TITLE: PROTOCOL FOR LARVICIDE BIOASSAYS
AUTHORS: Dr. Wanderli Pedro Tadei, Rochelly da Silva Mesquita

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed for the evaluation of the larvicidal activity of perspective insecticides on Anopheles larvae. The tests are performed according to the World Health Organization methodology (WHO, 2005).</div></div>

[STEPS]
?. Preparation of the stock solutionsDissolve accurately weighed quantity of the test substance in an appropriate volume of dimethylsulfoxide (DMSO) and/or distilled water (depends on the sample solubility) to obtain the concentration of .Final volume suggestion:   to , depending on the amount of the material available for the assay.Negative Control: Prepare  aqueous solution DMSO (the solution concentration should not exceed ).The solutions should be stored in a refrigerator in dark glass bottles, preferably wrapped in aluminium foil to prevent material degradation.
[(pg/mL)]
25 ml
50 ml
?. Selective BioassaysPurpose of Selective Bioassays: To screen the toxicity of substances exposed to larvae at different reading intervals (24, 48 and 72 h). Those substances showing mortality above 50 % will be considered active.Concentrations to be evaluated: In the case of substances with unknown larvicidal activity, the assays should start at the maximum concentration of 500 ppm. Other concentrations should be obtained by serial dilution as follows: 500, 250, 125, 62.5, 31.25, and 15.62 ppm. The bioassays should be performed in triplicate, with 3 disposable cups for each concentration:Each sequence of 3 cups corresponds to a concentration, where each cup should contain:  of distilled water;    Ten 3rd instar larvae (be careful when handling the larvae as they are sensitive to sudden movements);    Some powdered food (porosity of the food suitable for Aedes or Anopheles);    Test substance in the prescribed concentration (volume to be withdrawn from the stock solution varies with the concentration to be tested);Negative Control Composition:       of distilled water or   aqueous solution of the DMSO (if the test substance was solubilized in DMSO);    Ten 3rd instar larvae (be careful when handling the larvae as they are sensitive to sudden movements);    Some powdered food (porosity of the food suitable for Aedes or Anopheles);Negative Control Composition:  of distilled water or  aqueous solution of the DMSO (if the test substance was solubilized in DMSO);    Ten 3rd instar larvae (be careful when handling the larvae as they are sensitive to sudden movements);    Some powdered food (porosity of the food suitable for Aedes or Anopheles);Important: The experiment is performed only once. Thus, verify larval mortality after 24 and 48 hours of exposure by counting the number of larvae alive.
10 ml
10 ml
?. Dose BioassayPurpose of Dose Bioassay: The concentrations showing larval mortality above 50% in the selective bioassays will be selected for the dose bioassays and subsequent calculation of LC50 and LC90.Concentrations to be evaluated: Larvicidal bioassays should contain at least five different concentrations (chosen from the selective bioassays) and the negative control, whereas mortality in the control group should not exceed 10% if repeated assays should be performed.Assembly of the experiments:The bioassays must be performed in triplicate with 5 disposable cups for each concentration:Each sequence of 5 cups corresponds to a concentration, where each cup should contain: of distilled water;    Twenty 3rd instar anopheline larvae (be careful when handling the larvae, as they are sensitive to sudden movements);    Powdered food (porosity of food suitable for Aedes or Anopheles);    Concentration to be tested (volume to be withdrawn from the stock solution varies with the concentration to be tested);Negative Control Composition:      of distilled water or   aqueous solution of the DMSO (if the test substance was solubilized in DMSO);    Ten 3rd instar larvae (be careful when handling the larvae as they are sensitive to sudden movements);    Some powdered food (porosity of food suitable for Aedes or Anopheles);Negative Control Composition: of distilled water or  aqueous solution of the DMSO (if the test substance was solubilized in DMSO);    Ten 3rd instar larvae (be careful when handling the larvae as they are sensitive to sudden movements);    Some powdered food (porosity of food suitable for Aedes or Anopheles);Important: In the dose bioassays, it is necessary to repeat the experiment at three different times. Thus, verify larval mortality after 24 and 48 hours of exposure by counting the number of larvae alive.
50 ml
10 ml
?. Statistical analysisTo obtain the LC50, the mortality data are treated by the Polo plus software (Robertson, 2003) with a 95% confidence interval and p In cases when mortality in the control group exceeds 10%, the larval mortality has to be corrected by Abbott's formula (Abbott, 1925).(mortality % in the assay - mortality % in the control)/(100% - mortality % in the control)
?. [References]
Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925;18:265-7.Finney DJ. Probit analysis. Cambridge (UK): Cambridge University Press; 1971.Guidelines for laboratory and field testing of mosquito larvicides. Geneva: World Health Organization. Dept. of Communicable Disease Prevention, Control and Eradication WHO Pesticide Evaluation Scheme; 2005.Robertson J, Preisler H, Russel R. Poloplus probit and logit analysis. Parma (MO), USA: LeOra Software LLC; 2003. p. 1-36.