Jason B. Williams

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.

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

SUMMARY 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.

Partial link between the seasonal acquisition of cold-tolerance and desiccation resistance in the goldenrod gall fly Eurosta solidaginis (Diptera: Tephritidae).

SUMMARY Possible links between seasonal increases in cold-tolerance and desiccation resistance were examined in field-collected larvae of the goldenrod gall fly, Eurosta solidaginis. From 20 September to 30 October 2001, larvae exhibited a gradual increase in cold-tolerance culminating in 100% survival of freezing at –20°C for 24 h. The increase in cold-tolerance was probably due to a concomitant increase in cryoprotectants as measured by hemolymph osmolality (488–695 mOsmol kg–1). In contrast to the gradual increase in cold-tolerance, larvae exhibited two distinct phases of reduced rates of water loss. The first phase was an abrupt sixfold decrease to 0.57 μg mm–2 h–1 between 3 and 16 October. The first phase of reduced rates of water loss was not correlated with changes in cold-tolerance; nor was it correlated with hemolymph osmolality and body water content, which remained constant throughout the study. The reduction in rates of water loss during the first phase were probably the result of decreased respiratory water loss as the larvae entered diapause, and possibly reduced cuticular water loss as larvae increased the amount of their cuticular hydrocarbons. Interestingly, the first phase of reduced water loss was associated with, and may have been cued by, a reduction in the water potential of the gall tissues surrounding the larvae. The second phase was a more subtle fourfold reduction in rates of water loss occurring between 16 October and 11 December. In contrast to the first phase, the second phase of increased desiccation resistance correlated closely with increases in hemolymph osmolality (568–870 mOsmol kg–1). The correlation between seasonal increases in hemolymph osmolality and reduction in rates of water loss may represent a link between desiccation resistance and cold-tolerance in this species.

Plant senescence cues entry into diapause in the gall fly Eurosta solidaginis: resulting metabolic depression is critical for water conservation

SUMMARY Mechanisms and possible cues for seasonal increases in desiccation resistance in larvae of the goldenrod gall fly Eurosta solidaginis, were examined before and after natural and premature plant senescence, or after being removed from their gall and placed in either 100, 95 or 75% relative humidity (RH). Rates of water loss were 8.6-fold lower, averaging 0.7±0.2 μg mm–2 h–1, in larvae from senescent gall tissue and after all RH treatments than in control larvae from pre-senescent plants. Enhanced desiccation resistance occurred quickly, within 3 days of removal from their gall. Contrary to most previous reports, a large majority of the increased desiccation resistance (∼85%) was due to reduced respiratory transpiration with the remainder being the result of a lowered cuticular permeability. Rates of cuticular water loss were reduced by the presence of a vapor pressure gradient between the larval hemolymph and environmental water vapor and were probably due to increases in cuticular lipids and/or production of the cryoprotectant glycerol. Metabolic rate was reduced by over fourfold, averaging 0.07±0.01 μl CO2 g–1 h–1, in larvae from senescent gall tissue and all RH treatments compared to larvae from pre-senescent plants. The magnitude of the reduction in metabolic rates indicated that these larvae had entered diapause. In addition, larvae entered diapause in response to removal from, or degeneration of, the gall tissue they feed, on rather than seasonal changes in temperature or photoperiod. The low metabolic rates of the diapausing larvae probably allowed them to dramatically reduce their respiratory transpiration and total rate of water loss compared with non-diapausing controls. Thus, diapause, with its associated lowered metabolic rate, may be essential for conserving water in overwintering temperate insects, which may be dormant for six or more months of the year.

Repeated freezing induces oxidative stress and reduces survival in the freeze-tolerant goldenrod gall fly, Eurosta solidaginis

Freeze tolerant insects must not only survive extracellular ice formation but also the generation of reactive oxygen species (ROS) during oxygen reperfusion upon thawing. Furthermore, diurnal fluctuations in temperature place temperate insects at risk of being exposed to multiple freeze-thaw cycles, yet few studies have examined metrics of survival and oxidative stress in freeze-tolerant insects subjected to successive freezing events. To address this, we assessed survival in larvae of the goldenrod gall fly Eurosta solidaginis, after being subjected to 0, 5, 10, 20, or 30 diurnally repeated cold exposures (RCE) to -18°C or a single freeze to -18°C for 20days. In addition, we measured indicators of oxidative stress, levels of cryoprotectants, and total aqueous antioxidant capacity in animals exposed to the above treatments at 8, 32, or 80h after their final thaw. Repeated freezing and thawing, rather than time spent frozen, reduced survival as only 30% of larvae subjected to 20 or 30 RCE successfully pupated, compared to those subjected to fewer RCE or a single 20d freeze, of which 82% pupated. RCE had little effect on the concentration of the cryoprotectant glycerol (4.26±0.66μgglycerol·ngprotein(-1) for all treatments and time points) or sorbitol (18.8±2.9μgsorbitol·mgprotein(-1) for all treatments and time points); however, sorbitol concentrations were more than twofold higher than controls (16.3±2.2μgsorbitol·mgprotein(-1)) initially after a thaw in larvae subjected to a single extended freeze, but levels returned to values similar to controls at 80h after thaw. Thawing likely produced ROS as total aqueous antioxidant capacities peaked at 1.8-fold higher than controls (14.7±1.6mmoltrolox·ngprotein(-1)) in animals exposed to 5, 10, or 20 RCE. By contrast, aqueous antioxidant capacities were similar to controls in larvae subjected to 30 RCE or the single 20d freeze regardless of time post final thaw, indicating these animals may have had an impaired ability to produce primary antioxidants. Larvae lacking an antioxidant response also had elevated levels of oxidized proteins, nearly twice that of controls (21.8±3.2mmolchloramine-T·mgprotein(-1)). Repeated freezing also lead to substantial oxidative damage to lipids that was independent of aqueous antioxidant capacity; peroxides were, on average, 5.6-fold higher in larvae subjected to 10, 20 or 30 RCE compared to controls (29.1±7.3mmolTMOP·μgprotein(-1)). These data suggest that oxidative stress due to repeated freeze-thaw cycles reduces the capacity of E. solidaginis larvae to survive freezing.

Effect of freezing and dehydration on ion and cryoprotectant distribution and hemolymph volume in the goldenrod gall fly, Eurosta solidaginis

Extracellular freezing and dehydration concentrate hemolymph solutes, which can lead to cellular injury due to excessive water loss. Freeze tolerant larvae of the goldenrod gall fly, Eurosta solidaginis, may experience extreme cold and desiccation in winter. To determine whether larvae employ protective mechanisms against excessive cellular water loss we examined the effect of extracellular freezing and dehydration on hemolymph volume, and cryoprotectant and ion levels in the hemolymph. Dehydrated larvae or ones that had been frozen at -5 or -20°C had a significantly smaller proportion of their body water as hemolymph (26.0-27.4%) compared to controls (30.5%). Even with this reduction in water content, hemolymph osmolality was similar or only slightly higher in frozen or dehydrated individuals than controls (908 mOsm kg(-1)), indicating these stresses led to a reduction in hemolymph solutes. Hemolymph and intracellular content of ions remained largely unchanged between treatment groups; although levels of Mg(++) in the hemolymph were lower in larvae subjected to freezing (0.21±0.01 μg mg(-1)drymass) compared to controls (0.29±0.01 μg mg(-1)drymass), while intracellular levels of K(+) were lower in groups exposed to low temperature (8.31±0.21 μg mg(-1)drymass). Whole body glycerol and sorbitol content was similar among all treatment groups, averaging 432±25 mOsm kg(-1) and 549±78 mOsm kg(-1) respectively. However, larvae subjected to dehydration and freezing at -20°C had a much lower relative amount of cryoprotectants in their hemolymph (∼35%) compared to controls (54%) suggesting these solutes moved into intracellular compartments during these stresses. The correlation between reduced hemolymph volume (i.e. increased cellular water content) and intracellular movement of cryoprotectants may represent a link between tolerance of dehydration and cold in this species.