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Honey bees use a complex form of spatial referential communication. Their “waggle dance” communicates the direction, distance, and quality of a resource to nestmates by encoding celestial cues, retinal optic flow, and relative food value into motion and sound within the nest. We show that correct waggle dancing requires social learning. Bees without the opportunity to follow any dances before they first danced produced significantly more disordered dances with larger waggle angle divergence errors and encoded distance incorrectly. The former deficit improved with experience, but distance encoding was set for life. The first dances of bees that could follow other dancers showed neither impairment. Social learning, therefore, shapes honey bee signaling, as it does communication in human infants, birds, and multiple other vertebrate species.

Bee-collected pollen and nectar contain multiple phytochemicals that can have anti-pathogenic effects when ingested. For example, the plant alkaloid, nicotine, can reduce infections by the trypanosome gut parasite (Crithidia bombi) in bumblebees. Parasitized bumblebees may be drawn to nicotine and thereby self-medicate their infection. We tested the hypothesis that nicotine can reduce infections of a common microsporidian pathogen, Nosema ceranae, in the honey bee gut. We found, however, that that a field realistic exposure dose of 1 ppm nicotine was not preferentially consumed by Apis mellifera foragers fed live Nosema spores (5 × 104 spores per bee; N = 160). One-day-old bees infected with Nosema (1 × 104 spores per bee; N = 160) showed no repression of nosemosis over a chronically applied exposure gradient of 0, 10−2, 10−1, 100, 101, 102, 103 or 104 ppm nicotine. Since imbibed nicotine may not effectively reach the spores in the bee gut, we conducted an in vitro experiment, in which Nosema spores were exposed up to 104 ppm nicotine in vials, rinsed of nicotine, and then fed to 1 day old bees (2 × 104 spores per bee; N = 216). However, the in vitro nicotine-treated spores remained infectious. Nicotine did impair bee mortality at high concentrations. Dietary nicotine is evidently not a treatment for nosemosis, but future studies should continue to examine the role of phytochemicals and bee health.

Biological collectives, like honeybee colonies, can make intelligent decisions and robustly adapt to changing conditions via intricate systems of excitatory and inhibitory signals. In this study, we explore the role of behavioural plasticity and its relationship to network size by manipulating honeybee colony exposure to an artificial inhibitory signal. As predicted, inhibition was strongest in large colonies and weakest in small colonies. This is ecologically relevant for honeybees, for which reduced inhibitory effects may increase robustness in small colonies that must maintain a minimum level of foraging and food stores. We discuss evidence for size-dependent plasticity in other types of biological networks.

Antibiotic-resistant bacteria are a major threat to global public health, and there is an urgent need to find effective, antimicrobial treatments that can be well tolerated by humans. Hornet venom is known to have antimicrobial properties, and contains peptides with similarity to known antimicrobial eptides (AMPs), mastoparans. We identified multiple new AMPs from the venom glands of Vespa ducalis (U-VVTX-Vm1a, U-VVTX-Vm1b, and U-VVTX-Vm1c), Vespa mandarinia (U-VVTX-Vm1d), and Vespa affinis (U-VVTX-Vm1e). All of these AMPs have highly similar sequences and are related to the toxic peptide, mastoparan. Our newly identified AMPs have α-helical structures, are amphiphilic, and have antimicrobial properties. Both U-VVTX-Vm1b and U-VVTX-Vm1e killed bacteria, Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922, at the concentrations of 16μg/mL and 32μg/mL, respectively. None of the five AMPs exhibited strong toxicity as measured via their hemolytic activity on red blood cells. U-VVTX-Vm1b was able to increase the permeability of E. coli ATCC25922 and degrade its genomic DNA. These results are promising, demonstrate the value of investigating hornet venom as an antimicrobial treatment, and add to the growing arsenal of such naturally derived treatments.

Author(s): Bell, Heather Christine; Benavides, Jaime Edilberto; Montgomery, Corina Noelle; Navratil, Jennifer Rose Evershed; Nieh, James Charles | Abstract: BackgroundSublethal exposure to neonicotinoids, a popular class of agricultural pesticides, can lead to behavioral effects that impact the health of pollinators. Therefore, new compounds, such as flupyradifurone (FPF), have recently been developed as 'safer' alternatives. FPF is an excitotoxic nicotinic acetylcholine receptor agonist, similar to neonicotinoids. Given the novelty of FPF, what data exist are focused mostly on assessing the effect of FPF on pollinator mortality. One important avenue for investigation is the potential effect of FPF on the sensitivity of nectar foragers, such as Apis mellifera, to sucrose concentrations. Neonicotinoids can alter this sucrose responsiveness and disrupt foraging. Compounding this effect, neonicotinoid-containing solutions are preferred by A. mellifera over pure sucrose solutions. We therefore conducted four studies, administering FPF under both acute and chronic conditions, and at field-realistic and higher than field-realistic doses, to assess the influence of FPF exposure on sucrose responsiveness and sucrose solutions with FPF in A. mellifera nectar foragers.ResultsWe found no evidence that FPF exposure under acute or chronic field-realistic conditions significantly altered sucrose responsiveness, and we did not find that bees exposed to FPF consumed more of the solution. However, at the much higher median lethal dose (48 h), among bees that survived, FPF-exposed foragers responded to significantly lower concentrations of sucrose than controls and responded at significantly higher rates to all concentrations of sucrose than controls.ConclusionWe found no evidence that FPF alters the sucrose responsiveness of nectar foragers at field-realistic doses during winter or early spring, but caution and further investigation are warranted, particularly on the effects of FPF in conjunction with other stressors. © 2019 Society of Chemical Industry.

Referential communication provides a sophisticated way in which animals can communicate information about their environment. Previously, research demonstrated that honey bee stop signals encode predator danger in their fundamental frequency and danger context in their duration. Here, we show that these signals also encode danger in their vibrational amplitude. Stop signals elicited by the more dangerous predator, the large hornet (Vespa mandarinia) had significantly 1.5-fold higher vibrational amplitudes than those elicited by the small hornet predator (Vespa velutina). We measured the freezing vibrational response thresholds, and show that natural signals exceed these response thresholds. Finally, with artificial playbacks of the vibratory stop signal, we demonstrate that these signals referentially encode the danger that foragers experience at food source. Stop signals elicited by the larger and significantly more dangerous predator (V. mandarinia) were significantly 1.4-fold more inhibitory than stop signals elicited by the smaller and less dangerous predator (V. velutina).

Bees provide critical pollination services but are threatened by multiple stressors, including viral pathogens. Most studies of pollinator health focus on managed honey bees (Apis mellifera Linnaeus) (MHB) or native bee species, but a third player, the feral honey bee (FHB), requires further study. Spillover and spillback of viral pathogens between these managed, feral, and native bees is generating increasing interest. In this case study, we provide evidence suggesting that FHB colonies play an important role in viral pathogen dynamics of southern California pollinator communities because they act as reservoirs, of viral pathogens such as acute bee paralysis virus (ABPV), black queen cell virus (BQCV), and deformed wing virus (DWV). Surprisingly, even though FHB are not treated for diseases or parasites, they harbor similar pathogen loads to MHB, which are usually highly treated, suggesting the need for future studies to determine if FHB resist or are more resilient to viruses.

Simple Summary The co-evolution of attack and defense strategies between Apis and Vespa is a good model for studying arms races. Some honey bee species and subspecies can kill hornets with heat balls that generate heat and carbon dioxide. However, the role of stinging as a defense against hornets has been discounted, even though stings and venom are important honey bee weapons. No studies, to date, have tested the role of bee sting venom alone or in conjunction with elevated temperature on hornet survival. We found that bees can sting hornets but most hornets (87%) are able to remove bee stings less than 1 min after being stung, perhaps explaining why stinging is not considered a major anti-hornet defense. However, we show that such bee stings can kill hornets and demon-strate that the combination of sting venom and being heated is the most lethal to hornets. Abstract The heat ball defense of honey bees against their sympatric hornet predators is a classic and spectacular outcome of a co-evolutionary race. Hundreds of bees can encapsulate a hornet within a large ball that kills it with elevated heat. However, the role of stinging in this defense has been discounted, even though sting venom is an important weapon in bees. Surprisingly, no studies have tested the role of bee sting venom alone or in conjunction with elevated temperature on hornet survival. We surveyed dead Vespa velutina hornets found near and inside Apis cerana colonies and found stings retained in hornet bodies, most often in an intersegmental neck-like region, the veracervix. Experimentally stinging hornets in this region with A. cerana and Apis mellifera guards significantly increased hornet mortality. The combination of sting venom and elevated heat ball temperature (44 °C) was the most lethal, although there was no synergistic interaction between sting venom and temperature. As expected, hornet mortality increased when they were stung more often. The average amount of venom per insect species and the length of stinger lancets correlated with insect mass. Sting venom thus remains important in the arms race between bees and their hornet predators.

Bacterial resistance to drugs is a global problem requiring the urgent development of new antibiotics. Antimicrobial peptides (AMPs) are excellent candidates for the design of novel antibiotics to combat microbial resistance. In this research, we identified four new peptides (U-VVTX-Vp1a, U-VVTX-Vp1b, U-VVTX-Vp2a, and U-VVTX-Vp2b, respectively) from the venom of Vespa velutina, and tested their antimicrobial, antioxidant, and hemolytic effects. All four peptides showed scavenging ability against DPPH, ABTS+, and •OH free radicals. Of note, Vp1b strongly inhibited the growth of Staphylococcus aureus and Escherichia coli bacteria at concentrations of 60 and 120 μM. Due to their low hemolytic activity, all four peptides could be utilized in the development of new antioxidants and as candidates for the design of novel antimicrobial agents.

The co-evolutionary arms race between predators and their prey has led to complex signalling, especially in groups that benefit from the social transmission of alarm signals. In particular, pursuit deterrence signals can allow individuals and groups to indicate, at relatively low cost, that a predator's further approach is futile. Pursuit deterrence signals are usually more effective if amplified, for example, by becoming contagious and rapidly spreading among prey without requiring individual prey to confirm predator presence. However, this can also lead to runaway false signalling. We provide the first evidence of a contagious pursuit deterrence signal in social insects. The Asian honey bee Apis cerana, performs an I See You (ISY) signal that deters attacking hornets. We show that these signals enhance defensive signalling by also attracting guard bees and that the visual movements of appropriate stimuli alone (hornets and ISY signalling bees, but not harmless butterflies) provide sufficient stimuli. Olfaction and other potential cues are not necessary. The ISY signal is visually contagious and is buffered from runaway false signals because it is specifically triggered and by likely selection for honesty within the highly cooperative bee colony. These results expand our understanding of contagious signals and how they can be honestly maintained in highly cooperative collectives.

The assessment of pesticide risks to insect pollinators have typically focused on short-term, lethal impacts. The environmental ramifications of many of the world’s most commonly employed pesticides, such as those exhibiting systemic properties that can result in long-lasting exposure to insects, may thus be severely underestimated. Here, seven laboratories from Europe and North America performed a standardised experiment (a ring-test) to study the long-term lethal and sublethal impacts of the relatively recently approved ‘bee safe’ butenolide pesticide flupyradifurone (FPF, active ingredient in Sivanto®) on honey bees. The emerging contaminant, FPF, impaired bee survival and behaviour at field-realistic doses (down to 11 ng/bee/day, corresponding to 400 µg/kg) that were up to 101-fold lower than those reported by risk assessments (1110 ng/bee/day), despite an absence of time-reinforced toxicity. Our findings raise concerns about the chronic impact of pesticides on pollinators at a global scale and support a novel methodology for a refined risk assessment., Tosi and colleagues perform a large, multinational experiment on honey bees, encompassing multiple subspecies, to assess the short- and long-term effects of a next-generation pesticide, flupyradifurone, on this key pollinator species. Their findings indicate that this “bee safe” pesticide impairs survival and behaviour at real-world exposure levels.

Human environmental modifications have outpaced honey bees' ability to evolve adaptive regulation of foraging tactics, possibly including a tactic associated with extreme food shortage, honey robbing. Honey robbing is a high risk, high reward, and understudied honey bee tactic whereby workers attack and often kill neighboring colonies to steal honey. Humans have exacerbated the conditions that provoke such robbing and its consequences. We describe robbing as an individual-level and colony-level behavioral syndrome, implicating worker bees specialized for foraging, food processing, and defense. We discuss how colony signaling mechanisms could regulate this syndrome and then explore the ecological underpinnings of robbing-highlighting its unusual prevalence in the commonly managed Apis mellifera and outlining the conditions that provoke robbing. We advocate for studies that identify the cues that modulate this robbing syndrome. Additionally, studies that apply behavioral ecology modeling approaches to generate testable predictions about robbing could clarify basic bee biology and have practical implications for colony management.

The health of insect pollinators, particularly the honey bee, Apis mellifera (Linnaeus, 1758), is a major concern for agriculture and ecosystem health. In response to mounting evidence supporting the detrimental effects of neonicotinoid pesticides on pollinators, a novel ‘bee safe’ butenolide compound, flupyradifurone (FPF) has been registered for use in agricultural use. Although FPF is not a neonicotinoid, like neonicotinoids, it is an excitotoxic nicotinic acetylcholine receptor agonist. In addition, A. mellifera faces threats from pathogens, such as the microsporidian endoparasite, Nosema ceranae (Fries et al. 1996). We therefore sought 1) to increase our understanding of the potential effects of FPF on honey bees by focusing on a crucial behavior, the ability to learn and remember an odor associated with a food reward, and 2) to test for a potential synergistic effect on such learning by exposure to FPF and infection with N. ceranae. We found little evidence that FPF significantly alters learning and memory at short-term field-realistic doses. However, at high doses and at chronic, field-realistic exposure, FPF did reduce learning and memory in an olfactory conditioning task. Infection with N. ceranae also reduced learning, but there was no synergy (no significant interaction) between N. ceranae and exposure to FPF. These results suggest the importance of continued studies on the chronic effects of FPF.

There was an error published in [Journal of Experimental Biology (2015) 218, 3199-3205][1] ([doi: 10.1242/jeb.127472][2]). The concentration of imidacloprid is erroneously given as of 1.5 µg per bee day−1 in the Abstract; this should be 1.5 ng per bee day−1, as correctly stated in

Animal-pollinated plants face a common problem, how their defensive anti-herbivore compounds may impair or alter pollinator behavior. Evolution has tailored multiple solutions, which largely involve pollinator tolerance or manipulation, to the benefit of the plant, not the removal of these compounds from pollen or nectar. The tea plant, Camilla sinensis, is famous for the caffeine and tea polyphenols (TP) that it produces in its leaves. However, these compounds are also found in its nectar, which honey bees readily collect. We examined the effects of these compounds on bee foraging choices, learning, memory, and olfactory sensitivity. Foragers preferred a sucrose feeder with 100 µg or 10 µg TP/ml over a control feeder. Caffeine, but not TP, weakly increased honey bee learning. Both caffeine and TP significantly increased memory retention, even when tested 7 d after the last learning trial. In addition, TP generally elevated EAG responsiveness to alarm pheromone odors. These results demonstrate that other secondary plant compounds, not only caffeine, can attract pollinators and influence their learning and memory.

Learning is important for honey bee fitness and the pollination services that they provide. Neonicotinoid pesticides impair learning, fitness, colony health, and pollination, but most studies on how they affect bee learning have focused on olfactory learning. We tested the effects of field realistic doses of 0.8 ng/bee and 1.34 ng/bee of the neonicotinoid pesticide, thiamethoxam (TMX), on bee visual learning. We adapted a T-maze bioassay and classically conditioned bees to associate sugar reward with a simulated flower color (blue or yellow light) in a choice assay. At 1.34 ng/bee, TMX significantly reduced correct choices in the final learning trial as compared to the control treatment. There was no TMX effect in our 1-h memory test. We found stronger effects on decision time and abnormal behaviors. TMX decreased bee decision times, a potential byproduct of induced hyperactivity since bees walked to make choices. Behaviors (falling, trembling, and rapid abnormal movements) were significantly increased by both TMX doses as compared to the control treatment. These results suggest that the effects of neonicotinoids on bee visual learning should be further studied and incorporated into Risk Assessment protocols.

Eavesdropping is predicted to evolve between sympatric, but not allopatric, predator and prey. The evolutionary arms race between Asian honey bees and their hornet predators has led to a remarkable defence, heat balling, which suffocates hornets with heat and carbon dioxide. We show that the sympatric Asian species, Apis cerana (Ac), formed heat balls in response to Ac and hornet (Vespa velutina) alarm pheromones, demonstrating eavesdropping. The allopatric species, Apis mellifera (Am), only weakly responded to a live hornet and Am alarm pheromone, but not to hornet alarm pheromone. We observed typical hornet alarm pheromone-releasing behaviour, hornet sting extension, when guard bees initially attacked. Once heat balls were formed, guards released honey bee sting alarm pheromones: isopentyl acetate, octyl acetate, (E)-2-decen-1-yl acetate and benzyl acetate. Only Ac heat balled in response to realistic bee alarm pheromone component levels (

The Asian hornet, Vespa velutina, is an invasive, globally-distributed predator of European honey bees and other insects. To better under its reproductive biology and to find a specific, effective, and low-impact control method for this species, we identified and tested the key compounds in V. velutina sex pheromone. Virgin gynes (reproductive females) produced this sex pheromone in the sixth intersegmental sternal glands of their abdomens. The active compounds were 4-oxo-octanoic acid (4-OOA, 10.4 μg bee−1) and 4-oxo-decanoic acid (4-ODA, 13.3 μg bee−1) at a 0.78 ratio of 4-OOA/4-ODA. We synthesized these compounds and showed that male antennae were highly sensitive to them. Moreover, males were only strongly attracted to a 4-OOA/4-ODA blend at the natural ratio produced by gynes. These results provide the first demonstration of an effective way to lure V. velutina males, and the first chemical identification of a sex pheromone in the eusocial hornets.

Recent declines in the abundance and diversity of wild bee species have made it important to understand how introduced species such as honey bees contribute to pollinating native plants. In this study, we decoded the waggle dances of Apis mellifera pollen foragers and used DNA barcoding of bee-collected pollen over 17 months to characterize spatio-temporal patterns in recruitment behavior and dietary breadth in a diverse yet fragmented Mediterranean scrub environment. Foragers danced for small, nearby patches of pollen resources during the cool dormant season but danced for 1.6–2.7-fold more distant and more widely dispersed resources during the hot dry season, a time of seasonal drought and floral dearth. Individual foragers collected pollen from a wide variety of species, but colonies focused recruitment on a small subset of common species in flower during each season: 2.7, 6.3, and 10% of all species in bloom during the dormant, growth, and dry seasons, respectively. Pollen foragers danced almost exclusively for cold-adapted native species during the dormant season, equally for native and non-native plants during the short growth season, and primarily for non-native ornamental species during the dry season. Our results suggest that honey bee recruitment behavior and dietary breadth may be influenced by seasonal changes in pollen resources. Furthermore, they indicate that honey bees may be useful in pollinating common native plants, and that non-native ornamental species can provide a major source of colony protein during a seasonal dearth of flowering native species.

Ecological Entomology (2017), DOI: 10.1111/een.12406 Foraging at a safe distance: crab spider effects on pollinators S P E N C E R H U E Y and J A M E S C . N I E H Section of Ecology, Behavior, and Evolution, University of California San Diego, La Jolla, California, U.S.A. Abstract. 1. The ability of pollinating insects to discover and evade their predators can affect plant–pollinator mutualisms and have cascading ecosystem effects. Pollinators will avoid flowers with predators, but it is not clear how far away they will move to continue foraging. If these distances are relatively small, the impact of predators on the plant–pollinator mutualism may be lessened. The plant could continue to receive some pollination, and pollinators would reduce the time and energy needed to search for another patch. 2. A native crab spider, Xysticus elegans, was placed on one cluster in a small array of Baccharis pilularis inflorescence clusters, and the preferred short-range foraging distances of naturally visiting pollinators was determined. 3. Nearly all pollinator taxa (honey bees, wasps, other Hymenoptera, and non-bombyliid flies) spent less time foraging on the predator cluster. 4. The key result of this study is that inflorescences within 90 mm of the crab spider were avoided by visiting honey bees and wasps, which spent three- and 18-fold more time, respectively, foraging on more distant flower clusters. 5. Whether honey bees can use olfaction to detect spiders was then tested, and this study provides the first demonstration that honey bees will avoid crab spider odour alone at a food source. Key words. Crab spider, foraging, honey bee, olfaction, pollination, predation, public information. Introduction The ability of prey to detect and avoid predators plays a major role in structuring ecosystems, in part by altering the spatio-temporal distribution of prey within a landscape (Laundre et al., 2010; Wirsing et al., 2010). Prey decision-making is affected by information about predator location and should be tuned to allow prey to forage in a dangerous world (Krupa & Sih, 1998). For prey that are pollinators, such decisions have a broad importance, because pollination is a key ecosystem service (Fisher & Turner, 2008). Predators can disrupt the plant–pollinator mutualism by deterring pollinator visitation, resulting in reduced seed set (Suttle, 2003), fruit production (Dukas, 2005; Hanna et al., 2012) and fruit biomass (Antiqueira & Romero, 2016). However, in some cases, predators can attract pollinators and thereby increase seed weights (Welti et al., 2016) Correspondence: Spencer Huey, Section of Ecology, Behavior, and Evolution, University of California San Diego, La Jolla, California, U.S.A. E-mail: spencer.huey@gmail.com; James C. Nieh, Section of Ecology, Behavior, and Evolution, University of California San Diego, La Jolla, California, U.S.A. E-mail: jnieh@ucsd.edu © 2017 The Royal Entomological Society or increase the plant’s reproductive success (fruit and seed set) by deterring less effective pollinators (Gonzalvez et al., 2013). Thus, the ability of pollinators to detect predators and fine-tune where and for how long they forage has complex, cascading ecosystem effects (Knight et al., 2006). In general, predator presence decreases the rate of pollina- tor visitation (Elliott & Elliott, 1994; Dukas & Morse, 2003; Suttle, 2003; Dukas, 2005; Goncalves-Souza et al., 2008; Jones & Dornhaus, 2011) because insect pollinators usually avoid inflorescences occupied by predators (Romero et al., 2011; Antiqueira & Romero, 2016). For example, crab spiders (Thom- sidae) are common ambush predators that prey upon a wide vari- ety of insect pollinators (Lovell, 1915; Nentwig, 1986). Insect pollinators therefore decrease visitation when crab spiders are on inflorescences of milkweed (Dukas & Morse, 2003), slickspot peppergrass (Robertson & Maguire, 2005) or other plant species (Reader et al., 2006). Visitation rates are important because they influence the probability of pollination (Kearns & Inouye, 1993). In addition, the duration of pollinator visitation can be positively correlated with pollinia removal (Fishbein & Venable, 1996) and with increased pollen deposition on stigmas (Thomson, 1986).

There is growing concern about the impact of poor nutrition on honey bee health. With caged bee experiments and whole-colony field experiments, we examined the effects of supplementing bees with essential amino acids (EAA), or a control treatment of nonessential amino acids (NAA). Caged bees fed EAA developed significantly greater head weights than controls, weights that were similar to nurse bees. Caged bees fed EAA developed significantly greater thorax weights than controls, weights that were similar to foragers. Higher head and thorax weights may respectively reflect increased glandular development in nurse bees and higher flight muscle mass in forager bees. In our field study, 29% of the pollen collected by our honey bee colonies came from eucalyptus trees. Amino acid analyses revealed no EAA deficiencies for the bee-collected polyfloral pollen or for monofloral eucalyptus pollen. Colonies fed 29 g EAA supplement may have slightly increased individual bee growth and brood rearing, but this effect was not significant. A clear colony result was a correlation between nurse bee physiology and brood development: 17% increase in nurse bee weight corresponded to 100% more capped brood cells (R2 = 0.38). We suggest that colony supplementation should target nurse bee nutrition. Nurse bees eventually become forager bees. Hence, increased glandular development may support colony brood development and greater flight muscle mass may assist colony foraging.

Flupyradifurone (FPF, Sivanto®) is a new butenolide insecticide that, like the neonicotinoids, is a systemic nicotinic acetylcholine receptor (nAChR) agonist. However, FPF is considered bee-safe (according to standard Risk Assessment tests), and is thus a potential solution to the adverse effects of other pesticides on beneficial insects. To date, no studies have examined the impact of nutritional stress (decreased food diversity and quality) and FPF exposure on bee health although both stressors can occur, especially around agricultural monocultures. We therefore tested the effects of a field-realistic FPF concentration (4 ppm, FPFdaily dose = 241 ± 4 ng/bee/day, 1/12 of LD50) and nutritional stress (nectar with low-sugar concentrations) on honey bee (Apis mellifera L.) mortality, food consumption, thermoregulation, flight success (unsuccessful vs. successful), and flight ability (duration, distance, velocity). Flight and thermoregulation are critical to colony health: bees fly to collect food and reproduce, and they thermoregulate to increase flight efficiency and to rear brood. We studied the effects across seasons because seasonality can influence bee sensitivity to environmental stress. We demonstrate that, depending upon season and nutritional stress, FPF can reduce bee survival (-14%), food consumption (-14%), thermoregulation (-4%, i.e. hypothermia), flight success (-19%), and increase flight velocity (+13%). Because pesticide exposure and nutritional stress can co-occur, we suggest that future studies and pesticide risk assessments consider both seasonality and nutritional stress when evaluating pesticide safety for bees.

The ability of animals to learn and remember is an important adaptation for coping with environmental changes. The fitness benefits provided by these cognitive skills, in conjunction with social behaviours, contributes to the success of social insects. How these abilities are shared among the different castes and the long-term persistence of memory are now being elucidated in diverse systems, work that should shed light on general principles underlying cognitive evolution. Here we provide the first evidence of olfactory learning and long-term olfactory memory in all three castes of an Asian hornet, Vespa velutina. Using the first proboscis extension reflex assay developed for hornets or wasps, we found that all hornet castes could learn and remember odours associated with a food reward. Moreover, long-lasting memory was retained without significant decay in gynes (virgin queens) and drones even up to 30 d. Workers did not survive for 30 d. Drones learned and remembered simple odorant molecules and gyne sex pheromone with equal facility. These results increase our understanding of the outstanding cognitive abilities of social insects and suggest the likely importance of long-lasting memory in different castes of the same species.

Foraging is essential for honey bee colony fitness and is enhanced by the waggle dance, a recruitment behavior in which bees can communicate food location and quality. We tested if the consumption of nectar (sucrose solution) with a field-realistic concentration of 4ppm flupyradifurone (FPF) could alter foraging behavior and recruitment dancing in Apis mellifera. Foragers were repelled by FPF. They visited the FPF feeder less often and spent less time imbibing sucrose solution (2.5M, 65% w/w) with FPF. As a result, bees feeding on the FPF treatment consumed 16% less nectar. However, FPF did not affect dancing: there were no effects on unloading wait time, the number of dance bouts per nest visit, or the number of dance circuits performed per dance bout. FPF could therefore deter bees from foraging on contaminated nectar. However, the willingness of bees to recruit nestmates for nectar with FPF is concerning. Recruitment can rapidly amplify the number of foragers and could overcome the decrease in consumption of FPF-contaminated nectar, resulting in a net inflow of pesticide to the colony. FPF also significantly altered the expression of 116 genes, some of which may be relevant for the olfactory learning deficits induced by FPF and the toxicity of FPF.

Animals use diverse sensory stimuli to navigate their environment and to recognize rewarding food sources. Honey bees use visual attributes of the targeted food source, such as its color, shape, size, direction and distance from the hive, and the landmarks around it to navigate during foraging. They transmit the location information of the food source to other bees if the food source is highly rewarding. To investigate the relative importance of these attributes, we trained bees to feeders in two different experiments. In the first experiment, we asked whether bees prefer to land on (a) a similar feeder at a different distance on the same heading or on (b) a visually distinct feeder located at the exact same location. We found that, within a short foraging range, bees relied heavily on the color and the shape of the food source and to a lesser extent on its distance from the hive. In the second experiment, we asked if moving the main landmark or the feeder (visual target) influenced recruitment dancing for the feeder. We found that foragers took longer to land and danced fewer circuits when the location of the food source or a major landmark associated with it changed. These results demonstrate that prominent visual attributes of food sources and landmarks are evidently more reliable than distance information and foraging bees heavily utilize these visual cues at the later stages of their journey. This article is protected by copyright. All rights reserved.

There was an error published in J. Exp. Biol. (2018) 221 , [jeb168344][1] ([[doi:10.1242/jeb.168344][3]][3]). The terpenoid triptolide was mistakenly referred to as an alkaloid in the title and text. The title has been changed to ‘The reluctant visitor: a terpenoid in toxic nectar can reduce

As the primary source of colony reproduction, social insect queens play a vital role. However, the cognitive abilities of queens are not well understood, although queen learning and memory are essential in multiple species such as honey bees, in which virgin queens must leave the nest and then successfully learn to navigate back over repeated nuptial flights. Honey bee queen learning has never been previously demonstrated. We therefore tested olfactory learning in queens and workers and examined the role of DNA methylation, which plays a key role in long-term memory formation. We provide the first evidence that honey bee queens have excellent learning and memory. The proportion of honey bee queens that exhibited learning was 5-fold higher than that of workers at every tested age and, for memory, 4-fold higher than that of workers at a very young age. DNA methylation may play a key role in this queen memory because queens exhibiting remote memory had a more consistent elevation in Dnmt3 gene expression as compared with workers. Both castes also showed excellent remote memory (7 day memory), which was reduced by 14-20% by the DNA methylation inhibitor zebularine. Given that queens live approximately 10-fold longer than workers, these results suggest that queens can serve as an excellent long-term reservoir of colony memory.

Honey bees provide key ecosystem services. To pollinate and to sustain the colony, workers must walk, climb, and use phototaxis as they move inside and outside the nest. Phototaxis, orientation to light, is linked to sucrose responsiveness and the transition of work from inside to outside the nest, and is also a key component of division of labour. However, the sublethal effects of pesticides on locomotion and movement to light are relatively poorly understood. Thiamethoxam (TMX) is a common neonicotinoid pesticide that bees can consume in nectar and pollen. We used a vertical arena illuminated from the top to test the effects of acute and chronic sublethal exposures to TMX. Acute consumption (1.34 ng/bee) impaired locomotion, caused hyperactivity (velocity: +109%; time moving: +44%) shortly after exposure (30 min), and impaired motor functions (falls: +83%; time top: −43%; time bottom: +93%; abnormal behaviours: +138%; inability to ascend: +280%) over a longer period (60 min). A 2-day chronic exposure (field-relevant daily intakes of 1.42–3.48 ng/bee/day) impaired bee ability to ascend. TMX increased movement to light after acute and chronic exposure. Thus, TMX could reduce colony health by harming worker locomotion and, potentially, alter division of labour if bees move outside or remain outdoors.

Nosema ceranae is a pervasive and widespread honey bee pathogen that is associated with colony declines and has recently been shown to infect larval honey bees. In adult bees, Nosema infection is known to alter levels of a key protein, vitellogenin (Vg), which is necessary for egg-laying in queens, brood food production in workers, and proper immune function in all female bees. We therefore tested the effects of larval worker infection on hemolymph Vg titers. In 1-day old adult workers that were infected as larvae with 10,000 (10 K) or 40,000 (40 K) live N. ceranae spores/bee, Vg titers were significantly elevated by + 83% and + 73%, respectively, as compared to controls. At 7 days of adult age, Vg remained significantly elevated (+ 68%) in 10 K treated workers as compared to control workers. Nosema infection decreased total hemolymph protein titers in 1 and 7-day old adult bees (−50% in the 10 K and 40 K treated bees). Bees infected as larvae also had a more queen-like sting morphology. They developed slightly but significantly fewer barbs on their stings (−7% in the 40K-treated bees). Higher Vg levels are associated with younger bees. Thus, elevated Vg levels could delay normal age polyethism and disrupt colony balance.

The nectar of the thunder god vine, Tripterygium hypoglaucum, contains a terpenoid, triptolide (TRP), that may be toxic to the sympatric Asian honey bee, Apis cerana, because honey produced from this nectar is toxic to bees. However, these bees will forage on, recruit for, and pollinate this plant during a seasonal dearth of preferred food sources. Olfactory learning plays a key role in forager constancy and pollination, and we therefore tested the effects of acute and chronic TRP feeding on forager olfactory learning, using proboscis extension reflex conditioning. At concentrations of 0.5-10 µg TRP/ml, there were no learning effects of acute exposure. However, memory retention (1 h after the last learning trial) significantly decreased by 56% following acute consumption of 0.5 µg TRP/ml. Chronic exposure did not alter learning or memory, except at high concentrations (5 and 10 µg TRP/ml). TRP concentrations in nectar may therefore not significantly harm plant pollination. Surprisingly, TRP slightly increased bee survival, and thus other components in T. hypoglaucum honey may be toxic. Long term exposure to TRP could have colony effects, but these may be ameliorated by the bees’ aversion to T. hypoglaucum nectar when other food sources are available and, perhaps, by detoxification mechanisms. The co-evolution of this plant and its reluctant visitor may therefore likely illustrate a classic compromise between the interests of both actors.

While foraging, animals can form inter- and intraspecific social signalling networks to avoid similar predators. We report here that foragers of different native Asian honey bee species can detect and use a specialized alarm pheromone component, benzyl acetate (BA), to avoid danger. We analysed the volatile alarm pheromone produced by attacked workers of the most abundant native Asian honey bee, Apis cerana and tested the responses of other bee species to these alarm signals. As compared to nest guards, A. cerana foragers produced 3.38 fold higher levels of BA. In foragers, BA and (E)-dec-2-en-1-yl acetate (DA) generated the strongest antennal electrophysiological responses. BA was also the only compound that alerted flying foragers and inhibited A. cerana foraging. BA thereby decreased A. cerana foraging for risky sites. Interestingly, although BA occurs only in trace amounts and is nearly absent in sympatric honeybee species (respectively only 0.07% and 0.44% as much in A. dorsata and A. florea), these floral generalists detected and avoided BA as strongly as they did to their own alarm pheromone on natural inflorescences. These results demonstrate that competing pollinators can take advantage of alarm signal information provided by other species.

In highly social bees, queen mandibular pheromone (QMP) is vital for colony life. Both Apis cerana (Ac) and Apis mellifera (Am) share an evolutionarily conserved set of QMP compounds: (E)-9-oxodec-2-enoic acid (9-ODA), (E)-9-hydroxydec-2-enoic acid (9-HDA), (E)-10-hydroxy-dec-2-enoic acid (10-HDA), 10-hydroxy-decanoic acid (10-HDAA), and methyl p-hydroxybenzoate (HOB) found at similar levels. However, evidence suggests there may be species-specific sensitivity differences to QMP compounds because Ac workers have higher levels of ovarian activation than Am workers. Using electroantennograms, we found species-specific sensitivity differences for a blend of the major QMP compounds and three individual compounds (9-HDA, 10-HDAA, and 10-HDA). As predicted, Am was more sensitive than Ac in all cases (1.3- to 2.7- fold higher responses). There were also species differences in worker retinue attraction to three compounds (9-HDA, HOB, and 10-HDA). In all significantly different cases, Am workers were 4.5- to 6.2-fold more strongly attracted than Ac workers were. Thus, Ac workers responded less strongly to QMP than Am workers, and 9-HDA and 10-HDA consistently elicited stronger antennal and retinue formation responses [corrected].

Relatively little attention has focused on how pesticides may affect Asian honey bees, which provide vital crop pollination services and are key native pollinators. We therefore studied the effects of a relatively new pesticide, flupyradifurone (FLU), which has been developed, in part, because it appears safer for honey bees than neonicotinoids. We tested the effects of FLU on Apis cerana olfactory learning in larvae (lower dose of 0.033 µg/larvae/day over 6 days) and, in a separate experiment, adults (lower dose of 0.066 µg/adult bee/day) at sublethal, field-realistic doses given over 3 days. A worst-case field-realistic dose is 0.44 µg/bee/day. Learning was tested in adult bees. The lower larval dose did not increase mortality, but the lower adult dose resulted in 20% mortality. The lower FLU doses decreased average olfactory learning by 74% (larval treatment) and 48% (adult treatment) and reduced average memory by 48% (larval treatment) and 22% (adult treatment) as compared to controls. FLU at higher doses resulted in similar learning impairments. The effects of FLU, a pesticide that is reported to be safer than neonicotinoids for honey bees, thus deserve greater attention.

Pesticides can pose environmental risks, and a common neonicotinoid pesticide, thiamethoxam, decreases homing success in honey bees. Neonicotinoids can alter bee navigation, but we present the first evidence that neonicotinoid exposure alone can impair the physical ability of bees to fly. We tested the effects of acute or chronic exposure to thiamethoxam on the flight ability of foragers in flight mills. Within 1 h of consuming a single sublethal dose (1.34 ng/bee), foragers showed excitation and significantly increased flight duration (+78%) and distance (+72%). Chronic exposure significantly decreased flight duration (−54%), distance (−56%), and average velocity (−7%) after either one or two days of continuous exposure that resulted in bees ingesting field-relevant thiamethoxam doses of 1.96–2.90 ng/bee/day. These results provide the first demonstration that acute or chronic exposure to a neonicotinoid alone can significantly alter bee flight. Such exposure may impair foraging and homing, which are vital to normal colony function and ecosystem services.

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption and haemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (−50%). Nutritional and pesticide stressors reduced also food consumption (−48%) and haemolymph levels of glucose (−60%) and trehalose (−27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.

Nectar guides can enhance pollinator efficiency and plant fitness by allowing pollinators to more rapidly find and remember the location of floral nectar. We tested if a radiating nectar guide around a nectary would enhance the ability of naïve bumble bee foragers to find nectar. Most experiments that test nectar guide efficacy, specifically radiating linear guides, have used guides positioned around the center of a radially symmetric flower, where nectaries are often found. However, the flower center may be intrinsically attractive. We therefore used an off-center guide and nectary and compared "conjunct" feeders with a nectar guide surrounding the nectary to "disjunct" feeders with a nectar guide separated from the nectary. We focused on the innate response of novice bee foragers that had never previously visited such feeders. We hypothesized that a disjunct nectar guide would conflict with the visual information provided by the nectary and negatively affect foraging. Approximately, equal numbers of bumble bees (Bombus impatiens) found nectar on both feeder types. On disjunct feeders, however, unsuccessful foragers spent significantly more time (on average 1.6-fold longer) searching for nectar than any other forager group. Successful foragers on disjunct feeders approached these feeders from random directions unlike successful foragers on conjunct feeders, which preferentially approached the combined nectary and nectar guide. Thus, the nectary and a surrounding nectar guide can be considered a combination of two signals that attract naïve foragers even when not in the floral center. © 2014 Springer-Verlag Berlin Heidelberg.

In colonial organisms, alarm pheromones can provide a key fitness advantage by enhancing colony defence and warning of danger. Learning which species use alarm pheromone and the key compounds involved therefore enhances our understanding of how this important signal has evolved. However, our knowledge of alarm pheromones is more limited in the social wasps and hornets compared with the social bees and ants.

SUMMARY A nicotinic acetylcholine receptor agonist, imidacloprid, impairs memory formation in honey bees and has general effects on foraging. However, little is known about how this agonist affects two specific aspects of foraging: sucrose responsiveness (SR) and waggle dancing (which recruits nestmates). Using lab and field experiments, we tested the effect of sublethal doses of imidacloprid on (1) bee SR with the proboscis extension response assay, and (2) free-flying foragers visiting and dancing for a sucrose feeder. Bees that ingested imidacloprid (0.21 or 2.16 ng bee–1) had higher sucrose response thresholds 1 h after treatment. Foragers that ingested imidacloprid also produced significantly fewer waggle dance circuits (10.5- and 4.5-fold fewer for 50% and 30% sucrose solutions, respectively) 24 h after treatment as compared with controls. However, there was no significant effect of imidacloprid on the sucrose concentrations that foragers collected at a feeder 24 h after treatment. Thus, imidacloprid temporarily increased the minimum sucrose concentration that foragers would accept (short time scale, 1 h after treatment) and reduced waggle dancing (longer time scale, 24 h after treatment). The effect of time suggests different neurological effects of imidacloprid resulting from the parent compound and its metabolites. Waggle dancing can significantly increase colony food intake, and thus a sublethal dose (0.21 ng bee–1, 24 p.p.b.) of this commonly used pesticide may impair colony fitness.

Pollinators such as bees provide a critical ecosystem service that can be impaired by information about predation. We provide the first evidence for olfactory eavesdropping and avoidance of heterospecific alarm signals, alarm pheromones, at food sources in bees. We predicted that foragers could eavesdrop upon heterospecific alarm pheromones, and would detect and avoid conspicuous individual pheromone compounds, defined by abundance and their ability to persist. We show that Apis cerana foragers avoid the distinctive alarm pheromones of A. dorsata and A. mellifera, species that share the same floral resources and predators. We next examined responses to individual alarm pheromone compounds. Apis cerana foragers avoided isopentyl acetate (IPA), which is found in all three species and is the most abundant and volatile of the tested compounds. Interestingly, A. cerana also avoided an odor component, gamma-octanoic lactone (GOL), which is >150-fold less volatile than IPA. Chemical analyses confirmed that GOL is only present in A. dorsata, not in A. cerana. Electroantennogram (EAG) recordings revealed that A. cerana antennae are 10-fold more sensitive to GOL than to other tested compounds. Thus, the eavesdropping strategy is shaped by signal conspicuousness (abundance and commonality) and signal persistence (volatility).