The essential role of bursicon during Drosophila development

Background: The protective external cuticle of insects does not accommodate growth during development. To compensate for this, the insect life cycle is punctuated by a series of molts. During the molt, a new and larger cuticle is produced underneath the old cuticle. Replacement of the smaller, old cuticle culminates with ecdysis, a stereotyped sequence of shedding behaviors. Following each ecdysis, the new cuticle must expand and harden. Studies from a variety of insect species indicate that this cuticle hardening is regulated by the neuropeptide bursicon. However, genetic evidence from Drosophila melanogaster only supports such a role for bursicon after the final ecdysis, when the adult fly emerges. The research presented here investigates the role that bursicon has at stages of Drosophila development which precede adult ecdysis. Results: We addressed the mechanism and timing of hormonal release from bursicon-positive motor neurons at the larval neuromuscular junction. Our findings indicate that vesicle membrane proteins which are required for classical neurotransmitter release are also expressed at these peptidergic motor neuron terminals; and that these terminals secrete hormones including bursicon at the neuromuscular junction, coinciding with larval ecdysis. This release surprisingly occurs in two waves, indicating bursicon secretion preceding and following the ecdysis sequence. Next, we addressed the functional significance of bursicon signaling during development, by disrupting the expression of its receptor, rickets, in different target tissues. We determined that rickets is developmentally required in the epidermis and imaginal discs for proper formation of the prepupa. It is also required to harden the pharate adult cuticle before eclosion. Significantly, we have also found that the available rickets mutants are not genetic nulls as previously believed, which necessitated the use of targeted RNA interference to disrupt rickets expression. Conclusions: Our results are consistent with the view that bursicon is the insect tanning hormone. However, this is the first study to rigorously demonstrate both its release and function during development. Importantly, we provide new evidence that bursicon release can precede the initiation of larval ecdysis, and that bursicon tans the puparium. Our results firmly establish bursicon signaling as essential to insect growth and development.