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.
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 Culex quinquefasciatus Johannesburg (JHB) strain.
2. Adult Topical Assay (Single Point Dose or Dose Response Assay)
NOTE: The adult topical assay is conducted using acetone as a solvent. 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 48 h post-exposure. The assay may be performed as a single-point dose or dose-response assay and is used to determine lethal dose (LD). If performing the latter, it is recommended to test a range of concentrations (minimum of five), spanning the expected LD50.
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 female Aedes aegypti. Following treatment with chemistry or solvent, adult mosquitoes are placed in paper cups and transferred to a test chamber for the duration of the assay.
Figure 3. Stock and serial dilutions. Schematic diagram showing the procedure for preparation of (A) stock and (B) serial test solutions for the adult topical assay.
Table 1: Example score sheet used to record data from the adult topical dose-response assay
Mosquito Adult (3-5-day-old) Topical Assay | ||||||
Date initiated: | ||||||
Investigator: | ||||||
Species/Strain: | ||||||
Assay Type: Single point dose/dose response | ||||||
A. Mortality (No. dead mosquitoes) | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
0.5 | ||||||
1 | ||||||
1.5 | ||||||
2 | ||||||
2.5 | ||||||
B. Percent Mortality | ||||||
Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
0.5 | ||||||
1 | ||||||
1.5 | ||||||
2 | ||||||
2.5 |
The authors have nothing to disclose.
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 |