2.4. Essential Oils Repellent Activity

The repellence of the EOs was evaluated by the human-bait technique [28] with some modifications. The experiments were performed during the summer in the above-described cages. The cages contained about of 300 8–12 day-old adults (sex ratio 1:1). The mosquitoes were starved for 12 h and were not blood-fed or exposed to any form of repellent. The tests were performed by ten volunteers not allergic to mosquito bites, and that had no contact with perfumed products on the day of the bioassay. All volunteers were informed about the experiment and provided their written consent. After rinsing the hands in distilled water, the volunteer’s forearms were protected with thick fabric sleeves and the hands with latex gloves in which a dorsal square area 5 × 5 cm was cut open. The mosquito-exposed skin of one hand was treated with 100 μL of ethanol as a negative control. The other hand was treated with 100 μL of EO ethanolic solution at concentrations ranging from 0.02 to 200 nL cm⁻². After ethanol evaporation, the control hand was inserted inside the cage and exposed to mosquitoes for 3 min. Immediately after, the other hand was treated with the EO solution and, after ethanol evaporation, exposed to mosquitoes in the same cage. The number of probing mosquitoes was recorded by two observers. All the tests were performed between 8:00 and 10:00 am. The complete protection time (CPT) for the concentration of the EO of 0.2 µL cm⁻² of skin was calculated by tests performed every 15 min until either two bites occurred in a single exposure period or one bite occurred in each of two consecutive exposure periods. The complete protection times were calculated on an average of six replicates. To verify the mosquitoes’ readiness to bite, the control and the EOs treated hand were regularly interchanged during each test. We considered the test valid if at least 30 mosquitoes landed on the control hand and attempted to bite. If the number of probing was < 30, a new mosquito’s cage was used [11]. The study was approved by the ethical committee of the University of Pisa (Comitato Bioetico dell’Università di Pisa).