*4.8. In vitroA&K Repellent Bioassay*

Bioassays were conducted using Ali and Khan (A & K) bioassay system developed by Ali et al. [28] for quantitative evaluation of repellency against mosquitoes. Minimum effective dosage (MED) values in this bioassay were determined using a method described by Katritzky et al. [29]. Briefly, the bioassay system consists of a 30 × 30 × 30 cm collapsible aluminum cage having one penal of clear transparent acrylic sheet with 120 × 35 mm slit through which the blood box containing a removable feeding device was attached. The top of the blood box had a sliding door used to expose the females to the treatment during the bioassay. Rectangular areas of 4 × 7.5-cm were marked on the collagen sheet that matched the measurement of the rectangular liquid reservoirs. Treatments were applied in a volume of 107 μL using a micropipette. Treated collagen was secured on the feeding reservoir containing the feeding solution using a thin layer of grease (Dow Coming Corp., Midland, MI, USA). The feeding device was then pushed inside the blood box and the sliding door was opened to expose the females to the treatment. The numbers of females landing and biting were recorded visually for 1 min. To ensure proper landing and biting, we used 3-4 cages at a time and only one treatment replication of individual compounds was completed in a single cage. The data are presented as %age biting as a function of concentration. MED is ≤ 1% biting out of 200 females in the cage. A total of five replicates were conducted.

#### *4.9. Larval Bioassay*

Bioassays were conducted using the bioassay system described by Pridgeon et al. [30] to determine the larvicidal activity of essential oils from different parts of *Magnolia grandiflora* against *Ae. aegypti*. Eggs were hatched and larvae were held overnight in the hatching cup in a temperature-controlled room maintained at a temperature of 27 ± 2 ◦C and 60 ± 10% RH. Five 1-day-old larvae were transferred in each of 24-well tissue culture plates in a 40–50 μL droplet of water. Total of 50 μL of larval diet (2% slurry of 3:2 beef liver powder and brewer's yeast) and 1 mL of deionized water were added to each well by using a Finnpipette stepper (Thermo Fisher, Vantaa, Finland). All the essential oils and pure compounds were diluted in DMSO. After the treatment, the plates were swirled in clock-wise and counter-clockwise motions and front and back and side to side five times to ensure even mixing of the chemicals. Larval mortality was recorded 24-h post-treatment. Larvae that showed no movement in the well after manual disturbance were recorded as dead. A series of 4-5 dosages were used in each treatment to get a range of mortality. Treatments were replicated ten times for each extract/compound.
