Peptides, Vol. 5. pp. 585--592. 1984. ~ AnkhoInternational Inc. Printedin the U.S.A. 0196-9781/84$3.00 + .00 Presence and Distribution of Immunoreactive and Bioactive FMRFamide- Like Peptides in the Nervous System of the Horseshoe Crab, Limulus polyphemus W. H. WATSON, Ill, J. R. GROOME Department of Zoology, Spaulding L~['e Science Building University o.f New Hampshire, Durham. NH 03824 B. M. CHRONWALL, J. BISHOP AND T. L. O'DONOHUE Experimental Therapeutics Branch, National Institute of Neurological and Commttnicative Disorders and Stroke, National Institute of Health, Bethesda, MD 20205 Received l September 1983 WATSON, W. H., I11, J. R. GROOME, B. M. CHRONWALL, J. BISHOP AND T. L. O'DONOHUE. Presence and distribution ~["immumn'eactive and hioactive FMRFamide-like peptides in the nervous system of the horseshoe crab. Limulus polyphemus. PEPTIDES 5(3) 585-592, 1984.--FMRFamide immunoreactivity was detected in all regions of the Limulus nervous system, including the brain (6.5__0.6 pg FMRFamide/mg), cardiac ganglion (2.06-+0.67 pg FMRFamide/mg), and ventral nerve cord (5.8±0.7 pg FMRFamide/mg). The distribution of immunoreactive FMRFarnide firFMRFamide) was mapped by immunofluorescence and the distribution corresponded to regional RIA data. A good proportion of the CNS and cardiac ganglion neuropile contained irFMRFamide, and fluorescent cell bodies were observed in several areas. High performance liquid chromatography (HPLC) was employed to separate and characterize the FMRFamide-like peptides from extracts of Limulus brains. HPLC fractions were analyzed using coincidental radioim- munoassay and bioassay (the radula protractor muscle of Busycon contrarium). There appear to be at least three FMRFamide-like peptides in the Limulus brain, including one similar to clam FMRFamide. FMRFamide acts on Limulus heart in a biphasic manner at relatively high concentrations (10 -5 M), but has no effect on the activity of the isolated ventral nerve cord. These data suggest that in Limuhts FMRFamide-like peptides are acting as neurotransmitters, or neuromodula- lots. Horseshoe crab FMRFamide immunoreactivity Central nervous system FMRFamide was one of the first neuropeptides to be isolated from an invertebrate, the sunray venus clam Macrocallista ~imbosa [24]. During the next 6 years FMRFamide-like im- munoreactive material (irFMRFamide) and FMRFamide- like bioactive peptides have been identified in numerous other species, including another mollusc (Helix aspersa, [23]). several lower vertebrates [3,6], and mammals [4.6, 14, 19. 27]. To our knowledge this report is the first case in which FMRFamide-like peptides have been detected in a marine arthropod. It has been suggested that clam FMRFamide is only one member of a more extensive peptide family [10]. Present members of this family may include one of the-Helix FMRFamide peptides (p-Glu-Asn-Phe-Ile-Arg-Phe-NH~, [23]). CCK. c~-MSH, pancreatic polypeptide (PP), several opioid peptides such as MEAP (YGGFMRF), and others ~hose sequence has yet to be determined [10.19]. The question of how peptides and their receptors have evolved is very interesting, but peptide phylogeny is poorly understood at the present time due to the lack of compara- tive data. Although immunohistochemical techniques have allowed us to identify irFMRFamide in a number of species, it is difficult to draw any conclusions about the phylogenetic relationships between these peptides because we only know the amino acid sequence of a few. Greenberg et al. [l l] have suggested that many of the FMRFamide-like peptides could have evolved from a common ancestor with the sequence Tyr-Gly-Gly-Phe-Met-Arg-Phe-NH.,. If such a peptide ever existed, it may still be present in some species that have changed very little during the last 400-500 million years. The horseshoe crab, Limulus polyphemus, is therefore one of several species worth examining in this regard. In this paper, we present preliminary data from an ongoing study designed to identify and sequence FMRFamide-like peptides in 585