the health of western honey bee (Apis mellifera) colonies is challenged by the parasitic mite Varroa destructor and the numerous harmful pathogens it vectors. Selective breeding for the naturally occurring social immune trait "hygienic behavior" has emerged as one sustainable approach to reducing the mites' impact on honey bees. to expand our understanding of hygienic triggers and improve hygienic selection tools, we tested the hypothesis that the cuticular compounds (Z)-10-tritriacontene and (Z)-6-pentadecene, previously associated with unhealthy honey bee brood and/or brood targeted for hygiene, are triggers of honey bee hygienic behavior independent of brood health. in support of our hypothesis, application of synthetic (Z)-10-tritriacontene and (Z)-6-pentadecene onto brood and brood cell caps significantly increased hygienic behavior compared to application of similarly structured hydrocarbon controls (Z)-16-dotriacontene and (Z)-7-pentadecene. Furthermore, we demonstrate a significant positive correlation between colony-level hygienic responses to (Z)-10-tritriacontene and the traditional freeze-killed brood assay for selection of hygienic honey bee stocks. These results confirm biological activity of (Z)-6-pentadecene and reveal (Z)-10-tritriacontene as a novel hygiene trigger. They also support development of improved tools for honey bee colony monitoring and hygienic selection, and thus may accelerate development of honey bee stocks with greater resistance to Varroa and associated pathogens.As with other social insects, honey bees are highly susceptible to the horizontal spread of infectious diseases due to close contact, high coefficients of relatedness, and high frequency of social interactions among individuals within a colony 1-3 . Movement of parasites between bees within a colony can facilitate the spread of honey bee diseases 2 because ectoparasites such as the mite Varroa destructor, hereafter Varroa, vector numerous honey bee pathogens 4,5 . In addition, Varroa can amplify viral loads 6-8 , alter viral strain diversity 8 , and affect virus virulence 9,10 . In the western honey bee (Apis mellifera), the spread and proliferation of harmful pathogens such as Deformed Wing Virus (DWV) is especially problematic considering honey bees have fewer immune-related genes than non-social insects 11 .Combined with susceptibility to and spread of honey bee parasites and pathogens 12-14 , numerous anthropogenic threats such as pesticide exposure and poor nutrition have led to a shortage of colonies relative to an increasing global demand for crop pollinators [15][16][17][18] . Following pollinator population trends in much of the Northern Hemisphere 19-21 , managed honey bee colonies in the United States have declined more than 50% over the last six decades, from around 5.9 million colonies in 1947 to around 2.7 million colonies in 2015 22,23 . Early declines were likely due to reduced public demand for honey after the end of World War II, while the spread of pests and pathogens are responsible for more recent ...