CHARACTERIZATION OF 2 MYOTROPIC NEUROPEPTIDES IN THE FMRFAMIDE FAMILYFROM SEGMENTAL GANGLIA OF THE MOTH MANDUCA-SEXTA - CANDIDATE NEUROHORMONES AND NEUROMODULATORS

We have characterized two new members of the FMRFamide family of neuro peptides from the segmental ganglia of the tobacco hornworm Manduca se xta. Levels of peptides in ganglia used for purification were enhanced by manipulating their exposure to the steroid molting hormones. Expla nts of ganglia were cultured in the low-level ecdysteroid environment of diapausing pupae shown previously to evoke accumulation of FMRFamid e-like immunoreactivity (FLI). Sufficient material for sequencing was obtained from 180 explanted ganglia. Extracts of ganglia were fraction ated using two reverse-phase liquid chromatography procedures, and the immunoreactive fractions were subjected to sequence analysis using el ectrospray mass spectrometry. The sequences of the two peptides were d etermined to be GNSFLRFamide and DPSFLRFamide. These peptides have bee n named MasFLRFamide II and MasFLRFamide III, respectively; the previo usly characterized M. sexta FLRFamide (pEDVVHSFLRFamide) has been rena med MasFLRFamide I. The three peptides show distinctive tissue and dev elopmental distributions as determined from fractionated extracts of l arval and adult central nervous system structures and neurohemal organ s. In the retrocerebral corpora cardiaca/corpora allata, MasFLRFamide I was the predominant form, while in the segmental ganglia MasFLRFamid es II and III predominated. Higher levels of MasFLRFamide I and II wer e found in the adult, whereas there was little apparent change in the level of MasFLRFamide III upon metamorphosis. Determinations of peptid e levels in fractionated hemolymph of newly emerged moths revealed tha t levels of MasFLRFamide I and III could exceed 10 nmol l(-1). The act ions of the three peptides were tested on the moth ileum, MasFLRFamide s II and III were found to be stimulatory. At 1 nmol l(-1), these pept ides induced robust increases in the rate of rhythmic longitudinal and peristaltic waves of contractions. In contrast, MasFLRFamide I was in effective even at 20 nmol l(-1). Thus, while all three peptides have t he characteristics of neurohormones in M. sexta, the physiological fin dings show that the heptapeptide FLRFamides have properties distinct f rom those of the decapeptide.