Prospects of Ocimum Gratissimum Linn. (Lamiaceae) to Control Exorista SorbillansWiedemann (Diptera: Tachinidae) Menace of Silkworm in Seriecosystem

Tachinid parasitoids are in focus mostly as biocontrol agents to be released against lepidopteran pest. But surprisingly certain tachinid parasitoids attack economically benecial insects like silkworm and demands control measures against them. The uzi y Exoristasorbillans (Diptera: Tachinidae) infests all commercially important silkworm species including Antheraeaassamensis where it causes upto 80 percent crop loss. The control of such parasitoids is a dicult task as the larval stage is endoparasitic and chemical insecticides do not reach the targeted parasitoid without exposing the silkworm host.In the present study,we evaluated adulticidal activity of different solvent extracts and essential oil of Ocimumgratissimum. We found higher ecacy of essential oil in comparison to other solvent extracts. The effective fraction of oil was found to contain thymol or its isomer carvacrol as the major compound in GC-MS studies. Finally, O. gratissimum oil-based combination (MI) and carvacrol based combination of essential oil compounds (MII) were prepared, tested and found to be effective against the y. In silico positive interaction of essential oil compounds with acetylcholinesterase enzyme model of the y revealed that the said enzyme is one of the target proteins for these oil compounds to interrupt its function and subsequent lethal action.


Introduction
Tachinid ies have been gaining importance globally as promising biological control agent as many of them are natural predator of lepidopteran, coleopteran and some other insect pests. However, literature also reveals report on harmful effects of this group of insects on bene cial insects.The uzi y Exoristasorbillana (Diptera: Tachinidae) is such a tachinid y which is considered as one of the major constraints for the sericulture industry as they can parasitize all the commercial silkworm varieties along with some other lepidopteran caterpillars. The y infestation has been reported from all the silk producing countries of the world 1 . In India the incidence of this y infestation in mullberry silkworm Bombyx mori 2 , tasar silkworm Antheraeamylitta Drury 3 , eri silkworm Samia Cynthia riciniBoisduval 4

and muga silkworm
Antheraeaassama Westwood 5 has been reported at different times and the y menace still prevails. The overall damage caused by the y may reach upto 40%. For outdoor and semidomesticated silkworm crop like Antheraeaassamamensis Helfer the damage even extends upto 80% in seed broods. The parasitoid spends entire larval stages inside the silkworm body and when mature, the maggots come out by making hole in the silkworm cocoon case to form pupae on soil. The infested silkworm dies in late larval stage or pre-pupal or pupal stage. Thus, the crop fails to propagate further. The silk cocoons commercially unreelable as the y cut the cocoon case during emergence causing silk yield loss. To protect silkworm from E. sorbillans infestation, different methods viz. physical, mechanical, chemical and biological control have been suggested from time to time by earlier investigators. Use of chemical insecticides although give immediate and effective results but applications of chemical insecticides are not preferable at seri ecosystem as silkworms themselves are highly susceptible to the chemical insecticides and application of chemicals without exposing the silkworm is not feasible due to endoparasitic nature of the parasitoid. Similarly, microbial agents are also cannot be applied. Therefore, we hypothesize ecofriendly products most particularly herbal products having selective toxicity as one of the rational alternatives to synthetic counterparts for controlling such pests of silkworm.
Ocimumgratissimum L. (Lamiales:Lamiaceae) is a perennial herb with a long history of traditional medicinal uses in countries across the world especially in the Africa and Asia. Extracts and essential oils of this plant have been reported to possess medicinal, bactericidal, fungicidal, nematicidal activities.
Besides, the plant essential oil has been used safely in food, avor and fragrance industry. It is a potential candidate of pharmaceutical industry with report of low mammalian toxicity. The plant has been already exploited for the control and management of many insect pests and parasitoids belonging to order Coleoptera, Lepidoptera, Isoptera, Diptera, Thysanoptera, Heteroptera etc 6,7,8,9,10,11,12,13 but not found to be tested against uzi y. Earlier we reported about the e cacy of ethanolic extract of this plant species 14 .
We have also observed lesser toxicity of Ocimum species against silkworm as compared toExoristasorbillans 15,16 .Therefore in the present study an attempt was undertaken to assess the adulticidal potentiality of different solvent extracts and essential oil of Ocimumgratissimum and its combination with other plant essential oil and constituent compounds against E. sorbillans so as to incorporate this plant product in uzi y control program in future.

Preparation of crude plant extracts
Fresh leaves of Ocimum gratissimum were washed, shade dried and ground to powder using electric grinder and preserved in refrigerator at 4 0 C for future use until extraction. Crude extract of leaf powder was prepared in absolute ethanol solvent. 150g of ground leaves were dissolved in 350ml of the solvent and kept for 72h. Extracts were ltered by using Whatman Filter Paper No 1 after 72 h and the solvents were removed under vacuum below 40 0 C.

Fractionation of ethanol extract
Ethanol extracts of the leaves of Ocimum gratissimum was further fractionated by using a series of solvents (Merck) on the basis of polarity namely petroleum ether, chloroform, butanol and water. Initially ethanol extract was prepared by dissolving ground leaf powder in absolute alcohol for 72h. The extract was then ltered and solvent was removed. The dried ethanolic extract was then weighed and taken in a separating funnel (2.5 liter) and petroleum ether was added and shaked for 30 minutes. It was then allowed to settle for 3h and the supernatant was decanted and ltered. The residue was extracted thrice adding same solvent and the ltrate was taken as petroleum ether extract. Sequentially the chloroform, butanol and water were added and each solvent extract was prepared similarly as petroleum ether extract and stored in refrigerator for bioassay.

Collection and preparation of plant essential oils
Fresh leaves of the plant were collected, washed to remove dust, cut into small pieces and subjected for oil extraction by hydrodistillation method using Clevenger type apparatus 47 . Two hundred grams (200g) of fresh leaves of the selected plantwas put in 5 L round bottom ask per extraction of oil. The essential oil oating above water layer was collected after 4 hours of heating. Anhydrous sodium sulfate was added in the collecting vial to absorb traces of water. The oil was then stored in sealed screw cap vial in deep freezer (-20 0 C) for bioassay and analysis.
The percentage (v/w) of oil yield was calculated by using the following formula: Bioassay with fractionated ethanol extract against E. sorbillans Five percent concentration of petroleum ether extract, chloroform extract, butanol and water extracts derived from fractionation of ethanolic extracts were bioassayed against third day old adult uzi y using contact residual lm technique. 1.5ml of 5% concentration of each solution was applied into Whatman (110 mm) lter paper which was pasted in a petri dish and was allowed to dry in room temperature and then ies were released. The treated petri plates were covered by 110mm diameter glass funnel. The stem at terminal end of the funnel was covered with nylon net and thus aeration was facilitated to insects during treatment hours. For each experiment control was maintained where only the respective solvent was applied. Three replications were kept for each treatment and control set. Observation was taken at 1, 3, 6, 24, 30, 48 hours from the time of application. Abbot's correction factor 48 was applied in case of mortality of y in control if occurred.
Bioassay with effective solvent extract against E. sorbillans: The fractionated ethanolic extract which gave more than 50% death of E. sorbillans after 24h of treatment at 5% concentration was considered for further bioassay to get sub lethal concentration (LC50). For the purpose, different concentrations (0.01 to 15% at geometrically uniform interval) of the effective extract was prepared and subsequent bioassay against third day old adult uzi y was carried out by contact residual lm technique to determine LC50 value of the most effective solvent extract. Probit analysis was done for calculating LC50 using SPSS software.
Bioassay with essential oil of O.gratissimum Bioassay by using essential oil of Ocimum gratissimum against E. sorbillans was carried out by contact residual lm technique as described above. Essential oil solutions of O. gratissimum were prepared in acetone solvent. For control, only acetone solvent at an amount of 1.5ml was applied to Whatman No.1 Filter paper against each test concentration. Third day old adult uzi ies were subjected to treatments.
Three replications, each containing 10 insects were maintained for each treatment. Different concentrations of oil (0.01 to 0.5%) were prepared for determining LC50 value of essential oil of O. gratissimum. Lethal time for the essential oil against E. sorbillans was calculated by applying 1µl of crude essential oil topically, on thorax of the third day old uzi ies with the help of micropipette. Bioassay of each individual fraction obtained from TLC was carried out by contact residual lm technique using acetone (Merck) as solvent. Further GC-MS analysis of the best active fraction of oil was carried out to identify the compounds present in the active fraction of the oil.

Preparation of O.gratissimum essential oil based combination and bioassay against E.sorbillans:
Based on essential oil of O. gratissimum, initially six combinations were prepared and tested on 3 rd day old adult E. sorbillans using fumigant mode of application. Essential oil of some other locally available plants namely Ocimum sanctum (Lamiaceae), Eucalyptus maculata(Myrtaceae), Callistemon linearis(Myrtaceae), Citrus sinensis (Rutaceae) were considered to add as ingredient of combination on the basis of their e cacy obtained in our pilot studies. Topical application of each candidate essential oil at a dose 1µl per insect on thoracic region was done on third day old adult y and lethal time was recorded. The prepared combinations for fumigant application were 5:0G, 4:1GOs, 4:1GEm, 4:1GCl, 4:1GCs and M. Synthetic gum (GripFix Adhesive Paste) was used as control release agent. The and wrapped by muslin cloth from upper side and placed in conical ask of 500ml capacity. The muslin cloth prevented the ies to come in direct contact of the combination but allowed the vapor to come out and spread uniformly in the enclosed chamber (500ml volume). After keeping the formulation containing cap in the conical ask, 10 numbers of ies were released in each replication and the ask was sealed with the help of aluminium sheet and tightened by rubber band to keep the air blocked inside the ask. Response of the ies in terms of knock down and mortality was recorded from 1minute to 24hours at successive time interval.
Preparation of combination from essential oil compounds and bioassay against E.sorbillans: Based on the e ciencies of combinations prepared from crude essential oil, further combinations were prepared by selecting essential oil compounds which have been reported and identi ed as major constituent compound of the concerned plant essential oils. These were Carvacrol, Thymol, Eugenol, Eucalyptol and Citral. Carvacrol and Thymol are the constituent compounds of O.gratissimum, Eugenol is the constituent compound of O.sanctum, eucalyptol is the constituent compound of both E. maculata, and C.linearis, citral is the constituent compound of C. sinensis. Two combinations were prepared taking these compounds. Fumigant mode of application was used for bioassay studies. Synthetic gum was used as control release agent and plastic cap was used into which the prepared combination was poured Combination marked as MII was prepared by mixing equal amount of Carvacrol (1ul), Thymol(1ul), Eugenol(1ul), Eucalyptol(1ul) and Citral (1ul) into a total of 5ml synthetic gum. Three replications were set for each combination. Another formulation C1 was prepared by mixing 5ul of carvacrol into 5ml of synthetic gum and the prepared formulation was kept in conical ask having500ml capacity. Flies were then subjected to get exposure of the formulation and mortality data were recorded against time interval.
Docking performance of major essential oil compounds with acetylcholinesterase enzyme: Ligand Preparation: The 2 D structure of three ligands were drawn using pubchem search tool in NCBI and converted in to 3D structure with the help of Chimera software and subsequently saved in pdb format.

Receptor Protein Preparation:
Acetylcholinesterase enzyme of Exorista sorbillans was selected as targeted protein for the ligand. The protein sequence (ACHE) was downloaded in FASTA format from protein database in NCBI and pasted in notepad. Protein BLAST was performed and based on blast result four pdb ids were selected as template protein (1dx4; 1qo9;5ydi;6arx;6ary) to construct model of ACHE protein of E. sorbillans. The selected template protein structures were downloaded from the PDB site (http://www.rcsb.org). For preparation of ACHE protein model, the Modeller 9.21 was used following the steps of basic modelling using the commands like build_pro le.py, compare.py, align2d.py, evaluate_model.py. the generated models were uploaded in Procheck online service and Ramachandran plot for each model was obtained. Considering maximum percent value of amino acid residues present in most favored region and minimum in disallowed region, the most suitable model was selected for further docking with the selected ligands.

Docking:
Docking of the best model of ACHE protein was performed with essential oil compounds namely carvacrol, thymol, eugenol, eucalyptol, citral and the substrate acetylcholine in Python Molecular Viewer (PMV1.5.6) using AUTODOCK VINA 49 in MGL tools. Best nine mode of interactions were generated for each docking with respective a nity values and rmsd values. The best mode for each docking having least rmsd value and high binding a nity were considered for analyzing the mode of binding interactions. Pictorial representation of binding interactions was analyzed in PMV and ligplus software.

Results
In the present study the ethanol extract of the plant was initially prepared and further fractionated by eleutropic series of solvents, namely petroleum ether, chloroform, butanol and water to identify the active solvent fraction against adult E. sorbillans.The result of the four successive solvent extracts showed that the petroleum ether extract of the plant had greater toxicity in comparison to the other three solvent fractions at 5% concentration (Fig. 1) Supplementary Fig. 1).
Percentage of essential oil yield from fresh leaves of O.gratissimum was recorded as 0.63 ± 0.09 percent. A series of concentrations (0.01-0.5%) of the plant essential oil were applied to E. sorbillans by residual lm technique in order to determine LC 50 value which was calculated as 0.42% (Y = 6.07 + 2.81X; R.Sq = 0.941) ( Table 1, Supplementary Fig. 2).   Fumigant toxicity of six formulation which were prepared based on essential oil of O. gratissimum and its combination with some other plant essential oil showed that the combination MI comprising equal amount of essential oil of Ocimumgratissimum, Ocimum sanctum, Eucalyptus maculata, Callistemon linearis, Citrussinensis showed best result with sixty percent mortality at 24 h exposure period against 0-20 percent mortality caused by the rest of the prepared formulations (Table 3).  In our previous experiment we found higher toxicity of carvacrol than thymol 17 . Therefore, carvacrol (one of the major constituents of effective fraction of O. gratissimum oil) based formulations were prepared with synthetic gum and with other terpene compounds reported to present as dominant part in essential oil of Ocimum sanctum, Eucalyptus maculata, Callistemon linearis, Citrussinensis. The result showed that the formulation MII consisting of carvacrol, citral, eugenol, eucalyptol showed higher mortality with 60% mortality of ies after 6 h of treatment and 100% mortality after 24 h of treatment at 500 ml of air volume in comparison to the formulation CI comprising of the carvacrol alone with maximum 30 percent mortality after 24 h of exposure (Fig. 6). The formulation of essential oil compounds (MII) was more effective than the formulation of the mixture of the crude essential oils (MI) (Fig. 6).
For studying the mode of action, using ve template proteins (1dx4, 1qo9, 5ydi, 6arx, 6ary), a model of acetylcholinesterase enzyme of E. sorbillans having total amino acid residues 701 was constructed. The Ramachandran plot showed 90.5% amino acid residues of the model in most favored regions and with 0.3% residues in disallowed regions ( Supplementary Fig. 3).While docking was performed using autodock software taking ve essential oil compounds and the enzyme substrate acetylcholine as ligands with the modelled ACHE protein (Fig. 7), all the ligands were found to dock successfully to the protein molecule. Carvacrol with − 6.9 kcal/mol binding a nity showed ten hydrophobic interactions with amino acid residues Gly 569,  (Fig. 7, Table 4).

Discussion
The solvent like ethanol can easily penetrate the cellular membrane to extract the intracellular ingredients from the plant material 18 .In our earlier studies we found that the ethanolic extract ofO.gratissimum caused highest percent mortality over hot water,hydroalcoholand cold water extract 14 . It is also reported that ethanol is a potent solvent to extract both polar and nonpolar compounds 19 . Therefore, in the present investigation a stock of ethanolic extract was initially prepared to extract both polar and nonpolar compounds and further fractionated using eleutropic series of solvents to nd out active solvent extract. The results suggested that the petroleum ether extract of the plant was the most effective among the four solvent extract tested indicating that the solvent extract might contain potential active ingredients against E.sorbillans adult ies.E cacy of nonpolar extract of plants including petroleum ether extract of O.
gratissimum and some other plants on dipteran insects was earlier reported by several workers 20,21,22 . According to the principle of "like dissolves like", solvents would only extract those compounds which have similar polarity with that of the solvents 23,24,25,26 . Thus, the non-polar petroleum ether extract of the experimental plant under investigation extracted nonpolar compounds possibly phenylpropanoid, fatty acids, terpenes etc. 19,27,28 which might be responsible for giving the e cacy of the solvent extract against E. sorbillans.
As the O. gratissimum plant is aromatic and contains good quantity of essential oil which has been reported effective against different insect pest including dipteran ies by many earlier investigators and as the nonpolar fraction of the ethanolic extract i.e. the petroleum ether fraction was found effective against uzi y, we further focused on essential oil part of the plant for bioassay against E.sorbillans. Supporting our prediction, the result showed higher e cacy of O. gratissimum essential oil than the petroleum ether extract (Table1) indicating nonpolar compounds as the responsible factors for the lethal toxicity of the plant against the y. Although a considerable number of studies of earlier investigators highlighted effectiveness of this plant essential oil against many dipteran ies but the insecticidal potentiality of the plant oil was not found to be reported against E.sorbillans. We recorded 0.  30 . In response to essential oil of O.gratissimum, the adult ies exhibited fast walking behaviour followed by sudden knock down. At higher dosage the knocked down insects could not recover and death ensued which might imply interference of the toxicant with the insect's nervous system. Activity of the oil maybe attributed to action of a single major compound or synergistic actions of group of compounds 31 .In order to know the nature of the functional groups present in the constituent compound(s) of the petroleum ether extract and essential oil of O. gratissimum, FTIR spectra analysis was done. The spectral peak revealed that the compounds present in the petroleum ether extract and the essential oil part of the plant comprised almost similar functional groups (Fig2; Table2).
In the preparative thin layer chromatography, two fractions (F1, F2) were separated from the essential oil of O.gratissimum at solvent system comprising of petroleum ether and ethyl acetate (5:1). Effective fraction was determined by conducting bioassay of each fraction against 3 rd day old uzi y by using contact residual lm method. F2 fraction of O.gratissimum was found effective against uzi y.When the bioassay guided active TLC fraction (F2) of essential oil was subjected to GC/GC-MS analysis three peaks were detected at retention time 10.3 and 16.2 and 21.7 m (Fig.3, Fig. 4). The probable constituent compounds were identi ed as (i). thymol or phenol,2-methyl-5- Phenol 2-methyl-5-(1-methylethyl)-also known as Carvacrol is an isomer of thymol. Both thymol and carvacrol have antifungal and insecticidal potency 35,36,37 . We have reported potential activity of pure carvacrol compound (98-99% purity) against adult uzi y with LC 50 value of 194.5 ppm in contact residual mode of application 17 . The presence of carvacrol in the active oil fraction of the plant once again con rms its potential toxic affects against the y. However biological interactions of terpene compounds with other compounds in mixture could not be predicted as some of the associations may show synergism, some other association show antagonism and sometimes with no apparent differences in total toxic effects.
While six O. gratissimum essential oil-based combinations were prepared and tested for assessing fumigant toxicity then the composition M comprising equal parts of Ocimumgratissimum, Ocimum sanctum, Eucalyptus maculata, Calistemonlinearis, Citrus sinensis were found as the most effective one than the other combinations and O. gratiissimum oil alone. This might be due to the synergistic effects of the constituents present in the essential oils of the selected plants. The nding is in conformity with the nding of Alves et al. 38 who found mixture of essential oil of Cymbopogon citratus, Cedrusatlantica and Corymbiacitriodora as potential adulticide against Ceratitiscapitata (Diptera: Tephritidae). We have also recorded the combinations of essential oil of Allium sativum and Ocimum sanctum as potential larvicide against Culex quinquefasciatus 39 . In most of the earlier studies it is established that toxicity of a plant essential oil is mostly due to the activity of major constituent compounds. In the active fraction of O. gratissimum essential oil, the major compound detected was thymol or its isomer carvacrol. In our previous experiment as we found higher toxicity of carvacrol as compared to thymol against uzi y 17 , therefore, carvacrol based formulations were prepared taking some other major constituents namely eugenol, eucalyptol, citral reported to be present in essential oil of Ocimum sanctum, Eucalyptus maculata, Callistemon linearisand Citrussinensis. The result showed that the formulation MII comprising of carvacrol, citral, eugenol, eucalyptol showed higher mortality (60% mortality after 6h of treatment and 100% mortality after 24h of treatment) of ies at 500 ml of air volume in comparison to the formulation CI containing carvacrol alone with maximum 30 percent mortality after 24h of exposure in fumigant mode of application. The formulation of essential oil compounds (MII) was more effective than the formulation of the mixture of the crude essential oils (MI) (Fig6). Similar ndings were disclosed by Dean et al 40 where they described e cacy of aqueous formulation of nootkatone (5%) and carvacrol (5%) against Ixodes scapularis Say and Amblyommaamericanum L. (Acari: Ixodidae). The insecticidal e cacy of the combinations of major essential oil compounds such as 1,8-cineole plus camphor, thymol plus ρcymene and citral plus limonene were reported against Trichoplusiani 41 . We have also reported some of the combinations of major essential oil compounds viz. eucalyptol plus eudesmol and carvone plus limonene as effective combinations than the individual compounds against adult Aedes aegypti 42 .But no such reports of formulation or combination of plant products are found to be reported in literature against the silkworm parasitoid E. sorbillans. Therefore, the present nding can be very well explored further for developing commercial formulation to be used for the management of E. sorbillans in sericulture eld.
We also studied the target site of action of the primary essential oil constituents ofO. gratissimum and the others used in development of formulation. Findings of the previous researchers revealed that one of the primary targets of essential oils of plants and their terpene compounds as well as organophosphate and carbamate pesticides in insects are the acetylcholinesterase enzyme of nervous system 43 .The compounds inhibit AChE activity which subsequently lead to accumulation of the neurotransmitter acetylcholine at the neuronal synapse resulting in continuous stimulation and lack of coordination in the neuromuscular system and nal death of the treated insect. While studying the a nities of the selected terpene compounds used in preparation of combination to be treated against uzi y, we found positive interactions of all the selected compounds with the targeted enzyme. The highest a nity was recorded for the compound carvacrol (a nity -6.9 Kcal/mol) followed by eucalyptol (-6.1 kcal/mol), thymol (-5.1 kcal/mol), eugenol (-4.8 kcal/mol), citral (-4.1 kcal/mol). The lowest a nity was found for the enzyme substrate acetylcholine (-3.9 kcal/mol) among the all tested chemical compounds. The higher a nities of selected terpene compounds than the acetylcholine is a good indication that the compounds could effectively inhibit the enzyme. The result is consistent with our earlier ndings of in silico interactions of terpene compounds with acetylcholinesterase enzyme of Aedes aegypti where we recorded higher binding a nities of eugenol, eucalyptol and carvacrol along with three other terpene compounds than acetylcholine 44 . Among the compounds thymol and acetylcholine showed both hydrogen and hydrophobic interactions while other compounds showed hydrophobic interactions with amino acid residues of the target protein. Recently Hussain et al 45 reported positive binding interactions of twentyeight essential oil constituents of the medicinal plant Commiphoramyrrha which included eugenol, βeudesmol, caryophyllene, α-pinene, α-humulene etc. with more than -5 kcal/mol binding a nity with acetylcholinesterase enzyme. In another study Wang et al 46 showed invitro acetylcholinesterase activity of honokiol and nine structurally related phenolic compounds against larvae of Aedes aegypti. From this nding it can be inferred that one of the reasons of the insecticidal activity of the combination might be due to the inhibitory effect of the constituents on AChE enzyme. However further study on extraction and isolation of the enzyme and activity test will be needed to con rm the same.