Chemical composition and insecticidal activity of Aeollanthus pubescens Benth leaf essential oil on the malaria vector Anopheles gambiae (Diptera: Culicidae)

The use of synthetic insecticides is responsible for many cases of resistance in insects. Therefore, the use of natural molecules of ecological interest with insecticidal properties turns out to be an alternative approach to the use of synthetic insecticides. This study aims at investigating the larvicidal, adulticidal activity and the composition of the essential oil of Aeollanthus pubescens Benth on the major malaria vector Anopheles gambiae. The leaves of Aeollanthus pubescens were collected in the South of the Republic of Benin. Three reference strains of Anopheles gambiae s.s. such as Kisumu, Kiskdr and Acerkis were used. The chemical composition of the essential oil was analysed by gas chromatography coupled to mass spectrometry. Larvae were exposed to the essential oil extract for 24 h. Adult mosquitoes were exposed to the fragment nets coated with the essential oil for 3 min. Larval mortality and adult survivorship were monitored. Fourteen components were identified representing 98.31% of the total of oil. The major components were carvacrol (51.06 %), thymyle acetate (14.01 %) and γ-terpinene (10.60 %). The essential oil has remarkable larvicidal properties with LC50 of 29.26, 22.65, and 28.37 ppm respectively on Kisumu, Acerkis and Kiskdr strains. With the fragment net treated at 165 µg/cm2, the KDT50 of both Acerkis (1.71 s, p < 0.001) and Kiskdr (2.67 s, p < 0.001) individuals were significantly lower than that of Kisumu (3.77 s). The lifespan of the three mosquito strains decreased respectively to one day for Kisumu (p < 0.001), two days for Acerkis (p < 0.001) and three days for Kiskdr (p < 0.001) compared to their control. Our findings show that the Aeollanthus pubescens essential oil is an efficient larvicide and adulticide against malaria vector Anopheles gambiae. This bioinsecticidal activity is a promising discovery for the control of the resistant malaria-transmitting vectors.

139 of water and 1 mL of each prepared concentration was added to obtain test solutions of 10, 20, 140 30, 40, 50 and 60 ppm respectively. In each round of the experiment, larvae were exposed for 141 24 hours at 26 ± 2°C without any food provided to them. After exposure, the larval mortality 142 was recorded. Larvae were considered dead when they were not able to move or swim actively 143 when touched. For each strain, four replicates were performed for a total of 100 larvae per 144 concentration. The control group consisted of batches of larvae exposed to water and the solvent 145 alone (ethanol). In total, three different experiments were conducted on three different days. 153 Then, the essential oil which was yellow with a distinct and sharp odour, was incorporated into 154 the net fragment fibres at 55, 110 and 165 µg/cm 2 .
155 The masses of essential oil proportional to the net area (169 cm 2 ) per concentration were 156 determined: 9.3, 18.6 and 27.9 mg for the impregnation at 55, 110 and 165 ng/cm 2 respectively. 157 A volume of 1.5 mL of ethanol HPLC grade was poured into a Petri dish containing the mass 158 of essential oil corresponding to a given concentration. After complete dissolution, the fragment 159 of the mosquito net was soaked in the mixture. The impregnated fragment nets were left to dry 160 at room temperature for 5 minutes to allow the essential oil to adhere to the mosquito net and 161 to evaporate the ethanol completely. After drying treated fragment nets were stored in the dark 162 and used 2 to 4 hours later. All coated fragment nets used during the day were treated in the 163 morning at the same time. The net of the same size were also treated with 1.5 mL of solvent 164 and was used as control.

165
Cone test 166 The cone test was used to assess the effectiveness of the essential oil on the adult mosquitoes.
167 The cone test is an adaptation of the WHO cone bioassay with the following modification: 168 During the assay, the test operator holds a forearm behind the cone to provide a host (Fig 2).
169 Unfed 3-5 days old female mosquitoes of Kisumu, Acerkis and Kiskdr strains were used in the 170 test. On the day of testing, female were starved for 4 hours before testing. Groups of five female 171 mosquitoes were placed into plastic cups and moved into the testing room one hour before 172 testing begins to allow the mosquitoes to acclimatise to room conditions. The fragment nets for 173 test or control were placed over a dedicated hole on the Perspex boards and secured using a 174 clear tape. A second Perspex board was laid on the first board creating a test/control net 175 "sandwich" between the two boards. The cone was placed over the net and plugged above with 176 a piece of parafilm. A batch of 5 mosquitoes was transferred into the cone with the operator's 177 forearm in position. Mosquitoes were then exposed to the fragments for three (03) minutes. Ten 178 (10) replicates of batches of 5 mosquitoes of each strain were run per concentration of 179 impregnated nets. 183 After exposure, mosquitoes were removed from the cone, transferred into a recovery cups and 184 provided with 10% of honey solution soaked on a cotton pad. Mosquito knockdown was 209 carvacrol (51.06%), followed by other components thymol acetate (14.01%), ɣ-terpinene 210 (10.60%), O-cymene (8.40%) and thymol (5.46%). The percentage of composition of the 211 remaining nine compounds ranged from 0.19 to 2.02% (Table 1).
239 for Kiskdr (p < 0.001) in exposed groups (Fig 3C). With the net treated at 110 µg/cm 2 , Kisumu 252 females longevity was significantly reduced by 21 days compared to that recorded with the 55 253 µg/cm 2 treatment (by 14 days; p < 0.001) (Figure 3A and 3B). With each of these two 254 treatments, no significant effect was observed on the longevity of Kiskdr (p = 0.8).
255 380 interfere with the insect locomotor system and even the nervous system leading to the death in 381 the following days.
382 At the doses 55 and 110 µg/cm 2 of the essential oil, the lifespan of mosquitoes is fourteen (14) 383 days maximum for both resistant Acerkis and Kiskdr strains. During this time period, 384 mosquitoes might still be able to reproduce. Therefore, further studies are needed to assess the 385 blood-feeding success, the fecundity and the fertility of mosquitoes following exposure. This 386 could lead to highlight putative detrimental effects of the essential oil exposure that could also 387 hamper the vectorial competence of the mosquitoes.