Effects of copper sulphate on in vitro encystment of the cercariae of Echinostoma caproni

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.     

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 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.     

Studies on encystment of Echinostoma revolutum cercariae.

Cercariae of Echinostoma revolutum encysted in the kidney of the snail Physa heterostropha within 1 hr and on mucus trails from Helisoma trivolvis, P. heterostropha and Lymnaea sp. within 2 hr. Significantly, more normal cysts were formed in mucus of Helisoma than in mucus of Physa or Lymnaea. Optimal, in vitro encystment occurred within 24 h in either Locke's 1 : 1 or Locke's 1 : 1 + 1% glucose. Significantly more normal cysts occurred in the Locke's 1 : 1 medium. Both normal and abnormal cysts from Lock's media and snail mucus excysted in an alkaline bile trypsin medium. Cercariae did not encyst in Lock'e media supplemented with casein hydrolysate or in agar cultures containing various chemicals.     

Effects of copper sulfate toxicity on cercariae and metacercariae of Echinostoma caproni and Echinostoma trivolvis and on the survival of Biomphalaria glabrata snails

Copper in the form of copper sulfate (CuSO4) decreases the survival of Biomphalaria glabrata snails, but the effects of this molluscicide on Echinostoma caproni and Echinostoma trivolvis, 2 species of digeneans that use B. glabrata as intermediate hosts, are not known. Studies were done on the effects of various concentrations of CuSO4 in artificial spring water (ASW) on the survival and infectivity of E. caproni and E. trivolvis cercariae. Solutions containing 1.0, 0.1, and 0.01% CuSO4 were 100% lethal within 2 hr of exposure for both species. Time to 50% mortality in 0.001% CuSO4 was 8 hr for E. caproni and 16 hr for E. trivolvis; at 24 hr, the controls showed 50 and 65% mortality, respectively. Treatment of cercariae of both species for 0.5 hr in 0.001% CuSO4 had no effect on the ability of cercariae to form normal cysts in juvenile B. glabrata snails. However, treatment with 0.01% CuSO4 for 0.5 hr caused a significant reduction in the ability of cercariae of both species to encyst in snails. Treatment of encysted metacercariae of both species in 0.001% CuSO4 for I hr had no effect on subsequent excystation of these echinostomes in a trypsin-bile salts medium, whereas concentrations of 1.0, 0.1, and 0.01% CuSO4 and 1.0 and 0.1% CuSO4 decreased chemical excystation of E. caproni and E. trivolvis cysts, respectively. Survival studies on the effects of CuSO4 in Locke's solution on chemically excysted metacercariae of both species were also done. Excysted metacercariae of both species were killed by 2 hr in either 0.1 or 0.01% CuSO4 in Locke's solution. However, time to 50% mortality for both species of excysted metacercariae in 0.001% CuSO4 was approximately 5 hr. Time to 50% mortality for the controls was about 12 hr. Survival of juvenile B. glabrata snails was also examined. All B. glabrata snails were dead by 6 hr in 1 and 0.1% CuSO4 in ASW. Biomphalaria glabrata snails showed 50% mortality by about 6 hr in 0.01% CuSO4 and about 80% were still alive at 24 hr in 0.001% CuSO4. All controls were alive at 24 hr, at which time the experiment was terminated. Concentrations greater than 0.001% CuSO4 increased snail mortality, as well as that of the cercariae and excysted metacercariae of E. caproni and E. trivolvis. Our findings suggest that concentrations of copper sufficient to eliminate juvenile B. glabratta snails are also sufficient to kill the cercariae and excysted metacercariae of these digeneans but not the encysted metacercariae, which may be protected by their cyst walls.     

Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni

High performance thin-layer chromatography (HPTLC) was used to analyse the neutral lipids in the rediae, cercariae, and encysted metacercariae of Echinostoma caproni from Biomphalaria glabrata snails. Visual observations of the chromatograms showed that the most abundant lipid fraction in all stages was free sterol. Quantification of the free sterol revealed mean weights of 2.7 +/- 0.64 ng per redia, 0.53 +/- 0.023 ng per cercaria, and 0.081 +/- 0.0098 ng per encysted metacercaria. Oil Red O staining of the larval stages confirmed the presence of lipids within the rediae and cercariae but did not show lipids in the encysted metacercariae. The dimunition in neutral lipids from the cercarial to the encysted metacercarial stage does not support a previous observation that fat increases in successive phases of the digenean life cycle.     

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.     

Cercarial tail loss in Echinostoma caproni: the influence of in vivo encystment and copper sulphate

Echinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l(-1) in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg(-1) H2O, Ca(2+) 20 mg l(-1)) at 23 degrees C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l(-1), 1000 mg l(-1) and 100 mg l(-1) CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59+/-5% of cercariae had lost their tails and only 4+/-1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72+/-3% of the cercariae began to form cysts within the snails.     

Effects of a diet deficient in the B complex vitamins on infectivity, growth and distribution of Echinostoma caproni in ICR mice

The effects of a diet deficient in the B vitamins on infectivity, growth, and distribution of Echinostoma caproni in ICR mice were studied. The vitamin-deficient diet (experimental) was isocaloric to the control diet but lacked the B vitamins. Thirty-six female, 6- to 8-week-old ICR mice were each infected with 25 metacercarial cysts. From the day of infection to the day of necropsy, 18 mice were fed the experimental diet and the remaining mice received the control diet. Equal numbers of experimental and control mice were necropsied at 2, 3 and 4 weeks postinfection (p.i.). Mice on the experimental diet showed a significant loss in body weight between 2 and 4 weeks p.i. There was no significant difference in worm recovery at 2 to 4 weeks p.i. from mice on either diet. Worms from hosts on the experimental diet were more dispersed and located more posteriad in the small intestine than those from mice on the control diet. Worm dry weight was significantly less in hosts on the experimental diet at all weeks p.i. compared with that of hosts on the control diet. The body area of worms on the experimental diet was significantly less at 2 and 3 weeks p.i. than that of worms on the control diet. An isocaloric diet deficient in the B vitamins had a detrimental effect on the growth of E. caproni in ICR mice.     

Exposure of Dugesia tigrina (Turbellaria) to cercariae of Echinostoma trivolvis and Echinostoma caproni (Trematoda)

Laboratory-reared planarians, Dugesia tigrina, were exposed to cercariae of Echinostoma trivolvis or Echinostoma caproni. Of 100 D. tigrina exposed to 2,750 cercariae of E. trivolvis, 29 were infected with a total of 85 encysted metacercariae 24 hr postinfection (PI). None of 40 D. tigrina exposed to 1,100 cercariae of E. caproni was infected with metacercariae 24 hr PI. Cyst structure of E. trivolvis from the parenchyma of D. tigrina varied from normal to abnormal. Metacercariae removed from planarians could be excysted in an alkaline trypsin-bile salts medium. Cercariae of E. trivolvis were not attracted to dialysate material from D. tigrina. Cercariae of E. trivolvis that penetrated, but did not encyst in planarians, voided the contents of their paraesophageal glands. Cercariae of E. caproni lack paraesophageal glands. Secretions of those glands probably are involved in tissue penetration of D. tigrina by E. trivolvis cercariae.     

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.     

In vitro culture of rediae of Echinostoma caproni.

Rediae of Echinostoma caproni (Egyptian strain) were dissected from Biomphalaria glabrata snails at intervals from 13-34 days post-exposure and co-cultured for up to 51 days with cells of the B. glabrata embryonic (Bge) cell line. Rediae readily ingested Bge cells and survived longer when co-cultured with cells than in cell-free cultures. Rediae released mostly motile cercariae throughout the observation period when in Bge medium and cells. Rediae cultured in 199 medium with Bge cells also produced progeny throughout most of the observation period. In the latter medium, progeny were much more likely to include rediae as well as cercariae. Some cercariae produced in vitro encysted as metacercariae. Rediae consumed cercariae released into culture but were not observed to attack one another or rediae of a different echinostome species.     

Influence of intramolluscan larval stages of Echinostoma liei on the infectivity of Schistosoma mansoni cercariae

During co-infection of Biomphalaria glabrata with Schistosoma mansoni and Echinostoma liei, S. mansoni cercariae released before the complete resorption of S. mansoni sporocysts show a very strong decrease of their infectivity in mice. Under conditions of high interspecific competition (i.e. when the snails are infected by E. liei 8 days after infection by S. mansoni), the mean overall worm return is five times lower than that of the control experiment. A marked decrease of the infectivity of cercariae is also noted when snails, infected exclusively with either sporocysts of S. mansoni or rediae of E. liei, are associated in the same tank.     

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.     

Effect of ultraviolet radiation on survival, infectivity and maturation of Schistosoma mansoni cercariae

S. mansoni cercariae exposed to ultraviolet radiation for 1, 3, 5, 10 and 20 s as well as non-irradiated cercariae remained actively motile 30 min post-irradiation. Thereafter the activity decreased with increasing dose level of radiation and age of cercariae. There was no significant difference between the rates of attachment of the batches of cercariae. The recovery rates (0-49% of cercariae to which mice were exposed) of adult worms were, however, significantly different from the number of cercariae calculated to have attached to the mice (93.5-100% of cercariae to which mice were exposed). Maturation and penetration rates were dependent on radiation exposure levels. Numbers of eggs deposited in the liver of mice as well as hatchability rate of eggs varied significantly with the levels of exposure to radiation.     

Concurrent infections of Echinostoma caproni and Echinostoma trivolvis in ICR mice.

ICR female mice, 6- to 8-weeks old, were exposed concurrently to 25 metacercarial cysts of Echinostoma caproni and 25 metacercarial cysts of Echinostoma trivolvis and necropsied 10 and 14 days post-infection. Controls consisted of mice exposed singly to either 25 or 50 E. caproni or E. trivolvis cysts. All 23 mice exposed to E. caproni cysts were infected with a total of 331 worms (37.8%), whereas only 11 (37.9%) of 29 mice exposed to E. trivolvis cysts were infected with a total of 77 (6.4%) worms. In the concurrent infections, 13 (59.1%) of 22 mice were infected with both species and the percentage of worm recovery was 72.6% for E. caproni and 14.2% for E. trivolvis. There was no difference in worm distribution of either species in single vs concurrent infections. In concurrent infections at 14 days PI, there was a significant decrease in the body area of worms of both species, when compared to single worm species.     

Effects of Echinostoma caproni infections on metallic ions in the intestinal mucosa of ICR mice

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study metallic ions in the intestinal mucosa of ICR mice infected with Echinostoma caproni and the mucosa of uninfected control mice. Infected mucosa (n = 9 with about 100 mg wet weight per sample) were examined at 2 weeks p.i. in mice that were infected with about 25 worms per host. Uninfected mucosa (n = 9 with about 100 mg wet weight per sample) were examined in the same time frame as the infected mucosa. Five metals were measured in the mucosa by ICP-AES analysis, as follows: calcium, potassium, magnesium, sodium and zinc. There were no significant differences (Student's t-test, P > 0.05) in the concentrations of calcium, potassium or zinc in infected versus uninfected mucosa. The concentration of sodium was significantly greater (P < 0.05) in the mucosa of infected versus uninfected mucosa, but the situation was reversed in regard to magnesium.     

Effect of temperature on emergence, survival and infectivity of cercariae of the marine trematode Renicola roscovita (Digenea: Renicolidae)

Marine bivalves harbour a diversity of trematode parasites affecting population and community dynamics of their hosts. Although ecologically and economically important, factors influencing transmission between first (snail) and second (bivalve) intermediate hosts have rarely been studied in marine systems. In laboratory experiments, the effect of temperature (10, 15, 20, 25 degrees C) was investigated on (1) emergence from snails, (2) survival outside hosts and (3) infectivity in second intermediate hosts of cercariae of the trematode Renicola roscovita (Digenea: Renicolidae), a major parasite in North Sea bivalves. Emergence of cercariae peaked at 20 degrees C (2609 +/- 478 cercariae snail(-1) 120 h(-1)) and was considerably lower at 10 degrees C (80 +/- 79), 15 degrees C (747 +/- 384) and 25 degrees C (1141 +/- 334). Survival time decreased with increasing temperature, resulting in 50% mortality of the cercariae after 32.8 +/- 0.6 h (10 degrees C), 26.8 +/- 0.8 h (15 degrees C), 20.2 +/- 0.5 h (20 degrees C) and 16.6 +/- 0.3 h (25 degrees C ). Infectivity of R. roscovita cercariae in cockles Cerastoderma edule increased with increasing temperature and was highest at 25 degrees C (42.6 +/- 3.9%). However, mesocosm experiments with infected snails and cockle hosts in small aquaria, integrating cercarial emergence, survival and infectivity, showed highest infection of cockles at 20 degrees C (415 +/- 115 metacercariae host(-1)), indicating 20 degrees C to be the optimum temperature for transmission of this species. A field experiment showed metacercariae of R. roscovita to appear in C. edule with rising water temperature in April; highest infection rates were in August, when the water temperature reached 20 degrees C. Since another trematode species (Himasthla elongata; Digenea: Echinostomatidae) occurring at the experimental site showed a similar temporal pattern, trematode transmission to second intermediate bivalve hosts may peak during especially warm (> or = 20 degrees C) summers in the variable climate regime of the North Sea.     

In vivo and in vitro encystment of Echinochasmus liliputanus cercariae and biological activity of the metacercariae

In vivo and in vitro encystment of the cercariae of Echinochasmus liliputanus and biological activity of the metacercariae were studied. In vivo encystment of cercariae occurred in the gills of goldfish, the second intermediate host. However, the cercariae also encysted in vitro in Locke solution (0.6x to 1.2x strength), 0.7-1.2% NaCI, artificial gastric juice, and human gastric juice. Locke or NaCI solutions were shown to be appropriate for in vitro encystment to occur within 24 hr; however, full-strength Locke solution was shown to be optimal. The 1-day-old metacercariae formed in vivo and treated in 0.1% sodium deoxycholate excystation medium at 37 C for 1 hr showed 88.5% excystation. The metacercariae formed in vitro, however, showed 88.6% and 85.0% excystation for normal and abnormal ones, respectively. Abnormal cysts at room temperature usually die within 10 days. About 70% of the normal cysts, both in vivo and in vitro, can still excyst after being stored in Locke 0.5x solution at 4 C for 3 mo. Cysts formed in vivo and in vitro were equally infective. The encystment of the cercariae in vitro could be inhibited when the cercariae were treated with 1 micromol silver nitrate. Because silver nitrate binds to the papillae, especially to the ciliated papillae, on the cercaria surface, it is suggested that papillary chemoreceptors may be involved in encystment of the cercariae. The finding of E. liliputanus cercariae encysting in vitro, especially in human gastric juice, might be helpful in elucidating mechanisms of the definitive hosts that are directly infected by the cercariae.