8The mechanisms that maintain reproductive division of labor in social insects are still 9 incompletely understood. Most studies focus on the relationship between adults, overlooking 10 another important stakeholder in the gamethe juvenile offspring. Recent studies from various 11 social species show that not only the queen, but also the brood regulates reproductive division of 12 labor between females, but how the two coordinate to maintain reproductive monopoly remained 13 unexplored.14 Our study aims at disentangling the roles of the brood and the queen in regulating worker 15 reproduction in primitively eusocial bees. We examined the effects induced by the brood and 16 queen, separately and together, on the behavioral, physiological and brain gene expression of 17 Bombus impatiens workers. We found that young larvae induce a releaser effect in workers, 18 decreasing egg laying and aggressive behaviors, while the queen induces both releaser and primer 19 effects, modifying worker aggressive and egg laying behavior and reproductive physiology. The 20 expression of reproduction-and aggression-related genes was altered in the presence of both 21 queen and brood, but the effect was stronger or the same in the presence of the queen.
22We identified two types of interactions between the queen and the brood in regulating worker 23 reproduction: (1) synergistic interactions regulating worker physiology, where the combined 24 effect of the queen and the brood was greater than each of them separately; (2) additive 25 interactions regulating worker behavior, where the combined effects of the queen and the brood 26 are the gross sum of their separated effects. In these interactions the brood acted in a manner 27 similar to the queen but to a much smaller extent and improved the quality of the effect induced 28 by the queen. Our results suggest that the queen and the brood of primitively eusocial bees 29 coordinate synergistically, additively, and sometimes even redundantly to regulate worker 30 behavior and reproduction, and the interaction between them exists in multiple regulatory levels. 31 32