Supplementary Table 2. Complete data set from Chemical communication is not sufficient to explain reproductive inhibition in the bumblebee <i>Bombus impatiens</i>

Reproductive division of labour is a hallmark of eusociality, but disentangling the underlying proximate mechanisms can be challenging. In bumblebees, workers isolated from the queen can activate their ovaries and lay haploid, male eggs. We investigated if volatile, contact, visual or behavioural cues produced by the queen or brood mediate reproductive dominance in <i>Bombus impatiens.</i> Exposure to queen-produced volatiles, brood-produced volatiles and direct contact with pupae did not reduce worker ovary activation: only direct contact with the queen could reduce ovary activation. We evaluated behaviour, physiology and gene expression patterns in workers that were reared in chambers with all stages of brood and a free queen, caged queen (where workers could contact the queen, but the queen was unable to initiate interactions) or no queen. Workers housed with a caged queen or no queen fully activated their ovaries, whereas ovary activation in workers housed with a free queen was completely inhibited. The caged queen marginally reduced worker aggression and expression of an aggression-associated gene relative to queenless workers. Thus, queen-initiated behavioural interactions appear necessary to establish reproductive dominance. Queen-produced chemical cues may function secondarily in a context-specific manner to augment behavioural cues, as reliable or honest signal.生殖劳动分工是真社会性（eusociality）的标志性特征，但厘清其背后的近缘机制颇具挑战。在熊蜂中，与蜂后隔离的工蜂可激活卵巢并产下单倍体雄性卵。我们针对<i>Bombus impatiens</i>研究了蜂后或育幼群体产生的挥发性、接触性、视觉或行为信号，是否能介导<i>Bombus impatiens</i>的生殖优势地位。接触蜂后分泌的挥发性物质、育幼群体分泌的挥发性物质以及直接接触蛹，均未降低工蜂的卵巢激活水平；唯有直接接触蜂后，方可抑制工蜂的卵巢激活。我们对三种饲养环境下的工蜂行为、生理及基因表达模式展开了评估：饲养箱内包含各发育阶段的育幼群体且蜂后可自由活动、饲养箱内包含笼养蜂后（工蜂可接触蜂后，但蜂后无法主动发起互动），以及无蜂后的饲养箱。与笼养蜂后或无蜂后共同饲养的工蜂，其卵巢均完全激活；而与可自由活动的蜂后共同饲养的工蜂，其卵巢激活则被完全抑制。相较于无蜂后的工蜂，笼养蜂后仅能小幅降低工蜂的攻击行为及攻击相关基因的表达水平。由此可见，蜂后主动发起的行为互动，是确立生殖优势地位的必要条件。蜂后分泌的化学信号或可作为辅助信号，在特定情境下强化行为信号，以充当可靠或诚实的通讯信号。