Effects of a high cholesterol diet on chill tolerance are highly context-dependent in Drosophila.

Chill susceptible insects are thought to be injured through different mechanisms depending on the duration and severity of chilling. While chronic chilling causes "indirect" injury through disruption of metabolic and ion homeostasis, acute chilling is suspected to cause "direct" injury, in part through phase transitions of cell membrane lipids. Dietary supplementation of cholesterol can reduce acute chilling injury in Drosophila melanogaster (Shreve et al., 2007), but the generality of this effect and the mechanisms underlying it remain unclear. To better understand how and why cholesterol has this effect, we assessed how a high cholesterol diet and thermal acclimation independently and interactively impact several measures of chill tolerance. Cholesterol supplementation positively affected tolerance to acute chilling in warm-acclimated flies (as reported previously). Conversely, feeding on the high-cholesterol diet negatively affected tolerance to chronic chilling in both cold and warm acclimated flies, as well as tolerance to acute chilling in cold acclimated flies. Cholesterol had no effect on the ability of flies to remain active in the cold or recover movement after a cold stress. Our findings support the idea that dietary cholesterol reduces mechanical injury to membranes caused by direct chilling injury, and that acute and chronic chilling are associated with distinct mechanisms of injury. Feeding on a high-cholesterol diet may interfere with mechanisms involved in cold acclimation, leaving cholesterol augmented flies more susceptible to chilling injury under some conditions.

The digestive system of a cricket pulverizes polyethylene microplastics down to the nanoplastic scale.

Microplastics (MPs; <5 mm) are a growing concern and a poorly understood threat to biota. We used a generalist insect (a cricket; Gryllodes sigillatus) to examine whether individuals would ingest and physically degrade MPs in their food. We fed crickets a range of concentrations (0, 2.5, 5, and 10% w/w) of fluorescent polyethylene MPs mixed into a standard diet and dissected the gut regions to isolate the MPs within. Comparing plastic content and fragment size within gut regions, we sought to identify whether and where crickets can fragment ingested MP particles. Given the digestive tract morphology of this species, we expected that the crickets would both ingest and egest the MPs. We also predicted that the MPs would be fragmented into smaller pieces during this digestive process. We found that G. sigillatus egested much smaller pieces than they ingested, and this fragmentation occurs early in the digestive process of this insect. We found this for both sexes as well as across the range of concentrations of MPs. The degree of plastic breakdown relative to plastic feeding time suggests that the ability to fragment MPs is intrinsic and not altered by how much time crickets have spent eating the plastics. The amount of plastics found in each region of the gut in relation to feeding time also suggests that this size and shape of PE microplastic does not cause any physical blockage in the gut. This lack of evidence for blockage is likely due to plastic breakdown. We found a ∼1000-fold reduction in plastic size occurs during passage through the digestive system, yielding particles very near nanoplastics (NPs; <1 µm), and likely smaller, that are then excreted back into the environment. These findings suggest that generalist insects can act as agents of plastic transformation in their environment if/when encountering MPs.

A neurophysiological limit and its biogeographic correlations: cold-induced spreading depolarization in tropical butterflies.

The physiology of insects is directly influenced by environmental temperature, and thermal tolerance is therefore intrinsically linked to their thermal niche and distribution. Understanding the mechanisms that limit insect thermal tolerance is crucial to predicting biogeography and range shifts. Recent studies on locusts and flies suggest that the critical thermal minimum (CTmin) follows a loss of CNS function via a spreading depolarization. We hypothesized that other insect taxa share this phenomenon. Here, we investigate whether spreading depolarization events occur in butterflies exposed to cold. Supporting our hypothesis, we found that exposure to stressful cold induced spreading depolarization in all 12 species tested. This reinforces the idea that spreading depolarization is a common mechanism underlying the insect CTmin. Furthermore, our results highlight how CNS function is tuned to match the environment of a species. Further research into the physiology underlying spreading depolarization will likely elucidate key mechanisms determining insect thermal tolerance and ecology.

Quantifying microplastic ingestion, degradation and excretion in insects using fluorescent plastics.

Plastic pollution is a growing threat to our natural environment. Plastic waste/pollution results from high emissions of both macro (>5 mm) and microplastics (MPs; <5 mm) as well as environmental fractioning of macroplastics into MPs. MPs have been shown to have a range of negative impacts on biota. Harmonized methods to accurately measure and count MPs from animal samples are limited, but what methods exist are not ideal for a controlled laboratory environment where plastic ingestion, degradation and elimination can be quantified and related to molecular, physiological and organismal traits. Here, we propose a complete method for isolating and quantifying fluorescent MPs by combining several previously reported approaches into one comprehensive workflow. We combine tissue dissection, organic material digestion, sample filtering and automated imaging techniques to show how fluorescently labelled MPs provided to insects (e.g. in their diet) in a laboratory setting can be isolated, identified and quantified. As a proof of concept, we fed crickets (Gryllodes sigillatus) a diet of 2.5% (w/w) fluorescently labelled plastics and isolated and quantified plastic particles within the gut and frass.

Locust gut epithelia do not become more permeable to fluorescent dextran and bacteria in the cold.

The insect gut, which plays a role in ion and water balance, has been shown to leak solutes in the cold. Cold stress can also activate insect immune systems, but it is unknown whether the leak of the gut microbiome is a possible immune trigger in the cold. We developed a novel feeding protocol to load the gut of locusts (Locusta migratoria) with fluorescent bacteria before exposing them to -2°C for up to 48 h. No bacteria were recovered from the hemolymph of cold-exposed locusts, regardless of exposure duration. To examine this further, we used an ex vivo gut sac preparation to re-test cold-induced fluorescent FITC-dextran leak across the gut and found no increased rate of leak. These results question not only the validity of FITC-dextran as a marker of paracellular barrier permeability in the gut, but also to what extent the insect gut becomes leaky in the cold.

Cold-induced immune activation in chill-susceptible insects.

Chilling injuries in chill-susceptible insects, such as the model dipteran Drosophila melanogaster, have been well-documented as a consequence of stressful low-temperature exposures. Cold stress also causes upregulation of genes in the insect immune pathways, some of which are also upregulated following other forms of sterile stress. The adaptive significance and underlying mechanisms surrounding cold-induced immune activation, however, are still unclear. Here, we review recent work on the roles of reactive oxygen species, damage-associated molecular patterns, and antimicrobial peptides in insect immune signaling or function. Using this emerging knowledge, we propose a conceptual model linking biochemical and molecular causes of immune activation to its consequences during and following cold stress.

A cold and quiet brain: mechanisms of insect CNS arrest at low temperatures.

Exposure to cold causes insects to enter a chill coma at species-specific temperatures and such temperature sensitivity contributes to geographic distribution and phenology. Coma results from abrupt spreading depolarization (SD) of neural tissue in the integrative centers of the central nervous system (CNS). SD abolishes neuronal signaling and the operation of neural circuits, like an off switch for the CNS. Turning off the CNS by allowing ion gradients to collapse will conserve energy and may offset negative consequences of temporary immobility. SD is modified by prior experience via rapid cold hardening (RCH) or cold acclimation that alter properties of Kv channels, Na+/K+-ATPase, and Na+/K+/2Cl- cotransporter. The stress hormone octopamine mediates RCH. Future progress depends on developing a more complete understanding of ion homeostasis in and of the insect CNS.

Plasticity in Na+/K+-ATPase thermal kinetics drives variation in the temperature of cold-induced neural shutdown of adult Drosophila melanogaster.

Most insects can acclimate to changes in their thermal environment and counteract temperature effects on neuromuscular function. At the critical thermal minimum, a spreading depolarization (SD) event silences central neurons, but the temperature at which this event occurs can be altered through acclimation. SD is triggered by an inability to maintain ion homeostasis in the extracellular space in the brain and is characterized by a rapid surge in extracellular K+ concentration, implicating ion pump and channel function. Here, we focused on the role of the Na+/K+-ATPase specifically in lowering the SD temperature in cold-acclimated Drosophila melanogaster. After first confirming cold acclimation altered SD onset, we investigated the dependency of the SD event on Na+/K+-ATPase activity by injecting the inhibitor ouabain into the head of the flies to induce SD over a range of temperatures. Latency to SD followed the pattern of a thermal performance curve, but cold acclimation resulted in a left-shift of the curve to an extent similar to its effect on the SD temperature. With Na+/K+-ATPase activity assays and immunoblots, we found that cold-acclimated flies have ion pumps that are less sensitive to temperature, but do not differ in their overall abundance in the brain. Combined, these findings suggest a key role for plasticity in Na+/K+-ATPase thermal sensitivity in maintaining central nervous system function in the cold, and more broadly highlight that a single ion pump can be an important determinant of whether insects can respond to their environment to remain active at low temperatures.

Ingestion of Microplastic Fibres, But Not Microplastic Beads, Impacts Growth Rates in the Tropical House Cricket Gryllodes Sigillatus.

Microplastic is a growing concern as an environmental contaminant as it is ubiquitous in our ecosystems. Microplastics are present in terrestrial environments, yet the majority of studies have focused on the adverse effects of microplastics on aquatic biota. We hypothesized that microplastic ingestion by a terrestrial insect would have localized effects on gut health and nutrient absorption, such that prolonged dietary microplastic exposure would impact growth rate and adult body size. We further hypothesized that plastic form (fibres vs. beads) would influence these effects because of the nature of gut-plastic interactions. Freshly hatched tropical house crickets (Gryllodes sigillatus) were fed a standard diet containing different concentrations of either fluorescent polyethylene microplastic beads (75-105 µm), or untreated polyethylene terephthalate microfibers (< 5 mm) until they died or reached adulthood (approximately 8 weeks). Weight and body length were measured weekly and microplastic ingestion was confirmed through fluorescence microscopy and visual inspection of the frass. While, to our surprise, we found no effect of polyethylene bead ingestion on growth rate or final body size of G. sigillatus, females experienced a reduction in size and weight when fed high concentrations of polyethylene terephthalate microfibers. These results suggest that high concentrations of polyethylene beads of the 100 µm size range can pass through the cricket gut without a substantial negative effect on their growth and development time, but high concentrations of polyethylene terephthalate microfibers cannot. Although we report the negative effects of microplastic ingestion on the growth of G. sigillatus, it remains uncertain what threats microplastics pose to terrestrial insects.

A lack of repeatability creates the illusion of a trade-off between basal and plastic cold tolerance.

The thermotolerance-plasticity trade-off hypothesis predicts that ectotherms with greater basal thermal tolerance have a lower acclimation capacity. This hypothesis has been tested at both high and low temperatures but the results often conflict. If basal tolerance constrains plasticity (e.g. through shared mechanisms that create physiological constraints), it should be evident at the level of the individual, provided the trait measured is repeatable. Here, we used chill-coma onset temperature and chill-coma recovery time (CCO and CCRT; non-lethal thermal limits) to quantify cold tolerance of Drosophila melanogaster across two trials (pre- and post-acclimation). Cold acclimation improved cold tolerance, as expected, but individual measurements of CCO and CCRT in non-acclimated flies were not (or only slightly) repeatable. Surprisingly, however, there was still a strong correlation between basal tolerance and plasticity in cold-acclimated flies. We argue that this relationship is a statistical artefact (specifically, a manifestation of regression to the mean; RTM) and does not reflect a true trade-off or physiological constraint. Thermal tolerance trade-off patterns in previous studies that used similar methodology are thus likely to be impacted by RTM. Moving forward, controlling and/or correcting for RTM effects is critical to determining whether such a trade-off or physiological constraint exists.

Autumn larval cold tolerance does not predict the northern range limit of a widespread butterfly species.

Climate change is driving range shifts, and a lack of cold tolerance is hypothesized to constrain insect range expansion at poleward latitudes. However, few, if any, studies have tested this hypothesis during autumn when organisms are subjected to sporadic low-temperature exposure but may not have become cold-tolerant yet. In this study, we integrated organismal thermal tolerance measures into species distribution models for larvae of the Giant Swallowtail butterfly, Papilio cresphontes (Lepidoptera: Papilionidae), living at the northern edge of its actively expanding range. Cold hardiness of field-collected larvae was determined using three common metrics of cold-induced physiological thresholds: the supercooling point, critical thermal minimum, and survival following cold exposure. P. cresphontes larvae were determined to be tolerant of chilling but generally die at temperatures below their SCP, suggesting they are chill-tolerant or modestly freeze-avoidant. Using this information, we examined the importance of low temperatures at a broad scale, by comparing species distribution models of P. cresphontes based only on environmental data derived from other sources to models that also included the cold tolerance parameters generated experimentally. Our modeling revealed that growing degree-days and precipitation best predicted the distribution of P. cresphontes, while the cold tolerance variables did not explain much variation in habitat suitability. As such, the modeling results were consistent with our experimental results: Low temperatures in autumn are unlikely to limit the distribution of P. cresphontes. Understanding the factors that limit species distributions is key to predicting how climate change will drive species range shifts.

Chill coma onset and recovery fail to reveal true variation in thermal performance among populations of Drosophila melanogaster.

Species from colder climates tend to be more chill tolerant regardless of the chill tolerance trait measured, but for Drosophila melanogaster, population-level differences in chill tolerance among populations are not always found when a single trait is measured in the laboratory. We measured chill coma onset temperature, chill coma recovery time, and survival after chronic cold exposure in replicate lines derived from multiple paired African and European D. melanogaster populations. The populations in our study were previously found to differ in chronic cold survival ability, which is believed to have evolved independently in each population pair; however, they did not differ in chill coma onset temperature and chill coma recovery time in a manner that reflected their geographic origins, even though these traits are known to vary with origin latitude among Drosophila species and are among the most common metrics of thermal tolerance in insects. While it is common practice to measure only one chill tolerance trait when comparing chill tolerance among insect populations, our results emphasise the importance of measuring more than one thermal tolerance trait to minimize the risk of missing real adaptive variation in insect thermal tolerance.

One hundred research questions in conservation physiology for generating actionable evidence to inform conservation policy and practice.

Environmental change and biodiversity loss are but two of the complex challenges facing conservation practitioners and policy makers. Relevant and robust scientific knowledge is critical for providing decision-makers with the actionable evidence needed to inform conservation decisions. In the Anthropocene, science that leads to meaningful improvements in biodiversity conservation, restoration and management is desperately needed. Conservation Physiology has emerged as a discipline that is well-positioned to identify the mechanisms underpinning population declines, predict responses to environmental change and test different in situ and ex situ conservation interventions for diverse taxa and ecosystems. Here we present a consensus list of 10 priority research themes. Within each theme we identify specific research questions (100 in total), answers to which will address conservation problems and should improve the management of biological resources. The themes frame a set of research questions related to the following: (i) adaptation and phenotypic plasticity; (ii) human-induced environmental change; (iii) human-wildlife interactions; (iv) invasive species; (v) methods, biomarkers and monitoring; (vi) policy, engagement and communication; (vii) pollution; (viii) restoration actions; (ix) threatened species; and (x) urban systems. The themes and questions will hopefully guide and inspire researchers while also helping to demonstrate to practitioners and policy makers the many ways in which physiology can help to support their decisions.

Thermal acclimation alters Na+/K+-ATPase activity in a tissue-specific manner in Drosophila melanogaster.

Insects, like the model species Drosophila melanogaster, lose neuromuscular function and enter a state of paralysis (chill coma) at a population- and species-specific low temperature threshold that is decreased by cold acclimation. Entry into this coma is related to a spreading depolarization in the central nervous system, while recovery involves restoration of electrochemical gradients across muscle cell membranes. The Na+/K+-ATPase helps maintain ion balance and membrane potential in both the brain and hemolymph (surrounding muscles), and changes in thermal tolerance traits have therefore been hypothesized to be closely linked to variation in the expression and/or activity of this pump in multiple tissues. Here, we tested this hypothesis by measuring activity and thermal sensitivity of the Na+/K+-ATPase at the tagma-specific level (head, thorax and abdomen) in warm- (25 °C) and cold-acclimated (15 °C) flies by measuring Na+/K+-ATPase activity at 15, 20, and 25 °C. We relate differences in pump activity to differences in chill coma temperature, spreading depolarization temperature, and thermal dependence of muscle cell polarization. Differences in pump activity and thermal sensitivity induced by cold acclimation varied in a tissue-specific manner: While thermal sensitivity remained unchanged, cold-acclimated flies had decreased Na+/K+-ATPase activity in the thorax (mainly muscle) and head (mainly composed of brain). We argue that these changes may assist in maintenance of K+ homeostasis and membrane potential across muscle membranes, and discuss how reduced Na+/K+-ATPase activity in the brain may counterintuitively help insects delay coma onset in the cold.

A simple and dynamic thermal gradient device for measuring thermal performance in small ectotherms.

The body temperature of ectothermic animals is heavily dependent on environmental temperature, impacting fitness. Laboratory exposure to favorable and unfavorable temperatures is used to understand these effects, as well as the physiological, biochemical, and molecular underpinnings of variation in thermal performance. Although small ectotherms, like insects, can often be easily reared in large numbers, it can be challenging and expensive to simultaneously create and manipulate several thermal environments in a laboratory setting. Here, we describe the creation and use of a thermal gradient device that can produce a wide range of constant or varying temperatures concurrently. Conservatively, this system as designed can operate between -6 °C and 40 °C. This device is composed of a solid aluminum plate and copper piping, combined with a pair of refrigerated circulators. As a simple proof-of-concept, we completed single experimental runs to produce a low-temperature survival curve for flies (Drosophila melanogaster) and explore the effects of daily thermal cycles of varying amplitude on growth rates of crickets (Gryllodes sigillatus). This approach avoids the use of multiple heating/cooling water or glycol baths or incubators for large-scale assessments of organismal thermal performance. It makes static or dynamic thermal experiments (e.g., creating a thermal performance or survival curves, quantifying responses to fluctuating thermal environments, or monitoring animal behavior across a range of temperatures) easier, faster, and less costly.

Hyperkalaemia, not apoptosis, accurately predicts insect chilling injury.

There is a growing appreciation that insect distribution and abundance are associated with the limits of thermal tolerance, but the physiology underlying thermal tolerance remains poorly understood. Many insects, like the migratory locust (Locusta migratoria), suffer a loss of ion and water balance leading to hyperkalaemia (high extracellular [K+]) in the cold that indirectly causes cell death. Cells can die in several ways under stress, and how they die is of critical importance to identifying and understanding the nature of thermal adaptation. Whether apoptotic or necrotic cell death pathways are responsible for low-temperature injury is unclear. Here, we use a caspase-3 specific assay to indirectly quantify apoptotic cell death in three locust tissues (muscle, nerves and midgut) following prolonged chilling and recovery from an injury-inducing cold exposure. Furthermore, we obtain matching measurements of injury, extracellular [K+] and muscle caspase-3 activity in individual locusts to gain further insight into the mechanistic nature of chilling injury. We found a significant increase in muscle caspase-3 activity, but no such increase was observed in either nervous or gut tissue from the same animals, suggesting that chill injury primarily relates to muscle cell death. Levels of chilling injury measured at the whole animal level, however, were strongly correlated with the degree of haemolymph hyperkalaemia, and not apoptosis. These results support the notion that cold-induced ion balance disruption triggers cell death but also that apoptosis is not the main form of cell damage driving low-temperature injury.

Chilling induces unidirectional solute leak through the locust gut epithelia.

Chill-susceptible insects, like the migratory locust, often die when exposed to low temperatures from an accumulation of tissue damage that is unrelated to freezing (chilling injury). Chilling injury is often associated with a loss of ion balance across the gut epithelia. It has recently been suggested that this imbalance is at least partly caused by a cold-induced disruption of epithelial barrier function. Here, we aimed to test this hypothesis in the migratory locust (Locustamigratoria). First, chill tolerance was quantified by exposing locusts to -2°C and recording chill coma recovery time and survival 24 h post-cold exposure. Longer exposure times significantly increased recovery time and caused injury and death. Ion-selective microelectrodes were also used to test for a loss of ion balance in the cold. We found a significant increase of haemolymph K+ and decrease of haemolymph Na+ concentration over time. Next, barrier failure along the gut was tested by monitoring the movement of an epithelial barrier marker (FITC-dextran) across the gut epithelia during exposure to -2°C. We found a significant increase in haemolymph FITC-dextran concentration over time in the cold when assayed in the mucosal to serosal direction. However, when tested in the serosal to mucosal direction, we saw minimal marker movement across the gut epithelia. This suggests that while cold-induced barrier disruption is present, it is apparently unidirectional. It is important to note that these data reveal only the phenomenon itself. The location of this leak as well as the underlying mechanisms remain unclear and require further investigation.

Warm periods in repeated cold stresses protect Drosophila against ionoregulatory collapse, chilling injury, and reproductive deficits.

In some insects, repeated cold stresses, characterized by warm periods that interrupt a sustained cold period, have been found to yield survival benefits over continuous cold stresses, but at the cost of reproduction. During a cold stress, chill susceptible insects like Drosophila melanogaster suffer from a loss of ion and water balance, and the current model of recovery from chilling posits that re-establishment of ion homeostasis begins upon return to a warm environment, but that it takes minutes to hours for an insect to fully restore homeostasis. Following this ionoregulatory model of chill coma recovery, we predicted that the longer the duration of the warm periods between cold stresses, the better a fly will endure a subsequent chill coma event and the more likely they will be to survive. We also predicted, however, that this recovery may lead to reduced fecundity, possibly due to allocation of energy reserves away from reproduction. Here, female D.melanogaster were treated to a long continuous cold stress (25 h at 0 °C), or experienced the same total time in the cold with repeated short (15 min), or long (120 min) breaks at 22 °C. We found that warm periods in general improved survival outcomes, and individuals that recovered for more time in between cold periods had significantly lower rates of injury, faster recovery from chill coma, and produced greater, rather than fewer, offspring. These improvements in chill tolerance were associated with mitigation of ionoregulatory collapse, as flies that experienced either short or long warm periods better maintained low hemolymph [K+]. Thus, warm periods that interrupt cold periods improve cold tolerance and fertility in D. melanogaster females relative to a single sustained cold stress, potentially because this time allows for recovery of ion and water homeostasis.

Reframing conservation physiology to be more inclusive, integrative, relevant and forward-looking: reflections and a horizon scan.

Applying physiological tools, knowledge and concepts to understand conservation problems (i.e. conservation physiology) has become commonplace and confers an ability to understand mechanistic processes, develop predictive models and identify cause-and-effect relationships. Conservation physiology is making contributions to conservation solutions; the number of 'success stories' is growing, but there remain unexplored opportunities for which conservation physiology shows immense promise and has the potential to contribute to major advances in protecting and restoring biodiversity. Here, we consider how conservation physiology has evolved with a focus on reframing the discipline to be more inclusive and integrative. Using a 'horizon scan', we further explore ways in which conservation physiology can be more relevant to pressing conservation issues of today (e.g. addressing the Sustainable Development Goals; delivering science to support the UN Decade on Ecosystem Restoration), as well as more forward-looking to inform emerging issues and policies for tomorrow. Our horizon scan provides evidence that, as the discipline of conservation physiology continues to mature, it provides a wealth of opportunities to promote integration, inclusivity and forward-thinking goals that contribute to achieving conservation gains. To advance environmental management and ecosystem restoration, we need to ensure that the underlying science (such as that generated by conservation physiology) is relevant with accompanying messaging that is straightforward and accessible to end users.