Toxic effect of Croton rudolphianus leaf essential oil against Biomphalaria glabrata, Schistosoma mansoni cercariae and Artemia salina.

This research investigated the effect of the Croton rudolphianus leaf essential oil (EO) on Biomphalaria glabrata embryos (at different development stages) and adults, Schistosoma mansoni cercariae, and Artemia salina (non-target organism). It was possible to identify 31 compounds in the C. rudolphianus EO through GC-MS analysis. The major compounds from this oil were (E)-caryophyllene (17.33%), an unknown compound (16.87%), bicyclogermacrene (7.1%), δ-cadinene (6.62%) and germacrene D (5.38%). After incubation for 24 h, the EO of C. rudolphianus induced the occurrence of non-viable embryos (dead and malformed), with an LC50 value of 126.54, 133.51, 143.53 and 161.95 µg/mL and an LC90 value of 202.61, 216.48, 232.98 and 271.16 µg/mL to blastula, gastrula, trochophore and veliger embryonic stages, respectively. The EO was more effective against B. glabrata adults (LC50 and LC90 = 47.89 and 78.86 µg/mL, respectively), and S. mansoni cercariae (LC50 and LC90 = 14.81 and 22.15 after 120 mins of exposure, respectively) than against B. glabrata embryos. Concerning the micronucleus assay, the mean frequency of apoptosis, binucleation and micronucleus were 45.33 ± 3.51, 19.33 ± 1.53 and 0.67 ± 0.58 per 1000 cells at 25 µg/mL, which is the highest concentration tested. The oil killed A. salina with LC50 and LC90 values (68.33 and 111.5 µg/mL, respectively) higher than those determined for adult snails and S. mansoni cercariae. In conclusion, C. rudolphianus EO had a toxic effect against B. glabrata adults and embryos, and S. mansoni cercariae. Furthermore, this oil showed to be cytotoxic to hemocytes of B. glabrata. Concerning the non-target organism assay, C. rudolphianus EO was less toxic to A. salina then to adult snails and S. mansoni cercariae. Due to this, the EO from C. rudolphianus leaves is a potential alternative for schistosomiasis control.

Molluscicidal and parasiticidal activities of Eryngium triquetrum essential oil on Schistosoma mansoni and its intermediate snail host Biomphalaria glabrata, a double impact.

BACKGROUND: Freshwater snails are the intermediate hosts of a large variety of trematode flukes such as Schistosoma mansoni responsible for one of the most important parasitic diseases caused by helminths, affecting 67 million people worldwide. Recently, the WHO Global Vector Control Response 2017-2030 (GVCR) programme reinforced its message for safer molluscicides as part of required strategies to strengthen vector control worldwide. Here, we present the essential oil from Eryngium triquetrum as a powerful product with molluscicide and parasiticide effect against S. mansoni and the snail intermediate host Biomphalaria glabrata. METHODS: In the present study, we describe using several experimental approaches, the chemical composition of E. triquetrum essential oil extract and its biological effects against the snail B. glabrata and its parasite S. mansoni. Vector and the free-swimming larval stages of the parasite were exposed to different oil concentrations to determine the lethal concentration required to produce a mortality of 50% (LC50) and 90% (LC90). In addition, toxic activity of this essential oil was analyzed against embryos of B. glabrata snails by monitoring egg hatching and snail development. Also, short-time exposure to sublethal molluscicide concentrations on S. mansoni miracidia was performed to test a potential effect on parasite infectivity on snails. Mortality of miracidia and cercariae of S. mansoni is complete for 5, 1 and 0.5 ppm of oil extract after 1 and 4 h exposure. RESULTS: The major chemical component found in E. triquetrum oil determined by GC-FID and GC/MS analyses is an aliphatic polyacetylene molecule, the falcarinol with 86.9-93.1% of the total composition. The LC50 and LC90 values for uninfected snails were 0.61 and 1.02 ppm respectively for 24 h exposure. At 0.5 ppm, the essential oil was two times more toxic to parasitized snails with a mortality rate of 88.8 ± 4.8%. Moderate embryonic lethal effects were observed at the concentration of 1 ppm. Severe surface damage in miracidia was observed with a general loss of cilia that probably cause their immobility. Miracidia exposed 30 min to low concentration of plant extract (0.1 ppm) were less infective with 3.3% of prevalence compare to untreated with a prevalence of 44%. CONCLUSIONS: Essential oil extracted from E. triquetrum and falcarinol must be considered as a promising product for the development of new interventions for schistosomiasis control and could proceed to be tested on Phase II according to the WHO requirements.

Molluscicidal activity of polyvinylpyrrolidone (PVP)-functionalized silver nanoparticles to Biomphalaria glabrata: Implications for control of intermediate host snail of Schistosoma mansoni.

Silver nanoparticles (Ag NPs) have been applied in several commercial products due to their antimicrobial properties, while their molluscicide properties, mode of action and toxicity to snail species remain unclear. In this study, the comparative toxicity of polyvinylpyrrolidone (PVP)-functionalized Ag NPs and their dissolved counterpart (Ag ions) was analyzed during the early developmental stages of the freshwater snail Biomphalaria glabrata, intermediate host of Schistosoma mansoni. Ag NPs were synthesized and characterized by multiple techniques, and the snail embryotoxicity was analyzed in terms of mortality, hatching, developmental stages and morphological alterations, while the acute toxicity to newly-hatched snails was analyzed by mortality and behavioral impairments. Results showed that both Ag forms induced mortality, hatching delay and morphological alterations (especially hydropic abnormalities) in snail embryos in a concentration and exposure time dependent patterns. Ag NPs showed low embryotoxic effects and similar toxicity for newly-hatched snails when compared to their dissolved counterparts, indicating that the nanotoxicity was dependent of snail developmental stages. The knowledge about the Ag NP toxicity to different early development stages of B. glabrata contributes to its potential use as molluscicide and control of neglected tropical diseases, including schistosomiasis.

Toxicity evaluation of the active ingredient acetamiprid and a commercial formulation (Assail® 70) on the non-target gastropod Biomphalaria straminea (Mollusca: Planorbidae).

Neonicotinoids emerged as an environmentally safe alternative to previous generations of insecticides becoming one of the most widely applied in modern agriculture. Nevertheless, they have been reported to affect several non-target organisms. Most toxicity studies focus on the effects on pollinators or terrestrial invertebrates and evaluate either the active ingredient or the commercial formulation. In the present study, we aimed to assess the long-term effects of the active ingredient acetamiprid and a broadly used commercial formulation (Assail® 70) on the non-target freshwater gastropod Biomphalaria straminea using a battery of biomarkers. A 14 day-exposure of adult organisms to both active ingredient and commercial formulation increased carboxylesterase activity and glutathione content, inhibited superoxide dismutase activity and decreased reactive oxygen species levels. The commercial formulation additionally increased glutathione S-transferase activity and inhibited catalase activity. The results indicate a greater toxicity of the commercial formulation than that of the active ingredient alone. Cholinesterase activity, development and offspring survival of B. straminea were not impaired. We conclude that the toxicity of acetamiprid on this gastropod species is mainly related to effects on detoxification and oxidative metabolism responses. This study provides novel information about the adverse effects of the active ingredient and a commercial formulation of a widely used neonicotinoid on a non-target aquatic species.

Ocotea pulchellaas an alternative against schistosomiasis: chemical analysis, development of nanoemulsion and biological control activity / Ocotea pulchellacomo una alternativa ante esquistosomiasis: análisis químico, desarrollo de una nanoemulsión y actividad en control biológico

The aim of this work was to evaluate the potential of the essential oil (EO) from Ocotea pulchella leaves as an alternative in the control of schistosomiasis. It was tested O. pulchella EO nanoformulation to assess its activity against adult Biomphalaria glabrata, their spawning and Schistossoma mansoni cercariae. Additionally, the EO chemical composition was investigated by gas-chromatography. Nanoemulsion were elaborated by the low energy method. The adult mollusks, their spawning and cercariae were placed in contact with nanoemulsion to calculate lethal concentrations. Myristicin, bicyclogermacrene and α-Pinene were the main substances in the EO. Nanoemulsion caused mortality of adult B. glabrata, its egg embryos and S. mansoni. These results suggest the use of this nanoemulsion as an alternative in the control of the schistosomiasis cycle.

El objetivo de este trabajo fue evaluar el potencial de los aceites esenciales (AE) de las hojas de Ocotea pulchellacomo una alternativa en el control de esquistosomiasis. Se probó una nanoformulación de AE de O. pulchellapara evaluar su actividad ante adultos de Biomphalaria glabrata, sus huevos y cercarías de Schistossoma mansoni. La nanoemulsión fue elaborada por el método de baja energía. Los moluscos adultos, sus huevos y cercarías se colocaron en contacto con la nanoemulsión para calcular concentraciones letales. Los compuestos mayoritarios en el AE fueron miristicina, biciclogermacreno y α-pineno. La nanoemulsión causó mortalidad en adultos de B. glabrata, sus huevos y a S. mansoni. Los resultados sugieren el uso de esta nanoemulsión como una alternativa en el control del ciclo de esquistosomiasis.

The antioxidants resveratrol and N-acetylcysteine enhance anthelmintic activity of praziquantel and artesunate against Schistosoma mansoni.

BACKGROUND: Treatment of schistosomiasis has relied on the anthelmintic drug praziquantel (PZQ) for more than a generation. Despite its celebrated performance for treatment and control of schistosomiasis and other platyhelminth infections, praziquantel has some shortcomings and the inability of this drug to counteract disease sequelae prompts the need for novel therapeutic strategies. METHODS: Using a host-parasite model involving Biomphalaria glabrata and Schistosoma mansoni we established mechanical transformation of S. mansoni cercariae into newly transformed schistosomula (NTS) and characterized optimal culture conditions. Thereafter, we investigated the antischistosomal activity and ability of the antioxidants N-acetylcysteine (NAC) and resveratrol (RESV) to augment the performance of praziquantel and/or artesunate (AS) against larval stages of the parasite. Drug effects were evaluated by using an automated microscopical system to study live and fixed parasites and by transmission electron microscopy (TEM). RESULTS: Transformation rates of cercariae to schistosomula reached ~ 70% when the manipulation process was optimized. Several culture media were tested, with M199 supplemented with HEPES found to be suitable for S. mansoni NTS. Among the antioxidants studied, RESV alone or combined with anthelminthic drugs achieved better results rather N-acetylcysteine (NAC). TEM observations demonstrated that the combination of AS + RESV induced severe, extensive alterations to the tegument and subtegument of NTS when compared to the constituent compounds alone. Two anthelmintic-antioxidant combinations, praziquantel-resveratrol [combination index (CI) = 0.74] and artesunate-resveratrol (CI = 0.34) displayed moderate and strong synergy, respectively. CONCLUSIONS: The use of viability markers including staining with propidium iodide increased the accuracy of drug screening assays against S. mansoni NTS. The synergies observed might be the consequence of increased action by RESV on targets of AS and PZQ and/or they may act through concomitantly on discrete targets to enhance overall antischistosomal action. Combinations of active agents, preferably with discrete modes of action including activity against developmental stages and/or the potential to ameliorate infection-associated pathology, might be pursued in order to identify novel therapeutic interventions.

Biomphalaria alexandrina: a model organism for assessing the endocrine disrupting effect of 17ß-estradiol.

A wide range of endocrine disruptor compounds are routinely discharged to the ecosystem. Water contaminated with these compounds has a potential effect on the reproductive physiology of aquatic organisms as well as humans. In the present study, we tested the effect of the steroid estrogen, 17ß-estradiol, on Biomphalaria alexandrina, a snail species that is widely distributed in Egypt and that acts as an intermediate host for the human blood fluke, Schistosoma mansoni. The effects of exposure to 0.3 mg/L and 1 mg/L 17ß-estradiol on fecundity (MX) and reproductive rate (R0) of B. alexandrina were recorded. In addition, levels of steroid sex hormones and antioxidants in the hemolymph and ovotestis (OT) of exposed snails were measured. Histopathological changes in the OT of B. alexandrina were also investigated. Exposure to 0.3 mg/L and 1 mg/L 17ß-estradiol caused a significant increase in the number of egg masses per snail after 3 weeks and 1 week of exposure for the two tested concentrations compared with unexposed controls. An increase in the levels of progesterone hormone was recorded in the hemolymph of exposed snails in comparison with unexposed controls. Additionally, levels of the antioxidant enzyme glutathione (GSH) were increased in the hemolymph and OT tissues of snails after 2 and 4 weeks of exposure. Histopathological sections in the OT revealed an increase in the oocyte and a decrease in the sperm densities after 2 weeks and this effect was restored to normal conditions after 4 weeks of exposure to both tested concentrations. The current results indicate that B. alexandrina is sensitive to 17ß-estradiol and can therefore be used as bioindicator and model organism for the assessment of water pollution with endocrine disruptor compounds.

Nanoemulsion containing essential oil from Xylopia ochrantha Mart. produces molluscicidal effects against different species of Biomphalaria (Schistosoma hosts).

BACKGROUND: This work describes a chemical study of the essential oil from leaves of Xylopia ochrantha, an endemic Annonaceae species from Brazil, and its activity against Biomphalaria species. Considering its poor solubility in aqueous medium, the essential oil was nanoemulsified to evaluate its action on controlling some mollusc species of genus Biomphalaria, snail hosts of Schistosoma mansoni that causes schistosomiasis, which mainly affects tropical and subtropical countries. OBJECTIVES: The main aims of this work were to analyse the chemical composition of essential oil from X. ochrantha, and to evaluate the effect of its nanoemulsion on molluscs of genus Biomphalaria and their oviposition. METHODS: Chemical analysis was performed by gas chromatography coupled to mass spectrometry. Nanoemulsions were prepared by a low energy method and characterised by particle size and polydispersity index. Biological assays evaluating the mortality of adult species of B. glabrata, B. straminea and B. tenagophila and their ovipositions upon contact with the most stable nanoemulsion during 24 and 48 h were performed. FINDINGS: Chemical analysis by mass spectrometry revealed the majority presence of bicyclogermacrene and germacrene D in the essential oil. The formulation with a hydrophilic-lipophilic balance (HLB) of 9.26 was the most suitable for the oil delivery system. This nanoemulsion caused the mortality in B. tenagophila, B. straminea and B. glabarata of different sizes at levels ranging from 50 to 100% in 48 h. Additionally, the formulation could inhibit the development of deposited eggs. CONCLUSION: Thus, these results suggest the use of nanoemulsified essential oil from X. ochrantha as a possible alternative in controlling some Biomphalaria species involved in the schistosomiasis cycle.

Development of a natural molluscicide prototype kit (MoluSchall) for the control of schistosomiasis mansoni transmission

Abstract INTRODUCTION In Brazil, Biomphalaria glabrata, B. tenagophila, and B. straminea are intermediate hosts of Schistosoma mansoni, the etiological agent of schistosomiasis mansoni. Molluscicide use is recommended by the WHO for controlling the transmission of this parasite. Euphorbia milii latex has shown promising results as an alternative molluscicide. Thus, a natural molluscicide prototype kit based on freeze-dried E. milii latex was developed and evaluated against Biomphalaria spp. METHODS E. milii latex was collected, processed, and lyophilized. Two diluents were defined for freeze-dried latex rehydration, and a prototype kit, called MoluSchall, was produced. A stability test was conducted using prototype kits stored at different temperatures, and a toxicity assay was performed using Danio rerio. Additionally, MoluSchall was tested against B. glabrata under semi-natural conditions according to defined conditions in the laboratory. RESULTS MoluSchall was lethal to three Brazilian snail species while exhibiting low toxicity to D. rerio. Regardless of storage temperature, MoluSchall was stable for 24 months and was effective against B. glabrata under semi-natural conditions, with the same LD100 as observed under laboratory conditions. CONCLUSIONS MoluSchall is a natural, effective, and inexpensive molluscicide with lower environmental toxicity than existing molluscicides. Its production offers a possible alternative strategy for controlling S. mansoni transmission.

Biological screening of extracts from leaf and stem bark of Croton floribundus Spreng. (Euphorbiaceae) / Triagem biológica de extratos das folhas e caules de Croton floribundus Spreng. (Euphorbiaceae)

Abstract This work describes the preliminary evaluation of cytotoxic, antimicrobial, molluscicidal, antioxidant and anticholinesterase activities from leaf (LECF) and stem bark alcoholic extracts (BECF) of the species Croton floribundus Spreng. (Euphorbiaceae), popularly known as capixingui or tapixingui. BECF presented significant toxicity (LC50 = 89.6 μg/ml) in the Artemia salina Leach, 1819 (Crustacea: Branchiopoda) bioassay, whereas LECF did not show activity (LC50 > 1000 μg/ml). From DPPH method, the values of IC50 for the LECF and BECF were 61.2 μg/ml and 62.2 μg/ml, respectively, showing that C. floribundus has an expressive antioxidant activity. Antimicrobial susceptibility was evaluated by microdilution technique and only BECF was active against Staphylococcus aureus (MIC = 39.6 μg/ml). The extracts did not present molluscicidal activity against snail Biomphalaria glabrata Say, 1818 (Gastropoda: Planorbidae). Both extracts revealed the presence of several components with an inhibiting capacity of acetylcholinesterase enzyme on the bioautographic assay. C. floribundus showed to be a promising species considering that it exhibited good biological activity in the most assays performed.

Resumo Este trabalho descreve a avaliação preliminar das atividades citotóxica, antimicrobiana, moluscicida, antioxidante e anticolinesterásica de extratos alcoólicos das folhas (LECF) e das cascas do caule (BECF) da espécie Croton floribundus Spreng. (Euphorbiaceae), popularmente conhecida como capixingui ou tapixingui. No bioensaio com Artemia salina Leach, 1819 (Crustacea: Branchiopoda), BECF apresentou toxicidade significante (LC50 = 89,6 µg/ml), enquanto que LECF não apresentou atividade (LC50 > 1000 µg/ml). A partir do método de DPPH, os valores de IC50 para o LECF e BECF foram 61,2 µg/ml e 62,2 µg/ml, respectivamente, evidenciando que C. floribundus tem uma atividade antioxidante expressiva. A susceptibilidade antimicrobiana foi avaliada pela técnica de microdiluição e apenas BECF foi ativo contra Staphylococcus aureus (MIC = 39,6 mg/ml). Os extratos não apresentaram atividade moluscicida contra o caramujo Biomphalaria glabrata Say, 1818 (Gastropoda: Planorbidae). Ambos os extratos revelaram a presença de componentes com capacidade inibidora da enzima acetilcolinesterase no ensaio bioautográfico. C. floribundus mostrou ser uma espécie promissora considerando que exibiu boa atividade biológica na maioria dos ensaios testados.

The potential effects of silver and gold nanoparticles as molluscicides and cercaricides on Schistosoma mansoni.

Schistosomiasis seriously affects human health in tropical regions. Its prevention is more important than treatment, raising the need for effective control methods. Recently, the role of nanomaterials in medical science has been growing. The present study aimed to evaluate the potential effects of silver (Ag) and gold (Au) nanoparticles (NPs) on Biomphalaria alexandrina snails and Schistosoma mansoni cercariae in vitro and to assess their effects on the infectivity of cercariae in vivo. The in vitro study proved that Ag and Au NPs were effective in killing B. alexandrina snails, with 30 µg/ml Ag and 160 µg/ml Au causing 100% mortality. The LC50 of 9.68 µg/ml for Ag NPs and 133.7 µg/ml for Au NPs prevented snail infection with S. mansoni miracidia. Furthermore, Ag NPs at 50 µg/ml and Au NPs at 100 µg/ml increased the mortality of S. mansoni cercariae in a dose- and time-dependent manner, reaching 100% mortality after 1 h. The in vivo study found that Ag NPs prevented the occurrence of infection when cercariae were treated before the infection by either the tail immersion (TI) or subcutaneous (SC) route, as proven by parasitological parameters and by the absence of granuloma formation in hepatic tissue. Meanwhile, infection of mice by untreated cercariae followed by treatment with NPs 1 h post-infection (PI) caused a decrease in egg count/g intestine and egg count/g liver in the TI-infected group only. The oogram patterns and granuloma formation results were similar between infection control and the SC-infected group. On the other hand, Au NPs led to a decrease in total worm burden (TWB) in all tested groups, with a decrease in egg count/g intestine and egg count/g liver in TI-infected groups with either pre-treated or post-treated cercariae, in contrast to SC-infected groups. However, the oogram patterns and granuloma formation showed similar results to infection control. Ag and Au NPs have potential as molluscicides and cercaricides in vitro and can prevent or modulate the infectivity of cercariae in vivo.

Molluscicidal impacts of Anagallis arvensis aqueous extract on biological, hormonal, histological and molecular aspects of Biomphalaria alexandrina snails.

Controlling of Biomphalaria alexandrina snails by plant molluscicides is the cornerstone in treating schistosomiasis in Egypt. The objective of this study is, to evaluate the molluscicidal activity of the aqueous leaves extract of Anagallis arvensis against B. alexandrina snails. The present results showed that this aqueous extract was lethal for B. alexandrina snails at (LC50 37.9 mg/l; LC90 48.3 mg/l), and caused reduction in survival; reproductive rates and hormonal activity (testosterone (T) and 17ß-estradiol (E)) of these snails. Histopathological changes occurred in the hermaphrodite glands of snails exposed to the sub lethal concentrations of this aqueous extract are detected, where, there were degeneration in both eggs and sperms and there were losses of connective tissues between acini. The present investigation revealed that this plant had a genotoxic effect especially with its concentration (LC10 and LC25), where, the length of olive tail moment was significantly increased than control group. These observations prove the potent molluscicidal activity of aqueous leaves extract of A. arvensis against the intermediate hosts of Schistosoma mansoni and provide natural biodegradable resources for snails' molluscicidal agents.

Changes in energetic metabolism of Biomphalaria glabrata (Mollusca, Planorbidae) in response to exogenous calcium / Alterações no metabolismo energético de Biomphalaria glabrata em resposta ao cálcio exógeno

Abstract Calcium is considered an essential element for the metabolism of aquatic snail Biomphalaria glabrata (Say, 1818), intermediate host of Schistosoma mansoni Sambon, 1907 in Brazil, and represents a limiting factor to its distribution and adaptation to the environment. This study investigated the effect of different concentrations of exogenous CaCO3 on the energetic metabolism of B. glabrata for better understanding the physiological interference of chemical elements dissolved in the environment with the physiology of this species. Sixty-day-old snails were distributed into six groups, five exposed to different concentrations of CaCO3 (20, 40, 60, 80 and 100 mg/L) and a control group. The exposure to CaCO3 was assessed over time, with analysis of 15 snails of each group in the following intervals: 1, 14, 21 or 30 days for hemolymph extraction. Concentrations of calcium and glucose in the hemolymph were determined by commercial kits, and organic acids were extracted using an ion exchange column and analyzed by high-performance liquid chromatography. Concentration of calcium in the hemolymph showed no significant difference (p>0.05) from the control group and between the concentrations tested. Concentration of glucose decreased (p<0.05) in the treatments of exposure to 20 and 40 mg/L and increased when exposed to 80 and 100 mg/L CaCO3 compared to control and to other concentrations tested over 30 days. The organic acids pyruvate, oxaloacetate, citrate, succinate, fumarate, beta-hydroxybutyrate and lactate presented increased concentrations, while propionate and acetoacetate, decreased concentrations, when exposed to CaCO3 compared to control. Considering the influence of different periods of exposure to CaCO3, on the 14th day, there were stronger alterations in the metabolism of B. glabrata. In conclusion, exposure to CaCO3 reduced the concentration of glucose, which is metabolized into pyruvate, the final product of glycolysis, and also influenced the energetic metabolism pathways, indicating an aerobic or partially anaerobic functioning.

Resumo O cálcio é considerado um elemento essencial no metabolismo do molusco aquático Biomphalaria glabrata (Say, 1818), principal hospedeiro intermediário de Schistosoma mansoni Sambon, 1907 no Brasil e, tem sido descrito como um fator limitante na distribuição e adaptação desse molusco no ambiente. O presente trabalho avaliou o efeito de diferentes concentrações de carbonato de cálcio (CaCO3) exógeno ao metabolismo energético de B. glabrata, a fim de subsidiar uma melhor compreensão da interferência de elementos químicos dissolvidos no meio aquático na fisiologia destes moluscos. Foram utilizados moluscos com sessenta dias de vida, distribuídos em seis grupos, cinco expostos a diferentes concentrações de CaCO3 (20, 40, 60, 80 e 100mg/L) e um controle. A exposição ao CaCO3 foi avaliada em função do tempo, sendo retirados 15 moluscos de cada grupo nos seguintes intervalos: 1, 14, 21 ou 30 dias para extração da hemolinfa. As concentrações de cálcio e glicose na hemolinfa foram determinadas usando-se kits comercial e os ácidos orgânicos foram extraídos por meio da coluna de troca iônica e analisados através cromatografia líquida de alta eficiência. Os resultados demonstraram que a concentração de cálcio na hemolinfa não apresentou diferença significativa (p>0,05) em relação ao controle e nas concentrações testadas. A concentração de glicose diminuiu (p<0,05) nas exposições a 20 mg e 40 mg/L e aumentou nas exposições a 80 mg e 100 mg/L de CaCO3 em relação ao controle e demais concentrações testadas ao longo de 30 dias. Os ácidos orgânicos piruvato, oxaloaceato, citrato, succinato, fumarato, β-hidroxibutirato e lactato tiveram suas concentrações aumentadas, enqunato, propionato e acetoacetato tiveram suas concentrações diminuídas na exposição ao CaCO3 comparada ao controle. Quanto a influência dos diferentes períodos de exposição ao CaCO3, aos 14 dias, as alterações no metabolismo de B. glabrata foram mais expressivas. Conclui-se que as exposições ao CaCO3 influenciaram na redução de glicose, sendo esta metabolizada a piruvato, produto final da glicólise e alteraram as vias de metabolismo energético, indicando um funcionamento aeróbio ou parcialmente anaeróbio.

HSP70 expression in Biomphalaria glabrata snails exposed to cadmium.

In this study, the effects of the heavy metal cadmium on the stress protein HSP70 are investigated in freshwater mollusks Biomphalaria glabrata. Adult snails were exposed for 96h to CdCl2 at concentrations ranging from 0.09 to 0.7mgL-1 (LC50/96h=0.34 (0.30-0.37). Time and concentration-dependent increases in the expression of HSP70 were observed at sub-lethal levels in the immunoblotting assay. Further, an increased survival to a lethal heat shock was observed in animals pre-exposed to a nonlethal concentration of cadmium, evidencing the induction of acquired tolerance. The present study demonstrated the inducibility of B. glabrata HSP70 by cadmium, a relevant environmental contaminant, at non-lethal levels, providing evidences that the assessment of HSP70 in B. glabrata can be regarded as a suitable biomarker for ecotoxicological studies.

Alterations in the fatty acid profile, antioxidant enzymes and protein pattern of Biomphalaria alexandrina snails exposed to the pesticides diazinon and profenfos.

The use of pesticides is widespread in agricultural activities. These pesticides may contaminate the irrigation and drainage systems during agriculture activities and pests' control and then negatively affect the biotic and a biotic component of the polluted water courses. The present study aimed to evaluate the effect of the pesticides diazinon and profenfos on some biological activities of Biomphalaria alexandrina snails such as fatty acid profile, some antioxidant enzymes (thioredoxin reductase (TrxR), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT) as well as glutathione reductase (GR) and lipid peroxidation (LP)) and protein patterns in snails' tissues exposed for 4 weeks to LC10 of diazinon and profenfos. The results showed that the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Identification of fatty acid composition in snail tissues treated with diazinon and profenfos pesticides was carried out using gas-liquid chromatography (GLC). The results declared alteration in fatty acid profile, fluctuation in percentage of long chain and short chain fatty acid contributions either saturated or unsaturated ones, and a decrease in total lipid content in tissues of snails treated with these pesticides. The data demonstrate that there was a significant inhibition in the activities of tissues SOD, CAT, glutathione reductase (GR), TrxR, and SDH in tissues of treated snails, while a significant elevation was detected in LP as compared to the normal control. On the other hand, the electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands due to the treatment of snails. It was concluded that the residues of diazinon and profenfos pesticides in aquatic environments have toxic effects onB. alexandrina snails.

The endocrine disruptor effect of the herbicides atrazine and glyphosate on Biomphalaria alexandrina snails.

Atrazine (AZ) and glyphosate (GL) are herbicides that are widely applied to cereal crops in Egypt. The present study was designed to investigate the response of the snailBiomphalaria alexandrina(Mollusca: Gastropoda) as a bioindicator for endocrine disrupters in terms of steroid levels (testosterone (T) and 17ß-estradiol (E)), alteration of microsomal CYP4501B1-like immunoreactivity, total protein (TP) level, and gonadal structure after exposure to sublethal concentrations of AZ or GL for 3 weeks. In order to study the ability of the snails' recuperation, the exposed snails were subjected to a recovery period for 2 weeks. The results showed that the level of T, E, and TP contents were significantly decreased (p ≤ 0.05) in both AZ- and GL-exposed groups compared with control (unexposed) group. The level of microsomal CYP4501B1-like immunoreactivity increased significantly (p ≤ 0.05) in GL- and AZ-exposed snails and reach nearly a 50% increase in AZ-exposed group. Histological investigation of the ovotestis showed that AZ and GL caused degenerative changes including azoospermia and oocytes deformation. Interestingly, all the recovered groups did not return back to their normal state. It can be concluded that both herbicides are endocrine disrupters and cause cellular toxicity indicated by the decrease of protein content and the increase in CYP4501B1-like immunoreactivity. This toxicity is irreversible and the snail is not able to recover its normal state. The fluctuation of CYP4501B1 suggests that this vertebrate-like enzyme may be functional also in the snail and may be used as a biomarker for insecticide toxicity.

Altered Gene Expression in the Schistosome-Transmitting Snail Biomphalaria glabrata following Exposure to Niclosamide, the Active Ingredient in the Widely Used Molluscicide Bayluscide.

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.

EVALUATION OF THE MOLLUSCICIDAL POTENTIAL OF HYDROALCOHOLIC EXTRACTS OF Jatropha gossypiifolia Linnaeus, 1753 ON Biomphalaria glabrata (Say, 1818) / Avaliação do potencial moluscicida de extratos hidroalcoólicos do caule, folhas e frutos de Jatropha gossypiifolia Linnaeus, 1753 em Biomphalaria glabrata (Say, 1818)

The action of extracts from the stem, leaves, and fruit of Jatropha gossypiifolia on Biomphalaria glabrata was studied by analyzing survival, feeding capacity and oviposition ability. The extracts were obtained by macerating the plant parts in 92% ethanol, which were then evaporated until a dry residue was obtained and phytochemically studied. The molluscicidal activity on B. glabrata was investigated using the procedures recommended by WHO (1965). The amount of food ingested and oviposition were measured during each experiment. The extract of leaves from J. gossypiifolia was shown to be a strong molluscicidal agent, causing 100% mortality of B. glabrata, even in the lowest concentration tested, of 25 ppm. Regarding the fruit extract, there was variation in the mortality, depending on the concentration used (100, 75, 50 and 25 ppm). The snails that were in contact with the fruit extract had significant reduction in feeding and number of embryos in comparison to the control. The stem extract did not present molluscicidal activity nor had any influence on the feeding and oviposition abilities of B. glabrata, in the concentrations tested. In conclusion, the extracts of leaves and fruits of J. gossypiifolia investigated in this work show molluscicidal effect and may be sources of useful compounds for the schistosomiasis control.

Estudou-se a ação dos extratos do caule, folhas e frutos de Jatropha gossypiifolia (Pinhão-roxo) sobre Biomphalaria glabrata analisando a sobrevivência, capacidade alimentar e de oviposição. Os extratos foram obtidos pela maceração das partes do vegetal em álcool etílico 92%, evaporados até obter-se um resíduo seco e estudados fitoquimicamente. A atividade moluscicida em B. glabrata seguiu os procedimentos recomendados pela WHO (1965). A medida de quantidade de alimento ingerido e a oviposição foi realizada durante cada experimento. O extrato das folhas de J. gossypiifolia mostrou-se um forte agente moluscicida, promovendo 100% de mortalidade de B. glabrata mesmo na menor concentração testada, 25 ppm. Com o extrato dos frutos houve variação na mortalidade, de acordo com a concentração utilizada (100, 75, 50 e 25 ppm). Os caramujos em contato com o extrato dos frutos apresentaram quando comparado ao controle uma diminuição significativa nos comportamentos alimentares e de número de embriões. O extrato do caule não apresentou atividade moluscicida e nenhuma influência sobre a capacidade alimentar e de oviposição de B. glabrata nas concentrações testadas. Em conclusão, os extratos de folhas e frutos de J. gossypiifolia investigados neste trabalho apresentam efeito moluscicida e possivelmente podem ser fontes de compostos no controle da esquistossomose.

Usnic acid potassium salt: an alternative for the control of Biomphalaria glabrata (Say, 1818).

In Brazil, the snail Biomphalaria glabrata is the most important vector of schistosomiasis due to its wide geographical distribution, high infection rate and efficient disease transmission. Among the methods of schistosomiasis control, the World Health Organization recommends the use of synthetic molluscicides, such as niclosamide. However, different substances of natural origin have been tested as alternatives for the control or eradication of mollusks. The literature describes the antitumor, antimicrobial and antiviral properties of usnic acid as well as other important activities of common interest between medicine and the environment. However, usnic acid has a low degree of water solubility, which can be a limiting factor for its use, especially in aquatic environments, since the organic solvents commonly used to solubilize this substance can have toxic effects on aquatic biota. Thus, the aim of the present study was to test the potassium salt of usnic acid (potassium usnate) with regard to molluscicidal activity and toxicity to brine shrimp (Artemia salina). To obtain potassium usnate, usnic acid was extracted with diethyl ether isolated and purified from the lichen Cladonia substellata. Biological assays were performed with embryos and adult snails of B. glabrata exposed for 24 h to the usnate solution solubilized in dechlorinated water at 2.5; 5 and 10 µg/ml for embryos, 0.5; 0.9; 1;5 and 10 µg/ml for mollusks and 0.5; 1; 5; 10 µg/ml for A. salina. The lowest lethal concentration for the embryos and adult snails was 10 and 1 µg/ml, respectively. No toxicity to A. salina was found. The results show that modified usnic acid has increased solubility (100%) without losing its biological activity and may be a viable alternative for the control of B. glabrata.