulated social, economic, and person-related stressors due to unequal access to resources and opportunities, which increases a person's biological risk for disease. The purpose of this research was to develop an instrument tailored to women's experiences that had intervention and translational potential. In addition, we explored whether CD contributed to racial health disparities among black and white women.Methods:In-depth life course interviews were used to assess stressful experiences of 15 black and 15 white women. Using information from the interviews, we developed the Cumulative Stress Inventory of Women's Experiences (CSI-WE) as a quantitative instrument to measure stressful life experiences from childhood to adulthood. The CSI-WE was then administered to the original 30 women for validation and feedback.Results:Qualitative and quantitative assessments were highly correlated, which suggested that the CSI-WE reliably captured the experiences of the interviewed women. Black participants reported significantly higher numbers of childhood and adult stressors, more acute adulthood and lifetime stressors, and worse adult physical self-rated health.Conclusions:This study supports the preliminary validity of an instrument that once fully validated may be used in future studies to elucidate the experiences of CD among black and white women and examines how these experiences relate to perceived and objective health status.

Older adults during the pandemic: Mental health symptoms are predicted by childhood trauma

IntroductionIt has been broadly anticipated that COVID-19 pandemic-related experiences may constitute traumatic stressors in vulnerable populations, and that older adults’ might be especially at risk of experiencing mental health symptoms during the pandemic.ObjectivesThe present study aimed to examine older adults’ psychological distress: posttraumatic stress, Covid-related fears, anxiety, and depression during the pandemic, and the relationship between present distress, defensive functioning, and childhood trauma. We also explored potential differences between younger-older adults (between 65 and 74 years), and older-older adults (75 years and above).MethodsData was collected in a large-scale online survey during the early months of the pandemic, for the present study, we included participants above 65 years old (N = 1,225).Resultsshowed that age, adverse childhood experiences, and overall defensive functioning were all significantly related to posttraumatic stress, anxiety, and depression. Specifically, younger age and more reported childhood adversity were related to higher distress, whereas higher defensive functioning was related to less distress. Covid-related fears were not associated with age. Our final model showed that defensive functioning mediated the relationship between childhood trauma and distress.ConclusionsOur results support the relative resilience of older-older adults compared to younger-older adults, as well as the long-lasting impact of childhood adversity through defensive functioning later in life, specifically in times of heightened stress, such as the COVID-19 pandemic. Future studies are warranted to identify further factors affecting defensive functioning as adults age, as well as processes that are associated with resilience in response to stressors in older adulthood.DisclosureNo significant relationships.

Transcriptomic and functional genetic evidence for distinct ecophysiological responses across complex life cycle stages.

Organisms with complex life cycles demonstrate a remarkable ability to change their phenotypes across development, presumably as an evolutionary adaptation to developmentally variable environments. Developmental variation in environmentally sensitive performance, and thermal sensitivity in particular, has been well documented in holometabolous insects. For example, thermal performance in adults and juvenile stages exhibit little genetic correlation (genetic decoupling) and can evolve independently, resulting in divergent thermal responses. Yet, we understand very little about how this genetic decoupling occurs. We tested the hypothesis that genetic decoupling of thermal physiology is driven by fundamental differences in physiology between life stages, despite a potentially conserved cellular stress response. We used RNAseq to compare transcript expression in response to a cold stressor in Drosophila melanogaster larvae and adults and used RNA interference (RNAi) to test whether knocking down nine target genes differentially affected larval and adult cold tolerance. Transcriptomic responses of whole larvae and adults during and following exposure to -5°C were largely unique both in identity of responding transcripts and in temporal dynamics. Further, we analyzed the tissue-specificity of differentially expressed transcripts from FlyAtlas 2 data, and concluded that stage-specific differences in transcription were not simply driven by differences in tissue composition. In addition, RNAi of target genes resulted in largely stage-specific and sometimes sex-specific effects on cold tolerance. The combined evidence suggests that thermal physiology is largely stage-specific at the level of gene expression, and thus natural selection may be acting on different loci during the independent thermal adaptation of different life stages.