Protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels.

The stop signal is produced in response to negative experiences at the food source and inhibits honey bee (Apis mellifera) waggle dancing. Here, we present a protocol for measuring the effects of an inhibitory signal associated with danger on honey bee dopamine levels. We describe steps for observing honey bee colonies, training them with artificial nectar, and simulating hornet attacks. We then detail procedures for recording waggle dancing and stop signals and measuring brain dopamine levels during different treatments. For complete details on the use and execution of this protocol, please refer to Dong et al.1.

The Varroa paradox: infestation levels and hygienic behavior in feral scutellata-hybrid and managed Apis mellifera ligustica honey bees.

The Varroa destructor mite is a parasitic threat to managed and feral honey bee colonies around the world. Beekeepers use miticides to eliminate Varroa in commercial hives, but these chemicals can diminish bee health and increase miticide resistance. In contrast, feral honey bees have developed multiple ways to counteract mites without chemical treatment. We compared mite levels, grooming habits, and mite-biting behavior between feral Africanized honey bees (genomically verified Apis mellifera scutellata hybrids) and managed Italian honey bees (A. mellifera ligustica). Surprisingly, there was no difference in mite infestation levels between scutellata-hybrids and managed bees over one year despite the regular use of miticides in managed colonies. We also found no differences in the social immunity responses of the two groups, as measured by their hygienic habits (through worker brood pin-kill assays), self-grooming, and mite-biting behavior. However, we provide the first report that both scutellata-hybrids and managed honey bees bite off mite chemosensory forelegs, which the mites use to locate brood cells for reproduction, to a significantly greater degree than other legs (a twofold greater reduction in foreleg length relative to the most anterior legs). Such biting may impair mite reproduction.

A Method for Studying Social Signal Learning of the Waggle Dance in Honey Bees.

Honey bees use a complex form of spatial referential communication. Their waggle dance communicates to nestmates the direction, distance, and quality of a resource by encoding celestial cues, retinal optic flow, and relative food value into motion and sound within the nest. This protocol was developed to investigate the potential for social learning of this waggle dance. Using this protocol, we showed that correct waggle dancing requires social learning. Bees (Apis mellifera) that did not follow any dances before they first danced produced significantly more disordered dances, with larger waggle angle divergence errors, and encoded distance incorrectly. The former deficits improved with experience, but distance encoding was set for life. The first dances of bees that could follow other dancers had none of these impairments. Social learning, therefore, shapes honey bee signaling, as it does communication in human infants, birds, and multiple other vertebrate species. However, much remains to be learned about insects' social learning, and this protocol will help to address knowledge gaps in the understanding of sophisticated social signal learning, particularly in understanding the molecular bases for such learning. Key features It was unclear if honey bees (Apis mellifera) could improve their waggle dance by following experienced dancers before they first waggle dance. Honey bees perform their first waggle dances with more errors if they cannot follow experienced waggle dancers first. Directional and disorder errors improved over time, but distance error was maintained. Bees in experimental colonies continued to communicate longer distances than control bees. Dancing correctly, with less directional error and disorder, requires social learning. Distance encoding in the honey bee dance is largely genetic but may also include a component of cultural transmission.

Seasonal variation in defense behavior in European and scutellata-hybrid honey bees (Apis mellifera) in Southern California.

Nest defense in the honey bee (Apis mellifera) is a complex collective behavior modulated by various interacting social, environmental, and genetic factors. Scutellata-hybrid ("Africanized") honey bees are usually considered to be far more defensive than European honey bees which are therefore preferred for commercial and hobbyist beekeeping. In the most recent zone of scutellata hybridization, the southern USA, the degree to which this defensiveness differs among current strains, and the extent to which defensiveness varies across a season has not been measured. We quantified the levels of A. m. scutellata ancestry in colonies and conducted a seasonal assessment (May through November) of colony nest defensiveness in feral scutellata-hybrid and a popular lineage of European honey bee commonly used in managed environments (sold as A. mellifera ligustica) hives at two apiaries in Southern California. Standard measures of defensiveness were low in both scutellata-hybrid and European colonies during May. Defensiveness increased during the later months of the study in scutellata-hybrid colonies. Most measures of defensiveness did not increase in managed colonies. Defensiveness in the scutellata-hybrids appears lower than what has been previously documented in Brazil and Mexico, possibly due to their lower proportion of A. m. scutellata ancestry.

An inhibitory signal associated with danger reduces honeybee dopamine levels.

Positive and negative experiences can alter animal brain dopamine levels.1 When first arriving at a rewarding food source or beginning to waggle dance and recruit nestmates to food, honeybees have increased brain dopamine levels, indicating a desire for food.2 We provide the first evidence that an inhibitory signal, the stop signal, which counters waggle dancing and is triggered by negative events at the food source, can decrease head dopamine levels and dancing, independent of the dancer having any negative experiences. The hedonic value of food can therefore be depressed simply by the receipt of an inhibitory signal. Increasing the brain dopamine levels reduced the aversive effects of an attack, increasing the time that bees spent subsequently feeding and waggle dancing and decreasing their stop signaling and time spent in the hive. Because honeybees regulate food recruitment and its inhibition at the colony level, these results highlight the complex integration of colony information with a basic and highly conserved neural mechanism in mammals and insects.2 VIDEO ABSTRACT.

Social signal learning of the waggle dance in honey bees.

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.

Identification of giant hornet Vespa mandarinia queen sex pheromone components.

The Vespidae is a diverse family of wasps and hornets that are formidable predators of insects, including social bees1, and includes a number of invasive species2. Recently, the world's largest hornet, Vespa mandarinia Smith (Hymenoptera: Vespidae), which occurs naturally in the Indomalayan region, has been found in Canada and the United States2. Some simulations indicate that it could rapidly spread throughout Washington and Oregon in the western US, as well as some eastern parts of the country2,3, threaten native bees and honeybees, and harm bee-pollinated crop production worth over $100 million annually3. There is consequently an urgent need to learn more about V. mandarinia's reproductive biology and to develop trapping methods to locate its nests and to control its reproduction. We identified V. mandarinia queen-produced sex pheromone from the 5th and 6th intersegmental sternal glands of virgin queens. The major active compounds were hexanoic acid, octanoic acid, and decanoic acid. When placed in field traps, the synthetic compounds and a queen-equivalent mixture rapidly attracted hundreds of males but no females or other species.

Responsiveness to inhibitory signals changes as a function of colony size in honeybees (Apis mellifera).

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.

Functional characterization, antimicrobial effects, and potential antibacterial mechanisms of new mastoparan peptides from hornet venom (Vespa ducalis, Vespa mandarinia, and Vespa affinis).

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 16 µg/mL and 32 µg/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.

Lethality of Honey Bee Stings to Heavily Armored Hornets.

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.

Long-term field-realistic exposure to a next-generation pesticide, flupyradifurone, impairs honey bee behaviour and survival.

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.

New bioactive peptides from the venom gland of a social hornet Vespa velutina.

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.

Honey robbing: could human changes to the environment transform a rare foraging tactic into a maladaptive behavior?

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.

Flupyradifurone reduces nectar consumption and foraging but does not alter honey bee recruitment dancing.

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 4 ppm 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 (2.5 M, 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.

Floral tea polyphenols can improve honey bee memory retention and olfactory sensitivity.

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.

Visual contagion in prey defence signals can enhance honest defence.

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.

Effects of Nosema ceranae (Dissociodihaplophasida: Nosematidae) and Flupyradifurone on Olfactory Learning in Honey Bees, Apis mellifera (Hymenoptera: Apidae).

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.

Thiamethoxam impairs honey bee visual learning, alters decision times, and increases abnormal behaviors.

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.

Influence of visual targets and landmarks on honey bee foraging and waggle dancing.

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 it 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 that foraging bees heavily utilize these visual cues at the later stages of their journey.