Identification of a UDP-Gal:beta-galactoside beta 1----3-galactosyltransferase in the albumen gland of the snail Lymnaea stagnalis.

Detergent extracts of the albumen gland of the snail Lymnaea stagnalis contain an enzyme activity that transfers Gal from UDP-Gal to acceptor substrates with terminal non-reducing beta-galactose residues. The products formed with lactose (Gal beta 1----4Glc) as the acceptor were characterized by HPLC, and subjected to 400-MHz 1H-NMR and methylation analysis. The main product was shown to have the structure Gal beta 1----3Gal beta 1----4Glc. Therefore, the galactosyltransferase can be identified as a UDP-Gal:beta-galactoside beta 1----3-galactosyltransferase. In view of its linkage and acceptor specificity, the enzyme may be essential to the biosynthesis of galactogen, the main polysaccharide produced by albumen glands of 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)

Identification of free and conjugated ecdysteroids in cercariae of the schistosome Trichobilharzia ocellata.

Extracts of cercariae of the avian schistosome Trichobilharzia ocellata were analysed for the presence of ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and gas chromatography/mass spectrometry (selected ion monitoring). Both free ecdysteroids and polar conjugated ecdysteroids were detected in the cercarial extracts. The free ecdysteroid fraction, as well as the hydrolysed polar conjugated ecdysteroid fraction, contained both ecdysone and 20-hydroxyecdysone in approximately equal amounts. The amount of ecdysteroids detected is comparable to those found in other platyhelminths. A possible role for the ecdysteroids in the development of the parasite and/or the interactions between the parasite and its intermediate host, the freshwater snail Lymnaea stagnalis, is discussed.

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.

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.

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.

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.

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.

The occurrence of schistosomin, an antagonist of female gonadotropic hormones, is a general phenomenon in haemolymph of schistosome-infected freshwater snails.

In haemolymph of Lymnaea stagnalis, parasitized with the digenetic trematode parasite Trichobilharzia ocellata, a neuropeptide (schistosomin) occurs which antagonizes female gonadotropic hormones, e.g. calfluxin (CaFl). By means of an ultracytochemical hormone-assay, the CaFl assay, it was demonstrated that the occurrence of schistosomin is a general phenomenon in schistosome-infected freshwater snails. Haemolymph of the schistosomiasis-transmitting snail species Biomphalaria glabrata and B. pfeifferi, parasitized with Schistosoma mansoni, also appeared to contain an antagonizing factor, i.e. schistosomin. In contrast, in haemolymph of L. stagnalis parasitized with Diplostomum spathaceum (Diplostomatidae) no schistosomin could be found. This suggests that schistosomin may only occur in snails infected with parasites belonging to the Schistosomatidae. The effect of schistosomin is rather specific. Haemolymph of B. glabrata parasitized with S. mansoni had not the capacity to inhibit the response to CaFl in the target organs for CaFl, the albumen glands of L. stagnalis and Bulinus truncatus. The same holds true for haemolymph of infected L. stagnalis: it did not inhibit the CaFl response in glands of B. glabrata and B. truncatus and even not in those of a related species (L. ovata). Schistosomins in haemolymph of infected B. glabrata and B. pfeifferi, on the other hand, seem more related. Both appeared to inhibit the hormone response in glands of the two Biomphalaria species studied. The results indicate that schistosomin in haemolymph of schistosome-infected pulmonate snails, although functionally related, may differ structurally.

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.

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.

Trichobilharzia ocellata: influence of infection on the interaction between the dorsal body hormone, a female gonadotropic hormone, and the follicle cells in the gonad of the intermediate snail host Lymnaea stagnalis.

Activation of adenylate cyclase (AC)-cAMP system in follicle cells of Lymnaea stagnalis by the gonadotropic dorsal body hormone (DBH) is inhibited by schistosomin, an agent present in hemolymph of snails infected with Trichobilharzia ocellata. AC activation was determined enzyme cytochemically. This conclusion is based on the observation that the percentage of oocytes with AC-positive follicle cells in gonads incubated in the presence of schistosomin, i.e., in serum of infected snails (IS) with DBH, is significantly lower than that in gonads incubated in the absence of schistosomin, i.e., in serum of noninfected snails (NS) with DBH. Follicle cells in gonads preincubated in the absence of schistosomin, i.e., in NS, and subsequently incubated with freshly dissolved DBH showed a considerably lower response to DBH than those in not preincubated gonads. This indicates that the number of receptors for DBH on follicle cells had decreased during preincubation. The response to DBH also appeared to decrease when the hormone was preincubated in NS. This indicates that the activity of DBH decreases during preincubation. These data make it impossible to answer the question of whether or not schistosomin acts as an antagonist of DBH at the receptor level.

Isolation of schistosomin, a neuropeptide which antagonizes gonadotropic hormones in a freshwater snail.

The molecular mechanisms underlying parasite-induced inhibitory effects on host reproduction were studied in the freshwater snail, Lymnaea stagnalis, infected with the schistosome parasite Trichobilharzia ocellata. This combination is used as a model system for host-parasite interactions involved in schistosomiasis transmission. The female gonadotropic snail neuropeptide, calfluxin, was labelled with fluorescein isothiocyanate (FITC) and used as a ligand in receptor-binding studies on membranes of its target organ, the albumen gland. The binding of calfluxin to its receptor-guanyl-nucleotide-binding-protein (G-protein) complex was inhibited in vitro in the presence of haemolymph of schistosome-infected snails. This inhibition appeared to be established by a peptidergic factor called schistosomin. The receptor assay was used to identify schistosomin from haemolymph during subsequent purification and characterization steps. The peptide could also be purified from the central nervous systems of non-infected snails, indicating that it is produced by the snail itself and released into the haemolymph as a result of infection. Analysis by plasma-desorption mass spectrometry revealed that purified schistosomin has a molecular mass of 8780 Da.

Trichobilharzia ocellata: influence of infection on the fecundity of its intermediate snail host Lymnaea stagnalis and cercarial induction of the release of schistosomin, a snail neuropeptide antagonizing female gonadotropic hormones.

Subadult and adult specimens of the pond snail Lymnaea stagnalis were infected with the schistosome Trichobilharzia ocellata. Egg production and growth of the snails were monitored over an 8-week period post-infection (p.i.). Snail haemolymph was collected and analysed for the presence of schistosomin, a neuropeptide which antagonizes the action of the snails' female gonadotropic hormones. Snails infected as subadults showed an increase in fecundity during the first 4 weeks p.i. compared with non-infected controls. The possibility is discussed that this increase is caused by an accelerated maturation of the female sex organs due to elevated levels of Dorsal Body Hormone, a female gonadotropic hormone. No difference in fecundity was found between snails infected as adults and control snails during the first 4 weeks p.i. Snails infected as subadults and as adults showed a decrease in fecundity from week 5 p.i. and onwards. This decrease coincided with the appearance of schistosomin in the haemolymph of the snails and with that of differentiating cercariae in the daughter sporocysts. Cercariae are probably involved in the induction of schistosomin release from the snails' CNS into the haemolymph. Snails infected as subadults or as adults grew at approximately the same rate as uninfected snails.

Spermatogenesis and the role of Sertoli cells in the freshwater snail Biomphalaria glabrata.

The various stages of spermatogenesis and the Sertoli cells of Biomphalaria glabrata were studied with histochemical and electron microscope techniques. During spermatogenesis a manchette of microtubules is formed around the nucleus and the mid-piece of the spermatids. This manchette becomes helically coiled and probably plays an important role in the spiralisation of the nucleus and of the mitochondrial sheath. During spermatogenesis so-called chromatoid bodies (CB) occur, which consist of arginine-rich proteins. These CB disintegrate during the early spermatid stage. The results suggest that the CB are either involved in histone transition or in the formation of microtubules. The remaining cytoplasm of the spermatids is phagocytised by the Sertoli cells. Apparently this process of phagocytosis is an important part of the mechanism of spermiation. Morphological measurements of the Sertoli cells showed that the relative volume of most organelles decrease during spermatogenesis, indicating a general decrease in cell activity. Possible functions of the Sertoli cells, such as transportation and nutrition of spermatogenic cells and hormone production, are discussed. It is concluded on the basis of the histochemical and ultrastructural observations that the Sertoli cells are involved in the nutrition of spermatogenic cells. It seems unlikely that they are hormone producing cells.

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.

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.

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.