Factors determining the release of Nasonov pheromone by honeybees at the hive entrance

ABSTRACT. Worker bees recently denied access to their colony expose their Nasonov glands, thereby releasing pheromone, at the entrance to their hive. Odours of the following induced this response: empty comb, purified beeswax, honey, pollen, propolis, a live queen, the (E)-9-hydroxy-2-decenoic acid component of a queen's mandibular glands, live drones and workers, inert material on which workers had walked inside the hive, and synthetic Nasonov pheromone. The total odour of a foreign colony also induced worker bees to expose their Nasonov glands but was less effective than the odour of their own colony. Odours of the following were not effective: the (E)-9-oxo-2-decenoic acid component of a queen's mandibular glands, recently killed drones and workers, worker brood (eggs, larvae, pupae).     

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.     

Behavioral responses of the small hive beetle,Aethina tumida,to odors of three meliponine bee species and honey bees,Apis mellifera scutellata

Recent studies have shown that honey bees, bumble bees, and some meliponine bee species of the genera Trigona, Meliponula, and Dactylurina are hosts of the small hive beetle (SHB) Aethina tumidaMurray (Coleoptera: Nitidulidae), a pest of honey bee colonies in various regions of the world. Olfaction has been implicated in SHB infestations of honey bee and bumble bee colonies. We used olfactometer bioassays to investigate responses of adult male and female SHBs to odors from intact colonies and separate hive components (pot honey, pot pollen, cerumen, and propolis) of three African meliponine bee species, Meliponula ferruginea (Lepeletier) (black morphospecies), M. ferruginea (reddish brown morphospecies), and Meliponula bocandei (Spinola) (Hymenoptera: Apidae). Although both sexes of the beetle strongly preferred intact colony, pot honey, and pot pollen odors, there was no evidence of attraction to propolis and cerumen odors from the three meliponine bee species. Both sexes of SHB also strongly preferred odors from honey bees, Apis mellifera L. (Hymenoptera: Apidae), over odors from the three meliponine bee species. Our results provide substantial evidence of the host potential of African meliponine bees for the SHB, and we discuss this complex association of the SHB with species within the Apidae family.     

Honey Bees Modulate Their Olfactory Learning in the Presence of Hornet Predators and Alarm Component

In Southeast Asia the native honey bee species Apis cerana is often attacked by hornets (Vespa velutina), mainly in the period from April to November. During the co-evolution of these two species honey bees have developed several strategies to defend themselves such as learning the odors of hornets and releasing alarm components to inform other mates. However, so far little is known about whether and how honey bees modulate their olfactory learning in the presence of the hornet predator and alarm components of honey bee itself. In the present study, we test for associative olfactory learning of A. cerana in the presence of predator odors, the alarm pheromone component isopentyl acetate (IPA), or a floral odor (hexanal) as a control. The results show that bees can detect live hornet odors, that there is almost no association between the innately aversive hornet odor and the appetitive stimulus sucrose, and that IPA is less well associated with an appetitive stimulus when compared with a floral odor. In order to imitate natural conditions, e.g. when bees are foraging on flowers and a predator shows up, or alarm pheromone is released by a captured mate, we tested combinations of the hornet odor and floral odor, or IPA and floral odor. Both of these combinations led to reduced learning scores. This study aims to contribute to a better understanding of the prey-predator system between A. cerana and V. velutina.     

Old comb for nesting site recognition by <Emphasis Type="Italic">Apis dorsata?</Emphasis> Field experiments in China

The Asian giant honey bee, Apis dorsata, often conducts seasonal, long-distance migrations in southern China, between a preferred tree (having more than one nest) and alternate sites. Although worker bees cannot make a round-trip journey, colonies re-utilize preferred trees after an absence of several months. We performed comb experiments in which bases and all abandoned combs were entirely scraped off trees and their sites covered with plastic, or comb was moved to trees of the same species. Swarms of giant honey bees investigated trees where combs were removed and continued to nest on the same trees. In contrast, placing combs in nets on previously used trees, or on nearby trees of the same species, did not attract more swarms. The same number of colonies that left them returned to previously occupied trees. Our findings suggest that direct olfactory or sensory contact with old comb bases might regulate nest establishment, but individual trees, lacking normal visual or chemical cues of old nests, are relocated using behavioral devices that remain to be elucidated. Received 12 February 2007; revised 5 June 2007; accepted 13 September 2007.     

Longevity and reproductive success of Aethina tumida (Coleoptera: Nitidulidae) fed different natural diets

The longevity and reproductive success of newly emerged, unfed adult Aethina tumida Murray assigned different diets (control = unfed; honey-pollen; honey; pollen; empty brood comb; bee brood; fresh Kei apples; and rotten Kei apples) were determined. Longevity in honey-fed small hive beetle adults (average maximum: 167 d) was significantly higher than on other diets. Small hive beetles fed empty brood comb lived significantly longer (average maximum: 49.8 d) than unfed beetles (average maximum: 9.6 d). Small hive beetle offspring were produced on honey-pollen, pollen, bee brood, fresh Kei apples, and rotten Kei apples but not on honey alone, empty brood comb, or in control treatments. The highest reproductive success occurred in pollen fed adults (1773.8 +/- 294.4 larvae per three mating pairs of adults). The data also show that A. tumida can reproduce on fruits alone, indicating that they are facultative parasites. The pupation success and sex ratio of small hive beetle offspring were also analyzed. Larvae fed pollen, honey-pollen, or brood had significantly higher pupation success rates of 0.64, 0.73, and 0.65 respectively than on the other diets. Sex ratios of emerging adults fed diets of pollen or brood as larvae were significantly skewed toward females. Because small hive beetle longevity and overall reproductive success was highest on foodstuffs located in honey bee colonies, A. tumida are efficient at causing large-scale damage to colonies of honey bees resulting in economic injury for the beekeeper. Practical considerations for the control of A. tumida are briefly discussed.     

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.     

大蜂螨对一些气味物质的趋性

狄斯瓦螨Varroa destructor Anderson & Trueman是意大利蜜蜂Apis mellifera Spinola的主要外寄生螨。雌成螨在幼虫巢房封盖前不久侵入幼虫巢房,并开始繁殖为害。从雌成螨在一个很短的时间内进入蜜蜂幼虫巢房,以及雄蜂幼虫巢房蜂螨的寄生率明显高于工蜂幼虫巢房的现象,表明蜜蜂幼虫体表一些信息素(semiochemicals)可能起着重要的引诱作用。作者对与大蜂螨相关的19种气味物质进行筛选,并对封盖前工蜂幼虫和雄蜂幼虫表皮挥发物进行气谱及气-质联谱测定。结果表明:雄蜂6龄幼虫对大蜂螨的引诱作用显著高于丁香水等10种气味物质。工蜂和雄蜂末龄幼虫体表挥发物的共有组份是9-二十三烯(C23H46),但它在雄蜂幼虫中所占的比例要明显高于工蜂幼虫。工蜂幼虫的特有主要组分是十八烷(C18H38)和9-甲基十九烷(C19H40);而雄蜂幼虫的特有主要组分是二十五烷(C25H52)和二十三烷(C23H48)。     

Semiochemicals Influencing the Host-finding Behaviour of Varroa Destructor

Studies of Varroa destructor orientation to honey bees were undertaken to isolate discrete chemical compounds that elicit host-finding activity. Petri dish bioassays were used to study cues that evoked invasion behaviour into simulated brood cells and a Y-tube olfactometer was used to evaluate varroa orientation to olfactory volatiles. In Petri dish bioassays, mites were highly attracted to live L5 worker larvae and to live and freshly freeze-killed nurse bees. Olfactometer bioassays indicated olfactory orientation to the same type of hosts, however mites were not attracted to the odour produced by live pollen foragers. The odour of forager hexane extracts also interfered with the ability of mites to localize and infest a restrained nurse bee host. Varroa mites oriented to the odour produced by newly emerged bees (<16 h old) when choosing against a clean airstream, however in choices between the odours of newly emerged workers and nurses, mites readily oriented to nurses when newly emerged workers were <3 h old. The odour produced by newly emerged workers 18–20 h of age was equally as attractive to mites as that of nurse bees, suggesting a changing profile of volatiles is produced as newly emerged workers age. Through fractionation and isolation of active components of nurse bee-derived solvent washes, two honey bee Nasonov pheromone components, geraniol and nerolic acid, were shown to confuse mite orientation. We suggest that V. destructor may detect relative concentrations of these compounds in order to discriminate between adult bee hosts, and preferentially parasitize nurse bees over older workers in honey bee colonies. The volatile profile of newly emerged worker bees also may serve as an initial stimulus for mites to disperse before being guided by allomonal cues produced by older workers to locate nurses. Fatty acid esters, previously identified as putative kairomones for varroa, proved to be inactive in both types of bioassays.     

Distribution of Paenibacillus larvae spores inside honey bee colonies and its relevance for diagnosis

One of the most important factors affecting the development of honey bee colonies is infectious diseases such as American foulbrood (AFB) caused by the spore forming Gram-positive bacterium Paenibacillus larvae. Colony inspections for AFB clinical symptoms are time consuming. Moreover, diseased cells in the early stages of the infection may easily be overlooked. In this study, we investigated whether it is possible to determine the sanitary status of a colony based on analyses of different materials collected from the hive. We analysed 237 bee samples and 67 honey samples originating from 71 colonies situated in 13 apiaries with clinical AFB occurrences. We tested whether a difference in spore load among bees inside the whole hive exists and which sample material related to its location inside the hive was the most appropriate for an early AFB diagnosis based on the culture method. Results indicated that diagnostics based on analysis of honey samples and bees collected at the hive entrance are of limited value as only 86% and 83%, respectively, of samples from AFB-symptomatic colonies were positive. Analysis of bee samples collected from the brood nest, honey chamber, and edge frame allowed the detection of all colonies showing AFB clinical symptoms. Microbiological analysis showed that more than one quarter of samples collected from colonies without AFB clinical symptoms were positive for P. larvae. Based on these results, we recommend investigating colonies by testing bee samples from the brood nest, edge frame or honey chamber for P. larvae spores.     

Comparative reproduction of <Emphasis Type="Italic">Varroa destructor</Emphasis> in different types of Russian and Italian honey bee combs

Earlier studies showed that Russian honey bees support slow growth of varroa mite population. We studied whether or not comb type influenced varroa reproduction in both Russian and Italian honey bees, and whether Russian bees produced comb which inhibited varroa reproduction. The major differences found in this study concerned honey bee type. Overall, the Russian honey bees had lower (2.44 ± 0.18%) levels of varroa infestation than Italian honey bees (7.20 ± 0.60%). This decreased infestation resulted in part from a reduced number of viable female offspring per foundress in the Russian (0.85 ± 0.04 female) compared to the Italian (1.23 ± 0.04 females) honey bee colonies. In addition, there was an effect by the comb built by the Russian honey bee colonies that reduced varroa reproduction. When comparing combs having Russian or Italian colony origins, Russian honey bee colonies had more non-reproducing foundress mites and fewer viable female offspring in Russian honey bee comb. This difference did not occur in Italian colonies. The age of comb in this study had mixed effects. Older comb produced similar responses for six of the seven varroa infestation parameters measured. In colonies of Italian honey bees, the older comb (2001 dark) had fewer (1.13 ± 0.07 females) viable female offspring per foundress than were found in the 2002 new (1.21 ± 0.06 females) and 1980s new (1.36 ± 0.08 females) combs. This difference did not occur with Russian honey bee colonies where the number of viable female offspring was low in all three types of combs. This study suggests that honey bee type largely influences growth of varroa mite population in a colony.     

Orientation of comb building by honeybees

Summary Upon entering a new home site a honeybee swarm is faced with the task of organizing the building activities of thousands of component bees so that several straight and parallel vertically oriented combs can be quickly and efficiently built. As a part of this organization process it is necessary for the bees to select and agree upon a planar orientation for the new combs.This paper presents evidence that memory of a previously used comb direction influences the building of the new set of combs. Swarms which have recently moved into bait-hives (empty boxes placed in trees to attract feral swarms) tend to maintain the previously used comb direction when removed and forced to build new combs, whereas swarms which have occupied the bait-hives for a longer period (over 9 days) do not.Recent swarms predictably alter their comb building direction within the influence of an applied earthstrength magnetic field, indicating that honey bees are able to use the earth's magnetic field as a reference at the commencement of comb construction in a new hive.     

Effects of acute sublethal exposure to coumaphos or diazinon on acquisition and discrimination of odor stimuli in the honey bee (Hymenoptera: Apidae)

Two organophosphate compounds, coumaphos and diazinon, were examined for effects of sublethal exposure on odor learning and generalization in honey bees, Apis mellifera L. Using proboscis extension response training as a measure of odor learning and discrimination, a series of two experiments tested whether these compounds would inhibit bees from learning a new odor or discriminating between different odors. Bees were exposed to coumaphos or diazinon in acetone applied to the thorax, or to coumaphos or diazinon in hexane injected intracranially. At no dose tested or exposure method used was coumaphos shown to inhibit acquisition of a novel odor stimulus, although it was shown to slightly reduce discriminatory ability when given by intracranial injection. Diazinon had effects on odor learning at several small doses, and a small injected dose was shown to significantly inhibit learning of an odor stimulus paired with a sucrose reward. When bee head acetylcholineasterase activity was measured after dermal applications of both pesticides, only the higher doses of diazinon showed reduced activity, indicating that externally-applied coumaphos shows no significant effect on bee brain acetylcholinesterase activity. These data suggest that acute application of coumaphos has only slight nonlethal effects upon the behavior of honey bees and should have little effect upon bee tasks that involve odor learning.     

Reduction of tracheal mite parasitism of honey bees by swarming.

Based on population dynamics, tracheal mite (Acarapis woodi) parasitism of colonies of honey bees (Apis mellifera) appears to be, potentially at least, regulatory and stable. Empirical and theoretical considerations suggest, however, that intracolony population dynamics of mite-honey bee worker seem to be unstable in managed situations where honey bee worker population is allowed to grow unchecked. Experimental studies showed that tracheal mite population levels increased in a managed honey bee colony but were impaired in one in which brood rearing was interrupted by loss of the queen. Mite densities but not prevalence were lowered in experimental swarms kept from rearing brood. We propose that swarming reduces mite density within a colony, therefore implicating modern techniques of hive management in the sudden historical appearance of the mite on the Isle of Wight.     

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.     

Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)

Through the use of proboscis-extension reflex conditioning, we demonstrate that honey bees (Apis mellifera L.) bred for hygienic behavior (a behavioral mechanism of disease resistance) are able to discriminate between odors of healthy and diseased brood at a lower stimulus level than bees from a non-hygienic line. Electroantennogram recordings confirmed that hygienic bees exhibit increased olfactory sensitivity to low concentrations of the odor of chalkbrood infected pupae (a fungal disease caused by Ascosphaera apis). Three-week-old hygienic bees were able to discriminate between the brood odors significantly better than three-week old non-hygienic bees. However, the differential performance in brood odor discrimination was primarily genetically based, not a direct result of age, experience, or the temporary behavioral state of the bee. Lower stimulus thresholds for both the olfactory and behavioral responses of hygienic bees may facilitate their ability to detect, uncap and remove diseased brood rapidly from the nest. In contrast, non-hygienic bees, possessing higher response thresholds, may not be able to detect diseased brood as easily. Our results provide an example of how physiological and behavioral differences between the hygienic and non-hygienic honey bee lines, operating at the level of the individual, could produce colony-specific behavioral phenotypes.     

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.     

Selective modulation of task performance by octopamine in honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) division of labour

Octopamine treatment has previously been shown to increase honey bee foraging behaviour. We determined the effects of octopamine on other tasks to learn how octopamine affects division of labour in honey bee colonies. Octopamine treatment did not increase the rate of corpse removal from the hive, suggesting that elevated brain levels of octopamine do not act to increase the performance of all flight-related tasks. Octopamine treatment also did not increase attendance in the queen's retinue, suggesting that elevated brain levels of octopamine do not act to increase responsiveness to all olfactory stimuli. Consistent with these findings, octopamine treatment enhanced the foraging response to brood pheromone but not the cell capping response, a component of brood care. These results demonstrate a relatively specific form of neuromodulation by octopamine in the regulation of division of labour in honey bee colonies.     

The Significance of Odor Cues and Dance Language Information for the Food Search Behavior of Honeybees (Hymenoptera: Apidae)

Although several independent lines of evidence show that bees can make use of information provided by their dance language, there is an ongoing controversy about the significance of the dance information versus odor cues in the search behavior of recruited bees. A series of experiments was performed to assess the relative significance of dance information and odors for the site-specific search of recruit bees. In these experiments recruit bees were trapped automatically at arrays of artificial flowers at various distances from the hive. The distribution of directions in which the recruits searched for food was compared between recruitment by dancers performing well-oriented dances on the vertical side of the comb and dancers performing disoriented dances on a horizontal comb. The results show quantitatively that bees use both odor cues and the dance information. The greater the distance to the feeding site, the greater is the relative significance of the dance information.