M.Sc. Molecular biology and biotechnology(2012-2014)

Project work: Study of the induction level of Cytochrome p450 enzymes of Helopeltis theivora (a tea mosquito bug) upon insecticide susceptibility test using Synthetic and botanical insecticide.

Date: 3/6/2014

Submitted to:Dr. Tapas Medhi Assistant professor, MBBT, Tezpur University.

Submitted by:Kabita SharmaMBM 12016,M.Sc.(4th sem.), MBBT,2014,Tezpur University.

By: Kabita Sharma M.Sc. (4th SEM.), Roll no. : MBM 12016 Molecular biology and Biotechnology, Tezpur University, Napam, Assam (784028).

CERTIFICATE

Certified that the projectStudy of the induction level of Cytochrome p450 enzymes ofHelopeltis theivora (a tea mosquito bug) upon insecticide susceptibility test using synthetic and botanical insecticide is a bona fide work undertaken by Ms. Kabita Sharma, Roll No.: MBM12016, M.Sc. (4th sem.), in the partial fulfillment for the award of degree of M.Sc. in Molecular biology and Biotechnology, Department of MBBT, Tezpur University.

The project abides by the norms and criteria as laid down by the UGC-DBT for the said degree.

Signature (S. K. Ray) HOD, MBBT, Tezpur University, Napam, Assam (784028)

Dated- Place-

CERTIFICATE

This is to certify that Ms Kabita Sharma bearing Roll No. MBM12016, has completed her project work entitled Study of the induction level of Cytochrome p450 enzymes of Helopeltis theivora (a tea mosquito bug) upon insecticide susceptibility test using Synthetic and botanical insecticide for partial fulfillment for the award of degree of M.Sc. (Molecular biology and Biotechnology), in Tezpur University affiliated to UGC (DBT), Tezpur, Napam, Assam for the academic year of 2012-14.

Signature (Tapas Medhi ) Assistant professor, MBBT, T.U., Project guide Date- Place-

ACKNOWLEDGEMENT:

I want to convey my deep regards and gratitude to all the people who have helped me to complete my project work titled Study of the induction level of Cytochrome p450 enzymes of Helopeltis theivora (a tea mosquito bug) upon insecticide susceptibility test using Synthetic and botanical insecticide. This project would not have been success without them.

I thank Dr. S. K. Ray, Head of the Molecular Biology & Biotechnology Department , Tezpur University, Napam, Assam for his support and guidance.My heartfelt gratitude to preceding HOD, Dr. Shashi Baruah, for her precious guidance and support. My project guide Dr Tapas Medhi for being patient to teach me each and every step so perfectly and for his continuous and untiring support at each and every step of my work.

I would like to pay my gratitude to our Vice chancellor Dr. M.K. Chaudhary for immense support and encouragement and concern for the DBT funded project-work that has been introduced in our course.

And lastly, l would like to pay my gratitude to my laboratory mates for their support and the laboratory attendants for providing me with the required chemicals, equipment and glass-wares for the project. Special thanks go to all my friends and the professors who were always there for me at the time of need. Last but not the least, I believe that with blessings of my parents and with grace of God, I have been able to complete my work.

Kabita Sharma M.Sc. (4th SEM.), Roll no. : MBM 12016 Molecular biology and Biotechnology, Tezpur University, Napam, Assam (784028). Date: Place:

CONTENTS:

S. No. TitlesPage no.

1.Abstract, Introduction6

2.Introduction7

3.Review of literature8-16

4.Materials and methods17-28

5.Results29-35

6.Discussion36

ABSTRACT:

Use of synthetic pesticide Quinalphos against sap sucking tea (Camillia sinensis) pest Helopeltis theivora may cause residual toxicity and other related environmental problems. Persistent use of the same may even lead to development of resistance towards these insecticides. The development of resistance is reported to be associated with the enzymes like Cytochrome P450 mono-oxygenase, Glutathione-S-Transferases and General Esterases. In this study an in vitro trial was made to assess the pest response to increasing doses of insecticide application and subsequent induction of detoxifying enzymes Cyotchromep450, Glutathione-S-Transferases and General Esterases. Further insecticide susceptibility test was performed to determine the lethal dose of Synthetic insecticide (Quinalphos) and Natural insecticide (Duranta erecta methanol extract) along with insecticide residue analysis by GC-MS. The result shows: hot methanol extract (100%-260g/ml or 260ppm) was highly effective as botanical insecticide as compared to cold extract (100%-240ppm). Whereas for synthetic- Quinalphos, it was effective as an insecticide even at lower doses like 0.35ppm (7%), 0.45ppm (9%) along with the recommended lethal dose of 5ppm (100%) and showed mortality in less than 24 hours of incubation. Results of enzyme activity assay for the three detoxifying enzymes showed the induced level of the enzymes in live insect in comparison to the mortal once for each concentration of both the insecticides that were tested to check the insecticide susceptibility. Molecular docking shows interaction of flavonoids with the detoxifying enzymes.

INTRODUCTION:

Tea mosquito bug (Helopeltis theivora) a common sap sucking insect of tea(Camillia sinensis) plantation feeds on the young leaves and causes a great economic loss by reducing the production to a considerable extent to circumvent this problem planters have been spraying different concentration of synthetic insecticides which are effective but at the same time toxic not only to the environment but also to the humans. Apart from this due to excessive use of these insecticides these pests are developing resistance towards these insecticides.

The biochemical reasons for such resistance implicate induced expression of the detoxifying enzymes like Cytochrome P450 mono-oxygenase, Glutathione-S-Transferases and General Esterases. These enzymes are reported to be responsible for the detoxification of the xenobiotic ingested by the insect.

The critical dose for lethality and dose dependent induction of these enzymes are important aspects of sustainable pest management. The study may even help in understanding the extent and frequency of previous exposure to insecticide on the insect population in various agro climatic zones of North East. To address this problem and to find out the lethal concentration of the insecticides that need to be used such that it is not left in the leaves as residue but at the same time effective in killing and controlling the pest population the following objective were formulated to carry out in our laboratory

Objectives:

1. To check the insecticide susceptibility of Helopeltis theivora towards different concentrations of synthetic as well as botanical insecticide and hence find the lethal dose.2. To find out if botanical insecticide could be effective enough as an insecticide to be used against synthetic insecticides such as quinalphos, thus a more eco-friendly approach to check the bugs population.3. To find out to what extent the detoxifying enzymes get induced upon the insecticide susceptibility test through enzymatic assay.4. To perform molecular docking to find out the probable compounds from the plant extract that is interacting with the detoxifying enzymes and leading to their inactivation and hence mortality of the pest. CHAPTER 1REVIEW OF LITERATURE1.1 Introduction:The tea mosquito bug Helopeltis theivora (Waterhouse) (Heteroptera: Miridae) is one of the major sucking pests of tea throughout India. More than 80% of tea cultivation area is being affected by nymphs and adults of H. theivora , leading to dark brown shrunken spots in young foliage, no shoot formation, delayed flushing, stunted growth and die back of stems (Rahman et al. 2005 and 2007; Roy et al. 2008 and 2009). There exist many reports about H. theivora causing significant losses (10-50%) in tea cultivations, when mainly broad-spectrum pesticides-which eliminated natural enemy populations-were used (e. g. Gurusubramanian et al. 2009; Hazarika et al. 2009).A major problem in the control of H. theivora is its capability to develop resistance quickly to frequently used insecticides (Gurusubramanian and Bora 2008). Their high reproductive potential and numerous annual generations, combined with continued and repeated use of a variety of active substances of insecticides on tea cultivations for many years (7.5 L h-1), have limited the control of H. theivora populations by natural enemies and led to resurgences in bug populations with development of resistance and consequently control failure (Roy et al. 2009). Such failures are already known in case of organo-chlorine (OC), organo-phosphorus (OP) and synthetic pyrethroid (SP) insecticides and more recently for the newer compound such as neo-nicotinoids (NN) (e.g. Gurusubramanian and Bora 2007; Gurusubramanian et al. 2008; Roy et al. 2008).OC, OP and SP compounds are highly effective broad-spectrum insecticides. Insecticides like endosulfan, monocrotophos, quinalphos, deltamethrin and cypermethrin are being extensively used in many tea cultivating regions of the country, Instead of practicing integrated pest management in tea cultivations, producers prefer to use broad-spectrum insecticides at no threshold level, which are supposed to be effective against pest insects such as caterpillar pests, thrips, jassids, scale insects and aphids. Thus, H. theivora populations are frequently exposed to above mentioned broad-spectrum insecticides. The problem is more acute as it has rapid multiplication capacity and polyphagous in nature (Roy et al., 2009). It has attained the national importance in India and was estimated that 80% of the tea plantations area is affected resulting in crop loss to the tune of 10 - 50% (Bora and Gurusubramanian, 2007; Roy et al., 2009)In recent years, control failures have led the growers to question the efficacy of registered insecticides. Although there are bioassay results about the decreasing efficacy of some insecticides against H. theivora in tea cultivations of the eastern states(West Bengal, Assam, parts of Meghalaya among few others), the exact situation in respect to OC, OP SP and NN resistances in H. theivora is unknown (Roy et al. 2008). The present study focused on resistance of H. theivora populations collected from tea cultivations of Jorhat Assam to synthetic insecticide Quinalphos and Natural insecticide ( Duranta erecta) and hence check the dosage dependent induced expression of the detoxifying enzyme profile and hence determining their increased enzymatic levels through enzymatic activity assay.

Among the tea growing areas of North East India, the Jorhat tea plantations of Assam (Jorhat City is located at 26.75N 94.22E. It has an average elevation of 116 meters (381 feet). It had suffered badly by this pest attack and consumed highest quantity of insecticides.Climate-Semi-Arid, precipitation 500 mm (20 in), avg. annual temperature 26 C (79 F).Summer temperature (25 - 35 C)Winter temperature (22 - 10 C)With this point in the background, a study was made with three objectives as to determine susceptibility change in the field collected populations of H. theivora from Jorhat, Assam, check the level of induction of the detoxifying enzymes upon exposure to different concentrations of insecticides tested and find out the probable compounds of the plant extract that interact with these enzymes and hence render them inactive.

1.2 Use of synthetic insecticide and the problem of development of resistance:

Season of occurrence of Helopeltis theivora infestation (May-November) and monsoon coincides in the Jorhat condition. Use of insecticide has been the main controlling measure against this pest. All Tocklai (Tea Research Association, Jorhat, Assam) released tea clones, tea garden released clones and seed jats are found to be susceptible to H. theivora attack at varying degrees (Rahman et al., 2007; Roy et al., 2009).In spite of regular application of insecticides, H. theivora has turn into a menace all around the year. Decrease in the susceptibility to different classes of insecticides may be one of the causes for their resurgence and persistence on tea crop. Added to this, in recent days the situation further worsened as it developed resistance to commonly used insecticides. The resistance developed by H. theivora populations in North East India ranged from 1.47- 62.99 folds for males and 1.25 - 62.82 folds for females to different classes of insecticides (Gurusubramanian and Bora, 2008).Though the distribution of this pest is ubiquitous in all the North East tea plantations, yet no work has been undertaken to assess the level of resistance in this pest to the most commonly used insecticides for the Tezpur tea plantations, inspite of the fact that at many occasions in the past, planters have approached with information of insecticidal control failure against this pest even after spraying repeatedly at much higher dosage of endosulfan than recommended for its control and frequency of sprays, but this irrational use of this insecticide only speeded up the development of insecticide resistance in H. theivora. A need was, therefore, realized to quantify the potential of development of resistance to endosulfan of this pest.For over two decades, synthetic pyrethroid insecticides have been widely used by planters because of their efficacy in controlling a wide range of tea pests at low doses and at extremely low cost (Gurusubramanian et al., 2008). The use pattern of insecticides in tea was 5.21-7.49 l/ha, of which synthetic pyrethroids represent 36.6% (Sannigrahi and Talukdar, 2003; Roy et al., 2008), deltamethrin and cypermethrin being the most popular. In the regions surveyed, an average of 7-12 times of insecticide applications to tea are common (Roy et al., 2008). Of the synthetic pyrethroids, deltamethrin is the most widely used insecticide.

1.3 Enzymatic studies:In addition, qualitative and quantitative changes could be recorded in the enzyme patterns of insecticide-exposed H. theivora, due to formation of greater amounts of esterases (Sarker and Mukhopadhyay, 2003), glutathione S-transferases and acetylcholine-esterase (Sarker and Mukhopadhyay, 2006a, b), indicating an adaptation for higher insecticide tolerance. Though the distribution of this pest is ubiquitous in all tea growing regions of India, no work has been undertaken to assess the cumulative resistance acquired by the pest through generations against Quinalphos pesticide, resistance to insecticides has resulted in changes in the biological characteristics of the strains of different species.Most resistance management tactics involve the reduction of fitness of resistant genotypes relative to susceptible genotypes by either preserving susceptible homozygotes or eliminating heterozygotes and resistance homozygotes.The mitochondrial cytochrome oxidase-I (COX-1. Exhibits maternal inherited characteristics and reliable interspecific variation as compared to other markers (Savolainen et al. 2005), and therefore this same marker has been employed in molecular systematics of insects (Simon et al. 1994).2. The concept of DNA barcoding of species using mitochondrial cytochrome oxidase I gene was first reported by Hebert et al. (2003). 3. Apart from this, various molecular markers have been employed for species identification and molecular phylogeny studies of insects, viz., Cytochrome b, 16S rRNA (von Dohlen & Moran 2000), 18S rRNA, 28S rRNA, 5.8S rRNA, ITS-Internal Transcribed Spacers, and EF1 (Elongation Factor) (Ji et al. 2003). 4. Yet another method of resolving identification of closely related species is by developing species-specific primers producing specific amplicons.The development of quantitative techniques for measuring enzyme activity in individual species of Helopeltis theivora thus has become increasingly important.1. First measurement of enzyme activity in individuals collected from populations distributed throughout the range of a species can lead to understanding of the genetic and environmental factors that influence enzyme expression.These experiments can give information on the role of natural selection in determining the allele frequency. 2. Understanding the more applied but analogous involved in insecticide resistance management depends on the ability to measure resistant as well as susceptible allele frequency, in order to guide decisions on pesticides use and evaluate the effects of strategies designed to avoid delay the development of resistance.3. Measurement of resistance in the field population I clearly the method of choice for understanding the long term and short term effects of insecticide use on insect population.

Thus enzyme assays are the best of the techniques that not only quantifies in a fast and sensitive way by giving flexibility to replicate measurement of several different enzymes using single individual. This method gives the complete analysis of an individual insect's ability to metabolize a given class of insecticides. Synthetic Quinalphos are known to excel other insecticides in Helopeltis theivora abatement programs primarily due to their outstanding insecticidal potency coupled with marvelous ecological compatibility. Earlier studies have elucidated a preeminent role of mono-oxygenase -mediated degradation of deltamethrin in conferring deltamethrin resistance in the larvae of Aedes aegypti, Culex quinquefasciatus and Anopheles Stephensi (Kumar et al 1991). Since the role of esterases in pyrethroid-resistance in mosquitoes has not been established unlike in other insects (Jao and Casida 1974; Riskallah 1983) the possibility of ester hydrolysis as a mechanism of permethrin- and deltamethrin-resistance in strains of Aedes aegypti , Culex quinquefasciatus and An. stephensi has now been investigated. This was accomplished by estimating levels of non-specific -esterases in single larva/adult mosquitoes using the microplate assay method of Brogdon and Dickinson (1983) with certain modifications.Apart from the problem of development of resistance to various types and concentrations of synthetic insecticides that are being used to control the H. theivora menace are the potent health hazard to humans as subsequent amount of residue is left out in the leaves even after processing of the tea leaves and also pose great threat to the environment.One of the approaches to solve this problem would be to use some natural source which is insecticidal but at the same time environment friendly and less hazardous to humans.Detoxification of insecticides is an important mechanism for insect pests to tolerate regularly applied insecticides (Brown and Brogdon, 1987; Soderlund and Bloomquist, 1990; Yu, 1996; Scharf et al., 1998; Yu et al., 2003; Nehare et al., 2010;Changes in susceptibility level are mainly due to metabolic detoxification of insecticides through a higher level of activity of some detoxifying enzymes under the stress of different management practices.Generally, three principal enzymes (General Esterases (GEs), Glutathione S-Transferases (GSTs)and cytochrome P450-mediated mono-oxygenases (CYPs) are involved in the process of metabolic detoxification of insecticides (Soderlund and Bloomquist, 1990). Estimation of the activities of these metabolic defense-related detoxifying enzymes may give information on the degree of tolerance/resistance of the insect pest population to insecticides and may be useful tools in monitoring the tolerance/resistance levels to insecticides in the pest population. Early detection of pest specimens representing metabolic threats related to tolerance/ resistance levels to insecticides is important for devising control measures that can minimize the risk of subsequent build-up of the pest population and reduce environmental toxicity and wastage of insecticides.Literature also provides evidence of the extensive interaction of insect cytochrome p450 enzymes with plant flavonoids like rutin, quarcetin etc., which is thought to further enhance the detoxifying ability the enzymes.Hence it would be important to know which plant secondary metabolite of the botanical insecticide will be actually insecticidal and at what concentration.

1.4 Herbal pest control measures:Literature provides evidence of various medicinal plants and herbs having insecticidal property and hence could be used as a natural insecticide. Duranta erecta Linn. (Syn. Duranta plumieri, D. repens Linn. and Eng.: Golden dewdrop) is commonly known as pigeon berry and locally called Kata mehedi belongs to the family Verbenaceae. It is shrubs, herbs or small tree usually 1 to 3 m. in height 5. The plant is not browsed by cattle and is believed to be poisonous. The fruits are used in the treatment of malaria and intestinal worms. The leaves are used in the treatment of abscess. From the genus Duranta several iridoid glycosides as durantosides I, II, III, IV, and lamiide were isolated. Flavonoids and C-alkylated flavonoids and some alkaloids 14 were isolated. Phytochemical screening of plant and evaluation of the in vitro antifungal activity of the crude methanolic extracts (Leaf, stem and root), of Duranta erecta against some phytopathogenic fungi. Duranta repens Linn. var. variegate (syn: Duranta plumieri ) (Verbenaceae) is native to scrub and open woodlands in the West Indies and northern part of Pakistan and central and South America and north, north eastern and southern parts of India. The genus Duranta comprises about 35 species which are evergreen shrubs distributed in tropical and sub-tropical regions. It was introduced to Egypt as an ornamental plant in the 1920s. The fruits showed in vivo anti-malarial activity against Plasmodium berghei. Thrombin inhibitory coumarins were isolated from fruits. From the genus Duranta several iridoid glycosides as durantosides I, II, III, IV, and lamiide were isolated. Flavonoids and C-alkylated flavonoids and some alkaloids were isolated. Duranta repens (variegate) is one of the most common varieties of D. repens and little phytochemical work has been carried out on this plant till now. This plant and its isolated fractions showed strong antioxidant activity which is considered as a prerequisite for other biological activities. On searching for new antiviral agents from medicinal plants the crude methanol extract of D. repens showed 76% inhibition for the viral titer of Hepatitis A virus. This evidence encouraged the authors to carry out biologically guided fractionation for the plant which resulted in the isolation of the bioactive components; acteoside and lamiide, and other less active components like -sitoserol 1, naringenin 2, -glucopyranosyl, -fructopyranoside (sucrose), -galactopyranosyl, -glucopyranosy fructopyranoside (raffinose). As per the earlier studies, the larvicidal activity of crude extracts from the stem and fruits, their fractions and fresh fruit juice of Duranta repens were assayed against the larvae of Culex quinquefasciatus.

All these reports were enough to choose Duranta erecta as botanical insecticide and the extraction procedure followed was hot exhaustive process of methanol extraction.There has been a lot of literature regarding the insecticide susceptibility test in Helopeltis theivora against various insecticides. In conventionally managed (synthetic insecticide-treated) tea plantations, different organosynthetic insecticides of different functional groups (organochlorines, organophosphates, synthetic pyrethroids and, very recently, neonicotinoids) are routinely applied round the year to keep pest insect and mite populations under control (Sannigrahi and Talukdar, 2003; Gurusubramanian et al., 2008). This is a burden to planters as well as to the environment and can result in the resurgence of primary pests (Sivapalan, 1999), secondary pest outbreaks (Cranham, 1966), resistance development (Georghiou, 1972; Kawai, 1997; Hsu, 2004; Roy et al., 2010b) and environmental contamination including undesirable insecticide residues in tea (Chaudhuri, 1999). Continuous use of synthetic insecticides leads to the development of higher tolerance or resistance in many insects (Scharf et al., 1998; Martin et al., 2002; Yu et al., 2003; Yang et al., 2004; Markussaen and Kristensen, 2010; Komagata et al., 2010). Variations in relative toxicity to commonly used insecticides have been observed in H. theivora populations from Jorhat, Assam (Gurusubramanian and Bora, 2007), Darjeeling (Bora et al., 2007), and from the sub-Himalayan Dooars region of northeast India (Roy et al., 2008a). In tea plantations of Terai, Dooars and Darjeeling foothill regions, for instance, endosulphan, quinalphos, monocrotophos, deltamethrin and cypermethrin are extensively used (Roy et al., 2008b)

CHAPTER IIMATERIAL AND METHODS2.1 Collection of samples.(a) Collection of Helopeltis theivora and tea leaves. Tea mosquito bug populations: The susceptible strain (SS) of H. theivora was originally collected from Hathikhuli Tea Estate (Organic), Kaziranga, Jorhat, Assam (Guru Subramanian and Bora 2008). Field populations were collected from, commercial tea estates of Jorhat, Assam were cultured in separate cages on young tea foliage (variety - TV1, named after Tocklai vegetative clone) in a BOD at 27 2C, 80 % r.h., and a photoperiod of 16L/8D.

Synchronized cultures of H. theivora were produced from stock populations and the SS population for use in bioassays.

Adults were transferred from stock populations to young tea leaves in small plastic containers. Insecticidal compounds belonging to OC, OP, SP and NN groups used in this study along with their details, regarding trade name, formulations, manufacturers and field recommended dose (ppm).

Fig1: Jorhat, tea plantation (Assam)Tea leaves-Camellia sinensis was collected and stored until insecticide susceptibility test.

Fig2: Helopeltis theivora collected from Jorhat tea plantation in netted chambers with tea leaves and humid condition was maintained while keeping them alive at room temperature for 3-4 hr. till the susceptibility test was performed.

(b) Collection of young leaves of Duranta erecta (source of botanical insecticide) from Tezpur University campus).

Fig3: Duranta erecta

2.2 Extraction of plant flavonoids from Duranta erecta:

Plant materials- The plant part (young leaves) of Duranta erecta (Linn.) were collected from Tezpur University campus, April-May, 2014 and following extraction procedure was followed.

Extraction Duranta erecta leaves were shade dried (4-5 weeks) and pulverized into a coarse powder.The ground plant materials (1g) were then extracted in: cold with methanol in pre-chilled mortar and pestle at r.t. and hot methanol extraction using Soxhlet apparatus.Rotary evaporator at 40oC under reduced pressure to concentrate the solvents afforded a semisolid mass of methanol extract (0.5 gm)1.2.3.

2.3 Insecticidal bioassay:

The insecticidal effect of crude methanol extracts (young leaves) and their solvent fractions were determined by the WHO standard procedure. The stock solutions were prepared by dissolving the grounded leaves (10 mg of each) in 1 ml of methanol. After that twenty-five laboratory reared I, II, III and IV instars larvae were released into 100 ml glass beakers separately, containing 50 ml of distilled water to which 25%, 50% ,75%,100%(Hot) of the plant extract which corresponded to the following concentrations 25ppm,50ppm,75ppm and 100ppm. The following were the controls that were maintained for the two sets of insecticide susceptibility test i.e., quinalphos and Plant extract from D. erecta:i) Distilled water; ii) +ve control for both the hot and cold process of extractioniii) Three replicates were made for each concentration and the experiment was performed under laboratory conditions at 27 1oC and 40-60 % relative humidity. (Brewers yeast could be supplied as a larval food during the test periods for larval feeding.)Since the mosquitoes were used in laboratory conditions just for 2-3hr before performing the insecticide susceptibility test hence feeding on tea leaves while providing humid temperature and water was sufficient to give appropriate living conditions.

Leaf dipped assay for Plant extractLeaf dipped assay for Quinalphos Fig: 1 Control (water) 3 %( Quinalphos) Fig: 1 Control (water) 25 %( Plant extract) Fig:2 5%(Q) 7%(Q) Fig: 2 50%(PE) 75%(PE) Fig:3 9%(Q) 100%(Q) Fig: 3 100%(Hot)-PE 100%(C)-PEFig 4: Leaf dipped bioassay for synthetic insecticide- Quinalphos and botanical insecticide-plant extract of Duranta erecta.

2.3 Insecticide susceptibility tests:Materials and methods:i) Collection and rearing of Helopeltis theivora :ii) H. theivora were collected periodically from different conventional and organic tea plantations.iii) A laboratory culture of H. theivora was set up (at 25+/- 2C and 7080% relative humidity) on the TV1 clone, collected from organically maintained tea plantations and was maintained in the laboratory for five generations following the procedure of Roy et al. (2010a).iv) These were used as a control (synthetic insecticide unexposed).v) Synthetic insecticide-exposed specimens were collected from different synthetic insecticide-managed plantations.Both the laboratory-reared (control) and field-collected populations were subjected to relative toxicity and biochemical assays.Insecticides and chemicals:The two insecticides: quinalphos 25 EC (organophosphate: Flashw; Indofil India Ltd) and botanical insecticide (plant extract) were used for enzyme studies.Chemicals and stocks solutions required:Bovine serum albumin (BSA), -naphthyl acetate (-NA), -naphthol, 1-chloro-2,4-dinitrobenzene (CDNB), Reduced glutathione (GSH), 3,3 , 5,5 -tetra methyl benzidine (TMBZ), Fast Blue BB salt, acrylamide, bis-acrylamide (Sisco Research Laboratory, Mumbai) and Cytochrome C (Sigma-Aldrich, Mumbai) was used for enzyme studies.Solutions of -NA (30mM), CDNB (50mM) and TMBZ (6.3mM) and the staining solutions were prepared fresh just before use.

2.4 Relative toxicity bioassay: The insecticides were diluted with demineralized water in five selected concentrations for use and were expressed in parts per million. Adult H. theivora were exposed initially to a wide range of concentrations and on the basis of the resultant mortality, a series of concentrations within a narrow range were further tested. The second series was determined on the basis of the concentrations causing insect mortality above 20% and below100%. Toxicity assays were conducted using the standard leaf dipped method recommended by the Insecticide Resistance Action Committee of the International Group of National Associations of Manufacturers of Agrochemical Products. Thirty adult females of H. theivora were released using a camel-hair brush separately into each glass chimney containing insecticide treated tea shoots. For each concentration, there were three replications and a control. Five doses from the second series were tested for each of the insecticides and the demineralized water was used for the control. The entire experiment was replicated three times. Final assessment of the lethal effects of the insecticides was done after 48 h of insecticide exposure, and was expressed as per cent mortality of the insect at each dose, in relation tocontrol mortalities using Abbotts formula (Abbott,1925) as needed (mortality above 10%).

Fig5: Insecticide susceptibility test:a) Botanical insecticideb) Quinalphos(synthetic)

Fig: 6: Insect and the tea leaves after insecticide susceptibility test

To check the dosage dependent expression profile and enzymatic activity of the three detoxifying enzymes-Cytp450, GST and GE, the mosquitoes both live and mortal were collected from each concentration of both the insecticides tested in insecticide susceptibility test, then homogenized in Potassium phosphate buffer.To get the lethal dose to be applied for both the insecticides the tea leaves treated with insecticides (both natural and synthetic) were homogenized for residue analysis by GC-MS.

Homogenization of insects:a. Single adult (female) H. theivora was homogenized in 500 ml of ice-cold 0.1M sodium phosphate buffer (pH 7.0) and centrifuged at 12,000 g for 20 min at 4C in a high-speed refrigerated centrifuge (SIGMA 3K30). b. The resultant post-mitochondrial supernatant was aliquoted (100 ml each) into a 0.5 ml centrifuge tube and was stored at 280C as an enzyme source for GE, GSTand CYP activity assays and to estimate the amount of total protein. c. For the GST assay, the pH of the homogenization buffer was 6.5. For both the control and field-exposed specimens, 60 adult females were assayed for enzyme activities.

2.5 Enzymatic activity assay for the detoxifying enzymes: GE activity:b. A GE activity was measured using -NA as a substrate according to the method of van Asperen (1962), with minor modifications, using a microplate reader. c. Twenty l of the supernatant were taken in each well of the microplate reader (Opsys MR, DYNEX Technologies) in duplicate.d. Two hundred l of freshly prepared 30mM -NA were added to each well for the reaction to occur. e. The reaction was stopped after 15 min by adding 50 ml of staining solution prepared fresh by mixing two parts 0.1% Fast Blue BB salt with five parts of 5% sodium dodecyl sulphate (SDS).f. The plate was left for 5 min for equilibration and absorbance was recorded at 590 nm. g. The change in absorbance was converted to an end product (-naphthol) using the standard curve of -naphthol (0.051.00 mM). h. Blanks were set at the same time using a reaction mixture without protein extracts.

GST activity:a) GST activity was estimated using the method of Kao et al. (1989) with minor modifications. b) 2.50 of 50mM CDNB and 150 ml of 50mM GSH were added to 2.78 ml of sodium phosphate buffer (100mM, pH 6.5).c) 20 of the enzyme stock was then added. d) The contents were shaken gently, incubated for 23 min at 20 8C and then transferred to a cuvette in the sample cuvette slot of a UVvisual spectrophotometer (RayleighUV-2601). (The reaction was carried out in duplicate).e) The reaction mixture (3 ml) without the enzyme was placed in the reference slot for setting zero. Absorbance at 340nm was recorded for 1012 min employing a kinetics (time-scan) menu.f) (A unit of enzyme activity is defined as the amount of enzyme that catalyzes the formation of 1mmol 2, 4- Dinitrophenyl-glutathione/min at 30 C using 1mM concentrations of GSH and CDNB. Specific activity is defined as units of activity per mg protein.)g) At 340nm, the difference in mM extinction coefficient between the CDNBGSH conjugate and CDNB was calculated.Changes in absorbance/min were converted to mmol CDNB conjugated/min per mg protein using the formula (Habig et al., 1974; Nehare et al., 2010).

Cytochrome P450 activity:a. Most of the classic methods to evaluate oxidase activity with chromogenic substrate require purification of microsomal fractions. b. However, these methods cannot be used to estimate the differences in oxidase activity in single small insects (Martin et al., 2002).c. The alternative method was to measure the level of haem-containing enzymes, including thecytochrome oxidase enzymes (Brogdon et al., 1997). Peroxidation of TMBZ was catalyzed by microsomal proteins with hydrogen peroxide as a co-substrate. d. The amount of oxidase enzymes was correlated with the peroxidase activity of the haem groups. e. Cytochrome P450 activity was estimated by measuring haem peroxidase activity (Penilla et al., 2007;Tiwari et al., 2011).As haem constitutes the majority of cytochrome P450 in non-blood-fed (herbivorous) insects, quantification of haem activity can be expressed as cytochrome P450 (Brogdon et al., 1997).

For cytochrome P450 activity:

a. 20 ml of the supernatant was taken in the well of the microplate containing 200 ml of 6.3mM TMBZ solution (10mg TMBZ dissolved in 5ml absolute methanol mixed with 15 ml of 0.25M sodium acetate buffer (NaC2H3O2), pH 5.0, prepared fresh daily).b. Then, to each well of the microplate, 80ml of 0.0625M potassium phosphate buffer, pH 7.2, and 25 ml of 3% hydrogen peroxide were added.c. Two controls per plate were prepared each with 20 ml of homogenizing buffer and with all the ingredients except the enzyme source.d. Absorbance was recorded at 630nm after 30 min of incubation using a microplate reader (Opsys MR; DYNEX Technologies) against blanks at 25 8C.e. A standard curve for haem peroxidase activity was prepared using different concentrations of Cytochrome C (0.00250.02 nmol) from horse heart type VI (Sigma Aldrich).f. Total cytochrome P450 activity was expressed as nmol of cytochrome P450 equivalent unit-mg protein/min.

2.6 A fast multi-residue method for determining pesticides in tea:Materials required:a. Analytical standards of the pesticides.b. Analytical grade petroleum ether.c. Analytical grade ethyl acetate and acetone.d. Analytical grade acetonitrile.e. Instrumentation and GCFID analytical conditions:

StandardsA standard stock solution of the two insecticides was prepared in hexane.(a) Stock solution of the synthetic insecticide (Quinalphos) (5ppm, 5ml) prepared in hexane.(b) Stock of natural insecticide (Duranta erecta) (260g/ml-260ppm) prepared in n-hexane. A standard solution of the 2 pesticides for preparing a calibration graph (0.01, 0.02, 0.05, 0.1, 0.2 and 0.5 mg/L) were prepared from the stock solution using serial dilution with hexane). The mixture standard solutions of the pesticides (1 and 10 mg/L) used for sample spiking were to be prepared in acetone.Methods:Sample preparation:a) The tea samples were fortified at 3 concentration levels by adding appropriate amount of pesticides standard solution.b) Twenty milliliters of fortified tea samples were weighed into a graduated cylinder with stopper.c) 50-mL mixture of petroleum etherethyl acetate (3:1, v/v) was added, shaken vigorously for 1 min and allowed to stand for 20 min.d) The supernatant (25 mL) was concentrated to dryness using a rotary evaporator at 40C. e) Resulting residues were dissolved with petroleum ether (2 mL) for purification by Florisil cartridges.f) A Florisil cartridge was conditioned with petroleum ether (5 mL). A 2-mL extract solution was loaded onto the cartridge and collected in a flask.g) Analytes were eluted with 6 mL petroleum etheracetone (3:2, v/v), and the elute was collected and brought to dryness using a rotary evaporator at 40C.h) The dry residue was dissolved in 5 mL hexane for GCFID analysis.

2.7 Bioinformatics work:

1. Cyp6b8 gene protein sequence was retrieved from UniProtKB/Swiss-Prot and protein blast in NCBI gave the list of organisms showing maximum homology with the query sequence.2. The one showing maximum homology was chosen and the fasta sequence was submitted to I-TASSER server which is an on-line platform for protein structure and function predictions.3. Five models generated and the two models having the least c-value were submitted to I-Tasser- ModRefiner.(ModRefiner: It is an algorithm for atomic-level, high-resolution protein structure refinement, which can start from either C-alpha trace, main-chain model or full-atomic model.)4. Refined model generated after energy minimization-the PDB file of the two models were submitted to PDBsum generate to give detailed secondary structures of the two models.5. The library of the compounds found in the three peaks chosen from the GC-MS graph of the extract was made in CLC Drug discovery software (online software to perform molecular docking).6. The PDB file of the refined model obtained (I-Tasser online software for protein homology modelling the secondary and 3-D structure generation) was imported to CLC Drug discovery software and docking was performed.

CHAPTER 3 - RESULTS:3.1 Botanical insecticide (extraction) and characterization.

(a) Aluminium chloride colorimetric assay was done to determine the concentration of flavonoids in the plant extract. Hot methanol extraction (260g/ml-260ppm) and Cold methanol extraction (255g/ml-255ppm).

(b) GC-MS analysis of the extract gave the list of secondary metabolites, their structure, molecular weight and the retention time of the same in the column used (elite 9).

3.2 GC-MS analysis of both synthetic and botanical insecticides. (a) GC-MS analysis graph of the hot methanol extract.

Ascorbic acidBase peek. Z, Z-6,28-HEPTATRIACTONTADIEN-2-ONE (Z)-14-TRICOSENYL FORMATE STYRYLCARBAMIC ACID METHYL ESTER

(b) GC-MS analysis of the cold extract.

Base peek. Z, Z-6,28-HEPTATRIACTONTADIEN-2-ONE (Z)-14-TRICOSENYL FORMATEAscorbic acidSTYRYLCARBAMIC ACID METHYL ESTER

Three peaks with retention time:12.00, 11.54 and 11.46 were common in both the extracts suggesting that Z,Z-6,28-HEPTATRIACTONTADIEN-2-ONE, (Z)-14-TRICOSENYL FORMATE, ASCORBIC ACID and STYRYLCARBAMIC ACID METHYL ESTER are the major fraction of the extract and hence might play significant role in the insecticidal activity of the extract apart from others which were though present in small quantity but might have insecticidal activity.In the cold methanol extract there is quite a large library of compounds which are not present in hot extract henceforth can be concluded to heat labile and hence destroyed due to heat.

GC-MS analysis of quinalphos 25 ECThe retention time of quinalphos was checked in the column.

n-HexaneQuinalphos

3.4.Enzymatic assays of the three major detoxifying enzymes:(a) Glutathione-S-Transferases: The standard curve was plotted and the concentration of the substrate left out in the 96-well plate (reaction well for each concentration was plated to get the estimate of enzymatic activity and hence reduction in the substrate level.

(b) Cytochrome p450 activity: The standard curve was plotted and induced enzymatic activity of the detoxifying enzymes for both synthetic as well as natural insecticides fed mosquito samples.

General esterase activity was determined by taking the absorbance of the colored complex formed between CDNB-Glutathione at 560nm for enzyme samples from both the insecticide susceptibility test for varying time of incubation: 2min, 4min, 6min, 8min and 10min and the graphs were plotted as below. The trend is exponential for Plant extract reaction set but in case of quinalphos nothing can be clearly pointed out with just one round of insecticide susceptibility test.

3.5 Docking study:Docking result was prominent for flavonoids of the botanical extract.This suggested that though flavonoids interact with the cytochrome p450 enzymes the insecticidal property of the botanical insecticide was by virtue of some other secondary metabolite present in the plant extract which have to be particularly fractionated and then study their insecticidal property in detail.

CHAPTER 4 DISCUSSION:1. The purpose of the project was fruitful in terms of finding the lethal concentration for the insecticide that would not only kill the pest but also not be left out in the processed tea in large residual concentrations.2. The natural insecticide though proved to be effective at 260g/ml (100%-Hot methanol extraction), requires large amount of Duranta erecta leaves to produce the extract in substantial quantity to be used on large scale by the tea planters. 3. Insecticide susceptibility test: The mortality rate were- (a)For natural: 100 %( H) : >40% Mortality in less than 24hrs. (b)For synthetic insecticide: 7% and 9% >40% Mortality and 100%-100% mortality in less than 24 hrs. The enzymatic activity assay results for the three enzymes: CYPp450, General Esterase and Glutathione-S-Transferases also reflected the same as observed by insecticide susceptibility test: Irrespective of concentration variations, the living populations showed higher enzymatic activity than the dead counterpart one for both the insecticides (micro-plate reader assay) used in susceptibility test. This indicates positive correlation between enzyme activity and possible metabolic detoxification and development of resistance against the insecticides in Helopeltis theivora.4. The molecular docking studies also confirm the interaction of Cytochrome P 450 enzymes with the following specific flavonoids like quarcetin, rutin, 7-O-D-glucopyranosyl-3,5-dihydroxy-3-(4acetoxyl-3-methylbutyl)-6,4-dimethoxyflavone, 7-O-D-glucopyranosyl-3,5-dihydroxy-3-(4acetoxyl-3-methylbutyl)-5,6-dimethoxyflavone,3,7,4-trihydroxy-3-(8-acetoxy-7methyloctyl)-, etc. But use of Quinalphos on regular basis may again pose the similar problem of development of resistance which needs persistent exposure of the insect pest for several seasons even though it is used in prescribed doses to cause environment and health hazards. More detailed studies regarding dose dependent expression profile of cytochrome p450 genes for several generations is necessary to observe the phenomenon of resistance development. SDS-PAGE or western blotting studies of the expressed protein may provide better insight into insect pesticide interactions and resistance development to formulate effective pest management practices in terms of dose, frequency and duration of exposure.

CHAPTER 5 - REFERENCES: 1. Resistance to insecticides in field collected populations of Tea Mosquito Bug (Helopeltis theivora) Waterhouse from Doors (North Bengal, India) tea cultivation. -Somnath Roy, Ananda Mukhopadhyay and Guruswami Gurusubramanian.2. Insecticidal Resistance to tea mosquito bug, Helopeltis theivora , Waterhouse (Miridae: Heteroptera) in North East India. -G. Gurusubramanium and S. Bora.3. Variation of resistance to endusulfan in Tea Mosquito Bug, Helopeltis theivora Waterhouse (Heteroptera: Miridae) in the tea plantation of the Sub-Himalayan Doors, northern west Bengal, India. -Somnath Roy, G. Gurusubramanium and Ananda Mukhopadhyay.4. Development of Tolerant traits in tea mosquito bug (Helopeltis theivora Waterhouse (Heteroptera: Miridae) under Insecticide Stress. -Ananda Mukhopadhyay and Somnath Roy.5. Comparison of life cycle traits of Helopeltis theivora Waterhouse (Heteroptera: Miridae population infesting organic and conventional tea plantations, with emphasis on Deltamethrin resistance. -Somnath Roy and G. Gurusubramanium.6. Fast multi-residue screening for 84 pesticides in tea by gas chromatography with Dual-tower Auto-sampler, Dual column and dual detectors.7. Phytochemical analysis and antifungal potential of Duranta erecta against some of phyto-pathogenic fungi. - P. Sharma*, S. Khandelwal, T. Singh and R. Vijayvergia.8. Phytochemical Investigation and Antiviral Activity of Duranta repens. Lobna M. Abou-Setta, Naglaa M. Nazif, Abdelaaty A. Shahat Photochemistry Department, National Research Centre, 12311 Dokki, Cairo, Egypt.9. Larvicidal Effects of Stem and Fruits of Duranta repens against the Mosquito Culex quinquefasciatus. -Farjana Nikkon1*, Zahangir A. Saud1, Khaled Hossain1, Mst. Shahnaj Parvin2 and M. Ekramul Haque2.10. Insecticide susceptibility and activity of major detoxifying enzymes in female Helopeltis theivora (Heteroptera: Miridae) from sub-Himalayan tea plantations of North Bengal, India. -Dhiraj Saha*, Somnath Roy and Ananda Mukhopadhyay).

10

, 10-Mar-2014 + 15:08:04

2.50 4.50 6.50 8.50 10.50 12.50 14.50 16.50 18.50Time0

100

%

Duranta Scan EI+ TIC

1.31e812.90;55

11.5455

11.4655

5.2655

3.2957

2.0279

3.9391

10.682056.16

81

5.8981

7.56147

6.5457 8.88

1908.0579

12.5255

12.9355

14.8055

, 10-Mar-2014 + 16:17:55

2.50 4.50 6.50 8.50 10.50 12.50 14.50 16.50 18.50Time0

100

%

QUINOL Blue Scan EI+ TIC

1.37e93.55;105

3.24105

2.5191

3.1891

2.5691

12.28146

3.78105

4.07119

4.33119

4.68119

8.8157 9.40

97