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).

Molecular signatures of diapause in the Asian longhorned beetle: Gene expression.

Most previous studies on gene expression during insect diapause do not address among-tissue variation in physiological processes. We measured transcriptomic changes during larval diapause in the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae). We conducted RNA-seq on fat body, the supraesophageal ganglion, midgut, hindgut, and Malpighian tubules during pre-diapause, diapause maintenance, post-diapause quiescence, and post-diapause development. We observed a small, but consistent, proportion of genes within each gene expression profile that were shared among tissues, lending support for a core set of diapause-associated genes whose expression is tissue-independent. We evaluated the overarching hypotheses that diapause would be associated with cell cycle arrest, developmental arrest, and increased stress tolerance and found evidence of repressed TOR and insulin signaling, reduced cell cycle activity and increased capacity of stress response via heat shock protein expression and remodeling of the cytoskeleton. However, these processes varied among tissues, with the brain and fat body appearing to maintain higher levels of cellular activity during diapause than the midgut or Malpighian tubules. We also observed temperature-dependent changes in gene expression during diapause maintenance, particularly in genes related to the heat shock response and MAPK, insulin, and TOR signaling pathways. Additionally, we provide evidence for epigenetic reorganization during the diapause/post-diapause quiescence transition and expression of genes involved in post-translational modification, highlighting the need for investigations of the protein activity of these candidate genes and processes. We conclude that diapause development is coordinated via diverse tissue-specific gene expression profiles and that canonical diapause phenotypes vary among tissues.

Plasticity drives extreme cold tolerance of emerald ash borer (Agrilus planipennis) during a polar vortex.

Invasive species must often survive combinations of environmental conditions that differ considerably from their native range; however, for a given species it is unclear whether improved tolerance is the result of phenotypic plasticity or genetic adaptation (or both). Agrilus planipennis (Coleoptera: Buprestidae; the emerald ash borer) is an invasive pest of Fraxinus trees in North America and Europe. Previous studies in SW Ontario, Canada, showed that A. planipennis is freeze avoidant, preventing internal ice formation by accumulating Molar concentrations of glycerol in its hemolymph and depressing its supercooling point (SCP, the temperature at which it freezes). The cold tolerance of these SW Ontario animals was used to predict potential distribution, revealing that some Canadian cities should be too cold to allow populations to persist. However, a small population of A. planipennis has persisted in Winnipeg, Manitoba, Canada, through several severe 'polar vortex' events. In 2018/19, we collected A. planipennis larvae and prepupae from Winnipeg, MB and Southern Ontario, and found that individuals from Winnipeg were extremely cold tolerant - with SCPs as low as -52°C in prepupae (compared to -32°C in SW Ontario), and observed survival of unfrozen individuals exposed to -50°C for one hour. This cold tolerance was accompanied by higher hemolymph osmolality and glycerol concentration than in the SW Ontario individuals. To distinguish between phenotypic plasticity and local adaptation, in 2020/21 we overwintered Winnipeg-sourced individuals either outdoors in SW Ontario or in a simulated Winnipeg winter. Simulated Winnipeg winter individuals had cold tolerance similar to those overwintered in Winnipeg, while SW Ontario overwintered individuals had cold tolerance similar to those collected previously in the region. The simulated winter individuals had higher hemolymph glycerol concentrations than SW Ontario overwintered animals, at least in part due to greater dehydration. Thus, A. planipennis are cold-tolerant enough to survive some of the harshest winters where their host trees can grow, and most likely attain this cold tolerance via phenotypic plasticity. These findings raise the importance of delineating sensitivity of conclusions to unexpected phenotypic plasticity when predicting potential distributions of new invasives or responses to climate change.

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.

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.

The mitochondrial genomes of two Gryllus crickets (Grylloidea: Gryllidae) via RNA-seq.

Here, we used RNA-seq reads to assemble the complete mitochondrial genomes of the spring field cricket, Gryllus veletis, and the variable field cricket, Gryllus lineaticeps. The mitochondrial genomes of G. veletis (15,686 bp, MW322713) and G. lineaticeps (15,607 bp, MW315773) each contain the expected 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a large control (D-loop) region. The arrangements of these features were similar for both species and consistent with other closely related Orthoptera. A phylogenetic analysis of the mitochondrial genome sequences revealed that G. veletis and G. lineaticeps cluster with the other Gryllus species and all reside in a clade with the Gryllidae.

The Resilience of Polar Collembola (Springtails) in a Changing Climate.

Assessing the resilience of polar biota to climate change is essential for predicting the effects of changing environmental conditions for ecosystems. Collembola are abundant in terrestrial polar ecosystems and are integral to food-webs and soil nutrient cycling. Using available literature, we consider resistance (genetic diversity; behavioural avoidance and physiological tolerances; biotic interactions) and recovery potential for polar Collembola. Polar Collembola have high levels of genetic diversity, considerable capacity for behavioural avoidance, wide thermal tolerance ranges, physiological plasticity, generalist-opportunistic feeding habits and broad ecological niches. The biggest threats to the ongoing resistance of polar Collembola are increasing levels of dispersal (gene flow), increased mean and extreme temperatures, drought, changing biotic interactions, and the arrival and spread of invasive species. If resistance capacities are insufficient, numerous studies have highlighted that while some species can recover from disturbances quickly, complete community-level recovery is exceedingly slow. Species dwelling deeper in the soil profile may be less able to resist climate change and may not recover in ecologically realistic timescales given the current rate of climate change. Ultimately, diverse communities are more likely to have species or populations that are able to resist or recover from disturbances. While much of the Arctic has comparatively high levels of diversity and phenotypic plasticity; areas of Antarctica have extremely low levels of diversity and are potentially much more vulnerable to climate change.

Diapause differentially modulates the transcriptomes of fat body and flight muscle in the Colorado potato beetle.

Many temperate insects, such as the Colorado potato beetle, enter diapause in winter, during which they arrest their development, suppress their metabolic rate and have high stress tolerance. Diapause phenotypes can be transcriptionally regulated, however many studies to date report only whole animal gene expression rather than tissue-specific processes during diapause. We used RNA-seq to measure gene expression in fat body and flight muscle of diapausing and non-diapausing beetles. We used differential expression and GO enrichment analyses to evaluate longstanding hypotheses about the mechanisms that drive arrested development, changes in energy metabolism, and increased stress tolerance during diapause. We found evidence of G2/M cell cycle arrest, juvenile hormone catabolism, increased antioxidant metabolism, epigenetic modification, transposable element regulation, and cytoskeletal remodeling in both the fat body and flight muscle of diapausing beetles. Beetles differentially modulated the fat body and flight muscle transcriptomes during diapause with fat body playing a larger role in the hypoxia response and immunity, whereas flight muscle had higher abundance of transcripts related to the chaperone response and proteostasis. Our transcriptome provides evidence for distinct roles and responses of fat body and flight muscle during diapause in the Colorado potato beetle, and we provide testable hypotheses for biological processes that appear to drive diapause phenotypes in insects.

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.

Cold tolerance, water balance, energetics, gas exchange, and diapause in overwintering brown marmorated stink bugs.

Halyomorpha halys (Hemiptera: Pentatomidae) is an emerging pest which established in Ontario, Canada, in 2012. Halyomporpha halys overwinters in anthropogenic structures as an adult. We investigated seasonal variation in the cold tolerance, water balance, and energetics of H. halys in southwestern Ontario. We also induced diapause in laboratory-reared animals with short daylength at permissive temperatures and compared cold tolerance, water balance, energetics, and metabolism and gas exchange between diapausing and non-diapausing individuals. Halyomorpha halys that overwintered outside in Ontario all died, but most of those that overwintered in sheltered habitats survived. We confirm that overwintering H. halys are chill-susceptible. Over winter, Ontario H. halys depressed their supercooling point to c. -15.4 °C, and 50% survived a 1 h exposure to -17.5 °C. They reduce water loss rates over winter, and do not appear to significantly consume lipid or carbohydrate reserves to a level that might cause starvation. Overall, it appears that H. halys is dependent on built structures and other buffered microhabitats to successfully overwinter in Ontario. Laboratory-reared diapausing H. halys have lower supercooling points than their non-diapausing counterparts, but LT50 is not enhanced by diapause induction. Diapausing H. halys survive desiccating conditions for 3-4 times longer than those not in diapause, through decreases in both respiratory and cuticular water loss. Diapausing H. halys do not appear to accumulate any more lipid or carbohydrate than those not in diapause, but do have lower metabolic rates, and are slightly more likely to exhibit discontinuous gas exchange.

Dormancy in laboratory-reared Asian longhorned beetles, Anoplophora glabripennis.

An insect's capacity to survive winter is critical for range expansion in temperate regions. The Asian longhorned beetle (Anoplophora glabripennis) is a polyphagous wood-boring insect native to China and the Korean peninsula and poses a high risk of invasion in North America and Europe. It is unclear whether A. glabripennis enters diapause, which means that diapause cannot be included in assessments of the risk of this species invading forests in temperate regions. Using a laboratory colony, we examine larval developmental arrest, metabolic rates, gas exchange patterns, thermal sensitivity, and body composition to characterize larval dormancy. Chilled larvae entered a temperature-independent developmental arrest which usually required more than four weeks of chilling to break, decreased their metabolic rate by as much as 63%, and maintained energy stores throughout the chilling period - results consistent with an obligate diapause. We also observed a switch to discontinuous gas exchange at low temperatures. Thermal sensitivity of metabolic rate did not differ between chilled and non-chilled larvae. Taken together, we conclude that A. glabripennis enters a larval diapause during chilling and terminates diapause after a requisite chilling period. These results will enhance our ability to predict phenology and potential distribution of current and future invasions of A. glabripennis.

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.

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.

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.

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.

Gut yeasts do not improve desiccation survival in Drosophila melanogaster.

A healthy gut microbiota generally improves the performance of its insect host. Although the effects can be specific to the species composition of the microbial community, the role of gut microbiota in determining water balance has not been well explored. We used axenic and gnotobiotic (reared with a known microbiota) Drosophila melanogaster to test three hypotheses about the effects of gut yeasts on the water balance of adult flies: 1) that gut yeasts would improve desiccation survival in adult flies; 2) that larval yeasts would improve adult desiccation survival; 3) that the effects would be species-specific, such that yeasts closely associated with D. melanogaster in nature are more likely to be beneficial than those rarely found in association with D. melanogaster. We used Saccharomyces cerevisiae (often used in Drosophila cultures, but rarely associated with D. melanogaster in nature), Lachancea kluyveri (associated with some species of Drosophila, but not D. melanogaster), and Pichia kluyveri (associated with D. melanogaster in nature). Adult inoculation with yeasts had no effect on survival of desiccating conditions. Inoculation with P. kluyveri as larvae did not change desiccation survival in adults; however, rearing with L. kluyveri or S. cerevisiae reduced adult desiccation survival. We conclude that adult inoculation with gut yeasts has no impact on desiccation survival, but that rearing with yeasts can have either no or detrimental effect. The effects appear to be species-specific: P. kluyveri did not have a negative impact on desiccation tolerance, suggesting some level of co-adaptation with D. melanogaster. We note that S. cerevisiae may not be an appropriate species for studying the effects of gut yeasts on D. melanogaster.