Egg viability and worker policing in honey bees

In many species of social Hymenoptera, unmated workers can lay eggs that will produce males by parthenogenesis. Nevertheless, in queenright honey bee colonies (Apis mellifera), worker reproduction is low. One possible mechanism for this difference is worker policing, the removal of worker-laid eggs by other workers. This behavior can evolve in species in which queens are multiply mated, where workers are more closely related to the sons of their mother than those of their sisters. Another possible mechanism of the low level of worker reproduction is worker-laid eggs being less viable than queen-laid eggs. We show that this difference in quality is the case for honey bees.     

Regulation of behavioral maturation by a primer pheromone produced by adult worker honey bees

Previous research showed that the presence of older workers causes a delayed onset of foraging in younger individuals in honey bee colonies, but a specific worker inhibitory factor had not yet been identified. Here, we report on the identification of a substance produced by adult forager honey bees, ethyl oleate, that acts as a chemical inhibitory factor to delay age at onset of foraging. Ethyl oleate is synthesized de novo and is present in highest concentrations in the bee's crop. These results suggest that worker behavioral maturation is modulated via trophallaxis, a form of food exchange that also serves as a prominent communication channel in insect societies. Our findings provide critical validation for a model of self-organization explaining how bees are able to respond to fragmentary information with actions that are appropriate to the state of the whole colony.     

Evolution and mechanisms of long life and high fertility in queen honey bees

Honey bees (Apis mellifera) are eusocial insects that exhibit striking caste-specific differences in longevity. Queen honey bees live on average 1–2 years whereas workers live on average 15–38 days in the summer and 150–200 days in the winter. Previous studies of senescence in the honey bee have focused on establishing the importance of extrinsic mortality factors (predation, weather) and behavior (nursing and foraging) in worker bee longevity. However, few studies have tried to elucidate the mechanisms that allow queen honey bees to achieve their long lifespan without sacrificing fecundity. Here, we review both types of studies and emphasize the importance of understanding both proximate and ultimate causes of the unusual life history of honey bee queens.     

Chronic Bee Paralysis Virus and Nosema ceranae Experimental Co-Infection of Winter Honey Bee Workers (Apis mellifera L.)

Chronic bee paralysis virus (CBPV) is an important viral disease of adult bees which induces significant losses in honey bee colonies. Despite comprehensive research, only limited data is available from experimental infection for this virus. In the present study winter worker bees were experimentally infected in three different experiments. Bees were first inoculated per os (p/o) or per cuticle (p/c) with CBPV field strain M92/2010 in order to evaluate the virus replication in individual bees. In addition, potential synergistic effects of co-infection with CBPV and Nosema ceranae (N. ceranae) on bees were investigated. In total 558 individual bees were inoculated in small cages and data were analyzed using quantitative real time RT-PCR (RT-qPCR). Our results revealed successful replication of CBPV after p/o inoculation, while it was less effective when bees were inoculated p/c. Dead bees harbored about 1,000 times higher copy numbers of the virus than live bees. Co-infection of workers with CBPV and N. ceranae using either method of virus inoculation (p/c or p/o) showed increased replication ability for CBPV. In the third experiment the effect of inoculation on bee mortality was evaluated. The highest level of bee mortality was observed in a group of bees inoculated with CBPV p/o, followed by a group of workers simultaneously inoculated with CBPV and N. ceranae p/o, followed by the group inoculated with CBPV p/c and the group with only N. ceranae p/o. The experimental infection with CBPV showed important differences after p/o or p/c inoculation in winter bees, while simultaneous infection with CBPV and N. ceranae suggesting a synergistic effect after inoculation.     

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.     

Dynamics of persistent and acute deformed wing virus infections in honey bees, Apis mellifera

The dynamics of viruses are critical to our understanding of disease pathogenesis. Using honey bee Deformed wing virus (DWV) as a model, we conducted field and laboratory studies to investigate the roles of abiotic and biotic stress factors as well as host health conditions in dynamics of virus replication in honey bees. The results showed that temperature decline could lead to not only significant decrease in the rate for pupae to emerge as adult bees, but also an increased severity of the virus infection in emerged bees, partly explaining the high levels of winter losses of managed honey bees, Apis mellifera, around the world. By experimentally exposing adult bees with variable levels of parasitic mite Varroa destructor, we showed that the severity of DWV infection was positively correlated with the density and time period of Varroa mite infestation, confirming the role of Varroa mites in virus transmission and activation in honey bees. Further, we showed that host conditions have a significant impact on the outcome of DWV infection as bees that originate from strong colonies resist DWV infection and replication significantly better than bee originating from weak colonies. The information obtained from this study has important implications for enhancing our understanding of host‑pathogen interactions and can be used to develop effective disease control strategies for honey bees.     

Comb wax mediates the acquisition of nest-mate recognition cues in honey bees

Honey bees, Apis mellifera, acquire nest-mate recognition cues from wax, the predominant material used in nest construction. Exposure of a newly emerged worker bee to wax-comb substrate significantly reduced the acceptability of that worker to sister bees. Cues acquired from the comb provided colony-specific information about the identity of worker bees; moreover, the effect of comb exposure has been previously shown to override individually produced cues. Food odors (anise oil), when dissolved in paraffin wax, affected worker-recognition characteristics but food odors did not affect these characteristics when fed to bees in sugar candy. Paraffin wax alone did not affect the recognition cues of bees, showing that the wax can be a neutral medium for the transmission of cues. The wax comb in the colony and the hydrocarbon outer layer of the bee cuticle may be a continuous medium for any hydrocarbon-soluble substances used by honey bees in nest-mate recognition; if so, a mechanism by which environmental cues are acquired by honey bees is provided.     

Pleiotropy of segregating genetic variants that affect honey bee worker life expectancy

In contrast to many other complex traits, the natural genetic architecture of life expectancy has not been intensely studied, particularly in non-model organisms, such as the honey bee (Apis mellifera L.). Multiple factors that determine honey bee worker lifespan have been identified and genetic analyses have been performed on some of those traits. Several of the traits are included in a suite of correlated traits that form the pollen hoarding syndrome, which was named after the behavior to store surplus pollen in the nest and is tied to social evolution. Here, seven quantitative trait loci that had previously been identified for their effects on different aspects of the pollen hoarding syndrome were studied for their genetic influence on the survival of adult honey bee workers. To gain a more comprehensive understanding of the genetic architecture of worker longevity, a panel of 280 additional SNP markers distributed across the genome was also tested. Allelic distributions were compared between young and old bees in two backcross populations of the bi-directionally selected high- and low-pollen hoarding strain. Our results suggest a pleiotropic effect of at least one of the behavioral quantitative trait loci on worker longevity and one significant and several other putative genetic effects in other genomic regions. At least one locus showed evidence for strong antagonistic pleiotropy and several others suggested genetic factors that influence pre-emergence survival of worker honey bees. Thus, the predicted association between worker lifespan and the pollen hoarding syndrome was supported at the genetic level and the magnitude of the identified effects also strengthened the view that naturally segregating genetic variation can have major effects on age-specific survival probability in the wild.     

Cellular senescence in honey bee brain is largely independent of chronological age

Accumulation of oxidative stress-induced damage in brain tissue plays an important role in the pathogenesis of normal aging and neurodegenerative diseases. Neuronal oxidative damage typically increases with age in humans, and also in the invertebrate and vertebrate model species most commonly used in aging research. By use of quantitative immunohistochemistry and Western blot, we show that this aspect of brain senescence is largely decoupled from chronological age in the honey bee (Apis mellifera). The bee is a eusocial insect characterized by the presence of a reproductive queen caste and a caste of functionally sterile female workers that performs various alloparental tasks such as nursing and foraging. We studied patterns of oxidative nitration and carbonylation damage in the brain of worker bees that performed nurse tasks as 8- and 200-day-olds and foraging tasks as 20- and 200-day-olds. In addition, we examined 180-day-old diutinus bees, a stress-resistant temporal worker form that survives unfavorable periods. Our results indicate that nitration damage occurs only at low levels in vivo, but that a 60-kDa protein from honey bee brain is selectively nitrated by peroxynitrite in vitro. Oxidative carbonylation is present at varying levels in the visual and chemosensory neuropiles of worker bees, and this inter-individual variation is better explained by social role than by chronological age.     

Regulation of life history determines lifespan of worker honey bees (Apis mellifera L.)

Life expectancy of honey bees (Apis mellifera L.) is of general interest to gerontological research because its variability among different groups of bees is one of the most striking cases of natural plasticity of aging. Worker honey bees spend their first days of adult life working in the nest, then transition to foraging and die between 4 and 8 weeks of age. Foraging is believed to be primarily responsible for the early death of workers. Three large-scale experiments were performed to quantitatively assess the importance of flight activity, chronological age, extrinsic mortality factors and foraging specialization. Forager mortality was higher than in-hive bee mortality. Most importantly however, reducing the external mortality hazards and foraging activity did not lead to the expected strong extension of life. Most of the experimental effects were attributable to an earlier transition from hive tasks to foraging. This transition is accompanied by a significant mortality peak. The age at the onset of foraging is the central variable in worker life-history and behavioral state was found more important than chronological age for honey bee aging. However, mortality risk increased with age and the negative relation between pre-foraging and foraging lifespan indicate some senescence irrespective of behavioral state. Overall, honey bee workers exhibit a logistic mortality dynamic which is mainly caused by the age-dependent transition from a low mortality pre-foraging state to a higher mortality foraging state.     

Chronic Bee Paralysis Virus in Honeybee Queens: Evaluating Susceptibility and Infection Routes

Chronic bee paralysis virus (CBPV) is known as a disease of worker honey bees. To investigate pathogenesis of the CBPV on the queen, the sole reproductive individual in a colony, we conducted experiments regarding the susceptibility of queens to CBPV. Results from susceptibility experiment showed a similar disease progress in the queens compared to worker bees after infection. Infected queens exhibit symptoms by Day 6 post infection and virus levels reach 10¹¹ copies per head. In a transmission experiment we showed that social interactions may affect the disease progression. Queens with forced contact to symptomatic worker bees acquired an overt infection with up to 10¹¹ virus copies per head in six days. In contrast, queens in contact with symptomatic worker bees, but with a chance to receive food from healthy bees outside the cage appeared healthy. The virus loads did not exceed 10⁷ in the majority of these queens after nine days. Symptomatic worker bees may transmit sufficient active CBPV particles to the queen through trophallaxis, to cause an overt infection.     

Reproduction in worker honey bees is associated with low juvenile hormone titers and rates of biosynthesis.

Three experiments were performed to determine the role of juvenile hormone (JH) in worker reproduction in queenless colonies of honey bees. In Experiment 1, egg-laying workers had low hemolymph titers of JH, as did bees engaged in brood care, while foragers had significantly higher titers. Experiment 2 confirmed these findings by demonstrating that laying workers have significantly lower rates of JH biosynthesis than foragers do. In Experiment 3, ovary development was inhibited slightly by application of the JH analog methoprene to 1-day-old bees, but was not affected by application to older bees, at least some already displaying egg-laying behavior. These results, which are consistent with earlier findings for queen honey bees, are contrary to a common model of insect reproduction, in which elevated JH titers trigger ovary development, which then leads to oviposition. Previous experiments have demonstrated that JH regulates nonreproductive behavior in workers that is associated with colony division of labor; perhaps this function is incompatible with a traditional role for JH in reproduction.     

Metatranscriptome Analysis of Sympatric Bee Species Identifies Bee Virus Variants and a New Virus,Andrena-Associated Bee Virus-1

Bees are important plant pollinators in agricultural and natural ecosystems. High average annual losses of honey bee (Apis mellifera) colonies in some parts of the world, and regional population declines of some mining bee species (Andrena spp.), are attributed to multiple factors including habitat loss, lack of quality forage, insecticide exposure, and pathogens, including viruses. While research has primarily focused on viruses in honey bees, many of these viruses have a broad host range. It is therefore important to apply a community level approach in studying the epidemiology of bee viruses. We utilized high-throughput sequencing to evaluate viral diversity and viral sharing in sympatric, co-foraging bees in the context of habitat type. Variants of four common viruses (i.e., black queen cell virus, deformed wing virus, Lake Sinai virus 2, and Lake Sinai virus NE) were identified in honey bee and mining bee samples, and the high degree of nucleotide identity in the virus consensus sequences obtained from both taxa indicates virus sharing. We discovered a unique bipartite + ssRNA Tombo-like virus, Andrena-associated bee virus-1 (AnBV-1). AnBV-1 infects mining bees, honey bees, and primary honey bee pupal cells maintained in culture. AnBV-1 prevalence and abundance was greater in mining bees than in honey bees. Statistical modeling that examined the roles of ecological factors, including floral diversity and abundance, indicated that AnBV-1 infection prevalence in honey bees was greater in habitats with low floral diversity and abundance, and that interspecific virus transmission is strongly modulated by the floral community in the habitat. These results suggest that land management strategies that aim to enhance floral diversity and abundance may reduce AnBV-1 spread between co-foraging bees.     

Multitrophic interaction facilitates parasite-host relationship between an invasive beetle and the honey bee

Colony defense by honey bees, Apis mellifera, is associated with stinging and mass attack, fueled by the release of alarm pheromones. Thus, alarm pheromones are critically important to survival of honey bee colonies. Here we report that in the parasitic relationship between the European honey bee and the small hive beetle, Aethina tumida, the honey bee's alarm pheromones serve a negative function because they are potent attractants for the beetle. Furthermore, we discovered that the beetles from both Africa and the United States vector a strain of Kodamaea ohmeri yeast, which produces these same honey bee alarm pheromones when grown on pollen in hives. The beetle is not a pest of African honey bees because African bees have evolved effective methods to mitigate beetle infestation. However, European honey bees, faced with disease and pest management stresses different from those experienced by African bees, are unable to effectively inhibit beetle infestation. Therefore, the environment of the European honey bee colony provides optimal conditions to promote the unique bee-beetle-yeast-pollen multitrophic interaction that facilitates effective infestation of hives at the expense of the European honey bee.     

A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The "queen substance" [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queen's dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50=280+/-31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.     

Wild bees enhance honey bees' pollination of hybrid sunflower

Pollinators are required for producing 15-30% of the human food supply, and farmers rely on managed honey bees throughout the world to provide these services. Yet honey bees are not always the most efficient pollinators of all crops and are declining in various parts of the world. Crop pollination shortages are becoming increasingly common. We found that behavioral interactions between wild and honey bees increase the pollination efficiency of honey bees on hybrid sunflower up to 5-fold, effectively doubling honey bee pollination services on the average field. These indirect contributions caused by interspecific interactions between wild and honey bees were more than five times more important than the contributions wild bees make to sunflower pollination directly. Both proximity to natural habitat and crop planting practices were significantly correlated with pollination services provided directly and indirectly by wild bees. Our results suggest that conserving wild habitat at the landscape scale and altering selected farm management techniques could increase hybrid sunflower production. These findings also demonstrate the economic importance of interspecific interactions for ecosystem services and suggest that protecting wild bee populations can help buffer the human food supply from honey bee shortages.     

Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development

To investigate the possible consequences of brood-temperature regulation in honey bee colonies on the quality of behavioral performance of adults, we placed honey bee pupae in incubators and allowed them to develop at temperatures held constant at 32 degrees C, 34.5 degrees C, and 36 degrees C. This temperature range occurs naturally within hives. On emergence, the young adult bees were marked and introduced into foster colonies housed in normal and observation hives and allowed to live out their lives. No obvious difference in within-hive behavior was noted between the temperature-treated bees and the foster-colony bees. However, when the temperature-treated bees became foragers and were trained to visit a feeder 200 m from the hive, they exhibited clear differences in dance performance that could be correlated with the temperatures at which they had been raised: bees raised at 32 degrees C completed only approximately 20% of the dance circuits when compared with bees of the higher-temperature group. Also, the variance in the duration of the waggle phase is larger in 32 degrees C-raised bees compared with 36 degrees C-raised bees. All other parameters compared across all groups were not significantly different. One-trial learning and memory consolidation in the bees raised at different temperatures was investigated 1 and 10 min after conditioning the proboscis-extension reflex. Bees raised at 36 degrees C performed as expected for bees typically classified as "good learners," whereas bees raised at 32 degrees C and 34.5 degrees C performed significantly less well. We propose that the temperature at which pupae are raised will influence their behavioral performance as adults and may determine the tasks they carry out best inside and outside the hive.     

Reproductive protein protects functionally sterile honey bee workers from oxidative stress

Research on aging shows that regulatory pathways of fertility and senescence are closely interlinked. However, evolutionary theories on social species propose that lifelong care for offspring can shape the course of senescence beyond the restricted context of reproductive capability. These observations suggest that control circuits of aging are remodeled in social organisms with continuing care for offspring. Here, we studied a circuit of aging in the honey bee (Apis mellifera). The bee is characterized by the presence of a long-lived reproductive queen caste and a shorter-lived caste of female workers that are life-long alloparental care givers. We focus on the role of the conserved yolk precursor gene vitellogenin that, in Caenorhabditis elegans, shortens lifespan as a downstream element of the insulin/insulin-like growth factor signaling cascade. Vitellogenin protein is synthesized at high levels in honey bee queens and is abundant in long-lived workers. We establish that vitellogenin gene activity protects worker bees from oxidative stress. Our finding suggests that one mechanistic explanation for patterns of longevity in bees is that a reproductive regulatory pathway has been remodeled to extend life. This perspective is of considerable relevance to research on longevity regulation that builds largely on inference from solitary model species.     

Honey Bee Infecting Lake Sinai Viruses

Honey bees are critical pollinators of important agricultural crops. Recently, high annual losses of honey bee colonies have prompted further investigation of honey bee infecting viruses. To better characterize the recently discovered and very prevalent Lake Sinai virus (LSV) group, we sequenced currently circulating LSVs, performed phylogenetic analysis, and obtained images of LSV2. Sequence analysis resulted in extension of the LSV1 and LSV2 genomes, the first detection of LSV4 in the US, and the discovery of LSV6 and LSV7. We detected LSV1 and LSV2 in the Varroa destructor mite, and determined that a large proportion of LSV2 is found in the honey bee gut, suggesting that vector-mediated, food-associated, and/or fecal-oral routes may be important for LSV dissemination. Pathogen-specific quantitative PCR data, obtained from samples collected during a small-scale monitoring project, revealed that LSV2, LSV1, Black queen cell virus (BQCV), and Nosema ceranae were more abundant in weak colonies than strong colonies within this sample cohort. Together, these results enhance our current understanding of LSVs and illustrate the importance of future studies aimed at investigating the role of LSVs and other pathogens on honey bee health at both the individual and colony levels.