Molecular cloning, developmental expression, and tissue distribution of the gene encoding DH, PBAN and other FXPRL neuropeptides in Samia cynthia ricini

Abstract

We obtained a full-length cDNA encoding diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) in Samia cynthia ricini based on both reverse transciptase-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) strategies. The open reading frame (ORF) of this cDNA encodes a 198-amino acid precursor protein that contains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which share a common C-terminal pentapeptide motif FXPR/KL (X=G, T, S). Samia DH-like and PBAN show high homology to their counterpart in other Lepidoptera. Northern blots demonstrate the presence of a 0.8-kb transcript in the suboesophageal ganglion (SG). The DH-PBAN mRNA was detectable at much lower levels in other neural tissues, such as brain and thoracic ganglia (TG), but not in non-neural tissue, such as the midgut, silk gland, fat body or epidermis. The DH-PBAN mRNA content in the SG was measured using the combined method of quantitative RT-PCR and Southern blotting and was shown to vary with developmental stage. Using an antiserum against Helicoverpa armigera PBAN, PBAN-like immunoreactivity was detected in the SG, TG and terminal abdomen ganglion of S. cynthia ricini by whole-mount immunocytochemistry. The changes of PBAN-like immunoreactivity in the hemolymph are consistent with PBAN transcripts in the SG during pupal development. PBAN increases quickly at adult eclosion, an observation that is consistent with PBAN's key role in pheromone biosynthesis, and synthetic PBAN or brain-SG extracts successfully stimulates pheromone biosynthesis in decapitated moths.