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 α-, β-, γ-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-α-SGNP has a unique C-terminal FXPEL (the arginine or lysine at FXPR(or K)L is replaced by glutamic acid), Mas-γ-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.