Fruit Flies: Challenges and Opportunities to Stem the Tide of Global Invasions.

Global trade in fresh fruit and vegetables, intensification of human mobility, and climate change facilitate fruit fly (Diptera: Tephritidae) invasions. Life-history traits, environmental stress response, dispersal stress, and novel genetic admixtures contribute to their establishment and spread. Tephritids are among the most frequently intercepted taxa at ports of entry. In some countries, supported by the rules-based trade framework, a remarkable amount of biosecurity effort is being arrayed against the range expansion of tephritids. Despite this effort, fruit flies continue to arrive in new jurisdictions, sometimes triggering expensive eradication responses. Surprisingly, scant attention has been paid to biosecurity in the recent discourse about new multilateral trade agreements. Much of the available literature on managing tephritid invasions is focused on a limited number of charismatic (historically high-profile) species, and the generality of many patterns remains speculative.

Heatwaves are detrimental to fertility in the viviparous tsetse fly.

Heatwaves are increasing in frequency and intensity due to climate change, pushing animals beyond physiological limits. While most studies focus on survival limits, sublethal effects on fertility tend to occur below lethal thresholds, and consequently can be as important for population viability. Typically, male fertility is more heat-sensitive than female fertility, yet direct comparisons are limited. Here, we measured the effect of experimental heatwaves on tsetse flies, Glossina pallidipes, disease vectors and unusual live-bearing insects of sub-Saharan Africa. We exposed males or females to a 3-day heatwave peaking at 36, 38 or 40°C for 2 h, and a 25°C control, monitoring mortality and reproduction over six weeks. For a heatwave peaking at 40°C, mortality was 100%, while a 38°C peak resulted in only 8% acute mortality. Females exposed to the 38°C heatwave experienced a one-week delay in producing offspring, whereas no such delay occurred in males. Over six weeks, heatwaves resulted in equivalent fertility loss in both sexes. Combined with mortality, this lead to a 10% population decline over six weeks compared to the control. Furthermore, parental heatwave exposure gave rise to a female-biased offspring sex ratio. Ultimately, thermal limits of both survival and fertility should be considered when assessing climate change vulnerability.

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).

Exploring the connection between autophagy and heat-stress tolerance in Drosophila melanogaster.

Mechanisms aimed at recovering from heat-induced damages are closely associated with the ability of ectotherms to survive exposure to stressful temperatures. Autophagy, a ubiquitous stress-responsive catabolic process, has recently gained renewed attention as one of these mechanisms. By increasing the turnover of cellular structures as well as the clearance of long-lived protein and protein aggregates, the induction of autophagy has been linked to increased tolerance to a range of abiotic stressors in diverse ectothermic organisms. However, whether a link between autophagy and heat-tolerance exists in insect models remains unclear despite broad ecophysiological implications thereof. Here, we explored the putative association between autophagy and heat-tolerance using Drosophila melanogaster as a model. We hypothesized that (i) heat-stress would cause an increase of autophagy in flies' tissues, and (ii) rapamycin exposure would trigger a detectable autophagic response in adults and increase their heat-tolerance. In line with our hypothesis, we report that flies exposed to heat-stress present signs of protein aggregation and appear to trigger an autophagy-related homoeostatic response as a result. We further show that rapamycin feeding causes the systemic effect associated with target of rapamycin (TOR) inhibition, induces autophagy locally in the fly gut, and increases the heat-stress tolerance of individuals. These results argue in favour of a substantial contribution of autophagy to the heat-stress tolerance mechanisms of insects.

Through the looking glass: attempting to predict future opportunities and challenges in experimental biology.

To celebrate its centenary year, Journal of Experimental Biology (JEB) commissioned a collection of articles examining the past, present and future of experimental biology. This Commentary closes the collection by considering the important research opportunities and challenges that await us in the future. We expect that researchers will harness the power of technological advances, such as '-omics' and gene editing, to probe resistance and resilience to environmental change as well as other organismal responses. The capacity to handle large data sets will allow high-resolution data to be collected for individual animals and to understand population, species and community responses. The availability of large data sets will also place greater emphasis on approaches such as modeling and simulations. Finally, the increasing sophistication of biologgers will allow more comprehensive data to be collected for individual animals in the wild. Collectively, these approaches will provide an unprecedented understanding of 'how animals work' as well as keys to safeguarding animals at a time when anthropogenic activities are degrading the natural environment.

Does Host Plant Drive Variation in Microbial Gut Communities in a Recently Shifted Pest?

Biotic interactions can modulate the responses of organisms to environmental stresses, including diet changes. Gut microbes have substantial effects on diverse ecological and evolutionary traits of their hosts, and microbial communities can be highly dynamic within and between individuals in space and time. Modulations of the gut microbiome composition and their potential role in the success of a species to maintain itself in a new environment have been poorly studied to date. Here we examine this question in a large wood-boring beetle Cacosceles newmannii (Cerambycidae), that was recently found thriving on a newly colonized host plant. Using 16S metabarcoding, we assessed the gut bacterial community composition of larvae collected in an infested field and in "common garden" conditions, fed under laboratory-controlled conditions on four either suspected or known hosts (sugarcane, tea tree, wattle, and eucalyptus). We analysed microbiome variation (i.e. diversity and differentiation), measured fitness-related larval growth, and studied host plant lignin and cellulose contents, since their degradation is especially challenging for wood-boring insects. We show that sugarcane seems to be a much more favourable host for larval growth. Bacterial diversity level was the highest in field-collected larvae, whereas lab-reared larvae fed on sugarcane showed a relatively low level of diversity but very specific bacterial variants. Bacterial communities were mainly dominated by Proteobacteria, but were significantly different between sugarcane-fed lab-reared larvae and any other hosts or field-collected larvae. We identified changes in the gut microbiome associated with different hosts over a short time frame, which support the hypothesis of a role of the microbiome in host switches.

How plastic are upper thermal limits? A comparative study in tsetse (family: Glossinidae) and wider Diptera.

Critical thermal maximum (CTmax) describes the upper thermal tolerance of an animal where biological functions start to fail. A period of acclimation can enhance CTmax through plasticity, potentially buffering animals from extreme temperatures caused by climate change. Basal and acclimated CTmax vary within and between species and may be explained by traits related to thermal physiology, such as body size and sex. Differences in CTmax have not been established among species of tsetse fly (Glossina spp.), vectors of animal and human African trypanosomiasis. Here, we investigated basal CTmax and its plasticity for five tsetse species following adult acclimation at constant 25 or 30 °C for five days. We then set our findings in context using a meta-analysis on 33 species of Diptera. We find that, of the five tsetse species considered, only Glossina palpalis gambiensis and Glossina brevipalpis exhibited plasticity of CTmax, with an increase of 0.12 °C and 0.10 °C per 1 °C acclimation respectively. Within some species, higher basal CTmax values were associated with larger body size and being female, while variation in plasticity (i.e., response to the acclimation temperature) could not be explained by sex or size. Our broader meta-analysis across Diptera revealed overall CTmax plasticity of 0.06 °C per 1 °C acclimation, versus a similar 0.05 °C mean increase in tsetse. In contrast, there was greater CTmax plasticity in males compared to females in Diptera. Our study highlights that CTmax and its plasticity varies even among closely related species. Broader patterns across groups are not always reflected at a finer resolution; we thus emphasise the need for detailed experimental studies across a wide range of insect species to capture their capacity to cope with rapidly warming temperatures.

Arthropods on imported plant products: Volumes predict general trends while contextual details enhance predictive power.

Agricultural biosecurity interventions are aimed at minimizing introductions of harmful non-native organisms to new areas via agricultural trade. To prioritize such interventions, historical data on interceptions have been used to elucidate which factors determine the likelihood that a particular import is carrying a harmful organism. Here we use an interception data set of arthropod contaminants recorded on plant imports arriving in South Africa from 2005 to 2019, comprising 13,566 samples inspected for arthropod contaminants, of which 4902 were positive for the presence of at least one arthropod. We tested 29 predictor variables that have previously been used to explain variation in rates of detection and three variables describing possible sources of additional variation and grouped these into six mutually exclusive "factor classes." We used boosted regression trees as a non-parametric stochastic machine-learning method to build models for each factor class and interactions between them. We explored the influence of these variables with data split either randomly or chronologically. While we identified some specific patterns that could be explained post-hoc by historical events, only inspected volumes were reliably correlated with detection of arthropod contaminants across the whole data set. However, inspected volumes could not predict future interceptions of arthropods, which instead relied on contextual factors such as country, crop or year of import. This suggests that, although certain factors may be important in certain circumstances or for particular crops or commodities, there is little general predictive power in the current data. Instead, an idiographic approach would be most beneficial in biosecurity to ascertain the details of why a particular pest arrived on a particular pathway and how it might move (and be stopped) in future.

Understanding costs and benefits of thermal plasticity for pest management: insights from the integration of laboratory, semi-field and field assessments of Ceratitis capitata (Diptera: Tephritidae).

The relative costs and benefits of thermal acclimation for manipulating field performance of pest insects depend upon a number of factors including which traits are affected and how persistent any trait changes are in different environments. By assessing plastic trait responses of Ceratitis capitata (Mediterranean fruit fly) across three distinct operational environments (laboratory, semi-field, and field), we examined the influence of different thermal acclimation regimes (cool, intermediate [or handling control], and warm) on thermal tolerance traits (chill-coma recovery, heat-knockdown time, critical thermal minimum and critical thermal maximum) and flight performance (mark-release-recapture). Under laboratory conditions, thermal acclimation altered thermal limits in a relatively predictable manner and there was a generally positive effect across all traits assessed, although some traits responded more strongly. By contrast, dispersal-related performance yielded strongly contrasting results depending on the specific operational environment assessed. In semi-field conditions, warm- or cold-acclimated flies were recaptured more often than the control group at cooler ambient conditions suggesting an overall stimulatory influence of thermal variability on low-temperature dispersal. Under field conditions, a different pattern was identified: colder flies were recaptured more in warmer field conditions relative to other treatment groups. This study highlights the trait- and context-specific nature of how thermal acclimation influences traits of thermal performance and tolerance. Consequently, laboratory and semi-field assessments of dispersal may not provide results that extend into the field setting despite the apparent continuum of environmental complexity among them (laboratory < semi-field < field).

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.

Male Mediterranean fruit flies prefer warmer temperatures that improve sexual performance.

Females and males have divergent strategies of energy investment, so the thermal preference of each sex in insects may differ because energetic conversion of metabolic reserves is dependent on temperature. We determined the thermal preference of virgin, sexually mature Mediterranean fruit flies, Ceratitis capitata, and found that males preferred a significantly higher temperature (23.8 ± 0.3 °C) than that of females (22.1 ± 0.3 °C). We then tested predictions for the difference in thermal preference related to the energetic demands of reproduction over a range of temperatures. The frequency and duration of calling bouts by male C. capitata were optimal at 26 °C. Mating propensity and latency, and copula duration, were optimal over the range of 22-28 °C. When mating occurred, temperature had little effect on the incidence of sperm storage by females, but there was a notable decline in the number stored at 28 °C. Female lifespan was highest at 18 °C, but lifetime egg production was optimal at 24 °C. These results illustrate temperature-related differences in the reproductive fitness of the sexes in C. capitata, although the optima for male traits align best with their thermal preference. They also support the theoretical prediction that insect thermal preference should be lower than the optimum for fitness.

Interactions between developmental and adult acclimation have distinct consequences for heat tolerance and heat stress recovery.

Developmental and adult thermal acclimation can have distinct, even opposite, effects on adult heat resistance in ectotherms. Yet, their relative contribution to heat-hardiness of ectotherms remains unclear despite the broad ecological implications thereof. Furthermore, the deterministic relationship between heat knockdown and recovery from heat stress is poorly understood but significant for establishing causal links between climate variability and population dynamics. Here, using Drosophila melanogaster in a full-factorial experimental design, we assessed the heat tolerance of flies in static stress assays, and document how developmental and adult acclimation interact with a distinct pattern to promote survival to heat stress in adults. We show that warmer adult acclimation is the initial factor enhancing survival to constant stressful high temperatures in flies, but also that the interaction between adult and developmental acclimation becomes gradually more important to ensure survival as the stress persists. This provides an important framework revealing the dynamic interplay between these two forms of acclimation that ultimately enhance thermal tolerance as a function of stress duration. Furthermore, by investigating recovery rates post-stress, we also show that the process of heat-hardening and recovery post-heat knockdown are likely to be based on set of (at least partially) divergent mechanisms. This could bear ecological significance as a trade-off may exist between increasing thermal tolerance and maximizing recovery rates post-stress, constraining population responses when exposed to variable and stressful climatic conditions.

An unusually diverse genus of Collembola in the Cape Floristic Region characterised by substantial desiccation tolerance.

Trait-environment interactions have contributed to the remarkable plant radiations in the Cape Floristic Region (CFR) of southern Africa. Whether such interactions have also resulted in the diversification of the invertebrate fauna, independently of direct associations with plants is, however, not clear. One candidate where this may be the case is the unusually diverse Collembola genus Seira. Including 89 species in the CFR, many of which are localised habitat specialists, this genus includes many species inhabiting the warm, dry fynbos shrubland-a habitat atypical of usually desiccation-sensitive Collembola. Here, we investigate whether desiccation tolerance may have contributed to the considerable diversity of Seira in the CFR. First, we demonstrate, by measuring vapour pressure deficits (VPD) of the species' microhabitats (fynbos shrubland and moister Afrotemperate Forests), that the fynbos shrublands are dry environments (mean ± S.E. maximum VPD 5.2 ± 0.1 kPa) compared with the Afrotemperate Forest patches (0.3 ± 0.02 kPa) during the summer activity period of Seira. Then we show that Seira species living in these shrublands are more desiccation tolerant (mean ± S.E. survival time at 76% relative humidity: 74.3 ± 3.3 h) than their congeners in the cooler, moister Afrotemperate Forests (34.3 ± 2.8 h), and compared with Collembola species globally (3.7 ± 0.2 h). These results, and a previous demonstration of pronounced thermal tolerance in the fynbos shrubland species, suggest that the diversity of Seira in the CFR is at least partly due to pronounced desiccation and thermal tolerance, which has enabled species in the genus to exploit the hot and dry habitats of the CFR.

Geographic variation in acclimation responses of thermal tolerance in South African diving beetles (Dytiscidae: Coleoptera).

Understanding sources of variation in animal thermal limits is critical to forecasting ecological responses to climate change. Here, we estimated upper and lower thermal limits, and their capacity to respond to thermal acclimation, in several species and populations of diving beetles (Dytiscidae) from diverse geographic regions representative of variable climate within South Africa. We also considered ecoregions and latitudinal ranges as potential predictors of thermal limits and the plasticity thereof. For upper thermal limits, species showed significant variation and limited acclimation-related plasticity. Lower thermal limits responded to acclimation in some cases and showed marked variation among species that could be explained by taxonomic affiliation and ecoregion. Limited acclimation ability in the species included in this study suggest plasticity of thermal limits will not be a likely buffer for coping with climate change. From the present results for the Dytiscidae of the region, it appears the group may be particularly susceptible to heat and/or drought and may thus serve as useful indicator species of ecosystem change. Understanding how these climate-related impacts play out at different spatial and temporal scales will have profound implications for conservation management and functional responses, especially important in a region already showing a trend for warming and drying.

Comparative demography of Bactrocera dorsalis (Hendel) and Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) on deciduous fruit.

Bactrocera dorsalis (Hendel) and Ceratitis capitata (Wiedemann) are highly polyphagous fruit fly species and important pests of commercial fruit in regions of the world where they are present. In South Africa, B. dorsalis is now established in the north and northeastern parts of the country. B. dorsalis is currently absent in other parts of the country including the Western Cape Province which is an important area for the production of deciduous fruit. C. capitata is widespread in South Africa and is the dominant pest of deciduous fruit. The demographic parameters of B. dorsalis and C. capitata on four deciduous fruit types Prunus persica (L.) Batsch, Prunus domestica L., Malus domestica Borkh. and Pyrus communis L. were studied to aid in predicting the potential population establishment and growth of B. dorsalis in a deciduous fruit growing environment. All deciduous fruit types tested were suitable for population persistence of both B. dorsalis and C. capitata. Development was fastest and survival highest on nectarine for both species. B. dorsalis adults generally lived longer than those of C. capitata, irrespective of the fruit types that they developed from. B. dorsalis had a higher net reproductive rate (Ro) on all deciduous fruit tested compared to C. capitata. However, the intrinsic rate of population increase was estimated to be higher for C. capitata than for B. dorsalis on all fruit types tested primarily due to C. capitata's faster generation time. Provided abiotic conditions are optimal, B. dorsalis would be able to establish and grow in deciduous fruit growing areas.

A computing platform to map ecological metabolism by integrating functional mapping and the metabolic theory of ecology.

Whole-organism metabolic rate co-varies allometrically with body mass, and is also affected by temperature through different biochemical mechanisms. Here we implement a computational platform to map specific quantitative trait loci (QTLs) that govern the dependence of metabolic rate on size and temperature. The model was formulated within settings of genetic mapping or genome-wide association studies through a mapping population genotyped by a set of molecular markers throughout the genome and phenotyped for metabolic parameters over a range of temperature. The model, estimated by a maximum-likelihood approach, allows a genome-wide search for the underlying metabolic QTLs and the estimation of genotype-specific parameters that specify the metabolism of an organism. Our model provides a tool to detect pleiotropy and epistasis that cause the size- and temperature-dependent change of metabolic rate.

Environmental temperature alters the overall digestive energetics and differentially affects dietary protein and lipid use in a lizard.

Processing food (e.g. ingestion, digestion, assimilation) requires energy referred to as specific dynamic action (SDA) and is at least partially fuelled by oxidation of the nutrients (e.g. proteins and lipids) within the recently ingested meal. In ectotherms, environmental temperature can affect the magnitude and/or duration of the SDA, but is likely to also alter the mixture of nutrients that are oxidized to cover these costs. Here, we examined metabolic rate, gut passage time, assimilation efficiency and fuel use in the lizard Agama atra digesting cricket meals at three ecologically relevant temperatures (20, 25 and 32°C). Crickets were isotopically enriched with 13C-leucine or 13C-palmitic-acid tracers to distinguish between protein and lipid oxidation, respectively. Our results show that higher temperatures increased the magnitude of the SDA peak (by 318% between 32 and 20°C) and gut passage rate (63%), and decreased the duration of the SDA response (by 20% for males and 48% for females). Peak rate of dietary protein oxidation occurred sooner than peak lipid oxidation at all temperatures (70, 60 and 31 h earlier for 20, 25 and 32°C, respectively). Assimilation efficiency of proteins, but not lipids, was positively related to temperature. Interestingly, the SDA response exhibited a notable circadian rhythm. These results show that temperature has a pronounced effect on digestive energetics in A.atra, and that this effect differs between nutrient classes. Variation in environmental temperatures may thus alter the energy budget and nutrient reserves of these animals.

Oxygen limitation is not the cause of death during lethal heat exposure in an insect.

Oxygen- and capacity-limited thermal tolerance (OCLTT) is a controversial hypothesis claiming to explain variation in, and mechanistically determine, animal thermal limits. The lack of support from Insecta is typically argued to be a consequence of their high-performance respiratory systems. However, no studies have reported internal body oxygen levels during thermal ramping so it is unclear if changes in ambient gas are partially or fully offset by a compensatory respiratory system. Here we provide such an assessment by simultaneously recording haemolymph oxygen (pO2) levels-as an approximation of tissue oxygenation-while experimentally manipulating ambient oxygen and subjecting organisms to thermal extremes in a series of thermolimit respirometry experiments using pupae of the butterfly Pieris napi. The main results are that while P. napi undergo large changes in haemolymph pO2 that are positively correlated with experimental oxygen levels, haemolymph pO2 is similar pre- and post-death during thermal assays. OCLTT predicts that reduction in body oxygen level should lead to a reduction in CTmax. Despite finding the former, there was no change in CTmax across a wide range of body oxygen levels. Thus, we argue that oxygen availability is not a functional determinant of the upper thermal limits in pupae of P. napi.

Exploring thermal flight responses as predictors of flight ability and geographic range size in Drosophila.

Thermal flight performance curves (TFPCs) may be a useful proxy for determining dispersal on daily timescales in winged insect species. Few studies have assessed TFPCs across a range of species under standard conditions despite that they may be useful in predicting variation in performance, abundance or geographic range shifts with forecast climate variability. Indeed, the factors determining realized dispersal within and among flying insect species are generally poorly understood. To better understand how flight performance may be correlated with geographic range extent and potential latitudinal climate variability, we estimated the thermal performance curves of flight ability in 11 Drosophilidae species (in 4 °C increments across 16-28 °C) after standard laboratory rearing for two generations. We tested if key morphological, evolutionary or ecological factors (e.g. species identity, sex, body mass, wing loading, geographic range size) predicted traits of TFPCs (including optimum temperature, maximum performance, thermal breadth of performance) or flight ability (success/failure to fly). Although several parameters of TFPCs varied among species these were typically not statistically significant probably owing to the relatively small pool of species assessed and the limited trait variation detected. The best explanatory model of these flight responses across species included significant positive effects of test temperature and wing area. However, the rank of geographic distribution breadth and phylogeny failed to explain significant variation in most of the traits, except for thermal performance breadth, of thermal flight performance curves among these 11 species. Future studies that employ a wider range of Drosophilidae species, especially if coupled with fine-scale estimates of species' environmental niches, would be useful.

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.