Larvicidal Effect of Hyptis suaveolens (L.) Poit. Essential Oil Nanoemulsion on Culex quinquefasciatus (Diptera: Culicidae)

Mosquitoes can be vectors of pathogens and transmit diseases to both animals and humans. Species of the genus Culex are part of the cycle of neglected diseases, especially Culex quinquefasciatus, which is an anthropophilic vector of lymphatic filariasis. Natural products can be an alternative to synthetic insecticides for vector control; however, the main issue is the poor water availability of some compounds from plant origin. In this context, nanoemulsions are kinetic stable delivery systems of great interest for lipophilic substances. The objective of this study was to investigate the larvicidal activity of the Hyptis suaveolens essential oil nanoemulsion on Cx. quinquefasciatus. The essential oil showed a predominance of monoterpenes with retention time (RT) lower than 15 min. The average size diameter of the emulsions (sorbitan monooleate/polysorbate 20) was ≤ 200 nm. The nanoemulsion showed high larvicidal activity in concentrations of 250 and 125 ppm. CL50 values were 102.41 (77.5253–149.14) ppm and 70.8105 (44.5282–109.811) ppm after 24 and 48 h, respectively. The mortality rate in the surfactant control was lower than 9%. Scanning micrograph images showed changes in the larvae’s integument. This study achieved an active nanoemulsion on Cx. quinquefasciatus through a low-energy-input technique and without using potentially toxic organic solvents. Therefore, it expands the scope of possible applications of H. suaveolens essential oil in the production of high-added-value nanosystems for tropical disease vector control.


Introduction
Culex quinquefasciatus Say 1983 is cosmopolitan, synanthropic, and well adapted to anthropized and peridomestic environments [1]. It is an important vector of filariasis in the tropics [2] and can cause alarming socio-economic impacts in the affected regions [3]. Filariasis is a neglected tropical and subtropical disease, although there has been a decrease in cases since the 2000s [4]. It is estimated to affect more than 120 million people in 72 countries [5]. In 2020, more than 800 million people in 47 countries were still infected with filariasis and needed treatment because they live in risk areas for microfilariae infection [6]. In Brazil, Cx. quinquefasciatus is a vector of lymphatic filariasis, also known as

Hyptis Suaveolens Nanoemulsion
Nanoemulsions prepared with sorbitan trioleate/polysorbate 20 at HLB of 10, 11, 12, and 13 resulted in creaming after the preparation. Only at HLB of 14, 15, and 16.7 was a slightly blue reflection observed, typical of nanoemulsions due to the Tyndall effect ( Figure 2). The systems prepared with sorbitan monooleate/polysorbate 20 had a thin and homogeneous aspect at HLB of 11, 12, 13, 14, and 15, evidenced by a slightly blue reflection. After seven days of storage, few changes and a low creaming formation were observed at HLB of 11-14 ( Figure 3). Therefore, the nanoemulsion for the larvicidal assay had a determined rHLB of 15 (sorbitan monooleate/polysorbate 20) since it showed no signs of instability and preserved the initial characteristics after the evaluation period of storage. 25 29.

Hyptis Suaveolens Nanoemulsion
Nanoemulsions prepared with sorbitan trioleate/polysorbate 20 at HLB of 10, 11, 12, and 13 resulted in creaming after the preparation. Only at HLB of 14, 15, and 16.7 was a slightly blue reflection observed, typical of nanoemulsions due to the Tyndall effect ( Figure 2). The systems prepared with sorbitan monooleate/polysorbate 20 had a thin and homogeneous aspect at HLB of 11, 12, 13, 14, and 15, evidenced by a slightly blue reflection. After seven days of storage, few changes and a low creaming formation were observed at HLB of 11-14 ( Figure 3). Therefore, the nanoemulsion for the larvicidal assay had a determined rHLB of 15 (sorbitan monooleate/polysorbate 20) since it showed no signs of instability and preserved the initial characteristics after the evaluation period of storage.    Table 2 shows the droplet size, polydispersity index, and zeta potential measurements of the formulations prepared with H. suaveolens essential oil and the surfactant pair of sorbitan trioleate/polysorbate 20. Emulsions prepared with these surfactants at HLB of 10, 11, 12, and 13 had an average size diameter size above 200 nm. Each size was 793.0 ± 536.9, 664.5 ± 122.2, 263.9 ± 15.36, and 255.1 ± 33.26, respectively. Thus, measurements were taken on day 0 only. In the nanoemulsions prepared with these  Table 2 shows the droplet size, polydispersity index, and zeta potential measurements of the formulations prepared with H. suaveolens essential oil and the surfactant pair of sorbitan trioleate/polysorbate 20. Emulsions prepared with these surfactants at HLB of 10, 11, 12, and 13 had an average size diameter size above 200 nm. Each size was 793.0 ± 536.9, 664.5 ± 122.2, 263.9 ± 15.36, and 255.1 ± 33.26, respectively. Thus, measurements were taken on day 0 only. In the nanoemulsions prepared with these surfactants at HLB of 14, 15, and 16.7, droplet sizes below 200 nm, a polydispersity index below 0.300, and zeta potential below −20 mV were observed during storage. Table 3 shows the measurements for the nanoemulsions set made with the surfactant pair of sorbitan monooleate/polysorbate 20. All emulsions had measurements of ≤ 200 nm. The system prepared solely with polysorbate 20 resulted in the largest size and values were significant when compared between days 0 and 7 (p < 0.0001). The nanoemulsion prepared with this pair at HLB of 15 resulted in the smallest diameter (day 0 = 69.47 ± 0.6717; day 1 = 70.42 ± 0.2055; day 2 = 69.31 ± 0.1212; and day 7 = 67.79 ± 1.040) with little variation in the polydispersity index (day 0 = 0.179 ± 0.009; day 1 = 0.173 ± 0.004; day 2 = 0.177 ± 0.009; and day 7 = 0.206 ± 0.017). Moreover, there was no significant difference in size and polydispersity (p > 0.05), which validates the macroscopic analysis that this was the nanoemulsion with the highest stability. Size distribution and zeta potential graphs are presented in Figure 4. Therefore, the rHLB of the essential oil of H. suaveolens was determined as 15 and this nanoemulsion was chosen for the larvicidal assay.   Table 4 shows the results of the influence of the nanoemulsion of H. suaveolens on the mortality of Cx. quinquefasciatus larvae at 24 and 48 h. Low mortality was observed in the control with distilled water (2 ± 0.45%). The percentage of mortality was 100% for the 200 ppm concentration (p = 0.000000) in the first 24 h of the experiment. The H. suaveolens   Polydispersity index (PDI). Data are expressed as the mean and standard deviation (n = 3, mean ± SD).  Table 4 shows the results of the influence of the nanoemulsion of H. suaveolens on the mortality of Cx. quinquefasciatus larvae at 24 and 48 h. Low mortality was observed in the control with distilled water (2 ± 0.45%). The percentage of mortality was 100% for the 200 ppm concentration (p = 0.000000) in the first 24 h of the experiment. The H. suaveolens nanoemulsion exhibited significant larvicidal activity. Mortality rates increased alongside exposure time. The concentration of 125 ppm (p = 0.000000) showed a mortality rate of 78% after 48 h. The ANOVA test indicated significant differences between exposure periods (F 1 = 36.31; p ≤ 0.0001) and between treatments (F 20   Values are the mean of the five replicates and standard deviation (mean ± SD). Mean in the same row with different superscripts indicate significant difference (p < 0.05).

Larvicidal Activity of the Nanoemulsion
The mortality rate induced by the surfactant concentration was below 9%. The Chisquare values were X 2 (1) = 0.137867 (p = 0.7104) and X 2 (1) = 0.347238 (p = 0.5557) at 24 and 48 h, respectively, indicating that it is not possible to estimate the CL 50 . Furthermore, there was no significant difference between mortalities in the groups treated with surfactants under different concentrations (p > 0.05).

Morphological Study of Culex Quinquefasciatus Larvae
Electron micrograph images on Cx. quinquefasciatus larvae killed upon treatment with H. suaveolens oil nanoemulsion ( Figure 5D The mortality rate induced by the surfactant concentration was below 9%. The Chisquare values were X 2 (1) = 0.137867 (p = 0.7104) and X 2 (1) = 0.347238 (p = 0.5557) at 24 and 48 h, respectively, indicating that it is not possible to estimate the CL50. Furthermore, there was no significant difference between mortalities in the groups treated with surfactants under different concentrations (p > 0.05).

Morphological Study of Culex Quinquefasciatus Larvae
Electron micrograph images on Cx. quinquefasciatus larvae killed upon treatment with H. suaveolens oil nanoemulsion ( Figure 5D-F) demonstrated changes in the cuticle structure, bristle shortening, cephalic capsule flattening (H), and abdomen segments' disintegration (AB). The siphon (S) did not change noticeably. The results suggest the toxic effect of the H. suaveolens nanoemulsion. The control larvae remained normal in appearance ( Figure 5A-C).

Hyptis Suaveolens Essential Oil
The chromatographic analysis of H. suaveolens essential oil revealed similar main constituents to Kandpal et al. [38], who reported 1,8-cineole (17.61%) and sabinene (9.42%) among the major components. The same major compound, 1,8-cineole, was identified in three species of the genus Hyptis, with a higher percentage in H. suaveolens [39]. We observed that 1,8-cineole was the most representative substance in the H. suaveolens collected from the same region. However, others were either not identified or had a lower

Hyptis Suaveolens Nanoemulsion
Nanoemulsions are kinetic stable colloids constituted by two immiscible liquids often stabilized by surfactants. In the case of natural-product-based nanoemulsions, one would expect solubilization of the active secondary metabolite in inert oil or the utilization of a bioactive oil, as can be observed for essential oils [42]. The main mechanism for the destabilization of nanoemulsions is Ostwald ripening; however, an opposite thermodynamic force maintaining the droplets' composition (Compositional ripening) has been suggested to be responsible for the enhancement of stability of essential-oil-based nanoemulsions [43]. Studies of our group showed that the alteration of droplet size distribution may also be associated with a reduction in size [44], while the utilization of a blend of surfactants from the polysorbate/sorbitan series can dramatically remedy this behavior and even induce highly stable systems with stability higher than 12 months [45].
The H. suaveolens nanoemulsion demonstrated larvicidal potential against Cx. quinquefasciatus in this study. Studies supporting these results have been conducted, with some species of the genus Hyptis being tested on the vector mosquito larvae. In studies conducted with H. suaveolens extracts with different solvents on Ae. aegypti larvae, the lowest CL 50 [47], respectively. The Hyptis pectinata and H. fruticosa showed CL 50 values of 366 and 502, respectively, on Ae. aegypti [48]. In Pavela's [49] studies, the synergistic effect of compounds on the mortality of Cx. quinquefasciatus larvae was observed. 1,8-cineole, limonene, and camphor were some of the aromatic evaluated compounds.
The larvicidal effect of H. suaveolens essential oil was observed in Aedes albopictus, with a mortality rate of 65% at a concentration of 250 ppm and CL 50 of 240.3 ppm [31]. Additionally, other plant products have proven to be promising larvicides and repellents [50,51]. Nanoformulations with oil and different surfactant ratios have been studied for larvicidal activity on mosquito vectors [52]. The nanoemulsion with Rosmarinus officinalis essential oil containing polysorbate 20 had a mortality rate of Ae. aegypti larvae of 90% [53]. Results in Ae. aegypti (CL 50 = 34.75) and Cx. quinquefasciatus (CL 50 = 56.70 ppm) larvae were observed, with nanoemulsions of Pterodon emarginatus produced by the low-energy-input method [54,55]. In studies with nanomaterials, the lavender (Lavandula officinalis) nanoemulsion showed a mortality rate of 50% after 24 h and 90% after 24 h of exposure to Cx. quinquefasciatus larvae. The nanoemulsion was more effective than metal nanoparticles [56].
Nanoformulations of Azadirachta indica (neem)-at different proportions of oil and surfactant (polysorbate 20) (1:0.30, 1:1.5, and 1:3) and average sizes of 31.03 to 251.43 nm-were tested on Cx. quinquefasciatus and showed low values for CL 50 = 11. 75 mgL −1 [57]. Furthermore, Sugumar and co-workers [58] observed histological changes in the gut of Cx. quinquefasciatus larvae that were exposed to the Eucalyptus nanoemulsion. However, this study employed high-energy-input methods. Changes in the gut, body, and tissue of Cx. quinquefasciatus larvae were observed when they were exposed to different insecticides, which suggests cell toxicity [59].
The results found in this study validate the morphological study on Cx. quinquefasciatus larvae, in which the larvae showed flattening of the thorax (TH) and abdomen (AB) [55]. Some similar changes were caused in the anal papillae (AP) in Ae. aegypti larvae that were exposed to the ethanolic extract of three species of the genus Pipper [60]. Magonia pubescens extract showed changes in the epithelial cells of the A. aegypti larvae's digestive tract [61].
The nanoemulsion of Thymus vulgaris tested in the larvae of Anopheles stephensi, Ae. aegypti, and Cx. tritaeniorhynchus showed morphological changes in the cuticle of the larvae, destruction of the bristles, damage to the head, and abrupt rupture of the abdominal segments, as well as the shortening of the body [62]. Similar results were observed with Baccharis reticularia essential oil and d-limonene nanoemulsions. [63]. Nanostructured systems were evaluated in the Cx. quinquefasciatus larvae. Through scanning microscopy, changes and cuticle stiffening were observed in the larvae exposed to a lavender nanoemulsion and chitosan nanoparticles [56]. Changes in the integument can damage the larvae's internal organs. However, it is not possible to infer that it causes mortality. There are few studies with scanning images that show the effects of plant-based products on vector mosquito larvae. The changes are commonly evidenced by histology. According to Kasai and contributors [64], penetration through the cuticle is one of the action mechanisms of insecticides. There are other factors that can be related to larvicidal activity in mosquitoes, such as enzyme inhibition [65] and deterrent effects [66,67].

Plant material
Leaves of Hyptis suaveolens were collected in Macapá, AP, Brazil (00 • 01 33.7 N and 51 • 08 56.6 W) during the rainy season. Professor Rosângela S.F. Rodrigues Sarquis identified it. The specimen voucher was registered (18856) and filed in the herbarium of the Institute of Scientific and Technological Research of the State of Amapá (HAMAB).

Extraction of the Essential Oil
The essential oil of Hyptis suaveolens was extracted by hydrodistillating the leaves (1102.118 g) using a Clevenger-type apparatus for three hours. Then, the essential oil was collected and filtered with sodium sulfate anhydrous and stored at 4 • C.

Analysis of Gas-Chromatography
The essential oil of H. suaveolens was analyzed using a GCMS-QP5000 gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a mass spectrometer. The gas chromatograph (GC) conditions were [68] an injector temperature of 260 • C, a temperature (FID) of 290 • C, helium as the carrier gas, a flow rate of 1 mL/min, and an injection rate of 1:40. The temperature started at 60 • C and increased to 290 • C at a rate of 3 • C/min. One microliter of the sample was dissolved in dichloromethane (1:100 mg/µL) and injected into a DB-5 column (0.25 mm ID, 30 m in length, 0.25 µm of film thickness). The mass spectrometer (MS) (Shimadzu, Kyoto, Japan) conditions were electron impact ionization of 70 eV and a scan rate of 1 scan/s. The retention indices (RI) were calculated by interpolating the retention times of a mixture of aliphatic hydrocarbons (C9-C30) analyzed under the same conditions [69]. The identification of substances was performed by comparing the retention indices and mass spectra with those reported in the literature [70]. The MS fragmentation pattern of the compounds was also compared with NIST mass spectrum libraries. The relative abundance of the chemical constituents was performed through flame ionization gas chromatography (GC/FID) (Shimadzu, Kyoto, Japan) under the same GC/ES conditions. Analysis and percentages of these compounds were obtained through the FID peak area normalization method.  (10, 11, 12, 13, 14, and 15) associated with the most stable system. If polysorbate 20 allows the obtainment of the most stable system, the HLBr = HLB polysorbate20 = 16.7. Calculation of the HLB of a pair of surfactants can be performed as follows:

Characterization of Hyptis Suaveolens Nano-Emulsion
The particle size distribution was assessed through the dynamic light scattering technique using a Zetasizer ZS (Malvern, UK). The nanoemulsion was diluted in deionized water (1:25 v/v) [72]. The measurements were taken in triplicate. The droplet size and polydispersity index were expressed by the mean and standard deviation.

Larvicidal Bioassay
Culex quinquefasciatus larvae were obtained from the Laboratory of Arthropoda (Federal University of Amapá, Macapá, Brazil). The biological assay was performed under controlled conditions, having kept the larvae at 25 ± 2 • C, a relative humidity of 75 ± 5%, and a 12 h light/dark cycle. The experimental evaluation was performed according to the World Health Organization (WHO) [73] protocol with some modifications. The experimental design was completely randomized in five replicates, with 10 fourth-instar larvae in each sample. The nanoemulsions were diluted in distilled water at 15.625, 31.25, 62.25, 125, and 250 ppm (concentrations refer to H. suaveolens essential oil in an aqueous medium). Solutions of the surfactants used in the evaluated nanoemulsion were diluted in distilled water at the same concentrations for control. The negative control group consisted of distilled water. Mortality rates were registered after 24 and 48 h of exposure.

Morphological Study of Culex Quinquefasciatus Larvae
Larvae of the treatment and control group were fixed in formaldehyde (10%), and the external morphology was checked under a low vacuum using a scanning electron microscope (Tabletop Microscope TM3030Plus, Hitachi, Tokyo, Japan).

Statistical Analysis
Before the analyses, the mortality rate in the treatments was corrected to that of the controls using Abbott's formula [74]. Mortality data were subjected to Probit analysis to estimate CL 50 and CL 90 values using the software Statgraphics Plus V 5.1 (Stat Easy Co., Minneapolis, MN, USA). A two-way ANOVA test and a subsequent Tukey's HSD test were performed using the software R [75], and differences were considered significant when p ≤ 0.05.

Conclusions
Natural products can be considered alternatives to synthetic insecticides for vector control. Due to the low solubility in water of many substances from the secondary metabolism of plants, such as the constituents of essential oils, the preparation of oil-inwater nanoemulsions is promising. This study achieved an optimal nanoemulsion through the utilization of two blends of non-ionic surfactants that led to the determination of the required hydrophile-lipophile balance of the H. suaveolens essential oil. The active nanoemulsion against Culex quinquefasciatus, which presented a narrow size distribution after its preparation by a low-energy-input technique and without the use of potentially toxic organic solvents, expands the scope of possible applications of Hyptis suaveolens essential oil in the production of colloidal nanosystems with high added value for tropical disease vector control, contributing to the state of the art of phytopharmaceutical nanobiotechnology.