Flying, nectar-loaded honey bees conserve water and improve heat tolerance by reducing wingbeat frequency and metabolic heat production.

Heat waves are becoming increasingly common due to climate change, making it crucial to identify and understand the capacities for insect pollinators, such as honey bees, to avoid overheating. We examined the effects of hot, dry air temperatures on the physiological and behavioral mechanisms that honey bees use to fly when carrying nectar loads, to assess how foraging is limited by overheating or desiccation. We found that flight muscle temperatures increased linearly with load mass at air temperatures of 20 or 30 °C, but, remarkably, there was no change with increasing nectar loads at an air temperature of 40 °C. Flying, nectar-loaded bees were able to avoid overheating at 40 °C by reducing their flight metabolic rates and increasing evaporative cooling. At high body temperatures, bees apparently increase flight efficiency by lowering their wingbeat frequency and increasing stroke amplitude to compensate, reducing the need for evaporative cooling. However, even with reductions in metabolic heat production, desiccation likely limits foraging at temperatures well below bees' critical thermal maxima in hot, dry conditions.

Target for lipid-to-carbohydrate intake minimizes cost of growth.

Many theoretical treatments of foraging use energy as currency, with carbohydrates and lipids considered interchangeable as energy sources. However, herbivores must often synthesize lipids from carbohydrates since they are in short supply in plants, theoretically increasing the cost of growth. We tested whether a generalist insect herbivore (Locusta migratoria) can improve its growth efficiency by consuming lipids, and whether these locusts have a preferred caloric intake ratio of carbohydrate to lipid (C : L). Locusts fed pairs of isocaloric, isoprotein diets differing in C and L consistently selected a 2C : 1L target. Locusts reared on isocaloric, isoprotein 3C : 0L diets attained similar final body masses and lipid contents to locusts fed the 2C : 1L diet, but they ate more and had a ~12% higher metabolic rate, indicating an energetic cost for lipogenesis. These results demonstrate that some animals can selectively regulate carbohydrate-to-lipid intake and that consumption of dietary lipids can improve growth efficiency.

Synergistic negative effects between a fungicide and high temperatures on homing behaviours in honeybees.

Interactions between environmental stressors may contribute to ongoing pollinator declines, but have not been extensively studied. Here, we examined the interaction between the agricultural fungicide Pristine (active ingredients: 25.2% boscalid, 12.8% pyraclostrobin) and high temperatures on critical honeybee behaviours. We have previously shown that consumption of field-realistic levels of this fungicide shortens worker lifespan in the field and impairs associative learning performance in a laboratory-based assay. We hypothesized that Pristine would also impair homing and foraging behaviours in the field, and that an interaction with hot weather would exacerbate this effect. Both field-relevant Pristine exposure and higher air temperatures reduced the probability of successful return on their own. Together, the two factors synergistically reduced the probability of return and increased the time required for bees to return to the hive. Pristine did not affect the masses of pollen or volumes of nectar or water brought back to the hive by foragers, and it did not affect the ratio of forager types in a colony. However, Pristine-fed bees brought more concentrated nectar back to the hive. As both agrochemical usage and heat waves increase, additive and synergistic negative effects may pose major threats to pollinators and sustainable agriculture.

A thermal performance curve perspective explains decades of disagreements over how air temperature affects the flight metabolism of honey bees.

While multiple studies have shown that honey bees and some other flying insects lower their flight metabolic rates when flying at high air temperatures, critics have suggested such patterns result from poor experimental methods as, theoretically, air temperature should not appreciably affect aerodynamic force requirements. Here, we show that apparently contradictory studies can be reconciled by considering the thermal performance curve of flight muscle. We show that prior studies that found no effects of air temperature on flight metabolism of honey bees achieved flight muscle temperatures that were near or on equal, opposite sides of the thermal performance curve. Honey bees vary their wing kinematics and metabolic heat production to thermoregulate, and how air temperature affects the flight metabolic rate of honey bees is predictable using a non-linear thermal performance perspective of honey bee flight muscle.

Breaking the cycle: Reforming pesticide regulation to protect pollinators.

Over decades, pesticide regulations have cycled between approval and implementation, followed by the discovery of negative effects on nontarget organisms that result in new regulations, pesticides, and harmful effects. This relentless pattern undermines the capacity to protect the environment from pesticide hazards and frustrates end users that need pest management tools. Wild pollinating insects are in decline, and managed pollinators such as honey bees are experiencing excessive losses, which threatens sustainable food security and ecosystem function. An increasing number of studies demonstrate the negative effects of field-realistic exposure to pesticides on pollinator health and fitness, which contribute to pollinator declines. Current pesticide approval processes, although they are superior to past practices, clearly continue to fail to protect pollinator health. In the present article, we provide a conceptual framework to reform cyclical pesticide approval processes and better protect pollinators.

High carbohydrate consumption increases lipid storage and promotes migratory flight in locusts.

Migration allows animals to track favorable environments and avoid harmful conditions. However, migration is energetically costly, so migrating animals must prepare themselves by increasing their energy stores. Despite the importance of locust migratory swarms, we still understand little about the physiology of locust migration. During long-distance flight, locusts rely on lipid oxidation, despite the fact that lipids are relatively rare in their leaf-based diets. Therefore, locusts and other insect herbivores synthesize and store lipid from ingested carbohydrates, which are also important for initial flight. These data suggest that diets high in carbohydrate should increase lipid stores and the capacity for migratory flight in locusts. As predicted, locust lipid stores and flight performance increased with an increase in the relative carbohydrate content in their food. However, locust flight termination was not associated with complete lipid depletion. We propose potential testable mechanisms that might explain how macronutrient consumption can affect flight endurance.

Air sacs are a key adaptive trait of the insect respiratory system.

Air sacs are a well-known aspect of insect tracheal systems, but have received little research attention. In this Commentary, we suggest that the study of the distribution and function of air sacs in tracheate arthropods can provide insights of broad significance. We provide preliminary phylogenetic evidence that the developmental pathways for creation of air sacs are broadly conserved throughout the arthropods, and that possession of air sacs is strongly associated with a few traits, including the capacity for powerful flight, large body or appendage size and buoyancy control. We also discuss how tracheal compression can serve as an additional mechanism for achieving advection in tracheal systems. Together, these patterns suggest that the possession of air sacs has both benefits and costs that remain poorly understood. New technologies for visualization and functional analysis of tracheal systems provide exciting approaches for investigations that will be of broad significance for understanding invertebrate evolution.

Physiological responses to gravity in an insect.

Gravity is one of the most ubiquitous environmental effects on living systems: Cellular and organismal responses to gravity are of central importance to understanding the physiological function of organisms, especially eukaryotes. Gravity has been demonstrated to have strong effects on the closed cardiovascular systems of terrestrial vertebrates, with rapidly responding neural reflexes ensuring proper blood flow despite changes in posture. Invertebrates possess open circulatory systems, which could provide fewer mechanisms to restrict gravity effects on blood flow, suggesting that these species also experience effects of gravity on blood pressure and distribution. However, whether gravity affects the open circulatory systems of invertebrates is unknown, partly due to technical measurement issues associated with small body size. Here we used X-ray imaging, radio-tracing of hemolymph, and micropressure measurements in the American grasshopper, Schistocerca americana, to assess responses to body orientation. Our results show that during changes in body orientation, gravity causes large changes in blood and air distribution, and that body position affects ventilation rate. Remarkably, we also found that insects show similar heart rate responses to body position as vertebrates, and contrasting with the classic understanding of open circulatory systems, have flexible valving systems between thorax and abdomen that can separate pressures. Gravitational effects on invertebrate cardiovascular and respiratory systems are likely to be widely distributed among invertebrates and to have broad influence on morphological and physiological evolution.

The thermal performance curve for aerobic metabolism of a flying endotherm.

Performance benefits of stable, warm muscles are believed to be important for the evolution of endothermy in mammals, birds and flying insects. However, thermal performance curves have never been measured for a free-flying endotherm, as it is challenging to vary body temperatures of these animals, and maximal flight performance is difficult to elicit. We varied air temperatures and gas densities to manipulate thoracic temperatures of flying honeybees from 29°C to 44°C, with low air densities used to increase flight metabolic rates to maximal values. Honeybees showed a clear thermal performance curve with an optimal temperature of 39°C. Maximal flight metabolic rates increased by approximately 2% per 1°C increase in thoracic temperature at suboptimal thoracic temperatures, but decreased approximately 5% per 1°C increase as the bees continued to heat up. This study provides the first quantification of the maximal metabolic performance benefit of thermoregulation in an endotherm. These data directly support aerobic capacity models for benefits of thermoregulation in honeybees, and suggest that improved aerobic capacity probably contributes to the multiple origins of endothermic heterothermy in bees and other insects.

A desert bee thermoregulates with an abdominal convector during flight.

Flying endothermic insects thermoregulate, likely to improve flight performance. Males of the Sonoran Desert bee, Centris caesalpiniae, seek females at aggregations beginning at sunrise and cease flight near midday when the air temperature peaks. To identify the thermoregulatory mechanisms for C. caesalpiniae males, we measured tagma temperature, wingbeat frequency, water loss rate, metabolic rate and tagma mass of flying bees across shaded air temperatures of 19-38°C. Surface area, wet mass and dry mass declined with air temperature, suggesting that individual bees do not persist for the entire morning. The largest bees may be associated with cool, early mornings because they are best able to warm themselves and/or because they run the risk of overheating in the hot afternoons. Thorax temperature was high (38-45°C) and moderately well regulated, while head and abdomen temperatures were cooler and less controlled. The abdominal temperature excess ratio increased as air temperature rose, indicating active heat transfer from the pubescent thorax to the relatively bare abdomen with warming. Mass-specific metabolic rate increased with time, and air and thorax temperatures, but wingbeat frequency did not vary. Mass-specific water loss rate increased with air temperature, but this was a minor mechanism of thermoregulation. Using a heat budget model, we showed that whole-body convective conductance more than doubled through the morning, providing strong evidence that the primary mechanism of regulating thorax temperature during flight for these bees is increased use of the abdomen as a convector at higher air temperatures.

Social consequences of energetically costly nest construction in a facultatively social bee.

Social groups form when the costs of breeding independently exceed fitness costs imposed by group living. The costs of independent breeding can often be energetic, especially for animals performing expensive behaviours, such as nest construction. To test the hypothesis that nesting costs can drive sociality by disincentivizing independent nest founding, we measured the energetics of nest construction and inheritance in a facultatively social carpenter bee (Xylocopa sonorina Smith), which bores tunnel nests in wood. We measured metabolic rates of bees excavating wood and used computerized tomography images of nesting logs to measure excavation volumes. From these data, we demonstrate costly energetic investments in nest excavation of a minimum 4.3 kJ per offspring provisioned, an expense equivalent to nearly 7 h of flight. This high, potentially prohibitive cost of nest founding may explain why females compete for existing nests rather than constructing new ones, often leading to the formation of social groups. Further, we found that nest inheritors varied considerably in their investment in nest renovation, with costs ranging more than 12-fold (from 7.08 to 89.1 kJ energy), probably reflecting differences in inherited nest quality. On average, renovation costs were lower than estimated new nest construction costs, with some nests providing major savings. These results suggest that females may join social groups to avoid steep energetic costs, but that the benefits of this strategy are not experienced equally.

The active ingredients of a mitotoxic fungicide negatively affect pollen consumption and worker survival in laboratory-reared honey bees (Apis mellifera).

Recent observations of many sublethal effects of pesticides on pollinators have raised questions about whether standard short-term laboratory tests of pesticide effects on survival are sufficient for pollinator protection. The fungicide Pristine® and its active ingredients (25.2% boscalid, 12.8% pyraclostrobin) have been reported to have low acute toxicity to caged honey bee workers, but many sublethal effects at field-relevant doses have been reported and Pristine® was recently found to increase worker pollen consumption, reduce worker longevity and colony populations at field relevant concentrations (Fisher et al. 2021). To directly compare these whole-colony field results to more standard laboratory toxicology tests, the effects of Pristine®, at a range of field-relevant concentrations, were assessed on the survival and pollen consumption of honey bee workers 0-14 days of age. Also, to separate the effects of the inert and two active ingredients, bees were fed pollen containing boscalid, pyraclostrobin, or pyraclostrobin plus boscalid, at concentrations matching those in the Pristine® treatments. Pyraclostrobin significantly reduced pollen consumption across the duration of the experiment, and dose-dependently reduced pollen consumption on days 12-14. Pristine® and boscalid significantly reduced pollen feeding rate on days 12-14. Boscalid reduced survival in a dose-dependent manner. Consumption of Pristine® or pyraclostrobin plus boscalid did not affect survival, providing evidence against strong negative effects of the inert ingredients in Pristine® and against negative synergistic effects of boscalid and pyraclostrobin. The stronger toxic effects of Pristine® observed in field colonies compared to this laboratory test, and the opposite responses of pollen consumption in the laboratory and field to Pristine®, show that standard laboratory toxicology tests can fail to predict responses of pollinators to pesticides and to provide protection.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera).

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Plant carbohydrate content limits performance and lipid accumulation of an outbreaking herbivore.

Locusts are major intermittent threats to food security and the ecological factors determining where and when these occur remain poorly understood. For many herbivores, obtaining adequate protein from plants is a key challenge. We tested how the dietary protein : non-structural carbohydrate ratio (p : c) affects the developmental and physiological performance of 4th-5th instar nymphs of the South American locust, Schistocerca cancellata, which has recently resurged in Argentina, Bolivia and Paraguay. Field marching locusts preferred to feed on high carbohydrate foods. Field-collected juveniles transferred to the laboratory selected artificial diets or local plants with low p : c. On single artificial diets, survival rate increased as foods became more carbohydrate-biased. On single local plants, growth only occurred on the plant with the lowest p : c. Most local plants had p : c ratios substantially higher than optimal, demonstrating that field marching locusts must search for adequate carbohydrate or their survival and growth will be carbohydrate-limited. Total body lipids increased as dietary p : c decreased on both artificial and plant diets, and the low lipid contents of field-collected nymphs suggest that obtaining adequate carbohydrate may pose a strong limitation on migration for S. cancellata. Anthropogenic influences such as conversions of forests to pastures, may increase carbohydrate availability and promote outbreaks and migration of some locusts.

PO2 of the metathoracic ganglion in response to progressive hypoxia in an insect.

Mammals regulate their brain tissue PO2 tightly, and only small changes in brain PO2 are required to elicit compensatory ventilation. However, unlike the flow-through cardiovascular system of vertebrates, insect tissues exchange gases through blind-ended tracheoles, which may involve a more prominent role for diffusive gas exchange. We tested the effect of progressive hypoxia on ventilation and the PO2 of the metathoracic ganglion (neural site of control of ventilation) using microelectrodes in the American locust, Schistocerca americana. In normal air (21 kPa), PO2 of the metathoracic ganglion was 12 kPa. The PO2 of the ganglion dropped as air PO2 dropped, with ventilatory responses occurring when ganglion PO2 reached 3 kPa. Unlike vertebrates, insects tolerate relatively high resting tissue PO2 levels and allow tissue PO2 to drop during hypoxia, activity and discontinuous gas exchange before activating convective or spiracular gas exchange. Tracheated animals, and possibly pancrustaceans in general, seem likely to generally experience wide spatial and temporal variation in tissue PO2 compared with vertebrates, with important implications for physiological function and the evolution of oxygen-using proteins.

Metabolomics of anoxia tolerance in Drosophila melanogaster: evidence against substrate limitation and for roles of protective metabolites and paralytic hypometabolism.

Animals vary tremendously in their capacities to survive anoxia, and the mechanisms responsible are poorly understood. Adult Drosophila melanogaster are rapidly paralyzed and survive up to 12 h of anoxia, whereas larvae vigorously attempt escape but then die if anoxia exceeds 2 h. Here we use nuclear magnetic resonance methods to compare the metabolome of larvae and adult D. melanogaster under normoxic conditions and after various anoxic durations up to 1 h. Glucose increased during anoxia in both larvae and adults, so anoxic death by carbohydrate limitation is unlikely for either stage. Lactate and alanine were the primary anaerobic end products in both adults and larvae. During the first 30 min of anoxia, larvae accumulated anaerobic end products (predominately lactate) at a higher rate, suggesting that larvae may experience greater initial acid-base disruption during anoxic exposures. Adult Drosophila did not possess higher levels of putative protective metabolites; however, these increased during anoxia in adults and decreased in larvae. Metabolites that decreased during anoxia in larvae included mannitol, xylitol, glycerol, betaine, serine, and tyrosine, perhaps due to use as fuels, antioxidants, or binding to denatured proteins. Adults showed significant increases in glycine, taurine, and the polyols glycerol, mannitol, and xylitol, suggesting that adults upregulate protective metabolites to prevent damage. Our results suggest that lower initial metabolic demand due to paralytic hypometabolism and capacities to upregulate protective metabolites may assist the better anoxia tolerance of adult Drosophila.

Effects of anoxia on ATP, water, ion and pH balance in an insect (Locusta migratoria).

When exposed to anoxia, insects rapidly go into a hypometabolic coma from which they can recover when exposed to normoxia again. However, prolonged anoxic bouts eventually lead to death in most insects, although some species are surprisingly tolerant. Anoxia challenges ATP, ion, pH and water homeostasis, but it is not clear how fast and to what degree each of these parameters is disrupted during anoxia, nor how quickly they recover. Further, it has not been investigated which disruptions are the primary source of the tissue damage that ultimately causes death. Here, we show, in the migratory locust (Locusta migratoria), that prolonged anoxic exposures are associated with increased recovery time, decreased survival, rapidly disrupted ATP and pH homeostasis and a slower disruption of ion ([K+] and [Na+]) and water balance. Locusts could not fully recover after 4 h of anoxia at 30°C, and at this point hemolymph [K+] was elevated 5-fold and [Na+] was decreased 2-fold, muscle [ATP] was decreased to ≤3% of normoxic values, hemolymph pH had dropped 0.8 units from 7.3 to 6.5, and hemolymph water content was halved. These physiological changes are associated with marked tissue damage in vivo and we show that the isolated and combined effects of hyperkalemia, acidosis and anoxia can all cause muscle tissue damage in vitro to equally large degrees. When locusts were returned to normoxia after a moderate (2 h) exposure of anoxia, ATP recovered rapidly (15 min) and this was quickly followed by recovery of ion balance (30 min), while pH recovery took 2-24 h. Recovery of [K+] and [Na+] coincided with the animals exiting the comatose state, but recovery to an upright position took ∼90 min and was not related to any of the physiological parameters examined.

Functional Hypoxia in Insects: Definition, Assessment, and Consequences for Physiology, Ecology, and Evolution.

Insects can experience functional hypoxia, a situation in which O2 supply is inadequate to meet oxygen demand. Assessing when functional hypoxia occurs is complex, because responses are graded, age and tissue dependent, and compensatory. Here, we compare information gained from metabolomics and transcriptional approaches and by manipulation of the partial pressure of oxygen. Functional hypoxia produces graded damage, including damaged macromolecules and inflammation. Insects respond by compensatory physiological and morphological changes in the tracheal system, metabolic reorganization, and suppression of activity, feeding, and growth. There is evidence for functional hypoxia in eggs, near the end of juvenile instars, and during molting. Functional hypoxia is more likely in species with lower O2 availability or transport capacities and when O2 need is great. Functional hypoxia occurs normally during insect development and is a factor in mediating life-history trade-offs.

Approaches for testing hypotheses for the hypometric scaling of aerobic metabolic rate in animals.

Hypometric scaling of aerobic metabolism [larger organisms have lower mass-specific metabolic rates (MR/g)] is nearly universal for interspecific comparisons among animals, yet we lack an agreed upon explanation for this pattern. If physiological constraints on the function of larger animals occur and limit MR/g, these should be observable as direct constraints on animals of extant species and/or as evolved responses to compensate for the proposed constraint. There is evidence for direct constraints and compensatory responses to O2 supply constraint in skin-breathing animals, but not in vertebrates with gas-exchange organs. The duration of food retention in the gut is longer for larger birds and mammals, consistent with a direct constraint on nutrient uptake across the gut wall, but there is little evidence for evolving compensatory responses to gut transport constraints in larger animals. Larger placental mammals (but not marsupials or birds) show evidence of greater challenges with heat dissipation, but there is little evidence for compensatory adaptations to enhance heat loss in larger endotherms, suggesting that metabolic rate (MR) more generally balances heat loss for thermoregulation in endotherms. Size-dependent patterns in many molecular, physiological, and morphological properties are consistent with size-dependent natural selection, such as stronger selection for neurolocomotor performance and growth rate in smaller animals and stronger selection for safety and longevity in larger animals. Hypometric scaling of MR very likely arises from different mechanisms in different taxa and conditions, consistent with the diversity of scaling slopes for MR.