Effects of flight activity and age on oxidative damage in the honey bee, Apis mellifera.

Frequent and highly aerobic behaviors likely contribute to naturally occurring stress, accelerate senescence and limit lifespan. To understand how the physiological and cellular mechanisms that determine the onset and duration of senescence are shaped by behavioral development and behavioral duration, we exploited the tractability of the honey bee (Apis mellifera) model system. First, we determined whether a cause-effect relationship exists between honey bee flight and oxidative stress by comparing oxidative damage accrued from intense flight bouts to damage accrued from d-galactose ingestion, which induces oxidative stress and limits lifespan in other insects. Second, we experimentally manipulated the duration of honey bee flight across a range of ages to determine the effects on reactive oxygen species (ROS) accumulation and associated enzymatic antioxidant protective mechanisms. In bees fed d-galactose, lipid peroxidation (assessed by measuring malondialdehyde levels) was higher than in bees fed sucrose and age-matched bees with a high and low number of flight experiences collected from a colony. Bees with high amounts of flight experience exhibited elevated 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, relative to bees with less flight experience. Bees with high amounts of flight experience also showed increased levels of pro-oxidants (superoxide and hydrogen peroxide) and decreased or unchanged levels of antioxidants (superoxide dismutase and catalase). These data implicate an imbalance of pro- to anti-oxidants in flight-associated oxidative stress, and reveal how behavior can damage a cell and consequently limit lifespan.

Environmental effects on Drosophila brain development and learning.

Brain development and behavior are sensitive to a variety of environmental influences including social interactions and physicochemical stressors. Sensory input in situ is a mosaic of both enrichment and stress, yet little is known about how multiple environmental factors interact to affect brain anatomical structures, circuits and cognitive function. In this study, we addressed these issues by testing the individual and combined effects of sub-adult thermal stress, larval density and early-adult living spatial enrichment on brain anatomy and olfactory associative learning in adult Drosophila melanogaster In response to heat stress, the mushroom bodies (MBs) were the most volumetrically impaired among all of the brain structures, an effect highly correlated with reduced odor learning performance. However, MBs were not sensitive to either larval culture density or early-adult living conditions. Extreme larval crowding reduced the volume of the antennal lobes, optic lobes and central complex. Neither larval crowding nor early-adult spatial enrichment affected olfactory learning. These results illustrate that various brain structures react differently to environmental inputs, and that MB development and learning are highly sensitive to certain stressors (pre-adult hyperthermia) and resistant to others (larval crowding).

Hovering flight in the honeybee Apis mellifera: kinematic mechanisms for varying aerodynamic forces.

During hovering flight, animals can increase the wing velocity and therefore the net aerodynamic force per stroke by increasing wingbeat frequency, wing stroke amplitude, or both. The magnitude and orientation of aerodynamic forces are also influenced by the geometric angle of attack, timing of wing rotation, wing contact, and pattern of deviation from the primary stroke plane. Most of the kinematic data available for flying animals are average values for wing stroke amplitude and wingbeat frequency because these features are relatively easy to measure, but it is frequently suggested that the more subtle and difficult-to-measure features of wing kinematics can explain variation in force production for different flight behaviors. Here, we test this hypothesis with multicamera high-speed recording and digitization of wing kinematics of honeybees (Apis mellifera) hovering and ascending in air and hovering in a hypodense gas (heliox: 21% O2, 79% He). Bees employed low stroke amplitudes (86.7° ± 7.9°) and high wingbeat frequencies (226.8 ± 12.8 Hz) when hovering in air. When ascending in air or hovering in heliox, bees increased stroke amplitude by 30%-45%, which yielded a much higher wing tip velocity relative to that during simple hovering in air. Across the three flight conditions, there were no statistical differences in the amplitude of wing stroke deviation, minimum and stroke-averaged geometric angle of attack, maximum wing rotation velocity, or even wingbeat frequency. We employed a quasi-steady aerodynamic model to estimate the effects of wing tip velocity and geometric angle of attack on lift and drag. Lift forces were sensitive to variation in wing tip velocity, whereas drag was sensitive to both variation in wing tip velocity and angle of attack. Bees utilized kinematic patterns that did not maximize lift production but rather maintained lift-to-drag ratio. Thus, our data indicate that, at least for honeybees, the overall time course of wing angles is generally preserved and modulation of wing tip velocity is sufficient to perform a diverse set of vertical flight behaviors.

The effects of age and lifetime flight behavior on flight capacity in Drosophila melanogaster.

The effects of flight behavior on physiology and senescence may be profound in insects because of the extremely high metabolic costs of flight. Flight capacity in insects decreases with age; in contrast, limiting flight behavior extends lifespan and slows the age-related loss of antioxidant capacity and accumulation of oxidative damage in flight muscles. In this study, we tested the effects of age and lifetime flight behavior on flight capacity by measuring wingbeat frequency, the ability to fly in a hypo-dense gas mixture, and metabolic rate in Drosophila melanogaster. Specifically, 5-day-old adult flies were separated into three life-long treatments: (1) those not allowed to fly (no flight), (2) those allowed - but not forced - to fly (voluntary flight) and (3) those mechanically stimulated to fly (induced flight). Flight capacity senesced earliest in flies from the no-flight treatment, followed by the induced-flight group and then the voluntary flight group. Wingbeat frequency senesced with age in all treatment groups, but was most apparent in the voluntary- and induced-flight groups. Metabolic rate during agitated flight senesced earliest and most rapidly in the induced flight group, and was low and uniform throughout age in the no-flight group. Early senescence in the induced-flight group was likely due to the acceleration of deleterious aging phenomena such as the rapid accumulation of damage at the cellular level, while the early loss of flight capacity and low metabolic rates in the no-flight group demonstrate that disuse effects can also significantly alter senescence patterns of whole-insect performance.

The effects of artificial wing wear on the flight capacity of the honey bee Apis mellifera.

The wings of bees and other insects accumulate permanent wear, which increases the rate of mortality and impacts foraging behavior, presumably due to effects on flight performance. In this study, we investigated how experimental wing wear affects flight performance in honey bees. Variable density gases and high-speed videography were used to determine the maximum hovering flight capacity and wing kinematics of bees from three treatment groups: no wing wear, symmetric and asymmetric wing wear. Wing wear was simulated by clipping the distal-trailing edge of one or both of the wings. Across all bees from treatment groups combined, wingbeat frequency was inversely related to wing area. During hovering in air, bees with symmetric and asymmetric wing wear responded kinematically so as to produce wingtip velocities similar to those bees with no wing wear. However, maximal hovering flight capacity (revealed during flight in hypodense gases) decreased in direct proportion to wing area and inversely to wing asymmetry. Bees with reduced wing area and high asymmetry produced lower maximum wingtip velocity than bees with intact or symmetric wings, which caused a greater impairment in maximal flight capacity. These results demonstrate that the magnitude and type of wing wear affects maximal aerodynamic power production and, likely, the control of hovering flight. Wing wear reduces aerodynamic reserve capacity and, subsequently, the capacity for flight behaviors such as load carriage, maneuverability, and evading predators.

Effects of Flight on Gene Expression and Aging in the Honey Bee Brain and Flight Muscle.

Honey bees move through a series of in-hive tasks (e.g., "nursing") to outside tasks (e.g., "foraging") that are coincident with physiological changes and higher levels of metabolic activity. Social context can cause worker bees to speed up or slow down this process, and foragers may revert back to their earlier in-hive tasks accompanied by reversion to earlier physiological states. To investigate the effects of flight, behavioral state and age on gene expression, we used whole-genome microarrays and real-time PCR. Brain tissue and flight muscle exhibited different patterns of expression during behavioral transitions, with expression patterns in the brain reflecting both age and behavior, and expression patterns in flight muscle being primarily determined by age. Our data suggest that the transition from behaviors requiring little to no flight (nursing) to those requiring prolonged flight bouts (foraging), rather than the amount of previous flight per se, has a major effect on gene expression. Following behavioral reversion there was a partial reversion in gene expression but some aspects of forager expression patterns, such as those for genes involved in immune function, remained. Combined with our real-time PCR data, these data suggest an epigenetic control and energy balance role in honey bee functional senescence.

Synchrotron x-ray visualisation of ice formation in insects during lethal and non-lethal freezing.

Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3(rd) instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above -14 degrees C, and non-diapausing 3(rd) instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.

The effects of age and behavioral development on honey bee (Apis mellifera) flight performance.

A critical but seldom-studied component of life history theory is how behavior and age affect whole-organism performance. To address this issue we compared the flight performance of honey bees (whose behavioral development and age can be assessed independently via simple manipulations of colony demographics) between distinct behavioral castes (in-hive nurse bees vs out-of-hive foragers) and across lifespan. Variable-density gases and high-speed video were used to determine the maximum hovering flight capacity and wing kinematics of age-matched nurse bees and foragers sampled from a single-cohort colony over a period of 34 days. The transition from hive work to foraging was accompanied by a 42% decrease in body mass and a proportional increase in flight capacity (defined as the minimum gas density allowing hovering flight). The lower flight capacity of hive bees was primarily due to the fact that in air they were functioning at a near-maximal wing angular velocity due to their high body masses. Foragers were lighter and when hovering in air required a much lower wing angular velocity, which they were able to increase by 32% during maximal flight performance. Flight performance of hive bees was independent of age, but in foragers the maximal wingbeat frequency and maximal average angular velocity were lowest in precocious (7-14 day old) foragers, highest in normal-aged (15-28 day old) foragers and intermediate in foragers older than 29 days. This pattern coincides with previously described age-dependent biochemical and metabolic properties of honey bee flight muscle.

Cold rearing improves cold-flight performance in Drosophila via changes in wing morphology.

We use a factorial experimental design to test whether rearing at colder temperatures shifts the lower thermal envelope for flight of Drosophila melanogaster Meigen to colder temperatures. D. melanogaster that developed in colder temperatures (15 degrees C) had a significant flight advantage in cold air compared to flies that developed in warmer temperatures (28 degrees C). At 14 degrees C, cold-reared flies failed to perform a take-off flight approximately 47% of the time whereas warm-reared flies failed approximately 94% of the time. At 18 degrees C, cold- and warm-reared flies performed equally well. We also compared several traits in cold- and warm-developing flies to determine if cold-developing flies had better flight performance at cold temperatures due to changes in body mass, wing length, wing loading, relative flight muscle mass or wing-beat frequency. The improved ability to fly at low temperatures was associated with a dramatic increase in wing area and an increase in wing length (after controlling for wing area). Flies that developed at 15 degrees C had approximately 25% more wing area than similarly sized flies that developed at 28 degrees C. Cold-reared flies had slower wing-beat frequencies than similarly sized flies from warmer developmental environments, whereas other traits did not vary with developmental temperature. These results demonstrate that developmental plasticity in wing dimensions contributes to the improved flight performance of D. melanogaster at cold temperatures, and ultimately, may help D. melanogaster live in a wide range of thermal environments.

Age and natural metabolically-intensive behavior affect oxidative stress and antioxidant mechanisms.

Flying honey bees have among the highest mass-specific metabolic rates ever measured, suggesting that their flight muscles may experience high levels of oxidative stress during normal daily activities. We measured parameters of oxidative stress and antioxidant capacity in highly metabolic flight muscle and less active head tissue in cohorts of age-matched nurse bees, which rarely fly, and foragers, which fly several hours per a day. Naturally occurring foraging flight elicited an increase in flight muscle Hsp70 content in both young and old foragers; however catalase and total antioxidant capacity increased only in young flight muscle. Surprisingly, young nurse bees also showed a modest daily increase in Hsp70, catalase levels and antioxidant capacity, and these effects were likely due to collecting the young nurses soon after orientation flights. There were no differences in flight muscle carbonyl content over the course of daily activity and few differences in Hsp70, catalase, total antioxidant capacity and protein carbonyl levels in head tissue regardless of age or activity. In summary, honey bee flight likely produces high levels of reactive oxygen species in flight muscle that, when coupled with age-related decreases in antioxidant activity may be responsible for behavioral senescence and reduced longevity.

Thermal disruption of mushroom body development and odor learning in Drosophila.

Environmental stress (nutritive, chemical, electromagnetic and thermal) has been shown to disrupt central nervous system (CNS) development in every model system studied to date. However, empirical linkages between stress, specific targets in the brain, and consequences for behavior have rarely been established. The present study experimentally demonstrates one such linkage by examining the effects of ecologically-relevant thermal stress on development of the Drosophila melanogaster mushroom body (MB), a conserved sensory integration and associative center in the insect brain. We show that a daily hyperthermic episode throughout larval and pupal development (1) severely disrupts MB anatomy by reducing intrinsic Kenyon cell (KC) neuron numbers but has little effect on other brain structures or general anatomy, and (2) greatly impairs associative odor learning in adults, despite having little effect on memory or sensory acuity. Hence, heat stress of ecologically relevant duration and intensity can impair brain development and learning potential.

The effects of carbon dioxide anesthesia and anoxia on rapid cold-hardening and chill coma recovery in Drosophila melanogaster.

Carbon dioxide gas is used as an insect anesthetic in many laboratories, despite recent studies which have shown that CO(2) can alter behavior and fitness. We examine the effects of CO(2) and anoxia (N(2)) on cold tolerance, measuring the rapid cold-hardening (RCH) response and chill coma recovery in Drosophila melanogaster. Short exposures to CO(2) or N(2) do not significantly affect RCH, but 60 min of exposure negates RCH. Exposure to CO(2) anesthesia increases chill coma recovery time, but this effect disappears if the flies are given 90 min recovery in air before chill coma induction. Flies treated with N(2) show a similar pattern, but require significantly longer chill coma recovery times even after 90 min of recovery from anoxia. Our results suggest that CO(2) anesthesia is an acceptable way to manipulate flies before cold tolerance experiments (when using RCH or chill coma recovery as a measure), provided exposure duration is minimized and recovery is permitted before chill coma induction. However, we recommend that exposure to N(2) not be used as a method of anesthesia for chill coma studies.

Short-amplitude high-frequency wing strokes determine the aerodynamics of honeybee flight.

Most insects are thought to fly by creating a leading-edge vortex that remains attached to the wing as it translates through a stroke. In the species examined so far, stroke amplitude is large, and most of the aerodynamic force is produced halfway through a stroke when translation velocities are highest. Here we demonstrate that honeybees use an alternative strategy, hovering with relatively low stroke amplitude (approximately 90 degrees) and high wingbeat frequency (approximately 230 Hz). When measured on a dynamically scaled robot, the kinematics of honeybee wings generate prominent force peaks during the beginning, middle, and end of each stroke, indicating the importance of additional unsteady mechanisms at stroke reversal. When challenged to fly in low-density heliox, bees responded by maintaining nearly constant wingbeat frequency while increasing stroke amplitude by nearly 50%. We examined the aerodynamic consequences of this change in wing motion by using artificial kinematic patterns in which amplitude was systematically increased in 5 degrees increments. To separate the aerodynamic effects of stroke velocity from those due to amplitude, we performed this analysis under both constant frequency and constant velocity conditions. The results indicate that unsteady forces during stroke reversal make a large contribution to net upward force during hovering but play a diminished role as the animal increases stroke amplitude and flight power. We suggest that the peculiar kinematics of bees may reflect either a specialization for increasing load capacity or a physiological limitation of their flight muscles.

Muscle biochemistry and the ontogeny of flight capacity during behavioral development in the honey bee, Apis mellifera.

A fundamental issue in physiology and behavior is underlie major behavioral shifts in organisms as they transitions are common in nature and include the age-related switch from nest/hive work to foraging in social insects such as honey bees (understanding the functional and genetic mechanisms that adopt new environments or life history tactics. Such). Because of their experimental Apis mellifera tractability, recently sequenced genome and well understood biology, honey bees are an ideal model system for integrating molecular, genetic, physiological and sociobiological perspectives to advance understanding of behavioral and life history transitions. When honey bees (Apis mellifera) transition from hive work to foraging, their flight muscles undergo changes Apis mellifera that allow these insects to attain the highest rates of flight muscle metabolism and power output ever recorded in the animal kingdom. Here, we review research to date showing that honey bee flight muscles undergo significant changes in biochemistry and gene expression and that these changes accompany a significant increase in the capacity to generate metabolic and aerodynamic power during flight. It is likely that changes in muscle gene expression, biochemistry, metabolism and functional capacity may be driven primarily by behavior as opposed to age, as is the case for changes in honey bee brains.

Honey bees as a model for understanding mechanisms of life history transitions.

As honey bee workers switch from in-hive tasks to foraging, they undergo transition from constant exposure to the controlled homogenous physical and sensory environment of the hive to prolonged diurnal exposures to a far more heterogeneous environment outside the hive. The switch from hive work to foraging offers an opportunity for the integrative study of the physiological and genetic mechanisms that produce the behavioral plasticity required for major life history transitions. Although such transitions have been studied in a number of animals, currently there is no model system where the evolution, development, physiology, molecular biology, neurobiology and behavior of such a transition can all be studied in the same organism in its natural habitat. With a large literature covering its evolution, behavior and physiology (plus the recent sequencing of the honey bee genome), the honey bee is uniquely suited to integrative studies of the mechanisms of behavior. In this review we discuss the physiological and genetic mechanisms of this behavioral transition, which include large scale changes in hormonal activity, metabolism, flight ability, circadian rhythms, sensory perception and processing, neural architecture, learning ability, memory and gene expression.

Allometry of kinematics and energetics in carpenter bees (Xylocopa varipuncta) hovering in variable-density gases.

We assessed the energetic and aerodynamic limits of hovering flight in the carpenter bee Xylocopa varipuncta. Using normoxic, variable-density mixtures of O(2), N(2) and He, we were able to elicit maximal hovering performance and aerodynamic failure in the majority of bees sampled. Bees were not isometric regarding thorax mass and wing area, both of which were disproportionately lower in heavier individuals. The minimal gas density necessary for hovering (MGD) increased with body mass and decreased with relative thoracic muscle mass. Only the four bees in our sample with the highest body mass-specific thorax masses were able to hover in pure heliox. Wingbeat frequency and stroke amplitude during maximal hovering were significantly greater than in normodense hovering, increased significantly with body mass during normodense hovering but were mass independent during maximal hovering. Reserve capacity for wingbeat frequency and stroke amplitude decreased significantly with increasing body mass, although reserve capacity in stroke amplitude (10-30%) exceeded that of wingbeat frequency (0-8%). Stroke plane angle during normodense hovering was significantly greater than during maximal hovering, whereas body angle was significantly greater during maximal hovering than during normodense hovering. Power production during normodense hovering was significantly less than during maximal hovering. Metabolic rates were significantly greater during maximal hovering than during normodense hovering and were inversely related to body mass during maximal and normodense hovering. Metabolic reserve capacity averaged 34% and was independent of body mass. Muscle efficiencies were slightly higher during normodense hovering. The allometry of power production, power reserve capacity and muscle efficiency were dependent on the assumed coefficient of drag (C(D)), with significant allometries most often at lower values of C(D). Larger bees operate near the envelope of maximal performance even in normodense hovering due to smaller body mass-specific flight muscles and limited reserve capacities for kinematics and power production.

Dropping like flies: environmentally induced impairment and protection of locomotor performance in adult Drosophila melanogaster.

In Drosophila, heat shock (HS) during the pupal stage chronically hinders adult locomotor performance by disrupting wing development and cellular and/or tissue-level mechanisms that support walking and flight. Furthermore, heat pretreatment (PT) protects locomotor function against these disruptions. HS flies with abnormal wings were less able to alter trajectory in free fall relative to control, PT-only, and PT+HS wild-type flies. This deficit was less severe but still present in HS-only flies with wild-type wings. Transgenic increases in the copies of genes encoding the major inducible heat-shock protein of Drosophila melanogaster, Hsp70, also protected walking ability from disruption due to pupal HS. Walking velocity did not differ between excision (five natural hsp70 copies) and extra-copy (five natural and six transgenic hsp70 copies) flies in the control, PT, and PT+HS groups, nor did velocity vary among these thermal treatment groups. HS dramatically reduced walking velocity, however, but this effect occurred primarily in the excision flies. These results suggest that Hsp70 and other mechanisms protect against heat-induced locomotor impairment.

Effects of load type (pollen or nectar) and load mass on hovering metabolic rate and mechanical power output in the honey bee Apis mellifera.

In this study we tested the effect of pollen and nectar loading on metabolic rate (in mW) and wingbeat frequency during hovering, and also examined the effect of pollen loading on wing kinematics and mechanical power output. Pollen foragers had hovering metabolic rates approximately 10% higher than nectar foragers, regardless of the amount of load carried. Pollen foragers also had a more horizontal body position and higher inclination of stroke plane than measured previously for honey bees (probably nectar foragers). Thorax temperatures ranked pollen > nectar > water foragers, and higher flight metabolic rate could explain the higher thorax temperature of pollen foragers. Load mass did not affect hovering metabolic rate or wingbeat frequency in a regression-model experiment. However, using an analysis of variance (ANOVA) design, loaded pollen and nectar foragers (mean loads 27% and 40% of body mass, respectively) significantly increased metabolic rate by 6%. Mean pollen loads of 18% of body mass had no effect on wingbeat frequency, stroke amplitude, body angle or inclination of stroke plane, but increased the calculated mechanical power output by 16-18% (depending on the method of estimating drag). A rise in lift coefficient as bees carry loads without increasing wingbeat frequency or stroke amplitude (and only minimal increases in metabolic rate) suggests an increased use of unsteady power-generating mechanisms.

Effect of heat shock, pretreatment and hsp70 copy number on wing development in Drosophila melanogaster.

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.