Honeybee Brain Research Articles

MALDI-imaging analyses of honeybee brains exposed to a neonicotinoid insecticide.

Toxicological studies evaluating the possible harmful effects of pesticides on bees are important and allow the emergence of protection and pollinator conservation strategies. This study aimed to evaluate the effects of exposure to a sublethal concentration of imidacloprid (LC50/100 : 0.014651 ng imidacloprid µL-1 diet) on the distribution of certain proteins identified in the brain of Apis mellifera worker bees using a MALDI-imaging approach. This technique enables proteomic analysis of tissues in situ by monitoring the spatiotemporal dynamics of the biochemical processes occurring at a specific time in specific brain neuropils. For this purpose, foraging bees were exposed to an 8-day diet containing a sublethal concentration of imidacloprid corresponding to the LC50/100 . Bees were collected on day 8 of exposure, and their brains analyzed using protein density maps. The results showed that exposure to imidacloprid led to a series of biochemical changes, including alterations in synapse regulation, apoptosis regulation and oxidative stress, which may adversely impair the physiology of these colony bees. Worker bee contact with even tiny amounts of imidacloprid had potent effects leading to the overexpression of a series of proteins related to important cellular processes that were possibly damaged by the insecticide. © 2018 Society of Chemical Industry.

Comparison of RNAi knockdown effect of tyramine receptor 1 induced by dsRNA and siRNA in brains of the honey bee, Apis mellifera

RNA interference (RNAi) is a powerful tool for artificially manipulating gene expression in diverse organisms. In the honey bee, Apis mellifera, both long double stranded RNA (dsRNA) and small interference RNA (siRNA) have been successfully used to reduce targeted gene expression and induce specific phenotypes. However, whether dsRNA and siRNA have different effects and efficiencies in gene silencing has never been investigated in honey bees. Thus, we tested the effect of dsRNA and siRNA on the tyramine receptor 1 (tyr1), which encodes a receptor of neurotransmitter tyramine, in honey bee brains at mRNA and protein levels over time. We found that both dsRNA and siRNA achieved successful gene knockdown. The siRNA mixes affected tyr1 gene expression faster than dsRNA, and the duration of the knockdown between dsRNA and siRNA varied. We also found that the turnover rate of TYR1 protein was relatively fast, which is consistent with its role as a neurotransmitter receptor. Our study reveals the different efficiencies of dsRNA and siRNA in honey bee brains. We show that consideration of the gene regions targeted by RNAi, prior screening for RNAi molecules and combing siRNAs are important strategies to enhance RNAi efficiency.

Mass Spectrometric Quantification of Arousal Associated Neurochemical Changes in Single Honey Bee Brains and Brain Regions.

Honey bee foragers show a strong diurnal rhythm of foraging activity, and such behavioral changes are likely under the control of specific neuromodulators. To identify and quantify neuromodulators involved in regulating rest and arousal in honey bees, we established a mass spectrometric method for quantifying 14 different neurochemicals and precursor molecules. We measured forager type and brain region specific differences in amine levels from individual honey bee brains and brain regions. The observed differences in amine levels between resting and aroused foragers resemble findings in other species indicating a conserved molecular mechanism by glutamate and GABA in regulating arousal. Subesophageal ganglion specific changes in the histaminergic system and global increases in aspartate during arousal suggest a possible role of histamine and aspartate in feeding and arousal, respectively. More aminergic systems were significantly affected due to arousal in nectar foragers than in pollen foragers, implying that forager phenotypes differ not only in their food preference but also in their neuromodulatory signaling systems (brain states). Finally, we found that neurotransmitter precursors were better at distinguishing brain states in the central brain, while their end products correlated with arousal associated changes in sensory regions like the optic and antennal lobes.

Transcriptome Analysis of Newly Emerged Honeybees Exposure to Sublethal Carbendazim During Larval Stage.

There are increasing concerns regarding the impact of agrochemical pesticides on non-target organisms. Pesticides could cause honeybee abnormal development in response to neurotoxins such as neonicotinoid. However, knowledge of carbendazim, a widespread fungicide in beekeeping practice, influencing on honeybee (Apis mellifera L.) brain development is lacking. Large-scale transcriptome approaches were applied to determine the changes in global gene expression in the brains of newly emerged honeybees after carbendazim exposure during the larval stage. To further understand the effects of carbendazim on the brain development of honeybees, the functions of differentially expressed genes were compared between the treatment and control groups. We found that neuroregulatory genes were down-regulated after carbendazim exposure, which suggest the neurotoxic effects of this fungicide on honeybee nervous system. Carbendazim exposure also altered the expression of genes implicated in metabolism, transport, sensor, and hormone. Notably, larvae in the carbendazim-treated group observed longer time to shift into the dormant pupal state than the control group. Moreover, a low juvenile hormone and high ecdysone titers were found in the treatment group compared to control group. The data is the first report of neurotoxic effects on honeybee caused by carbendazim, and the sublethal carbendazim may disturb honeybee development and is a potential chemical threating the honeybee colonies.

Transcriptional and physiological effects of the pyrethroid deltamethrin and the organophosphate dimethoate in the brain of honey bees (Apis mellifera)

The pyrethroid deltamethrin and the organophosphate insecticide dimethoate are widely used in agriculture and in urban areas. Both plant protection products (PPPs) unintendedly result in adverse effects in pollinators. Currently, the sublethal effects of both compounds are poorly known, particularly on the molecular and biochemical level. Here we analysed effects of deltamethrin and dimethoate at environmental and sublethal concentrations in honey bee workers by focusing on transcriptional changes of target genes in the brain. In addition, expression of vitellogenin protein and activity of acetylcholinesterase were assessed upon dimethoate exposure to assess physiological effects. Deltamethrin resulted in induction of the cyp9q2 transcript at 0.53 ng/bee, while dimethoate led to induction of vitellogenin on the mRNA and protein level at 2 ng/bee. Transcripts of additional cytochrome P450-dependent monooxygenases (cyps) and genes related to immune system regulation were not differentially expressed upon PPP exposure. Dimethoate but not deltamethrin led to a strong and concentration-related inhibition of the acetylcholinesterase at 2 and 20 ng/bee. Our data demonstrate that deltamethrin and dimethoate exhibit transcriptional effects at environmental concentrations in the brain of honey bees. Dimethoate also strongly affected physiological traits, which may translate to adverse effects in forager bees.

Kenyon Cell Subtypes/Populations in the Honeybee Mushroom Bodies: Possible Function Based on Their Gene Expression Profiles, Differentiation, Possible Evolution, and Application of Genome Editing.

Mushroom bodies (MBs), a higher-order center in the honeybee brain, comprise some subtypes/populations of interneurons termed as Kenyon cells (KCs), which are distinguished by their cell body size and location in the MBs, as well as their gene expression profiles. Although the role of MBs in learning ability has been studied extensively in the honeybee, the roles of each KC subtype and their evolution in hymenopteran insects remain mostly unknown. This mini-review describes recent progress in the analysis of gene/protein expression profiles and possible functions of KC subtypes/populations in the honeybee. Especially, the discovery of novel KC subtypes/populations, the “middle-type KCs” and “KC population expressing FoxP,” necessitated a redefinition of the KC subtype/population. Analysis of the effects of inhibiting gene function in a KC subtype-preferential manner revealed the function of the gene product as well as of the KC subtype where it is expressed. Genes expressed in a KC subtype/population-preferential manner can be used to trace the differentiation of KC subtypes during the honeybee ontogeny and the possible evolution of KC subtypes in hymenopteran insects. Current findings suggest that the three KC subtypes are unique characteristics to the aculeate hymenopteran insects. Finally, prospects regarding future application of genome editing for the study of KC subtype functions in the honeybee are described. Genes expressed in a KC subtype-preferential manner can be good candidate target genes for genome editing, because they are likely related to highly advanced brain functions and some of them are dispensable for normal development and sexual maturation in honeybees.

A Segmentation Scheme for Complex Neuronal Arbors and Application to Vibration Sensitive Neurons in the Honeybee Brain.

The morphology of a neuron is strongly related to its physiological properties, application of logical product and thus to information processing functions. Optical microscope images are widely used for extracting the structure of neurons. Although several approaches have been proposed to trace and extract complex neuronal structures from microscopy images, available methods remain prone to errors. In this study, we present a practical scheme for processing confocal microscope images and reconstructing neuronal structures. We evaluated this scheme using image data samples and associated “gold standard” reconstructions from the BigNeuron Project. In these samples, dendritic arbors belonging to multiple projection branches of the same neuron overlapped in space, making it difficult to automatically and accurately trace their structural connectivity. Our proposed scheme, which combines several software tools for image masking and filtering with an existing tool for dendritic segmentation and tracing, outperformed state-of-the-art automatic methods in reconstructing such neuron structures. For evaluating our scheme, we applied it to a honeybee local interneuron, DL-Int-1, which has complex arbors and is considered to be a critical neuron for encoding the distance information indicated in the waggle dance of the honeybee.

Abstract concept learning in a simple neural network inspired by the insect brain.

The capacity to learn abstract concepts such as ‘sameness’ and ‘difference’ is considered a higher-order cognitive function, typically thought to be dependent on top-down neocortical processing. It is therefore surprising that honey bees apparantly have this capacity. Here we report a model of the structures of the honey bee brain that can learn sameness and difference, as well as a range of complex and simple associative learning tasks. Our model is constrained by the known connections and properties of the mushroom body, including the protocerebral tract, and provides a good fit to the learning rates and performances of real bees in all tasks, including learning sameness and difference. The model proposes a novel mechanism for learning the abstract concepts of ‘sameness’ and ‘difference’ that is compatible with the insect brain, and is not dependent on top-down or executive control processing.

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee.

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom body (MB) is a higher brain center proposed to be the neural substrate of complex honeybee behaviors. Although previous studies identified genes and proteins that are differentially expressed in the MBs and other brain regions, the activities of the proteins in each region are not yet fully understood. To reveal the functions of these proteins in the brain, pharmacologic analysis is a feasible approach, but it is first necessary to confirm that pharmacologic manipulations indeed alter the protein activity in these brain regions. We previously identified a higher expression of genes encoding phospholipase C (PLC) in the MBs than in other brain regions, and pharmacologically assessed the involvement of PLC in honeybee behavior. In that study, we biochemically tested two pharmacologic agents and confirmed that they decreased PLC activity in the MBs and other brain regions. Here, we present a detailed description of how to detect PLC activity in honeybee brain homogenate. In this assay system, homogenates derived from different brain regions are reacted with a synthetic fluorogenic substrate, and fluorescence resulting from PLC activity is quantified and compared between brain regions. We also describe our evaluation of the inhibitory effects of certain drugs on PLC activity using the same system. Although this system is likely affected by other endogenous fluorescence compounds and/or the absorbance of the assay components and tissues, the measurement of PLC activity using this system is safer and easier than that using the traditional assay, which requires radiolabeled substrates. The simple procedure and manipulations allow us to examine PLC activity in the brains and other tissues of honeybees involved in different social tasks.

Neuronal Response Latencies Encode First Odor Identity Information across Subjects.

Odorants are coded in the primary olfactory processing centers by spatially and temporally distributed patterns of glomerular activity. Whereas the spatial distribution of odorant-induced responses is known to be conserved across individuals, the universality of its temporal structure is still debated. Via fast two-photon calcium imaging, we analyzed the early phase of neuronal responses in the form of the activity onset latencies in the antennal lobe projection neurons of honeybee foragers. We show that each odorant evokes a stimulus-specific response latency pattern across the glomerular coding space. Moreover, we investigate these early response features for the first time across animals, revealing that the order of glomerular firing onsets is conserved across individuals and allows them to reliably predict odorant identity, but not concentration. These results suggest that the neuronal response latencies provide the first available code for fast odor identification.SIGNIFICANCE STATEMENT Here, we studied early temporal coding in the primary olfactory processing centers of the honeybee brain by fast imaging of glomerular responses to different odorants across glomeruli and across individuals. Regarding the elusive role of rapid response dynamics in olfactory coding, we were able to clarify the following aspects: (1) the rank of glomerular activation is conserved across individuals, (2) its stimulus prediction accuracy is equal to that of the response amplitude code, and (3) it contains complementary information. Our findings suggest a substantial role of response latencies in odor identification, anticipating the static response amplitude code.

MKast is dispensable for normal development and sexual maturation of the male European honeybee

The European honeybee (Apis mellifera L.) exhibits various social behaviors. The molecular and neural mechanisms underlying these behaviors have long been explored, but causal relations between genes or neurons and behaviors remain to be elucidated because effective gene manipulation methods in the honeybee have not been available until recently. We recently established a basic technology to produce mutant honeybee drones using CRISPR/Cas9. Here we produced mutant drones using CRISPR/Cas9 targeting mKast, which is preferentially expressed in a certain subtype of class I Kenyon cells that comprise the mushroom bodies in the honeybee brain. By immunoblot analysis, we showed that mKast protein expression was completely lost in the mutant drone heads. In addition, during the production process of homozygous mutant workers, we demonstrated that heterozygous mutant workers could be produced by artificial insemination of wild-type queens with the sperm of mutant drones, indicating that mKast mutant drones were sexually mature. These results demonstrate that mKast is dispensable for normal development and sexual maturation in drone honeybees, and allow us to proceed with the production of homozygous mutant workers for the analysis of a particular gene by gene knockout in the future.

Construction of standard brain map of honeybee brain region related to vibration stimulus processing

Construction of standard brain map of honeybee brain region related to vibration stimulus processing

Neural Organization of A3 Mushroom Body Extrinsic Neurons in the Honeybee Brain.

In the insect brain, the mushroom body is a higher order brain area that is key to memory formation and sensory processing. Mushroom body (MB) extrinsic neurons leaving the output region of the MB, the lobes and the peduncle, are thought to be especially important in these processes. In the honeybee brain, a distinct class of MB extrinsic neurons, A3 neurons, are implicated in playing a role in learning. Their MB arborisations are either restricted to the lobes and the peduncle, here called A3 lobe connecting neurons, or they provide feedback information from the lobes to the input region of the MB, the calyces, here called A3 feedback neurons. In this study, we analyzed the morphology of individual A3 lobe connecting and feedback neurons using confocal imaging. A3 feedback neurons were previously assumed to innervate each lip compartment homogenously. We demonstrate here that A3 feedback neurons do not innervate whole subcompartments, but rather innervate zones of varying sizes in the MB lip, collar, and basal ring. We describe for the first time the anatomical details of A3 lobe connecting neurons and show that their connection pattern in the lobes resemble those of A3 feedback cells. Previous studies showed that A3 feedback neurons mostly connect zones of the vertical lobe that receive input from Kenyon cells of distinct calycal subcompartments with the corresponding subcompartments of the calyces. We can show that this also applies to the neck of the peduncle and the medial lobe, where both types of A3 neurons arborize only in corresponding zones in the calycal subcompartments. Some A3 lobe connecting neurons however connect multiple vertical lobe areas. Contrarily, in the medial lobe, the A3 neurons only innervate one division. We found evidence for both input and output areas in the vertical lobe. Thus, A3 neurons are more diverse than previously thought. The understanding of their detailed anatomy might enable us to derive circuit models for learning and memory and test physiological data.

Metabolic enzymes in glial cells of the honeybee brain and their associations with aging, starvation and food response.

The honey bee has been extensively studied as a model for neuronal circuit and memory function and more recently has emerged as an unconventional model in biogerontology. Yet, the detailed knowledge of neuronal processing in the honey bee brain contrasts with the very sparse information available on glial cells. In other systems glial cells are involved in nutritional homeostasis, detoxification, and aging. These glial functions have been linked to metabolic enzymes, such as glutamine synthetase and glycogen phosphorylase. As a step in identifying functional roles and potential differences among honey bee glial types, we examined the spatial distribution of these enzymes and asked if enzyme abundance is associated with aging and other processes essential for survival. Using immunohistochemistry and confocal laser microscopy we demonstrate that glutamine synthetase and glycogen phosphorylase are abundant in glia but appear to co-localize with different glial sub-types. The overall spatial distribution of both enzymes was not homogenous and differed markedly between different neuropiles and also within each neuropil. Using semi-quantitative Western blotting we found that rapid aging, typically observed in shortest-lived worker bees (foragers), was associated with declining enzyme levels. Further, we found enzyme abundance changes after severe starvation stress, and that glutamine synthetase is associated with food response. Together, our data indicate that aging and nutritional physiology in bees are linked to glial specific metabolic enzymes. Enzyme specific localization patterns suggest a functional differentiation among identified glial types.

MALDI Imaging Analysis of Neuropeptides in Africanized Honeybee ( Apis mellifera) Brain: Effect of Aggressiveness.

Aggressiveness in honeybees seems to be regulated by multiple genes, under the influence of different factors, such as polyethism of workers, environmental factors, and response to alarm pheromones, creating a series of behavioral responses. It is suspected that neuropeptides seem to be involved with the regulation of the aggressive behavior. The role of allatostatin and tachykinin-related neuropeptides in honeybee brain during the aggressive behavior is unknown, and thus worker honeybees were stimulated to attack and to sting leather targets hung in front of the colonies. The aggressive individuals were collected and immediately frozen in liquid nitrogen; the heads were removed and sliced at sagittal plan. The brain slices were submitted to MALDI spectral imaging analysis, and the results of the present study reported the processing of the precursors proteins into mature forms of the neuropeptides AmAST A (59-76) (AYTYVSEYKRLPVYNFGL-NH2), AmAST A (69-76) (LPVYNFGL-NH2), AmTRP (88-96) (APMGFQGMR-NH2), and AmTRP (254-262) (ARMGFHGMR-NH2), which apparently acted in different neuropils of the honeybee brain during the aggressive behavior, possibly taking part in the neuromodulation of different aspects of this complex behavior. These results were biologically validated by performing aggressiveness-related behavioral assays using young honeybee workers that received 1 ng of AmAST A (69-76) or AmTRP (88-96) via hemocele. The young workers that were not expected to be aggressive individuals presented a complete series of aggressive behaviors in the presence of the neuropeptides, corroborating the hypothesis that correlates the presence of mature AmASTs A and AmTRPs in the honeybee brain with the aggressiveness of this insect.

Defense against territorial intrusion is associated with DNA methylation changes in the honey bee brain

BackgroundAggression is influenced by individual variation in temperament as well as behavioral plasticity in response to adversity. DNA methylation is stably maintained over time, but also reversible in response to specific environmental conditions, and may thus be a neuromolecular regulator of both of these processes. A previous study reported DNA methylation differences between aggressive Africanized and gentle European honey bees. We investigated whether threat-induced aggression altered DNA methylation profiles in the honey bee brain in response to a behavioral stimulus (aggression-provoking intruder bee or inert control). We sampled five minutes and two hours after stimulus exposure to examine the effect of time on epigenetic profiles of aggression.ResultsThere were DNA methylation differences between aggressive and control bees for individual cytosine-guanine dinucleotides (CpGs) across the genome. Eighteen individual CpG sites showed significant difference between aggressive and control bees 120 min post stimulus. For clusters of CpGs, we report four genomic regions differentially methylated between aggressive and control bees at the 5-min time point, and 50 regions differentially methylated at the120-minute time point following intruder exposure. Differential methylation occurred at genes involved in neural plasticity, chromatin remodeling and hormone signaling. Additionally, there was a significant overlap of differential methylation with previously published epigenetic differences that distinguish aggressive Africanized and gentle European honey bees, suggesting an evolutionarily conserved use of brain DNA methylation in the regulation of aggression. Lastly, we identified individually statistically suggestive CpGs that as a group were significantly associated with differentially expressed genes underlying aggressive behavior and also co-localize with binding sites of transcription factors involved in neuroplasticity or neurodevelopment.ConclusionsThere were DNA methylation differences in the brain associated with response to an intruder. These differences increased in number a few hours after the initial exposure and overlap with previously reported aggression-associated genes and neurobiologically relevant transcription factor binding sites. Many DNA methylation differences that occurred in association with the expression of aggression in real time also exist between Africanized bees and European bees, suggesting an evolutionarily conserved role for epigenetic regulation in aggressive behavior.

Effects of the novel pesticide flupyradifurone (Sivanto) on honeybee taste and cognition

Due to intensive agriculture honeybees are threatened by various pesticides. The use of one group of them, the neonicotinoids, was recently restricted by the European Union. These chemicals bind to the nicotinic acetylcholine receptor (nAchR) in the honeybee brain. Recently, Bayer AG released a new pesticide by the name of “Sivanto” against sucking insects. It is assumed to be harmless for honeybees, although its active ingredient, flupyradifurone, binds nAchR similar to the neonicotinoids. We investigated if this pesticide affects the taste for sugar and cognitive performance in honeybee foragers. These bees are directly exposed to the pesticide while foraging for pollen or nectar. Our results demonstrate that flupyradifurone can reduce taste and appetitive learning performance in honeybees foraging for pollen and nectar, although only the highest concentration had significant effects. Most likely, honeybee foragers will not be exposed to these high concentrations. Therefore, the appropriate use of this pesticide is considered safe for honeybees, at least with respect to the behaviors studied here.

Proteomics Reveals the Molecular Underpinnings of Stronger Learning and Memory in Eastern Compared to Western Bees

The eastern (Apis cerana cerana, Acc) and western (Apis mellifera ligustica, Aml) honeybee are two major honeybee species. Surprisingly, little is known about the fundamental molecular neurobiology of brain suborgans of Acc and Aml. We characterized and compared the proteomes of mushroom bodies (MBs), antennal lobes (ALs) and optical lobes (OLs) in the brain of both species, and biologically validated the functions related to learning and memory. Acc and Aml have evolved similar proteome signatures in MBs and OLs to drive the domain-specific neural activities. In MBs of both species, commonly enriched and enhanced functional groups related to protein metabolism and Ca2+ transport relative to ALs and OLs, suggests that proteins and Ca2+ are vital for consolidating learning and memory via modulation of synaptic structure and signal transduction. Furthermore, in OLs of both species, the mainly enriched ribonucleoside metabolism suggests its vital role as second messenger in promoting phototransduction. Notably, in ALs of both species, distinct proteome settings have shaped to prime olfactory learning and memory. In ALs of Acc, this is supported by the enriched cytoskeleton organization to sustain olfactory signaling through modulation of plasticity in glomeruli and intracellular transport. In ALs of Aml, however, the enriched functional groups implicated in hydrogen ion transport are indicative of their importance in supporting olfactory processes by regulation of synaptic transmission. The biological confirmation of enhanced activities of protein metabolism and signal transduction in ALs and MBs of Acc relative to in Aml demonstrates that a stronger sense of olfactory learning and memory has evolved in Acc. The reported first in-depth proteome data of honeybee brain suborgans provide a novel insight into the molecular basis of neurobiology, and is potentially useful for further neurological studies in honeybees and other insects.

Differential Processing by Two Olfactory Subsystems in the Honeybee Brain

Among insects, Hymenoptera present a striking olfactory system with a clear neural dichotomy from the periphery to higher order centers, based on two main tracts of second-order (projection) neurons: the medial and lateral antennal lobe tracts (m-ALT and l-ALT). Despite substantial work on this dual pathway, its exact function is yet unclear. Here, we ask how attributes of odor quality and odor quantity are represented in the projection neurons (PNs) of the two pathways. Using in vivo calcium imaging, we compared the responses of m-ALT and l-ALT PNs of the honey bee Apis mellifera to a panel of 16 aliphatic odorants, and to three chosen odorants at eight concentrations. The results show that each pathway conveys differential information about odorants’ chemical features or concentration to higher order centers. While the l-ALT primarily conveys information about odorants’ chain length, the m-ALT informs about odorants’ functional group. Furthermore, each tract can only predict chemical distances or bees’ behavioral responses for odorants that differ according to its main feature, chain length or functional group. Generally l-ALT neurons displayed more graded dose–response relationships than m-ALT neurons, with a correspondingly smoother progression of inter-odor distances with increasing concentration. Comparison of these results with previous data recorded at AL input reveals differential processing by local networks within the two pathways. These results support the existence of parallel processing of odorant features in the insect brain.

Cooperative defence operates by social modulation of biogenic amine levels in the honey bee brain.

The defence of a society often requires that some specialized members coordinate to repel a threat at personal risk. This is especially true for honey bee guards, which defend the hive and may sacrifice their lives upon stinging. Central to this cooperative defensive response is the sting alarm pheromone, which has isoamyl acetate (IAA) as its main component. Although this defensive behaviour has been well described, the neural mechanisms triggered by IAA to coordinate stinging have long remained unknown. Here we show that IAA upregulates brain levels of serotonin and dopamine, thereby increasing the likelihood of an individual bee to attack and sting. Pharmacological enhancement of the levels of both amines induces higher defensive responsiveness, while decreasing them via antagonists decreases stinging. Our results thus uncover the neural mechanism by which an alarm pheromone recruits individuals to attack and repel a threat, and suggest that the alarm pheromone of honey bees acts on their response threshold rather than as a direct trigger.