Effects of diet on the development of Schistosoma mansoni in Biomphalaria glabrata and on the neutral lipid content of the digestive gland-gonad complex of the snail

In a previous study, when the snail Biomphalaria glabrata was infected with Schistosoma mansoni and maintained on a diet of hen's egg yolk, it produced fully developed cercariae in about one-half the time taken by snails fed Romaine lettuce. Increased lipids were also noted in the snails fed the yolk diet. The purpose of the present study was to further investigate nutritional effects of a high-lipid diet on larval schistosome development and to reexamine the time to cercarial patency in infected snails maintained on either the yolk or lettuce diet and to use high-performance thin-layer chromatography (HPTLC) to analyze the neutral lipids in the digestive gland-gonad complex (DGG) of snails maintained on both diets. Infected snails maintained at 26 C and fed either diet produced fully developed cercariae by 4 wk postinfection (PI). Likewise, infected snails maintained at 23 C and fed either diet produced fully developed cercariae by 6 wk PI. The contention that the yolk diet enhanced the time to cercarial patency was not confirmed. The HPTLC analysis of neutral lipids showed that the DGG of infected snails fed the yolk diet contained significantly greater amounts of free sterols and cholesteryl esters but not triacylglycerols than that of the infected snails fed the lettuce diet.     

Free-pool amino acids in Biomphalaria glabrata infected with Echinostoma caproni as determined by thin-layer chromatography

Thin-layer chromatography was used to analyze the free-pool amino acids of the digestive gland-gonad complex (DGG) of Biomphalaria glabrata infected with Echinostoma caproni and uninfected (control) snails. Qualitative analysis revealed the presence of histidine, lysine, serine, alanine, valine, and isoleucine or leucine in all samples. Quantitative analysis of lysine and valine gave mean weight percentages of 0.00699 +/- 0.0022 and 0.00174 +/- 0.00056, respectively, in the DGG of uninfected snails, and 0.00504 +/- 0.0014 and 0.00254 +/- 0.00033, respectively, in the DGG of infected snails. The differences in values between infected and uninfected snails were not statistically significant (Student's t-test, P > 0.05).     

Emergence of cercariae of Echinostoma caproni and Schistosoma mansoni from Biomphalaria glabrata under different laboratory conditions

Release of Echinostoma caproni cercariae and Schistosoma mansoni from experimentally infected Biomphalaria glabrata snails maintained under different laboratory conditions was studied. Infected snails were isolated individually for 1 h in Stender dishes containing 5 ml of artificial spring water and the number of cercariae released during this time was recorded. Of numerous conditions tested, the addition of lettuce, the use of water conditioned by B. glabrata snails and a temperature of 35 degrees C significantly increased the release of E. caproni cercariae. A significant increase in cercarial release of S. mansoni was seen only in cultures fed lettuce. A temperature of 12 degrees C caused a significant decrease in cercarial release of both E. caproni and S. mansoni. Increased snail activity associated with feeding behaviour was probably responsible for the enhanced cercarial sheds observed in this study.     

Echinostoma paraensei: hemocytes of Biomphalaria glabrata as targets of echinostome mediated interference with host snail resistance to Schistosoma mansoni

Earlier in vivo work by Lie et al. (1977) indicated that the innate resistance of the 10R2 strain of Biomphalaria glabrata to PR1 Schistosoma mansoni could be interfered with if the snails were infected previously with another trematode, Echinostoma paraensei. We have studied this interference phenomenon using in vitro methods in an attempt to understand its mechanistic basis. Hemolymph, derived from 10R2 snails infected with E. paraensei for 14-28 days, killed 25% of S. mansoni sporocysts in vitro, significantly less (P less than 0.001) than the 90% killing rate observed with hemolymph from uninfected, control 10R2 snails. Hemolymph from the infected 10R2 snails and from schistosome susceptible M line snails did not differ significantly (P greater than 0.1) in their relative inability to kill S. mansoni sporocysts in vitro. The defect in sporocyst killing exhibited by echinostome infected 10R2 snails was traced to the cellular, rather than the humoral, component of the hemolymph. Preparations containing uninfected 10R2 snail hemolymph and echinostome daughter rediae exhibited significantly less (P less than 0.001) killing of S. mansoni sporocysts than did controls containing only 10R2 hemolymph and S. mansoni sporocysts. Our results suggest that echinostome larvae release factors that interfere with the ability of B. glabrata hemocytes to kill S. mansoni sporocysts.     

Effects of glucose on survival, infectivity and linear movement of the cercariae of Echinostoma caproni

The effects of glucose in artificial spring water (ASW) on the survival, infectivity, and linear movement of Echinostoma caproni cercariae were studied. Cercariae maintained at 23 degrees C in 1% glucose in ASW (ASWG) or ASW alone, reached 50% survival at 26 and 23 h, respectively. All cercariae in ASWG and ASW were dead by 50 and 32 h, respectively. Infectivity to juvenile Helisoma trivolvis (Colorado strain) snails was significantly less for cercariae aged 16 h in ASWG compared to cercariae aged 16 h in ASW. Linear movement, i.e. the ability of cercariae to traverse a 1-cm radius, ceased at 16 and 20 h for cercariae maintained in ASWG and ASW, respectively. Glucose added to ASW extended the survival time of E. caproni cercariae but decreased their ability to infect snails or move in a linear direction.     

Sterols in the plasma and digestive gland-gonad complex of Biomphalaria glabrata snails, fed lettuce versus hen's egg yolk, as determined by GLC

1. Gas-liquid chromatography studies were done on sterols in plasma and the digestive gland-gonad (DGG) complex of Biomphalaria glabrata snails fed lettuce vs hen's egg yolk. 2. The major sterols present in the DGG of both populations were cholesterol, stigmasterol, beta-sitosterol, campesterol, and desmosterol. 3. The percentage composition of cholesterol in the DGG of yolk vs lettuce fed snails was 82 and 51, respectively. 4. The elution profiles of sterols in the plasma of yolk vs lettuce fed snails were similar; both contained desmosterol, campesterol, and stigmasterol, negligible amounts of cholesterol, and unidentified sterols. 5. The high lipid diet increased the level of cholesterol in the DGG but not in the plasma.     

Effects of Schistosoma mansoni on the nutrition of its intermediate host, Biomphalaria glabrata

Biomphalaria glabrata infected with Schistosoma mansoni and maintained on an artificial diet of Spirulina alga displayed reduced growth during the 5 wk following patency. Food consumption per unit snail weight was unaffected. Infected snails also failed to lay eggs. No difference in percentage of assimilation was observed between control and infected individuals, but infected snails had significantly decreased gross conversion efficiencies. The effects of infection on nutrition of B. glabrata were similar to those observed in nutrient-deprived snails fed diets containing low Spirulina levels. Nutrient deprivation, however, did not alter conversion efficiency.     

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.     

Experimental double infection of Biomphalaria glabrata snails with Angiostrongylus cantonensis and Schistosoma mansoni

Two groups of Biomphalaria glabrata snails primarily infected with Angiostrongylus contonensis were secondarily exposed to infection with Schistosoma mansoni. To investigate any anatagonistic effect of the first infection on a superimposed one and to compare to singly and non-infected snails, a series of experiments was undertaken in which snails were individually exposed, variously, to 1,000 and 2,000 first-stage larvae of A. cantonensis and then to 5 and 10 miracidia of S. mansoni 1 day and 3 weeks later. Snails became infected with S. mansoni in both groups of snails with double infections and shed cercariae after the same incubation period as in the singly infected groups. The number of snails shedding cercariae simultaneously was similar in single and double infection groups during the first two weeks of shedding, after which this number decreased somewhat in doubly infected groups. Snails with double infection showed higher cumulative mortality rates than in snail groups with single infection with either A. cantonensis or S. mansoni. Therefore, initial infection of B. glabrata with A. cantonensis produced no inhibitory or retarding effect on subsequent infection of snails with S. mansoni.     

Effect of Schistosoma mansoni infection on fecundity and perivitelline fluid composition in Biomphalaria glabrata

Egg production in the snail, Biomphalaria glabrata, infected with Schistosoma mansoni declined on day 23 postinfection, and was significantly lower than uninfected control snails by day 28 and thereafter. Protein and galactogen content of eggs produced by infected snails did not change during the period of reduced fecundity. This suggests that decreased hemolymph nutrient levels (rather than depleted albumen gland reserves) are responsible for inhibition of snail egg production. Growth rates of infected and uninfected snails were indistinguishable from days 14 through 35 postinfection. The hatching success of eggs produced by infected snails decreased slightly beginning at day 21 postinfection.     

Altered feeding response as a cause for the altered heartbeat rate and locomotor activity of Schistosoma mansoni-infected biomphalaria glabrata

Biomphalaria glabrata infected with Schistosoma mansoni for 33 days fed more often than uninfected snails. Whereas uninfected snails had nocturnal increases in feeding, snails with a 33-day-old infection of S. mansoni fed as often during the day as in the night. Using direct observation and film analysis, we found that feeding increased the heartbeat rate and locomotor activity of B. glabrata. When snails were allowed to feed ad lib., infected snails had higher heartbeat rates than uninfected snails both during the day (P less than 0.01) and the night (P less than 0.001). However, when the snails were deprived of food for 24 hr, infected snails had slightly higher heartbeat rates than uninfected snails only during the day (P less than 0.05). There was no difference between the heartbeat rates of feeding, infected snails and the heartbeat rates of uninfected snails that were starved for 8 hr, and then allowed to feed. Uninfected snails had nocturnal increases in heartbeat rate regardless of feeding schedule, but infected snails had greater nighttime heartbeat rate than daytime heartbeat rate only when they were not allowed to feed. Infected snails had less nocturnal locomotor activity than uninfected snails when feeding, but there was no difference between the locomotor activity of infected and uninfected snails when the snails were deprived of food for 24 hr. Absence of food also resulted in an increased nighttime to daytime ratio of locomotor activity of infected snails. These results suggest that the increased heartbeat rate and altered rhythms of heartbeat rate and locomotor activity in B. glabrata infected with S. mansoni for 33 days were caused by the altered feeding response of these snails.     

Reversal of dietary-induced hyperlipidemia in Biomphalaria glabrata (Gastropoda).

1. To determine if dietary induced hyperlipidemia in snails could be reversed, thin-layer chromatography studies were done on lipids in the digestive gland-gonad (DGG) complex of Biomphalaria glabrata fed either hen's egg yolk for 2 weeks (Group A), yolk for 1 week followed by leaf lettuce for 1 week (Group B), or lettuce for 2 weeks (Group C). 2. The major lipid fractions in Group A were triacylglycerols, phosphatidylethanolamine, and phosphatidylcholine, along with lesser amounts of sterol esters, methyl esters, free fatty acids, free sterols and unidentified phospholipids. 3. Groups B and C had about one-half the amount of triacylglycerols than Group A and did not show sterol esters or methyl esters. 4. The triacylglycerol increase of the DGG resulting from the hen's egg diet could be reversed by returning the snails to the lettuce diet.     

In vitro and in vivo encystment of the cercariae of Echinostoma caproni

In vitro and in vivo studies were conducted on the cercariae of Echinostoma caproni. Of the 15 media tried, 2 resulted in effective in vitro encystment in petri dish cultures maintained at 23 +/- 1 C. They were a Locke's--artificial springwater (ASW) (1:1) medium (67% encystment) and a Biomphalaria glabrata embryonic cell line medium (23% encystment). To obtain large numbers of in vitro--formed cysts, finger bowl cultures containing 40 ml of the Locke's-ASW (1:1) medium were used at 23 +/- 1 C. Of 3,000 cercariae tested, 1,890 (63%) were encysted in this medium by 48 hr. Most of these cysts looked similar to those formed in vivo, although some showed abnormalities in the outer cyst wall and other malformations. A total of 200 in vitro-formed cysts treated in an alkaline trypsin-bile salts (TB) medium for 2 hr at 41 C showed 94% excystation. In vitro-formed cysts fed to mice produced ovigerous adults within 2 wk postinfection (PI). Eggs from these worms gave rise to miracidia that produced patent intramolluscan infections in B. glabrata snails. In vivo encystment was studied in lab-raised juvenile Helisoma trivolvis (Colorado strain) snails, 1-3 mm in shell diameter. From 6 to 24 hr PI, 93-100% of the cercariae were recovered as metacercarial cysts in the snail tissue. Treatment of these cysts in the TB medium resulted in 96% excystation within 2 hr at 41 C.     

Effects of temperature on survival,infectivity and in vitro encystment of the cercariae of Echinostoma caproni

The effects of temperature on survival, infectivity and in vitro encystment of Echinostoma caproni cercariae in artificial spring water (ASW) were studied. Effects of aging cercariae in ASW at various temperatures showed that at 23 degrees C cercariae achieved 50% survival in 24 h, compared to 92 h at 12 degrees C. Cercariae aged in ASW at 28 and 37.5 degrees C showed 50% survival at 16 and 10 h, respectively. Cercariae aged at different temperatures for various times were used to infect juvenile Helisoma trivolvis (Colorado strain) snails maintained in ASW at 23 degrees C. Index of infectivity was based on counting encysted metacercariae in the snails at 8 to 12 h post-infection. Cercariae aged at 23, 28 and 37.5 degrees C showed 50% encystment at 6, 8 and 4 h, respectively. Cercariae aged at 4 degrees C showed 50% encystment in 10 h and cercariae aged at 12 degrees C showed 50% encystment beyond 16 h. Cercariae showed maximal longevity and infectivity in snails when aged at 12 degrees C in ASW. For E. caproni, as in other digeneans, the infective period of cercariae is markedly shorter than the maximal life-span at any given temperature. Studies on in vitro encystment of E. caproni cercariae in Locke's solution:ASW (1:1) showed that encystment was optimal at 23 degrees C (78% encystment) and that it declined to 44% at 28 degrees C and became almost nil (0.02%) at 12 or 37.5 degrees C.     

Intraspecific variations in the hemolymph of Biomphalaria glabrata, a snail host of Schistosoma mansoni.

An attempt was made to characterize the hemolymph of Biomphalaria glabrata with reference to "normal" intra-specific variation, i.e., both inter- and intra-strain differences. Total protein concentration, per cent hemoglobin, pH, and osmolarity were studied. Seven geographic strains of B, glabrata were examined. In addition, observations were made on the hemolymph of Biomphalaria straminea, several strains of Helisoma caribaeum, and on B. glabrata subjected to infection with Schistosoma mansoni or to periods of starvation. Intra-strain differences in total protein concentration and total hemoglobin concentration in B. glabrata appeared to be more closely related with snail size than with absolute age. Inter-strain variation in B. glabrata was also noted, but the differences were of the same magnitude as those from intra-strain samples. Significant differences in total protein concentration were observed, however, between the means of similar size B. glabrata, B. straminea and H. caribaeum. The osmolatity of the hemolymph from different size B. glabrata was similar as were the osmolalities of the hemolymph from similar size snails of different strains. However, all B. glabrata strains exhibited hemolymph osmolalities lower than observed in strains of H. caribaeum. Infection with S. mansoni reduced the protein concentration of B. glabrata hemolymph. Differences were noted as early as 1.5-24 hr post-infection, with significant alterations occurring at about 11 days post-infection. To a lesser extent, starvation also depleted the protein content of the hemolymph.     

Resistance to desiccation of Biomphalaria glabrata adults infested by various miracidia of Schistosoma mansoni

1200 adult Biomphalaria glabrata were submitted during 6 weeks to anhydrobiosis condition. Some snails were healthy, some were previously infected 3 days or 12 days ago with 8 +/- 2 miracidia of Schistosoma mansoni, others were shedding cercariae. The snails were put on soil or buried into hermetically closed, or ventilated, plastic boxes. There was no survival of snails kept in sealed boxes, or among positive snails, but 44% of control healthy snails and 40.6% of infected (for 3 or 12 days) snails in ventilated boxes were living at the term of the desiccation stage. Survival was better for "on soil" snails than for "buried" snails, but no difference was shown between 3-days and 12-days infection. The surviving desiccated B. glabrata had a lesser death rate and a lesser cercarial production than infected snails kept in water. An inferior production of male cercariae comparatively to female and to "mixed" cercariae was demonstrated by statistical analysis of the cercarial sheddings. In all positive snails, periodic variations of cercarial production was shown, whatever the sex of those cercariae. In addition many pauses of the sheddings were established by the authors.     

Rescue of sporocysts of Schistosoma mansoni in nonsusceptible Biomphalaria by head-foot transplantation into susceptible snails

To measure the longevity of sporocysts of Schistosoma mansoni in nonsusceptible snails (13-16-R1 and Salvador strains of Biomphalaria glabrata, and Biomphalaria obstructa), the head-foot (HF) of miracidia-exposed snails was transplanted into the hemocoel of a susceptible NIH albino recipient at 1-36 days postexposure (DPE). Recipient snails which were not exposed to miracidia then were monitored for infection transferred by the implant, and infection prevalences in recipients of HF transplants from nonsusceptible donors were compared to those in snails implanted with an HF from NIH albino donors. Transplants from NIH albino snails between 1 to 15 DPE infected 98% of recipients. Similarly, at 1 DPE, 69-85% of transplants from nonsusceptible snails contained viable sporocysts, as shown by resulting patent infections in the recipients. Recipient infection prevalence, and presumably numbers of transplants containing viable sporocysts, declined as a function of DPE, and by 5-9 DPE this decrease was significant for all 3 types of nonsusceptible donors. However, viable sporocysts still occurred in B. obstructa and 13-16-R1 B. glabrata as late as 19 and 20 DPE, respectively, and in Salvador B. glabrata as late as 33 DPE. Thus, sporocysts persist in nonsusceptible snails considerably longer than suggested by results of previous histological studies.     

Effects of exogenous glucose on survival and infectivity of Schistosoma mansoni cercariae

The effects of exogenous glucose in artificial spring water (ASW) were studied on the survival and infectivity of Schistosoma mansoni cercariae. The mean percent survival of cercariae maintained in 1% glucose in ASW for 36 and 48 hr was significantly greater than that of cercariae maintained identically in ASW. Cercariae maintained in ASW with or without glucose for 24 hr, fixed in neutral buffered formalin, and stained in Oil Red O, showed an accumulation of neutral lipid in the tail. Cercariae maintained as described above and stained in periodic acid-Schiff exhibited depleted glycogen, mainly from the tail. Cercariae maintained in ASW with glucose for 24 hr did not resynthesize glycogen. Cercariae maintained in ASW with glucose for 24 hr were as capable of infecting male FVBN202 mice as were freshly emerged cercariae, and increased the percent of worm recovery. Exogeneous glucose added to ASW prolonged the survival of S. mansoni cercariae and increased infectivity in terms of worm recovery.     

Effects of alterations in the heartbeat rate and locomotor activity of Schistosoma mansoni-infected Biomphalaria glabrata on cercarial emergence

During a 27-hr period of food deprivation, Biomphalaria glabrata infected with Schistosoma mansoni had lower heartbeat rates and locomotor activities than did controls that were feeding ad lib. However, there was no difference between the cercarial emergence from control and experimental snails either before, during, or after the period of food deprivation. No correlation was found between the locomotor activity of the snail and the number of cercariae emerging from the snail in continuous light. Our results indicated that the emergence of S. mansoni cercariae from B. glabrata was not affected by the heartbeat rate or locomotor activity of the snail. The factors controlling the rhythm of S. mansoni cercarial emergence from B. glabrata may be independent of the snail.     

The effects of temperature change on the infection rate of Biomphalaria glabrata with Schistosoma mansoni

The aim of this study was to investigate the influence of temperature on the development of Schistosoma mansoni infections in Biomphalaria glabrata. The snails were infected at 15, 20, and 30 degrees C, and the cercarial release was analyzed after 30 and 60 days post-infection. Our results showed that a decrease in the temperature has a substantial influence on the development of S. mansoni infection in B. glabrata, with significant differences (p < 0.05) between 15 and 30 degrees C. These data could provide a better understanding of the epidemiological aspects of schistosomiasis.