Topical Testing Assay: A Technique to Evaluate the Insecticidal Potential of Dopamine Receptor Antagonists on Adult Female Mosquitoes by Topical Application

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 demonstrate the topical testing assay to determine the toxicity of a potential insecticide on adult female mosquitoes. Using a dopamine receptor antagonist that causes lethal inhibition of neurotransmission in mosquitoes, this method describes a way to evaluate the toxic effects of insecticides upon topical application.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board 1. Culture of Mosquito Adults NOTE: Insecticide susceptible strains of mosquitoes are available from the Malaria Research and Reagent Reference Repository. Recommended strains are as follows: Aedes aegypti Liverpool (LVP) strain, Anopheles gambiae Kisumu (KISUMU1) strain, and…

Representative Results

Figure 1: Culture of adult mosquitoes. Image showing 20 L plastic bucket used for culture of adult mosquitoes and use of an aspirator to remove mosquitoes. Figure 2. Adult topical assay performed using 4-5-day-old femal…

Materials

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
Amitriptyline	Sigma-Aldrich	CAS: 549-18-8	Can be diluted in acetone
Bifenthrin	Sigma-Aldrich	CAS: 51529-01-2	Synthetic pyrethorid used as positive control
Micro-applicator	Burkard Manufacturing Co.	–	Tool needed for topical application experiments
24-well cell culture plate with lid	Corning Incorporated	3526	–
Advantage rubber bands	Alliance Rubber Co.	–	Used to seal the paper cups
Glass syringe (1 ml)	Becton Dickinson and Co.	512004	Needed for the micro-applications. Glass is better than plastic
Disposable scintillation vials (20 ml)	Fisher Scientific	74505-20	Glass vials prevent evaporation
Tulle fabric, white	Walmart	–	–
Paper cups	Dixie Consumer Products LLC.	PF15675/13D	Used to keep adult mosquitoes in adult topical assays
Petri dishes (150 mm)	Corning Life Sciences	–	Used to maintain the mosquitoes "slept" on cold without direct contact with ice
Transfer pipettes	Fisher Scientific	13-711-7M	Used to sort larvae
Stereo microscope	Olympus	SZ6045	Used to score larval assays and perform micro-applications
Tweezers (fine)	Fontax	–	Used to handle adult mosquitoes