In 2019, a joint eight-variant model was published in which eight single nucleotide polymorphisms (SNPs) in seven Apis mellifera genes were associated with Varroa destructor drone brood resistance (DBR, i.e. mite non-reproduction in drone brood). As this model was derived from only one Darwinian Black Bee Box colony, it could not directly be applied on a population-overarching scale in the northern part of Belgium (Flanders), where beekeepers prefer the carnica subspecies. To determine whether these eight SNPs remained associated with the DBR trait on a Flemish colony-broad scope, we performed population-wide modelling through sampling of various A. mellifera carnica colonies, DBR scoring of Varroa-infested drone brood and variant genotyping. Novel eight-variant modelling was performed and the classification performance of the eight SNPs was evaluated. Besides, we built a reduced three-variant model retaining only three genetic variants and found that this model classified 76% of the phenotyped drones correctly. To examine the spread of beneficial alleles and predict the DBR probability distribution in Flanders, we determined the allelic frequencies of the three variants in 292 A. mellifera carnica queens. As such, this research reveals prospects of marker-assisted selection for Varroa drone brood resistance in honeybees.

Implementation of marker-assisted selection (MAS) in modern beekeeping would improve sustainability, especially in breeding programs aiming for resilience against the parasitic mite Varroa destructor. Selecting honey bee colonies for natural resistance traits, such as brood-intrinsic suppression of varroa mite reproduction, reduces the use of chemical acaricides while respecting local adaptation. In 2019, eight genomic variants associated with varroa non-reproduction in drone brood were discovered in a single colony from the Amsterdam Water Dune population in the Netherlands. Recently, a new study tested the applicability of these eight genetic variants for the same phenotype on a population-wide scale in Flanders, Belgium. As the properties of some variants varied between the two studies, one hypothesized that the difference in genetic ancestry of the sampled colonies may underly these contribution shifts. In order to frame this, we determined the allele frequencies of the eight genetic variants in more than 360 Apis mellifera colonies across the European continent and found that variant type allele frequencies of these variants are primarily related to the A. mellifera subspecies or phylogenetic honey bee lineage. Our results confirm that population-specific genetic markers should always be evaluated in a new population prior to using them in MAS programs.

The mechanisms of action behind decreased mite reproduction (DMR) are still unknown, but current hypotheses state that DMR is the result of brood-intrinsic and/or external disturbances in the V. destructor—honey bee pupa signal interactions. For accurate and precise DMR phenotyping, sufficient single infested honey bee brood cells are required (e.g., 35), which requires extensive labor and time and may exclude many samples not reaching the threshold. We defined a new comprehensive trait called the ‘mean V. destructor reproduction rate’ (mVR), which describes the mean number of offspring mites per infested cell in the sample while compensating for the reduced number of offspring with increasing multiple infested cells. We found a significant correlation between mVR and DMR, allowing for an estimation of DMR based on the mVR only. When the mVR was calculated with 10 infested cells, we found an average variation in mVR of 16.8%. For the same variation in DMR determination, 40 single infested cells are required. This broader look at V. destructor resistance phenotyping can improve the applicability and effectiveness of traits related to V. destructor reproduction in honey bee breeding programs.

Selective breeding for Varroa resistance is an important strategy to improve honey bee colony survival against Varroa destructor, which remains a major threat to their health. However, phenotyping resistance traits is often labor-intensive. Marker-assisted selection (MAS) offers an alternative by selecting colonies based on molecular markers associated with resistance traits. This study evaluated the implementation of MAS for drone brood resistance (DBR, i.e., mite non-reproduction in drone brood), using a three-SNP model within the ongoing Flemish honey bee breeding program. From 2022 to 2025, predominantly Apis mellifera carnica workers and drones were genotyped. Through targeted breeding and mating via instrumental insemination, we aimed to reconstruct the genetic profile with all three SNPs’ protective alleles. Over sampling years, SNP2 and SNP6 increased in frequency in both workers and drones, whereas SNP4 did not. The cumulative three-SNP score also increased over years and generations, with the greatest genetic gain achieved by combining targeted breeding and targeted mating. The ideal genetic profile was obtained in three queens. However, genetic progress was not reflected in the DBR phenotype. These findings provide insights into the potential and limitations of MAS, suggesting future efforts in breeding programs towards genomic selection.

Today, the deformed wing virus (DWV) can be considered as one of the major causes of global elevated western honey bee colony losses (Apis mellifera). Virus transmission may occur horizontally between individuals of the same generation, but also vertically from parents to offspring. The recently defined heritable suppressed in ovo virus infection (SOV) trait describes the absence of viruses in pooled drone eggs of a queen, associated with significant lower DWV prevalence and viral loads in the subsequent developmental offspring stages. By definition, the trait reflects the absence of vertical virus transmission from SOV-positive (SOV+) queens themselves to their offspring. However, the genetic basis influencing this heritable virus resilience has not been identified yet. In this study, we aimed to identify SOV-associated genetic marker(s) or loci in the honey bee genome through genome-wide variant comparison of 44 DWV-positive and 44 DWV-negative drone pupae descendent from an artificially created hybrid SOV+/SOV- colony. After whole genome sequencing (WGS), variant calling, and genotype-phenotype association analysis by means of single marker tests and elastic net regression, one variant in a locus of 241.246 bp on chromosome 7 that contained 17 other highly SOV-associated variants classified 68,2% of the drone phenotypes correctly. These results may support the potential application of marker-assisted selection (MAS) strategies targeting reduced vertical virus transmission in honey bees.