Identification of a cDNA encoding DH, PBAN and other FXPRL neuropeptides from the tobacco hornworm, Manduca sexta, and expression associated with pupal diapause

Abstract

We have cloned the diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) cDNA from the suboesophageal ganglion (SG) of Manduca sexta pupae using rapid amplification of cDNA ends. The Mas-DH-PBAN cDNA encodes a preprohormone of 194 amino acids that contains five peptides (PBAN, DH-like, and alpha-, beta-, gamma-SGNP), all of which share a common FXPRL sequence at the C-terminus. Yet, the sequences are rather distinct from those reported from other species: Mas-alpha-SGNP has a unique C-terminal FXPEL (the arginine or lysine at FXPR(or K)L is replaced by glutamic acid), Mas-gamma-SGNP is one amino acid shorter than its counterpart in other species, and Mas-PBAN contains two extra residues not seen in other species. Mas-DH-like peptide has the highest homology (83%) to Bombyx mori DH. Northern blot analysis shows a single mRNA corresponding in size to the Mas-DH-PBAN cDNA detected in brain-SG samples of pupae and adults, suggesting that these peptides are derived from a precursor through posttranslational processing. Using the more sensitive method of RT-PCR, DH-PBAN mRNA is also detectable in thoracic ganglia, although the expression is much lower than in the SG. Developmental profiles of DH-PBAN transcripts in the early pupal stage reveal different patterns in diapause and nondiapause individuals. While a conspicuous drop in expression of the DH-PBAN gene is noted in diapausing pupae 9 days after pupation, high expression persists in nondiapausing individuals. At earlier stages (wandering larva and day 3 pupae) expression is high in diapausing individuals but low in nondiapausing individuals. These observations suggest a possible contribution of the DH-like peptide to the induction phase of diapause in M. sexta.