Occurrence of tetrodotoxin in the flatworm Planocera multitentaculata.

The paralytic toxin which occurs in a flatworm Planocera multitentaculata was partially purified by column chromatography using Bio-Gel P-2 and Bio-Rex 70 (H+ form). Thin-layer chromatographic, electrophoretic and gas chromatography-mass spectrometric analyses demonstrated that the flatworm toxin is tetrodotoxin.

Tetrodotoxin and related substances in a ribbon worm Cephalothrix linearis (Nemertean).

A ribbon worm Cephalothrix linearis (Nemertean) showed a high toxicity, of up to 22,000 MU/g proboscis and 13,600 MU/g body, in terms of tetrodotoxin. This organism secreted the toxin from the skin when wiped with gauze. The toxin was partially purified from the secretion as well as the wiped body by ultrafiltration and Bio-Gel P-2 column chromatography. Thin-layer chromatographic, electrophoretic, high performance liquid chromatographic, and gas chromatography-mass spectrometric analyses showed that the secreted toxin was composed almost exclusively of a tetrodonic acidic-like substance, whereas the remaining toxin in the wiped body consisted of the said substance and tetrodotoxin.

Hoplonemertine worms -- a new source of pyridine neurotoxins.

Two pyridine bases were isolated from the marine hoplonemertine Amphiporus angulatus (Fabricius) and identified by mass and PMR-spectroscopy as 2, 3'-bipyridyl and 3, 2'; 3', 2"; 4", 3"'-tetrapyridyl (Nemertelline). Nemertelline, the first tetrapyridyl natural product to be reported, shows a structural resemblance to the tobacco constituent nicotelline. The crustacean toxicity of 2, 3' -bipyridyl is very similar to that of nicotine, but its mammalian toxicity is negligible.

Localization, quantification, and characterization of pancreatic polypeptide immunoreactivity in the parasitic flatworm Diclidophora merlangi and its fish host (Merlangius merlangus).

Pancreatic polypeptide (PP) immunoreactivity (IR) has been identified, quantified, and subsequently chemically characterised in the parasitic platyhelminth, Diclidophora merlangi, and its specific teleostean host the whiting, Merlangius merlangus. Immunocytochemistry demonstrated PP-IR throughout the central and peripheral nervous systems of the parasite and in open-type endocrine cells of the gastric mucosa of its host. Radioimmunoassay detected PP-IR in alcoholic extracts of whole parasites (39.2 ng/g) and in extracts of gastrointestinal tract (2.1 ng/g), brain (4.6 ng/g), and pancreas (12 ng/g) of the host. Chromatographic analysis of parasite extracts revealed a single immunoreactive species of PP in both high-performance gel permeation and reverse-phase systems. The molecular size of this peptide was similar to bovine PP standard. In contrast, whiting tissues contained two immunoreactive species of PP in both gel permeation and reverse-phase systems. The major species was similar in size to bovine PP standard and the minor species was smaller, with a molecular size comparable to bovine neurotensin. Reverse-phase HPLC revealed that parasite and host peptides were not identical.

A cytochemical study of the serotoninergic, cholinergic and peptidergic components of the reproductive system in the monogenean parasite, Diclidophora merlangi.

The reproductive system of the monogenean gill parasite, Diclidophora merlangi, was examined for the presence of cholinergic, serotoninergic and peptidergic innervation using cytochemical and immunocytochemical techniques. Cholinesterase activity and 5-hydroxytryptamine immunoreactivity (5-HT-IR) were confined to neural elements of the male reproductive system, being evident in the innervation of the cirrus, whereas only 5-HT was present in nerves and somata of the elongate seminal vesicle. Peptidergic innervation was localised to both the male and female reproductive systems of the worm. Within the female reproductive apparatus pancreatic polypeptide, peptide tyrosine tyrosine, neuropeptide Y, substance P, neurokinin A, eledoisin, FMRFamide and gastrin/cholecystokinin immunoreactive fibres and somata were observed in the oviduct, vitelline reservoir and ovovitelline duct. Intense peptide immunoreactivity was identified in fibres in the wall of the ootype and in a surrounding population (greater than 100) of somata that were situated beyond Mehlis' gland cells and all of which were connected to the ootype wall by fine cytoplasmic connectives. The strategic location of this peptidergic cell population infers its involvement in the egg-forming sequence in this platyhelminth parasite.

Tachykinin immunoreactivity in the parasitic flatworm Diclidophora merlangi and its fish host the whiting (Merlangius merlangus): radioimmunoassay and chromatographic characterisation using region-specific substance P and neurokinin A antisera.

1. Tachykinin immunoreactivity has been quantified and chemically characterised in extracts of the monogenean parasite, Diclidophora merlangi and its fish host, Merlangius merlangus, by means of four tachykinin radioimmunoassays interfaced with gel permeation chromatography and reverse-phase HPLC. 2. Of the two tachykinins identified in parasite tissue, one was SP-like and the other was NKA-like, although neither was identical to previously identified tachykinins. 3. Three tachykinins were identified in extracts of whiting GI tract, one of which was a neuropeptide and also occurred in whiting brain. 4. The parasite and fish tachykinins had different molecular weights and elution profiles in HPLC analyses, and were therefore chemically distinct.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

Purification and characterization of a new family of polypeptide neurotoxins from the heteronemertine Cerebratulus lacteus (Leidy).

Mucus secreted from the integument of the marine heteronemertine Cerebratulus lacteus (Leidy) contains two major types of polypeptide neurotoxin: the A toxins (11,000 daltons) which are lethal to a variety of animal species including mammals, and the B toxins (6,000 daltons) which appear to be selectively toxic for crustaceans. Both types of toxin were readily obtained from live worms by stimulation and collection of mucus with 1% acetic acid followed by batch adsorption of the basic polypeptide fraction upon CM-cellulose. Separate A and B toxin fractions were obtained by gel chromatography. Four toxins were purified from the B toxin fraction by CM-cellulose gradient elution chromatography. Each consists of a single polypeptide chain with NH2-terminal alanine and three (B-I) or four (B-II, B-III, B-IV) intrachain disulfide bonds. All four B toxins contain large proportions of lysine and exhibit identical gel electrophoretic mobilities at neutral pH. The B toxins lack cysteine and reducing sugar moieties. Toxin B-I differs considerably from the others in its amino acid composition and its shorter chain length (49 instead of 53 or 54 residues). The other three toxins have similar compositions, but lack proline, methionine, and phenylalanine. All four polypeptides show crustacean-selective toxicities which vary over a 30-fold range; B-II possesses the highest toxicity. As a working hypothesis it is suggested that the Cerebratulus B toxins are isotoxins which act via a common receptor present in crustacean axon membranes.