Identification and characterization of a novel target for eclosion hormone in the tobacco hornworm, Manduca sexta

Abstract

The insect neuropeptide eclosion hormone (EH) interacts in a positive feedback loop with two other hormones to trigger ecdysis behavior in the hawkmoth, Manduca sexta. There is also evidence that EH may act at other targets to coordinate physiological changes that must occur with ecdysis. Recently, the use of an antibody that recognizes the second messenger cyclic guanosine monophosphate (cGMP) in fixed tissue has allowed us to identify a novel target for EH in the proximal region of the abdominal transverse nerves of Manduca. Here we show that a significant increase in cGMP can be measured in vivo in the transverse nerves at a time when EH is normally released in the animal. Furthermore, in vitro experiments revealed that the ability of the transverse nerve to respond to EH is abruptly lost after the pupal ecdysis. Finally, it was found that up to 40% of the cGMP produced by the STNR in response to EH is exported out of the cells in probenecid-sensitive manner, and there is a significant increase in the hemolymph concentration of cGMP at the time of pupal ecdysis. Microscopic examination of transverse nerve sections revealed that the cGMP-positive filaments are the processes of intrinsic cells, and the area in which they reside was named the subtransverse nerve region (STNR). Ultrastructurally, the cells of the STNR fall into two groups: large-granule containing cells and ribosome rich cells. EH-stimulated cGMP immunoreactivity appears to be restricted to the ribosome rich cells. The discrete STNRs of the abdominal transverse nerves can be identified as early as the first instar and probably originates embryologically. After the time of wandering in the last instar, the cells of the STNR begin to divide and spread in a flat sheet between the transverse and dorsal nerves of each ganglion, and are positive for myosin by pupal stage 8. These STNR-derived cells appear to form the ventral diaphragm muscles of the adult. This is the first evidence in any insect that a reservoir of myoblasts exists in close proximity to the transverse nerves and the first evidence that myoblasts are a target for EH.