Preparation of EOs

Based on a review of traditional Chinese medicine products as mosquito repellents after safety evaluation by the Research Ethics Committees, seven EOs were chosen for further testing as potential mosquito repellents (Table 1). These EOs were selected because they were previously reported to have repellent properties against mosquitoes or their chemical structures were similar to those of other components with repellent properties (Chellappandian et al. 2018; Al-Sarar et al. 2020; de Oliveira et al. 2020; Esmaili et al. 2021).

Table 1 The seven essential oils (EOs) selected for testing as mosquito repellents in in vivo (GB2009, WHO2009) bioassaysTest insects

Disease-free Ae. albopictus and Ae. aegypti female mosquitoes were obtained from cultures maintained at the Institute of Insect Sciences, College of Agriculture & Biotechnology, Zhejiang University (Hangzhou, China), and the London School of Hygiene and Tropical Medicine (London, UK). Laboratory cultures of the mosquitoes were maintained in a controlled environment at 27 ± 2°C, 55–60% relative humidity (RH), and a light:dark photoperiod of 12 h:12 h. Adult mosquitoes were placed in plastic rearing cages and provided with a 10% sucrose solution on cotton wool. Females were blood-fed weekly on horse blood using a Hemotek® membrane feeding system (Discovery Workshops, UK) or a homemade system. For the bioassays of repellent activity, 5- to 12-day-old female mosquitoes not previously blood-fed were collected and placed in a fresh cage, without sucrose or water, in the bioassay room for 2 h, which allowed the insects to become acclimated to their environment.

Bioassays In vivo tests

Two different bioassays were used to assess mosquito repellency. In the first, the seven EOs were tested according to a standard method used in China (GB2009). In the second, the two most effective EOs were further evaluated according to the WHO’s human-arm in cage test (WHO2009). GB2009 is the first method developed for the screening of potential mosquito repellents against Ae. albopictus which is the major species of mosquito in China. The WHO guideline 2009 was used to assess the two most effective EOs to provide a reference for researchers in comparisons with other EOs tested as mosquito repellents against Ae. aegypti.

GB2009

In this test, a 16-cm2 area of skin on a human hand is treated with the test or control compound and placed in a mosquito-containing test arena (described below). The limited skin-exposure area significantly reduces the risk of side effects from the bites.

Test arena

Three hundred female adult (4–5-days-old) mosquitoes (Ae. albopictus) were used in the trial. The insects were given access to sugar water prior to their use in the test but did not receive a blood meal. The experiments were conducted in a 40 cm * 30 cm * 30 cm cage maintained at 25–27°C and a RH of 60–70%.

Test procedure

The mosquito repellence activity of the EOs was assayed as follows. The tester’s hands were cleaned with distilled water and his/her forearms protected with a thick fabric sleeve. The tester then donned latex surgical gloves with a 4 × 4 cm area cut out to expose the skin on the dorsal side. One hand served as the control and the other as the test hand. The tester’s control hand was rubbed with 37.5 μL of ethanol (70%) and then placed for 3 min in a cage containing 300 mosquitoes. The number of probing mosquitoes was recorded. If the recorded number was > 30, the test was continued; otherwise a new population of mosquitoes was placed in the cage. The test hand was then treated with 37.5 μL of EO in ethanol (see Table 2 for the dose) and exposed to mosquitoes in the same test cage. Dissolving the EOs in alcohol improved their skin permeability (Lupi et al. 2013). Each EO concentration was tested for up to 3 min during a 15-min period, until either two bites occurred in a single exposure or one bite occurred in each of two consecutive exposures. The time between repellent application and the first two bites in a single exposure or successive exposures was defined as the complete protection time (CPT). Each concentration was tested four times. The longest time for a test did not exceed 7 h.

Table 2 The tested EOs and their test concentrations

The efficacy of the EOs as repellents was assessed according to:

1.

The percentage protective efficacy (PE%), calculated as PE% = [(number of probing mosquitos in the untreated vs. the treated hand)/number of probing mosquitoes of the untreated hand] × 100 (Fradin and Day 2002)

2.

The CPT, calculated as the time elapsed between repellent application and the first confirmed mosquito bite. An effective mosquito bite of the treated skin of the volunteer was defined as the persistence of the mosquito on the skin for 10 s.

Human arm in cage test (WHO2009) Test arena

One hour before the start of the test, 50 Ae. aegypti female mosquitoes (5–12-days-old) were placed in a 30 * 30 * 30 cm cage inside the testing room. The insects were fed with sugar water prior to the test and did not receive a blood meal. The cage was held at 25°C ± 2°C and 80% RH.

Test procedure

The EO or the ethanol control is applied to the forearm skin (between the wrist and elbow) of three volunteers. The exposed area (~ 600 cm2) is determined using the WHO formula for testing skin repellents:

$$\mathrm=^\!\left/ \!_\right.\left(_+_\right) \times _$$

where CW is the circumference of the wrist, CE is the circumference of the elbow, and DEW is the distance between the wrist and elbow in centimeters (cm). The remaining skin area is covered by a rubber sleeve. The dose of the tested repellent is expressed as mg/cm2 or mL/cm2 and is measured using a micropipette or balance before being applied to the exposed forearm using a gloved finger. The volunteer is instructed to avoid contact with lotions, perfumes, oils, or perfumed soaps on the day of the assay.

Repellent activity in the WHO2009 testMinimum repellent dose

The two tested EOs, patchouli oil and clove bud oil, were applied at the doses (µg/cm2) shown in Table 3. The maximum amount of clove bud oil was 720 mg (1200 µg/cm2) and that of patchouli oil 56 mg (93 µg/cm2). The control consisted of ethanol alone. The test was conducted during the day, from 07:00 to 17:00. Each concentration was tested six times. The tester first inserted the control arm into the cages for 30 s, and then the arm treated with the first dose of EO for 30 s. The number of landing and probing mosquitoes was recorded. Successive doses of the EOs were tested until the mosquitoes stopped biting or the maximum safe dose had been reached, or the dose applied became uncomfortably high. As a final control, the untreated arm was again placed in the cage for 30 s and the number of landing and probing mosquitoes again recorded. Landing was defined as the mosquito arriving on the volunteer’s treated skin and remaining for 10 s. Biting was defined as the mosquito probing into the skin for > 10 s. If the mosquito did not land on or bite the volunteer’s treated skin, the respective dose of EO was considered to have provided 99.9% protection.

Table 3 Doses of clove bud oil and patchouli oil applied to human forearm skin (exposed area: ~ 600 cm2)

The percentage repellency (R) was calculated as follows:

$$\mathrm=\left[\left(\mathrm-\mathrm\right)/\mathrm\right]\times 100$$

where C is the mean number of mosquitoes of two control tests, i.e., C = (C Before + C After)/2, and T is the number of mosquitoes in the treated group.

Complete protection time

The time between repellent application and the first two mosquito bites during the same observation period or one bite in each of two consecutive intervals was defined as the CPT. Longevity was determined by estimating the CPT of a predetermined dose applied to the skin, either the dose conferring a 100% reduction at the ED99 (the effective dose of a repellent required to reduce biting by 99%) or the maximum safe dose as determined in the WHO’s standard application volume of 1.67 µL/cm2 (1.002 mL/600 cm2).

First, a control was conducted in which the volunteer inserted his/her bare left arm into a cage containing 50 mosquitoes and the number of mosquitoes probing the arm after 30 s was recorded. Immediately thereafter, the volunteer inserted the treated arm into the same cage for 30 s. The number of mosquitoes probing the arm was determined as described for the control.

The test was repeated at 30-min intervals for the first 3 h, followed by hourly observations until treatment failure or until 8 h had elapsed since EO application. The CPT was defined as the occurrence of one probing event by a mosquito in a 30-s test followed by a confirmed bite within 30 min. The test was repeated with six volunteers.

GC–MS analysis

The GC–MS analysis was carried out with an Agilent 5975 GC-MSD system. An Innowax fused silica capillary column (DB-5, 60 m * 0.25 mm* 0.25 μm film thickness) was used with helium as the carrier gas (1.5 mL/min). The GC oven temperature was held at 50°C for 2 min after injection and then ramped to 250°C at a rate of 10°C/min, and held at 250°C for 5 min. The split ratio was set at 100:1. The injector temperature was set at 250°C. Mass spectra were recorded using 70 eV electrons in election ionization (EI) mode. The mass analyzer was scanned from m/z 35–500 amu. MS was identified on the basis of computer matching of the mass spectra with those of the Wiley and MassFinder libraries and comparison with literature data.