Molecular analysis of two FMRFamide-encoding transcripts expressed during the development of the tropical abalone...

FMRFamide-related peptides (FaRPs) are involved in numerous neural functions across the animal kingdom and serve as important models for understanding the evolution of neuropeptides. Gastropod molluscs have proved to be particularly useful foci for such studies, but the developmental expression of FaRPs and the evolution of specific transcripts for different peptides are unclear within the molluscs. Here we show that FaRPs are encoded by two transcripts that appear to be splice variants of a single gene in the abalone, Haliotis asinina, which represents the basal vetigastropods. Has-FMRF1 comprises 1,438 nucleotides and encodes a precursor protein of 329 amino acids that can potentially produce two copies of FLRFamide, one copy each of TLAGDSFLRFamide, QFYRIamide, SDPDLDDVIRASLLAYSLDDSPNN, and SVATAPVEAKAVEAGNKDIE, and 13 copies of FMRFamide. The second 1,241-nucleotide transcript, Has-FMRF2, encodes a 206-amino acid precursor protein with single copies of FLRFamide and FMRFamide along with such extended forms as NFGEPFLRFamide, FDSYEDKALRFamide, and NGWLHFamide, in addition to SDPGEDMLKSILLRGAPSNNGLQY and DTUDETTUNDNAHSRQ. Both transcripts are present early in life and are expressed in different but overlapping patterns within the developing larval nervous system. Mass spectrometry and immunocytochemistry demonstrate that FaRPs are cleaved from larger precursors and localize to the developing nervous system. Our results confirm previous evidence that FaRPs are expressed early and potentially play many roles during molluscan development and suggest that the last common ancestor to living gastropods used alternative splicing of an FMRFamide gene to generate a diversity of FaRPs in spatially restricted patterns in the nervous system.