5-HYDROXYTRYPTAMINE-IMMUNOREACTIVITY IN THE MONOGENEAN PARASITE, ENTOBDELLA-SOLEAE

5-HT-immunoreactivity in Entobdella soleae was found to be extensive throughout both the central and peripheral nervous systems, with the strongest staining occurring in the innervation of the forebody, most notably in the paired cerebral ganglia, pharynx and adhesive pads. In the reproductive system, staining was evident throughout the numerous cell bodies and fibres innervating the musculature of the egg-assembly apparatus. The haptor contained an extensive array of serotoninergic fibres derived from the main longitudinal cords; this array was associated with the haptoral muscles and sclerites, and possibly with the ventral sensory papillae.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Proteomic analysis of the excretory-secretory proteins of the Trichinella spiralis L1 larva, a nematode parasite of skeletal muscle

Trichinella spiralis is an intracellular nematode parasite of mammalian skeletal muscle. Infection of the muscle cell leads to the formation of a host-parasite complex that results in profound alterations to the host cell and a re-alignment of muscle-specific gene expression. The role of parasite excretory-secretory (ES) proteins in mediating these effects is currently unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, a global proteomics approach was used to analyse the ES proteins from T. spiralis muscle larvae. Following 2-DE of ES proteins,MALDI-TOF-MS and LC-MS/MS were used to identify the peptide spots. Specific Trichinella EST databases were assembled and used to analyse the data. Despite the current absence of a Trichinella genome-sequencing project, 43 out of 52 protein spots analysed were identified and included the major secreted glycoproteins. Other novel proteins were identified from matches with sequences in the T. spiralis database. Our results demonstrate the value of proteomics as a tool for the identification of Trichinella ES proteins and in the study of the molecular mechanism underpinning the formation of the host-parasite complex during Trichinella infections.

Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

Neuromusculature of Macrogyrodactylus congolensis, a monogenean skin parasite of the Nile catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry, in conjunction with confocal scanning laser microscopy, were used to describe the neuromusculature of the monogenean skin parasite Macrogyrodactylus congolensis from the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of compactly arranged circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The central nervous system consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. Both central and peripheral nervous systems are bilaterally symmetrical and better developed ventrally than laterally and dorsally. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were examined. Results implicate acetylcholine, FMRFamide-related peptides and serotonin in sensory and motor function. The results were compared with those of Macrogyrodactylus clarii, a gill parasite of the same host fish C. gariepinus.

Neuromusculature of Macrogyrodactylus clarii, a monogenean gill parasite of the Nile catfish Clarias gariepinus in Egypt

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy have been used for the first time to describe the nervous and muscle systems of the viviparous monogenean gill parasite, Macrogyrodactylus clarii. The gross spatial arrangement of muscle and associated cholinergic, peptidergic and aminergic innervations has been examined. The central nervous system (CNS) consists of paired cerebral ganglia from which emanate three pairs of longitudinal ventral, lateral and dorsal nerve cords, connected at intervals by transverse connectives. The CNS is better developed ventrally than dorsally or laterally, and has the strongest reactivity for all neuroactive substances examined. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts have been examined. Results implicate acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function in this monogenean, although confirmatory physiological data are obviously required.

RNA interference in adult Ascaris suum - an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.

Parasite neuropeptide biology:Seeding rational drug target selection?

The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components - putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths.

The extracellular vesicles of the helminth pathogen, Fasciola hepatica: biogenesis pathways and cargo molecules involved in parasite pathogenesis.

Extracellular vesicles (EVs) released by parasites have important roles in establishing and maintaining infection. Analysis of the soluble and vesicular secretions of adult Fasciola hepatica has established a definitive characterisation of the total secretome of this zoonotic parasite. Fasciola secretes at least two sub-populations of EVs that differ according to size, cargo molecules and site of release from the parasite. The larger EVs are released from the specialised cells that line the parasite gastrodermus and contain the zymogen of the 37 kDa cathepsin L peptidase that performs a digestive function. The smaller exosome-like vesicle population originate from multivesicular bodies within the tegumental syncytium and carry many previously described immunomodulatory molecules that could be delivered into host cells. By integrating our proteomics data with recently available transcriptomic datasets we have detailed the pathways involved with EV biogenesis in F. hepatica and propose that the small exosome biogenesis occurs via ESCRT-dependent MVB formation in the tegumental syncytium before being shed from the apical plasma membrane. Furthermore, we found that the molecular machinery required for EV biogenesis is constitutively expressed across the intra-mammalian development stages of the parasite. By contrast, the cargo molecules packaged within the EVs are developmentally regulated, most likely to facilitate the parasites migration through host tissue and to counteract host immune attack.

Review of the genus Bursaphelenchus Fuchs, 1937 (Nematoda, Aphelenchida) with some conclusions on the host-parasite and vector-parasite evolution.

INTRODUCTION AND GOALS: Genus Bursaphelenchus includes several pests of the world importance for the rural economy, the most dangerous are the Bursaphelenchus xylophilus (the pinewood nematode caused decline of the pine trees in south Asia and in one spot area in Europe, Portugal, Peninsula de Setubal) and the Bursaphelenchus cocophilus, causing the decline of coco-palm plantations in Carribean and Latin American regions. The peculiarity of the host-parasite association of the genus that the nematode life cycle includes three trophic components: plant (mostly a tree), insect vector and a fungus. Goals of the presentation is to list all species of the world fauna and all efficient diagnostic characters, then create the identification tool and analyze the similarity of species and possible ways and causes of the host-parasite evolution of the group. RESULTS: Complete list of species with synonymy and a catalogue of all efficient diagnostic characters with their states, selected from papers of the most experienced taxonomists of the genus, are given for the genus Bursaphelenchus. List of known records of Bursaphelenchus species with names of natural vectors and plants and their families is given (for world pests the most important groups of trees and insects are listed). The tabular, traditional and computer-aided keys are presented. Dendrograms of species relationships (UPGMA, standard distance: mean character difference) based on all efficient taxonomic characters and separately on the spicule characters only, are given. Discussion whether the species groups are natural or purely diagnostic ones is based on the relationships dendrograms and the vector and associated plant ranges of Bursaphelenchus species; the xylophilus species group (B. xylophilus, B. abruptus, B. baujardi, B. conicaudatus, B. eroshenkii, B. fraudulentus, B. kolymensis, B. luxuriosae; B. mucronatus), the hunti group (B. hunti, B. seani, B. kevini and B. fungivorus) are probably the natural ones. CONCLUSIONS: The parasitic nematode association includes three trophic components: plant, insect vector and fungus. The initial insect-plant complex Scolytidae-Pinaceae is changeable and only in rare occasions the change of the preferred vector to Cerambycidae (the xylophilus group), Hymenoptera (the hunti group) led to formation of the natural species-groups. From the analysis it is clear that although the vector range is changeable it is comparatively more important for the evolution of the genus Bursaphelenchus than associations with plants at the family level. Data on the fungi species (3rd component in natural Bursaphelenchus associations) are insufficient for the detailed comparative analysis.