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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.

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.

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.

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 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.

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 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.

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.

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.

In the moth Manduca sexta 50% of the abdominal motor neurons and interneurons die during the first days after the adult emerges. Although the dying motor neurons innervate larval muscles that also die at this time, the death of the neurons has been shown to be a direct response to an endocrine signal, the decline in ecdysteroids that occurs at the end of metamorphosis. The response to the withdrawal of the hormone apparently requires new synthesis of RNA and protein, as actinomycin-D and cycloheximide can prevent neuronal death. This selective neuronal death probably results from changes in gene activation. The fixed spatial and temporal pattern of neuronal death, however, suggests that trophic interactions among neurons may also be involved. Current work is focused on the fate of a pair of motor neurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from the thoracic ganglia can prevent the death of these cells at the time when they would normally die in response to removal of ecdysteroids. The factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

You probably know that hard-working dung beetles keep the Serengeti Plain and cow pastures clean, but even most biologists are unaware of the efforts of another diligent group of beetles: the burying beetles of the genus Nicrophorus. Burying beetles play an important role in many ecosystems through their processing of small vertebrate carcasses. The reproductive cycle of such beetles is tied to the discovery of a recently deceased small animal (Scott, 1998; Trumbo, 1997). When beetles discover a carcass, they dig a hole beneath the body and cover it with dirt. They next remove the fur or feathers. The process is complete when the denuded corpse is covered with a layer of anal gland secretions. If more than two beetles find the carcass, males fight against males and females against females until a single pair is in control. Oviposition occurs within a day of the discovery of a carcass. The female lays her eggs in the soil; the larvae hatch about 4 days later and crawl to the carcass, which serves as nest and food source. For the first 2 days on the carcass, both parents feed the larvae regurgitated carrion and defend the carcass against other burying beetles. After this time, the larvae are able to feed themselves directly from the carcass, and the parents’ role is primarily defensive (Fetherston et al., 1990). The bout of parental behavior ends with the departure of the adult beetles from the nest: the female will remain until the larvae leave the carcass in preparation for pupation (6–8 days), while the male typically leaves a little sooner. A set of larvae can be raised successfully by a single parent as well as by a pair, with males able to compensate fully for the disappearance of the female after oviposition (Fetherston et al., 1994). Burying beetles therefore provide an uncommon example of extended, facultative biparental care in insects, of interest to entomologists, ecologists, and evolutionary biologists.

Insects have excellent color vision based on the expression of different opsins in specific sets of photoreceptive cells. Opsins are members of the rhodopsin superfamily of G-protein coupled receptors, and are transmembrane proteins found coupled to light-sensitive chromophores in animal photoreceptors. Diversification of opsins during animal evolution provided the basis for the development of wavelength-specific behavior and color vision, but with the exception of the recently discovered non-visual melanopsins, vertebrate and invertebrate opsins have generally been viewed as representing distinct lineages. We report a novel lineage of insect opsins, designated pteropsins. On the basis of sequence analysis and intron location, pteropsins are more closely related to vertebrate visual opsins than to invertebrate opsins. Of note is that the pteropsins are missing entirely from the genome of drosophilid flies. In situ hybridization studies of the honey bee, Apis mellifera, revealed that pteropsin is expressed in the brain of this species and not in either the simple or compound eyes. It was also possible, on the basis of in situ hybridization studies, to assign different long wavelength opsins to the compound eyes (AmLop1) and ocelli (AmLop2). Insect pteropsin might be orthologous to a ciliary opsin recently described from the annelid Platynereis, and therefore represents the presence of this vertebrate-like light-detecting system in insects.

We explored the neural basis of age- and task-related plasticity in circadian patterns of activity in the honeybee. To identify putative circadian pacemakers in the bee brain, we used antibodies against Drosophila melanogaster and Antheraea pernyi PERIOD and an antiserum to crustacean pigment-dispersing hormone (PDH) known to cross-react with insect pigment-dispersing factors (PDFs). In contrast to previous results from Drosophila, PDH and PER immunoreactivity (-ir) were not colocalized in bee neurons. The most intense PER-ir was cytoplasmic, in two groups of large neurons in the protocerebrum. The number of protocerebral PER-ir neurons and PER-ir intensity within individual cells were highest in brains collected during subjective night and higher in old bees than in young bees. These results are consistent with previous analyses of brain per mRNA in honeybees. Nuclear PER-ir was found throughout the brain, including the optic and antennal lobes. A single group of PDH-ir neurons (approximately 20/optic lobe) was consistently and intensely labeled at the medial margin of the medulla, independent of age or time of day. The processes of these neurons extended to specific neuropils in the protocerebrum and the optic lobes but not to the deutocerebrum. The patterns displayed by PER- and PDH-ir do not completely match any patterns previously described. This suggests that, although clock proteins are conserved across insect groups, there is no universal pattern of coexpression that allows ready identification of pacemaker neurons within the insect brain.

SUMMARY Three experiments were performed to determine why removal of the corpora allata (the glands that produce juvenile hormone) causes honey bees to fail to return to their hive upon initiating flight. In Experiment 1, the naturally occurring flights of allatectomized bees were tracked with radar to determine whether the deficit is physical or cognitive. The results indicated a physical impairment: allatectomized bees had a significantly slower ground speed than sham and untreated bees during orientation flights, but otherwise attributes such as flight range and area were normal. Flight impairment was confirmed in Experiment 2, based on observations of takeoff made in the field at the hive entrance. The allatectomized group had a significantly smaller percentage of flightworthy bees than did the sham and untreated groups. Experiment 3 confirmed the flight impairment in laboratory tests and showed that allatectomy causes a decrease in metabolic rate. Allatectomized bees had significantly lower metabolic rates than untreated and sham bees, while allatectomized bees receiving hormone replacement had intermediate values. These results indicate that allatectomy causes flight impairment, probably partly due to effects on metabolic rate. They also suggest that juvenile hormone plays an additional, previously unknown, role in coordinating the physiological underpinning of division of labor in honey bee colonies.

The extremely large number of insects and members of allied groups alive today suggests that molting--shedding of an old cuticle--may be one of the most commonly performed behaviors on our planet. Removal of an old cuticle in insects is associated with stereotyped, species-specific patterns of behavior referred to as ecdysis. It has been recognized for decades that the initiation of ecdysis is under hormonal control, but until recently many of the key peptides that regulate ecdysis were unknown. The report in 1996 of a new ecdysis-triggering hormone (ETH) sparked an era of significant advances in our understanding of the regulation of molting. This article summarizes the current model of peptide regulation of ecdysis, a model that is based on a positive feedback loop between ETH and a brain peptide, eclosion hormone. Then the relationship of these regulatory peptides to the neural circuitry that is the ultimate driver of the behavior are described. Because insects can undergo both status quo (larval-larval) and metamorphic (larval-pupal and pupal-adult) molts, differences in ecdysis behavior at different life stages are described and potential sources of these differences are identified. Most of the work described is based on studies of ecdysis in the hawkmoth, Manduca sexta, but results from studies of ecdysis in the fruit fly Drosophila melanogaster are also discussed.

Publisher Summary This chapter describes the proleg withdrawal reflex, the metamorphosis of leg motor neurons, gin trap reflex, and the initiation of wandering—all provide clear demonstrations of hormonal regulation of behavioral plasticity in insects. Substantial progress has been made because several different laboratories have concentrated their efforts on a single insect, the tobacco hornworm or Manduca sexta. Study of the neural bases of behavior in this insect has been facilitated by exploitation of identified cells in both neuroanatomical and electrophysiological studies. Instead of efforts focused on the identification of hormone target cells, the widespread distribution of the EcR gene product in the nervous system and associated structures has resulted in an emphasis on characterization of functional sites of hormone action within defined neural circuits. The field also retains a strong emphasis on behavioral assays.

The causes of the current global decline in honey bee health are unknown. One major group of hypotheses invokes the pesticides and other xenobiotics to which this important pollinator species is often exposed. Most studies have focused on mortality or behavioral deficiencies in exposed honey bees while neglecting other biological functions and target organs. The midgut epithelium of honey bees presents an important interface between the insect and its environment. It is maintained by proliferation of intestinal stem cells throughout the adult life of honey bees. We used caged honey bees to test multiple xenobiotics for effects on the replicative activity of the intestinal stem cells under laboratory conditions. Most of the tested compounds did not alter the replicative activity of intestinal stem cells. However, colchicine, methoxyfenozide, tetracycline, and a combination of coumaphos and tau-fluvalinate significantly affected proliferation rate. All substances except methoxyfenozide decreased proliferation rate. Thus, the results indicate that some xenobiotics frequently used in apiculture and known to accumulate in honey bee hives may have hitherto unknown physiological effects. The nutritional status and the susceptibility to pathogens of honey bees could be compromised by the impacts of xenobiotics on the maintenance of the midgut epithelium. This study contributes to a growing body of evidence that more comprehensive testing of xenobiotics may be required before novel or existing compounds can be considered safe for honey bees and other non-target species.

Juvenile hormone regulates metamorphosis in insects, and its effects on the nervous system during the larval-pupal transition have been studied primarily in the hawk moth, Manduca sexta. The effects of juvenile hormone on the nervous system of adult insects have been little studied. Elucidating the role of juvenile hormone during behavioral development in adult honey bees provides an opportunity to study hormone regulation of nervous system structure and function in an insect with a rich behavioral repertoire and social life. A worker honey bee typically lives 30-60 days. During this time, she performs a sequence of different tasks that sustain the colony. A striking behavioral transition typically occurs at about 3 weeks of age. At this time, worker bees stop performing within-hive tasks such as rearing brood and building comb and begin to forage outside the hive. This behavioral development is accompanied by a marked increase in the production of juvenile hormone. The mushroom bodies of the protocerebrum, the region of the insect brain most often associated with learning and memory, also undergo an internal reorganization during behavioral development. High titers of juvenile hormone and an increased volume of neuropil associated with the mushroom bodies are characteristic of the forager. Importantly, the time of the behavioral transition to foraging is not fixed. Individual bees can respond to changing colony or environmental conditions by accelerating or delaying the switch from within-hive tasks to foraging. For example, in the absence of older workers, some bees will undergo precocious development and may forage as early as 4 days of age. These workers also experience a precocious rise in juvenile hormone and an earlier reorganization of the mushroom bodies. Our current studies investigate the roles played by juvenile hormone and experience in shaping the mushroom bodies of the adult honey bee, and the relationship of these changes to the bee's ability to forage successfully. It is proposed that juvenile hormone may mediate neural plasticity in the brains of adult honey bees to support the demanding cognitive task of foraging.

The mushroom bodies in the protocerebrum are believed to be the structures of the insect brain most closely associated with higher-order sensory integration and learning. Drosophila melanogaster mutants with olfactory learning deficits have anatomically abnormal mushroom bodies or altered patterns of gene expression in mushroom body neurons. In addition, anatomical reorganization of the mushroom bodies occurs in adult flies, and possibly in adult honeybees; disturbance of electrical activity in this region disrupts memory formation in honeybees. Little is known, however, about the relationship of naturally occurring anatomical changes in the mushroom bodies to naturally occurring behavioural plasticity. We now report that age-based division of labour in adult worker honeybees (Apis mellifera) is associated with substantial changes in certain brain regions, notably the mushroom bodies. Moreover, these striking changes in brain structure are dependent, not on the age of the bee, but on its foraging experience, thus demonstrating a robust anatomical plasticity associated with complex behaviour in an adult insect.

The emergence of the adult Manduca sexta moth is followed by the loss of almost half of this insect's abdominal motoneurons and interneurons (Truman, 1983). This programmed cell death completes the transformation of the nervous system of the caterpillar into that of the moth. The death of these neurons has been previously shown to be a response to an endocrine signal: the decline in ecdysteroids that occurs at the end of metamorphosis (Truman and Schwartz, 1984). Our current research is focussed on the regulation of the fate of a pair of identified motoneurons, the MN-12 cells, in the third abdominal ganglion. Isolation of this ganglion from anterior parts of the nervous system can prevent the death of these cells at the time when they would normally die in response to the decline in ecdysteroids. Transection of the ventral nerve cord at various levels revealed that the source of this regulatory "death signal" is the fused pterothoracic ganglion and that it is transmitted via the interganglionic connectives. We hypothesize that the factors mediating this effect may act in concert with the ecdysteroid decline to specify the exact time of death for individual neurons.

The steroid hormone 20-hydroxyecdysone regulates many aspects of nervous system development in the moth Manduca sexta, including stage-specific neuronal morphology and stage-specific neuronal death. We have used steroid hormone autoradiography to study the distribution of cells that concentrate ecdysteroids in the ventral nervous system of this insect. The ligand was [3H]-ponasterone A, a bioactive phytoecdysone. Tissue was examined from three stages of development: the end of larval life (first day of wandering), the end of metamorphosis (pharate adult), and 4-day-old adults. In the abdominal ganglia of wandering larvae and pharate adults, a subset of neurons including both motoneurons and interneurons exhibited a nuclear concentration of radiolabeled hormone. The pattern of binding was reproducible but stage-specific, with a greater proportion of neurons showing binding in the larvae than in pharate adults. No labeled neurons were found in abdominal ganglia from mature (4-day-old) adults. In the case of the pharate adult ganglia, the ecdysteroid receptor content of specific, identified motoneurons was determined. These results are discussed in light of the responses of these neurons to physiological changes in levels of circulating ecdysteroids.