Apis mellifera Worker Bees Selected for Varroa-sensitive Hygiene Show Higher Specific Sensitivity and Perception Speed Towards Low Concentrations of Chemical Cues Emitted by the Brood

J Insect Behav https://doi.org/10.1007/s10905-023-09824-9 RESEARCH Apis mellifera Worker Bees Selected for Varroa‑sensitive Hygiene Show Higher Specific Sensitivity and Perception Speed Towards Low Concentrations of Chemical Cues Emitted by the Brood Ivelina Ivanova · Kaspar Bienefeld Received: 24 June 2022 / Revised: 2 March 2023 / Accepted: 16 March 2023 © The Author(s) 2023 Abstract Varroa-sensitive hygiene (VSH) is highly influenced by the worker bee’s olfactory ability. Workers bred for VSH and non-selected control line workers were tested for differences in their speed and perception ability when presented with highly diluted stimuli. Four different substances (citral – dilution 1:1300, linalool dilution 1:1300, Varroa-parasitized brood extract, isopropanol) were used as tactile stimuli for differential conditioning with the proboscis extension response (PER). Discrimination ability and generalization were assessed. In a second set of conditioning experiments differences in sensitivity to the highly diluted citral and the Varroa-parasitized brood Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10905-023-09824-9. I. Ivanova (*) · K. Bienefeld Institute of Bee Research Hohen Neuendorf, Hohen Neuendorf, Germany e-mail: K. Bienefeld e-mail: I. Ivanova Institute of Parasitology and Tropical Veterinary Medicine, Faculty of Veterinary Medicine, Free University of Berlin, Berlin, Germany K. Bienefeld Albrecht Daniel Thaer‑Institute for Agricultural and Horticultural Sciences, Humboldt University of Berlin, Berlin, Germany extract as reinforced stimuli (Cs +) were explored between workers from both lines. The worker bees were classified into three groups (Time points) depending on how long before they started correctly extending their proboscis to the Cs + , and results were examined separately for each of the two stimuli and group. While the VSH-selected line exhibited a significantly higher perception ability for the parasitized brood extract than the non-selected line, the two lines showed no differences when conditioned with the floral stimulus citral as Cs + . Furthermore, the VSH-selected line displayed a significantly higher number of worker bees that perceived the complex bouquet of the Varroa-parasitized brood extract at the earliest time grouping (Time point 1). The odds of perception at the earliest possible time point were 2.6-times higher for the VSH-selected line. Although no comparison was made between healthy and parasitized brood, the results indicate an enhanced specific sensitivity in VSH-selected workers towards chemical cues emitted by the brood, which might play a role in the detection of Varroa destructor. Keywords Varroa-sensitive hygiene · Olfactory sensitivity · Resistance breeding · Apis mellifera Introduction The European honey bee, Apis mellifera, is one of the most important agricultural pollinators worldwide. Vol.: (0123456789) 13 J Insect Behav However, since the parasitic mite Varroa destructor shifted hosts from the Asian honey bee Apis cerana to the European honey bee, a global increase in colony loses has been observed for the latter species (Genersch et al. 2010; Dietemann et al. 2012; Martin et al. 2012). While some populations appear to be Varroa-resistant (Locke 2016; Oddie et al. 2017, 2018), most of the honey bee colonies are still dependent on the Varroa-treatment administered by beekeepers (Jacques et al. 2017). However, recent breeding efforts to create bees with enhanced Varroa-sensitive hygiene (VSH) — a specialized type of hygienic behavior comprising the targeting and removal of Varroa-infested brood — have improved bee colonies’ survival in the face of parasitization (Mondet et al. 2020). Varroa destructor induces a shift in the cuticular hydrocarbon profile of parasitized brood (Nazzi et al. 2004; Wagoner et al. 2019; Mondet et al. 2021) which is detected through the cell cap by nursing bees. Compounds such as tricosan-2-one, pentacosan-2-one, tetracosyl acetate, heptacosan-2-one, hexacosyl acetate and nonacosan-2-one have been detected in extracts of parasitized pupae (Mondet et al. 2021). Furthermore, (Z)-pentadec-6-ene and (Z)-10-tritiacontene, the non-volatile oleic acid, as well as the increase of brood ester pheromone are also able to elicit a hygienic response (Nazzi et al. 2004; Mondet et al. 2016; Wagoner et al. 2020) and are associated with Varroa-parasitization (Wagoner et al. 2021). This odor change acts as a signal for the worker bees and a trigger for VSH (Harbo and Harris 2005; Wagoner et al. 2018). Subsequently, the brood cells are uncapped and the diseased pupae removed (Martin et al. 2002; Swanson et al. 2009). Mondet et al. (2021) observed that while all worker bees can perceive the compounds typical for a V. destructor parasitization at the level of the antennae, only those bees performing VSH can differentiate between these compounds and the odor of unparasitized healthy brood. Moreover, worker bees from colonies bred for VSH are more likely to uncap infested cells with more than one foundress mite (Kim et al. 2018) and brood severely affected by transmitted viruses (Schöning et al. 2012). The early detection of parasitized brood and the subsequent removal of the mites has been identified as being significantly genetically influenced (Spötter et al. 2012, 2016; Guarna et al. 2015). The differential expression of genes for the olfactory and sensory Vol:. (1234567890) 13 activity determines the perception ability and olfactory sensitivity of the single worker bee (Boutin et al. 2015; Hu et al. 2016; Gempe et al. 2016). Under laboratory conditions, olfactory ability can be tested with the help of differential conditioning using the proboscis extension response (PER). First described by Takeda in 1961, this method lies at the center of assessing olfactory discrimination abilities in bees (Takeda 1961; Bitterman et al. 1983; Giurfa and Malun 2004; Giurfa 2008; Matsumoto et al. 2012; Smith and Burden 2014). Through a series of trials, a bee learns to differentiate between two odors: Cs + (reinforced with a reward) and Cs- (unreinforced, or novel odor). In order to feed on the reward sugar solution, the bee displays a behavioral change by extending its mouthparts, or proboscis. PER conditioning can provide valuable information on the differences in perception ability towards various chemicals in lines bred for enhanced hygienic behavior including VSH and non-selected lines. Masterman et al. (2000) observed significantly better discrimination ability in hygienic bees when exposed to the odor of healthy and chalkbrood infested brood compared to non-hygienic bees. Flower odors, on the other hand, were perceived equally well by both groups of bees. Compared to a chalkbrood infection where the brood dies, the parasitization with V. destructor causes amongst others immunosuppression without killing the brood (Rosenkranz et al. 2010; (...truncated)