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.     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

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).     

Honeybee colony drone production and maintenance in accordance with environmental factors: an interplay of queen and worker decisions

Social insect colonies display a remarkable ability to adjust investment in reproduction (i.e., production of sexuals) in accordance with environmental conditions such as season and food availability. How this feat is accomplished by the colony’s queen(s) and workers remains a puzzle. Here, I review what we have learned about this subject in the European honeybee (Apis mellifera), specifically with regard to a colony’s production of males (drones). I identify five environmental conditions that influence colony-level patterns of drone production and then define five stages of drone rearing that are accomplished by the queen and workers. Using this framework, I detail our current understanding of how the queen or workers adjust their actions at each stage of drone rearing in response to each of the environmental conditions. Future investigations of this topic in honeybees and other social insect societies will lead to a better understanding of how colonies manage to flexibly and efficiently allocate their resources under changing environmental conditions.     

Quantitative trait loci influencing honeybee alarm pheromone levels.

Quantitative trait loci (QTL) mapping procedures were used to identify loci that influence the levels of alarm pheromones found in the stinging apparatus of worker honeybees. 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 European queens to produce 172 colonies. Samples of stings were taken from backcross workers of these colonies. Alarm pheromone levels were determined by gas chromatography. RAPD markers were scored from the haploid drone fathers of these colonies. The multiple-QTL model (MQM) of MapQTL was used to identify QTLs that influence the levels of four alarm pheromone components. Seven independent, potential QTLs were identified with LOD scores greater than two, and one at LOD 1.88. We identified one QTL for n-decyl acetate, three for n-octanol, four for isopentyl acetate, and one for hexyl acetate. One region of linkage group XI shows a strong influence on body size and the levels of three alarm pheromone components. This locus explained 40% of the variance for the amount of n-decyl acetate (LOD 6.57). In general, the QTLs influencing alarm pheromone levels were independent of previously identified loci that influenced the stinging behavior of these colonies. The only exception was a potential locus influencing levels of n-octanol, which was inversely correlated with stinging behavior.     

Relatedness among honeybees (Apis mellifera) of a drone congregation

The honeybee (Apis mellifera) queen mates during nuptial flights, in the so-called drone congregation area where many males from surrounding colonies gather. Using 20 highly polymorphic microsatellite loci, we studied a sample of 142 drones captured in a congregation close to Oberursel (Germany). A parentage test based on lod score showed that this sample contained one group of four brothers, six groups of three brothers, 20 groups of two brothers and 80 singletons. These values are very close to a Poisson distribution. Therefore, colonies were apparently equally represented in the drone congregation, and calculations showed that the congregation comprised males that originated from about 240 different colonies. This figure is surprisingly high. Considering the density of colonies around the congregation area and the average flight range of males, it suggests that most colonies within the recruitment perimeter delegated drones to the congregation with an equal probability, resulting in an almost perfect panmixis. Consequently, the relatedness between a queen and her mates, and hence the inbreeding coefficient of the progeny, should be minimized. The relatedness among the drones mated to the same queen is also very low, maximizing the genetic diversity among the different patrilines of a colony.     

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.     

Queen mating frequency and maternity of males in the stingless bee Trigona carbonaria Smith

Summary. Aspects of the sociobiology of the native Australian stingless bee Trigona carbonaria (Hymenoptera, Meliponini) were investigated using highly variable microsatellite markers. The queen mating frequency was estimated by examining genotypes of samples of workers from five colonies across three to five microsatellite loci. In each case, results were consistent with the workers being progeny of a queen mated with a single male. Microsatellite analysis of haploid males from two of the colonies suggested they predominantly arose from queen-laid eggs. As workers are more related to sons-of-workers than to sons-of-queens in monandrous colonies, this is somewhat surprising, and suggests that there may be colony-level costs associated with worker reproduction, or that queens are able to regulate worker reproduction via the ritualized cell provisioning and oviposition process. Males from another T. carbonaria colony were found to be diploid.     

Mimicking a Honeybee Queen?Vespa affinis indosinensis Pérez 1910 Hunts Drones of Apis cerana F. 1793

Hornets (Vespa affinis) flying in a drone congregation area attracted drones of Apis cerana. The drones followed the hornet and were ‘manoeuvred’ towards a leaf or a tree. The hornet then rushed at one of the drones. Many attempts by the hornet to catch a drone were unsuccessful and all drones fled. After failing, the hornet returned to centre of the drone congregation area and repeated the behaviour. Only after successfully seizing of a drone did the hornet leave the drone congregation area carrying its prey. In a two-choice test in the centre of the drone congregation area, free-flying A. cerana drones preferred a hornet model to a live A. cerana queen. V. affinis apparently ‘exploits’ the intraspecific communication between queen and drones of A. cerana. Hunting of drones in the drone congregation area by V. affinis may be an example of predatory mimicry.     

Further characterization of nuclear DNA RFLP markers that distinguish African and European honeybees

Two nuclear honeybee DNA probes, 12R1C1 and 2A2, were reported previously to detect restriction fragment patterns specific to African and neotropical African honeybee populations. Individual drones and workers from several additional Old and New World populations, African and European, were tested further with these probes. With probe 12R1C1, only two of several Hhal fragment patterns were seen among haploid drone progeny of each queen bee, indicating that the patterns represented alleles at a single locus. Four alleles detected by probe 12R1C1 were described previously, three of which had been found only in populations of African descent. In this study, one of the three was found at a low frequency among samples from western Europe, northern Mexico, and the United States. However, ten additional alleles were discovered in South African drones, six of which were seen also in neotropical African colonies. With probe 2A2, only one or the other of two Alul restriction fragments was detected in drones indicating that the fragments represented alles at a single locus. One of the two alleles, seen previously only in populations of African descent, was found at a very low frequency in bees from western Europe and northern Mexico.     

The honeybee queen influences the regulation of colony drone production

Social insect colonies invest in reproduction and growth, buthow colonies achieve an adaptive allocation to these life-historycharacters remains an open question in social insect biology.Attempts to understand how a colony's investment in reproductionis shaped by the queen and the workers have proved complicatedbecause of the potential for queen–worker conflict overthe colony's investment in males versus females. Honeybees,in which this conflict is expected to be minimal or absent,provide an opportunity to more clearly study how the actionsand interactions of individuals influence the colony's productionand regulation of males (drones). We examined whether honeybeequeens can influence drone regulation by either allowing orpreventing them from laying drone eggs for a period of timeand then examining their subsequent tendency to lay drone andworker eggs. Queens who initially laid drone eggs subsequentlylaid fewer drone eggs than the queens who were initially preventedfrom producing drone eggs. This indicates that a colony's regulationof drones may be achieved not only by the workers, who buildwax cells for drones and feed the larvae, but also by the queen,who can modify her production of drone eggs. In order to betterunderstand how the queen and workers contribute to social insectcolony decisions, future work should attempt to distinguishbetween actions that reflect conflict over sex allocation andthose that reflect cooperation and shared control over the colony'sinvestment in reproduction.     

Identification and mapping of random amplified polymorphic DNA (RAPD) markers linked to resistance against beet necrotic yellow vein virus (BNYVV) in Beta accessions

Molecular markers linked to resistance genes are useful to facilitate the introgression of one or more of these genes in breeding materials. Following the approach of bulked segregant analysis, RAPD markers linked to resistance genes against beet necrotic yellow vein virus were identified in the four Beta accessions Holly-1-4, R104, R128 and WB42. Two primers were found which generate RAPD markers tightly linked to resistance in segregating families of Holly-1-4, R104 and R128, indicating that the resistance genes in these accessions might be situated at the same locus. Other, specific, primers were identified which generate RAPD markers linked to resistance in each of these accessions. Short-range maps were established around the resistance locus in these accessions. For WB42, RAPD markers were only identified at a relatively large distance from the resistance gene. Conversion of three RAPD primers of Holly-1-4, R104 and R128 into STS primers resulted in STS markers which can be readily used for marker-assisted selection in breeding programmes. Received: 8 January 1996 / Accepted: 14 June 1996     

A linkage map of the turnip sawfly Athalia rosae (Hymenoptera: Symphyta) based on random amplified polymorphic DNAs

A linkage map was constructed for the sawfly, Athalia rosae (Hymenoptera), based on the segregation of random amplified polymorphic DNA (RAPD) markers and a visible mutation, yellow fat body (yfb). Forty haploid male progeny (20 yfb and 20+) from a single diploid female parent (yfb/+) were examined. Sixty-one of the 180 arbitrary primers tested by polymerase chain reaction (PCR) produced one or more RAPD bands. A total of 79 RAPD markers were detected. Of these, seven showed significant deviation from the expected 1:1 ratio, and were therefore excluded from further analysis. The remaining 72 RAPD markers and the marker mutation, yfb, were subjected to linkage analysis. Sixty RAPD markers and the yfb marker were organized into 16 linkage groups, spanning a distance of 517.2 cM. Twelve RAPD markers showed no linkage relationship to any group. Thirteen gel-purified RAPD bands were cloned and sequenced to generate the sequence-tagged sites (STSs). A single locus was represented by two markers, with one of them having a short internal deletion.     

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.     

Isolation and characterization of RAPD-based markers linked to the beet cyst nematode resistance locus (Hs1 pat-1) on chromosome 1 of B. patellaris

A beet cyst nematode (BCN)-resistant telosomic addition of B. patellaris chromosome 1 in B. vulgaris was used to isolate 6 RAPD markers linked to the BCN resistance locus Hs1 ^pat-1. Southern analysis showed that the analyzed RAPD products contain either low-, middle or high-repetitive DNA. The relative positions of the random amplified polymorphic DNA (RAPD) markers and of the restriction fragment length polymorphism (RFLP) loci corresponding to the low-repetitive RAPD products were determined by deletion mapping using a panel of seven nematode-resistant B. patellaris chromosome-1 fragment additions. One RAPD marker, OPB11₈₀₀, was found to be present in two copies on the long arm telosome of B. patellaris chromosome 1. These copies are closely linked to the BCN resistance gene and flank the gene on both sides. On the basis of the nucleotide sequence of OPB11₈₀₀, sequence-tagged site (STS) primers were developed that amplify specific fragments derived from the two OPB11₈₀₀ loci. These STS markers can be used in the map-based cloning of the BCN gene, as they define start and finishing points of a chromosomal walk towards the Hs1 ^pat-1 locus. Two copies of the middle-repetitive OPX2₁₁₀₀ marker were mapped in the same interval of the deletion mapping panel as the resistance gene locus and thereby belong to the nearest markers as yet found for the BCN gene in B. patellaris.     

A Game Theoretical Analysis of the Mating Sign Behavior in the Honey Bee

Queens of the honey bee, Apis mellifera (L.), exhibit extreme polyandry, mating with up to 45 different males (drones). This increases the genetic diversity of their colonies, and consequently their fitness. After copulation, drones leave a mating sign in the genital opening of the queen which has been shown to promote additional mating of the queen. On one hand, this signing behavior is beneficial for the drone because it increases the genetic diversity of the resulting colony that is to perpetuate his genes. On the other hand, it decreases the proportion of the drone??s personal offspring among colony members which is reducing drone fitness. We analyze the adaptiveness and evolutionary stability of this drone??s behavior with a game-theoretical model. We find that theoretically both the strategy of leaving a mating sign and the strategy of not leaving a mating sign can be evolutionary stable, depending on natural parameters. However, the signing strategy is not favored for most scenarios, including the cases that are biologically plausible in reference to empirical data. We conclude that leaving a sign is not in the interest of the drone unless it serves biological functions other than increasing subsequent queen mating chances. Nevertheless, our analysis can also explain the prevalence of such a behavior of honey bee drones by a very low evolutionary pressure for an invasion of the nonsigning strategy.     

Some reproductive characteristics of honeybee drones in relation to their ages

The number of spermatozoa, length of testis, length of mucus gland and weights of drones were estimated in caged and free honeybee drones in relation to their ages. For this purpose, three Carniolan colonies were used as sources of drones and two were used as nursery colonies. The drones were held either in cages or freely in the queenless part of the nursery colonies. Measurements were carried out on samples of drones taken at 3-day intervals from 1–3, 4–6, 7–9, 10–12 and 13–15 days old. While the number of spermatozoa in drone testes decreased in both caged and free drones as the drone age increased, it increased gradually in the seminal vesicles. The results also showed that weight of drones and length of testes decreased, but the length of the mucus gland did not significantly differ with age. Further, the drones' weight, length of mucus gland and total number of spermatozoa tend to be slightly larger in the caged than in free drones. The total number of spermatozoa in all parts of the reproductive organs was lower in the older than younger drones; thus, the number of spermatozoa in the newly emerged drones does not help to predict the real number reaching the queen during mating.     

Identification of a rice gene (Bph 1) conferring resistance to brown planthopper (Nilaparvata lugens Stal) using STS markers

This study was carried out to identify a high-resolution marker for a gene conferring resistance to brown planthopper (BPH) biotype 1, using japonica type resistant lines. Bulked segregant analyses were conducted using 520 RAPD primers to identify RAPD fragments linked to the BPH resistance gene. Eleven RAPDs were shown to be polymorphic amplicons between resistant and susceptible progeny. One of these primers, OPE 18, which amplified a 923 bp band tightly linked to resistance, was converted into a sequence-tagged-site (STS) marker. The STS marker, BpE18-3, was easily detectable as a dominant band with tight linkage (3.9cM) to Bph1. It promises to be useful as a marker for assisted selection of resistant progeny in backcross breeding programs to introgress the resistance gene into elite japonica cultivars.     

Genetic pollution and number of matings in a black honey bee (Apis mellifera mellifera) population

Summary In the south-east of France, local honey bees possess only the B allele at the MDH locus, whereas the races which are usually imported into this area do not have this allele. The proportion of non-B genes in a sample of drones was used to measure the genetic pollution in the local population. Within the course of a breeding scheme of local bees, 99 queens, whose genotypes are BB, were naturally mated between April 25 and June 10, 1985 at la Tave (Gard, France). Twenty daughters-workers of each queen were analysed at the MDH locus. The frequency of the B allele in drones that mated with these queens is estimated by the proportion of workers with genotype BB and the genetic pollution by the cumulated frequency of the other alleles. The sampling variances of these frequencies involve a coefficient which is a function of the average number of drones mated with a queen. This latter parameter is estimated through the maximum likelihood method. In addition to the three well-known alleles, a rare allele (frequency=0.0055), possibly equivalent to the S1 allele described by Badino et al. (1983), has been found in three different colonies. Cumulating the frequencies of the non-B alleles results in an estimation of the genetic pollution equal to 0.0394 (±0.0071). This low value allows us to proceed to the next step of the selection project. The mean number of drones mated to a queen is 12.4 with a (10.4–19.3) confidence interval at the 90% level.