T. Oumi

Annetocin, an annelid oxytocin‐related peptide, induces egg‐laying behavior in the earthworm, Eisenia foetida

Annetocin, an oxytocin-related peptide which we isolated from the earthworm Eisenia foetida, induced a series of egg-laying-related behaviors in the earthworms. These stereotyped behaviors consisted of well-defined rotatory movements, characteristic body-shape changes, and mucous secretion from the clitellum. Each of these behaviors is known to be associated with formation of the cocoon in which eggs are deposited. In fact, some of the earthworms injected with annetocin (> 5 nmol) laid eggs. Such egg-laying-related behaviors except for oviposition were also induced by oxytocin, but not by Arg-vasopressin and some other bioactive peptides isolated from E. foetida. Furthermore, annetocin also induced these egg-laying-like behaviors in the leech Whitmania pigra, but not in the polychaete Perinereis vancaurica. These results suggest that annetocin plays some key role in triggering stereotyped egg-laying behaviors in terrestrial or fresh-water annelids that have the clitella.

Possible functions of oxytocin/vasopressin-superfamily peptides in annelids with special reference to reproduction and osmoregulation

Annetocin is an earthworm oxytocin-related peptide that we previously isolated from the whole body of a lumbricid earthworm Eisenia foetida. We have reported that annetocin induces egg-laying-like behaviors in E. foetida and a gnathobdellid leech, Whitmania pigra, when it is injected into the respective animals. The present study was undertaken to probe physiological functions of invertebrate oxytocin-vasopressin-superfamily peptides with special reference to reproductive and osmoregulatory events in which vertebrate peptides of this superfamily are involved. Annetocin, Lys-conopressin (a leech vasopressin-related peptide) and two analog peptides, [Tyr(3)]-annetocin ((3)Y-annetocin) and [Phe(3)]-annetocin ((3)F-annetocin), were compared for their activities to induce egg-laying-like behavior and to change body weight as a measure of water balance in the leech W. pigra. Injection of annetocin, Lys-conopressin, and (3)F-annetocin caused both egg-laying-like behavior and reduction of body weight in the animals, but (3)Y-annetocin induced neither. Furthermore, leeches in the non-breeding season responded to peptides less conspicuously than those in the breeding season. Such a concomitant induction of egg-laying-like behavior and body-weight reduction suggests that these two phenomena are unitary and might be accounted for by the fact that egg-laying in leeches and earthworms is accompanied by secretion of a large quantity of mucus, which should significantly contribute to body-weight loss. J. Exp. Zool. 284:401-406, 1999.

A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida

A novel bioactive tetradecapeptide, GFKDGAADRISHGFamide, was isolated from the gut of the oligochaete annelid, Eisenia foetida, using the isolated anterior gut (crop-gizzard) as a bioassay system. A highly homologous peptide, GFRDGSADRISHGFamide, was also purified from the whole body of another species of earthworm, Pheretima vittata. These peptides were termed Eisenia tetradecapeptide (ETP) and Pheretima tetradecapeptide (PTP), respectively. Both the peptides showed a potent excitatory action on spontaneous contractions of the anterior gut with a threshold as low as 10(-10)-10(-9) M. These peptides were significantly homologous to molluscan tetradecapeptides and, to a lesser extent, to arthropodan tridecapeptides that have been reported to date. All these peptides seem to be evolutionally related to each other.

THE LEECH EXCITATORY PEPTIDE, A MEMBER OF THE GGNG PEPTIDE FAMILY : ISOLATION AND COMPARISON WITH THE EARTHWORM GGNG PEPTIDES

A member of the GGNG peptide family was isolated from Hirudo nipponia (leech). GGNG peptides had only been isolated previously from earthworms. The C‐terminus structure of the leech peptide, LEP (leech excitatory peptide), was –Gly–Gly–Asn–amide, while that of the earthworm peptides, EEP (earthworm excitatory peptide), was –Gly–Gly–Asn–Gly. LEP exerted 1000‐fold more potent activities on leech gut than did EEP‐2. On the other hand, EEP‐2 was 1000‐fold more potent than LEP on the crop‐gizzard of the earthworm. Analog peptides of LEP and EEP‐2 were synthesized, and the myoactive potency of each analog on the leech and earthworm tissues was compared.

Inhibitory pentapeptides isolated from the gut of the earthworm, Eisenia foetida☆

Abstract The isolated anterior gut (crop-gizzard) of the oligochaete annelid, Eisenia foetida, was used as a bioassay system to identify the bioactive peptides that regulate rhythmic spontaneous contractions of the gut. Three pentapeptides were purified from the extract of the gut (crop, gizzard and intestine) of the earthworm. These peptides showed inhibitory actions on gut motility and were termed Eisenia inhibitory pentapeptides (EIPPs). EIPPs had the most remarkable effect on the crop among the various parts of the gut with a threshold concentration as low as 10−10–10−9 M. From the pharmacological results, the inhibitory actions of EIPPs on the gut motility seem to be different from those of FMRFamide. The amino acid sequences of EIPPs were very similar to certain molluscan and echiuroid peptides previously identified. This is the first isolation of annelid peptides inhibiting gut motility except for FMRFamide.

Immunohistochemical Localization of Annetocin, an Earthworm Oxytocin-Related Peptide, and Identification and Ultrastructural Characteristics of the Annetocin-Secretory Cells in the Oligochaete Earthworm Eisenia foetida

Abstract Annetocin is an egg-laying-inducing oxytocin-related peptide which we have previously isolated from the earthworm, Eisenia foetida. Here we report the results of immunohistochemical and ultrastructural studies on annetocin-secretory cells in the earthworm. Annetocin-immunoreactive (IR) cell-somata were located mainly at the ventro-lateral side of the subesophageal ganglion. Only four annetocin-IR cells were seen in the cerebral ganglion. Some annetocin-IR cells displayed unipolar-like structure with a process directing to the core region (the neuropile) of the ganglion. Annetocin-IR fibers were also observed in the neuropile of the ventral ganglia and the ventral nerve cord between the 4th and the 30th segments including the clitellum, but not in the posterior segments (31-55th). The number of annetocin-IR fibers decreased from the 4th to the 30th segment. The annetocin-secretory cells were identified by the immunogold staining, and filled with gold-labeled vesicles, 200-250 nm in diameter, which included moderately electron dense material. The annetocin-secretory cells possessed a euchromatic nucleus, well-developed rough endoplasmic reticulum and Golgi apparatus. Some of the annetocin-secretory cells were found to form a neurohemal-like structure, where somata or fibers with loose glial investment came in contact with the coelomic space at the ventral side of the subesophageal ganglion. The results suggest that annetocin is a neuropeptide produced and secreted by the neuron in the cerebral and subesophageal ganglia of the earthworm.

Immunochemical demonstration of Eisenia tetradecapeptide, a bioactive peptide isolated from the gut of the earthworm Eisenia foetida, in tissues of the earthworm

Abstract.The quantity and localization of Eisenia tetradecapeptide which was isolated from the gut of the earthworm Eisenia foetida were examined in tissues of the same species by enzyme-linked immunosorbent assay and immunohistochemistry. Analysis by enzyme-linked immunosorbent assay showed that Eisenia-tetradecapeptide-like immunoreactivity was present in both the central nervous system (cerebral ganglion, subesophageal ganglion, ventral ganglia, and ventral nerve cord) and the gut (esophagus, crop, gizzard, and intestine). The central nervous system contained a higher amount of Eisenia-tetradecapeptide-like immunoreactivity (1.3 pmol/mg wet weight) than the gut (0.2–0.6 pmol/mg wet weight). Eisenia-tetradecapeptide-like immunoreactivity was scarcely detected in the body-wall muscle, nephridia, and sexual organs (testis, ovary, seminal vesicle, and ovisac). Immunohistochemical analysis demonstrated that intense Eisenia-tetradecapeptide-like immunopositive cells and nerve fibers were present in the central nervous system. Immunoreactivity was found in the epithelial cells lining the esophagus and in the submucous plexus in various parts of the gut. Thus, the present study suggests that Eisenia tetradecapeptide is a neuropeptide and/or peptide hormone present in both the central nervous system and the gut of the earthworm and that its role involves the regulation of gut motility.

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization.

Abstract We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10–8 M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.

Characterization of binding of the annelidan myoactive peptides, GGNG peptides, to tissues of the earthworm, Eisenia foetida

The GGNG peptides are myoactive peptides which we have isolated from several species of annelids. Two types of peptides have been isolated as GGNG peptides: earthworm excitatory peptides (EEP) and leech excitatory peptide (LEP). The EEP and LEP are highly homologous with each other but act specifically on earthworms and leeches, respectively. To approach the mechanism of the specific action, we attempted to characterize the receptors for EEP in the earthworm Eisenia foetida, from which EEP have been isolated, by a radioreceptor assay. To prepare a radiolabeled ligand, we first coupled EEP to Bolton-Hunter reagent and then labeled it with 125I ([125I] BH-EEP). Binding of the [125I] BH-EEP to the membrane preparation of the earthworm tissue (crop-gizzard) was reversible, saturable, and specific with a Kd of 4.9 ± 1.2 nM and a Bmax of 15.9 ± 2.0 fmol/mg wet tissue. For the earthworm membrane preparation, EEP showed a much greater potency than LEP in displacing [125I] BH-EEP, suggesting that the earthworm tissues contain EEP-specific receptors. This result would account for the specific biological activity of EEP and LEP on the earthworm and leech tissues, respectively. The binding capacity was high in the anterior part of digestive tract including the esophagus, crop and gizzard, and the nephridia. From these results, together with the observed myotropic activity of EEP on gut tissues, it is assumed that EEP is physiologically involved in regulation of the gut motility in the earthworm E. foetida.J. Exp. Zool. 279:562–570, 1997.© 1997 Wiley-Liss, Inc.