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.     

Studies on resistance in snails. 5. Tissue reactions to Echinostoma lincloense in naturally resistant Biomphalaria glabrata.

Laboratory-raised juvenile albino Biomphalaria glabrata snails show a wide range of natural resistance to a single infection with 50 or 100 miracidia of Echinostoma lindoense. In the most resistant snails all sporocysts are destroyed in peripheral tissues soon after miracidial penetration. In less resistant snails some sporocysts reach the heart where they are encapsulated. In fully susceptible snails, all sporocysts rapidly migrate to the heart, where they mature and continue to develop. The greater part of our B. glabrata colony consists of snails in which sporocysts reaching the heart will survive, but in which a varying number of sporocysts will be destroyed in the tissues. These snails are usually considered susceptible, as they do become infected. Tissue reactions induced by sporocysts following a single infection in naturally resistant snails are similar to reactions in snails with an acquired resistance. In fully susceptible snails, the amebocyte-producing organ remains small and inactive. It is slightly to moderately stimulated in partially resistant snails in which destruction of sporocysts occurs in the tissues and surviving larvae are found in the ventricle. In snails in which amebocyte aggregates or capsules develop in the ventricle, the organ becomes markedly enlarged. Migration of sporocysts in the snail appears not to be continuous, as periodic rests seem to occur. Migration follows intrusion of the sporocyst through the tissues, induced by bodily distension and contraction, and then proceeds within the arteries against the blood flow, passing from one endothelial attachment site to another, possibly aided by negative pressure during ventricular diastole.     

Effect of miracidial dose on adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host, Biomphalaria glabrata

Adoptively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata was measured as a function of miracidial challenge dose. Schistosome-susceptible snails implanted with the amebocyte-producing organ (APO) from resistant donors showed 29 and 39% prevalences of infection after challenge with 5 and 10 miracidia, respectively, but 68-83% prevalences when exposed to 25-200 miracidia. Prevalences in control (untampered) susceptible snails ranged from 97 to 100% at the different miracidial doses. Higher infection prevalences at elevated doses suggest that a range of transferred resistance occurs and possibly that low levels of APO-derived plasma factors or hemocytes in some recipients can be overwhelmed by larger numbers of parasites.     

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.     

Athletic aids: fact or fiction?

A major problem facing physicians involved in the daily care of amateur athletes in international competition is that of so-called doping. While there are many substances used that might be considered ineffectual or physiologic, the two main categories of substances considered as doping agents are stimulants and anabolic steroids. No substance is as yet known that will improve an athlete''s performance. Often such substances are taken in error rather than in a misguided attempt to improve one''s ability. These substances can be dangerous and, because of this, doping rules were established basically to protect the athlete. Anabolic steroids are particularly dangerous; they usually are taken by those engaged in lifting or throwing sports in an attempt to improve performance by increasing muscle bulk. There is as yet no scientific proof that performance is improved. Dope testing for stimulants should be carried out in a quantitative rather than qualitative manner so that the athlete who has taken a small amount of a so-called stimulant (such as an asthma or common cold preparation) would not be excluded from competition. Rigid testing for anabolic steroids should be continued.     

Effects of diet on the lipid composition of the digestive gland-gonad complex of Biomphalaria Glabrata (Gastropoda) infected with larval Echinostoma Caproni (Trematoda)

This study examined the effects of a larval Echinostoma caproni infection on the neutral lipid composition of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata snails fed hen's egg yolk supplemented with lettuce (Y-L) or lettuce supplemented with Tetramin (L-T). Snails were experimentally infected with the miracidial stage of this echinostome, and their DGGs containing daughter rediae were analyzed for neutral lipids five weeks post-infection by qualitative and quantitative thin-layer chromatography. Light microscopy using Oil Red O (ORO) staining and transmission electron microscopy (TEM) were used to localize neutral lipids in the rediae. The DGGs of infected snails maintained on the Y-L diet showed a significant increase in free sterols and a significant decrease in triacylglycerols compared to uninfected snails maintained on the Y-L diet. The DGGs of infected snails maintained on the L-T diet showed no significant difference in free sterols or triacylglycerols compared to uninfected snails maintained on the L-T diet. ORO staining and TEM showed the presence of lipid droplets in rediae from snails on the Y-L diet. The significant decrease in triacylglycerols in the DGGs of infected snails maintained on the Y-L diet suggests that triacylglycerols were utilized by the rediae.     

Schistosoma mansoni: influence of Biomphalaria glabrata size on susceptibility to infection and resultant cercarial production

Biomphalaria glabrata snails of the same age, but different sizes, were used to determine size-related susceptibility to Schistosoma mansoni miracidial infection and the influence of snail size on total cercarial production. Snails with shell diameters from less than 5 to greater than 17 mm were individually exposed to one or several miracidia, depending on the experiment. In snails exposed to multiple numbers of miracidia, the percentage of snails which developed patent infections was lower in snails with larger shell sizes. This was also reflected by fewer primary sporocysts per infected snail found in tissues of the larger snails. Upon determining cercarial production in these groups over a 1-month period there were no statistical differences between any groups in the numbers of cercariae produced per snail. However, upon determining the number of successful primary sporocysts found in cohort snails of each size group, cercarial production increased as a function of the number of successful primary sporocysts. This was verified by examining cercarial production in various size snails with known monomiracidial infections. Our data therefore confirm and extend earlier work using snails infected with unknown numbers of miracidia and clearly show that total S. mansoni cercarial development and decreased susceptibility of snails is a direct reflection of snail size and not necessarily age of the snail.     

Studies on resistance in snails: A specific tissue reaction to Echinostoma lindoense in Biomphalaria glabrata snails

In juvenile albino Biomphalaria glabrata snails exposed for the first time to Echinostoma lindoense miracidia, and observed to be resistant, the sporocysts migrated to the heart at the same speed as they did in susceptible snails. However, in resistant snails the sporocysts were soon destroyed in the heart by amebocyte clumps. When these snails were then re-exposed to miracidia of the same species of trematode, the sporocysts were quickly destroyed soon after miracidial penetration, chiefly in the head-foot region. This strongly accelerated tissue reaction appears to have been induced by the previous contact with the same parasite. The sensitization of the snail tissues was highly specific: the hosts remained susceptible to Schistosoma mansoni and Paryphostomum segregation (Echinostomatidae), although partial resistance was observed against Echinostoma paraensei and E. liei, which are closely related to E. lindoense.     

Schistosoma mansoni infections in neonatal Biomphalaria glabrata snails.

In areas endemic for schistosomiasis, the population dynamics of the snail intermediate hosts have a direct effect on parasite transmission. The present study focused on the potential for neonatal Biomphalaria glabrata snails to become infected with Schistosoma mansoni and to produce cercariae under various conditions. It was found that snails as small as 0.74 mm in shell diameter could survive miracidial penetration and could release cercariae when as small as 1.6 mm in diameter. Cercariae produced by small snails were equally infectious for mice when compared with those shed by larger snails. Likewise, histological examination of neonatally exposed snails revealed normally developing parasites at all stages of infection. It was found that in 2 snail populations expressing either high or low susceptibility to the parasite, peak susceptibility occurred at 25 days of age in both groups. Daily cercarial production for neonatally exposed snails was initially low but increased dramatically as the snails grew, eventually reaching values as high as 2,100 cercariae/snail/day. A moderate to high percentage of snails infected as neonates was eventually capable of simultaneously producing both eggs and cercariae. These studies emphasize the potential importance of neonatal and preadult snails in helping to maintain foci of S. mansoni infection in endemic areas.     

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.     

Influence of Echinostoma paraensei (Lie and Basch, 1967) infection on the calcium content in Biomphalaria glabrata (Say, 1818)

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4 weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used.     

Effects of environmental calcium on fecundity and cercarial production of Biomphalaria glabrata (Say) infected with Schistosoma mansoni Sambon

Biomphalaria glabrata were reared in stock culture and subjected to either 7-day or 60-day acclimation periods in complex CaCO3 media with calcium values ranging from 1.5 mg/L to 75 mg/L. Following 60-day acclimation, snails from series I were each exposed to 8 miracidia of Schistosoma mansoni. Snails of series II were each exposed to a single miracidium. Snails of both experimental regimens were observed for mortality, growth, rate of infection, and number of cercariae shed. Series I snails were also monitored for fecundity during acclimation and following miracidial exposure. Calcium levels of 1.5 and 75 mg/L resulted in significant snail mortality. Shell growth and rates of infection were positively correlated with calcium maintenance level. Snails with high fecundity prior to miracidial exposure subsequently shed more cercariae. In contrast, post-exposure (PE) fecundity of snails reared in media with up to 30 mg/L Ca++ were negatively correlated with calcium level, infection rate, and number of cercariae shed. Maximal cercarial emergence occurred at 30 mg/L Ca++. These results suggest that environmental calcium affects both the distribution patterns of snail hosts of human schistosomes and the productivity of intramolluscan schistosome infection.     

Experimental life history of Echinostoma cinetorchis

The life history of Echinostoma cinetorchis was completed in the laboratory using Hippeutis cantori as the first and second intermediate host. The incubation for maturation of eggs and hatching of miracidia took 24 days at 28 degrees C. On the 66th day after miracidial challenge, 16 snails were crushed and examined for the presence of E. cinetorchis larvae. The metacercariae were detected in all of the snails, and from three of them were found the rediae and cercariae. The morphological characteristics of the larvae and the experimentally obtained adults were compatible with that of E. cinetorchis.     

Sequential histological changes in Biomphalaria glabrata during the course of Schistosoma mansoni infection

Biomphalaria glabrata, highly susceptible to Schistosoma mansoni, were seen to shed less and less cercariae along the time of infection. Histological examination kept a close correlation with this changing pattern of cercarial shedding, turning an initial picture of no-reaction (tolerance) gradually into one of hemocyte proliferation with formation of focal encapsulating lesions around disintegrating sporocysts and cercariae, a change that became disseminated toward the 142nd day post miracidial exposure. Findings were suggestive of a gradual installation of acquired immunity in snails infected with S. mansoni.     

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.     

Characterization of chemoreceptive protein binding to an oviposition stimulant using a fluorescent micro-binding assay in a butterfly

A native female-specific chemoreceptive protein of a swallowtail butterfly [oviposition stimulant binding protein (OSBP)] was shown to specifically bind to aristolochic acid, a main stimulant for oviposition from its host plant. Oviposition stimulants are recognized by chemoreceptive organs of insects. OSBP isolated previously from the chemoreceptive organs was assumed to bind to an oviposition stimulant. Using a highly sensitive fluorescent micro-binding assay, we clarified OSBP bound to aristolochic acid. Three-dimensional molecular modeling revealed the structure of the OSBP-aristolochic acid complex. This is the first report of a native chemoreceptive protein binding to an oviposition stimulant as a ligand in insects.     

The Production of the Striga and Orobanche Germination Stimulants by Maize Roots: I. THE NUMBER AND VARIETY OF STIMULANTS

By means of paper partition chromatography and a suitable biologicaltest, the number and variety of the Orobanche and Striga germinationstimulants produced by maize roots have been assessed. It isshown that extracts of maize roots and aqueous solutions inwhich roots have been suspended (diffusate solutions) containa complex of stimulating substances consisting of, at least,one water-soluble and a number of ether-soluble stimulants.Germination of O. minor is promoted by the water-soluble stimulantand one of the ether-soluble stimulants (M_o). Germination ofS. hermonthica is promoted by another ether-soluble stimulant(M_s). Aqueous extracts are rich in M_s and the water-solublematerial, whereas the diffusate contains a higher proportionof M_o and only a trace of the water-soluble stimulant. The complex of ether-soluble stimulants, but not the water-solublestimulant, accumulates in a solution of glucose when it is exposedto fragments of maize roots. These solutions contain a highproportion of M_o which is synthesized by the fragments and passesout into the sugar solution. It is suggested that the water-solublestimulant is a precursor of M_o. The effects of the water-soluble stimulant and of M_s and M_oare greatly enhanced if a trace of the diffusate solution fromlinseed roots is mixed with them. The significance of the enhancementis discussed.     

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.     

Mechanisms underlying digenean-snail specificity: role of miracidial attachment and host plasma factors

Digenetic trematodes usually show a high degree of specificity for their molluscan intermediate hosts. A panel of 4 digenean 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 representing 3 gastropod families) was used to assess the relative contributions of miracidial behavior, host plasma osmolality, and host plasma factors in dictating specificity. Additional experiments were undertaken with a fifth digenean, Echinoparyphium sp. Expected patterns of compatibility were first confirmed; each parasite species produced patent infections in its known snail host, but not in the other snail species. One exception was S. douthitti, which unexpectedly did not infect L. stagnalis. As judged by direct observation and by noting their disappearance after exposure to snails, miracidia were generally less likely to attach to or penetrate incompatible than compatible hosts. However, over half of the miracidia of each parasite species attached to or attempted penetration of both compatible and incompatible hosts, suggesting that under the experimental conditions used, miracidial host location and attachment behaviors were not of overriding importance in dictating observed patterns of specificity. For each digenean species, the percentage of larvae that became immobile, rounded, showed tegumental damage, or died over a 6-hr interval in plasma of the various snails was assessed. In no case was plasma from a compatible host harmful to sporocysts or rediae. In contrast, in 8 of 16 (50%) incompatible combinations, snail plasma had a significant negative effect on sporocyst condition. In 4 of 12 (33%) incompatible combinations, plasma had a significant negative effect on rediae. In 9 of 10 combinations tested, lymnaeid plasma was toxic for the parasites of planorbid snails and in 2 of 4 combinations, planorbid plasma was toxic for the parasites of lymnaeid snails. Toxicity was not attributable to differences in plasma osmolality between snail species. The ability of plasma from incompatible snails to affect viability of both sporocysts and rediae was surprisingly strong, suggesting that humoral factors play a greater role in dictating patterns of digenean-snail specificity than previously appreciated.     

The effect of variations in host and parasite density on the level of parasitization of Lymnaea truncatula by Fasciola hepatica

Groups of the snail Lymnaea truncatula, maintained at a range of spatial densities, were exposed to different densities of miracidia of Fasiola hepatica. The resulting degree of parasitization was measured 3--4 weeks after infection. The relationship between parasite densities and parasitization appeared to be curvilinear. The lower than expected parasitization at high parasite densities could be explained by the multiple infection of some snails by two or more miracidia. The level of parasitization was not related exponentially to the temperature at which infection was carried out. This was thought to be due to the inverse relationship between miracidial longevity and swimming speed, with respect to temperature. A depth of free water overlying a mud surface was an absolute requirement for miracidia to successfully infect snails.