Improved adulticidal activity against Aedes aegypti (L.) and Aedes albopictus (Skuse) from synergy between Cinnamomum spp. essential oils

Improved natural adulticidal agents against mosquito vectors are in urgent need, and essential oils from Cinnamomum plants can assume this role quite readily. Cinnamomum verum, C. cassia, and C. loureiroi essential oils (EOs) were extracted from the barks and evaluated for their chemical composition by GC–MS. The major constituent of the three EOs was cinnamaldehyde. WHO susceptibility tests on individual and combined EOs as well as cinnamaldehyde were conducted against female adults of Aedes aegypti and Aedes albopictus. All EO combinations exhibited a synergistic effect, manifesting a higher toxicity, with a synergistic value ranging from 2.9 to 6.7. Their increasing mortality value was improved between 16.0 to 41.7%. The highest synergistic effect was achieved by an EO combination of 0.5% C. cassia + 0.5% C. loureiroi, while the highest insecticidal activity was achieved by 2.5% C. verum + 2.5% C. cassia and 1% cinnamaldehyde, with a knockdown and mortality rate of 100% and a KT50 between 0.7 and 2.1 min. This combination was more toxic to both mosquito species than 1% w/v cypermethrin. These findings demonstrate that cinnamaldehyde and synergistic combinations of C. verum + C. cassia EOs and C. cassia + C. loureiroi EOs have a high insecticidal efficacy against Aedes populations.


Results
Chemical compositions of the three Cinnamomum spp. EOs. Hydro-distillation of C. verum, C.
cassia, and C. loureiroi barks provided pale yellow and pale tan EOs. The highest essential oil yield was obtained from C. cassia (1.12% v/w), followed by C. verum (1.01% v/w) and C. loureiroi (0.82% v/w). The chemical compositions of the three Cinnamomum spp. EOs were analyzed by GC-MS. A total of 15, 15, and 11 chemical constituents were identified from C. verum, C. cassia, and C. loureiroi EOs, respectively, accounting for 98.24, 98.60 and 97.07% of their composition, respectively, as presented in Table 1. Cinnamaldehyde was the major constituent of these three Cinnamomum spp. EOs. Its chemical structure is displayed in Fig. 1. The highest cinnamaldehyde content of 73.21% was found in C. verum EO; the second highest was 72.93% in C. cassia EO; and C. loureiroi EO had the lowest cinnamaldehyde content at 72.38% of its chemical composition.
Toxicity of the three EOs on Ae. aegypti and Ae. albopictus females and their synergistic effect. The efficacies of individual EOs from C. verum, C. cassia, and C. loureiroi and several of their combinations against females of Ae. aegypti and Ae. albopictus were evaluated, at 60 min after treatment, in terms of knockdown rate (K), 50% knockdown time (KT 50 ), increasing knockdown value (IKV), effective knockdown index (EKI) and synergistic value (SV), presented in Tables 2, 3 Table 4. Regarding the outcomes of knockdown and mortality rate assays, for a treatment of an insecticidal agent, it is quite possible that the mosquitoes may be knocked down after a short period of time but may recover and did not die afterward, so the knockdown rate may be high but the mortality is low. This was not the case in this study: a higher knockdown rate also came with a higher mortality rate against both species (Tables 2, 3). Females of both species were more susceptible to the EO combinations (100% mortality) than the individual EOs (57.8-88.8% mortality). Individual Cinnamomum spp. EOs achieved a mortality rate of 69.6-88.8% against Ae. aegypti females and 57.8-84.0% against Ae. albopictus females. They were less toxic (EMI < 1) to both mosquito species than 1% w/v cypermethrin. Every combination with 0.5% Cinnamomum spp. EOs exhibited a mortality rate against Ae. aegypti females from 98.4 to 99.3%, an IMV of 21.1-29.9% and a mortality rate against Ae. albopictus females from 99.2 to 99.7% and an IMV of 27.4-41.7%. The highest IMV was achieved by the combination of 0.5% C. cassia + 0.5% C. loureiroi EOs. The IMV achieved by combinations of EOs was improved by 23.6-29.3% against Ae. aegypti females and by 27.4-41.7% against Ae. albopictus. Their adulticidal activities were higher than that of 1% w/v cypermethrin with an EMI < 1.0. The highest insecticidal activity was achieved by the combinations of 2.5% C. verum + 2.5% C. cassia EOs, 2.5% C. verum + 2.5% C. loureiroi EOs, and 2.5% C. cassia + 2.5% C. loureiroi EOs, with 100% mortality Table 1. Physical property, chemical constituents of C. verum, C. cassia, and C. loureiroi essential oils. a RI = Retention index analyzed with HP-5 MS column, experimentally determined using standard alkanes (C 7 -C 30 ). b KI = Kovats index from https ://pubch em.ncbi.nlm.nih.gov and NIST (https ://webbo ok.nist.gov). c IM = Identification methods; MS, mass spectrum matching with chemicals in the computer mass library of Adams 61 . Knockdown rates (K) at 60 min, mortality rates (M) at 24 h after exposure, KT 50 , effective knockdown index (EKI), and effective mortality index (EMI) of 0.25, 0.5 and 1.0% cinnamaldehyde against females of Ae. aegypti and Ae. albopictus are summarized in Table 5. At the highest concentration (1%), cinnamaldehyde showed the highest knockdown and mortality rates. All females of Ae. albopictus were more susceptible to cinnamaldehyde than Ae. aegypti females with a KT 50 value ranging of 0.7 to 6.8 min (KT 90 of 2.0-13.3 min) and 0.9 to 7.3 min (KT 90 of 2.8-14.0 min), respectively. One percent cinnamaldehyde achieved the highest 100% knockdown and 100% mortality rates against both mosquito species and a KT 50 of 0.7 to 0.9 min (KT 90 of 2.0-2.8 min). These mortality and knockdown rates were equivalent to those provided by 1% w/v cypermethrin (which showed an EKI of 0.31 to 0.32 and an EMI of 1).

Discussion
The essential oil yields from the barks of the three Cinnamomum species were in the range of 0.82-1.12% v/w. Several works reported a similar C. verum EO yield by steam distillation and hydro-distillation methods, such as 0.48% v/w 44 , 0.54% v/w 36 , and 1.14% v/w 49 . Some works also reported a similar C. cassia EO yield, for example, 0.72-2.38% v/w 49 , and 0.41-2.61% w/w [50][51][52] . The EO yields from several samples of Cinnamomum spp. showed some variations due to the different climates of different countries and different growth conditions (plant nutrition, soil fertilizer, and pest management, etc.) at the different cultivation sites. There are also other factors that influence yield such as harvesting time, growth stage of plant, age of bark, thickness of bark, density of oil cells in the bark 49 , and extraction method 50 . Moreover, extraction method also affects EO yield, and good cultivation management can increase EO yield 26,49,50 . www.nature.com/scientificreports/ Although the EO yield of cinnamon is not high and cinnamon EOs are 10 times more expensive than common insecticides for mosquito control such as permethrin, cypermethrin, a cinnamon EO is much safer to humans and non-target organisms since it has been used as food ingredient for global populations since ancient times. The most important reason for using natural products from cinnamon, though, is that mosquito vectors have not developed resistance to them 20,21,25 .
Cinnamaldehyde was the major compound found from the three Cinnamomum spp. EOs. The cinnamaldehyde content ranged from 72.38 to 73.21% of the chemical composition. Several works reported similar cinnamaldehyde percentages in the chemical composition of C. verum, such as 64.66% 36 , 74.49% 49 , and 90.17% 44 . Other researchers reported that the cinnamaldehyde percentage in the composition of C. cassia EO was in the range of   53,54 , and that the cinnamaldehyde percentage in C. loureiroi EO was 81.97% 50 . Cinnamaldehyde percentage in the composition of an extracted EO is a very important factor to consider because it is the main active constituent against mosquito vectors 53,54 ; hence, the higher the better. Cinnamaldehyde has already been successfully used for mosquito control as well as several medicine and pharmacological applications 53,54 . Several factors that influence the percentage of cinnamaldehyde in a cinnamon EO were good agricultural management as well as good climate and environment 55 . Moreover, the experimental conditions (temperature, relative humidity and photoperiod cycle) might affect the efficiency of EOs for mosquito control 56 . The temperature was 26 ± 2 °C, and the RH was 74 ± 4% RH with a photoperiod cycle of 12.5-h light: 11.5-h dark in this study. Under these conditions, the mortality rate exhibited by all Cinnamomum spp. EOs against females of Ae. aegypti and Ae. albopictus was in the range of 57.8-100%. These results agree well with a study by Soonwera and Sitthichock 26 . In that study, the post-application temperature of 25.3 ± 2.5 °C and an RH of 75.2 ± 3.4% of treatments of C. citratus and E. globulus EOs against Ae. aegypti and Ae. albopictus females yielded a mortality rate ranging from 59.2-100%. There have been reports that a high post-application temperature (30 °C) affected the efficacy of Thymus vulgaris EO against Cx. quinquefasciatus larvae: its LC 50 was lower than that provided by a lower post-application temperature (15 °C) 56 . At the time that the experiments were designed, we did not consider that the actual usage temperature in Thailand and other www.nature.com/scientificreports/ tropical Asian countries might be a lot higher than our laboratory temperature, and so we did not devise an experiment to test the post-application temperature effect. In our future research, we would conduct experiments at an extreme temperature that might happen in Thailand and checked the EOs' efficiency. All combinations of Cinnamomum spp. EOs exhibited high, synergistic adulticidal activity against females of Ae. aegypti and Ae. albopictus with 100% mortality. Their increasing mortality value was improved from 11.2 to 41.1% compared to those of the individual EOs. One percent cinnamaldehyde showed the highest toxicity against both species with 100% knockdown and mortality rates and a KT 50 and a KT 90 ranging from 0.7-0.9 and 2.0-2.8 min, respectively. Although the toxicity of several EOs against adults of Ae. aegypti and Ae. albopictus were reported in previous studies, but the data on the efficacy of combinations of EOs from Cinnamomum spp. against females of Ae. aegypti and Ae. albopictus are limited. EOs of C. verum and C. cassia as well as cinnamaldehyde were previously found to be toxic against Ae. aegypti adult and larvae of Ae. caspius and Cx. quinquefasciatus 44,57,58 . Cinnamaldehyde showed toxicity against Ae. aegypti female adult with an LD 50 of less than 3.5 µg/ mg female 44 . C. verum EO and cinnamaldehyde also showed some toxicity to other insect pests (M. domestica and Sitophilus oryzae) 58,59 .
All EO combinations in these study showed a highly synergistic effect against females of the two mosquito species. EO combinations from C. citratus + E. globulus showed a highly synergistic effect against Ae. aegypti females with an improvement of more than 33% mortality rate increase 26 . Combined EOs from C. cassia + Liex Table 4. Mortality rates (MR), increasing mortality value (IMV) and effective mortality index (EMI) of essential oils from C. verum, C. cassia, and C. loureiroi and their combinations against females of Ae. aegypti and Ae. albopictus. Mean percentage knockdown rates in each column followed by a different letter are significantly different (one way ANOVA and Duncan's multiple range test, P < 0.05). IMV (%) Increasing Mortality Value, EMI Effective Mortality Index. Treatment codes are defined in Table 1.  Table 5. Knockdown and mortality rates and KT 50 of cinnamaldehyde against females of Ae. aegypti and Ae. albopictus. KT 50 50% knockdown time, R 2 regression coefficient, LCL lower confidence limit, UCL upper confidence limit, EKI Effective Knockdown Index, EMI Effective Mortality Index, ns not significant (P < 0.05). Treatment codes are defined in Table 1. The high toxicity and synergistic effect of all combinations of EOs in this study appear to be associated with their major composition, cinnamaldehyde. The mode of action of Cinnamomum spp. EOs against insect pests was permeability inhibition of cell membrane and disruption of intracellular enzymes 59,60 . Cinnamaldehyde inhibits the respiratory system of insects by inhibiting the enzymes involved in cytokinesis and reducing the ATPase activity of cell membrane, causing decreased cell respiration, decreased membrane depolarization, reduced membrane integrity and eventual mortality [58][59][60] .
More importantly, the combination of 2.5% C. verum + 2.5% C. cassia and individual 1% cinnamaldehyde were more toxic to both species of mosquitoes than cypermethrin. Cypermethrin is a neurotoxic chemical insecticide with a low LD 50 10-13,26 that affects the nervous, immune, and reproductive systems of humans 10,11,13 . Much safer than cypermethrin, C. verum and C. cassia EOs as well as cinnamaldehyde provided a high toxicity against mosquitoes but are non-toxic to humans, other mammals, or beneficial insects 19,20,58 . Furthermore, they are easily degraded in the environment, and they have already been used for ages by Asian people as an antimicrobial agent in their local medicine [45][46][47][48] .
To conclude, our objective of determining the insecticidal efficacies of C. verum, C. cassia, C. loureiroi, and their major constituents was fully achieved. According to the results, the combinations of EOs from C. verum + C. cassia, C. cassia + C. loureiroi, and C. verum + C. loureiroi showed a highly synergistic insecticidal effect against Ae. aegypti and Ae. albopictus. They have a high potential to be developed and improved into a spray formulation of eco-friendly adulticides for controlling or eradicating populations of Ae. aegypti and Ae. albopictus mosquitoes in urban and rural areas as well as for controlling dengue diseases and other vector-borne diseases. EOs from three Cinnamomum spp. barks may be the best source of alternative adulticides for sustainable mosquito control and safe for the environment and human health. Cinnamaldehyde, the major composition of the three Cinnamomum spp. also showed a high potential to be developed and improved into a new formulation of adulticides for controlling Ae. aegypti and Ae. albopictus. Some further research and development tasks are needed before the Cinnamomum spp. EO combinations and cinnamaldehyde can be used as adulticides in rural and urban areas. For example, their cost, safety, stability, post-application temperature effect, and other factors that may limit their use should be thoroughly investigated first.

Methods
Plant materials and essential oil extraction method. Dried barks of C. verum, C. cassia, and C. loureiroi, purchased from Chao Krompoe pharmacy, Chakkrawat, Bangkok 10100, Thailand, were extracted of their essential oils. Images of the three plant species and the chemical structure of their major constituent are shown in Fig. 1. Specimens of all Cinnamomum spp. were positively identified by a botanist from the botanical center, King Mongkut's Institute of Technology Ladkrabang (KMITL), Bangkok, Thailand. All specimens were cleaned, crushed, and extracted of essential oils (EOs) by a hydro-distillation method [26][27][28][29] . After 6-7 h, the process was completed. Each EO was collected from the separating funnel, removed of water with anhydrous sodium sulfate (Na 2 SO 4 ), preserved in airtight vials, and kept at 4 °C for further chemical composition analysis and bioassays (Table 1). All EOs and their combinations were diluted with ethyl alcohol into several formulations shown in Table 6. www.nature.com/scientificreports/ SV > 1 indicated that the combined EOs were synergistic; SV < 1 indicated that the combined EOs were antagonistic; and SV = 1 indicated that the combined EOs did not show any synergistic or antagonistic effect 36 .
• The effective knockdown index (EKI) was calculated by the following formula: EKI < 1 indicated that the individual EO or combined EOs was more toxic than 1% w/v cypermethrin; EKI > 1 indicated that the individual EO or combined EOs was less toxic than 1% w/v cypermethrin; and EKI = 1 indicated that the individual EO or combined EOs was as toxic as 1% w/v cypermethrin.
• The effective mortality index (EMI) was calculated by the following formula: EMI = 0 or > 1 indicated that the individual EO or combined EOs was more toxic than 1%w/v cypermethrin, and EMI < 1 indicated that the individual EO or combined EOs was less toxic than 1% w/v cypermethrin.