Honey Bee Inhibitory Signaling Is Tuned to Threat Severity and Can Act as a Colony Alarm Signal

Alarm communication is a key adaptation that helps social groups resist predation and rally defenses. In Asia, the world’s largest hornet, Vespa mandarinia, and the smaller hornet, Vespa velutina, prey upon foragers and nests of the Asian honey bee, Apis cerana. We attacked foragers and colony nest entrances with these predators and provide the first evidence, in social insects, of an alarm signal that encodes graded danger and attack context. We show that, like Apis mellifera, A. cerana possesses a vibrational “stop signal,” which can be triggered by predator attacks upon foragers and inhibits waggle dancing. Large hornet attacks were more dangerous and resulted in higher bee mortality. Per attack at the colony level, large hornets elicited more stop signals than small hornets. Unexpectedly, stop signals elicited by large hornets (SS _{large hornet}) had a significantly higher vibrational fundamental frequency than those elicited by small hornets (SS _{small hornet}) and were more effective at inhibiting waggle dancing. Stop signals resulting from attacks upon the nest entrance (SS _nest) were produced by foragers and guards and were significantly longer in pulse duration than stop signals elicited by attacks upon foragers (SS _forager). Unlike SS _forager, SS _nest were targeted at dancing and non-dancing foragers and had the common effect, tuned to hornet threat level, of inhibiting bee departures from the safe interior of the nest. Meanwhile, nest defenders were triggered by the bee alarm pheromone and live hornet presence to heat-ball the hornet. In A. cerana, sophisticated recruitment communication that encodes food location, the waggle dance, is therefore matched with an inhibitory/alarm signal that encodes information about the context of danger and its threat level.     

Both olfactory and visual cues promote the hornet Vespa velutina to locate its honeybee prey Apis cerana

Predators use olfactory, visual and sometimes acoustic cues from the preys to assess food information. However, it is not known if the aggressive hornets (Vespa spp.) use olfactory, visual, or both types of information to find and recognize prey. In the present study, we trained hornet workers (Vespa velutina) to a feeding area. Once the hornets began consistently foraging at this feeding area, we determined whether they located prey (bees, Apis cerana) via olfactory or visual cues. We did this by testing whether hornets were attracted to a dummy bait (bee dummy bait or non-bee dummy bait) treated with extracts of honeybee cuticular hydrocarbons. We then tested whether hornets could distinguish between bee dummy bait and cotton ball dummy bait, both treated with bee odors. Hornets preferred the dummy treated with bee odors, and bee dummies (with bee images) were more attractive to the hornet than the cotton ball dummies with only bee odors. These results clearly indicate that a combination of olfactory and visual cues helps the hornet to locate its prey.     

Orchid Mimics Honey Bee Alarm Pheromone in Order to Attract Hornets for Pollination

Approximately one-third of the world's estimated 30,000 orchid species are deceptive and do not reward their pollinators with nectar or pollen [1]. Most of these deceptive orchids imitate the scent of rewarding flowers or potential mates [2] and [3]. In this study, we investigated the floral scent involved in pollinator attraction to the rewardless orchid Dendrobium sinense, a species endemic to the Chinese island Hainan that is pollinated by the hornet Vespa bicolor. Via chemical analyses and electrophysiological methods, we demonstrate that the flowers of D. sinense produce (Z)-11-eicosen-1-ol and that the pollinator can smell this compound. This is a major compound in the alarm pheromones of both Asian (Apis cerana) and European (Apis mellifera) honey bees [4] and [5] and is also exploited by the European beewolf (Philanthus triangulum) to locate its prey [6]. This is the first time that (Z)-11-eicosen-1-ol has been identified as a floral volatile. In behavioral experiments, we demonstrate that the floral scent of D. sinense and synthetic (Z)-11-eicosen-1-ol are both attractive to hornets. Because hornets frequently capture honey bees to feed to their larvae, we suggest that the flowers of D. sinense mimic the alarm pheromone of honey bees in order to attract prey-hunting hornets for pollination.     

Testing muzzle and ploy devices to reduce predation of bees by Asian hornets

Since its accidental introduction in 2004, the Asian yellow-legged hornet (Vespa velutina) has quickly spread in France. V. velutina specializes in the emblematic honeybee, Apis mellifera which are unable to protect their colonies efficiently against this fierce new predator. Here, we investigated whether two defence devices, a ploy and a muzzle, can protect bee colonies. Our results showed that neither device was able to reduce the number of hornets present in front of the hive or their predation efficiency (i.e. the number of captured bees). However, we found more flying bees and a smaller bee carpet in the presence of a muzzle, evidence that this very cheap method of protection can reduce the hornets’ impact on the activity of the bee colony and thus probably enabling its survival. We also showed that the number of agonistic interactions among V. velutina individuals increased with the number of hornets but without influencing the predation rate in our experimental conditions. The threat to honey bees is now well described, but the Asian yellow-legged hornet very likely has an impact on another insect species that has been the subject of far fewer studies: The native European hornet (Vespa cabro). Monitoring of several apiaries suggests that high densities of both V. velutina and V. crabro are exclusive and that the two species are engaged in an exploitative competition interaction. We are thus already in a position to underline the need for combined protection, as well as for further studies to better understand the ecology of V. velutina, to reduce the damage they cause.     

Imidacloprid Alters Foraging and Decreases Bee Avoidance of Predators

Concern is growing over the effects of neonicotinoid pesticides, which can impair honey bee cognition. We provide the first demonstration that sublethal concentrations of imidacloprid can harm honey bee decision-making about danger by significantly increasing the probability of a bee visiting a dangerous food source. Apis cerana is a native bee that is an important pollinator of agricultural crops and native plants in Asia. When foraging on nectar containing 40 µg/L (34 ppb) imidacloprid, honey bees (Apis cerana) showed no aversion to a feeder with a hornet predator, and 1.8 fold more bees chose the dangerous feeder as compared to control bees. Control bees exhibited significant predator avoidance. We also give the first evidence that foraging by A. cerana workers can be inhibited by sublethal concentrations of the pesticide, imidacloprid, which is widely used in Asia. Compared to bees collecting uncontaminated nectar, 23% fewer foragers returned to collect the nectar with 40 µg/L imidacloprid. Bees that did return respectively collected 46% and 63% less nectar containing 20 µg/L and 40 µg/L imidacloprid. These results suggest that the effects of neonicotinoids on honey bee decision-making and other advanced cognitive functions should be explored. Moreover, research should extend beyond the classic model, the European honey bee (A. mellifera), to other important bee species.     

High Concentrations of the Alarm Pheromone Component,Isopentyl Acetate,Reduces Foraging and Dancing in <Emphasis Type="Italic">Apis mellifera</Emphasis> Ligustica and <Emphasis Type="Italic">Apis cerana</Emphasis> Cerana

A honeybee colony is a superorganism that has evolved precise communication systems, which allow the colony to gather information from numerous individuals and coordinate its behavior. Alarm pheromones, such as isopentyl acetate (IPA), the main component of sting alarm pheromone, play a critical role in the coordination of individual behaviors as well as colony communication in honeybee colonies. In this study, honeybees (Apis mellifera ligustica and Apis cerana cerana) were exposed to relatively high levels of IPA at a foraging site (6–8 bee equivalents) and inside their colony (28–58 bee equivalents) to investigate the influence of alarm pheromones on foraging activity and hive flight activity. IPA reduced the number of bees that flew out the hive, foraged, and waggle danced. Under both contexts in the hive and at the food source, IPA can therefore inhibit honey bee foraging and foraging communication.     

Rearing techniques for hornets with emphasis on Vespa velutina (Hymenoptera: Vespidae): A review

Of the 34 vespid species recorded worldwide as invasive aliens, particular attention is currently being given to the yellow-legged hornet Vespa velutina that has invaded Europe, Japan, and Korea. The hornet is a voracious predator of bees and a serious threat to bee colonies and bee pollination. Control measures are needed, but their development has been challenging as biological and ecological studies are limited by the short field season and the cryptic nesting behavior of these hornets. A Vespa rearing process can generate the large numbers of workers, gynes, and males that are essential for studying chemical ecology and life history and for experimental testing of various hypotheses. We present a synthesis of suitable methods and techniques for year-round Vespa hornet rearing. Particular reference is given to Chinese know-how and experience with Vespa rearing for medicinal and culinary motivations. We also draw on observations with reared insects that have a similar life history as Vespa hornets, namely Bombus bumblebees and Vespula yellowjackets. Key challenges to optimizing Vespa hornet rearing are identified and discussed.     

Hierarchy of Attractants for Honey Bee Swarms

Chemical signals influence the selection of potential nest cavities by honey bee reproductive swarms. Attractants for swarms include the odors of old dark honey bee brood combs, odors from noncomb hive materials and propolis, and Nasonov pheromone, the odor released from the Nasonov glands of worker bees. Based on crossover and choice test experiments, swarms were shown to prefer, among otherwise identical cavities, those cavities containing Nasonov pheromone over cavities with only comb or other hive odors, cavities containing old comb over those with only noncomb odors or propolis, and cavities containing noncomb odors or propolis over those without bee or hive odor. Synergy between odors was not observed; that is, comb and/or noncomb hive odors did not enhance the attractiveness of Nasonov pheromone. The data support a model based on a hierarchy of olfactory attractants used by honey bee swarms, in order of highest to lowest: Nasonov pheromone, comb odor, noncomb and propolis odors, and, finally, absence of bee- or hive-produced odor.     

Floral volatile sesquiterpenes of Elsholtzia rugulosa (Lamiaceae) selectively attract Asian honey bees

There are few floral volatiles of compounds that have been properly assessed for attracting pollinators. The intense plant odour of Elsholtzia rugulosa (Lamiaceae) to humans may be attributed to the presence of high concentrations of β‐caryophyllene. In a previous study, Zhang, Yang, and Zhang (Scientific Reports, 6, 2016a, 276161) speculated that the presence of β‐caryophyllene attracts pollinators (e.g., honey bees) to its flowers, an assumption that was assessed through the exploration of the functional significance of specific floral volatile compounds and the evaluation of their effects on the behaviour of Apis cerana (Asian honey bees; a known pollinator) and two non‐pollinators, a hornet (Vespa velutina) and a bumblebee (Bombus sp.). The results from these behavioural experiments indicated that both β‐caryophyllene and β‐elemene (both naturally found in flowers of E. rugulosa) were individually attractive to the Asian honey bees. Moreover, the combination of the two was more effective than either of them separately. In contrast, neither compound, nor a blend of the two at different concentrations, was attractive to the hornet and bumblebee species. These results demonstrate that β‐caryophyllene and β‐elemene play a key signalling role in attracting Asian honey bees to E. rugulosa.     

Olfactory Attraction of the Hornet Vespa velutina to Honeybee Colony Odors and Pheromones

Since the beginning of the last century, the number of biological invasions has continuously increased worldwide. Due to their environmental and economical consequences, invasive species are now a major concern. Social wasps are particularly efficient invaders because of their distinctive biology and behavior. Among them, the yellow-legged hornet, Vespa velutina, is a keen hunter of domestic honeybees. Its recent introduction to Europe may induce important beekeeping, pollination, and biodiversity problems. Hornets use olfactory cues for the long-range detection of food sources, in this case the location of honeybee colonies, but the exact nature of these cues remains unknown. Here, we studied the orientation behavior of V. velutina workers towards a range of hive products and protein sources, as well as towards prominent chemical substances emitted by these food sources. In a multiple choice test performed under controlled laboratory conditions, we found that hornets are strongly attracted to the odor of some hive products, especially pollen and honey. When testing specific compounds, the honeybee aggregation pheromone, geraniol, proved highly attractive. Pheromones produced by honeybee larvae or by the queen were also of interest to hornet workers, albeit to a lesser extent. Our results indicate that V. velutina workers are selectively attracted towards olfactory cues from hives (stored food, brood, and queen), which may signal a high prey density. This study opens new perspectives for understanding hornets’ hunting behavior and paves the way for developing efficient trapping strategies against this invasive species.     

Alarm pheromone perception in honey bees is decreased by smoke (Hymenoptera: Apidae)

The application of smoke to honey bee(Apis mellifera) antennae reduced the subsequent electroantennograph response of the antennae to honey bee alarm pheromones, isopentyl acetate, and 2-heptanone. This effect was reversible, and the responsiveness of antennae gradually returned to that of controls within 10–20 min. A similar effect occurred with a floral odor, phenylacetaldehyde, suggesting that smoke interferes with olfaction generally, rather than specifically with honey bee alarm pheromones. A reduction in peripheral sensitivity appears to be one component of the mechanism by which smoke reduces nest defense behavior of honey bees.     

Task Specialization and Odor Effects on Proboscis Extension Conditioning in Bumblebees (Bombus huntii)

Workers in a social insect colony have distinct experiences that may affect their performance in a learning task. In this study using free-foraging and flight-cage bumblebee Bombus huntii colonies, the strength of olfactory proboscis extension conditioning (PEC) was affected by a bee’s task specialization as a nurse or forager and the stimulus odor. Nurses (n?=?26) learned to respond to the odors 1-hexanol and lavender, but foragers (n?=?25) had inhibited conditioning to both odors. More nurses (73 %) than foragers (48 %) had at least one conditioned response (CR), and nurses displayed significantly more CRs than foragers. As expected, a pseudoconditioned control group (n?=?9) showed very few CRs. Among bees that were given a second day of trials, nurses—but not foragers—showed modest improvement. Such strong inhibition of PEC has not been described in honeybee or bumblebee foragers. The stimulus odor also affected conditioning strength in nurses. Lavender, a familiar odor, elicited earlier and more CRs than 1-hexanol. We propose that learning floral odors in the context of foraging may inhibit PEC in bumblebee foragers, whereas exposure to odors in the honey stores may prime subsequent learning in nurses.     

Social Modulation of Stress Reactivity and Learning in Young Worker Honey Bees

Alarm pheromone and its major component isopentylacetate induce stress-like responses in forager honey bees, impairing their ability to associate odors with a food reward. We investigated whether isopentylacetate exposure decreases appetitive learning also in young worker bees. While isopentylacetate-induced learning deficits were observed in guards and foragers collected from a queen-right colony, learning impairments resulting from exposure to this pheromone could not be detected in bees cleaning cells. As cell cleaners are generally among the youngest workers in the colony, effects of isopentylacetate on learning behavior were examined further using bees of known age. Adult workers were maintained under laboratory conditions from the time of adult emergence. Fifty percent of the bees were exposed to queen mandibular pheromone during this period, whereas control bees were not exposed to this pheromone. Isopentylacetate-induced learning impairments were apparent in young (less than one week old) controls, but not in bees of the same age exposed to queen mandibular pheromone. This study reveals young worker bees can exhibit a stress-like response to alarm pheromone, but isopentylacetate-induced learning impairments in young bees are suppressed by queen mandibular pheromone. While isopentylacetate exposure reduced responses during associative learning (acquisition), it did not affect one-hour memory retrieval.     

Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana

Olfactory cues are important sensory modalities on individual discrimination, perception, and efficient orientation to food sources in most insects. In honey bees, which are well known as eusocial insects, olfactory cues are mainly used to maintain a colony. Although much research has been reported on olfactory systems in honey bee olfaction, little is known about the differences between two major honey bee species, the European honey bee Apis mellifera and the Asian honey bee Apis cerana. In order to understand the differences of olfactory characteristics in the two species, we compared the distribution of sensory hairs on the antennae and antennal olfactory responses, using electron microscopy, electrophysiological recording and molecular expression level of odorant receptors. Our present study demonstrated that the antennae of A. cerana have more olfactory sensilla than A. mellifera, responding more strongly to various floral volatile compounds. At the molecular level, olfactory co-receptor (Orco), which makes heterodimers with other conventional olfactory receptors, is more abundantly expressed in the antenna of A. cerana than of A. mellifera. These findings extend our understanding of the olfactory systems and behavioral responses to various ecological and biological signals in two closely related honey bee species.     

Native Prey and Invasive Predator Patterns of Foraging Activity: The Case of the Yellow-Legged Hornet Predation at European Honeybee Hives

Contrary to native predators, which have co-evolved with their prey, alien predators often benefit from native prey naïveté. Vespa velutina, a honeybee predator originating from Eastern China, was introduced into France just before 2004. The present study, based on video recordings of two beehives at an early stage of the invasion process, intends to analyse the alien hornet hunting behaviour on the native prey, Apis mellifera, and to understand the interaction between the activity of the predator and the prey during the day and the season. Chasing hornets spent most of their time hovering facing the hive, to catch flying honeybees returning to the hive. The predation pressure increased during the season confirming previous study based on predator trapping. The number of honeybee captures showed a maximum peak for an intermediate number of V. velutina, unrelated to honeybee activity, suggesting the occurrence of competition between hornets. The number of honeybees caught increased during midday hours while the number of hornets did not vary, suggesting an increase in their efficacy. These results suggest that the impact of V. velutina on honeybees is limited by its own biology and behaviour and did not match the pattern of activity of its prey. Also, it could have been advantageous during the invasion, limiting resource depletion and thus favouring colonisation. This lack of synchronization may also be beneficial for honeybee colonies by giving them an opportunity to increase their activity when the hornets are less effective.     

Dual Effect of Phenolic Nectar on Three Floral Visitors of Elsholtzia rugulosa (Lamiaceae) in SW China

Some plants secrete toxic nectar to appeal to most effective pollinators and deter non-pollinators or nectar thieves; however available information about ecological function of toxic nectar remains scarce. Elsholtzia rugulosa stands out as a plant with toxic nectar recorded in SW China. We focused on the functional significance of the phenolic compound that imparts toxic to the nectar of E. rugulosa. The effects of phenolic nectar were studied in three visitors of the flowers of the winter-blooming E. rugulosa Hemsl. (Lamiaceae) in SW China. The pollinating species Apis cerana Fabricius (Apidae; Asian honey bee) and two occasional visitors, Vespa velutina Lepeletier (Vespidae; yellow-legged Asian hornet) and Bombus eximius Smith (Apidae; a bumble bee) were tested for their preferences for low and high concentrations of 4-hydroxybenzoic acid in hexose and sucrose solutions. The pollinator is important for the plant, which is dependent on pollinator visits to attain a higher seed production and it is most likely that the combination of phenolic toxic nectar and the adaptation to phenolic nectar by A. cerana delivers an evolutionary advantage to both actors. The low and high concentrations of the phenolic acid were nearly totally refused by both occasional visitors V. velutina and B. eximius and were preferred by the pollinator A. cerana. E. rugulosa gains by having a much higher seed production and the pollinating honey bee by having an exclusive and reliable food source during the winter season at high altitudes in SW China. We found that the function of the toxic phenolic compound has dual roles by appealing to legitimate pollinators and deterring non-pollinators of E. rugulosa.     

Comparative Sucrose Responsiveness in Apis mellifera and A. cerana Foragers

In the European honey bee, Apis mellifera, pollen foragers have a higher sucrose responsiveness than nectar foragers when tested using a proboscis extension response (PER) assay. In addition, Africanized honey bees have a higher sucrose responsiveness than European honey bees. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should also have a higher responsiveness to sucrose than A. mellifera. To test this hypothesis, we compared the sucrose thresholds of pollen foragers and nectar foragers in both A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nectar foragers in both species, consistent with previous studies. However, contrary to our hypothesis, A. mellifera was more responsive than A. cerana. We also demonstrated that this higher sucrose responsiveness in A. mellifera was not due to differences in the colony environment by co-fostering two species of bees in the same mixed-species colonies. Because A. mellifera foragers were more responsive to sucrose, we predicted that their nectar foragers should bring in less concentrated nectar compared to that of A. cerana. However, we found no differences between the two species. We conclude that A. cerana shows a different pattern in sucrose responsiveness from that of Africanized bees. There may be other mechanisms that enable A. cerana to perform well in areas with sparse nectar resources.     

Deceptive orchids with Meliponini as pollinators

Visitation of orchids by Meliponini (stingless bees) is confirmed only in 13Melipona, Partamona andTrigona, forXylobium andMaxillaria, with the addition ofTrigona fulviventris visitingIonopsis. Some bees evinced multiple floral visitation by carrying several stipes and viscidia from pollinaria, thus may cause seed set. None foraged pseudopollen, nor is collection of this substance by bees verified. Meliponine-visited orchids had pollinia in quartets with emplacement on the bee's scutellum, possibly devices for pollinia survival on a social bee passing through its nest. Further, orchids produced no nectar, but bees repeatedly came to flowers. A testable basis for the orchid-meliponine relationship is mimicry of rewarding resources, or bee pheromone mimicry, recently documented for some honey bees. Meliponine pheromone analogs (nerol and 2-heptanol) are here noted forMaxillaria, but lack of foraging with pheromones byMelipona suggests multiple avenues of mimicry by orchids, including alarm pheromone and carrion mimicry.     

Metabolic response to alarm pheromone in honey bees

Exposure to alarm pheromone elicits increases in oxidative metabolism in Apis mellifera. The following quantitative results are reported: (1) The alarm response, measured by short-term elevated oxygen consumption in each bee, increases with increasing numbers of bees in the test groups. The rate of increase in response per bee shows a group effect which is greatest in small groups (1–100 bees) and remains constant in larger groups (up to 6,700 bees). (2) The pheromone concentration affects the reaction elicited. The greater the dose, the larger is the reaction up to a concentration of 2.4 μg/ml. Greater doses elicit no greater response. (3) Response to the pheromone by winter bees is maximum at 20°C and decreases at air temperatures above or below this value. (4) This metabolic reaction may provide a tool for quantitating temperament of honey bees.     

The pollination efficiency of a pollinator depends on its foraging strategy,flowering phenology,and the flower characteristics of a plant species

Honey bees and stingless bees are generalist visitors of several wild and cultivated plants. They forage with a high degree of floral fidelity and thereby help in the pollination services of those plants. We hypothesized that pollination efficiency might be influenced by flowering phenology, floral characteristics, and resource collection modes of the worker bees. In this paper, we surveyed the foraging strategies of honey bees (Apis cerana, Apis dorsata, and Apis florea) and stingless bees (Tetragonula iridipennis) concerning their pollination efficiencies. Bees showed different resource gathering strategies, including legitimate (helping in pollination as mixed foragers and specialized foragers) and illegitimate (serving as nectar robbers and pollen thieves) types of flower visitation patterns. Foraging strategies are influenced by the shape of flowers, the timing of the visitation, floral richness, and bee species. Honey bees and stingless bees mainly acted as legitimate visitors in most plants studied. Sometimes honey bees served as nectar robbers in tubular flowers and stingless bees as pollen thieves in large-sized flowers. Among the legitimate categories, mixed foragers have a comparatively lower flower visitation rate than the specialized nectar and pollen foragers. However, mixed foragers have greater abundance and higher values of the single-visit pollination efficiency index (PEi) than nectar and pollen foragers. The value of the combined parameter ‘importance in pollination (PI)’ was thus higher in mixed foragers than in nectar and pollen foragers.