Biology subjects

Overgaard, J.

Publications and source records attributed to Overgaard, J..

12 recordsLinked to original sources

bioRxiv2026

A conserved cold stress syndrome defines insect chill tolerance

The ability to withstand cold is arguably one of the most important traits defining the biogeographical distribution of insects. For most species, those classified as chill susceptible, cold tolerance is limited by the ability to maintain vital physiological functions at low temperature. However, there is currently no unified understanding of how physiological failure develops during cold stress or how this process explains variation in cold sensitivity within and among species. Although disruption of ion homeostasis has been implicated in chilling injury, it remains unclear whether it represents a common physiological limit across cold stress intensities and degrees of chill tolerance. Here, we tested whether ion balance disruption represents a unifying physiological syndrome of chill susceptibility by simultaneously quantifying cold mortality and hemolymph [K] in Drosophila. We examined this relationship across i) a broad range of cold stress intensities, ii) 25 species spanning extensive variation in cold sensitivity, biogeography and phylogeny, and iii) acclimation groups of D. melanogaster developed between 12 and 27{degrees}C. Across all comparisons, mortality was associated with the same cold-stress response, characterized by progressive hyperkalemia and mortality following a ~150% increase in hemolymph [K] above baseline ([K]crit). In contrast, adaptive and plastic variation in cold sensitivity was explained by differences in the critical temperature at which ion homeostasis failed (Tc). Extending this framework to published data from other insect orders revealed a remarkably similar [K]crit, suggesting that ionoregulatory collapse represents a conserved physiological constraint defining insect cold survival.

physiology↗

bioRxiv2026

Using thermal death time models to analyze cold stress resistance across Drosophila species

O_LIChill-susceptible insects such as Drosophila are vulnerable to progressive disruption of ion and water homeostasis during cold stress, and low temperature exposure is a key factor affecting their physiology and distribution. Comparative studies of cold tolerance traditionally use simple or single-condition assays for interspecific comparisons, but the emergence of thermal death time (TDT) models offers a comprehensive framework to assess cold tolerance across different stress intensities and durations. C_LIO_LIHere we construct TDT curves for six Drosophila species, spanning boreal to tropical habitats, using Lt50 estimates across a range of stressful low temperatures (Lt50 ranging from [~] 20 min to 2 days). For all species, the TDT curves provided good fits to the log(Lt50) vs. temperature data (R2 = 0.87 - 0.99). C_LIO_LITDT curves from all species had steep slopes demonstrating that cold injury rate has a high thermal sensitivity such that small changes in temperature have profound effects on survival duration. The interspecific similarity of TDT slopes indicates that a conserved physiological dysfunction underlies cold injury across species. Further, additive accumulation of cold-induced injury in split-dose experiments suggests that acute and moderate cold damage represent the same underlying physiological dysfunction occurring at different rates. C_LIO_LIThe TDT curve intercepts (species-specific tolerance thresholds) differed markedly between boreal, temperate, and tropical species and correlated strongly with their habitat temperature. Data from the present study and meta-analysis of published data find that the inherent species cold tolerance decreases by [~] 0.45 {degrees}C for each {degrees}C colder the winter environment of the species is. When also considering the cold acclimation cues in cold climates we argue the experienced level of cold stress intensity is similar across environments inhabited by the Drosophila genus. This suggests that cold tolerance is important in shaping the fundamental niche of both boreal and tropical species. C_LIO_LIOverall, the TDT analysis of Drosophila at low temperature provides a powerful and predictive tool for quantifying insect cold tolerance. This approach enables detailed cross-species comparisons that allows for both ecological and physiological inference. Thus, TDT curves offer relevant approximations of insect cold resistance that could help predict insect responses to climatic change. C_LI

ecology↗

bioRxiv2025

Using gas exchange measurements to monitor growth, energy expenditure, and body composition of black soldier fly larvae (Hermetia illucens) on diets varying in protein-to-carbohydrate ratio

Optimizing black soldier fly larvae (BSFL) production requires a better understanding of how diet composition shapes growth, nutrient allocation, and metabolic efficiency. This study investigated whether respiratory measurements could serve as predictive, non-invasive tools for BSFL performance on diets differing in protein-to-carbohydrate (P:C) ratio. Larvae were reared for seven days on four experimental diets, with growth, survival, gas exchange (O2 consumption, CO2 production, respiratory exchange ratio (RER)), and body composition (crude protein and lipid contents) measured across seven replicates per treatment. O2 consumption correlated strongly with final larval biomass, while RER was closely associated with protein and lipid deposition. Larvae on low-protein, high-carbohydrate diets accumulated proportionally more lipid, whereas protein-rich diets increased crude protein content but required higher energetic investment. This demonstrates that lipid-rich larvae grew more efficient in energetic terms per unit accumulated biomass than protein-rich larvae. Across diets, gas exchange variables reliably reflected growth dynamics, metabolic activity, and nutrient assimilation. Integrating respirometry into rearing systems could enable real-time monitoring of insect biomass yield and nutrient composition.

physiology↗

bioRxiv2025

Consequences of dietary amino acid imbalance on growth and gas exchange in black soldier fly (Hermetia illucens) larvae

Black soldier fly larvae (Hermetia illucens (L.); BSFL) production relies on efficient nutrient conversion into protein-rich biomass to ensure sustainability and economic viability. While the effects of dietary protein content and macronutrient ratios are well described, the metabolic costs of providing an imbalanced essential amino acid (EAA) supply in dietary proteins remain poorly understood. We examined how dietary EAA imbalance affects growth, metabolism, and energy allocation in BSFL. Larvae were reared on a reference diet (chicken feed) and three isonitrogenous, semi-synthetic diets: one balanced diet matching the EAA profile of the reference, and two unbalanced diets selectively deficient in either leucine, phenylalanine, and threonine or isoleucine, lysine, and tryptophan. Using open flow respirometry, daily growth monitoring, and biochemical analysis, we quantified gas exchange, body composition, and the energetic costs of biomass and protein deposition. EAA imbalance delayed development, reduced growth and protein content, and promoted lipid accumulation, while O2 consumption and CO2 production were reduced and respiratory exchange ratios exceeded 1.0, indicating elevated lipogenesis. Metabolic costs per unit protein deposited were higher on EAA-deficient diets (up to 3.63 kJ per kJ protein), reflecting inefficient amino acid utilisation. In contrast, the balanced diet supported more efficient protein deposition at a lower energetic cost (2.73 kJ per kJ protein). Our findings demonstrate that suboptimal dietary EAA profiles reduce metabolic efficiency in BSFL. Quantifying the physiological costs of amino acid imbalance provides benchmarks to guide feed formulations in insect production systems to minimise input costs and maximise the nutritional quality and quantity of larval output.

physiology↗

bioRxiv2025

Endogenous circannual cycles drive seasonal cold hardening in temperate ants

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 "Kipyatkovs 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 (CTmin) and the temperature causing 50% mortality during acute cold exposure (LTe50), 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.

evolutionary biology↗

bioRxiv2025

Chill tolerant Drosophila species maintain electrogenic muscle membrane potential to resist cold-induced depolarization

The ability to tolerate low temperature is among the most important traits defining the functional niche of insects and it clear that cold tolerance of most insects is intimately linked to their ability to defend membrane potential (Vm). Failure to maintain membrane polarization results in loss of neuromuscular function and may ultimately initiate cell death and organismal injury. Prolonged cold exposure challenges membrane polarization through loss of transmembrane ion balance; however, the insect muscle Vm is also dependent on a strong and temperature-dependent electrogenic effect driven by Na+/K+-ATPase activity. In the present study we investigate the electrogenic contribution of the Na+/K+-ATPase at benign (20{degrees}C) and low (0{degrees}C) temperature in ten Drosophila species representing a broad spectrum of chill tolerance. We find that the electrogenic effect of the Na+/K+-ATPase contributes a considerable component of the muscle Vm in all ten species at 20{degrees}C. This electrogenic contribution is reduced significantly at 0{degrees}C in the chill sensitive species, while tolerant species retain their electrogenic effect at low temperature. Thus, the initial cold-induced muscle depolarization, that is a hallmark of chill sensitive insects, is largely caused by loss of Na+/K+-ATPase-dependent electrogenic polarization. We hypothesized that maintenance of Na+/K+-ATPase activity in the cold would be energetically costly, but in contrast to our hypothesis we find no evidence for major energetic costs in the species that maintain membrane polarization at low temperature. On the basis of these observations we discuss how other adaptations at the protein or membrane level could explain the observed intraspecific differences.

physiology↗

bioRxiv2023

Cold Comfort: metabolic rate and tolerance to low temperatures predict latitudinal distribution in ants

Metabolic compensation has been proposed as a mean for ectotherms to cope with colder climates. For example, under the metabolic cold adaptation/metabolic homeostasis hypotheses (MCA/MHH), it has been formulated that cold-adapted ectotherms should display higher/more thermally sensitive metabolic rates (MRs) at lower temperatures. However, whether such compensation can truly be associated with distribution, and whether it interplays with cold-tolerance to support species climatic niches, remains largely unclear despite broad ecological implications thereof. Here, we teased apart the relationship between MRs, cold-tolerance, and distribution, to confront the MCA/MHH among 13 ant species. We report clear metabolic compensation effects, consistent with the MCA and MHH, where MR parameters strongly correlated with latitude and climatic factors across species distributions. The combination of both cold-tolerance and MR further upheld the best predictions of species climatic niches. To our knowledge, this is the first study showing that the association of metabolic data with cold-tolerance supports increased predictive value for biogeographical models in social insects. These results also highlight that adaptation to higher latitudes in ants involved adjustments of both cold-tolerance and MRs, potentially at the expense of metabolic performance at warmer temperatures, to allow this extremely successful group of insects to thrive under colder climates.

evolutionary biology↗

bioRxiv2022

Balanced mitochondrial function at low temperature is linked to cold adaptation in Drosophila species

The ability of ectothermic animals to live in different thermal environments is closely associated with their capacity to maintain physiological homeostasis across diurnal and seasonal temperature fluctuations. For chill-susceptible insects, such as Drosophila, cold tolerance is tightly linked to ion and water homeostasis obtained through a regulated balance of active and passive transport. Active transport at low temperature requires a constant delivery of ATP and we therefore hypothesize that cold-adapted Drosophila are characterized by superior mitochondrial capacity at low temperature relative cold-sensitive species. To address this, we investigated how experimental temperatures 19-1 {degrees}C affected mitochondrial substrate oxidation in flight muscle of seven tropical and temperate Drosophila species that represent a broad spectrum of cold tolerance. Mitochondrial oxygen consumption rates measured using a substrate-uncoupler-inhibitor-titration protocol showed that cooling generally reduced oxygen consumption of all steps of the electron transport system across species. Complex I is the primary consumer of oxygen at benign temperatures, but low temperature decreases complex I respiration to a much greater extent in cold-sensitive species than in cold-adapted species. Accordingly, cold-induced reduction of complex I correlates strongly with CTmin (the temperature inducing cold coma). The relative contribution of alternative substrates, proline, succinate and glycerol-3-phosphate increased as temperature decreased, particularly in the cold-sensitive species. At present it is unclear whether the oxidation of alternative substrates can be used to offset the effects of the temperature-sensitive complex I, and the potential functional consequences of such a substrate switch are discussed. Summary statementMitochondrial oxygen consumption decreases at low temperature, particularly in cold-sensitive Drosophila species, which turn to oxidation of alternative substrates as complex I-supported respiration is impaired.

physiology↗

bioRxiv2022

Mortality without springing a leak: Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown if the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2{degrees}C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

physiology↗

bioRxiv2020

Dramatic changes in mitochondrial substrate use at critically high temperatures: a comparative study using Drosophila

Ectotherm thermal tolerance is critical to species distribution, but at present the physiological underpinnings of heat tolerance remain poorly understood. Mitochondrial function is perturbed at critically high temperatures in some ectotherms, including insects, suggesting that heat tolerance of these animals is linked to failure of oxidative phosphorylation (OXPHOS) and/or ATP production. To test this hypothesis we measured mitochondrial oxygen consumption rates in six Drosophila species with different heat tolerance using high-resolution respirometry. Using a substrate-uncoupler-inhibitor titration protocol we examined specific steps of the electron transport system to study how temperatures below, bracketing and above organismal heat limits affected mitochondrial function and substrate oxidation. At benign temperatures (19 and 30{degrees}C), complex I-supported respiration (CI-OXPHOS) was the most significant contributor to maximal OXPHOS. At higher temperatures (34, 38, 42 and 46{degrees}C), CI-OXPHOS decreased considerably, ultimately to very low levels at 42 and 46{degrees}C. The enzymatic catalytic capacity of complex I was intact across all temperatures and accordingly the decreased CI-OXPHOS is unlikely to be caused directly by hyperthermic denaturation/inactivation of complex I. Despite the reduction in CI-OXPHOS, maximal OXPHOS capacities were maintained in all species, through oxidation of alternative substrates; proline, succinate and, particularly, glycerol-3-phosphate, suggesting important mitochondrial flexibility at temperatures exceeding the organismal heat limit. Interestingly, this compensatory oxidation of alternative substrates occurred at temperatures that tended to correlate with species heat tolerance, such that heat-tolerant species could defend "normal" mitochondrial function at higher temperatures than sensitive species. Future studies should investigate why CI-OXPHOS is perturbed and how this potentially affects ATP production rates.

physiology↗

bioRxiv2019

Neural dysfunction correlates with heat coma and CTmax in Drosophila but does not set the boundaries for heat stress survival

When heated, insects loose coordinated movement followed by the onset of heat coma (CTmax). These phenotypes are popular measures to quantify inter- and intraspecific differences in insect heat tolerance, and CTmax correlate well with current species distributions. Here we examined the function of the central nervous system (CNS) in five species of Drosophila with different heat tolerances, while they were exposed to either constant high temperature or a gradual increasing temperature (ramp). Tolerant species were able to preserve CNS function at higher temperatures and for longer durations than sensitive species and similar differences were found for the behavioral indices (loss of coordination and onset of heat coma). Furthermore, the timing and temperature (constant and ramp exposure, respectively) for loss of coordination or complete coma coincided with the occurrence of spreading depolarisation (SD) events in the CNS. These SD events disrupt neurological function and silence the CNS suggesting that CNS failure is the primary cause of impaired coordination and heat coma. Heat mortality occurs soon after heat coma in insects and to examine if CNS failure could also be the proximal cause of heat death, we used selective heating of the head (CNS) and abdomen (visceral tissues). When comparing the temperature causing 50% mortality (LT50) of each body part to that of the whole animal, we found that the head was not particularly heat sensitive compared to the abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in Drosophila. Summary statementHyperthermic failure of the Drosophila central nervous system causes heat coma, a phenotype varying in temperature between drosophilids, but neural failure is likely not the primary cause of heat mortality.

physiology↗

bioRxiv2019

Inter- and intraspecific differences in Drosophila cold tolerance are linked to hindgut reabsorption capacity

Maintaining extracellular osmotic and ionic homeostasis is crucial to maintain organismal function. In insects, hemolymph volume and ion content is regulated by the combined actions of the secretory Malpighian tubules and reabsorptive hindgut. When exposed to stressful cold, homeostasis is gradually disrupted, characterized by a debilitating increase in extracellular K+ concentration (hyperkalemia). In accordance with this paradigm, studies have found a strong link between the cold tolerance of insect species and their ability to maintain ion and water homeostasis at low temperature. This is also the case for drosophilids where studies have already established how inter- and intra-specific differences in cold tolerance are linked to the secretory capacity of Malpighian tubules. However, presently there is little information on the effects of temperature on the reabsorptive capacity of the hindgut in Drosophila. To address this question we developed a novel method that allows for continued measurements of hindgut ion and fluid reabsorption in Drosophila. Firstly we demonstrate that this assay is temporally stable (> 3 hours) and that the preparation is responsive to humoral stimulation and pharmacological intervention of active and passive transport in accordance with the current insect hindgut reabsorption model. Using this method at benign (24{degrees}C) and low temperature (3{degrees}C) we investigated how cold acclimation or cold adaptation affected the thermal sensitivity of osmoregulatory function. We found that cold tolerant Drosophila species and cold-acclimated D. melanogaster are innately better at maintaining rates of fluid and Na+ reabsorption at low temperature. Furthermore, cold adaptation and acclimation causes a relative reduction in K+ reabsorption at low temperature. These characteristic responses of cold adapted/acclimated Drosophila will act to promote maintenance of ion and water homeostasis at low temperature and therefore provide further links between adaptations in osmoregulatory capacity of insects and their ability to tolerate cold exposure.

physiology↗