Salima Guettal

Samir Tine
email: samir.tine@univ-tebessa.dz

Fouzia Tine-Djebbar

Noureddine Soltani

Repellency and toxicity of azadirachtin against granary weevil Sitophilus granarius L. (Coleoptera: Curculionidae)

Keywords: Sitophylus granarius, Azadirachtin, Toxicity, Repellent activity, Biomarkers, Nutrition index

The granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), is known as a primary pest; and is able to feed on whole and undamaged cereal grains. This pest is probably one of the most destructive stored-product insect pests throughout the world affecting the quantity as well as quality of the grains. We have evaluated the fumigant and contact toxicity and the repellent property of azadirachtin a neem-based insecticide against S. granarius adults. Azadirachtin was found to exhibit fumigant and contact toxicity and the mortality increased as function the concentration and exposure time. In addition, the obtained results revealed an increase in the percent repellency as a function of concentration.

Biomarker measurements in treated adult (LC 25 and LC 50 ) revealed, activation of detoxification system as showed by an increase in CAT and GST activity and also a decrease in GSH rate. Moreover, nutrition depletion index was found to be concentration dependent depicting maximum reduction at LC 50 concentration. The biochemical compositions show that azadirachtin affected the energy reserves of adult of S. granarius. The results of persistence testing of azadirachtin applied by fumigation showed that their toxicity decrease as function the time. This study has highlighted the bioinsecticide activity of azadirachtin against granary weevil.

Introduction

Insects are considered as the basis of problems in agricultural products storage since they affect the quality and quantity of the products. Due to the high potential and wide host range of products such as wheat, barley, rice and oats, granary weevil, Sitophilus granarius (L.) is ranked among the important stored grain pests. It was a primary pest in the past. [START_REF] Kučerová | Accumulation of pest-arthropods in grain residues found in an empty store[END_REF] Insect pest control in stored grain products heavily relies on the use of gaseous fumigants and residual contact insecticides. [START_REF] Udo | Evaluation of the potential of some local spices as stored grain protectants against the maize weevil Sitophilus zeamais Mots (Coleoptera: Curculionidae)[END_REF] Moreover, the use of potentially toxic synthetic insecticides lead to serious problems such as residue threats and health hazard. [START_REF] Ntonifor | Potency of chenopodium ambrosioides powders and its combinations with wood ash on Sitophilus zeamais in stored maize[END_REF][START_REF] Zikankuba | Pesticide regulations and their malpractice implications on food and environment safety[END_REF] Protection of agricultural products from pest infestations is in the concern of scientists and the agrochemical industries worldwide. Plant products are being used to control many insect pests in the field and also in storage. [START_REF] Yohannes | Evaluation of certain plant leaf powders and aqueous extracts against maize weevil, Sitophilus zeamais Motsch.(Coleoptera: Curculionidae)[END_REF][START_REF] Campolo | Essential oils in stored product insect pest control[END_REF] This highlights the importance to develop eco-friendly materials and methods with slight adverse effects on the environment and on consumers. [START_REF] Ebadollahi | A Review on recent research results on bio-effects of plant essential oils against major coleopteran insect pests[END_REF][START_REF] Ebadollahi | Essential Oils extracted from different species of the lamiaceae plant family as prospective bioagents against several detrimental pests[END_REF] Among the bioactive plant compounds, azadirachtin, abundantly found in Azadirachta indica A. Juss (Meliaceae) (a plant commonly known as neem), is the most studied and used plant species due to its high efficacy and very low toxicity to humans and antifeedant properties. [START_REF] Souza Martinez | Azadirachta Indica) Natureza, Usos Multiplos, ProduÇâo[END_REF][START_REF] Chaudhary | Progress on Azadirachta indica based biopesticides in replacing synthetic toxic pesticides[END_REF] It is demonstrated high potential for use against pests of agricultural importance in different production systems due to its high insecticide and acaricide activities and rapid degradation in the environment. [START_REF] Charbonneau | Effects of azadirachtin and of simpler epoxyalcohols on survival and behaviour of Galleria mellonella (Lepidoptera)[END_REF][START_REF] Grimalt | Foliar residue dynamics of azadirachtins following direct stem injection into white and green ash trees for control of emerald ash borer[END_REF][START_REF] Biondi | Using organic-certified rather than synthetic pesticides may not be safer for biological control agents: Selectivity and side effects of 14 pesticides on the predator Orius laevigatus[END_REF] In recent decades, A. indica has been extensively studied because it contains terpenoids with powerful insecticidal activity. [START_REF] Schmutterer | Properties and Potential of Natural Pesticides from the Neem Tree, Azadirachta Indica[END_REF] Azadirachtin, a limonoid with different modes of action, acts mainly in numerous species of economic pests such as antifeedancy, growth regulation, fecundity suppression and sterilization, oviposition repellency or attractancy, and changes in biological fitness. [START_REF] Weathersbee | Effect of a neem biopesticide on repellency, mortality, oviposition, and development of Diaphorina citri (Homoptera: Psyllidae)[END_REF][START_REF] Abedi | Lethal and sublethal effects of azadirachtin and cypermethrin on Habrobracon hebetor (Hymenoptera: Braconidae)[END_REF][START_REF] Sánchez-Ramos | Laboratory evaluation of alternative control methods against the false tiger, Monosteira unicostata (Hemiptera: Tingidae)[END_REF] Azadirachtin acts as a growth regulator with an antagonistic action of both juvenile hormone (JH) and moulting hormone (ecdysteroids) [START_REF] Chaudhary | Progress on Azadirachta indica based biopesticides in replacing synthetic toxic pesticides[END_REF][START_REF] Mordue | azadirachtin, a natural product in insect control[END_REF][START_REF] Mordue | ddendum: Azadirachtin, a natural product in insect control: An update[END_REF] but the mechanism of action of this pesticide remains unknown. [START_REF] Wang | Fumigant, contact, and repellent activities of essential oils against the darkling beetle, Alphitobius diaperinus[END_REF] In order to determine the action of the AZ on oxidative stress and to confirm the intervention of GST in the mechanism of its detoxication of azadirachtin [START_REF] Wang | Effects of spinosad on helicoverpa armigera (Lepidoptera: Noctuidae) from China: Tolerance status, synergism and enzymatic responses[END_REF] , we have chosen to follow the enzyme activities of two enzymes, CAT and GSTs and GSH rate.

Glutathione S-transferases (GST, EC 2.5. 1.18) are multifunctional enzymes involved in many cellular physiological activities, such as detoxification of endogenous and xenobiotic compounds, biosynthesis of hormones and protection against oxidative stress. [START_REF] Enayati | Insect glutathione transferases and insecticide resistance[END_REF] In insects, three classes of GSTs have been identified namely delta, sigma, and epsilon classes [START_REF] Sawicki | Cloning, expression and biochemical characterization of one epsilon-class (GST-3) and Ten delta-class (GST-1) glutathione s-transferases from Drosophila melanogaster, and identification of additional nine members of the epsilon class[END_REF] , and have GSH-dependent peroxidase activities, for the detoxification metabolism of insecticide. [START_REF] Hemingway | The molecular basis of two contrasting metabolic mechanisms of insecticide resistance[END_REF] Catalase (CAT, EC 1.11.1.6) plays a vital role in reducing reactive oxygen-free radicals and maintaining cellular homeostasis in organisms [START_REF] Nandi | Role of catalase in oxidative stress-and ageassociated degenerative diseases. Oxidative medicine and cellular longevity[END_REF] . It is the initial line of defense in antioxidant systems due to their significant function against oxidative stress. [START_REF] Ugokwe | Induction of genetic alterations and oxidative stress in giant african land snail (Limicolaria aurora) Exposed to Municipal Waste Leachate[END_REF] The aim of this study was to examine the insecticidal activity of azadirachtin and its repellency against S. granarius adults. Then, we investigated its effects on nutritional and biochemical profile of S. granarius adults and tested its residual activity. In order to give additional information on its mode of action, selected biomarkers (CAT, GST, GSH) were also measured.

Materials and methods

Insects rearing

The insect species used in this study i.e. granary weevil S. granarius was procured from a farmer (Tébessa, Algeria). The insects were not affected by any material primarily. Cube containers (60x60x60cm) covered by a fine mesh cloth were used for insect rearing. The rearing was conducted as described by Aref & Valizadegan [START_REF] Aref | Fumigant toxicity and repellent effect of three iranian eucalyptus species against the lesser grain beetle, Rhyzopertha dominica (F.)(Col.: Bostrichidae)[END_REF] , at 27 ± 1 °C and 65 ± 5% relative humidity. Experiments were done between January and May 2018, and adult insects aged as 7 to 14 old days were used.

Azadirachtin

Neem Azal-TS, a commercial formulation of azadirachtin (1% EC; Trifolio-M GmbH, Lahnau, Germany) was used in all experiments. Azadirachtin (AZ) is a triterpenoid isolated from the kernels of the neem tree, Azadirachta indica A. Juss.

Fumigant bioassay

The fumigant toxicity of azadirachtin on S. granarius adults was tested in glass vials (60 mL).

In each of them 10 adults (both sexes, male or female, 7-14 days old) were released. No.2 Whatman filter paper disks were cut to 2.5 cm in diameter and attached to the undersurface of glass vial screw caps. Filter papers were impregnated with series of pure concentrations of essential oil: 20, 40, 80, 100, 200 and 400 µl/l air. Control insects were kept under the same conditions without essential oil. Each dose was replicated five times. After 24, 48 and 72 hours from the beginning of exposure, numbers of dead and alive insects were counted. In these experiments, those insects incapable of moving their heads, antennae and body were considered as dead. Lethal concentrations (LC 10 , LC 25 and LC 50 ) with their respective confidence limits (95% FL) were determined by a non-linear regression.

Contact toxicity

Azadirachtin dissolved in acetone has been tested at different concentrations (4, 8, 16, 20, 30 and 60 µl/ml) on S. granarius adults in plastic vials with a capacity of 60 ml and containing 10 g of wheat. Five replicates were run for each concentration and for the control. Numbers of dead insects were also counted after 12 and 24 hours from the start of exposure treatment.

Control insects were kept under the same conditions with acetone. The lethal concentrations (LC 10 , LC 25 and LC 50 ) were determined together with their corresponding 95% fiducial limits (95% FL) by a non-linear regression.

Repellent activity

The repellent effect of azadirachtin against adults of S. granarius was evaluated using the method of the preferred area on filter papers as described by Jilani & Saxena [START_REF] Jilani | Repellent and feeding deterrent effects of turmeric oil, sweetflag oil, neem oil, and a neem-based insecticide against lesser grain borer (Coleoptera: Bostrychidae)[END_REF] Thus, the filter paper discs of 9 cm in diameter used for this purpose have been cut into two equal parts.

Four doses were prepared (1, 2, 4 and 8 μl/ml) and diluted with ethanol. Then, 0.5 mL of each solution thus prepared was spread evenly over one-half of the disc. After 15 min, the two halves of the discs were glued together using adhesive tape. The filter paper disc was restored and placed in a box and kneaded a batch of 10 adult insects was placed in the center of each disk. The percentages of insects present on treated (P) and control (G) areas were recorded after 30 min. The repulsion percentage (RP) was calculated using Mc Donald et al. [START_REF] Mcdonald | Preliminary evaluation of new candidate materials as toxicants, repellents, and attractants against stored-product insects[END_REF] formula: RP = [(P-G) / (P+G)] ×100

The average values were calculated and assigned as ranked by McDonald et al. [START_REF] Mcdonald | Preliminary evaluation of new candidate materials as toxicants, repellents, and attractants against stored-product insects[END_REF]

Biomarker assays

The LC 25 (15.26 μl/ml) and LC 50 (74.83 μl/ml) at 72h, were applied by fumigation on adult of S. granarius and its effects examined on CAT and GST activities and GSH concentration measured at various times (24, 48 and 72 h) following treatment.

CAT activity was measured by determining the decomposition of its substrate H 2 O 2 as described by Claiborne. [START_REF] Claiborne | Handbook of methods for oxygen radical research[END_REF] Each sample (3 pools each containing 10 individuals) was conserved in buffer phosphate (100 mM; pH 7.4). After sonication and centrifugation (15 000 rpm for 10 min), the supernatant was collected and used for the determination of the CAT activity. The protein amount in the total homogenate was quantified according to Bradford. [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] The absorbance was red at 240 nm. The assay was conducted with 6-8 repeats and data expressed as mmol/min/mg protein.

The assay of GST was carried out according to Habig et al. [START_REF] Habig | Glutathione s-transferases the first enzymatic step in mercapturic acid formation[END_REF] previously described [START_REF] Dris | Chemical composition and activity of an Ocimum basilicum essential oil on Culex pipiens larvae: Toxicological, biometrical and biochemical aspects[END_REF] with use of GSH (5 mM). Larvae decapitated body was homogenized in 1ml phosphate buffer (0.1 M, pH 6). The homogenate was centrifuged (14000 rpm for 30 min). 200μl of the resulting supernatant was added to 1.2 ml of the mixture GSH-CDNB in phosphate buffer (0.1, pH 7).

Changes in absorbance were measured at 340 nm every minute for a period of 5 min.

The assay of GSH was conducted according to the method of [START_REF] Weckbecker | Ribonucleotide reductase activity and growth of glutathionedepleted mouse leukemia L1210 cells in vitro[END_REF] previouly used. [START_REF] Maiza | Sublethal effects on reproduction and biomarkers by spinosad and indoxacarb in cockroaches Blattella germanica[END_REF] Larvae bodies were homogenized in 1ml of EDTA (0.02 M, pH 6). The homogenate was subjected to a deproteinisation with sulfosalysilic acid (SSA) at 0.25 %. The optical density was measured at 412 nm.

Extraction and estimation of energy reserves

Proteins, carbohydrates and lipids were extracted following the procedure of [START_REF] Shibko | A method for sequential quantitative separation and determination of protein, RNA, DNA, lipid, and glycogen from a single rat liver homogenate or from a subcellular fraction[END_REF] and quantified as previously described. [START_REF] Soltani-Mazouni | Sublethal effects of methoxyfenozide on reproduction of the mediterranean flour moth, Ephestia kuehniella Zeller[END_REF] Briefly, for body biochemical analyses, newly molted adults from were collected. Pooled samples (10 individuals per pool) were weighed and extracted in 1 ml of trichloracetic acid (20%). In brief, quantification of proteins was carried following the Coomassie Brilliant Blue G-250 dye-binding method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with bovine serum albumin as a standard. The absorbance was measured at 595 nm. Carbohydrates were determined following the anthrone method [START_REF] Duvh\a Ateau | Sur la tréhalosémie des insectes et sa signification[END_REF] using glucose as standard. Lipids were measured by the vanillin method. [START_REF] Goldsworthy | Studies on insect adipokinetic hormones[END_REF] Data were expressed in μg per individual. The amount of carbohydrate, lipid and protein in each sample was calculated in μg per adult by using standard graphs. The values of carbohydrate, lipid and protein in μg were converted into joules. [START_REF] Clements | Development, Nutrition and Reproduction[END_REF] Where: 1mg of carbohydrate or protein has an energy value = 16.74 J 1mg of lipid has an energy value = 37.65 J

Nutrition depletion index

The total nutrition (carbohydrates + lipids) depletion index (NDI) was calculated as follows:

NDI = [(C-T) / (C + T)] × 100

Where: C is the control total energy reserve and T is the total energy reserves present in treated adult. The NDI is considered important when it is greater than 75%, moderate when it is between 50 and 75%, and low when it is less than 50%.

Evaluation of the residual activity

Persistence of insecticidal activity of AZAD was evaluated as described by Ngamo et al. [START_REF] Ngamo | Persistence of insecticidal activities of crude essential oils of three aromatic plants towards four major stored product insect pests[END_REF] The fumigation LC 50 values of essential oils were pipetted onto filter paper discs (2.5 cm diameter) in plastic vials. Six hours later, 10 adults were introduced separately into vial and then numbers of dead insects were recorded 24h after commencement of the exposure. This procedure was also conducted at 6 h intervals (i.e. 12, 18, 24, 30h). For each interval, separate series were set up with ten replications.

Statistical analysis

Data are presented as the mean ± SEM. Repetitions and numbers of individuals were also cited. One-way analysis of variance (ANOVA at P ≤ 0.05) followed by a post-hoc honestly significant difference (HSD) Tukey's test were used to compare between the different series.

Results and discussion

Insecticidal activity

Azadirachtin, produced as secondary metabolite, is the principal active constituent in neem extracts. [START_REF] Mordue | azadirachtin, a natural product in insect control[END_REF] As reported by published reviews [START_REF] Mordue | azadirachtin, a natural product in insect control[END_REF][START_REF] Su | Activity and biological effects of neem products against arthropods of medical and veterinary importance[END_REF] , it is able to induce multiple effects in numerous species of economic pests such as antifeedancy, growth regulation, fecundity suppression and sterilization, oviposition repellency or attractancy, and changes in biological fitness. However, its effects depend on the species, stages of the insect, concentration and the method of application (contact, ingestion and injection. [START_REF] Mordue | Azadirachtin: An Update[END_REF][START_REF] Huang | Protein metabolism in Spodoptera litura (F.) Is influenced by the botanical insecticide azadirachtin[END_REF] Azadirachtin has been shown to exhibit insecticidal activity against >400 insect species such as Helicoverpa armigera, Spodoptera litura, Plutella xylostella, Sitophilus oryzae, Sitophilus zeamis, Earis vitella, Aphis gossypii, Bemicia tabaci, Pectiniphora gossypiella, nematodes like Cosmopilitisn sordidus etc. [START_REF] Schmutterer | List of Insect pests susceptible to neem products[END_REF] The toxicity of this growth regulator is related to its high retention and stability. [START_REF] Schneider | Action of insect growth regulator insecticides and spinosad on life history parameters and absorption in third-instar larvae of the endoparasitoid Hyposoter didymator[END_REF] Figure 1 shows the percent mortality of S. granarius after exposure to different concentrations of azadirachtin applied by fumigation method. The highest percentage mortality was seen at 100 µl/liter air concentrations of AZAD. We calculated LC 25 and LC 50 values of azadirachtin and their fudicial limits (Table 2). Otherwise, application of azadirachtin by contact with the highest dose induces a 100% mortality rate at 12h (Fig. 1). Indeed, the LC 25 and LC 50 values decrease as a function of time (Table 1).

Our results indicate that azadirachtin exhibit an interesting insecticidal activity with doseresponse relationship against S. granarius adults. Similar results were found with the same insecticide applied against Drosophila melanogaster [START_REF] Boulahbel | Insecticidal activity of azadirachtin on Drosophila melanogaster and recovery of normal status by exogenous 20-hydroxyecdysone[END_REF][START_REF] Anjum | Toxicological evaluation of chlorpyrifos and neem extract (Biosal B) against 3RD instars larvae of Drosophila melanogaster[END_REF] and Ceraeochrysa claveri [START_REF] Scudeler | Effects of neem oil (Azadirachta indica A. Juss) on midgut cells of predatory larvae Ceraeochrysa claveri (Navás, 1911)(Neuroptera: Chrysopidae)[END_REF][START_REF]tine-djebbar, f. fumigant toxicity and repellent effect of azadirachtin against the lesser grain beetle, Rhyzopertha dominica (F.)(Col.: Bostrichidae)[END_REF] reported that this compound presented fumigant toxicity against Rhyzopertha dominica. However, the lethal concentrations (LC 50 and CL 90 ) recorded in our study are higher to those found in this work (LC 25 =7.41 µl/liter air and LC 50 =21.33µl/liter air). Azadirachta indica showed high toxicity (35.61%, 29.31% and 34.48%) when applied by contact on R. dominica, Trogoderma granarium and Tribolium castaneum, respectively [START_REF] Hanif | Insecticidal and repellent activities of essential oils of three medicinal plants towards insect pests of stored wheat[END_REF] Various studies demonstrated the lethal effects of azadirachtin on different insect species. [START_REF] Mordue | Azadirachtin: An Update[END_REF][START_REF] Dere | Insecticidal and oxidative effects of azadirachtin on the model organism Galleria mellonella L.(Lepidoptera: Pyralidae)[END_REF][START_REF] Tomé | Azadirachtin Avoidance by larvae and adult females of the tomato leafminer Tuta absoluta[END_REF] Topical application of azadirachtin on G. mellonella induced lethal concentrations of 16,564 and 3191,307 ppm corresponding to the LC 50 and LC 90 , respectively. [START_REF] Dere | Insecticidal and oxidative effects of azadirachtin on the model organism Galleria mellonella L.(Lepidoptera: Pyralidae)[END_REF][START_REF] Izhar-Ul-Haq | Effect of neem (Azadirachta indica A. Juss) seed extracts against greater wax moth (Galleria mellonella L.) Larvae[END_REF] The toxicity of Azadirachtin (NeemAzal®) has been reported in different species of mosquito, Culex pipiens [START_REF] Rehimi | Efficacy of azadirachtin against mosquito larvae Culex pipiens (Diptera: Culicidae) under laboratory conditions[END_REF][START_REF] Alouani | Larvicidal activity of a neem tree extract (Azadirachtin) against mosquito larvae in the Republic of Algeria[END_REF][START_REF] Alouani | Ovicidal activity of Azadirachtin and Bacillus thuringiensis israelensis against eggs of mosquito Culex pipiens[END_REF] , Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi. [58] [19] showed the efficacy of Azadirachtin against Lepidoptera, such as Shistocerca gregaria, where the LC 50 has a very low value (0.007 ppm), whereas in the Hemiptera and Coleoptera species, the LC 50 is 100 ppm. The obtained results by Zhong et al. [START_REF] Zhong | Effectiveness of the botanical insecticide azadirachtin against Tirathaba rufivena (Lepidoptera: Pyralidae)[END_REF] indicated that AZ had a strong stomach and contact toxicity to Tirathaba rufivena (Lepidoptera: Pyralidae) larvae, and that the contact toxicity was greater than the stomach toxicity.

Repellent Activity

The repellent activity is a physiological phenomenon that occurs in insects as a defense mechanism against toxins secreted by plants. [START_REF]tine-djebbar, f. fumigant toxicity and repellent effect of azadirachtin against the lesser grain beetle, Rhyzopertha dominica (F.)(Col.: Bostrichidae)[END_REF] An insect repellent has been defined as a chemical substance that causes the insect to make oriented movements away from the source. [START_REF] Dethier | The designation of chemicals in terms of the responses they elicit from insects[END_REF] The strong repellency of azadirachtin and neem concentrates in Xie et al. [START_REF] Xie | Repellency and toxicity of azadirachtin and neem concentrates to three stored-product beetles[END_REF] study was reflected by reduced numbers of insects on treated wheat. This reduction is presumably caused by chemosensory effects of these products, either olfactory or gustatory.

In this study, this test was applied on S. granarius adult. The percent repellency of R. dominica adult after 30min of treatment with AZ (1, 2, 4 and 8 μl/ml) are presented in table 2.

The obtained results revealed an increase in the repellency percentage as a function the concentration. The maximum repellency rate is 60% recorded with a dose of 8µl/ml.

According to Mc Donald et al. [START_REF] Mcdonald | Preliminary evaluation of new candidate materials as toxicants, repellents, and attractants against stored-product insects[END_REF] , this product belongs to the repellent class III.

These pesticides have shown contact, fumigant, antifeedant, repellent activity and growth regulating properties against insects. [START_REF] Sankari | Bio-efficacy of flyash-based herbal pesticides against pests of rice and vegetables[END_REF] AZ is a powerful behavior-modifying agent for a number of phytophagous insect species. [START_REF] Mordue | ddendum: Azadirachtin, a natural product in insect control: An update[END_REF][START_REF] Mordue | Azadirachtin: An Update[END_REF][START_REF] Schmutterer | Properties and potential of natural pesticides from the neem tree, Azadirachta indica[END_REF] Various azadirachtin-based commercial formulations, applied at different concentrations, caused strong repellent and oviposition deterrent effects on T. urticae females. [START_REF] Dimetry | Laboratory evaluation of neem formulations with and without additive against the two spotted spider mite, Tetranychus urticae Koch[END_REF][START_REF] Sundaram | Effects of pure and formulated azadirachtin, a neembased biopesticide, on the phytophagous spider mite, Tetranychus urticae Koch[END_REF][START_REF] Kashenge | Comparative efficacy of neem (Azadirachta indica) formulations and amitraz (Mitac) against the two-spotted spider mites (Tetranychus urticae) on Tomatoes: Mortality, repellence, feeding deterrence and effects on predatory mites, Phytoseiulus persimilis[END_REF][START_REF] Knapp | Effects of different neem formulations on the two spotted spider mite,Tetranychus urticae Koch, on Tomato (Lycopersicon esculentum Mill.)[END_REF] Hanif et al. [START_REF] Hanif | Insecticidal and repellent activities of essential oils of three medicinal plants towards insect pests of stored wheat[END_REF] reported a repellent activity of azadirachtin against T. castaneum and Rhyzopertha dominica with maximum of 77.66% and 81.48% repulsive potential, respectively.

Biomarker assays

The lethal concentrations (LC 25 : 15.26 μl/ml and LC 50 : 74.83 μl/ml) of azadirachtin at 72h were applied by fumigation on adult of S. granarius and its effects examined on CAT and GST activities, and on GSH rate measured at various times (24, 48 and 72 h) following treatment (Table 6). Results show a significant increase in CAT activity for the two tested concentrations only at 72h (control vs LC 25 p= 0.0412 and control vs LC 50 p=0.0153) (Fig. 2A, B andC), while GST activity measurements revealed a significant increase in the treated series (LC 25 and LC 50 ) respectively compared to control at 48 (p= 0.0196 and p= 0.0015) and 72 h (p= 0.0178 and p= 0.0032) without dose-response relationship. Finally, a significant decrease of glutathione rate was observed in treated series (LC 25 and LC 50 ) (p= 0.0133 and 0.0035) respectively at 72h as compared to control series.

The present results revealed a significant induction in glutathione S-transferase activity in S. granarius adult treated with AZ. This is in accordance with the literature as reported in Choristoneura rasaceana [START_REF] Lowery | Toxicity of insecticides to obliquebanded leafroller, Choristoneura rosaceana, larvae and adults exposed previously to neem seed oil[END_REF] , in Xanthogaleruca luteola (Müller) (Coleoptera) [START_REF] Valizadeh | Effect of neem based insecticide achook® on mortality, biological and biochemical parameters of elm leaf beetle Xanthogaleruca luteola Mull (Col.: Chrysomelidae)[END_REF] , in Helicoverpa armigera larvae (Hübner) (Lepidoptera) [START_REF] War | Efficacy of a combined treatment of neem oil formulation and endosulfan against Helicoverpa armigera (Hub.)(Lepidoptera: Noctuidae)[END_REF] and in Drosophila melanogaster [START_REF] Boulahbel | Insecticidal activity of azadirachtin on Drosophila melanogaster and recovery of normal status by exogenous 20-hydroxyecdysone[END_REF] ;

Or various insecticides such as neem oil in Xanthogaleruca luteola. [START_REF] Valizadeh | Effect of neem based insecticide achook® on mortality, biological and biochemical parameters of elm leaf beetle Xanthogaleruca luteola Mull (Col.: Chrysomelidae)[END_REF] Sometimes, the GST activities could be not affected by azadirachtin in Choristoneura rasaceana. [START_REF] Smirle | Influence of neem oil on detoxication enzyme activity in the obliquebanded leafroller, Choristoneura rosaceana[END_REF] But the results of [START_REF] Walters | Evolution of the GST omega gene family in 12 Drosophila species[END_REF] have confirmed the intervention of GST in the mechanism of the detoxication of azadirachtin. Increased GST activity results in the detoxification process, is a form of insect defense against pesticide. [START_REF] Clark | Kinetic studies on a glutathione s-transferase from the larvae of Costelytra zealandica[END_REF] Glutathione (GSH) plays an important role in the detoxification and excretion of xenobiotics. [START_REF] Palanikumar | Biochemical response of anthracene and benzo [a] pyrene in milkfish Chanos chanos[END_REF] In our study, AZ induces a significant greater decrease in GSH rate in S. granarius adult. Similar effects observed by Kiran et al. [START_REF] Kiran | Assessment of toxicity and biochemical mechanisms underlying the insecticidal activity of chemically characterized Boswellia carterii essential oil against insect pest of legume seeds[END_REF] who mentioned that Boswellia carterii essential oil on Callosobruchus chinensis and C. maculatus increased significantly the concentration of GSH. This cofactor in S. oryzae and R. dominica was also increased after treatment (CL 50 ) with Gaultheria procumbens essential oil [START_REF] Kiran | Assessment of toxicity, antifeedant activity, and biochemical responses in stored-grain insects exposed to lethal and sublethal doses of Gaultheria procumbens L. Essential Oil[END_REF] , and in C. pipiens with T.

vulgaris. [START_REF] Bouguerra | Effect of Thymus vulgaris L. (Lamiales: Lamiaceae) essential oil on energy reserves and biomarkers in Culex pipiens L. (Diptera: Culicidae) from Tebessa (Algeria)[END_REF] In contrast, the adult of S. oryzae treated with anhydride 2,3-diméthylmaléique displayed an increase in the GSH activity. The decrease of glutathione could be explained by an increased consumption of this cofactor by the GSTs in order to detoxify the organism and a reduction of the non-enzymatic antioxidant system.

Our finding shows a significant increase of CAT activity. Similar results were found with azadirachtin applied in Drosophila melanogaster. [47] [76] was found also an increase in CAT levels of 30.29% and 38.82% after 24 h exposure to the LC 50 of Boswellia carterii essential oil on C. chinensis and C. maculatus respectively. The increase in activity of CAT reflects an establishment of the process of detoxification, which is a form of defense of the insect against the pesticide. [START_REF] Clark | The comparative enzymology of the glutathione s-transferases from nonvertebrate organisms[END_REF] In contrast, a decrease in CAT activity was observed in S. oryzae and R. dominica treated with Gaultheria procumbens [START_REF] Kiran | Assessment of toxicity, antifeedant activity, and biochemical responses in stored-grain insects exposed to lethal and sublethal doses of Gaultheria procumbens L. Essential Oil[END_REF] , which could be explained by an increased production of the radical superoxide anion. [START_REF] Kurutaş | Oxidative stress biomarkers in liver and gill tissues of spotted barb (Capoeta barroisi Lortet, 1894) living in Ceyhan river, Adana-Turkey[END_REF][START_REF] Sreejai | Studies on the changes in lipid peroxidation and antioxidants in fishes exposed to hydrogen sulfide[END_REF] This decrease in CAT activity results accumulation of toxic H 2 O 2 in the cell, leading to peroxidation of membrane lipids. [START_REF] Sharma | Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions[END_REF] The induction of the GST system in D. melanogaster is correlated with an increase in specific CAT activity after treatment with Neem Azal. [START_REF] Boulahbel | Insecticidal activity of azadirachtin on Drosophila melanogaster and recovery of normal status by exogenous 20-hydroxyecdysone[END_REF] This oxidative stress could be explained by the antagonist action of azadirachtin on endogenous 20E and its antioxidant activity. [START_REF] Boulahbel | Insecticidal activity of azadirachtin on Drosophila melanogaster and recovery of normal status by exogenous 20-hydroxyecdysone[END_REF]

Estimation of energy reserves and protein content

Changes in main biochemical components (carbohydrates, lipids and proteins) were estimated in the whole body of the control and treated adult of S. granarius at different times following treatment (Table 3). Results show a significant decrease (p <0.001) in the protein content in treated series (LC 25 and LC 50 ) as compared to controls during the tested period: 24 (control vs LC 25 : p<0.001 ; control vs LC 50 : p<0.001 ; LC 25 vs LC 50 : p= 0.006), 48 (control vs LC 25 : <0.001 ; control vs LC 50 : <0.001) and 72 hours (control vs LC 25 : <0.001 ; control vs LC 50 : <0.001 ; LC 25 vs LC 50 : p= 0.008).

Concerning the total energy (Table 3) , the results revealed a significant decrease in the treated series (LC 25

Nutrition depletion index

Nutrition depletion index (NDI %) in treated adult was determined in order to investigate the effectiveness of azadirachtin (Table 4). The decrease was concentration-dependent with a maximum depletion in LC 50 treated series at different periods after treatment: 24 (p=0.003), 48 (p<0.001) and 72hours (p=0.022). Azadirachtin induced a moderate nutritional depletion.

All types of insecticides have some negative impact on the growth and development of the insect, and also affect the metabolic and biochemical processes. [START_REF] Sharma | Status of carbohydrate, protein and lipid profile in the mosquito larvae treated with certain phytoextracts[END_REF] This investigation shows that after treatment of azadirachtin, the protein level and energy reserves of S. granarius larvae decreased during the tested period.

Protein synthesis is necessary particularly for the maintenance of body growth and reproduction. They enter in various reactions such as the hormonal regulation and they integrated in the cell as a structural element at the same time as the carbohydrates and the lipids. [START_REF] Cohen | Chapter 2 -Chitin Biochemistry: Synthesis, Hydrolysis and Inhibition[END_REF][START_REF] Sugumaran | Chapter 5 -Chemistry of cuticular sclerotization[END_REF] In the present investigation, after treatment of S. granarius adults with AZ, an inhibitory action on proteins was generally exhibited. [START_REF] Nath | Changes in protein metabolism in hemolymph and fat body of the silkworm, Bombyx mori (Lepidoptera: Bombycidae) in response to organophosphorus insecticides toxicity[END_REF] reported that stress due to insecticide exposure might interfere with insect physiology, consequently resulting in a decrease in total protein leading to low amino acids formation in Krebs cycle. This further leads to insufficient fatty acid required for synthesis of Adenosine Triphosphate (ATP) energy, thus reduction in ATP energy triggers stress in insects leading to death. [START_REF] Smirle | Influence of neem oil on detoxication enzyme activity in the obliquebanded leafroller, Choristoneura rosaceana[END_REF] Nevertheless, Ebadollahi et al. [START_REF] Ebadollahi | Toxicity and physiological effects of essential oil from Agastache foeniculum (Pursh) Kuntze against Tribolium castaneum herbst (Coleoptera: Tenebrionidae) Larvae[END_REF] reported a decrease in the carbohydrates, proteins, and lipids content in T. castaneum larvae treated with Agastache foeniculum EO. The same observations were reported by Tarigan and Harahap [START_REF] Tarigan | Toxicological and physiological effects of essential oils against Tribolium castaneum (Coleoptera: Tenebrionidae) and Callosobruchus maculatus (Coleoptera: Bruchidae)[END_REF] after treatment of Tribolium castaneum with Cinnamomum aromaticum, Elettaria cardamomum and Myristica fragrans EOs, with efficacy of the cinnamon oil. This depletion might be due to their degradation for metabolic purposes or to an impaired incorporation of amino acids into polypeptide chains or inhibition of protein synthesis [START_REF] Sharma | Status of carbohydrate, protein and lipid profile in the mosquito larvae treated with certain phytoextracts[END_REF] or to the breakdown of these proteins into amino acids used in the compensatory mechanism as energy source to compensate stress. [START_REF] Ali | biochemical response of malathion-resistant andsusceptible adults of Rhyzopertha dominica to the sublethal doses of deltamethrin[END_REF] Neem extract contains azadirachtin that has been known to affect protein amount and expression. For instance, azadirachtin have been known to interfere with protein synthesis in Schistocerca gregaria [START_REF] Annadurai | Azadirachtin a modulates the tissue-specific 2d polypeptide patterns of the desert locust, Schistocerca gregaria[END_REF] and Spodoptera litura. [START_REF] Li | Studies on the effects and mechanisms of azadirachtin and rhodojaponin-on Spodoptera litura (F.)[END_REF] Further, it is reported that protein expression in S. litura was significantly lowered under azadirachtin treatment. [START_REF] Huang | Protein metabolism in Spodoptera litura (F.) Is influenced by the botanical insecticide azadirachtin[END_REF] Rao & Subrahmanyam [START_REF] Rao | Azadirachtin induced changes in development, food utilization and haemolymph constituents of Schistocerca gregaria Forskal[END_REF] found disturbance in the hormone that regulates protein synthesis due to azadirachtin in Schistocerca gregaria. The decrease in total protein in the adult of S. granarius was postulated as an indicator of toxic exposure to insecticides. According to Mordue et al. [START_REF] Mordue | azadirachtin, a natural product in insect control[END_REF] , AZ alters or prevents the formation of new assemblages of organelles or cytoskeleton resulting in the disruption of cell division, blocked transport and release of neurosecretory peptides. It also inhibits protein synthesis in cells that are metabolically active.

The carbohydrates are considered as important energy elements playing a crucial role in the insect physiology, such as the molt process and the reproduction. [START_REF] Kaufmann | Regulation of carbohydrate metabolism and flight performance by a hypertrehalosaemic hormone in the mosquito Anopheles gambiae[END_REF] In the present study, AZ decreases the carbohydrate contents in S. granarius adults. Glucose level of the larvae treated with A. annua extract was decreased by 24.65%. The reduction in glucose content was more significant in larvae exposed to Az. indica extract by 58.96% decline over control. This depletion in glucose content may be due to utilization of the reserved glucose sources of larval tissues as a result of insecticidal stress. AZAD derivatives also lead to a decrease in the concentration of carbohydrates in Ctenoparyngodon idella. [START_REF] Hassanein | Toxicity determination and hypoglycaemic effect of neem biopesticide on the grass carp "Ctenopharyngodon idella[END_REF] Tine et al. [START_REF] Tine | Laboratory evaluation of azadirachtin against the oriental cockroach, Blatta orientalis L.(Dictyoptera, Blattellidae): Insecticidal activity and reproductive effects[END_REF] show a significant decrease in ovarian protein, lipid and carbohydrate contents in B. orientalis treated by azadirachtin. Treatment may have caused possible disturbance in the vitellogenesis process via the nervous, neuroendocrine and/or endocrine system. In another study, Tine et al. [START_REF]tine-djebbar, f. fumigant toxicity and repellent effect of azadirachtin against the lesser grain beetle, Rhyzopertha dominica (F.)(Col.: Bostrichidae)[END_REF] found that azadirachtin induced negative effects on energy contents compared with control in Ryzopertha dominica. The carbohydrate content was reduced in larvae of Spodoptera littoralis after treatment with essential oil of A. indica and Citrullus colocynthis methylene chloride extract and was increased with garlic and lemon Eos. [START_REF] Ali | Antifeedant activity and some biochemical effects of garlic and lemon essential oils on Spodoptera littoralis (Boisduval)(Lepidoptera: Noctuidae)[END_REF] Lipids are also an important source of acetyl groups needed to synthesize the enzymes from constitutive amino acids. [START_REF] Rivero | Energetic cost of insecticide resistance in Culex pipiens mosquitoes[END_REF] This reduction in lipid profile indicates a negative effect of the extract on lipid metabolism and peroxidation. This observation is similar to the findings of Lohar & Wright [START_REF] Lohar | Changes in the lipid content in haemolymph, fat body and oocytes of malathion treated Tenebrio molitor L. Adult Females[END_REF] , who found that Tenebrio molitor suffered lipid depletion in haemolymph, fat bodies and oocytes when exposed to malathion. Sak et al. [START_REF] Sak | Effects of cypermethrin on total body weight, glycogen, protein, and lipid contents of Pimpla turionellae (L.)(Hymenoptera: Ichneumonidae)[END_REF] reported the decline in lipid content due to shift in energy metabolism to lipid catabolism due to insecticidal stress induced by Pimpala turionellae.

Residual activity of azadirachtin

During the 30h treatment periods; the results of persistence testing of azadirachtin applied by fumigation showed that their toxicity decrease as function the time. The toxicity of AZAD decreased with time; after 6h its toxicity was 32 % and decreased to 6 % after 24 h to disappear after 30h of exposure (Fig. 3).

The biopotency is negatively correlated with time. Ngamo et al. [START_REF] Ngamo | Persistence of insecticidal activities of crude essential oils of three aromatic plants towards four major stored product insect pests[END_REF] and Heydarzade & Moravvej [START_REF] Heydarzade | Contact toxicity and persistence of essential oils from Foeniculum vulgare, Teucrium polium and Satureja hortensis against Callosobruchus maculatus (Fabricius)(Coleoptera: Bruchidae) Adults[END_REF] reported that the persistency of Lippia rugosa Hochs (Lamiales: Verbenaceae) and Satureja hortensis (L.) (Lamiales: Laminaceae) EOs were probably the result of its high content in oxygenated monoterpenes which attribute more stability in the biological activity of EOs. Securidaca longepedunculata has preserved toxicity while for B. grandifolia plant powder, the toxicity decreases rapidly. [START_REF] Habiba | Insecticidal properties and persistence of Berlinia grandifolia (J. Vahl) and Securidaca longepedunculata Fres., two aromatic plants from the far-north region of cameroon alone or in combination against Sitophilus oryzae L. (Coleoptera : Curculionidae)[END_REF] This decrease is similar to this obtained with Xylopia aethiopica against Callosobruchus maculatus. [START_REF] Habiba | Persistent effect of a preparation of essential oil from Xylopia aethiopica against Callosobruchus maculatus (Coleoptera, Bruchidae)[END_REF] These results can be explained through chemical active component of the species plant used. Investigations on the EO of several aromatics plants in Northern Cameroon [START_REF] Ngamo | Persistence of insecticidal activities of crude essential oils of three aromatic plants towards four major stored product insect pests[END_REF][START_REF] Goudoum | Persistence of active compounds of essential oils of Clausena anisata (Rutaceae) and Plectranthus glandulosus (Labiateae) used as insecticides on maize grains and flour[END_REF] had proven that plant species has more persistence toxic effect when they contained higher proportion of oxygenated molecules such as oxygenated monoterpens and sesquiterpens. The persistence of insecticidal activity was in relationship with the sensitivity of the major target pest to active compound. [START_REF] Ngamo | Persistence of insecticidal activities of crude essential oils of three aromatic plants towards four major stored product insect pests[END_REF][START_REF] Goudoum | Persistence of active compounds of essential oils of Clausena anisata (Rutaceae) and Plectranthus glandulosus (Labiateae) used as insecticides on maize grains and flour[END_REF][START_REF] Obeng-Ofori | Biological activity of 1, 8 cineole, a major component of essential oil of Ocimum kenyense (Ayobangira) against stored product beetles[END_REF] In the experiment of Akami et al. [START_REF] Akami | Persistence and comparative pesticidal potentials of some constituents of Lippia adoensis (Hochst. Ex Walp.)(Lamiales: Verbenaceae) essential oil against three life stages of Callosobruchus maculatus (Fab.)(Coleoptera: Bruchidae)[END_REF] , when tested individually, none of the isolated major constituents had produced as higher effects as the crude EO not even their complex mixture.

The crude EO is the most persistent. This situation could be the result of many factors: the high volatility of the compounds, the rapid degradation of low single compounds, and the potential oxidation of Sesquiterpene hydrocarbons. [START_REF] Akami | Persistence and comparative pesticidal potentials of some constituents of Lippia adoensis (Hochst. Ex Walp.)(Lamiales: Verbenaceae) essential oil against three life stages of Callosobruchus maculatus (Fab.)(Coleoptera: Bruchidae)[END_REF] Regnault-Roger et al. [START_REF] Regnault-Roger | Biopesticides d'origine végétale[END_REF] showed the lower volatility of oxygenated molecules because of their higher molecular weight.

Conclusion

Azadirachtin exhibited fumigant toxicity against S. granarius adults confirming its potential as a natural alternative to synthetic insecticides for the control of stored-product pests. In addition, a strong repellent activity. Moreover, azadirachtin was found to exhibit a residual toxicity on S. granarius. The bioinsecticide caused the activation of the system of detoxification, traduced by an increase of the specific activity of GST and Catalase and a decrease of GSH rate. Our results provide an interesting opportunity to develop bioinsecticides and repellent formulations.

Legends of Table and Figures

Table 1.

  by a repulsive different classes varying from 0 to V [Class 0 (RP < 0.1%), class I (RP = 0.1% -20.0%), class II (RP = 20.1% -40.0%), class III (RP =40.1% -60.0%), class IV (RP = 60.1% -80.0%) and class V (RP =80.1% -100.0%)].

  and LC 50 ) respectively compared to control at 24h (control vs LC 25 : p<0.001 ; control vs LC 50 : p<0.001 ; LC 25 vs LC 50 : p= 0.001), 48 (control vs LC 25 : <0.001 ; control vs LC 50 : <0.001; LC 25 vs LC 50 : p<0.001) and 72 hours (control vs LC 25 : <0.001 ; control vs LC 50 : <0.001 ; LC 25 vs LC 50 : p= 0.02).

  Fumigant (µl/l air) and Contact (µl/ml) toxicity of azadirachtin against the adult of S. granarius: Determination of lethal concentrations and their confidence intervals (95%), LCL -Lower confidence Limit, UCL -Upper confidence Limit)

Figure 1 .

 1 Figure 1. Efficacy of azadirachtin applied by fumigation (A) and by contact (B) on adult as function the exposure time (hours) (mean ± SEM, n = 5 replicates each containing 10 adults).

Figure 2 .

 2 Figure 2. Effect of azadirachtin (LC 25 and LC 50 ) on CAT (A) and GST (B) activities (μM/min/mg of protein) and GSH (C) rate (C) (μM/mg of protein) in S. granarius adults at different time after treatment (mean ± SEM, n= 3 pools each containing 10 individuals). The different lowercase letters indicate significant differences at the same time based on Tukey's HSD test (p <0.05).

Figure 3 .

 3 Figure 3. Residual activity of azadirachtin (LC 50 ) by fumigation against S. granarius adults for a delay of 30h (mean ± SD, n = 4 pools each containing 10 individuals)

Table 2 .

 2 Repellent Percentage (RP) of azadirachtin against S. granarius adults at different concentrations.

	Concentrations	RP (%)	Class
	1µl/ml	25	II
	2µl/ml	35	II
	4µl/ml	50	III
	8µl/ml	60	III

Table 3 .

 3 Effect of azadirachtin (LC 25 and LC 50 ) on proteins content and total energy (joule /individual) in S. granarius adults at different time after treatment (mean ± SE, n = 3 pools each containing 10 individuals).

	Times (hours)	Components	Control	LC 25	LC 50
	24	Proteins Total energy	3.61 ± 0.04 a 8.30 ± 0.17 a	0.92 ± 0.02 b 4.54 ± 0.13 b	0.74 ± 0.02 c 3.53 ± 0.05 c
	48	Proteins Total energy	3.61 ± 0.05 a 8.18 ± 0.11 a	0.83 ± 0.01 b 4.31 ± 0.08 b	0.73 ± 0.04 b 2.95 ± 0.00 c
	72	Proteins Total energy	3.61 ± 0.00 a 7.87 ± 0.27 a	0.78 ± 0.02 b 3.19 ± 0.06 b	0.65 ± 0.03 c 2.39 ± 0.19 c

The different lowercase letters indicate significant differences at the same time based on Tukey's HSD test (p <0.05).

Table 4 .

 4 Nutrition Depletion Index (%) of S. granarius adult after treatment with AZAD at different time after treatment. Data represented as mean ± SE (n =3 pools each containing 10 individuals).Different small letters indicate a significant difference between treated individuals in the same period.

	Times (hours)	LC 25	LC 50	P value
	24	29.26 ± 2.07 a	40.31 ± 0.93 b	0.003
	48	30.98 ± 0.58 a	46.93 ± 0.59 b	<0.001
	72	42.21 ± 2.21 a	53.36 ± 3.32 b	0.022

Acknowledgement

This work was supported by the National Fund for Scientific Research to Pr. N. Soltani (Laboratory of Applied Animal Biology) and the Ministry of High Education and Scientific Research of Algeria (PRFU Project to Dr. S. Tine).