Ecological comparison of native (Apis mellifera mellifera) and hybrid (Buckfast) honeybee drones in southwestern Sweden indicates local adaptation.

Honeybee drones' only known task is to mate with a virgin queen. Apart from their mating behaviour, their ecology has been little studied, especially in comparison to honeybee females. Previous knowledge is primarily based on short-term direct observations at single experimental hives, rarely, if ever, addressing the effect of drones' genetic origin. Here, Radio Frequency Identification Technology was utilised to gather drone and worker bee lifetime data of Apis mellifera mellifera and Apis mellifera x (hybrid Buckfast) colonies over one mating season (spring and summer) with the ultimate goal to investigate differences at subspecies level. This technique enabled continuous monitoring of tagged bees at the hive entrance and recording of individuals' movement directions. The results confirmed that spring-born drones survive longer than summer-born drones and that they generally live longer than worker bees. Drones' peak activity occurred in the afternoon while worker bees showed more even activity levels throughout the day. Earlier orientation flights than usually reported for drones were observed. In summer, mating flights were practiced before reaching sexual maturity (at 12 days of age). Differences were found between Apis m. mellifera and Buckfast drones, where Apis m. mellifera showed later drone production in spring, but significantly earlier first activities outside the hive in summer and a later peak in diurnal activity. Additionally, Apis m. mellifera flew more in higher light intensities and windy conditions and performed significantly longer flights than Buckfast drones. The observed differences in drone ecology indicate the existence of a local adaptation of the native subspecies Apis m. mellifera to environmental conditions in southwestern Sweden.

CRP and IHF act as host regulators in Royal Jelly's antibacterial activity.

Royal Jelly (RJ) is a natural substance produced by honeybees, serving not only as nutrition for bee brood and queens but also as a functional food due to its health-promoting properties. Despite its well-known broad-spectrum antibacterial activity, the precise molecular mechanism underlying its antibacterial action has remained elusive. In this study, we investigated the impact of RJ on the bacteria model MG1655 at its half-maximal inhibitory concentration, employing LC-MS/MS to analyze proteomic changes. The differentially expressed proteins were found to primarily contribute to the suppression of gene expression processes, specifically transcription and translation, disrupting nutrition and energy metabolism, and inducing oxidative stress. Notably, RJ treatment led to a marked inhibition of superoxide dismutase and catalase activities, resulting in heightened oxidative damage and lipid peroxidation. Furthermore, through a protein-protein interaction network analysis using the STRING database, we identified CRP and IHF as crucial host regulators responsive to RJ. These regulators were found to play a pivotal role in suppressing essential hub genes associated with energy production and antioxidant capabilities. Our findings significantly contribute to the understanding of RJ's antibacterial mechanism, highlighting its potential as a natural alternative to conventional antibiotics. The identification of CRP and IHF as central players highlights the intricate regulatory networks involved in RJ's action, offering new targets for developing innovative antimicrobial strategies.

Landscape crop diversity contributes to higher pollination effectiveness and positively affects rapeseed quality in Mediterranean agricultural landscapes.

Pollination is crucial for biodiversity and food security. Heterogeneous agricultural landscapes have a positive effect on pollinator abundance and enhance crop production and quality. In this study, we explored the effects of three landscape features (past crop diversity measured as the Equivalent Richness of crop functional Groups in the previous year [ERGp], semi-natural habitat percentage [SNH], and mean field size [MFS]) and pollinator densities (wild bees [WB] and honey bees [HB]) on pollination and seed quantity and quality in rapeseed crops. Surveying the pollinator density in 20 rapeseed fields revealed a positive relationship with ERGp in the landscape. A pollinator exclusion experiment compared bagged and open-pollinated self-compatible rapeseed plants and revealed insect pollination effectiveness (fruits per flower and number of seeds per pod) and seed quality (oil content). Seed parameters were evaluated in relation to pollinator density (WB-HB) and landscape characteristics. The ERGp emerged as a crucial landscape feature that positively impacted WB density. When insect pollinators were excluded, plants exhibited reduced pollination effectiveness and seed quality. Analysis of open-pollinated plants highlighted ERGp as the most influential variable, positively affecting both sets of parameters. The MFS and SNH showed different but important relationships. Total tocopherol and α-tocopherol were positively correlated with pollinator density in HB, whereas WB showed a positive correlation with γ-tocopherol levels. Increased ERGp positively affected pollinator density and pollination effectiveness, thereby improving oilseed rape production quantity and quality. This study provides new insights into agroecosystem management and pollinator-friendly practices.

Host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system.

Cophylogeny has been identified between gut bacteria and their animal host and is highly relevant to host health, but little research has extended to gut bacteriophages. Here we use bee model to investigate host specificity and cophylogeny in the "animal-gut bacteria-phage" tripartite system. Through metagenomic sequencing upon different bee species, the gut phageome revealed a more variable composition than the gut bacteriome. Nevertheless, the bacteriome and the phageome showed a significant association of their dissimilarity matrices, indicating a reciprocal interaction between the two kinds of communities. Most of the gut phages were host generalist at the viral cluster level but host specialist at the viral OTU level. While the dominant gut bacteria Gilliamella and Snodgrassella exhibited matched phylogeny with bee hosts, most of their phages showed a diminished level of cophylogeny. The evolutionary rates of the bee, the gut bacteria and the gut phages showed a remarkably increasing trend, including synonymous and non-synonymous substitution and gene content variation. For all of the three codiversified tripartite members, however, their genes under positive selection and genes involving gain/loss during evolution simultaneously enriched the functions into metabolism of nutrients, therefore highlighting the tripartite coevolution that results in an enhanced ecological fitness for the whole holobiont.

Dimethyl sulfoxide, an alternative for control of Nosema ceranae infection in honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that threatens current apiculture. N. ceranae-infected honey bees (Apis mellifera) exhibit morbid physiological impairments and reduced honey production, malnutrition, shorter life span, and higher mortality than healthy honey bees. In this study, we found that dimethyl sulfoxide (DMSO) could enhance the survival rate of N. ceranae-infected honey bees. Therefore, we investigated the effect of DMSO on N. ceranae-infected honey bees using comparative RNA sequencing analysis. Our results revealed that DMSO was able to affect several biochemical pathways, especially the metabolic-related pathways in N. ceranae-infected honey bees. Based on these findings, we conclude that DMSO may be a useful alternative for treating N. ceranae infection in apiculture.

Age-dependent hypopharyngeal gland size and protein content of stingless bee workers, Tetragonula pagdeni.

Eusocial insects, such as stingless bees (Meliponini), depend on division of labour, overlapping generations, and collaborative brood care to ensure the functionality and success of their colony. Female workers transition through a range of age-specific tasks during their lifespan (i.e., age-polyethism) and play a central role in the success of a colony. These age-specific tasks (e.g., brood care or foraging) often closely coincide with key physiological changes necessary to ensure optimal performance. However, our understanding of how nutrition, age, and polyethism may affect the development of such physiological traits in stingless bees remains limited. Here we show that pollen consumption and age-polyethism govern hypopharyngeal gland (HPG) acini size and protein content in Tetragonula pagdeni. By conducting a controlled laboratory experiment we monitored the effect of pollen consumption on worker bee survival as well as assessed how a pollen diet and age affected their HPG acini width and protein content. Further, we sampled nurses and foragers from field colonies to measure the effect of age-polyethism on HPG acini width. We found that pollen consumption enhanced survival and led to increased HPG acini width and protein content and that HPG acini were as expected largest in nurse bees. Our findings highlight the beneficial effects of an adequate diet for physiological development and health in stingless bees and reveal that age-polyethism is the key factor governing HPG size in worker bees. As HPGs are imperative for collaborative brood care-an essential component of eusociality-the data provide a foundation for future studies to investigate the impact of potential environmental stressors on a critical physiological trait in stingless bees which may serve as a proxy to understand the effects at the colony level.

Prevalence and molecular analysis of some important viruses in honey bee colonies in Türkiye: the status of multiple infections.

In this study, seven bee viruses of significant importance for bee health in Türkiye were investigated using one-step RT-PCR. For this purpose, larvae from 1183 hives and adult bees from 1196 hives were sampled from 400 apiaries in 40 provinces. The prevalence of viral infections in hives was as follows: acute bee paralysis virus (ABPV), 6.4%; black queen cell virus (BQCV), 77%; chronic bee paralysis virus (CBPV), 3.2%; deformed wing virus (DWV), 63.8%; Israel acute bee paralysis virus (IAPV), 7%; Kashmir bee virus (KBV), 2.7%; sacbrood virus (SBV), 49.7%. Moreover, 50 different combinations of viral infections were identified in the hives. While dual infections (36.1%) were the most common in hives, triple infections with BQCV, DWV, and SBV were found to have the highest prevalence (22.1%). At least one viral infection was detected in all of the apiaries tested. Phylogenetic analysis showed that the isolates from this study generally exhibited the highest similarity to previously reported Turkish isolates. When similarity ratios and the locations and types of amino acid mutations were analyzed, it was observed that the isolates from our study exhibited high similarity to isolates from various countries, including China, the United Kingdom, Syria, and Germany.

Honeybee (Apis mellifera L.) pollination enhances the yield and flavor quality of kiwifruit.

Pollination is essential for achieving high yields and enhancing the quality of kiwifruit cultivation, both of which significantly influence growers' interests and consumers' preferences. However, compared to studies on yield, there are fewer studies exploring the impact of pollination methods on the flavor of kiwifruit Actinidia chinensis Planchon. This study examined the effects of bee (Apis mellifera L.) pollination and artificial pollination on the yield and flavor of kiwifruit in the main producing areas of China. Compared with those pollinated artificially, bee-pollinated kiwifruit exhibited a greater fruit set rate, heavier fruit weight, and greater number of seeds. Notably, the number of seeds was positively correlated with fruit weight in bee-pollinated kiwifruit, whereas no such correlation was detected in artificially pollinated fruit. Bee pollination not only enhanced the yield but also improved the flavor of kiwifruit. Specifically, bee-pollinated kiwifruit contained higher levels of sucrose and lower concentrations of glucose and fructose, while the acid content was less affected by pollination methods. Furthermore, significant differences were observed in the volatile organic compound (VOC) levels in kiwifruit subjected to different pollination treatments, with bee-pollinated fruit exhibiting a superior flavor. Our findings provide new insights into the beneficial role of bee pollination in enhancing kiwifruit yield and quality, underscoring the crucial importance of bees in kiwifruit pollination.

Characterization of Antibiofilm and Antimicrobial Effects of Trigona Stingless Bee Honey Compared to Stinging Bee Centaurea hyalolepis and Citrus Honeys.

The antibiofilm and antimicrobial properties of tropical honey types including Malaysian stingless bee honey remain explicitly unexplored when compared with Apies honey. The antibiofilm and antimicrobial activities of the Malaysian Trigona honey were characterized with two stinging bee honey types (Centaurea hyalolepis and Citrus honeys) from Jordan. The antibiofilm and antimicrobial investigations were conducted on a set of seven microbial strains; five bacterial species of Pseudomonas aeruginosa ATCC 10145, Streptococcus pyogenes ATCC 19615, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, and two fungal strains Candida albicans ATCC 10231 and Candida krusei ATCC 14243. The antimicrobial investigations revealed a broad spectrum activity for Trigona honey against Gram-positive, Gram-negative, and fungal strains over the two honey types. One-way ANOVA showed a significant difference (p < 0.001) in the zone of inhibition ranging from 9 to 25 mm and minimum inhibition activity (MIC) ranged from 9.4-29.6% (w/v) against the microbial strains. Moreover, the addition of honey to established biofilms has induced a degradation activity in the biofilm mass. Two-way ANOVA showed a significant biofilm degradation proportion (p < 0.001) ranging from 1.3% to 91.3% following treatment with Trigona honey and the other honey types in relevance to the concentration ranging from 10% to 50% (w/v). Moreover, the antibiofilm activity was highly consistent with MIC affecting bacterial growth inhibition. In conclusion, a robust antimicrobial and antibiofilm activity for Trigona stingless bee honey over the stinging bee Centaurea hyalolepis and Citrus honeys is noticed which endows the usage of Trigona honey in the antimicrobial industry.

Effects of a supplemented diet containing 7 probiotic strains (Honeybeeotic) on honeybee physiology and immune response: analysis of hemolymph cytology, phenoloxidase activity, and gut microbiome.

BACKGROUND: In this study, a probiotic mixture (Honeybeeotic) consisting of seven bacterial strains isolated from a unique population of honeybees (Apis mellifera ligustica) was used. That honeybee population was located in the Roti Abbey locality of the Marche Region in Italy, an area isolated from human activities, and genetic contamination from other honeybee populations. The aim was to investigate the effects of this probiotic mixture on the innate immunity and intestinal microbiome of healthy common honeybees in two hives of the same apiary. Hive A received a diet of 50% glucose syrup, while hive B received the same syrup supplemented with the probiotics, both administered daily for 1 month. To determine whether the probiotic altered the immune response, phenoloxidase activity and hemolymph cellular subtype count were investigated. Additionally, metagenomic approaches were used to analyze the effects on gut microbiota composition and function, considering the critical role the gut microbiota plays in modulating host physiology. RESULTS: The results revealed differences in hemocyte populations between the two hives, as hive A exhibited higher counts of oenocytoids and granulocytes. These findings indicated that the dietary supplementation with the probiotic mixture was safe and well-tolerated. Furthermore, phenoloxidase activity significantly decreased in hive B (1.75 ± 0.19 U/mg) compared to hive A (3.62 ± 0.44 U/mg, p < 0.005), suggesting an improved state of well-being in the honeybees, as they did not require activation of immune defense mechanisms. Regarding the microbiome composition, the probiotic modulated the gut microbiota in hive B compared to the control, retaining core microbiota components while causing both positive and negative variations. Notably, several genes, particularly KEGG genes involved in amino acid metabolism, carbohydrate metabolism, and branched-chain amino acid (BCAA) transport, were more abundant in the probiotic-fed group, suggesting an effective nutritional supplement for the host. CONCLUSIONS: This study advocated that feeding with this probiotic mixture induces beneficial immunological effects and promoted a balanced gut microbiota with enhanced metabolic activities related to digestion. The use of highly selected probiotics was shown to contribute to the overall well-being of the honeybees, improving their immune response and gut health.

MSPB: a longitudinal multi-sensor dataset with phenotypic trait measurements from honey bees.

We present a one-year-long multi-sensor dataset collected from honey bee colonies (Apis mellifera) with rich phenotypic measurements. Data were collected non-stop from April 2020 to April 2021 from 53 hives located at two apiaries in Québec, Canada. The sensor data included audio features, temperature, and relative humidity. The phenotypic measurements contained beehive population, number of brood cells (eggs, larva and pupa), Varroa destructor infestation levels, defensive and hygienic behaviors, honey yield, and winter mortality. Our study is amongst the first to combine a wide variety of phenotypic trait measurements annotated by apicultural science experts with multi-sensor data, which facilitate a broader scope of analysis. We first summarize the data collection procedure, sensor data pre-processing steps, and data composition. We then provide an overview of the phenotypic data distribution as well as a visualization of the sensor data patterns. Lastly, we showcase several hive monitoring applications based on sensor data analysis and machine learning, such as winter mortality prediction, hive population estimation, and the presence of an active and laying queen.

Pollinator response to livestock grazing: implications for rangeland conservation in sagebrush ecosystems.

World food supplies rely on pollination, making this plant-animal relationship a highly valued ecosystem service. Bees pollinate flowering plants in rangelands that constitute up to half of global terrestrial vegetation. Livestock grazing is the most widespread rangeland use and can affect insect pollinators through herbivory. We examined management effects on bee abundance and other insect pollinators on grazed and idle sagebrush rangelands in central Montana, USA. From 2016 to 2018, we sampled pollinators on lands enrolled in rest-rotation grazing, unenrolled grazing lands, and geographically separate idle lands without grazing for over a decade. Bare ground covered twice as much area (15% vs. 7) with half the litter (12% vs. 24) on grazed than idle regardless of enrollment. Bee pollinators were 2-3 times more prevalent in grazed than idle in 2016-2017. In 2018, bees were similar among grazed and idled during an unseasonably wet and cool summer that depressed pollinator catches; captures of secondary pollinators was similar among treatments 2 of 3 study years. Ground-nesting bees (94.6% of total bee abundance) were driven by periodic grazing that maintained bare ground and kept litter accumulations in check. In contrast, idle provided fewer nesting opportunities for bees that were mostly solitary, ground-nesting genera requiring unvegetated spaces for reproduction. Managed lands supported higher bee abundance that evolved with bison grazing on the eastern edge of the sagebrush ecosystem. Our findings suggest that periodic disturbance may enhance pollinator habitat, and that rangelands may benefit from periodic grazing by livestock.

Spatiotemporal evolution of the distribution of Chronic bee paralysis virus (CBPV) in honey bee colonies.

Chronic bee paralysis virus (CBPV) is a Apis mellifera viral infectious disease, exhibiting dark and hairless abdomen in workers with tremors and ataxita. Clinical signs are also typically linked to adverse weather conditions and overcrowding in the hive. The disease occurs in spring but recently it has been observed cases increase and seasonality loss of the disease incidence. This study analyses the evolution of CBPV in Italy, through data collected from 2009 to 2023 within three monitoring projects comprising nationwide extended detection networks, aimed to investigate the evolution of the CBPV spatial distribution, identifying high-risk areas for the virus spread. This study highlights an increased risk over years. Prevalence increased from 4.3% during 2009-2010 to 84.7% during 2021-2023 monitoring years. CBPV outbreaks were irregular between investigated seasons, highlighting Spring and Autumn as the most susceptible seasons. Risk of CBPV infection has increased, reaching high-risk in last years of monitoring. Sequence analysis showed a high similarity to other isolated Italian CBPVs. The study offers an epidemiological insight into the aetiology of this disease. CBPV distribution is a prerequisite to predict its future spread and factors involved in its propagation not only in honey bees but also in other pollinators and environments.

Sugar-conditioned honey bees can be biased towards a nectarless dioecious crop.

The targeted pollination strategy has shown positive results in directing honey bees to crop flowers offering nectar along with pollen as reward. Kiwifruit is a functionally dioecious species, which relies on bees to transport pollen from staminate to pistillate nectarless flowers. Following the targeted pollination procedures recently validated, we first developed a mimic odor (KM) based on kiwifruit floral volatiles for which bees showed the highest level of generalization to the natural floral scent, although the response towards pistillate flowers was higher than towards staminate flowers. Then, in the field, feeding colonies KM-scented sucrose solution resulted in higher amounts of kiwifruit pollen collected by honey bees compared to control colonies fed unscented sucrose solution. Our results support the hypothesis that olfactory conditioning bees biases their foraging preferences in a nectarless crop, given the higher visitation to target flowers despite having provided the mimic odor paired with a sugar reward.

Acetylcholine and choline in honey bee (Apis mellifera) worker brood food are seasonal and age-dependent.

Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April-September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020-2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.

Targeted viromes and total metagenomes capture distinct components of bee gut phage communities.

BACKGROUND: Despite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey bee Apis mellifera and the eastern bumble bee Bombus impatiens. RESULTS: We find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments. CONCLUSIONS: Overall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage. Video Abstract.

Wing vein abnormality analysis in honeybee (Apis mellifera L. 1758) populations from Iran.

The insect wing is one of the most important characteristics that allowed insects to occupy most of the habitats on the planet. Honeybee wings has been the subject of studies on the venation abnormalities. A total of 424 honeybees from 14 locations were collected and all four wings were removed and examined for 19 abnormalities on the forewings and 6 abnormalities on the hindwings. In general, supernumerary veins were the most common abnormalities seen and abnormalities no. 23, 2, 6, 1, 5, 21, 10, 13 had the highest and abnormalities no. 11, 17, 18, 19, 20, and 25 had the lowest frequencies. All of the abnormalities had similar frequencies in the right and left wings in the population. In terms of correlation between 25 abnormalities, abnormality pairs AB3-AB13, AB6-AB7, AB7-AB8, AB10-AB12, AB16-AB17 on the forewing and AB2-AB23, AB12-AB20, AB12-AB24, AB13-AB21, AB16-AB25, and AB17-AB25 between the forewing and hindwing show significant positive correlations and abnormality pairs AB4-AB5, AB7-AB15 and AB8-AB9 on the forewing show significant negative correlations with each other. In terms of the differential occurrence of abnormalities , a few locations differed significantly from other locations. This study provides some insights into the nature of these abnormalities on the honeybee wings.

HBeeID: a molecular tool that identifies honey bee subspecies from different geographic populations.

BACKGROUND: Honey bees are the principal commercial pollinators. Along with other arthropods, they are increasingly under threat from anthropogenic factors such as the incursion of invasive honey bee subspecies, pathogens and parasites. Better tools are needed to identify bee subspecies. Genomic data for economic and ecologically important organisms is increasing, but in its basic form its practical application to address ecological problems is limited. RESULTS: We introduce HBeeID a means to identify honey bees. The tool utilizes a knowledge-based network and diagnostic SNPs identified by discriminant analysis of principle components and hierarchical agglomerative clustering. Tests of HBeeID showed that it identifies African, Americas-Africanized, Asian, and European honey bees with a high degree of certainty even when samples lack the full 272 SNPs of HBeeID. Its prediction capacity decreases with highly admixed samples. CONCLUSION: HBeeID is a high-resolution genomic, SNP based tool, that can be used to identify honey bees and screen species that are invasive. Its flexible design allows for future improvements via sample data additions from other localities.

The number of drones to inseminate a queen with has little potential for optimization of honeybee breeding programs.

BACKGROUND: Mating control is a crucial aspect of honeybee breeding. Instrumental insemination of queens gives the breeder maximum control over the genetic origin of the involved drones. However, in addition to the drones' descent, the breeder's control also extends over the number of drones to use for inseminations. Thus far, this aspect has largely been ignored in attempts to optimize honeybee breeding schemes. The literature provides some comparisons between single drone inseminations (SDI) and multi drone inseminations (MDI) but it is unclear whether the number of drones used in MDI is a relevant parameter for the optimization of honeybee breeding programs. METHODS: By computer simulations, we investigated the effect of the number of drones per inseminated queen in breeding programs that relied on best linear unbiased prediction (BLUP) breeding values. We covered a range of 1 to 50 drones per queen and observed the developments of genetic gain and inbreeding over a period of 20 years. Hereby, we focused on insemination schemes that take the drones for one queen from a single colony. RESULTS: SDI strategies led to 5.46% to 14.19% higher genetic gain than MDI at the cost of 6.1% to 30.2% higher inbreeding rates. The number of drones used in MDI settings had only a negligible impact on the results. There was a slight tendency that more drones lead to lower genetic gain and lower inbreeding rates but whenever more than five drones were used for inseminations, no significant differences could be observed. CONCLUSION: The opportunities to optimize breeding schemes via the number of drones used in inseminations are very limited. SDI can be a viable strategy in situations where breeders are interested in genetically homogeneous offspring or precise pedigree information. However, such strategies have to account for the fact that the semen from a single drone is insufficient to fill a queen's spermatheca, whence SDI queens will not build full-strength colonies. When deciding for MDI, breeders should focus on collecting enough semen for a succesful insemination, regardless of how many drones they need for this purpose.