Chemical Composition, Larvicidal and Repellent Activities of Wild Plant Essential Oils against Aedes aegypti

Simple Summary Mosquitoes are the deadliest insects alive due to the transmission of pathogens that cause diseases. Plant essential oils are considered an alternative to synthetic repellents for controlling mosquitoes. We have investigated the repellent and larvicidal activity of six plant essential oils against adult female mosquitos and the larvae of yellow fever mosquitos, Aedes aegypti. The essential oils extracted from Mentha longifolia, Zanthoxylum armatum, Erigeron bonariensis, and E. canadensis have the potential to manage Ae. aegypti at the larval stage. Moreover, M. longifolia, E. canadensis, E. bonariensis, and Salsola imbricata essential oils exhibited prolonged mosquito-repellent activity against adult female Ae. Aegypti; these oils might be used to develop formulations that are efficient and cost-effective as mosquito repellents without harming humans and the environment. Abstract Bio-degradable and eco-friendly essential oils (EOs) extracted from Mentha longifolia, Salsola imbricata, Erigeron bonariensis, E. canadensis, Ailanthus altissima, and Zanthoxylum armatum were investigated for their repellent and larvicidal potential against Aedes aegypti mosquitoes. The EOs of M. longifolia, S. imbricata, E. bonariensis, E. canadensis, A. altissima, and Z. armatum exhibited 99.0%, 96.8%, 40.2%, 41.7%, 29.1%, and 13.2% repellency against mosquitoes at a tested dose of 33.3 μg/cm2, respectively. In time span bioassays, the EOs of M. longifolia, S. imbricata, E. bonariensis, and E. canadensis showed more than 40% repellency for 60 min at a tested dose of 330 μg/cm2. Larvicidal bioassays revealed that larvae of Ae. aegypti were the most susceptible to M. longifolia (LC50, 39.3 mg/L), E. bonariensis (LC50, 26.0 mg/L), E. canadensis (LC50, 35.7 mg/L), and Z. armatum (LC50, 35.9 mg/L) EOs upon 48 h exposure. The most abundant constituents in the EOs of M. longifolia, S. imbricata, E. bonariensis, E. canadensis and A. altissima were piperitone oxide (45.5%), carvone (39.9%), matricaria ester (43.1%), (31.7%) and eugenol (24.4%), respectively. Our study demonstrates that EOs of M. longifolia, S. imbricata, E. bonariensis, and E. canadensis might be used to control Ae. aegypti mosquitoes without harming humans or the environment.


Introduction
Mosquitoes are the deadliest insects [1] due to the transmission of pathogens, causing diseases such as the West Nile virus, filariasis, dengue, chikungunya, Japanese encephalitis,

Rearing of Ae. aegypti
The Ae. aegypti colony was reared in the laboratory, using the method described by Johnson [30] and Zheng et al. [31]. The larval population of Ae. aegypti was taken from Health Department, Multan, Pakistan. Larvae were placed in a plastic container (20 × 16 × 4 cm) filled with water. A fish diet (Osaka green fish food) consisting of 3% crude fat, 4% crude fibre, and 28% crude protein was used for larval feeding. The pupae collected daily from the larval container were transferred to plastic cups (350 mL capacity) filled with distilled water (200 mL). They were then placed in a separate Plexiglass cage (30 × 30 × 30 cm), with 3 meshes (one on the upper side and one on each lateral side) as well as an opening hole (18 cm diameter) covered with a muslin cloth fastened by a rubber band. Cotton soaked with a 10% sugar solution was kept in the cages as a food for adults. Ae. aegypti were used to mate after 3-4 days of emergence, and therefore the adult females were fed on the blood of constrained pigeons. The plastic cups (350 mL capacity), lined with 10 cm long wax paper and filled with 120 mL of water, were placed in the adult cage of blood-fed female mosquitoes for oviposition. Egg laying was observed after 3-5 days of mating. The eggs laid by the female mosquitoes were either placed in a larval container filled with water for hatching or stored in a dry place for whenever eggs may be needed for use [32]. Only mated females and larvae of Ae. aegypti were used in the repellent and larvicidal bioassays, respectively. Rearing as well as experiments were performed in a climatic chamber where the relative humidity was 70 ± 10%, the temperature was 28 ± 2 • C, and a photoperiod of 14:10 h light: dark was maintained.

Mosquito Repellency Bioassay
The repellency of extracted essential oils was investigated by using the human bait method against female Ae. aegypti before the scotophase period. The essential oils solutions at concentrations of 1, 5, and 10% were prepared in ethanol to evaluate their repellency against adult female mosquitoes. Thirty mated and blood-starved four-to-five day-old female mosquitoes were separated in an adult cage (30 × 30 × 30 cm) 24 h before the repellency experiment. Before the start of the bioassay, the hands of the subject were washed with scent-free soap and then dried. Afterwards, gloves were worn on the hands to cover the entire hands, except for a circular area of 30 cm 2 on the dorsal side of the hands. A 100 µL aliquot of solvent or solution of the test substance was applied on the exposed area of the hand in each replication of treatments, giving doses of 33.3 µg/cm 2 , 166.5 µg/cm 2 , and 330 µg/cm 2 when 1%, 5%, and 10% concentrations of EO were used. After 3 min drying at room temperature, the solvent or substance-treated hands were exposed to female Ae. aegypti in the experimental units for five min. The number of female Ae. aegypti landings on the negative control and test-treated exposed area of hands were counted. The human subjects (volunteers) were informed about the test procedure and consent was obtained before conducting repellency bioassays, moreover, permission regarding human subject use was also obtained from the Ethical and Biosafety Committee of Bahauddin Zakariya University. The percentage repellency was calculated by adopting the formula reported by Azeem et al. [29]: percentage repellency = [(M c − M t )/M c ] × 100, where M c is the number of mosquito landings on the negative control (solvent) treated hand and M t is the number of mosquito landings on the test substance treated hand. The essential oils that showed more than 60% repellency against Ae. aegypti at each tested dose and were further investigated to evaluate the time span repellency (repellent longevity). The time span bioassay was carried out in the same way as the repellency bioassay described above, except this time using the same treated hand after each 15 min period and counting females landing for 5 min until the number of mosquito landings on the control and treated hands became equal. All the repellency bioassays were repeated five times, randomly, to minimize the error in the experiment.

Larvicidal Activity Bioassay
The larvicidal activity of selected essential oils was tested against the second-instar larvae of Ae. aegypti by following the protocol explained by Ali et al. [33], with some modifications. Briefly, five second-instar larvae of Ae. aegypti were added to each portion of the ice tray (50 mL capacity), which had 20 mL water. The larvicidal activity of the essential oils was evaluated at different concentrations where it remained effective. Different dilutions of essential oils were prepared in DMSO, and 50 µL essential oil solution or DMSO was added to each well. Thus, the final concentration of essential oil in wells was 6.25 mg/L to 1600 mg/L (6. 25, 12.5, 25, 50, 100, 200, 400, 800, 1600 mg/L) with twofold dilution at each step. DMSO was used as the negative control and its concentration in test media never exceed 0.25%. The larvae were exposed to essential oils or the negative control for 24 and 48 h to evaluate susceptibility. A fish diet was also provided to larvae during the exposure period. After the exposure period, larvae mortality was checked by using a camel hairbrush, and the larvae that did not show any movement were considered dead. At least seven replicates of each concentration of different essential oils and control were employed.

Chemical Analysis of the Essential Oils
Essential oils that showed good repellency against Ae. aegypti were investigated further, using a Hewlett Packard gas chromatography-mass spectrometry (GC-MS) system (Agilent Technologies Inc., Santa Clara, CA, USA), to detect the main components of the essential oils. The 6890N GC was equipped with a 30 m long capillary column with 0.25 mm internal diameter and 0.25 µm stationary phase film thickness. The stationary phase of the GC column was 95% dimethylpolysiloxane and 5% diphenyl (DB-5, Agilent Technologies Inc., Santa Clara, CA, USA). The injector of GC was constantly operated at a temperature of 235 • C. The temperature of the GC oven was set as follows: initially, it was kept constant at 40 • C for 2 min, then raised to 240 • C at a constant rate of 4 • C/min and finally was programmed isothermally at 240 • C for 8 min. Helium (gas) was used as a mobile phase at a constant flow of 1 mL/min. An aliquot of 1 µL dilute solution of essential oil was injected into GC in the splitless mode for 30 s. The mass spectrometer parameters were programmed as follows: electron energy for ionization was maintained at 70 eV, ion source temperature was set at 180 • C, and the range of mass spectra scan was set at 30-400 amu. To calculate the composition (%) of compounds in essential oils, a total ion chromatogram was used. Separated compounds were initially identified through the comparison of mass spectra with the NIST-2008 MS library. In addition, the retention indices of separated compounds were compared to published data and the NIST online library. To calculate retention indices of separated compounds, the standard mixture of n-alkanes (C 9 -C 24 ) was analyzed using the same GC-MS parameters as were used for essential oils. A final verification of the compounds was carried out by injecting the solution of pure available standard compounds at the same conditions used for essential oils analysis. The standard compounds were purchased from Sigma-Aldrich (St. Louis, MI, USA) or Alfa Aesar (Haverhill, MA, USA) chemical suppliers, or otherwise purified in a laboratory at the same parameters used for essential oils analysis.

Statistical Analysis
General Linear Model (GLM) was used to evaluate the EO type and dose effect on the repellency of mosquitoes in repellency bioassay as well as EO type and time effect on the repellency in time span experiments. In all models, experimental replication was treated as a random variable. If a significant effect was determined, pairwise comparisons of group means by Tukey's post hoc test at the significance threshold (alpha = 0.05) were used. The statistical analysis was performed by Statistica software version 14.0.1.25 (TIBCO Software Inc, Palo Alto, CA, USA). For larvae mortality data, the Abbott formula [34] was used to calculate corrected mortality. The different lethal concentrations LC 50 and LC 90 were calculated by using probit analysis through the Polo-Plus software. The LC 50 values of the two bioassays were considered significantly different when their fiducial limits did not overlap [35].

Yield of Essential Oils
The Z. armatum leaves were the richest in essential oil and yielded 0.76%, whereas the least amount of essential oil was obtained from S. imbricata and A. altissima, which produced yields of 0.01% and 0.04%, respectively (Table 1).

Repellency of Essential Oils
A statistical data evaluation by GLM revealed that the EO type (df = 6, F = 1178, p < 0.001) and dose (df = 2, F = 5423, p < 0.001) significantly affected the repellency of mosquitoes. Pairwise comparisons of group means by Tukey's post hoc test at the significance threshold (alpha = 0.05) revealed that the essential oil of M. longifolia showed high repellent activities, comparable to those of DEET, at the lowest dose of 33.3 µg/cm 2 ( Figure 1). EO of S. imbricate had a good repellent effect as well, while essential oils of E. bonariensis, E. canadensis, and A. altissima demonstrated from 30% to 40% of DEET efficiency. The essential oil of Z. armatum showed the lowest repellency compared to all tested essential oils. At the medium dose of 166.5 µg/cm 2 , the essential oils of A. altissima and Z. armatum were significantly weaker repellents compared to the rest of the samples, whose activities did not differ significantly from each other. The repellent activity of six essential oils did not differ significantly from DEET tested directly after application at a dose of 330 µg/cm 2 concentration ( Figure 1).

Time Span Repellency at a Dose of 33.3 µg/cm 2
The GLM model showed significant effects of EO type (df = 2, F = 2970, p < 0.001) and time (df = 2, F = 4239, p < 0.001) on the repellency effect. EO of M. longifolia and S. imbricata showed repellency comparable to DEET immediately after application. However, their repellency decreased drastically after 15 and 30 min of application ( Figure 2). S. imbricata exhibited 63% repellency after 15 min, which was significantly higher than the repellency of M. longifolia. However, after 30 min, both plants' essential oils exhibited similar activity ( Figure 2). Biology 2023, 11, x FOR PEER REVIEW 6 of 16 essential oils did not differ significantly from DEET tested directly after application at a dose of 330 μg/cm 2 concentration ( Figure 1). The GLM model showed significant effects of EO type (df = 2, F = 2970, p < 0.001) and time (df = 2, F = 4239, p < 0.001) on the repellency effect. EO of M. longifolia and S. imbricata showed repellency comparable to DEET immediately after application. However, their repellency decreased drastically after 15 and 30 min of application ( Figure 2). S. imbricata exhibited 63% repellency after 15 min, which was significantly higher than the repellency of M. longifolia. However, after 30 min, both plants' essential oils exhibited similar activity ( Figure 2).

Time Span
Repellency at a Dose of 166.5 μg/cm 2 GLM analyses revealed significant effects of EO type (df = 4, F = 8625, p < 0.001) and time (df = 4, F = 8559, p < 0.001) on the repellency effect. Four out of six plants' essential oils exhibited 100% repellency at a dose of 166.5 μg/cm 2 when tested immediately after application. However, their repellencies decreased when tested after 15 or 30 min. A. altissima and Z. armatum exhibited significantly lower repellency compared to all other essential oils or the positive control ( Figure 3). The essential oil of M. longifolia proved best in repellency at a dose of 166.5 μg/cm 2 and showed 33% repellency after 45 min of treatment, which was significantly higher than that of all other essential oils, but significantly lower than that of DEET ( Figure 3).    GLM analyses revealed significant effects of EO type (df = 4, F = 8625, p < 0.001) and time (df = 4, F = 8559, p < 0.001) on the repellency effect. Four out of six plants' essential oils exhibited 100% repellency at a dose of 166.5 µg/cm 2 when tested immediately after application. However, their repellencies decreased when tested after 15 or 30 min. A. altissima and Z. armatum exhibited significantly lower repellency compared to all other essential oils or the positive control ( Figure 3). The essential oil of M. longifolia proved best in repellency at a dose of 166.5 µg/cm 2 and showed 33% repellency after 45 min of treatment, which was significantly higher than that of all other essential oils, but significantly lower than that of DEET ( Figure 3).   aegypti. Bars having different letters depict significant differences (p < 0.05) among the repellency of different tested substances at different periods of post-treatment that were compared for each period independently by ANOVA post hoc Tukey test. Error bars denote the standard error (n = 5).

Time Span
Repellency at a Dose of 330 µg/cm 2 GLM analyses showed significant effects of EO type (df = 6, F = 2803, p < 0.001) and time (df = 5, F = 11813, p < 0.001) on the repellency effect. All the tested plants showed 100% repellency towards female Ae. aegypti when tested immediately after application, except for A. altissima essential oil, which showed 96.5% repellency (Figure 4). Furthermore, DEET provided complete protection for 45 min, while E. bonariensis, E. canadensis, and S. imbricata showed complete repellency until 30 min, against Ae. aegypti. However, A. altissima showed repellency for up to only 30 min (Figure 4). At this dose, the most active plant essential oils were M. longifolia, S. imbricata, E. bonariensis, and E. canadensis, which exhibited 70% or higher repellence for more than 45 min. After 75 min post-treatment, the repellency of DEET declined to 79%, M. longifolia, S. imbricata, E. bonariensis, and E. canadensis essential oils showed 7-22% repellency, whereas M. longifolia exhibited significantly higher repellence (p < 0.05) compared to all essential oils (Figure 4). A. altissima showed repellency for up to only 30 min (Figure 4). At this dose, the most active plant essential oils were M. longifolia, S. imbricata, E. bonariensis, and E. canadensis, which exhibited 70% or higher repellence for more than 45 min. After 75 min post-treatment, the repellency of DEET declined to 79%, M. longifolia, S. imbricata, E. bonariensis, and E. canadensis essential oils showed 7-22% repellency, whereas M. longifolia exhibited significantly higher repellence (p < 0.05) compared to all essential oils ( Figure  4).

Larvicidal Activity of Essential Oils
All the tested essential oils showed larvicidal effects against the second-instar larvae of Ae. aegypti. There was no statistically significant difference among toxicity of E. canadensis, Z. armatum, M. longifolia, and E. bonariensis but their toxicities were significantly different from that of S. imbricata and A. altissima based on non-overlapping of fiducial limits after 24 and 48 h of post-treatment (Table 2). Furthermore, there was also a significant difference between the toxicity of S. imbricata and A. altissima based on non-overlapping of the fiducial limits after 24 and 48 h of post-treatment. The LC50 value of E. bonariensis was 28.48 mg/L after 24 h of larvae exposure which decreased to 26.03 mg/L after 48 h of post-treatment ( Table 2). The tested larvae showed the least susceptibility, statistically, towards the exposure of A. altissima as compared to all the tested essential oils (Table 2).

Larvicidal Activity of Essential Oils
All the tested essential oils showed larvicidal effects against the second-instar larvae of Ae. aegypti. There was no statistically significant difference among toxicity of E. canadensis, Z. armatum, M. longifolia, and E. bonariensis but their toxicities were significantly different from that of S. imbricata and A. altissima based on non-overlapping of fiducial limits after 24 and 48 h of post-treatment (Table 2). Furthermore, there was also a significant difference between the toxicity of S. imbricata and A. altissima based on non-overlapping of the fiducial limits after 24 and 48 h of post-treatment. The LC 50 value of E. bonariensis was 28.48 mg/L after 24 h of larvae exposure which decreased to 26.03 mg/L after 48 h of post-treatment ( Table 2). The tested larvae showed the least susceptibility, statistically, towards the exposure of A. altissima as compared to all the tested essential oils (Table 2).

Composition of Essential Oils
Piperitone oxide (45.5%), piperitenone oxide (30.1%), and limonene (4.6%) were the most abundant compounds in the M. longifolia essential oil. The major compounds in S. imbricata essential oil were 20% camphor, 39.9% carvone, and 6.9% piperitone, which constituted about 70% of the oil ( Table 3). The E. bonariensis essential oil comprised trans-βfarnesene (10.2%), cis-lachnophyllum ester (24.9%), and matricaria ester (43.1%), whereas the major compounds of E. canadensis were limonene (28.4%), cis-lachnophyllum ester (16.3%), and matricaria ester (31.7%). The most abundant compounds in the essential oil of A. altissima were eugenol (24.4%) methylugenol (16.5%) and capillin (19.3%), comprising 60.2% of the essential oil (Table 3).  RI: retention index of a separated compound, which was calculated relative to the retention time of C 9 -C 26 hydrocarbons using DB-5 gas chromatographic column, and the same parameters were applied for analyses of essential oils. CAS Chemical Abstract Service.* Identification of compounds was verified by comparing mass spectrum and retention index with those recorded from the injection of standard compounds. # CAS number of this Piperitenone oxide was not found. The data shown in table are approximate relative compositions, expressed as %, where tr stands for trace amount < 0.1%.

Discussion
Products derived from plants can be used as repellents against mosquitoes. However, their potential varies, depending upon their chemical compounds [36,37]. In the present study, essential oils of six aromatic plants, including M. longifolia, S. imbricata, E. bonariensis, E. canadensis, A. altissima, and Z. armatum, were assessed for their repellent and larvicidal effects against Ae. aegypti. All the essential oils showed repellency and larvicidal effects against adult females and second-instar larvae of Ae. aegypti, respectively. The essential oils which showed strong repellency at a dose of 33.3 µg/cm 2 were further investigated for their longevity at tested at doses of 33.3 µg/cm 2 , 166.5 µg/cm 2 , and 330 µg/cm 2 .
The essential oil of M. longifolia showed the highest repellency at the lowest tested dose; moreover, these samples showed the most prolonged activity in the time span repellency bioassay. A previous study reported the repellent effect of M. longifolia essential oil for 65 min against Culex pipiens at a tested dose of 1 µL/cm 2 (approx. 1000 µg/cm 2 ), where the major compounds were 74.9% pulegone, 6.6% menthone, and 7.4% 1-8-cineole [38]. A previous study from Pakistan reported 68% repellent activity of M. longifolia against Sitophilus oryzae [39]. M. longifolia has also proven very effective against Sitophilus zeamais and showed 100% repellency [40]. Motazedian et al. [41] demonstrated that the essential oil of M. longifolia possessed killing and repellent ability against Tetranychus urticae. Koc et al. [42] reported the repellent effect (73.8%) of M. longifolia against Ochlerotatus caspius. The study of Saeidi and Moharramipour [43] also demonstrated the repellence activity of M. longifolia against Tribolium confusum.
In our study, 45.5% piperitone oxide and 30% piperitenone oxide were the major components of the M. longifolia essential oil and possibly contributedtowards the higher repellency of this EO against Ae. aegypti. Furthermore, the lower volatility of these compounds could be the reason behind the long-lasting repellency. Previously, essential oils with trans-piperitone oxide have shown toxic effects against Cx. pipiens [44]. In previous studies, piperitenone oxide has been proven as an excellent repellent against Anopheles stephensi [45] and Ae. albopictus [46]. Though the repellency of piperitone oxide against Ae. albopictus was moderate, its combined effect was significant in the case of essential oil, which contained 23% piperitone oxide and 41% piperitenone oxide [46]. A study from India reported the presence of 32.4% piperitone oxide and 41.5% piperitenone oxide in Plectranthus incanus essential oil that showed excellent repellency against Anopheles stephensi and Culex fatigans [47]. Thus, the synergetic effects of different components of M. longifiolia essential oil make it a potent repellent for Ae. aegypti.
The essential oil of E. bonariensis did not show good activity at the lowest tested dose. However, it showed 100% repellency against the tested population of mosquitoes at higher doses, such as 166.5 µg/cm 2 and 333 µg/cm 2 . Matricaria ester, cis-lachnophyllum ester, and trans-β-farnesene were the most abundant compounds in the essential oil of E.
bonariensis. The presence of these major compounds along with others could be the reason for prolonged repellency at the higher concentrations. Previously, matricaria ester has shown lethal effects on Heliothis virescens moths [48]. The presence of matricaria ester might contribute towards the repellency of E. bonariensis against Ae. aegypti.
The essential oil of E. canadensis showed excellent repellency at the tested doses of 166.5 µg/cm 2 and 333 µg/cm 2 . Interestingly, in our previous study, this plant's essential oil showed about 85% repellence at 33 µg/cm 2 [29], whereas in the present study the essential oil of this plant species showed about 42% repellence at a similar dose. The difference in bioactivity could be attributed to the chemistry of the essential oils, as the plant samples from each study were collected from different locations. In the current study, cis-lachnophyllum ester (16.3%), limonene (28.4%), and matricaria ester (31.7%) were the most abundant compounds in the essential oils of E. canadensis, whereas Azeem et al. [29] reported results of 41.3% limonene, 10.3% of each of germacrene D and matricaria ester, and 6.5% cis-lachnophyllum ester. From the comparison of both studies, it could be concluded that plants growing on different soil types could have different chemistries and hence, varied bioactivity. The essential oil of E. canadensis also showed strong larvicidal potential against Ae. aegypti having LC 50 of 35.75 mg/L. Another study from Vietnam described that E. canadensis essential oil possessed strong insecticidal activity against three different species of mosquitoes including Ae. aegypti (LC 50 9.80 mg/L) and Ae. albopictus (LC 50 = 18.0 mg/L), indicating the toxic effect of E. canadensis [49]. The difference in LC 50 values against Ae. aegypti in the previous and current studies might be due to a difference in the chemical composition of E. canadensis.
A. altissima showed quite good repellency against Ae. aegypti at higher tested doses, albeit for a shorter period. A previous study from China has also demonstrated the repellent effects of A. altissima against four stored grain pests: Tribolium castaneum, Oryzaephilus surinamensis, Sitophilus oryzae, and Liposcelis paeta [57]. A. altissima showed insecticidal properties against Sitophilus zeamaise [58]. The high volatility and absence of pungent smell in the components of A. altissima might contribute towards repellency for short period against Ae. aegypti. Furthermore, in the present study eugenole (24.4%), capillin (19.3%), and methyleugenole (16.5%) were the major constituents of A. altissima, while in a previous study the main constituents of A. altissima were apocarotenoids (17.2%), oxygenated sesquiterpenes (42.1%) caryophyllene oxide (22.7%) [59]. In another study, the main compounds of A. altissima were α-curcumene, α-gurjunene, γ-cadinene, α-humulene β-caryophyllene, caryophyllene oxide, and germacrene D [60]. The change in a major chemical compound of A. altissima in the present study and previous studies might be due to a change in the location of plants of A. altissima.
In the larvicidal bioassays, the LC 50 results depicted the second-instar larvae of Ae. aegypti to be more sensitive to the essential oils of E. bonariensis, M. longifolia, E. canadensis, and Z. armatum, as compared to those of S. imbricata and A. altissima. The LC 50 value for E. bonariensis was 28.28 mg/L and 26.03 mg/L after 24 h and 48 h exposure, respectively. The presence of major compounds, such as matricaria ester and cis-lachnophyllum ester with high toxicity, might be contribute to the highest larvicidal activity, but the effects of other major and minor compounds cannot be ruled out. In a previous study from Vietnam, the Conyza (Syn: Erigeron) bonariensis essential oil exhibited LC 50 values of 69.71 mg/L and 63.85 mg/L after 24 h and 48 h exposure, respectively [49], results which differ from the data presented here. The reason for this clear difference could be due to the difference in the chemical composition reported in the two different studies.
The LC 50 value of E. canadensis was 35.7 mg/L after 48 h, which demonstrated good larvicidal potential against Ae. aegypti. Both E. bonariensis and E. canadensis consisted of a similar ratio of major compounds, for example, matricaria ester and cis-lachnophyllum ester. However, there was the one exception of limonene that was present in abundance only in E. canadensis. The slight difference in their bioactivity could be attributed to the difference in this chemical composition. Hoi et al. [49] reported that the LC 50 of E. canadensis essential oil and pure limonene against Ae. aegypti was 6.09 mg/L and 17.43 mg/L, respectively. The larvicidal activity reported by Hoi et al. [49] is higher than that we found in the current study. The difference in bioactivity could be explained based on differences in the chemistry of the essential oils as well as differences in the mosquito populations. Another previous study demonstrated that E. canadensis essential oil exhibited quite good LD 50 of 14.42 mg/10 g rice against adult T. castaneum [28]. The relative proportions of limonene, determined in the studies carried out by Azeem et al. [28] and Hoi et al. [49], were similar.
The LC 50 values for S. imbricata and A. altissima were 124.2 and 333.6 mg/L, respectively. In a previous study, the essential oil of A. altissima proved toxic against aphids, having an LC 50 of 340.06 µg/cm [61]. However, it only showed good toxic effects against C. quinquefasciatus, and Ae. aegypti at the higher concentrations of extracts, like at 75 and 100% [62]. In our study, it showed rather good toxicity towards mosquitoes, as compared to the results of Wallace et al. [62]. This might be due to a change in the chemical composition of A. altissima.
The essential oil of M. longifolia showed strong larvicidal activity (LC 50 39.29 mg/L) against second-instar larvae, in addition to strong deterrence activity against adult female Ae. aegypti. The compounds in M. longifolia might be toxic, which might contribute to the larvicidal activity of M. longifolia. In a previous study, the essential oil of M. longifoliam having pipertenone (43.9%) as a major compound, showed insecticidal activity against T. castaneum (flour beetle) and Callosobruchus maculatus with LC 50 of 13.05 µL/L [63]. In another study, M. longifolia having trans-piperitone epoxide and piperitenone oxide as major compounds provided a toxic effect against the larvae of Cx. pipiens [44].
The essential oil of Z. armatum showed strong larvicidal activity against Ae. Aegypti, having LC 50 of 35.92 mg/L. In the previous study, Z. armatum (monoterpenes as major constituents) revealed insecticidal activity against three mosquito species, including Ae. aegypti (LC 50 54 mg/L), An. stephensi (LC 50 58 mg/L), and Cx. quinquefasciatus (LC 50 49 mg/L) [64]. Previously, Z. armatum (2-undecanone as a major compound) has shown larvicidal activity against An. anthropophagus (LC 50 36 mg/L), An. sinensis (LC 50 38.56) [62], T. castaneum with (LC 50 -25.64 mg/L) [65], and Lasioderma serricorn (LC 50 -13.3 mg/L) [66], showing toxic effects of Z. armatum similar to the toxic effects showed against Ae. aegypti in the present study. The essential oil of S. imbricata showed good larvicidal activity against Ae. Aegypti, having a LC 50 value of 124.2 mg/L, and previously also proved toxic against aphid with LC 50 340 µg/cm 2 [62], and Cx. pipiens with LC 50 = 79.1 µg/mL [67]. The change in the toxic effects of S. imbricata against Ae. aegypti and Cx. pipiens might be due to a difference in the chemical composition in the S. imbricata or due to a difference in the tested species of the mosquito.

Conclusions
The essential oils extracted from M. longifolia, Z. armatum, E. bonariensis, and C. canadensis have the potential to manage Ae. aegypti at the larval stage. M. longifolia, E. canadensis, E. bonariensis, and S. imbricata essential oils exhibited prolonged mosquito-repellent activity against adult female Ae. aegypti. These essential oils could be used to develop cost-effective and efficient mosquito-repellent formulations for personal protection, without harming humans and the environment.  Data Availability Statement: Data presented in this study are available on request from the corresponding authors.