Larval Contact Assay: A Procedure to Evaluate Mosquito Larval Mortality following Direct Contact with Test Larvicidal Compound

Abstract

Source: Brito-Sierra, C. A., et al. Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes J. Vis. Exp. (2019)

In this video, we describe the larval contact assay to evaluate the toxicity of the dopamine receptor antagonist amitriptyline on the third instar stage larvae of mosquitoes, known vectors of several human infectious diseases. Assessing the larval mortality upon incubation with different amitriptyline concentrations over different time intervals helps determine its lethal concentration 50.

Protocol

1. Larval Contact Assay (Single Point Dose or Dose Response Assay)

NOTE: The assay can be conducted with chemistries that are soluble in water or dimethyl sulfoxide (DMSO), provided that the final concentration of DMSO in test wells does not exceed 1%. Alternative solvents may be an option, but it is essential to first confirm that the final concentration does not cause more than 10% mortality at 72 h post-exposure. The assay may be performed as a single-point dose or dose-response assay. If performing the latter, it is recommended to test a range of concentrations (minimum of five), spanning the expected LC₅₀.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Insecticide susceptible mosquito strains	Malaria Research and Reagent Reference Repository		https://www.beiresources.org/MR4Home.aspx; Recommended strains: Aedes aegypti Liverpool (LVP) strain, Anopheles gambiae Kisumu (KISUMU1) strain and Culex quinquefasciatus Johannesburg (JHB) strain
Acetone	Mallinckrodt Chemical	CAS 67-64-1	Use for dilutions and control
Amitraz	Sigma-Aldrich	CAS 33089-61-1	Requires dilution in DMSO
Amitriptyline	Sigma-Aldrich	CAS: 549-18-8	Can be diluted in acetone
Cis-(Z)-flupenthixol	Sigma-Aldrich	CAS: 51529-01-2	Pharmacologically tested as disruptor of dop-like receptors
Dimethylsulphoxide (DMSO)	Sigma-Aldrich	MKBF2985	Organic solvent used to disolve amitraz
24-well cell culture plate with lid	Corning Incorporated	3526
Transfer pipettes	Fisher Scientific	13-711-7M	Used to sort larvae
Stereo microscope	Olympus	SZ6045	Used to score larval assays and perform micro-applications