Trichobilharzia ocellata: Quantification of effects on haemocytes of the pond snail Lymnaea stagnalis by morphometric means

To identify functionally different subpopulations, we quantified by morphometric means the spreading activity of circulating haemocytes of the pond snail Lymnaea stagnalis, recognized by monoclonal antibodies (5 surface and 5 cytoplasmic). The influence of snail age and of the different intramolluscan stages of the compatible avian schistosome Trichobilharzia ocellata on this activity were studied. The antibody-recognized cells could be separated into two groups, differing in their spreading activities. The probes detecting cytoplasmic markers recognized the majority of cells (78-95%). These were active (well spreading), differentiated cells. The surface probes recognized a smaller part (12-38%) of the total haemocyte population. These harmocytes were less active (less spreading), and were predominantly immature cells. The relative sizes of the antibody-detected subpopulations were not affected by snail age or infection with T. ocellata. The maximal size (planimetric area) attained after attaching to glass of all cells increased between day 0 (juvenile snails) and about 6 weeks post (sham) exposure and then decreased. Infection had little effect on the spreading activity of the more differentiated cells. The less differentiated cells showed a larger spreading activity when the parasite was present as mother sporocyst and also when the digestive gland area became colonized by growing daughter sporocysts.     

Cloning of Schistosoma mansoni sporocysts in vitro and detection of genetic heterogeneity among individuals within clones

The establishment of in vitro cultivation techniques to maintain larval and adult stages of the trematode Schistosoma mansoni has facilitated research on diverse aspects of the biology of this parasite. Because of the difficulty in obtaining defined intramolluscan stages of this parasite, one aim of this study was to develop an in vitro technique for the generation of defined clonal daughter sporocyst (DSp) generations that originate from a single mother sporocyst. Sporocysts died when cultured singly; however, when single sporocysts were cultured in inserts within wells with about 1,000 others, the single individuals produced daughters asexually. In recent years, evidence has been accumulating for variability among, and within, schistosome populations. Such variability has been seen in both larval and adult stages. Even within clonal cercariae, genomic and biochemical heterogeneity has been observed, indicating the existence of a yet unknown mechanism that generates variability during larval development. Therefore, another aim of this study was to examine clonal DSps generated in vitro for diversity regarding the presence or absence of a specific repetitive DNA element (W1). Such sporocysts were found by molecular analysis to be heterogeneous with respect to the occurrence of W1. This phenomenon had previously been observed in clonal schistosome populations and described as genomic instability. In this study, we provide the first molecular evidence that variability can be generated within sporocyst generations, supporting the hypothesis of mitotic recombination events during the asexual life stage of schistosomes.     

A comparative study of mechanisms underlying digenean-snail specificity: in vitro interactions between hemocytes and digenean larvae

A panel of 4 digenetic trematode species (Echinostoma paraensei, E. trivolvis, Schistosoma mansoni, and Schistosomatium douthitti) and 5 snail species (Biomphalaria glabrata, Helisoma trivolvis, Lymnaea stagnalis, Stagnicola elodes, and Helix aspersa) was examined to determine if known patterns of host specificity could be explained by the tendency of digenean larvae to be bound by snail hemocytes, or by the ability of larvae to influence the spreading behavior of hemocytes. In short-term (1 hr) in vitro adherence assays, there was no overall pattern to suggest that sporocysts were more likely to be bound by hemocytes from incompatible than compatible snails. Compared with the other parasites, sporocysts of E. paraensei were less likely to be bound by hemocytes from any of the snail species tested. All rediae examined, including those of another species Echinoparyphium sp., were also remarkably refractory to binding by hemocytes from any of the snails. Of all the larvae examined, only sporocysts and young daughter rediae of E. paraensei caused hemocytes to round up in their presence. This was true for hemocytes from the compatible species B. glabrata and the incompatible lymnaeid species S. elodes and L. stagnalis. The patterns of host specificity shown by this particular panel of parasites and snails were not predicted by either the extent of hemocyte adherence to digenean larvae or by the ability of larvae to affect hemocyte spreading behavior. The results of this study suggest that a role for hemocytes, although likely, may require different assays, possibly of a more prolonged nature, for its detection. Also, different parasite species (notably E. paraensei) and intramolluscan stages have distinctive interactions with host hemocytes, suggesting that the determinants of specificity vary with the host-parasite combination, and with the parasite life cycle stage.     

Search for shared antigens in the schistosome-snail combination Trichobilharzia ocellata-Lymnaea stagnalis

Mother and daughter sporocysts of Tricholbilharzia ocellata, developing in the snail host Lymnaea stagnalis, were searched for substances with antigenic similarities to the snail's haemolymph. Antisera to cell-free snail haemolymph and fractions thereof were used in three different immunocytochemical staining methods, applied on sections of parasitized snails. Snail tissue was consistently stained; cercariae were stained, indicating that the applied methods were successful. Most sections through mother and daughter sporocysts were completely unstained. It is concluded that neither mother nor daughter sporocysts are masked by the antigens studied or substances mimicking these. The relevance of the present observations is discussed.     

Axenic culture of Schistosoma mansoni sporocysts in low O2 environments.

Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata. To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed. When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring. Because Bge-derived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels. In an hypoxic environment, S. mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules. Sporocyst production occurs independently of host influence.     

Serotonin-induced muscular activity in Schistosoma mansoni larval stages: importance of 5-HT transport and role in daughter sporocyst production

Mother sporocysts of Schistosoma mansoni transport exogenously supplied serotonin (5-hydroxytrypamine; 5-HT), and respond to it with increases in motility. In the present study, we investigated the importance of 5-HT transporter activity in the manifestation of these 5-HT-induced motility changes, and further examined the role of 5-HT in the development of daughter sporocysts in vitro. Serotonin-induced motility of in vitro-derived sporocysts is not inhibited by antidepressant compounds, e.g., fluoxetine, that block 5-HT transport, suggesting that the receptors responsible for motility responses to 5-HT are surface exposed. Using a sporocyst in vitro culture system, we show that depletion of larval stores of 5-HT reduces production of daughter sporocysts, the second intramolluscan larval stage. Moreover, we demonstrate a strong correlation between endogenous 5-HT levels and basal mother sporocyst muscle activity. Overall, these data suggest that larval stages of S. mansoni can detect exogenous 5-HT via surface-exposed receptors, and they are consistent with the hypothesis that endogenous stores of 5-HT are important for the proper regulation of muscular contractions in mother sporocysts, and for the successful emergence of daughter sporocysts.     

Schistosoma mansoni sporocysts in culture: host plasma hemoglobin contributes to in vitro oxidative stress

The initiation and promotion of sporocyst propagation and subsequent production of cercariae by intramolluscan larval stages of digenic trematodes are thought to depend on mollusc-derived factors. The ability to investigate this using in vitro cultures of Schistosoma mansoni sporocysts has been impeded by the fact that plasma from the host, Biomphalaria glabrata, becomes toxic to the parasite in long-term cultures. The present study identifies hemoglobin as the plasma component responsible for this toxicity. The addition of the enzyme catalase to sporocyst cultures neutralized the toxic effects of both purified hemoglobin and whole plasma, suggesting that the generation of H2O2 as a consequence of hemoglobin oxidation is the mechanism of plasma toxicity. Furthermore, cultures incubated in unconditioned schistosome medium with plasma plus catalase yielded significantly higher numbers of daughter sporocysts than cultures with media or plasma alone, but not higher than cultures with catalase alone. These latter results suggest that the oxidative environment and the antioxidant capacity of the media are critical factors for in vitro propagation of S. mansoni sporocysts.     

Insights into the development of Notocotylus attenuatus (Digenea: Notocotylidae) in Lymnaea stagnalis: from mother sporocyst to cercariae

Notocotylus attenuatus (Digenea: Notocotylidae) is a monostome fluke parasitizing the intestinal caeca of waterfowl that uses an injection apparatus to infect its intermediate snail host. Morphology of the invading larva (a sporocyst), and the intramolluscan larval development of this fluke have not been characterized extensively. In this study, experimental infections of Lymnaea stagnalis using N. attenuatus eggs resulted in the development of sporocysts containing one germ ball or mother redia between 12 and 21 days post exposure (p.e.) within the hepatopancreas. Independent mother rediae and developing daughter rediae were present between day 25 and day 42 p.e. Cercariae, within the body of rediae, were detected 42 days p.e. The development of daughter rediae and cercariae started posteriorly in the body of parent redia and these larvae migrated anteriorly during development towards the birth pore. A cercaria was also observed emerging from the birth pore and released cercariae maturated further within the snail hepatopancreas prior to leaving the snail. The intramolluscan development was completed 45 days p.e. when the first fully formed cercariae were shed into the outer environment. These data detail the fascinating post-embryonic development of N. attenuatus and highlight the intricate nature of larval transitions within its snail host.     

Histochemical demonstration of acetylcholinesterase in sporocysts of Schistosoma mansoni (Trematoda).

The distribution of acetylcholinesterase in mother and daughter sporocysts of Schistosoma mansoni was studied histochemically. In young mother sporocysts derived from miracidia cultured in vitro the miracidial neural mass and flame cells were shown to persist. The nerve trunks and commissures, as well as papillae, are apparently lost in the transformation process. In young daughter sporocysts freshly dissected from mother sporocysts there was little enzyme activity except for a sparse distribution in the tegument. After cultivation, intense enzyme activity was associated with developing cercarial embryos. A similar distribution of activity was observed in older daughter sporocysts obtained from the digestive gland of snails. No evidence of flame cells, neural mass, or commissures was detected in daughter sporocysts by the methods employed.     

Differential expression of LacdiNAc,fucosylated LacdiNAc,and Lewis x glycan antigens in intramolluscan stages of Schistosoma mansoni

We report the expression of 3 well-characterized adult Schistosoma mansoni glycan antigens among molluscan stages of the parasite. These antigens are LacdiNAc (LDN; GalNAcbeta1-4GlcNAc-R), fucosylated LacdiNAc (LDNF; GalNAc[Fucal-3]beta1-4GlcNAc-R), and Lewis x (Le(x); Gal[Fucalpha1-3]beta1-4GlcNAc-R). The presence of the glycans was determined by both immunoblot and immunohistological methods using monoclonal antibodies that specifically recognize each glycan epitope. Immunoblot analyses reveal that LDN and LDNF epitopes are expressed on many different glycoproteins, including eggs, mother sporocysts, daughter sporocysts, and cercariae, although LDN expression among daughter sporocysts is greatly reduced. LDN and LDNF epitopes are localized on the tegument and in the intrasporocyst cell masses of both in vitro-derived and in vivo-derived mother sporocysts and in the daughter sporocysts derived on day 16 after infection. Unexpectedly, high levels of LDN and LDNF glycans were detected in the infected, but not in the uninfected, snail hemolymph, suggesting that the infecting larvae secrete LDN and LDNF glycoconjugates into the snail hosts. In contrast, the expression of Le(x) antigen among the molluscan stages is highly restricted. Le(x) is present on a few high-molecular weight glycoproteins in eggs and cercariae but is undetectable in mother and daughter sporocysts. Taken together with our earlier studies on vertebrate stages of S. mansoni, these results show that LDN and LDNF glycans are conserved during schistosome development. The study further extends the evidence that Le(x) is a developmentally regulated antigen in schistosomes.     

Detection of bird schistosomes in lakes by PCR and filter-hybridization

Many lakes around the world are contaminated with bird schistosome cercariae, which penetrate into human skin, causing an itching dermatitis called "swimmers' itch." Bathers could be forewarned from exposure to the larvae and ecological examinations could be performed, when a sensitive method to detect the parasites in aquatic systems, where lots of organisms hinder microscopic examinations, would be available. For this purpose we cloned, sequenced, and analyzed a 396 bp tandem repeated DNA sequence from Trichobilharzia ocellata (ToSau3A), and employed it for developing molecular detection assays. It hybridized with less than 100 pg DNA from different Trichobilharzia species (T. ocellata, Trichobilharzia franki, and Trichobilharzia regenti), but not with 10 ng DNA from other related or sympatric trematodes. A PCR assay, amplifying this sequence with the same specificity, detected 100 fg T. ocellata DNA, 1 cercaria in 0.5 g plankton, and 2 cercariae in 0.5 g host snail tissues.     

Development of Trichobilharzia australis Blair & Islam, 1983 in the snail, Lymnaea lessoni Deshayes and in an experimental definitive host, the Muscovy duck

The development of Trichobilharzia australis Blair & Islam, 1983 in the intermediate host, Lymnaea lessoni Deshayes and in experimental definitive hosts, Muscovy ducks is described. 24 hours after entry into the snail, miracidia had lost their cilia, epidermal plates and lateral processes and became young mother sporocysts. They were located in the tissues of the head-foot organ of the snail and were oval in cross section but still retained the shape of a miracidium, and measured 0.058-0.066 X 0.041-0.049 mm. From the seventh day onward young mother sporocysts were tubular, thin-walled, irregular in shape and were in the tissues of the lung and kidney of the snail. On the 24th day mature mother sporocysts and young daughter sporocysts were found in the digestive gland. Between the 24th and 29th day mature daughter sporocysts with fully developed cercariae ready to emerge, or already emerged, could be seen in the digestive gland of the snail. Cercariae emerged from the snail from the 29th to 46th day after exposure at 25 degrees +/- 1 degree C. The prepatent period of T. australis in the Muscovy ducks was 22 to 42 days after the beginning of exposure to cercariae.     

Effect of infection with Trichobilharzia ocellata and Schistosoma mansoni on the ultrastructure of the albumen gland of their respective hosts, Lymnaea stagnalis and Biomphalaria glabrata

The albumen gland, a female accessory sex gland of pulmonate snails, produces the perivitelline fluid. The ultrastructure of the albumen glands of control and infected specimens of Lymnaea stagnalis and Biomphalaria glabrata was studied. The albumen gland of L. stagnalis contains two types of secretory cells--light (active) and dark (inactive)--and two types of supporting cells--centroacinar and myoepithelial. The secretory cells apparently represent two activity stages of one type of cell. The gland B. glabrata possesses only one secretory cell type, which alternates with one type of supporting cell. The albumen glands of L. stagnalis and B. glabrata infected at a juvenile stage were studied 4 and 14 weeks (L. stagnalis) and 4 and 9 weeks (B. glabrata) after exposure. After four weeks' infection, B. glabrata produced some egg masses, but in subsequent stages egg mass production completely coased. Infected L. stagnalis never produced eggs. B. glabrata was apparently infected at a "physiologically" more mature stage than L. stagnalis. The morphology of the albumen glands four weeks after exposure (the daughter sporocyst stage) is in agreement with this hypothesis. At this interval the secretory cells of L. stagnalis appeared to be much more severely affected (inactive Golgi bodies and rough endoplasmic reticulum, crinophagy of the secretory granules) than the cells of B. glabrata. In the later stages studied (shedding of the cercariae), the glands of both species appeared to be completely inactive (reduced height of the epithelium, inactive organelles, crinophagy, absence of secretory granules). At this stage of infection, daughter sporocysts containing cercaria embryos were seen in the connective tissue of the albumen gland of B. glabrata, but not of L. stagnalis. The results thus indicate that the development and synthetic activity of the albumen gland are seriously affected by infection. These processes are known to be under the endocrine control of the female gonadotrophic hormones. Since it has been established that these hormones are normally present in the haemolymph of infected snails, the findings can be explained by assuming that the parasite interferes in some way or other with the snail's endocrine system.     

Flukes without snails: advances in the in vitro cultivation of intramolluscan stages of trematodes

In vitro cultivation of parasitic helminths, including the digenetic trematodes, has long been a valuable tool in medical and veterinary parasitology, permitting and/or facilitating the development of diagnostic reagents, chemotherapeutic agents, and vaccines and providing insights into naturally complex host-parasite interactions. In vitro cultivation of the intramolluscan stages of trematodes has been particularly challenging, given the ontogenic complexities involved in the production of multiple larval generations from germinal tissues through an asexual "budding" process. Recently, however, advanced larval development has been achieved by incorporating the Biomphalaria glabrata embryonic (Bge) cell line into cocultivation systems. Most notably, the entire intramolluscan cycle (from miracidium to cercaria) has been completed for the human blood fluke Schistosoma mansoni, while significant primary sporocyst development has been attained for several other digeneans including S. japonicum and Fascioloides magna. Here we review recent advances in the cultivation of several larval trematode species and discuss the potential use of this culture system for addressing fundamental questions of host-parasite compatibility.     

The organizational characteristics of the generative material and the the proliferative dynamics of trematode mother sporocysts

Analysis of miracidia germinal material organization and proliferation dynamics of mother sporocysts enabled us to divide them into three well-defined groups. The first one includes species whose miracidia possess only differentiated (mature) generative cells and embryos of earlier stages of cleavage. In this case during parasite phase of development of maternal sporocysts the generative function is not performed. To the first group therefore trematode species with pedogenetic larvae could be attributed also. The next group embraces species whose miracidia as well as mature generated cells have some undifferentiated cells; thus the parasitic phase of mother sporocyst development acquires restricted proliferative capacity. The third group consists of species with higher trematodes dominating. They perform generative function exclusively at parasitic phase of mother sporocyst development. Representatives of more archaic and ancient species are the bases of the first two groups on the contrary. Such type of distribution can not occur occasionally and apparently reflects first steps of emergenes of parthenogenetic generations of trematodes.     

Lectin and human blood group determinants of Schistosoma mansoni: alteration following in vitro transformation of miracidium to mother sporocyst.

A mixed agglutination assay method was employed to detect the presence of surface determinants for various lectins and human blood group antibodies on Schistosoma mansoni miracidia and cultured mother sporocysts. Miracidia were found to possess surface receptors for the lectins Con A (concanavalin A), anti-Heel (eel serum agglutinin), and anti-ADb (Dolichos seed extract), as well as human anti-A antibodies. Following in vitro transformation of the miracidium to mother sporocyst, anti-Heel and human anti-A receptors were no longer detectable on the sporocyst surface, while determinants for Con A and anti-ADb remained essentially unaltered. It is concluded that transition of the miracidium to the sporocyst results in the alteration of surface molecular structures on schistosome larve. Furthermore, since determinants for Con A, anti-Heel, anti ADb, and human anti-A have been found associated with macromolecules in the hemolymph of the snail Biomphalaria glabrata (Stnislawski et al., 1976), there is now evidence that miracidia and mother sporocysts of S. mansoni and their snail host share molecules with common lectin and human blood group determinants.     

Accumulation of lipids in Schistosoma mansoni sporocysts cultured in vitro

Neutral lipids were detected histochemically in mother and daughter sporocysts of Schistosoma mansoni cultured in vitro. These lipids progressively increased with prolonged culture. There was little phospholipid and no fatty acid, esterases, or lipases found in sporocysts by the methods employed. Mother and daughter sporocysts incorporated labeled acetate from the culture medium but no further information was obtained on the complex lipid-synthesizing capabilities of these organisms.     

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.     

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avian schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.