Malformations and mortality in the Asian Common Toad induced by exposure to pleurolophocercous cercariae (Trematoda: Cryptogonimidae)

Malformations and increased mortality due to infection by the digenetic trematode, Riberioa ondatrae have been reported for many species of amphibians. Severe malformations have also been reported in the Common Hourglass Tree Frog, Polypedates cruciger induced by pleurolophocercous cercariae in Sri Lanka in addition to the changes in the behaviour, development and survival of the host. We exposed pre-limb bud stage tadpoles (Gosner stages 25–26) of the Asian Common Toad, Duttaphrynus melanostictus to the same pleurolophocercous type cercariae under laboratory conditions. Molecular and morphological identification showed that these cercariae belonged Acanthostomum burminis infecting freshwater snakes as definitive hosts. These cercariae induced malformations (27.8%) and reduced survival to metamorphosis (53.8%). The magnitude of the effects increased with the dose of cercariae. Types of malformations were mainly axial, such as scoliosis and kyphosis. Severe limb malformations such as extra or missing limbs as reported for amphibians exposed to R. ondatrae were not observed in the D. melanostictus. Same authors reported a higher percentage of malformations previously when P. cruciger was exposed to the cercariae A. burminis compared to D. melanostictus. However, tadpoles of D. melanostictus, which are smaller compared to those of P. cruciger, experienced higher mortality than P. cruciger tadpoles. Trematode induced malformations and mortality in amphibians are highly variable and depend on multiple factors such as host species differences such as resistance to infection and tolerance, life-history characteristics such as size at metamorphosis and length of the metamorphosis period, and other factors such as size of the amphibian at the time of trematode exposure.     

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.     

A review of the currently recognised opecoelid cercariae,including the identification and emergence ecology of Cercaria capricornia XII (Digenea: Opecoelidae) from Nassarius olivaceus (Gastropoda: Nassariidae) in Central Queensland,Australia

A new opecoelid cercaria, Cercaria capricornia XII, is reported from Nassarius olivaceus in Capricornia, Central Queensland, Australia. Combined molecular and morphological data indicate that this cercaria is a member of the subfamily Opecoelinae. Cercaria capricornia XII is the first known opecoeline cercaria reported from a nassariid gastropod. Cercaria capricornia XII can be distinguished from other opecoelid cercariae by the combination of the presence of a 2-pointed stylet, body length and width, and the size of the tail. The emergence pattern for C. capricornia XII in captivity was erratic; rapid emergences of thousands of cercariae were interspersed by periods that sometimes exceeded a month in which no emergence occurred. There was no detectable pattern to or stimulus of the emergence. The molluscan host range of opecoelids is analysed in detail. Gastropods from the Buccinoidea, Cerithioidea and Rissooidea are hosts to both opecoeline and plagioporine cercariae, but the dominant subfamily infecting the Cerithioidea and Rissooidea is the Plagioporinae. The dominant marine host gastropod superfamily for opecoeline cercariae is the Buccinoidea; the family Nassariidae is contained in the Buccinoidea. The range of gastropod superfamilies known as hosts of plagioporines is much broader than that for opecoelines, which may be explained by the relative size of the two opecoelid families and perhaps by indications that the Plagioporinae is polyphyletic.     

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.     

The presence of antibody in mice chronically infected with Schistosoma mansoni which blocks in vitro killing of schistosomula

We have previously reported that IgM monoclonal antibodies (mAb) that recognize surface carbohydrate determinants shared between schistosomula, cercariae, and miracidia block antibody/complement dependent killing of schistosomula in vitro. Binding assays that make use of one of the IgM mAb labeled with 125I demonstrated that serum from chronically infected mice (CMS) contained high levels of competing antibody, whereas serum from mice vaccinated with irradiated cercariae (VMS) contained little antibody of this specificity. Absorption of CMS with cercariae that removed antibodies to schistosomulum surface carbohydrate determinants increased its ability to kill schistosomula in vitro; absorption of VMS with cercariae failed to alter the lethal activity of the serum. Furthermore, fractionation of CMS by protein A Sepharose chromatography demonstrated that the IgG fraction had an increased lethal activity compared with unfractionated serum; this result was not seen with VMS. Finally, the IgM fraction of CMS was shown to block in vitro killing of the IgG fractions of both CMS and VMS. These data suggest that the blocking activities observed with the IgM mAb are contained within the serum of chronically infected mice but not in the serum of mice vaccinated with irradiated cercariae.     

Schistosoma mansoni: cercarial eicosanoid production and penetration response inhibited by esculetin and ibuprofen

Cercariae of Schistosoma mansoni produce a wide variety of eicosanoids when stimulated by 3.3 mM linoleate. High-performance liquid chromatography indicated that 10(-5) M esculetin dramatically decreased eicosanoid production by cercariae. Ibuprofen (10(-4) M) also decreased eicosanoid production, but to a lesser extent. These results were confirmed by radioimmunoassay using time-dose curves for esculetin and time curves for ibuprofen. The results reported here indicated that, for cercariae, ibuprofen was neither a specific cyclo-oxygenase inhibitor, as has been reported for platelet and endothelial cells, nor was esculetin a specific inhibitor of lipoxygenase, as has been reported for platelets and mastocytoma cells. Rather, both drugs inhibited cyclo-oxygenase and lipoxygenase enzyme systems. Further, the data indicated that two forms of cyclo-oxygenase exist in cercariae (isozymes?), one initiating the conversion of gamma-dihomolinolenate into the 1-series prostaglandins and another acting on arachidonate forming the 2-series prostaglandins. The cyclo-oxygenase acting on arachidonate has a greater sensitivity to both ibuprofen and esculetin than the enzyme acting on gamma-dihomolinolenate. Cercarial lipoxygenases also varied greatly in their sensitivity to ibuprofen.     

The role of prostaglandins in cercarial (Schistosoma mansoni) response to free fatty acids

In examining the structure-activity relationship of a diverse group of chemicals reported to prevent cercarial penetration after topical application, we noticed a moiety that was common to free fatty acids and prostaglandins. Because unsaturated fatty acids have been reported to stimulate cercarial penetration, we hypothesized that cercarial stimulation by skin and fatty acids may invoke prostaglandin mechanisms in cercariae, skin, or both. Thus we compared the stimulation of cercariae by a series of essential and nonessential fatty acids and demonstrated an inhibition of this response by ibuprofen and aspirin, known cyclo-oxygenase inhibitors, and by 13-azaprostanoic acid, a potent antagonist of the thromboxane/endoperoxide receptor. These data led us to postulate a major role for prostaglandins in the cercarial penetration response.     

Adaptibility of Diplostomum cercariae in carp and the effect of a prior infestation

Experiments on the infection of carp fry with cercariae of Diplostomum have shown that cercariae of Diplostomum spathaceum can penetrate crystalline lens of larvae of these fishes in three days after their hatching and that the adaptability of cercariae of D. paracaudum to carp is much higher than that of cercariae of D. spathaceum. After the first infection with cercariae a relative postinfectious immunity against infection with cercariae of this or close species of this genus arises in carp fry.     

Argentophilic and scanning electron microscopic observations of the tegumentary papillae of Echinostoma revolutum (Trematoda) cercariae

Argentophilic staining and scanning electron microscopy (SEM) were used to study the tegumentary papillae of Echinostoma revolutum cercariae. The most abundant papilla was uniciliate and arranged bilaterally on the body and tail of the cercaria. The distribution of these papillae was similar to that seen in other 37-collar spined echinostome cercariae. SEM revealed a total of 18 multiciliate papillae--16 on the anterior end and 2 at the middle of the body. These papillae contained up to 18 cilia per papilla and have not been reported previously from any cercaria.     

Schistosoma mansoni: cercaricidal effect of 2-tetradecenoic acid, a penetration stimulant

Schistosoma mansoni cercariae are stimulated by 2-tetradecenoic acid (TDA) to penetrate agar substrates. TDA simultaneously causes tegumental transformation similar to that seen when cercariae transform to schistosomula, reduces the Cercarienhüllen reaction in immune human serum, and reduces larval tolerance to water. TDA damages cercariae that fail to penetrate or have no opportunity to do so. This damage apparently stems from increased tegumental permeability to water. Preincubation in TDA for 60 min reduces the percutaneous infectivity of cercariae to mice by from 95% at 0.2 ppm to 100% at 0.7 ppm TDA, but does not reduce the infectivity of subcutaneously injected cercariae. The interference with percutaneous infection seems to be entirely due to osmotic damage. TDA does not induce premature secretion of the acetabular glands or block host-recognition chemoreceptors. TDA may be a promising cercaricide for schistosomiasis control. It is highly specific for schistosome cercariae and is effective at low concentrations (0.2 to 0.7 ppm). Both cercariae and TDA tend to collect in the upper few millimeters of standing water. It is unlikely that cercariae can evolve resistance to a chemical that triggers the host penetration mechanisms.     

Respiration and motor activity of cercariae of three trematode species from the intertidal mollusc Littorina littorea L (Gastropoda) from the White sea

The age dynamics in oxygen consumption of the cercariae Himasthla elongata, Cryptocotyle lingua and Cercaria parvicaudata (Renicola sp.) was studied using modified Winkler's method. It was detected that under stable temperature and water salinity conditions the rate of oxygen uptake depends directly on cercariae size. The highest intensity of energetic metabolism was recorded in the first few hours of cercariae life when their movement activity was maximal. The following reduction of oxygen consumption passed unequally in three cercariae species studied. Large, long-lived H. elongata cercariae after relatively short period of active swimming turn to crawling on the bottom. The rate of oxygen uptake in such cercariae was two times less than in free-swimming ones and remained approximately invariable up to the cercariae death. The smaller sized, short-lived Renicola sp. cercariae swim actively in the water and during this time the level of their energetic metabolism remains more or less stable. It decreased drastically after cercariae sinking to the bottom, after that they perished very soon. Also relatively small C. lingua cercariae alternate the active and passive phases of swimming. Thanks to that they consume the energetic resources economically and prolong their longevity. During free-swimming period the rate of oxygen uptake of C. lingua cercariae remains more or less stable. As in the case of Renicola sp. cercariae, it decreased drastically after the cercariae sinking to the bottom. Apparently such cercariae lose their ability to infect the second intermediate host (fish).     

Longevity of marine cystophorous cercariae in the environment

Experiments were conducted on the duration of life in sea water of three species of cystophorous cercariae from the Barents sea. The expenditure of reserve nutritive substances (glycogene) in the process of the vital activity of cercariae was followed. The life duration of cystophorous cercariae is considerably longer than that cited in literature for other groups of cercariae that is associated with their morphological and biological peculiarities.     

Morphological peculiarities of schistosomatid cercariae of Trichobilharzia cf. ocellata group occuring in Moscow and Saint-Petersburg populations

Morphology of schistosomatid cercariae of the genus Trichobilharzia (ocellata group) is described based on original and reference data. The material (Trichobilharzia cercariae) was collected in 1999-2000 from naturally 'infected snails Lymnaea stagnalis from Moscow and Saint-Petersburg megalopolises. The more accurate flame cell formula for these cercariae, as well as some aspects of chaetotaxy and eye morphology are given. The differences between cercariae from these regions were found. Based on a comparison of nef data with the reference data, it was found that the cercariae examined differ from those from the Central Europe.     

Characteristic structural features of schistosome cercarial N-glycans: expression of Lewis X and core xylosylation

Schistosomal egg N-glycans are the only examples in nature that have been structurally shown to contain beta2-xylosylation, alpha6-fucosylation, and alpha3-fucosylation on the N,N'-diacetyl chitobiose core. We present evidence that core difucosylated and xylosylated N-glycans are characteristics of Schistosoma japonicum eggs but not of the cercariae and adults, for which neither core xylosylation nor alpha3-fucosylation could be readily detected. In contrast, a majority of the N-glycans from Schistosoma mansoni cercariae but not the adults are core xylosylated. Tandem mass spectrometry analysis coupled with chromatographic mapping, sequential exoglycosidase digestion, and methylation analysis were employed to unambiguously define the structures of core beta2-xylosylated, alpha6-fucosylated N-glycans from S. mansoni cercariae. Unexpectedly, a majority of these N-glycans were found to carry Lewis X determinant, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->, on the nonreducing termini of mono- and biantennary structures. The Lewis X-containing glycoproteins were found to be distinct from those carrying the complex, multifucosylated glycocalyx O-glycans reported previously. The corresponding N-glycans from S. japonicum cercariae are likewise dominated by Lewis X termini but without the core xylosylation. We concluded that the invading cercariae present an important and abundant source of Lewis X antigens, which may contribute to the induced humoral response upon infection. Following transformation and development into the adults, the N-glycans synthesized comprise a significantly larger amount of high mannose and fucosylated pauci-mannose structures in comparison with the cercarial N-glycans. A portion of the mono- and biantennary complex types were identified to carry Lewis X and fucosylated LacdiNAc termini, which could also be detected by mass spectrometry analysis on larger, complex-type structures.     

Small estuarine fishes feed on large trematode cercariae: lab and field investigations

In aquatic ecosystems, dense populations of snails can shed millions of digenean trematode cercariae every day. These short-lived, free-living larvae are rich in energy and present a potential resource for consumers. We investigated whether estuarine fishes eat cercariae shed by trematodes of the estuarine snail Cerithidea californica. In aquaria we presented cercariae from 10 native trematode species to 6 species of native estuarine fishes. Many of these fishes readily engorged on cercariae. To determine if fishes ate cercariae in the field, we collected the most common fish species, Fundulus parvipinnis (California killifish), from shallow water on rising tides when snails shed cercariae. Of 61 killifish, 3 had recognizable cercariae in their gut. Because cercariae are common in this estuary, they could be frequent sources of energy for small fishes. In turn, predation on cercariae by fishes (and other predators) could also reduce the transmission success of trematodes.     

Putative immune response of rainbow trout, Salmo gairdneri, to Diplostomum spathaceum infections

The immune response of rainbow trout to infection with the cercariae of the digenean parasite Diplostomum spathaceum was investigated. Rainbow trout infected with cercariae at weekly intervals, or injected with a suspension of dead cercariae, did not produce a specific humoral response against D. spathaceum cercariae, as tested using immunoperoxidase, agglutination and cytotoxicity assays. However, a significant difference occurred in the rate of infection of rainbow trout given weekly exposure to cercariae of D. spathaceum in winter and summer. Rainbow trout injected with a suspension of dead cercariae acquired significantly fewer metacercariae than controls when exposed to a challenge infection. This suggested a specific immune response by the host and is the first example of a reduction in the infection rate of rainbow trout immunized against a digenean parasite, when exposed to a challenge infection.     

Ribeiroia ondatrae cercariae are consumed by aquatic invertebrate predators

Trematodes amplify asexually in their snail intermediate hosts, resulting in the potential release of hundreds to thousands of free-living cercariae per day for the life of the snail. The high number of cercariae released into the environment undoubtedly increases the probability of transmission. Although many individual cercariae successfully infect another host in their life cycle, most fail. Factors that prevent successful transmission of cercariae are poorly understood. Microcrustaceans and fish have been observed to eat cercariae of some species, although the possibility that predation represents a significant source of mortality for cercariae has been largely unexplored. We tested the cercariophagic activity of several freshwater invertebrates on Ribeiroia ondatrae, a trematode that causes limb deformities in amphibians. Individuals of potential predators were placed into wells of multiwell plates with 10-15 cercariae, and numbers of cercariae remaining over time were recorded and compared with numbers in control wells that contained no predators. Of the species tested, Hydra sp., damselfly (Odonata, Coenagrionidae) larvae, dragonfly (Odonata, Libellulidae), larvae, and copepods (Cyclopoida) consumed cercariae. In some cases, 80-90% of the cercariae offered to damselfly and dragonfly larvae were consumed within 10 min. In most cases, predators continued to consume cercariae at the same average rates when offered cercariae together with individuals of an alternate prey item. Hydra sp. ate fewer cercariae in these trials. Our findings suggest the need for field and laboratory studies to further explore the effects of predators on transmission of R. ondatrae to amphibian larvae. In addition, the results suggest that conservation of the biodiversity and numbers of aquatic predators may limit adverse impacts of trematode infections in vertebrate hosts.     

Histopathology of experimental Schistosoma incognitum infection in mice following exposure to normal and irradiated cercariae.

The tissue reactions in mice, experimentally infected with normal and irradiated cercariae of S. incognitum were studied. The lesions observed in the skin, liver, lungs and the intestine of mice infected with normal cercariae are briefly described, and compared with those observed with cercariae irradiated at 3000 r of gamma rays. In general, the reactions in mice exposed to normal cercariae were more intense than in those infected with irradiated cercariae. The severity of the reactions appeared largely due to the deposition of eggs in the tissues of the mice infected with normal cercariae. The experimental evidence suggested that most of the flukes from the irradiated cercariae are destroyed in the liver by tissue reaction.     

Infectivity of Diplostomum spp. in Arctic charr: aspects of exposure duration and cercariae morphology

The diplostomid flukes, Diplostomum spp., infect fish and cause cataract opacities in the eye lens. The effect of exposure dose on abundance of Diplostomum spp. eye flukes in fish is known, but the effect of the duration of cercariae exposure has not been studied. However, under natural conditions, the temporal window for a successful cercaria attachment on fish is very short and, consequently, differences in infectivity of eye fluke cercariae, in the short-exposure durations of a few seconds, are probably biologically the most meaningful. We investigated infectivity of Diplostomum spp. cercariae originating from snail hosts in 3 lakes (3 Lymnaea stagnalis populations and 1 Radix balthica population) in 6 exposure times, ranging from 5 sec to 15 min, in young-of-the-year Arctic charr Salvelinus alpinus. In addition, we compared the infectivity to the cross-morphology of the cercariae. In the long-exposure duration, i.e., > or = 5 min, infectivity of Diplostomum spp. did not vary between the snail host species (L. stagnalis and R. balthica) of the same lake or across the L. stagnalis populations of 3 different lakes. However, in the short-exposure duration, i.e., < or = 60 sec, Diplostomum spp. cercariae shed from L. stagnalis had higher infectivity than did cercariae from R. balthica of the same lake. This indicates that that there is an interaction between length of cercariae exposure and origin of Diplostomum spp., and that the duration of exposure may influence the results when fish are experimentally infected. Within a lake, cercariae shed from L. stagnalis were also smaller than cercariae shed from R. balthica.     

A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: The influence of the ages of the cercariae and of the host

A study of the death of Schistosoma mansoni cercariae during penetration of mammalian host skin: the influence of the ages of the cercariae and of the host. International Journal for Parasitology 3: 789–794. The number of cercariae of S. mansoni which die during penetration of mouse abdominal skin steadily increases with age following emergence from the snail. An initial mortality level of about 30 per cent is observed for 2-h-old cercariae which rises to 50 per cent at 8 h and 85 per cent at 24 h. These increased losses in the skin are shown to account substantially for the known decrease in infectivity which accompanies ageing of cercariae. The number of cercariae which die in the skin of very young mice (2 days old) is less than one-third of the level in adult mice. Losses in the skin increase with age of the host up to about 28–35 days. This increased mortality in the skin is shown to account for the observed age resistance of mice, where fewer cercariae mature into adult worms in mice of 1 month or more, than in very young mice.