Chemical detection triggers honey bee defense against a destructive parasitic threat.

Invasive species events related to globalization are increasing, resulting in parasitic outbreaks. Understanding of host defense mechanisms is needed to predict and mitigate against the consequences of parasite invasion. Using the honey bee Apis mellifera and the mite Varroa destructor, as a host-parasite model, we provide a comprehensive study of a mechanism of parasite detection that triggers a behavioral defense associated with social immunity. Six Varroa-parasitization-specific (VPS) compounds are identified that (1) trigger Varroa-sensitive hygiene (VSH, bees' key defense against Varroa sp.), (2) enable the selective recognition of a parasitized brood and (3) induce responses that mimic intrinsic VSH activity in bee colonies. We also show that individuals engaged in VSH exhibit a unique ability to discriminate VPS compounds from healthy brood signals. These findings enhance our understanding of a critical mechanism of host defense against parasites, and have the potential to apply the integration of pest management in the beekeeping sector.

Changes in responsiveness to allatostatin treatment accompany shifts in stress reactivity in young worker honey bees.

Exposing honey bees to isopentylacetate (IPA) can cause stress-related changes in learning performance. In bees of foraging age, IPA's effects on learning are mimicked by C-type allatostatins (AstCC, AstCCC) injected into the brain. Here we ask whether allatostatins induce a similar response in young (6-day-old) bees and if so, whether their effects on learning performance are modulated by queen mandibular pheromone (QMP). We found that young bees exposed to IPA responded less to the conditioned stimulus during training than controls (Type 1-like stress response). AstCC treatment induced a similar response, but only in bees maintained without QMP. Bees exposed to QMP responded to AstCC with increased odour responsiveness and odour generalisation in the 1-h memory test (Type 2-like response). Type 2-like responses could be induced also by the A-type allatostatin, AstA. However, in bees exposed to QMP, AstA-induced odour generalisation was absent. Effects of AstCCC treatment in young bees were weak, indicating that responsiveness to this peptide changes with age. Our findings are consistent with the hypothesis that honey bee allatostatins play a role in stress reactivity, but suggest in addition that allatostatin signalling is age dependent and susceptible to modulation by pheromone released by the queen bee.

Viral infections alter antennal epithelium ultrastructure in honey bees.

Varroa destructor and its associated viruses, in particular deformed wing virus (DWV), have been identified as probable causes of honey bee (Apis mellif era L.) colony losses. Evidence suggests that elevated DWV titres in bees could compromise sensory and communication abilities resulting in negative consequences for hygienic behaviour. As antennae play a central role in this behaviour, we compared antennal ultrastructure in DWV-symptomatic and asymptomatic bees. The results show that virus capsids accumulate in the basal regions of the antennal epithelium, close to the haemolymph. No virus particles were detected at the level of sensory sensilla, such as pore plates, nor within the sensory cell dendrites associated with these sensilla. However, membranous structures appeared to be more prevalent in supporting cells surrounding the dendrites of DWV-symptomatic bees. Para-crystalline arrays containing large numbers of virus particles were detected in the antennae of DWV-symptomatic bees but not in asymptomatic bees.

On the front line: quantitative virus dynamics in honeybee (Apis mellifera L.) colonies along a new expansion front of the parasite Varroa destructor.

Over the past fifty years, annual honeybee (Apis mellifera) colony losses have been steadily increasing worldwide. These losses have occurred in parallel with the global spread of the honeybee parasite Varroa destructor. Indeed, Varroa mite infestations are considered to be a key explanatory factor for the widespread increase in annual honeybee colony mortality. The host-parasite relationship between honeybees and Varroa is complicated by the mite's close association with a range of honeybee viral pathogens. The 10-year history of the expanding front of Varroa infestation in New Zealand offered a rare opportunity to assess the dynamic quantitative and qualitative changes in honeybee viral landscapes in response to the arrival, spread and level of Varroa infestation. We studied the impact of de novo infestation of bee colonies by Varroa on the prevalence and titres of seven well-characterised honeybee viruses in both bees and mites, using a large-scale molecular ecology approach. We also examined the effect of the number of years since Varroa arrival on honeybee and mite viral titres. The dynamic shifts in the viral titres of black queen cell virus and Kashmir bee virus mirrored the patterns of change in Varroa infestation rates along the Varroa expansion front. The deformed wing virus (DWV) titres in bees continued to increase with Varroa infestation history, despite dropping infestation rates, which could be linked to increasing DWV titres in the mites. This suggests that the DWV titres in mites, perhaps boosted by virus replication, may be a major factor in maintaining the DWV epidemic after initial establishment. Both positive and negative associations were identified for several pairs of viruses, in response to the arrival of Varroa. These findings provide important new insights into the role of the parasitic mite Varroa destructor in influencing the viral landscape that affects honeybee colonies.

Association of Amine-Receptor DNA Sequence Variants with Associative Learning in the Honeybee.

Octopamine- and dopamine-based neuromodulatory systems play a critical role in learning and learning-related behaviour in insects. To further our understanding of these systems and resulting phenotypes, we quantified DNA sequence variations at six loci coding octopamine-and dopamine-receptors and their association with aversive and appetitive learning traits in a population of honeybees. We identified 79 polymorphic sequence markers (mostly SNPs and a few insertions/deletions) located within or close to six candidate genes. Intriguingly, we found that levels of sequence variation in the protein-coding regions studied were low, indicating that sequence variation in the coding regions of receptor genes critical to learning and memory is strongly selected against. Non-coding and upstream regions of the same genes, however, were less conserved and sequence variations in these regions were weakly associated with between-individual differences in learning-related traits. While these associations do not directly imply a specific molecular mechanism, they suggest that the cross-talk between dopamine and octopamine signalling pathways may influence olfactory learning and memory in the honeybee.

Measurements of Chlorpyrifos Levels in Forager Bees and Comparison with Levels that Disrupt Honey Bee Odor-Mediated Learning Under Laboratory Conditions.

Chlorpyrifos is an organophosphate pesticide used around the world to protect food crops against insects and mites. Despite guidelines for chlorpyrifos usage, including precautions to protect beneficial insects, such as honeybees from spray drift, this pesticide has been detected in bees in various countries, indicating that exposure still occurs. Here, we examined chlorpyrifos levels in bees collected from 17 locations in Otago, New Zealand, and compared doses of this pesticide that cause sub-lethal effects on learning performance under laboratory conditions with amounts of chlorpyrifos detected in the bees in the field. The pesticide was detected at 17 % of the sites sampled and in 12 % of the colonies examined. Amounts detected ranged from 35 to 286 pg.bee(-1), far below the LD50 of ~100 ng.bee(-1). We detected no adverse effect of chlorpyrifos on aversive learning, but the formation and retrieval of appetitive olfactory memories was severely affected. Chlorpyrifos fed to bees in amounts several orders of magnitude lower than the LD50, and also lower than levels detected in bees, was found to slow appetitive learning and reduce the specificity of memory recall. As learning and memory play a central role in the behavioral ecology and communication of foraging bees, chlorpyrifos, even in sublethal doses, may threaten the success and survival of this important insect pollinator.

Steroid hormone (20-hydroxyecdysone) modulates the acquisition of aversive olfactory memories in pollen forager honeybees.

Here, we examine effects of the steroid hormone, 20-hydroxyecdysone (20-E), on associative olfactory learning in the honeybee, Apis mellifera. 20-E impaired the bees' ability to associate odors with punishment during aversive conditioning, but did not interfere with their ability to associate odors with a food reward (appetitive learning). The steroid had a significant impact also on the expression of amine-receptor genes in centers of the brain involved in the formation and recall of associative olfactory memories (mushroom bodies). 20-E increased expression of the dopamine receptor gene, Amdop2, and reduced the expression of the putative dopamine/ecdysone receptor gene, Amgpcr19. Interestingly, Amgpcr19 tended to be highly expressed in the brains of foragers that exhibited strong aversive learning, but expressed at lower levels in bees that performed well in appetitive learning assays. In 2-d-old bees, transcript levels of the same gene could be reduced by queen mandibular pheromone, a pheromone that blocks aversive learning in young worker bees. As ecdysteroid levels rise to a peak ∼2 d after adult emergence and then fall to low levels in foragers, we examined aversive learning also in young worker bees. Aversive learning performance in 2-d-old bees was consistently poor. The results of this study indicate that learning in honeybees can be modulated by ecdysteroids. They highlight, in addition, a potential involvement of the putative dopamine/ecdysone receptor, AmGPCR19, in hormonal regulation of associative olfactory learning in the honeybee.

Characterisation of a functional allatotropin receptor in the bumblebee, Bombus terrestris (Hymenoptera, Apidae).

Allatotropins (ATs) are multifunctional neuropeptides initially isolated from the tobacco hornworm, Manduca sexta, where they were found to stimulate juvenile hormone synthesis and release from the corpora allata. ATs have been found in a wide range of insects, but appear to be absent in Drosophila. The first AT receptor (ATR) was characterised in 2008 in the lepidopteran Bombyx mori. Since then ATRs have been characterised in Coleoptera and Diptera and in 2012, an AT precursor gene was identified in hymenopteran species. ATRs show large sequence and structural similarity to vertebrate orexin receptors (OXR). Also, AT in insects and orexin in vertebrates show some overlap in functions, including modulation of feeding behaviour and reproduction. The goal of this study was to identify a functional ATR in a hymenopteran species. We used ATRs (insect sequences) and OXRs (vertebrate sequences) to search the genome of the bumblebee, Bombus terrestris. Two receptors (XP_003402490 and XP_003394933) with resemblance to ATRs and OXRs were found. Phylogenetic analysis provided the first indication that XP_003402490 was more closely related to ATRs than XP_003394933. We investigated the transcript level distribution of both receptors and the AT precursor gene by means of quantitative real-time reverse transcriptase PCR. XP_003402490 displayed a tissue distribution comparable with ATRs in other species, with high transcript levels in the male accessory glands. After pharmacological characterisation, it appeared that XP_003402490 is indeed a functional ATR. Activation of the receptor causes an increase in intracellular calcium and cyclic AMP levels with an EC50 value in the low nanomolar to picomolar range. XP_003394933 remains an orphan receptor.

Mushroom bodies of the honeybee brain show cell population-specific plasticity in expression of amine-receptor genes.

Dopamine and octopamine released in the mushroom bodies of the insect brain play a critical role in the formation of aversive and appetitive memories, respectively. As recent evidence suggests a complex relationship between the effects of these two amines on the output of mushroom body circuits, we compared the expression of dopamine- and octopamine-receptor genes in three major subpopulations of mushroom body intrinsic neurons (Kenyon cells). Using the brain of the honeybee, Apis mellifera, we found that expression of amine-receptor genes differs markedly across Kenyon cell subpopulations. We found, in addition, that levels of expression of these genes change dramatically during the lifetime of the bee and that shifts in expression are cell population-specific. Differential expression of amine-receptor genes in mushroom body neurons and the plasticity that exists at this level are features largely ignored in current models of mushroom body function. However, our results are consistent with the growing body of evidence that short- and long-term olfactory memories form in different regions of the mushroom bodies of the brain and that there is functional compartmentalization of the modulatory inputs to this multifunctional brain center.

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.

Age- and behaviour-related changes in the expression of biogenic amine receptor genes in the antennae of honey bees (Apis mellifera).

We recently identified changes in amine-receptor gene expression in the antennae of the honey bee that correlate with shifts in the behavioural responsiveness of worker bees towards queen mandibular pheromone. Here we examine whether variations in expression of amine-receptor genes are related to age and/or to behavioural state. Colonies with a normal age structure were used to collect bees of different ages, as well as pollen foragers of unknown age. Single- and double-cohort colonies were established also to generate nurses and pollen foragers of the same age. Amdop1 was the only gene examined that showed no significant change in expression levels across the age groups tested. However, expression of this gene was significantly higher in 6-day-old nurses than in pollen foragers of the same age. Levels of expression of Amdop2 were very variable, particularly during the first week of adult life, and showed no correlation with nursing or foraging behaviour. Amdop3 and Amtyr1 expression levels changed dramatically with age. Interestingly, Amtyr1 expression was significantly higher in 15-day-old pollen foragers than in same-age nurses, whereas the opposite was true for Amoa1. While Amoa1 expression in the antennae was lower in 6- and 15-day-old pollen foragers than in nurses of the same age, differences in gene expression levels between nurses and pollen foragers could not be detected in 22-day-old bees. Our data show dynamic modulation of gene expression in the antennae of worker bees and suggest a peripheral role for biogenic amines in regulating behavioural plasticity in the honey bee.

Peripheral modulation of worker bee responses to queen mandibular pheromone.

It is generally accepted that young worker bees (Apis mellifera L.) are highly attracted to queen mandibular pheromone (QMP). Our results challenge this widely held view. We have found that unless young workers are exposed to QMP early in adult life, they, like foragers, avoid contact with this pheromone. Our data indicate that responses to QMP are regulated peripherally, at the level of the antennal sensory neurons, and that a window of opportunity exists in which QMP can alter a young bee's response to this critically important pheromone. Exposing young bees to QMP from the time of adult emergence reduces expression in the antennae of the D1-like dopamine receptor gene, Amdop1. Levels of Amdop3 transcript, on the other hand, and of the octopamine receptor gene Amoa1, are significantly higher in the antennae of bees strongly attracted to QMP than in bees showing no attraction to this pheromone. A decline in QMP attraction with age is accompanied by a fall in expression in worker antennae of the D2-like dopamine receptor, AmDOP3, a receptor that is selectively activated by QMP. Taken together, our findings suggest that QMP's actions peripherally not only suppress avoidance behavior, but also enhance attraction to QMP, thereby facilitating attendance of the queen.

Age-related changes in the behavioural response of honeybees to Apiguard®, a thymol-based treatment used to control the mite Varroa destructor.

The parasitic mite Varroa destructor is responsible for heavy losses in honey bee colonies and represents a major threat to the beekeeping industry. Essential oils offer an attractive alternative to the use of synthetic chemicals for the control of varroa. Amongst them, thymol appears to be particularly promising. However, treatments using thymol as their active substance, such as the gel formulation Apiguard(®), are suspected to have adverse effects on honey bee colonies. In this study, laboratory assays are used to investigate the effects of Apiguard(®) exposure on honey bee behaviour. Our results reveal that honey bee responses to this anti-varroa treatment change with honey bee age. While 2-day-old bees respond neutrally to Apiguard(®), older bees generally avoid the Apiguard(®) gel. Responses of forager bees were particularly striking. Foragers appear to be repelled by Apiguard(®). Touching their antennae with Apiguard(®) induces robust fanning behaviour. Our data suggest, however, that forager bees exposed to Apiguard(®) in the hive can become habituated to this treatment. These results offer interesting new perspectives on the effects of Apiguard(®) on honey bee behaviour and serve to highlight age-related changes in honey bee responses to gustatory, as well as olfactory cues.

Dopamine release in mushroom bodies of the honey bee (Apis mellifera L.) in response to aversive stimulation.

In Drosophila melanogaster, aversive (electric shock) stimuli have been shown to activate subpopulations of dopaminergic neurons with terminals in the mushroom bodies (MBs) of the brain. While there is compelling evidence that dopamine (DA)-induced synaptic plasticity underpins the formation of aversive memories in insects, the mechanisms involved have yet to be fully resolved. Here we take advantage of the accessibility of MBs in the brain of the honey bee to examine, using fast scan cyclic voltammetry, the kinetics of DA release and reuptake in vivo in response to electric shock, and to investigate factors that modulate the release of this amine. DA increased transiently in the MBs in response to electric shock stimuli. The magnitude of release varied depending on stimulus duration and intensity, and a strong correlation was identified between DA release and the intensity of behavioural responses to shock. With repeated stimulation, peak DA levels increased. However, the amount of DA released on the first stimulation pulse typically exceeded that evoked by subsequent pulses. No signal was detected in response to odour alone. Interestingly, however, if odour presentation was paired with electric shock, DA release was enhanced. These results set the stage for analysing the mechanisms that modulate DA release in the MBs of the bee.

C-type allatostatins mimic stress-related effects of alarm pheromone on honey bee learning and memory recall.

As honey bee populations worldwide are declining there is an urgent need for a deeper understanding of stress reactivity in these important insects. Our data indicate that stress responses in bees (Apis mellifera L.) may be mediated by neuropeptides identified, on the basis of sequence similarities, as allatostatins (ASTA, ASTC and ASTCC). Effects of allatostatin injection are compared with stress-related changes in learning performance induced by the honeybee alarm pheromone, isopentylacetate (IPA). We find that bees can exhibit two markedly different responses to IPA, with opposing effects on learning behaviour and memory generalisation, and that strikingly similar responses can be elicited by allatostatins, in particular ASTCC. These findings lend support to the hypothesis that allatostatins mediate stress reactivity in honey bees and suggest responses to stress in these insects are state dependent.

Specific Cues Associated With Honey Bee Social Defence against Varroa destructor Infested Brood.

Social immunity forms an essential part of the defence repertoire of social insects. In response to infestation by the parasitic mite Varroa destructor and its associated viruses, honey bees (Apis mellifera L.) have developed a specific behaviour (varroa-sensitive hygiene, or VSH) that helps protect the colony from this parasite. Brood cells heavily infested with mites are uncapped, the brood killed, and the cell contents removed. For this extreme sacrifice to be beneficial to the colony, the targeting of parasitized brood for removal must be accurate and selective. Here we show that varroa-infested brood produce uniquely identifiable cues that could be used by VSH-performing bees to identify with high specificity which brood cells to sacrifice. This selective elimination of mite-infested brood is a disease resistance strategy analogous to programmed cell death, where young bees likely to be highly dysfunctional as adults are sacrificed for the greater good of the colony.

Honey Bee Allatostatins Target Galanin/Somatostatin-Like Receptors and Modulate Learning: A Conserved Function?

Sequencing of the honeybee genome revealed many neuropeptides and putative neuropeptide receptors, yet functional characterization of these peptidic systems is scarce. In this study, we focus on allatostatins, which were first identified as inhibitors of juvenile hormone synthesis, but whose role in the adult honey bee (Apis mellifera) brain remains to be determined. We characterize the bee allatostatin system, represented by two families: allatostatin A (Apime-ASTA) and its receptor (Apime-ASTA-R); and C-type allatostatins (Apime-ASTC and Apime-ASTCC) and their common receptor (Apime-ASTC-R). Apime-ASTA-R and Apime-ASTC-R are the receptors in bees most closely related to vertebrate galanin and somatostatin receptors, respectively. We examine the functional properties of the two honeybee receptors and show that they are transcriptionally expressed in the adult brain, including in brain centers known to be important for learning and memory processes. Thus we investigated the effects of exogenously applied allatostatins on appetitive olfactory learning in the bee. Our results show that allatostatins modulate learning in this insect, and provide important insights into the evolution of somatostatin/allatostatin signaling.

Stereological analysis reveals striking differences in the structural plasticity of two readily identifiable glomeruli in the antennal lobes of the adult worker honeybee.

The primary antennal sensory centers (antennal lobes) in the brain of the honeybee are highly compartmentalized into discrete spheres of synaptic neuropil called glomeruli, many of which can be identified according to their predictable size and location. Glomeruli undergo significant changes in volume during the lifetime of the adult worker bee, at least some of which are activity dependent. This study tests the commonly expressed assumption that increases in neuropil volume are accompanied by an underlying increase in the number of synapses present in the tissue. A combination of light and electron microscopy was used to determine total synapse number within two glomeruli, T1-44 and T4-2(1). The Cavalieri direct estimator of volume was applied to 1.5 microm sections of resin-embedded brains. Selected sections were then re-embedded and prepared for transmission electron microscopy. Synapse densities were determined using the physical disector method on electron micrographs. Synapse density and glomerulus volume were combined to give an unbiased estimate of the total number of synapses. In glomerulus T1-44, a significant increase in volume was accompanied by a significant increase in the total number of synapses. In contrast, synapse counts in T4-2(1) remained unchanged, despite a significant increase in the volume of this glomerulus. These results demonstrate that synapse proliferation in antennal lobes of the adult worker bee is highly site specific. Although volumetric changes and changes in synapse number both contribute to the structural plasticity of the antennal lobes, these two components of plasticity appear to be independent processes.

Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera.

This study reveals that the tyramine receptor gene, Amtyr1, is expressed in the developing brain, as well as in the brain of the adult worker honey bee. Changes in levels of Amtyr1 expression were examined using Northern analysis. Age-related increases in Amtyr1 transcript levels were observed not only during metamorphic adult development, but also in the brain of the adult worker bee. RNA in situ hybridization revealed the pattern of Amtyr1 expression. Cell bodies staining intensely for tyramine receptor-gene transcript were observed throughout the somata rind, with well-defined clusters of cells associated with developing mushroom bodies, optic lobes, and antennal lobes of the brain. Staining for Amtyr1 transcript was particularly intense within the three major divisions of mushroom body intrinsic neurons (outer compact, noncompact, and inner compact cells), suggesting that Amtyr1 is highly expressed in these structures. Activation of AmTYR1 receptors heterologously expressed in insect (Spodoptera frugiperda) cells led to a reduction in intracellular levels of cAMP similar to that reported for AmTYR1 receptors expressed in mammalian (HEK 293) cells (Blenau et al. [2000] J Neurochem 74:900-908). Taken together, these results suggest that AmTYR1 receptors may play a role in the developing brain as well as in the brain of the adult worker bee. The actions of tyramine are likely to be mediated, at least in part, via the cAMP-signaling pathway.