Novel Formula as Mosquito Larvicide †

: Background: Natural products derived from plants and secondary metabolites from microorganisms are promising in the discovery of synthetic analogs with improved efﬁcacy, potency, and safety. Our study attempts to examine the effect of a new formula as a mosquito larvicide. Methods: Isolation and characterization of prodigiosin and essential oil from Thuja orientalis and puriﬁcation of PDG. The dose response bioassay, the synergistic effect, and the mode of action are investigated for each preparation. Results: The treatment of the 3rd larva stage of Cx. pipiens reveals that the LC 50 of PDG and T. orientalis leaves’ E.O are 39.5 ± 0.341 ppm and 102.9 ± 0.46 ppm, respectively, after 24 h. The combination of LC10 of PDG with LC25 and LC50 of the E.O. shows a synergistic effect resulting in 33.3% and 100% death, respectively. Individual and combination treatment show a reduction in the activity of acetylcholine esterase, total protein, and AChE speciﬁc gravity as compared to the untreated 3rd larva stage of Cx. pipiens . PDG and E.O. result in a reduction in midgut pH leading to cellular respiration inhibition as compared to untreaded larvae that show alkaline medium. Conclusions: PDG and the T. orientails leaves’ oil combination show a promising synergistic potency against the 3rd larva stage of Cx. pipiens . Analysis Was Used to Determine the LC 10 , LC 25 , and LC 50 of PDG and E.O. of T. orientalis Leaves and no sharp peaks were observed after 4 min [26]. The application of TLC for the identiﬁcation of the puriﬁed red pigment in the present study revealed that the best mobile phase was n-hexan:ethyl acetate ( v / v ) (2:1) and the R f of the tested sample was equal to that of the standard; this conﬁrms that the tested sample is PDG. Mandal et al. reported that the R f values of the extracted pigments were found to be approximately 0.88 after using methanol, chloroform, and hexane in the ratio of 7:3:1 [26].


Introduction
Vector-borne diseases account for more than 17% of all infections, causing more than 1 million deaths annually [1]. Mosquitoes are responsible for the transmission of many medically important pathogens such as viruses, bacteria, and parasites, which cause serious diseases such as malaria, dengue, West Nile virus, yellow fever, encephalitis, filariasis, and Zika fever [2,3]. Mosquito-borne diseases can be prevented by several methods, including chemical and biological techniques, as well as genetic control, environmental management, and personal protection [4]. Pesticides pose a potential risk to humans and unwanted side effects to the environment [5]. Annually, approximately 1 million deaths worldwide and diagnoses of chronic diseases are due to the poisoning effect of pesticides [6]. Natural products represent one of the critical sources of chemical diversity and potential medicinal use [7].
Natural products derived from plants, animals, and microorganism fermentation have medicinal importance and pharmacological activity in treating different diseases [8,9]. Prodigiosin (PDG) is one of the most studied bioactive pigments of microbial origin normally produced by Serratia marcescens (SM), Pseudomonas magnesorubra, Vibrio psychroerythrous, and other bacteria. SM, a Gram-negative Entero bactericeae has received Biol. Life Sci. Forum 2022, 11, 54 2 of 11 attention because of triarylmethane, a naturally occurring dark red pigment [10]. Prodigiosin revealed a broad range of inhibitory activities against many bacterial, fungal, and protozoan species [11]. In addition, essential oils have an aromatherapy effect to cure or prevent diseases, infection, and indisposition by means of inhalation in controlling the central nervous system [12]. Furthermore, they have anti-parasitic, antibacterial, fungicidal, relaxant, stimulating, and antidepressant effects [13]. Thuja orientails (T. orientails) exhibits extensive biological activities including anticancer, antiepileptic, anti-inflammatory, antibacterial, antifungal activities, hair growth-promoting, antiviral, antiallergic, antioxidant, and molluscicidal [14][15][16]. Therefore, the present work aims to study a novel formula as mosquito larvicidal.

Stage 1: Preparation, Characterization, Purification, and Identification of Prodigiosin
Under aseptic conditions, S. marcescens was inoculated and incubated at shaking conditions for 24 h at 28-30 • C; then, it was inoculated in peanut media and kept in shaking condition for 48-72 h at 28-30 • C. It was finally subjected to Fermentor, with inoculum size 3%*30 = 90 mL. Furthermore, pH = 7, agitation = 400 rpm, and aeration was the maximum aeration [17,18]; then, PDG was extracted later by alkaline medium. The crude PDG was purified through n-hexane: ethyl acetate (2:1) as a solvent. The yield was identified by UVvisible spectrophotometry in the range 400-700 nm in absolute ethanol to find the maximum absorption spectra against methanol as a blank [19]. Then, the pigment was purified by preparative HPLC using C18 column (2.5 × 10 cm) with a flow rate of 0.8 mL/min and an injection volume of 10 µL. The mobile phase used was acetonitrile/HPLC water (60:40). Then the FT-IR spectrum of the pigment was recorded with a test case Shimadzu FT-IR spectrophotometer at 800-4000 nm. Finally, application of the purified PDG fractions and the standard on TLC.

Stage 2: Preparation and Characterization of the Essential Oil Isolated from T. orientalis
Fresh leaves of T. orientalis were collected from Anotoniadis Botanical garden in Alexandria, Egypt in August 2019. The plants were authenticated by Dr. Hesham Ali, Antoniadis Research Center, and specimens were deposited at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University. Essential oil (E.O.) was prepared by water-steam distillation [20]. An FT-IR spectrum of the E.O. has been recorded with a Tests can Shimadzu FT-IR spectrophotometer at 800-4000 nm. Then, its constituents were profiled by GC-MS. E.O. was diluted in diethyl ether and 0.5 L was injected into the gas chromatography (GC-MS) apparatus. The GC column was a 30 m (0.25 mm i.d., film thickness 0.25 _m) HP-5MS (5% diphenyl) dimethylpolysiloxane capillary column. The GC conditions were as follows: injector temperature, 240 • C; column temperature, isothermal at 70 • C and held for 2 min; then, it was programmed to 280 • C at 6•C/min and held at this temperature for 2 min; ion source temperature, 200 • C; detector temperature, 300 • C. Helium was used as the carrier gas at the rate of 1 mL/min. The effluent of the GC column was introduced directly into the ion source of the MS. Spectra were obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 400 amu for 5 s. The oil components were identified by comparison of their retention indices and mass spectra with the NIST Mass Spectral Library and the refractive index (RI) of the crude E.O. This was determined by a refractometer. Larvae and pupae of Cx pipiens were purchased two times from the Institute of Medical Insects in El-Dokki, Cairo-Egypt. Upon reaching the laboratory of Vector Control and Pesticide Risks, HIPH, the larvae, and the pupae were reared under laboratory conditions. The rearing of the aquatic stages was on bread. That of the adult males was on glucose 30%, and the females on blood meals from pigeons. All that was maintained under specific  25 and LC 50 from the E.O. was applied and replicated three times. The mortality of the larvae was recorded after 24 h. The larvae were observed for any movement and considered as dead when they did not show any movement even after pin press; they were then collected. The AChE was determined using the anticholinesterase activity Kit of Cholinesterase (BTC/DTNB), Biochemical Enterprise [21,22]. Total protein was determined using the Kit of VITRO SCIENT and Biuret colorimetric endpoint method [23]. Finally, the midgut pH medium and bromothymol blue dye, respectively. Figure 1 shows that the crude PDG was identified by UV-visible spectrophotometry in the range 700-400 nm and the maximum UV absorbance was observed at 530 nm for both batch scale and bioreactor samples; this was in agreement with previous studies done by Patil [19,24,25] for purified PDG from Serratia sp. KH −95. Higher absorption rates were detected for purified red pigment extracted from solutions of two bacteria strains (PNSRR and PNSHR), which had been run in the range of 200 to 700 nm. The maximum absorption peaks were detected at 540 nm and 541 nm for PNSRR and PNSHR, respectively [26]; that was equivalent to what was detected by Kumar and Aparna, 2014 [27].

Isolation, Purification, and Characterization of PDG from SM
The rearing of the aquatic stages was on bread. That of the adult males was on glucose 30%, and the females on blood meals from pigeons. All that was maintained under specific conditions including temperature (26 ± 2 °C) and RH (70 ± 5%), and water was replaced every two days.

Stage 5: Investigation of the Synergistic Effect of PDG with the E.O. as Mosquito Larvicidal Potential
A combination of LC10 from PDG with LC25 and LC50 from the E.O. was applied and replicated three times. The mortality of the larvae was recorded after 24 h. The larvae were observed for any movement and considered as dead when they did not show any movement even after pin press; they were then collected. The AChE was determined using the anticholinesterase activity Kit of Cholinesterase (BTC/DTNB), Biochemical Enterprise [21,22]. Total protein was determined using the Kit of VITRO SCIENT and Biuret colorimetric endpoint method [23]. Finally, the midgut pH medium and bromothymol blue dye, respectively. Figure 1 shows that the crude PDG was identified by UV-visible spectrophotometry in the range 700-400 nm and the maximum UV absorbance was observed at 530 nm for both batch scale and bioreactor samples; this was in agreement with previous studies done by Patil [19,24,25] for purified PDG from Serratia sp. KH −95. Higher absorption rates were detected for purified red pigment extracted from solutions of two bacteria strains (PNSRR and PNSHR), which had been run in the range of 200 to 700 nm. The maximum absorption peaks were detected at 540 nm and 541 nm for PNSRR and PNSHR, respectively [26]; that was equivalent to what was detected by Kumar and Aparna, 2014 [27].   The purity of the red pigment was identical to that of the standard PDG by TLC application (Figure 2). In addition, HPLC profiling showed a single peak at 536 nm for both the column chromatography purified pigment and the standard PDG, and retention times (R f ) were 4.827 min and 4.963 min, respectively ( Figure 3). In agreement with our results, Kumar and Aparna, 2014 showed that >95% purity of a single peak was observed at R f > 4 min (4.523 min) [27]. In contrast, Mandal et al. revealed that the R f went until Biol. Life Sci. Forum 2022, 11, 54 4 of 11 4 min, and no sharp peaks were observed after 4 min [26]. The application of TLC for the identification of the purified red pigment in the present study revealed that the best mobile phase was n-hexan:ethyl acetate (v/v) (2:1) and the R f of the tested sample was equal to that of the standard; this confirms that the tested sample is PDG. Mandal et al. reported that the R f values of the extracted pigments were found to be approximately 0.88 after using methanol, chloroform, and hexane in the ratio of 7:3:1 [26].

Isolation, Purification, and Characterization of PDG from SM
The purity of the red pigment was identical to that of the standard PDG by TLC ap plication ( Figure 2). In addition, HPLC profiling showed a single peak at 536 nm for bot the column chromatography purified pigment and the standard PDG, and retention time (Rf) were 4.827 min and 4.963 min, respectively ( Figure 3). In agreement with our results Kumar and Aparna, 2014 showed that >95% purity of a single peak was observed at Rf 4 min (4.523 min) [27]. In contrast, Mandal et al. revealed that the Rf went until 4 min, an no sharp peaks were observed after 4 min [26]. The application of TLC for the identifica tion of the purified red pigment in the present study revealed that the best mobile phas was n-hexan:ethyl acetate (v/v) (2:1) and the Rf of the tested sample was equal to that o the standard; this confirms that the tested sample is PDG. Mandal et al. reported that th Rf values of the extracted pigments were found to be approximately 0.88 after using meth anol, chloroform, and hexane in the ratio of 7:3:1 [26].  Figure 4 shows that the FT-IR absorption in ethanol for the studied red pigment wa dominated by very strong bands at 2921.5 cm −1 and 2851.1 cm −1 (aromatic CH). The fin gerprint region for the red pigment was characterized by medium intensity bands at νma 1609.3 cm −1 , 1362.4 cm −1 , 1265.1 cm −1 , 1040.9 cm −1 , and 955.1 cm −1 . In contrast, lower inten sity bands were observed at νmax 2373.9 cm −1 , 2341.99 cm −1 , 2292.8 cm −1 , and 2166.9 cm (alkyl C-H). A broad peak pyrrole was observed at 3105.841 cm −1 , 3274.8 cm −1 , and 3427. cm −1 , due to the presence of protonated nitrogen; this confirms that the pattern of the re pigment is identical to the standard. In addition, the FT-IR of the red pigment wa matched with that of the standard and this confirms that the red pigment is PDG (C20H25N3O). In agreement with our study, Patil et al. revealed that the absorption wa dominated by very strong bands at 2977 cm −1 (aromatic CH) and 1648 cm −1 (aromatic C=C Furthermore, he reported that PDG exhibits similar absorptions in CHCl3 at 1630 and 160 cm −1 , except that the relative intensities are reversed and the first band is possibly The purity of the red pigment was identical to that of the standard PDG by TLC application ( Figure 2). In addition, HPLC profiling showed a single peak at 536 nm for both the column chromatography purified pigment and the standard PDG, and retention times (Rf) were 4.827 min and 4.963 min, respectively ( Figure 3). In agreement with our results, Kumar and Aparna, 2014 showed that >95% purity of a single peak was observed at Rf > 4 min (4.523 min) [27]. In contrast, Mandal et al. revealed that the Rf went until 4 min, and no sharp peaks were observed after 4 min [26]. The application of TLC for the identification of the purified red pigment in the present study revealed that the best mobile phase was n-hexan:ethyl acetate (v/v) (2:1) and the Rf of the tested sample was equal to that of the standard; this confirms that the tested sample is PDG. Mandal et al. reported that the Rf values of the extracted pigments were found to be approximately 0.88 after using methanol, chloroform, and hexane in the ratio of 7:3:1 [26].  Figure 4 shows that the FT-IR absorption in ethanol for the studied red pigment was dominated by very strong bands at 2921.5 cm −1 and 2851.1 cm −1 (aromatic CH). The fingerprint region for the red pigment was characterized by medium intensity bands at νmax 1609.3 cm −1 , 1362.4 cm −1 , 1265.1 cm −1 , 1040.9 cm −1 , and 955.1 cm −1 . In contrast, lower intensity bands were observed at νmax 2373.9 cm −1 , 2341.99 cm −1 , 2292.8 cm −1 , and 2166.9 cm −1 (alkyl C-H). A broad peak pyrrole was observed at 3105.841 cm −1 , 3274.8 cm −1 , and 3427.0 cm −1 , due to the presence of protonated nitrogen; this confirms that the pattern of the red pigment is identical to the standard. In addition, the FT-IR of the red pigment was matched with that of the standard and this confirms that the red pigment is PDG (C20H25N3O). In agreement with our study, Patil et al. revealed that the absorption was dominated by very strong bands at 2977 cm −1 (aromatic CH) and 1648 cm −1 (aromatic C=C). Furthermore, he reported that PDG exhibits similar absorptions in CHCl3 at 1630 and 1602 cm −1 , except that the relative intensities are reversed and the first band is possibly a  In contrast, lower intensity bands were observed at νmax 2373.9 cm −1 , 2341.99 cm −1 , 2292.8 cm −1 , and 2166.9 cm −1 (alkyl C-H). A broad peak pyrrole was observed at 3105.841 cm −1 , 3274.8 cm −1 , and 3427.0 cm −1 , due to the presence of protonated nitrogen; this confirms that the pattern of the red pigment is identical to the standard. In addition, the FT-IR of the red pigment was matched with that of the standard and this confirms that the red pigment is PDG (C 20 H 25 N 3 O). In agreement with our study, Patil et al. revealed that the absorption was dominated by very strong bands at 2977 cm −1 (aromatic CH) and 1648 cm −1 (aromatic C=C). Furthermore, he reported that PDG exhibits similar absorptions in CHCl3 at 1630 and 1602 cm −1 , except that the relative intensities are reversed and the first band is possibly a pyrrolenine (C=N). In addition, the fingerprint region for the red pigment was characterized by medium intensity bands at νmax 1648 (C=O), 1087, 1047 (C-O and C-N), and 879 cm −1 . A strong and broad absorption for NH was evident at νmax 3403 cm −1 . This indicates that this pigment pattern is identical to that of PDG [19].
Biol. Life Sci. Forum 2022, 11, 54 5 of 11 pyrrolenine (C=N). In addition, the fingerprint region for the red pigment was characterized by medium intensity bands at νmax 1648 (C=O), 1087, 1047 (C-O and C-N), and 879 cm −1 . A strong and broad absorption for NH was evident at νmax 3403 cm −1 . This indicates that this pigment pattern is identical to that of PDG [19].

Preparation and Characterization of E.O. from Fresh Leaves of T. orientalis
Water-steam distillation of 750 gm from fresh leaves of T. orientalis revealed 3 mL of E.O. That oil was subjected to FT-IR spectroscopy and it revealed different bands with a diversity of intensities, as shown in Figure 5. The E.O. was characterized by GC-MS and it identified 25 constituents. The representative GC-MS revealed that the total chemical composition of E.O. of T. orientalis is 97.04%, as shown in Table 1

Preparation and Characterization of E.O. from Fresh Leaves of T. orientalis
Water-steam distillation of 750 gm from fresh leaves of T. orientalis revealed 3 mL of E.O. That oil was subjected to FT-IR spectroscopy and it revealed different bands with a diversity of intensities, as shown in Figure 5. The E.O. was characterized by GC-MS and it identified 25 constituents. The representative GC-MS revealed that the total chemical composition of E.O. of T. orientalis is 97.04%, as shown in Table 1       In this study, the yield of the EO was 0.3% and that was in agreement with what was reported by Nickavar et al. [24], where the hydrodistillation of T. orientalis leaves gave oils with a yield of 0.25%. Twenty-five compounds of the studied EO were identified by GC-MS, revealing that the total chemical composition of E.O. is 97.04%, constituting 64.98% of monoterpene hydrocarbons, followed by a lower percentage of oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes, which were 12.08%, 10.39%, and 9.59%, respectively. The percentages of the main monoterpene hydrocarbons In this study, the yield of the EO was 0.3% and that was in agreement with what was reported by Nickavar et al. [24], where the hydrodistillation of T. orientalis leaves gave oils with a yield of 0.25%. Twenty-five compounds of the studied EO were identified by GC-MS, revealing that the total chemical composition of E.O. is 97.04%, constituting 64.98% of monoterpene hydrocarbons, followed by a lower percentage of oxygenated monoterpenes, sesquiterpene hydrocarbons, and oxygenated sesquiterpenes, which were 12.08%, 10.39%, and 9.59%, respectively. The percentages of the main monoterpene hydrocarbons were 3-carene 30. The main oxygenated sesquiterpenes were Caryophyllene oxide, α-Acorenol, and cedrol, which were 0.24, 0.48, and 8.87, respectively. Ibrahim et al. [29] reported that the percent-ages of oxygenated compounds, hydrocarbons monoterpenes, and sesquiterpenes in the T. orientalis leaves' oil were 29.85%, 44.74%, and 24.35%, respectively. The major components were α -pinene (21.83%), benzyl benzoate (19.12%), caryophyllene (12.07%), and α -cedrol (6.86%). The refractive index of the E.O. was 1.482 nD, revealing high purity of the E.O. This is because its value was in the range of the typical value (1.4785 nD-1.4885 nD) [30]; the higher value (1.5) was detected in 2022 by Rehman et al. [31].

Dose Response Bioassay Separately of the Preparations
The result of log probit analysis (95% confidence level) recorded that the LC 50 value of PDG (39.5ppm) showed a high larvicidal rate after 24 as compared to the E.O. (102.9 ppm) ( Table 2). In 2002, Metacycloprodigiosin hydrochloride and bafilomycin A1 revealed a significant antimalarial activity; meanwhile, spectinabilin moderately inhibited the proliferation of P. falciparum K1 [32]. Jeon et al. [33] reported that the larvicidal activities of leaf oils prepared from T. orientalis were significantly higher than those of stem, fruit, and seed oils against 4th-instar larvae of Ae. aegypti and Cx. pipiens pallens. Leaf oils of T. orientalis leaves show promise as activity natural larvicides against Ae. aegypti and Cx. pipiens pallens. In India (2015), pure PDG showed LC 50 values 15.6 ± 1.48 and 24.7 ± 1.47 µg mL −1 against 3rd instars of Ae. Aegypti and An. Stephensi, respectively [34]. In 2016, it was reported that the larvicidal properties of Plectranthus barbatus leaves EO (40,80,120,160, and 200 µg/mL) and their components, such as eugenol, α-pinene, and β-caryophyllene (12-100 µg/mL each) were measured using WHO protocol. EO displayed considerable larvicidal properties with LC 50 values of 94.3 µg/mL for Cx tritaeniorhynchus. The three main components (eugenol, α-pinene, and β-caryophyllene) demonstrated potent larvicidal properties (LC50 = 30.8, 36.8, and 48.2 µg/mL, respectively) [35]. Two years later, Sanei-Dehkordi et al. [36] investigated that the dosage of 80 ppm from Platycladus orientalis oil was sufficient to cause 100% larval mortality against the larvae of Cx pipiens after 24 h. Forty-six components in leaves of P. orientalis were identified. The major components were α-Pinene (20.17%), 3-Carene (14%), and Cedrol (9.51%). The LC 50 value against Cx. pipiens larvae was 18.60 ppm after 24 h; hence, the authors considered E.O. as a natural larvicide for mosquito larval control. Table 3 shows that the combination between LC 10 PDG and LC 50 of E.O. had a high synergistic effect on the mortality rate after 24 h compared with its combination with LC 25 E.O. (100% and 33.3%, respectively). Clerodendrum inerme showed the highest toxicity when tested individually at 24 h against early 4th instar mosquito larvae, Aedes aegypti. In contrast, G. sepium showed low toxicity (LC 50 = 292 ppm and LC 50 = 564 ppm, respectively). The maximum synergistic activities were found in the combination extracts of Vitex negundo with Pongamia glabra (LC 50 = 191.73 ppm). These results are significantly more effective than the combination extract ratio of C. inerme with P. glabra (LC 50 = 195.02 ppm) and Gliricidia sepium with P. glabra (LC 50 = 328.72 ppm) [37].  Table 4 shows that the highest percentage of AChE arbitrary activity by unit/gm tissue was (5.8%) among untreated 3rd larvae of Cx. pipiens, followed by the treated ones with E.O. and PDG (3.5% and 2.5%, respectively). Next were those treated with combination LC 10 of PDG with LC 25 and LC 50 of E.O. (3.8% and 3.0%, respectively). Concerning the total protein in mg/gm tissue, the untreated larvae showed a high percentage (1.32%) compared with E.O. (1.12%), followed by those treated with PDG (0.72%) and those treated with a combination of LC 10 of PDG with LC 25 and LC 50 of E.O (0.92% and 0.52%, respectively). Regarding the AChE arbitrary specific activity, the untreated larvae showed the highest rate (4.39%) as compared to PDG, EO, and treated ones with a combination of LC 10 of PDG with LC 25 and LC 50 of E.O (3.47%, 3.13%, 4.13%, and 0.91%, respectively). Figure 7 shows that the midgut of untreated larvae showed a blue color after 12 h incubation in bromothymol dye; meanwhile, treated larvae showed a yellowish color, indicating reduction in the pH medium. PDG causes reduction in the AChE and the total protein content of the treated larvae. AChE breaks down the neurotransmitter Ach at the synaptic cleft so that the nerve impulse can be transported across the gap. Neurotransmitters must be cleaned immediately after the message is passed, and if not, it causes paralysis [38]. Larvicidal activities of leaf oils prepared from T. orientalis were significantly higher than those of stem, fruit, and seed oils against 4th-instar larvae of Ae. aegypti and Cx. pipiens pallens. Leaf oils of T. orientalis leaves show promise as activity natural larvicides against Ae. aegypti and Cx. pipiens pallens [33]. Purified PDG caused reduction in the activity of AChE and total protein content of the treated larvae by 70% and 43.4%, respectively, compared to the control among the 4th-instar larvae of Ae. Aegypti [34]. Bromothymol blue dye is a weak acid and a member of the class of 2,1-benzoxathioles, so it acts as a pH indicator. This reagent is blue in alkaline media, green in neutral media, and yellow in acidic media [39]. In the present study, untreated larvae showed a dark blue color by a stereomicroscope, in contrast to the midgut of the treated larvae with PDG, E.O, and their combination, which showed a reduction in the pH. In agreement with this, Suryawanshi et al. [34] reported that the larval midgut of PDG treated larvae showed a greenish yellow color, suggesting acidic pH; in contrast, untreated larvae showed up as blue, indicating the basic condition. As reported by Zhuang et al. [40], this may be attributed to the hydrophobicity of natural products including PDG and E.O. and their effect as carbonic anhydrase inhibitors, leading to a reduction in pH of the midgut that subsequently results in cellular respiration inhibition [41][42][43].

Conclusions
High LC50 was observed in essential oils' Thuja orientalis leaves. The combination between LC10 of prodigiosin and LC50 of T. orientalis leaves showed the highest synergistic effect (100%). The treated 3rd larval Cx. pipiens showed a reduction in the acetylcholine esterase, total protein content, and midgut pH as compared to the untreated ones.

Conclusions
High LC 50 was observed in essential oils' Thuja orientalis leaves. The combination between LC 10 of prodigiosin and LC 50 of T. orientalis leaves showed the highest synergistic effect (100%). The treated 3rd larval Cx. pipiens showed a reduction in the acetylcholine esterase, total protein content, and midgut pH as compared to the untreated ones.