Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait ‘low brood-viability’ resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait low brood-viability resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

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.     

Estimation of intracolonial worker relationship in a honey bee colony (Apis mellifera L.) using DNA fingerprinting

Summary The number and frequencies of subfamilies in a honey bee colony were determined by DNA fingerprinting. Queen and brood samples were taken from three colonies with artificially inseminated queens and from one colony with a naturally mated queen. UsingHae III restriction enzyme and (GATA)₄ oligonucleotide, the number of subfamilies in the colonies with artificially inseminated queens corresponded with the number of drones used for insemination. In the colony with the naturally mated queen, 12 subfamilies were found in a random sample of 104 workers. Considering that subfamily frequencies range from 1 to 26%, introcolonial worker relationship was estimated to be 0.328, corresponding to a genetical effective number of 6.4 matings.     

Caste, Sex and Strain of Honey Bees (Apis mellifera) Affect Infestation with Tracheal Mites (Acarapis woodi)*

Worker honey bees from genetic strains selected for being resistant (R) or susceptible (S) to tracheal mites typically show large differences in infestation in field colonies and in bioassays that involve controlled exposure to infested bees. We used bioassays exposing newly emerged individuals to infested workers to compare the propensity for tracheal mites to infest queens, drones and workers from R and S colonies. In tests with queens, newly emerged R and S queens were either simultaneously confined in infested colonies (n = 95 and 87 respectively), or individually caged with groups of 5–20 infested workers (n = 119 and 115 respectively). Mite prevalence (percentage of individuals infested) and abundance (foundress mites per individual) after 4–6 days did not differ between R and S queens. In another test, five newly emerged drones and workers from both an R and an S colony, and a queen of one of the two strains, were caged in each of 38 cages with 20 g of workers infested at 60–96% prevalence. Infestations of the R queens (n = 17) and S queens (n = 19) did not differ significantly, but R workers had half the mite abundance of S workers, while R drones received about a third more migrating mites than S drones. In tests to evaluate possible mechanisms, removal of one mesothoracic leg from R and S workers resulted in 2- to 10-fold increase in mite abundance on the treated side, but excising legs did not affect infestation of the corresponding tracheae in drones. This suggests that differences in infestation between R and S workers, but not drones, are largely determined by their ability to remove mites through autogrooming. If autogrooming is the primary mechanism of colony resistance to tracheal mites, selection for resistance to tracheal mites using infestation of hemizygous drones may be inefficient. *The U.S. Government’s right ot retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

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.     

Reproductive biology and morphology of Apis mellifera jemenitica (Apidae) queens and drones

The current study aimed to investigate the important reproductive biology and morphology of A.m. jemenitica queens and drones through measuring the weight of virgin and mated queens, size and weight of spermathecae, weight of ovaries, number of ovarioles, quantity and viability of semen in queen and drones. Accordingly, the average weights of 0.139 ± 0.01 g and 0.143 ± 0.013 g recorded for virgin and mated queens respectively. The sizes of spermathecae were 1.248 ± 0.103 mm and 1.25 ± 0.022 mm for virgin and mated queens respectively. The mean weight of ovaries was 0.013 ± 0.003 g and the numbers of ovarioles varied from 124 to 163 with the mean of 142.9 ± 9.47 and with no significant difference between virgin and mated queens. The average number of stored sperm per spermathecae of mated queen was estimated to be 4.202 ± 0.613 million with the viability of 80.39%. The average number of sperm per drone recorded was 8,763,950 ± 1,633,203.15 with viability of 79.54 ± 6.70%. In general, the current study revealed that the values recorded for reproductive biology and morphological characters of A. m. jemenitica queens and drones were relatively lower than values recorded for other Apis mellifera races. This mainly could be associated with the body size of the race which is known to be the smallest race among A. mellifera races. Moreover, the harsh environmental conditions of the regions, high temperature, low humidity and limited resources may have contributed for the smaller biological and morphological values. The information will serve as a base in future selection and breeding of program of the race.     

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.     

Quantifying honey bee mating range and isolation in semi-isolated valleys by DNA microsatellite paternity analysis

Honey bee males and queens mate in mid air and can fly many kilometres on their nuptial flights. The conservation of native honey bees, such as the European black bee (Apis mellifera mellifera), therefore, requires large isolated areas to prevent hybridisation with other subspecies, such as A. m. ligustica or A. m. carnica, which may have been introduced by beekeepers. This study used DNA microsatellite markers to determine the mating range of A. m. mellifera in two adjacent semi-isolated valleys (Edale and Hope Valley) in the Peak District National Park, England, in order to assess their suitability for native honey bee conservation and as isolated mating locations. Three apiaries were set up in each valley, each containing 12 colonies headed by a virgin queen and 2 queenright drone producing hives. The virgin queens were allowed to mate naturally with drones from the hives we had set up and with drones from hives owned by local beekeepers. After mating, samples of worker larvae were taken from the 41 queens that mated successfully and genotyped at 11 DNA microsatellite loci. Paternity analyses were then carried out to determine mating distances and isolation. An average of 10.2 fathers were detected among the 16 worker progeny. After correction for non-detection and non-sampling errors, the mean effective mating frequency of the test queens was estimated to be 17.2, which is a normal figure for honey bees. Ninety percent of the matings occurred within a distance of 7.5 km, and fifty percent within 2.5 km. The maximal mating distance recorded was 15 km. Queens and drones did occasionally mate across the borders between the two valleys, showing that the dividing mountain ridge Losehill does not provide complete isolation. Nevertheless, in the most isolated part of Edale sixty percent of all matings were to drones from Edale hives. The large majority of observed mating distances fell within the range of Hope Valley, making this site a suitable location for the long term conservation of a breeding population of black bees.     

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.     

意蜂（Apis mellifera）蜂王婚飞交尾机制的初探

通过氯气球悬挂意蜂（Apis mellifera）雌蜂及提取物等方法模拟蜂王婚飞交尾试验,比较不同生理状态的雌蜂及其提取物对雄蜂的性引诱力,结果表明：1．在工峰、处女王与产卵王及其提取物中,以产卵王及其提取物对雄峰性诱最大．平均分别为16只和14．3只雄蜂;2．不同数量的处女王提取物对雄蜂引诱力存在差异．以3只处女王提取物对雄蜂引诱力最大,平均引诱31.3只雄蜂;3．1000烛光以上的光照比400烛光以下的光照更有利于雄蜂的水分,且雄蜂集聚的个性体敏越多,相互激活力越强。     

“Spraying” Behavior During Queen Competition in Honey Bees

In honey bees (Apis mellifera), virgin queens may eject a liquid substance from their abdomens while they are engaged in fatal combat. We investigated the functional significance of spraying behavior by staging queen duels within colonies housed in observation hives. Spraying occurred in 39.7% of all interactions between rival queens and was recorded in 12 of 15 duels. Workers were highly attracted to the surfaces and individuals contaminated by spraying, forming tight clusters with hundreds of bees in which movement was severely restricted. One or both queens in a pair became immobilized by the workers in 37.5 and 29.2% of all spraying events, respectively, but the queens were never killed by the workers. Conversely, a mobile queen penetrated the worker aggregation and stung an immobilized queen in one-third of the observed bouts. Thus spraying may serve as a fighting tactic by virgin queens to increase their chances of winning by temporarily immobilizing their rivals.     

Genetic control and developmental expression of malate dehydrogenase in Apis mellifera

Starch gel electrophoresis of extracts of Apis mellifera indicates that genetic variability exists for the enzyme cytoplasmic malate dehydrogenase (E.C. 1.1.1.37). Analysis of individuals throughout development indicates that the isozyme patterns are identical for larvae and adults and suggests a dimeric structure for the molecule. The isozyme pattern observed in pupae is more complex than that of larvae and adults may be due to an additional pupal-specific MDH gene being expressed or to an epigenetic modification of the isozymes. Forty-three colonies with artificially inseminated queens were used to study the Mendelian pattern of inheritance. The data revealed that the MDH isozymes are encoded by three alleles, Mdh-1A, Mdh-1B, and Mdh-1C. The frequency of the Mdh-1 alleles is different in two analyzed subspecies, A. m. adansonii (African bees) and A. m. ligustica (Italian bees), with Mdh-1A and Mdh-1B in the African bees being 0-768 and 0.202, respectively. For the Italian bees, these frequencies are 0.136 and 0:154, respectively.     

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.     

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.     

Genetic variance of mating frequency in the honeybee (Apis mellifera L.)

Summary. The genetic variance of queen mating frequency was studied in honeybees (Apis mellifera carnica). Worker offspring (N = 966) of 28 naturally mated half sister-queens (r = 0.25) from seven unrelated breeding lines were genotyped at four DNA microsatellites. The mating frequencies of the queens were derived from the offspring genotypes. The number of observed matings per queen ranged from 10 to 28 with an average of 17.32 ± 1.10 (number of estimated matings: 24.94 ± 2.51; number of effective matings: 20.09 ± 1.73). Half-sib analyses of the breeding lines were used to estimate heritability. Heritability was h² = 0.449 ± 0.135 for the estimated number of matings and h² = 0.262 ± 0.103 for the number of effective males, which are both significantly different from zero. We conclude that a high genetic variance for polyandry in honeybees can be favored by balanced selection between individual queen and colony level.Received 16 October 2003; revised 4 May 2004; accepted 4 May 2004.     

Evolution of sex ratios in social hymenoptera: kin selection,local mate competition,polyandry and kin recognition

A model is constructed to study the effects of local mate competition and multiple mating on the optimum allocation of resources between the male and female reproductive brood in social hymenopteran colonies from the ‘points of view’ of the queen (parental manipulation theory) as well as the workers (kin selection theory). Competition between pairs of alleles specifying different sex investment ratios is investigated in a game theoretic frame work. All other things being equal, local mate competition shifts the sex allocation ratio in favour of females both under queen and worker control. While multiple mating has no effect on the queen’s optimum investment ratio, it leads to a relatively male biased investment ratio under worker control. Under queen control a true Evolutionarily Stable Strategy(ess) does not exist but the ‘best’ strategy is merely immune from extinction. A trueess exists under worker control in colonies with singly mated queens but there is an asymmetry between the dominant and recessive alleles so that for some values of sex ratio a recessive allele goes to fixation but a dominant allele with the same properties fails to do so. Under multiple mating, again, a trueess does not exist but a frequency dependent region emerges. The best strategy here is one that is guaranteed fixation against any competing allele with a lower relative frequency. Our results emphasize the need to determine levels of local mate competition and multiple mating before drawing any conclusions regarding the outcome of queen-worker conflict in social hymenoptera. Multiple mating followed by sperm mixing, both of which are known to occur in social hymenoptera, lower average genetic relatedness between workers and their reproductive sisters. This not only shifts the optimum sex ratio from the workers’ ‘point of view’ in favour of males but also poses problems for the kin selection theory. We show that kin recognition resulting in the ability to invest in full but not in half sisters reverts the sex ratio back to that in the case of single mating and thus completely overcomes the hurdles for the operation of kin selection.     

Sib mating without inbreeding in the longhorn crazy ant

Sib matings increase homozygosity and, hence, the frequency of detrimental phenotypes caused by recessive deleterious alleles. However, many species have evolved adaptations that prevent the genetic costs associated with inbreeding. We discovered that the highly invasive longhorn crazy ant, Paratrechina longicornis, has evolved an unusual mode of reproduction whereby sib mating does not result in inbreeding. A population genetic study of P. longicornis revealed dramatic differences in allele frequencies between queens, males and workers. Mother-offspring analyses demonstrated that these allele frequency differences resulted from the fact that the three castes were all produced through different means. Workers developed through normal sexual reproduction between queens and males. However, queens were produced clonally and, thus, were genetically identical to their mothers. In contrast, males never inherited maternal alleles and were genetically identical to their fathers. The outcome of this system is that genetic inbreeding is impossible because queen and male genomes remain completely separate. Moreover, the sexually produced worker offspring retain the same genotype, combining alleles from both the maternal and paternal lineage over generations. Thus, queens may mate with their brothers in the parental nest, yet their offspring are no more homozygous than if the queen mated with a male randomly chosen from the population. The complete segregation of the male and female gene pools allows the queens to circumvent the costs associated with inbreeding and therefore may act as an important pre-adaptation for the crazy ant's tremendous invasive success.     

The flight physiology of reproductives of Africanized, European, and hybrid honeybees (Apis mellifera)

Neotropical African honeybees (Apis mellifera scutellata), in the process of spreading throughout tropical and subtropical regions of the Americas, hybridize with and mostly replace European honeybees (primarily Apis mellifera mellifera and Apis mellifera ligustica). To help understand this process, we studied the effect of lineage (African, European, or hybrid) on the flight physiology of honeybee reproductives. Flight metabolic rates were higher in queens and drones of African lineage than in European or hybrid bees, as has been previously found for foraging workers. These differences were associated with higher thorax/body mass ratios and higher thorax-specific metabolic rates in African lineage bees. Queens were reared in common colonies, so these metabolic and morphological differences are likely to be genetic in origin. African drones had higher wing beat frequencies and thorax temperatures than European or hybrid bees. Hybrids were intermediate for many parameters, but hybrid queen mass-specific flight metabolic rates were low relative to Africans and were nonlinearly affected by the proportion of African lineage, consistent with some negative heterosis for this trait.     

Free amino acids in the haemolymph of honey bee queens (Apis mellifera L.)

In queen honey bees the free amino acid content in the haemolymph clearly depends on the physiological function and social environment of the individual. While in drones and workers the content of free amino acids increases after emergence until it reaches a peak in 5-day-old animals and decreases afterwards, the amino acid content in queens reaches its highest level (>60 nmol/ microl haemolymph) with the onset of egg laying (10 d of age). This level is about 2.5 times more than the highest level found in workers. Queens maintain this high level also when they are older (>30 d) and continue to lay eggs in average colonies. As in drones and workers, in queens the predominant amino acid is proline, which accounts for more than 50% of the total content of free amino acids in egg-laying individuals. When 10-day-old queens are prevented from mating and do not lay eggs, their amino acid content is significantly lower compared to laying queens of the same age. Also the social environment influences the contents of free amino acids in queens. When virgin queens were kept for 6 days with 20 worker bees and sufficient honey and pollen in an incubator, they had significantly lower concentrations of amino acids than virgin queens living for the same period with about 8000 workers in a colony. Most probably, the high amino acid concentration in the haemolymph is the basis for the high protein synthesis activity of laying queens.