First large-scale genomic prediction in the honey bee.

Genomic selection has increased genetic gain in several livestock species, but due to the complicated genetics and reproduction biology not yet in honey bees. Recently, 2970 queens were genotyped to gather a reference population. For the application of genomic selection in honey bees, this study analyzes the accuracy and bias of pedigree-based and genomic breeding values for honey yield, three workability traits, and two traits for resistance against the parasite Varroa destructor. For breeding value estimation, we use a honey bee-specific model with maternal and direct effects, to account for the contributions of the workers and the queen of a colony to the phenotypes. We conducted a validation for the last generation and a five-fold cross-validation. In the validation for the last generation, the accuracy of pedigree-based estimated breeding values was 0.12 for honey yield, and ranged from 0.42 to 0.61 for the workability traits. The inclusion of genomic marker data improved these accuracies to 0.23 for honey yield, and a range from 0.44 to 0.65 for the workability traits. The inclusion of genomic data did not improve the accuracy of the disease-related traits. Traits with high heritability for maternal effects compared to the heritability for direct effects showed the most promising results. For all traits except the Varroa resistance traits, the bias with genomic methods was on a similar level compared to the bias with pedigree-based BLUP. The results show that genomic selection can successfully be applied to honey bees.

Consequences of incorrect genetic parameter estimates for single-trait and multi-trait genetic evaluations in honeybees.

Genetic and residual variances of traits are important input parameters for best linear unbiased prediction (BLUP) breeding value estimation. In honeybees, estimates of these variances are often associated with large standard errors, entailing a risk to perform genetic evaluations under wrong premises. The consequences hereof have not been sufficiently studied. In particular, there are no adequate investigations on this topic accounting for multi-trait selection or genetic peculiarities of the honeybee. We performed simulation studies and explored the consequences of selection for honeybee populations with a broad range of true and assumed genetic parameters. We found that in single-trait evaluations, the response to selection was barely compromised by assuming erroneous parameters, so that reductions in genetic progress after 20 years never exceeded 21%. Phenotypic selection appeared inferior to BLUP selection, particularly under low heritabilities. Parameter choices for genetic evaluation had great effects on inbreeding development. By wrongly assuming high heritabilities, inbreeding rates were reduced by up to 74%. When parallel selection was performed for two traits, the right choice of genetic parameters appeared considerably more crucial as several incorrect premises yielded inadvertent negative selection for one of the traits. This phenomenon occurred in multiple constellations in which the selection traits expressed a negative genetic correlation. It was not reflected in the estimated breeding values. Our results indicate that breeding efforts heavily rely on detailed knowledge on genetic parameters, particularly when multi-trait selection is performed. Thus, considerable effort should be invested into precise parameter estimations.

Short-term effects of controlled mating and selection on the genetic variance of honeybee populations.

Directional selection in a population yields reduced genetic variance due to the Bulmer effect. While this effect has been thoroughly investigated in mammals, it is poorly studied in social insects with biological peculiarities such as haplo-diploidy or the collective expression of traits. In addition to the natural adaptation to climate change, parasites, and pesticides, honeybees increasingly experience artificial selection pressure through modern breeding programs. Besides selection, many honeybee breeding schemes introduce controlled mating. We investigated which individual effects selection and controlled mating have on genetic variance. We derived formulas to describe short-term changes of genetic variance in honeybee populations and conducted computer simulations to confirm them. Thereby, we found that the changes in genetic variance depend on whether the variance is measured between queens (inheritance criterion), worker groups (selection criterion), or both (performance criterion). All three criteria showed reduced genetic variance under selection. In the selection and performance criteria, our formulas and simulations showed an increased genetic variance through controlled mating. This newly described effect counterbalanced and occasionally outweighed the Bulmer effect. It could not be observed in the inheritance criterion. A good understanding of the different notions of genetic variance in honeybees, therefore, appears crucial to interpreting population parameters correctly.

Simulation studies to optimize genomic selection in honey bees.

BACKGROUND: With the completion of a single nucleotide polymorphism (SNP) chip for honey bees, the technical basis of genomic selection is laid. However, for its application in practice, methods to estimate genomic breeding values need to be adapted to the specificities of the genetics and breeding infrastructure of this species. Drone-producing queens (DPQ) are used for mating control, and usually, they head non-phenotyped colonies that will be placed on mating stations. Breeding queens (BQ) head colonies that are intended to be phenotyped and used to produce new queens. Our aim was to evaluate different breeding program designs for the initiation of genomic selection in honey bees. METHODS: Stochastic simulations were conducted to evaluate the quality of the estimated breeding values. We developed a variation of the genomic relationship matrix to include genotypes of DPQ and tested different sizes of the reference population. The results were used to estimate genetic gain in the initial selection cycle of a genomic breeding program. This program was run over six years, and different numbers of genotyped queens per year were considered. Resources could be allocated to increase the reference population, or to perform genomic preselection of BQ and/or DPQ. RESULTS: Including the genotypes of 5000 phenotyped BQ increased the accuracy of predictions of breeding values by up to 173%, depending on the size of the reference population and the trait considered. To initiate a breeding program, genotyping a minimum number of 1000 queens per year is required. In this case, genetic gain was highest when genomic preselection of DPQ was coupled with the genotyping of 10-20% of the phenotyped BQ. For maximum genetic gain per used genotype, more than 2500 genotyped queens per year and preselection of all BQ and DPQ are required. CONCLUSIONS: This study shows that the first priority in a breeding program is to genotype phenotyped BQ to obtain a sufficiently large reference population, which allows successful genomic preselection of queens. To maximize genetic gain, DPQ should be preselected, and their genotypes included in the genomic relationship matrix. We suggest, that the developed methods for genomic prediction are suitable for implementation in genomic honey bee breeding programs.

A theoretical derivation of response to selection with and without controlled mating in honeybees.

BACKGROUND: In recent years, the breeding of honeybees has gained significant scientific interest, and numerous theoretical and practical improvements have been made regarding the collection and processing of their performance data. It is now known that the selection of high-quality drone material is crucial for mid to long-term breeding success. However, there has been no conclusive mathematical theory to explain these findings. METHODS: We derived mathematical formulas to describe the response to selection of a breeding population and an unselected passive population of honeybees that benefits indirectly from genetic improvement in the breeding population via migration. This was done under the assumption of either controlled or uncontrolled mating of queens in the breeding population. RESULTS: Our model equations confirm what has been observed in simulation studies. In particular, we have proven that the breeding population and the passive population will show parallel genetic gain after some years and we were able to assess the responses to selection for different breeding strategies. Thus, we confirmed the crucial importance of controlled mating for successful honeybee breeding. When compared with data from simulation studies, the derived formulas showed high coefficients of determination [Formula: see text] in cases where many passive queens had dams from the breeding population. For self-sufficient passive populations, the coefficients of determination were lower ([Formula: see text]) if the breeding population was under controlled mating. This can be explained by the limited simulated time-frame and lower convergence rates. CONCLUSION: The presented theoretical derivations allow extrapolation of honeybee-specific simulation results for breeding programs to a wide range of population parameters. Furthermore, they provide general insights into the genetic dynamics of interdependent populations, not only for honeybees but also in a broader context.

The importance of controlled mating in honeybee breeding.

BACKGROUND: Controlled mating procedures are widely accepted as a key aspect of successful breeding in almost all animal species. In honeybees, however, controlled mating is hard to achieve. Therefore, there have been several attempts to breed honeybees using free-mated queens. In such breeding schemes, selection occurs only on the maternal path since the drone sires are random samples of the population. The success rates of breeding approaches without controlled mating have so far not been investigated on a theoretical or simulation-based level. METHODS: Stochastic simulation studies were carried out to examine the chances of success in honeybee breeding with and without controlled mating. We investigated the influence of different sizes of breeding populations (500, 1000, 2000 colonies per year) and unselected passive populations (0, 500, 1000, 2000, infinitely many colonies per year) on selection for a maternally (queen) and directly (worker group) influenced trait with moderate ([Formula: see text]) or strong ([Formula: see text]) negative correlation between the two effects. The simulations described 20 years of selection. RESULTS: Our simulations showed a reduction of breeding success between 47 and 99% if mating was not controlled. In the most drastic cases, practically no genetic gain could be generated without controlled mating. We observed that in the trade-off between selection for direct or maternal effects, the absence of mating control leads to a shift in favor of maternal effects. Moreover, we describe the implications of different breeding strategies on the unselected passive population that benefits only indirectly via the transfer of queens or drones from the breeding population. We show that genetic gain in the passive population develops parallel to that of the breeding population. However, we found a genetic lag that became significantly smaller as more breeding queens served as dams of queens in the passive population. CONCLUSIONS: We conclude that even when unwanted admixture of subspecies can be excluded in natural matings, controlled mating is imperative for successful breeding efforts. This is especially highlighted by the strong positive impact that controlled mating in the breeding population has on the unselected passive population.

Recognition of mite-infested brood by honeybee (Apis mellifera) workers may involve thermal sensing.

Hygienic behavior, i.e. the removal of diseased or damaged brood by worker honey bees (Apis mellifera), is seen as one of the principal behavioral elements of this species' social immunity. Identification of the stimuli that trigger it would be helpful in searching for biochemical and molecular markers of this important breeding trait. While many studies at the genomic, transcriptomic, and behavioral level have pointed to the implication of chemical cues, we here hypothesized that thermal cues are alternatively/additionally involved. To test this hypothesis, we first measured whether infestation by the mite Varroa destructor (a condition known to induce hygienic behavior) leads to a thermal gradient between affected and unaffected brood. We found that infested brood cells were between 0.03 and 0.19 °C warmer than uninfested controls. Next, we tested whether artificially heating an area of a brood comb would increase the removal of infested or uninfested brood as compared to an unheated control area, and found that this was not the case. Finally, we investigated whether the heating of individual brood cells, as opposed to comb areas, would influence brood removal from cells adjacent to the heated one. This was the case for uninfested, though not for infested cells. We conclude that infestation by V. destructor leads to a heating of brood cells that should be perceivable by bees, and that small-scale temperature gradients can influence brood removal. This makes it appear possible that thermal cues play a role in triggering hygienic behavior of honey bees directed at varroa-infested larvae/pupae, although our results are insufficient to prove such an involvement.

Proteome Analysis of the Hemolymph, Mushroom Body, and Antenna Provides Novel Insight into Honeybee Resistance against Varroa Infestation.

Varroa destructor has been identified as a major culprit responsible for the losses of millions of honeybee colonies. Varroa sensitive hygiene (VSH) is a suite of behaviors from adult bees to suppress mite reproduction by uncapping and/or removing mite infested pupae from a sealed brood. Despite the efforts to elucidate the molecular underpinnings of VSH, they remain largely unknown. We investigated the proteome of mushroom bodies (MBs) and antennae of adult bees with and without VSH from a stock selected for VSH based on their response to artificially Varroa-infected brood cells by near-infrared camera observation. The pupal hemolymph proteome was also compared between the VSH-line and the line that was not selected for VSH. The identified 8609 proteins in the hemolymph, MBs, and antennae represent the most depth coverage of the honeybee proteome (>55%) to date. In the hemolymph, the VSH-line adapts a unique strategy to boost the social immunity and drive pupal organogenesis by enhancing energy metabolism and protein biosynthesis. In MBs, the up-regulated proteins implicated in neuronal sensitivity suggest their roles to promote the execution of VSH by activation of synaptic vesicles and calcium channel activities. In antennae, the highly expressed proteins associated with sensitivity of olfactory senses and signal transmissions signify their roles by inputting a strong signal to the MBs for initiating VSH. These observations illustrate that the enhanced social immunities and olfactory and neuronal sensitivity play key roles in the combat against Varroa infestation. The identified candidate markers may be useful for accelerating marker-associated selection for VSH to aid in resistance to a parasite responsible for decline in honeybee health.

Genome-Wide Association Study of a Varroa-Specific Defense Behavior in Honeybees (Apis mellifera).

Honey bees are exposed to many damaging pathogens and parasites. The most devastating is Varroa destructor, which mainly affects the brood. A promising approach for preventing its spread is to breed Varroa-resistant honey bees. One trait that has been shown to provide significant resistance against the Varroa mite is hygienic behavior, which is a behavioral response of honeybee workers to brood diseases in general. Here, we report the use of an Affymetrix 44K SNP array to analyze SNPs associated with detection and uncapping of Varroa-parasitized brood by individual worker bees (Apis mellifera). For this study, 22 000 individually labeled bees were video-monitored and a sample of 122 cases and 122 controls was collected and analyzed to determine the dependence/independence of SNP genotypes from hygienic and nonhygienic behavior on a genome-wide scale. After false-discovery rate correction of the P values, 6 SNP markers had highly significant associations with the trait investigated (α < 0.01). Inspection of the genomic regions around these SNPs led to the discovery of putative candidate genes.

Secondary biomarkers of insecticide-induced stress of honey bee colonies and their relevance for overwintering strength.

The evaluation of pesticide side-effects on honeybees is hampered by a lack of colony-level bioassays that not only are sensitive to physiological changes, but also allow predictions about the consequences of exposure for longer-term colony productivity and survival. Here we measured 28 biometrical, biochemical and behavioural indicators in a field study with 63 colonies and 3 apiaries. Colonies were stressed in early summer by feeding them for five days with either the carbamate growth regulator fenoxycarb or the neurotoxic neonicotinoid imidacloprid, or left untreated. Candidate stress indicators were measured 8-64 days later. We determined which of the indicators were influenced by the treatments, and which could be used as predictors in regression analyses of overwintering strength. Among the indicators influenced by fenoxycarb were the amount of brood in colonies as well as the learning performance and 24h-memory of bees, and the concentration of the brood food component 10HDA in head extracts. Imidacloprid significantly affected honey production, total number of bees and activity of the immune-related enzyme phenoloxidase in forager bee extracts. Indicators predictive of overwintering strength but unrelated to insecticide feeding included vitellogenin titer and glucose oxidase-activity in haemolymph/whole body-extracts of hive bees. Apart from variables that were themselves components of colony strength (numbers of bees/brood cells), the only indicator that was both influenced by an insecticide and predictive of overwintering strength was the concentration of 10HDA in worker bee heads. Our results show that physiological and biochemical bioassays can be used to study effects of insecticides at the colony level and assess the vitality of bee colonies. At the same time, most bioassays evaluated here appear of limited use for predicting pesticide effects on colony overwintering strength, because those that were sensitive to the insecticides were not identical with those that were predictive of colony overwintering. Our study therefore illustrates the difficulties involved in evaluating the economic/ecological significance of pesticide-induced stress in honey bee field studies.

Effects of an insect growth regulator and a solvent on honeybee (Apis mellifera L.) brood development and queen viability.

Honeybee toxicology is complex because effects on individual bees are modulated by social interactions between colony members. In the present study, we applied high doses of the insect growth regulator fenoxycarb to honeybee colonies to elucidate a possible interplay of individually- and colony-mediated effects regarding honey bee toxicology. Additionally, possible effects of the solvent dimethyl sulfoxide (DMSO) were assessed. We conducted studies on egg hatching and brood development to assess brood care by nurse bees as well as queen viability. Egg hatching was determined by the eclosion rate of larvae from eggs originating from colonies (i) treated with sugar syrup only, (ii) treated with sugar syrup containing DMSO and (iii) treated with sugar syrup containing fenoxycarb (dissolved in DMSO). To evaluate brood development, combs with freshly laid eggs were reciprocally transferred between colonies, and development of brood was examined in the recipient hive. Brood reared inside DMSO- and fenoxycarb-treated colonies as well as brood from DMSO- and from fenoxycarb-exposed queens showed higher mortality than brood not exposed to the chemicals. No differences were found in egg hatching among the treatments, but there was a higher variability of eclosion rates after queens were exposed to fenoxycarb. We also observed queen loss and absconding of whole colonies. Based on our results we infer that fenoxycarb has queen- as well as nurse bee-mediated effects on brood quality and development which can lead to the queen's death. There also is an effect of DMSO on the nurse bees' performance that could disturb the colony's equilibrium, at least for a delimited timespan.

On the relevance of technical variation due to building pools in microarray experiments.

BACKGROUND: Pooled samples are frequently used in experiments measuring gene expression. In this method, RNA from different individuals sharing the same experimental conditions and explanatory variables is blended and their concentrations are jointly measured. As a matter of principle, individuals are represented in equal shares in each pool. However, some degree of disproportionality may arise from the limits of technical precision. As a consequence a special kind of technical error occurs, which can be modelled by a respective variance component. Previously published theory - allowing for variable pool sizes - has been applied to four microarray gene expression data sets from different species in order to assess the practical relevance of this type of technical error in terms of significance and size of this variance component. RESULTS: The number of transcripts with a significant variance component due to imperfect blending was found to be 4329 (23 %) in mouse data and 7093 (49 %) in honey bees, but only 6 in rats and none whatsoever in human data. These results correspond to a false discovery rate of 5 % in each data set. The number of transcripts found to be differentially expressed between treatments was always higher when the blending error variance was neglected. Simulations clearly indicated overly-optimistic (anti-conservative) test results in terms of false discovery rates whenever this source of variability was not represented in the model. CONCLUSIONS: Imperfect equality of shares when blending RNA from different individuals into joint pools of variable size is a source of technical variation with relevance for experimental design, practice at the laboratory bench and data analysis. Its potentially adverse effects, incorrect identification of differentially expressed transcripts and overly-optimistic significance tests, can be fully avoided, however, by the sound application of recently established theory and models for data analysis.

A successful new approach to honeybee semen cryopreservation.

Honeybee biodiversity is under massive threat, and improved methods for gamete cryopreservation could be a precious tool for both the in situ- and ex situ-conservation of subspecies and ecotypes. Recent cryoprotocols for drone semen have improved the viability and fertility of frozen-thawed semen by using increased diluent:semen-ratios, but there is still much room for progress. As semen cryopreserved after dilution often appeared hyperactive, we speculated that the disruption of sperm-sperm interactions during dilution and cryopreservation could reduce the fertile lifespan of the cells. We therefore developed protocols to reduce admixture, or abolish it altogether by dialyzing semen against a hypertonic solution of cryoprotectant. Additionally, we tested methods to reduce the cryoprotectant concentration after thawing. Insemination of queens with semen cryopreserved after dialysis yielded 49%, 59% and 79% female (= stemming from fertilized eggs) pupae in three separate experiments, and the numbers of sperm found in the spermathecae of the queens were significantly higher than those previously reported. Post-thaw dilution and reconcentration of semen for cryoprotectant removal reduced fertility, but sizeable proportions of female brood were still produced. Workers stemming from cryopreserved semen did not differ from bees stemming from untreated semen with regard to indicators of fluctuating asymmetry, but were slightly heavier. Cryopreservation after dialysis tended to increase the proportion of cells with DNA-nicks, as measured by the TUNEL-assay, but this increase appears small when compared to the baseline variations of this indicator. Overall, we conclude that cryoprotectant-addition through dialysis can improve the quality of cryopreserved drone semen. Testing of offspring for vitality and genetic integrity should continue.

New methods and media for the centrifugation of honey bee (Hymenoptera: Apidae) drone semen.

Centrifugation of Apis mellifera L. drone semen is a necessary step in the homogenization of semen pools for the enlargement of the effective breeding population, as well as in the collection of semen by the so-called washing technique. It is also of interest for the removal of cryoprotectants after cryopreservation. The adoption of methods involving semen centrifugation has been hampered by their damaging effect to sperm. Here, we tested four new diluents as well as three additives (catalase, hen egg yolk, and a protease inhibitor), using sperm motility and dual fluorescent staining as indicators of semen quality. Three of the new diluents significantly reduced motility losses after centrifugation, as compared with the literature standard. Values of motility and propidium iodide negativity obtained with two of these diluents were not different from those measured with untreated semen. The least damaging diluent, a citrate-HEPES buffer containing trehalose, was then tested in an insemination experiment with centrifuged semen. Most queens receiving this semen produced normal brood, and the number of sperm reaching the storage organ of the queen was not significantly different from that in queens receiving untreated semen. These results could improve the acceptance of techniques involving the centrifugation of drone semen. The diluent used in the insemination experiment could also serve as semen extender for applications not involving centrifugation.

Accuracy of the unified approach in maternally influenced traits--illustrated by a simulation study in the honey bee (Apis mellifera).

BACKGROUND: The honey bee is an economically important species. With a rapid decline of the honey bee population, it is necessary to implement an improved genetic evaluation methodology. In this study, we investigated the applicability of the unified approach and its impact on the accuracy of estimation of breeding values for maternally influenced traits on a simulated dataset for the honey bee. Due to the limitation to the number of individuals that can be genotyped in a honey bee population, the unified approach can be an efficient strategy to increase the genetic gain and to provide a more accurate estimation of breeding values. We calculated the accuracy of estimated breeding values for two evaluation approaches, the unified approach and the traditional pedigree based approach. We analyzed the effects of different heritabilities as well as genetic correlation between direct and maternal effects on the accuracy of estimation of direct, maternal and overall breeding values (sum of maternal and direct breeding values). The genetic and reproductive biology of the honey bee was accounted for by taking into consideration characteristics such as colony structure, uncertain paternity, overlapping generations and polyandry. In addition, we used a modified numerator relationship matrix and a realistic genome for the honey bee. RESULTS: For all values of heritability and correlation, the accuracy of overall estimated breeding values increased significantly with the unified approach. The increase in accuracy was always higher for the case when there was no correlation as compared to the case where a negative correlation existed between maternal and direct effects. CONCLUSIONS: Our study shows that the unified approach is a useful methodology for genetic evaluation in honey bees, and can contribute immensely to the improvement of traits of apicultural interest such as resistance to Varroa or production and behavioural traits. In particular, the study is of great interest for cases where negative correlation between maternal and direct effects and uncertain paternity exist, thus, is of relevance for other species as well. The study also provides an important framework for simulating genomic and pedigree datasets that will prove to be helpful for future studies.

Hexagonal comb cells of honeybees are not produced via a liquid equilibrium process.

The nests of European honeybees (Apis mellifera) are organised into wax combs that contain many cells with a hexagonal structure. Many previous studies on comb-building behaviour have been made in order to understand how bees produce this geometrical structure; however, it still remains a mystery. Direct construction of hexagons by bees was suggested previously, while a recent hypothesis postulated the self-organised construction of hexagonal comb cell arrays; however, infrared and thermographic video observations of comb building in the present study failed to support the self-organisation hypothesis because bees were shown to be engaged in direct construction. Bees used their antennae, mandibles and legs in a regular sequence to manipulate the wax, while some bees supported their work by actively warming the wax. During the construction of hexagonal cells, the wax temperature was between 33.6 and 37.6 °C. This is well below 40 °C, i.e. the temperature at which wax is assumed to exist in the liquid equilibrium that is essential for self-organised building.

Evidence for damage-dependent hygienic behaviour towards Varroa destructor-parasitised brood in the western honey bee, Apis mellifera.

The ectoparasitic mite Varroa destructor and honey bee pathogenic viruses have been implicated in the recent demise of honey bee colonies. Several studies have shown that the combination of V. destructor and deformed wing virus (DWV) poses an especially serious threat to honey bee health. Mites transmitting virulent forms of DWV may cause fatal DWV infections in the developing bee, while pupae parasitised by mites not inducing or activating overt DWV infections may develop normally. Adult bees respond to brood diseases by removing affected brood. This hygienic behaviour is an essential part of the bees' immune response repertoire and is also shown towards mite-parasitised brood. However, it is still unclear whether the bees react towards the mite in the brood cell or rather towards the damage done to the brood. We hypothesised that the extent of mite-associated damage rather than the mere presence of parasitising mites triggers hygienic behaviour. Hygienic behaviour assays performed with mites differing in their potential to transmit overt DWV infections revealed that brood parasitised by 'virulent' mites (i.e. mites with a high potential to induce fatal DWV infections in parasitised pupae) were removed significantly more often than brood parasitised by 'less virulent' mites (i.e. mites with a very low potential to induce overt DWV infections) or non-parasitised brood. Chemical analyses of brood odour profiles suggested that the bees recognise severely affected brood by olfactory cues. Our results suggest that bees show selective, damage-dependent hygienic behaviour, which may be an economic way for colonies to cope with mite infestation.

Simulating a base population in honey bee for molecular genetic studies.

BACKGROUND: Over the past years, reports have indicated that honey bee populations are declining and that infestation by an ecto-parasitic mite (Varroa destructor) is one of the main causes. Selective breeding of resistant bees can help to prevent losses due to the parasite, but it requires that a robust breeding program and genetic evaluation are implemented. Genomic selection has emerged as an important tool in animal breeding programs and simulation studies have shown that it yields more accurate breeding value estimates, higher genetic gain and low rates of inbreeding. Since genomic selection relies on marker data, simulations conducted on a genomic dataset are a pre-requisite before selection can be implemented. Although genomic datasets have been simulated in other species undergoing genetic evaluation, simulation of a genomic dataset specific to the honey bee is required since this species has a distinct genetic and reproductive biology. Our software program was aimed at constructing a base population by simulating a random mating honey bee population. A forward-time population simulation approach was applied since it allows modeling of genetic characteristics and reproductive behavior specific to the honey bee. RESULTS: Our software program yielded a genomic dataset for a base population in linkage disequilibrium. In addition, information was obtained on (1) the position of markers on each chromosome, (2) allele frequency, (3) χ(2) statistics for Hardy-Weinberg equilibrium, (4) a sorted list of markers with a minor allele frequency less than or equal to the input value, (5) average r(2) values of linkage disequilibrium between all simulated marker loci pair for all generations and (6) average r2 value of linkage disequilibrium in the last generation for selected markers with the highest minor allele frequency. CONCLUSION: We developed a software program that takes into account the genetic and reproductive biology specific to the honey bee and that can be used to constitute a genomic dataset compatible with the simulation studies necessary to optimize breeding programs. The source code together with an instruction file is freely accessible at http://msproteomics.org/Research/Misc/honeybeepopulationsimulator.html.

In vivo validation of in vitro quality tests for cryopreserved honey bee semen.

Development of cryopreservation protocols for honey bee semen is hampered by the lack of validated laboratory tests that allow the prediction of in vivo performance of frozen-thawed semen. Here we analyzed correlations between seven in vitro tests and indicators of semen performance after insemination. These tests included measures of motility, cell conformation, and membrane permeability before and after exposure to physiochemical stress. We show that the proposed protocol for motility measurement yields results that correlate well with the number of sperm reaching the storage organ of queens (correlation coefficient ρ=0.67) and the proportion of viable eggs in inseminated queens (ρ=0.48). The conventional live/dead assay of membrane permeability by dual fluorescent staining and a new test based on the leakage of the glycolytic enzyme glucose-phosphate-isomerase (GPI) from damaged cells were also correlated to the number of sperm reaching the spermatheca (ρ=0.54 and -0.61, respectively). We conclude that motility, live/dead-staining and the assay for GPI-leakage are valuable tools for the improvement of cryopreservation of honey bee semen.