Lifetime- and caste-specific changes in flight metabolic rate and muscle biochemistry of honeybees, Apis mellifera

Honeybees, Apis mellifera, who show temporal polyethism, begin their adult life performing tasks inside the hive (hive bees) and then switch to foraging when they are about 2–3 weeks old (foragers). Usually hive tasks require little or no flying, whereas foraging involves flying for several hours a day and carrying heavy loads of nectar and pollen. Flight muscles are particularly plastic organs that can respond to use and disuse, and accordingly it would be expected that adjustments in flight muscle metabolism occur throughout a bee’s life. We thus investigated changes in lifetime flight metabolic rate and flight muscle biochemistry of differently aged hive bees and of foragers with varying foraging experience. Rapid increases in flight metabolic rates early in life coincided with a switch in troponin T isoforms and increases in flight muscle maximal activities (V _max) of the enzymes citrate synthase, cytochrome c oxidase, hexokinase, phosphofructokinase, and pyruvate kinase. However, further increases in flight metabolic rate in experienced foragers occurred without additional changes in the in vitro V _max of these flight muscle metabolic enzymes. Estimates of in vivo flux (v) compared to maximum flux of each enzyme in vitro (fractional velocity, v/V _max) suggest that most enzymes operate at a higher fraction of V _max in mature foragers compared to young hive bees. Our results indicate that honeybees develop most of their flight muscle metabolic machinery early in life. Any further increases in flight metabolism with age or foraging experience are most likely achieved by operating metabolic enzymes closer to their maximal flux capacity.     

Methoprene does not affect juvenile hormone titers in honey bee (Apis mellifera) workers

Methoprene, a juvenile hormone (JH) analog, is a widely used insecticide that also accelerates behavioral development in honey bees (Apis mellifera). JH regulates the transition from nursing to foraging in adult worker bees, and treatment with JH or methoprene have both been shown to induce precocious foraging. To determine how methoprene changes honey bee behavior, we compared JH titers of methoprene‐treated and untreated bees. Behavioral observations confirmed that methoprene treatment significantly increased the number of precocious foragers in 3 out of 4 colonies. In only 1 out of 4 colonies, however, was there a significant difference in JH titers between the methoprene‐treated and control bees. Further, in all 4 colonies, there was no significant differences in JH titers between precocious and normal‐aged foragers. These results suggest that methoprene did not directly affect the endogenous JH secreted by corpora allata. Because methoprene caused early foraging without changing workers’ JH titers, we conclude that methoprene most likely acts directly on the JH receptors as a substitute for JH.     

Methoprene does not affect food preferences and foraging performance in honey bee workers

The fundamental determinants of division of labor among honey bee workers are age, genotype, and environment. These determinants work through intermediate physiological channels to realize particular patterns of division of labor. The change of juvenile hormone (JH) titer in worker bees is one such channel. Previous studies concentrated on the impact of JH on timing of in-hive and foraging activity. Here we examined the effects of JH on task specialization and the collection of pollen or nectar by same-age bees and we tested the possible impact on JH titer on foraging performance. Methoprene treatments were conducted after workers began to forage inside a flight room. We found that methoprene, a JH analogue, had no effect on preferences for pollen or nectar and, also, did not influence nectar foraging rate, nectar load size, and foraging span.     

Juvenile hormone and octopamine in the regulation of division of labor in honey bee colonies

Forager honey bees have high circulating levels of juvenile hormone (JH) and high brain levels of octopamine, especially in the antennal lobes, and treatment with either of these compounds induces foraging. Experiments were performed to determine whether octopamine acts more proximally than JH to affect the initiation of foraging behavior. Bees treated with octopamine became foragers more rapidly than bees treated with the JH analog methoprene. Bees treated with methoprene showed an increase in antennal lobe levels of octopamine, especially after 12 days. Bees with no circulating JH (corpora allata glands removed) treated with octopamine became foragers in similar numbers to bees with intact corpora allata. These results suggest that JH affects the initiation of foraging at least in part by increasing brain levels of octopamine, but octopamine can act independently of JH. Effects of JH that are not related to octopamine also are possible, as bees treated with both octopamine and methoprene were more likely to become foragers than bees treated with only octopamine or methoprene.     

Limits on volume changes in the mushroom bodies of the honey bee brain

The behavioral maturation of adult worker honey bees is influenced by a rising titer of juvenile hormone (JH), and is temporally correlated with an increase in the volume of the neuropil of the mushroom bodies, a brain region involved in learning and memory. We explored the stability of this neuropil expansion and its possible dependence on JH. We studied the volume of the mushroom bodies in adult bees deprived of JH by surgical removal of the source glands, the corpora allata. We also asked if the neuropil expansion detected in foragers persists when bees no longer engage in foraging, either because of the onset of winter or because colony social structure was experimentally manipulated to cause some bees to revert from foraging to tending brood (nursing). Results show that adult exposure to JH is not necessary for growth of the mushroom body neuropil, and that the volume of the mushroom body neuropil in adult bees is not reduced if foraging stops. These results are interpreted in the context of a qualitative model that posits that mushroom body neuropil volume enlargement in the honey bee has both experience-independent and experience-dependent components.     

Experience-dependent plasticity in the mushroom bodies of the solitary bee Osmia lignaria (Megachilidae)

All members of the solitary bee species Osmia lignaria (the orchard bee) forage upon emergence from their natal nest cell. Conversely, in the honey bee, days-to-weeks of socially regulated behavioral development precede the onset of foraging. The social honey bee's behavioral transition to foraging is accompanied by neuroanatomical changes in the mushroom bodies, a region of the insect brain implicated in learning. If these changes were general adaptations to foraging, they should also occur in the solitary orchard bee. Using unbiased stereological methods, we estimated the volume of the major compartments of the mushroom bodies, the neuropil and Kenyon cell body region, in adult orchard bees. We compared the mushroom bodies of recently emerged bees with mature bees that had extensive foraging experience. To separate effects of general maturation from field foraging, some orchard bees were confined to a cage indoors. The mushroom body neuropil of experienced field foragers was significantly greater than that of both recently emerged and mature caged orchard bees, suggesting that, like the honey bee, this increase is driven by outdoor foraging experience. Unlike the honey bee, where increases in the ratio of neuropil to Kenyon cell region occur in the worker after emerging from the hive cell, the orchard bee emerged from the natal nest cell with a ratio that did not change with maturation and was comparable to honey-bee foragers. These results suggest that a common developmental endpoint may be reached via different development paths in social and solitary species of foraging bees.     

Effects of experience and juvenile hormone on the organization of the mushroom bodies of honey bees

There is an age-related division of labor in the honey bee colony that is regulated by juvenile hormone. After completing metamorphosis, young workers have low titers of juvenile hormone and spend the first several weeks of their adult lives performing tasks within the hive. Older workers, approximately 3 weeks of age, have high titers of juvenile hormone and forage outside the hive for nectar and pollen. We have previously reported that changes in the volume of the mushroom bodies of the honey bee brain are temporally associated with the performance of foraging. The neuropil of the mushroom bodies is increased in volume, whereas the volume occupied by the somata of the Kenyon cells is significantly decreased in foragers relative to younger workers. To study the effect of flight experience and juvenile hormone on these changes within the mushroom bodies, young worker bees were treated with the juvenile hormone analog methoprene but a subset was prevented from foraging (big back bees). Stereological volume estimates revealed that, regardless of foraging experience, bees treated with methoprene had a significantly larger volume of neuropil in the mushroom bodies and a significantly smaller Kenyon cell somal region volume than did 1-day-old bees. The bees treated with methoprene did not differ on these volume estimates from untreated foragers (presumed to have high endogenous levels of juvenile hormone) of the same age sampled from the same colony. Bees prevented from flying and foraging nonetheless received visual stimulation as they gathered at the hive entrance. These results, coupled with a subregional analysis of the neuropil, suggest a potentially important role of visual stimulation, possibly interacting with juvenile hormone, as an organizer of the mushroom bodies. In an independent study, the brains of worker bees in which the transition to foraging was delayed (overaged nurse bees) were also studied. The mushroom bodies of overaged nurse bees had a Kenyon cell somal region volume typical of normal aged nurse bees. However, they displayed a significantly expanded neuropil relative to normal aged nurse bees. Analysis of the big back bees demonstrates that certain aspects of adult brain plasticity associated with foraging can be displayed by worker bees treated with methoprene independent of foraging experience. Analysis of the over-aged nurse bees suggests that the post-metamorphic expansion of the neuropil of the mushroom bodies of worker honey bees is not a result of foraging experience. © 1995 John Wiley & Sons, Inc.     

Light exposure leads to reorganization of microglomeruli in the mushroom bodies and influences juvenile hormone levels in the honeybee

Honeybees show a remarkable behavioral plasticity at the transition from nursing inside the hive to foraging for nectar and/or pollen outside. This plasticity is important for age‐related division of labor in honeybee colonies. The behavioral transition is associated with significant volume and synaptic changes in the mushroom bodies (MBs), brain centers for sensory integration, learning, and memory. We tested whether precocious sensory exposure to light leads to changes in the density of synaptic complexes [microglomeruli (MG)] in the MBs. The results show that exposure to light pulses over 3 days induces a significant decrease in the MG density in visual subregions (collar) of the MB. Earlier studies had shown that foragers have increased levels of juvenile hormone (JH) co‐occurring with a decrease of vitellogenin (Vg). Previous work further established that RNAi‐mediated knockdown of vg and ultraspiracle (usp) induced an upregulation of JH levels, which can lead to precocious foraging. By disturbing both Vg and JH pathways using gene knockdown of vg and usp, we tested whether the changes in the hormonal system directly affect MG densities. Our study shows that MG numbers remained unchanged when Vg and JH pathways were perturbed, suggesting no direct hormonal influences on MG densities. However, mass spectrometry detection of JH revealed that precocious light exposure triggered an increase in JH levels in the hemolymph (HL) of young bees. This suggests a dual effect following light exposure via direct effects on MG reorganization in the MB calyx and a possible positive feedback on HL JH levels. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1141–1153, 2014     

In-Hive Behavior of Pollen Foragers (Apis mellifera) in Honey Bee Colonies Under Conditions of High and Low Pollen Need

Pollen collection in honey bees is regulated around a homeostatic set-point. How the control of pollen collection is achieved is still unclear. Different feedback mechanisms have been proposed but little is known about the experience of pollen foragers in the hive. A detailed documentation of the behavior of pollen foragers in the hive under different pollen need conditions is presented here. Taking a broad observational approach, we analyze the behavior of individual pollen foragers in the hive between collecting trips and quantify the different variables constituting the in-hive stay. Comparing data from two colonies and 143 individuals during experimentally induced times of low vs. times of high pollen need, we show that individual foragers modulate their in-hive working tempo according to the actual pollen need of the colony: pollen foragers slowed down and stayed in the hive longer when pollen need was low and spent less time in the hive between foraging trips when pollen need by their colony was high. Furthermore, our data show a significant change in the trophallactic experience of pollen foragers with changing pollen need conditions of their colony. Pollen foragers had more short (< 3 s) trophallactic contacts when pollen need was high, resulting in an increase of total number of trophallactic contacts. Thus, our results support the hypothesis that trophallactic experience is one of the various information pathways used by pollen foragers to assess their colony's pollen need.     

Physiological correlates of division of labor among similarly aged honey bees

Hormone analyses and exocrine gland measurements were made to probe for physiological correlates of division of labor among similarly aged adult worker honey bees (Apis mellifera L.). Middle-age bees (ca. 2 weeks old) performing different tasks showed significant differences in both juvenile hormone (JH) biosynthesis rates and hemolymph titers; guards and undertakers had high JH, and wax producers and food storers, low JH. Guards and undertakers had similar hormone levels to foragers, even though they were 10 days younger than foragers. No differences in JH were detected among young bees (1-week-old queen attendants and nurses) or older bees (3–4 week-old pollen foragers, non-pollen foragers, and soldiers). Hypopharyngeal gland size was inversely correlated with worker age and rate of JH biosynthesis, but soldiers had significantly larger hypopharyngeal glands than did foragers, despite their similar age and JH level. Results from soldiers indicate that exocrine gland development is not always linked with age-related behavior and endocrine development; they also support the recent claim that soldiers constitute a group of older bees that are distinct from foragers. Hormonal analyses indicate that the current model of JH's role in honey bee division of labor needs to be expanded because high levels of JH are associated with several other tasks besides foraging. JH may be involved in the regulation of division of labor among similarly aged workers in addition to its role in age-related division of labor.Abbreviations JH Juvenile hormone - RIA radioimmunoassay - CA corpora allata - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Juvenile hormone paces behavioral development in the adult worker honey bee

Behavioral development in the adult worker honey bee (Apis mellifera), from performing tasks inside the hive to foraging, is associated with an increase in the blood titer of juvenile hormone III (JH), and hormone treatment results in precocious foraging. To study behavioral development in the absence of JH we removed its glandular source, the corpora allata, in 1-day-old adult bees. The age at onset of foraging for allatectomized bees in typical colonies was significantly older compared with that of sham-operated bees in 3 out of 4 colonies; this delay was eliminated by hormone replacement in 3 out of 3 colonies. To determine the effects of corpora allata removal on sensitivity to changes in conditions that influence the rate of behavioral development, we used "single-cohort" colonies (composed of only young bees) in which some colony members initiate foraging precociously. The age at onset of foraging for allatectomized bees was significantly older compared with that of sham-operated bees in 2 out of 3 colonies, and this delay was eliminated by hormone replacement. Allatectomized bees initiated foraging at significantly younger ages in single-cohort colonies than in typical colonies. These results demonstrate that JH influences the pace of behavioral development in honey bees, but is not essential for either foraging or altering behavioral development in response to changes in conditions.     

Persistence to Unrewarding Feeding Locations by Honeybee Foragers (Apis mellifera): the Effects of Experience,Resource Profitability and Season

Honeybee foragers that find a profitable food source quickly establish spatiotemporal memories, which allow them to return to this foraging site on subsequent days. The aim of this study was to investigate how the previous experience of honeybee foragers at a feeding location affects their persistence at that location once food is no longer available. We hypothesised that persistence would be greater to locations that were more rewarding (closer to the hive, higher concentration of sucrose solution), for which a bee had greater prior experience (0.5‐h vs. 2‐h training access), and at times of the year of lower nectar availability in the environment. We studied individually marked worker bees from four colonies trained to sucrose‐solution feeders. Our results support most of these predictions. Persistence, measured both in duration and number of visits, was greater to locations that previously offered sucrose solution of higher concentration (2 m vs. 1 m ) or were closer to the hive (20 m vs. 450 m). Persistence was also greater in bees that had longer access at the feeder before the syrup was terminated (2 h vs. 0.5 h). However, contrary to our prediction, persistence was not higher in the season of the lowest nectar availability in the environment in the study year. In summary, honeybees show considerable persistence at foraging sites that ceased providing rewards. The decision to abandon a foraging site depends on the profitability the forager experienced when the foraging site was still rewarding.     

Octopamine influences division of labor in honey bee colonies

Forager honey bees have higher brain levels of octopamine than do bees tending larvae in the hive. To test the hypothesis that octopamine influences honey bee division of labor we treated bees orally with octopamine or its immediate precursor tyramine and determined whether these treatments increased the probability of initiating foraging. Octopamine treatment significantly elevated levels of octopamine in the brain and caused a significant dose-dependent increase in the number of new foragers. This effect was seen for precocious foragers in single-cohort colonies and foragers in larger colonies with more typical age demographies. Tyramine treatment did not increase the number of new foragers, suggesting that octopamine was exerting a specific effect. Octopamine treatment was effective only when given to bees old enough to forage, i.e., older than 4 days of age. Treatment when bees were 1-3 days of age did not cause a significant increase in the number of new foragers when the bees reached the minimal foraging age. These results demonstrate that octopamine influences division of labor in honey bee colonies. We speculate that octopamine is acting in this context as a neuromodulator.     

period expression in the honey bee brain is developmentally regulated and not affected by light, flight experience, or colony type

Changes in circadian rhythms of behavior are related to age-based division of labor in honey bee colonies. The expression of the clock gene period (per) in the bee brain is associated with age-related changes in circadian rhythms of behavior, but previous efforts to firmly associate per brain expression with division of labor or age have produced variable results. We explored whether this variability was due to differences in light and flight experience, which vary with division of labor, or differences in colony environment, which are known to affect honey bee behavioral development. Our results support the hypothesis that per mRNA expression in the bee brain is developmentally regulated. One-day-old bees had the lowest levels of expression and rarely showed evidence of diurnal fluctuation, while foragers and forager-age bees (> 21 days of age) always had high levels of brain per and strong and consistent diurnal patterns. Results from laboratory and field experiments do not support the hypothesis that light, flight experience, and colony type influence per expression. Our results suggest that the rate of developmental elevation in per expression is influenced by factors other than the ones studied in our experiments, and that young bees are more sensitive to these factors than foragers.     

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.     

Recruitment of honeybees to non-scented food sources

Small groups of honeybees (five to nine individuals) were trained to forage at feeders 150 m, 300 m and 800 m from an observation hive. Their behaviour in the hive and at the feeder was recorded by observers that maintained continuous radio contact with one another. At low concentrations of sugar in the feeder (0.5 mol x l(-1)) foragers do not dance in the hives, their flights to the feeder are often undertaken alone, they land immediately after arrival at the site and no recruits from the hive landed on the feeder during 30 h of observation. Raising the concentration of sugar in the feeder to 2 mol x l(-1) leads to vigorous dancing by the foragers and the gradual (over 10-15 min) synchronisation of their flights so that they arrive in groups of up to five bees at the feeder and undertake circular "buzzing" flights before landing. Such behaviour of the foragers is associated with the appearance of recruits which were never seen to fly around the feeder and land alone or before the foragers. Recruits typically circle the feeder together with foragers and land with them or continue their circling flights to land about 10 s later. While circling the feeder recruits, but not foragers, will fly after a moving lure if the presentation of the lure is accompanied by the release of geraniol scent. We propose that recruits that have witnessed a waggle dance are unlikely to find a non-scented feeder unless the foragers continue their flights to that feeder and provide supplementary visual and/or olfactory cues, at least in the vicinity of the feeder. We propose that the synchronisation of the flights of foragers and their behaviour at the feeding site is a strategy designed to overcome a navigational gap in the recruiting process in which the dance can indicate the general area of a food source but not the precise position of a highly localised site.     

Effects of recent experience on foraging decisions by bumble bees

The temporal and spatial scales employed by foraging bees in sampling their environment and making foraging decisions should depend both on the limits of bumble bee memory and on the spatial and temporal pattern of rewards in the habitat. We analyzed data from previous experiments to determine how recent foraging experience by bumble bees affects their flight distances to subsequent flowers. A single visit to a flower as sufficient to affect the flight distance to the next flower. However, longer sequences of two or three visits had an additional effect on the subsequent flight distance of individual foragers. This suggests that bumble bees can integrate information from at least three flowers for making a subsequent foraging decision. The existence of memory for floral characteristics at least at this scale may have significance for floral selection in natural environments.     

Olfactory learning in the stingless bee Tetragonisca angustula (Hymenoptera, Apidae, Meliponini)

Tetragonisca angustula stingless bees are considered as solitary foragers that lack specific communication strategies. In their orientation towards a food source, these social bees use chemical cues left by co-specifics and the information obtained in previous foraging trips by the association of visual stimuli with the food reward. Here, we investigated their ability to learn the association between odors and reward (sugar solution) and the effect on learning of previous encounters with scented food either inside the hive or during foraging. During food choice experiments, when the odor associated with the food was encountered at the feeding site, the bees’ choice is biased to the same odor afterwards. The same was not the case when scented food was placed inside the nest. We also performed a differential olfactory conditioning of proboscis extension response with this species for the first time. Inexperienced bees did not show significant discrimination levels. However, when they had had already interacted with scented food inside the hive, they were able to learn the association with a specific odor. Possible olfactory information circulation inside the hive and its use in their foraging strategies is discussed.     

The ontogeny of immunity: development of innate immune strength in the honey bee (Apis mellifera)

Honey bees (Apis mellifera) are of vital economic and ecological importance. These eusocial animals display temporal polyethism, which is an age-driven division of labor. Younger adult bees remain in the hive and tend to developing brood, while older adult bees forage for pollen and nectar to feed the colony. As honey bees mature, the types of pathogens they experience also change. As such, pathogen pressure may affect bees differently throughout their lifespan. We provide the first direct tests of honey bee innate immune strength across developmental stages. We investigated immune strength across four developmental stages: larvae, pupae, nurses (1-day-old adults), and foragers (22-30 days old adults). The immune strength of honey bees was quantified using standard immunocompetence assays: total hemocyte count, encapsulation response, fat body quantification, and phenoloxidase activity. Larvae and pupae had the highest total hemocyte counts, while there was no difference in encapsulation response between developmental stages. Nurses had more fat body mass than foragers, while phenoloxidase activity increased directly with honey bee development. Immune strength was most vigorous in older, foraging bees and weakest in young bees. Importantly, we found that adult honey bees do not abandon cellular immunocompetence as has recently been proposed. Induced shifts in behavioral roles may increase a colony's susceptibility to disease if nurses begin foraging activity prematurely.     

Effect of Foraging Distance on the Thermal Behaviour of Honeybees during Dancing,Walking and Trophallaxis

By means of infrared thermography and without disturbing social interactions, the correlation between thoracic temperature in honeybees, Apis mellifera carnica, upon their return to the hive and their foraging distance was investigated. Thoracic temperature while dancing and walking and during trophallactic contact with hive bees decreased with increasing flight distance. In bees foraging 0.5, 1, 1.5 and 2 molar sucrose solutions from a distance of 120 m, dancing temperature amounted to 38.4, 40.1, 40.9 and 40.6 °C, respectively; while in bees foraging from a distance of 2950 m it amounted to 36.6, 38.4, 38.6 and 39.1 °C, respectively. The rate of decrease in dancing temperature per 1000 m increase in flight distance was 0.64, 0.47, 0.81 and 0.54 °C with a 0.5, 1, 1.5 and 2 molar sucrose solution, respectively. Both at short and at long flight distances, the relationship between thoracic temperature and sucrose concentration of the food followed a non-linear curve, which flattened at concentrations higher than 1 mol/1. The experiments showed that inside the hive the foragers' level of thermoregulation depends not only on the energy (sugar) content of the food; but rather, the level of thermoregulation corresponds to the general quality of the food source, which includes both energy content and distance from the hive. Because the thermal behaviour of foragers correlates with several behavioural parameters indicating the bees' foraging tendency and their eagerness to dance, thoracic temperature seems to be a correlate of the profitability of foraging.