Africanized honey bees (Apis mellifera) have low infestation levels of the mite Varroa destructor in different ecological regions in Mexico.

Honey bee (Apis mellifera) colonies of African and European descent were compared for levels of Varroa destructor infestation in 3 different ecological regions in Mexico. The 300 colonies that were studied were located in subtropical, temperate sub-humid, and temperate dry climates. The morphotype and mitotype of adult bees as well as their rates of infestation by varroa mites were determined. Additionally, the number of combs with brood and covered with bees was recorded for each colony. The highest frequency of colonies that were classified as African-derived was found in the subtropical environment, whereas the lowest occurred in the temperate dry region. Overall, the colonies of African genotype had significantly lower mite infestation rates (3.5±0.34%) than the colonies of European genotype (4.7±0.49%) regardless of the region sampled. Significant effects of genotype and region on Varroa infestation rates were evident, and there were no differences in bee population or capped brood between genotypes. Mite infestation levels were significantly lower in the colonies of the temperate dry region than in the colonies of the other 2 regions. These results are discussed within the context of results from studies that were previously conducted in Brazil. This is the first study that demonstrates the effects of Africanization and ecological environment on V. destructor infestation rates in honey bee colonies in North America.

Improving viability of cryopreserved honey bee (Apis mellifera L.) sperm with selected diluents, cryoprotectants, and semen dilution ratios.

This is the first study where the systematic application of theories and techniques used in mammalian sperm cryopreservation have been applied to honey bee (Apis mellifera L.) semen as a means to improve postthaw viability of cryopreserved sperm. Six newly designed diluents, three cryoprotectants (dimethyl sulfoxide, DMA, glycerol), and five diluent:semen ratios (1:1, 3:1, 6:1, 9:1, and 12:1) were tested. In addition, the sperm freezing tolerance of three honey bee strains was evaluated. Specific protocols were designed to control semen freezing and thawing rates. Sperm motility was assessed visually, whereas sperm viability was assessed using SYBR-14 and propidium iodide fluorescent stains. Diluent treatments did not affect fresh (nonfrozen) sperm viability yet affected fresh sperm motility (P<0.05). Based on these assessments, two diluents were chosen and used in all successive cryopreservation experiments. Using the selected diluents, semen was collected at various diluent:semen ratios, along with one of the three cryoprotectants. Semen collected at high dilution ratios, using a hypotonic antioxidant diluent containing catalase, in combination with dimethyl sulfoxide, provided higher postthaw sperm viability than that of all other combinations tested (68.3+/-5.4%; P<0.05). Using this combination of dilution ratio, diluent, and cryoprotectant, there were no differences among honey bee strains for postthaw sperm viability (P=0.805). Nevertheless, these new semen dilution and freezing methods improved postthaw viability of sperm to levels that could theoretically sustain worker populations in colonies, thus providing potential for further optimization of cryopreservation techniques for the genetic preservation and improvement of honey bee genotypes.

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.

New genomic resources for the honey bee(Apis mellifera L): development of a deep-coverage BAC library and a preliminary STC database

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2 empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99 probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/)

New genomic resources for the honey bee(Apis mellifera L.): development of a deep-coverage BAC library and a preliminary STC database.

We have constructed a bacterial artificial chromosome (BAC) library for a European honey bee strain using the cloning enzyme HindIII in order to develop resources for structural genomics research. The library contains 36,864 clones (ninety-six 384-well plates). A random sampling of 247 clones indicated an average insert size of 113 kb (range = 27 to 213 kb) and 2% empty vectors. Based on an estimated genome size of 270 Mb, this library provides approximately 15 haploid genome equivalents, allowing >99% probability of recovering any specific sequence of interest. High-density colony filters were gridded robotically using a Genetix Q-BOT in a 4 x 4 double-spotted array on 22.5-cm2 filters. Screening of the library with four mapped honey bee genomic clones and two bee cDNA probes identified an average of 21 positive signals per probe, with a range of 7-38 positive signals per probe. An additional screening was performed with nine aphid gene fragments and one Drosophila gene fragment resulting in seven of the nine aphid probes and the Drosophila probe producing positive signals with a range of 1 to 122 positive signals per probe (average of 45). To evaluate the utility of the library for sequence tagged connector analysis, 1152 BAC clones were end sequenced in both forward and reverse directions, giving a total of 2061 successful reads of high quality. End sequences were queried against SWISS-PROT, insect genomic sequence GSS, insect EST, and insect transposable element databases. Results in spreadsheet format from these searches are publicly available at the Clemson University Genomics Institute (CUGI) website in a searchable format (http://www.genome.clemson.edu/projects/stc/bee/AM__Ba/).

Genetic dissection of honeybee (Apis mellifera L.) foraging behavior.

We demonstrate the effects of a new quantitative trait locus (QTL), designated pln3, that was mapped in a backcross population derived from strains of bees selected for the amount of pollen they store in combs. We independently confirmed pln3 by demonstrating its effects on individual foraging behavior, as we did previously for QTLs pln1 and pln2 (Hunt et al. 1995). QTL pln2 is very robust in its effects on foraging behavior. In this study, pln2 was again shown to affect individual foraging behavior of workers derived from a hybrid backcross of the selected strains. In addition, pln2 was shown to affect the amount of pollen stored in combs of colonies derived from a wide cross of European and Africanized honeybees. This is noteworthy because it demonstrates that we can map QTLs for behavior in interstrain crosses derived from selective breeding and study their effects in unselected, natural populations. The results we present also demonstrate the repeatability of finding QTLs with measurable effects, even after outcrossing selected strains, suggesting that there is a relatively small subset of QTLs with major effects segregating in the population from which we selected our founding breeding populations. The different QTLs, pln1, pln2, and pln3, appear to have different effects, revealing the complex genetic architecture of honeybee foraging behavior.

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.

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).