Intracellular immunohistochemical detection of tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria).

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.

Immunoenzymatic visualization of tetrodotoxin (TTX) in Cephalothrix species (Nemertea: Anopla: Palaeonemertea: Cephalotrichidae) and Planocera reticulata (Platyhelminthes: Turbellaria: Polycladida: Planoceridae).

Tetrodotoxin (TTX) was localized as brown color in different tissues of an undescribed species of the nemertean genus Cephalothrix (phylum Nemertea) and a turbellarian Planocera reticulata (phylum Platyhelminthes) on light microscopy by means of a monoclonal anti-TTX antibody. In the Cephalothrix sp., TTX was recognized in the vesicles apically arranged in the bacillary cells in the epidermis, basal lamina, the granular cells in the proboscis epithelium, rhynchocoel epithelium, and the vesicles in the basal portion of the intestinal wall near the blood vessels and rhynchocoel. The excretory system and the ovum also showed positive reaction of TTX antigen-antibody. On the other hand, the hermaphrodite flatworm P. reticulata exhibited TTX antigen-antibody complex only in their ovum. To our knowledge, this is the first experimental effort on micro-distribution of TTX in invertebrates.

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei).

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.

The effects of FaRPs on the motility of isolated muscle strips from the liver fluke, Fasciola hepatica.

The effects of a range of FMRFamide-related peptides (FaRPs) on isometric contractility were tested using isolated muscle strips from the liver fluke, F. hepatica. The neuropeptides tested were the molluscan FaRPs, FMRFamide and FLRFamide, the turbellarian FaRPs, RYIRFamide and GYIRFamide, the cestode peptides, NPF and GNFFRFamide, and the nematode FaRPs, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-8 (KSAYMRFamide), and PF-4 (KPNFIRFamide). Dose-response experiments were undertaken at a concentration range of 5 nM-5 microM for all of the neuropeptides tested. FMRFamide and AF-8 caused statistically significant increases in the amplitude and frequency of contractions at concentrations of 0.5 microM and 5 microM. FLRFamide and AF-2 also caused significant increases in contraction frequency at concentrations of 0.5 microM and 5 microM, although a significant increase in amplitude of contraction was observed only at a concentration of 5 microM. GYIRFamide increased both amplitude and frequency significantly at concentrations of 50 nM, 0.5 microM and 5 microM. RYIRFamide significantly increased frequency of contractions at concentrations of 0.5 microM and 5 microM, but failed to have a significant effect on contraction amplitude. AF-1 at a concentration of 5 microM increased contraction amplitude, but failed to have an effect on frequency at any of the concentrations used. PF-4 caused a statistically significant increase in both the amplitude and frequency of contractions at a concentration of 5 microM. NPF and GNFFRFamide had no effect on the in vitro motility of F. hepatica over the range of concentrations tested. The results are discussed in the light of possible structure-activity relationships in the FaRPs tested.

Isolation, localization, and bioactivity of the FMRFamide-related neuropeptides GYIRFamide and YIRFamide from the marine turbellarian Bdelloura candida.

Two FMRFamide-related neuropeptides, GYIRFamide and YIRFamide, were isolated from the marine turbellarian Bdelloura candida. The peptides elicited a dose-dependent contraction of isolated turbellarian muscle fibers, and both were more potent than FMRFamide. Structure-activity studies, using a range of analogues of the tetrapeptide amide, indicated that the structure of the endogenous peptides was optimal for peak activity. Immunocytochemistry, using an autologous antiserum, revealed a widespread distribution of peptide immunoreactivity within central and peripheral neurons and their processes. This study indicates an important role for GYIRFamide and YIRFamide in the control of neuromuscular function in turbellarians.

Localisation, quantitation, and characterisation of neuropeptide F- and FMRFamide-immunoreactive peptides in turbellarians and a monogenean: a comparative study.

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a mere sedentary parasitic lifestyle.

RYIRFamide: a turbellarian FMRFamide-related peptide (FaRP).

FMRFamide was isolated originally from neural-tissue extracts of a bivalve mollusc, since when either authentic FMRFamide or a series of structurally-related peptides have been isolated from representative arthropods, annelids and many additional molluscs. However, to date no information exists as to the definitive presence and primary structure of a FaRP in a free-living flatworm. Here, we report the isolation and primary structure of a FaRP from the free-living turbellarian, Artioposthia triangulata, a species from which NPF has been previously structurally-characterised. Unlike molluscs and insects, in which several FaRPs are expressed, only a single member of this family was detected in this turbellarian. The primary structure of this turbellarian FaRP was established as Arg-Tyr-Ile-Arg-Phe-NH2 (RYIRFamide) and the molecular mass as 752.7 Da. These data have established unequivocally that FaRPs occur in the nervous systems of the most phylogenetically-ancient invertebrates which display bilaterally-symmetrical neuronal plans and that authentic FMRFamide is probably not the original member of the family in molecular evolutionary terms.