Mosquito Feeding Assay: A Method to Evaluate Test Compound Effects on Mortality and Fecundity of Adult Female Mosquitoes

Protocolo

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Adult Blood-Feeding Assay (Single Point Dose or Dose-Response Assay)

1. Collect approximately 150, 4-5-day old adult female mosquitoes and transfer to a separate cage. Repeat to obtain 6 cages if performing a dose-response assay. Remove the source of sugar 1-24 h prior to the feeding assay.
2. Prepare a stock solution of chemistry as described above, accounting for the purity of the chemistry (a typical stock solution is 80 mM in water or salt buffer), and subsequently prepare serial dilutions in water/buffer as required.
3. Add 40 µL of each of the dilutions to 960 µL of defibrinated rabbit blood to obtain the desired test concentration. Apply membrane film to a feeding unit and seal with a rubber ring. Transfer 1 mL of blood via pipette and attach the feeding unit to a heating unit.
4. Gently swab the membrane surface with freshly made 10% lactic acid solution and place the feeding unit into the cage of adult mosquitoes. Cover the cage with a dark cloth or black trash bag and allow mosquitoes to feed for one hour.
5. Repeat steps 1.2 - 1.4 for the remainder of test solutions and the negative (blood-only) control.
6. On completion of feeding, place buckets at 4 °C in a refrigerator for 5 min to anesthetize mosquitoes. Remove male mosquitoes and non-fed female mosquitoes, and count and record the total number of fed and partially fed mosquitoes by examination of the abdomen.
7. Retain partially and fully fed female mosquitoes, aiming for a minimum of 50 blood-fed mosquitoes per dose.
8. Transfer buckets to a plastic tub or growth chamber under constant conditions of 28 °C and 75 - 85% RH (on a 12 h day/12 h night cycle is preferable).
9. Record the total number of dead/non-responsive mosquitoes at 0.5, 1, 1.5, 2, 24, 48, and 72 h (or alternative time points as desired) using the score sheet (Table 1).
10. On the third day post blood feeding, introduce an egg cup into the cage and collect eggs over a 72-h period. Count the total number of eggs produced per treatment under a dissecting microscope.
11. Calculate the percent mortality and fecundity as a function of the total number of fed mosquitoes for each treatment group and the control. Correct for control mortality using the modified Abbott's formula if desired.
12. Display toxicity and fecundity data as a histogram plot or exponential curve (if performing a dose-response assay) using software of choice. If performing a dose-response assay, calculate the LD₅₀, LD₇₅, or LD₉₀ value for the test chemistry relative to the control.
13. Repeat the assay at least twice using separate batches of mosquitoes to obtain n = 3 or more biological replicates.

Table 1. Example score sheet used to record data from the adult blood-feeding assay. Typical doses used to test small molecule antagonist chemistries such as amitriptyline are 50 µM, 100 µM, 200 µM, and 400 µM.

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

Materiais

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
Bifenthrin	Sigma-Aldrich	CAS: 51529-01-2	Synthetic pyrethorid used as positive control
Defibrinated rabbit blood	Hemostat	DRB0100	Blood source for adapted mosquitoes to artificial feeding
Hemotek membrane feeding system	Hemotek Ltd	Serial no. 1303	Used for feeding mosquitoes
Transfer pipettes	Fisher Scientific	13-711-7M U
Stereo microscope	Olympus	SZ6045	Used to score larval assays and perform micro-applications
Tweezers (fine)	Fontax		Used to handle adult mosquitoes
Paper cups	Dixie Consumer Products LLC.	PF15675/13D	Used to keep adult mosquitoes in adult topical assays
Advantage rubber bands	Alliance Rubber Co.		Used to seal the paper cups