Response of Spodoptera litura Fab. (Lepidoptera: Noctuidae) larvae to Citrullus colocynthis L. (Cucurbitales: Cucurbitaceae) chemical constituents: Larval tolerance, food utilization and detoxifying enzyme activities

Accepted Manuscript Response of Spodoptera litura Fab. (Lepidopteran: Noctuidae) larvae to Citrullus colocynthis L. (Cucurbitales: Cucurbitales) chemical constituents: Larval tolerance, food utilization and detoxifying enzyme activities Athirstam Ponsankar, Prabhakaran Vasantha-Srinivasan, Annamalai Thanigaivel, Edward-Sam Edwin, Selvaraj Selin-Rani, Muthiah Chellappandian, Sengottayan Senthil-Nathan, Kandaswamy Kalaivani, Annamalai Mahendiran, Wayne B. Hunter, Rocco T. Alessandro, Veeramuthu Duraipandiyan, Naif Abdullah Al-Dhabi PII:

S0885-5765(16)30175-8

DOI:

10.1016/j.pmpp.2016.12.006

Reference:

YPMPP 1227

To appear in:

Physiological and Molecular Plant Pathology

Received Date: 15 November 2016 Revised Date:

15 December 2016

Accepted Date: 18 December 2016

Please cite this article as: Ponsankar A, Vasantha-Srinivasan P, Thanigaivel A, Edwin E-S, SelinRani S, Chellappandian M, Senthil-Nathan S, Kalaivani K, Mahendiran A, Hunter WB, Alessandro RT, Duraipandiyan V, Al-Dhabi NA, Response of Spodoptera litura Fab. (Lepidopteran: Noctuidae) larvae to Citrullus colocynthis L. (Cucurbitales: Cucurbitales) chemical constituents: Larval tolerance, food utilization and detoxifying enzyme activities, Physiological and Molecular Plant Pathology (2017), doi: 10.1016/j.pmpp.2016.12.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Title: Response of Spodoptera litura Fab. (Lepidopteran: Noctuidae) larvae to Citrullus

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colocynthis L. (Cucurbitales: Cucurbitales) chemical constituents: Larval tolerance, food

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utilization and detoxifying enzyme activities

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Running heading: Botanical insecticide inhibit enzyme production of polyphagous insect

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Authors: Athirstam Ponsankara, Prabhakaran Vasantha-Srinivasana, Annamalai Thanigaivela,

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Edward-Sam Edwina, Selvaraj Selin-Rania, Muthiah Chellappandiana, Sengottayan Senthil-

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Nathana*, Kandaswamy Kalaivanib, Annamalai Mahendirana,c, Wayne B. Hunterd, Rocco T.

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Alessandroe ,Veeramuthu Duraipandiyanf, Naif Abdullah Al-Dhabif

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Affiliations

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a

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Excellence in Environmental Sciences, Manonmaniam Sundaranar University,

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Alwarkurichi – 627 412, Tirunelveli, Tamil Nadu, India.

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b

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Courtrallam-627 802, Tirunelveli, Tamil Nadu, India.

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c

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d

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2001 South Rock Road, Fort Pierce, FL 34945, USA

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e

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USA

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f

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College of Science, King Saud University, P.O. Box.2455, Riyadh 11451, Kingdom of Saudi

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Arabia

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* Corresponding author, email- [email protected]; Phone & Fax: +91 4634 283066

Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for

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Post Graduate and Research Department of Zoology, Sri Parasakthi College for Women,

Crop Protection Division, NRRI, ICAR, Cuttack, Odisha-735006, India.

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United States Department of Agriculture, U.S. Horticultural Research Laboratory,

Treasure Coast Chemistry Consultants, LLC 107 Lakes End Drive, Apt. B Ft. Pierce, FL 34982,

Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies,

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Abstract Pest management has increased the alarm across researchers because of the possible risk from harmful insecticides dispersed in the natural environment. Plant derivatives are established

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from plant extracts and displays latent effects against damaging pests in a multiple ways.

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Citrullus colocynthis (L.) Schrad, largely dispersed across the world in the warm area. A

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purified, fractionated solvent extract of Citrullus colocynthis L. (bitter apple) has shown 90%

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lethality toward Spodoptera litura third instar larvae, and slightly lower lethality to the fourth

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and fifth instars. Based on the food utilization, the mechanism of lethality appears to be reduced

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digestive enzyme activity. The enzyme analysis of S. litura against fraction F5 demonstrated that

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the level of ACP, ALP, ATP and LDH decreased significantly based on their concentration. The

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gut histology of S. litura shows disturbance in the midgut columnar cells against the fraction F5.

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The fraction F5 was further eluted using column chromatography and the subfraction A3

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obtained and it was further characterized using FTIR and NMR. The subfraction A3 exhibits

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prominent mortality rate with 84% at 100 ppm concentration. The subfraction A3 was

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characterized and identified as stigmasterol.

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Key words: Bitter apple; GC-MS; S. litura; mortality; gut enzyme; FTIR; stigmasterol

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1. Introduction Chemical pesticides are broadly used to convince increased crop yield and to supply the frequently aggregating food demand across the nation [1,2]. Around five billion pounds of

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chemical pesticides were used in the year of 2006 and 2007 all over the world [3], but it targets

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around 1% of the target pests at lethal doses [4,5]. These effects leads to increasing the rigorous

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environmental regulation of pesticides [6]. Management of pest has elevated a abundant deal of

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concern in scientific communities because of the potential risk from various pesticide

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components being widely dispersed in the natural environment [7]. Acclimation due to repeated

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exposure to pesticide chemicals occurs at the pest species level leads to decline in pesticide

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effectiveness and increased the insect resistance [8,9]. Finding plant derived extracts, which are

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effective against agriculture pests, is considered crucial to meeting the demand for organic food

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by targeting specific activity towards natural enemies [10]. Different modes of action of the plant

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extracts may reduce the pesticide resistance and problems of resurgence of the pest while being

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secure and environmental friendly [11,12,13].

Spodoptera litura (Fabricius) is the critical lepidopteran pests distributed throughout

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Southeast Asian countries [14] and causing commercial damages to several demanding crops

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[15]. S. litura feeds over 290 plant species, corresponding to ninety nine families and creates

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yield loss of 26-100% in the agriculture field [16,17]. Moreover, this pest has developed

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resistance to synthetic pesticides [18,19]. Experiments with the botanical pesticides shows that

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they are more effective against S. litura and cause less risk to the environment when compare to

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the chemical pesticides [20]. Botanical pesticides are developed from plant extracts and function

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against harmful pests in a variety of ways [21]. Midgut enzymes plays a key role in passage and

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re-absorption of nutrients and secondary metabolites and also transports non-electrolytes and

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ions [20,22]. The midgut cell wall is connected with the neuroendocrine cells, which control

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enzyme levels towards gut lumen [23]. Previous findings suggests that plant derivatives can

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affects the endocrine cell secretions in various pests [20,24].

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The Cucurbitaceae family is one of the most flat or herbaceous climbers, annual plants covering over nine hundred species [25]. Citrullus colocynthis (L.) Schrad, broadly spreads

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across the warm area of the world including West Asia and Tropical Africa and is widely

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distributed in India, Pakistan and Saudi Arabia [26,27]. Bitter apple, bitter cucumber, chitrapala,

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egusimelon, ground melon are the common names of the fruits of C. colocynthis [26,28]. C.

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colocynthis are used in the human medicines to cure several diseases [27]. Active metabolites

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isolated from C. colocynthis shows potential activity against cowpea aphid and different

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mosquito vectors [29,30].

Thus the aim of current investigation is to evaluate entomotoxic effect of C. colocynthis

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against the lepidopteran pest S. litura by evaluating the normal nutritional index, enzymatic and

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histological changes in the midgut cells and also to identify the bioactive compound responsible

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for insecticidal activity using gas chromatography/mass spectrometry (GCMS), IR and NMR

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spectroscopy.

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2. Materials and Method

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2.1 Plant harvesting

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The fruits of C. colocynthis (Fig. 1A) were harvested in and around Southern Western

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Ghats India (Latitude: 8° 43’ 13.64” N; Longitude: 77° 37’ 21.50” E; Elevation: 221 feet) during

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summer and spring seasons. The collected plants were authenticated and voucher specimen

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(Collection number 966 and reference number BSI/CRC/Tech./ 2012–13) of this collection has

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been submitted to herbarium of SPKCESS, Manonmanium Sundaranar University, India.

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2.2. Preparation of crude extract The fruit powder (500 g) was extracted by soxhlet technique using the different solvents hexane, chloroform, ethyl acetate and methanol similar to Visetson et al. [31]. Vacuum

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evaporation was performed using rotary evaporator to remove the solvent and the residues were

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preserved at 4 ºC. The yields of various extracts (hexane, chloroform, ethyl acetate and

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methanol) were 3.93, 2.76, 4.02 and 4.31 g respectively. The preliminary bioassays of fourth

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instar larvae of S. litura showed the greatest mortality rate in ethyl acetate extract and it was used

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for chromatographic separation of the active components.

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2.3. Purification of active compound

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The extract of ethyl acetate was eluted through a column with silica gel packed (60-120 pore size) (Merck kGaA, Darmstadt, Germany). Through increased gradient the column was

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eluted with an hexane and ethyl acetate ratio from 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30,

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65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85 and 10:90 then lastly

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5% methanol was used to wash. The fractions eluted were represented as F1, F2, F3, F4, F5 till F20

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respectively. The rotary evaporator was used to evaporate the solvent and the fraction deposits

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were preserved at 4 °C. In the preliminary bioassay, fraction F5 displayed prominent mortality to

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the other eluted fractions. Consequently, F5 fractions was used for further experiments.

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2.4. C. colocynthis fraction F5 chemical characterization

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Two hundred micro liter of HPLC grade ethyl acetate was dissolved with 200 µl of

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fraction F5 and examined using a GC-MS spectrometer (Agilent Technologies, Santa Clara, CA).

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The HP5 column was merged with silica 50 m x 0.25 mm I.D. Examination settings were twenty

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minutes at 100 °C, and 300 °C for injector temperature, for carrier gas helium was used with 5:4

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split ratio. The sample (2 µl) was evaporated for twenty two minutes run time in a split less

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injector at 300 °C. The compound structure, molecular weight and molecular formula were

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identified by matching the sample spectrum to those in the database (NIST). The total ion

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chromatogram (TIC) peak areas were used to generate an area % report allowing an estimate of

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the concentrations of the eluted compounds.

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2.5. Insect culture

The S. litura culture has been maintained since 2007 in our lab without exposure to the

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pesticides. Moisten cotton were provided for separated pre-pupae were for pupation. The

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emerged moths were detached to the insect cages holding leaves of Ricinus communis L. with 1

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male: 2 female and 10% sucrose solution were fed for oviposition. For egg laying the oviposition

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cage were covered with sanitary black cloth. In in-situ conditions the eggs were surface sterilized

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in ten percentage formaldehyde for five minute, washing with double distilled water for two

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minutes then air dried and allowed to hatch.

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2.6. Mortality assays

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Larvicidal assays were executed with third, fourth and fifth instars larvae of S. litura with different concentrations of 50, 100, 150 and 200 ppm of fraction F5. Different concentration of

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fraction F5 were sprayed in the castor leaves and allowed to air dry. Methanol (0.1%) alone were

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used to treat the control. Four hour starved third to fifth instars were released to the leaves treated

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and kept in a sterilized room (27 ± 2 °C and 80% RH). A minimum of twenty larvae per

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concentration used for all the treatments, and the experiments were five times replicated (n=100)

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and one control was assigned for each replication. Mortality rate was observed for every 24h and

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recorded with control comparison. The mortality percentage was analyzed using the Abbott’s

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formula [32]. The percent corrected mortality data was analyzed to Probit analysis to calculate

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the lethal concentration (LC50 and LC90) [33]. The treatment doses were selected from the mean

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lethal concentration for biological studies.

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2.7. Food utilization studies

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Nutritional studies were determined by the adapted methodology of Senthil-

Nathan et al, [34] to analyze the effect of the F5 fraction of C. colocynthis on food utilization of

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fourth and fifth instar larva of S. litura. Freshly molted fourth and fifth instars were starved for

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three hours. After early weight measurement, the larvae were separately placed into individual

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containers (4 cm × 4 cm). Fresh castor leaves were dried and weighed were saturated with one

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ml of fraction F5 and fed to starved fourth and fifth instar larva. The similar procedure was

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carried for control treated with 0.1% methanol. For each set of experiments ten larvae were used.

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Post 24h treatments uneaten leaves were weighed and changed with fresh castor leaves. After

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24h, each larva was weighed, oven dried (48h at 60 °C) and reweighed to find out percentage dry

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weight. Correspondingly, fecal matter and dry weight of diet was documented under

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experimental conditions. Electronic balance (Shimadzu, Japan) were used for measuring the

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weight. Nutritional indices were calculated on dry weight basis by Waldbauer [35]. Post 24h

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larval growth and food utilization were calculated. Minitab® was used to calculate the linear

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regression between RCR and RGR.

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2.8. Enzyme extract preparation

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S. litura fourth and fifth instars treated larvae were used to analyze the gut enzymatic

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activity. Adapted methodology of Applebaum [36] and Applebaum et al. [37] were used for

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enzyme extract preparation.

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2.9. Acid (E.C.3.1.3.2) and alkaline phosphatases (E.C.3.1.3.1) estimation

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The enzyme assays of phosphatase were carried out based on the procedure followed by Bessey et al. [38].

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2.10. Estimation of adenosine triphosphatases

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Shiosaka et al. [39] described an assay to determine the definite activity of potassium and

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sodium-dependent gut ATPase. Fiske and Subbarow [40] assay was used to estimate the quantity

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of inorganic phosphorous liberated.

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2.11. Lactate dehydrogenase (EC 1.1.1.27) estimation

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The lactate dehydrogenase enzyme activity is stated as multi-international units (mIU)

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per milligram protein per minute [41]. A mIU is defined as the amount of enzyme that is required

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to catalyze the conversion of 1 µm lactate to pyruvate or pyruvate to lactate per minute per

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milliliter of the sample.

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2.12. Histological studies

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Sectioning procedure was performed based on the methodology of Senthil-Nathan et al.

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[34]. Control and fraction F5 (100 ppm) mid gut tissue from fourth instar of S. litura were used

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for tissue sectioning. Further the sections were examined and snapped under fluorescence

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microscope (model: B-600, Optika Flow series HBO, TiFl, Ponteranica, Italy).

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2.13. Separation of active compound from fraction F5

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The fraction F5 (5g) was further separated using column chromatography silica gel (60-

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120 pore size) (Merck kGaA, Darmstadt, Germany). and eluted with petroleum ether:ethyl

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acetate (3:1). Fractions (10 ml) with same Rf value were pooled together after TLC monitoring

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and bioassay were performed for all sub-fractions (A1 - A9) to find the toxicity against third

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instar larvae of S. litura.

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2.14. Mortality bioassay of subfraction A3

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Bioassays were executed with third instar larvae of S. litura using subfraction A3.

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Mortality was observed for every 24h and recorded with control comparison. Hence sub-fraction

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A3 was analyzed for toxicity and characterized through FTIR and NMR spectral analysis.

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2.15. Characterization of the active compound

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2.15.1 Fourier transform infrared (FTIR) spectroscopy

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FTIR spectra were recorded on a Perkin Elmer Spectrum One equipped with an ATR-

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FTIR unit. A few milligrams of sub-fraction A3 sample were placed in the head of ATR. The

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spectra were obtained with a resolution of 4 cm-1 and 16 co-addition scans in a wavelength range

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of 450-4000 cm-1. For each spectrum, 16 scans were accumulated and averaged. The spectra

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were collected and analyzed using Spectrum software (Perkin Elmer).

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2.15.2 NMR analysis

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The 1H and 13C NMR spectra of sub-fraction A3 were recorded at 300 MHz (1H NMR) and 75 MHz (13C NMR) carried out by Bruker (Advance) NMR instrument in CDCl3 solvent and

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few drops of DMSO. The chemical shifts referencing to TMS (Tetramethyl silane) and are given

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in ppm.

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2.15. Data analysis

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Bioassay and food utilization data were subjected to ANOVA of arcsine square root

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transformed percentages. Treatments difference were determined by Tukey’s family error rate by

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Minitab®17 statistical software (Minitab, Inc., State College, PA). Difference between means

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was considered significant at P≤ 0.05 [42]. The lethal concentrations required to kill 50% (LC50)

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of larvae in 24h were calculated by Probit analysis with a reliability interval of 95% using the

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Minitab®17 program.

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3. Results

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3.1. GCMS analysis of fraction F5

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The GCMS analysis showed nine major compounds were displayed in fraction F5 (Fig. 1B). Compounds were detected by comparing the each mass spectrum of each of the nine peaks

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with spectra in the NIST library. Nine compounds were identified and the major constituents

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were stigmasterol (26.69%), 2-Formyl-4-methylpentanoic acid ethyl ester (15.45%) and estra-

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1,3,5(10)-trien-17a-ol (12.81%). The retention time, compound names, molecular weight and

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formulas, peak area percentages and structure of the identified compounds are given in Table 1.

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3.2. Mortality rate of fraction F5 against S. litura instars

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Among the fractions (F1-F20), fraction F5 showed the prominent rate of mortality in preliminary bioassay, further we used fraction F5 for mortality bioassays. Active fraction F5

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treated with different dosage of 50, 100, 150 and 200 ppm against third, fourth and fifth instar

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larvae of S. litura respectively. Dose dependent mortality rate (200 ppm) was observed in third,

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fourth and fifth instars larvae (Fig. 2). Mortality rate was gradually increased with the higher

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concentrations of fraction F5, from 50 to 200 ppm. The estimated LC50 value of fraction F5 on

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third instar was determined as 91.20 ppm (Fig. 3). The predictable 90% (LC90) against third

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instar was observed in 229.08 ppm.

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The mortality rate was significantly higher in third, fourth and fifth instar larvae with 90

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(F4,20=47.08, P=0.001), 87 (F4,20=56.62, P=0.001) and 81% (F4,20=57.53, P=0.001) at 200 ppm

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respectively. The average mortality rate of 150 ppm shows 69, 66 and 61% for third to fifth

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instars of S. litura respectively. Despite, fraction F5 at 100 ppm showed reduced mortality rate

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(48, 44 and 41%) of all the treated instars of S. litura. Similar to 100 ppm, the mortality rate was

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also reduced at 50 ppm concentrations (27, 25 and 22%) when treated with the third, fourth and

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fifth instars of S. litura larvae respectively. All the treatment concentrations were significantly

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different against the control larvae of S. litura.

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3.3. Food utilization, consumption and nutritional indices

Nutritional indices proved fraction F5 treatments reduced relative growth and

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consumption rates with significant change in the efficiency of conversion of ingested food. As

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the fraction F5 dosage increased, consumption efficiency was decreased significantly.

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Results obtained from food consumption and its utilization, observed that fraction F5 triggered all nutritional indices of fourth instar larvae (Table 2). This activity was also dose

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dependent. The analysis of fourth instar larvae fed with different concentration of fraction F5 (50,

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75 and 100 ppm) showed a prominent reduction in relative growth rate parallel to the control

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(F4,20=32.41, P=0.001) but not significant to 25 ppm (F4,20=32.41, P=0.142). Larval

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consumption rate was highly depends on concentration of fraction F5. The relative consumption

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rate decreased from control and shows significant reduction after treatment with dosage of 50

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(F4,20=29.11, P=0.032), 75 (F4,20=32.41, P=0.001) and 100 ppm (F4,20=32.41, P=0.001). In

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contrast 25 ppm was not significant when compared with control (F4,20=32.41, P=0.996). The

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efficiency of conversion of ingested food (ECI) determines the ingested food which renewed into

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the body substance. The ECI in control larvae was different significantly post treatment with

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concentration of 50 (F4,20=13.92, P=0.006), 75 (F4,20=13.92, P=0.002) and 100 ppm

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(F4,20=13.92, P=0.001). But ECI value of control was not significant at 25 ppm (F4,20=13.92,

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P=0.095). Similarly ECD of control (F4,20=65.65, P≤0.001) was significantly different to

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treatment concentration of 50, 75 and 100 ppm. In contrast 25 ppm (F4,20=65.65, P=0.069) was

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not significant to control. The AD was increased in treatment concentration at 50, 75 and 100

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ppm and shows significant different compared to control (F4,20=32.30, P≤0.001). The AD after

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treatment with concentration of 25 ppm (F4,20=32.30, P=0.103) was not significant when

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compared with control. Fraction F5 treated fourth instar larvae (R2= 0.953) were significant

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different in the regression coefficients of the RCR–RGR relations for control (R2= 0.909) (Fig.

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4A).

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As found in the fourth instar, reduction observed in nutritional indices RGR, RCR, ECI, ECD and AD of fifth instar larvae when larvae fed with fraction F5. The inhibitory relative

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growth rate of different recommended doses (50, 75 and 100 ppm) were significant comparing to

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untreated control larvae. However, there was no significant difference between the lower

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concentration of 25 ppm (F4,20=25.36, P≤0.001) and control. In addition, relative consumption

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rate also decreased at higher concentration of 75 and 100 ppm parallel to the control

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(F4,20=14.46, P≤0.001). But, it has been noticed that fraction F5 at lower concentration of 25

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(F4,20=14.46, P=0.776) and 50 ppm (F4,20=14.46, P=0.183) was not significant to control.

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Furthermore, ECI of fraction F5 at all the concentration shows significant different when

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compared to the control (F4,20=51.27, P≤0.001). Parallel to this, ECD also decreased when

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compared to control. ECD in the control was 60.44% and decreased to 53.14, 50.18, 46.41 and

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41.29% with 25, 50, 100 and 200 ppm treatment of fraction F5 respectively. The results revealed

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that concentration of fraction F5 increased with decreasing the ECD and it illustrate significant

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different compared to control (F4,20=48.84, P≤0.001). Fraction F5 at all treatment concentration

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(25, 50, 75 and 100 ppm) was significantly increased the AD compared to untreated control

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(F4,20=50.92, P≤0.001). The regression coefficients of the RCR–RGR relations for control and

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treated larvae were significantly different (R2= 0.913, R2= 0.925) (Fig. 4B).

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3.4. Estimation of acid (E.C.3.1.3.2) and alkaline phosphatases (E.C.3.1.3.1) The effect of fraction F5 on the ACP activity of fourth instar larvae was examined. The result demonstrated that the specific activity was decreased low at lower concentration of 25 ppm

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(20.48%). On the other hand, higher concentration (50, 75 and 100 ppm) shows increased in

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decline activity of ACP (31.71, 39.81 and 50.93% respectively (R2 = 0.808) (Fig. 5A). Though,

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all the treated concentration illustrated significant different judge against control (F4,20 = 32.99, P

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= 0.001).

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As found in fourth instar, ACP activity was also declined in fifth instar larvae treated

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with fraction F5 (Fig. 5). ACP activity in control larvae was 19.43 ± 2.62 nmol/min/mg protein. When insects were exposed to fraction F5 (25, 50, 75 and 100 ppm), whose ACP activity was

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decline to 15.23, 26.55, 36.64 and 48.48% respectively (R2 = 0.833) (Fig. 5B). Fifth instar larvae

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shows significant difference in ACP activity in all the treatment concentration of fraction F5,

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compared to its control (F4,20 = 28.84, P = 0.001).

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The ALP activity in fourth instar control larvae was 23.27 ± 1.33 nmol/min/mg protein

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and decreased to 10.91, 20.62, 33.04 and 43.61% by the fraction F5 treatment concentration of

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25, 50, 75 and 100 ppm respectively (R2 = 0.893) (Fig. 5C). Fourth instar larvae shows

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significant difference in ALP activity in all the treatment concentration of fraction F5, compared

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to its control (F4,20 = 60.27, P = 0.001).

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Similar to fourth instar, ALP activity of fifth instar was significantly lower in all the

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treatment concentration of fraction F5. The ALP activity in control was 28.04 ± 1.90

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nmol/min/mg protein and decreased to 12.19, 19.90, 29.74 and 40.33% by the fraction F5

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treatment concentration of 25, 50, 75 and 100 ppm respectively (R2 = 0.811) (Fig. 5D).

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Compared to control (F4,20 = 63.39, P = 0.001), fraction F5 shows significant different in ALP

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activity of all the treatment concentration of (25, 50, 75 and 100 ppm).

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3.5. Estimation of adenosine triphosphatases

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Decreased in ATP activity achieved in fourth instar S. litura larvae where 10.97, 19.24, 27.45 and 33.87% were recorded (R2 = 0.986) (Fig. 6A) with following treatment concentration

6

of fraction F5 (25, 50, 75 and 100 ppm) respectively compared to 54.26 ± 3.34 nmol/min/mg

7

protein in the control (F4,20 = 65.10, P = 0.001).

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In addition, the fraction F5 treatments at 25, 50, 75 and 100 ppm against fifth instar are

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considered to be statistically significant (F4,20 = 68.91, P = 0.001) when compared to control (91.33 ± 2.59 nmol/min/mg protein). But, larvae treated with 75 ppm did not vary significantly

11

with 100 ppm (F4,20 = 68.91, P = 0.239). Treated larvae were gradually decreased with 10.29,

12

21.85, 30.20 and 35.10% by the treatment concentration of 25, 50, 75 and 100 ppm respectively

13

(R2 = 0.944) (Fig. 6B).

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3.6. Estimation of lactate dehydrogenase (EC 1.1.1.27) A reduction in LDH activity was observed in fourth instar larvae with the increase of

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fraction F5 concentration. Of those larvae exposed to fraction F5 (25, 50, 75 and 100 ppm), the

17

LDH activity rate had declined (16.49, 28.68, 39.10 and 50.12% respectively) compared with

18

control (R2 = 0.992) (Fig. 6C). The significant difference was noted in LDH activity between

19

fraction F5 treated larvae and control (F4,20 = 46.57, P = 0.001). According to the control (F4,20 =

20

56.81, P = 0.001), significant differences in fifth instar LDH activity were found in larvae

21

exposed to fraction F5. LDH activity was decreased to 14.72, 31.20, 42.17 and 50.01% by the

22

treatment concentration of 25, 50, 75 and 100 ppm (R2 = 0.928) (Fig. 6D). These results confirm

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that compounds present in fraction F5 was more toxic in inhibition of digestive enzyme and

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increasing of detoxification enzyme.

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3.7. Histological studies

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The fraction F5 (100 ppm) treated fourth instar larvae of S. litura shows disturbance in the

4

midgut columnar cells (Fig. 7B). In addition, were damaged and intercellular spaces were

6

increased due to columnar cells detachment from peritrophic membrane. In contrast, midgut

7

columnar cells, brush border membrane and epithelial layer are visible clearly in the untreated

8

larvae (Fig. 7A).

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3.8. FTIR analysis

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FTIR spectrum analysis of the sub-fraction A3 showed distinct peaks due to the

11

transmission of the IR rays. The peaks were formed at 3371, 2936, 1461, 1381, 1367, 970, 959,

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838 and 799 cm-1. These peaks arise due to the functional groups in the crude extract (Fig. 8). The peak observed around 3371 cm-1 in the fraction arises due to the O-H stretch of

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alcohols and phenol. The C-H stretch of alkanes was formed around 2936 cm-1. The peaks

15

observed at 1461, 1381 and 1367 cm-1 attributes the presence C-H bending planes of alkanes.

16

The peaks at 970, 959, 838 and 799 cm-1 correspond to the =C-H bend of alkenes.

17

3.9. NMR analysis

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Chemical shifts were given on a δ scale (ppm) with CDCl3 as an internal standard. The

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H-NMR and 13C-NMR of the sub-fraction A3 were analyzes and the spectra were given in

20

Figure 12. 1H-NMR (300 MHz, CDCl3/ DMSO): δH1.227-2.289 (m, aliphatic hydrogens), 3.493-

21

3.526 (m, aliphatic hydrogens), 5.021-5.360 (m, aliphatic hydrogens), 7.265 (s, 1H, OH) (Fig.

22

9B). 13C-NMR (75 MHz, CDCl3/ DMSO): δC 140.702, 138.278, 129.214, 121.640, 71.712,

23

56.814, 55.893, 51.192, 50.101, 42.227, 42.158, 40.467, 39.631, 37.210, 36.460, 31.841, 31.574,

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28.882, 25.366, 24.318, 21.183, 21.051, 19.354, 18.941, 12.213, 12.001. The molecular formula

2

of C29H48O was determined in the light of these 1H NMR and 13C NMR data (Fig. 9A).

3

3.10. Mortality rate of stigmasterol against S. litura

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The stigmasterol exhibited 64% mortality at 75 ppm concentration, which increased up to 84% at the concentration of 100 ppm (Fig. 10). Stigmasterol at all the treatment concentration

6

shows a significant difference compared to control (F4,20=50.08, P≤0.001).

7

4. Discussion

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Phytochemicals are generally secondary metabolites that defends the plants from the

9

predators and other environmental burdens. Large groups of chemical compounds including

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steroids, terpenoids, alkaloids, phenolics and volatile oils from two thousand plants have been

11

previously reported for their pesticidal actions [43]. At present, botanical chemicals covers

12

around 1% of world’s pesticide market [43,44].

The insecticidal potential of cucurbitacin E glycoside derived from C. colocynthis has

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earlier been reported against Aphis craccivora (Koch.) [29]. Similarly, active metabolites linoleic

15

and oleic acids extracted from C. colocynthis shows effective mosquitocidal activity towards

16

Aedes aegypti (Lin.), Anopheles stephensi (Lin.) and Culex quinquefasciatus (Lin.) [30].

17

However, to our knowledge, this is the first documentation of C. colocynthis fruit extracts

18

related with the polyphagus insect S. litura. Different concentration (50 - 200 ppm) of fraction F5

19

showed mortality (27.4 - 90.4%) against third instar larvae. Generally, fraction F5 at lower

20

concentration associated with negative effects on nutritional physiology thus may preclude

21

disruption changes in food utilization and gut enzyme, while higher level of fraction F5 are

22

associated with mortality.

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The nutritional indices showed decrease in the larval growth rate under the influence of toxic compounds present in fraction F5. Fraction F5 at the concentration of 50-100 ppm

3

significantly decreased the RGR and RCR of fourth and fifth instar of S. litura. The approximate

4

digestibility (AD) in the fraction F5 treated S. litura was significantly higher compared to control.

5

This result agree with the other studies using lepidopterous insect treated with phyto-components

6

and plant extracts, which showed increase in rate of AD [19,34]. ECI measures the utilization

7

capability of food ingested by the insect. Decreasing in the conversation of ingested food (ECI)

8

was observed in fraction F5 treated larvae of S. litura which indicated that function disturbance

9

occurred in the fat body of S. litura. Koul et al. [45] proclaimed that topical application of

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azadirachtin (0.1, 0.5 and 1µg/Ins) reduce both the ECI and ECD with a notable reduction in

11

growth rate.

12

Acid phosphatase (ACP) and alkaline phosphatase was a primary hydrolytic enzyme and they present in the gut of lepidopteran insect [46]. Under acidic or alkaline conditions, ACP

14

remove the phosphate group (dephosphorylation). Our present revealed that fourth instar larvae

15

treated with C. colocynthis fruit extract (75 and 100 ppm) showed a significant reduction in ACP

16

(39.81 and 50.93%) and ALP (33.04 and 43.61%). Similar results were observed to the S. litura

17

larvae treated with chitinase (6 µmol) reduced the ACP and ALP activity upto 39 and 46%

18

respectively [47].

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Adenosine triphosphatase (ATPase) are essential for glucose transportation and they are

20

situated in the midgut, malphigian tubules and nerve fibres of Lepitopteran pest [20]. Our present

21

finding revealed that changes observed in nutrional physiology of C. colocynthis treated S. litura.

22

Hence, ATPase also decreased (33.87%) in C. colocynthis (100 ppm) treated S. litura larvae. The

23

phytochemical compounds present in fraction F5 affects the treated larval metabolism hence

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1

enough energy was consumed. Parallel results were obtained by Babu et al. [48] with decreased

2

in ATPase activity when Helicoverpa armigera (Hub.) treated with azadirachtin. In toxicology, LDH are used to spot out the organ or tissue damage since LDH shows a

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critical role in carbohydrate metabolism and act as a chemical stress indicator [13,49]. A

5

significant reduction of LDH was noticed in fourth and fifth instar larvae of S. litura after treated

6

with C. colocynthis and suppression of LDH is a sign of insect toxic allelo-chemicals present in

7

fraction F5. These results supports the findings of Senthil-Nathan et al. [50] who observe,

8

decreased in LDH activity of Cnaphalocrocis medinalis (Guenée) when larvae treated with neem

9

limonoids.

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Our histological results also showed damage occurred in mid gut columnar cells and brush border membrane similar to our previous results [51,52]. This result proved that fourth

12

instars of S. litura was sensitive to compounds present in fraction F5. Similar effects are

13

suggested by Chandrasekaran et al. [53] when S. litura larvae exposed to chitinase. Rawi et al.

14

[54] found that C. colocynthis extract damaged the mid gut and body cell walls of Spodoptera

15

littoralis (Boisduval).

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Stigmasterol was the major constituent present in the fraction F5 of C. colocynthis and it shows mortality against S. litura. Our results concur with the study of Haung et al. [55] who

18

studied stigmasterol from Cacalia tangutica (Maxim) showed prominent cytotoxic effect on S.

19

litura cells compared to friedeline and rotenone. Similarly, Tandon et al. [56] proved

20

stigmasterol from Vernonia cinerea (Schreb) have feeding deterrency of 94.84 and 94.38%

21

against S. litura and Spilosoma obligua (Walker) respectively.

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Thus our results conclude, that C. colocynthis fractions is able to reduce the growth of S. litura at larval instars. The active constituents of C. colocynthis fraction F5 able to inhibit the

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enzymes ACP, ALP, ATP and LDH at 100 ppm concentrations. Future investigations are

2

required to examine the major constituents of C. colocynthis will be suitable for integration into

3

IPM programs aimed at reducing the lepidopteran pest.

4

Acknowledgement

5

The project was full financially supported by King Saud University, through Vice Deanship of

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Research Chairs

7

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[55] J.G. Huang, L.J. Zhou, H.H. Xu, W.O. Li, Insecticidal and Cytotoxic Activities of Extracts of Cacalia tangutica and Its Two Active Ingredients against Musca domestica and Aedes

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[56] M. Tandon, Y.N. Shukla, A.K. Tripathi, S.C. Singh, Insect Antifeedant Principles from

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Legends Table 1. GC-MS analysis of fraction F5 of C. colocynthis.

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Table 2. Nutritional indices of fourth instar larvae of S. litura after treatment with fraction F5 from C. colocynthis. ± standard deviation, RGR- relative growth rate, RCR- relative

consumption rate, ECI- efficiency of food ingestion, ECD- efficiency of food digestion, AD-

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approximate digestibility.

Table 3. Nutritional indices of fifith instar larvae of S. litura after treatment with fraction F5 from

M AN U

C. colocynthis. ± standard deviation, RGR- relative growth rate, RCR- relative consumption rate, ECI- efficiency of food ingestion, ECD- efficiency of food digestion, AD- approximate

AC C

EP

TE D

digestibility.

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Table 1.

1

2.98

Compound name

Emprical formula

Pyridinium mesylate

C12H13NO3S

Molecular Peak Structure weight area % 251.2939 10.83

RI PT

S.no RT

N

O

4

8.58

10.4

C9H16O3

Ascaridole epoxide

C10H16O3

SC

2-Formyl-4methylpentanoic acid ethyl ester

172.2215

184.2322

Methyl 2,3,4 tri-O-acetyl- C20H31N3O9S 393.4477 1-(3-(0aminophenyl)thioureido)1-deoxy-beta-dglucopyranuronate

13.1

Estra-1,3,5(10)-trien-17aol

6

15.67 3-Ethenylcholestan-3-ol

S O H

15.45

O O O

11.94 O

O-O

O

6.66 O

O

S

H N

O O

N H O

O O

O

C18H30O

262.4194

12.81

C29H51O

415.708

8.05

OH

AC C

EP

5

H

M AN U

3

6.68

TE D

2

O

OH

7

16.92 Tetrahydroaraucarolone

C20H34O4

338.4815

OH

3.58

OH O HO

NH2

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18.13 5HC24H37O5 cyclopropal(3,4)benz(1,2e)azulen-5-one, 1,1 a-a, 1 b-a, 4, 4a, 7a-a, 7b, 8, 9, 9a-decahydro-4a-a, 7b-a, 9a-a-trihydroxy-3(hydroxymethyl)-1, 1,6,8-a-tetramethyl-, 9aisobutyrate

405.533

3.99 O O

H OH

RI PT

8

OH

O

C29H48O

412.6908

26.69

EP

TE D

M AN U

SC

20.62 Stigmasta-5,24(28)-dien3-ol,(3a,24Z) (Stigmasterol)

AC C

9

HO

OH

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Table 2. Fraction VI (ppm)

RGR (mg/mg/day)

RCR (mg/mg/day)

ECI (%)

ECD (%)

AD (%)

1 2 3 4 5

25 50 75 100 Control

0.52±0.0015ab 0.45±0.0052bc 0.42±0.0029d 0.32±0.0047d 0.58±0.0038a

2.19±0.0158ab 2.03±0.1441bc 1.92±0.0721c 1.69±0.0897d 2.21±0.0711a

23.59±0.72ab 22.33±0.77b 21.92±0.71bc 19.08±1.41c 26.22±2.92a

46.37±0.72b 42.88±2.12c 36.20±2.92cd 30.25±2.12d 50.34±2.55a

55.02±0.71bc 57.13±0.71b 60.56±1.87a 63.09±2.83a 52.19±1.41c

AC C

EP

TE D

M AN U

SC

RI PT

S. no

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Table 3. Fraction VI (ppm)

RGR (mg/mg/day)

RCR (mg/mg/day)

ECI (%)

ECD (%)

AD (%)

1 2 3 4 5

25 50 75 100 Control

0.75±0.0019ab 0.70±0.0022bc 0.66±0.0022c 0.58±0.0072d 0.80±0.0022a

2.39±0.0158ab 2.33±0.0255ab 2.25±0.0791b 2.09±0.0149c 2.45±0.0644a

31.38±0.29b 30.04±0.21c 29.33±0.73c 27.75±0.72d 32.65±0.72a

53.14±2.12b 50.18±2.12bc 46.41±0.70c 41.29±2.92d 60.44±2.92a

57.11±1.09c 59.85±2.12c 63.21±1.41b 67.33±2.24a 54.02±0.71d

AC C

EP

TE D

M AN U

SC

RI PT

S. no

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Legends Fig. 1. Fruit of C. colocynthis (A); GC-MS analysis of C. colocynthis fraction F5(B).

RI PT

Fig. 2. Percentage mortality of S. litura after treatment with fractionF5against third, fourth and fifth instar of S. litura. Means (± (SE) standard error) followed by the same letters above bars indicate no significant difference (P≤0.05) according to a Tukey test. Fig. 3. Lethal concentrations (LC50 and LC90) of fraction F5against S. litura.

SC

Fig. 4. Regression equation and correlation between relative consumption rate and relative growth rate of fourth (A) and fifth (B) instar larvae of S. litura fed on leaves containing fraction F5 with different concentration and larvae with different quantities of control leaves.

M AN U

Fig. 5. ACP and ALP activities of fourth (A & C) and fifth (B & D) instar larvae of S. litura after treatment with C. colocynthis. The data were fitted on polynomial (regression) model, whereas vertical bars indicate standard error (±SEM). Fig. 6. ATP and LDH activities of fourth (A & C) and fifth (B & D) instar larvae of S. litura after treatment with C. colocynthis. The data were fitted on polynomial (regression) model, whereas vertical bars indicate standard error (±SEM). Fig. 7. Histological changes of S. litura treated with C. colocynthis. Control (A), Treated (B) ELepithelial layer, BBM- Brush border membrane,GL-Gut lumen, CC- columnar cells.

TE D

Fig. 8. FT-IR spectrum of sub-fraction A3.

Fig. 9. 13CNMR spectrum of stigmasterol (A) 1H NMR spectrum of stigmasterol (B) Structure of stigmasterol (C).

AC C

EP

Fig. 10. Percentage mortality of third instar larvae of S. litura after treatment with stigmasterol Means (± (SE) standard error) followed by the same letters above bars indicate no significant difference (P≤0.05) according to a Tukey test

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AC C

EP

TE D

M AN U

SC

RI PT

Fig. 1.

ACCEPTED MANUSCRIPT

RI PT

Fig. 2.

a

100

a

SC

Control 50 ppm 100 ppm 150 ppm 200 ppm

Mortality (%)

a

b

50 d

c

e

4th instar

EP

Larval instar

AC C

d

e

TE D

3rd instar

b

c

d

e 0

M AN U

c

b

5th instar

ACCEPTED MANUSCRIPT

RI PT

Fig. 3.

0.99

0.95

SC

0.8 0.7

LC = 1.96 χ2 = 24.715 CI: Lower = 1.93 Upper = 1.98 50

M AN U

0.6 0.5 0.4 0.3 0.2

LC = 2.36 χ2 = 24.715 CI: Lower = 2.32 Upper = 2.40 90

0.1

0.01 1.42

TE D

0.05

1.54

1.66

1.78

1.90

EP

Log concentration (ppm)

AC C

Probability of mortality

0.9

2.02

2.14

2.26

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Treated (25,50,75 and 100 ppm) Control

0.8

R e lati ve g r owth r ate (m g /m d/ day)

y = 0.3987 + 32.99x; Regression coefficient (r) = 0.953 0.5

0.3

0.2

0.7

y = 0.0104 + 0.0230x; Regression coefficient (r) = 0.913

0.6

0.5

M AN U

0.4

y = 0.7949 + 30.76x; Regression coefficient (r) = 0.909

0.4

y = 0.429 + 35.09x; Regression coefficient (r) = 0.925

0.3

0.1

0.2

0.0 1.5

2.0

2.5

TE D

Relative consumption rate(mg/mg/day)

EP

A

1.0

AC C

Relative g rowth rate(mg /md/day)

0.6

Treated (25,50,75 and 100 ppm) Control

SC

0.7

RI PT

Fig. 4.

1.2

B

1.4

1.6

1.8

2.0

2.2

Relative consumption rate(mg/mg/day)

2.4

2.6

2.8

ACCEPTED MANUSCRIPT

6.0

2

RI PT

Fig. 5.

2

5.0

4.5

M AN U

4.5

5.0

SC

Probit value of enzyme inhibition

5.5

4.0

3.5

3.5 1.0

1.5

2.0

Log concentration (ppm)

B

EP

TE D

A

AC C

Probit value of enzyme inhibition

5.5

4.0

2

Y = 9.049 - 7.684X + 2.815X R = 0.833

2

Y = 8.176 - 6.238X + 2.321X R = 0.808

C

D

1.0

1.5

Log concentration (ppm)

2.0

ACCEPTED MANUSCRIPT

Fig. 6.

2

Probit value of enzyme inhibition

4.5

4.0

3.5

RI PT

5.0

5.0

4.5

4.0

3.5

3.0

A

1.0

1.5

B

2.0

Log concentration (ppm)

2

Y = 4.2741 - 2.3073X + 1.3012X R = 0.9442

2

SC

2

Y = 4.4973 - 2.4897X + 1.3438X R = 0.9868

M AN U

Probit value of enzyme inhibition

5.5

1.0

1.5

2.0

Log concentration (ppm)

5.5

2

TE D

4.5

C

1.0

EP

4.0

1.5

Log concentration (ppm)

AC C

Probit value of enzyme inhibition

5.0

2

2

Y = 4.965 - 2.677X + 1.376X R = 0.928

Probit value of enzyme inhibition

2

Y = 4.475 - 1.728X + 0.992X R = 0.970

5.0

4.5

4.0

2.0

D

1.0

1.5

Log concentration (ppm)

2.0

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 7.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 8.

ACCEPTED MANUSCRIPT

Fig. 9.

EP

C

AC C

B

TE D

M AN U

SC

RI PT

A

HO

ACCEPTED MANUSCRIPT

100 a

Control 25 ppm 50 ppm 75 ppm 100 ppm

SC

80

40 d

20 e

0

EP

TE D

3rd instar

M AN U

60

AC C

Mortality (%)

b

c

RI PT

Fig. 10.

ACCEPTED MANUSCRIPT

Highlights •

Purified solvent extract of Citrullus colocynthis has shown toxicity against Spodoptera litura larvae. Survival, nutritional indices and gut enzymes of S. litura was negatively affected after

RI PT

•

treatment with C. colocynthis.

The gut histology of S. litura shows disruption in the midgut columnar cells against

EP

TE D

M AN U

SC

treatment with C. colocynthis.

AC C

•