Behavioral responses of Schistosoma mansoni miracidia in concentration gradients of snail-conditioned water.

Movement patterns of Schistosoma mansoni miracidia were examined in several concentrations and gradients of snail-conditioned water (SCW). Miracidia surrounded by uniform concentrations of SCW swam at the same speed and exhibited the same rate of turning (angular velocity) as did control miracidia swimming in spring water. However, miracidia in gradients of SCW exhibited a 3-fold increase in their angular velocity without altering their swimming speed. Miracidia ascending gradients of SCW did not increase their angular velocity and failed to orient to the gradient of the stimulant. In contrast, miracidia which encountered sufficiently abrupt decreases in SCW concentration, while descending the gradient, sharply increased their angular velocity. This behavior caused miracidia to remain in regions of high concentration of stimulant. The magnitude of decrease in SCW concentration needed to evoke this response depended on the absolute concentration of SCW. Thus, the miracidial response is a "boundary reaction", a form of chemoklinokinesis, and not a chemotaxis.     

The longevity and hatchability of Philophthalmus megalurus and P. gralli miracidia in different environmental conditions.

The effects of salinity, pH, and temperature on the longevity and hatchability of miracidia of Philophthalmus megalurus and P. gralli were determined. Miracidia of both species are able to hatch and survive at saline concentrations much above physiological levels, although these processes are reduced in 2.0--2.4% saline and completely inhibited at 2.6%. The greatest hatching rates for both species were found near neutrality (pH 6--8) but some miracidia hatched at the extreme pH levels of 3 and 12. Philophthalmus megalurus miracidia exhibited longer half-lives under acid conditions (pH 2--6) than P. gralli miracidia; conversely, P. gralli miracidia showed longer half-lives in alkaline conditions (pH 8--11). Hatching and longevity were much greater below room temperature (5--20 C) than above (30--50 C) for miracidia of both species. Temperatures above 50 C proved lethal for eyefluke eggs. Except in acid pH, P. gralli miracidia showed longer half-lives than miracidia of P. megalurus. Comparison to studies on schistosomes revealed that the inhibitory effects of physiological saline and host body temperature on the hatching process of schistosome eggs does not occur in these 2 species of eyeflukes.     

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.     

Chemosensitivity of Philophthalmus megalurus (Trematoda) miracidia

Miracidia of the eyefluke Philophthalmus megalurus were tested in phi-chambers to determine if they reacted similarly to chemicals found stimulative for miracidia of Philophthalmus gralli, a closely related species. Philophthalmus megalurus miracidia were less responsive than P. gralli to the dicarboxylic amino acids and showed a significantly positive response only to 10 mM glutamic acid. These P. megalurus miracidia were chemonegative to 10 mM HCl and H2SO4, chemicals to which P. gralli miracidia gave a significantly positive response. Ammonia and Mg2+ did not elicit any response from P. megalurus miracidia.     

The involvement of cyclic adenosine monophosphate in the control of schistosome miracidium cilia

This study examined the possible involvement of cyclic adenosine monophosphate (cAMP) in the control of ciliary action of Schistosoma mansoni miracidia. Miracidia immobilized in hypertonic NaCl solution were treated with 3 compounds that are known to increase intracellular cAMP concentrations. Forskolin, at a concentration of 50 microM, induced 50.1% of the miracidia to swim in hypertonic solution. The corresponding values obtained for 3-isobutyl-1-methylxanthine (IBMX) at 1 mM and 8-bromo-cAMP at 10 mM were 42.2 and 50.4%, respectively. The motility-enhancing effect of these compounds was dose dependent. Nevertheless, the swimming speed of miracidia activated in this way was only 10% of that observed in artificial pond water (APW). Cholera toxin had no apparent effect on miracidia swimming in hypertonic NaCl solution. Likewise, swimming in APW treated with forskolin at 50 microM, IBMX at 1 mM, or 8-bromo-cAMP at 10 mM did not induce any apparent change in motility. Miracidia swimming in APW were then treated with 3 compounds that decrease the intracellular concentration of cAMP. MDL-12,330A, at a concentration of 250 microM, caused a dramatic decrease in swimming over a period of 1 hr. Likewise, SQ22536 and imidazole, at concentrations of 20 and 50 mM, respectively, caused 36.5 and 73.4% decreases in swimming under the same conditions. Finally, inhibitors of cAMP-dependent protein kinase, i.e., PKI(14-22)amide, H89, and H88, completely inhibited miracidia swimming in APW at concentrations of 25, 50, and 100 microM, respectively. These results suggest that cAMP and cAMP-dependent protein kinase are involved in osmosis-controlled ciliary motion of schistosome miracidia.     

Echinostoma friedi: the effect of age of adult worms on the infectivity of miracidia

The effect of ageing of adults of Echinostoma friedi (Trematoda: Echinostomatidae) on the infectivity of miracidia yielded was analysed. Miracidia were obtained after hatching of eggs obtained from adult worms of E. friedi collected weekly during the course of experimental infections in golden hamsters. Miracidial infectivity, measured in terms of percentage of infection in Lymnaea peregra, was significantly influenced by the age of the adult worms from which the miracidia were derived. Infective miracidia only were obtained from adult worms in the age range from 4 to 9 weeks post-infection. Infectivity was maximal in those miracidia derived from adults collected 8 and 9 weeks post-infection. The results suggest that adult worms producing viable eggs require additional maturation to be able to yield eggs containing infective miracidia.     

Location of Biomphalaria glabrata (Say) by miracidia of Schistosoma mansoni Sambon in natural standing and running waters on the West Indian Island of St. Lucia

Upatham E. S. 1973. Location of Biomphalaria glabrata (Say) by miracidia of Schistosoma mansoni Sambon in natural standing and running waters on the West Indian Island of St. Lucia. International Journal of Parasitology3: 289–297. The ability of S. mansoni miracidia to locate B. glabrata in natural ditches and streams was investigated. Miracidia located and infected snails at distances of 9–14 and 97-54 in horizontally in standing and running waters respectively. In running water, no infection occurred above a velocity of 13.11 $\text{cm}\text{sec}$. In both types of habitat, infection rates in snails increased with increasing levels of miracidia but decreased as the location of caged snails moved away from the miracidial point of entry. Laboratory experi- ments showed that the number of daughter sporocysts was proportional to the number of miracidia. Judging by the number of daughter sporocysts recovered only a small percentage of miracidia succeeded in locating and penetrating snails (6.8–13-7 % and 1.4–6.2 % in standing and running waters respectively). In standing water, infection may be inhibited by the limited ability of miracidia to move horizontally. In running water, the flow extends significantly the effective scanning capacity of the miracidia, giving them a better chance of coming into contact with snails, which is of importance in the epidemiology of schistosomiasis. Owing to a con- siderable wastage of miracidia and the higher relative efficiency of miracidia at lower densities in detecting snails, control measures such as chemotherapy or provision of safe water supplies designed to lower egg output and reduce contamination may not seriously influence transmission unless S. mansoni egg production or contamination is massively reduced.     

Miracidia of an Egyptian strain of Schistosoma mansoni differentiate between sympatric snail species

The host-finding behavior of miracidia of 2 strains of Schistosoma mansoni from Egypt and Brazil was studied by recording their responses to snail-conditioned water (SCW) from the Egyptian sympatric snails, Biomphalaria alexandrina, Physa acuta, Lymnaea cailliudi, and Balinus truncatus, as well as from Biomphalaria arabica and Biomphalaria glabrata. Miracidia of the Egyptian strain significantly preferred SCW from their compatible hosts B. alexandrina and B. arabica and showed no or a weak response to SCW from the other sympatric species, whereas miracidia of the Brazilian strain did not differentiate between SCW from different snail species.     

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts

Miracidia (and adults) of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA encoding enhanced green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP70) promoter and termination elements were shown to express the reporter gene. Bombarded miracidia were able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post-infection by RT-PCR in snail tissues, but not by microscopy or Western blot which probably reflected sub-optimal expression levels of constructs. Our findings indicated that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.     

Schistosoma mansoni: degradation of host extracellular matrix by eggs and miracidia

A radioactively labeled in vitro model of the extracellular matrix of the mammalian intestinal wall and of snail tissue was used to determine whether proteolytic enzymes released by eggs and miracidia of Schistosoma mansoni could degrade connective tissue macromolecules in the type of interactive framework found in vivo. Eggs were collected and miracidia hatched in the presence of antibiotics to eliminate bacterial contamination. Uninfected livers were used as controls to ensure that the tissue dissociation and egg collection procedures did not produce proteolytic activity. One thousand live eggs incubated with the extracellular matrix for 72 hr at 37 C degraded 31% of the glycoprotein in the matrix; there was no degradation of elastin or collagen. Medium conditioned by incubation with eggs degraded 60% as much of the matrix as the live eggs themselves. The proteolytic activity of the egg-conditioned medium was greater in the presence of dithiothreitol. Miracidia incubated with the extracellular matrix in tissue culture medium at 27 or 37 C rapidly transformed to living sporocysts. This transformation was accompanied by a release of proteolytic activity, resulting in the degradation of 49 to 58% of the glycoprotein in the extracellular matrix by 1000 miracidia. Again, no elastin or collagen was degraded. The time course of degradation by miracidia was rapid over 24 hr and thus similar to that previously reported for cercariae. Degradation by eggs occurred more slowly over 72 hr. These data confirm that both eggs and miracidia secrete proteinases which are capable of degrading at least the glycoprotein components of extracellular matrix to facilitate their migration through intestinal wall or penetration of snail tissue.     

Escape of rediae from miracidia of Philophthalmus megalurus and Philophthalmus gralli during in vitro culture

Miracidia of the eyeflukes Philophthalmus megalurus and Philophthalmus gralli contain a preformed larval stage that was identified as a redia by electron microscopy and, on living forms, by the presence of ambulatory buds and an oral opening. This redial stage escaped and actively moved about when the miracidium stopped swimming in pond water. No redial escape was observed in NaCl solutions even though miracidia became immobile, but escape was noted in Hanks' balanced salt solution (HBSS) after 7 hr of exposure. When miracidia were placed in RPMI-1640 and Eagle's minimum essential medium, rediae escaped much earlier than in pond water and HBSS. Redial escape in vitro will provide a good source of material to initiate cultures of this larval stage.     

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.     

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.     

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.     

Schistosomatium douthitti: effects of Lymnaea catascopium age on susceptibility to infection

Laboratory-reared Lymnaea catascopium snails (1–269 days old) were exposed individually to different numbers of Schistosomatium douthitti miracidia. Increasing the exposure dosage from 3 to 10 miracidia generally increased infection rates, in some age classes up to 100%. Successful re-exposure of snails not infected after a primary exposure was possible. Neonatal snails were least likely to become infected, primarily because miracidia were not attracted to them. Snails 12–55 days old were most susceptible to infection. Miracidia were readily attracted to these snails, and many were ingested and subsequently penetrated the host esophageal wall. Miracidial penetration of external snail surfaces was rare. Susceptibility of older snails (65–269 days) progressively declined with age. Many miracidia were entangled and immobilized in mucus produced by these snails, and fewer were ingested. No conspicuous host cellular responses to mother sporocysts were observed in any of the snails sectioned. A comparison of susceptibility of deliberately stunted snails and comparably aged controls of normal size indicated that the former were more susceptible.     

Development of Fasciola hepatica in Lymnaea columella infected with miracidia derived from cattle and marmoset infections

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.     

RESULTS OF IRRADIATING SACCHAROMYCES WITH MONOCHROMATIC ULTRA-VIOLET LIGHT : II. THE INFLUENCE OF MODIFYING FACTORS

Possible variation in the probability that absorbed quanta of ultra-violet energy will produce observable inhibitory and lethal effects in the yeast cell, due to non-uniformity in sensitivity of the different regions of the cell, may be further modified by the reproductive stage of the cell at the time of irradiation. Tests of the survival of yeast cells of 15 day and 24 hour cultures indicate that the older resting cells are more resistant to ultra-violet irradiation effects than cells undergoing rapid cell division. The effects of temperature changes within the range of normal growth are evidently small, as judged from the temperature coefficient (1.10). Possible inhibitory effects due to the action of ultra-violet radiation on the malt agar medium and to toxic substances diffused from cells killed by irradiation were not found under the conditions of the experiments. Tests of the validity of the Bunsen-Roscoe reciprocity law for variation in the intensity of the incident ultra-violet radiation up to 30 per cent indicate that for this range the rate of absorption of quanta by the cell does not produce any marked change in the lethal effects observed.     

Single and multiple worm infections of Echinostoma caproni (Trematoda) in the golden hamster

Six of 10 hamsters fed a single metacercarial cyst of Echinostoma caproni (single-worm infections) and 13 of 19 hamsters fed either 2 or 5 cysts (multiple-worm infections) were infected with adult echinostomes at necropsy 22 days post-infection. Considerable histopathological changes to the small intestine occurred in hamsters carrying single-worm infections. There were no differences in either mean length, width or wet weight of echinostomes in single- versus multiple-worm infections. The mean number of eggs/worm from single-worm infections (525) was significantly greater than that from multiple-worm infections (288). The average percentage of fully developed miracidia/worm from single worms (94%) was similar to that from worms in multiple infections (92-95%). Single worms of E. caproni were capable of self-fertilization and production of viable eggs. Miracidia derived from single worms were as capable of infecting laboratory-reared Biomphalaria glabrata and producing patent rediae as were those from multiple infections.     

Scanning electron microscope observations on the miracidium of Schistosoma

Miracidia of two species of Schistosoma, viz. haematobium and japonicum, were studied with the scanning electron microscope to more clearly visualize what is seen with the light microscope and the transmission electron microscope, and more specifically, to relate the structure of the apical papilla to its function in snail penetration.The apical papilla of schistosome miracidia is composed of corrugated areas which form tiny suckerlike cups, presumably used by the miracidium to facilitate attachment to the snail during penetration. A lateral opening (secretory pore) on the apical papilla and short stubby apical cilia (tactile or sensory) are also demonstrated.     

Host choice and penetration by Schistosoma haematobium miracidia

Schistosome parasites commonly show specificity to their intermediate mollusc hosts and the degree of specificity can vary between parasite strains and geographical location. Here the role of miracidial behaviour in host specificity of Schistosoma haematobium on the islands of Zanzibar is investigated. In choice-chamber experiments, S. haematobium miracidia moved towards Bulinus globosus snail hosts in preference to empty chambers. In addition, miracidia preferred uninfected over patent B. globosus. This preference should benefit the parasite as patent snails are likely to have mounted an immune response to S. haematobium as well as providing poorer resources than uninfected snails. Miracidia also discriminated between the host B. globosus and the sympatric, non-host species Cleopatra ferruginea. In contrast, S. haematobium did not discriminate against the allopatric Bulinus nasutus. Penetration of the host by miracidia was investigated by screening snails 24 h after exposure using polymerase chain reaction (PCR) with S. haematobium specific DraI repeat primers. There was no difference in the frequency of penetration of B. globosus versus B. nasutus. These responses to different snail species may reflect selection pressure to avoid sympatric non-hosts which represent a transmission dead end. The distribution of B. nasutus on Unguja is outside the endemic zone and so there is less chance of exposure to S. haematobium, hence there will be little selection pressure to avoid this non-host snail.