Effects of Schistosomatium douthitti infection on the growth, survival, and reproduction of Lymnaea catascopium

Lymnaea catascopium snails infected with Schistosomatium douthitti grew faster than uninfected control snails during the first 2 months postexposure, but thereafter grew more slowly, and by 8 months postexposure were significantly smaller. When reared in isolation, uninfected snails survived significantly longer (mean survival time, 515 days) than snails exposed to three miracidia each (400 days), which in turn survived longer than snails exposed to 10 miracidia per snail (223 days). When maintained in aquaria in contact with other snails, snails exposed to three miracidia each survived longer (227 days), but not significantly longer, than control snails (198 days). Production of large numbers of eggs by control snails grown under the latter conditions may account for their reduced survival. The ovotestes and accessory genitalia of snails infected with S. douthitti were much reduced in size in comparison with uninfected control snails. These effects were most pronounced in snails which had been infected for over 100 days. Egg production was normally totally inhibited if snails were infected before the onset of sexual maturity. If infected after the onset of maturity, eggs were produced only during the prepatent period.     

Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata

Lai P. F. and Canning E. U. 1980. Infectivity of a microsporidium of mosquitoes (Nosema algerae) to larval stages of Schistosoma mansoni in Biomphalaria glabrata. International Journal for Parasitology10: 293–301. Nosema algerae derived from a closed colony of Anopheles stephensi was fed to Biomphalaria glabrata infected with Schistosoma mansoni. Mother and daughter sporocysts became hyperinfected but the snail tissues remained free of the microsporidia except for rare small aggregates of spores. These lay close to the sites occupied by mother or daughter sporocysts and were probably liberated from them. Irrespective of dose, fewer snails contained infected sporocysts when spores were given at 7 days post-miracidial infection than when given at 14 days. These periods corresponded respectively to stages when mother sporocysts only or daughter sporocysts as well were present in the snails. Infection of the sporocysts began in the tegumental cells, spread to the brood chamber and ultimately to the cercariae themselves. Heavily infected sporocysts contained fewer developing embryos. Doses of 10⁶ and 10⁷ spores/snail caused significant depression of cercaria output when given at 14 days but not at 7 days.     

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.     

Schistosomatium douthitti: Exposure of Lymnaea catascopium to irradiated miracidia

Irradiation of Schistosomatium douthitti miracidia (4000, 5000, or 6000 rad) did not substantially alter their behavior or ability to penetrate their snail host. Treatment with 4000 rad was not sufficient to prevent all miracidia from establishing patent infections in Lymnaea catascopium, although significantly fewer snails exposed to these miracidia shed cercariae than did controls exposed to normal miracidia. Irradiation of miracidia with either 5000 or 6000 rad totally prevented cercarial production. Although destruction of irradiated mother sporocysts by encapsulating amebocytes was occasionally observed, most expanded without concomitant multiplication of germinal cells and embryo production and then collapsed. They generally persisted in this state throughout the period of observation (32 days). Snails sensitized by exposure to irradiated miracidia and challenged 2 or 10 days later with normal miracidia were as likely to develop patent infections as were snails exposed only to normal miracidia. Double sensitization of snails with irradiated miracidia also failed to confer protection upon challenge with normal miracidia. Most challenge sporocysts developed normally, often in close proximity to collapsed irradiated sporocysts.     

Ribeiroia marini: Irradiated miracidia and induction of acquired resistance in Biomphalaria glabrata

Biomphalaria glabrata snails sensitized by exposure to X-irradiated miracidia of the trematode, Ribeiroia marini, acquired resistance to challenge with nonirradiated R. marini miracidia. Resistance was acquired within 1 day of sensitization; was strongest at 1 week, when infection rates of sensitized snails were 15% of the controls (i.e., $\text{S}\text{C}\text{= 0.15}$); and persisted for at least 3 weeks. By 30 days the difference between the infection rates of sensitized and control snails was no longer statistically significant. As in previous studies with echinostomes, acquired resistance to R. marini was characterized histologically by the destruction of irradiated sporocysts by host amoebocytes. Following destruction of all irradiated sporocysts, snails became resistant and encapsulated and destroyed nonirradiated challenge sporocysts within 1 day postchallenge. Associated with sporocyst destruction was an enlargement of the amoebocyte-producing organ, which showed intense mitotic activity. A proportion of the nonirradiated challenge sporocysts were also destroyed in most nonsensitized control snails, which consequently had a temporarily enlarged amoebocyte-producing organ. In contrast to acquired resistance reported to echinotomes, which is quite specific, acquired resistance to R. marini was associated with nonsusceptibility to both Echinostoma paraensei ($\text{S}\text{C}\text{= 0.19}$) and Schistosoma mansoni ($\text{S}\text{C}\text{= 0.81}$).     

Biomphalaria glabrata amoebocytes: Effect of Schistosoma mansoni infection on in vitro phagocytosis

The rate of phagocytosis by amoebocytes obtained from hemolymph of the pulmonate Biomphalaria glabrata infected with the trematode Schistosoma mansoni for 24 hr and 2, 4, and 6 weeks has been determined using the monolayer assay system. Amoebocyte preparations from snails infected for 4 and 6 weeks showed a gradual decrease in the phagocytic rates compared to those from uninfected controls. Snails harboring the parasite for 4 and 6 weeks also showed a significant increase in the number of amoebocytes in the hemolymph. No significant changes were detected in the rate of phagocytosis or number of amoebocytes in snails infected for 2 weeks or less. Alterations in the morphology and behavior of amoebocytes from infected snails were also noted.     

14C uptake by Schistosoma mansoni from Biomphalaria glabrata exposed to 14C-glucose

Exposure of Biomphalaria glabrata infected with Schistosoma mansoni to ¹⁴C-glucose results in a greater uptake of original total snail label by the parasitized digestive gland-gonad, site of the developing daughter sporocysts and cercariae, than by the digestive gland-gonad of control animals. As a consequence of this greater uptake by the infected digestive gland-gonad, the albumen gland and remainder of the carcass of parasitized snails receive less label than do those areas in normal snails. Emergence of cercariae from the snail and daughter sporocyst mass account for a diversion of 12.6% of original total label from the infected snail itself. This diversion of label from the snail to the parasite may explain carbohydrate depletion in parasitized snails.     

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.     

Search for shared antigens in the schistosome-snail combination Trichobilharzia ocellata-Lymnaea stagnalis

Mother and daughter sporocysts of Tricholbilharzia ocellata, developing in the snail host Lymnaea stagnalis, were searched for substances with antigenic similarities to the snail's haemolymph. Antisera to cell-free snail haemolymph and fractions thereof were used in three different immunocytochemical staining methods, applied on sections of parasitized snails. Snail tissue was consistently stained; cercariae were stained, indicating that the applied methods were successful. Most sections through mother and daughter sporocysts were completely unstained. It is concluded that neither mother nor daughter sporocysts are masked by the antigens studied or substances mimicking these. The relevance of the present observations is discussed.     

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

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

Schistosome sporocyst-killing Amoebae isolated from Biomphalaria glabrata.

Explants and swabs from the pericardium and mantle of three strains of Biomphalaria glabrata, two of them resistant to infection with Schistosoma mansoni, have yielded small amoebae, 3–5μm in diameter, in culture. These amoebae have been grown axenically through > 50 passages to date. The amoebae form cysts in dense cultures. When mixed with S. mansoni mother sporocysts in vitro, the amoebae adhere to and kill the trematodes within several hours. For 1–2 days thereafter, the amoebae proliferate rapidly at a generation time of about 5 hr, then return to normal growth. Sonically disrupted sporocysts also induce proliferation. Live sporocysts do not attract the amoebae or emit soluble substances which influence amoebal growth. Amoebae also adhered to and killed S. mansoni daughter sporocysts and cells derived from B. glabrata embryos; however, they did not harm S. mansoni cercariae or rediae of other trematode species. The proportion of mantle explants yielding amoebae was significantly higher (P<0.05) in one of the resistant snail strains than in the susceptible strain; however, whether amoebae contribute to snail resistance is unknown. Exposure of snails to S. mansoni miracidia did not influence the proportion of snails yielding amoebae.     

Accumulation of lipids in Schistosoma mansoni sporocysts cultured in vitro

Neutral lipids were detected histochemically in mother and daughter sporocysts of Schistosoma mansoni cultured in vitro. These lipids progressively increased with prolonged culture. There was little phospholipid and no fatty acid, esterases, or lipases found in sporocysts by the methods employed. Mother and daughter sporocysts incorporated labeled acetate from the culture medium but no further information was obtained on the complex lipid-synthesizing capabilities of these organisms.     

Hemocytes of Bulinus truncatus rohlfsi (Mollusca: Gastropoda)

Two basic cell types occur in the hemolymph of Bulinus truncatus rohlfsi: granulocytes and hyalinocytes. Granulocytes are divided into three subtypes: (1) Granulocytes I, which account for 19% of the hemocytes, are small, young amoebocytes with 1–20 filopodia and small numbers of cytoplasmic granules, including some lysosomes; (2) granulocytes II, which account for 78% of the cells, are large, fully developed amoebocytes that possess 1–20 filopodia and many granules, both acidophilic and basophilic, including numerous lysosomes, phagosomes, and mitochondria; and (3) spent granulocytes, which are rare, have few filopodia, large accumulations of glycogen granules and prominent vacuoles in addition to lysosomes in the cytoplasm. These three subtypes of granulocytes probably represent ontogenetic stages within a single cell line. In addition, granulocytes with 40 or more filopodia and little ectoplasm, found in only 1 of 45 snails examined, probably reflect a pathologic condition. Hyalinocytes, which account for 3% of all hemocytes, are similar in size to mature granulocytes, but have few or no cytoplasmic granules and lack filopodia and glycogen granules. Total hemocyte concentration in hemolymph is 328,000 ± 188,000 cells/ml.     

Studies on parasitic castration: Occurrence of a gametogenesis-inhibiting factor in extract of Zoogonus lasius (Trematoda)

Testes of nonparasitized Ilyanassa obsoleta were maintained in vitro in a modification of the medium originally devised by Burch and Cuadros [Nature (London)206, 637–638 (1965)] in order to ascertain whether a saline extract of the daughter sporocysts of Zoogonus lasius would cause the inhibition of spermatogenesis. This parasite is known to cause parasitic castration of I. obsoleta in nature. It has been ascertained that after 96 hr of incubation with the parasite extract the incorporation of [³H]thymidine is inhibited in some spermatogonia. This represents the first experimental evidence that some molecule(s) of parasite origin may be responsible for chemical parasitic castration.     

Studies on resistance in snails: Interference by nonirradiated echinostome larvae with natural resistance to Schistosoma mansoni in Biomphalaria glabrata

Most of the genetically selected juvenile Biomphalaria glabrata snails, normally strongly resistant to Schistosoma mansoni, lost their juvenile resistance to this parasite when other trematodes were concurrently present in the snail. Three echinostome species all were able to reduce this genetically controlled juvenile resistance: Echinostoma lindoense, E. paraensei, and e. liei. Subsequently, adult resistance to S. mansoni, clearly present in control snails of the same age and strain that were not doubly infected, failed to develop in most of the snails that also harbored echinostomes. Other snails, selected for resistance as adults to S. mansoni, also usually became susceptible to this parasite following infection with E. paraensei. The capacity of E. paraensei to interfere with the snails' resistance to S. mansoni was greater than that of E. lindoense. Destruction by predation of primary sporocysts of S. mansoni by echinostome rediae prevented completion of development of the S. mansoni infections. In a number of snails all primary S. mansoni sporocysts were consumed before secondary sporocysts could be formed. In most experimental snails, however, some of the schistosomes survived, often as a small number of degenerated secondary S. mansoni sporocysts. The capability of flukes to interfere with the natural defense of snails may be an important phenomenon whereby trematode species survive in their snail hosts.     

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.     

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.     

Plagiorchis elegans (Digenea: Plagiorchiidae) infections in Stagnicola elodes (Pulmonata: Lymnaeidae): host susceptibility, growth, reproduction, mortality, and cercarial production.

Eggs of Plagiorchis elegans were readily ingested by Stagnicola elodes of all ages, but were more infective to immature than mature snails. Infection enhanced the growth of the host in a dose-dependent manner. The number of cercariae released by immature snails increased with the age of the snail host; mature snails yielded fewer cercariae. Heavily infected snails tended to die prematurely, thereby reducing their total production of cercariae to levels below those of more lightly infected individuals. Even light infections castrated the snail host. Snails that acquired the infection as juveniles never produced eggs. Actively reproducing snails ceased egg laying within days of infection and never recovered. All parasite effects on the growth and reproduction of the snail host first manifested themselves during the early stages of infection, long before the development of daughter sporocysts and cercariae, and are therefore attributable to the effects of mother sporocysts. The study provides insight into how this natural enemy of mosquito larvae may be established in natural snail populations by means of strategically timed introductions of parasite eggs, with a goal of maximizing cercarial production for the biological control of sympatric mosquito larvae.     

Schistosoma haematobium in Bulinus guernei: Electron microscopy of hemocyte-sporocyst interactions

Electron microscopy has revealed that in Bulinus guernei (Gambian strain) snails infected with Schistosoma haematobium (Egyptian strain) daughter sporocysts and cercariae, two kinds of hemocytes called granulocytes and hyalinocytes are found associated with the sporocysts. Granulocytes are small, numerous, plumbophilic, and amoeboid. They contain lysosome-like granules. Hyalinocytes are large, sparse, less plumbophilic than granulocytes, and have intracellular microfilaments (about 9 nm wide), and few or no pseudopods. They are devoid of lysosome-like granules. Granulocytes and hyalinocytes infiltrate near sporocysts, but only granulocytes interact with sporocyst microvilli by contact. Granulocytes induce a restricted multilamellated encapsulation reaction. Extracellular microfilaments (about 12.5 nm wide), with a regular transverse structure pattern of about 50-nm periodicity, frequently are found along the outer surface of granulocytes located adjacent to sporocysts. Intracellular filamentous structures and a prominent glycocalyx also are features of the seemingly more active granulocytes contiguous with sporocysts. Cell adhesions may occur between surfaces of (1) granulocytes and sporocysts, (2) interdigitating pseudopodial processes of capsular granulocytes, and (3) granulocytes and hyalinocytes.     

Schistosoma mansoni: Long-term maintenance of clones by microsurgical transplantation of sporocysts

Schistosoma mansoni sporocysts originally derived from monomiracidially infected Biomphalaria glabrata snails were serially transplanted into the cephalopedal sinus of anesthetized snails by the microsurgical implantation of fragments of parasitized hepato-pancreas and ovotestis. Three to six passages each of five male and five female clones were maintained for as long as 2.0 years. Of the recipient snails which survived surgery, 87% released cercariae, usually beginning 5–7 weeks after surgery. The percentage of snails which released cercariae increased with successive passages. The mean survival time of surgically infected snails after cercarial emergence began was 9.2 ± 0.5 weeks, nearly the same as that of miracidially infected snails. Longevities of snails infected with male or female clones were similar. Recipient snail size and age did not influence cloning success. Beginning 5 weeks from the onset of cercarial emergence large numbers of cercariae (a mean of 3900/snail from male clones and 1300/snail from females) were obtained during each shedding period. These results clearly demonstrate that the microsurgical transplantation of sporocysts is a practical means of maintaining and expanding populations of genetically homogeneous schistosomes (clones).