Impact of odorants on perception of sweetness by honey bees.

Organic volatiles produced by fruits can result in overestimation of sweetness by humans, but it is unknown if a comparable phenomenon occurs in other species. Honey bees collect nectar of varying sweetness at different flowering plants. Bees discriminate sugar concentration and generally prefer higher concentrations; they encounter floral volatiles as they collect nectar, suggesting that they, like humans, could be susceptible to sweetness enhancement by odorant. In this study, limonene, linalool, geraniol, and 6-methyl-5-hepten-2-ol were tested for their ability to alter behaviors related to perception of sweetness by honey bees. Honey bees were tested in the laboratory using proboscis extension response-based assays and in the field using feeder-based assays. In the laboratory assays, 6-methyl-5-hepten-2-ol and geraniol, but neither linalool nor limonene, significantly increased responses to low concentrations of sucrose compared with no odorant conditions in 15-day and 25-day-old adult worker honey bees, but not in 35-day-old bees. Limonene reduced responding in 15-day-old bees, but not in the older bees. There was no odorant-based difference in performance in field assays comparing geraniol and limonene with a no odorant control. The interaction of the tested plant volatiles with sucrose concentration revealed in laboratory testing is therefore unlikely to be a major determinant of nectar choice by honey bees foraging under natural conditions. Because geraniol is a component of honey bee Nasonov gland pheromone as well as a floral volatile, its impact on responses in the laboratory may reflect conveyance of different information than the other odorants tested.

Comment on "Food wanting is mediated by transient activation of dopaminergic signaling in the honey bee brain".

Huang et al. (1) make an exciting claim about a human-like dopamine-regulated neuromodulatory mechanism underlying food-seeking behavior in honey bees. Their claim is based on experiments designed to measure brain biogenic amine levels and manipulate receptor activity. We have concerns that need to be addressed before broad acceptance of their results and the interpretation provided.

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

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

Synapsin-based approaches to brain plasticity in adult social insects.

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.

Xenobiotic effects on intestinal stem cell proliferation in adult honey bee (Apis mellifera L) workers.

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.

Use of primary cultures of Kenyon cells from bumblebee brains to assess pesticide side effects.

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.

Visual associative learning in restrained honey bees with intact antennae.

A restrained honey bee can be trained to extend its proboscis in response to the pairing of an odor with a sucrose reward, a form of olfactory associative learning referred to as the proboscis extension response (PER). Although the ability of flying honey bees to respond to visual cues is well-established, associative visual learning in restrained honey bees has been challenging to demonstrate. Those few groups that have documented vision-based PER have reported that removing the antennae prior to training is a prerequisite for learning. Here we report, for a simple visual learning task, the first successful performance by restrained honey bees with intact antennae. Honey bee foragers were trained on a differential visual association task by pairing the presentation of a blue light with a sucrose reward and leaving the presentation of a green light unrewarded. A negative correlation was found between age of foragers and their performance in the visual PER task. Using the adaptations to the traditional PER task outlined here, future studies can exploit pharmacological and physiological techniques to explore the neural circuit basis of visual learning in the honey bee.

Transcriptional response to foraging experience in the honey bee mushroom bodies.

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

Insect nuclear receptors.

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.

Rho GTPase activity in the honey bee mushroom bodies is correlated with age and foraging experience.

Foraging experience is correlated with structural plasticity of the mushroom bodies of the honey bee brain. While several neurotransmitter and intracellular signaling pathways have been previously implicated as mediators of these structural changes, none interact directly with the cytoskeleton, the ultimate effector of changes in neuronal morphology. The Rho family of GTPases are small, monomeric G proteins that, when activated, initiate a signaling cascade that reorganizes the neuronal cytoskeleton. In this study, we measured activity of two members of the Rho family of GTPases, Rac and RhoA, in the mushroom bodies of bees with different durations of foraging experience. A transient increase in Rac activity coupled with a transient decrease in RhoA activity was found in honey bees with 4 days foraging experience compared with same-aged new foragers. These observations are in accord with previous reports based on studies of other species of a growth supporting role for Rac and a growth opposing role for RhoA. This is the first report of Rho GTPase activation in the honey bee brain.

Histological estimates of ovariole number in honey bee queens, Apis mellifera, reveal lack of correlation with other queen quality measures.

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.

Muscarinic regulation of Kenyon cell dendritic arborizations in adult worker honey bees.

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.

Body size-related variation in Pigment Dispersing Factor-immunoreactivity in the brain of the bumblebee Bombus terrestris (Hymenoptera, Apidae).

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.

Coordinated responses to developmental hormones in the Kenyon cells of the adult worker honey bee brain (Apis mellifera L.).

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.

Pilocarpine improves recognition of nestmates in young honey bees.

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.

Hormone-dependent expression of fasciclin II during ganglionic migration and fusion in the ventral nerve cord of the moth Manduca sexta.

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.

Nuclear receptors of the honey bee: annotation and expression in the adult brain.

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.

Structure of the mushroom bodies of the insect brain.

The past decade has produced an explosion of new information on the development, neuroanatomy, and possible functions of the mushroom bodies. This review provides a concise, contemporary overview of the structure of the mushroom bodies. Two topics are highlighted: the volume plasticity of mushroom body neuropils evident in the brains of some adult insects and a possible essential role for the gamma lobe in olfactory memory.