Temporal variation in the genetic structure of a drone congregation area: an insight into the population dynamics of wild African honeybees (Apis mellifera scutellata)

The mating system of the honeybee ( Apis mellifera ) has been regarded as one of the most panmictic in the animal kingdom, with thousands of males aggregating in drone congregation areas (DCAs) that virgin queens visit to mate with tens of partners. Although males from many colonies gather at such congregations, the temporal changes in the colonies contributing drones remain unknown. Yet, changes in the DCAs' genetic structure will ultimately determine population gene flow and effective population size. By repeatedly sampling drones from an African DCA over a period of 3 years, we studied the temporal changes in the genetic structure of a wild honeybee population. Using three sets of tightly linked microsatellite markers, we were able to reconstruct individual queen genotypes with a high accuracy, follow them through time and estimate their rate of replacement. The number of queens contributing drones to the DCA varied from 12 to 72 and was correlated with temperature and rainfall. We found that more than 80% of these queens were replaced by mostly unrelated ones in successive eight months sampling intervals, which resulted in a clear temporal genetic differentiation of the DCA. Our results suggest that the frequent long-range migration of colonies without nest-site fidelity is the main driver of this high queen turnover. DCAs of African honeybees should thus be regarded as extremely dynamic systems which together with migration boost the effective population size and maintain a high genetic diversity in the population.     

Nest Defense Behavior in Colonies from Crosses Between Africanized and European Honey Bees (Apis mellifera L.) (Hymenoptera: Apidae)

Honey bee (Apis mellifera L.) colonies with either European or Africanized queens mated to European or Africanized drones alone or in combination were tested for defensive behavior using a breath test. The most defensive colonies were those with European or Africanized queens mated to Africanized drones. In colonies where both European and Africanized patrilines existed, most of the workers participating in nest defense behavior for the first 30 s after a disturbance were of African patrilines. Nest defense behavior appears to be genetically dominant in honey bees.     

Extensive population admixture on drone congregation areas of the giant honeybee,Apis dorsata (Fabricius, 1793)

The giant honeybee Apis dorsata often forms dense colony aggregations which can include up to 200 often closely related nests in the same location, setting the stage for inbred matings. Yet, like in all other Apis species, A. dorsata queens mate in mid‐air on lek like drone congregation areas (DCAs) where large numbers of males gather in flight. We here report how the drone composition of A. dorsata DCAs facilitates outbreeding, taking into the account both spatial (three DCAs) and temporal (subsequent sampling days) dynamics. We compared the drones’ genotypes at ten microsatellite DNA markers with those of the queen genotypes of six drone‐producing colonies located close to the DCAs (Tenom, Sabah, Malaysia). None of 430 sampled drones originated from any of these nearby colonies. Moreover, we estimated that 141 unidentified colonies were contributing to the three DCAs. Most of these colonies were participating multiple times in the different locations and/or during the consecutive days of sampling. The drones sampled in the DCAs could be attributed to six subpopulations. These were all admixed in all DCA samples, increasing the effective population size an order of magnitude and preventing matings between potentially related queens and drones.     

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.     

The Africanization of honeybees (Apis mellifera L.) of the Yucatan: a study of a massive hybridization event across time

Until recently, African and European subspecies of the honeybee (Apis mellifera L.) had been geographically separated for around 10,000 years. However, human-assisted introductions have caused the mixing of large populations of African and European subspecies in South and Central America, permitting an unprecedented opportunity to study a large-scale hybridization event using molecular analyses. We obtained reference populations from Europe, Africa, and South America and used these to provide baseline information for a microsatellite and mitochondrial analysis of the process of Africanization of the bees of the Yucatan Peninsula, Mexico. The genetic structure of the Yucatecan population has changed dramatically over time. The pre-Africanized Yucatecan population (1985) comprised bees that were most similar to samples from southeastern Europe and northern and western Europe. Three years after the arrival of Africanized bees (1989), substantial paternal gene flow had occurred from feral Africanized drones into the resident European population, but maternal gene flow from the invading Africanized population into the local population was negligible. However by 1998, there was a radical shift with both African nuclear alleles (65%) and African-derived mitochondria (61%) dominating the genomes of domestic colonies. We suggest that although European mitochondria may eventually be driven to extinction in the feral population, stable introgression of European nuclear alleles has occurred.     

MtDNA COI‐COII marker and drone congregation area: An efficient method to establish and monitor honeybee (Apis mellifera L.) conservation centres

Honeybee subspecies have been affected by human activities in Europe over the past few decades. One such example is the importation of nonlocal subspecies of bees which has had an adverse impact on the geographical repartition and subsequently on the genetic diversity of the black honeybee Apis mellifera mellifera. To restore the original diversity of this local honeybee subspecies, different conservation centres were set up in Europe. In this study, we established a black honeybee conservation centre Conservatoire de l'Abeille Noire d'Ile de France (CANIF) in the region of Ile‐de‐France, France. CANIF's honeybee colonies were intensively studied over a 3‐year period. This study included a drone congregation area (DCA) located in the conservation centre. MtDNA COI‐COII marker was used to evaluate the genetic diversity of CANIF's honeybee populations and the drones found and collected from the DCA. The same marker (mtDNA) was used to estimate the interactions and the haplotype frequency between CANIF's honeybee populations and 10 surrounding honeybee apiaries located outside of the CANIF. Our results indicate that the colonies of the conservation centre and the drones of the DCA show similar stable profiles compared to the surrounding populations with lower level of introgression. The mtDNA marker used on both DCA and colonies of the conservation centre seems to be an efficient approach to monitor and maintain the genetic diversity of the protected honeybee populations.     

Mitochondrial gene frequencies in Africanized honeybees (Apis mellifera L.): Theoretical model and empirical evidence

A population genetical model is used to investigate the effects of queen and drone fitness and swarming ability on nuclear and mitochondrial (mt) DNA variation in honeybees. The analysis of both types of DNA is particularly useful for a genetic study of the Africanized bee problem in the Americas. Both an analytical model and a Monte Carlo simulation show that even if mtDNA proves to be selectively neutral, Africanized mitotypes are expected at high frequencies because of the more frequent swarming of Africanized honeybees. Since the fitness of Africanized drones is higher than that of European drones, European and African mitotypes are expected to be polymorphic and consequently unreliable as diagnostic tools to discriminate between the two types. Samples of Africanized honeybees in Brazil reveal high mtDNA polymorphisms as predicted by the theoretical models.     

Quantitative trait loci for honey bee stinging behavior and body size.

A study was conducted to identify quantitative trait loci (QTLs) that affect colony-level stinging behavior and individual body size of honey bees. An F1 queen was produced from a cross between a queen of European origin and a drone descended from an African subspecies. Haploid drones from the hybrid queen were individually backcrossed to sister European queens to produce 172 colonies with backcross workers that were evaluated for tendency to sting. Random amplified polymorphic DNA markers were scored from the haploid drone fathers of these colonies. Wings of workers and drones were used as a measure of body size because Africanized bees in the Americas are smaller than European bees. Standard interval mapping and multiple QTL models were used to analyze data. One possible QTL was identified with a significant effect on tendency to sting (LOD 3.57). Four other suggestive QTLs were also observed (about LOD 1.5). Possible QTLs also were identified that affect body size and were unlinked to defensive-behavior QTLs. Two of these were significant (LOD 3.54 and 5.15).     

Parental Analysis of Introgressive Hybridization between African and European Honeybees Using Nuclear DNA Rflps

African honeybees, introduced into Brazil 33 years ago, have spread through most of South and Central America and have largely replaced the extant European bees. Due to a paucity of genetic markers, genetic interactions between European and African bees are not well understood. Three restriction fragment length polymorphisms (RFLPs), detected with random, nuclear DNA probes, are described. The polymorphisms are specific to bees of European descent, possibly specific to certain European races. Each European marker was found present at a high frequency in U.S. colonies but absent in South African bees. Previous mitochondrial DNA studies of neotropical bees have revealed negligible maternal gene flow from managed European apiaries into feral African populations. The findings reported here with nuclear DNA show paternal gene flow between the two but suggest asymmetries in levels of introgressive hybridization. Managed colonies in southern Mexico, derived from European maternal lines, showed diminished levels of the European nuclear markers, reflecting significant hybridization with African drones. The European alleles were present only at low frequencies in feral swarms from the same area. The swarms were of African maternal descent. In Venezuelan colonies, also derived from African maternal lines, the European markers were almost totally absent. The results point to limited paternal introgression from European colonies into the African honeybee populations. These findings dispute other views regarding modes of Africanization.     

The nearer the better? Drones (Apis mellifera) prefer nearer drone congregation areas

Summary. At 2 drone congregation areas (DCA) the relation between drone presence and distance to the apiary of origin was studied. Two methods were applied. First, drones were caught and marked on the DCA and later recovered in the colonies. Second, drones which were marked before at the apiary (in the colonies) were subsequently recaptured on both DCAs. The 2 methods led to identical conclusions. Consistently in each of 3 years the majority of the drones from each of the 3 apiaries was found at the nearer DCA. There was, however, no direct correlation between the flight distances and the ratio of drones visiting from each apiary. Thus some other factors (attractiveness of the DCA) may also have influenced the choice of the drones. Our findings support the idea that there is an orientation phase during which drones explore several DCAs before each drone stays at 1 DCA, and energetic choices made by drones in relation to flight distances seem to be important. The choice of the nearer DCA would permit the drone to prolong his presence at the DCA and increase his chances to mate: the nearer the better!The drones strategy to chose the nearer DCA would boost the genetic representation of local colonies and this drone clumping would increase genetic differences among the DCAs within an area. In this context the choice of virgin queens among DCAs is of great significance.Received 1 December 2003; revised 9 April 2004; accepted 7 June 2004.     

Genetic variation in worker temporal polyethism and colony defensiveness in the honey bee, Apis mellifera

To test the hypothesis that colonies of honey bees composedof workers with faster rates of adult behavioral developmentare more defensive than colonies composed of workers with slowerbehavioral development, we determined whether there is a correlationbetween genetic variation in worker temporal polyethism andcolony defensiveness. There was a positive correlation for thesetwo traits, both for European and Africanized honey bees. Thecorrelation was larger for Africanized bees, due to differencesbetween Africanized and European bees, differences in experimentaldesign, or both. Consistent with these results was the findingthat colonies with a higher proportion of older bees were moredefensive than colonies of the same size that had a lower proportionof older bees. There also was a positive correlation betweenrate of individual behavioral development and the intensityof colony flight activity, and a negative correlation betweencolony defensiveness and flight activity. This suggests thatthe relationship between temporal polyethism and colony defensivenessmay vary with the manner in which foraging and defense dutiesare allocated among a colony's older workers. These resultsindicate that genotypic differences in rates of worker behavioraldevelopment can influence the phenotype of a honey bee colonyin a variety of ways.     

Genetic dominance and worker interactions affect honeybee colony defense

Colonies of honeybees (Apis mellifera L.) were established thatvaried in the proportions of their workers that were of Europeanand hybrid (Africanized x European) descent. Colony defensiveresponses increased with higher proportions of hybrid workers.Colonies consisting exclusively of hybrid workers did not differin their response from "pure" Africanized colonies, suggestingthat the strong defensive behavior of Africanized workers isgenetically dominant. European workers became more defensivein colonies that also contained hybrid workers, whereas hybridworkers became less defensive in the same mixed colonies. Inmixed colonies hybrid workers were individually more likelythan Europeans to sting a leather target but not more likelyto guard the entrance.     

Paternal effects on the defensive behavior of honeybees

The defensive behavior of 52 hybrid honeybee (Apis mellifera L.) colonies from four sets of crosses was studied and compared with that of European and Africanized bee colonies. Colonies containing F(1) hybrid workers were obtained through reciprocal crosses between European and Africanized bees. The total number of stings deposited by workers in a moving leather patch in 1 min was recorded. In each of the four sets of crosses, bees from hybrid colonies of Africanized paternity left more stings in leather patches than bees from hybrid colonies of European paternity. Results strongly suggest paternal effects of African origin increasing the defensive behavior of hybrid colonies. Although some degree of dominance was observed for high-defensive behavior in one of the four sets of crosses involving European paternity, most of the dominance effects reported in the literature appear to be the result of paternal effects. Several hypotheses to explain this phenomenon, as well as the implications of these effects on the fitness and breeding of honeybees are discussed.     

Temporal pattern of africanization in a feral honeybee population from Texas inferred from mitochondrial DNA

The invasion of Africanized honeybees (Apis mellifera L.) in the Americas provides a window of opportunity to study the dynamics of secondary contact of subspecies of bees that evolved in allopatry in ecologically distinctive habitats of the Old World. We report here the results of an 11-year mitochondrial DNA survey of a feral honeybee population from southern United States (Texas). The mitochondrial haplotype (mitotype) frequencies changed radically during the 11-year study period. Prior to immigration of Africanized honeybees, the resident population was essentially of eastern and western European maternal ancestry. Three years after detection of the first Africanized swarm there was a mitotype turnover in the population from predominantly eastern European to predominantly A. m. scutellata (ancestor of Africanized honeybees). This remarkable change in the mitotype composition coincided with arrival of the parasitic mite Varroa destructor, which was likely responsible for severe losses experienced by colonies of European ancestry. From 1997 onward the population stabilized with most colonies of A. m. scutellata maternal origin.     

Suppression of queen rearing in European and Africanized honey beesApis mellifera L. by synthetic queen mandibular gland pheromone

Summary Queen rearing is suppressed in honey bees (Apis mellifera L.) by pheromones, particularly the queen's mandibular gland pheromone. In this study we compared this pheromonally-based inhibition between temperate and tropically-evolved honey bees. Colonies of European and Africanized bees were exposed to synthetic queen mandibular gland pheromone (QMP) for ten days following removal of resident queens, and their queen rearing responses were examined. Queen rearing was suppressed similarly in both European and Africanized honey bees with the addition of synthetic QMP, indicating that QMP acts on workers of both races in a comparable fashion. QMP completely suppressed queen cell production for two days, but by day six, cells containing queen larvae were present in all treated colonies, indicating that other signals play a role in the suppression of queen rearing. In queenless control colonies not treated with QMP, Africanized bees reared 30% fewer queens than Europeans, possibly due to racial differences in response to feedback from developing queens and/or their cells. Queen development rate was faster in Africanized colonies, or they selected older larvae to initiate cells, as only 1 % of queen cells were unsealed after 10 days compared with 12% unsealed cells in European colonies.     

Maternal influence on the acceptance of virgin queens introduced into Africanized honey bee (Apis mellifera) colonies

The oviposition potential of honey bee queens decreases with age, therefore it is important to replace old queens with younger ones on a periodic basis. However, queen replacement is problematic, especially in Africanized honey bee colonies, since many introduced queens are not accepted, and virgin queens are less easily accepted than are mated queens. We assessed the influence of genetic origin (queen mother) on the acceptance of queens, when they were introduced as virgins into Africanized honey bee colonies. For this purpose, 12 daughter queens from each of 11 mother queens with no degree of kinship among themselves were introduced. Introductions were made monthly, for 12 months, though the winter months of June and July were not included, as there is little brood and drones are rare in winter. There was some seasonal variation in the acceptance rates; generally there was greater acceptance in months with good honey flows. However, the acceptance of introduced queens was influenced by their origin. The rate of acceptance of daughter queens from the 11 different mother queens varied significantly, ranging from 33 to 75%. There appears to be a genetic influence of the mother queen on the introduced queen acceptance rate.     

The influence of brood comb cell size on the reproductive behavior of the ectoparasitic mite Varroa destructor in Africanized honey bee colonies

Africanized honey bees (Apis mellifera, Hymenoptera: Apidae) in Brazil are tolerant of infestations with the exotic ectoparasitic mite, Varroa destructor (Mesostigmata: Varroidae), while the European honey bees used in apiculture throughout most of the world are severely affected. Africanized honey bees are normally kept in hives with both naturally built small width brood cells and with brood cells made from European-sized foundation, yet we know that comb cell size has an effect on varroa reproductive behavior. Three types (sizes) of brood combs were placed in each of six Africanized honey bee colonies: new (self-built) Africanized comb, new Italian comb (that the bees made from Italian-sized commercial foundation), and new Carniolan comb (built naturally by Carniolan bees). About 100 cells of each type were analyzed in each colony. The Africanized comb cells were significantly smaller in (inner) width (4.84 mm) than the European-sized comb cells (5.16 and 5.27 mm for Italian and Carniolan cells, respectively). The brood cell infestation rates (percentage cells infested) were significantly higher in the Carniolan-sized comb cells (19.3%) than in the Italian and Africanized cells (13.9 and 10.3%, respectively). The Carniolan-sized cells also had a significantly larger number of invading adult female mites per 100 brood cells (24.4) than did the Italian-sized cells (17.7) and the natural-sized Africanized worker brood cells (15.6). European-sized worker brood cells were always more infested than the Africanized worker brood cells in the same colony. There was a highly significant correlation (P<0.01) between cell width and the rate of infestation with varroa in four of the six colonies. The small width comb cells produced by Africanized honey bees may have a role in the ability of these bees to tolerate infestations by Varroa destructor, furthermore it appears that natural-sized comb cells are superior to over-sized comb cells for disease resistance.     

Africanization in the United States: replacement of feral European honeybees (Apis mellifera L.) by an African hybrid swarm

The expansion of Africanized honeybees from South America to the southwestern United States in <50 years is considered one of the most spectacular biological invasions yet documented. In the American tropics, it has been shown that during their expansion Africanized honeybees have low levels of introgressed alleles from resident European populations. In the United States, it has been speculated, but not shown, that Africanized honeybees would hybridize extensively with European honeybees. Here we report a continuous 11-year study investigating temporal changes in the genetic structure of a feral population from the southern United States undergoing Africanization. Our microsatellite data showed that (1) the process of Africanization involved both maternal and paternal bidirectional gene flow between European and Africanized honeybees and (2) the panmitic European population was replaced by panmitic mixtures of A. m. scutellata and European genes within 5 years after Africanization. The post-Africanization gene pool (1998-2001) was composed of a diverse array of recombinant classes with a substantial European genetic contribution (mean 25-37%). Therefore, the resulting feral honeybee population of south Texas was best viewed as a hybrid swarm.     

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.     

Africanized honey bees (Hymenoptera: Apidae) have a greater fidelity to sunflowers than European bees

A study of sunflower, Helianthus annuus L., pollen collection by Africanized and European honey bees, Apis mellifera L., was conducted in a hybrid seed production field in Argentina. Africanized honey bees collected significantly larger proportions of sunflower pollen than did European honey bees. The result suggests that Africanized bees would be more efficient for commercial sunflower seed production.