Chemical composition and larvicidal activity of Moroccan Atlas Cedar ( Cedrus atlantica Manetti ) against Culex pipiens ( Diptera : Culicidae )

1 Regional Diagnostic Laboratory of Epidemiological and Environmental Health, Regional Health Directorate, El Ghassani Hospital, Fez 30000, Morocco. 2 Laboratory of Applied Organic Chemistry, faculty of sciences and technology, Sidi Mohamed Ben Abdellah University, po.box 2202 – route d’Imouzzer, Fez, Morocco. 3 Laboratory of Animal Physiology, Department of Biology, Faculty of Science, University Mohammed V, Rabat, Morocco. 4 Institute of Nursing Professions and Health Techniques of Fez (annex Meknes), Regional Health Directorate, El Ghassani Hospital, Fez 30000, Morocco.


INTRODUCTION
Culex pipiens (C.pipiens) (Diptera: Culicidae) is one of the most widely distributed mosquitoes in the world.The species commonly referred to as "house mosquito", can be found in urban and suburban areas and lives near people (Bernard et al., 2001).
Several commercially available insecticides (e.g.temephos, chlorpyrifos-methyl, diflubenzuron) can be effective to control the species at immature stages (Cetin et al., 2006a(Cetin et al., , 2006b)).However, many of these chemical insecticides are expensive and harmful to the environment as well as to humans (Huseyin et al., 2009).The WHO expert committee (Samuel et al., 2013;WHO, 1982) felt the resistance in vectors was probably the "biggest single obstacle in the struggle against vector-borne diseases".Many mosquito species are known to have developed resistance to temephos in many parts of the world and also in Morocco (El-akhal et al., 2016;El Ouali Lalami et al., 2014;Cui et al., 2006;Braga et al., 2004;Faraj et al. 2002).Natural plant products can be an excellent alternative source of novel insecticidal chemistries.With some exceptions, botanicals are considered to be less toxic to non-target species and more environmentally friendly because of their biodegradable nature (El Ouali Lalami et al., 2016;Copping 1996).In Morocco, several studies have been carried out on the larvicidal effect of plants against Culex pipiens, for exemple Origanum majorana (El-Akhal et al., 2014), Lavandula stoechas (El Ouali Lalami et al., 2016), Citrus sinensis and Citrus aurantium (El-Akhal et al., 2014).Currently, the genus Cedrus includes three extant species native to the Mediterranean mountains (Cedrus atlantica Manetti from Algeria and Morocco; Cedrus libani Rich. in Asia Minor; Cedrus brevifolia Henry in Cyprus) and in the Himalaya (Cedrus deodara Don) (Farjon, 2008).The Atlas cedar is an endemic species of North Africa Mountains (Morocco, Algeria).In Morocco, the Atlas cedar occupies an area of 132,000 hectares divided into two blocks of unequal importance: the Rif; the Moroccan Middle and High Atlas (Cheddadi et al., 2009;M'hirit et al., 1993), presenting an altitudinal ranging from 1500m to 2600m.The Middle Atlas in northern Morocco contains about the 80% of the world's Atlas cedar forest area (Linares et al., 2011;Benabid, 1994).
This raw material would be important for essential oil production to be used for its medicinal properties and perfumery (Boudarene et al., 2004).Indeed, essential oils are a part of several products such as drugs and perfumes (Adams, 1991).The essential oil of C. atlantica Manetti (C.atlantica) has been shown to possess antifungal (Bouchra et al., 2003), antimicrobial (Zrira and Ghanmi, 2016), antiviral (Monica et al., 2008), and antiinflammatory activities (Sugita et al., 2004), but its larvicidal activity is not reported against C. pipiens in the literature.
In this work, the chemical quality of the Atlas cedarwood oil obtained from the middle Atlas forest were studied and its insecticidal activity against larvae of C. pipiens was determined in the first time in Morocco.

Plant material and extraction of the Essential Oil (EO)
The aerial parts (leaves, stems and wood) of C. atlantica (Fig. 1) are collected in Boulmane region (Middle Atlas Mountains, Morocco), between April and May 2014.The botanical identification and authenticated voucher specimens have been deposited in the Herbarium of National Institute of Medicinal and Aromatic Plants, Sidi Mohamed Ben Abdellah University, Fez, Morocco.Samples of 100g of the fresh aerial parts of C. atlantica were subjected to hydrodistillation for 2 hours using a Clevenger apparatus; the obtained Essential Oil (EO) was stored at 4°C so that can be used in the upcoming experiments.

Chemical characterization of essential oil of C. atlantica
The chromatographic analysis of essential oil was conducted in ''Centre Universitaire Régionale d'Interface'' (CURI) in Fez city.The gas chromatography (GC) analysis were performed using a Hewlett-Packard (HP 6890) gas chromatograph (flame ionization detector, FID), equipped with a HP-5 capillary column (30m x 0.25mm x 0.25 μm).The temperature was programmed from 50°C after 5 min initial hold to 200°C at 4°C min -1 .GC conditions were as follows: N 2 as carrier gass (1.7ml min -1 ); split mode was used (flow: 66 ml min -1 , ratio: 1/50) and the injected volume was about 1 μl.The Gas Chromatography/Mass Spectrometry (GC/MS) analysis were performed by a Hewlett-Packard Gas Chromatographer (HP 6890) coupled with a mass spectrometer (HP 5973).Fragmentation was performed by electron impact at 70 eV.The carrier gas is helium whose flow is fixed at 1.5 ml/min.The injection mode was split (split ratio: 1/70).The apparatus was controlled by a "Chemstation" computer system.The identification of the components is based on the comparison of their mass spectra (GC/MS), respective with spectra of the library (NIST 98), of the bibliography (Adams, 2007), Kovats index for each compound on OPTIMA-5 column was calculated in reference to n-alkanes.

Characteristics of larval site
The collection of larvae of C. pipiens was performed in a breeding site located in the urban area of the city of Fez, called (Grand Canal : 402 m altitude, 30 ∘ 03'37" N and 5 ∘ 08'35"E).This site is characterized by a very high density of Culicidae larvae.

Collecting larvae of C. pipiens
Larvae were collected using a rectangular plastic tray in June 2014.The larvae gathered were maintained in breeding in rectangular trays at an average temperature of 21.7 ∘ C ± 2 ∘ C in the Entomology Unit at the Regional Diagnostic Laboratory Epidemiological and Environmental Health (RDLEH) falling within Regional Health Directorate of Fez.

Identification of larvae
The identification of morphological characters of larvae has been determined using the Moroccan key of identification of Culicidae (Himmi et al., 1995) and the identification software of mosquitoes of the Mediterranean Africa (Brunhes et al., 2000).

Protocol of larval susceptibility testing
The susceptibility tests were carried out in accordance with the standard protocol developed by WHO in 2005(WHO, 2005).From the initial essential oil (1 mL stock solution) of plant, concentrations of 250, 500, 750, 1000, 1250 and 1500 ppm were prepared.Preliminary experiments were used to select a range of concentrations for the tests previously mentioned.1mL of each solution prepared was placed in beakers containing 99mL of distilled water in contact with 20 larvae of stages 3 and 4; the same number of larvae was placed in a beaker containing 99mL of distilled water plus 1mL of ethanol.Three replicates were carried out for each dilution and for the control.After 24 hours of contact, we counted the living and dead larvae.The results of susceptibility testing were expressed in the percentage of mortality versus the concentration of plant extract used.If the percentage of mortality in control is greater than 5%, the percentage of mortality in larvae exposed to the essential oil shall be corrected by using Abbott's formula (Abbott, 1925): % Mortality Corrected = [(% Mortality Observed -% Mortality Control) / (100 -% Mortality Control)] × 100.
If the control mortality exceeds 20%, the test is invalid and must be repeated.

Data processing
For the data processing we used the log-probit analysis (Windl version 2.0) software developed by CIRAD-CA/MABIS (Giner et al., 1999).The analysis of the averages and standard deviation was also performed by using the test of analysis of variance ANOVA.

Percent yield and physical characters of C. atlantica
The percent yield of the hydro-distilled volatile oil from aerial parts of C. atlantica and its physical characters are summarized in Table 1.The average yield of the cedar essential oil of Eastern Middle Atlas was 1.12±0.2%.Our result is lower than that obtained for the origin of the High Atlas (Morocco) (2.6%) (Rhafouri et al., 2014) and also of that of Djurdjuran region in Algeria (1.7%) (Boudarene et al., 2004).On the other hand our result is superior to that found by Mathieu et al. (2011) about C. atlantica growing in Corsica (0.05 -0.49%).

Variation in mortality rate and Lethal Concentrations (LC 50 and LC 90 )
After exposing the larvae C. pipiens to different concentrations of EO of C. atlantica for 24 h, the percentage of mortality varied according to concentrations (Figure 2).The minimum concentration of C. atlantica EO required achieving 100% of C. pipiens larvae mortality was 1500 ppm. Figure 1 also shows the different concentrations used with their standard deviations and their larvicidal activity, we have found that the mortality rate varies according to the concentrations (Figure 2) and the larval mortality rate reached 100% at a concentration of 1500 ppm Table 3 demonstrates that C. pipiens EO remains effective while using concentrations of 782.43 ppm for LC 50 (which varies between a lower limit 554.77 ppm and an upper limit of 934.84 ppm) and 1253.93 ppm for LC 90 (which also varies between a minimum of 1042.8 ppm and a maximum value of 1900.82 ppm).Table 3 shows also the regression equation and the Chi-square (χ2) analyses results.The regression analysis indicates that the mortality rate is positively correlated with C. pipiens concentration.
Taking into account the absence of studies on C. atlantica essential oil against C. pipiens, we tried to compare the action of Cedrus family against the C. pipiens.A study realized by Huseyin et al. (2009) demonstrated that the larvicidal activity of Cedrus libani from Antalya (southwestern Turkey) on C. pipiens essential oil, with LC 50 values ranging from 47.8 to 116.0 ppm.The larvicidal activity observed in the essential oil of C. atlantica against C. pipiens could be explained by the chemical composition of this oil and the action or effect of compound majority.Indeed, Naples et al. (1992) reported that cedrol, in particular, a principal component of cedar wood oil, seems to have a high toxicity to cercariae (Schistosoma mansoni), a parasite for humans.Himachalenes and atlantones fractions essential oil of Himalayan cedar (C.deodara) showed insecticidal activity against Plutella xylostella (Chaudhary et 2011).
Our results suggest that essential oil of C. atlantica has potential to be used in the search for chemical components as new larvicides.Further studies are needed to determine and isolate major oil components that are most effective for larvicidal activity.

Fig. 1 :
Fig. 1: Cedrus atlantica Manetti of Morocco.Fig. 2: Percentage of mortality recorded in the test sensitivity by essential oil of C. atlantica on C. pipiens.

Table 2 :
Chemical composition of areal part of essential oil of C. atlantica from Morocco.Trace for percentages ≤ 0.07%.KI: Kovats Index determined on OPTIMA-5 non-polar column in reference to n-alkanes.

Table 1 :
Percent yield and physical characters of C. atlantica from Morocco.

Table 3 :
Lethal concentrations LC 50 and LC 90 of larvae of C. pipiens after 24h.