Worker sterility is a defining characteristic of eusociality. The existence of the sterile worker caste remains a fundamental question for evolutionary biology as it requires the existence of genes that reduce personal reproduction. Currently, little is known about the proximate mechanisms underpinning worker sterility. Studies into a mutant "anarchistic" strain (in which workers can activate their ovaries) of honey bee, Apis mellifera, identified a list of candidate genes that regulate ovary activation. We quantified the expression of the four most promising candidate genes (Anarchy, Pdk1, S6k, and Ulk3) in nonactivated and activated ovaries of wild-type workers. Ovarian expression of Anarchy, a peroxisomal membrane protein, predicts the ovary state of workers with 88.2% accuracy. Increased expression of Anarchy in the ovary is strongly associated with suppression of oogenesis and its expression is sensitive to the presence of the queen. Therefore, Anarchy satisfies key criteria for a "gene underlying altruism". When we knocked down expression of Anarchy in the ovary using RNA interference (RNAi) we altered the expression of Buffy, a gene that regulates programmed cell death. Whole-mount multiplex fluorescent in situ hybridization (mFISH) shows Anarchy transcripts localize to degenerating oocytes within the ovary. Our results suggest that Anarchy is involved in the regulation of oogenesis through programmed cell death. The evolution of facultative worker sterility most likely occurred when the conserved mechanism of programmed cell death was co-opted to regulate ovary activation. Anarchy may therefore be the first example of a gene that has evolved through kin selection to regulate worker sterility.

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In social insect colonies the presence of a queen, secreting her pheromones, is a key environmental cue for regulating the reproductive state of workers. However, until recently the proximate molecular mechanisms underlying facultative worker sterility were unidentified. Studies into worker oogenesis in the honey bee (Apis mellifera) have indicated that programmed cell death is central to the regulation of oogenesis. Here we investigate how queen pheromone, age of the worker and ovary state affect both programmed cell death and cell number in worker ovaries. We describe a novel method to simultaneously measure programmed cell death (caspase activity) and live cell number (estimated from the amount of adenosine triphosphate) in an insect tissue. Workers exposed to queen pheromone have higher levels of caspase activity in the ovary than those not exposed. Our results suggest that queen pheromone triggers programmed cell death at the mid-oogenesis checkpoint causing the abortion of worker oocytes and reproductive inhibition of the worker caste. Nonetheless, high caspase activity is present in activated ovaries from workers not exposed to queen pheromone. This caspase activity is most likely to be from the nurse cells undergoing programmed cell death, in late oogenesis, for normal oocyte development. Our study shows that the social environment of an organism can influence programmed cell death within a tissue.

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin‐signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non‐reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co‐opted to regulate behavioural differences between queens and workers.