Composition and fumigant protectant potential of Tunisian Citrus aurantium L. essential oils against Rhyzopertha dominica F. (Coleoptera: Bostrichidae) †

: The valorization of Citrus peels from agro-industries is an important issue since Tunisia is known by the production of such crops. This work aims to assess the insecticidal potential of essential oils extracted from the bitter orange C. aurantium as protectant against adults of the less grain borer R. dominica . Results revealed that Limonene was the major and characteristic compound. The fumigant bioassay showed that C. aurantium from Nabeul was more toxic with LC 50 and LC 95 values were 307.9 and 652.9 µL/L air, respectively within 48h of exposure.


Introduction
In many regions of the world, the lasser grain borer, R. dominica, is one of the major pests feeds in quality of grains as larva's and adults (lose weight and the market value) and they are become generally unacceptable for human consumption [1,2,3]. Besides, the use of biological insecticides increases production costs as well as leads to environmental and consumer health risks [4,5,6]. In fact, Essential oil extracted from genus Citrus, have been identified to have insecticidal properties several coleopteran species [7,8,9] whith a variation in the composition of secondary metabolites [10,11]. This research assesed the chemical composition and the fumigant toxicity of the essential oils of peels of Tunisian Citrus auranthium against the adults of Rhyzopertha dominica.

Insect breeding
Rearing of the adults of R. dominica was affected in the Laboratory of Biotechnology Applied to Agriculture in the breeding chambre at 30 °C, 60 RH and photoperiod cycle of L:D 12:12h.

Sampling
At mature stage of the trees, Fruits of C. aurantium were collected from Bir Merwah and acheichia (Nabeul and Boussalem, north Tunisia), respectively. In the laboratory, fruits were rinsed with distilled water and we are recovered the peels. the extraction of the peels essential oil of C. aurantium were affected by hydrodistillation with the using of Clevenger type apparatus for two hours at 100 °C.

chemical composition
The quantitative and qualitative analysis of the volatile compounds in the essential oils was determined by combination of gas chromatography (GC) and mass spectrometry (MS) according to a standard analytical procedure BY using a DB-5MS column (30 m, 0.25 mm and 0.25 μm film thickness). The initial oven temperature was held at 60°C for 4min. it was then increased by 3 °C/min to 250 °C; the injection port and ionizing source were kept at 250 and 280 °C, respectively [12].

Insecticidal activities (Fumigant toxicity)
For the determination of the fumigant toxicity of bitter orange essential, 3,5 cm in diametre of whatman N°1 filter paper were impregnated with oil at doses calculated to release fumigant bioessay (41.67, 83.33 and 166.67 µ L/L air), into, these filter paper impregnated was attached to the screw cap of the 24 mL plexiglas bottle that contains ten adults and these caps were screwed on tightly. Than, the number of dead and alive insects in each bottle was counted after 48h of exposure for the determination of LC50 and LC95..

Statistical analysis
analysis of variance of the data was affected by using Statistical Package for Social Sciences (SPSS) version 11.0 and LC values were estimated by using the Probit analysis.

yields and chemical composition of the essential oils
Yields of C. aurantium essential oil were 0,555 % and 0,851 % in peels from Nabeul and Bousselem, respectively. Table 1 showed the percentages of the chemical compounds identified in the essential oil extracted from the peels of C. aurantium of two locations. in fact, GC/MS identified 99.99 % of the total essential oil. Monoterpenes were the main components in essential oil from peels of C. aurantium with 97.63 and 98.29 %, respectively from Bousselem and Nabeul. In fact, limonene represented the major composant of the peels essential oils od C. aurantium (93.69 and 92.36 %), β-myrcene (1.77 and 1.7 %), βocimene (0.72 and 0.77 %), α-pinene 0.58 and 0.49%), β-pinene (0.29 and 0.93 %), sabinene (0.16 and 0.22%), β-fenchol (0.12 and 0.32%) and nerol (0 and 0.15 %) were the main components in essential oil of Bousselem and Nabeul, respectively and linalool (0.3 and 1.45 %) was the only oxygenated monoterpenes in peel essential oil of Bousselem and Nabeul, respectively. Furthermore, sesquiterpene hydrocarbons were represented by germacrene D (0.09 %) in only the essential oil of Nabeul. the fraction of the non terpenic compounds was essentialy represented by aldehydes (0.06%) and esters (0.69%). Zarrad et al. [12] reported that C. aurantium peel oils from Chott Mariem (Sousse, Tunisia) was caracterised essentially by limonene with a percentage of 87.523 %. Additionally, Moraes et al. [13] reported that peels essential oils from Brazilan C. aurantium consist mainly for Limonene and myrcene with a percentage of 98% and 1.45%. These differences in the compositions of essential oils may be depended to different factor, for exemple, seasonal, maturational stage and pedoclimatic variation [14,15], also, the extraction method and the age of the trees can influence the quantity and the quality of the essential oils [16,17]. Thid study shows also the sensitivity of adults of Rhyzopertha dominica to the peels essential oils of C. aurantium.

Insecticidal activities (Fumigant toxicity)
Results showed that Citrus aurantium peel oils were toxic to the adults of Rhyzopertha dominica and the percentage of mortality was related to the doses of the essential oils. Analysis of results showed also that C. aurantium from Bousselem was less toxic to R. dominica than essential oil from Nabeul. The corresponding LC50 and LC95 of Nabeul and Bousselem were respectively 307.89 and 440.55 µ L/L air and 652.91 and 1283 µ L/L air.
Furthermore, our results does not agree with the results of Souza et al. [18], hwo are shown that the esssential oils of bitter orange has an effect in the killing of adults of R. dominica with an LC50 and LC100 values equal to 69.36 µ L/L air and 130.68 µ L/L air, respectively. the insecticidal activities of C. aurantium essential oils have previously been evaluated against several insect species [19]. Moreover, Pala and Pathipati [20] reported that essential oils of C. aurantium were highly effective with 76% mortality of R. dominica at 8.5 mg/cm². Indeed, The toxicity of these oils could be explained by its richness in monoterpenoids compounds that possess insecticidal activity of the limonene against several insect species [21] and the toxic effective of the limonene are mediated by penetrating the insect bodyy through the resoiratory systeme [7]. Tripathi et al. [22] reported the insecticidal activities of limonene against R. dominica and other insects, for exemple, Sytophylus oryzae, Tribolium castaneum [18].

Conclusion
This study, proved the fumigant toxisity of two North Tunisian C. aurantium peel oils into adults of R. dominica. Indeed, these proceeding showed that C. auranthium essential oils can be a source for eco-friendly alternatives in whitefly integrated control program.