Olfactory responses of Aethina tumida murray (Coleoptera: Nitidulidae) to some major volatile compounds from hive materials and workers of Apis mellifera

The small hive beetle (Aethina tumida Murray) is an invasive pest affecting honey bee colonies. The beetles are known to be attracted to volatiles from hive products and honey bees like Apis mellifera L. Previously we reported the presence of five major compounds from the volatile extracts of hive materials; ethyl linolenate and ethyl palmitate from pollen dough, oleamide and tetracosane in fermenting honey, and oleamide and 5-methyl-2-phenyl-1H-indole from A. mellifera worker bees. This study tested the attractiveness of the aforementioned five volatile organic compounds to small hive beetles (SHB) by Y-tube olfactometric bioassay. Ethyl linolenate was highly attractive to both male and female adults of SHB. Ethyl palmitate was attractive to SHB only at higher concentration (0.01–01 mg/ml). Interestingly, tetracosane, 5-methyl-2-phenyl-1H-indole and oleamide were repellent for SHB of both sexes, but ethyl linolenate and ethyl palmitate as components of honey bee brood pheromone attracted SHB. The results highlight that SHB differentially utilizes volatile chemicals from hive materials and honey bees as cues to locate honey bee hives.     

Unique Honey Bee (Apis mellifera) Hive Component-Based Communities as Detected by a Hybrid of Phospholipid Fatty-Acid and Fatty-Acid Methyl Ester Analyses

Microbial communities (microbiomes) are associated with almost all metazoans, including the honey bee Apis mellifera. Honey bees are social insects, maintaining complex hive systems composed of a variety of integral components including bees, comb, propolis, honey, and stored pollen. Given that the different components within hives can be physically separated and are nutritionally variable, we hypothesize that unique microbial communities may occur within the different microenvironments of honey bee colonies. To explore this hypothesis and to provide further insights into the microbiome of honey bees, we use a hybrid of fatty acid methyl ester (FAME) and phospholipid-derived fatty acid (PLFA) analysis to produce broad, lipid-based microbial community profiles of stored pollen, adults, pupae, honey, empty comb, and propolis for 11 honey bee hives. Averaging component lipid profiles by hive, we show that, in decreasing order, lipid markers representing fungi, Gram-negative bacteria, and Gram-positive bacteria have the highest relative abundances within honey bee colonies. Our lipid profiles reveal the presence of viable microbial communities in each of the six hive components sampled, with overall microbial community richness varying from lowest to highest in honey, comb, pupae, pollen, adults and propolis, respectively. Finally, microbial community lipid profiles were more similar when compared by component than by hive, location, or sampling year. Specifically, we found that individual hive components typically exhibited several dominant lipids and that these dominant lipids differ between components. Principal component and two-way clustering analyses both support significant grouping of lipids by hive component. Our findings indicate that in addition to the microbial communities present in individual workers, honey bee hives have resident microbial communities associated with different colony components.     

A new threat to honey bees, the parasitic phorid fly Apocephalus borealis

Honey bee colonies are subject to numerous pathogens and parasites. Interaction among multiple pathogens and parasites is the proposed cause for Colony Collapse Disorder (CCD), a syndrome characterized by worker bees abandoning their hive. Here we provide the first documentation that the phorid fly Apocephalus borealis, previously known to parasitize bumble bees, also infects and eventually kills honey bees and may pose an emerging threat to North American apiculture. Parasitized honey bees show hive abandonment behavior, leaving their hives at night and dying shortly thereafter. On average, seven days later up to 13 phorid larvae emerge from each dead bee and pupate away from the bee. Using DNA barcoding, we confirmed that phorids that emerged from honey bees and bumble bees were the same species. Microarray analyses of honey bees from infected hives revealed that these bees are often infected with deformed wing virus and Nosema ceranae. Larvae and adult phorids also tested positive for these pathogens, implicating the fly as a potential vector or reservoir of these honey bee pathogens. Phorid parasitism may affect hive viability since 77% of sites sampled in the San Francisco Bay Area were infected by the fly and microarray analyses detected phorids in commercial hives in South Dakota and California's Central Valley. Understanding details of phorid infection may shed light on similar hive abandonment behaviors seen in CCD.     

Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera

Studies of newly emerged Apis mellifera worker bees have demonstrated that their guts are colonized by a consistent core microbiota within several days of eclosure. We conducted experiments aimed at illuminating the transmission routes and spatiotemporal colonization dynamics of this microbiota. Experimental groups of newly emerged workers were maintained in cup cages and exposed to different potential transmission sources. Colonization patterns were evaluated using quantitative real-time PCR (qPCR) to assess community sizes and using deep sequencing of 16S rRNA gene amplicons to assess community composition. In addition, we monitored the establishment of the ileum and rectum communities within workers sampled over time from natural hive conditions. The study verified that workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. Typical communities, resembling those of workers within hives, were established in the presence of nurse workers or nurse worker fecal material, and atypical communities of noncore or highly skewed compositions were established when workers were exposed only to oral trophallaxis or hive components (comb, honey, bee bread). The core species of Gram-negative bacteria, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara, were dependent on the presence of nurses or hindgut material, whereas some Gram-positive species were more often transferred through exposure to hive components. These results indicate aspects of the colony life cycle and behavior that are key to the propagation of the characteristic honey bee gut microbiota.     

Competition between honey bees and wild bees and the role of nesting resources in a nature reserve

The European honey bee exploits floral resources efficiently and may therefore compete with solitary wild bees. Hence, conservationists and bee keepers are debating about the consequences of beekeeping for the conservation of wild bees in nature reserves. We observed flower-visiting bees on flowers of Calluna vulgaris in sites differing in the distance to the next honey-bee hive and in sites with hives present and absent in the Lüneburger Heath, Germany. Additionally, we counted wild bee ground nests in sites that differ in their distance to the next hive and wild bee stem nests and stem-nesting bee species in sites with hives present and absent. We did not observe fewer honey bees or higher wild bee flower visits in sites with different distances to the next hive (up to 1,229 m). However, wild bees visited fewer flowers and honey bee visits increased in sites containing honey-bee hives and in sites containing honey-bee hives we found fewer stem-nesting bee species. The reproductive success, measured as number of nests, was not affected by distance to honey-bee hives or their presence but by availability and characteristics of nesting resources. Our results suggest that beekeeping in the Lüneburg Heath can affect the conservation of stem-nesting bee species richness but not the overall reproduction either of stem-nesting or of ground-nesting bees. Future experiments need control sites with larger distances than 500 m to hives. Until more information is available, conservation efforts should forgo to enhance honey bee stocking rates but enhance the availability of nesting resources.     

Exposure scheme separates effects of electric shock and electric field for honey bees, Apis mellifera L

Mechanisms to explain disturbance of honey bee colonies under a 765-kV, 60-Hz transmission line [electric (E) field = 7 kV/m] fall into two categories: direct bee perception of enhanced in-hive E fields, and perception of shock from induced currents. The same adverse biological effects previously observed in honey bee colonies exposed under a 765-kV transmission line can be reproduced by exposing worker bees to shock or E field within elongated hive entranceways (= tunnels). Exposure to intense E field caused disturbance only if bees were in contact with a conductive substrate. E-field and shock exposure can be separated and precisely defined within tunnels, eliminating dosimetric vagaries that occur when entire hives are exposed to E field.     

Mechanism of biological effects observed in honey bees (apis mellifera,L.) hived under extra-high-voltage transmission lines: Implications derived from bee exposure to simulated intense electric fields and shocks

This work explores mechanisms for disturbance of honey bee colonies under a 765 kV, 60-Hz transmission line [electric (E) field = 7 kV/m] observed in previous studies. Proposed mechanisms fell into two categories: direct bee perception of enhanced in-hive E fields and perception of shock from induced currents. The adverse biological effects could be reproduced in simulations where only the worker bees were exposed to shock or to E field in elongated hive entranceways (=tunnels). We now report the results of full-scale experiments using the tunnel exposure scheme, which assesses the contribution of shock and intense E field to colony disturbance. Exposure of worker bees (1,400 h) to 60-Hz E fields including 100 kV/m under moisture-free conditions within a nonconductive tunnel causes no deleterious affect on colony behavior. Exposure of bees in conductive (e.g., wet) tunnels produces bee disturbance, increased mortality, abnormal propolization, and possible impairment of colony growth. We propose that this substrate dependence of bee disturbance is the result of perception of shock from coupled body currents and enhanced current densities postulated to exist in the legs and thorax of bees on conductors. Similarly, disturbance occurs when bees are exposed to step-potential-induced currents. At 275–350 nA single bees are disturbed; at 600 nA bees begin abnormal propolization behavior; and stinging occurs at 900 nA. We conclude that biological effects seen in bee colonies under a transmission line are primarily the result of electric shock from induced hive currents. This evaluation is based on the limited effects of E-field exposure in tunnels, the observed disturbance thresholds caused by shocks in tunnels, and the ability of hives exposed under a transmission line to source currents 100–1,000 times the shock thresholds.     

The influence of colony population and brood rearing intensity on the activity of detoxifying enzymes in worker honey bees

Abstract. Cohorts of worker honey bees from a single parental hive, cross-fostered into colonies which differed in population and other colony parameters, were assessed for activities of enzymes involved in metabolic detoxication. Activities of glutathione S-transferase and mixed-function oxidase enzymes were negatively correlated with foster colony population, but positively correlated with the ratio of larvae (brood)/adult workers. Worker bees which had begun foraging had enzyme activity levels higher than any found in bees which were still performing in-hive duties. Elevated levels of detoxifying enzymes in colonies with low populations and high ratios of larvae/adults may be a protective mechanism to prevent poisoning of larvae by toxins brought to the colony by foragers.     

Retention of long-term memories in different age groups of honeybee (Apis mellifera) workers

Honeybees can learn food odors inside the nest during food sharing and use this information during flower choice, dance choice or choice of a trophallactic partner. We investigated for how long odors learned inside the hive are retained by bees of different age groups. In our study, bees retrieved food odors for up to 10–11 days. Our results suggest that olfactory information acquired by young bees while performing in-hive duties can be retrieved when performing foraging duties later in life.     

Temperature-Dependent Clustering Behavior of Aethina Tumida Murray in Apis Mellifera L. Colonies

Temperate races of honey bees (Apis mellifera) are able to survive cold temperatures by forming thermoregulatory clusters. Small hive beetles (Aethina tumida), which inhabit honey bee colonies in their native range of sub-Saharan Africa and in their introduced ranges of the United States and Australia, are able to endure temperate climates by entering the bee cluster when cold temperatures persist. We conducted an experiment to address the temporal aspects of the cluster-entering behavior of small hive beetles. We did this by exposing beetle-infested observation bee hives to different ambient temperatures and counting the number of beetles remaining in confinement sites on the hive’s periphery at each temperature. The resulting regression analyses suggest that the beetles enter the cluster more rapidly than they exit it, a behavior possibly linked to a colony’s decision to form and dismantle a cluster.     

Does the Earth's Magnetic Field Serve as a Reference for Alignment of the Honeybee Waggle Dance?

The honeybee (Apis mellifera) waggle dance, which is performed inside the hive by forager bees, informs hive mates about a potent food source, and recruits them to its location. It consists of a repeated figure-8 pattern: two oppositely directed turns interspersed by a short straight segment, the “waggle run”. The waggle run consists of a single stride emphasized by lateral waggling motions of the abdomen. Directional information pointing to a food source relative to the sun''s azimuth is encoded in the angle between the waggle run line and a reference line, which is generally thought to be established by gravity. Yet, there is tantalizing evidence that the local (ambient) geomagnetic field (LGMF) could play a role. We tested the effect of the LGMF on the recruitment success of forager bees by placing observation hives inside large Helmholtz coils, and then either reducing the LGMF to 2% or shifting its apparent declination. Neither of these treatments reduced the number of nest mates that waggle dancing forager bees recruited to a feeding station located 200 m north of the hive. These results indicate that the LGMF does not act as the reference for the alignment of waggle-dancing bees.     

Key factors influencing forager distribution across macadamia orchards differ among species of managed bees

To achieve maximised and sustainable crop productivity, it is critical that we develop crop-specific strategies for managing pollination. Honey bees (Apis mellifera) and stingless bees (Tetragonula carbonaria) are considered effective pollinators of macadamia (Macadamia integrifolia). The introduction of managed honey bee or stingless bee hives into orchards is likely to boost the numbers of these insects visiting flowers; however, there is a lack of published information and consensus regarding their management for pollination. Here, we identify factors that affect the distribution of both honey bees and stingless bees across cultivated macadamia, and establish whether increased flower visitation leads to higher nut set. A gradient of bee visitation rates was created by placing colonies on the ends of a four-hectare block, and mixed-effect models were applied to assess forager abundance and nut set with respect to distance from hive, time of day, cultivar, and floral display size. Distance from colony had a strong effect on stingless bee numbers, with >96% of individuals recorded within 100 metres of colonies, whereas the distribution of honey bees was more closely related to daily floral display: trees with greater numbers of flowers attracted more honey bees. Simplified surveys conducted in a further 17 macadamia blocks confirm that these are broadly occurring distribution patterns. Bee abundance alone did not significantly predict nut production; however, an indirect effect of bee visits to flowers is inferred, as nut production increased with size of floral display. To encourage a more even distribution of bees and uniform pollination, we recommend placement of stingless bee hives to maximise their distribution through a block (e.g. at 100-m intervals) and management practices that promote even distributions of flowers across trees.     

Return of Drones: Flight Experience Improves Returning Performance in Honeybee Drones

The effect of experience on the behavior of worker bees has been extensively investigated; however, few such studies have been conducted on male bees. Honeybee (Apis mellifera) males (drones), unlike the males of other social hymenopterans, return to their nest after performing a mating flight and have, therefore, an opportunity to learn from their experiences. This provides a chance to understand the significance of experience in social hymenopteran males. Here, we investigated whether experience improves the returning performance in drones (rate and time of return to the hive). We compared the returning performance of “Experienced” drones that were allowed to fly freely and thus had an opportunity to learn the position of the hive before the experiment with “Naive” drones that were not allowed to fly and therefore, had no opportunity to learn. We found that Experienced drones returned to the hive after a displacement, whereas Naive drones did not. Furthermore, time to return decreased with the age of drones. These results suggest that flight experience improves the returning performance, which should increase the possibility of mating success and overall colony fitness.     

Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.     

Increased attractiveness of honeybee hive product volatiles to adult small hive beetle,Aethina tumida,resulting from small hive beetle larval infestation

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a recent but significant pest of honeybee [Apis mellifera L. (Hymenoptera: Apidae)] hives in various regions throughout the world, including Eastern Australia. The larval stage of this beetle damages hives when they feed on brood, pollen, and honeycomb, leaving behind fermented wastes. In cases of extreme damage, hives collapse and are turned to an odorous mass of larvae in fermenting hive products. The yeast Kodamaea ohmeri (Etchells & Bell) Yamada et al. (Ascomycota) has been consistently isolated from the fermenting material as well as each life stage of this beetle. Various studies have noted that the small hive beetle is attracted to volatiles from hive products and those of the yeast K. ohmeri, although earlier studies have not used naturally occurring hive products as their source of fermentation. This study investigated changes through time in the attractiveness of natural honeybee hive products to the small hive beetle as the hive products were altered by the action of beetle larvae and fermentation by K. ohmeri. We used gas chromatography‐mass spectrometry and choice‐test behavioural assays to investigate these changes using products sampled from three apiaries. Attractiveness of the fermenting hive products (‘slime’) increased as fermentation progressed, and volatile profiles became more complex. Fermenting hive products remained extremely attractive for more than 30 days, significantly longer than previous reports. These results have strong implications for the development of an external attractant trap to assist in the management of this invasive pest.     

Behavioral defense strategies of the stingless bee, <Emphasis Type="Italic">Austroplebeia australis</Emphasis>, against the small hive beetle, <Emphasis Type="Italic">Aethina tumida</Emphasis>

Small hive beetle, Aethina tumida Murray, is a parasite of social bee colonies and has become an invasive species, raising concern of the potential threat to native pollinators in its new ranges. Here, we report the defensive behavior strategies used by workers of the Australian stingless bee, Austroplebeia australis Friese, against the small hive beetle. A non-destructive method was used to observe in-hive behavior and interactions between bees and different life stages of small hive beetle (egg, larva, and adult). A number of different individual and group defensive behaviors were recorded. Up to 97% of small hive beetle eggs were destroyed within 90 min of introduction, with a significant increase in temporal rate of destruction between the first and subsequent introductions. A similar result was recorded for 3-day-old small hive beetle larvae, with an increased removal rate from 62.5 to 92.5% between the first and second introductions. Of 32 adult beetles introduced directly into the 4 colonies, 59% were ejected, with the remainder being entombed alive in hives within 6 h. Efficiency of ejection also significantly increased between the first and third introductions. Our observations suggest that A. australis colonies, despite no previous exposure to this exotic parasite, have well developed hive defences that are likely to minimize entry and survival of small hive beetles.     

Pathogenesis of varroosis at the level of the honey bee (Apis mellifera) colony

The parasitic mite Varroa destructor, in interaction with different viruses, is the main cause of honey bee colony mortality in most parts of the world. Here we studied how effects of individual-level parasitization are reflected by the bee colony as a whole. We measured disease progression in an apiary of 24 hives with differing degree of mite infestation, and investigated its relationship to 28 biometrical, physiological and biochemical indicators. In early summer, when the most heavily infested colonies already showed reduced growth, an elevated ratio of brood to bees, as well as a strong presence of phenoloxidase/prophenoloxidase in hive bees were found to be predictors of the time of colony collapse. One month later, the learning performance of worker bees as well as the activity of glucose oxidase measured from head extracts were significantly linked to the timing of colony collapse. Colonies at the brink of collapse were characterized by reduced weight of winter bees and a strong increase in their relative body water content. Our data confirm the importance of the immune system, known from studies of individually-infested bees, for the pathogenesis of varroosis at colony level. However, they also show that single-bee effects cannot always be extrapolated to the colony as a whole. This fact, together with the prominent role of colony-level factors like the ratio between brood and bees for disease progression, stress the importance of the superorganismal dimension of Varroa research.     

Stingless bees (<Emphasis Type="Italic">Scaptotrigona pectoralis</Emphasis>) learn foreign trail pheromones and use them to find food

Foragers of several species of stingless bees (Hymenoptera, Apidae and Meliponini) deposit pheromone marks in the vegetation to guide nestmates to new food sources. These pheromones are produced in the labial glands and are nest and species specific. Thus, an important question is how recruited foragers recognize their nestmates’ pheromone in the field. We tested whether naïve workers learn a specific trail pheromone composition while being recruited by nestmates inside the hive in the species Scaptotrigona pectoralis. We installed artificial scent trails branching off from trails deposited by recruiting foragers and registered whether newly recruited bees follow these trails. The artificial trails were baited with trail pheromones of workers collected from foreign S. pectoralis colonies. When the same foreign trail pheromone was presented inside the experimental hives while recruitment took place a significant higher number of bees followed the artificial trails than in experiments without intranidal presentation. Our results demonstrate that recruits of S. pectoralis can learn the composition of specific trail pheromone bouquets inside the nest and subsequently follow this pheromone in the field. We, therefore, suggest that trail pheromone recognition in S. pectoralis is based on a flexible learning process rather than being a genetically fixed behaviour.     

The influence of gustatory and olfactory experiences on responsiveness to reward in the honeybee

Background

Honeybees (Apis mellifera) exhibit an extraordinarily tuned division of labor that depends on age polyethism. This adjustment is generally associated with the fact that individuals of different ages display different response thresholds to given stimuli, which determine specific behaviors. For instance, the sucrose-response threshold (SRT) which largely depends on genetic factors may also be affected by the nectar sugar content. However, it remains unknown whether SRTs in workers of different ages and tasks can differ depending on gustatory and olfactory experiences.

Methodology

Groups of worker bees reared either in an artificial environment or else in a queen-right colony, were exposed to different reward conditions at different adult ages. Gustatory response scores (GRSs) and odor-memory retrieval were measured in bees that were previously exposed to changes in food characteristics.

Principal Findings

Results show that the gustatory responses of pre-foraging-aged bees are affected by changes in sucrose solution concentration and also to the presence of an odor provided it is presented as scented sucrose solution. In contrast no differences in worker responses were observed when presented with odor only in the rearing environment. Fast modulation of GRSs was observed in older bees (12–16 days of age) which are commonly involved in food processing tasks within the hive, while slower modulation times were observed in younger bees (commonly nurse bees, 6–9 days of age). This suggests that older food-processing bees have a higher plasticity when responding to fluctuations in resource information than younger hive bees. Adjustments in the number of trophallaxis events were also found when scented food circulated inside the nest, and this was positively correlated with the differences in timing observed in gustatory responsiveness and memory retention for hive bees of different age classes.

Conclusions

This work demonstrates the accessibility of chemosensory information in the honeybee colonies with respect to incoming nectar. The modulation of the sensory-response systems within the hive can have important effects on the dynamics of food transfer and information propagation.