LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis.

We report the characterization of a cDNA encoding a novel -RFamide neuropeptide precursor that is up-regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C-terminal sequence -LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+-currents, and inhibited electrical activity. We conclude that up-regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.

Target-dependent differentiation and development of molluscan neurons and neuroendocrine cells: use of parasitisation as a tool.

Specimens of the freshwater snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata show a strongly inhibited development of their reproductive tract. We hypothesised that the effects of the underdevelopment of targets are reflected at the level of the neuronal development of (i) the motor neurons innervating the male copulation organ and (ii) neuroendocrine cells regulating the gonad. We determined the state of neuronal development by measuring cell number, cell size and neuropeptide gene expression. Our results show that the neuronal development of both copulation controlling anterior lobe motor neurons of the right cerebral ganglion and neuroendocrine caudodorsal cells, which produce neuropeptides regulating ovulation, egg laying and accompanying behaviour, are affected in parasitised animals in which their respective target organs were not developed. The cell bodies were smaller and fewer cells were found to express neuropeptide genes compared to those in non-parasitised animals. These effects were not observed in the appropriate controls. Backfills and lesions of the penis nerve have shown that the inhibited development of central motor neurons in parasitised snails is target dependent; neighbouring neurons that have no connection with the male copulation organ are not affected. Our data suggest that this effect is established by target-derived neurotrophic factors that need this connection for being transported to the innervating motor neurons. We propose that the effect on the neuroendocrine caudodorsal cells is mediated by a humoral factor, since they have no known connection with their target. We have shown that the size and gene expression of motor neurons controlling copulation behaviour in the pond snail Lymnaea stagnalis are related to the size of their target, the copulation organ, and depend on the connection with this target.

NPY in invertebrates: molecular answers to altered functions during evolution.

As in Lymnaea stagnalis NPY plays a key role in regulating energy flows but has no effect on food intake, two important questions arise: 1) How is the amount of food consumed related to energy storage? 2) Can we give a molecular explanation for this alteration in function of NPY during evolution? Recent data have shown that also in Lymnaea a leptin-like factor is produced by glycogen storing cells which inhibits food intake, a Lymnaea storage feedback factor (LySFF). So, food consumption seems in balance with the amount of energy stored in this animal. We suppose that NPY neurons in Lymnaea have receptors for LySFF so that their activity in regulating energy homeostasis reflects the amount of stored energy. By comparing the molecular structure of NPYs in invertebrates it became clear that only molluscan and arthropod NPY are synthesized from a prohormone similar to vertebrate NPYs and should be considered as real invertebrate homologs of NPY. Based on pharmacological data we suppose that the identified Lymnaea NPY receptor is a Y1 subtype. This might explain that LyNPY has no effect on food intake in Lymnaea as this function of NPY in mammals is regulated through the Y5 subtype receptor.

Structure, localization and potential role of a novel molluscan trypsin inhibitor in Lymnaea.

Eggs and egg masses of the freshwater gastropod mollusc Lymnaea provide a microenvironment for developing embryos. Secretions of the exocrine albumen gland of Lymnaea are packaged in the eggs of an egg mass before the eggs are laid externally. The perivitelline fluid that directly surrounds individual oocytes is the main source of nutrition for developing embryos. During early stages of development, the perivitelline fluid is initially internalized by pinocytosis and degraded by lysosomes; in later stages, the embryo ingests the fluid. We previously found that the albumen gland produces large amounts of Lymnaea epidermal growth factor. The albumen gland also appears to produce significant amounts of a novel Lymnaea trypsin inhibitor (LTI), a second peptide that was purified and characterized from Lymnaea albumen gland extracts. The primary structure was determined by microsequence analysis, mass spectrometry, and C-terminal sequence analysis, and showed that LTI is a 57-residue glycosylated peptide. Comparison of the LTI sequence with other known serine protease inhibitors indicates that LTI is a member of the bovine pancreatic trypsin inhibitor family. Reverse phase-high performance liquid chromatography, microsequence analysis, mass spectrometry, and immunocytochemistry demonstrated that abundant amounts of intact LTI are packaged in egg masses. The presence of a trypsin inhibitor in the perivitelline fluid compartment of the egg mass may minimize digestion of peptides and proteins in the perivitelline fluid that are important for the development of the embryo, for example, Lymnaea epidermal growth factor.

Multiple strategies of schistosomes to meet their requirements in the intermediate snail host.

The results of the studies on our model combination Trichobilharzia ocellata-Lymnaea stagnalis, presented in this review, lead to the conclusion that schistosomes use multiple strategies to reach their goals, i.e. to propagate and to continue their life cycle. They have to escape from being attacked by the internal defence system (IDS) of the snail host and to profoundly affect the host's energy flow, of which reproduction and growth are the main determinants, for their own benefit. These physiological changes they establish mainly by interfering with the two regulatory systems in the snail host, the IDS and the neuroendocrine system (NES). Moreover, these two regulatory systems clearly interact with each other. Parasitic E/S products affect the host's IDS both in a direct and an indirect way. The neuropeptides or neuropeptide-like substances that are secreted by parasite glands into the host directly suppress haemocyte activity in the snail. The indirect effects include effects of (1) peptides from connective tissue cells and (2) neuropeptides from NES and/or IDS. Parasitic E/S products also induce the effects on energy flow in the host. These E/S products act either directly on a target, as shown for the inhibiting effect of the parasite on the development of the male copulation organ, or on the NES regulating reproductive activity, e.g. on gene expression. Indirect effects of E/S products on the NES (hormone-receptor interaction, electrical activity) are mediated by a factor from connective tissue cells, presumably belonging to the IDS. The physiological changes in the snail host are obviously of vital importance for the parasites, since they make use of different strategies to bring them about.

Parasites flicking the NPY gene on the host's switchboard: why NPY?

It was investigated whether up-regulation of the NPY gene by the schistosome Trichobilharzia ocellata in its snail host Lymnaea stagnalis redirects the host's energy flows. We cloned the cDNA encoding Lymnaea NPY (LyNPY), purified and sequenced the peptide, and used synthesized peptide for physiological and morphological studies. Increasing the LyNPY titer in nonparasitized snails (mimicking parasitosis) by 1) implantation of slow-release pellets and 2) injections suppressed reproductive activity and reduced growth in a dose- and time-dependent manner without affecting food intake. When the LyNPY titer was back to normal, reproduction and growth were resumed, coinciding with a transient increase of food intake serving to replenish glycogen stores. Observations on double-immunostained whole mount preparations of brains support these data. A close association was found between LyNPY-positive axons and axons both from ovulation hormone-producing neurons and molluscan insulin-like peptide-producing neurons involved in regulation of growth. As no synaptic(-like) contacts were observed, it is supposed that LyNPY acts nonsynaptically. No morphological interaction was found between LyNPY-positive axons and motoneurons innervating the feeding apparatus. Our data explain why it is an advantageous strategy for endoparasites to up-regulate the highly conserved NPY gene in their host.

The suppressive excretory-secretory product of Trichobilharzia ocellata: a possible factor for determining compatibility in parasite-host interactions.

Factors which may determine trematode-snail interactions were assessed in the present study. Compatibility was examined using a bacterial clearance assay to detect the modulatory effects of both compatible and incompatible trematode infections on the activity of haemocytes from Lymnaea stagnalis, during the early stages of infection. Exposure to and injection with Trichobilharzia ocellata, a compatible trematode, or the incompatible Schistosoma mansoni, resulted in modulation of haemocyte activity. However, T. ocellata activated haemocytes 1.5 h post-infection (p.i.) and then suppressed activity 24-72 h p.i. whereas with S. mansoni no suppression, only activation of haemocytes was observed throughout the test period (1.5-72 h p.i.). In previous studies, modulation of the haemocyte clearance activity by T. ocellata was found to be mediated by 2 E-S fractions, an activating fraction and a suppressing one. Investigations to assess whether the lack of suppression of haemocyte activity, observed in the S. mansoni-L. stagnalis incompatible trematode-snail interaction studied, was due to either the absence or ineffectiveness of the suppressing E-S fraction, were performed on a second incompatible combination, T. ocellata-Planorbis corneus. Using this combination it was revealed that only the activating E-S fraction had modulatory effects on P. corneus haemocytes, indicating that the suppressing E-S fraction, which actively interferes with the clearance activity of haemocytes from L. stagnalis, appears to act in a host-specific manner. In conclusion, the suppressing E-S fraction determines, at least in part, compatibility in the trematode-snail association studied. This is also probably likely in other trematode-snail combinations.

Altered gene expression in the host brain caused by a trematode parasite: neuropeptide genes are preferentially affected during parasitosis.

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

A new Ig-superfamily member, molluscan defence molecule (MDM) from Lymnaea stagnalis, is down-regulated during parasitosis.

To survive the attacks of the internal defence system (IDS) of their host, parasites have developed various strategies to manipulate the IDS. We present evidence that the avian schistosome parasite Trichobilharzia ocellata affects gene expression in the granular cells, a cell type of the IDS of the intermediate host, the mollusc Lymnaea stagnalis. From a differential screening, a clone was isolated encoding a protein named molluscan defence molecule (MDM), which encompasses five C2-like immunoglobulin (Ig) domains. The protein shares a domain organization and high amino acid sequence identity with hemolin, an Ig-family member of the insect IDS. Interestingly, both MDM and hemolin have highest sequence identity with neural cell adhesion molecules, but lack the typical fibronectin repeats and motifs for membrane anchors. We find that the expression of the MDM gene is gradually down-regulated during the course of parasitosis to approximately 21% compared to the non-parasitized level, 8 weeks post-infection. Based on our findings, we suggest that MDM is involved in the proper function of the Lymnaea IDS, and that down-regulation of MDM is part of the parasite-induced disabling on non-self recognition.

In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by beta 2GlcNAc-transferase I is an essential prerequisite for the action of beta 2GlcNAc-transferase II and beta 2Xyl-transferase.

Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (beta 2Xyl-T), beta 1-2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N-acetylglucosaminyltransferase II (beta 2GlcNAc-T II) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = -4GlcNAc beta 1-4GlcNAc or O-(CH2)7CH3] with UDP-Xyl and connective tissue microsomes have been purified and characterized by 1H-NMR spectroscopy in conjunction with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specificity studies focused on connective tissue beta 2Xyl-T show that the minimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)7CH3. The enzyme activity can therefore be characterized as UDP-Xyl:Glc-NAc beta 1-2Man alpha 1-3Man beta-R (Xyl to Man beta) beta 1-2 xylosyltransferase. In substrate-specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xyl residue at C2 of beta Man totally abolishes the enzyme activity. Xylose-containing oligosaccharides are not acceptors for beta 2GlcNAc-T I. In combination with the substrate specificity of beta Xyl-T, this shows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc-T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective tissue is proposed.

Identification of a novel UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis.

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta 1-4 linkage to the terminal GlcNAc residue of GlcNAc beta-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc or GlcNAc beta 1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAc beta 1-OMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAc alpha 1-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAc beta 1-R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAc beta 1-OMe as acceptor. In all instances, products with GalNAc beta 1-4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stagnalis.

How schistosomes profit from the stress responses they elicit in their hosts.

Results obtained with the model Trichobilharzia ocellata-Lymnaea stagnalis have confirmed the hypothesis that the physiological effects evoked by schistosomes in their snail host--castration and giant growth--are brought about by them interfering with the neuroendocrine systems (NES) regulating the physiological processes concerned. As soon as differentiating cercariae are present in the daughter sporocysts a factor can be detected in the haemolymph of the snail host, called schistosomin, which acts both at the central and the peripheral parts of the NES involved in regulation of reproduction and growth. Schistosomin appears to be a host-derived factor, which is probably released by cells of the internal defence system, the haemocytes, and by connective tissue cells, the telo-glial cells. It meets the criteria of having a cytokine-like function although its molecular structure does not show sequence homology with any of the vertebrate-type cytokines identified to date. Its cytokine nature explains why schistosomin can interfere with different neuroendocrine regulatory systems both at the central and peripheral--target--level, namely after binding to its own receptor. Schistosomin is probably not only responsible for the effects exerted by the parasite on female reproduction but also for those on male reproduction and on growth so that energy and space become available for the continuous production of cercariae. The nature of the humoral cercarial factor, which induces schistosomin release, is as yet unknown. Based on its hydrophobic character and on the fact that it can pass through the wall of the daughter sporocyst, it is supposed to be a diffusible molecule or a protonephridial excretion product. It does not seem to be a vertebrate-type steroid, an ecdysteroid or an eicosanoid. Results obtained in vitro have indicated that schistosomin might have a suppressive effect on haemocyte activity. Plasma from snails 5-6 weeks post-exposure showed a tendency to inhibit phagocytic activity of haemocytes from non-infected snails, that is preparatory to the escape and migration of cercariae. Once shedding has started this effect of schistosomin is overrruled by a strong activation of haemocyte activity coinciding with the tissue damage that the cercariae cause in the host. The cercariae escape from being attacked by masking their surface coat with host molecules. As the physiological effects caused by schistosomes resemble those observed during stress in mammals, experiments were carried out to find out whether schistosomin is also released in non-parasitized snails during stress resulting in an inhibiting effect on reproduction.(ABSTRACT TRUNCATED AT 400 WORDS)

An immunocytochemistry study comparing the occurrence of neuroactive substances in the nervous system of cercariae and metacercariae of the eye fluke Diplostomum spathaceum.

The nervous system of two larval stages (cercariae, metacercariae) of eye fluke Diplostomum spathaceum was investigated immunocytochemically by the application of antisera to the amino acid glutamate and to neuropeptides isolated from invertebrates (Mollusca) and from vertebrates to whole-mount preparations. In cercariae, positive immunoreactivity (IR) was observed with antisera raised against Catch-relaxing peptide (CARP), FMRFamide, alpha-caudodorsal cell peptide (alpha-CDCP), substance P, vasotocin, and vasopressin. In metacercariae, in addition to positive staining with these antisera, the ones raised against glutamate, APGWamide, caudodorsal cell hormone I (CDCH-I), and small cardiac peptide B (SCPB) also gave positive IR in the nervous system. In the two larval stages the most extensive pattern of IR was observed with anti-FMRFamide and anti-CARP. In the nervous system of metacercariae the same immunoreactive neurosubstances appeared to be present as in that of cercariae. The increase in the variety of immunoreactive neurosubstances in the more complex nervous system of metacercariae is discussed in relation to parasite development and to host adaptation.

Modulation of the bacterial clearance activity of haemocytes from the freshwater mollusc, Lymnaea stagnalis, by the avian schistosome, Trichobilharzia ocellata.

The ability of haemocytes, from the haemolymph of the gastropod mollusc Lymnaea stagnalis, to recognize and eliminate the bacterium Aeromonas salmonicida was shown using an in vitro bacterial clearance assay. The assay employs a dye which is reduced by A. salmonicida in direct proportion to the number of viable bacteria resulting in a colour change which can be determined spectrophotometrically. Addition of cytochalasin B resulted in a marked decrease in bacterial clearance, implicating both intracellular and extracellular cytotoxicity of haemocytes. A comparison of haemocytes from uninfected snails and snails infected with the avian schistosome parasite Trichobilharzia ocellata showed that both juveniles and adults of L. stagnalis were susceptible to infection with T. ocellata. After exposure to the trematode for 1.5 h the haemocytes from these infected snails had an enhanced clearance capacity, whilst cells obtained from snails with 24-96 h infections showed decreased clearance of the bacteria, indicating suppression by the parasite. Haemocytes, as well as plasma, which was tested on haemocytes from uninfected snails, were used and hence a distinction was made between cell and humoral-associated effects. The results show that both cellular and humoral components of immunity were activated, then suppressed, following exposure to the parasite. Infection with T. ocellata seems to have a modulating effect on the bactericidal activity of the internal defence system of the snail host, L., stagnalis.

Immunocytochemical study on biologically active neurosubstances in daughter sporocysts and cercariae of Trichobilharzia ocellata and Schistosoma mansoni.

Immunocytochemical techniques applied to sections and whole-mount preparations of cercariae from two species of trematodes, Trichobilharzia ocellata and Schistosoma mansoni, revealed the occurrence of immunoreactivity (IR) to several neurosubstances in the nervous system (NS). Immunostaining was localized in cerebral ganglia, in the main commissure, in anterior and posterior nerve trunks, as well as in a pair of nerve fibres running along the tail. In T. ocellata, immunoreactivity (IR) was observed with antisera raised against: glutamate, FMRFamide, catch-relaxing peptide (CARP), small cardiac peptide B (SCPB), arg-vasotocin (AVT), arg-vasopressin (AVP), and substance P. In S. mansoni antisera raised against glutamate, FMRFamide, CARP, SCPB, alpha-caudodorsal cell peptides (alpha-CDCP), and cholecystokinin (CCK) showed neuronal IR. With the other 51 antisera tested no IR was observed. With anti-APGWamide, IR was observed outside the NS in cells of the wall of the daughter sporocyst and in flame cells of cercariae of T. ocellata. IR to FMRFamide was present in the escape glands of the intrasporocystic cercariae of T. ocellata and S. mansoni. IR to somatostatin was observed in subtegumental parenchymal cells of cercariae of S. mansoni. IR to met-enkephalin was present in cells of the cercarial embryos and in undifferentiated cells in developing cercariae. Trematodes are, together with cestodes, phylogenetically the oldest classes in which glutamate-like material and immunopositivity to a number of neuropeptides isolated from invertebrates has been demonstrated. The results are discussed in relation to immunocytochemical data obtained for other platyhelminths, to endogenous functions of the immunopositive materials, and to their possible role in parasite-host interactions.

Trichobilharzia ocellata infections in its snail host Lymnaea stagnalis: an in vitro study showing direct and indirect effects on the snail internal defence system, via the host central nervous system.

In this in vitro study we investigated whether previously described in vivo plasma-associated effects, that occurred in the period shortly after penetration of Trichobilharzia ocellata into the snail host Lymnaea stagnalis (1.5-72 h post-exposure; p.e.) were direct and/or indirect effects of parasite-derived factor(s). It was investigated whether the effect is mediated by the central nervous system (CNS) of the host. Phagocytic activity of the haemocytes was taken as a parameter for the activity of internal defence of the host. A number of preliminary experiments were performed. When the supernatant of in vitro cultured parasites (33 h; corresponding with their developmental stage in vivo when plasma-associated activation was found) was applied directly to monolayers of haemocytes, it appeared to enhance their phagocytic activity. No direct effect, however, was found with a supernatant of parasites cultured for a longer period of time (72 h; when, in vivo, a plasma-associated suppression was found). In this case, indirect suppression was detected: the parasites appeared to have released a factor that induced the CNS of the host to release material suppressing the activity of the internal defence system of the host. To date the nature of this factor is unknown.

In vitro release of the anti-gonadotropic hormone, schistosomin, from the central nervous system of Lymnaea stagnalis is induced with a methanolic extract of cercariae of Trichobilharzia ocellata.

Infection with digenetic trematodes causes an inhibition or complete cessation of fecundity in their intermediate hosts, freshwater snails. It has been demonstrated in the host-parasite combination Lymnaea stagnalis-Trichobilharzia ocellata that the action of the female gonadotropic hormones upon their target organs is inhibited by the peptide schistosomin. Schistosomin is produced in the central nervous system of the snail and released upon parasitic infection. In order to study the in vitro release of schistosomin, a bioassay was developed. Central nervous systems were incubated with either an acetic acid or a methanolic extract of larval stages of Trichobilharzia ocellata (miracidia, mother sporocysts, cercariae). The incubation media were chromatographed using HPLC and released schistosomin (-like material) was tested for bioactivity in the calfluxin bioassay. The in vitro release of schistosomin was only induced with a methanolic extract of cercariae. The nature of the cercarial factor is discussed.

The neuropeptide schistosomin and haemolymph from parasitized snails induce similar changes in excitability in neuroendocrine cells controlling reproduction and growth in a freshwater snail.

Infection of the snail Lymnaea stagnalis with the schistosome parasite Trichobilharzia ocellata results in inhibition of reproduction and in giant growth. Parasite-related effects on the neuroendocrine centres that control these processes were studied electrophysiologically. Haemolymph from infected snails reduced the excitability of the caudodorsal cells, which control egg laying. In contrast, the excitability of the growth-controlling Light Green Cells was increased under these conditions. The endogenous anti-gonadotropic neuropeptide schistosomin, the presence of which is strongly enhanced in parasitized snails, induced similar effects. Schistosomin apparently plays an important role in the balance between reproduction and growth in Lymnaea. This balance is severely disturbed during parasitic infection, probably as a result of the release of the peptide.

Modulation of the activity of the internal defence system of the pond snail Lymnaea stagnalis by the avian schistosome Trichobilharzia ocellata.

Effects of infection with the avian schistosome Trichobilharzia ocellata on the activity of the internal defence system of the intermediate snail host Lymnaea stagnalis were studied, utilizing an in vitro phagocytosis assay for determining haemocyte activity. A distinction was made between plasma- and cell-associated effects. The period immediately after penetration of the parasite into the snail host (1.5-72 h post-exposure (p.e.)) was extensively studied. In addition, several time-points coinciding with the later-successive-stages of parasite development (2, 4, 6, 8 and 10 weeks p.e.) were investigated. Plasma-associated enhancement of defence activity was found between 1.5 and 6 h p.e., followed by plasma-associated suppression between 12 and 72 h p.e. A cell-associated activation was found between 1.5 and 6 h p.e. and also at 8 and 10 weeks p.e. How these effects on the defence system may be related to phenomena observed in infected snails at these time-points is discussed.