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.

Inhibitory effect of indole analogs against Paenibacillus larvae, the causal agent of American foulbrood disease.

Paenibacillus larvae, a Gram-positive bacterium, causes American foulbrood (AFB) in honey bee larvae (Apis mellifera Linnaeus [Hymenoptera: Apidae]). P. larvae spores exit dormancy in the gut of bee larvae, the germinated cells proliferate, and ultimately bacteremia kills the host. Hence, spore germination is a required step for establishing AFB disease. We previously found that P. larvae spores germinate in response to l-tyrosine plus uric acid in vitro. Additionally, we determined that indole and phenol blocked spore germination. In this work, we evaluated the antagonistic effect of 35 indole and phenol analogs and identified strong inhibitors of P. larvae spore germination in vitro. We further tested the most promising candidate, 5-chloroindole, and found that it significantly reduced bacterial proliferation. Finally, feeding artificial worker jelly containing anti-germination compounds to AFB-exposed larvae significantly decreased AFB infection in laboratory-reared honey bee larvae. Together, these results suggest that inhibitors of P. larvae spore germination could provide another method to control AFB.

Comparison of in vitro methods for the production of Paenibacillus larvae endospores.

Paenibacillus larvae endospores are the infectious particles of the honey bee brood disease, American Foulbrood. We demonstrate that our previously published protocol (Alvarado et al., 2013) consistently yields higher numbers and purer preparations of P. larvae endospores, than previously described protocols, regardless of the strain tested (B-3650, B-3554 or B-3685).

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.

Requirements for in vitro germination of Paenibacillus larvae spores.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a disease affecting honey bee larvae. First- and second-instar larvae become infected when they ingest food contaminated with P. larvae spores. The spores then germinate into vegetative cells that proliferate in the midgut of the honey bee. Although AFB affects honey bees only in the larval stage, P. larvae spores can be distributed throughout the hive. Because spore germination is critical for AFB establishment, we analyzed the requirements for P. larvae spore germination in vitro. We found that P. larvae spores germinated only in response to l-tyrosine plus uric acid under physiologic pH and temperature conditions. This suggests that the simultaneous presence of these signals is necessary for spore germination in vivo. Furthermore, the germination profiles of environmentally derived spores were identical to those of spores from a biochemically typed strain. Because l-tyrosine and uric acid are the only required germinants in vitro, we screened amino acid and purine analogs for their ability to act as antagonists of P. larvae spore germination. Indole and phenol, the side chains of tyrosine and tryptophan, strongly inhibited P. larvae spore germination. Methylation of the N-1 (but not the C-3) position of indole eliminated its ability to inhibit germination. Identification of the activators and inhibitors of P. larvae spore germination provides a basis for developing new tools to control AFB.

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.

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.

Extreme thermotolerance and behavioral induction of 70-kDa heat shock proteins and their encoding genes in honey bees.

Foraging honey bees frequently leave the hive to gather pollen and nectar for the colony. This period of their lives is marked by periodic extremes of body temperature, metabolic expenditure, and flight muscle activity. Following ecologically relevant episodes of hyperthermia between 33 degrees C and 50 degrees C, heat shock protein 70 (Hsp70) expression and hsp70/hsc70-4 activity in brains of nonflying laboratory-held bees increased by only two to three times baseline at temperatures 46-50 degrees C. Induction was undetectable in thoracic-flight muscles. Yet, thorax hsp70 mRNA (but not hsc70-4 mRNA) levels were up to ten times higher in flight-capable hive bees and foraging bees compared to 1-day-old, flight-incapable bees, while brain hsp70/hsc70-4 mRNA levels were low and varied little among behavioral groups. These data suggest honey bee tissues, especially flight muscles, are extremely thermotolerant. Furthermore, Hsp70 expression in the thoraces of flight-capable bees is probably flight-induced by oxidative and mechanical damage to flight muscle proteins rather than temperature.

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.

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.

Larval juvenile hormone treatment affects pre-adult development, but not adult age at onset of foraging in worker honey bees (Apis mellifera).

Previous research has shown that juvenile hormone (JH) titers increase as adult worker honey bees age and treatments with JH, JH analogs and JH mimics induce precocious foraging. Larvae from genotypes exhibiting faster adult behavioral development had significantly higher levels of juvenile hormone during the 2nd and 3rd larval instar. It is known that highly increased JH during this period causes the totipotent female larvae to differentiate into a queen. We treated third instar larvae with JH to test the hypothesis that this time period may be a developmental critical period for organizational effects of JH on brain and behavior also in the worker caste, such that JH treatment at a lower level than required to produce queens will speed adult behavioral development in workers. Larval JH treatment did not influence adult worker behavioral development. However, it made pre-adult development more queen-like in two ways: treated larvae were capped sooner by adult bees, and emerged from pupation earlier. These results suggest that some aspects of honey bee behavioral development may be relatively insensitive to pre-adult perturbation. These results also suggest JH titer may be connected to cues perceived by the adult bees indicating larval readiness for pupation resulting in adult bee cell capping behavior.

Biogenic amines in the antennal lobes and the initiation and maintenance of foraging behavior in honey bees.

Previous findings showed that high levels of octopamine and serotonin in the antennal lobes of adult worker honey bees are associated with foraging behavior, and octopamine treatment induces precocious foraging. To better characterize the relationship between amines and foraging behavior in honey bees, we performed a detailed correlative analysis of amine levels in the antennal lobes as a function of various aspects of foraging behavior. Flight activity was measured under controlled conditions in a large outdoor flight cage. Levels of octopamine in the antennal lobes were found to be elevated immediately subsequent to the onset of foraging, but they did not change as a consequence of preforaging orientation flight activity, diurnal pauses in foraging, or different amounts of foraging experience, suggesting that octopamine helps to trigger and maintain the foraging behavioral state. In contrast, levels of serotonin and dopamine did not show changes that would implicate them as either causal agents of foraging, or as neurochemical systems affected by the act of foraging. Serotonin treatment had no effect on the likelihood of foraging. These results provide further support for the hypothesis that an increase in octopamine levels in the antennal lobes plays a causal role in the initiation and maintenance of the behavioral state of foraging, and thus is involved in the regulation of division of labor in honey bees.

Insect allatotropins belong to a family of structurally-related myoactive peptides present in several invertebrate phyla.

Originally named for its ability to stimulate juvenile hormone production by lepidopteran corpora allata, allatotropin has emerged as a neuropeptide with multiple neural, endocrine and myoactive roles. This paper describes the experimental evidence for allatotropin action, its localization in several species of insects, and its multiple effects on a variety of different tissues that lead to increased hemolymph circulation and gut motility. The overall physiological effects may also include species-specific effects such as the regulation of nutrient absorption, modulation of the circadian cycle and migratory preparedness. In addition, we present evidence suggesting that allatotropins are members of a family of myoactive peptides found in several invertebrate phyla. Finally, we speculate that the myoactive properties of allatotropins are basal and it is likely that the stimulatory action of allatotropins on juvenile hormone synthesis evolved secondarily.