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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.

Development of the mushroom bodies continues after adult eclosion in social insects. Synapsins, phosphoproteins abundant in presynaptic boutons, are not required for development of the nervous system but have as their primary function modulation of synaptic transmission. A monoclonal antibody against a conserved region of Drosophila synapsin labels synaptic structures called microglomeruli in the mushroom bodies of adult social insects, permitting studies of microglomerular volume, density, and number. The results point to multiple forms of brain plasticity in social insects: age-based and experience-based maturation that results in a decrease in density coupled with an increase in volume of individual microglomeruli in simultaneous operation with shorter term changes in density produced by specific life experiences.

International audience; AbstractTo test the hypothesis that exposure to queen mandibular pheromone (QMP) modulates ecdysteroid production in adult worker honey bees, ecdysteroids were measured in hemolymph and other tissues of individual adult worker honey bees reared with or without QMP in cages and field colonies. Ecdysteroid titers were higher in caged workers exposed to QMP continuously from the first day of adult life than in workers reared without QMP. Statistical cluster analysis suggested the possibility that a subgroup of workers (“responders”) is more sensitive to QMP in this regard than other workers. In 12-day-old workers, ecdysteroid titers in workers reared in queenright (QR) colonies were similar to those observed in cages with QMP, but lower than those in queenless (QL) colonies. Differences in number of ovarioles or degree of ovarian activation did not correlate with hemolymph ecdysteroids. Previous studies have demonstrated ecdysteroids in hemolymph in very young adult workers and in workers in QL colonies; the present study indicates that production of ecdysteroids occurs in older adult worker honey bees in the absence of morphological signs of ovarian activation, with cage studies revealing a modulatory role for QMP masked in the complex environment of the hive.

Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

This chapter is a revision of the previous edition chapter by S.E. Fahrbach, R.A. Velarde, volume 2, pp. 943–966, © 2009, Elsevier Inc.

The nuclear receptors (NRs) of metazoans are an ancient family of transcription factors defined by conserved DNA- and ligand-binding domains (DBDs and LBDs, respectively). The Drosophila melanogaster genome project revealed 18 canonical NRs (with DBDs and LBDs both present) and 3 receptors with the DBD only. Annotation of subsequently sequenced insect genomes revealed only minor deviations from this pattern. A renewed focus on functional analysis of the isoforms of insect NRs is therefore required to understand the diverse roles of these transcription factors in embryogenesis, metamorphosis, reproduction, and homeostasis. One insect NR, ecdysone receptor (EcR), functions as a receptor for the ecdysteroid molting hormones of insects. Researchers have developed nonsteroidal ecdysteroid agonists for EcR that disrupt molting and can be used as safe pesticides. An exciting new technology allows EcR to be used in chimeric, ligand-inducible gene-switch systems with applications in pest management and medicine.

Published estimates of the number of ovarioles found in the ovaries of honey bee, Apis mellifera L. (Hymenoptera: Apidae) queens range from 100 to 180 per ovary. Within the context of a large-scale study designed to assay the overall quality of queens obtained from various commercial sources, a simple histology-based method for accurate determination of ovariole number was developed and then applied to a sample of 75 queens. Although all 10 commercial sources evaluated provided queens with ovariole numbers within the expected range, ovariole number was found to vary significantly across sources. Overall, and within most of the individual samples, there was no correlation of ovariole number with other morphological attributes such as thoracic width, wing length, or wet weight. Queens from two of the sources, however, displayed a significant negative relationship between wet weight and ovariole number. This study provides baseline data on ovariole number in commercial honey bee queens in the United States at a time when honey bee populations are declining; the method described can be used in studies relating ovariole number in queens to egg production and behavior.

Large bumblebee (Bombus terrestris) workers typically visit flowers to collect pollen and nectar during the day and rest in the nest at night. Small workers are less likely to forage, but instead stay in the nest and tend brood around the clock. Because Pigment Dispersing Factor (PDF) has been identified as a neuromodulator in the circadian network of insects, we used an antiserum that recognizes this peptide to compare patterns of PDF-immunoreactivity (PDF-ir) in the brains of large and small workers. Our study provides the first description of PDF distribution in the bumblebee brain, and shows a pattern that is overall similar to that of the honey bee, Apis mellifera. The brains of large bumblebee workers contained a slightly but significantly higher number of PDF-ir neurons than did the brains of small sister bees. Body size was positively correlated with area of the PDF-ir somata and negatively correlated with the maximal staining intensity. These results provide a neuronal correlate to the previously reported body size-associated variation in behavioral circadian rhythmicity. These differences in PDF-ir are consistent with the hypothesis that body size-based division of labor in bumblebees is associated with adaptations of the morphology and function of the brain circadian system.

Honey bees can distinguish nestmates from non-nestmates, directing aggressive responses toward non-nestmates and rarely attacking nestmates. Here we provide evidence that treatment with pilocarpine, a muscarinic agonist, significantly reduced the number of aggressive responses directed toward nestmates. By contrast, treatment with scopolamine, a muscarinic antagonist, significantly increased attacks on nestmates. Locomotor activity was not altered by these pharmacological treatments. When interpreted in light of known cholinergic pathways in the insect brain, our results provide the first evidence that cholinergic signaling via muscarinic receptors plays a role in olfaction-based social behavior in honey bees.

The Drosophila genome encodes 18 canonical nuclear receptors. All of the Drosophila nuclear receptors are here shown to be present in the genome of the honey bee (Apis mellifera). Given that the time since divergence of the Drosophila and Apis lineages is measured in hundreds of millions of years, the identification of matched orthologous nuclear receptors in the two genomes reveals the fundamental set of nuclear receptors required to 'make' an endopterygote insect. The single novelty is the presence in the A. mellifera genome of a third insect gene similar to vertebrate photoreceptor-specific nuclear receptor (PNR). Phylogenetic analysis indicates that this novel gene, which we have named AmPNR-like, is a new member of the NR2 subfamily not found in the Drosophila or human genomes. This gene is expressed in the developing compound eye of the honey bee. Like their vertebrate counterparts, arthropod nuclear receptors play key roles in embryonic and postembryonic development. Studies in Drosophila have focused primarily on the role of these transcription factors in embryogenesis and metamorphosis. Examination of an expressed sequence tag library developed from the adult bee brain and analysis of transcript expression in brain using in situ hybridization and quantitative RT-PCR revealed that several members of the nuclear receptor family (AmSVP, AmUSP, AmERR, AmHr46, AmFtz-F1, and AmHnf-4) are expressed in the brain of the adult bee. Further analysis of the expression of AmUSP and AmSVP in the mushroom bodies, the major insect brain centre for learning and memory, revealed changes in transcript abundance and, in the case of AmUSP, changes in transcript localization, during the development of foraging behaviour in the adult. Study of the honey bee therefore provides a model for understanding nuclear receptor function in the adult brain.

Honey bees begin life working in the hive. At ≈3 weeks of age, they shift to visiting flowers to forage for pollen and nectar. Foraging is a complex task associated with enlargement of the mushroom bodies, a brain region important in insects for certain forms of learning and memory. We report here that foraging bees had a larger volume of mushroom body neuropil than did age-matched bees confined to the hive. This result indicates that direct experience of the world outside the hive causes mushroom body neuropil growth in bees. We also show that oral treatment of caged bees with pilocarpine, a muscarinic agonist, induced an increase in the volume of the neuropil similar to that seen after a week of foraging experience. Effects of pilocarpine were blocked by scopolamine, a muscarinic antagonist. Our results suggest that signaling in cholinergic pathways couples experience to structural brain plasticity.

Progress in the field of insect neuroendocrinology has been rapid despite the relatively small number of investigators working on insect systems. This progress, in part, reflects the ease of studying insect behavior in the laboratory, and a historical perspective reveals that insect neuroendocrinology has been dominated since its inception by laboratory studies. Recent advances in methodology and a renewed interest in the concept of behavioral state in insects suggest that it might be useful for insect neuroendocrinologists to spend a little more time in the field.

During metamorphosis of the insect brain, the optic lobe anlagen generate the proliferation centers for the visual cortices. We show here that, in the moth Manduca sexta, an 80 kDa Golgi complex protein (Ms-golgin80) is abundantly expressed in the cytoplasm of neuroblasts and ganglion mother cells in the optic lobe anlagen and proliferation centers. The predicted amino acid sequence for Ms-golgin80 is similar to that of several members of the GM130 subfamily of Golgi-associated proteins, including rat GM130 and human golgin-95. Homologs of Ms-golgin80 from Drosophila melanogaster, Caenorhabditis elegans, and Brugia malayi were identified through homology sequence search. Sequence similarities are present in three regions: the N-terminus, an internal domain of 89 amino acids, and another domain of 89 amino acids near the C-terminus. Structural similarities further suggest that these molecules play the same cellular role as GM130. GM130 is involved in the docking and fusion of coatomer (COP I) coated vesicles to the Golgi membranes; it also regulates the fragmentation and subsequent reassembly of the Golgi complex during mitosis. Abundant expression of Ms-golgin80 in neuroblasts and ganglion mother cells and its reduced expression in the neuronal progeny of these cells suggest that this protein may be involved in the maintenance of the proliferative state. Abbreviation: BrdU 5-bromo-2′-deoxyuridine COP I, II coatomer proteins that coat vesicles and direct protein and membrane trafficking between early compartments of the secretory pathway in eukaryotic cells GM130 Peripheral membrane proteins associated with the Golgi bodies. GMC ganglion mother cell Ms-golgin80 The Manduca sexta homolog of the GM130 protein. Other homologs include Rn-GM130 (Rattus norvegicus); Hs-GM130 (Homo sapiens), Dm-golgin90 (Drosophila melanogaster), Ce-golgin107 (Caenorhabditis elegans) and Bm-golgin (Brugia malayi) PBS phosphate buffered saline PBST phosphate buffered saline containing 0.05% Tween-20 TBST Tris buffered saline containing 0.05% Tween-20

A worker honeybee performs tasks within the hive for approximately the first 3 weeks of adult life. After this time, it becomes a forager, flying repeatedly to collect food outside of the hive for the remainder of its 5-6 week life. Previous studies have shown that foragers have an increased volume of neuropil associated with the mushroom bodies, a brain region involved in learning, memory, and sensory integration. We report here that growth of the mushroom body neuropil in adult bees occurs throughout adult life and continues after bees begin to forage. Studies using Golgi impregnation asked whether the growth of the collar region of the mushroom body neuropil was a result of growth of the dendritic processes of the mushroom body intrinsic neurons, the Kenyon cells. Branching and length of dendrites in the collar region of the calyces were strongly correlated with worker age, but when age-matched bees were directly compared, those with foraging experience had longer, more branched dendrites than bees that had foraged less or not at all. The density of Kenyon cell dendritic spines remained constant regardless of age or behavioral state. Older and more experienced foragers therefore have a greater total number of dendritic spines in the mushroom body neuropil. Our findings indicate that, under natural conditions, the cytoarchitectural complexity of neurons in the mushroom bodies of adult honeybees increases as a function of increasing age, but that foraging experience promotes additional dendritic branching and growth.

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.

Cognitive ethology focuses on the study of animals under natural conditions to reveal ecologically adapted modes of learning. But biologists can more easily study what an animal learns than how it learns. For example, honeybees take repeated ‘orientation’ flights before becoming foragers at about three weeks of age1. These flights are a prerequisite for successful homing.2 Little is known2,3 about these flights because orienting bees rapidly fly out of the range of human observation. Using harmonic radar, we show for the first time a striking ontogeny to honeybee orientation flights. With increased experience, bees hold trip duration constant but fly faster, so later trips cover a larger area than earlier trips. In addition, each flight is typically restricted to a narrow sector around the hive. Orientation flights provide honeybees with repeated opportunities to view the hive and landscape features from different viewpoints, suggesting that bees learn the local landscape in a progressive fashion. We also show that these changes in orientation flight are related to the number of previous flights taken instead of chronological age, suggesting a learning process adapted to changes in weather conditions, flower availability and the needs of bee colonies.&nbsp

To test the hypothesis that colonies of honey bees composed of workers with faster rates of adult behavioral development are more defensive than colonies composed of workers with slower behavioral development, we determined whether there is a correlation between genetic variation in worker temporal polyethism and colony defensiveness. There was a positive correlation for these two traits, both for European and Africanized honey bees. The correlation was larger for Africanized bees, due to differences between Africanized and European bees, differences in experimental design, or both. Consistent with these results was the finding that colonies with a higher proportion of older bees were more defensive than colonies of the same size that had a lower proportion of older bees. There also was a positive correlation between rate of individual behavioral development and the intensity of colony flight activity, and a negative correlation between colony defensiveness and flight activity. This suggests that the relationship between temporal polyethism and colony defensiveness may vary with the manner in which foraging and defense duties are allocated among a colony’s older workers. These results indicate that genotypic differences in rates of worker behavioral development can influence the phenotype of a honey bee colony in a variety of ways.

The mushroom bodies are paired neuropils in the insect brain that act as multimodal sensory integration centers and are involved in learning and memory. Our studies, by using 5-bromo-2-deoxyuridine incorporation and the Feulgen technique, show that immediately before pupation, the brain of the developing honey bee (Apis mellifera) contains approximately 2,000 neuroblasts devoted to the production of the mushroom body intrinsic neurons (Kenyon cells). These neuroblasts are descended from four clusters of 45 or fewer neuroblasts each already present in the newly hatched larva. Subpopulations of Kenyon cells, distinct in cytoarchitecture, position, and immunohistochemical traits, are born at different, but overlapping, periods during the development of the mushroom bodies, with the final complement of these neurons in place by the mid-pupal stage. The mushroom bodies of the adult honey bee have a concentric arrangement of Kenyon cell types, with the outer layers born first and pushed to the periphery by later born neurons that remain nearer the center of proliferation. This concentricity is further reflected in morphologic and immunohistochemical traits of the adult neurons, and is demonstrated clearly by the pattern of expression of Drosophila myocyte enhancer factor 2 (DMEF2)-like immunoreactivity. This is the first comprehensive study of larval and pupal development of the honey bee mushroom bodies. Similarities to patterns of neurogenesis observed in the mushroom bodies of other insects and in the vertebrate cerebral cortex are discussed.

The eclosion of the adult Manduca sexta moth is followed by a wave of cell death that eliminates up to 50% of the neurons of the central nervous system within the first few days of imaginal life. While the identity of some of the dying motoneurons has been established, that of most doomed neurons is unknown. Here, we show that the dying cells include peptidergic neurons involved in the control of ecdysis behavior. These cells belong to a small population of 50 neurons that express crustacean cardioactive peptide (CCAP), a potent regulator of the ecdysis motor program, and show increases in cyclic 3',5'-guanosine monophosphate at each ecdysis. First, we describe new markers for these neurons and show that they are expressed in these CCAP-immunoreactive neurons in a complex temporal pattern during development. We then show that these neurons die within 36 h after adult eclosion, the last performance of ecdysis behavior in the life of the animal, via the active, genetically determined process of programmed cell death. The death of these neurons supports the hypothesis that outmoded or unused neurons are actively eliminated.

The daily patterns of task performance in honey bee colonies during behavioral development were studied to determine the role of circadian rhythmicity in age-related division of labor. Although it is well known that foragers exhibit robust circadian patterns of activity in both field and laboratory settings, we report that many in-hive tasks are not allocated according to a daily rhythm but rather are performed 24 h per day. Around-the-clock activity at the colony level is accomplished through the performance of some tasks by individual workers randomly with respect to time of day. Bees are initially arrhythmic with respect to task performance but develop diel rhythmicity, by increasing the occurrence of inactivity at night, prior to becoming foragers. There are genotypic differences for age at onset of rhythmicity and our results suggest that these differences are correlated with genotypic variation in rate of behavioral development: genotypes of bees that progressed through the age polyethism schedule faster also acquired behavioral rhythmicity at an earlier age. The ontogeny of circadian rhythmicity in honey bee workers ensures that essential in-hive behaviors are performed around the clock but also allows the circadian clock to be engaged before the onset of foraging.

Worker honeybees (Apis mellifera) were reared in social isolation in complete darkness to assess the effects of experience on growth of the neuropil of the mushroom bodies (MBs) during adult life. Comparison of the volume of the MBs of 1-day-old and 7-day-old bees showed that a significant increase in volume in the MB neuropil occurred during the first week of life in bees reared under these highly deprived conditions. All regions of the MB neuropil experienced a significant increase in volume with the exception of the basal ring. Measurement of titers of juvenile hormone (JH) in a subset of bees indicated that, as in previous studies, these rearing conditions induced in some bees the endocrine state of high JH associated with foraging, but there was no correlation between JH titer and volume of MB neuropil. Treatment of another subset of dark-reared bees with the JH analog, methoprene, also had no effect of the growth of the MB neuropil. These results demonstrate that there is a phase of MB neuropil growth early in the adult life of bees that occurs independent of light or any form of social interaction. Together with previous findings showing that an increase in MB neuropil volume begins around the time that orientation flights occur and then continues throughout the phase of life devoted to foraging, these results suggest that growth of the MB neuropil in adult bees may have both experience-expectant and experience-dependent components.

Bumblebees are important pollinators in natural and agricultural ecosystems. The latter results in the frequent exposure of bumblebees to pesticides. We report here on a new bioassay that uses primary cultures of neurons derived from adult bumblebee workers to evaluate possible side-effects of the neonicotinoid pesticide imidacloprid. Mushroom bodies (MBs) from the brains of bumblebee workers were dissected and dissociated to produce cultures of Kenyon cells (KCs). Cultured KCs typically extend branched, dendrite-like processes called neurites, with substantial growth evident 24-48 h after culture initiation. Exposure of cultured KCs obtained from newly eclosed adult workers to 2.5 parts per billion (ppb) imidacloprid, an environmentally relevant concentration of pesticide, did not have a detectable effect on neurite outgrowth. By contrast, in cultures prepared from newly eclosed adult bumblebees, inhibitory effects of imidacloprid were evident when the medium contained 25 ppb imidacloprid, and no growth was observed at 2,500 ppb. The KCs of older workers (13-day-old nurses and foragers) appeared to be more sensitive to imidacloprid than newly eclosed adults, as strong effects on KCs obtained from older nurses and foragers were also evident at 2.5 ppb imidacloprid. In conclusion, primary cultures using KCs of bumblebee worker brains offer a tool to assess sublethal effects of neurotoxic pesticides in vitro. Such studies also have the potential to contribute to the understanding of mechanisms of plasticity in the adult bumblebee brain.

The multicatalytic proteinase complex is a multi-subunit, high molecular weight proteinase present in the nucleus and cytoplasm of eukaryotic cells. This catalytic complex is involved in diverse cellular functions as part of the ubiquitin proteolysis system, including non-lysosomal proteolysis, antigen presentation, cell cycle progression, and cell proliferation, and in the programmed death of intersegmental muscles after adult eclosion in the tobacco hornworm moth, Manduca sexta. We have investigated the distribution of the multicatalytic proteinase complex in the central nervous system of this moth. At all stages of post-embryonic development, most cell types exhibited consistent, low levels of cytoplasmic and nuclear immunoreactivity for the multicatalytic proteinase complex. High levels of cell-specific accumulation of the complex were, however, demonstrated in abdominal neurosecretory cells and in imaginal cells in the larval brain, the larval segmental ganglia, and the developing wing discs. Imaginal cells exhibited intense immunoreactivity for the multicatalytic proteinase complex only until the onset of terminal differentiation. Intersegmental muscles undergoing programmed cell death exhibited intense cytoplasmic immunoreactivity for the multicatalytic proteinase, while persisting flight muscles and dying neurons were characterized by basal levels of staining. These staining patterns suggest that the multicatalytic proteinase of Manduca sexta serves multiple functions and is associated with the period of developmental arrest displayed by imaginal cells prior to metamorphosis. © 1996 Wiley-Liss, Inc.

The mushroom bodies, the insect brain structures most often associated with learning, exhibit structural plasticity during adult behavioral development in honey bees. We have investigated whether adult neurogenesis contributes to the plasticity of the mushroom bodies by labeling the DNA of replicating cells with 5-bromo-2'-deoxyuridine (BrdU). Immunocytochemical analysis of brain sections from bees fed or injected with BrdU as well as from bees treated in vitro with BrdU revealed no labeled neuronal nuclei, regardless of age or behavioral status of the worker bee (1-day old, nurse, or forager). Our results demonstrate that neurogenesis in the adult bee brain is a rare event, if it occurs at all. Therefore, the structural changes observed in the bee brain during adult behavioral development must be explained by developmental processes other than neurogenesis.

Neurons die during the development of nervous systems. The death of specific, idenified motoneuros during metamorphosis of the tobacco hornworm, Manduca sexta, provides an accessible model system in which to study the regulation of postembryonic neuronal death. Hormones and descending neural inputs have been shown toinfluence the survival of abdominal motoneurons during the first few days of adult life in this insect. Motoneurons prevented from undergoing the normal process of developmental degeneration by removal of neural inputs were examined at the physiological and structural levels using several cell imaging techniques. Although these neurons lost their muscle targets and experienced the endocrine cue that normally triggers death, they showed no overt electrophysiological or morphological signs of degeneration. Thus, by appropriate intervention, the MN-12 motoneuron can be spared from developmental neuronal death and remain as a functioning supernumerary element in the mature nervous system. © 1995 John Wiley & Sons, Inc.

The volume of the mushroom bodies of the brains of honey bee queens (Apis mellifera) was estimated using the method of Cavalieri. Tissue sampled was obtained from queens in five different behavioral and reproductive states: 1-day-old virgin queens, 14-day-old virgin queens, 14-day-old instrumentally inseminated queens, 9- to 13-day old naturally mated queens, and 5-month-old naturally mated queens. There were significant volume changes within the mushroom bodies during the first 2 weeks of adult life. The volume occupied by the somata of the intrinsic neuronal population (Kenyon cells) of the mushroom bodies decreased by approximately 30% and the volume of the neuropil of the mushroom bodies increased between 25 and 50%. These volume changes are strikingly similar to those previously reported to occur for worker honey bees switching from hive activities to foraging (Withers, Fahrbach, and Robinson, 1993). However, in this study they were found even in queens that had no flight experience. In addition, queens exhibiting these volume changes were found to have low blood levels of juvenile hormone, while previous studies have shown that foraging worker honey bees have high hormone levels. These results suggest that some aspect of behavioral development common to both the queen and the worker castes is fundamental to protocerebral volume changes early in adulthood in honey bees. If juvenile hormone regulates this process, results from queens suggest that it may play an organizational role.

Half of the neurons in the abdominal nervous system of the moth Manduca sexta die after adult eclosion. Two physiological signals regulate post-eclosion neuronal death in adult moths. The first is endocrine: a decline in blood ecdysteroids is necessary for the death of neurons in the segmental ganglia. The second signal, which is highly specific for a pair of motoneurons found at the posterior midline in each of the three unfused abdominal ganglia, originates in the nervous system. It is transmitted from the fused pterothoracic ganglion to abdominal ganglion A3 via the intersegmental connectives. To characterize the signal of neural origin, we have developed an in vitro bioassay for neuron-killing factors (“neurocidins”). Aqueous extracts of pterothoracic ganglia were prepared and applied to cultured ventral nerve cords. These extracts exhibited concentration-dependent effectiveness in killing motoneurons. The active component of the extract was heat-stable and protease-sensitive. Size fractionation studies suggested that the active component has a molecular mass between 10 and 30 kD. This is the first report of an endogenous neuron-killing protein from an insect nervous system. © 1995 Wiley-Liss, Inc.

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.

Publisher Summary This chapter reviews the history of studies of programmed cell death (PCD) in two key models, the hawkmoth Manduca sexta and the fruit fly Drosophila melanogaster. PCD is a normal component of development and homeostasis in animals, plants, and even some single-celled organisms. While there appears to be multiple forms of PCD, the best characterized are apoptosis and autophagy. In insects, PCD has been observed in diverse tissues and is required for the normal completion of metamorphosis. This highly active field of research is built on a sturdy foundation of decades of studies of hormonally regulated PCD in neuromuscular systems in these two species. Major discoveries based on insect research include identification of the RHG protein apoptosis activators and IAP family proteins as well as the first demonstration of the role of ubiquitination in muscle PCD. Contemporary studies of PCD in neuromuscular systems and dying larval tissues (salivary gland, midgut) have demonstrated the co-occurrence of apopotic and autophagic gene expression in individual cells fated to die. The study of PCD during metamorphosis in insects is a mature field of inquiry that offers numerous opportunities for study of mechanisms related both to insect development and human disease.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissue undergoing programmed cell death (e. g., larval intersegmental muscles inManduca sextamoths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathethic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells commited to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the adbominal gaglia of Manduca sextasuggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated. © 1994 Wiley-Lisx, Inc.

In insects, the ecdysteroids act to transform the CNS from its larval to its adult form. A key gene in this response is the ecdysone receptor (EcR), which has been shown in Drosophila to code for 3 protein isoforms. Two of these isoforms, EcR-A and EcR-B1, are prominently expressed in the CNS and we have used isoform-specific antibodies to examine their fluctuations through postembryonic life. EcR expression at the onset of metamorphosis is extremely diverse but specific patterns of EcR expression correlate with distinct patterns of steroid response. Most larval neurons show high levels of EcR-B1 at the start of meta-morphosis, a time when they lose larval features in response to ecdysteroids. Earlier, during the larval molts, the same cells have no detectable receptors and show no response to circulating ecdysteroids; later, during the pupal-adult transformation, they switch to EcR-A expression and respond by maturing to their adult form. During the latter period, a subset of the larval neurons hyperexpress EcR-A and these cells are fated to die after the emergence of the adult. The stem cells for the imaginal neurons show prominent EcR-B1 expression during the last larval stage correlated with their main proliferative period. Most imaginal neurons, by contrast, express only EcR-A when they sub-sequently initiate maturation at the start of metamorphosis. The imaginal neurons of the mushroom bodies are unusual amongst imaginal neurons in expressing the B1 isoform at the start of metamorphosis but they also show regressive changes at this time as they lose their larval axons. Imaginal neurons of the optic lobe show a delayed expression of EcR-B1 through the period when cell-cell interactions are important for establishing connections within this region of the CNS. Overall, the appearance of the two receptor isoforms in cells correlates with different types of steroid responses: EcR-A predominates when cells are undergoing maturational responses whereas EcR-B1 predominates during proliferative activity or regressive responses. The heterogeneity of EcR expression at the start of metamorphosis presumably reflects the diverse origins and requirements of the neurons that nevertheless are all exposed to a common hormonal signal.

The experience of foraging under natural conditions increases the volume of mushroom body neuropil in worker honey bees. A comparable increase in neuropil volume results from treatment of worker honey bees with pilocarpine, an agonist for muscarinic-type cholinergic receptors. A component of the neuropil growth induced by foraging experience is growth of dendrites in the collar region of the calyces. We show here, via analysis of Golgi-impregnated collar Kenyon cells with wedge arborizations, that significant increases in standard measures of dendritic complexity were also found in worker honey bees treated with pilocarpine. This result suggests that signaling via muscarinic-type receptors promotes the increase in Kenyon cell dendritic complexity associated with foraging. Treatment of worker honey bees with scopolamine, a muscarinic inhibitor, inhibited some aspects of dendritic growth. Spine density on the Kenyon cell dendrites varied with sampling location, with the distal portion of the dendritic field having greater total spine density than either the proximal or medial section. This observation may be functionally significant because of the stratified organization of projections from visual centers to the dendritic arborizations of the collar Kenyon cells. Pilocarpine treatment had no effect on the distribution of spines on dendrites of the collar Kenyon cells.

The extreme diversity of this large group of animals not only poses challenges in that the physiology of most invertebrate species is unknown, but also offers opportunities in the form of narrow and broad comparative studies. Research has focused on insects, in reflection of their economic importance. Most classic and molecular techniques developed for studies of hormone–behavior relationships in vertebrates can be adapted for use in insects, although the small size of many species limits determination of the temporal profile of hormone secretion. The sequencing and annotation of invertebrate genomes has led to the recognition that many invertebrate endocrine signals (such as the insulin-like peptides) and their receptors are homologs of signals and receptors found in vertebrates.

The emergence of the adult Manduca sexta moth is accompained by the death of half of the neurons present in the pupal abdominal nervous system (Truman, 1983). This developmental neuronal death is highly selective, so that the same neurons die at the same time relative to emergence in every moth. In the case of the MN-12 motoneurons, this cell death is regulated both by hemolymph concentrations of a steroid hormone, 20-hydroxyecdysone, and by actions exerted by adjacent ganglia (Truman and Schwartz, 1984; Fahrbach and Truman, 1987). This latter effect, which has been previously described in isolated abdomens and in moths with transected ventral nerve cords, has now been reproduced under controlled culture conditions in which the selectivity and extent of postemergence neuronal death is comparable to that seen in vivo. With respect to the MN-12 neurons found in the most anterior unfused abdominal ganglion, A3, the pterothoracic ganglion appears to be the source of a factor that permits these neurons to die according to their usual developmental schedule. © 1992 John Wiley & Sons, Inc.

Two classes of developmental hormones, the ecdysteroids and the juvenile hormones, regulate metamorphosis in insects. The actions of these hormones on the nervous system during metamorphosis have been extensively described in two insects: a dipteran, the fruit fly Drosophila melanogaster, and a lepidopteran, the tobacco hornworm moth Manduca sexta. Current research on hormone-mediated neurometamorphosis is focused on discovering the molecular mechanisms that account for the cell- and stage-specific details of responses to these hormones. Yet these developmental hormones, and the receptors through which they signal, are also present in adults. In this chapter we provide the necessary background and document the feasibility of developing the honeybee, Apis mellifera, as a new model for the study of hormones, brain, and behavior in adult insects. Such studies have already significantly extended our knowledge of the expression of members of the nuclear receptor protein superfamily in the adult insect brain.

Changes in the structure and function of identified neurons during insect metamorphosis that are linked to the gain or loss of specific elements of behavior are controlled by the steroid hormone, 20-hydroxyecdysone. Three approaches are being used to determine which cells are primary targets for steroid hormones: localized hormone manipulations, direct exposure of identified neurons to ecdysteroids in vitro and steroid hormone autoradiography. Identified neurons display an array of responses to steroids, which may have different thresholds and be subject to other regulatory influences. The current challenge is to link our understanding of the hormonally dependent cellular changes in identified neurons to emerging information about the molecular basis of ecdysteroid action and the regulation of cell fate in insects.

The brains of experienced forager honey bees exhibit predictable changes in structure, including significant growth of the neuropil of the mushroom bodies. In vertebrates, members of the superfamily of nuclear receptors function as key regulators of neuronal structure. The adult insect brain expresses many members of the nuclear receptor superfamily, suggesting that insect neurons are also likely important targets of developmental hormones. The actions of developmental hormones (the ecdysteroids and the juvenile hormones) in insects have been primarily explored in the contexts of metamorphosis and vitellogenesis. The cascade of gene expression activated by 20-hydroxyecdysone and modulated by juvenile hormone is strikingly conserved in these different physiological contexts. We used quantitative RT-PCR to measure, in the mushroom bodies of the adult worker honey bee brain, relative mRNA abundances of key members of the nuclear receptor superfamily (EcR, USP, E75, Ftz-f1, and Hr3) that participate in the metamorphosis/vitellogenesis cascade. We measured responses to endogenous peaks of hormones experienced early in adult life and to exogenous hormones. Our studies demonstrate that a population of adult insect neurons is responsive to endocrine signals through the use of conserved portions of the canonical ecdysteroid transcriptional cascade previously defined for metamorphosis and vitellogenesis.

The ventral nerve cord of holometabolous insects is reorganized during metamorphosis. A prominent feature of this reorganization is the migration of subsets of thoracic and abdominal larval ganglia to form fused compound ganglia. Studies in the hawkmoth Manduca sexta revealed that pulses of the steroid hormone 20-hydroxyecdysone (20E) regulate ganglionic fusion, but little is known about the cellular mechanisms that make migration and fusion possible. To test the hypothesis that modulation of cell adhesion molecules is an essential component of ventral nerve cord reorganization, we used antibodies selective for either the transmembrane isoform of the cell adhesion receptor fasciclin II (TM-MFas II) or the glycosyl phosphatidylinositol-linked isoform (GPI-MFas II) to study cell adhesion during ganglionic migration and fusion. Our observations show that expression of TM-MFas II is regulated temporally and spatially. GPI-MFas II was expressed on the surface of the segmental ganglia and the transverse nerve, but no evidence was obtained for regulation of GPI-MFas II expression during metamorphosis of the ventral nerve cord. Manipulation of 20E titers revealed that TM-MFas II expression on neurons in migrating ganglia is regulated by hormonal events previously shown to choreograph ganglionic migration and fusion. Injections of actinomycin D (an RNA synthesis inhibitor) or cycloheximide (a protein synthesis inhibitor) blocked ganglionic movement and the concomitant increase in TM-MFas II, suggesting that 20E regulates transcription of TM-MFas II. The few neurons that showed TM-MFas II immunoreactivity independent of endocrine milieu were immunoreactive to an antiserum specific for eclosion hormone (EH), a neuropeptide regulator of molting.

Honeybees (Apis mellifera L.) display a pronounced natural aging plasticity. The differences in aging rates between the alternative phenotypes and behavioral classes could reflect differences in protection against damage or in the ability to repair vulnerable tissues. As in other animals, including humans, the gut is continually exposed to environmental insults and harbors a large population of replicating stem cells that maintain the intestinal epithelium. Through studies of the major internal organs using incorporation and immunodetection of the mitotic marker bromo-deoxyuridine, the intestine was determined to be the main site of tissue renewal in adult honeybees. Proliferative activity of the intestinal stem cells was compared among queens, workers, and males of different ages. Simultaneous attempts to assess intestinal cell loss via apoptosis yielded inconclusive results. The relationship between intestinal cell proliferation and worker life-history was evaluated in greater depth by studying diutinus winter workers, reproductive workers, and by decoupling worker behavioral status from chronological age in a single-cohort colony. Intestinal cell proliferation was abundant in all groups and showed an age-related decline in workers, queens, and males. At young ages, workers exhibited relatively more intestinal cell proliferation than did queens and queens more than drones, but the caste and sex differences decreased with age. Cell proliferation did not decrease beyond 6 weeks of age in older queens and in diutinus workers. Ovary activation did not correlate with the amount of intestinal stem cell proliferation in workers, although the queenless hive condition was associated with lower overall counts. In the single-cohort colony, nurse bees exhibited more cell proliferation than foragers, regardless of age. The overall results do not support our hypothesis that longer-lived phenotypes exhibit increased somatic repair in the form of higher replicative activity of intestinal stem cells. Instead, the observed proliferation patterns reflect differential demands for digestive activity in the different groups, which result in different requirements for replacement of lost intestinal cells. The maintenance of proliferative capacity for over 1 year suggests that queen intestinal stem cells have a relatively high replicative potential, but further studies are needed to relate honeybee lifespan differences to cellular aging.

Two-dimensional gel electrophoresis was used to locate potential neuronal death-related proteins in the moth Manduca sexta. Protein patterns of ganglia of pharate adult moths (taken prior to adult ecdysis) compared with protein patterns of one-day-old adults revealed reproducible changes in protein patterns. An acidic protein of approximately 40000 Da was present in all samples from adult moths undergoing neuronal death and essentially absent from pharate adult samples.