Added Weights Lead to Reduced Flight Behavior and Mating Success in Polyandrous Honey Bee Queens (Apis mellifera)

Honey bee queens are exceptionally promiscuous. Early in life, queens perform one to five nuptial flights, mating with up to 44 drones. Many studies have documented potential benefits of multiple mating. In contrast, potential costs of polyandry and the sensitivity of queens to such costs have largely been ignored because they are difficult to address experimentally. To consider one aspect of mating costs to queens, the difficulty of flight, we compared flight behavior and success among a group of control queens and two experimental groups of queens that carried lead weights of two different sizes. For each queen, we assessed the number and duration of all flights and, after egg-laying commenced, the amount of stored sperm and the number of mates in terms of the offspring's patrilineal genetic diversity. Added weights quantitatively decreased the number of flights, the mean duration of flights and consequently the total time spent flying. Mating success in terms of sperm quantity and patrilines detected among the queens' offspring was also negatively impacted by the experimental manipulation. Thus, it can be concluded that the flight effort of honey bee queens during their mating period is adjusted in response to an experimentally increased cost of flying with multiple consequences for their mating success. Our results suggest that queen behavior is flexible and mating costs deserve more attention to explain the extreme polyandry in honey bees.     

Ecological Adaptation of Diverse Honey Bee (Apis mellifera) Populations

Background

Honey bees are complex eusocial insects that provide a critical contribution to human agricultural food production. Their natural migration has selected for traits that increase fitness within geographical areas, but in parallel their domestication has selected for traits that enhance productivity and survival under local conditions. Elucidating the biochemical mechanisms of these local adaptive processes is a key goal of evolutionary biology. Proteomics provides tools unique among the major ‘omics disciplines for identifying the mechanisms employed by an organism in adapting to environmental challenges.

Results

Through proteome profiling of adult honey bee midgut from geographically dispersed, domesticated populations combined with multiple parallel statistical treatments, the data presented here suggest some of the major cellular processes involved in adapting to different climates. These findings provide insight into the molecular underpinnings that may confer an advantage to honey bee populations. Significantly, the major energy-producing pathways of the mitochondria, the organelle most closely involved in heat production, were consistently higher in bees that had adapted to colder climates. In opposition, up-regulation of protein metabolism capacity, from biosynthesis to degradation, had been selected for in bees from warmer climates.

Conclusions

Overall, our results present a proteomic interpretation of expression polymorphisms between honey bee ecotypes and provide insight into molecular aspects of local adaptation or selection with consequences for honey bee management and breeding. The implications of our findings extend beyond apiculture as they underscore the need to consider the interdependence of animal populations and their agro-ecological context.     

A Honey Bee (Apis mellifera) Light Phase Response Curve

The authors report a phase response curve (PRC) for individual honey bees (Apis mellifera) to single 1-h light pulses (1000 lux) using an Aschoff Type 1 protocol (n?=?134). The bee PRC is a weak (Type 1) PRC with a maximum advance of 1.5?h between circadian time (CT) 18 and 3 and a maximum delay of 1.5?h between CT 12 and 18. This is the first published honey bee light PRC and provides an important resource for chronobiologists and honey bee researchers. It may also have practical applications for what is an economically important species frequently transported across different time zones. (Author correspondence: G.warman@auckland.ac.nz)     

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 Bacterial Communities Associated with Honey Bee (Apis mellifera) Foragers

The honey bee is a key pollinator species in decline worldwide. As part of a commercial operation, bee colonies are exposed to a variety of agricultural ecosystems throughout the year and a multitude of environmental variables that may affect the microbial balance of individuals and the hive. While many recent studies support the idea of a core microbiota in guts of younger in-hive bees, it is unknown whether this core is present in forager bees or the pollen they carry back to the hive. Additionally, several studies hypothesize that the foregut (crop), a key interface between the pollination environment and hive food stores, contains a set of 13 lactic acid bacteria (LAB) that inoculate collected pollen and act in synergy to preserve pollen stores. Here, we used a combination of 454 based 16S rRNA gene sequencing of the microbial communities of forager guts, crops, and corbicular pollen and crop plate counts to show that (1) despite a very different diet, forager guts contain a core microbiota similar to that found in younger bees, (2) corbicular pollen contains a diverse community dominated by hive-specific, environmental or phyllosphere bacteria that are not prevalent in the gut or crop, and (3) the 13 LAB found in culture-based studies are not specific to the crop but are a small subset of midgut or hindgut specific bacteria identified in many recent 454 amplicon-based studies. The crop is dominated by Lactobacillus kunkeei, and Alpha 2.2 (Acetobacteraceae), highly osmotolerant and acid resistant bacteria found in stored pollen and honey. Crop taxa at low abundance include core hindgut bacteria in transit to their primary niche, and potential pathogens or food spoilage organisms seemingly vectored from the pollination environment. We conclude that the crop microbial environment is influenced by worker task, and may function in both decontamination and inoculation.     

Orientation-sensitive Neurons in the Brain of the Honey Bee (Apis mellifera)

Recent behavioural experiments have shown that bees are able to distinguish vertically presented patterns with orientation cues, although the locations of areas of black are randomized. To discriminate between two orientations, the bees must possess more than one orientation-sensitive neuron type. Therefore, the aim is to search for different types of orientation-sensitive cells of the honey bee, and measure their receptive field, velocity sensitivity and contrast sensitivity. Orientation-sensitive cells with two different types of orientation tuning-curves were recorded intracellularly in the mid-brain of the honey bee when the stimulus was a narrow bar (bar width = 5 degrees ). These cells are sensitive to bar movement within their large receptive field, which covers the visual field of one eye. They are quite distinct from the well-known directional motion detectors. The contrast sensitivity of the orientation-sensitive cells recorded in this study corresponds to results from behavioural experiments. The velocity-sensitivity curves of the orientation-sensitive cells differ from those of the direction-sensitive cells. Measurements of orientation sensitivity and contrast sensitivity when the stimulus is a wide bar (bar width = 10 degrees ), done in different eye regions, suggest that each orientation-sensitive cell receives visual signals from an array of orientational subunits within its receptive field. The correspondence between these physiological results and the results of recent behavioural experiments are discussed. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Landscape Learning during Flight Ensures Homing in Honey Bee (Apis mellifera) Drones

Journal of Insect Behavior - The role of learning to navigate toward a specific location has been extensively studied in the foraging context of social hymenopteran females, which bring resources...     

Effects of Brood Pheromone Modulated Brood Rearing Behaviors on Honey Bee (Apis mellifera L.) Colony Growth

A hallmark of eusociality is cooperative brood care. In most social insect systems brood rearing labor is divided between individuals working in the nest tending the queen and larvae, and foragers collecting food outside the nest. To place brood rearing division of labor within an evolutionary context, it is necessary to understand relationships between individuals in the nest engaged in brood care and colony growth in the honey bee. Here we examined responses of the queen, queen-worker interactions, and nursing behaviors to an increase in the brood rearing stimulus environment using brood pheromone. Colony pairs were derived from a single source and were headed by open-mated sister queens, for a total of four colony pairs. One colony of a pair was treated with 336 μg of brood pheromone, and the other a blank control. Queens in the brood pheromone treated colonies laid significantly more eggs, were fed longer, and were less idle compared to controls. Workers spent significantly more time cleaning cells in pheromone treatments. Increasing the brood rearing stimulus environment with the addition of brood pheromone significantly increased the tempo of brood rearing behaviors by bees working in the nest resulting in a significantly greater amount of brood reared.     

Mating Frequencies of Honey Bee Queens (Apis mellifera L.) in a Population of Feral Colonies in the Northeastern United States

Across their introduced range in North America, populations of feral honey bee (Apis mellifera L.) colonies have supposedly declined in recent decades as a result of exotic parasites, most notably the ectoparasitic mite Varroa destructor. Nonetheless, recent studies have documented several wild populations of colonies that have persisted. The extreme polyandry of honey bee queens—and the increased intracolony genetic diversity it confers—has been attributed, in part, to improved disease resistance and may be a factor in the survival of these populations of feral colonies. We estimated the mating frequencies of queens in feral colonies in the Arnot Forest in New York State to determine if the level of polyandry of these queens is especially high and so might contribute to their survival success. We genotyped the worker offspring from 10 feral colonies in the Arnot Forest of upstate New York, as well as those from 20 managed colonies closest to this forest. We found no significant differences in mean mating frequency between the feral and managed queens, suggesting that queens in the remote, low-density population of colonies in the Arnot Forest are neither mate-limited nor adapted to mate at an especially high frequency. These findings support the hypothesis that the hyperpolyandry of honey bees has been shaped on an evolutionary timescale rather than on an ecological one.     

A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

A major hindrance to the study of honey bee pathogens or the effects of pesticides and nutritional deficiencies is the lack of controlled in vitro culture systems comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell culture techniques that supports sustained growth of honey bee cells in vitro. We used honey bee eggs mid to late in their embryogenesis to establish primary cultures, as these eggs contain cells that are progressively dividing. Primary cultures were initiated in modified Leibovitz’s L15 medium and incubated at 32^°C. Serial transfer of material from several primary cultures was maintained and has led to the isolation of young cell lines. A cell line (AmE-711) has been established that is composed mainly of fibroblast-type cells that form an adherent monolayer. Most cells in the line are diploid (2n = 32) and have the Apis mellifera karyotype as revealed by Giemsa stain. The partial sequence for the mitochondrial-encoded cytochrome c oxidase subunit I (Cox 1) gene in the cell line is identical to those from honey bee tissues and a consensus sequence for A. mellifera. The population doubling time is approximately 4 days. Importantly, the cell line is continuously subcultured every 10–14 days when split at a 1:3 ratio and is cryopreserved in liquid nitrogen. The cell culture system we have developed has potential application for studies aimed at honey bee development, genetics, pathogenesis, transgenesis, and toxicology.     

Reduced SNP Panels for Genetic Identification and Introgression Analysis in the Dark Honey Bee (Apis mellifera mellifera)

Beekeeping activities, especially queen trading, have shaped the distribution of honey bee (Apis mellifera) subspecies in Europe, and have resulted in extensive introductions of two eastern European C-lineage subspecies (A. m. ligustica and A. m. carnica) into the native range of the M-lineage A. m. mellifera subspecies in Western Europe. As a consequence, replacement and gene flow between native and commercial populations have occurred at varying levels across western European populations. Genetic identification and introgression analysis using molecular markers is an important tool for management and conservation of honey bee subspecies. Previous studies have monitored introgression by using microsatellite, PCR-RFLP markers and most recently, high density assays using single nucleotide polymorphism (SNP) markers. While the latter are almost prohibitively expensive, the information gained to date can be exploited to create a reduced panel containing the most ancestry-informative markers (AIMs) for those purposes with very little loss of information. The objective of this study was to design reduced panels of AIMs to verify the origin of A. m. mellifera individuals and to provide accurate estimates of the level of C-lineage introgression into their genome. The discriminant power of the SNPs using a variety of metrics and approaches including the Weir & Cockerham’s F_ST, an F_ST-based outlier test, Delta, informativeness (I_n), and PCA was evaluated. This study shows that reduced AIMs panels assign individuals to the correct origin and calculates the admixture level with a high degree of accuracy. These panels provide an essential tool in Europe for genetic stock identification and estimation of admixture levels which can assist management strategies and monitor honey bee conservation programs.     

Racial Differences in Division of Labor in Colonies of the Honey Bee (Apis mellifera)

We measured the age at onset of foraging in colonies derived from three races of European honey bees, Apis mellifera mellifera, Apis mellifera caucasica and Apis mellifera ligustica , using a cross-fostering design that involved six unrelated colonies of each race. There was a significant effect of the race of the introduced bees on the age at onset of foraging: cohorts of A. m. ligustica bees showed the earliest onset, regardless of the race of the colony they were introduced to. There also was a significant effect of the race of the host colony: cohorts of bees introduced into mellifera colonies showed the earliest onset of foraging, regardless of the race of the bees introduced. Significant inter-trial differences also were detected, primarily because of a later onset of foraging in trials conducted during the autumn (September–October). These results demonstrate differences among European races of honey bees in one important component of colony division of labor. They also provide a starting point for analyses of the evolution of division of labor under different ecological conditions.     

Morphometric Identification of Queens,Workers and Intermediates in In Vitro Reared Honey Bees (Apis mellifera)

In vitro rearing is an important and useful tool for honey bee (Apis mellifera L.) studies. However, it often results in intercastes between queens and workers, which are normally are not seen in hive-reared bees, except when larvae older than three days are grafted for queen rearing. Morphological classification (queen versus worker or intercastes) of bees produced by this method can be subjective and generally depends on size differences. Here, we propose an alternative method for caste classification of female honey bees reared in vitro, based on weight at emergence, ovariole number, spermatheca size and size and shape, and features of the head, mandible and basitarsus. Morphological measurements were made with both traditional morphometric and geometric morphometrics techniques. The classifications were performed by principal component analysis, using naturally developed queens and workers as controls. First, the analysis included all the characters. Subsequently, a new analysis was made without the information about ovariole number and spermatheca size. Geometric morphometrics was less dependent on ovariole number and spermatheca information for caste and intercaste identification. This is useful, since acquiring information concerning these reproductive structures requires time-consuming dissection and they are not accessible when abdomens have been removed for molecular assays or in dried specimens. Additionally, geometric morphometrics divided intercastes into more discrete phenotype subsets. We conclude that morphometric geometrics are superior to traditional morphometrics techniques for identification and classification of honey bee castes and intermediates.     

In-Hive Behavior of Pollen Foragers (Apis mellifera) in Honey Bee Colonies Under Conditions of High and Low Pollen Need

Pollen collection in honey bees is regulated around a homeostatic set-point. How the control of pollen collection is achieved is still unclear. Different feedback mechanisms have been proposed but little is known about the experience of pollen foragers in the hive. A detailed documentation of the behavior of pollen foragers in the hive under different pollen need conditions is presented here. Taking a broad observational approach, we analyze the behavior of individual pollen foragers in the hive between collecting trips and quantify the different variables constituting the in-hive stay. Comparing data from two colonies and 143 individuals during experimentally induced times of low vs. times of high pollen need, we show that individual foragers modulate their in-hive working tempo according to the actual pollen need of the colony: pollen foragers slowed down and stayed in the hive longer when pollen need was low and spent less time in the hive between foraging trips when pollen need by their colony was high. Furthermore, our data show a significant change in the trophallactic experience of pollen foragers with changing pollen need conditions of their colony. Pollen foragers had more short (< 3 s) trophallactic contacts when pollen need was high, resulting in an increase of total number of trophallactic contacts. Thus, our results support the hypothesis that trophallactic experience is one of the various information pathways used by pollen foragers to assess their colony's pollen need.     

Crithidia mellificae n. sp. an Acidophilic Trypanosomatid of the Honey Bee Apis mellifera

SYNOPSIS. Crithidia mellificae n. sp. is described from the honey bee Apis mellifera in apiaries of Victoria, Australia. Organisms were isolated and cultured in a modified NNN medium with Cowper-thwaite's medium as an overlay. In contrast to other isolates of Crithidia, C. mellificae grows best at a pH between 4.5 and 5.5 with optimum near pH 5. In Cowperthwaite's medium alone, buffered at pH 4.5–5.5, the organism will survive apparently indefinite passages. The organism has so far as known, no pathologic effects in the hymenopteran host.     

Ethanol-Induced Effects on Sting Extension Response and Punishment Learning in the Western Honey Bee (Apis mellifera)

Acute ethanol administration is associated with sedation and analgesia as well as behavioral disinhibition and memory loss but the mechanisms underlying these effects remain to be elucidated. During the past decade, insects have emerged as important model systems to understand the neural and genetic bases of alcohol effects. However, novel assays to assess ethanol''s effects on complex behaviors in social or isolated contexts are necessary. Here we used the honey bee as an especially relevant model system since bees are typically exposed to ethanol in nature when collecting standing nectar crop of flowers, and there is recent evidence for independent biological significance of this exposure for social behavior. Bee''s inhibitory control of the sting extension response (SER) and a conditioned-place aversion assay were used to study ethanol effects on analgesia, behavioral disinhibition, and associative learning. Our findings indicate that although ethanol, in a dose-dependent manner, increases SER thresholds (analgesic effects), it disrupts the ability of honey bees to inhibit SER and to associate aversive stimuli with their environment. These results suggest that ethanol''s effects on analgesia, behavioral disinhibition and associative learning are common across vertebrates and invertebrates. These results add to the use of honey bees as an ethanol model to understand ethanol''s effects on complex, socially relevant behaviors.     

A Pragmatic Approach to Assess the Exposure of the Honey Bee (Apis mellifera) When Subjected to Pesticide Spray

Plant protection spray treatments may expose non-target organisms to pesticides. In the pesticide registration procedure, the honey bee represents one of the non-target model species for which the risk posed by pesticides must be assessed on the basis of the hazard quotient (HQ). The HQ is defined as the ratio between environmental exposure and toxicity. For the honey bee, the HQ calculation is not consistent because it corresponds to the ratio between the pesticide field rate (in mass of pesticide/ha) and LD₅₀ (in mass of pesticide/bee). Thus, in contrast to all other species, the HQ can only be interpreted empirically because it corresponds to a number of bees/ha. This type of HQ calculation is due to the difficulty in transforming pesticide field rates into doses to which bees are exposed. In this study, we used a pragmatic approach to determine the apparent exposure surface area of honey bees submitted to pesticide treatments by spraying with a Potter-type tower. The doses received by the bees were quantified by very efficient chemical analyses, which enabled us to determine an apparent surface area of 1.05 cm²/bee. The apparent surface area was used to calculate the exposure levels of bees submitted to pesticide sprays and then to revisit the HQ ratios with a calculation mode similar to that used for all other living species. X-tomography was used to assess the physical surface area of a bee, which was 3.27 cm²/bee, and showed that the apparent exposure surface was not overestimated. The control experiments showed that the toxicity induced by doses calculated with the exposure surface area was similar to that induced by treatments according to the European testing procedure. This new approach to measure risk is more accurate and could become a tool to aid the decision-making process in the risk assessment of pesticides.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Sex-Specific Differences in Pathogen Susceptibility in Honey Bees (Apis mellifera)

Sex-related differences in susceptibility to pathogens are a common phenomenon in animals. In the eusocial Hymenoptera the two female castes, workers and queens, are diploid and males are haploid. The haploid susceptibility hypothesis predicts that haploid males are more susceptible to pathogen infections compared to females. Here we test this hypothesis using adult male (drone) and female (worker) honey bees (Apis mellifera), inoculated with the gut endoparasite Nosema ceranae and/or black queen cell virus (BQCV). These pathogens were chosen due to previously reported synergistic interactions between Nosema apis and BQCV. Our data do not support synergistic interactions between N. ceranae and BQCV and also suggest that BQCV has limited effect on both drone and worker health, regardless of the infection level. However, the data clearly show that, despite lower levels of N. ceranae spores in drones than in workers, Nosema-infected drones had both a higher mortality and a lower body mass than non-infected drones, across all treatment groups, while the mortality and body mass of worker bees were largely unaffected by N. ceranae infection, suggesting that drones are more susceptible to this pathogen than workers. In conclusion, the data reveal considerable sex-specific differences in pathogen susceptibility in honey bees and highlight the importance of ultimate measures for determining susceptibility, such as mortality and body quality, rather than mere infection levels.