Chemical Composition, Repellent, and Oviposition Deterrent Potential of Wild Plant Essential Oils against Three Mosquito Species

In this study, the chemical composition, repellent, and oviposition deterrent effects of five plant essential oils (EOs) extracted from Lantana camara (Verbenaceae), Schinus terebinthifolia (Anacardiaceae), Callistemon viminalis (Myrtaceae), Helichrysum odoratissimum (Asteraceae), and Hyptis suaveolens (Lamiaceae) were evaluated against Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus. When tested at 33.3 µg/cm2, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum were effective repellents against Ae. aegypti (89%, 91%, 90%, and 51% repellency, respectively), but they were less repellent against An. gambiae (66%, 86%, 59%, and 49% repellency, respectively). Interestingly, L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum exhibited 100% repellency against Cx. quinquefasciatus at 33.3 μg/cm2. In time-span bioassays performed at 333 μg/cm2, the EO of L. camara exhibited 100% repellence against Ae. aegypti and An. gambiae for up to 15 min and against Cx. quinquefasciatus for 75 min. The oviposition bioassays revealed that L. camara exhibited the highest activity, showing 85%, 59%, and 89% oviposition deterrence against Ae. aegypti, An. gambiae, and Cx. quinquefasciatus, respectively. The major compounds of L. camara, S. terebinthifolia, and C. viminalis were trans-β-caryophyllene (16.7%), α-pinene (15.5%), and 1,8-cineole (38.1%), respectively. In conclusion, the L. camara and S. terebinthifolia EOs have the potential to be natural mosquito repellents.


Introduction
Mosquitoes are a significant public health concern because they can serve as vectors of dengue fever, filariasis, Japanese encephalitis, chikungunya, malaria, and yellow fever [1].The Aedes mosquitos are known for their painful and persistent bites and can transmit pathogens causing dengue, chikungunya, Zika, and yellow fever diseases [2].The Anopheles mosquitos are considered the primary vectors of diseases such as filariasis and malaria, with an estimated 300-500 million malaria cases yearly [3].The Culex mosquitoes are the vectors of harmful diseases affecting birds and humans.These can serve as vectors of lymphatic filariasis, Saint Louis encephalitis, West Nile virus in humans, and avian malaria in birds [4].
Synthetic chemicals, while commonly used to control vector mosquitoes, have shown a decrease in efficacy against many species of mosquitoes due to their long-term use.Moreover, they pose risks to mammals, human health, and the environment [5,6].In addition to chemical insecticides, mosquito repellents are also used to prevent mosquito bites.Since the late 1950s, DEET (N, N-diethyl-meta-toluamide) has been widely used as a common mosquito repellent [7], and some adverse effects, including skin irritation, encephalopathy in children, and allergic reactions have been reported [8,9].Considering the harmful effects of synthetic insecticides and repellents, there is a need to develop phytochemical-based products to control these vectors of deadly diseases.
Plant-based products are considered safe to use and have either no harmful effects or low harmful effects on humans and the environment [10].Several plant-based materials have proven extremely effective against blood-sucking insects, showing good repellent and insecticidal potential against mosquitoes [11,12].Previously, several studies demonstrated the effectiveness of plant-derived essential oils (EOs) against mosquitoes (Table S1).Java citronella oil has been registered by the United States Environmental Protection Agency as an insect repellent due to its efficacy and low toxicity [13].Another example is a repellent formulation derived from the leaves of the Australian lemon-scented gum tree Corymbia citriodora, in which one of the active ingredients is a monoterpene p-menthane-3,8diol (PMD).In April 2005, the U.S. Centers for Disease Control and Prevention endorsed two non-DEET mosquito repellents, including PMD [14].The estimated market value for plant-based chemicals is $700 million, or 45,000 tons, of the global pesticide production.Furthermore, these can be used without harming people or the environment because of their biodegradable nature [15].Given the activity of plant EOs against harmful insects, the current study aimed to examine the repellent and oviposition deterrent potential of EOs from five plant species, including Lantana camara L. (Lamiales: Verbenaceae), Schinus terebinthifolia Raddi (Sapindales: Anacardiaceae), Callistemon viminalis Don.(Myrtales: Myrtaceae), Helichrysum odoratissimum (L.) (Asterales: Asteraceae), and Hyptis suaveolens (L.) (Lamiales: Lamiaceae), against adult female mosquitoes of three species (Diptera: Culicidae): Aedes aegypti (L.), Anopheles gambiae s. l.Giles, and Culex quinquefasciatus Say.

Yield (%) of EOs
The aerial parts of H. odoratissimum yielded the highest amount of EO, while the S. terebinthifolia leaves consisted of the least amount (Table 1).

Repellency Results
An ANOVA revealed that the EO type significantly affected repellency against Ae.aegypti (df = 7, F = 586, p < 0.001), An. gambiae s. l. (df = 7, F = 973, p < 0.001), and Cx.quinquefasciatus (df = 7, F = 1634, p < 0.001) females when tested at a dose of 33.3 µg/cm 2 .DEET showed 100% repellence towards mosquitoes of all three species.The EOs of L. camara, S. terebinthifolia, and C. viminalis showed similar repellency (p > 0.05) of around 90% and were the most efficient after DEET, followed by the EO of H. odoratissimum, which displayed 52% repellent activity (Figure 1).The EO of H. suaveolens revealed significantly lower repellency (5.6%) against Ae.aegypti females.The EO of S. terebinthifolia was the second most repellent formulation against An.gambiae after DEET.The EOs of L. camara, S. terebinthifolia, C. viminalis, H. odoratissimum, and the positive control DEET showed 100% repellency against Cx.quinquefasciatus, while the EO distilled from H. suaveolens was the least efficient against all the tested mosquito species (Figure 1).Overall, the strongest repellent response was observed for Cx.quinquefasciatus compared to Ae. aegypti and An.gambiae females.
S. terebinthifolia, C. viminalis, H. odoratissimum, and the positive control DEET showed 100% repellency against Cx.quinquefasciatus, while the EO distilled from H. suaveolens was the least efficient against all the tested mosquito species (Figure 1).Overall, the strongest repellent response was observed for Cx.quinquefasciatus compared to Ae. aegypti and An.gambiae females.
In the screening bioassay with a 1% w/v solution (33.3 µg/cm 2 tested dose), the EOs of L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum showed more than 50% repellency against all mosquito species and were further investigated to determine the maximum period of repellent activity.

Figure 1.
Repellency of positive control DEET and EOs at 33.3 µg/cm 2 against Aedes aegypti, Anopheles gambiae s. l., and Culex quinquefasciatus mosquito females.Lowercase letters above the columns represent significant differences (p < 0.05) among tested EOs against particular mosquito species, while uppercase letters represent the significant difference (p < 0.05) among mosquitoes towards a particular EO, according to the ANOVA post-hoc Tukey test for each mosquito species and each EO separately.Error bars represent the standard error (n = 5).

Time-Span Repellency against Ae. aegypti Mosquito Females
The statistical analysis of the data revealed a significant impact of the type of EO on repellency against Ae.aegypti at doses of 33.3 µg/cm 2 (df = 4, F = 138, p < 0.001), and 333 µg/cm 2 (df = 4, F = 187, p < 0.001).DEET was the most efficient repellent at all tested time points.The EOs of L. camara and S. terebinthifolia were active over 45 min, displaying an extended repellent activity compared to the EO of C. viminalis, which was active for over 30 min.The EO of H. odoratissimum showed 50% repellency, which registered for 15 min (Figure 2A).At the dose of 333 µg/cm 2 , DEET, as well as the EOs of L. camara, S. terebinthifolia, and C. viminalis, showed statistically similar repellency (p > 0.05) when tested immediately after application (Figure 2B).The EOs of L. camara and S. terebinthifolia remained repellent above the 50% level for over 60 min and exhibited over 10% activity for 105 min and 90 min, respectively, while the repellent activity of the positive control DEET remained at 34% after 105 min.The shortest repellency period was observed for the EO of H. odoratissimum (Figure 2B).In the screening bioassay with a 1% w/v solution (33.3 µg/cm 2 tested dose), the EOs of L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum showed more than 50% repellency against all mosquito species and were further investigated to determine the maximum period of repellent activity.

Time-Span Repellency against Ae. aegypti Mosquito Females
The statistical analysis of the data revealed a significant impact of the type of EO on repellency against Ae.aegypti at doses of 33.3 µg/cm 2 (df = 4, F = 138, p < 0.001), and 333 µg/cm 2 (df = 4, F = 187, p < 0.001).DEET was the most efficient repellent at all tested time points.The EOs of L. camara and S. terebinthifolia were active over 45 min, displaying an extended repellent activity compared to the EO of C. viminalis, which was active for over 30 min.The EO of H. odoratissimum showed 50% repellency, which registered for 15 min (Figure 2A).At the dose of 333 µg/cm 2 , DEET, as well as the EOs of L. camara, S. terebinthifolia, and C. viminalis, showed statistically similar repellency (p > 0.05) when tested immediately after application (Figure 2B).The EOs of L. camara and S. terebinthifolia remained repellent above the 50% level for over 60 min and exhibited over 10% activity for 105 min and 90 min, respectively, while the repellent activity of the positive control DEET remained at 34% after 105 min.The shortest repellency period was observed for the EO of H. odoratissimum (Figure 2B).

Figure 2. Repellent longevity of DEET and four EOs against
Aedes aegypti females at the tested doses of 33.3 µg/cm 2 (A) and 333 µg/cm 2 (B).Different letters on bars indicate significant differences (p < 0.05) between the repellency of different substances tested after specified time intervals independently, according to the ANOVA post-hoc Tukey test."SE" stands for standard error (n = 5).

Time-Span Repellency against An. gambiae s. l. Mosquito Females
An ANOVA revealed that the EO type significantly affected repellency at doses of 33.3 µg/cm 2 (df = 4, F = 331, p < 0.001), and 333 µg/cm 2 (df = 4, F = 210, p < 0.001).The EO of S. terebinthifolia showed over 50% repellent activity, while the efficiency of the second most repellent EO, distilled from the L. camara plants, decreased from 66.2% to 8.8% 30 min after post-treatment (Figure 3A).The EOs of C. viminalis and H. odoratissimum showed the shortest repellent effect and were active for only up to 15 min.The positive control DEET displayed 100% repellency, which lasted for over 45 min.
At the higher dose of 333 µg/cm 2 , the EOs of L. camara, S. terebinthifolia, and C. viminalis showed statistically similar repellence (p > 0.05) just after the treatment compared to  An ANOVA revealed that the EO type significantly affected repellency at doses of 33.3 µg/cm 2 (df = 4, F = 331, p < 0.001), and 333 µg/cm 2 (df = 4, F = 210, p < 0.001).The EO of S. terebinthifolia showed over 50% repellent activity, while the efficiency of the second most repellent EO, distilled from the L. camara plants, decreased from 66.2% to 8.8% 30 min after post-treatment (Figure 3A).The EOs of C. viminalis and H. odoratissimum showed the shortest repellent effect and were active for only up to 15 min.The positive control DEET displayed 100% repellency, which lasted for over 45 min.
that of DEET, while after a 15 min period, the efficiency of the C. viminalis and H. odoratissimum EOs dropped significantly (p < 0.05) compared to those of DEET and L. camara.The EOs of L. camara and S. terebinthifolia exhibited an active time span of 60 min, whereas the repellent effect of the C. viminalis EO lasted for 45 min (Figure 3B).At the higher dose of 333 µg/cm 2 , the EOs of L. camara, S. terebinthifolia, and C. viminalis showed statistically similar repellence (p > 0.05) just after the treatment compared to that of DEET, while after a 15 min period, the efficiency of the C. viminalis and H. odoratissimum EOs dropped significantly (p < 0.05) compared to those of DEET and L. camara.The EOs of L. camara and S. terebinthifolia exhibited an active time span of 60 min, whereas the repellent effect of the C. viminalis EO lasted for 45 min (Figure 3B).

Time-Span Repellency against Cx. quinquefasciatus Mosquito Females
There was a significant difference in the repellent effects of essential oils against Cx.quinquefasciatus at the doses of 33.3 µg/cm 2 and 333 µg/cm 2 .At 33.3 µg/cm 2 , L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum EOs showed a repellent effect similar to that of DEET (p > 0.05) immediately after application.The EOs of L. camara and S. terebinthifolia exhibited long-lasting repellent effects until 120 min and 90 min, respectively, and statistically similar repellent efficiency (p > 0.05) to that of DEET for 45 min and 30 min, respectively.The EOs of C. viminalis and H. odoratissimum had a lower repellent effect that lasted up to 60 min and 45 min, respectively (Figure 4A).The application of EOs at the higher dose of 333 µg/cm 2 extended the active time span of L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum for up to 180 min, 120 min, 90 min, and 90 min, respectively.The positive control DEET and the EO of L. camara showed 100% repellency for 75 min, after which the efficiency of both substances started to decrease at different rates, resulting in a significantly higher repellency of DEET (Figure 4B).

Time-Span Repellency against Cx. quinquefasciatus Mosquito Females
There was a significant difference in the repellent effects of essential oils against Cx quinquefasciatus at the doses of 33.3 µg/cm 2 and 333 µg/cm 2 .At 33.3 µg/cm 2 , L. camara, S terebinthifolia, C. viminalis, and H. odoratissimum EOs showed a repellent effect similar t that of DEET (p > 0.05) immediately after application.The EOs of L. camara and S. tere binthifolia exhibited long-lasting repellent effects until 120 min and 90 min, respectively and statistically similar repellent efficiency (p > 0.05) to that of DEET for 45 min and 3 min, respectively.The EOs of C. viminalis and H. odoratissimum had a lower repellent effec that lasted up to 60 min and 45 min, respectively (Figure 4A).The application of EOs a the higher dose of 333 µg/cm 2 extended the active time span of L. camara, S. terebinthifolia C. viminalis, and H. odoratissimum for up to 180 min, 120 min, 90 min, and 90 min, respec tively.The positive control DEET and the EO of L. camara showed 100% repellency for 7 min, after which the efficiency of both substances started to decrease at different rates resulting in a significantly higher repellency of DEET (Figure 4B).

Oviposition Deterrence of EOs
An ANOVA revealed that the EO type significantly affected the oviposition of Ae. aegypti (df = 4, F = 360, p < 0.001), An. gambiae s. l. (df = 4, F = 348, p < 0.001), and Cx.quinquefasciatus (df = 4, F = 968, p = 0) females.The EOs of L. camara, S. terebinthifolia, C. viminalis, and H. odoratissimum showed the same pattern of oviposition deterrence towards the females of Ae. aegypti, An. gambiae s. l., and Cx.quinquefasciatus, and the efficiency of these substances differed significantly from each other (Figure 5).The L. camara EO showed a higher oviposition deterrence against Cx.quinquefasciatus, followed by Ae. aegypti and An.gambiae.In the case of the S. terebinthifolia, C. viminalis, and H. odoratissimum EOs, a higher oviposition deterrence was observed against Ae.aegypti, and Cx.quinquefasciatus, as compared to An. gambiae (Figure 5).The EO of H. suaveolens showed the least oviposition deterrence towards the tested species of mosquitoes (Figure 5).

Discussion
Plant-based products can be used as repellents against mosquitoes.Repellent formulations derived from EOs have gained the attention of consumers and are considered safe to use compared to synthetic repellents.In the present study, five aromatic plant EOs were investigated for their mosquito repellent and oviposition deterrent activities against the females of three mosquito species.Overall, the host-seeking and oviposition behaviour of Cx. quinquefasciatus females was highly affected when exposed to the different EOs compared to Ae. aegypti and An.gambiae s. l. females, with An. gambiae s. l. showing the most resistance towards the tested EOs.The difference in the behavioural response of different mosquito species towards the same substance could be explained based on the presence of mismatched types of chemoreceptors in mosquito species.The findings of the current study are consistent with previously published reports, where mosquito species behaved differently when tested against the same substances [16,17].Similar results were also found when three different stored-grain pest insects were exposed to Artemisia sieberi essential oil [18].Previously, Amer and Mehlhorn [16] observed that the Cx.quinquefasciatus mosquitoes were found to be more sensitive to EOs compared to Ae. aegypti.Kweka, et al. [19] also demonstrated that the arms of volunteers treated with propylene glycol carbonate and DEET had a higher protective efficacy against Cx.quinquefasciatus compared to An. gambiae.A previous study demonstrated that the differential behavioural responses of different mosquito species towards the same test substances might be due to distinct sensitivities of their chemoreceptors [16].
Among all tested EOs, L. camara EO showed the highest repellency against all three mosquito species for an extended period of time.The effective and prolonged repellent activity of this EO could be attributed to the additive effect of β-caryophyllene, sabinene, 1,8-cineole, α-humulene, and nerolidol.However, the synergistic effects of the abovementioned compounds with the minor compounds could not be ruled out.Similarly, a previous study reported that β-caryophyllene and nerolidol showed some repellency towards female Ae.aegypti when tested individually, whereas the combination of β-caryophyllene and nerolidol exhibited an excellent mosquito-repellent activity for an extended period of time [20].To our knowledge, very few studies have described the mosquito-repellent activity of the L. camara EO or extracts.For example, L. camara flowers extracted in coconut oil exhibited 94% repellence against Ae.aegypti and Ae.albopictus; however, the concentration of the test substance was not given [21].Keziah, et al. [22] reported that the hexane, ethyl acetate, and methanolic extracts of L. camara leaves exhibited 100% repellency towards Ae.aegypti females for 60 min when tested at 8000 µg/cm 2 .In another study, the solvent extracts and EO of L. camara showed repellent activity against Ae.aegypti, lasting for 225 min at a dose of 1660 µg/cm 2 [23].In the current study, the L. camara EO exhibited 100% protection for 15 min and 75 min against Ae.aegypti and Cx.quinquefasciatus, respectively, when tested at 333 µg/cm 2 .Here, we used a concentration five times lower compared to that of Bhargava et al. [23] and 24 times lower than that of Keziah et al. [22] but still, the bioactivity was comparable to the previous results.The difference in the bioactivity of the plant species could be explained based on the extraction method, the specific chemistry of the extracts due to the chemical variation within species, and the concentration of the tested products.Many studies have shown that the concentration of a substance plays a significant role in determining its bioactivity [24][25][26][27].
The EO of S. terebinthifolia provided complete protection against Ae.aegypti and Cx.quinquefasciatus for 15 and 45 min, respectively; however, it did not show complete protection against An.gambiae at any tested concentration.The observed repellent activity of S. terebinthifolia EO could be associated with the synergistic effect of diverse types of compounds, including α-pinene, limonene, α-phellandrene, p-cymene, spathulenol, elixene, and β-elemene.There is no previous study reporting the mosquito repellent activity of the S. terebenthifolia leaves EO.However, a previous study from Saudi Arabia demonstrated that the EO extracted from S. terebinthifolia fruits provided complete protection against Cx.pipiens for 120 min at a concentration of 0.50 µL/cm 2 (~500 µg/cm 2 ), whereas the nanoemulsion made from the same EO provided complete protection against Cx.pipiens for 150 min [28].The major compounds in the S. terebinthifolia fruit EO were 31.2%3-carene, 15.3% α-pinene, 11.2% α-phellandrene, 9.3% limonene, and 3.7% α-terpineol [28].This composition was quite different from the chemical composition of the EO reported in the current study.The difference in the efficacy of S. terebinthifolia EOs reported in the current study and previously could be explained based on their different chemistry, probably due to the EOs being extracted from different parts of the plant, as well as the plants being grown under different conditions [29].In addition, mosquito species respond differently to the same substance, which might explain the difference in bioactivity of S. terebinthifolia against the mosquito species tested in the current and previous studies.The fact that a substance can have different biological effects against different organisms is well documented [18].
The EO of C. viminalis showed the third-best repellent activity after L. camara and S. terebinthifolia against the Ae.aegypti, An. gambiae, and Cx.quinquefasciatus.The repellent activity of the C. viminalis EO was found to be dose-dependent and exhibited the best activity against all mosquito species at higher concentrations.In addition, its repellency against Cx.quinquefasciatus was long-lasting compared to the other two mosquito species.The most abundant compounds in the EO of C. viminalis were 1,8-cineole, α-pinene, pcymene, and α-terpineol, which could be the reason for the good, but short-term repellent activity of C. viminalis.1,8-cineole, α-pinene, and p-cymene are quite volatile by nature, so they would evaporate faster from the subject's hand, leading to a loss of repellency after some time.The repellent activity of the C. viminalis EO against mosquitos can be deduced from work where the major compounds of C. viminalis were studied for their repellency against different mosquito species.For example, Klocke, et al. [30] reported that 1,8-cineole was a mild mosquito-feeding repellent against Ae.aegypti.Other studies showed that α-pinene possessed moderate repellent activity against An.gambiae [31] and Ae.albopictus [32].A thorough literature search showed that C. viminalis has not been investigated for its repellent activity against mosquitoes; however, there are some reports of the insecticidal activities of the C. viminalis EO or extracts against pest insects.Yadav, et al. [33] showed that isopropanol, methanol, and acetone extracts of C. viminalis dried leaves acted as larvicides, with an LC 50 of 71-115 ppm against the early third instar larvae of Ae. albopictus.In another study, the EO of C. viminalis decreased the growth rate of Tribolium confusum [34].Other work on the EO of C. viminalis showed insecticidal activity against aphids [35].In addition, a study from South Africa reported the insecticidal activity (LC 50 = 0.019 µL/cm 3 ) of the C. viminalis EO against Callosobruchus maculatus [36].
In the current study, the H. suaveolens EO showed the lowest repellence against the tested species of mosquitoes.Some studies reported the repellency activity of leaves EOs against Ae.albopictus [41], An. gambiae, and Cx.quinquefasciatus [42] mosquitoes.
Oviposition deterrents are considered an important tool in controlling mosquitoes.The current study's results revealed that the EO of L. camara proved to be an excellent oviposition deterrent against Ae.aegypti, An. gambiae, and Cx.quinquefasciatus, compared to other tested EOs.The L. camara EO exhibited the highest deterrent effect against Cx.quinquefasciatus and the lowest effect against An.gambiae.The difference in oviposition deterrence towards different species of mosquitoes could be explained based on their chemoreceptor's responses.Previously, the oviposition deterrent activity of L. camara has been observed against An.gambiae, where the EO of L. camara showed 98% oviposition deterrence when the egg laying media was treated with a 1 mL of 1000 ppm (1000 µg/mL) EO solution [53].In the current study, however, 59% oviposition deterrence was observed against An.gambiae when a 300 µg/cm 2 concentration was employed.The difference in the oviposition deterrent activity reported in the current and previous studies might be due to differences in the chemical compounds of EOs, differences in conducting the bioassay, as well as the tested concentration.It is well-documented that concentration plays an important role in determining the bioactivity of any substance.The EOs of S. terebinthifolia, C. viminalis, and H. odoratissimum also showed promising oviposition deterrence against Ae.aegypti, An. gambiae, and Cx.quinquefasciatus.Interestingly, all these EOs significantly affected the oviposition behaviour of Cx. quinquefasciatus, compared to other mosquito species, whereas An. gambiae was found to be less affected by the presence of these EOs.We know of no previous study on the oviposition deterrence effect of S. terebinthifolia, C. viminalis, and H. odoratissimum.However, there are studies where the major compounds of these EOs were tested for oviposition deterrence against some mosquito species.For example, limonene, β-pinene, and 1,8-cineole are proven oviposition deterrents of Cx. quinquefasciatus, Spodoptera litura, and Chilo partellus [54,55].Female mosquitoes select the most suitable oviposition sites for the best fitness of their offspring [56,57].The antennae of mosquitoes are replete with chemoreceptors that enable the insect to detect air-borne stimuli and assist in locating suitable sites for oviposition [58].This is why female mosquitoes might prefer control cups compared to EOs-treated cups for oviposition.The results of our oviposition deterrence experiments showed that the tested EOs can prevent gravid mosquito females of Ae. aegypti, An. gambiae, and Cx.quinquefasciatus species from laying eggs in treated water.

Collection of Plant Material
Selected parts of L. camara, S. terebinthifolia, C. viminalis, and H. suaveolens were collected from natural populations in different areas of Pakistan.The aerial parts of H. odoratissimum were collected from Masaka, Uganda, during the flowering season.The details of plant material collection are presented in Table 2.The identity of the plants was authenticated by Dr. Abdul Nazir, a plant taxonomist at the Department of Environmental Sciences, COM-SATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan.The plant material was shade-dried and stored at room temperature until used for essential oil extraction.

Extraction of Essential Oils
The steam distillation method was used to extract EOs from the collected plant material by using a Clevenger-type apparatus, as previously described [24].A stainless-steel vessel was loaded with plant material (400 g) and two litres of distilled water was added to the bottom of the vessel to prevent direct contact of the plant material with water.The distillation vessel was heated by using an electric hotplate.Volatile compounds released from the plant material along with steam were cooled using a condenser fitted on the head of the vessel and the distillate was collected in a separating funnel for 3 h.The essential oil above the water layer was decanted and dried over anhydrous MgSO 4 .The extracted EO was weighed, and the percentage yield was calculated using the dry mass of the plant.

Rearing of Mosquitoes
Cultures of Ae. aegypti, An. gambiae s. l., and Cx.quinquefasciatus were reared in the laboratory using methods described in previous studies [12,24,[59][60][61].The larval population of mosquitoes was collected from the Health Department of Multan, Pakistan.Larvae were placed in a plastic container (20 × 16 × 4 cm) filled with 1 L water and fed with fish food (Osaka green fish food, India) containing 3% crude fat, 4% crude fibre, and 28% crude protein.Pupae were collected daily from the larval container and transferred to plastic cups containing 200 mL of tap water.The plastic cups were placed in Plexiglass cages (30 × 30 × 30 cm) for the emergence of adults.Cotton soaked with a 10% sucrose solution was placed in cages as an adult diet.After 4-5 days, females (Ae.aegypti and Cx.quinquefasciatus) were fed blood from a constrained pigeon placed in the adult cage while An.gambiae were fed human arm blood.Wax paper was placed on the inner side of the plastic jar filled with tap water, which was kept in the adult cage for oviposition.After oviposition, the immersed wax paper with eggs was transferred to the larval container, with 1000 mL of tap water for hatching.The rearing process continued until enough adults were obtained for the repellence and oviposition bioassays.The rearing of mosquito species was carried out in separate rooms.All rearing was carried out in a controlled room maintained at 25 ± 2 • C for Ae.aegypti and Cx.quinquefasciatus, while for An.gambiae, the room was maintained at 30 ± 3 • C. The relative humidity in the rearing room was maintained 80 ± 10%, with a photoperiod of 12 h:12 h light/dark.

Mosquito Repellency Bioassay
A human bait technique was used to test the repellence potential of EOs using a 1% w/v solution (33.3 µg/cm 2 dose) and a 10% w/v solution (333 µg/cm 2 dose) against Ae.aegypti, An. gambiae s. l., and Cx.quinquefasciatus females [24].Solutions of each EO and DEET (99% purity, Sigma-Aldrich, St. Louis, MO, USA) were prepared using ethanol as a solvent.The DEET solution was used as a positive control and ethanol was used as a negative control.Twenty mated and blood-starved 4-5 days old female Ae.aegypti were released from the laboratory-reared colony in the experimental cage (30 × 30 × 30 cm) about 12 h before the start of the repellency bioassay.The hands of the subject (volunteer) were washed with scent-free liquid soap and allowed to dry for about 10 min before starting the bioassay.Plastic gloves were used to cover the subject's hand except for the 30 cm 2 circular area on the dorsal side of the hand.A 100 µL aliquot of the above-mentioned solution of the test substance or pure solvent as a negative control was evenly applied on the exposed area of the hand and dried in air for three min before exposing the hand to female Ae.aegypti.The subject's hand was exposed to the female mosquitoes in the experimental cage, and mosquito landings were counted for 5 min.The experiment was repeated randomly five times for both the test samples and the negative control.The same procedure was followed to evaluate the repellency of EOs against An.gambiae and Cx.quinquefasciatus.The human volunteers were informed about the test procedure and consent was obtained before conducting repellence bioassays.The repellency percentage was calculated using the formula: percentage repellency = [(Mc − Mt)/Mc] × 100, where Mc is the number of mosquito landings on the negative control (solvent)-treated hand and Mt is the number of mosquito landings on the test substance-treated hand.The 10th generation of Asian strains of all three mosquito species were employed in this study.

Time-Span Bioassays
Plant EOs that showed at least 50% repellence were further investigated to determine their repellent longevity against all three mosquito species.Time-span repellent bioassays were performed by following the same protocol as mentioned above in the repellency bioassay, except for the exposure of the same treated hand to the females of Ae. aegypti, An. gambiae s. l., or Cx.quinquefasciatus for 5 min after each 15 min time interval until the number of landings on control and treatment did not differ significantly.Time-span bioassays were conducted by using test samples at the dosages of 33.3 µg/cm 2 , and 333 µg/cm 2 .The experiments were repeated five times, and fresh female mosquitoes were employed for each replicate.A repellency bioassay for each mosquito species was conducted in separate climate-controlled rooms.

Oviposition Deterrence
The oviposition deterrence bioassay was conducted by adopting a method described by Soonwera [62].Briefly, sixty 5-7 days old and blood-fed female mosquitoes were placed in a bioassay cage.Two plastic cups filled with 100 mL of distilled water were placed diagonally in the corners of the bioassay cage.One cup (250 mL) served as a test treatment while the other as a control.An aliquot of 300 µL of 10% ethanolic solution of an EO (w/v) was evenly sprayed on one side the wax paper strip (10 × 20 cm), air dried for 2 min, and then wrapped along the inner walls of the water-filled plastic cup, in such a way that the EO-treated area (10 × 10 cm) remained above the water level.The overall concentration of an EO on the treated wax paper was 300 µg/cm 2 .In the control cup, the solvent-treated filter paper was wrapped in the same way described for the test cup.After the application of a sample or solvent, the cups were left outside the cages so that the solvent could evaporate before the commencement of the experiment.The control and sample-treated cups were left in the adult mosquito cage for 48 h for oviposition.Afterwards, the eggs laid were counted in each cup.The position of the control and test cups was changed randomly to avoid the position effects on oviposition.We conducted oviposition tests five times by using a fresh mosquito population in each experiment.

Chemical Analysis of EOs
EOs that showed about 60% or higher repellency against all tested species of mosquitoes were analysed using a Hewlett Packard gas chromatograph connected to a mass spectrometer (GC-MS).The GC was equipped with a 30 m capillary column (DB-5, Agilent Technologies Inc., Santa Clara, CA, USA) with a 0.25 mm internal diameter and a stationary phase film thickness of 0.25 µm.The GC injector temperature was maintained isothermally at 235 • C throughout the sample analysis.The GC oven temperature was programmed as follows: an initial temperature of 40 • C set for 2 min, then increased to 240 • C at a rate of 4 • C per min, and finally maintained at 240 • C for 8 min.Helium was used as the mobile phase, at a constant flow rate of 1 mL/min through the column.An aliquot of 1 µL of dilute essential oil solution was injected into the GC, and the injector was operated in a splitless mode for 30 s.The MS was operated using the following parameters: an electron energy of 70 eV for ionization, an ion source temperature of 180 • C, and a mass spectrum scan range of 30-400 m/z.The total ion chromatogram was used to calculate the percent composition of compounds in EOs.A solution of a series of straight-chain alkanes (C 9 -C 24 ) was injected in the GC-MS using the same parameters as used for the EOs analyses.The retention times of unknown compounds and alkanes were used to calculate the retention indices of separated compounds.The mass spectra and retention indices of separated compounds were initially compared to those available in the NIST-2008 MS library and webbook NIST online library to identify the separated compounds.Finally, identifications were verified by injecting the available pure standard compounds purchased from Sigma-Aldrich (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA, USA).

Statistical Analysis
A one-way ANOVA was used to evaluate statistical differences in the repellence and oviposition deterrent activity of the various EOs, followed by Tukey tests, at a significant threshold of alpha = 0.05 for pairwise comparisons of group means.Statistical analyses were performed using Statistica 8.1 software version 14.0.1.25(TIBCO Software Inc., Palo Alto, CA, USA).

Conclusions
The EOs distilled from L. camara and S. terebinthifolia leaves displayed prolonged repellent activity, as well as greater oviposition deterrent activities against adult females of Ae. aegypti, An. gambiae, and Cx.quinquefasciatus.Cx. quinquefasciatus was found to be the most susceptible to repellency, as compared to Ae. aegypti and An.gambiae.In conclusion, the L. camara and S. terebinthifolia EOs have the potential to be natural mosquito repellents.
Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Bahauddin Zakariya University, Multan.
Informed Consent Statement: Informed consent was obtained from all subjects involved in this study.

Figure 3 .Figure 3 .
Figure 3. Repellent longevity of DEET and four EOs against Anopheles gambiae s. l. females at the tested doses of 33.3 µg/cm 2 (A) and 333 µg/cm 2 (B).Different letters on bars indicate significant difference (p < 0.05) between the repellency of different samples tested after specified time intervals independently, according to the ANOVA post-hoc Tukey test."SE" stands for standard error (n = 5).

Molecules 2024 , 18 Figure 5 .
Figure 5.Oviposition behaviour-modifying effect of five EOs at the tested concentration of 300 µg/cm 2 against Aedes aegypti, Anopheles gambiae s. l., and Culex quinquefasciatus mosquitos.Different lowercase letters above the columns represent significant differences (p < 0.05) between the tested EOs against particular mosquito species independently.Different uppercase letters on the bars represent significant differences (p < 0.05) between the oviposition behaviour of different mosquito species in the presence of a particular EO independently.An ANOVA post-hoc Tukey test was employed for each type of comparison separately.Error bars represent the standard error (n =5).

Figure 5 .
Figure 5.Oviposition behaviour-modifying effect of five EOs at the tested concentration of 300 µg/cm 2 against Aedes aegypti, Anopheles gambiae s. l., and Culex quinquefasciatus mosquitos.Different lowercase letters above the columns represent significant differences (p < 0.05) between the tested EOs against particular mosquito species independently.Different uppercase letters on the bars represent significant differences (p < 0.05) between the oviposition behaviour of different mosquito species in the presence of a particular EO independently.An ANOVA post-hoc Tukey test was employed for each type of comparison separately.Error bars represent the standard error (n =5).

Table 1 .
Percentage yield of EOs.

Table 2 .
Chemical composition of EOs based on the total ion chromatogram of GC-MS.

Table 2 .
Chemical composition of EOs based on the total ion chromatogram of GC-MS.