Effects of insemination quantity on honey bee queen physiology

Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony.     

Population structure of honey bees in the Carpathian Basin (Hungary) confirms introgression from surrounding subspecies

Carniolan honey bees (Apis mellifera carnica) are considered as an indigenous subspecies in Hungary adapted to most of the ecological and climatic conditions in this area. However, during the last decades Hungarian beekeepers have recognized morphological signs of the Italian honey bee (Apis mellifera ligustica). As the natural distribution of the honey bee subspecies can be affected by the importation of honey bee queens or by natural gene flow, we aimed at determining the genetic structure and characteristics of the local honey bee population using molecular markers. All together, 48 Hungarian and 84 foreign (Italian, Polish, Spanish, Liberian) pupae and/or workers were used for mitochondrial DNA analysis. Additionally, 53 sequences corresponding to 10 subspecies and the Buckfast hybrid were downloaded from GenBank. For the nuclear analysis, 236 Hungarian and 106 foreign honey bees were genotyped using nine microsatellites. Heterozygosity values, population‐specific alleles, FST values, principal coordinate analysis, assignment tests, structure analysis, and dendrograms were calculated. Haplotype and nucleotide diversity values showed moderate values. We found that one haplotype (H9) was dominant in Hungary. The presence of the black honey bee (Apis mellifera mellifera) was negligible, but a few individuals resembling other subspecies were identified. We proved that the Hungarian honey bee population is nearly homogeneous but also demonstrated introgression from the foreign subspecies. Both mitochondrial DNA and microsatellite analyses corroborated the observations of the beekeepers. Molecular analyses suggested that Carniolan honey bee in Hungary is slightly affected by Italian and black honey bee introgression. Genetic differences were detected between Polish and Hungarian Carniolan honey bee populations, suggesting the existence of at least two different gene pools within A. m. carnica.     

Honey Bee Queens Do Not Count Mates to Assess their Mating Success

The mating system of honey bees (genus Apis) is extremely polyandrous, where reproductive females (queens) typically mate with 12 or more males (drones) during their mating flight(s). The evolutionary implications for hyperpolyandry have been subject to considerable debate and empirical testing because of the need to understand the proximate mechanisms that drive such extreme mating behavior despite the potential costs. The ability of queens to gauge and adjust their reproductive success is therefore important for selection to act on queen mating number at both the evolutionary (colony-level) and proximate (individual-level) timescales. We observed the mating flight activities of 80 queens in their respective mating nucleus hives each with a modified entrance that restricts flight attempts. We also attached a small weight (0, 16, or 38 mg) onto each queen’s thorax as a means of imposing additional flight costs. We then compared queens that were restricted from taking multiple mating flights to those that started oviposition after a single flight for their mating numbers as quantified by microsatellite analyses of their respective worker offspring. We found that neither additional weight nor restricted mating attempts had any significant effect on the effective mating frequencies of the experimental queens during their single mating flight. This observation suggests that queens are not adjusting their nuptial flight activity according to their precise mating number during their flight. These findings provide insights into the proximate regulation of honey bee queen mating behavior and the fitness consequences of hyperpolyandry at the colony level.     

Paternity and maternity frequencies in Apis mellifera sicula

Summary: Honey bee queens have been shown to mate with a high number of males, but the evolutionary advantage of this high degree of polyandry is still unclear. Mating data from a number of different Apis species and subspecies are needed to help explain polyandry in honey bees. Pupae of four colonies of Apis mellifera sicula from Sicily were genotyped on three polymorphic microsatellite loci. The genotypes of the queens and fathering drones from these colonies were deduced from the genotypes of the pupae. We found no evidence for polygyny, at least we can exclude more than one functional queen, even super-sister queens, if maternity contributions are equal. The four queens mated with at least 5 to 12 (mean: 9.3 - 3.0 SE) drones. We estimate the error in our determination of the mating frequency that is caused by limited genetic resolution of the marker loci to be less than 1 mating given that Hardy-Weinberg assumptions are satisfied. However, the drones the single queens mated with may be a non-random sample of the whole population, so that detection error may be more severe. The average pedigree relatedness among workers within the colonies was estimated to be 0.341. These results are within the range of those found in other A. mellifera subspecies and Apis species except A. dorsata. We speculate that mating frequency may be positively correlated with drone density.     

Assessing the mating 'health' of commercial honey bee queens

Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices.     

Inheritance of resistance to Acarapis woodi (Acari: Tarsonemidae) in first-generation crosses of honey bees (Hymenoptera: Apidae)

The tendency of honey bees, Apis mellifera L, to become infested with tracheal mites, Acarapis woodi (Rennie), was measured in six different types of F1 colonies. The colonies were produced by mating a stock (Buckfast) known to resist mite infestation to each of five commercially available stocks and to a stock known to be susceptible to mites. Young uninfested bees from progeny and parent colonies were simultaneously exposed to mites in infested colonies, then retrieved and dissected to determine resultant mite infestations. Reduced infestations similar to but numerically greater than those of the resistant parent bees occurred in each of the six crosses made with resistant bees regardless of the relative susceptibility of the other parental stock. Reciprocal crosses between resistant and susceptible queens and drones proved equally effective in improving resistance. Therefore, allowing resistant stock queens to mate naturally with unselected drones, or nonresistant queens to mate with drones produced by pure or outcrossed resistant queens, can be used for improving resistance of production queens.     

Melissopalynology in an area of Atlantic Forest (northeast region,Brazil)

Apis mellifera is a bee that was introduced to Brazil and has adapted very well to the climate conditions and vast diversity of plants that exist in the country. In the northeast region of Brazil, beekeepers make use of the association between bees and plants by selling various bee products, notably honey. One way to identify species visited by bees in an area is by the pollen in its products. Based on this, 16 samples of honey were analysed, which were collected over a period of two years and obtained from an apiary in the Atlantic Forest biome in the municipality of Entre Rios (Bahia). In addition, climatic data (precipitation and temperature) of the region were obtained for the months sampled. The average temperature of the region during the collection months varied from 22 to 28 °C. The highest precipitation recorded in the region was 133.7 mm³ and the lowest was 0.3 mm³. Seventy pollen types were found. The family Fabaceae was notable, with ten pollen types, of which Mimosa pudica was the most important with a high frequency of occurrence and distribution. The Eucalyptus (Myrtaceae) pollen type also had a high frequency of occurrence and distribution. In addition, pollen types corresponding to species that supply nectar to bees were identified, which contributes to the large diversity of bee plants for Apis mellifera in the study area.     

Age and Behavior of Honey Bees,Apis mellifera (Hymenoptera: Apidae), that Perform Vibration Signals on Queens and Queen Cells

The vibration signal of the honey bee (Apis mellifera) may play a central role in the regulation of queen behavior during reproductive swarming and supersedure. We examined honey bee workers that performed vibration signals on queens and developing queen cells in three observation hives, each containing a population of marked bees of known age. In all three colonies, workers of all ages greater than 2 d old could perform vibration signals on queens and queen cells. However, most signals were performed by a small proportion of the bees of greater than 10 d of age. Relatively few workers less than 10 d old vibrated queens and queen cells, even though this age-group is typically associated with queen care. Thus, the regulation of queen behavior by the vibration signal may occur primarily through a relatively small subset of older workers that, under most circumstances, have only limited involvement with queens. It is unclear what triggers the vibrating of queens. Workers producing vibration signals did not differ from same-age non-vibrating controls in rate of locomotion in the hive or in task performance, and they rarely engaged in foraging, even though the majority of observed bees were of foraging age; vibrators also did not spend more time with queens and queen cells compared with controls. Vibration signals performed on queens and queen cells therefore do not appear to be influenced by task performance or increased contact with queens.     

Western honey bee (Apis mellifera L.) attraction to commercial pollen substitutes and wildflower pollen in vitro

Many beekeepers feed their western honey bee (Apis mellifera) colonies artificial pollen substitutes to provide colonies with adequate nutrition during times of limited pollen quantity or quality. We provided caged worker bees commercially available pollen substitutes (AP23, MegaBee, UltraBee) and wildflower pollen in a choice-test to determine their relative attraction to/preference for the diets. We measured diet consumption by honey bees and observed honey bee behaviour to evaluate bee preferences for certain diets. The bees interacted with and consumed more wildflower pollen than they did any of the commercially available pollen substitutes. Our data suggest that bees have a strong preference for wildflower pollen over commercially available pollen substitutes.     

PVC nest boxes are less at risk of occupancy by feral honey bees than timber nest boxes and natural hollows

Feral European Honey Bee (Apis mellifera) has been identified as a potential nest competitor for Australian hollow nesting species, but few studies have investigated the impact of feral honey bee competition on Threatened species. Our study used data from Glossy Black‐cockatoo (Calyptorhynchus lathami halmaturinus) nests on Kangaroo Island, monitored and managed over an 11‐year period, and found 12% of nests became occupied by feral honey bees during that period. Our results indicate that feral honey bees were less likely to occupy nest boxes made of PVC (5%) compared with wooden nest boxes (24%) or natural hollows in Eucalyptus trees (14%). The removal of feral honey bee hives from nests is a priority for long‐term conservation of glossy black‐cockatoos on Kangaroo Island. We recommend that PVC nest boxes are chosen for future nesting habitat restoration, due to the more frequent use of wooden nest boxes by feral honey bees.     

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

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

Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera)

Nosema ceranae, a microsporidian parasite originally described in the Asian honey bee Apis cerana, has recently been found to be cross-infective and to also parasitize the European honey bee Apis mellifera. Since this discovery, many studies have attempted to characterize the impact of this parasite in A. mellifera honey bees. Nosema species can infect all colony members, workers, drones and queens, but the pathological effects of this microsporidium has been mainly investigated in workers, despite the prime importance of the queen, who monopolizes the reproduction and regulates the cohesion of the society via pheromones. We therefore analyzed the impact of N. ceranae on queen physiology. We found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).     

Determining colony densities in wild honeybee populations (<Emphasis Type="Italic">Apis mellifera</Emphasis>) with linked microsatellite DNA markers

Estimating the population size of social bee colonies in the wild is often difficult because nests are highly cryptic. Because of the honeybee (Apis mellifera) mating behaviour, which is characterized by multiple mating of queens at drone congregation areas (DCA), it is possible to use genotypes of drones caught at these areas to infer the number of colonies in a given region. However, DCAs are difficult to locate and we assess the effectiveness of an alternative sampling technique to determine colony density based on inferring male genotypes from queen offspring. We compare these methods in the same population of wild honeybees, Apis mellifera scutellata. A set of linked microsatellite loci is used to decrease the frequency of recombination among marker loci and therefore increase the precision of the estimates. Estimates of population size obtained through sampling of queen offspring is significantly larger than that obtained by sampling drones at DCAs. This difference may be due to the more extensive flying range of queens compared with drones on mating flights. We estimate that the population size sampled through queen offspring is about double that sampled through drones.     

Resistance to Varroa destructor (Mesostigmata: Varroidae) when mite-resistant queen honey bees (Hymenoptera: Apidae) were free-mated with unselected drones.

This study demonstrated (1) that honey bees, Apis mellifera L, can express a high level of resistance to Varroa destructor Anderson & Trueman when bees were selected for only one resistant trait (suppression of mite reproduction); and (2) that a significant level of mite-resistance was retained when these queens were free-mated with unselected drones. The test compared the growth of mite populations in colonies of bees that each received one of the following queens: (1) resistant--queens selected for suppression of mite reproduction and artificially inseminated in Baton Rouge with drones from similarly selected stocks; (2) resistant x control--resistant queens, as above, produced and free-mated to unselected drones by one of four commercial queen producers; and (3) control--commercial queens chosen by the same four queen producers and free-mated as above. All colonies started the test with approximately 0.9 kg of bees that were naturally infested with approximately 650 mites. Colonies with resistant x control queens ended the 115-d test period with significantly fewer mites than did colonies with control queens. This suggests that beekeepers can derive immediate benefit from mite-resistant queens that have been free-mated to unselected drones. Moreover, the production and distribution of these free-mated queens from many commercial sources may be an effective way to insert beneficial genes into our commercial population of honey bees without losing the genetic diversity and the useful beekeeping characteristics of this population.     

Reproductive interference between honeybee species in artificial sympatry

Reproductive isolation between closely related species is often incomplete. The Western honeybee, Apis mellifera, and the Eastern hive bee, Apis cerana, have been allopatric for millions of years, but are nonetheless similar in morphology and behaviour. During the last century, the two species were brought into contact anthropogenically, providing potential opportunities for interspecific matings. Hybrids between A. mellifera and A. cerana are inviable, so natural interspecific matings are of concern because they may reduce the viability of A. cerana and A. mellifera populations – two of the world's most important pollinators. We examined the mating behaviour of A. mellifera and A. cerana queens and drones from Caoba Basin, China and Cairns, Australia. Drone mating flight times overlap in both areas. Analysis of the spermathecal contents of queens with species‐specific genetic markers indicated that in Caoba Basin, 14% of A. mellifera queens mated with at least one A. cerana male, but we detected no A. cerana queens that had mated with A. mellifera males. Similarly, in Cairns, no A. cerana queens carried A. mellifera sperm, but one‐third of A. mellifera queens had mated with at least one A. cerana male. No hybrid embryos were detected in eggs laid by interspecifically mated A. mellifera queens in either location. However, A. mellifera queens artificially inseminated with A. cerana sperm produced inviable hybrid eggs or unfertilized drones. This suggests that reproductive interference will impact the viability of honeybee populations wherever A. cerana and A. mellifera are in contact.     

Potential negative effects of exotic honey bees on the diversity of native pollinators and yield of highland coffee plantations

• 1 The honey bee Apis mellifera is native to Eurasia and Africa, although it is commonly introduced into crop fields of different parts of the world because of the assumption that it improves yield. This bee is, however, a poor pollinator of several crops compared with native insects. Indeed, honey bees can displace native pollinators and reduce their diversity. The present study evaluated the potential impacts of A. mellifera on the diversity of native pollinators of highland coffee (Coffea arabica) and its putative consequences for coffee production at the state of Veracruz, Mexico.
• 2 The abundance of A. mellifera and diversity of native pollinators were assessed during blooming at 12 shade coffee plantations and pollination experiments were conducted to determine the impacts of pollinators on coffee fruit production. Regression analyses were used to assess whether the abundance of honey bees was related to native pollinator diversity, and whether fruit production was influenced by both the diversity of pollinators and the abundance of A. mellifera.
• 3 Native pollinator diversity decreased as the number of honey bees increased. Furthermore, although coffee fruit production was positively related to the diversity of native pollinators, an increasing abundance of A. mellifera was correlated with a decrease in fruit production.
• 4 Highland shade coffee plantations are considered as reservoirs of the Mexican insect fauna. Thus, native pollinator diversity could be better preserved if beekeepers reduced the number of managed hives that they brought into plantations. This may also help to increase coffee yield by decreasing the putative negative effects of A. mellifera on native pollinators.

     

Impact of the introduced honey bee (Apis mellifera) (Hymenoptera: Apidae) on native bees: A review

Abstract Interspecific competition for a limited resource can result in the reduction of survival, growth and/or reproduction in one of the species involved. The introduced honey bee (Apis mellifera Linnaeus) is an example of a species that can compete with native bees for floral resources. Often, research into honey bee/native bee competition has focused on floral resource overlap, visitation rates or resource harvesting, and any negative interaction has been interpreted as a negative impact. Although this research can be valuable in indicating the potential for competition between honey bees and native bees, to determine if the long‐term survival of a native bee species is threatened, fecundity, survival or population density needs to be assessed. The present review evaluates research that has investigated all these measurements of honey bee/native bee competition and finds that many studies have problems with sample size, confounding factors or data interpretation. Guidelines for future research include increasing replication and using long‐term studies to investigate the impact of both commercial and feral honey bees.     

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.     

PCR-based detection of a tracheal mite of the honey bee Acarapis woodi

The effects of the tracheal mite Acarapis woodi on the health of honey bees have been neglected since the prevalence of Varroa mites to Apis mellifera colonies. However, tracheal mite infestation of honey bee colonies still occurs worldwide and could impose negative impact on apiculture. The detection of A. woodi requires the dissection of honey bees followed by microscopic observation of the tracheal sacs. We thus developed PCR methods to detect A. woodi. These methods facilitate rapid and sensitive detection of A. woodi in many honey bee samples for epidemiologic surveys.     

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.