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Background: Schistosomiasis is one of the world's most common NTDs. Successful control operations often target snail vectors with the molluscicide niclosamide. Little is known about how niclosamide affects snails, including for Biomphalaria pfeifferi, the most important vector for Schistosoma mansoni in Africa. We used Illumina technology to explore how field-derived B. pfeifferi, either uninfected or harboring cercariae-producing S. mansoni sporocysts, respond to a sublethal treatment of niclosamide. This study afforded the opportunity to determine if snails respond differently to biotic or abiotic stressors, and if they reserve unique responses for when presented with both stressors in combination. We also examined how sporocysts respond when their snail host is treated with niclosamide., Principal Findings: Cercariae-producing sporocysts within snails treated with niclosamide express ~68% of the genes in the S. mansoni genome, as compared to 66% expressed by intramolluscan stages of S. mansoni in snails not treated with niclosamide. Niclosamide does not disable sporocysts nor does it seem to provoke from them distinctive responses associated with detoxifying a xenobiotic. For uninfected B. pfeifferi, niclosamide treatment alone increases expression of several features not up-regulated in infected snails including particular cytochrome p450s and heat shock proteins, glutathione-S-transferases, antimicrobial factors like LBP/BPI and protease inhibitors, and also provokes strong down regulation of proteases. Exposure of infected snails to niclosamide resulted in numerous up-regulated responses associated with apoptosis along with down-regulated ribosomal and defense functions, indicative of a distinctive, compromised state not achieved with either stimulus alone., Conclusions/significance: This study helps define the transcriptomic responses of an important and under-studied schistosome vector to S. mansoni sporocysts, to niclosamide, and to both in combination. It suggests the response of S. mansoni sporocysts to niclosamide is minimal and not reflective of a distinct repertoire of genes to handle xenobiotics while in the snail host. It also offers new insights for how niclosamide affects snails., Competing Interests: The authors have declared that no competing interests exist.

In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are naturally infected by the trematode Schistosoma mansoni, the causative agent of schistosomiasis. Despite decades of governmental efforts through official control programs, schistosomiasis remains an important public health problem in the country: thousands of people are infected with the trematode each year and millions live in endemic areas. The World Health Organization recommends using a combination of molluscicide (niclosamide) and mass chemotherapy to control the transmission of schistosomiasis, with this treatment successfully reducing the morbidity of the disease. In the past, niclosamide has been used in official schistosomiasis control programs in Brazil. However, as B. glabrata recolonizes even after molluscicide application, the use of molluscicides has gradually decreased in the country until they were discontinued in 2002, mainly due to the rising global pressure to preserve the environment and the difficulties of obtaining licenses from the Brazilian Ministry of Environment to use toxic substances in aquatic ecosystems. Therefore, the discovery of new molluscicides, which could be more selective to Biomphalaria species and less harmful to the aquatic ecosystem, is necessary. In addition, political efforts to sensitize funders to provide grants for this field of research are required. In this context, this article aims to make a critical analysis of molluscicide application in schistosomiasis control programs in Brazil.

In view of the call by the World Health Organization (WHO) for elimination of schistosomiasis as a public health problem by 2025, use of molluscicides in snail control to supplement chemotherapy-based control efforts is likely to increase in the coming years. The mechanisms of action of niclosamide, the active ingredient in the most widely used molluscicides, remain largely unknown. A better understanding of its toxicology at the molecular level will both improve our knowledge of snail biology and may offer valuable insights into the development of better chemical control methods for snails. We used a recently developed Biomphalaria glabrata oligonucleotide microarray (31K features) to investigate the effect of sublethal exposure to niclosamide on the transcriptional responses of the snail B. glabrata relative to untreated snails. Most of the genes highly upregulated following exposure of snails to niclosamide are involved in biotransformation of xenobiotics, including genes encoding cytochrome P450s (CYP), glutathione S-transferases (GST), and drug transporters, notably multi-drug resistance protein (efflux transporter) and solute linked carrier (influx transporter). Niclosamide also induced stress responses. Specifically, six heat shock protein (HSP) genes from three super-families (HSP20, HSP40 and HSP70) were upregulated. Genes encoding ADP-ribosylation factor (ARF), cAMP response element-binding protein (CREB) and coatomer, all of which are involved in vesicle trafficking in the Golgi of mammalian cells, were also upregulated. Lastly, a hemoglobin gene was downregulated, suggesting niclosamide may affect oxygen transport. Our results show that snails mount substantial responses to sublethal concentrations of niclosamide, at least some of which appear to be protective. The topic of how niclosamide's lethality at higher concentrations is determined requires further study. Given that niclosamide has also been used as an anthelmintic drug for decades and has been found to have activity against several types of cancer, our findings may be of relevance in understanding how both parasites and neoplastic cells respond to this compound.

As a new suspension concentrate of niclosamide (SCN) was more effective in controlling Oncomelania snails than a wettable powder of the same drug, it was tested against Biomphalaria glabrata. There were no differences in the effect of the suspension concentrate, the wettable powder of niclosamide and ball-milled pure niclosamide against the adult snails, but at after 48h 0.125mg/l SCN killed 100% of eggs compared with 84.3% for WPN and 17.7% for pure niclosamide. Because of the improved handling characteristics of SCN over the other formulations, further field tests on Biompharia and Bulinus species are warranted., (Crown Copyright 2009. Published by Elsevier Ltd. All rights reserved.)

Schistosomiasis is one of the most important public health problems in many developing countries. The present study was conducted to investigate the effect of the polymeric niclosamide formulations against Biomphalaria alexandrina snails, the intermediate host of Schistosoma mansoni in Egypt. Three new polymeric formulations were prepared for the molluscicide niclosamide. The formulations were prepared either by the chemical modifications of poly(glycidyl methacrylate) or by physical entrapment of the niclosamide in calcium alginate beads. The release of the niclosamide from the polymeric formulations was investigated. The activity of the prepared formulations against Biomphalaria alexandrina was investigated. The results obtained revealed higher potency for polymerized niclosamide B3 than B1; the lowest potency was revealed for B2. After an exposure period of 24 hours, LC(50) values were 0.073, 0.098 and 1.09 ppm for B3, B1 and B2, respectively. In addition, the molluscicidal potency of the test polymeric niclosamide was age-dependent, where old snails were more tolerant to the test solutions than young and newly hatched snails. The results also indicated that the molluscicidal activity of B3 was extended for 21 days and 17 days for B1, compared with 5 days for free niclosamide. However, the molluscicidal potency of the polymerized niclosamide was increased after boiling for one hour, and was increased with increasing the pH of the medium to pH 9. In addition, their potency was increased with decreasing the water hardness concentrations (CaCO(3)).Molluscicidal activity of free niclosamide and its polymeric formulations vs. exposure time.

The aim of this study was to determine the toxicity of niclosamide (Bayluscide (R)) on Melanoides tuberculata and Biomphalaria glabrata under laboratory conditions. The latter species is the intermediate host of Schistosoma mansoni (Sambon 1917). M. tuberculata was successfully used as competitor of B. glabrata in biological control programs in French West Indies. Both molluscicide and biological control using M. tuberculata have proved to be successful in reducing the population density of B. glabrata. The associated use of molluscicide in this area would be an effective measure if M. tuberculata were less susceptibility to the molluscicide than B. glabrata. Three hundreds individuals each of B. glabrata and of M. tuberculata, collected in Sumidouro, State of Rio de Janeiro, were used in the experiment. The molluscs were exposed to 14 different concentrations of niclosamide as recommended by the World Health Organization. Probit analysis was used to determine the LC 50 and LC 90. The LC 50 and LC 90 values for B. glabrata were 0.077 mg/l and 0.175 mg/l, respectively and the LC 50 and LC 90 values for M. tuberculata were 0.082 mg/l and 0.221 mg/l respectively. As the lethal concentrations of niclosamide were approximately the same to both species, this could be a disadvantage when controlling B. glabrata with niclosamide in an area of M. tuberculata occurrence. It might therefore be preferable to utilize the latex extracted from the Euphorbia splendens, which presented a much higher efficiency for B. glabrata than to M. tuberculata.

The toxicity of Euphorbia milii molluscicidal latex and niclosamide (NCL) to target snails (Biomphalaria glabrata and Biomphalaria tenagophila) and nontarget aquatic organisms is evaluated. Planorbidae snails were killed by very low concentrations of lyophilized latex (48-h LC(50), mg/L: B. glabrata, 0.12; B. tenagophila, 0.09; Helisoma duryi, 0.10). Latex was less toxic (48-h LC(50) or EC(50), mg/L) to oligochaeta (Tubifex tubifex, 0.31), planktonic crustacea (Daphnia similis, 0.38; C. dubia, 1.07; Artemia sp., 0.93), and fishes (Danio rerio, 0.96; Poecilia reticulata, 1. 39), and considerably less toxic to Ampullariidae snails (Pomacea sp. , 10.55) and frog tadpoles (Rana catesbeiana, 7.50). Latex (up to 100 mg/L) was not toxic to bacteria (P. putida and V. fischeri), algae (Selenastrum capricornutum and Chlorella vulgaris), and mosquito larvae (Anopheles albitarsis, Aedes aegypti, Aedes fluviatilis). NCL was very toxic (48-h LC(50) or EC(50), mg/L) to Planorbidae snails (B. glabrata, 0.15, B. tenagophila, 0.13; H. duryi, 0.10), T. tubifex (0.11), crustacea (D. similis, 0.19; Ceriodaphnia dubia, 0.47; Artemia sp. 0.18), fishes (D. rerio, 0.25; P. reticulata, 0.29), R. catesbeiana (0.16), and Pomacea sp. (0.76). NCL was toxic to bacteria, algae (96-h IC(50), mg/L: S. capricornutum, 0.34; C. vulgaris, 1.23) and slightly toxic to mosquito larvae. In conclusion, E. milii latex, as compared with the reference molluscicide niclosamide, presents a higher degree of selectivity toward snails which are intermediate hosts of Schistosoma trematodes., (Copyright 2000 Academic Press.)

The toxic and behavioural effects of niclosamide (Bayluscide WP 70) on Biomphalaria straminea from a highly endemic area of schistosomiasis in northeastern Brazil were investigated through laboratory bioassays. The LD50 and LD90 were 0.114 mg/l and 0.212 mg/l, respectively. Water-leaving behaviour occurred among 14% to 30% of the snails in the presence of sublethal doses of niclosamide and among 16% of the controls. It was concluded that both the relatively low susceptibility to niclosamide and water-leaving behaviour of local B. straminea may be responsible for the recolonization of transmission foci after mollusciciding. It was suggested that recently improved measures of snail control, such as controlled-release formulations of niclosamide and plant molluscicides should be considered in areas where snail control is recommended.

Laboratory studies were conducted to assess the conditions under which the use of malathion in ricelands of Cameroon may impact the transmission of schistosomiasis. Helisoma trivolvis and Biomphalaria havanensis were selected as test organisms due to the lack of intermediate snail hosts in the U.S. Using Bayluscide as a reference molluscicidal compound, malathion was tested against snail eggs, juveniles, and adults. Snail eggs were more susceptible to Bayluscide and malathion than juvenile snails which in turn were more susceptible than adult snails. A Bayluscide concentration of 0.200 mg/L caused 100% mortality to adults of both snail species after 24 h exposure. This relatively high toxicity of Bayluscide to freshwater snails is one of the reasons why it has been recommended by the World Health Organization as the molluscicide of choice for control of schistosome-bearing snails. The concentrations of malathion resulting in 100% kill of adult snails after 24 h exposure were 1,200 mg/L for H. trivolvis and 500 mg/L for B. havanensis. After 48 h exposure, these concentrations were reduced to 500 mg/L and 300 mg/L, respectively. Therefore it is expected that the use of malathion for insect control in ricelands of Cameroon may affect the survival of freshwater snails including the intermediate hosts of bilharziasis.

Experiments were undertaken both in the laboratory and in the field between 1980-1984 to evaluate the causes of repopulation of breeding places of Biomphalaria glabrata following treatment with Niclosamide. Laboratory bioassays showed that the susceptibility to emulsifiable Niclosamide of B. glabrata collected monthly from an irrigation ditch system varied during the year. Lethal concentrations (LC90) ranged between 0.15 mg/l-1 and 0.60 mg/l-1. Statistically significant differences (alpha = 0.01) were evident between the months of May/82 and January/83 and December/82 and January/83, and were related to snail nutrition. In the field two types of foci of B. glabrata were treated with 10 ppm of Niclosamide. The first one consisted of a reservoir of 12000 1 of water in which 14.5% of snails were infected with Schistosoma mansoni. One application of molluscicide followed by cleaning of the reservoir eliminated all the snails. The second one consisted of an irrigation system in which 5.6% of the snails were infected with S. mansoni. One application of molluscicide without cleaning the ditches reduced the density of snails by 98%. The causes of the survival of 2.0% of the snails in the ditches are discussed in relation to the substratum of the breeding places and the treatment technique.

Treatment of 3-day-old Biomphalaria glabrata eggs with aridanin caused a knock-down effect on the prehatch snails, making this group the most susceptible. The development and hatching of 0- to 1-day-old eggs could not be prevented but was prolonged by continuous exposure to aridanin (0.5-10 mg/l). Prehatch snails were less susceptible than juvenile and adult snails. Niclosamide (0.625-0.35 mg/l) arrested the development of B. glabrata. The results predict a poor action of aridanin as an ovicidal agent in the control of snail intermediate hosts of schistosomiasis.

Successive generations of the PR-79 and M-line laboratory stocks of Biomphalaria glabrata were exposed in the LC90 of niclosamide or copper sulfate. Survivors from each generation produced the succeeding generation by self fertilization. The PR-79 stock showed no evidence of tolerance to either molluscicide following five generations of selection. On the other hand, M-line F5 snails demonstrated approximately two-fold higher tolerance to both molluscicides than did non-selected M-line snails.

Tests were conducted with gelatin granules containing Bayluscide (niclosamide) w.p., 23-3% w/w a.i. Granules rendered a simulated field habitat (banana drain) lethal to Biomphalaria glabrata for 40 days when applied at a rate calculated to give 5 mg/1 in the water. At this dose, Bayluscide e.c. was effective for 8-12 days. Granules exposed to various combinations of natural or simulated tropical climatic factors for up to 56 days gave variable kills of snails, apparently unrelated to the factors tested. The rate at which the molluscicide was released from the granules was affected by climatic factors: high temperatures and low humidities slowed the rate, probably by hardening the granules; rainfall and high humidities accelerated the rate, probably by softening or disintegrating the granules. There was no apparent loss of molluscicidal activity in granules exposed for 56 days to various combinations of climatic factors. The results suggest that this formulation may be applied during droughts to temporary habitats when these are dry and accessible. The molluscicide would then be released at the unpredictable start of the next wet season and kill any snails which survive the drought by aestivation.