Sustainable Pest Management Using Novel Nanoemulsions of Honeysuckle and Patchouli Essential Oils against the West Nile Virus Vector, Culex pipiens, under Laboratory and Field Conditions

Essential oils are natural plant products that are very interesting, as they are important sources of biologically active compounds. They comprise eco-friendly alternatives to mosquito vector management, particularly essential oil nanoemulsion. Therefore, the aim of this study is to evaluate the effectiveness of 16 selected essential oils (1500 ppm) in controlling mosquitoes by investigating their larvicidal effects against the larvae and adults of the West Nile virus vector Culex pipiens L. (Diptera: Culicidae); the best oils were turned into nanoemulsions and evaluated under laboratory and field conditions. The results show that honeysuckle (Lonicera caprifolium) and patchouli (Pogostemon cablin) essential oils were more effective in killing larvae than the other oils (100% mortality) at 24 h post-treatment. The nanoemulsions of honeysuckle (LC50 = 88.30 ppm) and patchouli (LC50 = 93.05 ppm) showed significantly higher larvicidal activity compared with bulk honeysuckle (LC50 = 247.72 ppm) and patchouli (LC50 = 276.29 ppm) oils. L. caprifolium and P. cablin (100% mortality), followed by Narcissus tazetta (97.78%), Rosmarinus officinalis (95.56%), and Lavandula angustifolia (95.55%), were highly effective oils in killing female mosquitoes, and their relative efficacy at LT50 was 5.5, 5.3, 5.8, 4.1, and 3.2 times greater, respectively, than Aloe vera. The results of the field study show that the honeysuckle and patchouli oils and their nanoemulsions reduced densities to 89.4, 86.5, 98.6, and 97.0% at 24 h post-treatment, respectively, with persistence for eight days post-treatment in pools. Nano-honeysuckle (100% mortality) was more effective than honeysuckle oils (98.0%). Our results show that honeysuckle and patchouli oils exhibited promising larvicidal and adulticidal activity of C. pipiens.


Introduction
Mosquitoes are widely distributed in temperate, semi-tropical, tropical, and Arctic regions.Globally, there are 41 genera containing more than 3600 different species that have been recognized [1].Because of their high adaptation and sensitivity, mosquitoes quickly adapt to new habitats, climatic change, the structure of forests, and residential developments.Additionally, because of these features or conditions, some species can thrive in environments that were not previously suitable for them, such as artificial containers, pools, or new locations, which helps spread mosquito reproduction and therefore increases the possibility of disease and death rates [2,3].
Some species of mosquito are dangerous to public health because they carry pathogens that make people and animals sick, like the ones that cause malaria, dengue, Zika, chikungunya, Japanese encephalitis, West Nile fever, and yellow fever in people as a result of the bites of a female mosquito [4]; moreover, their bites can result in red rashes, pain, and itching, and scratching them can result in bacterial infection [5].Less than 5% of all mosquitoes in the world are dangerous carriers, and the majority are important components of aquatic and terrestrial ecosystems [5,6].
Culex pipiens (northern house mosquito) is the vector of the West Nile virus (WNV) that causes encephalitis and meningitis.The disease affects the brain tissue, and the most serious cases can result in permanent neurological damage and be fatal [7].There is no vaccine to prevent this infection, nor are there drugs to combat the disease in infected persons; thus, vector control is the most prevalent solution available so far for reducing morbidity.The most widely used vector interruption methods are synthetic and based on insecticides [8].
Synthetic pesticides have helped substantially in ridding areas of insects that spread diseases, but they may cause some very serious negative effects on people and the environment, such as the emergence of dangerous chemical residues and groundwater and soil pollution [9].Moreover, pesticide-resistant insects are becoming more common, which has made it important to look for safer natural alternatives, such as microbial insecticides and botanical pesticides that are effective in controlling many insect populations and that are simultaneously cheap and easily biodegradable, easy to obtain, and do not hurt organisms that are not their targets [10].
Botanical pesticides (essential oils, flavonoids, alkaloids, glycosides, esters, and fatty acids) are natural chemicals and a great alternative to chemical or synthetic pesticides, and they have different chemical properties, modes of action, and effects on insects; they are insect repellents, feed inhibitors or antifeedants, toxic substances, growth inhibitors, chemical enhancers, and attractants [11,12].Therefore, botanical pesticides are used instead of synthetic insecticides in many pest control programs [13][14][15].
Plants and their derivatives have acquired great importance for humanity due to their different pharmacological properties and have been used in curing and treating disease; moreover, they have numerous other applications in different fields.Terpenes are volatile compounds that do not dissolve in water because they are hydrophobic [16].The nano-encapsulation protocol for suitable biopolymers, lipid nanoparticles, and PLGA nanoparticles is one of the best ways to solve this problem, making it easy for natural oils to spread in water.In the presence of an emulsifying agent, oil and water are also mixed to create a nanoemulsion.Due to its small size (less than 100 nm) and good physicochemical properties, nanoemulsion is one of the most useful and widely used nano-systems [17][18][19].Due to their numerous benefits and applications, essential oils and their nano-formulations have recently received substantial attention [16].
One of the promising aromatic plants is honeysuckle (Lonicera caprifolium), which is found in North America and Eurasia, China, and southern Asia [20].It is a perennial flowering plant used in traditional herbal medicine for its antiviral, antibacterial, and antioxidant activities and as a source of ecofriendly larvicides for mosquito control, and it is also used in pharmaceutical industries and medicinal and cosmetic preparations [21][22][23].
The other significant fragrant and therapeutic plant is patchouli (Pogostemon cablin).It is a perennial bushy herb or undershrub that originates in Southeast Asia, India, China, Indonesia, Malaysia, Thailand, and West Africa [24].Patchouli oil has been used as a botanical insecticide on a limited scale against insect pests and possesses antibacterial, antiinflammatory, and anticancer properties, and anti-insecticidal, insect repellant, antifungal, and antibacterial activities [25,26].
The focus is on L. caprifolium and P. cablin plants due to their high efficiency, and due to the lack of extensive information about their chemical compositions, little is known about their insecticidal capabilities.Therefore, our study supports the exploration of their chemical compositions and tests the larvicidal and adulticidal activity of two essential oils in the lab and field before and after their nano-formulation against the West Nile vector C. pipiens.The results of this study provide important information that could lead to the development of new mosquito-killing biopesticides that are safe for the environment.

Screening of Larvicidal Activity for 16 Essential Oils
The larvicidal effects of 16 essential oils were screened against the late third-instar larvae of C. pipiens at 1500 ppm using the dipping technique.The results show that all plant oils had larvicidal activity (60-100% mortality, 24 h PT; 74.4-100% mortality at 48 h PT), and their lethal time (LT 50 ) values ranged from 16.26 h (L.sativa) to 2.86 h and 3.01 h (L.caprifolium and P. cablin, respectively) (Table 1 and Figure 1).The efficacy of the oils could be classified based on the mortality rate (94-100%) at 24 h post-treatment (PT) as a highly effective group, which included four oils: L. caprifolium, P. cablin, and R. officinalis and C. aurantium at 100%, 100%, and 96%, respectively, 24 h PT (Table 1).The LT 50 values of the highly effective oils were 2.86, 3.01, and 3.89 h for L. caprifolium, P. cablin, and R. officinalis, respectively, and the LT 95 values were 13.56, 14.10, and 19.22 h, respectively.The relative effects (REs) of the highly effective group of oils according to LT 50 values were 5.7, 5.4, and 4.2 times greater, respectively, than those of L. sativa (Figure 1).
The other five oils (P.rubra, M. officinalis, A. vera, F. virginiana, and L. sativa) were the least effective.The least effective ones were Fragaria virginiana and L. sativa, which killed 64 and 60% of the flies at 24 h PT, and their LT 50 values were 11.15 and 16.26 h, respectively (Table 1 and Figure 1).
The other five oils (P.rubra, M. officinalis, A. vera, F. virginiana, and L. sativa) were the least effective.The least effective ones were Fragaria virginiana and L. sativa, which killed 64 and 60% of the flies at 24 h PT, and their LT50 values were 11.15 and 16.26 h, respectively (Table 1 and Figure 1).
The results show that most of the oils used in this study exhibited a highly toxic effect, especially after 48 h of treatment, with mortality reaching 100% for L. caprifolium, P. cablin, R. officinalis, C. aurantium, C. schoenanthus, O. majorana, and R. communis and high levels for N. The results show that most of the oils used in this study exhibited a highly toxic effect, especially after 48 h of treatment, with mortality reaching 100% for L. caprifolium, P. cablin, R. officinalis, C. aurantium, C. schoenanthus, O. majorana, and R. communis and high levels for N. tazetta (97.6%),P. graveolens (96.8%),L. angustifolia (95.2%), and P. rubra (94.4%) oils (Table 1).
In addition to one-way analysis of variance (ANOVA) and Duncan's multiple range tests, the Mann-Whitney U test was used to compare the mean differences across groups after the Kruskal-Wallis test to compare the mean differences of more than two groups.However, the Kruskal-Wallis and Friedman's tests revealed that there were meaningful differences between the three groups at various points in time (Table S1).

Larvicidal Activity of Two Effective Oils
The larvicidal effects of oils and their nano-formulations were evaluated against the late third-instar larvae of C. pipiens.The mortality percentage post-treatment at 1500 ppm for 24 h with respect to honeysuckle and patchouli oils and nanoemulsions reached 100%.The LC 50 and LC 95 values were calculated for honeysuckle oil (247.72 and 1254.77ppm, respectively), nano-honeysuckle (88.30 and 282.71 ppm, respectively), patchouli oil (276.29 and 1355.99 ppm, respectively), and nano-patchouli (93.05 and 321.52 ppm, respectively) (Tables 2 and 3).Furthermore, the data show that nano-honeysuckle outperformed honeysuckle oil in terms of mortality, with 95.2% mortality at 250 ppm versus 36.8% mortality relative to the oil.Similarly, the data show that nano-patchouli was more effective than patchouli oil, with mortality rates of 91.2% and 34.4% at 250 ppm, respectively, after 24 h of treatment (Tables 2 and 3).

Adulticidal Activity for Two Effective Oils
The adulticidal effects of oils and their nano-formulations were evaluated against female mosquitoes.The data show that nano-honeysuckle and nano-patchouli were more effective than oils, with 100% mortality at 5% for 24 PT.The LT 50 (50%, median time concentration) values were calculated for nano-honeysuckle and nano-patchouli (13.04 and 14.34 min, respectively) and the corresponding oils (22.93 and 23.93 min, respectively) (Table 8).

A. Particle size distribution and polydispersity measurements
The particle size measurement in a nanoemulsion is a crucial parameter that confirms the particle distribution and motility, which reflect the nanoparticle's stability and half-life [27].The results of dynamic light scattering confirmed that the prepared nanoemulsions were within 100-300 nm, which is very good for drug delivery applications [28].The DLS values of both honeysuckle and patchouli essential oils were 183 and 250 nm, respectively.Such comparable results are consistent with similar results obtained by similar studies [29,30].For a more homogenous solution, the polydispersity index should not exceed 0.5 [31].The results of the polydispersity index of the prepared nanoemulsions were 0.285 and 0.420 for honeysuckle and patchouli essential oils, respectively.For honeysuckle, the prepared nanoparticles presented some type of homogeneity, whereas the borderline polydispersity value of the patchouli nanoemulsion seemed to be completely broad (Figures 2 and 3).
sion system stable without aggregation or flocculation is challenging, which is the main monitor of the stability of the nanoemulsion system.The zeta potential is a measure of the charges of the particles and their stability in colloidal systems.The results obtained by the two nanoemulsion systems were 49.8 mV and 23.5 mV, respectively.The results presented by honeysuckle nanoemulsion were very interesting and expressed excellent stability.The zeta potential presented by the patchouli nanoemulsion did not differ from that of honeysuckle even though it had a lower negative charge compared to the honeysuckle nanoemulsion, and it still exhibited a very good stability profile (Figures 2 and 3).

C. Internal morphology via transmission electron microscopy (TEM)
Along with the particle size and charge mobility, the TEM analysis of the internal structure of the prepared nanoparticles is one of the most important factors.This is because it confirms the stability and checks for clusters.The prepared nanoemulsion of patchouli and honeysuckle furnished regular spherical and semispherical particles with widely variant particle sizes, as was previously proven by the higher polydispersity values.Figure 4 depicts a wide range of particle sizes ranging from less than 100 nm to 350 nm, as well as some agglomeration, particularly in the patchouli nanoemulsion.

B. Charge distribution and stability (zeta potential)
The preparation of nanoemulsion is not truly the big dilemma, but keeping oil droplets far away from each other in the aqueous medium is; similarly, keeping the nanoemulsion system stable without aggregation or flocculation is challenging, which is the main monitor of the stability of the nanoemulsion system.The zeta potential is a measure of the charges of the particles and their stability in colloidal systems.The results obtained by the two nanoemulsion systems were 49.8 mV and 23.5 mV, respectively.The results presented by honeysuckle nanoemulsion were very interesting and expressed excellent stability.The zeta potential presented by the patchouli nanoemulsion did not differ from that of honeysuckle even though it had a lower negative charge compared to the honeysuckle nanoemulsion, and it still exhibited a very good stability profile (Figures 2 and 3).

C. Internal morphology via transmission electron microscopy (TEM)
Along with the particle size and charge mobility, the TEM analysis of the internal structure of the prepared nanoparticles is one of the most important factors.This is because it confirms the stability and checks for clusters.The prepared nanoemulsion of patchouli and honeysuckle furnished regular spherical and semispherical particles with widely variant particle sizes, as was previously proven by the higher polydispersity values.Figure 4 depicts a wide range of particle sizes ranging from less than 100 nm to 350 nm, as well as some agglomeration, particularly in the patchouli nanoemulsion.

C. Internal morphology via transmission electron microscopy (TEM)
Along with the particle size and charge mobility, the TEM analysis of the internal structure of the prepared nanoparticles is one of the most important factors.This is because it confirms the stability and checks for clusters.The prepared nanoemulsion of patchouli and honeysuckle furnished regular spherical and semispherical particles with widely variant particle sizes, as was previously proven by the higher polydispersity values.Figure 4 depicts a wide range of particle sizes ranging from less than 100 nm to 350 nm, as well as some agglomeration, particularly in the patchouli nanoemulsion.Similar to honeysuckle, patchouli contained some important polyphenols, like apigenin, quercetin, gallic acid, daidzein, coumaric acid, and vanillin, with concentrations of 174.82, 170.25, 47.45, 18.66, 10.77, and 6.32 µg/g, respectively.At first glance, we can conclude that the honeysuckle essential oil was enriched with highly concentrated polyphenols compared to the patchouli essential oil (Figure 5).At first glance, we can conclude that the honeysuckle essential oil was enriched with highly concentrated polyphenols compared to the patchouli essential oil (Figure 5).b.
The identification of the chemical composition of L. caprifolium and P. cablin oils via GC-MS revealed 21 and 24 compounds representing 98.53% and 99.34% of the total oil, respectively.The chemical profiles from the analysis of the chromatograms of L. caprifolium and P. cablin varied in terms of qualitative and quantitative components.

Larvicidal Field Evaluation
Honeysuckle and patchouli oils and their nanoemulsions were utilized in LC 95 X2 doses for the field evaluation of larvicides (2509.6 and 2711.9 ppm and 565.4 and 643.0 ppm, respectively), whereas only dechlorinated water was used at the control sites.The results show that there were not many larvae in the treated pools in the village of Kafr Saad PT.Treatments with honeysuckle and patchouli oils and their nanoemulsions reduced the larval density to 89.4, 86.5, 98.6, and 97.0%, respectively, at 24 h PT, with a persistence of five days for oils and eight days for nanoemulsions PT in pools (Figure 6).The Student's T-test showed a significant difference between honeysuckle oil and nano-honeysuckle (F = 25.52,df = 3.84, p < 0.01); moreover, the T-test revealed a significant difference between nano-patchouli and patchouli oil (F = 27.23,df = 3.64, p < 0.01).

Adulticidal Field Evaluation
The adulticidal effects of the applied materials were evaluated against C. pipiens adults in some houses in Kafr Saad village PT, and the results show that nano-honeysuckle (100% mortality) was more effective than honeysuckle oil (98.0%); their LT 95 values were 183.54 and 201.63 min, respectively (Figure 7).Furthermore, the data show that nanopatchouli was more persistent (7 days) than nano-honeysuckle (6 days).Similarly, the results show that patchouli oil (5 days) was more stable than honeysuckle oil (4 days) in the selected homes (Figure 7).

Adulticidal Field Evaluation
The adulticidal effects of the applied materials were evaluated against C. pipie adults in some houses in Kafr Saad village PT, and the results show that nano-honeysuck (100% mortality) was more effective than honeysuckle oil (98.0%); their LT95 values we 183.54 and 201.63 min, respectively (Figure 7).Furthermore, the data show that nan

Discussion
Culex mosquito-borne diseases are a global concern.C. pipiens is a species that is known as a pest in urban environments.Female C. pipiens mosquitoes are the main vectors

Discussion
Culex mosquito-borne diseases are a global concern.C. pipiens is a species that is known as a pest in urban environments.Female C. pipiens mosquitoes are the main vectors for the West Nile virus (WNV).They also transmit other arboviruses and act as a vector of filarial parasites, human lymphatic filariasis [32].Due to the scarcity of treatment options, disease control relies on targeting the mosquitoes that transmit the disease in order to eliminate them.Therefore, it is important to use green pesticides, including essential oils, as environmentally friendly natural products for the manufacture of sustainable commercial pesticides.In order to achieve this, our study focused on studying 16 essential oils extracted from plants; then, we highlighted 2 essential oils, L. caprifolium and P. cablin, and their nanoemulsions, which produced the highest efficiency against C. pipiens.
Essential oils are natural chemical substances extracted from plants with different insecticidal chemical properties and different effective modes of action.They are used instead of synthetic or chemical pesticides to protect the environment and avoid the negative effects of synthetic pesticides [13].Essential oils contain effective compounds that have the ability to combat mosquitoes by acting as insecticides, repellents, antifeedants, growth inhibitors, oviposition inhibitors, and ovicides, and they have growth-reducing effects that are larvicidal and toxic, thus reducing the abundance of disease vectors and the risk of disease [13,33].These plant compounds are effective, safe, and biodegradable [30].
Preliminary screening demonstrated a wide range of toxicities among the 16 essential oils tested, and most oils demonstrated high toxicity at the screening concentration of 1500 ppm.R. officinalis, C. aurantium, C. schoenanthus, O. majorana, and R. communis were among the oils with a clear effect on C. pipiens larvae in the screening tests, though their effect was less than that of the honeysuckle and patchouli oils.In contrast, the data show that the A. vera, F. virginiana, and L. sativa oils were among the least toxic oils for C. pipiens.
In general, there is a scarcity and significant lack of studies on the plants examined herein and their effect on C. pipiens.The following are the results from previous studies of essential oils extracted from the plants under study against C. pipiens.C. aurantium essential oil has a major limonene content (90%) and insecticidal activity on C. pipiens larvae.In addition, the essential oil showed 100% mortality with respect to larval stages 3 and 4 of C. pipiens at 300 ppm.The LC 50 and LC 90 values were 139.48 and 212.04, respectively [34,35].Theochari et al. [36] found that limonene had larvicidal properties against two mosquito species, Aedes albopictus and C. pipiens.
On the same topic, R. officinalis essential oil exhibited larvicidal activity against the fourth-instar larvae of C. pipiens at 24 h (its LC 25 , LC 50 , and LC 90 values were 39.47, 51.33, and 86.77 ppm, respectively).In addition, the oil contained 1,8-cineole (44.34%), camphor (16.74%), α-pinene (10.07%), and borneol (5.02%) as components [41].Aouinty et al. [42] reported that the aqueous extract of R. communis leaves exhibited toxic action against the mosquito larvae of C. pipens, where significant histological changes, toxic action, hypertrophy, and the lysis of epithelium intestinal cells were observed, as well as musculature and external teguments of larvae and tissue lysis.With respect to C. schoenanthus, Wangrawa et al. [43] concluded that its essential oil exhibited larvicidal activity against Anopheles funestus and C. quinquefasciatus.Likewise, the essential oils of P. granatum and R. communis were shown to have larvicidal effects against mosquitoes [44,45].
Concerning N. tazita, methyl dihydrojasmolate is a major component and exhibited mosquito-repelling activity [46,47].Also, methyl anthranilate, as one of the components of the essential oil, showed repellent properties against Aedes aegypti [48].Limonene and α-pinene are effective inhibitors of acetylcholinesterase in various insects and larvae due to their ability to alter the activity of the insect's acetylcholinesterase, which is required for neuro-neuronal and neuromuscular junctions in insects [49,50].Terpineol is very effective in inhibiting insect reproduction, and the occurrence of monoterpenes and sesquiterpenes leads to fumigant toxicity [51].
Despite the superior properties of honeysuckle oil from our findings, honeysuckle essential oil has not been studied before or researched in terms of C. pipiens.There is a scarcity of research on honeysuckle and patchouli oils against mosquitoes.The only study was conducted on L. caprifolium essential oils by Muturi et al. [23], which proved its insecticidal property against Aedes aegypti and verified it as a source of ecofriendly larvicides for mosquito control.We also found one study conducted on patchouli essential oil and its larvicidal activity against C. pipiens [52].
Our work is somewhat consistent with or similar to the study of Hazarika [53] on P. cablin oil and its toxicity on Aedes aegypti.The essential oil was most effective in killing A. aegypti larvae, with an LC 50 value of 25.14 mg/L.Similarly, the significant larvicidal potential of P. cablin essential oil on A. aegypti was reported, with high toxicity and an LC 50 value of 24.25 µg•mL −1 [54].Also, P. cablin oil showed repellent and insecticidal properties against Aedes aegypti [55].The 16 µL/mL concentration of P. cablin essential oil displayed 100% repellency for A. aegypti [56].
Variations in the composition of essential oils are influenced by different plant parts and their different stages of development and modifications due to the environment [60].These factors influence the plant's biosynthetic pathways and, consequently, the relative proportion of the main constituents.The insecticidal activity is probably due to the presence of monoterpenes, sesquiterpenes, and aromatic compounds with known biological properties [61]; moreover, various components of the essential oil and their roles in preventing or decreasing the activity of the enzyme acetylcholinesterase (AChE) and the effects on neurotransmitter receptors have been reported [62,63].Insecticidal activity could be attributed to the action of specific compounds within the essential oil or the synergistic action of several molecules [64].Furthermore, the diversity of oil components reduces insect resistance and behavioral habituation to deterrents.The toxic effects of essential oils can upregulate physiologically important proteins and enzymes in insects and synergize insecticide toxicity by inhibiting detoxification enzymes [62,63].
Our results indicate that L. caprifolium and P. cablin oils exhibited larvicidal and adulticidal activity, and their nanoemulsions showed increased efficacy and persistence.Honeysuckle and patchouli oils were found to be two of the most toxic essential oils to C. pipiens larvae and adults (100% mortality).Our data reveal that novel L. caprifolium oil effectively controlled the larvae and adults of C. pipiens (LC 50 = 247.72 ppm and 22.13 min, respectively).The honeysuckle nanoemulsion was more effective than the crude oil against larvae and adults (LC 50 = 88.30ppm and LT 50 = 13.04 min).Also, patchouli oil effectively controlled the larvae and adults of C. pipiens (LC 50 = 276.29 ppm and LT 50 = 22.93 min).The patchouli nanoemulsion was more effective than the crude oil against larvae and adults (LC 50 = 93.05ppm and LT 50 = 23.93 min).
There is a paucity of research published on L. caprifolium and P. cablin regarding the effects of their essential oil nanoemulsions.Furthermore, there are no previous studies on L. caprifolium and P. cablin essential oil nanoemulsions and their effects on C. pipiens.This study is, to our knowledge, the first research study on the effects of essential oil nanoemulsions on C. pipiens.
There are no reports on the effect of L. caprifolium and P. cablin essential oils on mosquitoes, especially C. pipiens, and there are no published studies on the effect of a nanoemulsion of L. caprifolium essential oil on any living organism.
Regarding P. cablin, Roshan et al. [59] demonstrated that a P. cablin essential oilencapsulated chitosan nanoemulsion showed broad-spectrum antifungal and antimycotoxin activities and protected stored maize seeds from mold-induced biodeterioration and aflatoxin.Adhavan et al. [65] concluded that essential oil nanoemulsions from two wild patchouli species, P. heyneanus and P. plectranthoides, showed better antibacterial and anticandida activities in comparison with commercial patchouli essential oils.Another study evaluated the biological activity of pure patchouli essential oil compared with an emulsion containing 18% of the oil against tomato leafminer (Tuta absoluta) [66].They added that the LD 50 values were 10.06 and 2.57 µg of patchouli per mg of insect for the essential oil (EO) and emulsion, respectively.Oviposition was reduced in adults derived from the second instar treated with LD 10 by 78.5% (EO) and 85.4% (emulsion) [66].
There are some published reports on the effects of essential oils and their nanoemulsions on C. pipens.Ocimum bascilicum and Cuminum cyminum essential oils showed larvicidal effects on C. pipiens, with LC 50 values of 81.07 ug/mL and 96.29 ug/mL, respectively.Also, nanoemulsion forms have higher efficiency, as evidenced by a reduction in the LC 50 to 65.19 ug/mL for O. bascilicum and 64.50 ug/mL for C. cyminum [68].Muturi et al. [69] demonstrated that Commiphora erythraea essential oil is a promising source of mosquito larvicide; LC 50 values for the whole essential oil were 19.05 ppm for C. restuans, 22.61 ppm for C. pipiens, and 29.83 ppm for Aedes aegypti.Using C. horaerythraea essential oil emulsions enhanced the insecticidal properties of C. erythraea essential oil, and they were more toxic than the whole essential oil.In another study, Cirtus sinensis essential oil nanoemulsion was recommended for the control of vector-borne C. pipiens larvae disease; the LC 50 values for the nanoemulsion and bulk emulsion were 27.4 and 86.3 ppm, respectively, and the larvicidal activity of the essential oil nanoemulsion was greater [70].
The goal has always been to search for environmentally friendly natural pesticides that are safer and more efficient than the synthetic pesticides that were once used to control mosquitoes, which are now seriously harming human health and, more significantly, breeding resistant mosquitoes.Phytochemicals have pesticidal effects and other harmful effects on mosquito physiology at different stages of development [71,72].In addition, insects have a greater chance of developing resistance to a single chemical component compared to a range of chemicals.Therefore, the use of combined phytochemicals would prevent the emergence of mosquito resistance.Also, phytochemicals have a short residual half-life, and their use in combination with other biological control agents can be beneficial [73,74].It is promising and encouraging that these phytochemical properties are present in essential oils.However, because they are volatile substances, their long-term applications in mosquito control have problems.The effectiveness and duration of essential oils have increased in recent years thanks to new technologies like microencapsulation and nanoemulsion [31,75].
The mixture of two phases of water and oil is called an emulsion; if the droplet size is at the nanoscale, it is called a nanoemulsion [76].Emulsions are divided into two types: oil-in-water (O/W) and water-in-oil (W/O); in the former, oil droplets are dispersed in water, whereas in the latter, the opposite is true.In both cases, surfactants or surface tension-reducing agents are used to mix the two phases.In particular, O/W nanoemulsions are much more frequently used because most drugs and all essential oils are lipophilic and should be solubilized in blood flow or water [77].
In a similar study, Radwan et al.
[78] discovered that nano-clays intercalated with green tea and fennel, Mg-LDH-GT, and Mg-LDH-F were the best-loaded systems, with relatively good desorption release to their active ingredients, and significantly affected C. pipiens larvae and adults in both laboratory and field conditions; moreover, the stability of the oil was improved.
Surfactants are crucial components of nanoemulsions.Surfactants come in four different varieties: cationic, anionic, amphoteric, and nonionic.Nonionic surfactants are typically encapsulated in the nanoemulsion when creating nanoemulsion-based pesticide applications because they are less sensitive to pH and ionic strength.Due to the cohesiveness between the anionic surfactant and the solution, this extra component has the potential to change the stability and size of the nanoemulsion [79].The most used pesticide formulations consist of active compounds that can kill weeds, insects such as mosquitoes and ticks, and other organisms such as snails, slugs, and fungi (fungicides).The nanoemulsion acts as a "carrier" that carries and delivers bioactive ingredients to pests on plants.This is carried out to obtain the most effective pesticide delivery [80].
Our data obtained via dynamic light scattering confirmed that oil nanoemulsions were within 100-300 nm, whereas the DLS values of both honeysuckle and patchouli essential oils were 183 and 250 nm, respectively.Also, data show nanoparticle homogeneity for honeysuckle and patchouli via the polydispersity value, which indicates greater particle stability [81,82] Our findings are consistent with those of Nuchuchua et al. [83], who studied citronella (Cymbopogon nardus), hairy basil (Ocimum Americanum), and vetiver (Vetiveria zizanioides) oil nanoemulsions against A. aegypti; nanoemulsions were prepared using a high-pressure homogenization technique and had mean droplet sizes ranging from 150 to 220 nm.This result could prolong mosquito protection times by up to 4.7 h due to the higher release rate and better physical stability of the nanoemulsion [83].
Copaiba (Copaifera duckei) oleoresin oil-in-water nanoemulsions had the lowest concentration at 200 ppm.The nanoemulsion oil killed 70% of A. aegypti larvae after 24 h and 90% after 48 h.The average size of the droplets was 145.2 nm [84].The essential oil of sweet oranges (Cirtus sinensis) was used to create a larvicidal nanoemulsion with a mean droplet size of 78.8 nm and a polydispersity index (PDI) of 0.28.After 24 h against C. pipiens, the nanoemulsion's LC 50 value was 27.4 ppm and that of the bulk emulsion was 86.3 ppm [85].Our data indicate, via the analysis of the studied oils using HPLC to determine the concentration of polyphenol contents, the presence of polyphenols in honeysuckle and patchouli oils.Polyphenols are abundant in honeysuckle oil through multiple organic compounds, such as vanillin, daidzein, and cinnamic acid, in addition to good concentrations of others, such as syringic acid, gallic acid, quercetin, catechin, methyl gallate, and naringenin.Patchouli oil also contains polyphenols such as gallic acid, coumaric acid, and vanillin.
Our data agree with recent findings with respect to Italian honeysuckle essential oil, which showed that the whole essential oil (LC 50 of 34.4 mg/L) was two times less toxic to A. aegypti larvae than four of its five fractions (LC 50 = 20.6,19.7, 18.6, and 17.7 mg/L for fractions B, C, D, and E, respectively) and some L. scoparium essential oils.Similar results were also shown to be true for the essential oil of parsley (Petroselinum crispum), where the LC 50 values for fractions 1, 3, and 4 against A. aegypti larvae were 0.49, 0.88, and 0.01 mg/L, respectively, as opposed to 4.19 mg/L for the entire essential oil [91].
Patchouli essential oil-loaded hydrogel activities may be due to the synergistic effects of the present associated compounds, such as patchoulene, patchoulol, pogostol, and patchouli alcohol, which has been reported in the literature to have potential antiinflammatory and antimicrobial activity [80].
With almost half of the doses applied in the field, the larval reduction percentage of honeysuckle and nanoemulsion reduced larval densities (91% and 98.6%, respectively, 24 h PT), and their effect lasted (reduction % > 50%) for five and eight days PT, respectively, in pools.In ditches, reduced larval density reached 90% and 95.0% at 24 h PT, and the effect lasted (reduction % > 50%) for 4 and 6 days PT, respectively.Those of patchouli oil and nanoemulsion were 89.0 and 96.0%24 h PT and persisted for six days 8 days PT, respectively.Similarly, the reduction was 91% and 98.6%, 24 h PT and their effect lasted (reduction % > 50%) for five and eight days PT, respectively, in ditches.
On the other hand, the reduction in adult density PT with respect to nano-honeysuckle and its oil and nano-patchouli and its oil reached 84.00, 100, 77.4, and 98.00%, respectively, and it was effective for five and three days relative to the nanoemulsions and oils.To the best of our knowledge, there was no previously filed application for the use of L. caprifolium and P. cablin oil nanoparticles against mosquitoes.Our search demonstrates that both L. caprifolium and P. cablin oils are highly effective essential oils that can be used against C. pipiens.
Once more, some essential oils have the ability to repel a variety of insects, including vectors [56].Some insects were subjected to the neurotoxic effects of essential oils, which led to hyperactivity, followed by hyperexcitation, and they were quickly knocked down and rendered immobile [58]; moreover, suffocation occurred due to the detrimental effects of bioactive plant products on neurotransmitter receptors [92].Bioactive plant products, according to Baz et al. [70], have anti-insect effects that are used to eradicate insects, such as feeding deterrents/antifeedants, toxicants, growth retardants, repellents, chemosterilants, and attractants [93].
Many pesticides that are widely used in agricultural applications are highly hydrophobic particles with low water solubility; therefore, they must be loaded within appropriate delivery systems prior to use.The advantages of nano-formulations include the increased apparent solubility of poorly soluble active ingredients, slow/targeted release of the active ingredient, and protection against premature degradation.By inserting essential oils into the structure of nanoemulsions, the oil's stability and ability to resist pests are high [94].

Chemicals and Plant Oils
Sixteen essential oils were purchased from the Nefertari Company (Fayoum, Cairo, Egypt) for natural plant oils and cosmetics.The oils were 100% pure and natural and were extracted from natural plants via hydrodistillation (Table 12; Figure 8).De-ionized water, butanol, polysorbate 20, sodium glycocholate, and oleic acid were purchased from Alfa Esar (Thermo Fisher GmbH, Kandel, Germany) and used without further purification.

Mosquito Colony
C. pipiens mosquito larvae were used for all investigations and have been reared and maintained in an insectary at the Department of Entomology, Faculty of Science, Benha University, for many generations (F12).Mosquito larvae were reared in enamel plates (30 × 25 × 15 cm), filled with 2 L of stored dechlorinated tap water, and given powdered dog biscuits and Tetramin ® fish food (w/w) every 2 days.The colony was kept in good condition at 27 ± 2 °C, 75-80% RH, and a 12:12 h (L/D) photoperiod.Pupae were transferred to a mosquito cage (30 cm × 25 cm × 25 cm).Mosquito adults received an 8% sucrose solution as food.Both larvae and adults were kept in identical laboratory settings and were continually available for the tests [95].

Larvicidal Efficacy In Vitro
Sixteen oils were screened for their larvicidal efficacy [94] against the last third-instar larvae of C. pipiens.Oils were added to a solvent consisting of dechlorinated water plus 0.5 mL of 0.5% Tween 20 (an emulsifier) using a shaker plate to yield a homogenous

Mosquito Colony
C. pipiens mosquito larvae were used for all investigations and have been reared and maintained in an insectary at the Department of Entomology, Faculty of Science, Benha University, for many generations (F12).Mosquito larvae were reared in enamel plates (30 × 25 × 15 cm), filled with 2 L of stored dechlorinated tap water, and given powdered dog biscuits and Tetramin ® fish food (w/w) every 2 days.The colony was kept in good condition at 27 ± 2 • C, 75-80% RH, and a 12:12 h (L/D) photoperiod.Pupae were transferred to a mosquito cage (30 cm × 25 cm × 25 cm).Mosquito adults received an 8% sucrose solution as food.Both larvae and adults were kept in identical laboratory settings and were continually available for the tests [95].

Larvicidal Efficacy In Vitro
Sixteen oils were screened for their larvicidal efficacy [94] against the last third-instar larvae of C. pipiens.Oils were added to a solvent consisting of dechlorinated water plus 0.5 mL of 0.5% Tween 20 (an emulsifier) using a shaker plate to yield a homogenous solution [14].Twenty larvae were placed in a 250 mL glass beaker containing 200 mL of 1000 ppm.Larval mortality for each oil concentration was recorded 24 h post-treatment (PT).After determining which oils were the most preferred for controlling mosquito larvae, two oils were chosen (mortality more than 95%), and different concentrations of two of the essential oils and their nanoemulsion formulations (50, 100, 250, 500, 1000, and 1500 ppm) were tested according to the WHO's guidelines [96].The control group was treated with the solvent only.The experiment was replicated five times.For two selected essential oils and their nanoemulsions, the larval mortality for each oil concentration was recorded at 24 h and 48 h post-treatment (PT).

Adulticidal Efficacy In Vitro
Using CDC bottle bioassays, adult mosquito susceptibility testing for the promising oils was carried out based on Vatandoost et al. [97].Oils were placed in three bottles, with one for each concentration.Using pure ethanol as a solvent, different concentrations of each oil (0.5, 1, 2, 4, and 5%) were created.The requisite concentrations were applied to the bottles, and they were left to evaporate for 20 min at 28 ± 2 • C. To fill each bottle, a hand aspirator was used to remove 15 adult mosquitoes (aged 3-4 days) from the cage.The mosquitoes were taken out of the bottles after one hour of exposure and placed in separate paper cups with a 10% sucrose solution.Mortality was then calculated after 24 h using three replicates.

Preparation of the Essential Oil Nanoemulsion
The nanoemulsion preparation protocol for essential oil was carried out using the homogenization method by Radwan et al. [33], with equivalent amounts of the essential oil and oleic acid, as follows: In a 50 mL beaker (B1), amounts of 2.5 mL of essential oil and 2.5 mL of oleic acid were mixed and warmed to 45 • C; in another 50 mL beaker (B2), 10 mL of distilled water, 0.2 g of sodium glycocholate, 0.25 mL of butanol, and 3 mL of polysorbate 20 were added and mixed very well using a hotplate stirrer until the final solution became homogenous.The temperature was monitored using an infrared thermometer; 45 • C was reached, and the same temperature was retained.To obtain the primary emulsion, the contents of the two beakers, B1 and B2, were mixed together at the same temperature under stirring, and the mixture was quenched rapidly with the addition of ice-cold water to obtain a final volume of 40 mL, which was reserved in a 50 mL Falcon tube (Figure 9).solution [14].Twenty larvae were placed in a 250 mL glass beaker containing 200 mL of 1000 ppm.Larval mortality for each oil concentration was recorded 24 h post-treatment (PT).After determining which oils were the most preferred for controlling mosquito larvae, two oils were chosen (mortality more than 95%), and different concentrations of two of the essential oils and their nanoemulsion formulations (50, 100, 250, 500, 1000, and 1500 ppm) were tested according to the WHO's guidelines [96].The control group was treated with the solvent only.The experiment was replicated five times.For two selected essential oils and their nanoemulsions, the larval mortality for each oil concentration was recorded at 24 h and 48 h post-treatment (PT).

Adulticidal Efficacy In Vitro
Using CDC bottle bioassays, adult mosquito susceptibility testing for the promising oils was carried out based on Vatandoost et al. [97].Oils were placed in three bottles, with one for each concentration.Using pure ethanol as a solvent, different concentrations of each oil (0.5, 1, 2, 4, and 5%) were created.The requisite concentrations were applied to the bottles, and they were left to evaporate for 20 min at 28 ± 2 °C.To fill each bottle, a hand aspirator was used to remove 15 adult mosquitoes (aged 3-4 days) from the cage.The mosquitoes were taken out of the bottles after one hour of exposure and placed in separate paper cups with a 10% sucrose solution.Mortality was then calculated after 24 h using three replicates.

Preparation of the Essential Oil Nanoemulsion
The nanoemulsion preparation protocol for essential oil was carried out using the homogenization method by Radwan et al. [33], with equivalent amounts of the essential oil and oleic acid, as follows: In a 50 mL beaker (B1), amounts of 2.5 mL of essential oil and 2.5 mL of oleic acid were mixed and warmed to 45 °C; in another 50 mL beaker (B2), 10 mL of distilled water, 0.2 g of sodium glycocholate, 0.25 mL of butanol, and 3 mL of polysorbate 20 were added and mixed very well using a hotplate stirrer until the final solution became homogenous.The temperature was monitored using an infrared thermometer; 45 °C was reached, and the same temperature was retained.To obtain the primary emulsion, the contents of the two beakers, B1 and B2, were mixed together at the same temperature under stirring, and the mixture was quenched rapidly with the addition of ice-cold water to obtain a final volume of 40 mL, which was reserved in a 50 mL Falcon tube (Figure 9).Polyphenol content concentration determination via HPLC High-performance liquid chromatography for polyphenol detection analyses was accomplished using the Agilent 1260 series on both honeysuckle and patchouli oils.The separation process was carried out using an Eclipse C18 column (4.6 mm × 250 mm i.d., 5 µm).The mobile phase consisted of two solutions, a mixture of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B), injected at a flow rate of 0.9 mL/min.The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (82% A), 0-5 min (80% A), 5-8 min (60% A), 8-12 min (60% A), 12-15 min (82% A), 15-16 min (82% A), and 16-20 min (82% A).The multi-wavelength detector was adjusted for a wavelength of 280 nm.For each sample solution, a volume of 5 µL was injected.The column temperature was adjusted to be maintained at 40 • C.

b.
Volatile content identification via GC-MS Analyses of the selected essential oils were performed via GC-MS (gas chromatographymass spectrometry).Thermo Scientific Trace GC Ultra/ISQ Single Quadrupole MS and TG-5MS fused silica capillary columns at 0.1 mm, 0.251 mm, and 30 m thick were utilized for GC-MS, which was employed for biochemical analyses.This was achieved using an electronic ionizer with 70 eV of ionization energy.As a carrier gas, helium was used (flow rate: 1 mL/min).The MS transmission line and injector were both set to 280 • C. The oven was preheated to 50 • C and then increased to 150 • C at a rate of 7 • C per minute, 270 • C at a rate of 5 • C per minute (pause for two minutes), and finally 310 • C at a rate of 3.5 • C per minute (continued for 10 min).A relative peak area was employed to explore the quantification of all components discovered.The chemicals were at least partially identified by comparing the retention times and mass spectra of the chemicals to those of NIST and Willy Library data from the GC-MS instrument.Identification was carried out using the aggregate spectrum of user-generated reference libraries.To evaluate peak homogeneity, single-ion chromatographic reconstructions were performed.To verify GC retention periods, co-chromatographic analyses of reference substances were used whenever practical [98].

a.
Average droplet size and surface charge The droplet size, radius, and polydispersity index (PDI) were measured using the dynamic light scattering (DLS) technique.The measuring conditions were set at room temperature with an angle of 173 • .The net surface charge, or zeta potential (z.p.), was monitored by measuring the change in the frequency shift of the scattered light at a scattering angle of 12 • due to laser beam irradiation.The average size, PDI, and zeta potential measurements were carried out using Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) at the Egyptian Petroleum Research Institute (EPRI).The sample was prepared by dispersing about 5-10 mg of the solution under investigation in 10 mL of distilled water at a temperature of 25 • C, with homogenization for two minutes before measurements.

b. Nanoemulsion droplet morphology via Transmission Electron Microscopy (TEM)
Visualization of the shape of the nanoemulsion droplets and their internal structure was carried out via field transmission microscopy (HR-TEM and JSM-7100F) in the central labs of the Egyptian Petroleum Research Institute (EPRI), Cairo.Images were taken using a JEOL JEM-2100-115 high-resolution transmission electron microscope system with an accelerating voltage that varied from 100 to 200 kV.The sample was prepared by diluting 1 µL of N.E.with distilled water at a dilution factor of 1:200 and placing it on a 200-mesh carbon-coated grid for 2 min before removing the excess liquid via absorption through a cellulose filter.One to two drops of 2% (w/w) phosphotungstic acid (PTA) were dropped onto the grid for 10 s to achieve negative staining, and the excess PTA was disposed of via filter paper by absorption.

Larvicidal Field Evaluation of Patchouli and Honeysuckle Nanoemulsions
In November 2022, honeysuckle and patchouli oils and their nanoemulsions were tested on mosquito larvae in small, still-water ditches in Kafr Saad village, Egypt (283304200 N, 335605700 E, altitude 2624).The ditches averaged 75 to 120 m in length, with 1.30 m 2 water surfaces and a depth of 0.55 m.The larval breeding ditches were selected to be adjacent to the population, contain steady water, and have high mosquito stage density.The honeysuckle and patchouli oils and their nanoemulsions (500 mL/m 2 ) at a dose of LC 95 X2 (8932.68,1659.34,11,729.2ppm, and 2251.24ppm, respectively) were applied to the breeding sites [31,68].The essential oils were dissolved in Tween 20 prior to the application to ditches (about 400 mL of oil were mixed with 0.2 mL of a solution of 0.05% Tween 20 v/v).For each treatment, three replicates were performed.Before and after treatment, mosquito larvae were sampled from each site every day for a week.To examine the efficacy of the selected larvicides on the mosquito population, larval instars were collected from field water at each site using an enamel pad (450 mL), and each larvicide was treated and transported to the laboratory to count the mosquito larvae in order to determine mortality; live larvae were counted until adulthood to determine the persistence of the tested materials [99].

Adulticidal Field Evaluation of Patchouli and Honeysuckle Nanoemulsions
The efficacy and stability testing of honeysuckle and patchouli oils and their nanoemulsions on adult mosquitoes was carried out in some homes in Kafr Saad village, and these homes contained humans and sometimes their animals.According to the guidelines of the WHO [71], honeysuckle and patchouli oils and their nanoemulsions (LC 95 X2) were sprayed into three rooms in each selected home for 5 min; then, the room's door was closed.Three rooms were sprayed with dechlorinated water as a control group.The reduction in adult mosquitoes was calculated according to the WHO's guidelines [99].Before spraying the tested materials in the rooms, white cloth was spread on the surface of the room's floor to collect dead mosquitoes after spraying in order to determine the density of adult mosquitoes; in untreated rooms, the adult mosquitoes were collected using CDC light traps, and the nets of the traps were placed in the freezer (20 • C) for 10 min to count the mosquitoes.

Data Analyses
The data were analyzed using the software SPSS V23 (IBM, Armonk, NY, USA), and probit analyses were carried out to calculate the lethal concentration (LC) values and a one-way analysis of variance (ANOVA) (Duncan's MRT).The significance level was set at p < 0.05.The non-parametric Kruskal-Wallis test was performed to compare the mean differences of more than two groups, followed by the Mann-Whitney U test to compare the mean differences between the active oil groups.The relative efficacies (REs) were calculated according to the following formula [13]: RE for LC = LC 50 (LC 90 or LC 99 ) for reference oil/LC 50 (LC 90 or LC 99 ) for EO.

Conclusions
This study revealed for the first time the efficacy of honeysuckle and patchouli essential oils against the larvae and adults of C. pipiens mosquitoes due to the large diversity and high efficacy of the phytochemical compounds that are found in honeysuckle and patchouli essential oils.The use of these natural oils as environmentally benign pesticides is a direction that must be followed, and our research shows the potential of these oils against insects that pose a threat to human health.Therefore, we recommend the use of honeysuckle oil in mosquito vector management as a green insecticide because it is inexpensive, safe, environmentally friendly, and highly efficient.Finally, further studies can be directed toward the effect of these oils against non-target organisms.

Figure 1 .
Figure 1.Lethal time values of oils applied at 1500 ppm against Culex pipiens larvae.

Figure 1 .
Figure 1.Lethal time values of oils applied at 1500 ppm against Culex pipiens larvae.
a. Polyphenol content concentration determination using HPLC

2. 6 .
Phytochemical Analysis of Honeysuckle and Patchouli Essential Oils a. Polyphenol content concentration determination using HPLC Polyphenol identification of honeysuckle and patchouli was carried out via HPLC using 18 standards by comparing the standards to the essential oils; the identified components are presented in Table 9, and the structures of the tested polyphenol active components identified via HPLC are shown in Figure S1.The highly abundant polyphenols presented in the honeysuckle were vanillin, daidzein, and cinnamic acid, with concentrations of 8152.26,3116.16, and 2447.91 µg/g, respectively, in addition to other good concentrations of syringic acid, gallic acid, quercetin, and catechin of 141.62, 70.95, 67.97, and 60.96 µg/g, respectively.

Figure 5 .
Figure 5. Chromatogram of the polyphenol concentration determination of patchouli (a) and honeysuckle (b) essential oils and standard polyphenols (c).

Figure 5 .
Figure 5. Chromatogram of the polyphenol concentration determination of patchouli (a) and honeysuckle (b) essential oils and standard polyphenols (c).

Figure 6 .
Figure 6.Field efficacy of nano-honeysuckle and its oil (A) and nano-patchouli and its oil (B) trea at a dose of LC95 X2 (2509.5, 565.4,2711.9, and 643.0 ppm, respectively) in larval breeding sites.

Figure 6 .
Figure 6.Field efficacy of nano-honeysuckle and its oil (A) and nano-patchouli and its oil (B) treated at a dose of LC 95 X2 (2509.5, 565.4,2711.9, and 643.0 ppm, respectively) in larval breeding sites.

Figure 7 .
Figure 7. Persistence of honeysuckle oil and its nanoemulsion (a) and patchouli oil and its nanoemulsion (b) against adult mosquitoes in treated houses 30 min post-exposure for 7 days.

Figure 7 .
Figure 7. Persistence of honeysuckle oil and its nanoemulsion (a) and patchouli oil and its nanoemulsion (b) against adult mosquitoes in treated houses 30 min post-exposure for 7 days.

Table 2 .
Efficacy of larvicidal activity of honeysuckle oil and its nanoemulsion against Culex pipiens 24 h post-treatment.

Table 3 .
Efficacy of larvicidal activity of patchouli oil and its nanoemulsion against Culex pipiens 24 h post-treatment.

Table 4 .
Efficacy of larvicidal activity of honeysuckle oil and its nanoemulsion against Culex pipiens 48 h post-treatment.

Table 5 .
Efficacy of larvicidal activity of patchouli oil and its nanoemulsion against Culex pipiens 48 h post-treatment.

Table 6 .
Knockdown time and mortality rate of Culex pipiens mosquitoes exposed to 1% essential oils, 60 min.

Table 7 .
Knockdown time and mortality rate of Culex pipiens mosquitoes exposed to 5% essential oils, 60 min.
bcNumbers in the same column followed by the same superscript letter are not significantly different (one-way ANOVA, Duncan's MRT, p > 0.05); RE: relative efficacy.

Table 8 .
Lethal time and mortality rate of female Culex pipiens mosquitoes exposed to 5% honeysuckle and patchouli oils and their nanoemulsions.

Table 9 .
Injected polyphenol standards (structure and concentration) and polyphenol contents and the concentration of honeysuckle and patchouli essential oils.

Table 10 .
The major chemical constituents of Lonicera caprifolium essential oil.

Table 11 .
The major chemical constituents of Pogostemon cablin essential oil.

Table 12 .
Plant species screened (No. of oils = 16) for use for larvicidal activity.oils purchased from the Nefertari Company for extracting natural essential oils and body care products. Plant

Table 12 .
Plant species screened (No. of oils = 16) for use for larvicidal activity.