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.     

Recipe for a Busy Bee: MicroRNAs in Honey Bee Caste Determination

Social caste determination in the honey bee is assumed to be determined by the dietary status of the young larvae and translated into physiological and epigenetic changes through nutrient-sensing pathways. We have employed Illumina/Solexa sequencing to examine the small RNA content in the bee larval food, and show that worker jelly is enriched in miRNA complexity and abundance relative to royal jelly. The miRNA levels in worker jelly were 7–215 fold higher than in royal jelly, and both jellies showed dynamic changes in miRNA content during the 4^th to 6^th day of larval development. Adding specific miRNAs to royal jelly elicited significant changes in queen larval mRNA expression and morphological characters of the emerging adult queen bee. We propose that miRNAs in the nurse bee secretions constitute an additional element in the regulatory control of caste determination in the honey bee.     

Women Use Red in Order to Attract Mates

Among certain non‐human primates, the red‐colored genitalia of females are a sexual ornament and attract males. The preference for red clothes among women is at times explained as being a parallel. We used here a within‐individual design to investigate the signaling role of the color red with a sample of Slovak participants. As expected, women preferred red clothing both in real‐life and would‐be situations more than men. The preference for red (but not for other colors) in mating game scenarios was only significant for women, but not for men. A preference for the color red was shown in particular for clothes on the upper parts of the participants' bodies, irrespective of gender. Women who were actually involved in a romantic sexual relationship had a preference for red in would‐be situations more than single women, although the menstrual cycle, the total number of lifetime sexual partners, and self‐perceived attractiveness were not associated with the preference for the color red. Our results support the sexual signaling hypothesis which suggests that women use the color red to attract potential mates in a similar way as non‐human primates.     

Varroa-Virus Interaction in Collapsing Honey Bee Colonies

Varroa mites and viruses are the currently the high-profile suspects in collapsing bee colonies. Therefore, seasonal variation in varroa load and viruses (Acute-Kashmir-Israeli complex (AKI) and Deformed Wing Virus (DWV)) were monitored in a year-long study. We investigated the viral titres in honey bees and varroa mites from 23 colonies (15 apiaries) under three treatment conditions: Organic acids (11 colonies), pyrethroid (9 colonies) and untreated (3 colonies). Approximately 200 bees were sampled every month from April 2011 to October 2011, and April 2012. The 200 bees were split to 10 subsamples of 20 bees and analysed separately, which allows us to determine the prevalence of virus-infected bees. The treatment efficacy was often low for both treatments. In colonies where varroa treatment reduced the mite load, colonies overwintered successfully, allowing the mites and viruses to be carried over with the bees into the next season. In general, AKI and DWV titres did not show any notable response to the treatment and steadily increased over the season from April to October. In the untreated control group, titres increased most dramatically. Viral copies were correlated to number of varroa mites. Most colonies that collapsed over the winter had significantly higher AKI and DWV titres in October compared to survivors. Only treated colonies survived the winter. We discuss our results in relation to the varroa-virus model developed by Stephen Martin.     

Chemical Profiles of Two Pheromone Glands Are Differentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.)

Pheromones mediate social interactions among individuals in a wide variety of species, from yeast to mammals. In social insects such as honey bees, pheromone communication systems can be extraordinarily complex and serve to coordinate behaviors among many individuals. One of the primary mediators of social behavior and organization in honey bee colonies is queen pheromone, which is produced by multiple glands. The types and quantities of chemicals produced differ significantly between virgin and mated queens, and recent studies have suggested that, in newly mated queens, insemination volume or quantity can affect pheromone production. Here, we examine the long-term impact of different factors involved during queen insemination on the chemical composition of the mandibular and Dufour''s glands, two of the major sources of queen pheromone. Our results demonstrate that carbon dioxide (an anesthetic used in instrumental insemination), physical manipulation of genital tract (presumably mimicking the act of copulation), insemination substance (saline vs. semen), and insemination volume (1 vs. 8 µl) all have long-term effects on mandibular gland chemical profiles. In contrast, Dufour''s gland chemical profiles were changed only upon insemination and were not influenced by exposure to carbon dioxide, manipulation, insemination substance or volume. These results suggest that the chemical contents of these two glands are regulated by different neuro-physiological mechanisms. Furthermore, workers responded differently to the different mandibular gland extracts in a choice assay. Although these studies must be validated in naturally mated queens of varying mating quality, our results suggest that while the chemical composition of Dufour''s gland is associated with mating status, that of the mandibular glands is associated with both mating status and insemination success. Thus, the queen appears to be signaling both status and reproductive quality to the workers, which may impact worker behavior and physiology as well as social organization and productivity of the colony.     

The Shaking Signal of the Honey Bee Informs Workers to Prepare for Greater Activity

One of the most conspicuous activities of worker bees inside a hive is the shaking of other workers. This shaking has long been suspected to be a communication behavior, but its information content and function have until recently remained mysterious. Prior studies of the colony-level patterns of the production of the shaking signal suggest strongly that this signal serves to arouse workers to greater activity, such as at times of good foraging. Data from our observations of individual bees bolster the hypothesis that the shaking signal informs workers to prepare for a higher level of activity. We followed foragers in a colony whose only source of ‘nectar’ was a sugar-water feeder and discovered that when the feeder was left empty for 1–3 d and then refilled, the first bees to find the food initially produced only shaking signals upon return to the hive. It was not until they had completed several trips to the feeder that they began to produce waggle dances. Evidently, the shaking signal and the waggle dance function together to stimulate a colony's foragers to activity.     

Using DNA Metabarcoding to Identify the Floral Composition of Honey: A New Tool for Investigating Honey Bee Foraging Preferences

Identifying the floral composition of honey provides a method for investigating the plants that honey bees visit. We compared melissopalynology, where pollen grains retrieved from honey are identified morphologically, with a DNA metabarcoding approach using the rbcL DNA barcode marker and 454-pyrosequencing. We compared nine honeys supplied by beekeepers in the UK. DNA metabarcoding and melissopalynology were able to detect the most abundant floral components of honey. There was 92% correspondence for the plant taxa that had an abundance of over 20%. However, the level of similarity when all taxa were compared was lower, ranging from 22–45%, and there was little correspondence between the relative abundance of taxa found using the two techniques. DNA metabarcoding provided much greater repeatability, with a 64% taxa match compared to 28% with melissopalynology. DNA metabarcoding has the advantage over melissopalynology in that it does not require a high level of taxonomic expertise, a greater sample size can be screened and it provides greater resolution for some plant families. However, it does not provide a quantitative approach and pollen present in low levels are less likely to be detected. We investigated the plants that were frequently used by honey bees by examining the results obtained from both techniques. Plants with a broad taxonomic range were detected, covering 46 families and 25 orders, but a relatively small number of plants were consistently seen across multiple honey samples. Frequently found herbaceous species were Rubus fruticosus, Filipendula ulmaria, Taraxacum officinale, Trifolium spp., Brassica spp. and the non-native, invasive, Impatiens glandulifera. Tree pollen was frequently seen belonging to Castanea sativa, Crataegus monogyna and species of Malus, Salix and Quercus. We conclude that although honey bees are considered to be supergeneralists in their foraging choices, there are certain key species or plant groups that are particularly important in the honey bees environment. The reasons for this require further investigation in order to better understand honey bee nutritional requirements. DNA metabarcoding can be easily and widely used to investigate floral visitation in honey bees and can be adapted for use with other insects. It provides a starting point for investigating how we can better provide for the insects that we rely upon for pollination.     

Learning and Task Interference by Corpse-removal Specialists in Honey Bee Colonies

Undertakers are considered to be among the most specialized of pre-foraging honey bee (Apis mellifera L.) workers. In this study we examined a possible benefit and a cost of the corpse-rem oval specialty, the improvement in performance with experience, and interference by individuals attempting to perform the same task in the same location, respectively. Experienced bees removed corpses significantly faster than less experienced bees and also were less likely to drop corpses while exiting the hive (5.5% vs. 14.3% of attempts). Superior performance by experienced undertakers might occur as a consequence of learning, or by greater ability from the outset. Because active undertakers (≥ 3 corpse removals) did not improve with experience over their own careers, learning was not demonstrated. An extreme specialist, Yellow 54, removed a total of 114 corpses (33.8% of experimentally introduced dead bees) from the hive over a 13-day period. This is the longest recorded tenure of undertaking to date and demonstrates how a few individuals can dominate this task in a honey bee colony-Yellow 54 removed corpses significantly faster than other active bees, but she demonstrated no obvious improvement in performance over her undertaking career. This suggests the possibility that active undertakers were more talented than less active undertakers, irrespective of learning. When two undertakers worked together to remove a corpse from the hive, they took longer to complete the task than did single individuals. When multiple undertakers flew together from the hive, they were less likely to clear a nearby obstruction than single undertakers and were more likely to drop the corpse within 1 m of the hive. Thus, mutual interference exacted a measurable cost as a result of the undertaking specialization while learning provided few benefits.     

Genotypical Variability for the Tasks of Water Collecting and Scenting in a Honey Bee Colony

The polyandrous mating behaviour of the honey bee queen increases the genotypical variability amongst her worker offspring. Microsatellite DNA analyses revealed a total of 16 subfamilies in one colony of honey bees. The subfamilies were represented in significantly different proportions in two subgroups of bees, water collecting bees and scenting bees, indicating a genetic component in task choice.     

Modeling the Adaptive Role of Negative Signaling in Honey Bee Intraspecific Competition

Collective decision making in the social insects often proceeds via feedback cycles based on positive signaling. Negative signals have, however, been found in a few contexts in which costs exist for paying attention to no longer useful information. Here we incorporate new research on the specificity and context of the negative stop signal into an agent based model of honey bee foraging to explore the adaptive basis of negative signaling in the dance language. Our work suggests that the stop signal, by acting as a counterbalance to the waggle dance, allows colonies to rapidly shut down attacks on other colonies. This could be a key adaptation, as the costs of attacking a colony strong enough to defend itself are significant.     

Comparing Alternative Methods for Holding Virgin Honey Bee Queens for One Week in Mailing Cages before Mating

In beekeeping, queen honey bees are often temporarily kept alive in cages. We determined the survival of newly-emerged virgin honey bee queens every day for seven days in an experiment that simultaneously investigated three factors: queen cage type (wooden three-hole or plastic), attendant workers (present or absent) and food type (sugar candy, honey, or both). Ten queens were tested in each of the 12 combinations. Queens were reared using standard beekeeping methods (Doolittle/grafting) and emerged from their cells into vials held in an incubator at 34C. All 12 combinations gave high survival (90 or 100%) for three days but only one method (wooden cage, with attendants, honey) gave 100% survival to day seven. Factors affecting queen survival were analysed. Across all combinations, attendant bees significantly increased survival (18% vs. 53%, p<0.001). In addition, there was an interaction between food type and cage type (p<0.001) with the honey and plastic cage combination giving reduced survival. An additional group of queens was reared and held for seven days using the best method, and then directly introduced using smoke into queenless nucleus colonies that had been dequeened five days previously. Acceptance was high (80%, 8/10) showing that this combination is also suitable for preparing queens for introduction into colonies. Having a simple method for keeping newly-emerged virgin queens alive in cages for one week and acceptable for introduction into queenless colonies will be useful in honey bee breeding. In particular, it facilitates the screening of many queens for genetic or phenotypic characteristics when only a small proportion meets the desired criteria. These can then be introduced into queenless hives for natural mating or insemination, both of which take place when queens are one week old.     

High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health

Background

Recent declines in honey bees for crop pollination threaten fruit, nut, vegetable and seed production in the United States. A broad survey of pesticide residues was conducted on samples from migratory and other beekeepers across 23 states, one Canadian province and several agricultural cropping systems during the 2007–08 growing seasons.

Methodology/Principal Findings

We have used LC/MS-MS and GC/MS to analyze bees and hive matrices for pesticide residues utilizing a modified QuEChERS method. We have found 121 different pesticides and metabolites within 887 wax, pollen, bee and associated hive samples. Almost 60% of the 259 wax and 350 pollen samples contained at least one systemic pesticide, and over 47% had both in-hive acaricides fluvalinate and coumaphos, and chlorothalonil, a widely-used fungicide. In bee pollen were found chlorothalonil at levels up to 99 ppm and the insecticides aldicarb, carbaryl, chlorpyrifos and imidacloprid, fungicides boscalid, captan and myclobutanil, and herbicide pendimethalin at 1 ppm levels. Almost all comb and foundation wax samples (98%) were contaminated with up to 204 and 94 ppm, respectively, of fluvalinate and coumaphos, and lower amounts of amitraz degradates and chlorothalonil, with an average of 6 pesticide detections per sample and a high of 39. There were fewer pesticides found in adults and brood except for those linked with bee kills by permethrin (20 ppm) and fipronil (3.1 ppm).

Conclusions/Significance

The 98 pesticides and metabolites detected in mixtures up to 214 ppm in bee pollen alone represents a remarkably high level for toxicants in the brood and adult food of this primary pollinator. This represents over half of the maximum individual pesticide incidences ever reported for apiaries. While exposure to many of these neurotoxicants elicits acute and sublethal reductions in honey bee fitness, the effects of these materials in combinations and their direct association with CCD or declining bee health remains to be determined.     

Down-Regulation of Honey Bee IRS Gene Biases Behavior toward Food Rich in Protein

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees'' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect''s foraging choice between protein and carbohydrate sources.     

Brood Pheromone Modulation of Pollen Forager Turnaround Time in the Honey Bee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Foraging for pollen is an important behavior of the honey bee because pollen is their sole source of protein. Through nurse bees, larvae are the principal consumers of pollen. Fatty acid esters extractable from the surface of larvae, called brood pheromone, release multiple colony-level and individual foraging behaviors increasing pollen intake. In this study pollen forager turnaround time was measured in observation hives supplemented with brood pheromone versus a blank control treatment. Treatment with brood pheromone significantly decreased pollen forager turnaround time in the hive between foraging bouts by approximately 72%. Concurrently, brood pheromone increased the ratio of pollen to non-pollen foragers entering colonies. Brood pheromone has been shown to release most of the mechanisms known to increase pollen intake by colonies acting as an important regulator of colony foraging decisions and growth.