Toxic effects of cadmium and zinc on the transmission of Echinoparyphium recurvatum cercariae

The toxicity of cadmium, zinc and Cd/Zn mixtures to the transmission of Echinoparyphium recurvatum (Digenea: Echinostomatidae) cercariae into the snail second intermediate hosts was investigated at concentrations ranging from 100 microg l-1 to 10 000 microg l-1 in both soft and hard water. A differential response in the infectivity of metal-exposed cercariae into Lymnaea peregra and Physa fontinalis was demonstrated which was dependent on the snail species being infected. Exposure of L. peregra, P. fontinalis, and L. stagnalis to heavy metals caused a differing susceptibility to E. recurvatum cercariae depending on the snail species being exposed. The mechanism and effects of metal toxicity, together with the importance of the parasite/host strain on cercarial transmission are discussed.     

Changes in survival characteristics of Diplostomum spathaceum cercariae emerged from cadmium-exposed Lymnaea stagnalis

The effect of exposing Lymnaea stagnalis (Gastropoda: Pulmonata), infected with Diplostomum spathaceum (Trematoda: Diplostomatidae), to 100 microg l(-1) cadmium for 7 days on survival characteristics (survival, tail loss, decaudized cercarial life-span) of emerged cercariae was investigated. Exposure of L. stagnalis to cadmium resulted in significantly increased D. spathaceum cercarial survival and an inhibited tail loss compared to controls. The normal parallel relationship which exists over time between decreasing cercarial survival and increasing tail loss in controls was changed in cercariae from cadmium-exposed hosts with an increased proportion of cercarial deaths occurring without tail loss. The decaudized cercarial life-span over the survival period of the cercarial population did not significantly change. However comparisons between individuals decaudized during the initial 24 h time period with those which were decaudized during the final period of cercarial survival showed a significantly altered life span which did not occur in the control population. As a potential indicator of penetration 'fitness' comparisons were also undertaken between control and exposed cercariae decaudized during the initial 24 h time period, which revealed that the decaudized cercarial life-span from the exposed hosts was significantly different from controls. This may have important implications for the ability of cercariae to migrate through the tissues of their target host. The importance and relevance of these results to parasite transmission are discussed.     

Diplostomum spathaceum (Rud. 1819): effect of miracidial age and lifespan on miracidial infectivity

Experiments were performed to study the effect of age and lifespan on the infection success of Diplostomum spathaceum miracidia. Specimens of Lymnaea peregra and L. stagnalis were individually exposed to single miracidia of D. spathaceum of different ages under conditions of constant illumination at 20 degrees C. The life-span of the free-swimming miracidium of D. spathaceum is 24 h at 20 degrees C. It was observed that as the miracidium ages, its ability to successfully infect snails declines rapidly.     

Influence of cadmium exposure on the incidence of first intermediate host encystment by Echinoparyphium recurvatum cercariae in Lymnaea peregra

The effect of cadmium exposure of the snail first intermediate host Lymnaea peregra on the incidence of encystment of Echinoparyphium recurvatum (Digenea: Echinostomatidae) cercariae without emergence from the snail was investigated. Exposure to 100 microg l(-1) Cd for 72 h caused a significant increase in the incidence of first host encystment when compared to controls. In addition, autometallographic staining of E. recurvatum daughter rediae and developing cercariae showed that there was metal accumulation within their body tissues. The significance of these findings to parasite transmission in metal-polluted environments is discussed.     

Toxicity of cadmium and zinc to Diplostomum spathaceum (Trematoda: Diplostomidae) cercarial survival

The toxicity of cadmium and zinc at concentrations ranging from 0.1 to 10000 microg/l was investigated with cercariae of Diplostomum spathaceum at three temperatures (12, 20, 25 degrees C) and three levels of water hardness (distilled water, soft water, hard water). Under most environmental conditions survival of cercariae was reduced by increasing metal concentration. Increasing water hardness and decreasing water temperature caused an increase in the survival of both control and metal exposed cercariae. However, with decreasing temperature increased survival of metal-exposed cercariae above the control occurred at a number of low metal concentrations. Differences in the relative toxicity of cadmium and zinc to cercariae was dependent on the environmental conditions of exposure. The two heavy metals demonstrated little toxicity to cercariae during the period of maximum cercarial infectivity (0-5 h).The mechanisms of metal toxicity and their effects on cercarial survival are discussed.     

Effect of water temperature on the ability of Diplostomum spathaceum miracidia to establish in lymnaeid snails

The effects of water temperature on the ability of Diplostomum spathaceum miracida to infect and establish patent infections in Lymnaea peregra and L. stagnalis were investigated. Snails were infected over a range of temperatures (6-20 degrees C) and kept thereafter at 20 degrees C or were infected at 20 degrees C and kept at either 14, 20, or 25 degrees C. Infection success was determined after 8 weeks by either observing cercarial shedding or examining snail viscera for sporocysts. The establishment of miracidia declined at lower water temperatures despite maintenance for 8 weeks at 20 degrees C while exposure of snails to miracidia at 20 degrees C and maintenance at different temperatures had little apparent effect. Infection success under these conditions was related more to the numbers of miracidia to which the snails were exposed. However, under this latter experimental regime, the time taken for the infection to become patent clearly depended upon maintenance temperature.     

Phenotypic variation in infectivity of Diplostomum spathaceum cercariae within a population

The present study examined phenotypic variation in infectivity of Diplostomum spathaceum (Trematoda) cercariae within a natural population. Twelve infected Lymnaea stagnalis were collected from the field, and the infectivity of cercariae from individual snails was assessed under constant laboratory conditions. At a water temperature of 16.3 C, the mean infectivity of cercariae from the snails varied between 55.5% and 87.5%. Depending on the source of variation, this may have important ecological and evolutionary implications for both natural parasite populations and those occurring in aquaculture.     

Metacercarial utilization of a naturally infected single species (Lymnaea peregra) snail community by Echinoparyphium recurvatum

Utilization of a single-species molluscan community of Lymnaea peregra by metacercariae of Echinoparyphium recurvatum over a summer (July-September) period in south-east England showed an increase in the mean number of cysts per host with host size and time of exposure. Aggregation resulting from host and habitat-related factors increased with host size and time of exposure. Encystment within the host was restricted to the peripheral organs in smaller juvenile snails but as snails increased in size, metacercariae were distributed throughout the tissues.     

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.     

Effects of the herbicide atrazine's metabolites on host snail mortality and production of trematode cercariae

Environmental stressors have the potential to greatly impact the transmission of parasites with complex, multi-host life cycles such as those of trematodes. The commonly used herbicide atrazine has been shown to affect the susceptibility of second intermediate hosts (such as larval amphibians) to trematode infection, as well as the longevity and infectivity of the free-swimming cercariae, but not eggs or the free-swimming miracidia that infect the gastropod first intermediate hosts. However, we do not know if this pesticide influences the survival of infected snails or whether it affects cercariae production within, or emergence from, these hosts. In addition, previous studies of host-parasite dynamics have only examined the parent atrazine compound, not any of the long-lasting metabolites commonly present in water bodies. Here, we report that a concentration of 0.33 μg/L of an atrazine metabolite, desethyl atrazine, increased the mortality of freshwater gastropods ( Stagnicola elodes ) infected with a gymnocephalus type of cercaria but not that of uninfected snails or those harboring a mature or dormant infection of Echinoparyphium sp. In contrast, 2 wk of exposure to desethyl atrazine did not affect the emergence of gymnocephalus cercariae from snails, although a trend for a decrease in the emergence of Echinoparyphium sp. cercariae was observed. We suggest that simultaneous trematode infection and exposure to contaminants may represent a significant combined stress to gastropods, but this is likely parasite species-specific as well as dependent on whether cercariae are being actively produced.     

Effects of environmental calcium on fecundity and cercarial production of Biomphalaria glabrata (Say) infected with Schistosoma mansoni Sambon

Biomphalaria glabrata were reared in stock culture and subjected to either 7-day or 60-day acclimation periods in complex CaCO3 media with calcium values ranging from 1.5 mg/L to 75 mg/L. Following 60-day acclimation, snails from series I were each exposed to 8 miracidia of Schistosoma mansoni. Snails of series II were each exposed to a single miracidium. Snails of both experimental regimens were observed for mortality, growth, rate of infection, and number of cercariae shed. Series I snails were also monitored for fecundity during acclimation and following miracidial exposure. Calcium levels of 1.5 and 75 mg/L resulted in significant snail mortality. Shell growth and rates of infection were positively correlated with calcium maintenance level. Snails with high fecundity prior to miracidial exposure subsequently shed more cercariae. In contrast, post-exposure (PE) fecundity of snails reared in media with up to 30 mg/L Ca++ were negatively correlated with calcium level, infection rate, and number of cercariae shed. Maximal cercarial emergence occurred at 30 mg/L Ca++. These results suggest that environmental calcium affects both the distribution patterns of snail hosts of human schistosomes and the productivity of intramolluscan schistosome infection.     

Effect of infection with Trichobilharzia ocellata and Schistosoma mansoni on the ultrastructure of the albumen gland of their respective hosts, Lymnaea stagnalis and Biomphalaria glabrata

The albumen gland, a female accessory sex gland of pulmonate snails, produces the perivitelline fluid. The ultrastructure of the albumen glands of control and infected specimens of Lymnaea stagnalis and Biomphalaria glabrata was studied. The albumen gland of L. stagnalis contains two types of secretory cells--light (active) and dark (inactive)--and two types of supporting cells--centroacinar and myoepithelial. The secretory cells apparently represent two activity stages of one type of cell. The gland B. glabrata possesses only one secretory cell type, which alternates with one type of supporting cell. The albumen glands of L. stagnalis and B. glabrata infected at a juvenile stage were studied 4 and 14 weeks (L. stagnalis) and 4 and 9 weeks (B. glabrata) after exposure. After four weeks' infection, B. glabrata produced some egg masses, but in subsequent stages egg mass production completely coased. Infected L. stagnalis never produced eggs. B. glabrata was apparently infected at a "physiologically" more mature stage than L. stagnalis. The morphology of the albumen glands four weeks after exposure (the daughter sporocyst stage) is in agreement with this hypothesis. At this interval the secretory cells of L. stagnalis appeared to be much more severely affected (inactive Golgi bodies and rough endoplasmic reticulum, crinophagy of the secretory granules) than the cells of B. glabrata. In the later stages studied (shedding of the cercariae), the glands of both species appeared to be completely inactive (reduced height of the epithelium, inactive organelles, crinophagy, absence of secretory granules). At this stage of infection, daughter sporocysts containing cercaria embryos were seen in the connective tissue of the albumen gland of B. glabrata, but not of L. stagnalis. The results thus indicate that the development and synthetic activity of the albumen gland are seriously affected by infection. These processes are known to be under the endocrine control of the female gonadotrophic hormones. Since it has been established that these hormones are normally present in the haemolymph of infected snails, the findings can be explained by assuming that the parasite interferes in some way or other with the snail's endocrine system.     

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.     

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

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

Toxicity of cadmium and zinc mixtures to the decaudized cercarial life span of Diplostomum spathaceum

The effects of cadmium and zinc mixtures at concentrations ranging from 0.1 to 10,000 microg l(-1) on the life-span of decaudized cercarial bodies (cercariae that have shed their tails) of Diplostomum spathaceum (Trematoda: Diplostomatidae) was investigated. Cercariae were exposed to metal mixtures of equal and unequal concentrations, and a low-dose pre-treatment followed by a high-dose exposure mixtures. Metal mixtures demonstrated variable effects on decaudized cercariae either by increasing or reducing their life-span compared to single metal exposures dependent on concentration and the type of mixed metal treatment. Prolonged exposure to equal metal mixtures at low concentrations (0.1-100 microg l(-1)) resulted in a reduction in the life-span of decaudized cercariae at 0.1 and 100 microg l(-1) in those individuals decaudized during the initial 24 h exposure period compared with those decaudized during the final 24 h period of cercarial survival, whilst in controls there was no significant life-span change between the two time periods. Decaudized cercariae which were exposed to low concentrations (0.1-100 microg l(-1)) of equal metal mixtures were also evaluated for their role as an indicator of larval 'fitness' for migrating through the tissues of their target fish host for those individuals decaudized during the initial 24 h exposure period, and demonstrated only a limited change in their life-span compared to control and single metal exposures. The importance of metal mixtures in parasite establishment in the fish host is discussed.     

Effect of light intensity on Opisthorchis viverrini cercarial shedding levels from Bithynia snails--a preliminary study

Opisthorchis viverrini requires Bithynia snails as the first intermediate host and cyprinid fish as the second intermediate host. Very low natural infection rates have been reported in Bithynia snails, but very high rates have been found in cyprinid fish in the same endemic region. This study investigated the effect of light intensity, the most important stimulus, on the quantity of O. viverrini cercariae shed from naturally infected Bithynia (Digoniostoma) siamensis goniomphalos snails. Snails were evaluated for cercariae output every hour after exposure to various light intensities for a total period of 7 h. The same infected snail was tested under different intensities of light: in the dark, and at 1000, 3000 and 5000 lx. The data showed that under exposure to 1000 and 3000 lx of light, the average percentage and number of cercariae released were higher than that exposed to 5000 lx during the first 2 h of the experiment. In contrast, under higher illumination (5000 lx) a longer time (6 h) was required to stimulate the peak emergence of cercariae. Darkness was not able to induce O. viverrini cercariae emergence. Among the three intensities of light, exposure at 1000 lx induced the highest average number of released cercariae per snail and the highest percentage of cercarial emergence within the first 2 h (125, 54.86%), followed by exposure at 3000 lx (69, 25.58%) and 5000 lx (12, 7.78%). The results suggest that the light intensity of 1000 lx for 2 h would be optimal for O. viverrini cercarial shedding from naturally infected B. (D.) siamensis goniomphalos snails.     

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 effects of the slug biological control agent, Phasmarhabditis hermaphrodita (Nematoda), on non-target aquatic molluscs

The nematode Phasmarhabditis hermaphrodita is used as a commercial biological control agent of slugs in the UK. Although it is known to affect other terrestrial mollusc species, its effects on freshwater molluscs are not known. The present study investigated the effects of P. hermaphrodita on the survival of juvenile Lymnaea stagnalis and Physa fontinalis, two common freshwater snails, at 'spray tank' concentration and a 50% diluted 'spray tank' concentration over a 14-day period. Survival of L. stagnalis was significantly reduced at both application levels but P. fontinalis suffered no mortalities over the experimental period. The possible differential mechanisms of pathology between the two host species are discussed.     

The toxicity and physiological effects of copper on the freshwater pulmonate snail, Lymnaea stagnalis

Several recent studies have demonstrated that the freshwater pulmonate snail Lymnaea stagnalis is extremely sensitive to metals (Co, Ni, Pb) in chronic exposures. The objective of the current study was to evaluate the acute and chronic sensitivity of L. stagnalis to Cu and investigate the underlying mechanism(s) of toxic action. A 96-h LC50 of 31μg L(-1) Cu was estimated indicating L. stagnalis was moderately acutely sensitive to Cu relative to other aquatic organisms. However, in a 30-day chronic exposure using juvenile snails an EC20 of 1.8μg L(-1) Cu was estimated for snail growth making L. stagnalis the most sensitive organism tested to date for Cu. Hardness-based and BLM-based water quality criteria for Cu at the water quality conditions used in this study were 7.8 and 1.5μg L(-1), respectively, indicating L. stagnalis is significantly under-protected by hardness-based WQC. Investigations into the mechanism(s) of toxic action for Cu were conducted on young adult snails necessitating higher Cu exposures. Exposure to Cu at 12μg L(-1) resulted in no detectable effects on hemolymph osmolality, net Ca(2+) uptake, titratable acid excretion, or ammonia excretion. Exposure to 48μg L(-1) Cu was shown to significantly reduce (91%) net Ca(2+) uptake which is strongly correlated with shell deposition and corresponding snail growth. Snails exposed to 48μg L(-1) Cu also exhibited reduced ammonia excretion, a marked hemolymph acidosis, and a compensatory increase in titratable acid excretion. The reduction in net Ca(2+) uptake was hypothesized to be a secondary effect of Cu-induced inhibition of carbonic anhydrase, but no reduction in carbonic anhydrase activity was detected. Overall, it remains unclear whether inhibition of Ca(2+) uptake is a direct result of Cu exposure or, along with the other observed physiological effects, is secondary to an unidentified primary mode of toxic action. Given the hypersensitivity of L. stagnalis to Cu, further study into the mechanisms of action and effects of varying water chemistry on Cu toxicity is clearly warranted.     

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.