Supplementary Table 3. Power analysis results 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.繁殖劳动分工是真社会性（eusociality）的标志性特征，但解析其背后的近因机制（proximate mechanisms）仍颇具挑战。在熊蜂中，与蜂王隔离的工蜂可激活卵巢并产下单倍体雄性卵。本研究针对印地安熊蜂（*Bombus impatiens*），探究了蜂王或蜂子代（brood）产生的挥发性信号（volatile cues）、接触信号（contact cues）、视觉信号（visual cues）与行为线索（behavioural cues）是否可介导其繁殖支配地位（reproductive dominance）。实验结果显示，暴露于蜂王释放的挥发性信号、蜂子代产生的挥发性信号，以及直接接触蛹体，均未降低工蜂的卵巢激活（ovary activation）水平；仅直接接触蜂王可抑制工蜂的卵巢激活。本研究进一步对三种饲养环境下的工蜂开展了行为、生理及基因表达（gene expression）模式分析：三种环境分别为包含所有发育阶段蜂子代与自由活动蜂王的饲养舱、仅允许工蜂接触但蜂王无法主动发起互动的笼养蜂王饲养舱，以及无蜂王的饲养舱。结果表明，饲养于笼养蜂王或无蜂王环境中的工蜂，其卵巢均完全激活；而饲养于自由蜂王环境中的工蜂，卵巢激活则被完全抑制。相较于无蜂王的工蜂，笼养蜂王仅小幅降低了工蜂的攻击行为及攻击相关基因（aggression-associated gene）的表达水平。由此可见，蜂王主动发起的行为互动，是确立繁殖支配地位的必要条件。蜂王释放的化学信号或可在特定情境下次要性地发挥作用，作为可靠或诚实信号（honest signal）强化行为线索的效应。