Parasitic castration in the snail-trematode relationship can be understood as any change in the reproductive function of the snail that is due to interference by the developing larvae inside the snail that leads to the reduction or complete disruption of egg-laying activity. This study was designed to observe the parasitic castration of Biomphalaria glabrata infected with Schistosoma mansoni during both the pre-patent and patent periods. The effect of infection on snail fecundity and fertility, growth rate and survival was studied during the 62 days following miracidia exposure. An integrated approach was employed that used biochemical and histological tools over the same period. To study the effect of infection on reproduction, we individually exposed 30 snails to 5 miracidia each and tracked their fertility and fecundity. For our histopathological studies, 50 snails were exposed to 20 miracidia each, and for our histochemical studies, 50 snails were exposed to 5 miracidia each. An equal number of uninfected snails were used as a control for each group. The B. glabrata exposed to the BH strain of S. mansoni showed 50% positivity for cercarial shedding. Both the experimental and control groups showed 100% survival. The pre-patent period lasted until 39 days after exposure to miracidia. Exposed snails that showed cercarial shedding exhibited higher growth rates than either exposed snails that did not demonstrate cercarial shedding or uninfected controls. Exposed snails without cercarial shedding and uninfected controls showed no differences in the reproductive parameters evaluated during the patent period; snails experiencing cercarial shedding showed a reduction in fecundity and fertility. These snails began to lay eggs only after the 50th day post miracidia exposure. The haemolymph glucose levels showed an oscillating pattern that decreased during periods of greater mobilisation of energy by the larvae and was accompanied by a depletion of glycogen in the cephalopodal mass and digestive gland. Histopathological examination at 55 days showed that the ovotestis was highly atrophied. There was almost complete disappearance of germ cells, and the supporting stroma formed a nearly empty net. At day 45, the infected digestive gland showed a high cylindrical epithelium with little preserved cytoplasm. The contents of the secretory granules of the albumen gland of infected animals stained with Alcian blue (AB), pH 1.0, indicating the presence of sulphated carbohydrates. Thus, parasitic castration in the B. glabrata-S. mansoni model may be regulated directly and indirectly by the developmental stage of the trematode and the biochemical and histopathological alterations during the patent period of infection.

Biomphalaria glabrata is the main intermediate host involved in schistosomiasis in Brazil. Studies have shown that physiological stress conditions, such as infection with Schistosoma mansoni, starvation, aestivation and exposure to molluscicides, can alter its carbohydrate metabolism (Schwartz & Carter 1982, Becker 1983, Bezerra et al. 1999, Alcanfor 2001, Mello-Silva et al. 2006a). These changes can alter the glycogenesis, gluconeogenesis and glycolysis in the snail.One of most promising Brazilian molluscicides is the crude extract of Euphorbia splendens (Sin. Euphorbia milii), which under laboratory and field conditions meets the recommendations of the WHO for use as a natural molluscicide (Vasconcellos & Amorim 2003). Mello-Silva et al. (2006a, 2007 studied the influence of sublethal doses of the latex of this plant on the carbohydrate and protein metabolism and reproductive biology of B. glabrata and found strong metabolic changes leading to a reduction in its population and consequently in the transmission of the parasite. In spite of this potential, there are no studies of the influence of this product on the physiology of Biomphalaria infected with S. mansoni.The present paper examines the action of the latex of E. splendens var. hislopii on the glucose content of the haemolymph and on the carbohydrate (glycogen) deposits of B. glabrata (BH strain) infected with S. mansoni (BH strain). MATERIALS AND METHODSObtaining the latex of E. splendens var. hislopiiSamples of E. splendens var. hislopii latex were collected in the autumn from plants cultivated in plots near the Biology Department, Fiocruz, in Rio de Janeiro, Brazil. The latex was collected as described by Vasconcellos and Amorim (2003) on the same day the tests were conducted.Lethal dose experiment -Using the recently collected latex, an aqueous stock solution at a concentration of 100 mg/L was prepared and, from this, solutions of different concentrations were prepared for use in the bioassays. The lethal (LC 90 ) and sublethal (LC 50 ) concentrations were determined according to Vasconcellos and Amorim (2003), as recommended by the World Health Organization (1983) and Mott (1987). The LC 50 and LC 90 values were, respectively, 1.0 mg/L and 2.3 mg/L (Mello-Silva et al. 2006a).Balloon flasks (1000 mL) were used and the latex solution was divided into two 500 mL glass beakers. The groups of B. glabrata (BH lineage), infected and uninfected, respectively, were placed in LC 50 solutions and exposed for 24 h (Vasconcellos & Amorim 2003) at 21ºC. Two glass beakers received 500 mL of distilled water without latex as a control group. None of the snails was fed during this period.After the latex exposure period, the snails were removed from the flasks and rinsed in distilled water to remove the residues. The number of dead specimens was noted. (1990). The snails were grouped according to their infection stage (1 day and 1, 2, 3 and 4 weeks post exposure). In each period analysed, 100 infected and 20 uninfected snails (control) were used. Sixty infected sn...

BackgroundSchistosomiasis has been reported in 78 endemic countries and affects 240 million people worldwide. The digenetic parasite Schistosoma mansoni needs fresh water to compete its life cycle. There, it is susceptible to soluble compounds that can affect directly and/or indirectly the parasite’s biology. The cercariae stage is one of the key points in which the parasite is vulnerable to different soluble compounds that can significantly alter the parasite’s life cycle. Molluscicides are recommended by the World Health Organization for the control of schistosomiasis transmission and Euphorbia milii latex is effective against snails intermediate hosts.Methodology/Principal findingsWe used parasitological tools and electron microscopy to verify the effects of cercariae exposure to natural molluscicide (Euphorbia milii latex) on morphology, physiology and fitness of adult parasite worms. In order to generate insights into key metabolic pathways that lead to the observed phenotypes we used comparative transcriptomics and proteomics.Conclusions/SignificanceWe describe here that the effect of latex on the adult is not due to direct toxicity but it triggers an early change in developmental trajectory and perturbs cell memory, mobility, energy metabolism and other key pathways. We conclude that latex has not only an effect on the vector but applies also long lasting schistosomastatic action. We believe that these results are of interest not only to parasitologists since it shows that natural compounds, presumably without side effects, can have an impact that occurred unexpectedly on developmental processes. Such collateral damage is in this case positive, since it impacts the true target of the treatment campaign. This type of treatment could also provide a rational for the control of other pests. Our results will contribute to enforce the use of E. milii latex in Brazil and other endemic countries as cheap alternative or complement to mass drug treatment with Praziquantel, the only available drug to cure the patients (without preventing re-infection).

Molluscides have been used as one of the strategies to control schistosomiasis. Many plant extracts with molluscidal effects have been tested, but the action of the latex of Euphorbia splendens var. hislopii is considered the most promising because it meets the recommendations of the World Health Organization (WHO). The objective of this study was to determine the lethal dose and identify the effects of the different doses of latex of E. splendens var.hislopii on the physiology of Biomphalaria glabrata submitted to treatment for 24 h. The concentrations of glucose, uric acid and total proteins in the hemolymph and of glycogen in the digestive gland and cephalopodal mass were determined. The LD 50 value was 1 mg/l. The highest escape index was found to be at a concentration of 0.6 mg/l. The results showed that the latex of E. splendens var. hislopii caused a sharp reduction in the reserves of glycogen in the digestive gland and elevation of the protein content in the hemolymph of B. glabrata. Key words: plant molluscide -Euphorbia splendens var. hislopii -Biomphalaria glabrataThe use of molluscides as one of the strategies to control schistosomiasis began in Brazil in 1976, with the creation of the Special Schistosomiasis Control Program by the National Health Foundation (Machado 1982). The product used in the program was niclosamide, an ethanolamine salt of 2',5-dichloro-4'-nitrosalicylanilide, manufactured under the trade name Bayluscide  , whose efficacy had previously been established (Gonnert 1961). Application of this product caused biocidal action on non-target plants and animals (Andrews et al. 1983), besides causing genotoxicity and carcinogenic effects (Vega et al. 1988). The high cost (Pieri 1995), the possibility of recolonization of breeding grounds (Sarquis et al. 1997(Sarquis et al. , 1998, and the ecological toxicity of this product were limitations on its use as an official molluscide in government programs to control schistosomiasis.At the same time, various plants were tested as natural molluscides. Jurberg et al. (1989) (Mendes et al. 1984). Of these, E. splendens stands out for its molluscidal activity in doses under 0.5 mg/l, eight times smaller than the lethal dose for fish (Vasconcellos & Schall 1986).Some questions have arisen in the selection of plant molluscides, such as: toxicity, availability, annual growth, adaptability to different local conditions, and location of molluscidal activity in parts of the plant that easily regenerate, such as the leaves. To be eligible for use, a product must be storable and remain viable for at least one year; be physically and chemically stable; have ethnobotanical value; and be easy to extract and apply, preferably in aqueous extracts (Kloos & McCullough 1982). Considering all these criteria, in 1998 the Oswaldo Cruz Institute (IOC, Fiocruz, RJ) obtained a biotechnology patent on a method to collect, extract and apply E. splendens var. hislopii latex as a molluscide (Vasconcellos 2000).In the published works on plants with molluscidal action and ...

The aim of this work was to analyze the content of total protein and nitrogen degradation products in Biomphalaria glabrata infected with Schistosoma mansoni and exposed to Euphorbia splendens var. hislopii latex. The LC(50) of this latex was 1.0mg/l. Concentrations of uric acid, urea and total proteins were determined in the hemolymph of B. glabrata infected with five S. mansoni miracidia and exposed to a sublethal concentration of E. splendens var. hislopii latex for 24h. The exposure to this molluscicide caused total depletion of the alterative sources of energy (total proteins) and significant variation in the nitrogen degradation products. The urea content increased while the uric acid level decreased. These results reflect a disturbance in the snails regulation of their metabolism due to intoxication caused by the latex exposure.

The effect of infection by Echinostoma paraensei on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated after exposure to five or 50 miracidia. The biochemical concentrations were measured weekly until the end of the fourth week after exposure. There was a significant decrease in the concentrations of total proteins in the snails exposed both to five and 50 miracidia, as well as an increase in the nitrogenous products of excretion, ALT and AST activities. The higher ALT activity in the hemolymph of the snails after infection with 50 miracidia suggests highest energetic requirement in these snails in relation to snails exposed to five miracidia. The results also suggest an increase in the use of total proteins, since there was increased formation of nitrogenous catabolites, in conformity with an increase in the aminotransferase activities, frequently associated with tissue damages. This can be explained by damage due to penetration by the miracidia and subsequent development of intramolluscan sporocysts and rediae.

The reproductive activity of Biomphalaria glabrata exposed to Euphorbia splendens var. hislopii latex was evaluated. Parameters related to fecundity and fertility were observed. The snails were exposed to the LD 50 (1 mg/l) of crude latex. At the first week post exposure (p.e.) (Magalhães & Lucca 1971, Kawazoe 1977.The physiological stress factors, as temperature variations (Pimentel-Souza et al. 1990), lightness (Barbosa et al. 1987), starvation (Livingstone & Zwaan 1983), and parasitism by larval trematodes (Looker & Etges 1979, Sullivan et al. 1985, Cooper et al. 1994, Cousin et al. 1995 have been pointed as able to influence the reproductive biology of the snails.There are few studies on the interference of plants that exhibit moluscicide action with the reproductive activity of the snails, intermediate host of helminthes. The reproductive capacity of B. alexandrina is reduced when it was exposed to Allium sativum, A. cepa, and Atriplex halimus (Mantawy 2001, Tanatawy 2002.Many molluscicide have been used to schistosomiasis control, and one of the most promising and widely studied in Brazil, is the crude latex of Euphorbia splendens var. hislopii. The latex obtained from this plant, under laboratory and field conditions, showed that it attends to queries needed to be used as a natural molluscicide (Schall et al. 2001, Vasconcelllos & Amorim 2003.The lethal effects on the embryos in the egg masses were obtained using elevated concentrations of the latex (870 a 1500 mg/l) (Schall et al. 1998), but, until the moment, there were no studies focusing the latex effects on the reproductive biology of B. glabrata.The objective of this study was evaluating the influence of sub lethal dose (LD 50 ) of latex of E. splendens var. hislopii on reproductive parameters of B. glabrata. MATERIALS AND METHODSCollection of E. splendens var. hislopii latex -Samples of E. splendens var. hislopii latex were collected in the autumn from plants cultivated in plots near Departamento de Biologia, Instituto Oswaldo Cruz-Fiocruz, Rio de Janeiro, RJ, Brazil. The latex was collected as described by Vasconcellos and Amorim (2003a), on the same day of tests conduction.Rearing of B. glabrata in the laboratory -Specimens of B. glabrata (Belo Horizonte, BH lineage) were reared at the Laboratório de Esquistossomose Experimental, Instituto Oswaldo Cruz-Fiocruz, RJ, Brazil. The snails were placed in 30 l polyethylene aquaria, with dechlorinated water. The average water temperature was 28 ± 1 o C and relative humidity varied from 70 to 78% throughout the experiment.Three times a week the aquaria were cleaned and the snails were fed ad libitum with lettuce leaves (Lactuca sativa L.). All the specimens of B. glabrata used in the experiments had shell diameters between 8-10 mm and exhibit ovipository activity.Determination of sublethal concentrations of latex of E. splendens var. hislopii -The determination of LD 50 were made according to Vasconcellos and Amorim (2003a), in accordance to WHO recommendations (WHO 1983) and Mott (1987) Reproductive ...

The egg-laying rate, number of egg masses, number of eggs/mass, number of eggs hatched/snail and egg viability of Biomphalaria glabrata exposed to different doses (5 and 50) of Echinostoma paraensei miracidia were analyzed as indicators of reproductive activity. Polystyrene plates were placed in aquariums containing the snails and every other day for four weeks after infection the plates were removed to count the number of egg masses and eggs laid. After this, the plates were numbered individually and placed in new aquariums free of snails and the egg masses were observed daily to determine the hatching rate. On average there was an increase in the parameters evaluated in the infected snails in relation to the controls (uninfected snails), except for egg viability, which was significantly lower in the groups infected with 50 miracidia. These findings indicate that when infected, this host snail is able to increase its reproductive activity, suggesting an ecological strategy to maintain the species.