Isolation of NEB-LFamide, a novel myotropic neuropeptide from the grey fleshfly

Abstract

A methanolic extract of 350 000 adult grey fleshflies Neobellieria bullata, was prepared and screened for myotropic activity. After fractionation on the first column, all fractions were screened in two heterologous ( Locusta oviduct and Leucophaea hindgut) and one homologous ( Neobellieria hindgut) myotropic bioassay. We here report the purification of one fraction, which stimulates the contractions of the Locusta oviduct. Electrospray Mass Spectrometry of the peptide revealed a molecular mass of 1395.82. The primary structure has been determined as AYRKPFNGSLF-amide. This novel peptide was designated Neb-LFamide. This sequences is different from the other known myotropic peptides in insects. The threshold concentration of the synthetic peptide is 1 × 10 −7 M on the Locusta oviduct. On the hindgut of Neobellieria or Leucophaea, the synthetic peptide is not active. By use of a polyclonal antiserum raised against the synthetic peptide, immunoreactivity was localized in median neurosecretory cells in the pars intercerebralis of the fly brain, indicating that Neb-LFamide is a neuropeptide.