Miracidial survival.

Studies on the toxic action of pollutants to miracidia have, in comparison to studies on cercariae, been limited. Principally species of Schistosoma and Fasciola¹

However, miracidia possess a period of maximum infectivity upon hatching which is only a fraction of their total lifespan. For S. mansoni this is approximately up to 4 h post hatching at 25 °C, dependent on a number of environmental parameters (Sturrock, 1993). It is unfortunate that few studies have attempted to analyse survival statistically during this period, as this would provide a more accurate assessment of any effective toxicity on miracidial transmission to their snail host. For instance, Morley et al. (2001 a) found that the survival of S. mansoni exposed to Cd/Zn mixtures was significantly reduced at concentrations of 10 μg/l or higher. However, after 4 h post-hatching, only a toxic concentration of 10000 μg/l significantly reduced survival.

The mechanisms of toxicity to miracidial survival are not really understood but are likely to be similar to those described for cercariae below. Nevertheless work by Moczon (1976 a,b) suggests that pesticide toxicity is associated with a wide-ranging inhibition of enzymatic function of miracidia which appears to influence respiration and the associated utilisation of glycogen (Bonieka & Guttowa, 1975) leading to an altered life span.

Cercarial survival.

There have been extensive studies on survival of cercariae exposed to toxic pollutants. Most previous investigations, however, have been confined to either Schistosoma or Diplostomum³

The majority of survival studies have utilised heavy metals as the selected toxicant and it can be tentatively interpreted from those investigations that have utilised two or more metals⁴

Although it is difficult to accurately compare data on toxicant-affected cercarial survival from the published literature, due to most studies having utilised different species exposed to the same toxicant under different environmental and experimental conditions, we have attempted to highlight species-specific differences using our own standard experimental protocols (Morley et al. 2001 c). Fig. 2 shows the effects of cadmium exposure to a furcocercaria (D. spathaceum) and an echinostome cercaria (E. recurvatum). At 100 μg/l D. spathaceum survival is significantly affected by cadmium (Lee-Desu test P<0·0001) but at this concentration there is no difference in the survival of E. recurvatum between control and toxicant-exposed cercariae (Lee-Desu test P=0·1780). Using a simple survival index (see figure legend for details) this difference can be graphically presented (Fig. 2b). Diplostomum spathaceum shows an increased vulnerability to cadmium compared with E. recurvatum at both exposure concentrations. It has been suggested that inhibition of the enzymes involved in glycogen utilisation is responsible for changes in survival of metal-exposed cercariae (Siddall & des Clers, 1994; Morley et al. 2001 c, 2002 b). The metal-exposed survival shown in this study may therefore be related to differences in the glycogen distribution within the cercariae of each species and the way such glycogen is utilised.

Fig. 2. Comparison of the toxicity of cadmium to a furcocercaria (Diplostomum spathaceum) and an echinostome cercaria (Echinoparyphium recurvatum). Cercariae were exposed using protocols devised by Morley et al. (2001 c) at 20 °C in soft water (25 mg/l CaCO3, pH 7·85) (HMSO, 1969). a. Toxicity over time. ([bull ] – Control D. spathaceum, [utrif ] – 100 μg/l Cd exposure of D. spathaceum, [squf ] – 10000 μg/l Cd exposure of D. spathaceum, ○ – Control E. recurvatum, [utri ] – 100 μg/l Cd exposure of E. recurvatum, □ – 10000 μg/l Cd exposure of E. recurvatum.) b. Comparison of the relative susceptibility of D. spathaceum ([bull ]) and E. recurvatum (○) using a survival index – Survival index<1 (-----) is reduced survival compared to control.

It is well known that cercariae possess a period of maximum infectivity which is much shorter than their life span, but there have been few attempts to analyse toxicity over this period. Morley et al. (2001 c) found that D. spathaceum cercariae, with maximum infectivity extending up to 5 h post emergence and declining to zero by 23 h (Whyte, Secombes & Chappell, 1991), had only a limited susceptibility to cadmium and zinc during this period. The available literature indicates that only acute concentrations can induce any effect on cercariae during this initial crucial period. Indeed when the infectivity of a cercarial population is in decline, following the period of maximum infectivity, it is difficult to interpret the impact of pollution toxicity on transmission due to the fluctuations in the degree of infectivity experienced in experimental studies conducted under unpolluted conditions. Therefore, in isolation, survival studies are of limited value in assessing pollutant toxicity and this led Morley and co-workers to investigate not only the survival of cercariae of D. spathaceum exposed to cadmium and zinc over a range of environmental conditions (Morley et al. 2001 c), but also the concurrent effects on cercarial tail loss (Morley et al. 2002 c) and decaudised cercarial life span (Morley, unpublished observations). Tail loss increased in parallel with a decrease in survival in controls but cadmium and zinc exposure, at high concentrations especially, induced stimulation or inhibition depending upon individual toxicant exposure. Although during the period of maximum cercarial infectivity metal exposure had only a limited influence on tail loss, this was of greater toxic effect than that induced by metals on cercarial survival (Morley et al. 2002 c). Therefore tail loss, which has been previously studied by Asch & Dresden (1977), Oshima et al. (1992) and Hara et al. (1993 a), may prove to be an important measurable parameter for ecotoxicological assessment. Nevertheless, some cercariae such as the echinostome E. recurvatum show no pattern of tail loss, with the majority of cercariae appearing to die with their tails still attached (Morley, unpublished observations).

In contrast, under many environmental conditions the life span of decaudised cercariae is independent of both survival and tail loss, increasing above and decreasing below control levels dependent on specific metal concentrations and environmental parameters (Morley, unpublished observations). This may prove to be an important feature, as the life span of decaudised cercariae is related to glycogen utilisation and, after shedding their tails, cercariae will use between 24% and 35% of their glycogen reserves whilst penetrating their target host (Lawson & Wilson, 1983).

The overall mechanisms of toxicity to survival of cercariae have been poorly studied with most interpretations being highly speculative and based on the actions of specific named toxicants, although it is likely that toxicant uptake occurs across the tegument. As in most aquatic invertebrates, diffusion has been suggested as the route of uptake of metal ions (Cross et al. 2001). Although this possibility cannot be entirely discounted, other studies suggest the uptake of ions occurs via active transport using specialised ‘channels’ (Fusco et al. 1991), with more complex molecules such as glucose taken up via facilitated diffusion (Arme, 1988). Indeed Morley and co-workers studied the toxicity of cadmium to the survival of E. recurvatum in a 1% glucose solution and found clear differences occurred (Fig. 3). Under normal conditions 1 μg/l Cd shows no difference in survival compared to controls (Lee-Desu test P=0·6402). In the glucose solution control survival increases whilst exposure to cadmium causes a significantly reduced survival compared not only to glucose control but also to cercarial survival in non-glucose solutions (Lee-Desu test P<0·0001) (Fig. 3). As cercariae are known to use external glucose in preference to their own glycogen reserves (Uglem, 1980), under the glucose conditions of this study, the free cadmium ions present in the water would be highly attracted to bond with the glucose molecules, leading to a more efficient and rapid uptake and availability of cadmium into the cercariae to inhibit metabolic enzyme function associated with glucose utilisation. The relatively inefficient uptake of low concentrations of metal ions across specialised channels may be associated with other aspects of cercarial functional biology. Calcium channels have been suggested to increase the uptake of calcium mainly during host penetration (Fusco et al. 1991; Hara et al. 1993 b). As cadmium is known to enter a variety of aquatic animals via calcium channels (Depledge, Weeks & Bjerregaard, 1994), this suggests that cercariae may be more vulnerable to toxic metals during penetration of the target host, but this requires further investigation. The present results (Fig. 3) also suggest that survival in metal-polluted environments is likely to be associated with enzymatic function of glycogen utilisation as previously proposed by Siddall & des Clers (1994) and Morley et al. (2001 a, c, 2002 b).

Fig. 3. Comparison of the survival of Echinoparyphium recurvatum cercariae exposed to cadmium in either soft water (25 mg/l CaCO3, pH 7·85)(HMSO, 1969) or soft water and 1% glucose at 20 °C. Cercariae were exposed using protocols devised by Morley et al. (2001 c). a. Survival over time ([bull ] – Control, [squf ] – 1 μg/l Cd, ○ – Glucose control, [utri ] – Glucose+1 μg/l Cd). b. Comparison of the relative survival of cadmium exposed cercariae in control and 1% glucose conditions using a survival index (see Fig. 2 for details). Note Survival index<1 (-----) is reduced survival compared to control.

Metacercarial survival.

Studies on metacercarial survival have been limited and have exclusively focused on metacercariae residing in protective multi-layered cysts in invertebrate intermediate hosts.

Experimental studies undertaken by Morley and co-workers with Echinoparyphium recurvatum within the second intermediate host Lymnaea peregra have shown that a five-day exposure to 100 μg/l of either cadmium or zinc did not significantly influence metacercarial viability and only an exposure of 100 μg/l Cd/Zn mixture (Fig. 4) induced a reduction of mean survival from 100% in controls to 74·3% (Mann–Whitney test P=0·047).

Fig. 4. Viability of Echinoparyphium recurvatum metacercarial cysts exposed to 100 μg/l Cd/Zn mixture for 5 days in laboratory-bred Lymnaea peregra. Snails were exposed to 50 cercariae for 1 hour in soft water (25 mg/l CaCO3, pH 7·85) (HMSO, 1969). After 72 h acclimation period, to ensure full cyst development, snails were exposed to toxicant in 500 ml of soft water for five days at 20 °C with daily water changes. Unviable cysts were identified by their granular and opaque appearance. A – viable cysts, D – dead cyst (Bar=125 μm).

A high tolerance to toxic substances by metacercariae within a protective cyst in an exposed host is not unexpected. Siddall (1992) found that the survival of metacercarial cysts of the marine digenean Zoogonoides viviparus in its second intermediate host Ophiocomina nigra was not affected after exposure to sewage sludge for 6 weeks. Brown & Pascoe (1989) noted no mortalities of the cystacanth of the acanthocephalan Pomphorhynchus laevis in Gammarus pulex exposed to cadmium.

Mixtures of cadmium and zinc in the present study (Fig. 4) are synergistic to each other, generating significant deaths of E. recurvatum cysts compared to the negligible toxicity of single metals, although the mechanism of toxicity to the parasite is unknown. Siddall (1992) suggested that metacercariae are not exposed directly to the toxicant but only to those contaminants that accumulate in adjacent tissue. However, if cysts are only likely to suffer exposure to toxicants in adjacent tissues the ability of E. recurvatum to encyst in a number of sites within the snail second intermediate host (Adam & Lewis, 1992) may prove influential in the overall viability of the parasite population, as differing levels of toxicant accumulation can occur within the body of the snail (Presing, Balogh & Salanki, 1993). Siddall (1992) also suggested that the complex, multilayered cyst wall acted as an effective barrier against the uptake of toxic chemicals by metacercariae. The effective nature of the cyst wall in protecting larval stages of parasites from heavy metal toxicity has been demonstrated with the cystacanth of acanthocephalans in crustaceans. Cadmium levels (Sures & Taraschewski, 1995) and lead levels (Siddall & Sures, 1998) were shown to be up to 16 times lower in the parasites than in the exposed host tissues (Sures & Taraschewski, 1995). However, metacercariae of more ‘advanced’ digenean families which use their host as a nutrient source for morphogenesis e.g. Microphallidae, may be more vulnerable to toxicant effects as the cyst wall, by definition, will not be impervious to the absorption of various substances.

Miracidia.

A range of pollution conditions has been demonstrated to have a toxic effect on the embryonation and hatching of miracidia from the egg. These have included heavy metals, pesticides, fertilisers, and acidification.⁵

Moczon (1976 a,b) undertook extensive histochemical tests on F. hepatica miracidia to elucidate the mechanisms of pesticide toxicity on enzyme activity. A wide range of oxidoreductases were inhibited by a number of pesticides suggesting that the toxic action damages the biocatalytic structures of the mitochondria and endoplasmic reticulum of the cell (Moczon, 1976 a). Additional studies on pesticide toxicity to phosphatase, glucosidase and acetylcholinesterase activity in the miracidia of F. hepatica found that inhibition and activation of enzymes could occur, which may be a product of pesticide damage to the lysosmes and disturbance of cellular energetic metabolism (Moczon, 1976 b).

Extensive morphological damage at the ultrastructural level has been reported in Schistosoma haematobium miracidia following exposure to chromium (Wolmarans et al. 1988). Chromium induced widespread internal morphological damage leading to degeneration of much of the tissue. External morphological damage included substantial deformation of shape, due to contraction of the underlying longitudinal muscle tissue, and aggregates of chromium attached to the outer surface. Selective binding of metals to the surface of miracidia has also been found for cadmium and zinc by Morley and co-workers using the histochemical staining method (autometallography) of Danscher (1984) and Danscher et al. (1987). Schistosoma mansoni miracidia demonstrated two distinct types of metal accumulation (Fig. 5d). Large ‘aggregates’ of metals and smaller ‘pin-point’ accumulations occurred on the tegument which were associated mainly with the anterior and posterior extremities of the parasite. This accumulation pattern may be associated with areas containing sensory structures as they correlate with some of the specific receptor sites mapped out by Albaret (1984) using silver nitrate stain. In addition, metal accumulation appears to be associated with glandular openings and excretory pores on the tegumental surface. This selective tegumental binding could have important consequences for sensory-assisted host location and infection using penetration glands.

Fig. 5. Autometallographic localisation of zinc on free-living digeneans using protocols devised by Danscher et al. (1987) and modified by Morley et al. (2003 c). Parasites were exposed to 10000 μg/l Zn for 30 min. Zinc is visualised as black deposits. a. Diplostomum spathaceum cercariae showing accumulation of zinc in the cephalic region (arrow) (bar=50 μm). b. D. spathaceum cercariae showing accumulation of zinc in areas associated with sensory receptors on the body (arrows) (bar=50 μm). c. D. spathaceum cercariae showing accumulation of zinc within the ducts of the penetration glands (arrow) (bar=50 μm). d. Schistosoma mansoni miracidia showing accumulation of zinc on the tegumental surface as large deposits (big arrow) or smaller ‘pin-point’ deposits (small arrows) (bar=50 μm).

Cercariae.

A range of pollutants, in particular heavy metals, has been shown to have an effect on the functional biology of cercariae. Cross et al. (2001) demonstrated that the mean horizontal swimming rate of C. lingua is reduced in the presence of a number of heavy metals. A decrease in cercarial mobility with exposure to the pesticide tributyltin was also reported by Ritchie et al. (1974) for S. mansoni. Cercariae that had emerged from snails collected from a field site polluted with heavy metals also had a reduced swimming rate compared to unpolluted controls (Cross et al. 2001). Cercariae of Gigantobilharzia sturniae exposed to Cartap hydrochloride (an insecticide) (Oshima et al. 1992) and Diplostomum spathaceum exposed to cadmium and zinc (Morley et al. 2003 b) showed a reduced activity over time. Morley et al. (2003 b) found that heavy metals at concentrations as low as 0·1 μg/l inhibited activity of D. spathaceum over 24 h. Indeed both Oshima et al. (1992) and Morley et al. (2003 b) found cercarial activity was significantly reduced after only 3 h exposure, and this may have implications for successful transmission as this falls within the crucial period of maximum cercarial infectivity. Consequently cercarial activity may be just as important a parameter as infectivity for interpreting digenean transmission in polluted environments.

Free encystment of cercariae is impaired in the presence of toxic pollutants for both Notocotylus attenuatus (Evans, 1982 b; Morley et al. 2002 d) and Parorchis acanthus (Morley et al. 2001 b, 2003 d). Both metals (Evans, 1982 b; Morley et al. 2001 b, 2002 d, 2003 d) and pesticides (Morley et al. 2003 d) reduced the successful encystment of cercariae, with water hardness significantly influencing metal toxicity (Evans, 1982 b). Morley et al. (2003 d) found that the rate of encystment of P. acanthus was influenced by toxic exposure. Tributyltin, Irgarol 1051 (irgarol) and copper inhibited the encystment rate, but irgarol and copper were only effective at high concentrations (>1000 μg/l). In addition exposure of the host snail, Nucella lapillus, to 10 μg/l for seven days was found to have no effect on subsequent encystment of emerged cercariae by tributlytin and copper, although emergence of cercariae was inhibited by irgarol (Morley et al. 2003 d). Morley et al. (2002 d) found that associated encystment behaviour (cyst associations, floating metacercariae) of N. attenuatus was influenced by exposure to cadmium and zinc. The number of metacercariae forming cyst associations was reduced and, at low concentrations, an increase in the number of floating metacercarial cysts occurred. Such changes in encystment strategy are likely to be related to either behavioural changes by the parasite in response to the toxic environment or a product of metal toxicity on cercarial physiology and thus having a detrimental effect on parasite transmission (Morley et al. 2002 d). Evans (1982 b) studied the ultrastructural effects of copper and zinc exposure on cyst formation of N. attenuatus and found that the metals inhibited the complete extrusion and unrolling of the ‘keratin-like’ granules from the cystogenous glands, with mortalities occurring frequently during the latter stage of encystment. This may have been the product of an increase in tegumental permeability during this period thereby enhancing the metal uptake (Evans, 1982 b). Indeed, Hsu (1986) investigated the uptake of foreign substances (radioactive isotopes) and their distribution in freeliving metacercarial cysts of F. hepatica using radioselenium (⁷⁵Se-M). In vivo uptake via exposure of the host snail showed that only 5% of ⁷⁵Se-M was incorporated into the metacercariae, the remainder being incorporated into the inner and outer cyst wall, suggesting that most of the isotope was incorporated into the cystogenic cells of the cercariae. In contrast, in vitro exposure of fully formed metacercarial cysts resulted in 92% and 7·6% uptake by the outer and inner cyst wall with only 0·4% uptake by the metacercariae, although metacercarial uptake increased over longer incubation times. Interestingly, additional infectivity studies on in vitro labelled metacercariae found that all radioactivity was lost upon entry to the host, suggesting that ⁷⁵Se-M was attached to, or had diffused into, the outer periphery of the tegument and was subsequently lost as a product of tegumental turnover after the metacercariae entered the liver (Hsu, 1986). The incorporation of radioselenium may have implications for the mechanisms of uptake of toxic substances, in particular heavy metals. Indeed, comparable findings by Morley (unpublished observations) noted that autometallography staining of P. acanthus metacercariae excysted from cysts exposed to cadmium or zinc had no visible accumulation of metals on the tegument compared to controls.

Host location behaviour of cercariae is influenced by heavy metals. Korner & Haas (1998) found that silver nitrate changed the chemo-orientation of Pseudechinoparyphium echinatum and Echinostoma revolutum. However, on extensive washing in dechlorinated tap water, cercariae largely regained their chemo-orientation abilities, suggesting that metal toxicity did not permanently inhibit cercarial behaviour. Morley et al. (2003 c) found that cadmium and zinc exposure changed the pattern of phototactic and geotactic behaviour of Echinoparyphium recurvatum over time. Under control conditions cercariae initially show positive phototactic and negative geotactic behaviour which changes after 2–3 h to a negative phototactic and positive geotactic behavioural responses. This is correlated with changes in the infectivity of cercariae to prevent superinfection of the first intermediate host. At concentrations as low as 10 μg/l the metals induced a more rapid change to negative phototaxis and positive geotaxis than controls, which was considered to be either a reflex response to the toxic environment or a by-product of the toxic action of the metals (Morley et al. 2003 c). These changes in behaviour have been attributed to the binding of metals to areas associated with sensory receptors (Korner & Haas, 1998; Morley et al. 2003 c), and histochemical studies on E. recurvatum appear to support this (Morley et al. 2003 c). Furthermore, metals may also bind to other structures on the tegumental surface including glandular openings in the cephalic region, excretory pores and the mouth opening. Fig. 5a,b shows some of the tegumental areas where selective binding of zinc occurred on D. spathaceum cercariae. Indeed on rare occasions dense internal metal accumulation within the ducts of the penetration glands occurs (Fig. 5c).

Miracidial infectivity.

The relatively few studies on the effects of toxicants on miracidial infectivity to date have focused on the effects of short duration acute concentrations. Most toxicants, including pesticides, heavy metals, sewage water and acidification,⁶

Cercarial infectivity.

The effects of toxicants on cercarial infectivity have been extensively studied. Most studies have focused on the toxicity of acute short-duration exposure on cercariae with S. mansoni being the preferred species of choice. Heavy metals, pesticides, acidification and inorganic fertilisers⁷

Morley et al. (2002 a) reported that different species of target hosts may have differing susceptibilities to toxicant-exposed cercariae. In addition, the exposure of the target host to toxicants can induce an increase or decrease in susceptibility to cercariae, probably via immunosuppression or immunostimulation, and dependent on the host species being exposed. Recent work by Kiesecher (2002) found reduced immunocompetency as a factor in the increased susceptibility of Rana sylvatica tadpoles exposed to cercariae of Ribeiroia sp. and Telorchis sp. after prior host exposure to pesticides. This increase in prevalence was correlated with a decrease in the number of circulating eosinophils and such a reduced immunocompetency may be linked with increased amphibian limb deformities found under field conditions (Kiesecher, 2002). Morley et al. (2002 a) contrasted their study on E. recurvatum transmission with the earlier study of Evans (1982 a), undertaken with the same species using the same synthetic water medium. It was found that the cercariae in the two studies showed different susceptibility to zinc. This may be due to differences in the host/parasite strains used (Fig. 6), which in turn may have a wide reaching impact on predicting and interpreting the effects of pollution on host/parasite systems. Indeed Salice & Roesijadi (2002) demonstrated that cadmium toxicity in Biomphalaria glabrata was more rapid in a parasite-resistant snail strain than in a parasite-susceptible strain. The authors considered that increased cadmium sensitivity was the cost of being able to resist parasitic infection. These results suggest that when environments are contaminated with toxic chemicals, this may lead to an increase in the survival of potential hosts which are more likely to propagate disease.

Fig. 6. Comparison of the infectivity of Echinoparyphium recurvatum cercariae to Lymnaea peregra from two distinct parasite/host strains (Bushy Park and Kelsey Park/Great Ouse strains) after exposure to zinc in the same synthetic hard water medium (250 mg/l CaCO3, pH 8·00) (HMSO, 1969). a. Mean number of metacercarial cysts per snail (□ – Bushy Park strain, [squf ] – Kelsey Park/Great Ouse strain). Direct comparison of differing effects using a transmission index (T.I.), ○ – Bushy Park strain, [bull ] – Kelsey Park/Great Ouse strain. T.I.>1=increased transmission, T.I.<1=reduced transmission. Transmission index calculated according to Morley et al. (2002 a). * – Significantly reduced transmission compared to controls. Error bars are standard error. Data derived from Evans (1982 a) (Kelsey Park/Great Ouse strain) and Morley et al. (2002 a) (Bushy Park strain).

There is limited evidence to relate some of the mechanisms of heavy metal toxicity with infectivity of cercariae. King & Higashi (1992) demonstrated that silver nitrate binding to cercarial sensory receptors significantly inhibited lipid induced penetration of S. mansoni, but inhibition was reversed after extensive washing of the cercariae. Also calcium uptake by S. mansoni cercariae is critical for the release of proteolytic enzymes from the preacetabular glands (Fusco et al. 1991), and for muscular contractions and tail loss (Matsumura et al. 1991). As previously mentioned, heavy metals, especially cadmium, can utilise calcium channels for uptake across the tegument. Therefore, if active uptake of toxic metals by cercariae into the preacetabular glands occurs during penetration, it is likely to inhibit proteolytic enzyme function as metals are known to be inhibitory to enzymatic function even at low concentrations (Lewert & Lee, 1956; Dresdin & Edlin, 1974). Cercariae may therefore be at their most vulnerable to toxic metals shortly before and during penetration of their target hosts.

Metacercarial infectivity.

Data on the infectivity of toxicant-exposed metacercariae is limited to species that form free-living cysts. Evans (1982 b), who investigated the toxicity of copper and zinc to the infectivity of exposed N. attenuatus metacercariae, found no significant difference in the recovery of adult worms from control or toxicant-exposed metacercariae. In contrast, Krupicer, Velebny & Legath (1996) found that lambs experimentally exposed to emissions from a mercury-treating metallurgical works and subsequently infected with F. hepatica metacercariae show an increase in parasite prevalence, which was attributed to a metal-impaired host immune system. Exposure of F. gigantica metacercarial cysts to silage waste (Buffalo sewage, paddy straw and molasses) for 20 days resulted in a complete inhibition of infectivity to guinea pigs (Gupta & Kamra, 1987) although other factors such as the pH (pH 4·5) and fermentation temperature (35·2–38·4 °C) may also be involved. Exposure of either cercariae or encysted metacercariae of F. hepatica to fertilizers or copper caused a reduction in worm establishment in guinea pigs (Brglez & Wikerhauser, 1968). It was concluded that this was due to an increase in the mortalities of the cercarial and metacercarial stages, which occurred with more frequency with increasing exposure to the toxicants. Morley et al. (2001 b), on the other hand, studied in vitro excystment of Parorchis acanthus metacercariae prior exposed to cadmium or zinc either as encysting cercariae or as metacercariae in fully formed cysts. Although only exposure of encysting cercariae to 25 mg/l Cd/Zn mixture significantly reduced in vitro excystment, a wide fluctuation in the rate of excystment was recorded in both types of experimental exposure. This was attributed to metal inhibition of either the formation of some of the cyst layers from exposed cercariae or an alteration in the structural integrity of the same layers in exposed cysts. Impairment of the protective function of the cyst may therefore influence the viability of metacercariae over more long-term and chronically exposed pollution incidents.

Toxicity to the snail host.

The toxicity of heavy metals to parasitised snails has been investigated in a number of studies. The reduced survival of infected snails compared to controls has been recorded by Massoud, Arfaa & Chu (1973), Yescott & Hansen (1976), Guth, Blankespoor & Cairns (1977) – who noted that naturally infected snails demonstrated a much higher degree of susceptibility to zinc than those that were laboratory infected, Sullivan & Palmieri (1979), Evans (1982 b), Misechko & Stadnichenko (1988) – who found that snails collected from polluted sites were less susceptible to copper than those collected from unpolluted sites, Stadnichenko & Golovacheva (1990), Stadnichenko et al. (1987, 1988, 1991, 1996 a, 1998), Siddall (1992), Abd Allah et al. (1997) – who found that snail growth as well as survival was affected and Morley et al. (2003 a) – who found a correlation between host survival and size for naturally infected Lymnaea spp. and reduced survival for overwintered compared to seasonally infected L. peregra. Most studies on the survival of snail populations infected by a range of digeneans have found a differential survival between specific parasite species infections (e.g. Guth et al. 1977; Siddall, 1992; Morley et al. 2003 a). However, little is known about the tolerance of parasitised bivalve molluscs to polluted conditions. Heinonen, Kukkonen & Holopainen (1999, 2000, 2001) and Heinonen et al. (2003) showed how trematode infections could affect the susceptibility of Pisidium amnicum to toxic exposure under different temperature conditions. Heinonen et al. (2001) found that parasitised P. amnicum had a greater tolerance to PCP than uninfected bivalves and suggested that the high lipid content of the parasites changed the internal distribution of PCP in the host. Additional studies have found that parasitised bivalves bioaccumulate organic chemicals less quickly than unparasitised individuals (Heinonen et al. 1999). Kraak & Davids (1991) found, in contrast, an increased accumulation of heavy metals in parasitised Dreissena polymorpha compared to unparasitised individuals.

Detailed studies of the response of parasite infected snails to acute concentrations of toxic pollutants have been undertaken by Stadnichenko and co-workers. Stadnichenko et al. (1985, 1986, 1987, 1993, 1995, 1996 a, 1999 b, 2001), Misechko & Stadnichenko (1988), Stadnichenko & Golovacheva (1990) and Stadnichenko & Kirichuk (2000) found that changes in the physiological parameters of the host haemolymph during toxicant exposure were often more quickly and significantly effected in parasitised than unparasitised snails. Respiration of parasitised snails was affected with a more rapid lengthening and shortening of each air intake occurring on exposure to toxicants (Stadnichenko et al. 1992, 1996 b,c). Stadnichenko & Kotsyuk (1990) also found that exposure to detergent affected the feeding of Lymnaea stagnalis infected with Echinostoma revolutum with a decrease in food consumption accompanied by an increase in digestion time. It was concluded that the presence of the parasite inhibits the adaptability of the snail to unfavourable conditions. Exposure to a fertiliser (nitroammophoska) resulted in a rise in aldolase activity in parasitised compared to uninfected snails (Stadnichenko et al. 1988). This high rate of carbohydrate expenditure led to an increase in the mortality of parasitised snails. In contrast, potassium chloride and ‘natrium’ were found to slow the physiological and behavioural reactions of infected L. stagnalis compared to uninfected controls (Stadnichenko et al. 1999 a). Cardiac activity has been demonstrated to be detrimentally effected in parasitised bivalve and gastropod molluscs compared to controls (Stadnichenko et al. 1994; Vyskushenko, 2001), and this may be due to the defensive and adaptive reactions of parasitised molluscs being insufficient to overcome the toxic conditions (Stadnichenko et al. 1994).

Intramolluscan stages.

Little is known about the effect of toxic substances on cercarial maturation and emergence into the snail host. Yescott & Hansen (1976) exposed Biomphalaria glabrata infected with S. mansoni to manganese and found that the sporocysts contained mature cercariae which appeared sluggish or immobile, while 83% of snails contained no emerged cercariae within haemolymph samples.

Both Ibrahim et al. (1992) and Morley et al. (2003 d) have shown that pesticides (chlorpyifos and irgarol respectively) may completely inhibit the emergence of cercariae from exposed snail hosts but, when chlorpyifos exposure ended emergence begins again. Ibrahim et al. (1992) considered that this was because the pesticide specifically targets the nearly mature cercariae, leaving the sporocyst unharmed. In contrast, Stpiczynska et al. (1978) found that certain pesticides actually stimulated asexual reproduction of the intramolluscan stages of F. hepatica whilst the miracidia were developing within the egg. However, Wolmarans et al. (1988) found that a 1 h exposure to chromium reduced the ability of S. haematobium miracidia to form sporocysts in vitro over a range of concentrations.

Morley (unpublished observations) found that a 72 h cadmium exposure of L. peregra containing a mature infection of Echinoparyphium recurvatum caused selective binding of metals to the tegumental surface of daughter rediae. In addition, daughter rediae fed on metal-contaminated host tissue which was absorbed into the parasite body, thereby exposing developing cercariae to the metal. In addition, the incidence of first host encystment by cercariae increased in the metal exposed snails compared to controls, and this was considered to be a reaction to unfavourable environmental conditions.

Cercarial emergence.

Little is known about the effects of pollutant toxicity on cercarial emergence. An early study by Yescott & Hansen (1976) found that manganese treatment of Biomphalaria glabrata infected with S. mansoni resulted in the termination of cercarial emergence when snails were exposed to only a short pulse (0·5 h) of light. However, with a 6 h light period a large number of cercariae emerged, suggesting that a long photoperiod can override manganese inhibition.

Additional studies using both acute and chronic toxicant concentrations have reported a reduction in cercarial emergence following exposure to metals, pesticides, and acidification.⁸

Fig. 7. Cercarial emergence of Diplostomum spathaceum from Lymnaea stagnalis exposed to zinc in soft water (25 mg/l CaCO3, pH 7·85) (HMSO, 1969) at 20 °C. ([bull ] – Control, [utrif ] – 1000 μg/l Zn, [squf ] – 100 μg/l Zn.) Exposure was undertaken using protocols devised by Morley et al. (2003 a). Error bars are standard error.

The functional biology of cercariae emerged from exposed hosts has been only briefly investigated. Cross et al. (2001) found that cercariae had a reduced swimming rate and survival compared to cercariae collected from unpolluted sites. In contrast, Morley (unpublished observations) found that D. spathaceum cercariae emerged from a L. stagnalis exposed to 100 μg/l cadmium for 7 days showed an increase in survival and a decrease rate of tail loss compared to controls. Interestingly, Knight et al. (1968) found that the uptake of radioselenium by S. mansoni cercariae from exposed snail hosts accumulated in areas in the cercariae associated with penetration glands. If a similar uptake is demonstrated by toxic substances, and in particular heavy metal ions, which inhibit enzymatic activity of these glandular secretions (Dresden & Edlin, 1974), this may have implications for the successful penetration of target hosts. However, no effect on the functional biology of cercariae was found by either Massoud & Webbe (1969), who reported no difference between the infectivity of S. mansoni cercariae from N-tritylmorpholine exposed snails and controls, or Morley et al. (2003 d), who found no difference in the encystment of Parorchis acanthus cercariae from copper and tributyltin exposed snails and controls. Chernogorenko (1982) considered that cercariae retain their ability to accumulate glycogen whilst developing within the snail host regardless of the detrimental conditions in the snails habitat. Consequently changes in the functional biology of cercariae from pollutant exposed hosts may be due to absorption of toxicants into their bodies whilst resident within the host, which directly effects their metabolism independent of normal glycogen accumulation.