Snail-conditioned Water Research Articles

Biomphalaria glabrata: Respiration, calcium and end products of carbohydrate metabolism

1. 1. Lactic acid and succinic acid (end products of anaerobiosis) also occur under aerobic conditions in the haemolymph and excretion products of Biomphalaria glabrata. This phenomenon has been investigated in more detail. 2. 2. Experiments on oxygen uptake, and analyses of organic acid, amino acid and calcium were carried out under various aerating conditions, various temperatures and in various water qualities. 3. 3. No differences were found in the concentrations of the organic acids and calcium in the haemolymph under different aerating conditions. 4. 4. Neither snail-conditioned water, nor artificial crowding effects played a role in the initiation of anaerobic respiration. 5. 5. A low exposure temperature (4°C) initiated anaerobic respiration in spite of the aeration.

Behavior of Schistosoma mansoni Miracidia in Gradients and in Uniform Concentrations of Glucose

Roberts et al. (1979, Journal of Parasitology 65: 41-49) showed that Schistosoma mansoni miracidia alter their behavior in response to a gradient of snail conditioned water (SCW), but not in response to uniform concentrations of SCW. Even though SCW is useful for studies of responses of miracidia to chemical stimuli, it often does not provide clear and replicable results. Several attractants have been found in SCW, including short-chain fatty acids (Wilson and Denison, 1970, Comparative Biochemistry and Physiology 32: 511-517), amino acids (Wright and Ronald, 1972, Canadian Journal of Zoology 50: 855-860), ions (Sponholtz and Short, 1976, Journal of Parasitology 62: 155-157) and other substances. However, it is not known which of these substances are important in a natural setting. Furthermore, the concentration and composition of SCW may vary between batches (MacInnis et al., 1974, Nature 248: 361-363; Saladin, 1979, Zeitschrift fur Parasitenkunde 60: 197-210). For more precision than is possible with this natural system, the present study uses a compound of known structure and concentration to confirm and extend the work of Roberts et al. (1979, loc. cit.). Miracidia were obtained from mouse livers and hatched in artificial pond water (APW) as described earlier (Plorin and Gilbertson, 1981, Journal of Parasitology 67: 45-49). D-glucose was chosen for an independent testing of miracidial response, since it has been found in snail mucus (Wilson, 1968, Comparative Biochemistry and Physiology 24: 629-633). To establish the effective range, the testing method of Roberts et al. (1978, Journal of Parasitology 64: 277-282) was used, except that the circular phi-chamber was replaced by a 1.75-inch 10? ellipse (Pickett ellipse guide No. 1225-10) and miracidia were left in the chamber for 60 sec before bisecting the chamber across its minor axis. This provided the same depth and volume as the earlier authors' circular phi-chamber, but with better separation of the 2 sides. A total of 1,783 miracidia were used in this step. The proportion of miracidia on the glucose side of the elliptic phi-chamber was significantly different from 0.5 at concentrations of 8, 10, 12, 14, and 16 mM glucose, but not at concentrations of 4 or 6 mM glucose (significance level is P = 0.05). Linear, logit, and probit regression each produced a significant slope across this range. This demonstrates a miracidial response to gradients of D-glucose, with a threshold level near 7.0 mM glucose. To determine the effect of uniform concentrations, miracidia were hatched in APW containing 2, 4, 6, 7, 8, and 10 mM D-glucose. Within 90 min of the placement of eggs in the glucoseAPW, darkfield photography was used (Plorin and Gilbertson, 1981, loc. cit.) to measure speed, turning frequency, and turning angles for 281 miracidia. Linear, logit, and probit regression were performed on each behavioral parameter, but no significant slopes were found. Although some accommodation to glucose stimulation may be present, the works of Shift and Kriel (1970, Journal of Parasitology 56:281286), and of Mason and Fripp (1976, Journal of Parasitology 62: 721-727) suggest that accommodation would not be entirely complete within the time allowed here. As a result, it can be concluded that Schistosoma mansoni miracidia do not respond to uniform solutions of glucose at concentrations known to elicit responses to gradients. The conclusions of Roberts et al. (1979, loc. cit.) are therefore confirmed for a substance of known composition and concentration, and glucose is added to the list of miracidial attractants.

Amino acid metabolism in the freshwater pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni

1. 1. Free amino acids (FAA) content of hemolymph, digestive gland and remaining tissues as well as the FAA release by individual Biomphalaria glabrata were measured by means of high performance liquid chromatography (HPLC). 2. 2. Primary amine concentrations in hemolymph, digestive gland, remaining tissues and snail conditioned water (SCW) were determined by means of fluorimetric measurements. 3. 3. As compared with uninfected individuals the FAA and primary amine content in hemolymph, digestive gland and remaining tissues of snails infected 33 days prior to sampling was significantly reduced. 4. 4. No significant difference was found between FAA and primary amine losses of parasitized and normal B. glabrata. 5. 5. In hemolymph and tissues of infected snails concentrations of serine and glutamine were particularly reduced whereas levels of lysine, citrulline, arginino-succinate, aspartic acid and glutamic acid remained constant or were slightly increased as compared with uninfected B. glabrata.

Hatching, Chemokinesis, and Transformation of Miracidia of Schistosoma mansoni

Hatching, chemokinesis, and transformation of miracidia of Schistosoma mansoni were examined with a light microscope equipped with a video recording system. Saline, linearly and reversibly, inhibited miracidial hatching and swimming. Both hatching and swimming were inhibited at 4 C and 12 C and accelerated at 34 C relative to rates at 22 C. Hatching was an explosive event that began with ciliary beating when the egg was placed in artificial pond water (APW) and culminated in the parasite's escape from the shell in 100 to 300 msec. Broken egg shells had sharp, complementary edges. Neither miracidia nor eggs swelled prior to hatching. Accumulation of miracidia in a spot of snail conditioned water (SCW) occurred rapidly due to a 60-75% decrease in the exit rate from the spot, rather than by an increase in the entry rate. The turning rate in SCW increased tenfold and the time spent in the spot was 6 times that of controls. Eserine sulfate inhibited miracidial turning and accumulation in SCW. Parasites accumulated in a spot of serotonin by increasing their rate of turning. Miracidia transformed to sporocysts in either complex media containing serum, RPMI-1640, Hanks' salts or phosphate buffered saline, but not in amino acids or vitamins. Transformation was inhibited when miracidia were incubated with serotonin or when miracidia had not been exposed previously to APW.

Philophthalmus gralli: Chemosensitivity of Miracidia

The responses of Philophthalmus gralli miracidia to various chemicals stimulative to other miracidial species were tested by contact and return (CR) to agar blocks containing chemicals and aggregation near a point inoculum in phi-chambers. Of a series of amino acids, glutamic and aspartic acid elicited the greatest responses in both tests, which were similar to that observed with snail-conditioned water. Sialic, acetic, hydrochloric, and sulfuric acids gave high percent CR at all concentrations tested but required higher levels to stimulate a significant miracidial response in phi-chambers. Miracidia were slightly responsive to Mg++ and ammonia in CR tests but only showed a significant reaction to 1.0 mM ammonia in phi-chambers. Attachment responses to the agar blocks by miracidia were noted at the higher concentrations of all chemicals tested except MgSO4 and NH4Cl. At 25 and 50 mM H2SO4 and HCl, contact with the agar block proved lethal to the miracidia. Miracidial behavior was klinokinetic rather than chemotactic in both test systems.

Echinostoma liei miracidia and Biomphalariaglabrata snails: Effect of egg age, habitat heterogeneity, water quality and volume on infectivity

Infectivity of Echinostoma liei miracidia to NIH albino Biomphalaria glabrata declines significantly from 62% with eggs incubated for 10–24 days to 3% for eggs incubated for 30–42 days. In mass exposures of 25 snails to 125 miracidia in 1 liter of water infectivity was high (54–66 %) and not affected by the presence of lettuce, plastic sheets, chalk, detritus or snail-conditioned water. In distilled water or snail-conditioned water the proportion of infected snails exposed singly to five miracidia per snail in 5 ml was not significantly different from the results of mass exposures of 25 snails in 1 liter to the same snail: miracidia ratio. Some evidence is presented suggesting that infected snails are less likely to suffer mortality than uninfected snails during the first 7–10 days post-exposure. The results suggest that Echinostoma liei miracidial searching efficiency is robust in volumes of at least 1 liter and in a heterogeneous habitat. These aspects enhance the competitive potential of echinostomes as possible biological control agents for Schistosoma mansoni.

Effector mechanism in the response of Schistosoma mansoni miracidia to snail‐conditioned water

Schistosoma mansoni miracidia respond to snail-conditioned water (SCW) by sharply increasing their rate of turning when they encounter abrupt decreases in stimulant concentration (Roberts et al., '79). We examined the role of the cilia and the subepithelial muscles in the turning behavior of stimulated miracidia. Several lines of evidence indicated that miracidia do not turn by altering their ciliary beat. Ciliary beating on detergent-treated miracidia was reactivated using solutions containing ATP and Mg2+. These organisms were unable to turn spontaneously, a characteristic of live miracidia. Several divalent cations which stimulate increased turning of intact miracidia failed to support ciliary reactivation of detergent-treated organisms. Further, intact miracidia increased their rate of turning in gradients of Mg2+, but detergent-treated organisms swimming in reactivation solution did not turn in Mg2+ gradients. High speed cinematography of intact miracidia swimming in gradients of SCW or Mg2+ illustrated that turning is invariably accompanied by flexion of the body. This bending occurred only at the transverse interciliary ridges between the first and second, and second and third tiers of ciliated plates. Flexion was not observed at the interciliary ridge between the third and fourth tiers of plates, suggesting that miracidia turn by contracting specific portions of their subepithelial musculature.

The Effects of Miracidial 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.

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.

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.

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.

Analysis of the Movements of Schistosoma mansoni Miracidia Using Dark-Ground Photography

A dark-ground photographic technique was used to study the effects of several physical and chemical conditions on the behavior of Schistosoma mansoni miracidia. Miracidial speed was affected by miracidial aging, temperature, light intensity, and to a small extent by snail conditioned water (SCW). Rates of change of direction were influenced by miracidial aging, sudden increases in light intensity, and by SCW at 20%, 60%, and 100% concentrations. Adaptation to 20% SCW was apparent within 40 min of miracidial contact with the solution.

Responses of Megalodiscus temperatus Miracidia to Amino and Sialic Acids Found in Snail-Conditioned Water

Analysis of snail-conditioned water (SCW) from Helisoma trivolvis revealed 17 free amino acids. Those in great concentration were glycine, serine, and alanine. The concentration of sialic acid was found to be twice that of the most abundant amino acid. The behavior of miracidia of Megalodiscus temperatus, measured by the contact with return method, to agar cylinders containing single amino acids and sialic acid indicated greater responses to polar molecules charged either positively or negatively at neutral pH. The molecules elicting the greatest response were aspartic, glutamic, and sialic acid. No correlation was found between concentration of amino acids in H. trivolvis SCW and response of M. temperatus miracidia.

Schistosoma mansoni Miracidial Behavior: An Assay System for Chemostimulation

A new system for evaluating the responses of miracidia to chemostimulants is described. The apparatus consists of a translucent plastic block with a center well and a hole in the edge leading to the well. One end of a glass tube, covered with a dialysis membrane, was inserted into the hole. Experimental solutions to be tested were put into the tube and Schistosoma mansoni miracidial behavior was observed in the well on the other side of the permeable membrane. Miracidia were released near the membrane; those which contacted the membrane were scored as to whether they returned (contact with return) or did not return (contact without return) before leaving the field of view. Materials eliciting significantly more contact with return responses than did controls were considered to be stimulatory. In this assay system, snail (Biomphalaria glabrata) conditioned water elicited 75% contact with return as compared to 8% for well water control (P less than 0.05). Tracings from motion pictures showed swimming behavior of miracidia toward snail-conditioned water to be different from behavior toward well water controls. This system permits generation of dilution response curves for chemicals and provides generally quantitative results.

Identification of chemicals of snail origin that attract Schistosoma mansoni miracidia.

MEASURES to control schistosomiasis (bilharziasis) aimed at either the molluscan vector or the trematode's larval stages might be enhanced by a better understanding of the intricacies of how miracidia and cercariae find and penetrate hosts. Previous work from our laboratory identified chemicals from mammalian skin that initiated penetration responses by Schistosoma mansoni cercariae1. Other earlier studies implicated amino acids and additional compounds as attractants of S. mansoni miracidia to the molluscan host, but did not show that these substances were derived from the snail2. Subsequently, based on Neuhaus'3 and C. A. Wright's4 indications that snails release substances into water that stimulate miracidia, several investigators5–8 have confirmed that snails ‘condition’ water making it stimulatory or attractive to miracidia. Wright and Ronald9 recently demonstrated that water conditioned by the snail Lymnaea palustris attracted miracidia of the rodent blood fluke, Schistosomatium douthitti, and that this snail-conditioned water (SCW) contained amino acids. Here we show that Biomphalaria glabrata, the molluscan vector of S. mansoni, the important parasite of humans, contributes amino acids to the aqueous environment, and that these, and possibly other substances elicit chemotactic and chemokinetic responses by miracidia of this parasite.

Penetrative Activity of Schistosoma mansoni Miracidia Stimulated by Exposure to Snail-Conditioned Water

Penetrative Activity of Schistosoma mansoni Miracidia Stimulated by Exposure to Snail-Conditioned Water