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.     

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.     

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.     

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.     

Experimental infection of Physa heterostropha, Helisoma trivolvis, and Biomphalaria glabrata (Gastropoda) with Echinostoma revolutum (Trematoda) Cercariae

Gross and histologic studies were done on laboratory-raised Physa heterostropha, Helisoma trivolvis, and Biomphalaria glabrata snails exposed individually to 100 cercariae of Echinostoma revolutum. Cercariae showed a predisposition for the kidneys of snails. They entered the nephridiopore, migrated up the tubular kidney, and encysted in the saccular kidney within 2 hr. Considerably more cysts were in the kidney of B. glabrata at 24 hr than in H. trivolvis or P. heterostropha kidneys. Encysted metacercariae were not infective to domestic chicks at 2 hr, but were infective by 4 hr. Biomphalaria glabrata exposed to about 1,000 cercariae/snail and necropsied either 10 or 16 wk postexposure contained 300-500 cysts/kidney; about one-half the cysts were viable and infective to chicks. Biomphalaria glabrata is an excellent second intermediate host for the laboratory propagation of E. revolutum.     

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.     

Cultivation of excysted metacercariae of Echinostoma caproni to ovigerous adults in the allantois of the chick embryo.

Chemically excysted metacercariae of Echinostoma caproni inoculated into the allantois of domestic chick embryos became ovigerous in that site within 9 days postinoculation. The egg preparation technique of Saville and Irwin was markedly better than that of a modified Zwilling procedure for obtaining large numbers of postinoculation embryos with worm infections. Adults of E. caproni from the allantois were larger and became ovigerous sooner than worms grown on the chorioallantois. Only worms from the allantois produced eggs with fully developed miracidia. Miracidia were released from these eggs, but an insufficient number was available to attempt infections in Biomphalaria glabrata snails.     

The influence of Echinostoma caproni metacercariae (Trematoda) on the survival of biomphalaria molluscs (Pulmonata)

The infrapopulation of the Echinostoma caproni partenites has a development of prolong character (Ataev et al, 2005). However, in laboratory conditions, Biomphalaria molluscs infested with this parasite die within 1--3 weeks after the beginning of cercariae emission. It has been suggested that autoinvasion of the mollusc host with the cercariae, which use it as second intermediate host, is the cause of this phenomenon. Studying the dynamics of metacercariae accumulation in the host (both infected and non-infected with the Echinostoma caproni rediae) and experiments where quantity of cercariae around molluscs reduced by different ways, confirmed this hypothesis. Evidently, pathogenicity of metacercariae for molluscs is lesser in nature, because the concentration of cercariae reduces to the values, which do not result in lethal effect: some part of cercariae dies, but another part uses other animals as a host (Haas, 2000).     

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.     

Cipangopaludina chinensis malleata (Gastropoda: Viviparidae): a new second molluscan intermediate host of a human intestinal fluke Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Two species of the viviparid snails have been reported in Korea, e.g., Cipangopaludina chinensis malleata and Cipangopaludina japonica. Cipangopaludina chinensis malleata collected at 3 of 12 localities were found to be infected with metacercariae of Echinostoma cinetorchis, one of the snail-borne human intestinal trematodes in Korea. Metacercariae from these snails were fed to rats (S/D strain), and adult worms of E. cinetorchis, characterized by 37-38 collar spines on the head crown, were recovered from the ileocecal regions. However, no C. japonica collected from 2 localities harbored the metacercariae. In experiments with laboratory-bred viviparid snails, all viviparids were not susceptible to miracidia of E. cinetorchis. To confirm the identity of second intermediate hosts of E. cinetorchis experimentally, 2 species of viviparid snails were exposed to the cercariae from Segmentina hemisphaerula that had been infected with miracidia of E. cinetorchis. Both species of snails were susceptible to cercariae of E. cinetorchis. This is the first report of Cipangopaludina spp. serving as the second intermediate host of E. cinetorchis and as a potential source of human infection.     

Austropeplea ollula (Pulmonata: Lymnaeidae): a new molluscan intermediate host of a human intestinal fluke, Echinostoma cinetorchis (Trematoda: Echinostomatidae) in Korea

Three freshwater snail species of the family Lymnaeidae have been reported from Korea, Radix auricularia coreana, Austropeplea ollula and Fossaria truncatula. Out of 3 lymnaeid snail species, A. ollula was naturally infected with the Echinostoma cinetorchis cercariae (infection rate = 0.7%). In the experiments with the laboratory-bred snails, F. truncatula as well as A. ollula was also susceptible to the E. cinetorchis miracidia with infection rates of 25% and 40%, respectively. All of three lymnaeid snail species exposed to the E. cinetorchis cercariae were infected with the E. cinetorchis metacercariae. It is evident that A. ollula acts as the first molluscan intermediate host of E. cinetorchis in Korea, and F. truncatula may be a possible candidate for the first intermediate host of this intestinal fluke. Also, three lymnaeid snail species targeted were experimentally infected with E. cinetorchis metacercariae.     

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.     

Larval trematodes (Digenea) of the great pond snail, Lymnaea stagnalis (L.), (Gastropoda, Pulmonata) in Central Europe: a survey of species and key to their identification

A survey of cercariae and metacercariae (Trematoda, Digenea) from the great pond snail (Lymnaea stagnalis) in Central Europe (Austria, Czech Republic, South-East Germany, Poland and Slovak Republic) is presented, based on a study of 3,628 snails examined from 1998 to 2005. A total of 953 (26.3%) L. stagnalis were infected with 24 trematode species comprising 19 species of cercariae and 11 species of metacercariae (six species occurred both as cercarie and metacercarie) of eight families. The dominant cercariae were those of Opisthioglyphe ranae (159 hosts infected), Plagiorchis elegans (141) (both family Plagiorchiidae) and Echinoparyphium aconiatum (153) (Echinostomatidae); 14 double infections were found. The most frequent metacercariae were those of Neoglyphe locellus (71) (Omphalometridae), E. aconiatum (66), Echinostoma sp. (59) and Moliniella anceps (48) (Echinostomatidae). In the previous studies carried out in Central Europe, a very similar spectrum of nine trematode families of 22 cercariae determined to species level and 43 types of cercariae reported under generic or provisional names, which can be in many cases conspecific with the previous taxa, were found. A simple key to identification of cercariae and metacercariae, together with their illustrations, is provided.     

Larval trematodes (Digenea) of planorbid snails (Gastropoda: Pulmonata) in Central Europe: a survey of species and key to their identification

A survey of the larval stages (cercariae and metacercariae) of trematodes (Digenea) found in planorbid snails in Central Europe (Austria, Czech Republic, south-east Germany, Hungary, Poland and the Slovak Republic) is presented based on a study of 7,628 snails of 12 species examined between 1998-2006. A total of 34 trematode larval stages, comprising cercariae of 28 species and metacercariae of seven species (one species occurred both as cercaria and metacercaria) of nine families were found in 898 (11.5%) snails of eight species. The dominant cercariae were those belonging to the Rubenstrema exasperatum (Rudolphi, 1819)/Neoglyphe locellus (Kossack, 1910) species complex, Tylodelphys excavata (Rudolphi, 1803) and Echinostoma spiniferum (La Valette, 1855) sensu Nasincová (1992), all from Planorbarius corneus (Linnaeus). Almost the same spectrum of cercariae of the families Echinostomatidae, Plagiorchiidae and Omphalometridae was found in the present study as in previous reports; however, a considerably lower spectrum of cercariae of the families Diplostomidae and Strigeidae was recorded. The most frequent metacercariae were those of Echinoparyphium aconiatum Dietz, 1909, Neoglyphe locellus and Moliniella anceps (Molin, 1859), all occurring mainly in P. corneus. The most heavily infected snail species was P. corneus, followed by Planorbis planorbis (Linnaeus) and Segmentina nitida (Müller). The widest spectrum of trematode species was found in P. planorbis and P. corneus. Forty-two cercariae identified to the species level belonging to 15 families, plus an additional 43 taxa recorded under generic or provisional names, were reported from 11 species of planorbids in previous studies carried out in Central Europe. However, the actual number of trematode species occurring in the planorbid snails is probably much lower, because many, if not most, larval stages reported under provisional names or unidentified to the species level may be conspecific with identified adult forms. A key to the cercariae and metacercariae recorded from planorbids in Central Europe, together with illustrations of those species encountered most frequently in the field, is provided to facilitate identification.     

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.     

Haplometra cylindracea (Zeder, 1800) (Trematoda:Plagiorchiidae): variation in the dates of cercarial shedding for overwintering Galba truncatula

Natural infections of Galba truncatula with Haplometra cylindracea were followed from 2001 to 2009 to determine if their characteristics were similar when snails came from water collections frequented by Bufo bufo or by frogs and newts for their egg-laying. Snail samples were collected from both types of sites to count shed cercariae for three days and also free cercariae when snails were dissected. In sites only frequented by B. bufo, cercarial shedding occurred earlier than in those colonized by frogs and newts (March instead of April-May). In contrast, the number of cercariae shed during three successive days was significantly higher in May. This variation in the dates of cercarial shedding might be due, either to a synchronism between cercaria-releasing snails and the presence of the definitive host (tadpoles) in water collections, or to an earlier infection of overwintering snails in autumn by H. cylindracea miracidia in the case of toad-frequented sites.     

Life cycle of Coitocoecum plagiorchis (Trematoda: Digenea: Opecoelidae)

The life cycle of Coitocoecum plagiorchis Ozaki, 1926 (Coitocaecum is an incorrect subsequent spelling) was studied in the field and laboratory. The study was conducted at the Futatsu River and the Chikugo River, Kyushu, Japan. Adults of C. plagiorchis were obtained from fishes Coreoperca kawamebari, Rhinogobius spp. and Odontobutis obscura. Cotylomicrocercous cercariae with a two-point stylet (= Cercaria distyloides Faust, 1924) were detected in pleurocerid snails (Semisulcospira spp.). Metacercariae with a cyclocoel were obtained from shrimp (Neocaridina denticulata). The cercariae were experimentally exposed to the shrimp, and subsequently metacercariae with a cyclocoel were recovered from the shrimp within 24 days after exposure. These metacercariae were identified as C. plagiorchis. The sporocysts, cercariae, metacercariae and adults are briefly described.     

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.     

In vivo efficacy of praziquantel against Echinoparyphium aconiatum (Trematoda: Echinostomatidae) parasitizing the great pond snails Lymnaea stagnalis (Gastropoda: Lymnaeidae)

The present study had a practical goal. I aimed to determine whether praziquantel could reduce the production of Echinoparyphium aconiatum (Trematoda: Echinostomatidae) cercariae in infected snails Lymnaea stagnalis (Gastropoda: Lymnaeidae) without killing the hosts. Praziquantel is a broad-spectrum antihelminth agent. It caused a total cessation of cercaria shedding when the praziquantel concentration in the treatment bath was 10 mg/L and the treatment time was 30 h or longer. A next research step which has to be taken before giving detailed recommendations about using praziquantel for ceasing production of E. aconiatum cercariae in parasitized snails is to follow the survivorship and performance of treated snails after a praziquantel exposure for longer than in this medium-term (3 days) experiment.