Neuromuscular system of the causative agent of dicrocoeliosis, Dicrocoelium lanceatum. II. Neuropeptide FMRFamide immunoreactivity in nervous system.

The parasitic flatworm, trematoda Dicrocoelium lanceatum or lancet fluke is the causative agent of a widespread parasite disease of grazing ruminants, dicrocoeliosis. The aim of this work is the study of the presence and localization of neuropeptide FMRFamide immunoreactive elements in the nervous system of D. lanceatum using immunocytochemical technique and confocal scanning laser microscopy. For the first time the data on the presence and distribution of the FMRFamide-immunopositive components in the central and peripheral departments of the nervous system of D. lanceatum has been obtained. FMRFamidergic neurons and neurites were identified in paired brain ganglia, in the brain commissure, longitudinal nerve cords and connective nerve commissures. The innervation of the oral and ventral suckers by peptidergic nerve structures was revealed. The distal part of the reproductive system is innervated by FMRFamide immunopositive neurites. The data obtained suggest that the neuropeptides of FMRFamide family can be involved in the regulation of functions of the attachment organs and the reproductive system in D. lanceatum. The study of neurotransmitters and their functions in flatworms expand our knowledge on the structure and function of the nervous system of trematodes of various taxonomic groups. The results obtained on the morphological organization of D. lanceatum nervous system support the exploitation of the FMRFamidergic components as an anthelmintic target.

Musculature and neurotransmitters of internal organs of trematodes (the digestive, reproductive and excretory systems).

The article analyzes the results on the presence and organization of the muscle elements in the visceral organs of parasitic flatworms, trematodes, as well as their innervations. The different regions of the digestive, reproductive and excretory systems of trematodes contain circular, longitudinal and diagonal muscle fibers. The results of immunocytochemical investigations and confocal scanning laser microscopy show the presence of serotonin and FMRFamide-like immunoreactivity in the nervous system elements in various parts of the digestive, reproductive and excretory systems of trematodes. The data suggest that serotonergic and FMRFamide-immunopositive components of parasite's nervous system are involved in the regulation of the muscle activity of the digestive, reproductive and excretory systems. Comparative analysis of the results presented for trematodes from different taxonomic groups indicates that the organization of muscle elements in the visceral organs in trematodes and their innervation by serotonergic and peptidergic components are highly conserved.

Serotonin Signalling in Flatworms: An Immunocytochemical Localisation of 5-HT7 Type of Serotonin Receptors in Opisthorchis felineus and Hymenolepis diminuta.

The study is dedicated to the investigation of serotonin (5-hydroxytryptamine, 5-HT) and 5-HT7 type serotonin receptor of localisation in larvae of two parasitic flatworms Opisthorchis felineus (Rivolta, 1884) Blanchard, 1895 and Hymenolepis diminuta Rudolphi, 1819, performed using the immunocytochemical method and confocal laser scanning microscopy (CLSM). Using whole mount preparations and specific antibodies, a microscopic analysis of the spatial distribution of 5-HT7-immunoreactivity(-IR) was revealed in worm tissue. In metacercariae of O. felineus 5-HT7-IR was observed in the main nerve cords and in the head commissure connecting the head ganglia. The presence of 5-HT7-IR was also found in several structures located on the oral sucker. 5-HT7-IR was evident in the round glandular cells scattered throughout the larva body. In cysticercoids of H. diminuta immunostaining to 5-HT7 was found in flame cells of the excretory system. Weak staining to 5-HT7 was observed along the longitudinal and transverse muscle fibres comprising the body wall and musculature of suckers, in thin longitudinal nerve cords and a connective commissure of the central nervous system. Available publications on serotonin action in flatworms and serotonin receptors identification were reviewed. Own results and the published data indicate that the muscular structures of flatworms are deeply supplied by 5-HT7-IR elements. It suggests that the 5-HT7 type receptor can mediate the serotonin action in the investigated species and is an important component of the flatworm motor control system. The study of the neurochemical basis of parasitic flatworms can play an important role in the solution of fundamental problems in early development of the nervous system and the evolution of neuronal signalling components.

The neuroactive substances and associated muscle system in Rhipidocotyle campanula (Digenea, Bucephalidae) from the intestine of the pike Esox lucius.

We report about the muscular system and the serotonergic and FMRFamidergic components of the nervous system of the Bucephalidae trematode, Rhipidocotyle campanula, an intestinal parasite of the pike. We use immunocytochemical methods and confocal scanning laser microscopy (CLSM). The musculature is identified by histochemical staining with fluorescently labeled phalloidin. The body wall musculature of R. campanula contains three layers of muscle fibres - the outer thin circular, intermediate longitudinal and inner diagonal muscle fibres running in two opposite directions. The digestive system of R. campanula possess of a well-developed musculature: radial, longitudinal and circular muscle elements are detected in the pharynx, circular and longitudinal muscle filaments seen in the oesophagus, and longitudinal and the circular muscle fibres were found in the intestinal wall. Specific staining indicating the presence of actin muscle filaments occurs in the cirrus sac localized in the posterior body region. The frontal region of anterior attachment organ, the rhynchus, in R. campanula is represented by radial muscle fibres. The posterior part of the rhynchus comprise of radial muscles forming the organ's wall, and several strong longitudinal muscle bundles. Serotonergic and FMRFamidergic structures are detected in the central and peripheral compartments of the nervous system of R. campanula, that is, in the paired brain ganglia, the brain commissure, the longitudinal nerve cords, and connective nerve commissures. The innervations of the rhynchus, pharynx, oesophagus and distal regions of the reproductive system by the serotonergic and FMRFamidergic nervous elements are revealed. We compare our findings obtained on R. campanula with related data for other trematodes.

Some aspects of the immunolocalization of FMRFamide in the nervous system of turbellarians, Polycelis tenuis and Girardia tigrina. Short communication.

The details of the morphology of the nervous system has been investigated in two turbellarian species Polycelis tenuis and Girardia tigrina using confocal laser scanning microscopy and immunostaining to neuropeptide FMRFamide. Abundant FMRFamide immunoreactivity (FMRF-IR) has been observed in central and peripheral nervous systems of both species. Intensive staining has been found in the sensory elements: cells and fibres surrounded the mouth opening, in the fibres enclosed the photoreceptors, triangular auricles in the head region of G. tigrina. The possible function of FMRF-IR neurons in the realization of sensory function in turbellarians is discussed.

Study of planarian stem cell proliferation by means of flow cytometry.

The stem cells in freshwater flatworms (planarian) are called neoblasts. Neoblasts are capable of proliferation and differentiation into every cell type, including the gametes. For the investigation of the mechanisms of stem cells proliferation and differentiation the proper evaluation of changes in the cell cycle of neoblasts in different physiological conditions of planarian is necessary. In the present study the possibility of qualitative and quantitative characteristics of the neoblasts population were investigated using flow cytometry. In the cell suspension prepared from planarian tissue proliferating neoblasts have been observed in heterogenic cell population. Quantitative estimation of the cell cycle related changes of planarian stem cells system have been performed in various physiological conditions (intact and regenerating animals) and under the influence of physical (ionizing radiation) and chemical (melatonin and colchicine) factors. The modified protocol for planarian stem cells isolation proved to be effective and reproducible and can be recommended for flow cytometry analyses of human and animal proliferating cells.

Functions of flatworm neuropeptides NPF, GYIRF and FMRF in course of pharyngeal regeneration of anterior body fragments of planarian, Girardia tigrina.

The development of the new pharynx in anterior body fragments of G. tigrina was followed for 7 days by immunocytochemical (ICC) study using antiserum to neuropeptide F (NPF) and muscle staining with phalloidin. ICC investigation revealed the presence of NPF in pharyngeal nervous system, peripheral nerve plexuses, in central nervous system of intact planarians. NPF-immunoreactive (IR) nerve fibres were found at the site of regeneration surrounding pharyngeal rudiment. Restoration of the pharynx function during regeneration was analysed by appearance of food response in anterior fragments. Stimulating effects of NPF and FMRF on the pharyngeal regeneration has been observed. The data indicates an important role of neuropeptides in morphogenetic processes.

The pattern of serotonin and FMRFamide in cercaria from different taxonomic groups--a preliminary study.

Serotoninergic and FMRFamidergic components of the nervous system were examined in cercaria from different types using immunocytochemical techniques interfaced with confocal scanning laser microscopy. Cercariae from 9 families were studied - Opisthorchis felineus, Parafasciolopsis fasciolaemorpha, Echinochasmus coaxatus, Echinoparyphium aconiatum, Notocotylus attenuatus, Psilotrema tuberculata, Plagiorchis sp., Cyathocotyle bithyniae, Diplostomum chromatophorum. The results show that 5-HT-IR and FMRFamide-IR occur in all types of cercariae, regardless of what morphological, taxonomic and biological group they belong to. Small differences in the patterns of 5-HT-IR and FMRFamide-IR were observed.

Effects of neuropeptide F on regeneration in Girardia tigrina (Platyhelminthes).

The effects of neuropeptide F (NPF; from Moniezia expansa) on the regeneration of Girardia tigrina were studied. The animals were decapitated and incubated in water (control) or NPF. The dynamics of the proliferation of the neoblasts in the developing tissue were studied during the course of regeneration by monitoring the mitotic index (MI). The effects of incubation in FMRFamide and GYIRFamide on the MI were also tested. The course of cephalic regeneration was followed with in vivo computer-assisted morphometry for up to 7 days. The development of the regenerating nervous system and the musculature was visualised by immunostaining with a primary antiserum to the C-terminal decapeptide of NPF (YFAIIGRPRFa) and tetramethylrhodamine-isothiocyanate-conjugated phalloidin, which stains F-actin in muscle filaments. The study showed that NPF had a stimulatory effect on the mitotic activity of the neoblasts. FMRFamide and GYIRFamide did not have this effect. NPF also stimulated the growth of the regenerating head and the growing nervous system and musculature. NPF is postulated to have a morphogenetic action in the regenerating animals.

Fasciola hepatica cathepsin L-like proteases: biology, function, and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.