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.     

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.     

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.     

Transcriptional response to foraging experience in the honey bee mushroom bodies

Enriched environmental conditions induce neuroanatomical plasticity in a variety of vertebrate and invertebrate species. We explored the molecular processes associated with experience-induced plasticity, using naturally occurring foraging behavior in adult worker honey bees (Apis mellifera). In honey bees, the mushroom bodies exhibit neuroanatomical plasticity that is dependent on accumulated foraging experience. To investigate molecular processes associated with foraging experience, we performed a time-course microarray study to examine gene expression changes in the mushroom bodies as a function of days foraged. We found almost 500 genes that were regulated by duration of foraging experience. Bioinformatic analyses of these genes suggest that foraging experience is associated with multiple molecular processes in the mushroom bodies, including some that may contribute directly to neuropil growth, and others that could potentially protect the brain from the effects of aging and physiological stress.     

Volume and density of microglomeruli in the honey bee mushroom bodies do not predict performance on a foraging task

The mushroom bodies (MBs) are insect brain regions important for sensory integration, learning, and memory. In adult worker honey bees (Apis mellifera ), the volume of neuropil associated with the MBs is larger in experienced foragers compared with hive bees and less experienced foragers. In addition, the characteristic synaptic structures of the calycal neuropils, the microglomeruli, are larger but present at lower density in 35‐day‐old foragers relative to 1‐day‐old workers. Age‐ and experience‐based changes in plasticity of the MBs are assumed to support performance of challenging tasks, but the behavioral consequences of brain plasticity in insects are rarely examined. In this study, foragers were recruited from a field hive to a patch comprising two colors of otherwise identical artificial flowers. Flowers of one color contained a sucrose reward mimicking nectar; flowers of the second were empty. Task difficulty was adjusted by changing flower colors according to the principle of honey bee color vision space. Microglomerular volume and density in the lip (olfactory inputs) and collar (visual inputs) compartments of the MB calyces were analyzed using anti‐synapsin I immunolabeling and laser scanning confocal microscopy. Foragers displayed significant variation in microglomerular volume and density, but no correlation was found between these synaptic attributes and foraging performance. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1057–1071, 2017     

Some cetoniinae (Coleoptera,Scarabaeidae) have structurally different medial and lateral calyces of mushroom bodies

Structural differences between the medial and lateral calyces of mushroom bodies in insects are described for the first time. In two cetoniine scarab beetles, Cetonia aurata and Oxythyrea funesta, the lateral calyces are subdivided into two portions showing a different neuropil structure. This feature is not reflected in the structure of the pedunculus and lobes, as well as in the relative neuropil volume occupied by transformed lateral calyx as compared with unmodified lateral calyx of related scarab beetles. The lateral calyx modification is considered to be related to changes in dendritic arborizations of central Kenyon cells. The subdivision of lateral calyx occurs only in adults and was not observed in larvae.     

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.     

Juvenile hormone profiles of worker honey bees,Apis mellifera,during normal and accelerated behavioural development

Juvenile hormone III (JH) haemolymph titres were quantified in adult worker honey bees under colony conditions conducive to either typical or accelerated behavioural development. JH titres of bees under conditions of accelerated behavioural development were significantly higher than same-aged bees under more typical conditions, even before the onset of foraging. These results are consistent with previous findings indicating that JH plays a causal role in timing the onset of foraging behaviour in honey bees. We also detected a peak of JH in 2-3 day old adult bees, the significance of which is unknown.     

Flower choice by honey bees (Apis mellifera L.): sex-phase of flowers and preferences among nectar and pollen foragers

Bees foraging for nectar should choose different inflorescences from those foraging for both pollen and nectar, if inflorescences consist of differing proportions of male and female flowers, particularly if the sex phases of the flowers differ in nectar content as well as the occurrence of pollen. This study tested this prediction using worker honey bees (Apis mellifera L.) foraging on inflorescences of Lavandula stoechas. Female flowers contained about twice the volume of nectar of male flowers. As one would predict, bees foraging for nectar only chose inflorescences with disproportionately more female flowers: time spent on the inflorescence was correlated with the number of female flowers, but not with the number of male flowers. Inflorescence size was inversely correlated with the number of female flowers, and could be used as a morphological cue by these bees. Also as predicted, workers foraging for both pollen and nectar chose inflorescences with relatively greater numbers of both male and female flowers: time spent on these inflorescences was correlated with the number of male flowers, but not with the number of females flowers. A morphological cue inversely associated with such inflorescences is the size of the bract display. Choice of flowers within inflorescences was also influenced predictably, but preferences appeared to be based upon corolla size rather than directly on sex phase.     

Juvenile hormone levels in honey bee (Apis mellifera L.) foragers: foraging experience and diurnal variation

A rising blood titer of juvenile hormone (JH) in adult worker honey bees is associated with the shift from working in the hive to foraging. We determined whether the JH increase occurs in anticipation of foraging or whether it is a result of actual foraging experience and/or diurnal changes in exposure to sunlight. We recorded all foraging flights of tagged bees observed at a feeder in a large outdoor flight cage. We measured JH from bees that had taken 1, 3-5, or >100 foraging flights and foragers of indeterminate experience leaving or entering the hive. To study diurnal variation in JH, we sampled foragers every 6h over one day. Titers of JH in foragers were high relative to nurses as in previous studies, suggesting that conditions in the flight cage had no effect on the relationship between foraging behavior and JH. Titers of JH in foragers showed no significant effects of foraging experience, but did show significant diurnal variation. Our results indicate that the high titer of JH in foragers anticipates the onset of foraging and is not affected by foraging experience, but is modulated diurnally.     

Hemolymph juvenile hormone titers in worker honey bees under normal and preswarming conditions

Swarming is an important mechanism by which honey bee, Apis mellifera L., colonies reproduce, yet very little is known about the physiological changes in workers that are preparing to swarm. In this study, we determined the endocrine status of worker honey bees in preswarming colonies and in normal (nonswarming) colonies. Juvenile hormone (JH) titers in worker bees were similar in both groups before queen cells were present, but they became significantly lower in preswarming colonies compared with normal colonies when queen cells occurred in preswarming colonies. The lower JH titers in the preswarming colonies suggest that behavioral development is delayed in these colonies, consistent with previous reports that preswarming colonies have reduced foraging activities. Understanding the endocrine status of bees preparing for swarming will help us to better understand the biology of swarming.     

Worker-worker inhibition of honey bee behavioural development independent of queen and brood

Summary. Foragers inhibit the behavioural development of young adult worker honey bees, delaying the age at onset of foraging. But the similar effect caused by pheromones produced by both the queen and brood raised the possibility that some of the previously attributed forager effects might be due to queen, brood, or both. Here we studied whether physical contacts between young bees and old foragers can inhibit behavioural development while controlling for queen and brood effects. Results demonstrated that foragers inhibit the behavioural development of young adult worker bees independent of the queen and brood, via a mechanism that requires physical contact.Received 24 November 2003; revised 27 March 2004; accepted 21 April 2004.     

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.     

Dendritic reorganization of an identified neuron during metamorphosis of the moth Manduca sexta: The influence of interactions with the periphery

During metamorphosis of the moth, Manduca sexta, an identified leg motor neuron, the femoral extensor motor neuron (FeExt MN) undergoes dramatic reorganization. Larval dendrites occupy two distinct regions of neuropil, one in the lateral leg neuropil and a second in dorsomedial neuropil. Adult dendrites occupy a greater volume of lateral leg neuropil but do not extend to the dorsomedial region of the ganglion. The adult dendritic morphology is acquired by extreme dendritic regression followed by extensive dendritic growth. Towards the end of larval life, MN dendrites begin to regress, but the most dramatic loss of dendrites occurs in the 3 days following pupation, such that only a few sparse dendrites are retained in the lateral region of leg neuropil. Extensive dendritic growth occurs over the subsequent days such that the MN acquires an adult-like morphology between 12 and 14 days after pupation. This basic process of dendritic remodeling is not dependent upon the presence of the adult leg, suggesting that neither contact with the new target muscle nor inputs from new leg sensory neurons are necessary for triggering dendritic changes. The final distribution of MN dendrites in the adult, however, is altered when the adult leg is absent, suggesting that cues from the adult leg are involved in directing or shaping the growth of MN dendrites to specific regions of neuropil. © 1993 John Wiley & Sons, Inc.     

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera l)

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed.     

Neurons with dopamine-like immunoreactivity target mushroom body Kenyon cell somata in the brain of some hymenopteran insects

The mushroom bodies of the insect brain are centers for olfactory and multimodal information processing and they are involved in associative olfactory learning. They are comprised of numerous (340,000 in the bee brain), small (3–8 μm soma diameter) local interneurons, the Kenyon cells. In the brain of honeybees (Apis mellifera) of all castes (worker bees, drones and queens), wasps (Vespula germanica) and hornets (Vespa crabro) immunostaining revealed fibers with dopamine-like immunoreactivity projecting from the pedunculus and the lip neuropil of the mushroom bodies into the Kenyon cell perikaryal layer. These fibers terminate with numerous varicosities, mainly around the border between medial and lateral Kenyon cell soma groups. Visualization of immunostained terminals in the transmission electron microscope showed that they directly contact the somata of the Kenyon cells and contain presynaptic elements. The somata of the Kenyon cells are clearly non-immunoreactive. Synaptic contacts at the somata are unusual for the central nervous systems of insects and other arthropods. This finding suggests that the somata of the Kenyon cells of Hymenoptera may serve an integrative role, and not merely a supportive function.     

Nectar distribution and its relation to food quality in honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) colonies

In honeybees (Apis mellifera), the process of nectar collection is considered a straightforward example of task partitioning with two subtasks or two intersecting cycles of activity: (1) foraging and (2) storing of nectar, linked via its transfer between foragers and food processors. Many observations suggest, however, that nectar collection and processing in honeybees is a complex process, involving workers of other sub-castes and depending on variables such as resource profitability or the amount of stored honey. It has been observed that food processor bees often distribute food to other hive bees after receiving it from incoming foragers, instead of storing it immediately in honey cells. While there is little information about the sub-caste affiliation and the behaviour of these second-order receivers, this stage may be important for the rapid distribution of nutrients and related information. To investigate the identity of these second-order receivers, we quantified behaviours following nectar transfer and compared these behaviours with the behaviour of average worker hive-bees. Furthermore, we tested whether food quality (sugar concentration) affects the behaviour of the second-order receivers. Of all identified second-order receivers, 59.3% performed nurse duties, 18.5% performed food-processor duties and 22.2% performed forager duties. After food intake, these bees were more active, had more trophallaxes (especially offering contacts) compared to average workers and they were found mainly in the brood area, independent of food quality. Our results show that the liquid food can be distributed rapidly among many bees of the three main worker sub-castes, without being stored in honey cells first. Furthermore, the results suggest that the rapid distribution of food partly depends on the high activity of second-order receivers. Received 31 August 2006; revised 8 December 2006; accepted 11 December 2006.     

Bumble bee abundance and richness improves honey bee pollination behaviour in sweet cherry

Pollination service in agricultural crops increases significantly with pollinator diversity and wild pollinator abundance. Differences in the foraging behaviour of pollinating insects are one of the reasons why pollinator diversity and abundance enhances crop pollination. Here, we focused on the foraging behaviour of honey bees and bumble bees in sweet cherry orchards. In addition, we studied the influence of bee diversity and abundance on the foraging behaviour of honey bees and bumble bees. Honey bees were found to visit fewer flowers than bumble bees. Bumble bees also showed a higher probability of changing trees between rows than honey bees. Both visitation rate and probability of row changes of honey bees increased with bumble bee diversity and with bumble bee abundance. We also found that the probability of row changes of honey bees increased with increasing bumble bee abundance. These effects of bumble bee richness and abundance on the pollination behaviour of honey bees can improve the pollination performance of honey bees in crops that depend on cross pollination. Our results highlight the higher pollination performance of bumble bees and the facilitative effect of wild pollinators to crop pollination.     

Colony Integration in Honey Bees: Mechanisms of Behavioral Reversion

After confirming that worker honey bees (Apis mellifera) can revert from foraging to brood care, we determined whether juvenile hormone (JH) mediates this form of plasticity in behavioral development and whether worker age and genotype influence the probability of its expression. Measurements of JH titers support the hypothesis that plasticity in honey bee behavioral development is a consequence of modulation of JH by extrinsic factors. Observations of individually marked bees in a colony composed of two phenotypically distinguishable subfamilies revealed that the likelihood of undergoing behavioral reversion was influenced by worker age but not by worker genotype. The effect of worker age on reversion is consistent with a previously formulated model for the regulation of age polyethism in honey bees that predicts that workers of different ages have different response thresholds for task-associated stimuli. The lack of a genotypic effect on reversion is in contrast to results for other forms of behavioral plasticity.     

Reproduction of Varroa destructor in worker brood of Africanized honey bees (Apis mellifera)

Reproduction and population growth of Varroa destructor was studied in ten naturally infested, Africanized honeybee (AHB) (Apis mellifera) colonies in Yucatan, Mexico. Between February 1997 and January 1998 monthly records of the amount of pollen, honey, sealed worker and drone brood were recorded. In addition, mite infestation levels of adult bees and worker brood and the fecundity of the mites reproducing in worker cells were determined. The mean number of sealed worker brood cells (10,070 ± 1,790) remained fairly constant over the experimental period in each colony. However, the presence and amount of sealed drone brood was very variable. One colony had drone brood for 10 months and another for only 1 month. Both the mean infestation level of worker brood (18.1 ± 8.4%) and adult bees (3.5 ± 1.3%) remained fairly constant over the study period and did not increase rapidly as is normally observed in European honey bees. In fact, the estimated mean number of mites fell from 3,500 in February 1997 to 2,380 in January 1998. In May 2000 the mean mite population in the study colonies was still only 1,821 mites. The fertility level of mites in this study was much higher (83–96%) than in AHB in Brazil(25–57%), and similar to that found in EHB (76–94%). Mite fertility remained high throughout the entire study and was not influenced by the amount of pollen, honey or worker brood in the colonies. This revised version was published online in July 2006 with corrections to the Cover Date.