Thermal limits of survival and reproduction depend on stress duration: A case study of Drosophila suzukii.

Studies of ectotherm responses to heat extremes often rely on assessing absolute critical limits for heat coma or death (CTmax), however, such single parameter metrics ignore the importance of stress exposure duration. Furthermore, population persistence may be affected at temperatures considerably below CTmax through decreased reproductive output. Here we investigate the relationship between tolerance duration and severity of heat stress across three ecologically relevant life-history traits (productivity, coma and mortality) using the global agricultural pest Drosophila suzukii. For the first time, we show that for sublethal reproductive traits, tolerance duration decreases exponentially with increasing temperature (R2 > 0.97), thereby extending the Thermal Death Time framework recently developed for mortality and coma. Using field micro-environmental temperatures, we show how thermal stress can lead to considerable reproductive loss at temperatures with limited heat mortality highlighting the importance of including limits to reproductive performance in ecological studies of heat stress vulnerability.

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 and the metabolic homeostasis hypotheses (MCA and MHH), it has been formulated that cold-adapted ectotherms should display both higher (MCA) and more thermally sensitive (MHH) metabolic rates (MRs) at lower temperatures. However, whether such compensation can truly be associated with distribution, and whether it interplays with cold tolerance to predict species' climatic niches, remains largely unclear despite broad ecological implications thereof. Here, we teased apart the relationship between MRs, cold tolerance and distribution, to test the MCA/MHH among 13 European 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 MRs further upheld the best predictions of species' environmental temperatures and limits of northernmost distribution. To our knowledge, this is the first study showing that the association of metabolic data with cold tolerance supports better predictive models of species' climate and distribution in social insects than models including cold tolerance alone. These results also highlight that adaptation to higher latitudes in ants involved adjustments of both cold tolerance and MRs, to allow this extremely successful group of insects to thrive under colder climates.

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 whether 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°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.

Interspecific differences in thermal tolerance landscape explain aphid community abundance under climate change.

A single critical thermal limit is often used to explain and infer the impact of climate change on geographic range and population abundance. However, it has limited application in describing the temporal dynamic and cumulative impacts of extreme temperatures. Here, we used a thermal tolerance landscape approach to address the impacts of extreme thermal events on the survival of co-existing aphid species (Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi). Specifically, we built the thermal death time (TDT) models based on detailed survival datasets of three aphid species with three ages across a broad range of stressful high (34-40 °C) and low (-3∼-11 °C) temperatures to compare the interspecific and developmental stage variations in thermal tolerance. Using these TDT parameters, we performed a thermal risk assessment by calculating the potential daily thermal injury accumulation associated with the regional temperature variations in three wheat-growing sites along a latitude gradient. Results showed that M. dirhodum was the most vulnerable to heat but more tolerant to low temperatures than R. padi and S. avenae. R. padi survived better at high temperatures than Sitobion avenae and M. dirhodum but was sensitive to cold. R. padi was estimated to accumulate higher cold injury than the other two species during winter, while M. dirhodum accrued more heat injury during summer. The warmer site had higher risks of heat injury and the cooler site had higher risks of cold injury along a latitude gradient. These results support recent field observations that the proportion of R. padi increases with the increased frequency of heat waves. We also found that young nymphs generally had a lower thermal tolerance than old nymphs or adults. Our results provide a useful dataset and method for modelling and predicting the consequence of climate change on the population dynamics and community structure of small insects.

Chilled, starved or frozen: insect mitochondrial adaptations to overcome the cold.

Physiological adaptations to tackle cold exposure are crucial for insects living in temperate and arctic environments and here we review how cold adaptation is manifested in terms of mitochondrial function. Cold challenges are diverse, and different insect species have evolved metabolic and mitochondrial adaptations to i) energize homeostatic regulation at low temperatures ii) stretch energy reserves during prolonged cold exposure, and iii) preserve the structural organization of organelles following extracellular freezing. While the literature is still sparse, our review suggests that cold-adapted insects preserve ATP production at low temperatures by maintaining preferred mitochondrial substrate oxidation, which is otherwise challenged in cold-sensitive species. Chronic cold exposure and metabolic depression during dormancy are linked to reduced mitochondrial metabolism and may involve mitochondrial degradation. Finally, adaptation to extracellular freezing could be associated with the superior structural integrity of the mitochondrial inner membrane following freezing which is linked to cellular and organismal survival.

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 to cold-sensitive species. To address this, we investigated how experimental temperatures from 1 to 19°C affected mitochondrial substrate oxidation in flight muscle of seven Drosophila species and compared it with a measure of species cold tolerance (CTmin, the temperature inducing cold coma). Mitochondrial oxygen consumption rates measured using a substrate-uncoupler-inhibitor titration (SUIT) protocol showed that cooling generally reduced oxygen consumption of the electron transport system across species, as was expected given thermodynamic effects. Complex I respiration is the primary consumer of oxygen at non-stressful 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 respiration correlates strongly with CTmin. The relative contribution of other 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.

Finding the right thermal limit: a framework to reconcile ecological, physiological and methodological aspects of CTmax in ectotherms.

Upper thermal limits (CTmax) are frequently used to parameterize the fundamental niche of ectothermic animals and to infer biogeographical distribution limits under current and future climate scenarios. However, there is considerable debate associated with the methodological, ecological and physiological definitions of CTmax. The recent (re)introduction of the thermal death time (TDT) model has reconciled some of these issues and now offers a solid mathematical foundation to model CTmax by considering both intensity and duration of thermal stress. Nevertheless, the physiological origin and boundaries of this temperature-duration model remain unexplored. Supported by empirical data, we here outline a reconciling framework that integrates the TDT model, which operates at stressful temperatures, with the classic thermal performance curve (TPC) that typically describes biological functions at permissive temperatures. Further, we discuss how the TDT model is founded on a balance between disruptive and regenerative biological processes that ultimately defines a critical boundary temperature (Tc) separating the TDT and TPC models. Collectively, this framework allows inclusion of both repair and accumulation of heat stress, and therefore also offers a consistent conceptual approach to understand the impact of high temperature under fluctuating thermal conditions. Further, this reconciling framework allows improved experimental designs to understand the physiological underpinnings and ecological consequences of ectotherm heat tolerance.

Extreme escalation of heat failure rates in ectotherms with global warming.

Temperature affects the rate of all biochemical processes in ectotherms1,2 and is therefore critical for determining their current and future distribution under global climate change3-5. Here we show that the rate of biological processes maintaining growth, homeostasis and ageing in the permissive temperature range increases by 7% per degree Celsius (median activation energy Ea = 0.48 eV from 1,351 rates across 314 species). By contrast, the processes underlying heat failure rate within the stressful temperature range are extremely temperature sensitive, such that heat failure increases by more than 100% per degree Celsius across a broad range of taxa (median Ea = 6.13 eV from 123 rates across 112 species). The extreme thermal sensitivity of heat failure rates implies that the projected increase in the frequency and intensity of heatwaves can exacerbate heat mortality for many ectothermic species with severe and disproportionate consequences. Combining the extreme thermal sensitivities with projected increases in maximum temperatures globally6, we predict that moderate warming scenarios can increase heat failure rates by 774% (terrestrial) and 180% (aquatic) by 2100. This finding suggests that we are likely to underestimate the potential impact of even a modest global warming scenario.

Phylogenetic and environmental patterns of sex differentiation in physiological traits across Drosophila species.

Sex-based differences in physiological traits may be influenced by both evolutionary and environmental factors. Here we used male and female flies from >80 Drosophila species reared under common conditions to examine variance in a number of physiological traits including size, starvation, desiccation and thermal tolerance. Sex-based differences for desiccation and starvation resistance were comparable in magnitude to those for size, with females tending to be relatively more resistant than males. In contrast thermal resistance showed low divergence between the sexes. Phylogenetic signal was detected for measures of divergence between the sexes, such that species from the Sophophora clade showed larger differences between the sexes than species from the Drosophila clade. We also found that sex-based differences in desiccation resistance, body size and starvation resistance were weakly associated with climate (annual mean temperature/precipitation seasonality) but the direction and association with environment depended on phylogenetic position. The results suggest that divergence between the sexes can be linked to environmental factors, while an association with phylogeny suggests sex-based differences persist over long evolutionary time-frames.

Acclimation, duration and intensity of cold exposure determine the rate of cold stress accumulation and mortality in Drosophila suzukii.

The spotted wing drosophila (SWD), Drosophila suzukii, is a major invasive fruit pest. There is strong consensus that low temperature is among the main drivers of SWD population distribution, and the invasion success of SWD is also linked to its thermal plasticity. Most studies on ectotherm cold tolerance focus on exposure to a single stressful temperature but here we investigated how cold stress intensity affected survival duration across a broad range of low temperatures (-7 to +3 °C). The analysis of Lt50 at different stressful temperatures (Thermal Death Time curve - TDT) is based on the suggestion that cold injury accumulation rate increases exponentially with the intensity of thermal stress. In accordance with the hypothesis, Lt50 of SWD decreased exponentially with temperature. Further, comparison of TDT curves from flies acclimated to 15, 19 and 23 °C, respectively, showed an almost full compensation with acclimation such that the temperature required to induce mortality over a fixed time decreased almost 1 °C per °C lowering of acclimation temperature. Importantly, this change in cold tolerance with acclimation was uniform across the range of moderate to intense cold stress exposures examined. To understand if cold stress at moderate and intense exposures affects the same physiological systems we examined how physiological markers/symptoms of chill injury developed at different intensities of the cold stress. Specifically, hsp23 expression and extracellular [K+] were measured in flies exposed to different intensities of cold stress (-6, -2 and +2 °C) and at various time points corresponding to the same progression of injury (equivalent to 1/3, 2/3 or 3/3 of Lt50). The different cold stress intensities all triggered hsp23 expression following 2 h of recovery, but patterns of expression differed. At the most intense cold stress (-6 and -2 °C) a gradual increase with time was found. In contrast, at +2 °C an initial increase was followed by a dissipating expression. A gradual perturbation of ion balance (hyperkalemia) was also found at all three cold stress intensities examined, with only slight dissimilarities between treatment temperatures. Despite some differences between the three cold intensities examined, the results generally support the hypothesis that intense and moderate cold stress induces the same physiological perturbation. This suggests that cold stress experienced during natural fluctuating conditions is additive and the results also illustrate that the rate of injury accumulation increases dramatically (exponentially) with decreasing temperature (increasing stress).

A unifying model to estimate thermal tolerance limits in ectotherms across static, dynamic and fluctuating exposures to thermal stress.

Temperature tolerance is critical for defining the fundamental niche of ectotherms and researchers classically use either static (exposure to a constant temperature) or dynamic (ramping temperature) assays to assess tolerance. The use of different methods complicates comparison between studies and here we present a mathematical model (and R-scripts) to reconcile thermal tolerance measures obtained from static and dynamic assays. Our model uses input data from several static or dynamic experiments and is based on the well-supported assumption that thermal injury accumulation rate increases exponentially with temperature (known as a thermal death time curve). The model also assumes thermal stress at different temperatures to be additive and using experiments with Drosophila melanogaster, we validate these central assumptions by demonstrating that heat injury attained at different heat stress intensities and durations is additive. In a separate experiment we demonstrate that our model can accurately describe injury accumulation during fluctuating temperature stress and further we validate the model by successfully converting literature data of ectotherm heat tolerance (both static and dynamic assays) to a single, comparable metric (the temperature tolerated for 1 h). The model presented here has many promising applications for the analysis of ectotherm thermal tolerance and we also discuss potential pitfalls that should be considered and avoided using this model.

Quantitative model analysis of the resting membrane potential in insect skeletal muscle: Implications for low temperature tolerance.

Abiotic stressors, such as cold exposure, can depolarize insect cells substantially causing cold coma and cell death. During cold exposure, insect skeletal muscle depolarization occurs through a 2-stage process. Firstly, short-term cold exposure reduces the activity of electrogenic ion pumps, which depolarize insect muscle markedly. Secondly, during long-term cold exposure, extracellular ion homeostasis is disrupted causing further depolarization. Consequently, many cold hardy insects improve membrane potential stability during cold exposure through adaptations that secure maintenance of ion homeostasis during cold exposure. Less is known about the adaptations permitting cold hardy insects to maintain membrane potential stability during the initial phase of cold exposure, before ion balance is disrupted. To address this problem it is critical to understand the membrane components (channels and transporters) that determine the membrane potential and to examine this question the present study constructed a mathematical "charge difference" model of the insect muscle membrane potential. This model was parameterized with known literature values for ion permeabilities, ion concentrations and membrane capacitance and the model was then further developed by comparing model predictions against empirical measurements following pharmacological inhibitors of the Na+/K+ ATPase, Cl- channels and symporters. Subsequently, we compared simulated and recorded membrane potentials at 0 and 31 °C and at 10-50 mM extracellular [K+] to examine if the model could describe membrane potentials during the perturbations occurring during cold exposure. Our results confirm the importance of both Na+/K+ ATPase activity and ion-selective Na+, K+ and Cl- channels, but the model also highlights that additional electroneutral flux of Na+ and K+ is needed to describe how membrane potentials respond to temperature and [K+] in insect muscle. While considerable further work is still needed, we argue that this "charge difference" model can be used to generate testable hypotheses of how insects can preserve membrane polarization in the face of stressful cold exposure.

Osmoregulatory capacity at low temperature is critical for insect cold tolerance.

At low temperature many insects lose extracellular ion homeostasis and the capacity to mitigate homeostatic imbalance determines their cold tolerance. Extracellular homeostasis is ensured by the osmoregulatory organs and recent research has emphasized key roles for the Malpighian tubules and hindgut in modulating insect cold tolerance. Here, we review the effects of low temperature on transport capacity of osmoregulatory organs and outline physiological processes leading from cold exposure to disruption of ion homeostasis and cold-injury in insects. We show how cold adaptation and cold acclimation are associated with physiological modifications to transport capacity in Malpighian tubules and hindgut. These responses mitigate loss of homeostasis and we highlight how further study of molecular and cellular mechanisms are critical to fully appreciate the adaptations that facilitate insect cold tolerance.

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 rate 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 affect mitochondrial function and substrate oxidation. At benign temperatures (19 and 30°C), complex I-supported respiration (CI-OXPHOS) was the most significant contributor to maximal OXPHOS. At higher temperatures (34, 38, 42 and 46°C), CI-OXPHOS decreased considerably, ultimately to very low levels at 42 and 46°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 capacity was 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 failure of CI-OXPHOS and compensatory oxidation of alternative substrates occurred at temperatures that correlated 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.

Cold acclimation preserves hindgut reabsorption capacity at low temperature in a chill-susceptible insect, Locusta migratoria.

Cold acclimation increases cold tolerance of chill-susceptible insects and the acclimation response often involves improved organismal ion balance and osmoregulatory function at low temperature. However, the physiological mechanisms underlying plasticity of ion regulatory capacity are largely unresolved. Here we used Ussing chambers to explore the effects of cold exposure on hindgut KCl reabsorption in cold- (11 °C) and warm-acclimated (30 °C) Locusta migratoria. Cooling (from 30 to 10 °C) reduced active reabsorption across recta from warm-acclimated locusts, while recta from cold-acclimated locusts maintained reabsorption at 10 °C. The differences in transport capacity were not linked to major rearrangements of membrane phospholipid profiles. Yet, the stimulatory effect of two signal transduction pathways were altered by temperature and/or acclimation. cAMP-stimulation increased reabsorption in both acclimation groups, with a strong stimulatory effect at 30 °C and a moderate stimulatory effect at 10 °C. cGMP-stimulation also increased reabsorption in both acclimation groups at 30 °C, but their response to cGMP differed at 10 °C. Recta from warm-acclimated locusts, characterised by reduced reabsorption at 10 °C, recovered reabsorption capacity following cGMP-stimulation at 10 °C. In contrast, recta from cold-acclimated locusts, characterised by sustained reabsorption at 10 °C, were unaffected by cGMP-stimulation. Furthermore, cold-exposed recta from warm-acclimated locusts were insensitive to bafilomycin-α1, a V-type H+-ATPase inhibitor, whereas this blocker reduced reabsorption across cold-exposed recta from cold-acclimated animals. In conclusion, bafilomycin-sensitive and cGMP-dependent transport mechanism(s) are likely blocked during cold exposure in warm-acclimated animals while preserved in cold-acclimated animals. These may in part explain the large differences in rectal ion transport capacity between acclimation groups at low temperature.

Cold acclimation increases depolarization resistance and tolerance in muscle fibers from a chill-susceptible insect, Locusta migratoria.

Cold exposure depolarizes cells in insects due to a reduced electrogenic ion transport and a gradual increase in extracellular K+ concentration ([K+]). Cold-induced depolarization is linked to cold injury in chill-susceptible insects, and the locust, Locusta migratoria, has been shown to improve cold tolerance following cold acclimation through depolarization resistance. Here we investigate how cold acclimation influences depolarization resistance and how this resistance relates to improved cold tolerance. To address this question, we investigated if cold acclimation affects the electrogenic transport capacity and/or the relative K+ permeability during cold exposure by measuring membrane potentials of warm- and cold-acclimated locusts in the presence and absence of ouabain (Na+-K+ pump blocker) or 4-aminopyridine (4-AP; voltage-gated K+ channel blocker). In addition, we compared the membrane lipid composition of muscle tissue from warm- and cold-acclimated locust and the abundance of a range transcripts related to ion transport and cell injury accumulation. We found that cold-acclimated locusts are depolarization resistant due to an elevated K+ permeability, facilitated by opening of 4-AP-sensitive K+ channels. In accordance, cold acclimation was associated with an increased abundance of Shaker transcripts (gene encoding 4-AP-sensitive voltage-gated K+ channels). Furthermore, we found that cold acclimation improved muscle cell viability following exposure to cold and hyperkalemia even when muscles were depolarized substantially. Thus cold acclimation confers resistance to depolarization by altering the relative ion permeability, but cold-acclimated locusts are also more tolerant to depolarization.

Comparison of Static and Dynamic Assays When Quantifying Thermal Plasticity of Drosophilids.

Numerous assays are used to quantify thermal tolerance of arthropods including dynamic ramping and static knockdown assays. The dynamic assay measures a critical temperature while the animal is gradually heated, whereas the static assay measures the time to knockdown at a constant temperature. Previous studies indicate that heat tolerance measured by both assays can be reconciled using the time × temperature interaction from "thermal tolerance landscapes" (TTLs) in unhardened animals. To investigate if this relationship remains true within hardened animals, we use a static assay to assess the effect of heat hardening treatments on heat tolerance in 10 Drosophila species. Using this TTL approach and data from the static heat knockdown experiments, we model the expected change in dynamic heat knockdown temperature (CTmax: temperature at which flies enter coma) and compare these predictions to empirical measurements of CTmax. We find that heat tolerance and hardening capacity are highly species specific and that the two assays report similar and consistent responses to heat hardening. Tested assays are therefore likely to measure the same underlying physiological trait and provide directly comparable estimates of heat tolerance. Regardless of this compliance, we discuss why and when static or dynamic assays may be more appropriate to investigate ectotherm heat tolerance.

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

When heated, insects lose coordinated movement followed by the onset of heat coma (critical thermal maximum, CTmax). These traits are popular measures to quantify interspecific and intraspecific differences in insect heat tolerance, and CTmax correlates well with current species distributions of insects, including Drosophila 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 gradually 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 behavioural 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; to examine whether 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 versus that of the whole animal, we found that the head was not particularly heat sensitive compared with the abdomen. Accordingly, it is unlikely that nervous failure is the principal/proximate cause of heat mortality in Drosophila.

Maintenance of hindgut reabsorption during cold exposure is a key adaptation for Drosophila cold tolerance.

Maintaining extracellular osmotic and ionic homeostasis is crucial for organismal function. In insects, hemolymph volume and ion content is regulated by 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). Accordingly, studies have found a strong link between species-specific cold tolerance and the ability to maintain ion and water homeostasis at low temperature. This is also true for drosophilids where inter- and intra-specific differences in cold tolerance are linked to the secretory capacity of Malpighian tubules. There is, however, little information on the reabsorptive capacity of the hindgut in Drosophila To address this, we developed a novel method that permits continuous measurements of hindgut ion and fluid reabsorption in Drosophila We demonstrate that this assay is temporally stable (∼2 h) and responsive to cAMP stimulation and pharmacological intervention in accordance with the current insect hindgut reabsorption model. We then investigated how cold acclimation or cold adaptation affected hindgut reabsorption at benign (24°C) and low temperature (3°C). Cold-tolerant Drosophila species and cold-acclimated D. melanogaster maintain superior fluid and Na+ reabsorption at low temperature. Furthermore, cold adaptation and acclimation caused a relative reduction in K+ reabsorption at low temperature. These characteristic responses of cold adaptation/acclimation will promote maintenance of ion and water homeostasis at low temperature. Our study of hindgut function therefore provides evidence that adaptations in the osmoregulatory capacity of insects are critical for their ability to tolerate cold.

Temperature preference across life stages and acclimation temperatures investigated in four species of Drosophila.

Ectotherms can use microclimatic variation and behavioral thermoregulation to cope with unfavorable environmental temperatures. However, relatively little is known about how and if thermoregulatory behavior is used across life stages in small ectothermic insects. Here we investigate differences between three specialized Drosophila species from temperate, tropical or desert habitats and one cosmopolitan species by estimating the preferred temperature (Tpref) and the breadth (Tbreadth) of the distribution of adults, adult egg-laying, and larvae in thermal gradients. We also assess the plasticity of thermal preference following developmental acclimation to three constant temperatures. For egg-laying and larvae, we observe significant species differences in preferred temperature but this is not predicted by thermal ecology of the species. We corroborated this with previous studies of other Drosophila species and found that Tpref for egg laying and larvae have no relationship with annual mean temperature of the species' natural habitat. While adults have the greatest mobility, they show the greater variation in preference compared to juveniles contradicting common assumptions. We found evidence of developmental thermal acclimation in adult egg-laying preferred temperature, Tpref increasing with acclimation temperature, and in the breadth of the temperature preference distributions, Tbreadth decreasing with increasing acclimation temperature. Together, these data provide a high resolution and comprehensive look at temperature preferences across life stages and in response to acclimation. Results suggest that thermal preference, particularly in the early life stages, is relatively conserved among species and unrelated to temperature at species origin. Measuring thermal preference, in addition to thermal performance, is essential for understanding how species have adapted/will adapt to their thermal environment.