Honey bee colonies are dying at alarming rates, yet management and medical interventions remain limited, leaving beekeepers largely defenseless. Extensive research has been conducted to uncover the causes of honey bee colony winter losses. However, a review of these studies shows that certain factors, such as age- and season-related patterns, have rarely been considered. Moreover, standard research methodologies often make it difficult to distinguish true infections from contamination and only study a predetermined set of pathogens, leaving an incomplete understanding of honey bee pathogen dynamics. Lastly, while the honey bee immune system plays a central role in countering stressors, limited attention has been given to one key component: hemocytes. As scientific techniques advance, new tools now offer opportunities to further unravel this phenomenon. This thesis applies innovative research techniques to deepen our understanding of this phenomenon, with a particular focus on honey bee hemolymph as a valuable resource for studying pathogen dynamics and immune parameters across age groups and seasons.

Chapter 1 provides an overview of reported colony mortality rates in Belgium and Europe. It outlines the different possibilities through which honey bee colony losses occur and the confusing nomenclature in literature. Importantly, this chapter introduces the concept of the honey bee winter mortality complex, a term adapted from veterinary medicine to describe multifactorial diseases. The potential causes of this complex and additional research gaps were described in detail. Standard research approaches and the limited preventive tools available to beekeepers are discussed. This chapter concludes with an introduction to the honey bee immune system, emphasizing hemocytes, an underexplored component of honey bee immunity.

Chapter 2 summarizes the main objectives, research questions, and methodologies employed throughout this thesis.

In Chapter 3, the honey bee winter mortality complex is investigated by employing third-generation nanopore sequencing on hemolymph samples from both young and old bees collected over the course of a year on one apiary. To collect hemolymph, a newly developed flushing technique was used. This provided a comprehensive overview of viral and bacterial dynamics throughout the production season and bee lifespan, excluding interference from the gut microbiome or surface contaminants. Several viruses and bacteria were identified, which are not routinely screened for. A stark contrast was observed between the pathogen load of young and old bees. This difference was not surprising but has its implementation for future research. Secondly, a pathogen peak was identified. This peak was surprising as the lack of an adaptive immunity should result in a continuous infection of the colony. This peak in viral infection has been reported before, but this was not delved into further by other researchers. The involvement of transgenerational immunity was first suggested. This process has been studied mostly for bacteria but in this chapter, we suggest its involvement in a colony wide immunity to protect the vulnerable winter bees until next spring.

Chapter 4 expands on this. Pathogen dynamics, including Nosema spp. and Varroa were further studied across three apiaries, alongside monitoring hemocyte dynamics. The pathogen peak was further divided into two peaks, associated with the main flowering periods, and new viral detections in Belgium and Europe were reported. Surprisingly, even though high viral loads could be present, this did not influence hive health or production but it did affect apiary health. Dead and sick bees contained similar pathogens as were present previously in the pathogen peak of the healthy hives suggesting a failure in controlling this peak and thus a failure in their transgenerational immune priming response. Hubei-partiti-like virus 34 in healthy hives was able to escape this priming effect so further research on this virus was recommended. Again, a stark contrast was observed between the pathogen load of young and old bees. Taken together with the striking differences in immune capacity between young and old bees the question was raised whether hemocyte decline is age-related or infection-driven. Additionally, this chapter is the first to describe seasonal hemocyte dynamics in honey bees. Lastly, the added value of our developed flushing technique but also its pitfalls was highlighted.

In Chapter 5, a controlled cage experiment was used to disentangle age-related hemocyte decline from pathogen effects, as was seen in Chapter 4. This decrease has been reported before but contradicting studies exist. A sharp reduction in hemocyte counts was observed in both caged “sterile” bees and field bees, suggesting that hemocyte decline is primarily age-related rather than pathogen-induced. However, differences between caged and field bees emphasized the influence of environmental factors. Implications for colony management were also briefly discussed as it is unknown if honey bees can compensate for this hemocyte decline. Hemocyte subtypes were further characterized through morphological analysis, flow cytometry, and the novel application of image flow cytometry. Several known and previously unidentified subtypes were identified, although subtype classification based solely on morphology proved challenging, this method still resulted in the best resolution. The presence of certain unknown subtypes raised the question if our flushing technique also enables us to sample the sessile reservoir which would give a more complete overview of hemocyte capacity in adult honey bees.

Building on the findings of Chapters 3-5, Chapter 6 translated the central hypothesis developed during chapters 3-5 into a practical field application. In short, this hypothesis entails that colonies are exposed to several pathogens during the production season which enables them to build-up a transgenerational immunity against these pathogens and thus eliminating them. As a result, a strong immune priming is present during winter season. When this priming is insufficient or pathogens are too pathogenic these pathogens will not be eliminated and be present during autumn and eventually multiple secondary pathogens will replicate, causing mortality in winter. We tested whether autumn sampling of winter bees could identify colonies with insufficient immune priming and thus are at risk of collapse. This case study confirmed the hypothesis: all colonies that later collapsed showed viral presence in their hemolymph, specifically Deformed Wing Virus (including recombinants). This stark contrast between the presence and the total absence of viral infections was attributed to the sampling of hemolymph which will not include contaminating viruses. Interestingly, hemocyte loads did not differ significantly, underlining the complex role of immune parameters in colony health. The role of Deformed wing virus recombinants was highlighted.

Finally, Chapter 7 integrates all results into a cohesive narrative, situating the newly generated data, conclusions, and hypotheses within the broader framework of the honey bee winter mortality complex. Our central hypothesis is further explored and substantiated using existing literature. The role of Deformed wing viruses and its recombinants is further explored. The (sometimes) contradicting literature reports on honey bee winter colony mortality are explained via our hypothetical model and suggestions for future research are provided. Additionally, our sampling size (apiary-hive-bee) is critically evaluated. This chapter concludes with future research plans and perspectives.