(201920592 Page 2 of 15 As in many social insects,honey bee (Apis mellifera) pollen forag ging within an hour of exposure and lasting for 3 a form of age- based ta prod oping shift over the coure fetime 231.This hat FRo is duced early in laryal。 while br called age-based polyethism- -is regulated iust before pupation Both larv enetically and vironmer ally,and stem rs.In fact,bro the first weeks of their lives performing tasks within the including modulation of sucros relative safety of the hive,including tending to the needs ovary dev opment,foraging ontoe developing larvae (i.e oning and hypopharyngea forage,workers may further specialize by collectin ically.brood pheromones cause an increase in the number of ominantly one flora resource type (either pollen aging trip and size of pollen loads【24 and pr ty fo e xhibit dis behavioral,physiological,and trans criptional traits.For the transition of bees from performing within-hive roles t example, he colony,nectar foragers 6. some componen EBO is d b nto hon comb themselves 28 291 Nectar and pollen gers produce ethyl oleate.a co of BP both oragers lso differ ior [48,49.Queen 30]an 32 an comp od i 9.33361.ph are ime scal which they induc beh avioral changes in re arvae, and nent pheromone blends often have ca pheromon hav eral Prime rate long (ie hours)and the are also in d in regulating the size of the foraging labor force in the long especially in the brai 361 er m (i.e weeks】 queen hat。oni of h bee to phe ne ession of large numbers of genes in worker brains 33. 6.In contrast,releaser pheromones elicit rapid behavioral and chromatin remodeling 50).However,it is unclear if nges eith by activa n pheromone ng pa the ala m be (BD honey bees elicits aggressive behaviors against intruders late fora y activating the expression of imm diate early gen nes in the How the behavioral tran sitions acros s different temporal 13 ponent of que or heir unde ing genetic,epigen orkers by binding to an olfact or in the anten tobe dete ined nae,activating dopamine receptors in the brain,and regu In previous studies.the effects of bP on gene expression lating brain gene expr ession[33,40,41. brain expre arv r an 360 age, which provides a fascinating rtunity to unders regulation of behavior acros time scales Two larvae ing their foraging prefer ence.Consequently,we seek to more produced pheromon brood pheron e (BP nd (E)-beta precisely ch e the tr ces assoc cimene (EBO),have been sho toelicit rapi increases in with rapid changes in honeyAs in many social insects, honey bee (Apis mellifera) workers exhibit a form of age-based task allocation in which behavioral repertoires incrementally expand or shift over the course of an individual’s lifetime [23]. This phenomenon—called age-based polyethism—is regulated both genetically and environmentally, and provides a tractable system in which to investigate temporal dimen￾sions of behavioral plasticity [24, 25]. Honey bees spend the first weeks of their lives performing tasks within the relative safety of the hive, including tending to the needs of developing larvae (i.e., nursing), before transitioning to increasingly dangerous tasks near the nest entrance and beyond, including foraging [26]. Once they begin to forage, workers may further specialize by collecting pre￾dominantly one floral resource type (either pollen or nectar [27]), and their proclivity for pollen vs. nectar for￾aging can persist throughout their lives. Bees that specialize on nectar vs. pollen foraging exhibit distinct behavioral, physiological, and transcriptional traits. For example, upon returning to the colony, nectar foragers regurgitate collected nectar to nestmates waiting to process it, while pollen foragers pack their pollen loads into honeycomb themselves [28, 29]. Nectar and pollen foragers also differ in their neural and sensory responses to sugar [30] and pheromones [31, 32]. Pheromone communication in honey bees plays a key role in mediating behavioral transitions across time scales [9, 33–36]. Pheromones are typically categorized by the time scale at which they induce behavioral changes in re￾ceivers: primer pheromones cause slow, enduring changes in physiology, while releaser pheromones cause rapid, ephemeral responses. Primer pheromones generate long￾term changes in behavior and physiology by altering pat￾terns in gene expression, especially in the brain [9, 33–36]. For example, brood and queen pheromones delay the be￾havioral transition from nurses to foragers by altering the expression of large numbers of genes in worker brains [33, 36]. In contrast, releaser pheromones elicit rapid behavioral changes either by activating or modulating neural circuits, triggering molecular signaling pathways, or regulating gene expression [34, 37–39]. For example, the alarm pheromone in honey bees elicits aggressive behaviors against intruders by activating the expression of immediate early genes in the brain [34], while one component of queen pheromone, homovanillyl alcohol, elicits grooming behavior from workers by binding to an olfactory receptor in the anten￾nae, activating dopamine receptors in the brain, and regu￾lating brain gene expression [33, 40, 41]. Honey bee larval pheromones cause primer and releaser effects that blur the distinction between these categories, which provides a fascinating opportunity to understand regulation of behavior across time scales. Two larvae￾produced pheromones, brood pheromone (BP) and (E)-beta￾ocimene (EBO), have been shown to elicit rapid increases in pollen foraging within an hour of exposure and lasting for 3 hours [42]. Both pheromones are produced by developing larvae but differ in the timing of their peak production, such that EBO is produced early in larval development while BP is produced later on, just before pupation [42]. Both larval pheromones cause additional behavioral and physiological ef￾fects in honey bee workers. In fact, brood pheromone in￾duces the greatest number of known primer responses in honey bees, including modulation of sucrose response thresholds, ovary development, foraging ontogeny, foraging choice behavior, and hypopharyngeal gland development [43]. The effect of brood pheromones on forager behavior seems to be driven by an increase in pollen foraging. Specif￾ically, brood pheromones cause an increase in the number of foraging trips and the size of pollen loads [42, 44], and this effect is not driven by task-switching from nectar to pollen foraging [42]. Both pheromones also increase the size of the foraging force of the colony in the long term, accelerating the transition of bees from performing within-hive roles to foraging [44–46]. Interestingly, some components of EBO and BP are also produced by honey bee adults as well. For example, EBO is also produced by mated queens [47], and foragers produce ethyl oleate, a component of BP [48]; both impact the ontogeny of foraging behavior [48, 49]. Queens and larvae both produce another BP component, ethyl palmitate, which inhibits ovarian development [37]. Al￾though BP components are also produced in adults, the full blend of BP and EBO has only been described in honey bee larvae, and multi-component pheromone blends often have synergistic effects [37]. Overall, larval pheromones have a strong effect on pollen foraging but not nectar foraging in the short term (i.e., hours), and they are also involved in regulating the size of the foraging labor force in the long term (i.e., weeks). Chronic exposure of honey bee adults to pheromones that cause primer effects, including BP, have been shown to affect the expression of genes involved in methylation and chromatin remodeling [50]. However, it is unclear if similar epigenetic effects are observed when pheromones act at the short-term, releaser time scale. This is a fascinat￾ing system because both pheromones (BP and EBO) regu￾late foraging behavior, but at different temporal scales. How these behavioral transitions across different temporal scales are related, or how their underlying genetic, epigen￾etic, and physiological mechanisms interact to regulate foraging behavior, remains to be determined. In previous studies, the effects of BP on gene expression were evaluated on whole brain expression patterns from bees collected at five and fifteen days of age, after life-long expos￾ure to brood pheromone [36]. However, in that study, the bees were collected without regard to their behavior, includ￾ing their foraging preference. Consequently, we seek to more precisely characterize the transcriptional differences associ￾ated with rapid, pheromonally-regulated changes in honey Ma et al. BMC Genomics (2019) 20:592 Page 2 of 15