Reversible mitophagy drives metabolic suppression in diapausing beetles.

Many insects enter a state of dormancy (diapause) during winter in which they lower their metabolism to save energy. Metabolic suppression is a hallmark of diapause, yet we know little about the mechanisms underpinning metabolic suppression in winter or how it is reversed in the spring. Here, we show that metabolic suppression in dormant Colorado potato beetles results from the breakdown of flight muscle mitochondria via mitophagy. Diapausing Colorado potato beetles suppress their metabolism by 90%, and this lowered metabolic rate coincides with a similar reduction in flight muscle mitochondrial function and density. During early diapause, beetles increase the expression of mitophagy-related transcripts (Parkin and ATG5) in their flight muscle coincident with an increase in mitophagy-related structures in the flight muscle. Knocking down Parkin expression with RNA interference in diapausing beetles prevented some mitochondrial breakdown and partially restored the whole animal metabolic rate, suggesting that metabolic suppression in diapausing beetles is driven by mitophagy. In other animals and in models of disease, such large-scale mitochondrial degradation is irreversible. However, we show that as diapause ends, beetles reverse mitophagy and increase the expression of PGC1α and NRF1 to replenish flight muscle mitochondrial pools. This mitochondrial biogenesis is activated in anticipation of diapause termination and in the absence of external stimuli. Our study provides a mechanistic link between mitochondrial degradation in insect tissues over the winter and whole-animal metabolic suppression.

Integrating water balance mechanisms into predictions of insect responses to climate change.

Efficient water balance is key to insect success. However, the hygric environment is changing with climate change; although there are compelling models of thermal vulnerability, water balance is often neglected in predictions. Insects survive desiccating conditions by reducing water loss, increasing their total amount of water (and replenishing it) and increasing their tolerance of dehydration. The physiology underlying these traits is reasonably well understood, as are the sources of variation and phenotypic plasticity. However, water balance and thermal tolerance intersect at high temperatures, such that mortality is sometimes determined by dehydration, rather than heat (especially during long exposures in dry conditions). Furthermore, water balance and thermal tolerance sometimes interact to determine survival. In this Commentary, we propose identifying a threshold where the cause of mortality shifts between dehydration and temperature, and that it should be possible to predict this threshold from trait measurements (and perhaps eventually a priori from physiological or -omic markers).

Harnessing thermal plasticity to enhance the performance of mass-reared insects: opportunities and challenges.

Insects are mass-reared for release for biocontrol including the sterile insect technique. Insects are usually reared at temperatures that maximize the number of animals produced, are chilled for handling and transport, and released into the field, where temperatures may be considerably different to those experienced previously. Insect thermal biology is phenotypically plastic (i.e. flexible), which means that there may exist opportunities to increase the performance of these programmes by modifying the temperature regimes during rearing, handling, and release. Here we synthesize the literature on thermal plasticity in relation to the opportunities to reduce temperature-related damage and increase the performance of released insects. We summarize how and why temperature affects insect biology, and the types of plasticity shown by insects. We specifically identify aspects of the production chain that might lead to mismatches between the thermal acclimation of the insect and the temperatures it is exposed to, and identify ways to harness physiological plasticity to reduce that potential mismatch. We address some of the practical (especially engineering) challenges to implementing some of the best-supported thermal regimes to maximize performance (e.g. fluctuating thermal regimes), and acknowledge that a focus only on thermal performance may lead to unwanted trade-offs with other traits that contribute to the success of the programme. Together, it appears that thermal physiological plasticity is well-enough understood to allow its implementation in release programmes.

Metabolic cost of freeze-thaw and source of CO2 production in the freeze-tolerant cricket Gryllus veletis.

Freeze-tolerant insects can survive the conversion of a substantial portion of their body water to ice. While the process of freezing induces active responses from some organisms, these responses appear absent from freeze-tolerant insects. Recovery from freezing likely requires energy expenditure to repair tissues and re-establish homeostasis, which should be evident as elevations in metabolic rate after thaw. We measured carbon dioxide (CO2) production in the spring field cricket (Gryllus veletis) as a proxy for metabolic rate during cooling, freezing and thawing and compared the metabolic costs associated with recovery from freezing and chilling. We hypothesized that freezing does not induce active responses, but that recovery from freeze-thaw is metabolically costly. We observed a burst of CO2 release at the onset of freezing in all crickets that froze, including those killed by either cyanide or an insecticide (thiacloprid), implying that the source of this CO2 was neither aerobic metabolism nor a coordinated nervous system response. These results suggest that freezing does not induce active responses from G. veletis, but may liberate buffered CO2 from hemolymph. There was a transient 'overshoot' in CO2 release during the first hour of recovery, and elevated metabolic rate at 24, 48 and 72 h, in crickets that had been frozen compared with crickets that had been chilled (but not frozen). Thus, recovery from freeze-thaw and the repair of freeze-induced damage appears metabolically costly in G. veletis, and this cost persists for several days after thawing.

Thermal acclimation has limited effect on the thermal tolerances of summer-collected Arctic and sub-Arctic wolf spiders.

High-latitude ectotherms contend with large daily and seasonal temperature variation. Summer-collected wolf spiders (Araneae; Lycosidae) from sub-Arctic and Arctic habitats have been previously documented as having low temperature tolerance insufficient for surviving year-round in their habitat. We tested two competing hypotheses: that they would have broad thermal breadth, or that they would use plasticity to extend the range of their thermal performance. We collected Pardosa moesta and P. lapponica from the Yukon Territory, Canada, P. furcifera, P. groenlandica, and P. hyperborea from southern Greenland, and P. hyperborea from sub-Arctic Norway, and acclimated them to warm (12 or 20 °C) or cool (4 °C) conditions under constant light for one week. We measured critical thermal minimum (CTmin) or supercooling point (SCP) as a measure of lower thermal limit, and critical thermal maximum (CTmax) as a measure of upper thermal limit. We found relatively little impact of acclimation on thermal limits, and some counterintuitive responses; for example, warm acclimation decreased the SCP and/or cool acclimation increased the CTmax in several cases. Together, this meant that acclimation did not appear to modify the thermal breadth, which supports our first hypothesis, but allows us to reject the hypothesis that spiders use plasticity to fine-tune their thermal physiology, at least in the summer. We note that we still cannot explain how these spiders withstand the very cold winters, and speculate that there are acclimatisation cues or processes that we were unable to capture in our study.

Cold tolerance of laboratory-reared Asian longhorned beetles.

Low winter temperatures in temperate climates can limit the success of non-native species. The Asian longhorned beetle, Anoplophora glabripennis, is an invasive wood-boring pest of hardwood trees in North America and Europe. Native A. glabripennis populations are spread across several climate zones in China and the Korean Peninsula and are likely to encounter low temperatures in at least some of this range. Understanding the lethal limits of the overwintering life stages of A. glabripennis is essential for accurately modeling the risk that invasive populations pose to non-native environments. In this study, we provide the first systematic characterization of the cold tolerance strategy and lower lethal limits of A. glabripennis eggs, larvae, and pupae. In diapausing larvae, the most common overwintering stage in this species, we measure hemolymph glycerol and osmolality and identify the effects of prolonged low temperature exposure. In developing pupae, we identify sublethal effects caused by low temperature exposure before freezing. Eggs and larvae were the most cold-tolerant life stages; eggs were freeze-avoidant with an average supercooling point of -25.8 °C and larvae were freeze tolerant with an LT90 of -25 °C. Hemolymph osmolality of freeze-tolerant larvae, on average, increased to 811 mOsm during chilling. This increase was primarily driven by a concurrent, average increase of 232 mM hemolymph glycerol. Pupae died upon exposure to freezing temperatures, but accumulate strong sublethal effects prior to freezing, indicating that they are chill susceptible. Taken together, these data will be useful to inform species distribution modeling in A. glabripennis.

Thermal Variability and Plasticity Drive the Outcome of a Host-Pathogen Interaction.

Variable, changing climates may affect each participant in a biotic interaction differently. We explored the effects of temperature and plasticity on the outcome of a host-pathogen interaction to try to predict the outcomes of infection under fluctuating temperatures. We infected Gryllus veletis crickets with the entomopathogenic fungus Metarhizium brunneum under constant (6°, 12°, 18°, or 25°C) or fluctuating (from 6° to 18°C or from 6° to 25°C) temperatures. We also acclimated crickets and fungi to constant or fluctuating conditions. Crickets acclimated to fluctuating conditions survived best under constant conditions if paired with warm-acclimated fungus. Overall, matches and mismatches in thermal performance, driven by acclimation, determined host survival. Mismatched performance also determined differences in survival under different fluctuating thermal regimes: crickets survived best when fluctuating temperatures favored their performance (from 6° to 25°C), compared with fluctuations that favored fungus performance (from 6° to 18°C). Thus, we could predict the outcome of infection under fluctuating temperatures by averaging relative host-pathogen performance under constant temperatures, suggesting that it may be possible to predict responses to fluctuating temperatures for at least some biotic interactions.

Help, there are 'omics' in my comparative physiology!

'Omics' methods, such as transcriptomics, proteomics, lipidomics or metabolomics, yield simultaneous measurements of many related molecules in a sample. These approaches have opened new opportunities to generate and test hypotheses about the mechanisms underlying biochemical and physiological phenotypes. In this Commentary, we discuss general approaches and considerations for successfully integrating omics into comparative physiology. The choice of omics approach will be guided by the availability of existing resources and the time scale of the process being studied. We discuss the use of whole-organism extracts (common in omics experiments on small invertebrates) because such an approach may mask underlying physiological mechanisms, and we consider the advantages and disadvantages of pooling samples within biological replicates. These methods can bring analytical challenges, so we describe the most easily analyzed omics experimental designs. We address the propensity of omics studies to digress into 'fishing expeditions' and show how omics can be used within the hypothetico-deductive framework. With this Commentary, we hope to provide a roadmap that will help newcomers approach omics in comparative physiology while avoiding some of the potential pitfalls, which include ambiguous experiments, long lists of candidate molecules and vague conclusions.

Reversing sodium differentials between the hemolymph and hindgut speeds chill coma recovery but reduces survival in the fall field cricket, Gryllus pennsylvanicus.

Chill-susceptible insects enter the reversible state of chill coma at their critical thermal minimum (CTmin). During chill coma, movement of Na+ and water from the hemolymph to the gut lumen disrupt ion and water balance. Recovery from cold exposure requires re-establishment of this balance, and failure to do so results in chilling injury or death. We hypothesized that the passive leak of Na+ and consequently water during cold exposure is driven by the [Na+] differential between the gut and hemolymph. To determine the extent to which this [Na+] differential affects cold tolerance, we used artificial diets to load the guts of fall field crickets (Gryllus pennsylvanicus) with various concentrations of Na+. Manipulating [Na+] differentials had no effect on the CTmin, agreeing with recent studies demonstrating that chill coma onset precedes loss of ion balance in the cold. A high [Na+] diet reversed the direction of the [Na+] differential between the gut and hemolymph. Crickets fed a high [Na+] diet recovered from 12 h of chill coma nearly twice as fast as those fed low [Na+] diets. However, the high [Na+] diet was detrimental to survival after prolonged cold exposure (three days at 0 °C). Therefore, while a reduced [Na+] differential helps crickets recover from short-term cold exposure, an increased gut Na+ load itself appears to carry longer-term costs and promotes irreversible chilling injury.

A comparison of low temperature biology of Pieris rapae from Ontario, Canada, and Yakutia, Far Eastern Russia.

Low temperatures limit the distribution and abundance of ectotherms. However, many insects can survive low temperatures by employing one of two cold tolerance strategies: freeze avoidance or freeze tolerance. Very few species can employ both strategies, but those that do provide a rare opportunity to study the mechanisms that differentiate freeze tolerance and freeze avoidance. We showed that overwintering pupae of the cabbage white butterfly Pieris rapae can be freeze tolerant or freeze avoidant. Pupae from a population of P. rapae in northeastern Russia (Yakutsk) froze at c. -9.3 °C and were freeze-tolerant in 2002-2003 when overwintered outside. However, P. rapae from both Yakutsk and southern Canada (London) acclimated to milder laboratory conditions in 2014 and 2017 froze at lower temperatures (< -20 °C) and were freeze-avoidant. Summer-collected P. rapae larvae (collected in Yakutsk in 2016) were partially freeze-tolerant, and decreased the temperature at which they froze in response to starvation at mild low temperatures (4 °C) and repeated partial freezing events. By comparing similarly-acclimated P. rapae pupae from both populations, we identified molecules that may facilitate low temperature tolerance, including the hemolymph ice-binding molecules and several potential low molecular weight cryoprotectants. Pieris rapae from Yakutsk exhibited high physiological plasticity, accumulating cryoprotectants and almost doubling their hemolymph osmolality when supercooled to -15 °C for two weeks, while the London P. rapae population exhibited minimal plasticity. We hypothesize that physiological plasticity is an important adaptation to extreme low temperatures (i.e. in Yakutsk) and may facilitate the transition between freeze avoidance and freeze tolerance.

Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect.

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis. Cold-acclimated G. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis. Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo, with implications for choosing new molecules for cryopreservation.

Loss of ion homeostasis is not the cause of chill coma or impaired dispersal in false codling moth Thaumatotibia leucotreta (Lepidoptera: Tortricidae).

Dispersal is a central requirement of a successful sterile insect release programme, but field-released false codling moth (FCM) typically suffer from poor dispersal ability, especially at low ambient temperatures. Here we test the hypothesis that poor activity and dispersal in FCM is caused by delayed or perturbed recovery of ion and/or water homeostasis after chilling for handling and transport prior to field release. Hemolymph and flight muscle were collected from two treatment groups at three time points that targeted thermal conditions above and below the chill coma induction threshold of ~ 6 °C: 1) control moths kept at 25 °C, 2) moths exposed to 3 °C or 9 °C for 4 h, and 3) moths allowed to recover at 25 °C for 24 h after exposure to either 3 °C or 9 °C. We measured concentrations of Na+, K+ and Mg2+ in the hemolymph and muscle collected at each time point. Exposure to a chill-coma inducing temperature had little effect overall on ion balance in the hemolymph and flight muscle of false codling moth, but hemolymph [Na+] decreased from 10.4 ±â€¯0.4 mM to 6.9 ±â€¯0.7 mM as moths were chilled to 3 °C and then increased to 10.4 ±â€¯0.9 mM after the 24 h recovery period. In the 9 °C cooling treatment, [K+] increased from 8.2 ±â€¯0.5 mM during chilling to 14.1 ±â€¯1.9 mM after the 24 h recovery period. No changes were seen in equilibrium potentials in either of the ions measured. Thus, we did not find evidence that water and ion homeostasis are lost by the moths in chill coma and conclude that reduced dispersal in field-released moths is not direct a consequence of the costs of re-establishment of homeostasis.

The many roles of fats in overwintering insects.

Temperate, polar and alpine insects generally do not feed over winter and hence must manage their energy stores to fuel their metabolism over winter and to meet the energetic demands of development and reproduction in the spring. In this Review, we give an overview of the accumulation, use and conservation of fat reserves in overwintering insects and discuss the ways insects modify fats to facilitate their selective consumption or conservation. Many insects are in diapause and have depressed metabolic rates over winter; together with low temperatures, this means that lipid stores are likely to be consumed predominantly in the autumn and spring, when temperatures are higher but insects remain dormant. Although there is ample evidence for a shift towards less-saturated lipids in overwintering insects, switches between the use of carbohydrate and lipid stores during winter have not been well-explored. Insects usually accumulate cryoprotectants over winter, and the resulting increase in haemolymph viscosity is likely to reduce lipid transport. For freeze-tolerant insects (which withstand internal ice), we speculate that impaired oxygen delivery limits lipid oxidation when frozen. Acetylated triacylglycerols remain liquid at low temperatures and interact with water molecules, providing intriguing possibilities for a role in cryoprotection. Similarly, antifreeze glycolipids may play an important role in structuring water and ice during overwintering. We also touch on the uncertain role of non-esterified fatty acids in insect overwintering. In conclusion, lipids are an important component of insect overwintering energetics, but there remain many uncertainties ripe for detailed exploration.

Repeated freezing induces a trade-off between cryoprotection and egg production in the goldenrod gall fly, Eurosta solidaginis.

Internal ice formation leads to wholesale changes in ionic, osmotic and pH homeostasis, energy metabolism, and mechanical damage, across a small range of temperatures, and is thus an abiotic stressor that acts at a distinct, physiologically relevant, threshold. Insects that experience repeated freeze-thaw cycles over winter will cross this stressor threshold many times over their lifespan. Here, we examined the effect of repeatedly crossing the freezing threshold on short-term physiological parameters (metabolic reserves and cryoprotectant concentration) as well as long-term fitness-related performance (survival and egg production) in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis We exposed overwintering prepupae to a series of low temperatures (-10, -15 or -20°C) with increasing numbers of freezing events (3, 6 or 10) with differing recovery periods between events (1, 5 or 10 days). Repeated freezing increased sorbitol concentration by about 50% relative to a single freezing episode, and prompted prepupae to modify long-chain triacylglycerols to acetylated triacylglycerols. Long-term, repeated freezing did not significantly reduce survival but did reduce egg production by 9.8% relative to a single freezing event. Exposure temperature did not affect any of these measures, suggesting that threshold crossing events may be more important to fitness than the intensity of stress in overwintering E. solidaginis.

Eco-immunology in the cold: the role of immunity in shaping the overwintering survival of ectotherms.

The effect of temperature on physiology mediates many of the challenges that ectotherms face under climate change. Ectotherm immunity is thermally sensitive and, as such, environmental change is likely to have complex effects on survival, disease resistance and transmission. The effects of temperature on immunity will be particularly profound in winter because cold and overwintering are important triggers and regulators of ectotherm immune activity. Low temperatures can both suppress and activate immune responses independent of parasites, which suggests that temperature not only affects the rate of immune responses but also provides information that allows overwintering ectotherms to balance investment in immunity and other physiological processes that underlie winter survival. Changing winter temperatures are now shifting ectotherm immunity, as well as the demand for energy conservation and protection against parasites. Whether an ectotherm can survive the winter will thus depend on whether new immune phenotypes will shift to match the conditions of the new environment, or leave ectotherms vulnerable to infection or energy depletion. Here, we synthesise patterns of overwintering immunity in ectotherms and examine how new winter conditions might affect ectotherm immunity. We then explore whether it is possible to predict the effects of changing winter conditions on ectotherm vulnerability to the direct and indirect effects of parasites.

Substantial heat tolerance acclimation capacity in tropical thermophilic snails, but to what benefit?

The theory for thermal acclimation of ectotherms suggests that (1) heat tolerance is traded off for thermal acclimation in thermophilic species and that (2) plasticity is constrained in tropically distributed ectotherms, which commonly experience relatively thermally stable environments. We observed substantial heat tolerance plasticity in a test of this theory using tropical, thermophilic marine intertidal snails that inhabit extremely hot and highly variable thermal environments. The implication of these results is that plasticity selection is largely driven by habitat temperature conditions irrespective of basal heat tolerance or latitude. However, heat tolerance of field-fresh snails was comparable with that of laboratory warm-acclimated snails, suggesting that snails in the field may often be unable to improve heat hardening with further environmental warming. These findings suggest that field referencing is crucial to using laboratory-measured acclimation capacity when inferring climate warming vulnerability in ectotherms, and overall they question how well current thermal biology theory predicts the outcomes of global change in intertidal environments.

The effect of cold acclimation on active ion transport in cricket ionoregulatory tissues.

Cold-acclimated insects defend ion and water transport function during cold exposure. We hypothesized that this is achieved via enhanced active transport. The Malpighian tubules and rectum are likely targets for such transport modifications, and recent transcriptomic studies indicate shifts in Na+-K+ ATPase (NKA) and V-ATPase expression in these tissues following cold acclimation. Here we quantify the effect of cold acclimation (one week at 12°C) on active transport in the ionoregulatory organs of adult Gryllus pennsylvanicus field crickets. We compared primary urine production of warm- and cold-acclimated crickets in excised Malpighian tubules via Ramsay assay at a range of temperatures between 4 and 25°C. We then compared NKA and V-ATPase activities in Malpighian tubule and rectal homogenates from warm- and cold-acclimated crickets via NADH-linked photometric assays. Malpighian tubules of cold-acclimated crickets excreted fluid at lower rates at all temperatures compared to warm-acclimated crickets. This reduction in Malpighian tubule excretion rates may be attributed to increased NKA activity that we observed for cold-acclimated crickets, but V-ATPase activity was unchanged. Cold acclimation had no effect on rectal NKA activity at either 21°C or 6°C, and did not modify rectal V-ATPase activity. Our results suggest that an overall reduction, rather than enhancement of active transport in the Malpighian tubules allows crickets to maintain hemolymph water balance during cold exposure, and increased Malpighian tubule NKA activity may help to defend and/or re-establish ion homeostasis.

Effects of cold-acclimation on gene expression in Fall field cricket (Gryllus pennsylvanicus) ionoregulatory tissues.

BACKGROUND: Cold tolerance is a key determinant of temperate insect distribution and performance. Chill-susceptible insects lose ion and water homeostasis during cold exposure, but prior cold acclimation improves both cold tolerance and defense of homeostasis. The mechanisms underlying these processes are mostly unknown; cold acclimation is thought to enhance ion transport in the cold and/or prevent leak of water and ions. To identify candidate mechanisms of cold tolerance plasticity we generated transcriptomes of ionoregulatory tissues (hindgut and Malpighian tubules) from Gryllus pennsylvanicus crickets and compared gene expression in warm- and cold-acclimated individuals. RESULTS: We assembled a G. pennsylvanicus transcriptome de novo from 286 million 50-bp reads, yielding 70,037 contigs (~44% of which had putative BLAST identities). We compared the transcriptomes of warm- and cold-acclimated hindguts and Malpighian tubules. Cold acclimation led to a ≥ 2-fold change in the expression of 1493 hindgut genes (733 downregulated, 760 upregulated) and 2008 Malpighian tubule genes (1009 downregulated, 999 upregulated). Cold-acclimated crickets had altered expression of genes putatively associated with ion and water balance, including: a downregulation of V-ATPase and carbonic anhydrase in the Malpighian tubules and an upregulation of Na+-K+ ATPase in the hindgut. We also observed acclimation-related shifts in the expression of cytoskeletal genes in the hindgut, including actin and actin-anchoring/stabilizing proteins, tubulin, α-actinin, and genes involved in adherens junctions organization. In both tissues, cold acclimation led to differential expression of genes encoding cytochrome P450s, glutathione-S-transferases, apoptosis factors, DNA repair, and heat shock proteins. CONCLUSIONS: This is the first G. pennsylvanicus transcriptome, and our tissue-specific approach yielded new candidate mechanisms of cold tolerance plasticity. Cold acclimation may reduce loss of hemolymph volume in the cold by 1) decreasing primary urine production via reduced expression of carbonic anhydrase and V-ATPase in the Malpighian tubules and 2) by increasing Na+ (and therefore water) reabsorption across the hindgut via increase in Na+-K+ ATPase expression. Cold acclimation may reduce chilling injury by remodeling and stabilizing the hindgut epithelial cytoskeleton and cell-to-cell junctions, and by increasing the expression of genes involved in DNA repair, detoxification, and protein chaperones.

CRISPR-induced null alleles show that Frost protects Drosophila melanogaster reproduction after cold exposure.

The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. We have used CRISPR-mediated homologous recombination to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction.

Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures?

Thermal performance curves (TPCs), which quantify how an ectotherm's body temperature (Tb ) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key - but often biologically unreasonable - assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced Tb. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with Tb , to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.