Localization of bursicon in CCAP-immunoreactive cells in the thoracic ganglia of the cricket Gryllus bimaculatus

Abstract

Bursicon is a neuropeptide that induces tanning of the cuticle in freshly moulted insects. In an earlier investigation, we demonstrated that bursicon activity can be detected throughout the ventral nerve cord of the cricket Gryllus bimaculatus. This study aims at identifying the neurosecretory cells within the thoracic ganglia that produce bursicon. When homogenates of anterior pieces of thoracic ganglia were separated using SDS gel electrophoresis, proteins with bursicon activity could be eluted only from a slice of the gel spanning the 28-33 kDa region. In the anterior lateral cortex of the thoracic ganglia, there are two bilaterally paired neurosecretory cells with large vacuoles that project contralaterally to neurohaemal release sites associated with segmental nerves N5 and N6. These cells and their processes in N5 and N6 were labelled using antisera against crustacean cardioactive peptide (CCAP). The cell projecting into N6 showed a Tyndall effect (i.e. appeared opaque under oblique illumination) in older adults, and single isolated somata contained bursicon activity. Homogenates of nerves N5 and N6 also showed bursicon activity, but neither bursicon activity nor CCAP-immunoreactive processes were found in segmental nerve N4. The thoracic connectives, which contain three major CCAP-immunoreactive processes, also showed bursicon activity. Homogenates of posterior pieces of the thoracic ganglia did not contain bursicon activity. Western blots demonstrated that the anti-CCAP serum does not recognize the 30 kDa bursicon-active protein fraction. These results suggest that a CCAP-like neuropeptide and a protein with bursicon activity are co-localized in the anterior lateral neurosecretory cells of the thoracic ganglia and in their segmental homologues in the other ganglia. Additionally, we have shown using western blots that a monoclonal antibody raised against a 56 kDa protein from the housefly Musca domestica, a protein thought to be bursicon, does not label the 30 kDa bursicon-active protein of crickets. However, this antibody does label an unidentified 56 kDa protein isolated from anterior as well as posterior pieces of thoracic ganglia.