Uninfected Snails Research Articles

On Macdonald's model for schistosomiasis

An attempt is made to reconstruct the specifics of the model in Macdonald's 1965 paper based on some manuscripts. The resulting model is not conclusive as discrepancies occur between this formulation and Macdonald's results. Assuming the formulation is adequate, the values of parameters yielding Macdonald's standard situation are provided. A way of studying the equilibrium behaviour of the model is outlined. The mathematical nature of the properties observed by Macdonald is studied. The model is altered to admit aggregated distributions of worms in hosts and to permit different death rates of infected and uninfected snails. The dynamics are found to depend strongly on the death rate of infected snails.

Studies on Resistance in Snails. 4. Induction of Ventricular Capsules and Changes in the Amebocyte-Producing Organ during Sensitization of Biomphalaria glabrata Snails

Formation of an amebocyte aggregate in the ventricular cavity of Biomphalaria glabrata can be induced 30 hr or more after a single infection by irradiated or (less frequently) by normal Echinostoma lindoense miracidia. The resulting amebocyte mass frequently encapsulated and destroyed the developing E. lindoense sporocysts within the ventricle. The constituent amebocytes of the capsule correspond in vitro and by staining characteristics to circulating amebocytes of uninfected snails, but with additional inclusion bodies, increased mucopolysaccharide, acid phosphatases, and lipid-positive staining reactions. Mitotic activity, rapid growth, and later regression of the amebocyte-producing organ (located between the posterior mantle epithelium and anterior pericardial endothelium) follow the growth and regression sequence of the ventricular capsule. Though peripheral foci of secondary amebocyte production have been found and were previously known, the amebocyte-producing organ appears to be the primary amebocyte source responsible for the snails' rapid intraventricular sporocyst encapsulation.

Echinostome rediae: Effect of pretreatment with cold on transplantation to uninfected snails

When donor snails, Lymnaea stagnalis, infected with Echinoparyphium aconiatum were held at 13 C for at least 24 days, transplantation of newborn rediae from them to uninfected snails was 2.5 times as successful as when donor snails were held at 23 C. The difference does not seem to be do to the fact that rediae transferred from 13 C-snails were slightly larger than those from 23 C. This effect of temperature was not observed in analogous experiments with another echinostome, Isthmiophora melis. The mechanism of this phenomenon found in the experiments with E. aconiatum is still unknown.

Carbohydrate metabolism in uninfected and trematode-infected snails Biomphalaria alexandrina and Bulinus truncatus

1. 1. Schistosome infection of the snails Biomphalaria alexandrina and Bulinus truncatus affected their citric acid cycle pathways; especially those concerned with succinic and cytochrome oxidases. 2. 2. The rate of gluconeogenesis was decreased in schistosome-infected snails to 62 per cent for Bulinus truncatus and to 45 per cent for Biomphalaria alexandrina. 3. 3. Lactic acid production was increased in schistosome-infected snails to 52 and 73 per cent that of uninfected B. alexandrina and B. truncatus respectively. 4. 4. Schistosome infection caused a marked decrease in the glycogen content of the tissues of both snail species. 5. 5. The effect of schistosome infection on the intermediary metabolism of both snails is discussed.

Isthmiophora melis (Trematoda, Echinostomatidae): Normal adults after 41 snail passages in 7 years of redial transplantation

A strain of Isthmiophora melis rediae has been kept for 8 years by transplantation from infected to uninfected snails. This strain has passed with undiminished redial reproductive ability in 45 transplantation steps through 46 Lymnaea stagnalis specimens which corresponds to a minimum of 93 successive redial generations. It was possible to start a normal sexual cycle of the trematode with cercariae deriving from rediae of the 41st snail passage, corresponding at least to the 83rd redial generation. Generative adults and infective miracidia were obtained. Hence the sexual reproduction potentiality of Isthmiophora melis has not been impaired by this artificial extension of the intramolluscan development for more than 7 years.

Schistosoma mansoni: Calcium Content of Cercariae and Its Effects on Protease Activity In vitro

The localization and quantitation of calcium in the protease-containing preacetabular glands of Schistosoma mansoni cercariae were determined by electron probe analysis and atomic absorption spectroscopy. Levels of calcium in these glands appear to exceed 8 to 10 M. In vitro, these high levels of calcium inhibit the cercarial proteases reversibly. It is suggested that calcium functions to control protease activity in situ in these organisms. The infective stage of the human blood fluke, Schistosoma mansoni, infects its host by penetrating the skin. The penetration process is thought to be mediated by proteolytic enzymes present in the preacetabular glands of this organism (Lewert and Lee, 1956; Stirewalt and Fregeau, 1966; Gazzinelli et al., 1966; Dresden and Asch, 1972). Moreover, it has been demonstrated that these glands contain large amounts of calcium (Stirewalt, 1959; Lewert et al., 1966). Lewert and Lee (1956) originally reported that these proteases are stimulated at low concentrations of these cations and we have confirmed this observation (Dresden and Edlin, 1974). Recently we determined that the proteolytic enzymes in extracts of this organism are inhibited by high concentrations of calcium and magnesium (Dresden and Edlin, 1974). We suggested that calcium might play a significant role in controlling the activity of these proteases by acting as an inhibitor of protease activity in situ. The results described in this report provide qualitative and quantitative evidence on the levels of calcium in these glands and its effects on protease activity. MATERIALS AND METHODS Infected snails, Biomphalaria glabrata (Puerto Rican strain), were allowed to shed their cercariae at room temperature in beakers containing dechlorinated tap water or 1 mM Tris-HCl, pH 7.8. Using a depression slide and a dissecting microscope, individual cercariae were transferred by disposable pipette. For electron probe analysis single cercariae were dried on silica discs at room temperature as rapidly as possible, aided by withdrawal of water with a micropipette. For calcium analysis, individual cercariae (50 Received for publication 10 October 1974. t 200) were counted and transferred to appropriate tubes. Spectrophotometric analysis of calcium was by the method of Williams and Wilson (1961). CaCl2 and CaCO3 (0.5 to 100 ,ug) were used as standards. Atomic absorption spectroscopy for calcium was determined following extraction of 100 to 200 cercariae in 5% lanthanum chloride-1% HC1 at 100 C for 10 min. Calcium contributed by sources other than the cercariae was corrected for by using uninfected snails. Protease activity in cercarial extracts was measured using Azocoll substrate as described previously (Dresden and Edlin, 1974). RESULTS AND DISCUSSION The results of the electron probe X-ray analysis and scanning electron microscopy are shown in Figure la-c. These figures represent a single cercaria examined by scanning electron microscopy (Fig. la) and by X-ray probe analysis for calcium (Fig. lb) and phosphorus (Fig. lc). A number of individual cercariae were examined by these combined techniques. The results demonstrate dramatically a concentration of calcium in the region of the preacetabular glands, and a fairly uniform distribution of phosphorus throughout the body of the cercariae. Using the tail region as a control, scanning X-ray analysis was carried out for 20to 40-sec intervals at 25 kv with a 0.185-,uamp sample current. These experiments demonstrated that calcium is at least 1,000 to 2,000 times more abundant in the preacetabular gland region than in the tail region. Magnesium levels in the gland region were not significantly elevated over those in the tail region. While these experiments established the localization of calcium, it would have been necessary to determine the thickness and composition of the cercariae in order to determine

Autoradiographic studies on the uprake of (14C)-glucose by Bithynia tentaculata (mollusca : Gastropoda) and its larval digeneans.

AbstractAutoradiographs revealed that sporocysts of Cercaria helvetica XII and rediae of Sphaeridiotrema globulus, incubated in a medium containing [14C]-glucose, exhibited slight radioactivity in their body wall after only 30 seconds incubation. Activity in sporocysts and rcdiae gradually increased as the incubation period was extended until after 4 minutes, a heavy reaction was observed in the parasite body wall as well as in the germ balls and developing cercariae within the parasite brood chambers. The redial caecum appeared to incorporate the labelled compound much more intensely than did the redial body wall.When uninfected Bithynia tentaculata, as well as B. tentaculata infected with either S. globulus or C. helvetica XII, were maintained in pond water containing [14C]-glucose, activity could be detected in the gills of both infected and uninfected snails after 5 hours incubation. After 12 hours a much heavier labelling was apparent in the gills, together with moderate activity in the haemocoelic spaces, foot musculature, stomach wall, and gut contents. After 20 hours a more intense labelling was apparent in these regions of the snail body and, in addition, some activity could be detected in the digestive gjand tubules of the host and, in infected snails, in the bodies of developing parasites. After 30 hours incubation the labelled compound had become incorporated into most of the host tissues but was most intense in the digestive gland tubules. Heavy activity was also evident in the parasites at this stage although certain cystogenous gland cells of the cercariae of both parasite species did not incorporate the labelled compound.

Alterations in total serum proteins and protein fractions in Biomphalaria glabrata parasitized by Schistosoma mansoni

Samples of serum from healthy Biomphalaria glabrata and from those infected with the larval stages of Schistosoma mansoni were subjected to quantitative electrophoretic analysis and total protein determinations. In the case of the infected snails, samples of serum were taken at 14 time intervals post-exposure to 5 miracidia ranging from 1 hr to 70 days. A total of 34 serum protein fractions have been identified in the hemolymph of uninfacted B. glabrata, although all these fractions usually do not occur in every snail. In infected snails, there is a gradual reduction in the staining intensity of the serum protein fractions until day 35 post-exposure to miracidia, and this is correlated with a reduction in the total serum protein concentration. By day 70 post-exposure to the parasite, the total protein concentration had declined to one-third of that in uninfected snails. Although the decrease in the amount of serum protein fractions as a result of parasitization by S. mansoni is generally nonselective; i.e., almost all the fractions are reduced, three fractions, 7, 9, and 10, do not appear to be reduced in quantity. These remain essentially unaltered in their intensity to staining for up to 70 days, the duration of this experiment. It is postulated that the reduction of serum protein is due to utilization by sporocysts of S. mansoni.

Life Cycle of Pseudodiplodiscoides pilai (Trematoda: Diplodiscidae) from the Gut of the Apple Snail, Pila globosa (Swainson)

Stages in the life cycle of Pseudodiplodiscoides pilai are described. Cercariae emerge from Indoplanorbis exustus and encyst in the open. Metacercariae excyst in the esophagus of the apple snail, Pila globosa, and grow to maturity in the stomach. In a previous communication (Murty, 1970) the author described Pseudodiplodiscoides pilai from the stomach and intestine of the apple snail, Pila globosa (Swainson). In the same collection of snails, Indoplanorbis exustus (Deshayes) shed an undescribed amphistome cercaria which resembled young flukes re- covered from the esophagus of P. globosa and was suspected to be the larval form of the trematode maturing in the gut of that snail. During further collections from Kolleru Lake, the adult trematode and the cercaria were encountered a second time. Uninfected apple snails were allowed to feed on the leaves of

Schistosoma mansoni infection in Biomphalaria glabrata: Alterations in heart rate and thermal tolerance in the host

It has been demonstrated that there is a reduction of heart rate (beats/min) in nonparasitized Biomphalaria glabrata as a function of increased shell diameter. When the heart rates of noninfected B. glabrata and those of specimens infected with larval Schistosoma mansoni are compared over a period of 8 weeks post-infection, it has been determined that there is a significant increase from the 4th week on in the latter. The influence of ambient temperatures on the heart rates of infected and noninfected B. glabrata has also been studied. It has been ascertained that, with the exception of 33°C, the heart rates of infected snails are consistently greater than those of control snails within the 23–33°C range. Infected snails demonstrate a lowered higher thermal tolerance limit than uninfected ones, with 40% dying within 5 days when maintained at 33°C. Furthermore, there is a rapid decline in their heart rates prior to death. When maintained at 28°C for 10 weeks, 75% mortality has been recorded among snails each infected with 10 miracidia, while the mortality rate during the same period among identically maintained, uninfected snails is only 40%.

Shell Abnormalities inBiomphalaria glabrataInfected withSchistosoma mansoniand their Significance in Field Transmission Studies

1. A distortion of the shell aperture ofBiomphalaria glabratainfected withSchistosoma mansonireported in earlier work was found to be due to a reversal from ventral to dorsal of the deflection found in uninfected snails.2. The most sensitive way of measuring this distortion was to record the height of the dorsal deflection. The difference in this figure from uninfected and infected snails of the same diameter is termed the relative distortion.3. A longitudinal study showed that the relative distortion first appeared at about the time when cercarial shedding started, and that it increased in size linearly as the infection progressed.4. Temperatures between 20 and 30°C. controlled the length of the prepatent period and thus the first appearance of the relative distortion, but they did not significantly alter its subsequent rate of development.5. Histological changes were observed in the mantle collar in the region where the relative distortion was most pronounced.6. Possible mechanisms causing the development of the distortion are discussed.7. Relative distortions were observed in some naturally infected field snails and the laboratory results were used to estimate how long these snails had been shedding cercariae.8. Naturally infected St. Lucian snails appeared to have shed cercariae for an average of less than three weeks in the field.9. A survival curve was drawn up for one collection of field snails, based on the frequency among them of relative distortions of different sizes. Another curve was obtained from an independent technique. The similarity of the two curves gives confidence in both methods.10. The findings in St. Lucia are discussed in relation to similar studies elsewhere in the world.

The potential number of redial generations in echinostomatids (trematoda)

By carrying out ‘chain-transplantations’ of rediae of Isthmiophora melis from infected to uninfected snails ( Lymnaea stagnalis) it has been shown to date that these rediae have passed through a minimum of 42, but possibly 102, successive generations. It is suggested that a high mortality due to transplantation procedures is responsible for the fact that only 11·94 per cent of transplanted rediae survive and give rise to further generations of rediae. Since rediae of different generation numbers do not differ significantly with regard to (a) their infectivity to recipient snails; (b) the time from their transplantation to the emergence of cercariae; and (c) the infectivity of nmetacercariae derived from them it is argued that the multiplication potential of daughter rediae of I. melis is unlimited. Similar results have been obtained with the rediae of Echinoparyphium aconiatum. Preliminary data for the rediae of Echinostoma revolutum suggest similar trends to the other species, although rediae of increasing generation number show a progressive decrease in their infectivity to recipient snails. It is concluded that the limiting factors for redial multiplication in the intermediate host must be the size and life span of the intermediate host.

Parthenogenesis in Parasitic Helminths

SYNOPSIS. In neinatodes, parthenogenesis, meiotic or more often mitotic with polyploidy, occurs in the orders Tylenchida and Rhabditida. It has evolved independently within the various families and even certain genera, replacing the usual amphimixis in some species and supplementing it in others with one or more parthenogenetic generations. Polyploidy, haploidy, abnormal spennatogenesis and absence of sperms have been cited as evidence of parthenogenesis in some adult cestodes and digenetic trematodes, especially dioecious species in those predominantly hermaphroditic groups. Evidence now favors interpretation of reproduction by germinal sacs in trematodes as diploid parthenogenesis which can be continued through an indefinite number of generations in some species by transplanting germinal sacs from infected to uninfected snails.

Host-Parasite Interrelationships Between the Freshwater Pulmonate Biomphalaria Pfeifferi and the Trematode Schistosoma Mansoni

The influence of infection with S. mansoni on reproduction, growth, food consumption and survival in B. pfeifferi was studied experimentally (Ch. II). The prepatent period in infected snails was shown to last 22-24 days under the conditions prevailing in the relevant experiment. The number of eggs laid by infected snails as compared with controls was significantly reduced from 7-10 days postinfection onwards. After day 13 postinfection it was completely suppressed in the majority of the infected snails. The increase in shell diameter in infected specimens was larger than that of the controls between 7 and 18 days, that in weight between 11 and 21 days postinfection. Gigantism did not occur. There was no difference in mortality between infected and control snails prior to the 11th week after the beginning of the experiment. After that, the mortality among infected specimens showed a sharp rise. Uninfected and infected snails consumed similar amounts of food (dried lettuce) in the first two weeks of the experiment. After that, infected snails consumed less food than controls. The digestive gland of B. pfeifferi was studied with histological, histochemical and ultrastructural methods (Ch. III). The highly folded epithelium of the ducts connecting the digestive gland with the pyloric part of the stomach, consists of cilia and/or microvilli bearing columnar cells with interspersed mucous cells. In the epithelium of the digestive gland two main cell types are distinguished: the digestive cell and the secretory cell. In addition some mucous cells occur. It is concluded that the main functions of the digestive cell are absorption and endocytosis of predigested food material, followed by intracellular digestion. Indigestible residues are accumulated in a large vacuole, which eventually is excreted. The secretory cell produces and secretes proteinaceous substances, presumably digestive enzymes. It does not store calcium. The so-called excretory cells, which are in fact degenerating secretory cells, have vacuoles containing yellow globules. These yellow globules together with cell debris are finally released into the lumen of the digestive gland. The observations indicate that the digestive gland is not important as a storage organ for reserve material. The lobules of the digestive gland are covered by a thin connective tissue sheath, consisting of ground substance, collagen-like fibrils, smooth muscle cells and cells with the appearance of pigment cells. The connective tissue between the lobules of the digestive gland and the acini of the ovotestis, and between these organs and the mantle, consists mainly of vesicular connective tissue cells. These cells are supposed to play an important metabolic role as storage cells for glycogen. The tegument of the daughter sporocyst of S. mansoni (Ch. IV), consisting of an outer syncytial anucleate layer which is joined by cytoplasmic connections to nucleated cell bodies (tegumental cells), has the same basic architecture as that described for various stages of trematodes and cestodes. Well-developed mitochondria, GER cisternae and tubules, free ribosomes and polysomes, and lipid droplets occur throughout the tegument. The ultrastructural features suggest that the outer layer is involved in uptake of nutrients by absorption and endocytosis. The poorly developed musculature is loosely arranged in two layers beneath the outer tegumental layer. The nucleated portions of the muscle fibres are situated between the tegumental cells. The protonephridial system consists of flame cells with efferent tubules. The bundle of cilia, implanted in the flame cell, beats in the cilia chamber or barrel. The proximal part of the barrel consists of alternating rib-like extensions of the flame cell and of the first tubule cell, respectively. Ultrafiltration probably takes place in the ribbed part of the barrel. The development of cercariae from germinal cells is briefly described. The pathological effects of infection with S. mansoni on the digestive gland epithelium of B. pfeifferi (Ch. V) are limited. From about 12 weeks after infection slight changes occur, but they are difficult to distinguish from changes due to ageing of the cells. The effect of starvation on the digestive gland epithelium is much more marked than the adverse influence of infection. Compression of digestive gland lumina by daughter sporocysts was not observed. Changes in the connective tissue of the digestive gland due to the presence of daughter sporocysts were: development of muscle cells into cells with the appearance of pigment cells and/or degeneration of these muscle cells, and an increase in the number of amoebocytes, which become involved in the elimination of cell debris and in the formation of a loose layer over the parasites.

Cotylurus lutzi sp. n. (Trematoda: Strigeidae) and Its Life Cycle

Snails Biomphalaria glabrata (Say), collected in Bahia, Brazil released furcocercariae that developed into tetracotyles in uninfected snails of the same species or as hyperparasites in rediae or sporocysts of other trematodes. Tetracotyles fed to canaries or finches become adults that usually survive 3 to 4 weeks, and are distinguished principally by numerous vitelline follicles in the holdfast lobes, size of pharynx, and size of eggs. Eggs appear in bird feces 1 week after feeding of tetracotyles, and usually hatch in 3 to 4 weeks at 28 C. In laboratory-infected albino B. glabrata, mother sporocysts may attain 28 mm in length and survive for more than 6 months. Daughter sporocysts are filiform and motile. Cercariae are released from 25 days postinfection. All stages, particularly the tetracotyle, are described in detail. This paper is one of a series describing the life cycles and biology of trematodes whose larval stages are passed in snails of the genus Biomphalaria that transmit schistosomiasis in the New World. The sporocyst stage of the strigeid described here produces furcocercariae that can easily be confused with those of Schistosoma mansoni Sambon. Several hundred Biomphalaria glabrata (Say), collected in the vicinity of Caetite, Bahia State, Brazil, by Dr. W. Lobato Paraense and co-workers in April 1967, were sent by air to our laboratory in California. All snails in the shipment harbored tetracotyle metacercariae, and two were shedding furcocercariae of the Cotylurus type. These two naturally infected snails provided the starting point for the cycle described here. MATERIALS AND METHODS The hyperparasitic habit of the metacercarial (or tetracotyle) stage was utilized for routine maintenance of the Cotylurus life cycle. Biomphalaria glabrata with advanced infections of the echinostome Paryphostomum segregatum Dietz were placed in aquaria with snails shedding cercariae of the Cotylurus type. After exposure, usually for about 24 hr, the Paryphostomum-infected snails were separated and maintained 3 weeks or more to ensure complete development of the tetracotyle Received for publication 25 October 1968. * This work was supported by Research Grant AI07054 from the NIAID, and by the University of California International Center for Medical Research and Training (UC ICMRT, Hooper Foundation, University of California San Francisco Medical Center) with Research Grant TW 00144 from the Office of International Research, both from the NIH, U. S. Public Health Service. larvae within the echinostome rediae. Hyperparasitized snails were crushed and rediae containing tetracotyles were fed to avian hosts to obtain adult worms. Other kinds of larval trematodes developing within Biomphalaria also serve equally well for incubation of tetracotyle stages. Sporocysts of S. mansoni have been used, and yield viable tetracotyles that develop into adult worms, but the handling of snails shedding S. mansoni entails unnecessary risks. Various other echinostomes being maintained in our laboratory, such as Echinostoma paraensei Lie and Basch, E. lindoense Sandground and Bonne, and E. barbosai Lie and Basch, have also been used, but cercariae of these species will encyst within the pericardial sacs of other B. glabrata, including those infected with Cotylurus sporocysts. Hence, the snails shedding Cotylurus cercariae, if exposed to echinostome cercariae over long periods, will accumulate many echinostome metacercariae in their pericardial sacs and may be adversely affected. Cercariae of P. segregatum, on the other hand, encyst only in fish and tadpoles. Snails shedding Cotylurus cercariae may be used repeatedly for several months to produce many thousands of tetracotyles within rediae of Paryphostomum without apparent ill effects to themselves. Avian hosts used in this study included young domestic ducks, pigeons, and chicks, and several types of smaller cage birds: canaries and finchesTaeniopygia castanotus (Gould), Estrilda troglodytes (Lichtenstein), and Uroloncha striata (Brisson). Four ducklings and 2 pigeons remained uninfected after ingesting large numbers of tetracotyles. Chicks became infected only when 500 or more tetracotyles were fed, but were generally not good hosts. The smaller cage birds, particularly the finches, proved to be excellent hosts highly suitable for use in the laboratory, producing many eggs of Cotylurus. The small birds were maintained in rectangular glass aquaria 14 by 24 by 17 cm high, each aquarium provided with a perch, seed cup, and cover of plastic insect screening held in place by a stout rubber band. About 1 cm of water was maintained in the aquaria at all times.

The effect of sublethal doses of the molluscicide (N-tritylmorpholine) on the development of Schistosoma mansoni in Biomphalaria glabrata (Say).

1. The effects of a sublethal concentration (0·0165 p.p.m.) of N-tritylmorpholine on the development of S. mansoni in B. glabrata are described.2. A total of 160 snails were infected with S. mansoni and then divided into 4 groups. Each group was treated separately with N-tritylmorpholine for a period of 24 hours.3. The first group was treated 10 days after infection (early prepatent stage); the second group 20 days after infection (late prepatent stage); the third group 42 days after infection (patent stage); and the fourth group was left untreated and kept as an infected but untreated control group.4. Three groups of uninfected snails were treated simultaneously with the infected groups, and a fourth uninfected group was left untreated and maintained as an uninfected and untreated control group.5. In the early and late prepatent groups treated with N-tritylmorpholine a relatively short incubation period was observed compared with those in the patent and untreated infected groups.6. No significant difference was observed between the cercarial infection rates of the 4 infected groups of snails (x2 = 0·744; p > 0·5).7. In all infected groups peak cercarial production was observed about 15–20 days after the emergence of cercariae was first noted, after which numbers declined. No significant difference was observed between the total cercarial production of the early prepatent, late prepatent or patent groups and that of the untreated infected control group (p > 0·4).8. The mortality rate of snails in the patent group was significantly higher than in the corresponding uninfected control group (x2 = 6·04; p <0·02).9. Treated infected snails in all three groups ultimately had a lower survival rate than did treated uninfected snails.10. The survival rate of untreated infected snails while initially as good as that of the untreated and uninfected group, was ultimately much poorer.

Reproduction of Echinostoma revolutum Rediae When Transferred to Uninfected Snails of Different Sizes

Mother rediae of Echinostoma revolutum were transplanted individually from infected to uninfected Stagnicola palustris ranging in length from 10 to 25 mm. The number of daughter rediae present at 30, 40, and 50 days after transfer was related to the size of the snail, progressively so with the age of the infection. There was also a direct relationship between the size of the snail and the maximum size attained by the rediae which in general was greater than that seen in naturally infected snails. When daughter rediae were transplanted in groups of five into snails 20 to 23 mm long, and examined 40 or 60 days later, none of the snails contained additional rediae and none of the rediae contained rediae and cercariae together, as is occasionally seen in natural infections. Transplantation of trematode larvae from infected to uninfected snails has been useful in studies on various aspects of larval development. Donges (1963) artificially infected snails with individual rediae of an echinostome (Isthmiophora spiculator) to determine the exact sequence of larval stages. Chernin (1966, 1967) developed a technique for transferring Schistosoma mansoni sporocysts and echinostome rediae into the cephalopedal sinus of Australorbis glabratus, and Heyneman (1966) successfully established echinostome infections by introducing either miracidia or rediae into the tissues of Lymnaea rubiginosa and Indoplanorbis exustus. Changes in the transplanted rediae and metacercariae of Metagonimoides oregonensis were studied by Meade and Pratt (1966). In a previous study the relationship between the size of the host and the size of the redial population of Echinostoma revolutum was examined (Zischke, 1967). Although it was established that the number of mother rediae produced by the sporocyst was related to the size of the snail host, the number of daughter rediae generated by mother rediae could only be indirectly estimated. Moreover, in laboratory Received for publication 23 June 1967. *Present address: Department of Biology, St. Olaf College, Northfield, Minnesota. From a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The study was supported in part by Grant 5-TI-AI-02 from the NIH, U. S. Public Health Service. infections only two generations of rediae were found whereas in naturally infected snails a third generation of rediae occasionally was seen in daughter rediae that were producing cercariae. The present study was undertaken to determine the number of daughter rediae produced when mother rediae of E. revolutum are transplanted into uninfected snails (Stagnicola palustris) of various sizes, and to establish whether transplanted daughter rediae produce an additional generation of rediae. MATERIALS AND METHODS Mature mother rediae, obtained from snails (S. palustris) which were previously exposed to 10 miracidia, were introduced through holes, drilled with a 20-gauge hypodermic needle, into the region of the body whorl of laboratory-reared snails. Mature daughter rediae were similarly introduced, either between the body whorl and the first whorl of the spire or between the first and second whorls of the spire. The rediae, together with a small amount of balanced salt solution (Dulbecco and Vogt, 1954) were placed in the hemocoel by means of a capillary tube (inner diameter, 0.4 to 0.8 mm). In vitro survival tests proved the balanced salt solution to be a suitable medium for maintaining rediae outside of the host. Care was taken to rupture the mantle so that the larvae would not be entrapped between the mantle and the shell. The opening was sealed with silicone and over-sealed with melted paraffin applied with a small artist's brush. The wound healed completely in 4 to 6 days. All procedures were carried out under a hood and with sterile instruments and glassware. Reports published by several authors subsequent to the completion of this study suggest that the latter precautions may not greatly increase transplant success. All snails were maintained at 20 to 25 C.

Histopathological and Histochemical Effects of the Trematode Nanophyetus salmincola (Chapin) on the Hepatopancreas of Its Snail Host, Oxytrema siliqua (Gould)

The histopathological and histochemical effects on the hepatopancreas of the snail Oxytrema siliqua, caused by the larvae of Nanophyetus salmincola, have been studied. The parasite causes damage to the organ by increased pressure, active ingestion of the hepatic tubules, and by the increased burden placed upon the snail in disposing of the waste of the parasite. Glycogen and lipid uptake from the hepatopancreas by the parasite were tested for and found to occur via two routes: active ingestion of hepatic cells by the parasites, and absorption across the body membranes of the parasite. Acid and alkaline phosphatases were found to be in greater quantities in snails infected with N. salmincola when compared to uninfected snails. Alkaline phosphatase was found to be associated with the parasite and is thought to function in the metabolism of glycogen. ABSTRACT: The histopathological and histochemical effects on the hepatopancreas of the snail Oxytrema siliqua, caused by the larvae of Nanophyetus salmincola, have been studied. The parasite causes damage to the organ by increased pressure, active ingestion of the hepatic tubules, and by the increased burden placed upon the snail in disposing of the waste of the parasite. Glycogen and lipid uptake from the hepatopancreas by the parasite were tested for and found to occur via two routes: active ingestion of hepatic cells by the parasites, and absorption across the body membranes of the parasite. Acid and alkaline phosphatases were found to be in greater quantities in snails infected with N. salmincola when compared to uninfected snails. Alkaline phosphatase was found to be associated with the parasite and is thought to function in the metabolism of glycogen.

Free Amino Acids of Australorbis glabratus Hemolymph: Comparison of Four Geographic Strains and Effect of Infection by Schistosoma mansoni

Concentrations of free amino acids in the hemolymph of uninfected Australorbis glabratus from Surinam, Venezuela, Puerto Rico, and Brazil were determined by automatic recording column chromatography. Additionally, the hemolymphs of normal and of Schistosoma mansoni-infected Puerto Rican A. glabratus were compared with respect to concentration of free amino acids and protein. The concentrations of certain free amino acids varied considerably in the different strains of uninfected snails, but similar variations also occurred in duplicate samples prepared from one of the strains. Total free amino acids of the hemolymph of infected snails varied only slightly. Levels of all free amino acids decreased during the course of infection with S. mansoni. The total free amino acid concentration in the hemolymph of snails with mature infections was approximately one-half that of normal snails. Total hemolymph protein varied among individual uninfected snails and was significantly decreased among infected snails. Several recent studies have dealt with the biochemical nature of snail hemolymph. Qualitative studies of amino acids of Australorbis glabratus hemolymph have been made (Dusanic and Lewert, 1963; Targett, 1962), and the hemolymph proteins have been characterized (Dusanic and Lewert, 1963; Wright and Ross, 1963; Michelson, 1966). This study was designed to determine quantitatively the hemolymph amino acids in four geographic strains of A. glabratus, one of which is highly refractory to infection by Schistosoma mansoni. Additionally, the free amino acid concentrations of the hemolymph of one of the strains were determined during the course of infection by S. mansoni, and the total protein concentrations of the hemolymph of infected and normal A. glabratus were compared. MATERIALS AND METHODS Maintenance and infection of snails A. glabratus from Venezuela (Ag-Ven) and Surinam (Ag-Sur) were obtained from the Tropical Research Medical Laboratory in San Juan, Puerto Rico, in 1962; the Puerto Rican strain of A. glabratus (Ag-PR) was obtained in 1958 from the National Institutes of Health; the Brazilian strain Received for publication 23 December 1966. * This study was supported by a NASA Predoctoral Traineeship to the senior author. t Present address: Department of Tropical Public Health, Harvard School of Public Health, 25 Shattuck Street, Boston, Massachusetts 02115. (Ag-Bah) originated in Bahia and was supplied to us in 1964 by Dr. W. L. Paraense. The Puerto Rican NIH strain of S. mansoni was used to infect Ag-PR. Exposures en masse to 20 to 40 miracidia per snail resulted in infection rates of over 90%. Infected and normal snails were maintained at 27 C in groups of 25 or less in 1-gal glass jars. Water was aerated continuously and changed every 10 to 14 days, leaf lettuce was provided as food, and the snails were fed a supplement once a week (Etges and Gresso, 1965). Collection and preparation of hemolymph Hemolymph for amino acid analysis was obtained from snails 14 to 17 mm in diameter. Snails were starved for 24 hr, rinsed with tap water, and allowed to air-dry. Hemolymph, obtained by puncturing the heart, was collected in 1-ml centrifuge tubes in ice water. Debris was removed by centrifugation (1,000 g/10 min), and the hemolymph was stored in sealed glass vials at -10 C for use within 6 weeks. Samples of 6 ml each, representing the hemolymph pooled from 175 to 225 snails, were used for each analysis. Single pools from uninfected Ag-Bah, Ag-Sur, and Ag-Ven, and duplicate pools from Ag-PR, were analyzed. Hemolymph was drawn from separate groups of snails (Ag-PR) 6, 16, and 32 days postexposure to determine the effect of infection. In addition, fresh hemolymph samples from 50 normal and 20 infected (shedding) Ag-PR, each 15 mm in diameter, were withdrawn for protein determinations. Hemolymph for amino acid analysis was deproteinized by dialysis. Each sample was filtered through a Millipore filter (0.45 J porosity) into a cellophane dialysis bag and allowed to dialyze at 3 to 5 C for 3 days against 50 ml of distilled water, which was changed daily. The total dialysate was

Transplantation of Larval Schistosoma mansoni from Infected to Uninfected Snails

Schistosoma mansoni was transferred from infected Australorbis glabratus to normal A. glabratus by transplanting parasitized tissues or parasites into the cephalopedal sinus. Transplants of liver fragments from donors with mature infections resulted in erratic low-level cercarial emergence from recipients during the next 2 weeks; thereafter cercarial emergence rose sharply and became sustained. Cercariae of the early phase were apparently those already present in the transplant, while those of the later phase apparently derived from young daughter sporocysts which migrated from the transplant to the recipient's liver. Transplantation of tentacles bearing mother sporocysts resulted in sustained cercarial emergence after an incubation period of about 2 weeks; mature daughter sporocysts, producing cercariae, were found in the livers of recipients. Similar findings occurred when mature mother sporocysts were ruptured and the liberated immature daughter sporocysts were transplanted. Snails infected by transplants were susceptible to infection by miracidia which developed into mother sporocysts containing viable offspring. Transplanted daughter sporocysts produced infection in 77% of recipient A. glabratus; infection rates varied from 0% to 96% among eight other strains or species of snails receiving similar transplants. No reference has been found in the literature regarding transplantation of larval trematodes from infected to uninfected snails. Observations are now presented that larval Schistosoma mansoni derived from infected Australorbis glabratus can be transplanted into normal A. glabratus, and into certain other snails, and that infections thus established may proceed to maturity. MATERIALS AND METHODS 1. Infection and maintenance of donor snails Stock snails were a Puerto Rican strain (PR-2) of A. glabratus (see Cherin and Schork, 1959). Groups of 50 to 100 snails, 4 to 8 mm in diameter, were exposed in finger bowls to miracidia of a Puerto Rican strain of S. mansoni (see Chemin and Dunavan, 1962) and then maintained on lettuce, in darkness, in covered, aerated, stainlesssteel pans. These infected snails were the source of transplants. All donor and recipient snails were manipulated and maintained at 25 to 27 C. 2. Securing and processing transplants Dissections were done under a stereomicroscope. Sterile precautions were not taken although transplants were handled in an initially sterile antibioticfree solution (BSS) described elsewhere (Chernin, 1963). Three types of transplants were used. A. Parasitized liver (= digestive gland): Using a snail from which cercariae were emerging at Received for publication 29 October 1965. * These studies were supported in part by Research Grant AI-00513 and by a Research Career Award from the NIAID, U. S. Public Health Service. 40 to 42 days postinfection, the liver was excised (Cherin, 1963), placed in BSS, and cut into fragments about 2 by 1 by 1 mm (Fig. 1). These tissues were used within an hour; each snail received one fragment. Fragments used in an experiment were usually obtained from a single donor. B. Intact mother sporocysts: Snails with sporocysts in the tentacles were selected 18 days postinfection. (Mother sporocysts in other sites were not used in order to avoid dissections in dense tissues.) The snail body was pinned in a paraffinfilled dish flooded with BSS, and the infected tentacle was excised with iris scissors and placed in BSS in a depression slide. An untrimmed tentacle (Fig. 5) was transplanted at once into a recipient snail. C. Immature daughter sporocysts: Sporocyst-bearing tentacles were removed from snails infected for 14 to 20 days. Mother sporocysts were ruptured individually using fine needles, snail tissue was discarded, and the liberated daughter sporocysts were transplanted at once. Each recipient received the entire contents of one mother sporocyst. 3. Transplantation procedures Recipient A. glabratus (PR-2) from normal stock (Table I) were narcotized for 2 to 5 hr in 1.25 to 1.50% urethane. Each snail was clamped in a dish under a stereomicroscope, and, except as noted, the hook of a micro-retractor was inserted into the male genital opening and the snail extended by gentle traction (Michelson, 1958). Excess fluid was removed from the dish. The transplant was introduced into the extended neck of the snail, i.e., into the cephalopedal sinus, midway between the male genital opening and the anterior margin of the mantle. A fragment of liver or a tentacle was taken up in a small volume of BSS into the bore of a blunted 18or 19-gauge needle attached to a 0.25-ml syringe An incision was made into the sinus with