SIFamide peptides in clawed lobsters and freshwater crayfish (Crustacea, Decapoda, Astacidea): A combined molecular, mass spectrometric and electrophysiological investigation

Recently, we identified the peptide VYRKPPFNGSIFamide (Val1-SIFamide) in the stomatogastric nervous system (STNS) of the American lobster Homarus americanus using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry (MALDI-FTMS). Given that H. americanus is the only species thus far shown to possess this peptide, and that a second SIFamide isoform, Gly1-SIFamide, is broadly conserved in other decapods, including another astacidean, the crayfish Procambarus clarkii, we became interested both in confirming our identification of Val1-SIFamide via molecular methods and in determining the extent to which this isoform is conserved within other members of the infraorder Astacidea. Here, we present the identification and characterization of an H. americanus prepro-SIFamide cDNA that encodes the Val1 isoform. Moreover, we demonstrate via MALDI-FTMS the presence of Val1-SIFamide in a second Homarus species, Homarus gammarus. In contrast, only the Gly1 isoform was detected in the other astacideans investigated, including the lobster Nephrops norvegicus, a member of the same family as Homarus, and the crayfish Cherax quadricarinatus, P. clarkii and Pacifastacus leniusculus, which represent members of each of the extant families of freshwater astacideans. These results suggest that Val1-SIFamide may be a genus (Homarus)-specific isoform. Interestingly, both Val1- and Gly1-SIFamide possess an internal dibasic site, Arg3-Lys4, raising the possibility of the ubiquitously conserved isoform PPFNGSIFamide. However, this octapeptide was not detected via MALDI-FTMS in any of the investigated species, and when applied to the isolated STNS of H. americanus possessed little bioactivity relative to the full-length Val1 isoform. Thus, it appears that the dodeca-variants Val1- and Gly1-SIFamide are the sole bioactive isoforms of this peptide family in clawed lobsters and freshwater crayfish.