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.
- Label the wells of a clear 24-well tissue plate.
- Prepare a stock solution of the test chemistry in a 1.5 mL tube.
- Weigh each chemistry using an analytical balance, and depending on solubility characteristics, re-suspend in sterile ddH2O, DMSO, or another suitable organic solvent of choice.
NOTE: Calculations should take into account the purity of the chemistry. For example, for a chemistry that is 99% pure, dissolve 10.1 mg in 1,000 μL of acetone to obtain a 1% solution. Seal with paraffin film and store at -20 °C.
- Determine the final concentration of the test chemistry that will be evaluated and prepare serial dilutions from the stock solution accordingly, using the appropriate solvent.
NOTE: The concentration and volume of chemistry and solvent can be calculated using the formula C1*V1=C2*V2 where C1=concentration of sample 1, C2=concentration of sample 2, V1=volume of sample 1 and V2=volume of sample 2.
- Prepare a 10 mM stock solution and serial dilutions as required to obtain desired test concentrations (Table 1B).
- Using a wide-bore plastic transfer pipette, transfer five L3 larvae to the well of a tissue culture test plate; L3 larvae can be recognized by length (~ 2.5 - 3.5 mm) and head capsule width of ~ 0.025 mm. Gently remove the water with a 1 mL pipette and replace it with the desired volume of ddH2O. Repeat to obtain n = 4 technical replicates per treatment (i.e., 20 larvae total per treatment).
- Add the appropriate volume of test chemistry to each of the four replicate wells in the 24-well test plate and gently swirl the plate to ensure uniform mixing of chemistry. Place the plate in a test or growth chamber under constant conditions (e.g., 25 °C and ~ 75-85% RH is recommended, and a 12 h light/12 h dark cycle if possible).
NOTE: For reproducibility between experiments, ensure subsequent assays are performed under the same environmental conditions.
- Record the number of dead/non-responsive larvae in each well at 30 min, 1, 1.5, 2, 3, 24, 48, and 72 h post-exposure (or alternative time points) on the score sheet (Table 2). Gently tap the side of the plate. If no movement is observed, gently touch the larva with a sterile toothpick. Score larvae that do not respond to tapping/touch as "dead".
NOTE: Other morphological and behavioral phenotypes may be observed and can be recorded.
- Correct for control mortality using the modified Abbott's formula if desired, as follows:
Mortality (%) = (X-Y) *100/(100-Y),
where X = the percent mortality in the treated sample, and Y = the percent mortality in the control.
- Graph results as a histogram or logarithmic curve using software of choice such as Graph-pad Prism 6 or similar and calculate Lethal Concentration (LC) values relative to the control.
- Repeat the assay using a separate batch of mosquitoes to obtain n = 3 or more biological replicates.
Table 1. Example calculations for Aedes aegypti L3 larval (A) single point dose assay and (B) dose-response assay.
| A. Single point dose assays with three chemistries |
| Chemistry | Concentration (400 uM) |
| Amitriptyline | 80 mM Stock: 2 mg of amitriptyline X 98% (purity of powder) X 0.001 g/1 mg X 1 mol /313.86 g X 1 L/0.08 mol X 106 μL/L = 78.06 μL of water. |
| Serial Dilutions: Dilute stock 1:2 with ddH2O and then 1:4 to achieve 10 mM. Add 40 µL of 10 mM stock to the well containing 960 µL of ddH20 to achieve a final concentration of 400 µM/well. Prepare serial dilutions of 10 mM stock 1:2 to achieve 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM stocks. |
| Cis-(z)-flupenthixol | 80 mM Stock: 2 mg of cis(z)flupenthixol X 98% (purity of powder) X 0.001 g/1 mg X 1 mol/507.44 g cis(z)flupenthixol X 1L/0.08 mol X 106 μL/L = 48.28 μL of water. |
| Serial Dilutions: As above. |
| Amitraz | 80 mM Stock: 2 mg of Amitraz X 0.001 g/1 mg X 1 mol/293.4 g of amitraz X 1 L/0.08 mol X 106 μL/1 L = 85.21 μL of DMSO (note: Amitraz is only soluble in DMSO). |
| Serial Dilutions: As above. |
| B. Dose-response assay |
| Chemistry | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 |
| 400 μM | 200 μM | 100 μM | 50 μM | 25 μM |
| e.g., Amitriptyline, cis-(Z)-flupenthixol oramitraz | 40 μL | 40 μL | 40 μL | 40 μL | 40 μL |
| 10 mM stock | 5 mM stock | 2.5 mM stock | 1.25 mM stock | 0.625 mM stock |
Table 2. Example score sheet used to record data from the mosquito larval dose-response assay. Control, water-only, or water with 1% DMSO or other solvent only. Typical doses used to test small molecule antagonist chemistries such as amitriptyline are 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM.
| Mosquito L3 Larval Assay |
| Date initiated: |
| Investigator: |
| Species/Strain: |
| Assay Type: Single point dose/dose response |
- 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 | | | | | | |
| 3 | | | | | | |
| 12 | | | | | | |
| 24 | | | | | | |
| 48 | | | | | | |
| 72 | | | | | | |
- Percent Mortality
|
| Time (hours) | Dose 1 | Dose 2 | Dose 3 | Dose 4 | Dose 5 | Control |
| 0.5 | | | | | | |
| 1 | | | | | | |
| 1.5 | | | | | | |
| 2 | | | | | | |
| 2.5 | | | | | | |
| 3 | | | | | | |
| 12 | | | | | | |
| 24 | | | | | | |
| 48 | | | | | | |
| 72 | | | | | | |
