Studies with solitary bees, facultatively social bees, and the primitively eusocial bees showing a positive correlation between JH levels and oocyte development lend credence this premise. There is also some evidence that treatment with JH or its analogs augmented oogenesis in both the sweat bee Lasioglossum zephyrum and the bumblebee B. terrestris. In B. terrestris, JH treatment accelerated oogenesis even in the presence of the queen that typically inhibits GSK2118436 Raf inhibitor worker reproduction. Treatment with JH-I, which is not the natural JH of bumblebees, enhanced Vg biosynthesis in the fat body of ovariectomized bumblebee gynes. On the other hand, treatment with either JH-I or the JH analogue methoprene did not influence task specialization in the bumblebees B. terrestris and B. impatiens. These studies suggest that in bumblebees JH influences oogenesis but has no or little influence on division of labor. These studies however, are not sufficient to establish JH as a gonadotropin that is necessary for oogenesis and reproduction. In this study we combined allatectomy and replacement-therapy with JH-III, the natural JH of bumblebees to rigorously test the hypothesis that JH has gonadotropic functions in the bumblebee B. terrestris. Our results show that JH is necessary for oocyte development and maturation and is involved in the regulation of vitellogenesis and several additional physiological processes that are associated with reproduction. We further compared our findings for B. terrestris to those resulting from similar JH manipulations in the honey bee, and discuss the evolution of JH signaling and sociality in bees. We collected newly emerged worker bees from several source colonies. At this age the cuticle of adult bumblebees is relatively soft and easy to manipulate. The bees had free access to sugar syrup and pollen ad libitum both before and after the allatectomy operation. We first anesthetized the bees on ice for 5–30 min and then fixed them with molded modeling clay on an icechilled metal stage under a stereoscopic microscope. The bees were fixed with the dorsal side up and the head bent down to expose the thin neck cuticle connecting the thorax and the head. We used a fine scalpel to open a latitudinal incision in the posterior part of the head capsule, and moved the inner membrane and trachea to expose the CA glands. Using fine forceps we gently grasped each one of the corpus allatum and detached it. The entire procedure took between 2–5 minutes and the cuticle resumed its original shape and the incision appeared self-sealed within few hours after the operation. Sham-operated bees were handled and dissected in a similar way but the CA were only touched gently and not detached. Control bees were anesthetized and handled similarly, but were not operated. After treating the bees we placed them in a small cage with the other similarly manipulated workers, and let them recover overnight in an incubator. On the second day the surviving bees from each treatment group were assigned to groups of three, each transferred to a fresh wooden cage. The groups were kept in the incubator for six days and then collected.