Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.

Gross anatomy of the muscle systems and associated innervation of Apatemon cobitidis proterorhini metacercaria (Trematoda: Strigeidea), as visualized by confocal microscopy.:Strigeidea), as visualized by confocal microscopy

The major muscle systems of the metacercaria of the strigeid trematode, Apatemon cobitidis proterorhini have been examined using phalloidin as a site-specific probe for filamentous actin. Regional differences were evident in the organization of the body wall musculature of the forebody and hindbody, the former comprising outer circular, intermediate longitudinal and inner diagonal fibres, the latter having the inner diagonal fibres replaced with an extra layer of more widely spaced circular muscle. Three orientations of muscle fibres (equatorial, meridional, radial) were discernible in the oral sucker, acetabulum and paired lappets. Large longitudinal extensor and flexor muscles project into the hindbody where they connect to the body wall or end blindly. Innervation to the muscle systems of Apatemon was examined by immunocytochemistry, using antibodies to known myoactive substances: the flatworm FMRFamide-related neuropeptide (FaRP), GYIRFamide, and the biogenic amine, 5-hydroxytryptamine (5-HT). Strong immunostaining for both peptidergic and serotoninergic components was found in the central nervous system and confocal microscopic mapping of the distribution of these neuroactive substances revealed they occupied separate neuronal pathways. In the peripheral nervous system, GYIRFamide-immunoreactivity was extensive and, in particular, associated with the innervation of all attachment structures; serotoninergic fibres, on the other hand, were localized to the oral sucker and pharynx and to regions along the anterior margins of the forebody.

The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Inter-phyla studies on neuropeptides: the potential for broad-spectrum anthelmintic and/or endectocide discovery

Flatworm, nematode and arthropod parasites have proven their ability to develop resistance to currently available chemotherapeutics. The heavy reliance on chemotherapy and the ability of target species to develop resistance has prompted the search for novel drug targets. In view of its importance to parasite/pest survival, the neuromusculature of parasitic helminths and pest arthropod species remains an attractive target for the discovery Of novel endectocide targets. Exploitation of the neuropeptidergic system in helminths and arthropods has been hampered by a limited Understanding of the functional roles of individual peptides and the structure of endogenous targets, such as receptors. Basic research into these systems has the potential to facilitate target characterization and its offshoots (screen development and drug identification). Of particular interest to parasitologists is the fact that selected neuropeptide families are common to metazoan pest species (nematodes, platyhelminths and arthropods) and fulfil specific roles in the modulation of muscle function in each of the three phyla. This article reviews the inter-phyla activity of two peptide families, the FMRFamide-like peptides and allatostatins, on motor function in helminths and arthropods and discusses the potential of neuropeptide signalling as a target system that could uncover novel endectocidal agents.