A New Technique Using Low Volumes: A New Technique to Assess the Molluscicidal Activity Using Low Volumes

Abstract

Schistosomiasis is a disease endemic in several states of Brazil. The population control of the transmitter mollusks is done with Bayluscide WP 70®, in the control programs. OMS preconize molluscicidal assays using Becker with 500 mL of final volume, restringing the number of natural products and synthetic drugs to be tested in function of high quantity of material necessary. A new technique to assess the toxic effects for Biomphalaria sp. is the purpose of this work, for developing adaptation for this aquatic organism, using a low volume of test solution in 24-well plates. We used Biomphalaria glabrata (10-12 mm size) in a static system, consisting of the following components: Becker containing 10 snails or 24-well plates where snails were individualized for a volume of 2 mL per well for 24 and 48 hours. For the assays, we added aqueous solutions of Bayluscide WP 70, at a concentration of 1-5 mg/L, distilled water, and 1% dimethyl sulfoxide. Data were evaluated using Kappa's coefficient, Z factor validation, and comparison study. This technique to assess the toxic effect has proven to be a useful tool to detect lethal and sublethal effects, which could be used as a new evaluation protocol.

Figure 1

Molluscicidal evaluation models with niclosamide on Biomphalaria glabrata under laboratory conditions. (a) Becker of 1000 mL, containing the control groups, water, and 1% aqueous dimethylsulfoxide, 500 mL of aqueous solutions of niclosamide (1 mg/L, 2 mg/L, 3 mg/L, 4 mg/L, and 5 mg/L), and 10 Biomphalaria glabrata, with a size between 10 and 12 mm in diameter by Becker, created and kept in the laboratory. (b) Methodology with 24-well plates, containing the control groups and the doses of niclosamide and 1 snail per well with 2 mL of solution. Exposure of 10 animals per dose as described in (a). (c) Methodology with 24-well plates, containing the control groups and the doses of niclosamide and 1 mollusk per well with 2 mL of solution as described above. Exposure of 3 animals per dose.

Figure 2

Niclosamide induced death of adult B. glabrata snails. (a) Dose-response graph representing niclosamide concentrations (24- or 48-hour stimulus) in the function of death of mollusks measured in the Becker methodology with 10 snails. (b) Dose-response graph representing niclosamide concentrations (24- or 48-hour stimulus) in the function of death of mollusks measured in the 24-well plate methodology with 10 snails. (c) Dose-response graph representing niclosamide concentrations (24- or 48-hour stimulus) in the function of death of mollusks measured in the 24-well plate methodology with 3 snails. The concentrations of the added blockers are given in the text. Experiments were performed for at least three independent days.

Figure 3

Comparison of the methodologies in relation to dose of 2% niclosamide against adult B. glabrata snails. Treatment for 24 hours with 2% niclosamide. These results are representative of three distinct days.

Figure 4

Dose-response effect of crude ethanol extract of stems of Manilkara subsericea against Biomphalaria glabrata. We treated the snails with crescent doses of the extract for 48 hours. These experiments were realized in three distinct days.