Artificial Spring Water Research Articles

Changes in snail chemical profiles through host-parasite interactions

Host behavior may be modified by their parasites to increase the likelihood of transmission, but mechanisms underlying these interactions are not well understood. Hosts and parasites release chemical signaling molecules, like oxylipins, that may affect transmission. Oxylipins are oxygenated metabolites of fatty acids that function as signaling molecules and have essential physiological and functional roles. Yet, the limited taxonomic and contextual scope of these studies constrains our ability to understand their role in parasite‐modified behavior. We characterized oxylipins in field-collected File Ramshorn snails, Planorbella pilsbryi. We tested for differences in oxylipin profiles based on infection status (infected with the trematode Echinostoma trivolvis lineage a and uninfected) and parasite activity (high and low). Snail-conditioned water samples were produced by placing five snails into artificial spring water for four hours. Oxylipins were extracted from snail-conditioned water samples and quantified using high performance liquid chromatography-tandem mass spectrometry. Infected snails emitted 69 oxylipins in higher amounts, with 37 only released by this group. Within infected snails, 18 oxylipins were emitted in higher amounts in snails with increased parasite activity. Oxylipins emitted in higher amounts by infected snails with increased parasite activity were predominantly derived from the cytochrome P450 pathway. As infected snails emit different oxylipin profiles than uninfected snails, their production may play a role in altering transmission success. By characterizing the oxylipins produced by snails, and how they are altered by infection, we can test their physiological and ecological roles in freshwater systems.

Variability in environmental persistence but not per capita transmission rates of the amphibian chytrid fungus leads to differences in host infection prevalence.

Heterogeneities in infections among host populations may arise through differences in environmental conditions through two mechanisms. First, environmental conditions may alter host exposure to pathogens via effects on survival. Second, environmental conditions may alter host susceptibility, making infection more or less likely if contact between a host and pathogen occurs. Further, host susceptibility might be altered through acquired resistance, which hosts can develop, in some systems, through exposure to dead or decaying pathogens and their metabolites. Environmental conditions may alter the rates of pathogen decomposition, influencing the likelihood of hosts developing acquired resistance. The present study primarily tests how environmental context influences the relative contributions of pathogen survival and per capita transmission on host infection prevalence using the amphibian chytrid fungus (Batrachochytrium dendrobatidis; Bd) as a model system. Secondarily, we evaluate how environmental context influences the decomposition of Bd because previous studies have shown that dead Bd and its metabolites can illicit acquired resistance in hosts. We conducted Bd survival and infection experiments and then fit models to discern how Bd mortality, decomposition and per capita transmission rates vary among water sources [e.g. artificial spring water (ASW) or water from three ponds]. We found that infection prevalence differed among water sources, which was driven by differences in mortality rates of Bd, rather than differences in per capita transmission rates. Bd mortality rates varied among pond water treatments and were lower in ASW compared to pond water. These results suggest that variation in Bd infection dynamics could be a function of environmental factors in waterbodies that result in differences in exposure of hosts to live Bd. In contrast to the persistence of live Bd, we found that the rates of decomposition of dead Bd did not vary among water sources, which may suggest that exposure of hosts to dead Bd or its metabolites might not commonly vary among nearby sites. Ultimately, a mechanistic understanding of the environmental dependence of free-living pathogens could lead to a deeper understanding of the patterns of outbreak heterogeneity, which could inform surveillance and management strategies.

Asymmetric cross-strain protection for amphibians exposed to a fungal-metabolite prophylactic treatment.

Chytridiomycosis, an infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), poses an imminent conservation threat. The global spread of Bd has led to mass mortality events in many amphibian species, resulting in at least 90 species' extinctions to date. Exposure to Bd metabolites (i.e. non-infectious antigenic chemicals released by Bd) partially protects frogs during subsequent challenges with live Bd, suggesting its use as a prophylactic treatment and potential vaccine. However, we do not know whether Bd metabolite exposure protects against strains beyond the one used for treatment. To address this knowledge gap, we conducted a 3 × 2 experiment where we exposed adult Cuban treefrogs, Osteopilus septentrionalis, to one of three treatments (Bd metabolites from California-isolated strain JEL-270, Panamá-isolated strain JEL-419, or an artificial spring water control) and then challenged individuals with live Bd from either strain. We found that exposure to Bd metabolites from the California-isolated strain significantly reduced Bd loads of frogs challenged with the live Panamá-isolated strain, but no other treatments were found to confer protective effects. These findings demonstrate asymmetric cross-protection of a Bd metabolite prophylaxis and suggest that work investigating multiple, diverse strains is urgently needed.

Freshwater snails and the green algae Cladophora are probably not hosts of Batrachochytrium dendrobatidis

Abstract Batrachochytrium dendrobatidis (Bd) is a pathogenic fungus that has devastated amphibian populations globally by causing the disease chytridiomycosis. Batrachochytrium dendrobatidis is capable of infecting non‐amphibian hosts, such as crayfish, and has been detected on reptile and bird species. Given the taxonomic heterogeneity in the known hosts and vectors of Bd, it is likely that there is a diversity of undiscovered non‐amphibian hosts of the fungus. Here, we investigated whether Bd could survive on freshwater snails (Physella acuta) and Cladophora algae. We exposed small and large snails (n = 15 snails/size category), Cladophora algae (n = 5), and artificial spring water controls (ASW; n = 5) to live Bd. We also maintained Bd‐free control snails (n = 5 snails/size category) in ASW. All treatments were maintained for 7 weeks at 18°C. Mortality was checked three times a week, snails were weighed every 2 weeks, and 7 weeks after exposure, the snails, algae, and water were tested for Bd using quantitative polymerase chain reaction. We found that Bd did not grow on live snails, algae, or ASW long term. Additionally, live snails (n = 20) collected from Bd‐positive ponds in California were all negative for Bd, as well. Given that we found no Bd on the experimentally exposed or field swabbed snails, snails are probably not a reservoir host of Bd. While negative results are often not published, Bd is one of the deadliest pathogens on earth; it is essential to know what is and is not capable of maintaining Bd for well‐designed disease models.

Metabolites produced by Batrachochytrium dendrobatidis alter development in tadpoles, but not growth or mortality.

The mass decline of amphibian populations poses a serious threat to global biodiversity and ecosystem stability. The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has contributed to the extirpation and extinction of hundreds of amphibian species worldwide. Bd produces potentially damaging metabolites during the host infection process that may affect amphibian growth and development, even in the absence of infection. In this experiment, Cuban tree frog Osteopilus septentrionalis tadpoles and adults were exposed once to either artificial spring water (ASW) or Bd metabolites (n = 31 tadpoles per treatment and n = 30 and 20 adults per treatment, respectively). Tadpoles exposed to Bd metabolites alone developed faster than those exposed to ASW; however, there was no difference in tadpole length, weight change, or mortality between treatments. Despite the faster developmental speed, metabolite exposure did not reduce tadpole weight or length (compared at Gosner stages 27, 29, and 31). There was no effect of treatment on adult size or mortality. These results indicate that both tadpole and adult O. septentrionalis do not appear to be negatively impacted by exposure to non-infectious Bd-contaminated water. In fact, tadpoles developed faster when exposed to metabolites and were of equal size as those in their stage cohort, implying a potential long-term benefit if faster development allows them to leave Bd-infected waters sooner.

Effects of starvation for 4–20 days on the neutral lipid and phospholipid content of Biomphalaria glabrata as determined by high performance thin layer chromatography-densitometry

ABSTRACTThe effects of 4, 7, 10, 14, and 20 days of starvation on the neutral and phospholipid levels of Biomphalaria glabrata were determined by high performance thin layer chromatography (HPTLC)-densitometry. Snails were maintained in artificial spring water in jars at 25 ± 1°C. The digestive gland–gonad complex was extracted with chloroform–methanol (2:1), and Analtech laned preadsorbent silica gel HPTLC plates were used with petroleum ether–diethyl ether–glacial acetic acid (80:20:1) mobile phase, phosphomolybdic acid detection reagent, and scanning at 610 nm for neutral lipid analysis, and chloroform–methanol–water (65:25:4.5) mobile phase, cupric sulfate–phosphoric acid reagent, and scanning at 370 nm for phospholipids. Significant differences were observed in the triacylglycerol and steryl ester levels in the starved versus the control snails.

Effects of Coexposure with Echinostoma caproni and Schistosoma mansoni Miracidia on Glucose and Maltose Levels in Biomphalaria glabrata as Determined by High-Performance Thin-Layer Chromatography–Densitometry

High-performance thin-layer chromatography (HPTLC)–densitometry was used to determine maltose and glucose in Biomphalaria glabrata snails subjected to either Echinostoma caproni and Schistosoma mansoni miracidia coexposure or single exposure to each of these trematode parasites. Each culture contained 800 mL of artificial spring water; snails were maintained at 25 ± 1°C and fed boiled romaine lettuce ad libitum. A sample of each population was necropsied at 2, 4, and 6 weeks post-exposure to E. caproni. The whole body of each snail was extracted with 70% ethanol for sugar analysis. Sugars were separated on 10 × 20 cm EMD Millipore Corp. HPTLC silica gel plates with a preabsorbent zone, the mobile phase was 1-butanol–glacial acetic acid–diethyl ether–deionized water (27:18:5:3), and the detection reagent was naphthol–sulfuric acid. Quantitative densitometric analysis was performed using a CAMAG TLC Scanner 3 in the absorbance–reflectance mode at 515 nm. Significant differences in maltose concentrations were shown among the four groups at 2, 4, and 6 weeks post-exposure (analysis of variance, P < 0.0125). Significant differences in glucose were found only at 2 and 4 weeks post-exposure.

No Effects of Two Anesthetic Agents on Circulating Leukocyte Counts or Resistance to Trematode Infections in Larval Amphibians

Abstract Pharmacological anesthetics are used frequently in aquatic animal husbandry and research. Although several studies have investigated the effects of these anesthetic agents on immune responses in fish, none have assessed such effects in amphibians. To address this disparity, we exposed Osteopilus septentrionalis tadpoles to 0.005% benzocaine, 0.1% tricaine methanesulfonate (MS-222), or artificial spring water, and quantified circulating white blood cells and susceptibility to infection by larval trematodes. Exposure to neither MS-222 nor benzocaine elevated circulating white blood cells relative to water-exposed tadpoles. Furthermore, anesthetic treatment did not affect resistance to larval trematode infection. These results indicate that the anesthetics tested here probably do not affect tadpole immune function and should aid researchers in determining anesthetic usage.

EFFECTS OF COEXPOSURE WITH ECHINOSTOMA CAPRONI AND SCHISTOSOMA MANSONI MIRACIDIA ON NEUTRAL AND POLAR LIPIDS OF BIOMPHALARIA GLABRATA AS DETERMINED BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY AND OBSERVATIONS ON SNAIL SURVIVAL AND FECUNDITY

High performance thin-layer chromatography (HPTLC)-densitometry was used to characterize and quantify neutral and polar lipids in Biomphalaria glabrata snails subjected to either Echinostoma caproni and Schistosoma mansoni miracidia coexposure, or single exposure to each of these trematode parasites. Observations on survival and fecundity were made on an unexposed population (2 cultures of 25 snails each); a population exposed exclusively to S. mansoni(2 cultures of 25 snails, each exposed to 6 miracidia per snail); a population exposed exclusively to E. caproni (2 cultures of 25 snails, each exposed to 10 miracidia per snail); and a population exposed to S. mansoni and one week later exposed to E. caproni (2 cultures of 25, each exposed to 10 E. caproni miracidia and 6 S. mansonimiracidia per snail). Each culture contained 800 mL of artificial spring water; snails were maintained at 25 ± 1°C and fed boiled romaine lettuce ad libitum. A sample of each population was necropsied at 2, 4, 6, and 8 weeks post exposure to E. caproni. The digestive-gland gonad complex (DGG) of each snail was extracted in chloroform-methanol for lipid analysis. Lipids were separated on 10 × 20 cm Analtech channeled HPTLC-HLF silica gel plates with a preadsorbent zone. For neutral lipids, the mobile phase and detection reagent used were petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and 5% ethanolic phosphomolybdic acid, respectively. For polar lipids, a chloroform-methanol-deionized water (65:25:4) mobile phase and a 10% cupric sulfate in 8% phosphoric acid detection reagent were used. Quantitative densitometric analysis was performed using a CAMAG TLC Scanner 3 with the tungsten light source set at 610 nm for neutral lipids and the deuterium light source set to 370 nm for polar lipids. Significant differences in lipid concentrations were only observed for the free fatty acids (ANOVA, P<0.0125). Survival of snails declined mainly in the group exposed only to E. caproni miracidia and in the coexposed group. Fecundity (based on egg laying) increased in the group exposed only to E. caproni miracidia, but declined in the other groups.

Effects of increased salinity on glucose and maltose composition ofBiomphalaria glabratasnails infected withSchistosoma mansonias determined by high-performance thin-layer chromatography-densitometry

Biomphalaria glabrata snails were infected with Schistosoma mansoni and maintained at different dilutions of artificial ocean water for up to 4 weeks. Glucose and maltose concentration of the digestive gland-gonad complex were analyzed by high-performance thin-layer chromatography at different stages of the infection. B. glabrata snails were divided into three experimental groups: Group A, snails with early prepatent infection (10 days post-infection); Group B, snails with late prepatent infection (22 days post-infection); and Group C, snails with patent infection (45 days post-infection). Infected snails in A were maintained at different salinities for 2 weeks and then necropsied, and their two main simple sugars, i.e., glucose and maltose, were analyzed. Groups B and C contained two subgroups: the first subgoups were analyzed after 2 weeks, and the second after 4 weeks. Controls for these experiments were maintained identically in either deionized water or artificial spring water. Maltose and glucose we...

Maintenance of Biomphalaria Glabrata Infected with Schistosoma Mansoni in Artificial Spring Water, Deionized Water, or Conditioned Tap Water

This study investigated the effects of artificial spring water (ASW), conditioned tap water (CTW), and deionized (DI) water on Biomphalaria glabrata snails infected with the trematode parasite Schistosoma mansoni. Biomphalaria glabrata snails infected with S. mansoni were placed in cultures containing ASW, CTW, or DI for 3 wk during which snail survival and the number of egg masses were recorded. Snails in ASW and DI showed similar survival, whereas all snails in CTW cultures died within 2 d. However, snails in ASW showed better fecundity then did those in DI and had a significantly greater number of egg masses. Despite decreased fecundity, we noted that DI may serve as an adequate cost- and labor-effective replacement for ASW in the laboratory maintenance of B. glabrata infected with S. mansoni.

EFFECTS OF INCREASED SALINITY ON GLUCOSE AND MALTOSE COMPOSITION OF BIOMPHALARIA GLABRATA SNAILS AS DETERMINED BY HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY

High performance thin-layer chromatography was used to analyze the glucose and maltose concentrations of the digestive gland-gonad complex of mature adult uninfected Biomphalaria glabrata snails maintained in laboratory cultures containing various dilutions of artificial ocean water (AOW) as well as the whole bodies of young adult B. glabrata snails maintained in laboratory cultures at the same dilutions of AOW for up to 4 weeks. Controls for both groups consisted of snails of the same size maintained identically in deionized water and artificial spring water. Sugars were extracted from the snails in ethanol-water (70:30). Random samples from both experimental groups were tested to detect the presence of different types of carbohydrates; the major sugars detected in the snails were glucose and maltose. These sugars were separated on EMD Millipore silica gel preadsorbent plates with mobile phase 1-butanol-glacial acetic acid-diethyl ether-deionized water (27:18:5:3), detected using α -naphthol-sulfuric acid reagent, and quantified by densitometry with a CAMAG TLC Scanner 3 at 515 nm. The mature adult snails showed high survival rates in most salinities, and only in 25% salinity was the survival lower than in the other cultures. In the experimental group of young adult snails, the survival was lower, especially in 25% salinity where only 40% of the snails survived up to 4 weeks. No significant statistical differences in the carbohydrate composition as a function of salinity were found in either of the two experimental groups.

EFFECTS OF CROWDING ON THE NEUTRAL AND POLAR LIPIDS OF BIOMPHALARIA GLABRATA AS DETERMINED BY HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY AND OBSERVATIONS ON SNAIL SURVIVAL AND FECUNDITY

High performance thin-layer chromatography-densitometry was used to characterize and quantify neutral and polar lipid classes present in Biomphalaria glabrata snails subjected to crowding. Observations on survival and fecundity were made on a noncrowded population (10 snails per culture), a moderately crowded population (20 snails per culture), and a heavily crowded population (40 snails per culture). Each culture contained 800 mL of artificial spring water, and snails were maintained at 25 ± 1°C and fed romaine lettuce ad libitum. After 4 weeks of culture, the snails were necropsied and the digestive-gland gonad complex (DGG) of each snail was extracted in chloroform-methanol for lipid analysis. Lipids were separated on 10 × 20 cm Analtech channeled HPTLC-HLF silica gel plates with a preadsorbent zone. For neutral lipids, the mobile phase petroleum ether-diethyl ether-glacial acetic acid (80:20:1) and 5% ethanolic phosphomolybdic acid detection reagent were used to analyze the DGG of each snail. Chloroform-methanol-deionized water (65:25:4) mobile phase and 10% cupric sulfate in 8% phosphoric acid detection reagent were used to determine the polar lipids in the DGG of each snail. Quantitative densitometric analysis was performed using a CAMAG TLC Scanner 3 with the tungsten light source set at 610 nm for neutral lipids and the deuterium source at 370 nm for polar lipids. No significant differences in any of the neutral and polar lipid classes were found among all three of the aforementioned snail populations (ANOVA, p < 0.017).

Longevity of Zygocotyle lunata in Balb/c mice and long-term survival of digeneans in vertebrate hosts

This study used Balb/c mice to examine the longevity of Zygocotyle lunata in a murine host. Of 11 mice, each exposed to 20 Z. lunata cysts, six were infected with a total of 12 worms from 11 to 24 weeks postinfection (PI). Live worms recovered at 24 weeks PI had a mean body area of about 25mm2. These worms produced viable eggs with well-developed miracidia following embryonation in artificial spring water for 2 weeks at 28°C. The Balb/c mouse is a useful model to study longevity of this paramphistomid trematode for at least 6 months PI. An additional aspect of this article is a review of the pertinent literature published from 1937 to 2007 on ageing and longevity of digeneans.

Infectivity, Development, and Growth of Zygocotyle lunata (Trematoda) in Balb/c Mice

Infectivity, development, and growth of Zygocotyle lunata in 18 male Balb/c mice, each exposed to 20 metacercariae cysts, were examined 1 to 6 wk post-infection (PI). Metacercariae cysts were obtained from naturally infected Helisoma trivolvis snails. At necropsy, 11 of 18 mice were found infected with a total of 32 worms. The range of recovery was 1 to 6 worms per host. The mean body area of worms, fixed in 10% neutral-buffered formalin and mounted in glycerin jelly, was found to be 4.0 mm(2) at 2 wk PI, increased to 6.8 mm(2) at 4 wk, and reached 11.4 mm(2) by 6 wk PI, at which time the study was terminated. Worms were ovigerous by 3 wk PI. Eggs were collected from 4-wk-old worms and incubated at 28 C in artificial spring water (ASW). Eggs were fully developed by 3 wk post-incubation and miracidia hatched in the ASW. Laboratory-reared H. trivolvis snails were not available to attempt miracidia infections with Z. lunata .

Effects of prolonged worm storage on the viability of Echinostoma caproni eggs

This study examined the effects of long-term worm storage on the viability of Echinostoma caproni eggs. Gravid adults (referred to as worm carcasses or WCs) were maintained in Locke's 1:1 at 4 degrees C for 2, 4, 6, 8, 10, and 12 months prior to use. When used, about 100 eggs at each time interval were tested for viability by embryonating them at either 22 +/- 1 degrees C for 14 days or 28 degrees C for 10 days in artificial spring water. Viability was determined by detecting fully developed miracidia in the eggs or miracidia in the cultures at the above-mentioned time periods. There was a high percentage of fully developed eggs from WCs stored in Locke's 1:1 at 4 degrees C for 2 and 4 months, 92% and 71%, respectively. Beyond these times, there was a marked decline in egg viability. We suggest that WCs should not be stored for longer than 4 months in Locke's 1:1 at 4 degrees C to obtain optimal embryonation and hatching of E. caproni.

The effects of various chemical and physical factors on encystment and excystment of Zygocotyle lunata

The effects of various chemical and physical treatments on the cercariae and metacercariae of Zygocotyle lunata were studied. Cercariae that actively emerged from Helisoma trivolvis snails were examined in 4 ml of test solution in a 3-cm diameter Stender dish. Observations were usually made on encystment and excystment from 2 to 24 h after cercariae were placed in the Stender dishes. Preferences for certain substrates for encystment were noted, such as smooth glass rather than rough glass, the macrophyte Eleocharis acicularis, and plastic Petri dishes. Many of the solutions tested allowed for encystment, i.e., artificial spring water (ASW), 10% artificial ocean water (AOW), and saline (0.85% NaCl). Certain hypertonic solutions allowed for both in vitro encystment and subsequent excystment, i.e., Locke's solution, 25% AOW, and Locke's 1:1. Serial dilutions of ammonium hydroxide, neutral buffered formalin, potassium permanganate, hydrochloric acid, and acetic acid killed cercariae at all concentrations. Ethanol was effective in killing cercariae at all concentrations except at 3.1%, which allowed for encystment. Cercariae were able to encyst at a low temperature of 16 degrees C and a high temperature of 41 degrees C. Morphometric analysis was performed on both live and fixed cercariae of Z. lunata as well as metacercarial cysts formed in Locke's, Locke's 1:1, and ASW. Live cercariae had larger body dimensions than cercariae fixed in hot 5% neutral buffered formalin and mounted in glycerin jelly. Cysts formed in ASW had very similar length and width measurements. Cyst dimensions became less similar to one another as the tonicity of the medium increased. The phenomenon of spontaneous excystation in Z. lunata after in vitro encystment in a hypertonic medium appears to be reported for the first time in digeneans.

EFFECTS OF AESTIVATION AND STARVATION ON THE NEUTRAL LIPID AND PHOSPHOLIPID CONTENT OF BIOMPHALARIA GLABRATA INFECTED WITH SCHISTOSOMA MANSONI

The effects of aestivation or starvation on the neutral lipid and phospholipid content of Biomphalaria glabrata patently infected with Schistosoma mansoni were determined by high-performance thin-layer chromatography-densitometry. Infected-aestivated snails were maintained in a moist chamber at 24 +/- 1 C and a relative humidity of 98 +/- 1%. Infected-starved snails were maintained in artificial spring water (ASW) at 23 +/- 1 C without exogenous food. Infected snails (the controls) were maintained in ASW at 23 +/- 1 C and fed lettuce ad libitum. The 3 groups were maintained in the laboratory for 7 days, and then the lipids from the digestive gland-gonad complex (DGG) were extracted and analyzed by class. Infected-aestivated snails exhibited greater mortality rate and weight loss after 7 days than did the infected-starved snails. The steryl ester concentration in the infected-starved snails was significantly increased (P = 0.010) compared with the controls but not compared with infected-aestivated snails; the concentration of phosphatidylcholine in infected-aestivated snails was significantly decreased (P = 0.007) compared with the controls but not when compared with the infected-starved snails. Aestivation or starvation had a significant effect on the concentration of certain lipid classes in the DGG of B. glabrata infected with S. mansoni.

Effects of Estivation on Lutein and β‐Carotene Concentrations in Biomphalaria glabrata (NMRI Strain) and Helisoma trivolvis (Colorado Strain) Snails as Determined by Quantitative High Performance Reversed Phase Thin Layer Chromatography

High performance thin layer chromatrography (HPTLC) on C‐18 chemically bonded silica gel layers with petroleum ether‐acetonitrile‐methanol (1:2:2) mobile phase and visible mode slit‐scanning densitometry was used to examine the effects of estivation on β‐carotene and lutein in the medically and economically important snails Biomphalaria glabrata (NMRI strain) and Helisoma trivolvis (Colorado strain). Snails were estivated for 2 weeks in moist chambers at 24°C and a relative humidity of 98%. Controls were maintained at 24°C for the same period in aerated aquaria containing artificial spring water and fed Romaine lettuce leaves ad libitum. As determined by HPTLC analysis, estivation had no significant effect on the weight percentage of β‐carotene in the whole body or digestive gland‐gonad complex (DGG) of both snail species. A significant reduction (Student's t‐test, P<0.5) was found in the percentage of lutein in the DGG of H. trivolvis. Whereas published and unpublished studies have demonstrated marked reductions of carbohydrates and lipids in estivated pulmonate snails, the effects of estivation on lipophilic pigments in these snails does not appear to be as pronounced.

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

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