Chemical Composition and Larvicidal Properties of Essential Oils from Wild and Cultivated Artemisia campestris L., an Endemic Plant in Morocco

The Asteraceae family is well known for its toxic and repellent activity against mosquitoes. In this study, essential oils (EOs) extracted from the aerial parts of both wild and cultivated Artemisia campestris L. plants were tested for larvicidal activity against Culex pipiens (Diptera: Culicidae), a pest mosquito widely suspected to be the vector responsible for West Nile virus transmission. The research aims at comparing the chemical composition and insecticidal activity of cultivated and wild A. campestris EOs. The EOs were obtained by hydrodistillation from the plant's aerial parts and were analyzed using GC-MS. Furthermore, the larviciding experiment was carried out following the standard WHO protocol. The result showed that wild and cultivated plant EOs differed only quantitatively, while the qualitative profile revealed a nearly identical chemical composition. Camphor (18.98%), car-3-en-5-one (11.25%), thujone (6.36%), chrysanthenone (6.24%), filifolone (4.56%), and borneol (3.56%) dominate the wild plant EO. Camphor (21.01%), car-3-en-5-one (17%), chrysanthenone (10.15%), filifolone (7.90%), borneol (3.38%), and thujone (3.08%) are the major compounds of the cultivated plant. Cultivation did not affect the EO production since the yield of the cultivated plant was 0.5 ± 0.1% and 0.6 ± 0.2% for the wild plant. The cultivated A. campestris EO had the highest insecticidal activity (LC50 = 9.79 µg/ml), and no significant difference was noticed between wild and cultivated A. campestris EO in terms of LC90. These findings could pave the way for a new method of producing biocides to control major disease vectors and offer a potential alternative for pest control.


Introduction
Mosquitoes are a major threat to millions of people worldwide.Tey transmit various diseases, including malaria, yellow fever, dengue fever, West Nile fever, and chikungunya [1].Te West Nile virus (WNV) (family Flaviviridae and genus Flavivirus) can infect humans, birds, and horses, and it is primarily transmitted by the genus Culex [2].
Te Culex genus is the most important vector of viruses causing Japanese encephalitis, St. Louis encephalitis, and West Nile fever [3].Te most common mosquito insect in rural and urban areas is Culex pipiens (Diptera: Culicidae) [4].Tis insect, also known as the house mosquito, is one of the most widely distributed mosquitoes worldwide, and several studies have implicated C. pipiens in West Nile virus transmission [5,6].Moreover, it is an important pest for humans, causing allergic reactions such as local skin reactions and systemic reactions such as angioedema and urticaria [7].Also, it has been linked to disease spread in several countries [8], including Morocco in 1996 [9] and more recently in 2010 [10].
Besides, the most efcient way to avoid mosquito bites [11,12] is to use efective vector management strategies to control and prevent the propagation of mosquito-borne diseases [13].
Several methods (physical, genetic, and chemical) have been studied to control this mosquito in both its larval and adult forms.Over the last few decades, genetic control, a term that refers to a variety of methodologies such as sterile insect technique (SIT) or chemosterilization, the release of hybrids or insects with translocations, has been used as a mosquito control method [14,15].Furthermore, the most efective of these strategies is chemical control, but it poses serious risks to the environment and human health [16], one of which is pesticide resistance [17,18].
As a result, replacing pesticides with biological alternatives based on natural products, especially aromatic plants, and EOs, is the ideal solution [19,20].Previous research on the plant's essential oils (EOs) against C. pipiens larvae has been conducted [21][22][23].
Artemisia is a large and important member of the Asteraceae family, with over 500 species found in Europe, North America, Asia, and South Africa [24].Tese species are known for their diverse medicinal and therapeutic properties and include a variety of biologically active plants [25,26], including Artemisia herba-alba, Artemisia annua L., Artemisia judaica L., and Artemisia arborescens [27,28].Tese plants are well known for their ability to treat a variety of ailments, including infammation, hepatitis, cancer, malaria, infections, and diabetes [29][30][31].A. herba-alba, commonly known as desert mugwort, found in arid regions, has traditionally been used to treat respiratory and parasitic infections (including helminths) and digestive issues, among other things [32][33][34].A. annua L. is notable for its artemisinin content [35], whereas A. judaica L., commonly known as "Beithran" in Arabic, is used to treat stomach upset, heart disease, and diabetes [36].Anti-infammatory, antihistaminic, and antiviral properties are provided by A. arborescens [37].Furthermore, Artemisia maritima and Artemisia nilagirica have powerful pharmacological efects and are efective against disease-carrying mosquitoes such as Aedes albopictus and C. pipiens [38][39][40].
Morocco is known for its diverse plant life, with a wide variety of species found throughout the country.Many of these plants have been traditionally used in Moroccan medicine, cooking, and other daily activities.Te genus Artemisia contains 14 species, eight of which are endemic [40].Artemisia campestris L is one of these endemic species, commonly known as "degoufet," "tgouft," or "alala.".It is known for its strong, pungent aroma and has many medicinal properties, including biological properties such as antileishmania [41], antivenom, anticancer, antidiabetic, antihypertensive, anthelmintic, antimicrobial, antifungal, and insecticidal properties [42,43], and it has been used to treat a variety of disorders, including digestive, respiratory, cutaneous, and genital diseases [44].
Te valorization of EO from A. campestris refers to the process of utilizing the plant's EOs for practical applications, such as vector control.Indeed, EOs generally contain a high concentration of monoterpenes and sesquiterpenes, as well as favonoids, phenolic acids, coumarins, and fatty acids [45]. A. campestris EO has been found to have numerous pharmacological activities, including antioxidant, antifungal, insecticidal, antibacterial, antimutagenic, antitumor, anthelmintic, and antihypertensive properties [46].Over the years, EOs have been extracted from a wide range of plant species, and several types of research have concentrated on their biological properties [47], particularly their insecticidal activity [48,49].Furthermore, multiple studies in the pharmaceutical [50], agricultural [51,52], and food [53] sectors have highlighted their potential use as alternatives to synthetic compounds.Also, several studies have found that EOs extracted from A. campestris have strong insecticidal activity [54,55], specifcally larvicidal activity against C. pipiens [56,57].Some of the compounds that have been identifed in the EO of A. campestris include terpenes such as 1,8-cineole, borneol, and terpinen-4-ol and sesquiterpenes such as betacaryophyllene, alpha-humulene, and germacrene D, as well as other compounds such as p-cymene, thujone, and caryophyllene oxide [46,[58][59][60].
Te current study was carried out for the frst time in Morocco in order to valorize the Moroccan endemic species A. campestris through the cultivation and to highlight the chemical components of EOs extracted from wild and cultivated A. campestris populations in Morocco, as well as to evaluate the larvicidal toxicity of these EOs against C. pipiens (Diptera: Culicidae).To the best of our knowledge, no study has been conducted to investigate the efect of the cultivation of A. campestris EOs on larvicidal activity against C. pipiens.Furthermore, our fndings could serve as preliminary data for researchers interested in the valorization of other endemic plants and aid in the development of a biolarvicide, ofering a natural and eco-friendly alternative to chemical insecticides.Additionally, it may play a crucial role in integrated vector management programs, paving the way for a natural product-based pest control method.

Plant Material. Te aerial parts of two endemic
Moroccan plants, wild and cultivated, were harvested at the fowering stage, during the period of May and July of 2022, and their entire details are listed in Table 1.Professor Badr Satrani, a botanist at the Forest Research Center in Rabat, made the identifcation.Both species' aerial parts were dried in a ventilated environment (in the shade in an airy space) for ten days before being extracted.We successfully cultivated this plant under the following conditions: we used the cutting propagation method, with the cuttings originating from a natural specimen at the Lmarija site (Guercif Te Scientifc World Journal Province).Tese cuttings (stems), which were about 10 cm long and had two to three leaves, were planted directly in the feld.To reduce transpiration, we covered the cuttings with plastic bottles.We provided regular watering at an ambient temperature and allowed the plant to grow in soil with a slightly alkaline limestone profle.

Extraction of Essential Oils and Chemical
Characterization.Te aerial part of each plant was divided into small plots and placed in a fask with 1 L of distilled water for a total of 100 g.Tereafter, EOs were extracted using a Clevenger apparatus [61] through a three-hour hydrodistillation process.After removing any remaining water with anhydrous sodium sulfate, they were kept at 4 °C.Gas chromatography-mass spectrometry (GC-MS) was used to chemically analyze the EOs.

GC-MS Analysis.
Gas chromatography-mass spectrometry (GC-MS) was used to determine the precise composition of EOs.Tis technique allows for compounds to be identifed by their mass-to-charge ratio.Te GC-MS analysis was performed using a Trace GC Ultra apparatus equipped with a triple quadrupole detector, a splitless injector, and an RTxi-5 Sil MS capillary apolar column (30 m × 0.25 mm ID × 0.25 m).Te operational conditions were as follows: the column was held at 50 °C for 2 minutes and then heated at a rate of 5 °C/min to 160 °C for 2 minutes and then to 280 °C for 2 minutes.Te connection to the Polaris QMS mass spectrometer was made at 280 °C.Te injection temperature was 250 °C, the injection volume was 1 µl, the pressure was 37.1 kPa•mL/min, the carrier gas was helium, and the solvent was hexane.Te identifcation of the diferent phytochemical components of the essential oil was obtained by determining their retention indices and comparing them with literature data [62,63].
2.3.Mosquito Larvae Collection.C. pipiens larvae were collected from the Oued El-Mehraz region (altitude: 423 m; 34 °02′13.74″N,4 °59′59.279″W).Te larvae were gathered in a rectangular plastic dish and then kept in the entomology unit at the Regional Public Health Laboratory of Fez under consistent conditions, in the breading site water, with a water temperature of 24.6 °C ± 2 °C and a relative humidity of 50%-70%.Te third and fourth-instar larvae were used in the experiments.We used two tools to identify mosquito larvae based on their morphological characteristics: the Moroccan identifcation key for Culicidae [64] and the African Mediterranean mosquito identifcation software [65].

Larvicidal Bioassays.
Te larviciding tests were carried out in accordance with the WHO recommendations, with minor modifcations [66].A sequence of exploratory experiments was carried out in order to establish an appropriate range of EO concentrations for both wild and cultivated A. campestris.Ethanol was used as a solvent, and the concentrations tested for wild A. campestris were 4, 8, 12.5, 20, 25, 30, and 50 µg/ml, while the concentrations tested for cultivated A. campestris were 2.5, 10, 20, 30, and 40 µg/ml.For each concentration, three replicates were prepared.In the experiment, one milliliter of each produced suspension was placed in a beaker containing 99 milliliters of distilled water and twenty third-and fourth-instar larvae of C. pipiens.A control test was also conducted by combining 1 ml of ethanol with 99 ml of distilled water and 20 C. pipiens larvae.Each larvicidal experiment, as well as the control test, received three replicates.Mortality of all concentrations was determined after 24 hours of treatment.If the control mortality test exceeds 5%, the mortality rate of larvae exposed to EOs must be corrected using Abbott's formula [67], and if the control mortality test exceeds 20%, the larvicide tests are invalid and must be repeated.% mortality corrected � ⌊ (% mortality observed − % mortality control 100 − % mortality control ⌋ × 100. (1) 2.5.Statistical Data Processing.Statistical tests were performed using the log-probit program from CIRAD-CA/ MABIS [68].Finney's mathematical procedures were used to calculate lethal concentration levels, along with 95 percent confdence limits and the Chi2 test (LC 50 and LC 90 ), and a Tukey post hoc test was used with Origin Pro 2021 software to examine signifcant diferences between average values (a probability of p ≤ 0.05 was considered statistically signifcant).

Results and Discussion
3.1.Yield of Essential Oils.Te yield obtained from the samples of A. campestris was 0.6 ± 0.2% and 0.5 ± 0.1% for wild and cultivated plants (Figure 1), respectively.No signifcant diferences (p > 0.05) were noticed between the wild and cultivated plants in terms of the average yield.Te results indicated that cultivation did not afect EO production.Te Scientifc World Journal Our fndings are consistent with those obtained in Morocco by Aljaiyash et al. [24], who demonstrated that there is no signifcant diference in the yield of EO from cultivated and wild A. herba-alba and that cultivation does not afect oil production.
Our discovery supports previous research that found camphor in the majority of A. campestris EOs [72,73].Tis bicyclic compound has numerous biological activities, including insecticidal, analgesic, antimicrobial, antiviral, anticancer, and antitussive properties [50].
Furthermore, several studies have been carried out and have revealed the presence of other chemical profles in the EO of A. campestris.Te EO of A. campestris from Algeria [58] revealed the presence of β-pinene (25.6%), sabinene (17%), and α-pinene (9.9%) as major compounds.
Tis disparity could be explained in part by qualitative and/or quantitative variations in the chemical composition of the EOs, which are primarily related to the cultivation conditions such as geographical factors, climate, and physical and chemical properties of the soil [77].EO production is infuenced by a variety of factors, including genetics and plant developmental stage.Environmental conditions also play an important role, causing biochemical and physiological changes that afect both the quantity and quality of EOs [78].Tese changes can adversely afect aromatic plant production and subsequently reduce the overall quality of the obtained EOs.Terefore, it becomes imperative to explore and implement efective agronomical management techniques to enhance EO production and optimize compound concentration.Another critical factor infuencing EO production is the presence of plant growth regulators or hormones.Tese substances, whether naturally occurring within plants or applied externally, can signifcantly infuence both the production and chemical composition of EOs.By comprehending and controlling these variables, it becomes possible to achieve more consistent and improved EO products [79].
Te synthesis of secondary metabolites is genetically controlled, but their production is greatly infuenced by environmental conditions, harvesting, and postharvest factors.Precipitation, temperature, light, and humidity also afect the volatile oil yield and the content of principal components [80].Plant development stage and specifc organ development, as well as exogenous factors (biotic and abiotic), all play a role in shaping essential oil production [81].Sangwan et al. [78] conducted research on EO production regulation and identifed several infuential factors afecting both the quantity and quality of these compounds.Such factors include ontogeny, photosynthetic rate, photoperiod, light quality, climatic and seasonal variations, nutritional availability, humidity, salinity, temperature, storage conditions, and growth regulators.Farooqi and 4 Te Scientifc World Journal Shukla's study [82] also confrmed the impact of growth regulators, or plant hormones, on the quality and quantity of EO.

Larvicidal Activity of Essential Oils against Culex pipiens.
Te two plant EOs were tested to evaluate their larvicidal activity against C. pipiens (Table 3), and the tested oils revealed various mortality percentages at diferent concentrations.Results are represented as mean ± standard error (SE) after 24 h of exposure.Te data obtained from the EO of wild A. campestris show that the highest percentage mortality was recorded at 50 µg/ml and was evaluated at 100 ± 0.00%, and 40 µg/ml for the EO of the cultivated plant with a mortality rate of 100 ± 0.00%, after 24 h of treatment (Figures 2  and 3).Our fndings were consistent with those found in the literature, which show that mortality increases with dose and contact time [83].Te most efective activity was obtained by the cultivated A. campestris EO (LC 50 � 9.79 μg/ml).Te results of the experiment revealed that both oils had interesting insecticidal properties against C. pipiens larvae.Te insecticidal efcacy of both EOs is due to the high concentration of camphor, an oxygenated monoterpene well known for its strong insecticidal activity [84].
Almost 2,000 plant species with insecticidal activity have already been identifed [85].Tis activity could be explained by the development of chemical substances such as (phenols, polyphenols, terpenoids, and alkaloids) by plants [86].In general, several authors state that products with LC 50 values less than 100 μg/mL are considered active [87][88][89].

6
Te Scientifc World Journal As far as we know, no previous studies have specifcally investigated the larvicidal efects of EO from wild and cultivated A. campestris against the C. pipiens mosquito.As a result, we conducted a comparison of our fndings with previous research on Artemisia's insecticidal properties against various Culicidae family species.
Several studies have investigated the insecticidal properties of EOs from wild and cultivated plants [93][94][95].Also, several studies have been conducted to investigate the larvicidal activity of plants of the genus Artemisia [24,96,97].
Our results corroborate with those found in Morocco by Aljaiyash et al. [24], who proved that cultivated A. herbaalba EO was more toxic than the wild plants against adults of the insect pest T. castaneum.In the same context, EOs from both wild and cultivated Ruta chalepensis plants were found to have very high toxicity against Aedes albopictus larvae with LC 50 values of 35.66 ppm and 33.18 ppm, respectively [95].
Also, both EOs present remarkable toxicity similar to that found by Ammar et al. [54] against C. quinquefasciatus with a value of LC 50 of 45.8 mg/L.
From the presented results, we can conclude that the insecticidal activity could be expressed by the variety of bioactive molecules that defne each EO.
Even if the two plants are of the same origin, the difference in chemical composition and insecticidal properties could be explained by the infuence of several factors that act on cultivation conditions, including climatic factors, soil properties, and altitude [24].

Conclusion
Tis study focused on the larvicidal efect of essential oils from cultivated and wild populations of A. campestris, which are endemic to Morocco, against the mosquito Culex pipiens, which transmits the West Nile virus.Te results revealed the efcacy of essential oils, as the chemical components within these oils displayed potent larvicidal activity.Te cultivated A. campestris revealed the highest larvicidal activity.Tese active compounds are recommended as natural, plant-based alternatives with notable insecticidal properties.However, further studies should be conducted to explore the efect of essential oils from other species of the same genus, as well as their synergistic efects, to optimize their larvicidal potential.Additionally, the potential toxicity of these products on wildlife should also be studied.Te Scientifc World Journal

Figure 1 :
Figure 1: Average yield of A. campestris essential oil for the wild and cultivated plants.According to the Tukey test, each column represented by a diferent letter demonstrated a signifcant difference (p < 0.05).

Figure 2 :Figure 3 :
Figure 2: Mortality rates of C. pipiens larvae after 24 h of exposure to wild A. campestris EO at diferent concentrations.

Figure 4 :
Figure 4: Average insecticidal activity of A. campestris EO for wild and cultivated plants.According to the Tukey test, each column represented by a diferent letter (A, B, and C) illustrated a significant diference (p < 0.05).

Table 1 :
Te main information on the two-plant species whose larvicidal activity was tested in this study.

Table 2 :
Chemical compounds derived from both wild and cultivated A. campestris aerial parts (%).Te larvicidal activities against C. pipiens obtained from the wild samples of A. campestris were 19.07 ± 2.57 µg/ml and 35.63 ± 03.37 µg/ml for LC 50 and LC 90 , respectively.Regarding lethal concentrations, the cultivated samples of A. campestris reported 9.79 ± 1.47 µg/ml and 38.57± 03.69 µg/ml for LC 50 and LC 90 , respectively.Figure 4 illustrates the signifcant diferences (p < 0.05) in LC 50 of wild and cultivated A. campestris EOs.However, no signifcant diference (p > 0.05) was noticed among wild and cultivated A. campestris EOs in terms of LC 90 .

Table 3 :
Lethal concentrations of EOs (LC 50 and LC 90 ) from wild and cultivated A. campestris against C. pipiens.