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

Salima Guettal, Samir Samir Bio Tine, Fouzia Tine-Djebbar, Noureddine Soltani

► To cite this version:

Salima Guettal, Samir Samir Bio Tine, Fouzia Tine-Djebbar, Noureddine Soltani. Repellency and toxicity of azadirachtin against granary weevil Sitophilus granarius L. (Coleoptera: Curculionidae). Agriculture International, 2021. hal-03169471

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# Repellency and toxicity of azadirachtin against granary weevil *Sitophilus granarius* L.

## (Coleoptera: Curculionidae)

Salima GUETTAL¹,², Samir TINE¹,², **Fouzia TINE-DJEBBAR** ¹,²*, & Noureddine SOLTAN²

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¹Laboratory of water and Environment, Larbi Tebessi University, Tébessa, Algeria 5

² Laboratory of Applied Animal Biology, University Badji Mokhtar, Annaba, Algeria 6

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Email: samir.tine@univ-tebessa.dz

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**Abstract:** 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 (LC₂₅ and LC₅₀) 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 LC₅₀ 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.

**Keywords:** *Sitophilus granarius*, Azadirachtin, Toxicity, Repellent activity, Biomarkers, Nutrition index.

## Introduction

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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.[1] Insect pest control in stored grain products heavily relies on the use of gaseous fumigants and residual contact insecticides.[2] Moreover, the use of potentially toxic synthetic insecticides lead to serious problems such as residue threats and health hazard.[3,4] 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.[5,6] This highlights the importance to develop eco-friendly materials and methods with slight adverse effects on the environment and on consumers.[7,8]

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.[9,10]
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.[11-13]

In recent decades, *A. indica* has been extensively studied because it contains terpenoids with powerful insecticidal activity.[14] 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.[15-17] Azadirachtin acts as a growth regulator with an antagonistic action of both juvenile hormone (JH) and moulting hormone (ecdysteroids)[10,18,19] but the mechanism of action of this pesticide remains unknown.[20]

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[21], 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.[22] In insects, three classes of GSTs have been identified namely delta, sigma, and epsilon classes[23], and have GSH-dependent peroxidase activities, for the detoxification metabolism of insecticide.[24] Catalase (CAT, EC 1.11.1.6) plays a vital role in reducing reactive oxygen-free radicals and maintaining cellular homeostasis in organisms[25]. It is the initial line of defense in antioxidant systems due to their significant function against oxidative stress.[26]

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[27], 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 (LC₁₀, LC₂₅ and LC₅₀) with their respective confidence limits (95% FL) were determined by a non-linear regression.

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## 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 (LC₁₀, LC₂₅ and LC₅₀) were determined together with their corresponding 95% fiducial limits (95% FL) by a non-linear regression.

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## 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[28]. 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.[29] formula: $RP = [(P-G) / (P+G)] \times 100$
The average values were calculated and assigned as ranked by McDonald et al.[29] 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%)].

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## Biomarker assays

The LC₂₅ (15.26 µl/ml) and LC₅₀ (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 H₂O₂ as described by Claiborne.[30] 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
131 activity. The protein amount in the total homogenate was quantified according to Bradford.[31]

132 The absorbance was red at 240 nm. The assay was conducted with 6–8 repeats and data
133 expressed as mmol/min/mg protein.

134 The assay of GST was carried out according to Habig et al.[32] previously described[33] with
135 use of GSH (5 mM). Larvae decapitated body was homogenized in 1ml phosphate buffer (0.1
136 M, pH 6). The homogenate was centrifuged (14000 rpm for 30 min). 200µl of the resulting
137 supernatant was added to 1.2 ml of the mixture GSH-CDNB in phosphate buffer (0.1, pH 7).
138 Changes in absorbance were measured at 340 nm every minute for a period of 5 min.

139 The assay of GSH was conducted according to the method of[34] previously used.[35] Larvae

140 bodies were homogenized in 1ml of EDTA (0.02 M, pH 6). The homogenate was subjected to
141 a deproteinisation with sulfosalysilic acid (SSA) at 0.25 %. The optical density was measured
142 at 412 nm.

143 Extraction and estimation of energy reserves

144 Proteins, carbohydrates and lipids were extracted following the procedure of[36] and quantified
145 as previously described.[37] Briefly, for body biochemical analyses, newly molted adults from
146 were collected. Pooled samples (10 individuals per pool) were weighed and extracted in 1 ml
147 of trichloracetic acid (20%). In brief, quantification of proteins was carried following the
148 Coomassie Brilliant Blue G-250 dye-binding method[31] with bovine serum albumin as a
149 standard. The absorbance was measured at 595 nm. Carbohydrates were determined following
150 the anthrone method[38] using glucose as standard. Lipids were measured by the vanillin
151 method.[39] Data were expressed in µg per individual. The amount of carbohydrate, lipid and
152 protein in each sample was calculated in µg per adult by using standard graphs. The values of
153 carbohydrate, lipid and protein in µg were converted into joules.[40]

154 Where: 1mg of carbohydrate or protein has an energy value = 16.74 J

155 1mg of lipid has an energy value = 37.65 J

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

$$
159 \qquad \mathrm{NDI} = \left[ \frac{(C - T)}{(C + T)} \right] \times 100
$$

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

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164 **Evaluation of the residual activity**
165 Persistence of insecticidal activity of AZAD was evaluated as described by Ngamo et al.[41]

166 The fumigation LC₅₀ 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.

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174 Statistical analysis

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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.

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180 Results and discussion

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182 Insecticidal activity

183 Azadirachtin, produced as secondary metabolite, is the principal active constituent in neem extracts.[18] As reported by published reviews[18,42], 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).[43,44] 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 *Cosmopilitis sordidus* etc.[45] The toxicity of this growth regulator is related to its high retention and stability.[46]

194 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<sub>25</sub> and LC<sub>50</sub> 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<sub>25</sub> and LC<sub>50</sub> values decrease as a function of time (Table 1).

200 Our results indicate that azadirachtin exhibit an interesting insecticidal activity with dose-response relationship against *S. granarius* adults. Similar results were found with the same insecticide applied against *Drosophila melanogaster*[47,48] and *Ceraeochrysa claveri*[49,50] reported that this compound presented fumigant toxicity against *Rhyzopertha dominica*. However, the lethal concentrations (LC<sub>50</sub> and CL<sub>90</sub>) recorded in our study are higher to those found in this work (LC<sub>25</sub> =7.41 µl/liter air and LC<sub>50</sub>=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[51] Various studies demonstrated the lethal effects of azadirachtin on different insect species.[43,52,53] Topical application of azadirachtin on *G. mellonella* induced lethal concentrations of 16,564 and 3191,307 ppm corresponding to the LC<sub>50</sub> and LC<sub>90</sub>, respectively.[52,54] The toxicity of Azadirachtin (NeemAzal®) has been reported in different species of mosquito, *Culex pipiens*[55-57], *Aedes aegypti*, *Culex quinquefasciatus* and *Anopheles stephensi*.[58] [19] showed the efficacy of Azadirachtin against Lepidoptera, such as *Shistocerca gregaria*, where the LC<sub>50</sub> has a very low value (0.007 ppm), whereas in the Hemiptera and Coleoptera species, the LC<sub>50</sub> is 100 ppm. The obtained results by Zhong et al.[59] 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.
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219 **Repellent Activity**

220 The repellent activity is a physiological phenomenon that occurs in insects as a defense
221 mechanism against toxins secreted by plants.[50] An insect repellent has been defined as a
222 chemical substance that causes the insect to make oriented movements away from the
223 source.[60] The strong repellency of azadirachtin and neem concentrates in Xie et al.[61] study
224 was reflected by reduced numbers of insects on treated wheat. This reduction is presumably
225 caused by chemosensory effects of these products, either olfactory or gustatory.

226 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.
227 The obtained results revealed an increase in the repellency percentage as a function the
228 concentration. The maximum repellency rate is 60% recorded with a dose of 8µl/ml.
229 According to Mc Donald et al. [29], this product belongs to the repellent class III.
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231 These pesticides have shown contact, fumigant, antifeedant, repellent activity and growth
232 regulating properties against insects.[62] AZ is a powerful behavior-modifying agent for a
233 number of phytophagous insect species.[19,43,63] Various azadirachtin-based commercial
234 formulations, applied at different concentrations, caused strong repellent and oviposition
235 deterrent effects on *T. urticae* females.[64-67] Hanif et al.[51] reported a repellent activity of
236 azadirachtin against *T. castaneum* and *Rhyzopertha dominica* with maximum of 77.66% and
237 81.48% repulsive potential, respectively.

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239 **Biomarker assays**

240 The lethal concentrations (LC<sub>25</sub>: 15.26 µl/ml and LC<sub>50</sub>: 74.83 µl/ml) of azadirachtin at 72h
241 were applied by fumigation on adult of *S. granarius* and its effects examined on CAT and
242 GST activities, and on GSH rate measured at various times (24, 48 and 72 h) following
243 treatment (Table 6). Results show a significant increase in CAT activity for the two tested
244 concentrations only at 72h (control vs LC<sub>25</sub> p= 0.0412 and control vs LC<sub>50</sub> p=0.0153) (Fig.
245 2A, B and C), while GST activity measurements revealed a significant increase in the treated
246 series (LC<sub>25</sub> and LC<sub>50</sub>) respectively compared to control at 48 (p= 0.0196 and p= 0.0015) and
247 72 h (p= 0.0178 and p= 0.0032) without dose-response relationship. Finally, a significant
248 decrease of glutathione rate was observed in treated series (LC<sub>25</sub> and LC<sub>50</sub>) (p= 0.0133 and
249 0.0035) respectively at 72h as compared to control series.
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251 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
252 *Choristoneura rasaceana*[68], in *Xanthogaleruca luteola* (Müller) (Coleoptera)[69], in
253 *Helicoverpa armigera* larvae (Hübner) (Lepidoptera)[70] and in *Drosophila melanogaster* [71];
254 Or various insecticides such as neem oil in *Xanthogaleruca luteola*. [69]

255 Sometimes, the GST activities could be not affected by azadirachtin in *Choristoneura rasaceana*. [72] But the results of [73] have confirmed the intervention of GST in the mechanism
256 of the detoxication of azadirachtin. Increased GST activity results in the detoxification
257 process, is a form of insect defense against pesticide.[74]
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260 Glutathione (GSH) plays an important role in the detoxification and excretion of
261 xenobiotics.[75] In our study, AZ induces a significant greater decrease in GSH rate in S.

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granarius adult. Similar effects observed by Kiran et al.[76] 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 (LC₅₀) with *Gaultheria procumbens* essential oil[77], and in *C. pipiens* with *T. vulgaris*. [78] 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 LC₅₀ 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.[79] In contrast, a decrease in CAT activity was observed in *S. oryzae* and *R. dominica* treated with *Gaultheria procumbens*[77], which could be explained by an increased production of the radical superoxide anion.[80,81] This decrease in CAT activity results accumulation of toxic H₂O₂ in the cell, leading to peroxidation of membrane lipids.[82] The induction of the GST system in *D. melanogaster* is correlated with an increase in specific CAT activity after treatment with Neem Azal.[47] This oxidative stress could be explained by the antagonist action of azadirachtin on endogenous 20E and its antioxidant activity.[47]

## 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 (LC₂₅ and LC₅₀) as compared to controls during the tested period: 24 (control vs LC₂₅: p < 0.001; control vs LC₅₀: p < 0.001; LC₂₅ vs LC₅₀: p = 0.006), 48 (control vs LC₂₅: <0.001; control vs LC₅₀: <0.001) and 72 hours (control vs LC₂₅: <0.001; control vs LC₅₀: <0.001; LC₂₅ vs LC₅₀: p = 0.008).

Concerning the total energy (Table 3), the results revealed a significant decrease in the treated series (LC₂₅ and LC₅₀) respectively compared to control at 24h (control vs LC₂₅: p < 0.001; control vs LC₅₀: p < 0.001; LC₂₅ vs LC₅₀: p = 0.001), 48 (control vs LC₂₅: <0.001; control vs LC₅₀: <0.001; LC₂₅ vs LC₅₀: p < 0.001) and 72 hours (control vs LC₂₅: <0.001; control vs LC₅₀: <0.001; LC₂₅ vs LC₅₀: p = 0.02).

## 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 LC₅₀ treated series at different periods after treatment: 24 (p = 0.003), 48 (p < 0.001) and 72 hours (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.[83] This investigation shows
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that after treatment of azadirachtin, the protein level and energy reserves of *S. granarius* larvae decreased during the tested period.

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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.[84,85] In the present investigation, after treatment of *S. granarius* adults with AZ, an inhibitory action on proteins was generally exhibited.[86] 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.[72] Nevertheless, Ebadollahi et al.[87] 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[88] 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[83] or to the breakdown of these proteins into amino acids used in the compensatory mechanism as energy source to compensate stress.[89]

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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*[90] and *Spodoptera litura*. [91] Further, it is reported that protein expression in *S. litura* was significantly lowered under azadirachtin treatment.[44] Rao & Subrahmanyam[92] 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.[18], 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.

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The carbohydrates are considered as important energy elements playing a crucial role in the insect physiology, such as the molt process and the reproduction.[93] 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.

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AZAD derivatives also lead to a decrease in the concentration of carbohydrates in *Ctenoparyngodon idella*. [94] Tine et al.[95] 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.[50] 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*.
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indica and *Citrullus colocynthis* methylene chloride extract and was increased with garlic and lemon Eos.[⁹⁶]

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Lipids are also an important source of acetyl groups needed to synthesize the enzymes from constitutive amino acids.[⁹⁷] 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[⁹⁸], who found that *Tenebrio molitor* suffered lipid depletion in haemolymph, fat bodies and oocytes when exposed to malathion. Sak et al.[⁹⁹] reported the decline in lipid content due to shift in energy metabolism to lipid catabolism due to insecticidal stress induced by *Pimpala turionellae*.

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## Residual activity of azadirachtin

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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).

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The biopotency is negatively correlated with time. Ngamo et al.[⁴¹] and Heydarzade & Moravvej[¹⁰⁰] 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.[¹⁰¹] This decrease is similar to this obtained with *Xylopia aethiopica* against *Callosobruchus maculatus*. [¹⁰²] 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[⁴¹,¹⁰³] had proven that plant species has more persistence toxic effect when they contained higher proportion of oxygenated molecules such as oxygenated monoterpenes and sesquiterpenes. The persistence of insecticidal activity was in relationship with the sensitivity of the major target pest to active compound.[⁴¹,¹⁰³,¹⁰⁴] In the experiment of Akami et al.[¹⁰⁵], 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.[¹⁰⁵] Regnault-Roger et al.[¹⁰⁶] showed the lower volatility of oxygenated molecules because of their higher molecular weight.

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## Conclusion

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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

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decrease of GSH rate. Our results provide an interesting opportunity to develop bioinsecticides and repellent formulations.

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).

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Legends of Table and Figures

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**Table 1.** 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)

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<table><thead><tr><th rowspan="2">Toxicity</th><th rowspan="2">Times (hours)</th><th rowspan="2">R<sup>2</sup></th><th colspan="2">Lethal concentrations (LCL-UCL)</th></tr><tr><th>LC<sub>25</sub> (95% FL)</th><th>LC<sub>50</sub> (95% FL)</th></tr></thead><tbody><tr><td rowspan="3">Fumigation (µl/l air)</td><td>24</td><td>0.93</td><td><strong>113.70</strong><br>(74.10 - 164.60)</td><td><strong>549.10</strong><br>(343.30 - 1480)</td></tr><tr><td>48</td><td>0.91</td><td><strong>57.71</strong><br>(27.34 - 92.94)</td><td><strong>335.70</strong><br>(213.20 - 852.90)</td></tr><tr><td>72</td><td>0.75</td><td><strong>17.29</strong><br>(0.02 - 55.15)</td><td><strong>72.01</strong><br>(12.52 - 185.90)</td></tr><tr><td rowspan="2">Contact (µl/ml)</td><td>12</td><td>0.96</td><td><strong>5.84</strong><br>(4.15 - 7.60)</td><td><strong>30.35</strong><br>(22.72 - 48.07)</td></tr><tr><td>24</td><td>0.94</td><td><strong>1.04</strong><br>(0.36 - 1.83)</td><td><strong>3.16</strong><br>(1.92 - 4.38)</td></tr></tbody></table>

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

<table><thead><tr><th>Concentrations</th><th>RP (%)</th><th>Class</th></tr></thead><tbody><tr><td>1µl/ml</td><td>25</td><td>II</td></tr><tr><td>2µl/ml</td><td>35</td><td>II</td></tr><tr><td>4µl/ml</td><td>50</td><td>III</td></tr><tr><td>8µl/ml</td><td>60</td><td>III</td></tr></tbody></table>

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**Table 3.** Effect of azadirachtin (LC<sub>25</sub> and LC<sub>50</sub>) 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).

<table><thead><tr><th>Times (hours)</th><th>Components</th><th>Control</th><th>LC<sub>25</sub></th><th>LC<sub>50</sub></th></tr></thead><tbody><tr><td rowspan="2">24</td><td><strong>Proteins</strong></td><td>3.61 ± 0.04 a</td><td>0.92 ± 0.02 b</td><td>0.74 ± 0.02 c</td></tr><tr><td><strong>Total energy</strong></td><td>8.30 ± 0.17 a</td><td>4.54 ± 0.13 b</td><td>3.53 ± 0.05 c</td></tr><tr><td rowspan="2">48</td><td><strong>Proteins</strong></td><td>3.61 ± 0.05 a</td><td>0.83 ± 0.01 b</td><td>0.73 ± 0.04 b</td></tr><tr><td><strong>Total energy</strong></td><td>8.18 ± 0.11 a</td><td>4.31 ± 0.08 b</td><td>2.95 ± 0.00 c</td></tr><tr><td rowspan="2">72</td><td><strong>Proteins</strong></td><td>3.61 ± 0.00 a</td><td>0.78 ± 0.02 b</td><td>0.65 ± 0.03 c</td></tr><tr><td><strong>Total energy</strong></td><td>7.87 ± 0.27 a</td><td>3.19 ± 0.06 b</td><td>2.39 ± 0.19 c</td></tr></tbody></table>

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The different lowercase letters indicate significant differences at the same time based on Tukey's HSD test (p < 0.05).
<table><caption>**Table 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).</caption><thead><tr><td>Times (hours)</td><td>LC<sub>25</sub></td><td>LC<sub>50</sub></td><td>P value</td></tr></thead><tbody><tr><td>24</td><td>29.26 ± 2.07<sup>a</sup></td><td>40.31 ± 0.93<sup>b</sup></td><td>0.003</td></tr><tr><td>48</td><td>30.98 ± 0.58<sup>a</sup></td><td>46.93 ± 0.59<sup>b</sup></td><td>&lt;0.001</td></tr><tr><td>72</td><td>42.21 ± 2.21<sup>a</sup></td><td>53.36 ± 3.32<sup>b</sup></td><td>0.022</td></tr></tbody></table>

Different small letters indicate a significant difference between treated individuals in the same period.

<figure><img src="image_1.png" /><figcaption>**Figure 1.** Efficacy of azadirachtin applied by fumigation (A) and by contact (B) on adult as function of the exposure time (hours) (mean ± SEM, n = 5 replicates each containing 10 adults).</figcaption></figure>

<figure><img src="image_2.png" /></figure>

Different small letters indicate a significant difference between treated individuals in the same period.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Efficacy of azadirachtin applied by fumigation (A) and by contact (B) on adult as function of the exposure time (hours) (mean ± SEM, n = 5 replicates each containing 10 adults).</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Effect of azadirachtin (LC<sub>25</sub> and LC<sub>50</sub>) 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).*</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Residual activity of azadirachtin (LC<sub>50</sub>) by fumigation against *S. granarius* adults for a delay of 30h (mean ± SD, n = 4 pools each containing 10 individuals).</figcaption></figure>