In the present study, 40 queens were each inseminated with sperm from one drone. A total of 5072 bees from the F1-generation were individually examined for their VSH. We aimed to display the connection between VSH and the drone's olfactory sensitivity by regrouping the data multiple times.
Masterman et al. [34] observed a difference in the discrimination abilities of hygienic and non-hygienic worker bees for brood odours. However, this does not seem to apply to drones. While the drones were able to perceive the extract of Varroa-parasitised pupae and be successfully conditioned to it, neither one of the groups (selection/control line) was more prone to learning the CS + than the other one. Moreover, the results from the PER-test did not deliver any advantage to the F1-generation. To our knowledge, this is the first conditioning experiment with drones using an extract from Varroa-parasitised pupae.
The drones' olfactory sensitivity to extract-low was not represented in the VSH of the drones' offspring. Moreover, the group with the highest results contained the sperm of drones which did not learn the conditioning stimulus (SelQ x SenD-). When the results from the video observation were displayed for two groups on the base of the PER-test – SenD + and SenD- - the PER-test showed no significance for the manifestation of VSH.
When mated with queens from the selection line, the successfully conditioned drones produced colonies with more active beginner and helper bees compared to the reference group ConQ x SenD-. Yet, those results were significant only for the helper activity (GLMM, p < 0,001; CI: 0,394; 0,947). Provided that the single drone's perception ability is crucial for the manifestation of VSH in the next generation, we would have expected groups SelQ x SenD + and ConQ x SenD + to exhibit the highest results in the observation. Contrary to our thesis, group SelQ x SenD produced the most active offspring in the three repetitions of the experiment. Further, group ConQ x SenD + scored lower than the reference group, although the differences were not significant. These observations led us to believe that the negative outcome from the conditioning with both extracts -high and -low did not prove to be a reliable exclusion instrument.
From our observations, drones proved to be much more sensitive to the conditioning temperature and length than bees. Vareschi [45] described differences between bees' and drones' conditioning, stating that drones are more "nervous" than bees. We, too, observed such a tendency. The PER-success depended on the physiological state of the tested individuals - factors such as the drone's disposition during the conditioning, hunger state, and environmental influences. Once the drones had ingested enough sugar, they stopped responding to the stimuli.
We ensured the same nursing conditions for all test subjects through the drones' collective upbringing in one hive. The laboratory conditions were as uniform as possible. Nevertheless, all bee species are known to be highly conscious of their natural surroundings [46], and the change in weather conditions [47] could trigger a physiological response. Li et al. [47] believed that high temperatures and humidity could influence oxidative stress in bees. The role of humidity as a factor is not yet clear; however, it might explain the unwillingness of some drones to respond to the CS+, for example, on cold or rainy days. Because of the PER-test drawbacks mentioned earlier, some drones with favourable genes for enhancing VSH might have potentially been excluded from the experiment or labelled with a "negative outcome", leading to the group outcomes we observed.
Brockmann et al. [48] conducted an electroantennogram (EAG) on bees and drones to study their reaction to the queen mandibular pheromone components. Drones exhibited an increased sensitivity towards 9-ODA, which acts as a sex pheromone and attracts drones during mating flights. Such specialisation is also typical for other species like moths [49, 50], bark beetles [51], cockroaches [52], ants [53, 54]. Brockman et al. [48] argued that while the sensitivity for 9-ODA in the honeybee drone was enhanced, the sensitivity to the other components was reduced. Whereas drones were specialised in detecting only one component, worker antennae showed a "generalised" antennal tuning. Bees exhibited no differences in the neurons sensitive for either individual pheromone components and those detecting other pheromonal or non-pheromonal components. These findings are compatible with the observations of Esslen and Kaissling [55] on the number of sensory cells in the antennae of bees (64 889) and drones (338 859). The worker bee exhibits a large number of secondary neurons relative to the number of sensory cells. The authors argued that these anatomical features could be the basis for a greater differentiation ability for scents. On the other hand, drones are assumed to be highly specialised in distinguishing only certain odours connected with their biological function. According to Arnold et al. [44], a well-pronounced sexual dimorphism in the glomeruli of the antennal lobe can be observed between bees and drones. While the worker bees displayed only two structural types of glomeruli, drones exhibited a third one. These large glomerular complexes (macroglomeruli) of the drone were responsible for the detection of queen pheromones.
Plant odours were processed in the ordinary glomeruli of the antennal lobe [56]. Having this in mind, it is likely that the extract of Varroa-parasitised brood is processed in the drone's ordinary glomeruli. When it comes to VSH, Mondet et al. [28] strengthened the view that all worker bees can detect the Varroa-parasitisation-compounds, but only VSH bees can distinguish the difference between healthy and diseased brood. The VSH is a sex-specific function. While we proved that drones can perceive the parasitised pupae extract, this ability is probably as unimportant to the drone's mating success as the distinction between two floral odours.
Eusocial insects are known for their caste systems and behaviours performed by each cast (foraging, nursing, mating). Different epigenetic mechanisms like DNA methylation and histone posttranslational modifications regulate these behaviours in ants [57], bumblebees [58], honeybees [59]. Flores et al. [59] suggested that certain environmentally induced non-heritable methylations of DNA could lead to better survival outcomes and in time become permanent, heritable methylations. Kucharski et al. [60] examined the expression of one Odorant binding protein (OBP) gene – obp11 – on the antennae of bees. OBP11 is also found in the sensilla basiconica of female ants [61]. It is involved in the accurate perception of cuticular hydrocarbons and pheromones, enabling workers to interact with each other and fulfil their social duties. While obp11 is expressed in female bees' sensilla basiconica, it is silenced through methylation on drones' antennae [60]. This observation is not surprising since the drones' only role is mating with a virgin queen. Epigenetic changes connected to labour division between the sexes could explain why the drones' PER-conditioning results did not prove useful for enhancing the VSH of the F1-generation.
Our experiments also add new information on the inheritance of VSH. When the group results were analysed with the hygienic status in mind, the number of beginner and helper actions increased when drones or/and queens of the selection line were used. The origin of the queen proved to play an even bigger role than that of the drone. This observation was in accord with the substantial effect of the queen origin (selection/control line) on the beginner activity when the results were analysed based on the PER-test. The Sel queens produced offspring with a higher VSH-activity when inseminated with sperm from Con drones compared to Con queens inseminated with sperm from Sel drones. The odds of commencing a beginner activity compared to the reference group were as followed: 1,5 times higher for ConQ x SelD (OR; CI:0,791; 2,732), 2,7-times higher for SelQ x ConD (OR; CI: 1,548; 4,657), 5,5-times higher for SelQ x SelD (OR; CI: 3,305; 9,093). The same tendency was observed in the helper activity: 3,0-times higher than the reference group for ConQ x SelD (OR; CI: 1,706; 5,378), 3,9-times higher for SelQ x ConD (OR; CI: 2,237; 6,858) and 7,4-times higher for SelQ x SelD (OR; CI: 4,327; 12,528). These results lead us to believe that maternal effects play a significant role in the manifestation of VSH.
Maternal effects affect behaviour and help offspring better adapt to changes in the environment. Maternal effects have been observed in many species [62–65], including honeybees. Dloniak, French and Holekamp [65] described rank-related maternal effects on offspring's phenotype in spotted hyenas (Crocuta crocuta). Dominant females exhibited higher androgen concentrations in late pregnancy, which shaped the new generation's behaviour and social structure. Storm and Lima [66] described an "adaptive transgenerational maternal effect on offspring antipredator behaviour" in crickets. The offspring of mothers exposed to Hogna helluo spiders survived longer than the offspring of naive mothers. The forewarned crickets exhibited a behavioural change that manifested in a mobility reduction. Such behavioural changes have been described by bees as well. Unger and Guzmán-Novoa [67] experimented with crossbreeding of highly hygienic Russian-bee strains and less hygienic Ontario-bee strains. The hybrid bees with a "hygienic mother" and "control father" exhibited higher results for individual bees uncapping cells, as well as the removal of brood. On the other hand, "control queens" and "hygienic drones" produced an F1-generation with weaker hygienic behaviour. Spivak and Reuter [68] assessed colonies with queens from a VSH-selection line naturally mated with unselected drones. Compared to unselected colonies, the hygienic colonies displayed a reduced mite-load. Our findings further strengthen these observations.
This research demonstrated drones' ability to sense odours connected to the distress signalling of brood infested with V. destructor. The PER-test proved a non-suitable selection tool for the enhancement of VSH. While an additive genetic effect was observed when drones from the selection line were paired with queens from the selection line, there was a tendency that maternal effects also played an important role. Since both genders inherit the same genes from their mother, it would be a big step towards creating a breeding strategy against V. destructor if a worker bees' odour sensitivity could be measured on her fathering gametes (drones). The odour sensitivity to parasitised brood of worker bees is the key factor in Varroa-resistance. Therefore, further research is necessary to find odours and suitable test methods to phenotype the drones' unspecific odour sensitivity. If the heritability of such test results is sufficient, VSH can be improved more efficiently by the breeding use of such individually tested drones.