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Immunohistochemical and physiological studies were carried out on Nereis (Hediste) diversicolor OF Müller in order to obtain evidence concerning the neuroendocrine control of polychaete osmoregulation. The occurrence in this animal of peptides immunologically related to mammalian angiotensin II and I (AII and AI) and oxytocin (OT) was demonstrated in the brain and the ventral nerve cord (VNC) perikarya and nerve fibres as well as in a few peripheral structures (peripheral nerves, epithelial cells, nuchal organ, intestine and nephridia). The exact localization of immunoreactive cells was achieved by serial sections of brain and ventral nerve cord followed by a three-dimensional reconstruction of brain ganglionic nuclei using the CATIA ('Conception Assistée Tridimensionnelle Inter Active') Dassault system program. Injections of polyclonal antisera against AII or OT provoked a partial inhibition of the increase in body weight in Nereis exposed to hypo-osmotic medium. The effect of a-AII seemed more pronounced than that of a-OT. In a subsequent test, injections of synthetic AII and AII-amide (peptide recently isolated from an achaete (Salzet et al (1995) J Biol Chem 270, 1575-1582) enhanced the increase in body weight and, therefore, strengthened the hypothesis of the neuroendocrine control of Nereis osmoregulation. The antidiuretic effect of both synthetic peptides in this study was indicative of the exact role of Nereis endogenous molecule(s). AII was less potent than its amidated form. If AI-like can easily be struck off the list of putative endogenous osmoregulatory factors, the role of OT-like substance in Nereis osmoregulation, which is partially demonstrated in this study, needs to be clarified by further physiological experiments using injection of synthetic peptide(s) or endogenous substance(s). All these results are discussed and compared to those recently obtained in an achaete annelid (Salzet et al (1993) Brain Res 631, 247-255; Salzet et al (1993) Brain Res 601, 173-184; Salzet et al (1995) J Biol Chem 270, 1575-1582.

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The primary sequence of the low-molecular-mass cadmium-binding protein metalloprotein II of Nereis diversicolor (Hediste diversicolor, recent denomination) has been determined. This protein is composed of 119 amino acids and has 80.8% identity with the N. diversicolor myohemerythrin [Takagi, T. & Cox, J. A. (1991) FEBS Lett. 285, 25-27]. The fact that iron, which normally binds to myohemerythrin, is not found to be associated with the cadmium-binding protein metalloprotein II in cadmium-exposed animals could be the result of the complete abolition of the iron-binding capacity of the protein due to the binding of cadmium.

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Total mRNA, extracted from brain of the marine worm, Nereis diversicolor (Annelida, Polychaeta), was translated either in vitro using a rabbit reticulocyte lysate or in ovo (Xenopus laevis oocyte). The synthesized polypeptides were analyzed by electrophoresis and Western blotting techniques using polyclonal antisera raised against three peptides: sodium influx stimulating peptide (SISP) sequences 10-19 and 67-76 and a monoclonal antibody raised against purified native SISP (1-77) of Lymnaea stagnalis. Among the products translated in vitro, three polypeptides of 80, 72, and 64 kDa were recognized by the anti-SISP (10-19) polyclonal antiserum and by the monoclonal antiserum, but not by anti-SISP (67-76). Some of the in ovo translated products showed almost identical immunoreactivity to both the anti-SISP (10-19) and the monoclonal antibody. These polypeptides have molecular masses of 80, 72, and 43 kDa. No polypeptides were recognized by anti-SISP (67-76). Western blotting analysis of brain extracts revealed a number of proteins that reacted with the antiserum raised against SISP (10-19) and the monoclonal antiserum. Several perikarya of brain ganglionic nuclei and ventral nerve cord were immunoreactive to anti-SISP (10-19). The monoclonal antiserum gave similar results, although with a less intense immunoreaction. The infracerebral region was also stained, suggesting that the immunoreactive material is released as a true neurohormone into the hemolymph. The largest polypeptides, in particular those translated from brain mRNA, could be neuropeptide precursors containing a SISP-related sequence.

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1. One Cd-binding peak was detected after gel filtration chromatography on Sephadex G75 in an extract from Allolobophora caliginosa contaminated with Cd. 2. Two subsequent cation-exchange chromatographies allowed the isolation of a Cd-binding protein which was called Cd-BP14. This protein is a monomer with a molecular weight of 14 kDa and has an isoelectric point of 6.5. 3. Amino acid analysis showed the presence of a high level of aromatic amino acids and a lack of cysteine. 4. On the basis of these results we conclude that Cd-BP14 is different from metallothioneins described in mammals or other invertebrates.

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Immunocytochemical studies have shown that peptides like Phe-Met-Arg-Phe-NH2 (FMRFamide) are widely distributed throughout the nervous system of three Nereidae. In Nereis diversicolor we have isolated these peptides from an extract of total worms by affinity chromatography and two steps of reversed-phase high-performance liquid chromatography. The sequences of the purified peptides have been determined by amino acid sequencing and on the basis of their reactivity with an anti-FMRFamide serum specific for the determinant Arg-Phe-NH2. Two primary structures have been established: Phe-Thr-Arg-Phe-NH2 (FTRFamide) and PHe-Met-Arg-Phe-NH2 (FMRFamide). Furthermore a methionine sulfoxide derivative of the FMRFamide has been identified. We have synthesized the FTRFamide peptide and in all cases, the native peptides were indistinguishable from the synthetic counterparts. The structure of the two native peptides and of the methionyl sulfoxide derivate have been confirmed by fast-atom-bombardment and tandem mass spectrometry.

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The determination of the first 33 amino acids of the Cd-binding-protein (MP II) of Nereis diversicolor (Annelida, Polychaeta) shows a homology of 79 and 61% with 2 respiratory proteins of sipunculids, respectively the myohemerythrin and the hemerythrin. The positive reaction obtained by immunocytochemistry over the hemerythrocytes of Sipunculus nudus using antibodies raised against MP II and the presence of iron on the MP II reinforce this similarity.

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Total mRNA extracted from the brain of a marine worm, Nereis diversicolor, were in vitro translated using 2 cell-free systems: rabbit reticulocyte lysate and wheat germ extract. Among numerous products newly translated in both systems, only one class of 70 kDa polypeptides immunoprecipitated when we used a mixture of 3 well defined antibodies raised against known sequences of the human prepro-CCK. At the cellular level, using immunocytochemistry techniques, strong and moderate immunoreactivities were seen in perikarya located in various ganglionic nuclei of the worm brain. Immunoreactive nerve fibres were visible in the neuropile but not in the infracerebral region, a neurohemal area. Immunoreactions also appeared on perikarya located in the anterior and medial groups of the ventral nerve cord. Furthermore, immunolabeled cells were observed in the midgut. Interestingly, several co-localizations of materials immunologically related to human prepro-CCK and CCK/gastrin were observed in the brain and the ventral nerve cord both in perikarya and in nerve fibres. We propose that, in Nereis a polypeptide (molecular mass 70 kDa) is the large precursor of molecules related to those of the CCK/gastrin peptide family.

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The presence in the marine worm Nereis diversicolor of a low molecular mass protein with the capacity to bind cadmium has been previously demonstrated. Poly(A)(+)-mRNA were extracted from coelomocytes of Nereis diversicolor and were translated either in vitro, using a rabbit reticulocyte lysate, or in vivo into Xenopus laevis oocytes. Analysis of synthesized polypeptides by enzyme-linked immunosorbent assay (ELISA) and by Western blotting, using a specific monoclonal anti-MP II antibody, showed that this metalloprotein was translated both in in vitro and in vivo translation systems, with an apparent molecular mass of 11-13 kDa. Two other products, with 26.5 and 28 kDa molecular mass, cross-reacted with the monoclonal anti-MP II antibodies. The present work confirms that coelomocytes are sites of important synthesis of MP II-mRNA.

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Comparison of the localization of immunoreactivities which were obtained with antibodies to ?-endorphin, ?-neoendorphin, Leu- and met-enk and dynorphin with those obtained earlier using antisera to ?-endorphin, somatostatin, CRF, GRF, ?-MSH, CCK/gastrin, VIP and substance P (Dhainaut-Courtois et al., 1985a,b) show the particular status of opiate-like substances in Nereis. Indeed, immunoreactivities for vertebrate opiates were observed quite exclusively in the brain, on paraffin embedded as well as on frozen tissue sections. Furthermore, immunoreactive neurones for dynorphin occur only in light green cells (C II cells) located in nuclei 20; axons may be followed to the infracerebral region (ICR), a neurohemal area where immunoreactive nerve fibres for Leu-enk and met-enk are also present. The question to be answered now is whether the compounds immunologically related to vertebrate opiates are authentic Nereis neurohormones released in the blood vessel or the coelom or if they act only as neuromodulators at the ICR level by controlling the activity of classical neurosecretory cells or infracerebral cells. At present, a coexistence in a single neurone of an uncharacterized Nereis neurohórmone and an opiate-like substance may also be postulated. In this study, colocalization of materials immunologically related to CCK/gastrin and GRF or CCK/gastrin and 5-HT were also demonstrated. In addition to this, the use of antibody to 5-HT allowed us to confirm previous results which were obtained with radioautography and histofluorescence methods (Dhainaut-Courtois et al. 1979a,b) and to specify the distribution of this biogenic amine in the whole CNS and in the peripheral nervous system.

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Numerous immunochemical and immunohistochemical studies have shown a wide distribution of several families of neuropeptides in invertebrates as well as vertebrates. There are relatively few data available for Annelida: Polychaeta. Therefore, we undertook an immunohistochemical investigation in the marine worm Nereis. Among the vertebrate type antibodies tested, those against met-enkephalin, LH-RH, vasopressin, oxytocin and ACTH had negative or only very slight effects. Slight to moderate reactions were obtained for VIP, SRIF, CRF, GRF, and leu-enkephalin. Moderate to very strong responses were found with anti-CCK/gastrin, -substance P, and -beta-MSH sera. Immunopositive reactions were usually observed in the entire CNS (except, until now, in neurosecretory cells, type II, in nuclei 20, and in nerve fibres located in the infracerebral neurohemal area). The immunoreactivity was, however, more or less abundant according to different CNS regions. For example, it appeared that the immunostaining for CRF is more important in the VNC while the leu-enkephalin family is more abundant in the brain (particularly in fuchsinophilic neurosecretory cells, type I, in nuclei 20). Moreover, several vertebrate type peptides (such as CRF/GRF and CCK/gastrin) may coexist in a single neurone. Several antisera may elicit a positive reaction in some specific area (for example, substance P in the nuchal organ; SRIF in oocytes; CCK/gastrin in the gastrointestinal tract). Nothing is known about the role of the different substances immunologically detected in Nereis. It is suggested that CCK/gastrin-, beta-MSH- and substance P-like materials transmit external stimuli to neurosecretory centres located in the caudal part of the brain.

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Perikarya and nerve fibers containing a substance immunologically related to CCK-8 were detected in the nervous system of Nereis, a marine annelid worm. The most noteworthy immunostaining was seen in cell bodies, localized at the periphery of the brain, within nuclei 5, 6, 7, 9, 10, 13, 14, 15, 17, 20, 23-24. Immunoreactive fibers were also found in the neuropile without any particular grouping. Numerous other "positive" perikarya occur in the medioventral portion of the ventral nerve cord, and in the ventral and dorsal parts of the suboesophageal ganglion. In addition to the cell bodies in the cerebral external layer, immunoreactive axons were abundantly observed in the connectives between the ganglia. Moreover, our results demonstrate CCK-like staining in neurons showing variations in size and shape, and in affinity for paraldehyde fuchsin. The present results support the hypothesis that this peptide may exert a role as neurotransmitter or neuromodulator in annelids.

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This minireview of aminergic neurosecretions of invertebrates deals with technics of detection and localization of these substances and considers their mode of action at cellular or organ level. A comparison with vertebrates is given. Some examples are analyzed as models. It is concluded that besides a phylogenetic interest, the studies done on invertebrates constitute an important contribution to the understanding of neurobiological mechanisms.

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This study provides details concerning the localization and role of 5-HT and DA in Schistosoma: 1) 3H-5HT is incorporated selectively by the nerve fibers containing dense granules measuring 820 +/- 20 A (type 2 granules), combined at times with other structures; the presence of "cold" dopamine in the environment does not alter the incorporation; 2) 3H-DA is also incorporated in vitro by the nerve fibers and occasionally by the muscle fibers of the adult Schistosoma; 3) neither the intraovular miracidium, the first and second generation sporocystes, nor the intrasporocystic cercariae appear to incorporate the 3H-5-HT or the 3H-DA, under our working conditions. The two amines are, however, incorporated by the hepatopancreas of Biomphalaria glabrata and the 3H-5-HT produced marking on certain neuromuscular zones of the tentacle.

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High specific and nonspecific cholinesterase activities have been demonstrated in the whole nervous system of Nereis. These might reveal the existence of a cholinergic nervous system whose control would be exerted in different sites (central nervous centers, muscular junctions...).

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