Papers by Heath Macmillan
Proceedings of the Royal Society B: Biological Sciences, 2015
Chilling injury and death are the ultimate consequence of low temperature exposure for chill susc... more Chilling injury and death are the ultimate consequence of low temperature exposure for chill susceptible insects, and low temperature tolerance is considered one of the most important factors determining insect distribution patterns. The physiological mechanisms that cause chilling injury are unknown, but chronic cold exposure that causes injury is consistently associated with elevated extracellular [K(+)], and cold tolerant insects possess a greater capacity to maintain ion balance at low temperatures. Here, we use the muscle tissue of the migratory locust (Locusta migratoria) to examine whether chill injury occurs during cold exposure or following return to benign temperature and we specifically examine if elevated extracellular [K(+)], low temperature, or a combination thereof causes cell death. We find that in vivo chill injury occurs during the cold exposure (when extracellular [K(+)] is high) and that there is limited capacity for repair immediately following the cold stress. Further, we demonstrate that that high extracellular [K(+)] causes cell death in situ, but only when experienced at low temperatures. These findings strongly suggest that that the ability to maintain ion (particularly K(+)) balance is critical to insect low temperature survival, and highlight novel routes of study in the mechanisms regulating cell death in insects in the cold.
The Journal of experimental biology, 2015
Chill-susceptible insects enter a reversible paralytic state, termed chill coma, at mild low temp... more Chill-susceptible insects enter a reversible paralytic state, termed chill coma, at mild low temperatures. Chill coma is caused by neuromuscular impairment, allegedly triggered by cold-induced depolarization of muscle resting membrane potential (Vm). We used five Drosophila species that vary in cold tolerance (chill coma temperature spanning ∼11°C) and repeatedly measured muscle Vm during a downward temperature ramp (20 to -3°C). Cold-tolerant species were able to defend their Vm down to lower temperatures, which is not explained by species-specific differences in initial Vm at 20°C, but by cold-tolerant drosophilids defending Vm across a broad range of temperatures. We found support for a previously suggested 'critical threshold' of Vm, related to chill coma, in three of the five species. Interestingly, the cold-tolerant Drosophila species may enter coma as a result of processes unrelated to muscle depolarization as their Vm was not significantly depolarized at their chill ...
Journal of Insect Physiology, 2015
Most insects are chill susceptible and will enter a coma if exposed to sufficiently low temperatu... more Most insects are chill susceptible and will enter a coma if exposed to sufficiently low temperature. This chill coma has been associated with a failure of the neuromuscular system. Insect heart rate (HR) is determined by intrinsic regulation (muscle pacemaker) with extrinsic (nervous) input. By examining the continually active heart of five Drosophila species with markedly different cold tolerance, we investigated whether cardiac performance is related to the whole animal critical thermal minimum (CTmin). Further, to separate the effects of cold on extrinsic and intrinsic regulators of HR, we measured HR under similar conditions in decapitated flies as well as amputated abdomens of D. montana. Cardiac performance was assessed from break points in HR-temperature relationship (Arrhenius break point, ABP) and from the HR cessation temperature. Among the five species, we found strong relationships for both the HR ABP and HR cessation temperatures to whole animal CTmin, such that temperate Drosophila species maintained cardiac function at considerably lower temperatures than their tropical congeners. Hearts of amputated abdomens, with reduced central nervous input, had a higher thermal sensitivity and a significantly lower break point temperature, suggesting that central neuronal input is important for stimulating HR at low temperatures.
Journal of insect physiology, Jan 3, 2015
Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanism... more Insect cold tolerance is both phenotypically-plastic and evolutionarily labile, but the mechanisms underlying this variation are uncertain. Chill-susceptible insects lose ion and water homeostasis in the cold, which contributes to the development of injuries and eventually death. We thus hypothesized that more cold-tolerant insects will better maintain ion and water balance at low temperatures. We used rapid cold-hardening (RCH) and cold acclimation to improve cold tolerance of male Gryllus pennsylvanicus, and also compared this species to its cold-tolerant relative (Gryllus veletis). Cold acclimation and RCH decreased the critical thermal minimum (CTmin) and chill coma recovery time (CCR) in G. pennsylvanicus, but while cold acclimation improved survival of 0°C, RCH did not; G. veletis was consistently more cold-tolerant (and had lower CCR and CTmin) than G. pennsylvanicus. During cold exposure, hemolymph water and Na(+) migrated to the gut of warm-acclimated G. pennsylvanicus, whi...
Functional Ecology, 2014
ABSTRACT Thermal tolerance may limit and therefore predict ectotherm geographic distributions. Ho... more ABSTRACT Thermal tolerance may limit and therefore predict ectotherm geographic distributions. However, which of the many metrics of thermal tolerance best predict distribution is often unclear, even for drosophilids, which constitute a popular and well-described animal model.Five metrics of cold tolerance were measured for 14 Drosophila species in order to determine which metrics most strongly correlate with geographic distribution. The species represent tropical to temperate regions but all were reared under similar (common garden) conditions (20 °C). The traits measured were: chill coma temperature (CTmin), lethal temperature (LTe50), lethal time at low temperature (LTi50), chill coma recovery time (CCRT) and supercooling point (SCP).Measures of CTmin, LTe50 and LTi50 proved to be the best predictors to describe the variation in realized latitudinal distributions (R² = 0.699, R² = 0.741 and 0.550, respectively) and estimated environmental cold exposure (R² = 0.633, R² = 0.641 and 0.511, respectively). Measures of CCRT also correlated significantly with estimated minimum temperature (R² = 0.373), while the SCP did not. These results remained consistent after phylogenetically independent analysis or when applying non-linear regression. Moreover, our findings were supported by a similar analysis based on existing data compiled from the Drosophila cold tolerance literature.Trait correlations were strong between LTe50, LTi50 and CTmin, respectively (0.83 > R² > 0.55). However, surprisingly there was only a weak correlation between the entrance into coma (CTmin) and the recovery from chill coma (CCRT) (R² = 0.256).Considering the findings of the present study, data from previous studies, and the logistical constraints of each measure of cold tolerance, we conclude that CTmin and LTe50 are superior measures when estimating the ecologically relevant cold tolerance of drosophilids. Of these two traits, CTmin requires less equipment, time and animals and thereby presents a relatively fast, simple and dynamic measure of cold tolerance.This article is protected by copyright. All rights reserved.
Journal of Experimental Biology, 2011
To predict the effects of changing climates on insect distribution and abundance, a clear underst... more To predict the effects of changing climates on insect distribution and abundance, a clear understanding of the mechanisms that underlie critical thermal limits is required. In insects, the loss of muscle function and onset of cold-induced injury has previously been correlated with a loss of muscle resting potential. To determine the cause of this loss of function, we measured the effects of cold exposure on ion and water homeostasis in muscle tissue, hemolymph and the alimentary canal of the fall field cricket, Gryllus pennsylvanicus, during an exposure to 0°C that caused chilling injury and death. Low temperature exposure had little effect on muscle osmotic balance but it dissipated muscle ion equilibrium potentials through interactions between the hemolymph and gut. Hemolymph volume declined by 84% during cold exposure whereas gut water content rose in a comparable manner. This rise in water content was driven by a failure to maintain osmotic equilibrium across the gut wall, which resulted in considerable migration of Na + , Ca 2+ and Mg 2+ into the alimentary canal during cold exposure. This loss of homeostasis is likely to be a primary mechanism driving the cold-induced loss of muscle excitability and progression of chilling injury in chill-susceptible insect species.
Proceedings of the National Academy of Sciences, 2012
The time required to recover from cold-induced paralysis (chill-coma) is a common measure of inse... more The time required to recover from cold-induced paralysis (chill-coma) is a common measure of insect cold tolerance used to test central questions in thermal biology and predict the effects of climate change on insect populations. The onset of chill-coma in the fall field cricket (Gryllus pennsylvanicus, Orthoptera: Gryllidae) is accompanied by a progressive drift of Na + and water from the hemolymph to the gut, but the physiological mechanisms underlying recovery from chill-coma are not understood for any insect. Using a combination of gravimetric methods and atomic absorption spectroscopy, we demonstrate that recovery from chill-coma involves a reestablishment of hemolymph ion content and volume driven by removal of Na + and water from the gut. Recovery is associated with a transient elevation of metabolic rate, the time span of which increases with increasing cold exposure duration and closely matches the duration of complete osmotic recovery. Thus, complete recovery from chill-coma is metabolically costly and encompasses a longer period than is required for the recovery of muscle potentials and movement. These findings provide evidence that physiological mechanisms of hemolymph ion content and volume regulation, such as ion-motive ATPase activity, are instrumental in chill-coma recovery and may underlie natural variation in insect cold tolerance.
PLoS ONE, 2012
Increases in thermal variability elevate metabolic rate due to Jensen's inequality, and increased... more Increases in thermal variability elevate metabolic rate due to Jensen's inequality, and increased metabolic rate decreases the fitness of dormant ectotherms by increasing consumption of stored energy reserves. Theory predicts that ectotherms should respond to increased thermal variability by lowering the thermal sensitivity of metabolism, which will reduce the impact of the warm portion of thermal variability. We examined the thermal sensitivity of metabolic rate of overwintering Erynnis propertius (Lepidoptera: Hesperiidae) larvae from a stable or variable environment reared in the laboratory in a reciprocal common garden design, and used these data to model energy use during the winters of 1973-2010 using meteorological data to predict the energetic outcomes of metabolic compensation and phenological shifts. Larvae that experienced variable temperatures had decreased thermal sensitivity of metabolic rate, and were larger than those reared at stable temperatures, which could partially compensate for the increased energetic demands. Even with depressed thermal sensitivity, the variable environment was more energy-demanding than the stable, with the majority of this demand occurring in autumn. Autumn phenology changes thus had disproportionate influence on energy consumption in variable environments, and variable-reared larvae were most susceptible to overwinter energy drain. Therefore the energetic impacts of the timing of entry into winter dormancy will strongly influence ectotherm fitness in northern temperate environments. We conclude that thermal variability drives the expression of metabolic suppression in this species; that phenological shifts will have a greater impact on ectotherms in variable thermal environments; and that E. propertius will be more sensitive to shifts in phenology in autumn than in spring. This suggests that increases in overwinter thermal variability and/or extended, warm autumns, will negatively impact all non-feeding dormant ectotherms which lack the ability to suppress their overwinter metabolic thermal sensitivity.
Journal of Insect Physiology, 2010
Journal of Insect Physiology, 2009
Journal of Insect Physiology, 2013
Thermal sensitivity of metabolism (estimated by the temperature coefficient, Q10) is important fo... more Thermal sensitivity of metabolism (estimated by the temperature coefficient, Q10) is important for understanding ectotherm responses to temperature, but can only be measured empirically. Several strategies can be used to estimate thermal sensitivity. Static temperature respirometry uses measurements of metabolic rate taken at a series of temperatures, either by using different individuals at each temperature (independent STR, iSTR), or the same individual at several different temperatures (repeated STR, rSTR). Q10 can also be estimated from measurements of metabolic rate during a monotonic change in temperature (dynamic temperature respirometry, DTR), using either upwards (uDTR) or downwards (dDTR) temperature ramps. We compared estimates of Q10 of metabolic rate in adult females of the fall field cricket, Gryllus pennsylvanicus, derived from measurements made between 8 and 35°C, using iSTR, rSTR, dDTR and uDTR. We also controlled for aging effects during rSTR, and for ramp rate during DTR. We found that all measurement methods yielded statistically comparable measures of Q10. However, DTR provided higher absolute estimates of metabolic rate than STR. Thus, it appears that the different methods provide comparable estimates of Q10, allowing meta-analyses to utilize estimates of Q10 derived from different methods, and for the measurement strategy to be tailored to the characteristics of the organism.
Insect Science, 2009
Cross tolerance, whereby tolerance to one environmental stress is correlated with tolerance to ot... more Cross tolerance, whereby tolerance to one environmental stress is correlated with tolerance to other stressors, is thought to be widespread in insects. We used lines of Drosophila melanogaster Meigen (Diptera: Drosophilidae) selected for survival at a 1-h exposure to −5 • C to examine the extent to which this selection results in increased tolerance to other stresses, including high and low temperatures, desiccation and starvation. While selection improved tolerance to acute cold exposure and survival at −5 • C, there was little effect of selection regime on tolerance to other stressors. There was no correlation between tolerances to any of the stressors, suggesting different mechanisms of tolerance. This supports arguments that correlations between stress tolerances during selection experiments with D. melanogaster may be coincidental. The magnitude of heat-hardening was apparently constrained by basal tolerance among lines, but the magnitude of the rapid cold-hardening response was not correlated with basal cold tolerance, implying that the relationship between inducible and basal tolerances differs at high and low temperatures.
Journal of Insect Physiology, 2011
Triacylglycerides (TAGs) are the most important stored energy reserve in eukaryotes and are regul... more Triacylglycerides (TAGs) are the most important stored energy reserve in eukaryotes and are regularly measured in insects. Quantitative analysis of TAGs is complicated by their diversity of structure, and there are concerns with the quantitative accuracy of commonly used analytical methods. We used thin layer chromatography coupled to a flame ionisation detector (TLC-FID), an accurate method that is not sensitive to saturation or chain length of fatty acids, to quantify TAG content in small amounts of insect tissue, and used it to validate three high-throughput lipid assays (gravimetric, vanillin, and enzymatic). The performance of gravimetric assays depended on the solvent used. Folch reagent (chloroform: methanol 2:1 v/v) was a good index of TAG content, but overestimated lipid content due to the extraction of structural lipid and non-lipid components. Diethyl ether produced reasonable quantitative measurements but lacked precision and could not produce a repeatable rank-order of samples. The vanillin assay was accurate both as a quantitative method and as an index, preferably with a standard of mixed fatty acid composition. The enzymatic assay did not accurately or precisely quantify TAGs under our assay conditions. We conclude that the vanillin assay is suitable as a high-throughput method for quantifying TAG providing fatty acid composition does not change among treatment groups. However, if samples contain significant quantities of di-or mono-acylglycerides, or the fatty acid composition differs across treatment groups, TLC-FID is recommended.
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Papers by Heath Macmillan