Stimulation of the activity of Schistosoma mansoni miracidia by snail-conditioned water.

A dark-ground photographic technique was used to analyse the reactions of Schistosoma mansoni miracidia to homogeneous solutions of snail-conditioned water (SCW). The most significant effect of this water was to increase miracidial turning. This effect was maintained under both acid and alkaline conditions, after passage of the SCW through a mixed bed resin and after chelation of either calcium or both calcium and magnesium ions. The stimulant in the water was unaffected by trypsin but was protease-sensitive, suggesting its possible identity as a peptide. The importance of 'active spaces' rather than concentration gradients in miracidial host-location was emphasized.     

The reactions of Schistosoma mansoni miracidia to light.

The reactions of Schistosoma mansoni miracidia to artificial light were studied by direct observations, choice-chambers and a photographic technique, at room temperature and at 15 C. The miracidia responded positively to directed illumination, but did not aggregate in a lighted dish. Photography indicated an orthokinetic response to light intensity, but sharp changes in light intensity had no detectable effect on miracidial behavior. At the lower temperature the response to a directed light beam was almost completely inhibited and the orthokinetic response could no longer be demonstrated.     

A simple quantitative technique for testing behavioral responses of Schistosoma mansoni miracidia to chemicals.

We describe a new technique for testing responses of Schistosoma mansoni miracidia to chemicals. Miracidia in spring water were placed in a chamber shaped like the Greek letter phi. Small volumes of test chemicals were inoculated into one side of the chamber. After 30 sec a dam was inserted to bisect the chamber and the percentage of miracidia on the inoculated side was calculated. Reproducible quantitative results were obtained using the known miracidial stimulants, snail-conditioned water (Biomphalaria glabrata) and Mg2+; up to 80% of the miracidia were recovered on the inoculated side of the chamber. Other substances also stimulated miracidia: several inorganic cations, 4 neurotransmitters, 3 acetycholine antagonists, and 1 acetycholine agonist. Modifying the technique by testing stimulants in altered chemical "backgrounds" allowed us to test for inhibitors of miracidial responses. Assays of the Mg2+ content of several of the test solutions indicated that their stimulatory or inhibitory effects could not be ascribed to Mg2+ contamination. However, results obtained with neurotransmitters and drugs were not sufficiently consistent to implicate specific neurotransmitters in the mechanism by which miracidia detect and respond to stimulants in snail-conditioned water.     

The effects of miracidal aging and dilution of snail-conditioned water on responses of miracidia of Megalodiscus temperatus.

Miracidia of Megalodiscus temperatus from newly hatched until 10 hr old were tested for their ability to react to Helisoma trivolvis snail-conditioned water (SCW) by contact with return (CR) to agar blocks and by percentage of miracidia reacting to a point inoculation of SCW as determined by a photographic time exposure method. CR to agar blocks containing 1:50 SCW was greatest during the first 6 hr after hatching but declined thereafter. The reaction during the first hour to a point inoculation was lower than during the 2nd and 3rd hr. Results were variable from 4 to 10 hr after hatching with the lowest response recorded from 9 to 10 hr. Miracidial responses to dilutions of SCW were assessed by the same two methods. CR to agar blocks containing decreasing concentrations of SCW declined until at a dilution of 1:500 CR was only slightly above the controls. On the other hand, miracidial reactions to point inoculations of SCW as determined by the photographic method were still apparent at a dilution of 1:25,000, when 12% of the miracidia tested reacted. Thus, the photographic time exposure method gives a sensitive means for detecting altered miracidial behavior to various intrinsic and extrinsic factors.     

Location of Biomphalaria pfeifferi by Schistosoma mansoni miracidia in stagnant and running water under field conditions

The efficiency of S. mansoni miracidia in locating and infecting Biomphalaria pfeifferi in Gezira canals has been studied under field conditions. When S. mansoni eggs were introduced into clean stagnant water in small field channels, the miracidia hatched to infect 100% of 30 snails in cages at the release point. Fifteen metres upstream and downstream 13% of caged snails were infected but no infections were found in snails 20 m away.When eggs were released into the same canal in flowing water (8.3 cm · s^–1), no infections were detected in any of the caged snails placed 0–100 m downstream. Releasing hatched miracidia instead of eggs resulted in infections in all cages at 5 m intervals from 0-100 m. The release of eggs into flowing water was likened to the method by which S. haematobium eggs are deposited during urination. The 0% infection suggests that eggs will be swept away from the point of contamination by the flow. Thus only urination into stagnant water will lead to heavy snail infection rates.When eggs were released into a small pond-like minor canal tail end snail infection rates were only 3%. This was probably due to the larger water volume, smaller number of caged snails, and the presence of vegetation and other fauna which may be decoys or predators.The results highlight how very high snail infection rates can be produced under ideal conditions but also show how large snail and miracidia numbers are required in natural situations.     

Schistosoma mansoni: activity responses in vitro to praziquantel.

The effects of praziquantel, a novel antischistosomal compound, on the activity of adult Schistosoma mansoni (Liverpool strain) in vitro were investigated. Worm activity was modified at all concentrations of praziquantel. High concentrations (above 0.5 microgram/ml) produced rapid paralysis, whilst low concentrations of praziquantel stimulated worm activity. It is concluded that such activity modification could lead to worm displacement in vivo.     

Ultrastructural study of the in vitro interaction between Biomphalaria glabrata hemocytes and Schistosoma mansoni miracidia

Biomphalaria glabrata and Schistosoma mansoni relationship was studied by light microscopy (LM) and freeze-fracture replica technique (FFR). We observed very thin cytoplasmic extensions of hemocytes in the LM, which then surround immobilize the miracidia. FFR images showed that the contact site between hemocytes cytoplasmic extensions and the external tegumentary coat involved only superficial layers of miracidia. Numerous vacuoles and filopodia were observed in the hemocyte cytoplasm, the latter binding with those from neighboring cells. In spite of the close interfilopodia contact, no cellular junctions were seen at these sites nor between filopodia-miracidia contact areas. The observed migration of hemocytes and their disposition in layers surrounding the miracidia in vitro correspond to previous studies.     

Mitotic responses to extracts of miracidia and cercariae of Schistosoma mansoni in the amebocyte-producing organ of the snail intermediate host Biomphalaria glabrata

Suspensions of miracidia and cercariae of Schistosoma mansoni were subjected to repeated freeze-thaw cycles and then injected into resistant Salvador strain Biomphalaria glabrata snails. A pronounced increase in the number of mitotic figures, relative to uninjected, sham-injected, or diluent (water)-injected controls, was observed in the amebocyte-producing organ (APO) at 3 days postinjection (PI). After centrifugation of miracidia freeze-thaw extract (FTE), the resulting supernatant (FTS) and pellet possessed equal stimulatory activity that was approximately half that seen with FTE. Ultracentrifugation of miracidia FTS resulted in a supernatant that retained full activity, indicating a soluble molecule. Heat treatment of miracidia FTE reduced but did not eliminate activity, suggesting a nonprotein active component. Concentration or dilution of FTS by a factor of 10 gave a nonlinear dose-response relationship. Susceptible NIH albino snails injected with miracidia FTE had increased mitotic activity in the APO, which was much less than that seen in Salvador snails, whereas injection of miracidia FTE into Helisoma duryi had no discernable effect. Measurement of mitotic activity as a function of time PI showed no increase in numbers of mitotic figures in the APO at 18 hr but a large increase at 24 hr PI. Mitotic activity returned to preinjection levels by 96 hr PI, although a subsequent increase occurred at 120 hr PI.     

Resistance to desiccation of Biomphalaria glabrata adults infested by various miracidia of Schistosoma mansoni

1200 adult Biomphalaria glabrata were submitted during 6 weeks to anhydrobiosis condition. Some snails were healthy, some were previously infected 3 days or 12 days ago with 8 +/- 2 miracidia of Schistosoma mansoni, others were shedding cercariae. The snails were put on soil or buried into hermetically closed, or ventilated, plastic boxes. There was no survival of snails kept in sealed boxes, or among positive snails, but 44% of control healthy snails and 40.6% of infected (for 3 or 12 days) snails in ventilated boxes were living at the term of the desiccation stage. Survival was better for "on soil" snails than for "buried" snails, but no difference was shown between 3-days and 12-days infection. The surviving desiccated B. glabrata had a lesser death rate and a lesser cercarial production than infected snails kept in water. An inferior production of male cercariae comparatively to female and to "mixed" cercariae was demonstrated by statistical analysis of the cercarial sheddings. In all positive snails, periodic variations of cercarial production was shown, whatever the sex of those cercariae. In addition many pauses of the sheddings were established by the authors.     

The invasion of Biomphalaria pfeifferi by Schistosoma mansoni miracidia and the development of daughter sporocysts

Laboratory experiments have been carried out to determine the susceptibility of Gezira Biomphalaria pfeifferi snails to S. mansoni miracidia and the relationship between miracidia and daughter sporocyst production at the 10–17 day development stage. The relationship between snail numbers, miracidia numbers and water volume has also been studied. Two non susceptible snails, Bulinus truncatus and Cleopatra bulimoides, both of which occur naturally in Gezira canals, were tested to see if they act as decoys for S. mansoni miracidia.The results showed that the B. pfeifferi are 100% susceptible to S. mansoni invasion, at least to the daughter sporocyst development stage. The more miracidia that penetrated the more daughter sporocysts were produced, however individual variation and overlap were great. When one miracidium was released to find one snail it succeeded in low water volumes (5 m, 50 ml), but failed in 5 litres. When 100 miracidia were released mortality of snails was high suggesting superinfection particularly when only one or five snails were available. Among survivors daughter sporocyst counts were very high. Cleopatra and Bulinus snails do have a decoy effect when present in large numbers. In their presence the number of infected snails was marginally reduced and the number of daughter sporocysts greatly reduced. However, if superinfection is reduced by decoy effect, it is conceivable that Biomphalaria may be protected by decoy snails in circumstances where miracidia counts are high.     

IgM antibodies recognizing carbohydrate epitopes shared between schistosomula and miracidia of Schistosoma mansoni that block in vitro killing

A series of monoclonal antibodies (mAb) was raised in mice against Schistosoma mansoni, which recognized a carbohydrate determinant on a major Mr greater than 200,000 schistosomulum surface antigen. These mAb cross-reacted with the surface of cercariae and miracidia and with schistosomula of S. haematobium and S. bovis. Other mAb were generated that only recognized a Mr 20,000 schistosomulum surface antigen; they did not cross-react with eggs or miracidia and were species specific. The anti-Mr 20,000 mAb of the IgG1 isotype exhibited high levels of complement-dependent cytotoxicity to schistosomula in vitro. IgM mAb that recognized carbohydrate epitopes of the Mr greater than 200,000 surface antigen blocked the lethal activity of the anti-Mr 20,000 mAb. The IgM anti-Mr greater than 200,000 mAb also reduced complement-dependent cytotoxicity of serum from mice vaccinated with irradiated cercariae.