/ 2x10 2281 Mp 27 Tuesday Mar 18 05:47 AM EL–PEP (v.18#1) 2281 27 Peptides, Vol. 18, No. 1, pp. 27–40, 1997 Copyright  1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 / .00 PII S0196-9781( 96 ) 00245-8 Localization and Neurohemal Release of FMRFamide-Related Peptides in the Stick Insect Carausius morosus 1 SHARON MIKSYS, ANGELA B. LANGE, IAN ORCHARD 2 AND VICTOR WONG Department of Zoology, University of Toronto, Toronto, Ontario M5S 3G5 Canada Received 1 May 1996; Accepted 5 August 1996 MIKSYS, S., A.B. LANGE, I. ORCHARD AND V. WONG. Localization and neurohemal release of FMRFamide-related peptides in the stick insect Carausius morosus. PEPTIDES 18 (1) 27–40, 1997.—FMRFamide-like immunoreactivity was localized im- munohistochemically in the central and stomatogastric nervous systems, visceral tissues, and the neurohemal corpora cardiaca, transverse, and segmental nerves. Each of these neurohemal areas contains one morphologically distinct type of immunoreactive neurosecretory granule. The hemolymph level of FMRFamide-like peptides, quantified by RIA, is higher in animals sampled 2 h into the dark cycle, relative to those sampled at mid-light cycle or 9 h into the dark cycle. High potassium depolarization evokes the calcium-dependent release of FMRFamide-like peptides from neurohemal areas in vitro and HPLC fractionation of hemolymph, corpora cardiaca, and their bathing medium suggests that these organs contribute a single peptide to the FMRFamide-related peptides circulating in the hemolymph of active animals.  1997 Elsevier Science Inc. FMRFamide Neuropeptide Insect Immunohistochemistry Immunogold labeling Neurohemal release 1 Taken in part from a paper presented at a satellite symposium on Insect Neuropeptides during the Seventh Annual Neuropeptide Conference, February 1–6, 1996, Breckenridge, CO. 2 Requests for reprints should be addressed to Ian Orchard, Department of Zoology, 25 Harbord Street, University of Toronto, Toronto, Ontario, Canada, M5S 3G5. THE tetrapeptide FMRFamide ( Phe-Met-Arg-Phe-amide ) was first isolated and sequenced from the clam Macrocallista nim- bosa (29) and is now known to belong to a growing family of structurally related neuropeptides, the FMRFamide-related pep- tides (FaRPs). These peptides are found throughout the animal kingdom (30), and immunohistochemical studies have shown FMRFamide-like immunoreactivity ( FLI ) to be widespread in the nervous system of insects (8,21,22,25,33,37). Very little is known of their function, although they have been implicated in a broad range of physiological processes such as those associated with feeding (7,10) and modulation of contraction of visceral (14,28) and skeletal muscle (11,39), and host-seeking behavior in the mosquito (3). In insects, FMRFamide-like immunoreactivity is often found associated with neurosecretory cells and neurohemal areas, and FaRPs are thought to be released into the hemolymph (4,9,20,21,33,35). However, except for the control of salivary glands in Calliphora vomitoria (7) and in relation to feeding in Rhodnius prolixus (10), there is little direct evidence for FaRPs acting as neurohormones. The stick insect Carausius morosus has a simple and easily accessible nervous system. The ganglia of the ventral nerve cord have well-developed transverse nerves on which are found prom- inent neurohemal organs known as perisympathetic organs ( 31 ) . The ultrastructure of these organs and the peripheral nerves in the abdominal segments has been well documented ( 13 ) and our preliminary investigations showed FLI in these areas. This article reports an extensive investigation of FLI in the nervous system and visceral tissues of C. morosus , and the stimulation of release of FaRPs from neurohemal areas in vitro. It is hoped that C. morosus will provide a useful system in which to study the action of endogenous FaRP(s) on their target tissues. METHOD Animals Adult female C. morosus were used from a parthenogenic colony maintained at room temperature, high relative humidity under a 12L:12D regime, and fed a variety of green foliage in- cluding varieties of ivy and Ficus. C. morosus is nocturnal, and animals were taken during the day when they are inactive, except for the hemolymph measurements (see later). Immunohistochemistry All dissections were performed under physiological saline ( 15 m M NaCl, 18 m M KCl, 50 m M MgCl 2 , 7.5 m M CaCl 2 ,2m M Tris buffer, 184 m M glucose, pH 6.6). Tissues were fixed 1.5 h