Impacts of thermal fluctuations on heat tolerance and its metabolomic basis in <i>Arabidopsis thaliana, Drosophila melanogaster</i>, and <i>Orchesella cincta</i>

Temperature varies on a daily and seasonal scale and thermal fluctuations are predicted to become even more pronounced under future climate changes. Studies suggest that plastic responses are crucial for species' ability to cope with thermal stress including variability in temperature, but most often laboratory studies on thermal adaptation in plant and ectotherm organisms are performed at constant temperatures and few species included. Recent studies using fluctuating thermal regimes find that thermal performance is affected by both temperature mean and fluctuations, and that plastic responses likely will differ between species according to life strategy and selective past. Here we investigate how acclimation to fluctuating or constant temperature regimes, but with the same mean temperature, impact on heat stress tolerance across a plant (Arabidopsis thaliana) and two arthropod species (Orchesella cincta and Drosophila melanogaster) inhabiting widely different thermal microhabitats and with varying capability for behavioral stress avoidance. Moreover, we investigate the underlying metabolic responses of acclimation using NMR metabolomics. We find increased heat tolerance for D. melanogaster and A. thaliana exposed to fluctuating acclimation temperatures, but not for O. cincta. The response was most pronounced for A. thaliana, which also showed a stronger metabolome response to thermal fluctuations than both arthropods. Generally, sugars were more abundant across A. thaliana and D. melanogaster when exposed to fluctuating compared to constant temperature, whereas amino acids were less abundant. This pattern was not evident for O. cincta, and generally we do not find much evidence for similar metabolomics responses to fluctuating temperature acclimation across species. Differences between the investigated species' ecology and different ability to behaviorally thermoregulate may have shaped their physiological responses to thermal fluctuations

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 &times; temperature interaction from &ldquo;thermal tolerance landscapes&rdquo; (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

Rapid Adjustments in Thermal Tolerance and the Metabolome to Daily Environmental Changes – A Field Study on the Arctic Seed Bug Nysius groenlandicus

International audienceLaboratory investigations on terrestrial model-species, typically of temperate origin, have demonstrated that terrestrial ectotherms can cope with daily temperature variations through rapid hardening responses. However, few studies have investigated this ability and its physiological basis in the field. Especially in polar regions, where the temporal and spatial temperature variations can be extreme, are hardening responses expected to be important. Here, we examined diurnal adjustments in heat and cold tolerance in the Greenlandic seed bug Nysius groenlandicus by collecting individuals for thermal assessment at different time points within and across days. We found a significant correlation between observed heat or cold tolerance and the ambient microhabitat temperatures at the time of capture, indicating that N. groenlandicus continuously and within short time-windows respond physiologically to thermal changes and/or other environmental variables in their microhabitats. Secondly, we assessed underlying metabolomic fingerprints using GC-MS metabolomics in a subset of individuals collected during days with either low or high temperature variation. Concentrations of metabolites, including sugars, polyols, and free amino acids varied significantly with time of collection. For instance, we detected elevated sugar levels in animals caught at the lowest daily field temperatures. Polyol concentrations were lower in individuals collected in the morning and evening and higher at midday and afternoon, possibly reflecting changes in temperature. Additionally, changes in concentrations of metabolites associated with energetic metabolism were observed across collection times. Our findings suggest that in these extreme polar environments hardening responses are marked and likely play a crucial role for coping with microhabitat temperature variation on a daily scale, and that metabolite levels are actively altered on a daily basis

Responses of terrestrial polar arthropods to high and increasing temperatures

Terrestrial arthropods in the Arctic and Antarctic are exposed to extreme and variable temperatures, and climate change is predicted to be especially pronounced in these regions. Available studies on insect ecophysiology typically focus on the ability of species to tolerate the extreme low temperatures that can occur in these regions, whereas studies investigating species' plasticity and the importance of evolutionary adaptation to periodically high and increasing temperatures are limited. Here, we provide an overview of current knowledge on thermal adaptation to high temperatures of terrestrial arthropods in Arctic and Antarctic regions. Firstly, we summarize the literature on heat tolerance for terrestrial arthropods in these regions, and discuss variation in heat tolerance across species, habitats and polar regions. Secondly, we discuss the potential for species to cope with increasing and more variable temperatures through thermal plasticity and evolutionary adaptation. Thirdly, we summarize our current knowledge of the underlying physiological adjustments to heat stress in arthropods from these regions. It is clear that very little data are available on the heat tolerance of arthropods in polar regions, but that large variation in arthropod thermal tolerance exists across polar regions, habitats and species. Further, the species investigated show unique physiological adjustments to heat stress in, for example, being able to respond quickly to increasing or extreme temperatures. To understand the consequences of climate change on terrestrial arthropods in polar regions, we suggest that more studies on the ability of species to cope with stressful high and variable temperatures are needed.Funding provided by: CarlsbergfondetCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100002808Award Number: CF17-0415Funding provided by: Natur og Univers, Detict Frie ForskningsrådCrossref Funder Registry ID: http://dx.doi.org/10.13039/100008394Award Number: DFF-8021-00014BUpper thermal tolerance limits (CTmax) were measured on nine species collected at two locations in Narsarsuaq, Greenland. The individuals used for thermal assays were collected in the field using species-specific catch methods (Table S1). Adults of unknown age and gender were tested. To measure CTmax, a dynamic ramping assay was used. Field-caught individuals were placed in 15 ml plastic vials with screw caps with a droplet of 2% agar to prevent desiccation during exposure. The vials were mounted to a rack and lowered into a water bath with a temperature of 25°C. Subsequently, the temperature was increased by 0.2 ± 0.01 °C min-1 using an immersion circulator (Polyscience MX Immersion Circulator model: MX-CA12E). Individuals in each vial were continuously stimulated with a flashlight and tapping on the screw cap with a rod until reaching a temperature at which movement ceased (heat coma). The temperature of heat coma was recorded for each individual. Individuals were stored in 70% ethanol after the thermal assay for later identification. Species were identified based on morphological features using a species identification key by Böcher et al. (2015)

Rapid induction of the heat hardening response in an Arctic insect

The ability to cope with increasing and more variable temperatures, due to predicted climate changes, through plastic and/or evolutionary responses will be crucial for the persistence of Arctic species. Here, we investigate plasticity of heat tolerance of the Greenlandic seed bug Nysius groenlandicus, which inhabits areas with widely fluctuating temperatures. We test the heat resistance and hardening capacity (plasticity) of N. groenlandicus using both static (heat knock down time, HKDT) and dynamic (critical thermal maximum, CTmax) assays. We find that N. groenlandicus is able to tolerate short-term exposure to temperatures up to almost 50°C and that it can quickly increase heat resistance following heat hardening. Further, we find that this hardening response is reversible within hours after hardening. These findings contrast with common observations from temperate and tropical insects and suggest that more attention should be given to species in extreme and thermally variable environments such as Arctic and subarctic regions.Hardening during temperature ramping (Figure 1) Heat hardening and recovery time (Figure 2a) Thermal exposure time (Figure 2b) Figure S2 (Heat hardening and recovery time) Supplementary data Figure S3 (Heat hardening and recovery time) Supplementary dat

Contrasting Manual and Automated Assessment of Thermal Stress Responses and Larval Body Size in Black Soldier Flies and Houseflies

International audienceWithin ecophysiological and genetic studies on insects, morphological and physiological traits are commonly assessed and phenotypes are typically obtained from manual measurements on numerous individuals. Manual observations are, however, time consuming, can introduce observer bias and are prone to human error. Here, we contrast results obtained from manual assessment of larval size and thermal tolerance traits in black soldier flies (Hermetia illucens) and houseflies (Musca domestica) that have been acclimated under three different temperature regimes with those obtained automatically using an image analysis software (Noldus EthoVision XT). We found that (i) larval size estimates of both species, obtained by manual weighing or by using the software, were highly correlated, (ii) measures of heat and cold tolerance using manual and automated approaches provided qualitatively similar results, and (iii) by using the software we obtained quantifiable information on stress responses and acclimation effects of potentially higher ecological relevance than the endpoint traits that are typically assessed when manual assessments are used. Based on these findings, we argue that automated assessment of insect stress responses and largescale phenotyping of morphological traits such as size will provide new opportunities within many disciplines where accurate and largescale phenotyping of insects is required