A Study On Certain Hydrolases and Oxidoreductases of Major Arthropod Pests of Tea from Darjeeling foothill and its Adjoining Plain
Thesis suBmitted to tfie ZJniversity ofO^orth (BengaCforpaitiaf fiUfiOment o f the (Degree o f(Doctor cfM lbsophy in Science (Zootogy)
By
M a y u ^ S a r ^
Department of Zoology University of North Bengal India 2007 mi 13010
f)0/Sf;r
WW60?S%?i'££9 UNIVERSITY OF NORTH BENGAL P.O. North Bengal university, Raja Rammoliunpur, Dt. Darjeeling, DEPARTMENT OF ZOOLOGY West Bengal, India, P IN -734 013
'From; ®r. JLmncCa Mu^fiopacffiyay, TZS (CdC) (professor
Supervisor's Certificate
‘Tfiis is to certify that 9/Lr. M ayu^ Scir^r, M.Sc. has w or^d on a programme entitkd “Ji Study On Certain HydroCases and Oxjdoreductases of Major Arthropod ^ests of from (Daijeefmg foothill and its Adjoining ^ in ” under my supervision and guidance and that he has fulfiCkd the requirements related to suSmission of since 2002 with a financiaC support of the 9(ationa[ T^ea (^search foundation. His research wor^emSodies originalfindings Based on a well- planned investigation. The dissertation suSmitted herewith is for the partial fulfillm ent o f the degree o f (Doctor o f (Philosophy in Science (Zoology) o f the Vniversity o f !Nbrth (Bengal T^e thesis material has not Been suSmitted to any other Ijniversityfo r any degree whatsoever 6y him or any one else. I sincerely wish Mr. S a r^ r and his endeavour success. (Date: j?? 3^ S.crhJ- (place: a !srlK JLnandaMu^opadhyay ‘E-mail: dr_amu^erjee_n6u@rediffrnailcotn Phone: (0353) 2699 124 Fax: (0353) 2699 001 e-mail: [email protected]. Visit us at: www.nbu.ac.in Contents no. 1. Introduction------6-21 1 .1 . Early history of tea cultivation ------7 1.2. Present scenario of tea cultivation and its arthropod pest problem ------7 1.3. Location and Physiognomy of the study area ------10 1.4. Major arthropod pests of tea from Darjeeling foothill andits adjoining plain — 12 1.4.1. Looper caterpillar, Buzura suppressaria Guenee ------16 1.4.2. Redslug caterpillar, £ferus/a magn/f/ca Butler ------17 1.4.3. Tea mosquito bug,/-/e/ope/f/s f/?e/Vora Waterhouse ------18 1.4.4. Red spider mite, O/Zgonyc/ius coffeae (Nietner) ------19 2. Objectives and Scope of S tudy------22-23 2 .1 . To detect and quantify the major hydrolytic (digestive) enzymes occurring in the salivary and midgut tissue of major arthropod tea pests viz. Buzura suppressaria (Looper), Eterusia magnifica (Red slug caterpillar), Helopeltis theivora (Tea mosquito bug) and whole body extract of Oligonychus coffeae (Red spider mite). 2.2. To identify and quantify certain oxidoreductase enzymes ofthese major tea pests. 2.3. To quantitatively and qualitatively assess the detoxifying enzymes viz. general esterases, glutathione S-transferases and acetylcholinesterases of these major pests, 2.4. To find out the inhibitory activity of pesticides to general esterases and acetylcholinesterases of these pests. -2- 3. Review of Literature ------24-42 3.1.Major lepidopteran defoliators and sucking pestsof tea and related crop loss — 25 3.2. Pesticide use patterns in tea ------30 3.3. Hydrolytic and oxidoreductase enzymes of lepidopteran, sucking and mite pests 31 3.4. An overview of enzyme based biochemical resistance ------35 3.5. Specific role of esterases, glutathione S-transferases and acetylcholinesterases in insecticide resistance ------37 4. Materials and Methods ------43-57 4.1. Collection of pest specimens: Buzura suppressaria, Eterusia magnified, Helopeltis theivora and Oligonychus coffeae from teaplantations------44 4.2. Laboratory rearing (culture) of the pests forexperimentation ------45 4.3. Dissection of salivary glands, midgut and cerebral ganglia ------46 4.4. Biochemical analysis ------47 4.4.1. Isolation of salivary and midgut enzymes of 4.4.1.1. B.suppressana ------47 AAA.2. Et.magnifica ------47 4AA.3. H.theivora ------48 4.4.1.4. O.coffeae (whole body) ------48 4.4.2. Gel diffusion assay of salivary and midgut digestive enzymes (amylase, protease) of AA.2A. B.suppressaria ------49 4.4.2.2. Et.magnifica ------50 4.4.2.3. H.theivora ------50 4.4.2.4. O.coffeae (whole body) ------50 4.4.3. Quantitative assay of digestive enzymes 4.4.3.1. Amylase ------51 4.4.3.2. Protease ------51 4.4.3.3. Lipase ------51 4.4.4. Assay of oxidoreductases 4.4.4.1. Catalase------51 4.4.4.2. Peroxidase ------52 4.4.4.3. Polyphenol-oxidase------52 -3- 4.4.5. Quantitative assay of detoxifying enzymes 4.4.5.1. General esterases ------52 4.4.5.2. Glutathione S-transferases ------53 4.4.5.3. Acetylcholinesterases ------53 4.4.6. Qualitative analysis of detoxifying enzymes of B.suppressaria, Etmagnifica, H.theivora and O.coffeae collected from pesticide-exposed (conventional plantation) and un-exposed (laboratory-reared) populations by Native Polyacrylamide Gel Electrophoresis 4.4.6.1. Esterase isozymes of salivary, midgut, or whole body extracts ------54 4.4.6.2. Isozymes of Glutathione S-transferase ------55 4.4.6.3. Acetylcholinesterase isozymes ------56 4.4.6.4. Inhibition tests of general esterases and acetylcholinesterases of the pests —56 4.5. Statistical analysis and computer application ------57 5. Results and Discussion------58-136 5.1. Results of quantitative assay of major hydrolases (digestive enzymes) and oxidoreductases of B.suppressaria, Etmagnifica, H.theivora and O.coffeae 5.1.1. Results of hydrolases of the four arthropod tea pests 5.1.1.1. Amylase ------59 5.1.1.2. Protease ------63 5.1.1.3. Lipase ------67 5.1.2. Discussion on hydrolytic enzymes ------70-76 5.2.1. Results of oxidoreductases of the four arthropod tea pests 5.2.1.1. Catalase ------77 5.2.1.2. Peroxidase — ------81 5.2.1.3. Polyphenol-oxidase ------85 5.2.2. Discussion on oxidoreductases ------89-92 -4- 5.3.1. Results of quantitative assay on detoxifying enzymes of the four- -arthropod tea pests 5.3.1.1. General esterases —— ------93 5.3.1.2. Glutathione S-transferases - — ------96 5.3.1.3. Acetylcholinesterases ------98 5.3.2. Results of qualitative analysis of detoxifying enzymes of tlie four- -arthropod tea pests 5.3.2.1. Esterase isozymes — ------101 5.3.2.2. Glutathione S-transferase isozymes ------107 5.3.2.3. Acetylcholinesterase isozymes ------108 5.3.3. Discussion on detoxifying enzyme ------112-122 5.4.1. Results of inhibition tests of isozymes for detection of insecticlde- -resistance / tolerance status of the four arthropod tea pests 5.4.1.1. Midgut esterase isozymes ------123 5.4.1.2. Acetylcholinesterase from cerebral ganglia ------127 5.4.2. Discussion on resistance I tolerance status of the four pests ------131-136 6. Summary ------137-142 7. Highlights ------143-148 8. References ------149-185 9. Acknowledgements------186-188 10. Annexure------189-195 I. List of abbreviations used ------190-191 11. List of published / accepted papers related to tea pests------192-194 III. Reprints of the published papers relevant to the topic of the thesis — 195 -5- 1. Introduction 1.1. Early history of tea cultivation The first attempt of tea cultivation in Darjeeling dates back to January 1834 when Lord Wiliam Bentlnck proposed, to the Council of the East India Company, setting up of a tea committee to investigate and make recommendations on suitability of tea cultivation in India. The tea committee decided to send their secretary G.J. Gordon to China in order to acquire tea seeds and some tea workmen familiar to tea cultivation and manufacture. From this original consignment of China seed around 42,000 young plants could be raised which were allocated to three main areas, 20,000 to the hill districts in the Kumaon in North India, 2,000 to the hills of South India and the remaining 20,000 to the then North-East (N.E.) frontier (Weatherstone, 1992). Out of this initial trial, seed tried in Darjeeling grew well. As per the available records one Dr. Campbell, a civil surgeon, planted tea seeds in his garden at Beechwood, Darjeeling 2100 m above mean sea level (amsi) as an experiment with reasonable success. Subsequently the government, in 1847, selected the area to raise tea nurseries. With the plants raised in the government nurseries, the first commercial tea gardens in Darjeeling hill area were Tukvar, Steinthal and Aloobari tea estates in 1852 (Pathak, 2004). 1.2. Present scenario of tea cultivation and its arthropod pest problem Tea plantation of North Bengal is spread over three regions - the Darjeeling hills, its Terai region, and the plains of the Dooars. North Bengal produced some 10,853 Th kgs of tea in March 2006. According to the statistics of Tea Board of India, there are 308 big and 1232 small tea gardens in North Bengal. Total area under tea is 5,19, 700 hectares and in 2005, total production of tea was 927.98 million kilograms. -7- At present there are 86 running gardens producing ‘Darjeeling Tea’ on a total land of 19,000 hectares. The cool and moist climate, the soil, the rainfall and the sloping terrain all combine to give Darjeeling tea its unique "Muscatel" flavour which is regarded as the "Champagne of Teas". The total annual production of such tea is in the range of about 10 to 12 million kilograms. Tea grown in the Darjeeling foothills, Terai and the Dooars plains are mostly high yielding clones. Each tea growing region has its own distinctive pest fauna, though many species have been recorded from more than one country. About 300 species of arthropod pests are known to attack tea in India (Banerjee, 1993; Muraleedharan et a!., 2001). During last three decades several changes have taken place in the agronomic practices, which have also magnified our pest problems. Every part of the tea plant is subject to attack of pests. A steady loss of 10% due to overall pest attack is a generally accepted figure though it could be 40% in devastating attacks by defoliators (Banerjee, 1993). In addition to direct crop loss, pest damage can adversely affect the quality of processed tea. General observation and planters’ experience indicate that looper {Buzura suppressaria), red slug [Eterusia magnifica), tea mosquito bug {Helopeltis theivora) and red spider mite {Oligonychus coffeae) are the most common tea pests of Darjeeling foothills, Terai, the Dooars areas with their incidence also in the plantations of North-East (N.E.) India. Planters of Terai and the Dooars are facing serious problems in combating the outbreaks of these folivores and sucking pests. Oligonychus coffeae breeds throughout the year and subsists on mature sustenance leaves. Helopeltis theivora causes extensive damage by attacking the tender leaves and the growing shoot. The defoliators like Buzura suppressaria and Eterusia magnifica have their share and defoliate tea in Terai, the Dooars and North-Eastern plantations (Muraleedharan, 1993). In the past, all tea phytophages were not active simultaneously and a well-marked seasonal appearance for each was evident with the seasonal cycle of the growth and productivity of the plants (Banerjee, 1977). Now a day, the planter’s experiences indicate that most of the pest species remain active throughout year with overlapping seasonal cycle of the growth and productivity of the tea plants. The appearance of the pests needs timely management mostly by use of synthetic pesticides. Since 1962, increasing use of pesticides in protection of tea plantations has been the common and popular practice. As the four major pest species have a wide occurrence attacking different tea cultivars in the plantations of the Darjeeling foothills and their adjoining plains of Terai and the Dooars, an assay of the major hydrolases (digestive enzymes) and oxidoreductases of the four common tea pests in question has been undertaken. The findings would likely furnish an understanding of the trophic strategies of these pests in exploiting and utilizing the tea plant. These pests have been exposed to conventional pesticide sprays such as organophosphates and synthetic pyrethroids for long, so there is possibility of development of different levels of pesticide resistance. This often makes the pests to a greater or less degree unmanageable by synthetic pesticides. Attempts have, therefore, been made to study the variation based on certain biochemical parameters of the four pests. Profiles of three main isozymes related to resistance and their quantities were studied from the local populations. As very little work has been done on the resistance I tolerance status of these pests, the contemplated study on three detoxifying enzymes is expected to reveal for the first time the biochemical nature and level of pesticide resistance / tolerance in the population of the four concerned pests occurring in this region. -9- 1.3. Location and Physiognomy of the study area Tea, the most ancient non-alcoholic beverage, is still a popular drink in the world. Tea, Camellia sinensis (L.) O. Kuntze, cultivation in India has a long history which is found to spread over approximately 5.19 lakh hectares. The Districts of Darjeeling and Jalpaiguri of West Bengal and a large part of Assam of N.E, India (Fig. 1) has gained importance both for quality tea and high yield. The tea growing regions of Darjeeling district is located between 26°31' and 27°13' north latitude and between 87°59' and 88°53' east longitude. The Northern part of the district has the distribution of eastern Himalayan range while the southern part consists of a stretch of alluvial plain at the base of the hills and is known as the Terai. The Terai is situated 91 meter above sea level with an average rainfall of 350 cm and an average temperature of maximum 30°C and minimum 12°C. The soil is moderately acidic, rich in organic material and is suitable for tea plantation. Besides heavy rainfall, Terai and its adjacent Dooars regions are well watered by a number of major rivers and a host of rivulets. The major rivers from east to west of Terai and the Dooars are; Siltorsha, Torsha, Jaldhaka, Teesta, Mahananda, Balason and Mechi. - 10- ASSAM Fig. 1, Tea growing areas of Darjeeling and Jalpaiguri districts of West Bengal (Not to scale) -11 - 1.4. Major arthropod pests of tea from Darjeeling foothill and its adjoining plain Tea, as a persnnial jea mosquito bug Looper monoculture crop caterpillar Red slug provides an Red spider mite caterpillar inexhaustible resource for colonization by several guilds of insects and mites (Fig. 2) many of Fig. 2. A Tea bush showing distribution of major which easily attain pests (four) attacl(ing leaves the pest status in such a stable ecosystem. Each tea-growing region has its own distinctive pest fauna though they may be found in other areas as well. More than a thousand species of arthropods are known to attack tea all over the world though only about 300 insects and mites are recorded from India (Muraleedharan et al., 2001). Out of them Lepidoptera form the major component (31.53%) followed by Hemiptera (26.29%) (Chen and Chen, 1989). Insects may consume every anatomical part of the plant but show specialization with regard to the feeding sites they occupy (Schoonhoven et al., 1998). Every part of the tea plant has a specific guild of pests. They may be grouped as per their feeding activity on stems, roots and foliage. Some sucking insects such as thrips, jassids and aphids cause extensive damage to the plant by making the shoot unproductive. The tea mosquito bug {Helopeltis theivora) is a serious pest both in South and N.E. India. H.theivora primarily feeds on leaves and new flushes and to a lesser extent on tender stem of tea plants. The feeding damage by this pest appears as a discoloured -12- necrotic area or a lesion around the point of entry of tiie stylets into the plant tissue. The lesion can be elongate and becomes darker with age as the tissue around the stylet puncture dies, presumably in response to the enzymatic action of the insects salivary secretions (Stonedahl, 1991). In the salivary gland of H.antonii, hydrolytic enzymes (protease, lipase), oxido-reductase enzymes (catechol oxidase, catalase and peroxidase) were detected. The salivary enzymes were implicated for the cause of phytoxaemia on various host plants as well as detoxification of defensive chemicals (Sundararaju and Sundara Babu, 1996). Among the leaf attackers, mites are most widespread. Distribution of foiivores, such as flushworms, leaf rollers, bunch caterpillars are more in the lower elevations of Darjeeling. In the Terai and the Dooars regions of Darjeeling foothills and plains, there are often outbreaks of defoliating pests such as Buzura (Biston) suppressaria Guenee (Looper caterpillar) and Eterusia magnified Butler (Red Slug caterpillar) causing extensive damage of the foliage. Hence, the need for introduction of Integrated Pest Management in tea (Barbora eta!., 1994) in N.E. India. The damage potential and recurrence of many pests are well known from tea plantations for long and many region specific new pests are now being recorded, but due to inappropriate measures taken for their control, the pest problem has further intensified. Although about Rs.15 crores is being spent in N.E. India alone for the control of pests and diseases, it has been found that about 6 to 14% of tea is lost due to insects, mites and weeds (Banerjee, 1976). Normally 10% of the total crop is lost annually due to pests but this could rise to 40% in devastating attack by lepidopteran defoliators (Banerjee, 1993). Sivapalan (1999) also reported that various assessments on crop loss by respective tea pest had been done from time to time in the different tea growing countries. This loss ranges from 5 to 10% to as high -13- as over 50%. But, these differences are also dependent on the prevailing climate, genetic variation / uniformity (seed /clone), age of tea, soil type and the prevailing fertility status etc. as such, it is difficult to estimate the crop loss accurately caused by a particular species. Among the tea growing regions of North-East India, pest activity has always been reported to be high in the Dooars and Terai regions. The average use pattern of the synthetic pesticides was estimated in the year 1990 to 1994 to be 11.5 kg / I / hectare / year in Assam Valley and Cachar; 16.75 kg /1 / hectare I year in the Dooars and Terai, and 7.35 kg / 1 / hectare / year in Darjeeling. Further the average annual consumption of insecticide and acaricide in the Dooars and Terai was 7.05 and 3.49 kg / I / hectare / year respectively (Barbora and Biswas, 1996). A recent survey, 1998-2000 suggested that the pesticide (insecticide + acaricide) consumption increase to 24.076 kg /1 / hectare / year in Terai region and around 14.16 kg / 1 / hectare / year in the Dooars region and average pest management cost is about Rs 7,000-10,000 / year / hectare in these regions. The average pesticide consumption was highest in the Dalgaon (19.56 kg / I / hectare / year) and lowest in Binnaguri (11.27 kg / I / hectare / year) sub districts of tea located in the Dooars area of Jalpaiguri district. Survey also reveals that on an average, the synthetic acaricides accounted for nearly 25% and synthetic insecticides around 60% while the rest 15% constituted safer pesticides. Organophosphates accounted for nearly 64%. The use of synthetic pyrethroid was also found to be alarmingly high at 0.73 kg / 1 / hectare / year (approx. 9%). Conversion of the quantity of pesticide used into number of effective spraying rounds on the basis of standard recommended dilution showed that, on an average nearly 18 rounds of pesticide application were resorted to by the estates in the region (Sannigrahi and Talukder, 2003). - 14- Routine application of chemical pesticides for the protection of tea crops has been a common and effective practice since the last 50 years but with growing usage of pesticides, the resistance of the arthropod tea pests to these chemicals has likely increased (Banerjee, 1968; Sarker and Mukhopadhyay, 2003, 2006a, b, c). Therefore extreme precautionary measures must be adopted before a pesticide is introduced to tea for pest control (Das, 1962), which will also save residue problem. In 1934, there were 10 insect species known to be resistant to pesticides whereas by 1980 the figure rose to 432 and by 1990 about 500 cases were reported (Schulten, 1990). High incidence of pests in tea has led to the indiscriminate use of pesticides leading to problems such as killing of non-target organisms including natural enemies of pests (Anonymous, 2003), human health hazards, enhanced environmental hazards and above all the problems of insecticide resistance. The need for conserving natural enemies of pest (Banerjee, 1967) and an integrated approach for controlling mite pests of tea has been emphasized (Banerjee, 1975). Further, the role of Integrated Pest Management has been stressed by Muraleedharan and Selvasundaram (2002) for the ever-growing pest problems in agricultural crops. It goes without saying that in the same spirit Integrated Pest Management (IPM) in tea requires planning to cover all the serious arthropod pests. The major folivores, B.suppressaria (Looper caterpillar), Et.magnifica (Red slug caterpillar) and major sucking pests, H.theivora (Tea mosquito bug) and O.coffeae (Red spider mite) that are important tea pests of Darjeeling foothills, Terai and the Dooars areas and the State of Assam in North-East India have been considered in this study. A brief introduction to these pests, the damage symptoms and the synthetic pesticides usually applied for their control are provided herewith: - 15- 1.4.1. Looper caterpillar: Buzura suppressaha Guenee (Geometrldae: Lepidoptera) Description Moth usually gray, finely speckled with black, forewing with yellow wavy bands, indistinct irregular yellow median lines and an ill defined median-postmedian maculate black and marginal series of yellow spots. Wing expanse 5- Fig. 3. An advanced stage of 7cm, female wings larger than male. Moths have looper caterpillar of tea high degree of melanism ranging from different shades of brown to creamy white. 1®’ and 2"*^ instar looper caterpillars are black with ring like thin white bands (c.7) at certain gaps, spread over the entire length of body. All larval stages have 3 pairs of thoracic legs, a pair of prolegs and a pair of claspers at the end of the body forcing the animal to make looping movement. The body colour of larva changes in advanced stages to green then to brown and finally taking the colour of twig or stem of the tea bush. There are five larval stages. It grows from the average length of 2.9 mm in the 1®‘ instar to a length of 30.55 mm in the fifth instar (Fig. 3). Freshly laid eggs are bluish green, cylindrical 0.27 mm length, laid in clusters (300-500) covered with buff coloured hair preferably on shade tree, sometime on axil of the tea leaf. Damage symptoms 1®* and 2"'* instar caterpillar nibble edge of young leaves of tea bush making tiny holes. S"'* instar chews the margin of a leaf making small cuts and nicks. 4”^ and 5*'’ instars can eat up the whole of the leaf resulting in large-scale defoliation of a tea bush. - 16- Control measures Insecticides usually applied for control of loopers are: Quinalphos 25EC @ 500 ml/ ha; Chlorpyriphos 20EC @ 500 ml/ha; Cypermethrin 10EC @ 250 ml/ha; Fenvalerate 20EC @180 ml/ha; Deltamethrin 2.8EC @ 180 ml/ha. 1.4.2. Red slug caterpillar: Eterusia magnifica Butler (Zygaenldae: Lepldoptera) Description Brightly coloured moths with an average wingspan of 50.6 mm in male and 56.46 mm in female. Body length of male is slightly smaller than that of female. Head, thorax and two basal segments of abdomen black and remaining abdomen except black tip in males pale yellow. Male antennae unipectinate, female antenna filiform. Forewing purple brown with a greenish tinge, Fig. 4. A fifth instar of red slug caterpillar a basal spot, medium white bands broken up usually into five spots. A white spot at the end of the cell and an irregular row of submarginal white spot. Hind wing black at the base followed by a yellow band, wide on the inner margin and with a few subtropical white spot. The apical area of hind wing marked with brilliant blue. Eggs oval. 0.9 mm in length and 0.4 mm breadth, yellow but turning green before hatching. 1®' instar brown with tubercles on the back bearing hairs, three pairs of thoracic legs and five pairs of prolegs, last pair acting as clasper. Prolegs end with disc like structure for attachment to the plant surface. 3''* instar larva develops a brown coloured ring like band on dorsal surface. A full-grown larva is brick red with well-developed tubercles on the back (Fig. 4), on disturbance a thick clear fluid -17- Jf)b7l)9 .V 0 1 OCT 2007 ^ exudes from the tubercles. I arval body length ranges from 1.32 mm for the 1^‘ instar to 20.54 mm for the 5*^ instar. Damage symptoms 1®’ and instar larvae nibble the epidermal surface of the leaf. instar feeds on the side of the leaf blade. Advanced larval instars eat entire leaf blade and parts of stem including the bark. Larvae usually attack the mature leaves of a tea bush. Control measures Insecticides applied for control of red slug caterpillars are; Endosulfan 35EC @ 750 ml/ha; Quinalphos 25EC @ 500 ml/ha; Chlorpyriphos 20EC @ 500 ml/ha; Cypermethrin 25EC @ 120 ml/ha; Fenvalerate 20EC @180 ml/ha. 1.4.3. Tea mosquito bug: Helopeltis f/ie/Vora Waterhouse (Miridae : Heteroptera : Hemiptera) Description The egg is white, cylindrical and slightly curved. ' Two unequal silvery filaments, the respiratory horns, arise laterally on either side of the operculum. Tea mosquito bug have five nymphal stages. The newly hatched nymph, light orange in colour, measured about 1.49 mm. Second instar Fig. 5. Helopeltis bug resting nymph measured about 2.00 mm and their abdomen on a tea leaf deep orange in colour. Body reddish green and measured about 3.00 mm length in third instar nymph, scutellar horn distinct. Wing pads became dark and body greenish yellow measure about 4.17 mm in fourth instar. Fifth nymphal instar measured about 5.00 mm and resembled adults with a reddish green thorax and - 18- green abdomen. The newly emerged yellowish brown adult turned metallic black (Fig. 5) with in an hour. Head black, eyes oval, wings black and abdomen green. Damage symptoms Feeding on the leaf results in the formation of water-soaked spot, which becomes distinct within minutes after the commencement of feeding. Subsequently these spots become circular and pale green and then gradually turned dark brown within an hour. These circular areas later become dark brown sunken spots. Gradually, they dry up and hole appear in their place. Feeding near the petiole and on the midrib cause an elongated lesion resulting in the curling of leaves. Severely infested leaves become deformed and curled, which lead to retardation of shoot grov^rth. Control measures The insecticides applied for control of mosquito bugs are: Quinalphos 25EC @750 ml/ha; Chlorpyriphos 20EC @ 750 ml/ha; Fenthion 80EC @ 200 ml/ha; Endosulfan 35EC@ 1000 ml/ha. 1.4.4. The Red spider mite: Oligonychus coffeae (Nietner) (Tetranychidae: Acarina) Description Eggs red, spherical with 0.1 mm diameter and with filament. Immature stages include roundish six-legged larva, 0.15mm in length, protonymph with 4 pairs of leg. Fig. 6. Adult Red spider mite oval shape, 0.2 mm long, anterior part pale red and abdomen deep reddish brown; deutonymph similar to pronymph but larger about 0.25 mm in length, female deutonymph slightly larger than male and resembles adult. Anterior part of adult female elliptical and crimson, while posterior part purplish brown (Fig. 6). Female measures, 0.35 mm to 0.45 mm in length. Males smaller with tapering abdomen. - 19- Damage symptoms Increase in red spider mite population is mainly related to the dry season and rise in temperature during spring and early summer in Nortii-East India. The mites attack the mature leaves on their dorsal side starting from the midrib ancj vein areas and gradually spreading on the whole of the leaf. The feeding impacts leave the leaf coppery-red coloured. Mites leave their cast skin and empty eggs as white spakes on the leaf surface. Heavy infestation leads to dropping of the leaf. Several factors like drought, unprunned tea, sunny days, unsmooth leaf surface texture and dust accumulation allow easy proliferation of the mite population. Further the increase in mite population may be influenced by organic manuring, poor drainage and may be influenced by the weeds acting as alternate host in the plantation areas. Control measures Chemical used for control of red spider mites are: Ethion 50EC @ 750 ml/ha; Quinalphos 25EC @ 750 ml/ha; lime sulphur or wettable sulphur (1 kg / ha), Dicofol 18.SEC @ 1000 ml/ha; Fenpropathrin 10EC @ 500 ml/ha. What prompted to undertake the present study? In view of the growing pest problem in tea and increased resurgence of pests it was felt necessary to undertake the present study on “certain hydrolases and oxidoreductases of the major arthropod pests of tea from Darjeeling foothill and its adjoining plain”. In the lower elevation of Darjeeling foothills and adjoining tea plantations lepidopteran larvae and sap-feeding bugs have been the subject of the study largely as far as their crop damage activities are concerned. In severe attacks by lepidopteran caterpillars, entire leaf as well as woody parts of the bush may be eaten, while young shoots and leaves dry up under attack of sap feeders. The digestive enzymes commonly found in the salivary and midgut of these pests are -20- therefore of interest in understanding their feeding relation with host (tea) as well as in devising methods of non-conventional pest management. Herbivore insects also possess an assemblage of enzymes that constitute their defense against chemical toxicants. These defense enzymes work by oxidation, reduction, hydrolysis or conjugation of molecules. The oxidoreductase enzymes are of great value because of their involvement not only in defensive but also in processing the secondary metabolites of the host plant. Many such enzymes involved in detoxification pathways act on a broad array of substrates found in plant allelochemicals and chemical pesticides. To characterize the array of digestive, oxidoreductase and detoxifying enzymes of major arthropod pests of tea, the present investigation is contemplated. This knowledge-base may be utilized in designing control programmes of these tea pests in future through use of enzyme inhibitors, host plant resistance programmes and by detection of pesticide resistance status of tea pests from this region. Moreover, no literature is available on biochemical identity of the tea-pest species from foothills and plains of Darjeeling and also on their pesticide resistance status as “strains”, or “biotype”. To fulfill these gaps and to usher in newer approaches of pest management, the following objectives of study have been laid down. -21 - 2. Objectives and Scope of Study 2.1. To detect and quantify the major hydrolytic (digestive) enzymes occurring in the salivary and midgut tissue of major arthropod tea pests viz. Buzura suppressaria (Looper), Eterusia magnifica (Red slug caterpillar), Helopeltis theivora (Tea mosquito bug) and whole body extract of Oligonychus coffeae (Red spider mite). 2.2. To identify and quantify certain oxidoreductase enzymes of these major tea pests. 2.3. To quantitatively and qualitatively assess the detoxifying enzymes viz. general esterases, glutathione S-transferases and acetylcholin - esterases of these major pests. 2.4. To find out the inhibitory activity of pesticides to general esterases and acetylcholinesterases of these pests. -23- 3. Review of Literaliire 3.1. M^or lepidopteran deloliators and sucking pests of tea and related crep toss In the world more than a thousand species of arthropod are known to be associated with different parts of tea plants. Out of them Lepidoptera form the largest order containing 31.53% of the pest species followed by Hemiptera 26.29% (Chen and Chen, 1989). But, only around 30Q species of insects and mites are recorded in India as tea pests under Acarina, Lepidoptera, Hemiptera and Coleoptera. A few minor pests have also been reported under Diptera, Hymenoptera and Orthoptera (Muraleedharan et a!., 2001). The monographs by Green (1890), Watt and Mann (1903) are the earliest contributions to the study of tea pests. Information on tea pest biology in N.E. India is given by Hainsworth (1952), Das (1965), Banerjee (1983a, b) and that of South India and Sri Lanka by Muraleedharan (1983) and Cranham (1966) respectively. Cramer (1967) estimated that tea in Asia suffers 8% crop loss due to pests. Glover et al. (1961) reported 13% crop loss, where as Banerjee (1993) reported a steady loss of 10% due to overall pest attack as a generally accepted figure, which may at times go up to 40% in devastating attack by defoliators. Sivapalan (1999) also reported that various assessments on crop loss by respective tea pest had been done from time to time in the different tea growing countries. This loss ranges from 5 to 10 to as high as over 50%. Reports from N.E. India suggested that in tea 6 to 12% crop loss annually may be due to red spider mite, Oligonychus coffeae alone (Sen and Chakraborty, 1964; Banerjee, 1971). Scarlet mites Brevipalpus phonicis and red spider mites are reported to cause 0.56 to 4.79% decrease in yield in Sri Lanka (Danthanarayana and Ranaweera, 1970). Some 8 to 17% increase in tea crop could be obtained following efficient mite control in South India (Rao and -25- Subramaniam, 1968). The crop loss due to Helopeltis theivora in south Incfia varied from 11-100% high cropping season (Rao and Murthy, 1976). Avespe crop loss due to Hetopeltis was 150 kg made tea per ha in Bangladesh (Ahmed, 1996). Damage to cocoa by H.theivora resulted in almost 95% crop loss if no biological or chemical control methods were adopted (Way and Khoo, 1989). The economic threshold level (ETL) for H.theivora was almost similar to that recommended for H.schoutedeni affecting tea in Central Africa during main cropping season (Rattan, 1987) and H.schoutedeni in Malawi was capable of causing a phenomenal loss in crop upto 55% (Rattan, 1984). The common looper Buzura (Biston) suppressaria Guen. caterpillar is one of the most destructive pests of tea and in recent years its activities have greatly increased becoming endemic to many gardens where it was unknown in the past (Anonymous, 1994). In the earliest record of tea pests by Cotes (1895), this pest was reported to have been collected first from Nowgong district of Assam. Subsequently Antram (1911) had reported looper caterpillar as one of the common pests of tea. Beeson (1941) in The Ecology and control of the forest insects of India and the neighbouring countries’ recorded looper caterpillar on alternate hosts such as Acacia modesta, A.catechu, Aleurites montana, Bauhinia variegata, Cassia auricuiata, Carissa diffusa, Dodonaea viscosa, Lagerstroemia indica, Daibergia assamica, Deris robusta, Aibizzia chinensis, A.odorotissima, A.iebbei<, Cajanus indicus and Priotropis cytisoides. Danthanarayana and Kathiravetpillai (1969) related the outbreaks of looper (Ectropis bhurmitra) with the presence of shade trees and use of dieldrin which killed many species of parasitic hymenopterans insects in Sri Lanka. Looper caterpillar was not considered to be a pest of major importance in North-East India -26- until 1990, when it caused considerable damage to tea in the Dooars and Cachar. Since then it has occurred from time to tirre and has been responsible for considerable losses in many estates of Upper Assam and the Dooars (specially Eastern and Central Dooars) (Anonymous, 1994). Borthakur (1975) had also reported looper caterpillar as one of the major pests of tea. Banerjee (1983b) outlined that the absenrce of natural enemies was the major contributing factor in looper outbreaks. Hill (1983) in ‘Agricultural insect pests of the tropics and their control’ had mentioned looper as an active defoliator of Indian and South-East Asian tea. Das and Gope (1987) had also suggested control measures against defoliators of tea and shade trees. Growing resurgence of pest and their resistance in N.E. India had prompted Das et at. (1988) to suggest non-conventionaI approach in tea pest management. Chen and Kunshan (1988) in China, were successful in controlling of tea looper by the field release of the laboratory-reared parasitic wasps, Trichogramma dendrolini. Singh et at. (1990) had reported olfactory behavioural response in both sexes of the moth, B.suppressaria possibly mediated by pheromone. Muraleedharan (1991) has a given account of the biology of looper caterpillar and their occurrence in shade trees in the book ‘Pest Management in Tea’. Further, Muraleedharan (1993) had recorded rare occurrence of looper caterpillar in the tea growing areas of south India. A sketchy account of natural occurrence and control measures of looper caterpillar is given in the book ‘Pests of tea in North-East India and their control’ (Anonymous, 1994). The book also includes names of other members of the family Geometridae that are known to occur on tea; these are Buzura (Biston) bengaliaria, Boarmia sclenaria, B.acaciaria, Medasina strixaria, etc, but none of them has attained the -27- status of a pest ^onym ous, 1994). Chakravartee (1995) had mentioned that looper infestations might become devastating within a short period, so tirrKly control of looper is very important. Berusia magnified Butl. (Red slug caterpillar) is one of the major pests of tea in the Terai, the Dooars, and North-East India. Early in 1906, Mann and Antram had reported approximate time of occurrence of different broods of caterpillars and adults of red slug. Hutson in 1932 had reported the occurrence of different species, Et.singala from Sri Lanka and Rau (1952) reported occurrence of Et.virescens Butl. on tea from South India. During nineteen fifties and sixties this pest was recorded to be a sporadic pest (Anonymous, 1994). Hill (1983) in ‘Agricultural insect pests of the tropics and their control’ has reported Eterusia magnifica as defoliators from India and South-East Asia. Muraleedharan (1991) has given brief biology of this pest and suggested its control measures. Again in 1993, Muraleedharan had mentioned about rare occurrence of this foiivore in the tea growing areas of South India. An account of biology of red slug is available in the treatise ‘Pests of tea in North-East India and their control’ (Anonymous, 1994). Chakravartee (1995) had mentioned that a destructive pest like red slug should not be allowed to grow to an epidemic stage and hence control measures should be undertaken once the pest is seen in tea. Of the 41 recognised species of Helopeltis, 26 are restricted to Africa and 15 are distributed in Austrasian region (Stonedahl, 1991; Stonedahi et a!., 1995). Helopeltis spp. are serious pests of various cultivated plants in old world tropics. The damaging effects of these insects on tea plants in India was documented over a century ago by Peal (1873) and Wood-Mason (1884). Subsequently, the species responsible for causing damage to tea was identified as H.theivora Waterhouse, -28- 1886. In south India, its outbreak was reported around 1920 on tea (Shaw, 1928; Rao, 1970). Tea shoots were damaged by H.antonii in Sri Lanka (Mann, 1907; Ballard, 1921), H.bradyi 'm Indonesia and Malaysia (Leefmans, 1916; Lever, 1949), H.bergrothi In Malawi-Africa (Leach and Smee, 1933), H.cinchonae in Malaysia (Lever, 1949), H.clavifer in New Guinea (Smith, 1978), H.fasciaticollis in China (Xie, 1993), H.sumatranus in Sumatra (Miller, 1941), H.schoutedeni in Malawi (Rattan, 1988) and H.theivora in India and Indonesia (Mann, 1907; Leefmans, 1916; Ballard, 1921). H.schoutedeni in Malawi was capable of causing a phenomenal loss in crop, up to 55% and short-term loss can be considerably higher (Rattan, 1984). Due to its infestation, an average of 25% of the total crop was lost. The loss due to the attack of Helopeltis theivora in south India varied from 11-100% during the high cropping season (Rao and Murthy, 1976). In North-Eastern India, a miximum loss of 7.5 lakhs kg. of made tea / year has been reported (Das, 1984). Prior to use of modern chemical pesticides, crop losses on tea plantations due to H.theivora in India sometimes reached 100% (Muraleedharan, 1987). The tea red spider, Oiigonychus coffeae is a serious pest of tea throughout the world. Out of the 12 species recorded, the red spider mite is the major one (Banerjee, 1988, 1993). The genetics of mite reproduction (Banerjee, 1979) also underlines the interplay of biological and physical factors that have made this species ubiquitous on tea the world over (Banerjee and Cranham, 1985). Although it primarily attacks tea, the mite causes considerable damage to jute and is also known to attack cotton, rubber, citrus, mango, oil palm and many other tropical plants and weeds (Das, 1959). The mite attacks Grevillea robusta and Albizzia falcata in tea plantations of Sri Lanka (Cranham, 1966); has also been reported -29- from Deris robusta a id Tephrosia Candida in India (Andrews, 1928). In s e ^ e damage the leaves get dried, leading eventually to defoliation (Das, 19B9; Banerjee, 1965, 1980). Red spider mite causes considerable loss in tea production (Lima et a!., 1977; Das, 1983; Mkwaila, 1983; Kilavuka, 1990). Red spider mite has gained economic importance because of crop failure for a period of two or more months due to complete defoliatron. It is reported to cause crop loss between 6-11% in the Dooars and 5-7% in Assam (Sen and Chakraborty, 1964). Crop loss also varies between 5-20% in North-East and south India (Aswathy and Venkatakrishnan, 1977; Muraleedharan and Radhakrishnan, 1989). 3.2. Pesticide use patterns in tea Sustainable and cost effective crop protection requires the optimum use of chemicals and non-chemical pest control techniques (Chakravartee, 1995). It has been estimated that annual loss in agriculture due to pests, diseases and weeds in India is around Rs. 6,000-9,000 crores. The consumption of pesticide is estimated to be 90,000 tonnes and around 0.336 kg / hectare as against 10.75 kg or more in developed countries (Atwal, 1986). The annual loss of tea crop in N.E. India due to pest, diseases and weeds is estimated to be about 85 million kg valued at Rs. 425 crores (Barbora and Biswas, 1996). Of the total pesticide applications, only 3.5 rounds were acaricides and the rest were other insecticides. It was also indicated that nearly 65% of the pesticide is used during the first half of the year (January- June) and balance 35% between July and December. 80% of the acaricides was used between January and June and in case of insecticide, the seasonal use was observed to be almost uniform which apparently means that the activities of insect pests remain consistent in the region throughout the year (Sannigrahi and Talukder, 2003). -30- It has been estimated that tea in India harbours atxjut 125 species of pests and in order to tackle these, extreme care must be taken befc^ a pesticide is introduced to tea for pest control (Das, 1962) specially to avoid residue build up. The problems of pest resurgence, pest resistance and secondary pest outbreak were reported in the past (Das, 1959). He also inferred that poor control of Helopeltis with chlorinated hydrocarbon (DDT) m ightte due to the outbreak of resistant strains of Helopeltis. De Bach (1974) in his classical book “Biological control by natural enemies” mentioned the cause of upsets and outbreaks of tea tortrix and leaf-eating caterpillars during late fifties in Sri Lanka due to insecticide use. The continuous use of copper fungicides against blister blight {Exobasidium vexans) disease had been proved to be responsible for mites build up in Sri Lanka, India and Indonesia (Cranham, 1966; Venkata Ram, 1966; Oomen, 1982). Compounds such as methoxychlor and carbaryl were found to increase red spider mite infestation (Cranham, 1966). 3.3. Hydrolytic and oxidoreductase enzymes of lepidopteran, sucking and mite pests Digestive enzymes are nearly universal in Animalia, but a means of delivering them efficiently to an external food source requires special modifications. The source of the digestive enzymes may be specialized structures, such as maxillary or salivary glands found in many insects and mites, or the midgut, as in some beetles and lepidopteran larvae (Evans, 1992; Snodgrass, 1935; Christeller et al., 1992). The fundamental objective of digestion is to render macromolecules into simple compounds that can be absorbed and circulated (Gilmour, 1961; House, 1974). The digestive enzymes are all hydrolases (Baldwin, 1967), including proteinases, lipases, carbohydrases and nucleases (Gilmour, 1961; House, 1974). Proteinases probably are the most important liquefaction enzymes for insect predators (Cohen, 1993; Miles, 1972). The digestion mainly seems to occur in the midgut where a variety of -31 - enzymes are available in ^ n d a n c e (Hori et al., 1981). Proteinases and peptidases from tfie intestinal tract oTSBi-instar larvae of Heliothis zea have been identified by Lenz et al. (1991). Herbivores possess various physiological and morphological traits that enable them to exploit their host plants. All herbivores deal with chemicals that are potentially damaging to their cellular processes, these come from various sources including secondary chemicals of plants that can be toxic or antinutritive to them (Duffey and Stout, 1996). The morphological traits of the gut have been described for most insect groups, physiological traits of the digestive tract are less well known (Appel, 1993). Some herbivores maintain extreme physiochemical conditions in the digestive tract, presumably to enhance digestion and inhibit the activity of some allelochemicals. In the larval Lepidoptera, the midgut is the primary site of digestion and absorption and midgut epithelial cells are modified to generate pHs depending on the species (Berenbaum, 1980; Dow, 1984, 1986; Appel, 1993). In addition physiochemical conditions of the midgut in caterpillars are likely to have a major impact on nutrient digestion and allelochemical activity (Appel and Maines, 1995). Lepidopteran larvae have been the subjects of study largely due to their impact on economically important plants. The digestive enzymes of these larvae are of interest both as a target for insect control and because of their unusual ability to function in the alkaline lepidopteran midgut (Christeiler et al., 1992). Digestive proteases catalyse the release of peptides and amino acids from dietary protein and they are found most abundantly in the midgut region of the insect digestive tract (Jongsma and Bolter, 1997). Most of the midgut proteolytic enzymes in lepidopteran larvae have been shown to be extracellular proteases with high pH optima which are well suited to the alkaline conditions of the midgut (Applebaum, 1985). Herbivores also produce salivary enzymes constitutively, prior to ingestion, that minimize the effectiveness of plant defenses, such enzymes are applied to leaf wounds as the herbivores chew -32- and these may reduce the activation of induced deferses in plants (Karban and ^ra w a l, 2002). Herbivore saliva is also known to c o rto i oxidative enzymes like peroxidase and polyphenol-oxidase (Feltor> and Eichenseer, 1999). Relatively little attention has been focused on the antioxidant defenses in the gut lumens of insects. However in the gut lumen, the ingested phenolic compounds may become extensively oxidized (Barbehenn and Martin, 1994; Barbehenn et al., 1996). A variety of antioxidant enzymes protect caterpillar tissues and extracellular fluids from oxidative damage. Among the most widely studied enzyme is catalase. The catalase activity was detected in the midgut tissues and regurgitate of larval Helicoverpa zee, Spodoptera exigua, Manduca sexta, and Heliothis virescens (Felton and Duffey, 1991). Like caterpillars, other herbivores may also secrete saliva that interferes with plant defenses. The chemical composition of the saliva of heteropteran insects is crucial for effective feeding. These insects rely heavily on saliva for extra-oral digestion (Cohen, 1998) and detoxification of defensive chemicals (Miles and Oertli, 1993). Sucking bugs deposit salivary secretion in or on plants when feeding, which significantly influences the physiology of the affected plant tissues. Some of the secretions result in phytotoxemia (Gopalan, 1976). The ability of insects to use plant materials as food is indicated by the presence of specific digestive enzymes in their saliva (Zeng and Cohen, 2000). In many heteropterans specific digestive enzymes for phytophagy include amylase and pectinase (Cohen, 1996). Proteolytic activity has also been detected in salivary glands of mirid bugs such as Lygus rugulipennis (Laurema et al., 1985) and Creontiades dilutus (Colebatch et al., 2001). Mirids use their digestive enzymes through the salivary canal to liquefy food into nutrient-rich slurry (Miles, 1972; Hori, 2000; Wheeler, 2001). The food slurry is ingested through -33- the food canal and is passed ^to alimentary canal where it is further digested and absorbed (Cohen, 2000). Salivary secretions of phytophagous Hemiptera contain various organic and inorganic compounds (Miles, 1972). An important group of them are proteins, mainly various enzymes, playing a fundamental role in food digestion of sucking-piercing insects (Miles, 1968; Miles and Sioviak, 1970; Peng and Miles, 1988a; Baumann and Baumann, 1995). Among these, polyphenol-oxidase uses molecular oxygen to catalyze two different types of reaction that are hydroxylation of monophenols to o- diphenols and oxidation of polyphenols to quinones and further dark brown or black pigments, melanins (Robinson et ai, 1991). Peroxidases use hydrogen peroxide to oxidize phenols and other aromatic derivatives (Deimann et ai, 1991). Both these oxidoreductases have been identified in the salivary secretions of aphid species (Miles and Peng, 1989; Madhusudhan and Miles, 1993) and they were found to be involved in overcoming the plant defenses by neutralizing phenolics and their derivatives (Miles, 1969; Urbanska and Leszczynski, 1992). Digestion is the process by which food molecules are broken down into smaller molecules that can be absorbed by the gut tissue. As a general adaptation of the digestive apparatus of stored product mites to starch digestion and utilization (Bowman and Childs, 1982; Bowman, 1984), amylases catalyze the initial hydrolysis of starch into oligosaccharides, an important step towards transforming sugar polymers into simpler units that can be assimilated by the organism. The high level of activity of the digestive a-amylases in the whole body extract from Acarus siro was detected by Hubert et at. (2005). In Dermatophagoides pteronyssinus amylase, protease and lipase activity was associated with digestive processes (Stewart et ai, 1992). The endogenous enzymes of protective systems (EEPS) including catalase, peroxidase were found in a number of organisms (Fridovich, 1977). It has been - 34- shEHA/n that the etevated EEPS activities were closely relaaed to the resistance of orgaffTtans to unfavourable environments (Packer, 1984). The enhanced anti oxidation enzymes (catalase and peroxidase) activities could reduce the effects of the loxic products on mites, resulting in defensive power strengthened, and survivorship and reproduction power of mites increased, thus density of mites on the host plant increased (Zhang et a!., 2004). Grubor-Lajsic et a/. (1997) reported that exposure to cold conditions significantly increased the activities of the antioxidant enzymes of two larval Lepidoptera, Ostrinia nubilalis and Sesamia cretica. Aucoin et at. (1991) reported that catalase might be inducible defenses against phototoxins by insects. 3.4. An overview of enzyme based biochemical resistance Nearly 40 years of studies, all over the world, suggest that insecticide resistance could be correlated with quantitative and/or qualitative changes in insecticide metabolizing enzymes or a change in target site or appearance of a protein in cuticle which retards the penetration of an insecticide of easy sequestration of insecticides by binding on a protein. The increased detoxification capabilities occur more frequently than the alteration of the insecticide target sites. The resistance associated enzymes could easily be assayed in laboratory. Efforts have been made to estimate the resistance associated enzyme activity to surrogate substrate usually naphtholic esters, whose products can be measured either in solution or cellulose filter paper or on nitrocellulose membranes. Further more, these surrogate substrates can be used to probe the isozymes and their mobility variance following electrophoresis and electrofocussing. The esterase banding patterns was proved very successful in the diagnosis of insecticide resistance in aphids and jassids. This technique is straightfonward to use when resistance is due to the production of a detoxifying enzymes (Mehrotra and Phokela, 1996). There are three major types of detoxtfication -35- enzymes; 1) broad-spectrum oaodases such as mixed function oxidases or monoxygenases that include cytocttfome P-450 enzyme system, 2) hydrolases that breaks up esters, ethers and epoxides and 3) conjugation systems such as glutathione S-transferase, which are mediated to cover up the reactive part of the toxic chemical and further facilitate its removal. Every type of detoxification enzyme has been documented to play a role in the development of some form of resistance against various classes of insecticides. Organophosphorous and pyrethroid insecticides are mainly degraded by hydrolases and the involvement of an increased hydrolytic enzyme activity may be suspected when insects develop resistance against these chemicals (Matsumura, 2003). Incresed carboxylesterase and acetylcholinesterase are often associated with insecticide resistance (Abdel-Aal ef a/., 1993). Esterases, in general have been noted to play a number of significant role in the hydrolysis of various chemicals that contain ester linkages and degrading various pesticides with carboxyl or amide-groups. These pesticides include organophosphates and synthetic pyrethroids (Kmeger and O’ Brien, 1959; Needam and Sawicki, 1971; Miyamoto and Suzuki, 1973; Motoyama and Dauterman, 1974; Hama, 1976; Yu and Terriere, 1977; Hughes and Devonshire, 1982; Oppenoorth, 1982; de Malkenson et al„ 1984). Differences in the amount of esterase activity between two strains of the same insect species is considered an indicator of relative sensitivity to certain insecticides, subsequently, various biochemical assays have been used for insect populations as possible indicators of insecticide resistance (Brown and Brogdon, 1987). One of the enzyme assays uses alpha-napthyl acetate (a-NA) as a model substrate for general esterase activity in wide variety of insects (Devonshire, 1977; Georghiou and Pasteur, 1978, 1980; Ferrari ef a/., 1993). Either high a-NA activity or extra esterase bands stained with a-NA have been associated with resistance (Lalah et al., 1995). A polyacrylamide gel electrophoresis assay has - 36 - been ised to identify and cliaracterize greenbug resistance (Si\aicumaran and Mayo, 1992; Siftrfran et al., 1993; Shufran and Wilde, 1996>. Electrc^jhcretic studies on organophosphate resistance of the cotton aphid, Aphis gossypii have indicated clearly the involvement of particular distinct isozynnatic forms of carboxylesterase in resistant strains (Owusu et al., 1996>. The greenbug, Schizaphis graminum produced a single esterase band that was either absent or undetectable in susceptible green bug (Ono etal., 1994). 3.5. Specific role of esterases, glutathione S-transferases and acetylchoiin- -esterases in insecticide resistance Esterases are the most significant enzymes in insect causing insecticide detoxication. Organophosphate (OP), carbamate and pyrethroids contain carboxylester and bonds that are subject to attack by esterase enzymes. These esterases can often be separated into isozymes Vi^ith different substrate specificities. Insect esterases are very diverse and can include monomers, dimmers and multimers, which mean that their relative molecular mass can cover a wide range. Polymorphism is a notable characteristic of insect esterases. Multiple forms of esterases are present in the soluble, cytosolic fraction of insect (Brattsten, 1992; Dauterman, 1985). Of the multiple forms of esterase isozymes that exist in insects, few participate in insecticide metabolism (Maa and Terrier, 1983). Each isozymes probably has a certain range of substrates. Unlike the monooxygenase reaction, esterases use low energy co-factors (Dauterman, 1985). Different types of esterases (Al, B1, A2, 82) have been recognized in organophosphate (OP) insecticide resistant populations of Culex pipiens complex throughout the world (Poirie et al., 1992) and overproduction of nonspecific esterases is a common mechanism of -37- resistance. For B1, resistance to C3P insecticides has been shown to be due to sequestration of insecticide and ovaptiduction of esterase B. This production at enhanced scale is due to gene anrtpBfication (Callaghan et al., 1994). The current work of Jayawardena ef al. (1994) on Cx.quinquefasciatus revealed that a strain with elevated A2 and B2 is resistant to a broad range of organophosphate insecticides. Enzymatic assays suggested that sequestration rather than metabolism is the primary mode of operation of these esterases on malathion. The basis of malathion resistance in the adults of Anopheles arabiansis from Sudan was a carboxylesterase (Hemingway, 1983) Malathion resistance due to an increase in degradation at the carboxylester linkage is a common detoxication pathway that has been implicated in An.culicifacies (Herath and Davidson, 1981); An.stephensi (Hemingway, 1982); Blattella germanica (Heuval and Cochran, 1965); Cx.tarsalis (Matsumura and Brown, 1963): Tetranychus urticae (Matsumura and Voss, 1964); Tribolium castaneum (Dyte and Rowlands, 1968). Esterase cleavage has been implicated in OP and pyrethroid resistance in Musca domestica (Funaki et al., 1994). Resistance to pyrethroids in Blattella germanica was partly due to elevated esterases (Atkinson et al., 1991). Esterase dependent cross-resistance between OPs and pyrethroids have been detected in several species. In OP resistant M.domestlca and Culex mosquitoes, the esterases responsible for cross-resistance are thought to be involved in pyrethroid hydrolysis (Soderlund and Bloomquist, 1990). The best documented example of esterase based metabolic resistance to OPs, carbamate and pyrethroid insecticides is that found in Aphid, Myzus perslcae (Devonshire and Moores, 1982) in which the overproduction of esterases FE4 and E4 responsible for insecticide hydrolysis and sequestration have been shown to be caused by amplification of a structural gene. The massive overproduction of any esterase protein by resistant M.persicae may - 38 - result in !he detoxication of insecticidal esters first by sequestration and then by hydrolysis p^vonshire and Field, 1991,1995). Gel eiectropFhoresis techniques have been used extensively in the investigation of the taxonomy, systematics and population genetics of a wide range of animals and plants. The techniques involve the electrophoretic separation and specific histochemical staining of enzymes and allow exaTnination of the variation of immediate protein products of genes (Ayala, 1983; Easteal and Boussy, 1987). The genes at different loci, giving rise to families of enzymes called isozymes and these may be of different molecular weight and electric charge and hence show mobility variation when separated on an electrophoretic gel (Loxdale, 1993). Electrophoresis has also aided the monitoring and investigation of insecticide resistance in populations of some aphid species like Myzus persicae (Devonshire, 1975a; Sawicki et a!., 1978; Baker, 1979) and in Aphis gossypii (Owusu et a!., 1996). In Schizaphis graminum, using a-napthyl acetate as substrate for nondenaturing polyacrylamide gel electrophoresis (PAGE) showed several strongly stained esterase bands with faster electrophoretic mobility from the organophosphate resistant greenbugs and these esterases were absent or expressed at low levels in the organophosphate susceptible greenbugs (Ono et a!., 1994; Zhu and Gao, 1998; Zhu and He, 2000). Glutathione S-transferase (GST) is a family of multifunctional isozymes found in all eukaryotes. One of the main functions of GST is to catalyse xenobiotics, including pesticides in the marcapturic acid pathway leading to the elimination of toxic compounds (Hayes and Pulford, 1995). In insects, this family of enzyme has been implicated as one of the major factors in neutralizing the toxic effects of insecticides (Clark et al., 1986; Grant et a!., 1991; Salinas and Wong, 1999). The majority of studies on insect GSTs have focused on their role in detoxifying foreign compounds, in -39- particular insecticides and plant allelochemicais and more recently, their role in mediating oxidative stress responses {Clsitf, el a!., 1986; Wang et al., 1991; Fournier et a!., 1992a; Ranson et al., 2001; Vontas et al., 2001; Sawicki et al., 2003). GSTs are important in phase I metabolism of organophosphorous and organochlorine compounds and play a significant role in resistance to these insecticides in insects (Yu, 1996; Clark and Shamaan, 1984; Ranson etal., 1997). Many studres have shown that insecticide resistant insects have elevated levels of GST activity in crude homogenates, and have suggested the possible role of GSTs in insecticide resistance (Armstrong and Suckling, 1990; Reidy et al., 1990; Smirle, 1990; Kao et al., 1989; Clark et al., 1986; Ottea and Plapp, 1984). In insects, GST isozymes are present in three to four forms in house flies and at least two in Australian sheep blowflies (Clark and Dauterman, 1982; Kotze and Rose, 1987). Several insecticide resistant strains of housefly have been reported to have elevated GST activity in crude homogenates against organophosphates (Clark etal., 1986; Wei etal., 2001). The GSTs are involved in 0-dealkylation or dearylation of organophosphates (OPs) (Hayes and Wolf, 1988). High frequencies of profenofos resistance were correlated with GST activity toward 1-chloro-2, 4-dinitrobenzene in larvae of H.virescens that were collected in Louisiana cotton fields during the 1995 cotton growing season (Harold and Ottea, 1997). Moreover, GSTs are involved in the dehydrochlorination of DDT in M.domestica (Clark and Shamaan, 1984) and are the primary metabolic mechanism of resistance to this insecticide. Finally, a recent study suggests that GSTs act as antioxidant- defense agents and confer pyrethroid resistance in Nilaparvata lugens and possibly in other insects (Vontas et al., 2001). Enhanced activities of GSTs that confer insecticide resistance result from both quantitative and qualitative alterations in gene expression. First, there is evidence for over-expression of one or more GST isoforms -40- in resistant insects. For example, tiie tiigii activity found in an insecticaie-resistant strain of M.donmsSca is correlated with high level of GST1 transcript (Fcaanier et al., 1992a). Similar phenomena were also found in insecticide-resistant Aeties aegypti (Grant et al., 1991), Anopheles gambiae (Prapanthadara ef al., 1993; Ranson et al., 2001), and Plutella xyostella (Ku et al., 1994). Moreover, qualitative differences of GSTs were also present between susceptible and resistant insects. For example, most subcellular fractions from susceptible M.domestica had higher conjugation activities toward 1-chloro-2, 4-dinitrobenzene than the fractions from the Cornell-R strain, but all fractions from the susceptible strain had lower conjugation activities toward 1, 2-dichloro-4-nitrobenzene than fractions from the Cornell-R strain (Chien et al., 1995). In addition, quantitative and qualitative alterations can be co^xpressed and confer resistance in one resistant strain. For example, GSTs from a DDT- resistant strain of An.gambiae had an altered GST profile and one of the GSTs was increased 8-fold in a resistant strain compared with the susceptible strain (Prapanthadara et al., 1993). The major target site for both OPs and carbamates (O’Brien, 1960) is acetylcholinesterase, which acts by binding to the neurotransmitter (acetylcholine) in some synapses of the nervous system. Reduced sensitivity of acetylcholinesterase (AChE) to these insecticides is well-studied and has been expressed in a number of insects (Oppenoorth, 1985), such as M.domestica (Walsh et al., 2001), the aphid, Nasonovia ribisnigri (Rufingier et al., 1999), the Colorado potato beetle, Leptinotarsa decemlineata (Zhu et al., 1996), and the fnjit fly. Drosophila melanogaster {Mu\ero et al., 1992). Reduced sensitivity of AChEs to OPs and carbamates is also expressed in H.vlrescens as a major resistance mechanism. For example, compared with a susceptible strain, AChE activity from a methyl parathion resistant strain of H.virescens was 22-fold less sensitive to inhibition by methyl paraoxon in both larvae -41 - and adults. This resistant ACliE was also le s sensitive to structurally analogous OPs, and to the N-methyl carbamates (Brown a id Bryson, 1992). Similar results were demonstrated in thiodicarb-selected larvae (Zhao et a!., 1996) and OP and carbamate-resistant adults of H.virescens (Kanga and Plapp, 1995). Reduced sensitivity of AChE to OPs and carbamates is conferred by point mutations. Although genetic and biochemical bases of reduced sensitivity of AChE have been examined in a number of insects, they have been well-studied in D.melanogaster, M.domestica and L.decemlineata. in the nucleotide sequence of AChE in D.melanogaster, Tyr^°® was first reported as a potential site of a point mutation conferring resistance to insecticides (Mutero et ai, 1992). Subsequently, amino acid substitutions at four different sites (Phe"® to Ser, lleu^®® to Val, Gly^°^ to Ala, and Phe^®® to Tyr) were found in OP-resistant D.melanogaster (Fournier et ai, 1993) and at five different sites (Var®° to Leu, Gl/®^ to Ala or Val, Phe®^^ to Tyr and Gly^®® to Ala) in M.domestica (Walsh et ai, 2001). The expression of varied combinations of mutations in a single AChE gene resulted in different patterns of resistance among OPs, and levels of resistance increased in an additive way. To date, more mutations in AChEs of D.melanogaster have been reported as being possibly involved in OP and carbamate resistance, but their toxicological significance has not been demonstrated (Villate et ai, 2000). interestingly, reduced sensitivity of AChEs to OPs and carbamates also occurs by replacement of a different amino acid (Ser^^® to Giy) in an azinphosmethyl-resistant strain of the Colorado potato beetle, L.decemlineata (Zhu etai, 1996). -42- 4. Materials and Methods 4.1. Collection of pest specimens: Buzura suppressaria, Eterusia magnifica, Helopeltis theivora and Oligonychus coffeae from tea plantations Periodic surveys were undertaken at different organic and conventional plantations of Terai, the Dooars and Darjeeling foothills located in the North Bengal, to name some of them are Singell Tea Estate (T.E.), Castleton T.E. of Darjeeling foothills, Matigara T.E., Maruti T.E., Kamalpur T.E., Chandmani T.E., Atal T.E., Gangaram T.E., Dagapur T.E. and North Bengal University campus plantation of Terai, and Debpara, Lakhipara, Dalgaon, Nagrakata and Nangdala T.E. of the Dooars. These TEs had mostly matured tea bushes of Tocklai vegetative clones that produced vigorous flush in Terai and the Dooars agro-climate. Four important tea pests of this region namely Buzura (Biston) suppressaria Guenee (Looper Caterpillar), Eterusia magnifica Butler (Red Slug Caterpillar), and Tea mosquito bug, Helopeltis theivora Waterhouse and Red spider mite, Oligonychus coffeae (Nietner) have been considered in the present study. Usually caterpillar stages and adults of loopers and red slug were collected (handpicked) from tea bushes and trunks of shade trees in the morning hours. Adult stages of tea mosquito bug and red spider mites were also collected in the morning and during dusk. The collected larvae and adults of sucking pests were brought to the laboratory in polythene packets and containers, along with the leafy twigs of tea. The specimens that were collected from conventional plantation, managed by regular spray of pesticides, have been referred to in the text as “pesticide-exposed" or “field-collected", and the specimens maintained in laboratory without pesticide exposure have been mentioned in the text as “laboratory-reared”. -44- 4.2. Laboratory rearing (culture) of the pests for experimentation Natural food (tea leaves), which was provided for rearing and for various experiments was collected from the experimental tea plot of the Department of Zoology at North Bengal University campus. The plantation was about 14 years old. The Tocklai vegetative clones, TV 1, TV 18, TV 25 and TV 26. The experimental garden was mainained under usual cultural practices with organic manure and no pesticide application. TV 1: It is one of the earliest clones released by Tocklai Experimental Station’, Assam (India) in 1949. TV 1 is a standard clone, having high yield potential and high quality. It has a compact frame with acute branch angle (<50°). Leaves are erect, medium sized with pubescence on lower surface and sunken stomata. Surface matty in nature. Fairly draught tolerant. It is a hybrid of Assam x China origin. TV 18: It is of Cambod hybrid origin. More or less of compact frame with glossy medium sized leaf. Leaf axil with an angle of 50° to 70°. It has a high yield potential but of average quality. Leaf has pinkish pigmentation in the petiole. TV 25 and TV 26: These are high yield clones of Cambod type. Morphologically both the clones are similar in nature, having compact frame and glossy medium sized leaf. Both the clones are fairiy drought tolerant having high yield potential but of average quality. Leaf axil being >70°. Larvae of B.suppressaria and Et.magnifica were randomly collected from conventional plantations (maintained by synthetic pesticide spray) of eastern Dooars and western Terai and Darjeeling foothills of West Bengal State. These larvae were reared separately on the Tocklai vegetative clones, TV 1, TV 18 and TV 25, for two generations at 27 ± 2° C and 72 ± 2 % RH with a photoperiod of 12:12 hrs (L: D), in -45- transparent containers (30 x 30 cm) with a supply of fresli tea twigs, obtained from tlie experimental tea plot of Department. Field populations of Helopeltis theivora were reared in laboratory on Tocklai vegetative clones (TV 1) for two generations at 25 ± 1° C; 85 ± 5 % RH with a photoperiod of 12:12 hrs (L: D) in transparent containers (20 x 20 cm) with a regular supply of fresh tea twigs. Oligonychus coffeae populations were collected from organic and conventional tea plantations of Darjeeling foothills areas. These were maintained in laboratory on Tocklai vegetative clones (TV) at 25 ± 2°C with 70 ± 5 % RH. 4.3. Dissection of salivary glands, midgut and cerebral ganglia The 5*^ instar larvae of B.suppressaria and Et.magnifica were under ice-cold dissection buffer in a paraffin tray. Incision was given along the mid dorsal line of the larva. Gut was also slit and the gut contents were removed by gentle stroke of brush. Fat bodies and food particles were scraped out. The salivary gland, midgut and cerebral ganglia were removed and were immediately preserved in ice-cold homogenization buffer (placed in an ice bath). In case of Helopeltis, the insects were placed at -20° C for 4 min and dissected in ice-cold phosphate-buffered saline (pH 7.2) under a dissecting microscope. The salivary gland complex, including all lobes, accessory glands and tubules was exposed by gently pulling the head and prothorax away from the abdomen with fine forceps. Subsequently the midgut and cerebral ganglia was removed by dissecting the body. The whole body of Oligonychus coffeae was taken for enzyme analysis, due to its minute size. -46- 4.4. Biochemical analysis 4.4.1. Isolation of salivary and midgut enzymes of 4.4.1.1. B.suppressaria Enzyme extraction was done from laboratory-reared 5'^ instar larvae of B.suppressaria, and from larvae of the same stage collected from natural populations occurring in conventionally managed plantations that were subjected to routine spraying of synthetic pesticide. Each larva was dissected and its salivary gland, midgut and cerebral ganglia were removed. Dissections were carried out in ice-cold sodium phosphate buffer, 0.1 M, pH 7.0 using sterilized scissors and needles. Salivary glands and midguts were homogenized separately in fresh sodium phosphate buffer containing 0.01 IVI each of EDTA (Ethylene Diamine Tetra Acetic Acid) and 0.5% Triton X-100. The volume of the buffer was adjusted to produce similar protein concentrations for the homogenates of each individual. The homogenate was centrifuged at 10,000 x g for 15 min at 4° C. The supernatant of this preparation was stored at -20° C for future use. 4.4.1.2. Et.magnifica The 5'^ instar larvae of Et.magnifica were collected from laboratory colonies and from natural populations occurring in conventionally managed plantation (with synthetic pesticide spraying). Each larva was dissected and its salivary gland and midgut were collected. Dissections were carried out with the help of sterilized scissors and needle in ice-cold sodium phosphate buffer (0.1 M, pH 7.0). After removing the fat bodies and food particles from the midgut it was homogenized in fresh sodium phosphate buffer containing 0.01 M each of EDTA (Ethylene Diamine Tetra Acetic Acid) and 0.5% Triton X-100. Similarly the salivary gland was also processed. The volume of the buffer was adjusted to produce similar protein concentration for the homogenates -47- of each individual. Eacii liomogenate was centrifuged at 10,000 x g for 15 min at 4°C and tile supernatant of tinis preparation was stored at -20° C for future use. 4.4.1.3. H.theivora Adults were used for enzyme extraction following slightly modified method of Cohen (1993). The insects were placed for immobilization at -20° C for 4 min and dissected in ice-cold phosphate buffer (pH 7.2) under dissecting microscope. The salivary gland complex, including all lobes, accessory glands and tubules was exposed by gently pulling the head and prothorax away from the abdomen with fine forceps. Subsequently the midgut and cerebral ganglia were removed by dissecting the body. The salivary glands of 10 insects were removed, placed in 1 ml of phosphate buffer, homogenized and centrifuged at 12, 000 x g for 10 min at 4° C. The supernatant was placed in a 1.5 ml centrifuge tube and kept at -20° C for use (within 48 h). The midgut and cerebral ganglia of the same 10 insects were homogenized and processed in the same way as the salivary glands. The enzyme extraction was done from three separate batches of insects collected from different conventional plantations and laboratory rearing. 4.4.1.4. O.coffeae (whole body) Oligonychus coffeae were collated by using camel hair brush from the surface of the tea leaves and about 100 mg of its fresh weight were placed in a homogenizer in 1 ml of phosphate buffer. This was then homogenized and centrifuged at 15, 000 x g for 10 min at 4° C. The supernatant was placed in a 1.5 ml centrifuge tube and kept at -20° C for use (within 48 h). -48- 4.4.2. Gel diffusion assay of salivary, midgut digestive enzymes (amylase, protease) of 4.4.2.I. B.suppressaria The 5'^ instar larvae of B.suppressaria was dissected out with the help of sterilized scissors and needle in ice-cold sodium phosphate buffer (0.1 M, pH 7.0). Each pair of glands and midgut was homogenized in a glass tissue grinder separately. The homogenate was then centrifuged at 10,000 x g for 5 minutes at 4°C. Aliquots of supernatants were used for tests of different digestive enzymes. Amylase Amylase was measured by modified Somogyi (1960) method. A solution of starch (Igm / litre) in phosphate buffer (pH 7.0) was mixed with 0.5 % agar (0.005 gm/ 10ml) and heated for 10 mins in a boiling water bath. The slurry was poured into pettry plates, and well of 2.5 mm diameter was cut into the gel. 10 pi aliquots of enzyme extract was dispensed in each well. A postitive control of commercial amylase (0.001 g/ml) was used concurrently. Plates were kept at 30°C for 20 hrs and developed with 0.002 N iodine in 2 % potassium iodide for 3 min. Rings of clear area (against a distinctly blue background) were considered as positive. Protease Proteases were determined by casein-gel method of Bjerrum (1975). 10 pi of both salivary and midgut homogenate were separately dispensed in wells of 2.5 mm diameter cut in the gel on a pettry plate. The gel was composed of 1 % casein mixed with 0.5 % agar, buffered to 7.8 with phosphate. The mixture was heated for 10 mins in a boiling water bath for gelling to take place. Sterilized double distilled water with commercial protease (0.001 g/ml) used as positive control. Plates were then covered with 3% acetic acid solution for 10 mins to stop the reaction and rings of diffusion -49- were observed for enzyme activity. Positive tests were those in wiiich clear rings appeared against a wiiite background. A.4.2.2. Etmagnifica Same procedure as B.suppressaria was followed for detecting amylase and protease of salivary and gut enzymes of Et.magnifica. 4.4.2.S. H.theivora In case of Helopeltis, the salivary glands and midgut of 10 insects were removed and placed in 1 ml of phosphate buffer, homogenized and centrifuged at 12, 000 X g for 10 min at 4° C. The supernatant was placed in a 1.5 ml centrifuge tube and kept at -20° C for future testing. Same procedure as described for B.suppressaria was followed to test the amylase and protease of the salivary and gut of H.theivora. 4.4.2.4. O.coffeae (whole body) 100 mg of fresh weight of O.coffeae was placed in 1 ml of phosphate buffer. This was then homogenized and centrifuged at 15, 000 x g for 10 min at 4° C. The supernatant was placed in a 1.5 ml centrifuge tube and kept at -20° C for use (within 48 h). Same procedure as described for B.suppressaria was followed for conducting the tests for amylase and protease enzymes present in O.coffeae. - 50- 4.4.3. Quantitative assay of digestive enzymes 4.4.3.1. Amylase Amylase activity in the salivary gland and midgut was determined after the method of Madhusudhan et al. (1994) combined with the method of Sadasivam and Manickam (1996) using dinitrosalicylic acid reagent. Quantification of enzyme product was deduced from a standard curve prepared using various concentration of maltose at 520 nm using UV-Vis spectrophotometer. The enzyme activity was expressed as |jM. mg protein"'', min 4.4.5.2. Protease Proteolytic activity was assayed after methods of Kunitz (1947) subsequently modified by Jayaraman (1981). 1% (w/v) casein was used as the substrate. 1 ml of casein prepared in 0.1 N NaOH was incubated with equal volume of enzyme. After incubation for one hour, the reaction was terminated by the addition of 10% TCA and the acid-soluble peptides were quantified using the biuret reagent at 520 nm using UV-Vis spectrophotometer. The enzyme activity was expressed as pg I mg of protein. 4.4.3.S. Lipase Lipase activity was measured following the method of Sadasivam and Manickam (1996). The enzyme activity was calculated as milliequivalent (meq) activity of free fatty acid / min/ g sample, 4.4.4. Assay of oxidoreductases 4.4.4.1. Catalase 3 ml of Hydrogen peroxide and phosphate buffer was allowed to stabilize at 25 ± 2 ° C for 10 min. After incubation period, 0.2 ml of salivary gland and midgut -51 - homogenate was separately added to cuvettes, and the change in absorbance was measured at 240 nm over a 10 sec period upto 4 min using UV-Vis spectrophotometer. Cataiase activity was expressed as decrease in OD of hydrogen peroxide I min I mg of protein (Laurema and Varis, 1991). 4.4.4.2. Peroxidase Peroxidase activity was measured by monitoring the increase in absorbance (OD) by 0.1 and noting the time required (in min) by using the slightly modified method prescribed by Hampton (1963). The enzyme reaction was started by adding 3 ml of 0.1 M phosphate buffer (pH 7.0), 0.05 ml guiacol solution, 0.1 ml enzyme extract and 0.03 ml hydrogen peroxide in a cuvette. The activity of peroxidase was determined by increase in OD / min / mg of protein at 436 nm. 4.4.4.S. Polyphenol-oxidase Polyphenol-oxidase activity was estimated after the method of Hampton (1963) at 495 nm for 4 min after the start of the reaction. The reaction was initiated by adding 2.5 ml of phosphate buffer (pH 6.5) and 0.3 ml of catechol solution (0.01 M) and then 0.2 ml of enzyme extract. Enzyme activity was calculated as the increase In the absorbance/ min / mg of protein. 4.4.5. Quantitative assay of detoxifying enzymes 4.4.5.1. General esterases The esterase activity was determined according to the procedure of van Asperen (1962). 1 ml of tissue supernatant (salivary glands or midgut homogenate) was mixed with 5 ml of substrate solution, 3.0 x 10 M a-napthy( acetate [in 0.04 M, phosphate -52- buffer, pH 7.0, containing 1% (v/v) acetone]. After 20 min of incubation at 30° C with siiaking, 1 mi of a fresliiy prepared solution of tlie dye, containing two parts of 1 % (w/v) diazo dye and five parts (w/v) of sodium laurylsulphate was added to the incubated mixture. A red colour immediately developed that quickly changed into a fairly stable blue colour, which was measured at 590 nm spectrophotometrically. The quantity of napthoi produced was determined from the standard curve of a-napthol. The nonenzymatic reaction (control samples) was accounted for by mixing 1 ml of buffer with the substrate solution and incubating as above. All samples and controls were read against the blanks. The experiments were repeated five times. 4.4.S.2. Glutathione S-transferases GST activity was measured according to Habig et at. (1974) using 1-chloro-2, 4- dinitrobenzene (CDNB). The reaction mixture, consisting of 50|xl of SOmlVI CDNB and 150 |il 50 mM reduced glutathione, was added to 2.77 ml phosphate buffer (lOOmM, pH 6.5). The enzyme solution (30 ^il) was then added to the above mixture. The content was shaken gently and incubated at 25° C for 2-3 minutes. A blank was run concurrently in the reference slot of the spectrophotometer. Absorbance was recoded for 6-7 minutes at 340 nm (e 340 = 9.6 mM'^ cm‘^). The increase in absorbance over 5 minutes period was considered for calculation. The GST activity assay was replicated five times. 4.4.5.S. Acetylcholinesterases Acetylcholinesterase activity was determined by the method of Ellman et al. (1961) with some modification. 100 )il of the enzyme solution was added to a test tube containing 2.86 ml of 0.1 M sodium phosphate buffer (pH 7.5) and the mixture was then incubated at room temperature for 5 min. To this 10 nl of 0.01 M 5,5' - dithiobis -53- (2-nitrobenzoic add) (DTNB) was mixed. After 10 min incubation of the above mixture 30 ^li of tiie acetyithiociioiine iodide in piiospliate buffer was added and the change in absorbance was determined at 412 nm. The change in absorbance was taken every 1 min for a period of 12 min. The increase in absorbance over 5 minutes period was considered for calculation. The test was replicated five times. The protein concentrations of the ail the above tissue supernatants were determined by the method of Lowry et al. (1951). Bovine serum albumin was used as standard. 4.4.6. Qualitative analysis of detoxifying enzymes of B.suppressaria, Et.magnifica, H.theivora and O.coffeae collected from pesticide-exposed (conventional plantation) and un-exposed (laboratory-reared) populations by Native Polyacrylamide Gel Electrophoresis 4.4.6.1. Esterase isozymes of salivary, midgut, or whole body extracts Native discontinuous polyacrylamide gel electrophoresis (5% stacking and 8% separating gels) was carried out. A continuous buffer system (Tris-glycine, pH 8.3) was used. Electrophoresis was conducted at 4° C with 10mA for 1.5 hrs (Davis, 1964) using vertical gel apparatus with power pack (Biotech make). The electrophoresed gel was stained for esterase according to Murphy et al. (1996). The gel was first pre-incubated in 100 ml of phosphate buffer (40 mM, pH 6.5) containing 0.02% a-napthyl acetate (0.02 gm of a-napthyl acetate in 2 mi of acetone) and the gel was then transferred to 100 ml of sodium phosphate buffer (40 mlVI, pH 6.5). After the pre-incubation step, 2.5% (w/v) Fast Blue BB salt was added. The gel was incubated in dark at room temperature for 20-30 mins with occasional shaking. The reaction was stopped after 15-20 minutes of incubation in distilled water.The gel -54- was then fixed in a fixative solution (glacial acetic acid: methanol: water = 1; 2: 7) for 30 min. The fixed gel was stored in 10% glycerol solution. The relative migration of esterase bands in the zymograms was determined by the Kodak digital science 1D Image Analysis Software, version 2.0.3. Relative mobility (Rm) was calculated as: distance migrated by the specific bands (cm) I distance migrated by the marker dye (cm). 4.4.6.2. Isozymes of Glutathione S-transferase The electrophoresed gel was stained according to Manchenko (1994) for GST. After electrophoresis, gels were preincubated in 20 ml of 4.5 mM reduced glutathione, 1 mM 1-chloro-2,4-dinitrobenzene (CDNB), and 1 mM nitro blue tetrazolium (NBT) in 0.1 M phosphate buffer (pH 6.5). The pre-incubations were carried out at 37°C for 10 min with gentle shaking. The gel was then transferred to a second solution and incubated for a period from 3 to 5 min with periodic agitation. The second solution consisted of Tris-HCI (pH 9.6) and 3 mM phenazine methosulphate (PMS). Blue insoluble formazan appeared on the gel surface, except in the areas with glutathione S-transferase activity. Because the superoxide dismutase also caused tetrazolium salt (i.e., NBT) reduction, a control gel was simultaneously processed with all reagents except CDNB to show the superoxide dismutase activities. Thus, glutathione S-transferase activity could be identified by comparing the control (without CDNB) and test (with CDNB) gels. The negatively stained gels were then photographed and the relative migration of glutathione S-transferase bands in the zymograms was determined by the Kodak digital science 1D Image Analysis Software, version 2.0.3. Relative mobility (R^) was calculated as: distance migrated by the specific bands (cm) / distance migrated by the marker dye (crti). -55- 4.4.6.3. Acetylcholinesterase isozymes Native polyacrylamide gel electrophoresis was performed in a vertical electrophoresis unit by using 8% separating and 5% stacking gel with a discontinuous Tris-glycine buffer system. 15 |jl of sample homogenate prepared from head region (brain) of the pests was loaded in each lane. Acetylcholiesterase activity was marked according to the method of Lewis and Shute (1966). The gel was preincubated with a mixture of 65 mi 0.1 M sodium phosphate buffer (pH 6.0) and 0.05 g acetyl thiocholine iodide. Then 5 ml 0.1 M sodium citrate was added to the gel buffer and the same was shake well. After that 10 ml of 30 mM copper sulphate (CUSO4) was added. Finally 10 ml 5 mM potassium ferricyanide was added in the reaction mixture and was shaken well. The incubation was completed when the background of the gel turned a yellowish brown. After incubation, the gel was washed for 1 hr with three changes of distilled water and then the gels were photographed and the relative migration of acetylcholinesterase bands in the zymograms was determined by the Kodak digital science ID Image Analysis Software, version 2.0.3. Relative mobility (Rm) was calculated as: distance migrated by the specific bands (cm) / distance migrated by the marker dye (cm). 4.4.6.4. Inhibition tests of general esterases and acetylcholinesterases of the pests Electrophoregrams of general esterase and aceylcholinesterase were prepared after Davis (1964) from the midgut, cerebral ganglia or whole body homogenates. After electrophoresis the same gel slab was vertically divided into two parts for the control and for inhibition tests. For inhibition tests of esterases and acetylcholiesterases, the gels were treated with substances regarded as inhibitors (Holmes and Masters, 1967). The inhibitors used were insecticides commonly used against tea pests i.e. organophosphate, -56- Quinalphos (Ekalux 25EC) (1:400) (v/v). i.e. 0.1 ml in 40 ml of water. In these tests, eiectrophoresed polyacrylamide gels were preincubated in dark at room temperature for 30 minutes in sodium phosplnate solution containing the inhibitor and then treated with the staining solution (which also contained the inhibitor). The other half of the same gel was treated with staining solution without any inhibitor. Using these tests, the isozyme bands of esterases and acetylcholinesterases of susceptible got blocked by the pesticides and could not be visualized in the stained and differentiated gel slabs, 4.5. Statistical analysis and computer application Documentation and analysis of zymograms were done by the software package of Kodak digital science ID Image Analysis version 2.0.3. Relative mobility (Rm) was calculated by: distance migrated by the specific bands (cm) / distance migrated by the marker dye. Smith’s statistical package was used to analyze the data where necessary. Excel and photoshop programmes were used for preparing tables and shaping photographs. The details of statistical analysis of the data have been mentioned under results and discussion. -57- 5. Results and Discussion 5.1. Results of quantitative assay of major liydrolases (digestive enzymes) and oxidoreductases of B.suppressaria, Et.magnifica, H.theivora and O.coffeae 5.1.1. Results of hydrolases of the four arthropod tea pests 5.1.1.1. Amylase In starch digestion, iodine is a good indicator witli a blue-black background in the agar gel plate. As starch gets digested, the blue-black colour disappears. In the positive control amylase activity was evident through ring formation whereas in the negative control no formation of halo was observed. In case of amylase detection test, rings of cleared areas (halo) against a distinctly blue-black background were observed. This indicated the presence of amylase in the saliva and midgut of B.suppressaria. The diameter of the halo in salivary amylase was 4.57 ± 0.14 mm vis-a-vis in midgut the halo was 6.60 ± 0.10 mm which indicated presence of a higher quantity of amylase in the midgut region of B.suppressaria (Table 1). Table 1. Measurement of halo diameter of digestive enzyme (amylase) of the salivary- -gland (SG) and the midgut (MG) homogenate of Buzura suppressaria (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial amylase 5.17 ±0.29 Amylase Salivary gland (SG) homogenate 4.5710.14 Midgut (MG) homogenate 6.60 ±0.10 The higher amylase activity in the midgut homogenate of B.suppressaria indicated the possibility of greater digestion of polysaccharides in midgut than its break down by saliva at the time of ingestion in the oral cavity (Table 2). -59- Table 2. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Buzura suppressaria (mean ± SD) Amylase activity (|jM. mg protein min Salivary gland Midgut B.suppressaria 0.318 ±0.006 a 0.405 ± 0.005 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test Formation of clear rings against a distinctly blue background in the agar gel plate indicated the presence of amylase both in the saliva and midgut of Et.magnifica. The diameter of the halo in the agar gel plate in case of saliva was 4.13 ± 0.12 mm and in the midgut was 4.52 ± 0.06 mm. Similar ring formation was also evident in the positive control while in the negative control no formation of halo was observed (Table 3). Table 3. Measurement of halo diameter of digestive enzyme (amylase) of the salivary- -gland (SG) and the midgut (MG) homogenate of Eterusia magnifica (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial amylase 5.17 ±0.29 Amylase Salivary gland (SG) homogenate 4.13±0.12 Midgut (MG) homogenate 4.52 ±0.06 In Et.magnifica amylase activity of equal quantity indicated almost similar polysaccharide digestion at salivary and midgut levels (Table 4). -60- Table 4. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Eterusia magnifica (mean ± SD) Amylase activity (fjM. mg protein min ~^) Salivary gland Midgut Et.magnifica 0.331 ±0.003 a 0.348 ± 0.005 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test The presence of amylase was observed both at salivary and midgut level in case of i-l.theivora. The gel diffusion assay showed that salivary amylase (halo diameter 4.18 ± 0.27 mm) was marginally lower than that of the midgut (halo diameter 4.92 ± 0.49 mm) (Table 5). The positive control showed the similar ring formation while in negative control no formation of halo was observed. In quantitative study also amylase was found to be slightly lower in salivary gland (0.0679 ± 0.0003 |jM . mg protein min ~^) than in midgut (0.0861 ± 0.0004 |jM . mg protein "V min "^) (Table 6). Table 5. Measurement of halo diameter of digestive enzyme (amylase) of the salivary- -gland (SG) and the midgut (MG) homogenate of Helopeltis theivora (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial amylase 5.17 ±0.29 Amylase Salivary gland (SG) homogenate 4.18 ±0.27 Midgut (MG) homogenate 4.92 ±0.49 -61 - Table 6. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Helopeltis theivora (mean ± SD) Amylase activity iu VI. mg protein min Salivary gland Midgut H.theivora 0.0679 ± 0.0003 a 0.0861 ± 0.0004 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test The agar gei diffusion assay showed the presence of amylase in the O.coffeae. The diameter of the halo was measured 3.55 ± 0.08 mm (Table 7). The positive control also showed similar ring formation vis-a-vis the negative control no formation of halo. The amylase activity was also evident in the whole body homogenate of O.coffeae (Table 8). Table 7. Measurement of halo diameter of digestive enzyme (amylase) of the whole- -body homogenate of Oligonychus coffeae (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial amylase 5.17 ±0.29 Amylase Whole body homogenate 3.55 ±0.08 -62- Table 8. Digestive enzyme activity of whole body homogenate of ■ -Oligonychus coffeae (mean ± SD) Amylase activity (MVI. mg protein min"'') Whole body homogenate 0 .coffeae 0.0243 ± 0.004 5.1.1.2. Protease In case of protease test, clear rings appeared against a white background. In B.suppressaria the diameter of halo developed for salivary was 3.99 ± 0.10 mm and for midgut homogenate 4.54 ± 0.05 mm respectively. A positive and negative control was run concurrently. Positive control gave a similar ring formation and negative no formation of halo (Table 9). The protease activity in oral as well as midgut of B.suppressaria possibly ensured an active protein digestion at both the levels (Table 10). Table 9. Measurement of halo diameter of digestive enzyme (protease) of the salivary- -giand (SG) and the midgut (MG) homogenate of Buzura suppressaria (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial Protease 4.69 ±0.51 Protease Salivary gland (SG) homogenate 3.99 ±0.10 Midgut (MG) homogenate 4.54 ±0.05 -63- Table 10. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Buzura suppressaria (mean ± SD) Protease activity (Amount of casein, utilized) (|jg /mg) Salivary gland Midgut B.suppressaria 38.22 ±0.19 a 44.81 ±0.38 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test In the agar gel plate, cleared ring of protease activity (halo) against white bacl 4.36 ± 0.04 mm and in midgut 3.94 ± 0.06 mm respectively (Table 11). The protease activity in oral and midgut of Et.magnifica possibly ensured an active protein digestion at both the levels (Table 12). Table 11. Measurement of halo diameter of digestive enzyme (protease) of the salivary- -gland (SG) and the midgut (MG) homogenate oi Eterusia magnifica (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial Protease 4.69 ±0,51 Protease Salivary gland (SG) homogenate 4.36 ±0.04 Midgut (MG) homogenate 3.94 ±0.06 -64- Table 12. Digestive enzyme activity of salivary gland (SG) and midgut (IVIG)- -homogenate of Eterusia magnifica (mean ± SD) Protease activity (Amount of casein, utilized) (|jg /mg) Salivary gland Midgut Et. magnifica 44.45 ± 0.46 a 43.49 ± 0.22 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test The gel diffusion assay of IH.theivora siiowed the protease activity both at salivary and midgut levels. The diameter of the halo in salivary protease was 3.95 ± 0.06 mm and in midgut 4.34 ± 0.09 mm respectively (Table 13). Proteolytic enzyme activity was also higher in midgut than in salivary homogenate (Table 14). The presence of general protease activity both in salivary gland and midgut of H.theivora indicated that this pest can well utilize the protein source of the tea leaf. Table 13. Measurement of halo diameter of digestive enzyme (protease) of the salivary- -gland (SG) and the midgut (MG) homogenate of Helopeltis theivora (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial Protease 4.69 ±0.51 Protease Salivary gland (SG) homogenate 3.95 ±0.06 Midgut (MG) homogenate 4.34 ±0.09 -65- Table 14. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Helopeltis theivora (mean ± SD) Protease activity (Amount of casein, utilized) (|jg /mg) Salivary gland Midgut H.theivora 17.54 ±0.4 a 19.06 ± 0.5 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test The whole body homogenate of O.coffeae showed presence of protease in the agar gel plate. The diameter of the halo measured 3.19 ± 0.06 mm (Table 15). The protease activity was also evident in the whole body homogenate of O.coffeae (Table 16). Table 15. Measurement of halo diameter of digestive enzyme (protease) of the- -whole body homogenate of Oligonychus coffeae (mean ± SD) Enzyme Enzyme source Observed value (mm) Commercial protease 4.69 ±0.51 Protease Whole body homogenate 3.19 ±0.06 - 66 - Table 16. Digestive enzyme activity of wlioie body homogenate of -Oligonychus coffeae (mean ± SD) Protease activity (Amount of casein, utilized) (pg /mg) Whole body homogenate 0. coffeae 5.5810.37 5.1.1.3. Lipase In B.suppressaria amount of lipase was much reduced than other two digestive enzymes. The quality of lipase measured in case of salivary gland homogenate was 0.0076 ± 0.0002 and in midgut 0.0328 ± 0.0013 meq/min/g of sample respectively (Table 17). Table 17. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Buzura suppressaria (mean ± SD) Lipase activity (Activity meq / min / g sample) Salivary gland Midgut B.suppressaria 0.0076 ± 0.0002 a 0.0328 ± 0.0013 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test -67- The activity of lipase was observed in both the salivary and midgut homogenate of Et.magnifica. It measured 0.0043 ± 0.0001 in case of salivary gland and 0.0127 ± 0.0009 (meq/ min/g sample) in midgut respectively (Table 18). Table 18. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Eterusia magnifica (mean ± SD) Lipase activity (Activity meq / min / g sample) Salivary gland Midqut Et.magnifica 0.0043 ± 0.0001 a 0.0127 ± 0.0009 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test The lipase activity could be detected in H.theivora but at a very low key both in salivary [0.0314 ± 0.002 milliequivalent (meq) of free fatty acid / min/ g sample] and midgut (0.0524 ± 0.004 milliequivalent of free fatty acid I min/ g sample) homogenate (Table 19). Table 19. Digestive enzyme activity of salivary gland (SG) and midgut (MG)- -homogenate of Helopeltis theivora (mean ± SD) Lipase activity (Activity meq / min / g sample) Salivary gland Midgut i-i.theivora 0.0314 ±0.002 a 0.0524 ± 0.004 b Different letters in a row indicate significance difference of mean at p> 0.001 using t-test -68- The lipase activity was found to be very low in the whole body homgenate of O.coffeae as compared with the other major tea pests in question. The amount of lipase occurring was 0.0022 ± 0.0005 milliequivalent (meq) of free fatty acid / min/ g sample (Table 20). Table 20. Digestive enzyme activity of whole body homogenate of- -Oligonychus coffeae (mean ± SD) Lipase activity (Activity meq / min / g sample) Whole body homogenate O.coffeae 0.0022 ±0.0005 -69- 5.1.2. Discussion on hydrolytic enzymes Lepidopteran caterpillars are continuous feeders. The presence of food in the midgut is necessary to stimulate synthesis and secretion of hydrolyic (digestive) enzymes. Digestive enzymes that participate in primary digestion (cleavage of polymers lii In case of Et.magnifica, preference of the larvae for mature tea leaves of middle tier of a bush with relatively lower food quality (than young leaves) (Mukhopadhyay et al., 2001) called for an effective carbohydrate digestion by salivary as well as migut secretions. Starch is the main reserve polysaccharide in tea (Banerjee, 1993). The amylase activity found both in the salivary and midgut homogenate of B.suppressaria indicated greater digestion of polysaccharides in midgut than its break down at the time of ingestion in the oral cavity (Table 1, 2) vis-a-vis in Et.magnifica amylase activity almost of equal quantity (Table 3, 4) ensured similar polysaccharide digestion at salivary and midgut levels. Deficiencies in the quality of a food resource can be balanced by various mechanisms of nutritional compensation, as was evident in Et.magnifica that overcame poor food quality by increasing their feeding period and by effecting better food conversion efficiency (Sarker etal., 2007). A higher food ingestion by B.suppressaria was possibly due to the nature of softer leaves of a - 70- high nutritional quality in which the percentage of nitrogen and moisture is more. As was apparent, the quicker intake of the host leaf by B.suppressaria possibly did not leave much scope of starch digestion in the oral cavity as compared to the passage of food for a much longer time in the midgut. This feeding behaviour commensurate with a higher secretion of amylase in the midgut than in salivary gland. In many insects with chewing mouthparts, salivary enzymes are mixed with the food during chewing and swallowing, that initiates digestion. However the midgut usually is the main site of production and secretion of digestive enzymes, and salivary enzymes in these insects play only a secondary role in digestion. The higher activity of digestive enzymes like amylase in relation to the amount food consumption have also been documented by Lazarevic and Mataruga (2003) in 5'^ instar of Lymantria dispar larvae. The amylase activity has also been well established in insect orders, Lepidoptera and Hemiptera (Mendiola-Olaya et ai, 2000; Zeng and Cohen, 2000). In Hemiptera, the ability to use plant materials as food is much dependent on presence of specific digestive enzymes include amylases and proteases that are advantageous for phytophagy (Cohen, 1996; Gopalan, 1976). The plant feeding mirids usually have high quantity of amylase in their salivary gland complex (Agusti and Cohen, 2000). The hydrolytic salivary enzymes in mirids may be involved in the liberation of osmotically active substances into the intercellular space causing an outflow of liquid (containing nutrients) that would be sucked back by the insect (Miles, 1987). The feeding mechanism of the bugs is evidently such that most of the enzymes injected into plant sap, play a role in digestion in the gut (Baptist, 1941; Nuorteva, 1954; Hori, 1970b; Miles, 1972). The present study showed that the H.theivora had enhanced levels of amylase in the salivary gland and midgut that possibly implied an effective digestion of leaf starch both extraorally and after ingestion. Complete breakdown of starch probably took place in the midgut where an increased amount of amylase existed (Table 5, 6). Similar results were also observed in the -71 - hemipteran bug. Eurygaster integriceps by Kazzazi et al. (2005). Mirids use their digestive enzymes througli tlie salivary canal to liquefy food into nutrient-rich slurry (Miles, 1972; Hori, 2000; Wheeler, 2001). The food slurry is ingested through the food canal and is passed into alimentary canal where it is further digested and absorbed (Cohen, 2000). Hence the presence of hydrolyzing enzyme in the salivary gland of H.theivora may be responsible for initial breakdown and dissolution of plant tissue facilitating penetration of the stylets and removal of the cell contents, whereas the amylase in the midgut may help further breakdown of carbohydrates though luminal digestion. The a-amylases are hydrolytic enzymes that are widespread in nature. These enzymes catalyze the hydrolysis of a-D-(1, 4)-glucan linkage in starch components, glycogen ahd various other related carbohydrates (StrobI et al., 1998; Franco et al., 2000). In O.coffeae the adults, nymphs and larvae actively feed on mature leaves. Since the tea leaf contains large quantities of starch, the presence of amylase enzyme in O.coffeae is of great significance in host plant utilization (Table 7, 8). In a similar finding, amylase mediated hydrolysis of starch is also reported in stored-product mites (Bowman and Childs, 1982; Bowman, 1984). In unprocessed tea, protein makes upto 20% of the dry weight (Mulky, 1993). The protease activity in oral as well as midgut of B.suppressaria ensured an active protein digestion at both the levels (Table 9, 10). For sequestration and digestion of protein from plant food active role of proteases is essential in most cases protein in diet is the limiting factor for optimal growth of insects (Bernays and Chapman, 1994). The digestive proteases catalyze the release of peptides and amino acids from dietary protein which are found most abundantly in the midgut region of the insect digestive tract (Jongsma and Bolter, 1997). In case of B.suppressaria the midgut protease activity was significantly higher than that of the salivary gland whereas in Et.magnifica almost equal protease activity in oral and midgut regions ensured an active protein digestion at both the levels. Nevertheless a slightly higher -72- protease activity in salivary secretion of Et.magnifica possibly ascertains a better digestion of the available protein of mature tea leaves in the oral cavity followed by that in the midgut (Table 11, 12). Lenz et al. (1991) have identified the proteinases and peptidases from the intestinal tract of fifth instar larvae of Heliothis zea. Midgut protease activity was also evident in caterpillars of pierid butterflies (Lecadet and Dedonder, 1966; Broadway, 1989), pyralids (Larocque and Houseman, 1990) and noctuid moths (Ahmad etal., 1980; Teo etal., 1990). Deficiency in the quality of food obtained from mature tea leaves (compared to young leave) is balanced by various mechanisms of nutritional compensation as is evident in Et.magnifica. Poor food quality was possibly overcomed by increase in their feeding period, better food conversion efficiency and higher digestive protease activity both in oral as well as midgut levels (Sarker et al., 2007). The presence of protease activity both in salivary gland and midgut of H.theivora indicated that this pest can well utilize the protein source of the tea leaf (Table 13, 14). In an earlier study, protease was detected in Helopeltis corbisieri (Kumar, 1970), and subsequently, the protease was found responsible for extra-intestinal digestion in Lygus disponsi (Hori, 1970a). Proteolytic activity has also been detected in salivary glands of mirid bugs such as Lygus rugulipennis (Laurema et al., 1985), Creontiades dilutus (Colebatch et al., 2001) and Ragmus importunitas (Gopalan, 1976). The feeding biology of Heteroptera is a remarkable integration of morphological, behavioural and physiological adaptations that lend themselves to feeding diversity and efficiency (Miles, 1972; Cohen, 1990). Heteropteran workers lament the lack of a thorough understanding of heteropteran feeding habits, especially that of Miridae (Wheeler, 2001).In general, heteropterans are known to use their digestive enzymes through the salivary canal to liquefy food into nutrient-rich slurry (Miles, 1972; Hori, 2000; Wheeler, 2001). The food slurry is ingested through the food canal and is passed into alimentary canal where it is further digested and absorbed (Cohen, 2000). As the mirid, H.theivora causes serious damage to tea leaves resulting in cessation of growth, curl up and -73- die back, the phenomenon may largely be related to the occurrence of the hydrolyzing enzymes in its salivary glands. The protease activity was also found in the whole body homogenate of O.coffeae (Table 15, 16). O.coffeae that attacked the mature tea leaves, containing upto 20% protein (Mulky, 1993), possibly could well utilize the protein sources of the leaf with help of the protease enzymes. Similar studies showed that the mite extracts contained a variety of active enzymes including trypsin, chymotrypsln (Stewart eta!., 1992). The activity of lipase in all the pests In question was much reduced than other two digestive enzymes. In B.suppressaria the lipase activity is relatively higher than that of Et.magnifica both at salivary and midgut levels (Table 17, 18). This is possibly due to the feeding preference of the former on young tea leaves in which lipid is present at an enhanced level i.e. upto 9% of the dry matter (Roberts, 1974; Mahanta ef a/., 1985). The lipase activity has also been reported in the mldgut of Manduca sexta (Rubiolo ef a/., 2000) and Spilosoma obliqua (Anwar and Saleemuddin, 1997). Lipase is also reported to occur in heteropterans at large (Nuorteva, 1954; Bronskill et al., 1958; Feir and Beck, 1961). While in H.theivora a very low activity of lipase was registered (Table 19), the enzyme was found to be absent in the mirid bug, Lygus sp. However in the saliva of heteropteran predators lipases commonly occur to digest storage lipids localized in the fat body and reproductive system of the prey (Cohen, 1995), it may also be involved in enlargement of pore size in the protein-lipid layer of plasma membrane of the host cells (Branton, 1969). The lipase activity was low in O.coffeae possibly because they attacked the mature leaves that contained low lipid (Table 20). As the literature is scanty, information on biochemistry of the lipase activity of O.coffeae is not available, but in case of predatory mites, salivary secretion contains the digestive enzymes including lipases to liquefy the prey (Legendre, 1978). -74- The present study on feeding biology and digestive enzyme activities revealed different strategies of the two folivores to exploit two qualities of tea leaves (young and mature). Et.magnifica appeared to have a better adaptive flexibility than that of B.suppressaria because of its greater efficiency in converting both ingested and digested food (Sarker et a!., 2007). The results showed that in the B.suppressaria larvae the amylase, protease and lipase activity was higher in the midgut than that of the salivary gland. This indicated the possibility of this species of leaf chewer to better utilize the starch, protein and lipid rich diets through its midgut digestive enzymes, in Et.magnifica larvae, the amylase activity was found to be almost equal in the saliva and gut, implying similar starch digestion at foregut and midgut levels. The protease activity was found to be higher in salivary secretion which possibly ensured a better breal The two sucking pests, H.theivora and O.coffeae showed different quantity of digestive enzymes. The former bug dispensed its digestive enzymes through the salivary canal to liquefy leaf tissue into nutrient-rich slurry. The food slurry was then ingested and was passed into alimentary canal for further digestion and absorbtion. In O.coffeae amylase, protease and lipase activity was much lower than that of the H.theivora. This might be related to the nature of feeding and quality of food consumed. These mites chiefly fed on the protoplasm of the epidermal tissue of mature tea leaves that in general contained low amount of all the three basic nutrients (Banerjee, 1993). Lepidopteran larvae and plant sucking pests have been the subjects of study as they cause severe economic loss to tea crop. Nothing much was known till date about the place occurrence (saliva/gut), and quantity of digestive enzymes of the chewing caterpillar stages of B.suppressaria and Et.magnifica, and the sucking pests like IH.theivora and O.coffeae. An -75- understanding of the levels and function of digestive enzymes is essential in designing methods of insect control based on enzyme inhibitors and transgenic plants (Bandani et al., 2001; Ghoshal et al., 2001; Maqbool et al., 2001). For developing these strategies, it is imperative to have a fair understanding of the target pests’ feeding biology besides biochemistry and physiology of feeding. The present findngs throw-up future research opportunities in non-conventional control of the four pests in question, based on digestive- enzyme inhibitors and other host-piant resistance strategies. Such strategies may prove useful in developing IPM-programme of tea. -76- 5.2.1. Results of oxidoreductases of the four arthropod tea pests 5.2.1.1. Catalase Insects and mites possess a battery of oxidoreductases of which catalase, peroxidase and polyphenol-oxidase perform important catabolic functions. Catalase assay was found to be almost similar both in salivary and midgut homogenate in B.suppressaria (Fig. 7). Considering the difference in OD, catalase assay was showed marginally higher in salivary gland homogenate as compared to that of midgut. Fig. 7 Catalase assay of the salivary gland (SG) and the midgut (MG) homogenate of- -B.suppressaria observed at an interval of 10 sec 77- Almost similar catalase assay was detected in the salivary gland and midgut homogenate of Et.magnifica (Fig. 8). Fig. 8 Catalase assay of the salivary gland (SG) and the midgut (MG) homogenate of- -Et.magnifica observed at an interval of 10 sec -78- Catalase in salivary gland homogenate of H.theivora initially showed more activity as compared to that of midgut (Fig. 9). But after about 140 sec the enzyme activities became as low as that of midgut homogenate. Fig. 9 Catalase assay of the salivary gland (SG) and the midgut (MG) homogenate of- -H.theivora observed at an interval of 10 sec -79- In case of O.coffeae the catalase was detected in the whole body homogenate (Fig. 10). The curve indicated a higher initial activity followed by low key at a later stage. c ■<1> 2 Q. O O) £ c E Q O < Fig. 10 Catalase assay of the whole body homogenate of O.coffeae observed at an interval of 10 sec - 8 0 - 5.2.1.2. Peroxidase The peroxidase was found to be active in both the salivary and midgut level of B.suppressaria (Fig. 11). The salivary gland homogenate showed less activity than that of the midgut throughout the period of observation. Fig. 11 Peroxidase assay of the salivary gland (SG) and the midgut (MG) homogenate of S.suppressar/a observed at an interval of 10 sec -81 - In case of Et.magnified the peroxidase was present both in salivary and midgut homogenate (Fig. 12). The mIdgut activity was higher at the initial phase, where as in saliva it increased in the later stage. Fig. 12 Peroxidase assay of the salivary gland (SG) and the midgut (MG) homogenate of Et.magnifica observed at an interval of 10 sec - 8 2 - Similar and overlapping peroxidase assay was recorded both in the salivary and midgut homogenate of H.theivora (Fig. 13). 0 .0 1 5 SG homogenate 0 .0 1 4 MG homogenate 0 .0 1 3 0 .0 1 2 c 0.011 a> 2 0.01 Q. 0 .0 0 9 “o 05 0 .0 0 8 E "c 0 .0 0 7 1 0 .0 0 6 Q O 0 .0 0 5 < 0 .0 0 4 0 .0 0 3 0 .0 0 2 0.001 0 Time ( seconds) Fig. 13 Peroxidase assay of the salivary gland (SG) and the midgut (MG)- -homogenate of H.theivora observed at an interval of 10 sec -83- Peroxidase constitutes an endogenous enzyme of protective systems (EEPS). The whole body homogenate of O.coffeae showed a fair activity of peroxidase (Fig. 14) that increased with time function. Time (seconds) Fig. 14 Peroxidase assay of the whole body homogenate of O.coffeae- -observed at an interval of 10 sec -84- 5.2.1.3. Polyphenol-oxidase Polyphenol-oxidase could not be apparently detected both in the saliva and midgut tissue homogenate of B.suppressaria (Fig. 15). SG homogenate 0 .0 2 5 MG homogenate 0.02 I Q. 0 0 .0 1 5 01 E c E 0.01 Q O < 0 ,0 0 5 Time (seconds) Fig. 15 Polyphenol-oxidase assay of the salivary gland (SG) and the midgut (MG) homogenate of B.suppressaria observed at an interval of 10 sec -85- In case of Et.magnified polyphenol-oxidase was detected in both saliva and midgut homogenate. At most of the time polyphenol-oxidase In the midgut showed a higher level than that of the salivary homogenate (Fig.16), nevertheless, the latter showed an ever increasing trend during the period observation. Fig. 16 Polyphenol-oxidase assay of the salivary gland (SG) and the midgut (MG) homogenate of Et.magnifica observed at an interval of 10 sec -86- In H.theivora level of polyphenol-oxidase assay was apparently more in the midgut than in the saliva (Fig.17). -SG homogenate - MG homogenate 0.03 0.028 0.026 0.024 0.022 0.02 0.018 0,016 c E 0.014 - Q 0.012 O < 0.01 0.008 0.006 0.004 0.002 0 Time (seconds) Fig. 17 Polyphenol-oxidase assay of the salivary gland (SG) and the midgut (MG)- -homogenate of H.theivora observed at an interval of 10 sec - 87- In case of O.coffeae polyphenol-oxidase assay was detected in whole body homogenate (Fig. 18). The curve showed a gradual rise in the activity at the initial stage followed by stable state during the period of observation. Fig. 18 Polyphenol-oxidase assay of the whole body homogenate of O.coffeae- -observed at an interval of 10 sec - 8 8 - 5.2.2. Discussion on oxidoreductases Antioxidant enzymes that are present in tiie gut lumen of caterpillars mainly scavange the oxidative component of the ingested phenolic compounds (Barbehenn and Martin, 1994; Barbehenn et ai, 1996). A variety of antioxidant enzymes protect caterpillar tissues and extracellular fluids from oxidative damage. Among the most widely studied enzymes that counteract reactive oxygen, catalase is well established (Barbehenn, 2002). The diminution of peroxidase activity and removal of hydrogen peroxidase by catalase are important adaptations to leaf feeding (Felton and Duffey, 1991). The catalase assay of B.suppressaria was found to be marginally higher in salivary gland homogenate as compared with the midgut homogenate (Fig. 7). As this enzyme is involved in inhibiting the action of plant phenolics besides removing of toxic hydrogen peroxide, an active blocking of these oxidants possibly takes place at the salivary gland level followed by that at gut level. In larvae of Et.magnifica almost similar catalase assay was observed both at salivary and midgut levels (Fig, 8) indicating by and large similar defensive strategy like that of the B.suppressaria. The secretion of catalase in salivary fluid during insect feeding was suggested to be potential mechanism for reducing hydrogen peroxide formation and for overcoming plant defense chemicals. Catalase assay has been detected in midgut tissues and salivary secretion of several lepidopteran pests of tomato plant, and its enhanced activity in the midgut was recorded in the larvae of Helicoverpa zea, Spodoptera exigua, Manduca sexta and Heliothis w'rescens (Felton and Duffey, 1991). Of the oxidoreductase enzymes, catalase present in salivary gland homogenate of H.theivora initially showed more activity as compared to that of midgut (Fig. 9). Catalase in the saliva has the ability to prevent the formation of plant protective compounds such as quinone (Laurema and Varis, 1991). The salivary gland of the hemipteran bug, Eoscarta carnifex is found to contain high level of catalase which is known to inhibit plant peroxidase (Rodman and Miller, 1992). Thus the salivary and midgut catalase in H.theivora may be of -89- importance in also scavenging the plant peroxidase and thereby suppressing the host defence. In O.coffeae the catalase was detected in whole body homogenate (Fig. 10), could possibly remove the toxic hydrogen peroxide and thus inhibit the oxidation of plant phenolics to quinone and other compounds of plant defence. Besides catalase, saliva of insect herbivores is also known to contained oxidative enzymes like peroxidase (Felton and Eichenseer, 1999). In B.suppressaria, the peroxidase in salivary gland homogenate showed less quantity than that of the midgut, (Fig. 11) which indicated that more plant phenolic compounds were oxidized in midgut than in the oral cavity by saliva. In Et.magnifica the peroxidase quantity in the midgut was higher at the initial phase (Fig. 12), than that in saliva. This possibly implied that oxidation of phenolic compound in the midgut occurred at an enhanced level than that in the foregut region by saliva. Peroxidases in conjuction with hydrogen peroxide is able to oxidize a variety of defensive phytochemicals (Miles, 1999) thus making host plants more acceptable as food. As an example of such anti-defence mechanism the salivary regurgitate of Leptinotarsa decemlineata was noted to contain high amont of peroxidase (Steinite et al, 2004). In H.theivora the peroxidase assay was found to be similar both in the salivary and midgut homogenate (Fig. 13) thus indicating the oxidative activity as a defence measure at both levels. The oxidoreductases, specially peroxidases, are known to cause phytotoxaemia as well as detoxification of secondary metabolites of the host plant. Peroxidases use hydrogen peroxide to oxidize phenols and other aromatic derivatives (Deimann et al., 1991). These oxidoreductases have been identified in the salivary secretions of aphid species (Miles and Peng, 1989; Madhusudhan and Miles, 1993) and these were found to be involved in overcoming the plant defenses by neutralizing phenolics and their derivatives (Miles, 1969; (Jrbanska and Leszczynski, 1992). The enzyme peroxidase is also reported to degrade chlorophyll (Mantile, 1980). So, the presence of peroxidase in the saliva and -90- midgut of H.theivora possibly enabled the bug to oxidize a wide range of tea plant phenolic compounds. In O.coffeae the enhanced peroxidase level (Fig. 14) could possibly minimise the effects of the toxic products that the mites ingest or experience while colonizing the tea leaves, as has also been known in case of carmine spider mite, Tetranychus cinnabarinus (Zhang et ai, 2004). The saliva of insect herbivore shows polyphenol-oxidase activity (Felton and Eichenseer, 1999). Polyphenol-oxidase uses molecular oxygen to catalyze two different types of reaction that are hydroxylation of monophenols to o-diphenols, and oxidation of polyphenols to quinones and further to darl< brown or black pigments, melanins (Robinson etal., 1991). The apparent lack of activity of polyphenol-oxidase in both saliva and midgut homogenate (Fig. 15) indicated that B.suppressaria could possibly use peroxidases as the only enzyme to metabolize tea phenolics and convert them into less toxic substances. In Et.magnifica presence of polyphenol-oxidase was detected in both saliva and midgut homogenate (Fig. 16). Polyphenol-oxidase in the saliva showed a higher titer at a later stage over midgut. So the occurrence of polyphenol-oxidase both in saliva and midgut homogenate of Et.magnifica enables the species to oxidize a wide range of tea phenolic compounds leading to to nutralization the defence allelochemicals of the host leaves ingested along with food. The saliva of most hemipteran insects contained polyphenol-oxidase (Miles, 1972). In H.theivora level of polyphenol-oxidase was apparently less than that of the peroxidase (Fig.17). This possibly implies that l-l.theivora depends much on peroxidase than poylphenol-oxidase to oxidize potentially toxic phenolics already ingested to non-toxic end products at the midgut. Polyphenol-oxidase occuring in the saliva of aphids (Miles and Peng, 1989) has been suggested to subserve the function of detoxification of phenolics such as catechin (Miles, 1985) and other potential toxins in the natural diets (Miles and Peng, 1989). In the healthy plant, phenolics are maintained in the reduced non-toxic state but -91 - when ingested by an insect, tlie antioxidants in the diet are not renewed and hence phenolics become free to autoxidize and combine with proteins in the gut. iVloreover, once plants are attacked by insects, the titre of ingested phenolics may rise due to defensive reactions of the plant (Peng and Miles, 1988b; Jiang and Miles, 1993). The small amount of polyphenol-oxidase and peroxidase in the saliva and midgut of H.theivora (Fig. 17) are able to possibly oxidize a wide range of phenolic compounds with a double efficiency as far as defence against tea plant phenolic compounds are concerned. The occurrence of salivary phenolase suggests that the enzyme, whether secreted alone or along with a substrate, may enable the insects to overcome the natural defences of host plants (Miles, 1972; Sengupta and Miles, 1975; Urbanska et al., 1998; Peng and Miles, 1988b). The catalase, peroxidase and polyphenol-oxidase in the salivary glands and midgut homogenate are important since these are considered by Miles (1964) to counter host plant’s defence chemicals. Hence the presence of these oxido-reductase enzymes in the salivary gland and midgut endows H.theivora to become one of the most destructive pests by depredating of the young leaves and growing shoot of tea specially with a phytotoxic effect. In O.coffeae polyphenol-oxidase was detectable in the whole body homogenate (Fig. 18). Little is known about the polyphenol-oxidase activity of O.coffeae. In this study the occurrence of high quantity of polyphenol-oxidase in O.coffeae possibly indicated that the mite could easily overcome the plant defenses by neutralizing toxic phenolics to non-toxic end products. In Tetranychus cinnabarinusihe enhanced level of phenolases was related to an increase in their defensive power against toxic substances (Zhang et a!., 2004). -92- 5.3.1. Results of quantitative assay of detoxifying enzymes of the four arthropod -tea pests 5.3.1.1. General esterases General esterase quantity was always significantly higher in the midgut than that in the salivary gland irrespective of tea clone on which the B.suppressaria were reared or source of their supply from field or laboratory culture. Table 21. Quantities of general esterases of B.suppressaria (mean ± SD) reared on- -TV 1 and TV 25 variety of tea in laboratory and those exposed to pesticides in field B.suppressaria Esterase quantity [j mol min . mg of protein Salivary gland Midgut TV 1 reared 14.39 ± 1.88 aA 51.63 ±1.54 aB TV 25 reared 20.12 ±1.49 bA 56.62 ±1.07 bB Pesticide-exposed 32.57 ± 1.62 cA 81.33 ±1.97 cB Means followed by the different lower case letters in columns and upper case letters in rows are significantly different at p>0.001 using t-test in the pesticide-exposed larvae, the salivary and midgut homogenate showed a higher esterase activity than that of the laboratory-reared ones on two Tocklai clonal varieties (TV 1 and TV 25) (Table 21). -93- In Et.magnifica a much enhanced level of the general esterases than that of the pesticide- exposed larvae over laboratory-reared ones could be registered (Table 22). Table 22. Quantities of general esterases of Etmagnifica (mean ± SD) reared on- -TV 18 and TV 25 variety of tea in laboratory and those exposed to pesticides in field Et.magnifica Esterase quantity |j m ol" ^. min . mg of protein Salivary gland Midgut TV 18 reared 19.24 ± 0.34 aA 24.73 ± 0.57 aB TV 25 reared 19.45 ± 0.42 aA 25.16 ±0.61 aB Pesticide-exposed 27.40 ± 0.66 bA 32.46 ± 0.43 bB Means followed by the different lower case letters In columns and upper case letters in rows are significantly different at p>0.001 using t-test Further, significant difference of esterase quantity was observed for salivary gland homogenate and midgut homogenate of pesticide-exposed and laboratory-reared larvae with a consistently higher midgut titer. Comparison of esterase quantity in laboratory-reared (pesticide un-exposed) and field- collected (pesticide-exposed) specimens of H.theivora showed that the quantity of esterases was always significantly higher in the midgut than that in the salivary gland independent of tea clone on which the Helopeltis were reared (Table 23). -94- Table 23. Quantities of general esterases of H.theivora (mean ± SD) reared on- -TV 1 and TV 25 variety of tea in laboratory and those exposed to pesticides in field H.theivora Esterase quantity |j m ol' ^. min . mg of protein Salivary gland Midgut TV 1 reared 1.99 ± 0.84 aA 3.64 ± 0.57 aB TV 25 reared 2.26 ±0.65 aA 3.94 ± 0.68 aB Pesticide-exposed 3.67 ±0.61 bA 7.27 ±0.82 bB Means followed by the different lower case letters In columns and upper case letters In rows are significantly different at p>0.001 using t-test In O.coffeae the whole body homogenate showed a significantly high quantity of general esterase in the pesticide-exposed populations than that of the laboratory-reared (TV clone reared) ones (Table 24), Table 24. Quantities of general esterases of O.coffeae (mean ± SO) reared on- -TV clone of tea in laboratory and those exposed to pesticides in field O.coffeae Esterase quantity jj mol min . mg of protein Whole body homogenate TV clone reared 0.84 ± 0.41a Pesticide-exposed 3.21 ± 0.96 b Means followed by the different lower case letters in the column are significantly different at p>0.001 using t-test -95- 5.3.1.2. Glutathione S-transferases In B.suppressaria no differences in glutathione S-transferase quantity were found between tiie laboratory-reared and pesticide-exposed individuals (Table 25). Table 25. Quantities of glutathione S-transferase of B.suppressaria (mean ± SD)- •reared on TV clone of tea in laboratory and those exposed to pesticides in field B.suppressaria Glutathione S-transferase quantity |j m o r \ nnin . mg of protein Salivary gland Midgut Laboratory-reared 278,75 ±15.64 a 279.37 ± 16.41a Pesticide-exposed 281.22 ±17.95 a 283.75 ±18.20 a Means followed by the same lower case letters in the columns are not significantly different Tlie quantity of glutathione S-transferase in the larvae of Et.magnifica was found to be similar in both laboratory-reared and pesticide-exposed individuals (Table 26), Table 26. Quantities of glutathione S-transferase of Et.magnifica (mean + SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field Et.magnifica Glutathione S-transferase quantity p mol “ \ min . mg of protein Salivary gland Midgut Laboratory-reared 157.50 ±19.72 a 160.60 ± 16.41a Pesticide-exposed 161.27 ±16.90 a 163.75 ±15.25 a Means followed by the same lower case letters in the columns are not significantly different -96- Glutathione S-transferase quantity was significantly higher in H.theivora that were collected from the field as compared to laboratory-reared ones (Table 27). The result also suggested that the midgut homogenate always showed a higher glutathione S-transferase quantity than the salivary gland. Table 27. Quantities of glutathione S-transferase of H.theivora (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field H.theivora Glutathione S-transferase quantity |j m o l"\ min . mg of protein Salivary gland Midgut Laboratory-reared 46.25 ± 7.33 aA 75.59 ± 9.04bB Pesticide-exposed 120.00 ±8.22 bA 179.34 ±7.82 aB Means followed by the different lower case letters in columns and upper case letters In rows are significantly different at p>0.001 using t-test In O.coffeae the whole body homogenate showed no differences in the quantity of glutathione S-transferase collected from field and those reared in laboratory (Table 28). -97- Table 28. Quantities of glutathione S-transferase of O.coffeae (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field O.coffeae Glutathione S-transferase quantity p m o r ’'. min . nng of protein Whole body homogenate Laboratory-reared 59.37 ± 8.96a Pesticide-exposed 61.87 ± 12.99 a Means followed by the same lower case letters in the column are not significantly different 5.3.1.3. Acetylcholinesterases In B.suppressaria aceylcholinesterase quantity in the liomogenate of cerebral ganglia showed a significant difference between the laboratory-reared and pesticide-exposed individuals of the field (Table 29). Table 29. Quantities of acetylcholinesterase of B.suppressaria (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field B.suppressaria Acetylcholinesterase quantity p nnorV min . nng of protein Homogenate of cerebral ganglia Laboratory-reared 0.051 ±0.009 a Pesticide-exposed 0.095 ±0.01 b Means followed by the different letters in the column are significantly different at p>0.001 using t-test -98- The acetylcholiesterase in the homogenate of cerebral ganglia of Et.magnifica showed a higher quantity of enzyme present in the pesticide-exposed individual than that of the laboratory-reared ones (Table 30). Table 30. Quantities of acetylcholinesterase of Et.magnifica (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field Et.magnifica Acetylcholinesterase quantity |j mol"\ min . mg of protein Homogenate of cerebral ganglia Laboratory-reared 0.053 ± 0.007a Pesticide-exposed 0.078 ± 0.008 b Means followed by the different letters in the column are significantly different at p>0.001 using t-test In H.theivora the acetylcholinesterase quantity was significantly higher in the specimens collected from field compared to the laboratory-reared ones (Table 31). -99- Table 31. Quantities of acetylcholinesterase of H.theivora (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field H.theivora Acetylcholinesterase quantity (j mol - ^. min . mg of protein Homogenate of cerebral ganglia Laboratory-reared 0.050 ± 0.005a Pesticide-exposed 0.077 ± 0.006 b Means followed by the different letters in the column are significantly different at p>0.001 using t-test The quantity of acetycholiesterase in tlie O.coffeae was significantly liiglier in tine specimens collected from field compared to the laboratory-reared ones (Table 32). Table 32. Quantities of acetylcholinesterase of O.coffeae (mean ± SD)- -reared on TV clone of tea in laboratory and those exposed to pesticides in field O.coffeae Acetylcholinesterase quantity p m o r\ min . mg of protein Whole body homogenate Laboratory-reared 0.022 ±0.005 a Pesticide-exposed 0.045 ± 0.007 b Means followed by the different letters In the column are significantly different at p>0.001 using t-test - 100- 5.3.2. Results of qualitative analysis of detoxifying enzymes of the four arthropod -tea pests 5.3.2.1. Esterase isozymes In the salivary gland homogenate of looper, B.suppressaria eight esterase (EST) bands with relative mobility (Rm) values of 0.332, 0.381, 0.430, 0.458, 0.481, 0.538, 0.672 and 0.684 were observed whereas in nnidgut homogenate, thirteen bands were observed with valuesof 0.161, 0.184, 0.201, 0.332, 0.381, 0.430, 0.458, 0.481, 0.538, 0.614, 0.632, 0.672 and 0.684. On the basis of relative mobility, the general esterase pherogram appeared in three groups: fast moving bands (FM or EST-1), slow moving bands (SM or EST-2), and very slow moving bands (VSM or EST-3). These groups were distinctly separate from one another (Fig. 19 a, b, c). The first three bands of the VSM group were absent in the salivary homogenates of all three categories of B.suppressaria populations. In the midgut homogenates, the VSM group was present as five bands in TV 25-reared and field-collected ones. However, the first three bands of VSM were not present in the midgut homogenate of TV 1 reared populations. Among four SM bands of esterases, bands 1, 2 and 3 were prominent, and were consistently present both in salivary and midgut homogenates. These appeared deeply stained and with uniform mobility in all groups of the B.suppressaria populations. A similar pattern, parallel to TV 25, was observed in field-collected B.suppressaria populations, in which the bands of VSM, SM and FM were deeply stained, indicating a larger quantity of esterases (Fig. 19 a, b, c). - 101 - VSM (EST-3) SM (EST-2) FM (EST-1) a) Looper reared on TV-1 b) Looper reared on TV-25 c) Field -collected looper (Pesticide-exposed) SG; Salivary gland homogenate VSM (EST-3): Very Slow moving bands MG: Midgut homogenate SM (EST-2): Slow moving bands FM (EST-1): Fast-moving bands Fig. 19 Zymograms of esterases of loopers, B.suppressaria fed and maintained on tea- clones (a) TV 1, and (b) TV 25, and from (c) conventional plantations exposed to pesticide- sprays (Each lane represents the pherogram of a single looper) - 102- Comparison of isozyme profiles for laboratory-reared Vth instar caterpillars of Et.magnifica on two different cultivars of tea (TV 18 and TV 25) showed a common basic pattern. In Et.magnifica the salivary gland homogenates showed two esterase bands with Rm values of 0.43 and 0.47 whereas midgut homogenates showed eight bands with values of 0.04, 0.12, 0.22, 0.30, 0.43, 0.47, 0.52 and 0.57. The intensity and number (EST-1- EST-8) of the isozyme bands from midgut homogenates were similar in all the individuals (larvae) reared on TV 18 and TV 25 but in the field-collected larvae all bands were deeply stained indicating an intensive formation of esterases (Fig. 20 a, b, c) EST Lane 1: Salivary gland homogenate Lane 2 and 3: Midgut homogenate Fig. 20(a) Tissue specific PAGE profile- -of esterase isozymes of Et.magnifica TV 25 reared: Lane 1: Midgut homogenate TV 18 reared: Lane 2: Midgut homogenate Fig. 20(b) Esterase isozymes of Et.magnifica- -reared on TV 25 and TV 18 - 103 - 1 2 3 Lane 1; Salivary gland homogenate Lane: 2 and 3; Midgut homogenate Fig. 20(c) Zymograms of Et.magnifica- -from pesticide-exposed plantations The esterase zymograms were developed from salivary, midgut homogenates of three discrete populations of H.theivora i.e. i) specimens reared in laboratory on TV 1 and TV 25 clones ii) specimens collected from organic and iii) specimens collected from conventional plantations. All of them by and large showed a common banding pattern. Two faint esterase bands were detectable in salivary gland homogenate with low-staining intensity, vis-a-vis in the midgut homogenates three prominent esterase bands were apparent. These bands were slow moving, medium slow moving, and fast moving with the values of 0.16, 0.20 and 0.31 respectively (Fig. 21 a). Esterase bands of the specimens exposed to pesticide sprays, in conventionally managed tea plantations, showed a higher staining intensity than the ones either collected from organic plantations (free from synthetic pesticide sprays) or reared in laboratory (Fig. 21 b). - 104- Rm values 1 2 3 4 5 0.16 0.20 0.31 Fig. 21 (a) Tissue specific PAGE profile of esterase isozymes of the Helopeltis theivora Salivary gland homogenate Midgut homogenate TV 1 reared; Lane 1 TV 1 reared; Lane 4 TV 25 reared; Lane 2 TV 25 reared; Lane 3, 5 1 2 3 Fig. 21 (b) Zymograms of esterase isozymes of midgut homogenate Helopeltis theivora- collected from various sources Lane 1; Organic plantation Lane 2; Pesticide-exposed plantation Lane 3; Laboratory-reared specimens on TV 1 tea clone - 105 - Results of analysis of esterase bands on polyacrylamide gel showed that the pesticide- exposed female O.coffeae possessed 3 major co-migrating bands (EST-1. EST-2, EST-3) whereas the pesticide unexposed female O.coffeae possessed only one (EST-1). Major esterase band with value of 0.264 (EST-1) was present both in pesticide-exposed and unexposed female mites (Fig. 22). On close inspection of lanes, two additional bands were apparent in pesticide-exposed female [R^ values 0.3403 (EST-2) and 0.4833 (EST-3)]. These bands (EST-2, EST-3) were however absent in the unexposed female O.coffeae (Fig. 22) EST-1 EST-2 EST-3 Pesticide-exposed Pesticide unexposed Fig. 22 PAGE profile of esterase isozymes of the Oligonychus coffeae (female) - 106- S.3.2.2. Glutathione S-transferase isozymes In B.suppressaria no band formation of glutathione S-transferase was observed both in laboratory-reared and pesticide-exposed populations. The phoregrams of gel electrophoresis of Et.magnifica also did not show any band formation of glutathione S-transferase both in laboratory-reared and pesticide-exposed ones. The glutathione S-transferase banding pattern of H.theivora when reared in laboratory showed a single low-intensity band with a Rm value 0.098 vis-a-vis field-collected specimens registered a parallel band with high intensity at both salivary and midgut homogenates (Fig. 23). 0.098 Fig. 23 Zymogram of glutathione S-transferase isozyme of midgut homogenate of H.theivora (bands appear as transparent negative impression) Salivary gland homogenate MIdgut homogenate Laboratory-reared: Lane 2 Laboratory-reared: Lane 4 Pesticide-exposed: Lane 1 Pesticide-exposed: Lane 3 - 107- In O.coffeae polyacrylamide gel electrophoregram did not show any glutathione S- transferase band both in laboratory-reared and pesticide-exposed individuals. S.3.2.3. Acetylcholinesterase Isozymes The electrophoregram developed from the homogenate of cerebral ganglia of B.suppressaria showed formation of a single band (Rm value 0.168) (Fig. 24). The band- intensity was notably high in the pesticide-exposed specimens and low in laboratory-reared ones. 1 2 0.168 Fig. 24 Zymogram of acetylcholinesterase developed from homogenate of cerebral ganglia- -of B.suppressaria Homogenate of cerebral ganglia of specimens: Laboratory-reared; Lane 1 Pesticide-exposed: Lane 2 - 108- The zymogram of acetylcholinesterase developed from the homogenate of cerebral ganglia of Et.magnifica indicated a single band formation (Rm value 0.073) (Fig. 25). The intensity of band was higher in pesticide-exposed specimen in comparison with the laboratory-reared ones. 1 2 T ■ * > - ...... 0.073 Fig. 25 Zymogram of acetylcholinesterase developed from homogenate of cerebral ganglia- -of Et.magnifica Homogenate of cerebral ganglia of specimens: Laboratory-reared: Lane 1 Pesticide-exposed: Lane 2 - 109- r f 0.13 Fig. 26 Zymogram of acetylcholinesterase developed from the homogenate of cerebral- ganglia of H.theivora Laboratory-reared: Lane 1 Pesticide-exposed; Lane 2 Electrophoregram pattern of acetylcholinesterase of H.theivora showed a single band formation (Rm value 0.13) (Fig. 26). The band-intensity was notably high in the pesticide- exposed specimens and low in unexposed ones. - 110- 1 r i----- iki 0.1 0.18 0.28 Fig. 27 Zymogram of acetylcholinesterase isozyme of whole body homogenate of- -O.coffeae Laboratory-reared: Lane 1 Pesticide-exposed: Lane 2 The acetylcholinesterase bands on polyacrylamide gel showed that the pesticide-exposed female O.coffeae possessed 3 major bands whereas the pesticide unexposed female O.coffeae possessed only one. Major acetylcholinesterase band with Rm value of 0.1 was present both In pesticide-exposed and unexposed female mites (Fig. 27). Close inspection of lanes showed two additional bands were apparent in pesticide-exposed female (Rm values 0.18 and 0.28). These bands were however absent in the unexposed female O.coffeae. - I l l - 5.3.3. Discussion on detoxifying enzymes The extent to which insects can metabolize and thereby degrade toxic or otherwise detrimental chemicals is of considerable importance to their survival in a chemically unfriendly environment. While all insects probably possess detoxifying capacity, the mode, mechanism and amount can be expected to vary among species, with developmental stage, and with the nature of the insect’s immediate environment. Studies on detoxication processes in insects have revealed the versatility in their adaptation to the changing environment. This is largely mediated by the process of induction in which a chemical stimulus of the environment enhances the activity of the detoxication system by the production of additional enzymes (Terriere, 1984). Esterases have been implicated as detoxifying enzymes responsible for insecticide resistance in a number of species. In many cases the implication has come from biochemical tests comparing the esterase activity of resistant and susceptible insects using substrates such as a-napthyl acetate (Hughes and Raftos, 1985). An enhanced metabolism by esterases is a major mechanism for countering pesticide stress that has been detected in lepidopterans (Beeman and Schmidt, 1982). In the present study the general esterase quantity was always significantly higher in the midgut than that in the salivary gland independent of tea clone on which B.suppressaria were reared or place of their collection i.e. field or laboratory (Table 21). The quantity of general esterases of B.suppressaria tangibly differed when reared on two different Tocklai varieties of tea, namely TV 1 (an early release) and TV 25 (a relatively late release), and it was also found significantly higher in pesticide-exposed specimen (Table 21). Isozyme analysis have been applied to identify species, biotypes and host-specific populations in many insects such as aphids, egg parasitoids {Trichogramma spp.) and others (Loxdale and Hollander, 1989). In the present study, zymograms of esterases failed to show major differences in banding patterns, except for three additional VSM bands which - 112 - were present in the midgut jiomogenate of the B.suppressaria populations reared on TV 25 and the field-collected ones (Fig. 19 a, b, c). Mullin and Croft (1983) found large differences in general esterase activity of Tetranychus urticae on snapbean varieties, ranging from 0,4 fold on a mint to 2.4-fold on umbellifers. Moreover, herbivorous insects metabolize and detoxify insecticides using the same set of enzymes that are involved in the metabolism of ingested plant allelochemicals (Brattsten, 1979; Ahmad eta!., 1986). The VSM (EST-3) and SM (EST-2) bands of B.suppressaria showed intense staining in the pesticide-exposed field specimens collected from conventional plantation. Such band intensity may be related to greater pesticide tolerance of B.suppressaria populations {vis-a- vis resistance). In a similar finding, higher midgut esterase activity has been reported in the pesticide-exposed lepidopteran pest, Plutella xylostella (Mohan and Gujar, 2003), along with a higher activity of slow moving esterases bands (Maa and Liao, 2000). Resistant aphids display a high level of non-specific esterase activity represented by intense esterase bands (Ono et a!., 1994) as is evident in maiathion resistant P.xylostella (Maa and Chuang, 1983; Doichuanngam and Thornhill, 1989). The VSM (EST-3) bands appeared to be crucial in utilizing relatively recent tea clone (TV 25) and for development of greater pesticide tolerance/ resistance in B.suppressaria populations. So the occurrence and intensity of VSM and SM bands can be used as markers in screening the populations of the pest on one hand for their trophic relation to a tea cultivar as well as on the other hand for their pesticide resistance / tolerance status. Metabolic resistance to organophosphorous compounds in insects is mainly due to quantitative and/or qualitative differences in carboxylesterases (Hemingway and Karunaratne, 1998). In many examples of esterase-mediated insecticide resistance, the resistant insects display high levels of non-specific esterase activity as it has been found in case Et.magnifica where quantity of esterase in the midgut homogenate was significantly higher than that of salivary gland homogenate irrespective of tea clones; and the pesticide- -113- exposed larvae showed a higher esterase quantity than the laboratory-reared ones (Table 22). A significantly high band activity of the general esterases in the pesticide-exposed Et.magnifica larvae over un-exposed ones, possibly was indicative of a greater esterase- based detoxifying activity in the former larvae (Fig. 20 a, b, c). The higher nnidgut esterase activity was reported in Plutella xylostella exposed to pesticides by Mohan and Gujar (2003). Resistant insects display a high level of non-specific esterase activity represented by intense esterase bands (Ono et a!., 1994), which are evident in malathion-resistant P.xylostella (Maa and Chuang, 1983; Doichuanngam and Thornhill, 1989). Further, in peach potato aphid, Myzus persicae, resistance is conferred by amplification of esterase genes, resulting in the higher production of esterases that can hydrolyse insecticides (Field etal., 1999). Two soluble esterase isozymes, designated as EST-3 and EST-4 due to their prominent presence in the pesticide-exposed larvae of Et.magnifica specimens appeared to be related to pesticide detoxification. Therefore these bands may be marked as useful indicator in screening populations of the pest for their resistance / tolerance (Fig. 20 c). The present study exhibited only minor difference in the activity of general non-specific esterases between host (clone) specific Et.magnifica populations, as their zymograms failed to show any major differences in banding patterns (Fig. 20 b). Lack of this variability in Et.magnifica in natural population may be due to a high interbreeding in the populations occurring in Darjeeling foothills, Terai and the Dooars areas. A similar absence of major variation of esterase bands is evident in the populations of “Kissing bug”, Triatoma infestans due to high interbreeding (Tavares etal., 1998). So, an understanding of the status of detoxifying enzyme (esterase) of Et.magnifica, as has been revealed in the present study, would be helpful in future planning of their insecticide resistance management. - 114- Carboxylesterases are ubiquitous nonspecific enzymes that hydrolyse an array of diverse esters of carboxyiic acids. Due to the frequent association of insecticide resistance and elevated carboxyiesterase activity toward surrogate substrates, detection methodology of esterase mediated resistance has become increasingly analytical. The napthol-based esterase assay has been accepted by toxicologists to study the linkage between insecticide resistance and esterase activity. In this assay, the hydrolysed products of naptholic esters react with diazo dyes to give a coloured precipitate (a napthyl-diazo complex), and measured colorimetrically (Abdel-Aal etal., 1992). Among insect species, carboxylesterases of the Myzus persicae aphid (Devonshire, 1977), Culex pipiens mosquito (Georghiou and Pasteur, 1980), housefly (Oppenoorth, 1965), brown planthopper(Ozaki and Kassai, 1970), and tobacco cutworm (Bull and Whiten, 1972) have been studied extensively because of their involvement in resistance to organophosphates (OP) or other kind of insecticides. In the present study H.theivora showed that the quantity of esterases was always significantly higher In the midgut than that in the salivary gland independent of tea clone (Table 23) as was evident in the peach potato aphid, Myzus persicae that has evolved resistance to organophosphorous, carbamate and pyrethroid insecticides by producing large amounts of an enzyme, carboxyiesterase, that both degrades and sequesters these insecticidal esters (Devonshire and Moores, 1982). The qualitative study the esterase zymograms, developed from salivary and midgut homogenates of H.theivora showed a common banding pattern. Two faint esterase bands were detectable In salivary gland homogenate with low-staining intensity, vis-a-vis three prominent esterase bands in the midgut homogenate. These bands were slow moving, medium slow moving, and fast moving (Fig. 21 a, b). Esterase bands of female specimens exposed to pesticide sprays, in conventionally managed tea plantations, showed a higher staining intensity than the ones either collected from organic plantations (free from synthetic pesticide sprays) or reared in laboratory (Fig. 21 b). - 115- Isozyme forms of Carboxylesterase occur in different tissues (Afimaci et a!., 1986). Tissue specific expression of esterases related to their functional roles has been recorded in Triatoma infestans (Tavares et a/., 1998). H.theivora when reared on two different clonal varieties (TV 1 and TV 25) did not show any major differences in the banding pattern, thus excluding any major influence of the host-variety on the variation of esterase isozymes in Helopeltis] in a similar case, lack of host influence has been observed in the fall army worm where host-plant allelochemicals marginally affected its esterase activity (Yu, 1984), Direct measurement and quantification of detoxifying enzyme activities in sucking insects is insufficient. So, in this maiden study on general esterase isozymes of the sucking bug, H.theivora at different tissue levels a generalized higher midgut esterase activity was found in specimens from conventional plantations, possibly endowing the bug with a greater insecticide tolerance {vis-a-vis resistance). In resistant green bug, Schizaphis graminum, high levels of non-specific and intensely stained esterase bands were obtained (Ono et ai, 1994). Myzus persicae has been reported to develop resistance to organophosphate, carbamate and synthetic pyrethroids insecticides through an increased amount of esterases (Sawicki et ai, 1978; Devonshire and Moores, 1982). In the above case, esterase-based resistance was conferred either by gene amplification or enhanced gene expression. Amplification of esterase genes, often in combination with altered gene regulation results in the production of more esterases that hydrolyze insecticides (Field and Devonshire, 1998; Field et ai, 1999). Therefore, the high staining intensity of esterase bands observed in the present zymograms could reasonably be due to greater production of esterases meant for hydrolyzing the insecticides to which the l-i.theivora was exposed in the conventional tea plantations (Fig. 21 b). In sheep blow fly, Lucilia cuprina, esterase band (EST-3) was present both in susceptible and resistant forms, but the esterase activity was higher in resistant forms due to amplification of esterase genes (Hughes and Raftos, 1985). Similar banding patterns (Rm values) of esterases of H.theivora - 116- populations from Darjeeling foothills, Dooars and laboratory-reared samples indicate stability of esterase loci and absence of genetic variability. Lack of such genetic variability for esterase loci is also known in the populations of “Kissing bug”, Triatoma infestans due to high interbreeding (Tavares etal., 1998). So, the isozyme pattern of esterases and their banding intensity in various H.theivora populations provide a knowledge base and state of art for screening the tea mosquito bug populations of different tea plantations as regard to their insecticide tolerance / resistance status. Differences in the amount of esterase activity between two strains of the same insect species is considered an indicator of relative sensitivity to certain insecticides. Subsequently, various biochemical assays have been used for insect populations as possible indicators or insecticide resistance (Brown and Brogdon, 1987). One of the enzyme assays uses a-naptyhyl acetate (a-NA) as a model substrate for general esterase activity in a wide variety of insects (Devonshire, 1977; Ferrari et ai, 1993). Either high a-NA activity or extra enzyme bands stained with a-NA have been associated with resistance (Lalah et al, 1995). In the present study the general esterase quantity in the whole body homogenate significantly differed between the conventional (pesticide-exposed) and laboratory-reared (pesticide unexposed) female O.coffeae (Table 24). Results of analysis of esterase bands on polyacrylamide gel showed that the pesticide- exposed female O.coffeae possessed 3 major co-migrating bands (EST-1, EST-2, EST-3) whereas the pesticide unexposed female O.coffeae possessed only one (EST-1). Major esterase band (EST-1) was present both in pesticide-exposed and unexposed female mites (Fig. 22). Two additional bands (EST-2 and EST-3) were apparent in pesticide-exposed female contrastingly were absent in the unexposed female O.coffeae (Fig. 22). Ethlon is an organophosphate used as mitlcide in tea. Its high LCso value indicates, its least toxicity to O.coffeae when compared with synthetic pyrethroid (fenproperthrin) (Sahoo et al., - 117- 2003). This implies tliat O.coffeae has developed resistance I higher tolerance to ethion. Elevated esterase activity is known to be involved in organophosphate resistance in Aphis gossypii (Owusu et ai, 1996). Further, different band numbers and varying esterase intensities on PAGE are evident in resistant, as compared to susceptible strains of the mite, Tetranychus urticae (Capua et ai, 1990; Sundukov et a!., 1989). Kuwahara (1984) related malathion resistance of Tetranychus kanzawai to increased esterase activity at EST-3 and EST-4 bands. In the present study a maiden attempt is made to evaluate non-specific (general) esterases of female O.coffeae occurring in tea plantations that are sprayed by synthetic pesticides and those occurring in unsprayed plantations. It may be interpreted from the present findings that enhance quantity of esterases as well as the additional bands (isozymes) of O.coffeae of the conventional plantation are possibly involved in the detoxification of synthetic acaricides and insecticides. Further, such enhancements must be endowing the pesticide-exposed mites to have a greater pesticide tolerance I resistance. In a similar finding Yang et a i (2002) cited high general esterase activity in pyrethroid resistant strains of Oligonychus pratensis and T.urticae and inferred that esterases may be involved in the detoxification and /or sequestration of pyrethroid insecticides in these mites. Elevated general esterase activity has been reported in bifenthrin-resistant silverleaf white fly, Bemisia argentifolii (Riley et ai., 2000) and several other pyrethroid-resistant insects (Delorme et a!., 1988; Lee and Clark, 1996; Zhao et ai, 1996). The practical significance of the present finding may be realized in designing a method for easy detection of the pesticide resistance / tolerance status of O.coffeae using esterases. Moreover, preparation of a database on the status of the red spider populations / strains from Darjeeling foothills and plains shall be useful in deciding upon the future strategy of resistance management of the pest. The glutathione S-transferases (GST) are the large family of multifunctional enzymes involved in the detoxification of a wide range of xenobiotics including insecticides (Salinas - 118- and Wong, 1999). GSTs prinnarily catalyse the conjugation of electrophillic compounds with the thiol group of reduced glutathione (GSH), generally making the resultant products more water soluble and excretable than the non-GSH conjugated substrates (Habig et a!., 1974). Glutathione S-transferases are important in phase I metabolism of organophosphorous and organochlorine compounds and play a significant role in resistance to these insecticides (Clark and Shamaan, 1984). Many studies have shown that insecticide resistant insects have elevated levels of GST activity in crude homogenates, suggesting the possible role of GSTs in insecticide resistance (Armstrong and Suckling, 1990). In B.suppressaria no significant difference in the quantity of glutathione S-transferase was observed in laboratory- reared specimen and pesticide-exposed ones (Table 25). In the qualitative assay, no band formation in PAGE was detected both in laboratory-reared and pesticide-exposed ones thus suggesting absence of any role of GST in pesticide detoxification in this species. In case of Et.magnifica the quantity of GST was found to be similar both at laboratory- reared as well as pesticide-exposed individuals (Table 26). The phoregram of gel electrophoresis failed to show any formation of GST band thus indicating that GST was less likely to be important in the detoxification of insecticides in this pest species. In H.theivora significantly high glutathione S-transferase quantity in specimens from the field-collected population as compared to laboratory-reared specimens (Table 27) convincingly suggested that GST might be involved in detoxification of pesticides besides esterases in the field specimens. The role of GST in the degradation of xenobiotics and in development of organophosphate resistance has been reported in Plutella xylostella (Ku et al., 1994) and housefly (Motoyama and Dauterman, 1975; Clark and Dauterman, 1982). With regard to GST banding pattern in PAGE, laboratory-reared specimen showed a single low-intensity band with a Rm value 0.098 vis-a-vis field-collected specimens registered a parallel band with high intensity (Fig. 23). Several insecticide resistant strains of housefly - 119- have been reported to have elevated GST activity in crude homogenates against organophosphates (Clark etal., 1986). In O.coffeae no significant difference in the quantity of glutathione S-transferase was observed in laboratory-reared specimen as well as those exposed to pesticide in the field (Table 28). The zynnogrann of GST did not show any band formation both in laboratory- reared specimen as well as in pesticide-exposed ones. These suggested that the metabolic enzyme like glutathione S-transferases were less important in the detoxification of insecticides. In a similar finding, the two spotted spider mite, Tetranychus urticae also failed to show the involvement of GST in detoxification of insecticides (Yang et a!., 2002). Acetylcholinesterase (AChE) is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in the nervous system. Organophosphorous insecticides, target AChE and irreversibly inhibit the enzyme by phosphorylating a serine hydroxyl group within the enzyme active site (Wang et al., 2004). In the present study the quantity of acetylcholinesterase in the homogenate of cerebral ganglia of B.suppressaria showed a significant difference between the laboratory-reared and pesticide-exposed ones (Table 29). The zymogram of the acetylcholinesterase of B.suppressaria showed a single band formation with a higher intensity in the pesticide- exposed larvae as compared to the laboratory-reared individuals (Fig. 24). The difference in the AChE quantity of the pesticide-exposed larvae clearly indicated a higher concentration of AChE molecules in the exposed larvae. Based on the finding that a high level of AChE occurs in the pesticide-exposed larvae we might predict that a different mechanism may be associated with resistance. A large number of available catalytic sites may bind and scavenge toxic molecules, still leaving enough free AChE for proper functioning of the nervous system. In Et.magnifica the quantity of acetylcholinesterase was significantly higher in pesticide-exposed individuals than in the laboratory-reared ones (Table 30). In a similar finding, high acetylcholinesterase activity was also observed in the head of the - 120- German cockroaches resistant to pesticides (Park and Kamble, 2001). The PAGE analysis also showed single AChE band formation in both the laboratory-reared and pesticide- exposed individuals with a higher band intensity in the former (Fig. 25). Many studies have revealed that organophosphate (OP) and carbamate insecticides act by inhibiting acetylcholinesterase in vertebrates and invertebrates (Aldridge and Reiner, 1972; Silver, 1974; Devonshire, 1975b; Oppenoorth, 1985; Siegfried and Scott, 1990). AChE is an important regulatory enzyme responsible for the termination of synaptic nerve impulse transmission in cholinergic nerve synapses in animals. In insects, AChE is the target site of organophosphate and carbamate insecticides immobilizing the formers’ function and causing death of the exposed insect (Eto, 1974). In H.theivora it was also evident that quantities of acetylcholinesterase that bind with the organophosphates and carbamates, was significantly higher in the specimens collected from field compared to the laboratory- reared ones (Table 31). Electrophoregram pattern of acetylcholinesterase also indicated the band-intensity was notably high in the pesticide-exposed specimens and low in unexposed ones (Fig. 26). So, enhanced occurrence of this enzyme, even at band level, speaks for development of a greater tolerance in the H.theivora populations that are exposed to pesticide spray in plantations. In a similar finding higher level of acetylcholinesterase was recorded in the resistant strains of red scale {Aonidiella aurantii) (Levitin and Cohen, 1998). Smissaert (1964) was the first to demonstrate that resistance of the spider mite Tetranychus urticae to organophosphorous compounds was related to a structural change in AChE that rendered an enzyme less sensitive to OP compounds. Since this original observation, resistance mechanism based on the insensitivity of AChE to OP and carbamate insecticides has been documented in a variety of insect pests (Byrne et al., 1994; Devonshire and Moores, 1984; Hama, 1976; Zhu et al., 1996). Additional mechanism of OP resistance in other arthropod pests were related to metabolic detoxification by glutathione S-transferases - 121 - (Armstrong and Suckling, 1988; Fournier etal., 1987) and nonspecific esterases (Abdel-Aal et a!., 1992, Dary et al., 1990; Zliu and Brindley, 1992). An interesting mode of insect resistance demonstrated in aphids and mosquitoes is apparently mediated via extensive OP binding to higin levels of circulating non-cholinergic esterases. Amplification of genes encoding detoxifying esterases were associated with resistance to OP compounds in aphids (Field etal., 1988) and mosquitoes (Mouches et al., 1986). In the present study, the quantitative and qualitative analysis of the whole body homogenate of O.coffeae, as evident from the Table 32 and Figure 27, showed that the quantity of acetylcholinesterase in the pesticide-exposed specimen was significantly higher as compared with the laboratory-reared ones. In a similar finding the involvement of acetylcholinesterase in resistance of California red scale, Aonidiella aurantii to organophosphorous pesticides showed that the resistant strains of Hulda and Shiller were higher as compared to the susceptible laboratory strain (Levitin and Cohen, 1998). Detoxification enzymes are important determinants of growth and survival in herbivorous insects (Brattsten, 1979). These enzymes metabolize plant allelochemicals and insecticides. Characterizing and understanding the mechanisms of induction and regulation of the enzymes are important for development of successful pest management strategies. Many enzymes involved in detoxification pathways act on broad array of substrates, including both naturally occurring plant allelochemicals and synthetic pesticides (Gordon, 1961). Therefore, physiological response of herbivores to host plants may lead to enhanced metabolism of pesticides because mechanism of detoxification of host-plant allelochemicals may also be effective in detoxifying pesticides (Yang et al., 2001). These implications and interaction of host chemicals, pesticides and the detoxifying enzymes have to be thoroughly understood before planning management of pesticide-resistant / tolerant pest populations in tea. - 122- 5.4.1. Results of inhibition tests of isozymes for detection of insecticide resistance I tolerance status of the four arthropod tea pests 5.4.1.1. Midgut esterase isozymes In Buzura suppressaria thirteen midgut esterase bands were detected on native PAGE in all the laboratory-reared larvae and those collected from conventional tea plantation exposed to the pesticide spray in the field (field-collected). Bands of VSM (EST-3), SM (EST-2) and FM (EST-1) were deeply stained in the field-collected B.suppressaria [Fig. 28b (I)] when compared with those of the laboratory-reared one [Fig. 28a (I)]. When subjected to organophosphate, Quinalphos (1:400) (v/v) inhibition, the EST-2 and EST-3 esterase bands were partially or not inhibited while EST-1 was almost inhibited in all the pesticide-exposed larvae [Fig. 28b (ii)] vis-a-vis the esterase bands completely disappeared (inhibited) in the laboratory-reared larvae after inhibition treatment [Fig. 28a (ii)]. Laboratory-reared Field-collected VSM (EST-3) SM (EST-2) FM (EST-1) Fig. 28a (i) Reference (ii) Treated: Inhibited b (i) Reference (ii) Treated: Partially inhibited Fig. 28a Esterase bands of midgut homogenate of (i) laboratory-reared B.suppressaria with (ii) complete inhibition of esterase bands after 30 min incubation with organophosphate b Esterase bands of midgut homogenate of (i) pesticide-exposed B.suppressaria with (ii) lack of inhibition of esterase bands after 30 min incubation with organophosphate - 123 - In Eterusia magnified midgut homogenates of Vth instar larva showed eight bands. The intensity and number (EST-1-EST-8) of the isozyme bands from midgut homogenates were similar in all the individuals (larvae) reared on laboratory [Fig. 29a (i)] but in the field- collected larvae all bands were deeply stained indicating an intensive formation of esterases [Fig. 29b (i)]. After organophosphate inhibition treatment no major inhibition of bands were apparent in pesticide-exposed larvae [Fig. 29b (ii)] while in laboratory-reared individual, all esterase bands were inhibited [Fig. 29a (ii)]. Laboratory-reared Field-collected EST 7 6 Fig. 29a (i) Reference (ii) Treated: Inhibited b (I) Reference (ii) Treated: no major inhibition Fig. 29a Esterase bands of midgut homogenate of (i) laboratory-reared Et.magnifica with (ii) complete inhibition of esterase bands after 30 min incubation with organophosphate b Esterase bands of midgut homogenate of (i) pesticide-exposed Et.magnifica with (ii) lack of inhibition of esterase bands after 30 min incubation with organophosphate - 124- The midgut homogenates of Helopeltis theivora showed three prominent esterase bands. These bands were slow moving, medium slow moving, and fast moving. Esterase bands of the specimens exposed to pesticide sprays showed a higher staining intensity [Fig. 30b (i)] than the ones reared in laboratory [Fig. 30a (i)]. After inhibition test with organophosphate the pesticide-exposed specimen showed no apparent inhibition [Fig. 30b (ii)] and bands disappear in the laboratory-reared ones [Fig. 30a (ii)]. EST Laboratory-reared Field-collected Fig. 30a (i) Reference (ii) Treated: Inhibited b (I) Reference (ii) Treated: no major inhibition Fig. 30 a Esterase bands of midgut homogenate of (i) laboratory-reared H.theivora with (ii) complete inhibition of esterase bands after 30 min incubation with organophosphate b Esterase bands of midgut homogenate of (i) pesticide-exposed H.theivora with (ii) lack of inhibition of esterase bands after 30 min incubation with organophosphate - 125- Polyacrylamide gel electrophoresis showed that the whole body homogenate of pesticide- exposed female Oligonychus coffeae collected from plantations possessed 3 major esterase bands (EST-1, EST-2 and EST-3) [Fig. 31b (i)] whereas the laboratory-reared showed only one (EST-1) [Fig. 31a (i)]. The bands (EST-2, EST-3) were however absent in the laboratory-reared female O.coffeae. Esterases of both the individual was sensitive to organophosphate. At 1:400 volume/ volume field application doses, the esterase band inhibited in laboratory-reared ones [Fig. 31a (ii)] and almost no inhibition of bands in pesticide-exposed one [Fig. 31b (ii)]. Laboratory-reared Field-collected EST 1 2 Fig. 31a (i) Reference (ii) Treated: Inhibited b (i) Reference (ii) Treated: no inhibition Fig. 31 a Esterase bands of whole body homogenate of (i) laboratory-reared O.coffeae with (ii) complete inhibition of esterase bands after 30 min incubation with organophosphate b Esterase bands of whole body homogenate of (i) pesticide-exposed O.coffeae with (ii) lack of inhibition of esterase bands after 30 min incubation with organophosphate - 126- 5.4.1.2. Acetylcholinesterase from cerebral ganglia Zymogram of acetylcholinesterase (AChE) developed from homogenate of cerebral ganglia of Buzura suppressaria showed formation of single band. The band-intensity was notably high in the pesticide-exposed specimens collected from conventional plantation (field) [Fig. 32b (i)] and low in laboratory-reared ones [Fig. 32a (i)]. After organophosphate inhibition test the AChE band disappear in laboratory-reared specimen [Fig. 32a (ii)] vis-a-vis partial /or no effect in the band intensity in pesticide-exposed one collected from field [Fig. 32b (ii)]. Laboratory-reared Field-collected 1 2 3 4 Flg. 32a (I) Reference (ii) Treated: Inhibited b (I) Reference (ii) Treated: no inhibition Fig. 32. Electrophoregram of acetylcholinesterase developed from homogenate of cerebral ganglia of B.suppressaria Homogenate of cerebral ganglia of specimens: Lane 1: Laboratory-reared Lane 2: Laboratory-reared with organophosphate inhibition Lane 3: Collected from conventional plantation (field: pesticide-exposed) Lane 4: Collected from conventional plantation (field: pesticide-exposed) with organophosphate inhibition - 127- Electrophoregram of acetylcholinesterase developed from the homogenate of cerebral ganglia of Eterusia magnifica showed single band formation. In pesticide-exposed specimen collected from conventional plantation (field) [Fig. 33b (i)] the band intensity was higher than the laboratory-reared ones [Fig. 33a (i)]. Organophosphate inhibition test showed no inhibitory effect in field-collected (pesticide-exposed) larvae [Fig. 33b (ii)] but band disappear in laboratory-reared one with the same doses of organophosphate [Fig. 33a (il)]. Laboratory-reared Field-collected 1 2 3 4 Fig. 33a (I) Reference (ii) Treated: Inhibited b (I) Reference (ii) Treated; no inhibition Fig. 33. Electrophoregram of acetylcholinesterase developed from homogenate of cerebral ganglia of Et.magnifica Homogenate of cerebral ganglia of specimens: Lane 1: Laboratory-reared Lane 2: Laboratory-reared with organophosphate inhibition Lane 3: Collected from conventional plantation (field: pesticide-exposed) Lane 4: Collected from conventional plantation (field: pesticide-exposed) with organophosphate inhibition -1 2 8 - The single acetylcholinesterase band intensity was notably high in pesticide-exposed Helopeltis theivora collected from field [Fig. 34b (i)] as compared with laboratory-reared ones [Fig. 34a (i)]. In the pesticide-exposed individuals the activity zone could not be inhibited by the field-applicatlon dose [Fig. 34b (ii)] of organophosphate but band was disappear in laboratory-reared ones [Fig. 34a (ii)]. Laboratory-reared Field-collected 1 2 3 4 ■3BT Fig. 34a (I) Reference (ii) Treated: Inhibited b (i) Reference (ii) Treated: no inhibition Fig. 34. Electrophoregram of acetylcholinesterase developed from homogenate of cerebral ganglia of H.theivora Homogenate of cerebral ganglia of specimens: Lane 1: Laboratory-reared Lane 2: Laboratory-reared with organophosphate inhibition Lane 3: Collected from conventional plantation (field: pesticide-exposed) Lane 4: Collected from conventional plantation (field: pesticide-exposed) with organophosphate inhibition - 129- Three major acetylcholinesterase band was detected in the pesticide-exposed female Oligonychus coffeae collected from conventional plantation (field) [Fig. 35b (i)] whereas laboratory-reared ones possessed only one [Fig. 35a (I)]. Field-application dose of organophosphate showed no inhibition of band in pesticide-exposed specimen [Fig. 35b (il)] while in the laboratory-reared one the band was inhibited by organophosphate [Fig. 35a (ii)]. Laboratory-reared Field-collected 1 2 K Fig. 35a (i) Reference (ii) Treated: Inhibited b (i) Reference (ii) Treated: no inhibition Fig. 35. Electrophoregram of acetylcholinesterase isozyme of whole body homogenate of O. coffeae Lane 1: Laboratory-reared Lane 2; Laboratory-reared with organophosphate inhibition Lane 3: Collected from conventional plantation (field: pesticide-exposed) Lane 4: Collected from conventional plantation (field: pesticide-exposed) with organophosphate inhibition - 130 - 5.4.2. Discussion on resistance I tolerance status of the four pests Detection of the status of resistance / tolerance to pesticides is very much necessary for management of pests and also for minimizing the development of further resistance to pesticides. Detection methods need to be simple, provide reproducible results, and simulate field treatment conditions as closely as possible (Walker et a!., 1973). The monetary and human costs of resistance are difficult to assess, but loss of pesticide effectiveness almost invariably entails increased application frequencies and doses and finally, more expensive replacement compounds, as new pesticides become increasingly more difficult to discover, develop, register and manufacture (Metcalf, 1980). Therefore it is essential to develop strategies to delay or minimize the probability of resistance evolution. This will be possible only if resistance monitoring is considered which is essential for insecticide and acaricide resistance management (Dennehy and Granett, 1984). Resistance is often based upon increased enzymatic detoxication of an insecticide (Brown and Brogdon, 1987). The development of quantitative and qualitative techniques for measuring enzyme activity in individual insects is becoming important for several reasons. Insecticide resistance management depends on the ability to measure resistant, as well as susceptible strain, in order to guide decisions on pesticide use and evaluate the effects of strategies designed to avoid or delay the development of resistance (Brent, 1986; Knight and Norton, 1989). Monitoring of resistance in the field populations is clearly the method of choice for understanding the long and short term effects of insecticide use on populations structure (Brown and Brogdon, 1987). Increased esterase detoxification is a common mechanism of resistance to organophosphorous insecticides in insects. In Culex pipiens mosquitoes it is due to the overproduction of esterases that have a high binding affinity with insecticides and the high titer of esterases present that serves as an “insecticide sink” and delays or prevents - 131 - interactions iaetween toxin and target site. Resistance due to this mechanism is called sequestraticffj and this mechanism mainly act in insects befotK they reach their acetylcholinesterase target (Cuany et al., 1993; Wheelock et ai, 2005). Biochemical mechanisms that confer malathion resistance have been studied in Homoptera (Abdel-Aal et al., 1990, 1992; Field et al., 1994) and Lepidoptera (Beeman and Schmidt, 1982; Halliday, 1988; Doichuanngam and Thornhill, 1992). In nearly every case in these insect orders, the resistance are probably mediated by enhanced metabolic detoxification or sequestration through the increased activity of a malathion-specific carboxylesterase. Despite extensive use of organophosphate nearly about 64%, in the tea agroecosystem (Sannigrahi and Talukder, 2003), comparable studies on resistance or tolerance status are lacking in the four major tea pests in question. Resistance studies of these pests are important in order to carry out the future management practices and take decisions on pesticide use. PAGE and inhibition tests for biochemical characterization of esterases from midgut homogenate of B.suppressaria showed thirteen esterase bands in both pesticide-exposed field (collected from conventional plantation) and laboratory-reared ones [Fig. 28a, b]. Close inspection of lanes indicated that the band activity were more intense in the pesticide-exposed larvae than laboratory-reared ones. When subjected to organophosphate, Quinalphos (1:400) (v/v) inhibition, the EST-2 and EST-3 esterase bands were only partially inhibited in all the pesticide-exposed larvae [Fig.28b(ii)] vis-a-vis the esterase bands disappeared in the laboratory-reared larvae after inhibition [Fig.28a(ii)]. Partial inhibition of all the carboxylesterase bands in the field-collected (pesticide-exposed) larvae in our study, suggest that all the isozymes of esterase specially EST-2 and EST-3 in B.suppressaria were probably related to organophosphate resistance or higher tolerance. In a similar finding overall activity of the esterase enzyme have been positively related with the degree of resistance / or higher tolerance in the cotton aphid. Aphis gossypii (Saito and Hama, 2000) - 132- and Myzus persicae (Devonshire and Moores, where all the carboxylesterase bands were responsible for organophosphate resistance. Elevated esterase activity has often been reported as a basis of resistance mechanism of Blattella germanica (Siegfried and Scott, 1992; Anspaugh et al., 1994). In Et.magnifica the native PAGE results indicated eight esterase bands in midgut homogenate and these bands were more intense in^pesticrde-exposed larvae when compared with those oHhe laboratory- reared ones [Fig. 29a, bj. After organophosphate treatment, the EST-3 and EST-4 bands were not inhibited in pesticide-exposed field larvae [Fig. 29b (ii)], while in laboratory-reared individual, all esterase bands were inhibited [Fig. 29a (ii)]. It implied that the pesticide- exposed larvae showed an overall lower sensitivity to organophosphate possibly due to a larger amount of esterase present in the exposed larvae. In a similar finding elevated esterase activity was detected in resistant strains of German cockroaches when compared to that of the susceptible strain. This may be resulting from an increased catalytic efficiency or overproduction of esterases through gene amplification or over-expression (Hemingway and Karunaratne, 1998). In the current study, there is no direct evidence to show which esterase isozymes are involved in insecticide resistance, although EST-3 and EST-4 showed more intense banding patterns in the pesticide-exposed field larvae appeared to be related to pesticide detoxification in Et.magnifica specimens. In a similar finding Lee et al. (2000) also showed EST-1, EST-5 and EST-7 esterase bands with more intense banding patterns in the resistant strains of Blattella germanica. Insecticide breakdown by metabolism is the common mechanism that has evolved to protect insects. Role of increased esterase activity in resistance was confirmed by enhanced hydrolysis of several insecticides by the resistant insects such as Myzus persicae (Devonshire, 1977), Culex quinquefasciatus (Georghiou and Pasteur, 1978) and Musca domestica (Kao et al., 1984). In the present finding the midgut homogenate of H.theivora showed three major esterase bands in the pesticide-exposed field specimen with a higher -133- staining intensity [Fig. 30b H}] as connparecl to the much reduced staining intensity in laboratory-reared ones [Fig. 3Ba ^)]. Esterases of both the categories of specimens vsspb sensitive to organophosphate. At 1:400 (v/v), all the esterases were inhibited in laboratory- reared ones [Fig. 30a (ii)] and partial inhibited in pesticide-exposed field individuals [Fig. 30b (ti)]. The results are comparable to the other sucking pests like Aphis gossypii, where all the bands of carboxylesterase was inhibited by higher dose offenitroxon suggesting that all the isozymes of carboxylesterase were related to organophosphate resistance (Saito and Hama, 2000). The soluble esterases present after separation by native PAGE in the whole body homogenate of pesticide-exposed female O.coffeae collected from field showed three esterase bands [Fig. 31b (i)] while the laboratory-reared ones showed only one esterase band [Fig. 31a (i)]. Esterases of both the individual was sensitive to organophosphate. At 1:400 volume/ volume, the esterase band completely inhibited in laboratory-reared ones [Fig. 31a (ii)] while almost no inhibition of bands in pesticide-exposed one was observed [Fig. 31b (il)]. In a similar finding field-collected strain (MR-VL) of Tetranychus urticae showed enhanced detoxification by increased activity (both quantitative and qualitative) of esterases, contrastingly the susceptible laboratory strain (LS-VL) showed much less activity than the former one. More esterases were inhibited in LS-VL strain at 0.04 pM paraoxon in comparison to MR-VL strain showing an overall lower sensitivity to paraoxon possibly due to presence of large amount of esterase in the resistant strain (Leeuwen et al., 2005). Acetylcholinesterase (AChE) terminates nerve impulse by catalyzing the hydrolysis of the neurotransmitter acetylcholine. It is a key enzyme in the cholinergic insect nervous system. Insecticides such as organophosphates and carbamates covalently bind to the active site of AChE and cause the death of the insect (Fournier et al., 1992b). In the present study the electrophoregrams of acetylcholinesterase (AChE) developed from homogenate of cerebral ganglia of B.suppressaria and Et.magnifica showed only one AChE band. This result might - 134- be ccRTBlated with the increased esterase activity in the midgut rromogenate that are able to hydrotyzE insecticides and which is also protect acetylcholinesterase by offering a large number of alternative sites of phosphorylation and therefore reduce the amount of OP available to bind AChE. The inhibition studies with organophosphate at 1 ;400 (v/v) dose showed partial/ or no inhibitory effect in the pesticide-exposed field-collected B.suppressaria and Et.magnifica larvae but the band on organophosphate treatment disappeared in laboratory-reared ones [Fig. 32a, b; 33a, bj. This indicated that a large number of available catalytic sites may bind scavenge toxic molecules, still leaving enough free AChE for proper functioning of the nervous system in pesticide-exposed field larvae. This phenomenon is well established in the sheep blowfly (Whyard et a!., 1994) and esterase E-4 in Myzus persicae (Devonshire and Moores, 1982), when treated with malathion. The band activity of acetylcholinesterase developed from homogenate of cerebral ganglia of H.theivora and whole body homogenate of O.coffeae showed a higher concentration of AChE molecules in the pesticide-exposed individuals than the laboratory-reared ones [Fig. 34a, b; 35a, b], AChE of the both the species were apparently sensitive to organophosphate (Ekalux). At 1:400 (v/v) dose usually recommended in field application, all the AChE bands were inhibited in laboratory-reared O coffeae and H.theivora but in the pesticide-exposed individuals the activity zone could not be inhibited by the field application dose showing an overall lower sensitivity of the organophosphate or indicating the presence of larger amount of AChE in the pesticide-exposed individuals. An elevated AChE activity in the resistant strain of Aonidiella aurantii to organophosphates has been well described by Levitin and Cohen (1998) where high level of AChE occurs in resistant strains besides a large number of available catalytic sites of AChE molecules that scavenges toxic molecules, leaving free AChE for proper functioning of nervous system. So, in the insecticide inhibition studies with organophosphate, all the pesticide-exposed specimen of the above mentioned tea pests collected from the field showed partial /or no - 135- inhibition of esterase bands vis-a-vis all the equivalent bands mostly disappeared in the PAGE of the latx>ratory-reared speciniEris when treated with the field application doses of organophosphate. The finding suggests the roie of esterases in pesticide detoxification in these pest species besides their possibly in endowing these species with a greater insecticide tolerance or in other word partial resistance. Theinhibitiorr tests with the same pesticide also showed partiah/orno inbitbn of AChE band in the pesticide-exposed field individual whereas they disappeared in the laboratory-reared ones which further confirmed their greater tolerance to organophosphate pesticide in all the four major tea pests in question. - 136- 6. Summary • The defoliating caterpillars, Buzura ssppressaria (looper), Eterusia magnifica (red slug) (Lepidoptera) and the sucking bug, Helopeltis theivora (tea mosquito) (Hemiptera) and the mite, Oligonychus coffeae (red spider) (Acari; Tetranychidae) are reported as major tea pests of the plantations of Darjeeling foothills, Terai and the Dooars, with their regular incidence in the tea growing areas of North-East India. • Herbivore insects possess an assemblage of enzymes that on one hand regulate their digestive activity as hydrolyzing enzymes and on the other hand constitute defence against chemical toxicants as oxidoreductases and detoxifying enzymes. • Among the digestive enzymes the quantity of amylase detected in the salivary and midgut homogenate was found to be higher in midgut of B.suppressaria as compared to almost equal quantity of amylase at salivary and midgut levels in Et.magnifica. • H.theivora showed enhanced levels of amylase both in salivary gland and midgut. Amylase could also be detected in the whole body homogenate of O.coffeae. • In 8. suppressaria the midgut protease activity was significantly higher than that of the salivary gland whereas in Et.magnifica almost equal protease activity in oral and midgut regions was recorded. The protease activity was evident in salivary gland and midgut of H.theivora. The protease activity was found in the whole body homogenate of O.coffeae. - 138- • In B.suppre^ma the lipase activity is relatively higher than that of Et.nsgnifica both at salivary and r a ^ u t levels and in H.theivora a very low activity of lipase was registered. In O.coffeae the li|iElse activity was also found at a low key. • Among the oxidoreductases, the catalase quantity of B.suppressaria was found to be marginally higher in salivary gland homogenate as compared with the midgut homogenate but in larvae of Et.magnifica almost similar quantity of catalase was observed both at salivary and midgut levels. • The catalase present in salivary gland homogenate of H.theivora initially showed more activity as compared to that of midgut, and in O.coffeae the catalase was detected in whole body homogenate. • In B.suppressaria as well as in Et.magnifica the peroxidase in salivary gland homogenate showed less quantity than that of the midgut. • The peroxidase quantity was found to be similar both in the salivary and midgut homogenate of H.theivora. Peroxidase could also be detected in O.coffeae at an enhanced level. • The apparent lack of activity of polyphenol-oxidase in both salivary and midgut homogenate indicated that B.suppressaria could possibly use peroxidases as the only enzyme to metabolize tea phenolics, however in Et.magnifica polyphenol-oxidase was detected in both saliva and midgut homogenate indicating a different strategy of oxidizing than the former lepidopteran pest species. In H.theivora, level of polyphenol- - 139- oxidase was apparently less than that of the {Kroxidase and in O.coffeae polyphenol- oxidase could also be detected in the whole bodynomogenate. • Among the detoxifying enzymes, a significantly high quantity of the general esterases (EST) in salivary gland homogenate and midgut homogenate of the pesticide-exposed B.suppressaria and Et.magnifies larvae, collected from conventional plantation (field), over unexposed ones could be registered. This difference possibly indicated a greater esterase-based detoxifying activity in the field-collected specimens. • Comparison of isozyme profiles for fifth instar caterpillars of laboratory-reared B.suppressaria and Et.magnifica showed that in the salivary gland homogenate of B.suppressaria eight esterase bands were present whereas in midgut homogenate, thirteen bands were present; whereas in Et.magnifica the salivary gland homogenates showed two esterase bands and midgut homogenates showed eight bands. • In H.theivora the quantity of esterases was always significantly higher in the midgut than that in the salivary gland independent of tea clone on which they were reared. A marked difference in general esterase quantity was noted between the conventional (pesticide- exposed) and laboratory-reared (pesticide unexposed) female O.coffeae. • The esterase bands developed from midgut homogenate of H.theivora were three and could be identified as slow-moving, medium-moving, and fast-moving and bands with a higher staining intensity in pesticide-exposed specimens than the laboratory-reared ones. Analysis of esterase bands showed that the pesticide-exposed female O.coffeae - 140- possessed 3 major co-fnigrating bands whereas the laboratory-reared ones showed only one. • In B.suppressaria, Et.magnifica and O.coffeae no significant difference in the quantity of glutathione S-transferase (GST) was observed between laboratory-reared specimens and pesticide-exposed ones. Moreover, in the qualitative assay, no band formation was detected in laboratory-reared and pesticide-exposed ones, thus suggesting absence of any role of GST in pesticide detoxification in these species. • In H.theivora significantly high glutathione S-transferase quantity in specimens from the field-collected population was estimated as compared to laboratory-reared specimens. The banding pattern in PAGE also showed a single low-intensity band in laboratory- reared specimen as compared to a parallel band with high intensity in field-collected specimens. • Acetylcholinesterase (AChE) quantified from the homogenate of cerebral ganglia of four major pests in question showed a significant difference between the laboratory-reared and pesticide-exposed ones. The zymogram of the acetylcholinesterase of B.suppressaria and Et.magnifica showed a single band formation with a higher intensity in the pesticide-exposed larvae as compared to the laboratory-reared individuals. • In H.theivora it was also evident that quantity of acetylcholinesterase that binds with the organophosphates and carbamates, was significantly higher in the specimens collected from field compared to the laboratory-reared ones. Electrophoregram pattern of acetylcholinesterase indicated notably high band-intensity in the pesticide-exposed field specimens and low intensity in unexposed ones. Both quantitative and qualitative - 141 - aialysis of the homogenate of O.coffeae showed tfiat the quantity of affityteholinesterase in the pesticide-exposed specimen viss significantly higher as compared with the laboratory-reared ones. The insecticide inhibition studies with organophosphate showed that all the pesticide- exposed specimen of the tea pests in the field showed partial or no inhibition of esterase bands vis-a-vis in the laboratory-reared individuals all the bands were completely or largely blocked resulting in disappearance of the electrophoretic bands when treated with field-recommended doses of organophosphate pesticides. The inhibition studies with the same pesticide also showed partial or no inhibition of AChE band in the pesticide-exposed individual while they disappeared in the laboratory- reared ones. The digestive enzymes commonly found in the salivary and mid gut of the tea pests in question are therefore of interest in understanding their feeding relation to host (tea) as well as in devising methods of non-conventional pest management. The defense enzymes work by oxidation, reduction, hydrolysis or conjugation of molecules. The oxidoreductase enzymes are of great value because of their involvement not only in defensive but also in processing the secondary metabolites of the host plant. Many such enzymes involved in detoxification pathways act on a broad array of substrates found as plant allelochemicals and chemical pesticides. The present investigation is contemplated to evaluate the character and quantity of digestive, oxidoreductase and detoxifying enzymes of the four arthropod pests of tea so that the knowledge-base may be utilized in designing control and resistance management programmes of these tea pests in future. - 142- 7. Highlights > The four major pests of tea, looper {Buzura suppressaria), red slug (Eterusia magnified), tea mosquito bug (Helopeltis theivora) and red spider mite {Oligonychus coffeae) have a wide occurrence in both organic and conventional plantations of the Darjeeling foothills and their adjoining plains of Terai and the Dooars. ^ Greater amount of amylase activity found in the midgut than in the salivary gland of B. suppressaria indicated greater digestion of polysaccharides of young leaves in midgut than their break down at the time of ingestion in the oral cavity. > The presence of protease activity both in salivary gland and midgut of H.theivora indicated that this pest can well utilize the protein source of the tea leaf by extra oral and gut digestion. > The activity of lipase in all the pests in question was much reduced than other two digestive enzymes. ^ Of the oxidoreductases, the catalase assay of B.suppressaria and Et.magnifica was found to be marginally higher in salivary gland homogenate as compared with the midgut homogenate. As this enzyme is involved in inhibiting the action of toxic plant phenolics besides removing the hydrogen peroxide, an active blocking of these oxidants possibly takes place at the salivary gland level followed by that at gut level. - 144- > Catalase present in salivary gtand homogaiate of H.theivora initially showed more activity as compared to that of midgut. CataJase rn the saliva has the ability to prevent the formation of plant protective compounds such as quinone. ^ In H.theivora the peroxidase assay was found to be similar both in the salivary and midgut homogenate, thus indicating the oxidative activity as a defence measure at both levels. In O.coffeae the enhanced peroxidase level could possibly minimise the effects of the toxic products that the mites ingest or experience while colonizing the tea leaves. ^ In Et.magnifica presence of polyphenol-oxidase was detected in both saliva and midgut homogenate, but the enzyme was lacking in B.suppressaria. Occurrence of high quantity of polyphenol-oxidase in O.coffeae suggested that the mite could easily overcome the plant defenses. > The enzyme possibly enabled the species to oxidize a wide range of tea phenolic compounds leading to nutralization the defence allelochemicals of the host leaves ingested along with food. > Among the detoxifying enzymes, a significantly high quantity of the general esterases (EST) in salivary gland homogenate and midgut homogenate of the pesticide-exposed field specimens of B.suppressaria and Et.magnifica larvae over laboratory-reared un exposed ones possibly indicated a greater esterase-based detoxifying activity in the former ones. - 145- > The EST-2 and EST-3 bands showed intense staining in B.suppressaria specaDens coHected from conventtC8»i plantations. Such high intensity may be related to gESsater pesticide tolerance / resistance of B.suppressaria populations. > Two soluble esterase isozymes, designated as EST-3 and EST-4 due to their prominent presence in the pesticide-exposed larvae of Et.magnifica specimens appeared to be related to pesticide detoxification. > Higher midgut esterase activity was found in H.theivora specimens of conventional plantations, possibly endowing the bug with a greater insecticide tolerance {vis-a-vis resistance). ^ Esterase bands on polyacrylamide gel showed that the pesticide-exposed female O.coffeae possessed 3 major co-migrating band whereas the pesticide unexposed female O.coffeae possessed only one. Enhance quantity of esterases as well as the additional bands (isozymes) of O.coffeae of the conventional plantation were possibly involved in the detoxification of synthetic acaricides and insecticides. > Significantly high glutathione S-transferase (GST) quantity in field-collected specimens of H.theivora as compared to laboratory-reared specimens was noted. The banding pattern also showed a single low-intensity band in laboratory-reared specimen where as field-collected specimens registered a parallel but a high intensity band. - 146- > Acstylcholinesterase (AChE) quantified in the homogenate of cerebral ganglia of four m a^r pests in question showed a significant difference b^»een the laboratory-reared and pesticide-exposed field-collected ones. 5^^ The zymogram of the acetylcholinesterase of B.suppressaria and Et.magnifica showed a single band formation with a higher intensity in the pesticide-exposed larvae as compared to the laboratory-reared individuals. ^ it was also evident that quantities of acetylcholinesterase that bind with the organophosphates and carbamates, was significantly higher in the specimens of H.theivora collected from field compared to the laboratory-reared ones. Electrophoregram pattern of acetylcholinesterase also indicated high band-intensity in the pesticide-exposed specimens and low in unexposed (laboratory-reared) ones. > Both quantitative and qualitative analysis of the homogenate of O.coffeae showed that the quantity of acetylcholinesterase in the pesticide-exposed specimen was significantly higher as compared with the laboratory-reared ones. > In insecticide inhibition test, treatment with field-recommended doses of organophosphate showed partial or no inhibition of esterase gel bands in all the pesticide-exposed field specimen of the above tea pests. Where as in the laboratory- reared specimens all the bands were largely blocked as evident through their disappearance, suggesting the role of esterase-based pesticide detoxification in these species. The uninhibited or over produced of esterase might confer a greater insecticide tolerance / resistance to these pest species. - 147- 8. 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Biochemical mechanisms and diagnostic microassays for pyrethroid, carbamate, and organophosphate insecticide resistance / cross-resistance in the tobacco budworm, Heliothis virescens. Pestic. Biochem. Physiol. 56(3): 183-195. Zhang, J.P., Wang, J.J., Zhao, Z.M., Dou, W. and Chen, Y. 2004. Effects of simulated acid rain on the physiology of carmine spider mite, Tetranychus cinnabarinus (Boisduvals) (Acri: Tetranychidae). J. Appl. Entomol. 128(5): 342-347. Zhu, K. Y., Lee, S.H. and Clark, J.M. 1996. A point mutation of acetylcholinesterase associated with azinphosmethyl resistance and reduced fitness in Colorado potato beetle. Pestic. Biochem. Physiol. 55(2): 100-108. - 184- Zhu, K.Y. and Brindley, W.A.1992. Significance of carboxs^terases and insensitive aiSF^ylcholinesterase conferring organophospliate resistsriCE in Lygus hesperus papulations. Pestic. Biochem. Physiol. 43; 223-231. Zhu, K.Y. and Gao, J-R. 1998. Kinetic properties and variability of esterases in organophosphate-susceptible and resistant greenbugs Schizaphis graminum (Homoptera: Aphididae). Pestic. Biochem. Physiol. 62; 135-145. Zhu, K.Y. and He, F. 2000. Elevated esterases exhibiting arylesterase-like characteristics in an organophosphate-resistant clone of the greenbug, Schizaphis graminum (Homoptera: Aphididae). Pestic. Biochem. Physiol. 67; 155-167. - 185- 9. Acknowledgements 1 express my deep sense of gratitude to Dr. Ananda Mukhopadhyay, FZS (Cal.), Professor, Department of Zoology and Former Director, Centre for Life Sciences, University of Nortin Bengal, under whose guidance the work was carried out. I have been inspired by his diligence, his attention to detail and energetic application to any problem and for his invaluable tutelage, constant encouragement, and sustained interest throughout this scientific study. I would like to extend my wholehearted appreciation to Dr. D.C. Deb, Professor, Department of Zoology, NBU for his generous advice in my research work. I am also grateful to Prof. J. Pal, Head, Deptt. of Zoology, Prof. A.K. Chakravarty, Prof. T.K. Choudhuri, Dr. S. Baratand Dr. M. Bahadur for providing laboratory facilities and invaluable counseling. My heart-felt thanks are due to Dr. G.T. Gujar, Head, Division of Entomology, Dr. V.V. Ramamurthy, Dr. A. Phokela, Dr. D. Dey and Dr. B. Paul of the same division lARI, New Delhi and Dr. K.R. Kranthi, CCRl, Nagpur for their bountiful help of literature and encouragement. I am also extremely grateful to Dr. S. Sannigrahi, Tea Research Associatloh, Dlbrugarh, Dr. S.K. Pathak, TRA, Dikom, Assam and Dr. S.E. Kabir, tea expert for providing me with literatures and timely information on the occurrence of the pests under my study. I extend my thankfulness to the Managers and Assistant Managers of the tea estates, to name some of them are: Singell Tea and Agricultural Industries limited, Goodricke Group limited, Seeyok T.E. of Darjeeling hills; KamalpurT.E., Atal T.E., Azamabad T.E., DagapurT.E. of Terai, and Debpara, Dalgaon T.E., Nangdala T.E. of the Dooars for giving me the opportunity to visit their gardens for research study. I sincerely acknowledge the Vice-Chancellor, and University authority for providing the valuable online e-journals through Local Area Network, VSAT and UGC-INFONET Consortium. - 187- I wish to express my thanks to all of my colleagues of the Post Graduate Diploma in Tea Management, Institute of Plantation Science and Management, University of North Bengal, and Dr. U. Gupta, Sr. Scientist, Spices Board, Gangtok, Sikkim for his encouragement about this study. I am also thankful to my friends specially, Ms. S. Das, Ms. D. De, Mr. S. Roy of Entomology Research Unit and Mr. S. Bagchi, Mr. A. Das, Ms. H.Yasmin and Mr. T. Majumder of the Department of Zoology and Dr. S. Dasgupta, Department of Botany for their immense help and cheerful cooperation from time to time. The assistance rendered by Mr. B. Barman, for the field studies are also gratefully acknowledged. I owe my gratitude to Dr. Kun Yan Zhu, Kansas State University, Manhattan, USA, Dr. William Can-Jen Maa, Academia Sinica, Taipei, Taiwan, R.O.C. and Dr. Thomas Van Leeuwen, Ghent University, Belgium and many other workers for their help of valuable literature and suggestion. My indebtedness and unfathomable thanks to Mr. B. Banerjee, Chairman, Tea Board, Dr. T.C. Choudhuri, Director, Research and Dr. B. Banerjee, Advisor, National Tea Research Foundation, Tea Board, Kolkata, for their encouragement and providing me the Junior and subsequently Senior Research Fellowship through a project sanctioned to my supervisor, Dr, A. Mukhopadhyay. I wish to acknowledge the support extended to me by all my family members and my sister, without whose love and patience throughout, this work would not be possible. Finally, I would like to express my deep indebtedness to my mother and father for their endless inspiration, affection and encouragement that has served as the fuel throughout my work and in appreciation of their love I dedicate this work to their sacred feet. Date: 29‘^ January 2007 Place; Siliguri !Mayuf^ Sar/(er - 188- }lnne~ure - 189 - 10. Annexure L L ist o f aSSreviaHons used AmsI: Above mean sea level ANOVA: Analysis of variance AChE: Acetylcholinesterase a: Alpha At the rate of cm: Centimetre ®C: Degree Celsius CDNB: 1-chloro-2, 4-dinitrobenzene CUSO4: Copper sulphate D: Darl< DTNB: 5,5' - dithiobis (2-nitrobenzoic acid) E: East EC: Emulsifiable concentrate EDTA: Ethylene Diamine Tetra Acetic Acid EEPS: Endogenous enzymes of protective systems EST: Esterase ETL: Economic threshold level Fig: Figure g: Gram GST: Glutathione S-transferase ha: Hectare h: Hour(s) kg: Kilogram I: Litre L: Light m: Meter M: Molarity |jl: Microlitre pM: Micro mole |jg; Microgram mg: Milligram - 190- ml: Millilitre mM: Millimole mA: Milliampere meq: Miiliequivalent min: Minutes nm: Nanometre N: Normality NaOH: Sodium hydroxide NBT: Nitro blue tetrazolium N.E: North-East OP: Organophosphate OD: Optical density pH: Negative logarithm of hydrogen ion concentration %: Percent PAGE: Polyacrylamide Gel Electrophoresis PMS: Phenazine methosulphate R„: Relative mobility RH: Relative humidity s: Seconds sp.: Species spp.: Species (plural) SD: Standard deviation SE: Standard error TCA: Trichloro acetic acid T.E: Tea estate Th: Thousand TV: Tocklai vegetative clone Tris- HCI: Tris (hydroxymethyl) aminomethane- hydrochloric acid UV: Ultraviolet (v/v): Volume / volume Vis: Visible (w/v): Weight / volume -191 - II. List ofpuBCishecC/ acceptedpapers reCatecC to Ua pests ofM ayul^ Sarl^r YEAR 2007 1. Sarker, M., Pradhan, B. and Mukhopadhyay, A. 2007. Digestive enzymes and feeding biology of Buzura suppressaria Guen. and Eterusia magnifica Butl., two major defoliating pests of Camellia sinensis from Darjeeling plains, India. Mun. Ent. Zool. 2(1): 29-38. YEAR 2006 2. Sarker, M. and Mukhopadhyay, A. 2006. A preliminary study on Host plant related changes of general esterases in Buzura suppressaria (Lepidoptera; Geometridae), a major defoliator of tea in the Darjeeling foothills and adjoining plains in India. Sri Lanka Journal of Tea Science. 71 (1): 1 -7. 3. Sarker, M. and Mukhopadhyay, A. 2006. Studies on salivary and midgut enzymes of a major sucking pest of tea, Helopeltis theivora (Heteroptera: Miridae) from Darjeeling plains, India. J. Ent. Res. Soc. 8(1): 27-36. 4. Sarker, M. and Mukhopadhyay, A. 2006. Tissue level variation in esterases of red slug caterpillar, Eterusia magnifica Butl. (Lepidoptera : Zygaenidae), exposed and unexposed to pesticide spray of tea plantations of Darjeeling plains. Journal of Plantation Crops. 34(2): 94-97. 5. Sarker, M. and Mukhopadhyay, A. 2006. General esterases of Oligonychus coffeae (Acarina: Tetranychidae) occurring in pesticide-treated and untreated tea plantations of Darjeeling plains, India. J. Appl. Zool. Res. 17(1): 67-71. 6. Pradhan, B., Sarker, M. and Mukhopadhyay, A. 2006. Development, reproduction and nutritional ecology of Euproctis latifascia (Lepldoptef-a: Lymantriidae) on artificial diet and natural host plant. Camellia sinensis. International Journal of Tropical Insect Science. 26(3):190-196. - 192- 7. Sarker, M. and Mukhopadhy^, A. 2006. Studies on some enzymes related to insecticide resistance in Atefcpe/f/s theivora Waterhouse (Insecta: Heteroptera: Miridae) from Darjeeling fc«3ttiills and plains. Journal of Plantation Crops. 34(3): (In press). 8. De, D., Sarker, M., Das, S. and Mukhopadhyay, A. 2006. Evaluation of kilting efficacy of the polyhedrosis virus isolated from Buzura suppressaria Guen. (Lepidoptera; Geometridae), a defoliating pest of tea from Darjeeling foothills. Journal of Plantation Crops. 34(3): (In Press). 9. Das, S., Sarker, M., De, D. and Mukhopadhyay, A. 2006. Exploring the potential of insect enemies in controlling red slug and looper caterpillar, two major lepidopteran defoliators of tea from Darjeeling foothill regions. Journal of Plantation Crops. 34(3): (In press). 10. Mukhopadhyay, A., De, D., Sarker, M. and Bambawale, O.M. 2006. New report of Baculovirus in Buzura suppressaria in India. Natural Product Radiance. (In Press). 11. Sarker, M. and Mukhopadhyay, A. 2006. A comparison of dynamics of tea aphid population from Darjeeling hills and terai plains with a note on its arthropod natural enemies. J. of Aphidology. 20; (In Press). YEAR 2005 12. Das, S. Sarker, M. and Mukhopadhyay, A. 2005. Changing diversity of hymenopteran parasitoids from organically and conventionally managed tea - ecosystem of North Bengal, India. Journal of Environmental Biology. 26(3); 505- 509. - 193- YEAR 20B4 13. Mukhopadhyay, A., Das. S. and Sarker, M. 2004. Laboratory rearing of Sycanus croceovrttatus (Dohrn) (Heteroptera: Reduviidae), a predator of nsd slug caterpillar of tea, on termite as food. J. Appl. Zool. Res. 15(2); 168-170. YEAR 2003 14. Sarker, M. and IVIul 15. Pathak, S. K., Srilal, S., Dukpa, N., Sarker, M. and Mukhopadhyay, A. 2003. Biological studies of a defoliator of Darjeeling tea, Orygia sp. (Lymantriidae: Lepidoptera) under laboratory conditions. Two and a Bud. 50: 31-34. 16. Sarker, M. and Mukhopadhyay, A. 2003. Variation in esterase isozymes of the red spider mite from Darjeeling- Terai Tea plantations, p.532. In: Proceedings of the National Symposium on Frontier Areas of Entomological Research. Division of Entomology, Indian Agricultural Research institute, New Delhi. 17. Mukhopadhyay, A. and Sarker, M. 2003. Detection and assay of amylase and protease in two major lepidopteran defoliators of tea from Darjeeling Terai. p.561. In: Proceedings of the National Symposium on Frontier Areas of Entomological Research. Division of Entomology, Indian Agricultural Research Institute, New Delhi. MONOGRAPH: YEAR 2006 18. Mukhopadhyay, A. and Sarker, M. 2006. Natural Enemies of some Tea Pests with special reference to Darjeeling, Terai and the Dooars, National Tea Research Foundation, Tea Board, Kolkata (Both in CD and paper form). - 194- I I I. ~nts of tlie pu6fislietfpapers refevant to tlie topic of tlie tliesis - 195- _ Mun. Ent. Zool. Vol. 2, No. 1, January 2007______2 9 FEEDING BIOLOGY AND DIGESTIVE ENZYMES OF BUZURA SUPPRESSARIA GUEN. AND ETERUSIA MAGNIFICA BUTL., TWO MAJOR DEFOLIATING PESTS OF CAMELLIA SINENSIS FROM DARJEELING PLAINS, INDIA Mayukh Sarker*, Bina Pradhan** and Ananda Mukhopadhyay*** * Postgraduate Diploma in Tea Management, University of North Bengal, Daijeeling, 734 013, INDIA. ** Department of Zoology, Sikkim Govt. College, Gangtok, Sikkim, 737102, INDIA. *** Entomology Research Unit, Department of Zoology, University of North Bengal, Darjeeling, 734 013, INDIA, e-mail: [email protected] [Sarker, M., Pradhan, B. & Mukhopadhyay, A. 2007. Feeding biology and digestive enzymes of Buzura suppressaria Guen. and Eterusia magnifica Butl., two major defoliating pests of Camellia sinensis from Darjeeling plains, India. Munis Entomology & Zoology2(l): 29-38] ABSTRACT: The common looper caterpillar, Buzura suppressaria and the red slug caterpillar, Eterusia magnifica are serious defoliators of tea bushes (Camellia sinensis) of the Terai and Dooars areas of Darjeeling and N.E. India. While the former species prefers young leaves, the latter feeds on more mature leaves. This study aims to find the difference of the nutritional indices for the two folivores, such as relative consumption rate (RCR), relative growth rate (RGR), gross growth efficiency (ECI), net growth efficiency (ECD) and approximate digestibility (AD) and relate the same with their maintenance cost and production index (body mass). B. suppressaria has an edge over Et. magnifica as far as RCR and AD values are concerned. However, Et. magnifica could make up for the poor food quality (as they feed on mature tea leaves) by increasing their feeding period and better food conversion efficiencies. Higher value of AD in B. suppressaria may be due to higher quantity of the digestive enzymes in the midgut of this caterpillar. Significant differences in the activities of amylase, protease and lipase could be detected at salivary and midgut levels in the two folivores. The adaptive strategies in exploiting the different qualities of leaves, from two hampers of tea bushes is important for optimal food utilization by the two folivores with niche segregation. KEY WORDS: B. suppressaria, Et. magnifica, Camellia sinensis, nutritional indices, digestive enzymes, Darjeeling The common looper caterpillar, Buzura suppressaria Guen. and the red slug caterpillar, Eterusia magnifica Butl. are serious defoliating pests of tea, Camellia sinensis (L) 0. Kuntze from Terai and the Dooars areas of Darjeeling and N.E. India (Anonymous, 1994). Of these folivores the former exercises preference for young and the latter for mature tea leaves. In case of severe infestation however, they may eat the entire leaf, as well as the woody parts of the bush. In order to have a better understanding of feeding biology of both the pests the present study was undertaken on their food consumption, utilization and digestive enzymes. The nutritional requirements of an insect change throughout development and such changes are typically reflected in changes of its food consumption and feeding behaviour (Barton 2 Q ______Mun. Ent. Zool. Vol. 2, No. 1, January 2007______ Browne, 1995). Numerous studies in the field of nutritional physiology have reviewed the effects of nutritive compounds (Mattson, 1980; Felton, 1996) on insect responses. Some of the nutritional responses are adaptive, such as preingestive increase in consumption of nutritionally poor food (Taylor, 1989; Woods, 1999) or postingestive increase in activity of digestive enzymes (Hinks & Erlandson, 1994; Lazarevic, 2000). As the ability of B. suppressaria and Et. magnifica to utilize leaves of C. sinensis is largely dependent on three basic digestive enzymes viz. amylase, protease and lipase, these have been quantified in the salivary secretions and midgut of the larvae of both the pests. Further, an attempt has been made to relate and compare the enzyme quantity with the nutritional indices of these pests. Such information on digestive enzymes vis a vis food utilization can help contemplation of control of these pests through use of enzyme inhibitors and allelochemicals under host-plant resistance programmes. MATERIAL AND METHODS A commonly planted high yielding tea clone of Assam x Cambod origin was provided as food for the rearing of the pest larvae in a transparent container (27.5x 27cm) in aseptic conditions. Freshly emerged adults in laboratory were sexed, paired and allowed to mate in glass chimneys (19.5 cm x 8.5 cm), containing a twig with tea plant immersed in water of a conical flask to elicit oviposition. Larvae hatched from these eggs were reared at 28 ± 2°C, 75 ± 5% relative humidity and 12 hours L: D. Nutritional ecology: In order to find out the daily food consumption and weight changes in final larval instar freshly ecdysed instar stages, 10 replicates each of B. suppressaria and Et magnifica were monitored under controlled conditions (as mentioned earlier) in BOD incubator. Daily-preweighed fresh food (tea leaves with twig) was offered to each individual kept in (26cmx8.5cm) plastic containers. After 24 hours of feeding, leftover food and excrement were removed, oven dried and weighed. Dry weight of the actual food consumed was calculated by subtracting the dry weight of the leftover food from the dry weight of an equivalent amount of the food offered. Dry weight change of larva was calculated by drying a larva of similar weight in the oven at 50° C for 72 hours. Control was run concurrently by keeping tea leaves with their twig immersed in water of a conical flask having its mouth plugged with a cotton ball. Gravimetric (diy mass) technique was used to determine food consumption, and post ingestive food utilization efficiencies after Waldbauer (1968), Slansky & Scriber (1985), Petrusewicz & MacFadyen (1970), Muthukrishnan & Pandian (1987) and Farrar et al. (1989). _ M un. Ent. Zool. Vol. 2, No. 1, January 2007______3 J Activity of digestive enzymes: Enzyme extraction was made from laboratory-reared instar larvae of B. suppressaria and Et. wagnifica. The dissections were carried out in an ice-cold sodium phosphate buffer (0.1 M, pH 7.0). Salivary gland and midgut were homogenized individually in fresh sodium phosphate buffer containing 0.01 M each of EDTA (Ethylene diamine tetra acetic acid) and 0.5% Triton X-100. The homogenate was centrifuged at lO.OOOg for 15 min at 4° C. The supernatant of this preparation were used for measuring enzyme activities and stored at - 20° C for future use. Amylase assay: Amylase activity in the salivary gland and midgut was determined after the method of Madhusudhan et al. (1994) followed by the method of Sadasivam & Manickam (1996) using dinitrosalicylic acid reagent; and quantification of enzyme product was deducted from a standard curve prepared using various concentration of maltose alone at 520 nm using UV-Vis spectrophotometer. The enzyme activity was expressed as pM / min/ mg of protein. PROTEASE ASSAY: Proteolytic activity was assayed after the methods of Kunitz (1947) modified % Jayaraman (1981). 1% (w /v) casein was used as the substrate. 1 ml of casein prepared in 0.1 N NaOH was incubated with equal volume of enzyme. After incubation for one hour, the reaction was terminated by the addition of 10% TCA and the acid-soluble peptides were quantified using the biuret reagent at 520 nm using UV- Vis spectrophotometer. The enzyme activity was expressed as pg / mg of protein. LIPASE ASSAY; Lipase activity was measured following the method of Sadasivam & Manickam (1996). The enzyme activity was calculated as milliequivalent activity of free fatty acid / min/ g sample. RESULTS AND DISCUSSION B. suppressaria and Et. magnifica showed considerable changes in the quantity of food ingested and development of body mass but with similar trends. Despite a greater quantity of leaf consumed (in total) by Et magnifica, the relative consumption rate (RCR) value of B. suppressaria was recorded to be higher. Such a difference may be due to quality of leaf consumed. Leaves of different plants / varieties differ 2 2 ______Mun. Ent. Zool. Vol. 2, No. 1, January 2007______in their suitability as insect food because of variations in nutrient content, water content, type and concentration of secondary plant compounds and degree of sclerophyll (toughness / fibre) (Gullan & Cranston, 1994). B. suppressaria consumed younger leaves of upper tier and Et. magnifica preferably fed more on mature leaves of middle tier of a tea bush. A better consumption rate of B. suppressaria is possibly due to consumption of leaves of higher nutritional quality, in which the percentage of nitrogen and moisture is more, than the mature leaves consumed by Et. magnifica. In a similar finding Scriber & Fenny (1979) showed that Swallowtails had a higher consumption rate on nitrogen and moisture-rich forbs than when feeding on tree foliage having relatively less values of nitrogen and moisture. In the two species, efficiencies of ingested (ECI) and digested food (ECD), showed that B. suppressaria had lower ECI and ECD values as compared to Et. magnifica (Table 1). This could be explained by a higher metabolic cost of processing the young leaves, which contain more allelochemicals. The young leaf of tea plants contains high levels of plant allelochemicals like polyphenolic compounds, caffeine (Roberts, 1962; Banerjee, 1993). These secondary plant compounds are associated with induction mechanisms at the level of digestion and detoxification. A reduction in ECD associated with allelochemical ingestion is a common phenomenon (Koul et al., 1990; Appel & Martin, 1992). Secondary plant compounds often inhibit growth and development of insects (Todd et al., 1971; Lindroth et al., 1988; Ayres et al., 1997). Secondary plant substances also frequently act at the behavioural level of insects as deterrents and feeding inhibitors (Kraft & Denno, 1982; Kelly & Curry, 1991; Van Dam et al., 1995). The above hypothesis is tested by a comparison of the life histories of two folivores in question on young and mature tea leaves and their adaptations to the different leaf quality and quantity. The maintenance cost of B. suppressaria was higher in comparison with Et magnifica. The increase in food consumption rate that enhanced the cost of maintenance of B. suppressaria than Et. magnifica may be due to its food quality. In B. suppressaria a large part of the ingested food is presumably utilized in maintaining of basal metabolism, resulting in low conversion for growth. In Pseudaletia unipuncta, similar phenomenon was observed by Mukerji & Guppy (1970). The suboptimal availability of nutrient often nitrogen or water reduces growth rate, increases maintenance costs and causes a lower metabolic efficiency (Schoonhoven et al., 1998). The production index of Et magnifica was found to be higher than B. suppressaria and this might be due to the better suitability of the mature tea leaf as food in supporting the advanced life stages of the former species. Study on approximate digestibility (AD) showed a higher value in B. suppressaria as compared with that of Et magnifica. A higher AD and assimilation are known to be influenced by quality, specially of nitrogen, water and toxin contents of the plant food (Muthukrishnan & _Mun. Ent. Zool. Vol. 2, No. 1, January 2007______^ 3 Pandian, 1987) . The increased AD in response to tea leaf quality could also be as a result of changes at the levels of digestive enzymes. Higher activity of digestive enzymes in relation to food composition have been reported by Hinks & Erlandson (1994) and Ishaaya & Swirski (1976). Deficiencies in the quality of a food resource can be balanced by various mechanisms of nutritional compensation as is evident in Et magnifica that overcome poor food quality by increase in their feeding period and better food conversion efficiency (Fig. 1 and Table 1). Starch is the main reserve polysaccharide in tea (Banerjee, 1993). The amylase activity found both in salivary and midgut of B. suppressana indicates greater digestion of polysaccharides in midgut than its break down at the time of ingestion in the oral cavity vis a vis in Et. magnifica amylase activity of equal quantity indicates almost similar polysaccharide digestion at salivary and midgut levels. This is possibly an adaptation for better digestion of starch through an increase of the feeding period and higher conversion efficiencies (Table 2). In unprocessed tea, protein makes upto 20% of the dry weight (Mulky, 1993). The protease activity in oral as well as midgut of B. suppressaria and Et. magnifica ensure an active protein digestion at both the levels. Nevertheless a higher protease activity in salivary secretion of Et. magnifica possibly ascertains a better digestion of the available protein of mature leaves, starting in the oral cavity followed by midgut (Table 2). The activity of lipase is much reduced than the other two digestive enzymes. In B. suppressaria the lipase activity is significantly higher than that of Et. magnifica both at salivary and midgut levels possibly because the former feeds on young tea leaves in which lipid make up 4% to 9% of the dry matter (Roberts, 1974; Mahanta et al., 1985). The Hpase activity has also been reported in the midgut of Manduca sexta (Rubiolo et al., 2000) and Spilosoma obliqua (Anwar & Saleemuddin, 1997). The digestive enzymes are mainly reported from the midgut of different insects (Hori et al., 1981; Lenz et al., 1991). The present study on feeding biology and digestive enzyme activities reveals different exploitation strategies by the two folivores of two qualities of tea leaves (young and mature). Further, it establishes that Et. magnifica has a better adaptive flexibility than that of B. suppressaria because of its greater efficiency in converting both ingested and digested food. The study throws-up future research opportunities in non-conventional management of these two pests based on digestive enzyme inhibitors and other HPR strategies, which would be a necessity in developing IPM - programme of tea. ______Mun. Ent. Zool. Vol. 2, No. 1, January 2007______ LITERATURE CITED Anonymous. 1994. Pests of tea in North-East India and their control. Memorandum 27 Tea Research Association, Tocklai Experimental Station, Jorhat, Assam, India, 43-44 pp. Anwar, A. & Saleemuddin, M. 1997, Alkaline-pH-acting digestive enzymes of the polyphagous insect pest Spilosoma obliqua: stability and potential as detergent additives. Biotechnololy and Applied Biochemistry, 25:43-46. Appel, H. M. & Martin, M. M. 1992. Significance of metabolic load in the evolution of host specificity of Manduca sexta. Ecology, 73: 216-228. Ayres, M. P., Clausen, T. P., MacLean, S. F. Jr., Redman, A. M. & Reichardt, P. B. 1997. Diversity of structure and antiherbivore activity in condensed tannins. 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Compensation for variable dietary nitrogen by larvae of the salvinia moth. Functional Ecology, 3: 407-416. Van Dam, N. M., Vuister, L. W. M., Bergshoeff, C., De Vos, H. & Van der Meijden E. 1995. The “raison d’etre” of pyrrolizidine alkaloids in Cynoglossum officinale-. Deterrent effect against generalist herbivores. Journal of Chemical Ecology, 21; 507-523. Waldbauer, G. P. 1968. The consumption and utilization of food by insects. Advances in Insect Physiology, 5; 229-288, Woods, H, A. 1999. Patterns and mechanisms of growth of fifth-instar Manduca sexta caterpillars following exposure to low-or high protein food during early instars. Physiology Biochemistry and Zoology, 72: 445-454. . Mun. Ent. Zool. Vol. 2, No. 1, Jamtaiy 2007_ 37 I— JLsfval mass change of B. suppressaria Larval mass change ofEt magnifica - Food consumpt. of B. suppressaria ■Foodconsumpt of Et.magnitica D-2 D-3 D4 DS DS D-7 D-8 D-9 Fig. 1.Relation of dry mass (mg) changes (left ordinate) and daily food consumed (mg) (right ordinate) during development of V"’ instar larvae of Buzura suppressaria and Eterusia magnifica (D = day) Table 1. Nutritional Indices of Buzura suppressaria and Eterusia magnifica (V'^ instars) on tea leaf (Mean ± SE). V*** instars RCR RGR ECI ECD AD Ment. Cost Prodn. Index 0.630a 0.078a 12.355a 24.761a 49.904a 3.056a 0.247a + + ± ±0.453 ±0.441 ± ± B. 0.008 0.002 0.246 0.070 0.005 suppressaria 0.574b 0.080a 13.879b 31.241b 44.453b 2.212b 0.312b E. magnifica ± ± ±0.212 ±0.509 ±0.243 ± ± 0.004 0.001 0.051 0.005 Means followed by the same letter are not significantly different using t- test at p > 0.05 38 . Mun. Ent. Zool. Vol. 2, No. 1, January 2007_ Table 2. Digestive enzymes of salivary gland (SG) and midgut (MG) homogenate of Buzura suppressaria and Eterusia magnifica (Mean ± SE) (n = 10) Am ylase Protease Lipase (pM. mg protein - ^ min -*) (Amount of protein, (Activity meq. / min casein, utilized) /g of sample) Salivary Midgut Salivary Midgut Salivary Midgut gland gland gland B. 0.318± 0.405 ± 38.22 ± 44.81 + 0.0076 ± 0.0328 suppressaria 0.71 aA 0.62 bB 0.19 aA 0.38 bB 0.0002 ± aA 0.0013 E. 0.331 + 0.348 44.45 ± 43.49 ± bB magnifica 0.37 bA ±0.63 0.46 bA 0.22 aB 0.0043 aB ± 0.0001 0.0127 bA ± 0.0009 aB Difference in lower case letters in columns indicate significance difference of mean using t-test at p> 0.001; Difference in upper case letters for each enzyme in rows indicate significance difference of mean using t-test at p> 0.001 S .U . Tea Sci. 71(1), 1-7, 2006, Printed in Sri Lanka A Preliminary Study on Host Plant Related Changes of General Esterases In Buzura Suppressaria (Lepidoptera: Geometridae), A Major Defoliator of Tea In The Darjeeling Foothills And Adjoining Plains In India Mayukh Sarker and Ananda Mukhopadhyay (Entomology Research Unit, Department o f Zoology, University of North Bengal, Darjeeling, 734 430, India) ABSTRACT The looper, Buzura suppressaria Guen. (Lepidoptera: Geometridae) is the major defoliating pest of tea in the Darjeeling foothills and plains in north- east India. Esterase isozymes were found to differ in looper populations maintained on TV-1 and TV-25 tea clones. The major clone-based difference was located in the ‘vei7 slow moving band’ group (VSM). While the three VSM bands were absent in the phoregrams of the midgut and salivary glands of TV-1 looper populations, they were prominent in the midgut of TV-25-reared specimens. In looper populations of pesticide-treated plantations, the esterase bands of the VSM group were more elaborate, along with deep staining of the slow moving (SM) and the fast moving (FM) bands. Such an enliancement of band intensity possibly reflects a greater tolerance or resistance of the loopers to plant allelochemicals and pesticides. The isozyme profiles of esterases of B. suppressaria can therefore be used as a tool for detecting the resistance associated with the tea clones (variety) as well as that which develops in the species on exposure to pesticide spray under field conditions (plantation). Key words: Esterase isozymes, looper caterpillars, tea clones INTRODUCTION The looper caterpillar, Buzura suppressaria Guen. (Lepidoptera: Geometridae), attacks mature leaves and defoliates tea {Camellia sinensis) bushes causing heavy crop loss. This major defoliator has a wide distribution in Indian subcontinent. While the pest is more prevalent in Darjeeling Terai—Dooars (foothills & plains) and north-east India, its occasional outbreaks are reported from tea plantations of South India (Tamil Nadu and Kerala) but, not at present from Sri Lanka. The species is recorded even from Indonesian tea (Muraleedharan, 1991; Muraieedharan and Selvasundarani, 2002; Danthanarayana, 1967). To control the depredations of various tea clones and the multiple generations of B. suppiessaria, regular insecticide spraying is required. The difference in susceptibility of insect pests to insecticides when maintained on specific plants has been related to different levels of metabolizing enzymes, presumably induced by the plants (Yu, 1982; Ambrose and Regupathy, 1992; Tan and Guo, 1996). Many enzymes involved in detoxification pathways act on a broad array of substrates, including both naturally occurring plant allelochemicals and synthetic pesticides (Gordon, 1961). An enhanced metabolism by esterases is a major mechanism for countering pesticide stress that has been detected in lepidopterans (Beeman and Schmidt, 1982). The objective of the present study was to determine the differences in the general esterase profiles of looper caterpillars when reared on two different Tocklai (Tea Research Association) varieties of tea, namely TV-1 (an early release) and TV-25 (a relatively late release). A further aim was to compare the banding pattern and staining density o f esterase bands of looper populations exposed and unexposed to pesticide sprays. MATERIALS AND METHODS Insect collection and maintenance 200-250 larvae of B. suppressaria were collected from tea plantations, mostly comprising TV-1 and TV-25 varieties, in the Darjeeling foothills, Terai and Dooars in West Bengal State, India. These larvae were reared separately on the Tocklai clonal varieties, TV-1 and TV-25, for two generations at 27 ± 2“ C and 72 ±2 % RH with a photoperiod o f L:D, 13.T 1, in transparent containers (30 x 30 cm) with a supply of fresh tea twigs, obtained from the tea plantation of the North Bengal University campus, culturally maintained without application of pesticides. Enzyme extraction and gel electrophoresis Enzyme extraction was done from laboratory-reared fifth instar larvae o f B. suppressaria, and from larvae of the same stage collected from natural populations occurring in conventionally managed plantations that were subjected to routine synthetic pesticide spraying. Each larva was dissected and its salivary gland and midgut were removed. Dissections were carried out in ice-cold sodium phosphate buffer, 0.1 M, pH 7.0 using sterilized scissors and needles. Salivary glands and midguts were homogenized individually in fresh sodium phosphate buffer containing 0.01 M each of EDTA (ethylene diamine tetra acetic acid) and 0.5% Triton X-100. The volume of the buffer was adjusted to produce similar protein concentrations in the homogenates of each individual. The homogenate was centrifuged at 10,000 g for 15 min at 4° C. The supernatant of this preparation was stored at -20° C for liiture use. Using the technique of Davis (1964), fifteen microlitres of homogenate was dispensed into each well of the electrophoresis gel. Electrophoresis was carried out at constant current (10 mA) for about 1.5 hr on 8% native polyacrylamide vertical slab gel, using tris -glycine (pH 8.3) as running buffer. The gel was then stained for 30 min at 36® C for esterase with Fast Blue BB salt in 0.1 M phosphate buffer, pH 7.0, containing 0.03 M alpha napthyl acetate solution dissolved in acetone. The relative migration of esterase bands in the zymograms was determined by the Kodak digital science ID Image Analysis Software, version 2.0.3. Relative mobility (R^^) was calculated as; distance migrated by the specific bands (cm) / distance migrated by the marker dye (cm). RESULTS AND DISCUSSION The general esterase phoregram appeared in three groups; fast moving bands (FM or EST-1), slow moving bands (SM or EST-2), and very slow moving bands (VSM or EST- 3), on the basis ol'relative mobility. These groups were distinctly separate from one another. The first three bands of the VSM group were absent in the salivai-y homogenates of all three categories o f looper populations. In the midgut homogenates, the VSM group was present as five bands in TV-25-reared and field-collected loopers. However, the first three bands of V SM were not present in the m idgut homogenate of TV-1 reared populations. Among four SM bands of esterases, bands 1, 2 and 3 were prominent, and were consistently present both in salivary and midgut homogenates. These appeared deeply stained and with uniform mobility in all the looper populations. A similar pattern, parallel to TV-25, was observed in field-collected looper populations, in which the bands of VSM, SM and FM were deeply stained, indicating a larger quantity of esterases (Table 1 and Fig.l a, b, c). Isozyme analysis has been applied to identify species, biotypes and host-specific populations in many insects such as aphids, egg parasitoids {Trichograntma spp.) and others (Loxdale and Hollander, 1989). In the present study, zymograms of esterases failed to show major differences in banding patterns, except for three additional VSM bands which were present in the gut homogenate of the looper populations reared on TV-25 and the field collected loopers. Mullin and Croft (1983) found large differences in general esterase activity of Tetranychiis urticae on snapbean varieties, ranging from 0.4 fold on a mint to 2.4 fold on umbellifers. Moreover, herbivorous insects metabolize and detoxify insecticides using the same set of enzymes that are involved in the metabolism of ingested plant allelochemicals (Brattsten, 1979; Ahmad e ta l, 1986). The bands parallel to the three V SM bands showed intense staining in loopers of field populations exposed to pesticide treatments in conventional plantations. Such band intensity niay be related to looper populations with greater pesticide tolerance (vis-a-vis resistance). Ill similar findings, a liigher midgut esterase activity was reported in Plutellaxylostella exposed to pesticides (Mohan and Gujar, 2003), and also a higher activity of slow moving esterases as apparent in PAGE for the same species (Maa and Liao, 2000). Resistant aphids display a high level of non-specific esterase activity represented by intense esterase bands (Ono et al., 1994) as is evident in malathion resistant P. xylostella (Maa and Chuang, 1983; Doicluianngam and Thornhill, 1989). Thus EST-3 or VSM bands appeared to be crucial in utilizing a relatively recent tea clone (TV-25) and development of greater pesticide tolerance / resistance in B. siippressan'a population. As such, these bands could be used as markers in screening the populations of the pest for resistance/tolerance status as well as screening the clonal varieties that sufficiently differ biochemically (allelochemically). ACKNOWLEDGEMENTS We thank the Head, Department of Zoology, University of North Bengal for providing research facility including the organic tea plantation on tlie University campus. We are grateful to Dr. K .R. Kranthi, Sr. Scientist, Central Cotton Research Institute, Nagpur, India for his suggestions and literature. REFERENCES Ahmad S, Brattsten L B, Mullin C A, and Yu S J 1986 Enzymes involved in the metabolism o f plant allelochemicals. In Molecular Aspects of Insect- Plant Associations, Eds. L. B. Brattsten and S. Ahmad, pp. 73-151. Plenum Press, New York. Ambrose H J and Regupathy A 1992 Influence of host plants on the susceptibility of Myzus persicae (Sulz.) to certain insecticides. Insect. Sci. Applic. 13, 79-86. Beeman R W and Schmidt B A 1982 Biochemical and genetic aspects of malathion- specific resistance in the Indian meal moth (Lepidoptera: Pyralidae). J. Econ. Entomol. 75,945 -949. Brattsten L B 1979 Biochemical defense mechanisms in herbivores against plant Allelochemicals. In Herbivores; Their Interaction with Secondaiy Plant Metabolites, Eds. G A Rosenthal and D Janzen. pp 199-270, Academic Press, New York. Danthanarayana W 1967 Tea Entomology in perspective. Tea Quarterly 38, 153- 177. Davis B J 1964 Disc electrophoresis II Method and application to human serum protein. Ann. B TAcad. Sci. 121, 404-427. Doichuanngam K and Thornhill R A 1989 The role of non-specific esterase insecticide resistance to malathion in the diamond-backmoth'Plutellaxylostella L. Comp. Biochem. Physiol. 93, 81 -86. Gordon H T 1961 Nutritional factors in insect resistance to chemicals. Annu. Rev. Entomol. 6, 27-54. Loxdale H D and Hollander J D 1989 Electrophoretic studies on agricultural pests. Oxford Univ. Press, New York. 497 p. Maa C J W and Chuang M L 1983 Esterase of diamond-backmoth, {PJutella xylostclla L.h Enzymatic properties of larval esterases. Bull. Inst. Zool. Acad. Sinica. 22,123-131. Maa W C J. and Liao S C 2000 Culture dependent variation in esterase isozymes and malathion susceptibility of diamond-backmoth, Plutellaxylostella (Linnaeus). Zool. Stud. 39,375-386. Mohan M and Gujar G T 2003 Local variation in susceptibility of the diamond-backmoth Plutella xylostella (Linnaeus) to insecticides and role of detoxification enzymes. Crop Protection. 22, 495 -504. Mullin C A and Croft B A 1983 Host-related alterations of detoxification enzymes in Tetranychus urticae (Acri: Tetranychidae). Environ. Entoinol. 12, 1278-1282. Muraleedharan N 1991 Pest Management in Tea. The United Planters’ Association of South India, TRl, Valparai, pp 130. Muraleedharan N and Selvasundaram R 2002 An IPM package ibr tea in hidia. Planters’ chronicle. 107-124. Ono M, Jonathan S R and Siegfried B D 1994 Characterization of general esterases from susceptible and parathion-resistant strains of the gi'cen bug (Homoptera: Aphididae). J. Econ. Entomol. 87, 1430-1436. Tan W J and Guo Y Y 1996 Effects of host plant on susceptibility to deltamethrin and detoxification enzymes of Heliothis armigera (Lepidoptera: Noctuidae). J. Econ. Entomol. 89,11-14. Yu S J 1982 Host plant induction of glutathiones-transferase in the fall armyworm. Pestic. Biochem .Physiol. 18, 101-106. Table 1. Electrophoretic variation in relative mobility (R^) (n =15) of esterase isozyme bands of salivary and midgut homogenate of Buzura suppressaria larvae on TV-1, TV-25 and field-collected populations from the Darjeeling foothills and adjoining plains Tea clone Field-collected Esterases population TV-1 TV -25 SG MG SG MG SG MG ______0.161 __ 0.161 VSM ------0.184 ---- 0.184 (EST- 3) ------0.201 ---- 0.201 0.332 0.332 0.332 0.332 0.332 0.332 0.381 0.381 0.381 0.381 0.381 0.381 SM 0.430 0.430 0.430 0.430 0.430 0.430 (EST-2) 0.458 0.458 0.458 0.458 0.458 0.458 0.481 0.481 0.481 0.481 0.481 0.481 0.538 0.538 0.538 0.538 0.538 0.538 FM __ 0.614 ___ 0.614 0.614 0.614 (EST-1) ---- 0.632 ---- 0.632 0.632 0.632 0.672 0.672 0.672 0.672 0.672 0.672 0.684 0.684 0.684 0.684 0.684 0.684 SG: Salivary gland homogenate VSM (EST-3); Very Slow moving MG: Midgut homogenate SM (EST-2); Slow moving bands FM(EST-l); Fast-moving bands VSM (EST-3) SG MG a) Looper reared on TV-1 b) Looper reared on TV-25 c) Field -collected looper (Pesticide exposed) SG: Salivary gland homogenate VSM (EST-3); Very Slow moving bands MG: Midgut homogenate SM(EST-2): Slow moving bands FM(EST-l); Fast-moving bands Fig. 1: Zymograms of esterases of loopers, B. suppressaria fed and maintained on tea clones (a) TV-1, and (b) TV-25, and from (c) conventional plantations exposed to pesticide sprays (Each lane represent the phoregram of a single looper) Journal of Plantation Crops, 2006, 34 (2): 94-97 Tissue level variation in esterases of red slug caterpillar, Eterusia magnifica Butl. (Lepidoptera: Zygaenidae), exposed and unexposed to pesticide spray of tea plantations of Darjeeling plains Mayukh Sarker and Ananda Mukhopadhyay* Entomology Research Unit. Department of Zoology, University o f North Bengal, Darjeeling, 734 430, India. (Maiiuscripl received : 14/03/05; Revised : 15/07/05 Accepted ; 07/06/06) Abstract Red slug caterpillar, Eterusia magnifica is a major defoliating pest of tea showing recurrence despite synthetic pesticide applications. In PAGE, general esterases were expressed in nine bands designated Est-1 to Est-9. Out of these, in the whole body homogenate Est-8, and in midgut Est-5 bands were absent. In salivary gland homogenate only two bands, Est-3 and Est-4 were detectable. Caterpillars reared in laboratory on two Tocklai clonal varieties, TV-18 and TV-25 and caterpillars exposed to pesticidal sprays in plantations from Darjeeling plains showed similar banding pattern. The pesticide exposed larvae registered a significantly high quantity of general esterases both in salivary and midgut tissue along with a significantly higher quantity of general esterases both in salivary gland and midgut tissue along with an intense staining of two bands (Est-3 and Est-4) in all the different homogenates, thus suggesting an involvement of these esterase isozymes in pesticide detoxification. Key words: Eterusia magnifica, esterases, tea, Darjeeling, pesticide Introduction (Yu, 1982; Ambrose and Regupathy, 1992; Tan and Guo, Red slug caterpillar, Eterusia magnifica Butl. 1996). Many enzymes involved in detoxification (Lepidoptera: Zygaenidae) is one of the widely pathways act on broad array of substrates, including both distributed major insect pests of tea. Camellia sinensis naturally occurring plant allelochemicals and synthetic (L.) O. Kuntze from North-Eastern part of India (Anon., pesticides (Gordon, 1961). Therefore, physiological 1994). To control depredation of various tea clones and response,of herbivores to host plants may lead to multiple generations of the E. magnifica, insecticides enhanced metabolism of pesticides because mechanism like organophosphates and pyrethroids are regularly of detoxification of host-plant allelochemicals may also applied. Host plant can influence the degree of be effective in detoxifying pesticides. insecticide susceptibility of herbivorous insects Importance of esterases in detoxification of indirectly by inducing higher activities of insecticide- insecticides especially ester-based organophosphates, detoxifying enzymes or inhibiting these enzymes by carbamates and pyrethroids is well established in many limiting the energy availability to the insects to perform pests, imparting insecticide resistance to many of them detoxification reactions (Brattsten, 1979). Different (Krueger and O’Brien, 1959; Yu and Terriere, 1977; susceptibilities of insect pests maintained on specific Devonshire er ai., 1986, 1993; Fournier ef a/., 1993). plants have been related to different levels of The objective of this study was to investigate the metabolizing enzymes, presumably induced by the plants tissue-specific differences in the general esterase profiles *Author for coirespondence E -mail: [email protected] 94 Mayuk Saraker and Ananda Mukhopadhyay *^of red slug caterpillars reared in laboratory on two Quantitative and qualitative assay of esterase different Tocklai Tea varieties viz., TV-18 and TV-25, General esterase activity was determined by the and then compare the esterase pattern of laboratory- method of van Asperen (1962). reared with the pesticide-exposed larvae collected from Quantification of enzyme activity was done using conventional plantations of Darjeeling foothills and standard curve prepared with a - naphthol and the data plains. were subjected to analysis of variance (ANOVA) with Materials and Methods the help of SPSS version 11.0. Insect material Relative migration of esterase bands and zymogram Larvae (200-250) of E. magnifica were randomly were determined by the Kodak digital science ID Image collected from conventional plantations (maintained by Analysis Software version 2.0.3. Relative mobility (RJ synthetic pesticide spray) of eastern Dooars and western was calculated by : Terai of West Bengal State, India. Separate batch of Distance migrated by the specific bands (cm) / specimens for laborarory rearing were collected from Distance migrated by the marker dye. bio-organic tea plantations of Darjeeling foothills and Results and Discussion maintained on TV-18 and TV-25 Tocklai variety of Tea Comparison of isozyme profiles for laboratory-reared Research Association for two generations. The rearing V"’ instar caterpillars of E. magnifica on two different was done at 27 ± 2® C; 72 ± 2 % RH with a photoperiod cultivars of tea (TV-18 and TV-25) showed a common of (L: D) 12:12 in transparent containers (30 x 30 cm) basic pattern. In the salivary gland homogenate two with supply of fresh tea twigs from the experimental tea esterase bands (Est-3 and Est-4) were apparent with R_^ plot maintained organically (without pesticide sprays) values of 0.43 and 0.47 respectively, whereas in midgut on the University campus. homogenate, eight bands (Est-1 to Est-9 except Est-5) Enzyme extraction and Gel Electrophoresis were observed with R^ values of 0.04, 0.12, 0.22, 0.30, Fifth instar larvae of E. magnifica, on an average 0.43, 0.47, 0.52 and 0.57 respectively. Whole body weighing 0.50 g were collected from laboratory colonies homogenate showed almost similar pattern as that of that were maintained separately on TV-18 and TV-25 midgut with less intense bands reflecting reduced varieties, and from natural populations occurring in esterase activity (Fig.l.a, b, c). conventionally managed plantation (with synthetic A significantly high activity of the general esterases pesticide spraying). Each larva was dissected and its in the pesticide-exposed red slug larvae over un exposed salivary gland and midgut were collected. Dissections ones, possibly was indicative of a greater esterase-based were carried out with the help of a sterilized scissors detoxifying activity in the former larvae (Table 1). and needle in ice-cold sodium phosphate buffer (0.1 M, Table 1. Activities of general esterases in red slug larvae (Mean ± SD) (n=10) pH 7.0). Fat bodies and food particles were removed reared on TV -18 and TV -25 variety of tea in lab and those ex posed to pesticides in field from the midgut, which were then homogenized individually in fresh sodium phosphate buffer containing Red slug caterpillar min mg of protein 0.01 M each of EDTA and 0.5% Triton X-100. The Salivary gland Midgut volume of the buffer was adjusted to produce similar TV-18 reared 19.24 ±0.34 a A 24,73 ±0.57 a B protein concentration in the homogenates of each TV-25 reared 19.45 ±0.42 a A 25.16 ±0.61 a B individual. The homogenate was centrifuged at 10,000 Pesticide-exposed 27.40 ± 0.66 b A 32.46 +0,43 bB g for 15 min at 4'’C. The supernatant of this preparation Means followed by the different lower case letters in the columns are significantly different (p>0.001); Means followed by the different upper case was stored at -20° C for future use. 15 /xl of homogenate letters in the rows indicate significant difference (p>0.001) was dispensed into each well in the gel. Electrophoresis Isozyme analysis have been applied to identify was earned out at constant current (10 mA) for about species, biotypes and host-specific populations in many 1.5 hr on 8% native polyacrylamide vertical slab gel, insects such as aphids, egg parasitoids, Trichogramma using Tris -glycine (pH 8.3) as running buffer (Davis, spp. and others (LoMale and Hollander, 1989). In the 1964). present study intensity and number (Est-1 to Est-9) Gel was then stained for 30 min at 36'’ C for esterase except Est-5 of the isozyme bands from gut homogenates with Fast Blue BB salt in 0.1 M phosphate buffer adding were similar in all the individuals (larvae) reared on TV- 0.03 M alpha napthyl acetate solution dissolved in 18 and TV-25 but in the field-collected larvae all bands acetone. were deeply stained indicating an intensive formation 95 Tissue specific expression of esterase isozynes in tlie tea pest Etenisia magnifica Butle. of esterases (Fig. l.b, c). This may perhaps be due to a 3 4 higher esterase-based detoxification activity in the larvae exposed to pesticidal spray in the field (conventional plantation) (Ahmad et al., 1986). The higher midgut esterase activity was reported in Plulella xylostella exposed to pesticides by Mohan and Gujar (2003). Resistant insects display a high level of non-specific esterase activity represented by intense esterase bands (Ono et a l, 1994), which are evident in malathion- resistant P. xylostella (Maa and Chuang, 1983; Doichuanngam and Thornhill, 1989). Further, in peach potato aphid, Myzus persicae resistance is conferred by amplification of esterase genes, resulting in the higher production of esterases that can hydrolyse insecticides (Field et a l, 1999). Two soluble esterase isozymes, designated as Est-3 and Est-4 due to their prominent presence in the pesticide-exposed larvae appeared to be pesticide detoxification in E. magnifica populations. Therefore these bands could be marked as useful indicator in screening populations of the pest for their resistance / tolerance (Fig.l c). Fig. 1(b). Esterase isoiymes of £. magnifica reared on TV-25 and TV-18 TV-25 reared Lane 1 Midgut homogenate The present study exhibited only minor difference in Lane 3 Wholebody homogenate TV-18 reared Lane 2 Midgut homogenate Lane 4 Wholebody homogenate Est 9 8 7 6 5 4 3 3 4 2 1 Fig. 1(a). Tissue specific PAGE profile of esterase isozymes of E. magnifica Lane 1 Salivary gland homogenate Fig. 1(c). Zymograms of E. magnifica from insecticide treated plantations Lane 2 & 3 Midgut homogenate Lane 1 Salivary gland homogenate Lane 4 & 5 Whole body (without salivary and gut) Lane 2 & 3 Midgut homogenate homogenate Lane 4 & 5 Whole body homogenate 96 Mayuk Saraker and Ananda Mukhopadhyay the activity of general non-specific esterases between Doichuanngam, K. and Thornhill, R.A. 1989. The role of non host (clone) specific E. magnifica populations, as their specific esterase insecticide resistance to malathion in the diamondback moth, Plutella .xylostella L. Comp. Biochem. zymograms failed to show any major differences in Physiol. 93C (1): 81 -86. banding patterns (Fig. 1 b). Lack of this variability in E. Field, L.M., Blackman, R.L., Tyler, S.C. and Devonshire, A.L. 1999. magnifica may be due to a high interbreeding in the Relationship between amount of esterase and gene copy number populations of Darjeeling foothills, Terai and Dooars in insecticide-resistant Myzus perjicae(Sulzer). J. Biochem. areas. A similar absence of major variation of esterase 339(3): 737-742. bands is evident in the populations of “Kissing bug”, Fournier, D., Mutero, A. and Rungger, D. 1993. Drosophila Triatoma infestans due to high interbreeding (Tavares acetylcholinesterase: expression of a functional precursor in et a i, 1998). Xenopits oocytes. Eur. J. Biochem. 203: 513-519. Gordon, H. T. 1961. Nutritional factors in insect resistance to So, an understanding of the status of detoxifying chemicals. Annu. Rev. Entoinol. 6: 27-54 enzyme (esterase) of E. magnifica, as has been revealed Krueger, H.F. and O’Brien, R.D. 1959. Relationship between in the present study, would be helpful in future planning metabolism and differential toxicity of malathion in insects and of their chemical control on different tea clones. mice. J. Econ. Entomol. 52: 1063-1067. Acknowledgments Loxdale, H. D. and Hollander, J. D. 1989. Electrophoretic studies on agricultural pests. Oxford Univ. Press, New York, pp 497. Authors are thankful to Dr. G.T. Gujar, Principal Maa, C.J.W. and Chuang, M.L. 1983. Esterase o f diamondback moth Scientist, Entomology Division, Indian Agricultural {Plutella xylostella L.I: Enzymatic properties o f larval esterases. Research Institute, New Delhi for reviewing the Bull. Inst. Zool. Acad Sinica. 22:123-131. manuscript and offering useful suggestions. Mohan, M. and Gujar, G.T. 2003. Local variation in susceptibility References of the diamondbackmoth, Plutella xylostella (Linnaeus) to insecticides and role of detoxification enzymes. Crop Protection. Ahmad, S., Brattsten, L.B., Mullin, C.A. and Yu, S.J. 1986. Enzymes 22: 495 -504. involved in the metabolism of plant allelochemicals. In: Molecular aspects of insect-plant associations. (Eds) Brattsten, Ono, M., Jonathan, S.R. and Siegfried, B.D. 1994. Characterization L.B. and Ahmad, S.; Plenum, New York, pp. 73-151. of general esterases from susceptible and parathion-resistant strains of the green bug (Homoptera: Aphididae). J. Econ. Ambrose, H. J, and Regupathy, A. 1992. Influence of host plants on Entomol. 87(6): 1430-1436. the susceptibility of Myziis persicae (Sulz.) to certain insecticides. Insect. Sci. Applic. 13: 79 -8 6 . Tan, W. J. and Guo, Y. Y. 1996. Effects o f host plant on susceptibility to deltamethrin and detoxification enzymes of Anonymous. 1994. Pests o f Tea in North-East India and their control. Heliothis armigera Memorandum No. 27, Tocklai Experimental Station, Jorhat, (Lepidoptera: Noctuidae). J. Econ. Entomol. 89: 11-14. Assam, pp.6-11. Tavares, G.M., Oliveira, M.T.V.A. and Ceron, C.R. 1998. Tissue- Brattsten, L. B. 1979. Biochemical defense mechanisms in herbivores specific expression of esterases in Triatoma infestans against plant allelochemicals. In: Herbivores: Their Interaction (Triatominae: Heteroptera). Genet. Mol. Biol. Vol. 21 n 4. with Secondary Plant Metabolites. (Eds) Rosenthal, G A. and van Asperen, K. 1962. A study of house fly esterases by means of a Janzen, D.; Academic Press, New York, pp. 199-270. sensitive colorimetric method. J. Insect. Physiol. 8: 401-416. Davis, B. J. 1964. Disc electrophoresis II. Method and application Yu, S. J. 1982. Host plant induction of glutathione S -transferase in to human serum protein. Ann. B. T. Acad. Sci. 121: 404-427. the fall armyworm. Pestic. Biochem. Physiol. 18: 101-106. Devonshire, A.L., Searle, L. M. and Moores, G. D. 1986. Yu, S.J. and Temere, L.C. 1977. Metabolism o f ('^C) hydroprene Quantitative and qualitative variation in the mRNA for (ethyl 3,7, 11 - trimethyI-2,4- dodecadienoate) by microsomal carboxylesterase in insecticide-susceptible and resistant Myzus oxidase and esterase from three species of diptera. J. Agric. Food persicae. J. Insect. Biochem. 16: 659-665. Chem. 25(5): 1076-1080. Devonshire, A.L., Williamson, M.S., Moores, GD. and Field, L.M. Zhu, K.Y. and Gao, J-R. 1998. Kinetic properties and variability of 1993. Analysis of the esterase genes conferring insecticide esterases in organophosphate-susceptible and resistant greenbugs resistance in peach-potato aphid, Myziis persicae. J. Biochem. Schizaphisgraminum (Homoptera: Aphididae). Pestic. Biochem. 294: 569-574. Physiol. 62: 135-145. 97 J. Appl. Zool.Res. (2006) 17( I ): 67-71 GENERAL ESTERASES OF OLIGONYCHUS COFFEAE (ACARINA; TETRANYCHIDAE) OCCURRING IN PESTICIDE-TREATED AND UNTREATED TEA PLANTATIONS OF DARJEELING PLAINS, INDIA MAYUKH SARKERand ANANDA MUKHOPADHYAY Department of Zoology, University of North Bengal, Darjeeling, 734 013, India. ABSTRACT: Red spider mite (RSM) Oligonychus coffeae is a major pest of tea, Camellia sinensis, showing resurgence despite the use of synthetic pesticides from the plains of Darjeeling, India. Quantitative and qualitative changes were recorded in the general esterases of the RSM. Activity of esterases was significantly higher in the whole body homogenates of the female specimens collected from conventional (synthetic pesticide treated) plantation than that of the organic (synthetic pesticide free) plantation. Three deeply stained esterase bands (E-1, E-2, E-3) were observed in the female RSM of conventional plantation vis a vis only one faint esterase band (E-1) with low staining intensity in RSM of organic plai;itation. These results suggest that the mites of conventional plantations have developed a greater tolerance to synthetic pesticides, possibly due to a higher quantity and activity of general esterases connected with detoxification. Keywords: Tea, Oligonychus coffeae, esterase, Darjeeling, pesFicide INTRODUCTION Oligonychus coffeae Nietner, the red spider mite (RSM) is a major arthropod pest that attacks most tea cultivars of Darjeeling foothills and their plains comprising Terai and Dooars plantations of North-East India (ANONYMOUS, 1994). The average annual consumption of insecticide and acaricide in Dooars and Terai is 7.05 and 3.49,kg/lt/ha., respectively (BARBORA and BISWAS, 1996). In spite of the use of synthetic pesticides, such as organochlorides, phosphates and synthetic pyrethroids, RSM remains a serious problem of tea and a difficult pest to control. Many w/orkers have reported that management of O. coffeae has become a challenge apparently due to its higher tolerance to pesticides (DAS, 1959; BANERJEE, 1968). The intensive use of acaricides such as, dicofol and ethion in tea for more than a decade has probably led to pesticide resistance in RSM (SAHOO et al., 2003). Pesticide resistance in two-spotted mite, Tetranycfius urficae is known to develop very quickly because of its numerous generations every year and exposure to high frequency of pesticide spray applications (CRANHAM and HELLE, 1985). General esterases are known as common detoxification enzymes, capable of degrading pesticides like organophosphate and pyrethroids (ANSPAUGH et al., 1995; VALLES, 1998). Specifically, carboxylesterase production has been implicated in organophosphate resistance of the green peach aphid, Myzus persicae (DEVONSHIRE, 1975). Electrophoretic studies on organophosphate resistance of the cotton aphid, A phis gossypii, have indicated clearly the involvement of particular distinct isozymatic forms of carboxylesterase in resistant strains (SUDDERUDDIN, 1973; DEVONSHIRE, 1975). YAN G et al. (2002) observed that the resistance in spider mite to miticides was poorly understood as compared to insecticide resistance in other arthropods. Similarly our 68 JOURNAL OF APPLIED ZOOLOGICAL RESEARCHES knowledge on the status of acaricide resistance/tolerance status of O. coffeae from the tea-plantations of Darjeeling foothill regions is meager, hence we evaluated their status based on studies of esterases, comparing the quantitative and qualitative changes of general esterases in O. coffeae populations collected from conventional (synthetic pesticide treated) and organic (synthetic pesticide free) tea plantations. MATERIAL AND ftflETHODS RSM populations were collected from organic and conventional tea plantations of Darjeeling foothills and plains.. These were maintained in laboratory on Tocklai clonal varieties (TV) at 25 ± 2‘^C with 70 ± 5 % RH. Biochemical studies were undertaken in the months of March to May during the years 2003 to 2005. General esterase activity was determined using the method of VAN ASPEREN (1962) modified by ZHU and GAO (1998), Ten female mites of a population were individually homogenized in ice-cold 0.1 M phosphate buffer. pH 7.5 containing 0.3 % (vol: vol) Triton X-100. The volume of buffer was 2 0 pi per female, adjusted to produce similar protein concentrations in the homogenates for each mite. Homogenates were centrifuged at 15,000g at 4 ° C for 15 min. The pellets were discarded and the supernatants were collected as enzyme sources. Non-denaturing polyacrylamide gel electrophoresis was done after DAVIS (1964). The gel system comprised a 4 % stacking and 8 % separating gels. Electrophoresis was conducted at a constant current of 10 mA for 2.0 hr at 4° C. The gel was then stained for 60 min'in dark at 37° C with Fast Blue BB salt in 0.1 M phosphate buffer adding 0.03 M alpha napthyl acetate dissolved in acetone. A microplate enzyme assay was conducted with slight modification of the technique of DEVONSHIRE et al. (1986). An enzyme (15 pi) was incubated at 37 “ C for 30 min in a final reaction volume of 150 pi containing 0.27 mM alpha napthyl acetate as substrate. The reaction was stopped by adding 50 pi of freshly prepared Fast Blue BB - SDS solution. The absorbance was determined 15 minutes later at 600 nm using microplate reader (Merck MIOS Junior 2100). The enzyme activities were analyzed by analysis of variance (ANOVA) using SPSS version 11.0. Relative mobility (R,„) of esterase bands in the zymogram was determined and quantified by the Kodak digital science 1 D image analysis software version 2.0.3. R^ = Distance migrated by the specific bands (cm) / Distance migrated by the marker dye. RESULTS AND DISCUSSION Activities of general esterases- differed significantly^ among the conventional (pesticide treated) and organic (pesticide untreated) field co'llected female O. coffeae (Table). Results of analysis of esterase bands on polyacrylamide gel showed that the pesticide expo^pd female RSM possessed 3 major co-migrating bands (E-1, E-2, E-3) whereas the pe^i^cide unexposed femaje RSM possessed only one (E-1), Major esterase band with R,,, value of 0.264 (E-1) was present both in pesticide-exposed and unexposed female mites (Fig. 1). On close inspection of lanes two additional bands were apparent in pesticide-exposed female [R,„ values 0.3403 (E-2) and 0.4833 (E-3)]. These bands (E-2, E-3) were however absent in the unexposed female'o. coffeae (Fig.1). JOURNAL OF APPLIED ZOOLOGICAL RESEARCHES 69 Table; Activities of esterase in female red spider mite (Mean ± SD) collected from conventional (pesticide treated) and organic (pesticide untreated) tea plantations "1“ O. coffeae OD/mg protein min df Conventional (pesticide treated) o'470T'±o'o^^^ 1,18 Organic (Pesticide untreated) 0.3639 ±0.01 1,18 a,b values are significantly different at p > 0 .0 0 1 For the first time we evaluated non-specific (general) esterases of female 0. coffeae occurring in tea plantations sprayed by synthetic pesticides and unsprayed plantations. High LC 50 value for ethion, an organophosphate, indicated its least toxicity to RSM on tea when compared with synthetic pyrethroid (fenproperthrin) (SAHOO e t a!., 2003), which implied that O. coffeae showed resistance/higher tolerance to ethion. Elevated esterase activity is known to be involved in organophosphate resistance in A. gossypii (O W U S U e t al., 1996). Further, different band numbers and varying esterase intensities on PAGE are evident in resistant, as compared to susceptible strains of Tetranychus urticae (C A P U A et al., 1990; SUNDUKOV e t a/.. 1989). OSAKABE and SAKAGAMI (1993) related the resistance of Tetranychus kanzawai to malathion with increased esterase activity at E-3 and E-4 bands. E-1 -> E -2 -> E -3^ Pesticide exposed Pesticide unexposed Fig. 1; PAGE profile of esterase isozyrnSi of Oligonychus coffeae (female); Each lane represents bands,of single specimen; Rm values: E-1: 0.264, E-2: 0.3403, and E-3: 0.4833 '81 The §)7hance quantity of esterases as well as the additional bands (isozymes) of O. coffeae of the conventional plantation are possibly involved in the detoxification of synthetic acaricides and insecticides. Further, such enhancements must be endowing the pesticide-exposed mites to have a greater pesticide tolerance / resistance. In a similar finding YANG e t al. (2002) cited high general esterase activity in pyrethroid resistant 70 JOURNAL OF APPLIED ZOOLOGICAL RESEARCHES strains of Oligonychus pratensis and T. uiHcae and Inferred that esterases may be involved in the detoxification and /or sequestration of pyrethroid insecticides in these mites. Elevated general esterase activity was reported in bifenthrin-resistant in Bem isia argentifolii (R ILE Y et a/., 2000) and several other pyrethroid-resistant insects (D E LO R M E et al., 1988; LEE and CLARK, 1996; ZHAO et al., 1996). The practical significance of the present finding may be realized in designing a method for easy detection of the pesticide resistance/ tolerance status of O, coffeae using esterases. Moreover, the pest status of the red spider populations/strains from Darjeeling foothills and plains shall be useful in deciding upon the future strategy of resistance management of the pest. ACKOWLEDGEMENT: Authors are thankful to Dr. Kun Yan Zhu, Department of Entomology, Kansas State University, Manhattan, USA for reviewing earlier version of the manuscript and offering useful suggestions. REFERENCES ANONYMOUS, 1994. Pests of Tea in North- East India and their control. Memorandum No 27, Tocklal Experimental Station, Jorhat, Assam, India, pp 169-180. ANSPAUGH, D. D.; KENNEDY, G.G. and ROE. R.M. 1995. Purification and characterization of a resistance-associated esterase from Leptinotarsa decemlineata (Say). Pestic. Biochem. Physioi. 53: 84-96. BANERJEE, B. 1968. Insect resistance. Two and a Bud. 16(1): 13-14. BARBORA, B.C. and BISWAS, A.K. 1996. Use pattern of pesticides in tea estates of North- East \nd\a. Two and a Sue/. 43(2): 4-14. CAPUA, S.; COHEN, E. and GERSON. U. 1990. Non-specific esterase in mites- a comparative study. Comp. Phaim. Toxicol. 96: 125-130. CRANHAM, J.E. and HELLE. W. 1985. Pesticide resistance in Tetranychidae. In: Spider mites, their biology, natural enemies & control, Vol. 1A, W. tielle & /W. W. Sabelis (Eds),Elsevier,Amsterdam, pp. 405-421. DAS, G.M. 1959. Problems of pest control in tea. Science and Culture. 24: 493-498. DAVIS, B. J. 1964. Disc electrophoresis II Method and application to human serum protein. Ann. B. T. Acad. Sci. 121: 404-427. DELORME, R.; FOURNIER, D.; CHAUFAUX, J., CUANY, A.; BRIDE, J.M.: AUGE, D. and BERGE, J.B. 1988. Esterase metabolism and reduced penetration are causes of resistance to deltamethrin in Spodoptera exigua Hubn. Pestic. Biochem. Physiol. 32: 240-246. DEVONSHIRE, A.L. 1975. Studies of the carboxylesterases of Myzus persicae resistant and susceptible to organophosphorus insecticides. Proc. of 8th British Insecticide and Fungicide Conference, pp. 67-73. DEVONSHIRE, A.L.; MOORES, G.D. and FFRENCH-CONSTANT, R.H. 1986. Detection of insecticide resistance by immunological estimation of carboxylesterase activity in IsAyzus persicae (sulzer) and cross-reS^i'tion of the antiserum with Phrodon humuli (Aphididae). Bull.Entomol. Res. 76: 97-^07. -'tl' LEE, S. and CLARK, J.M. 1996. Tissue distribution and biochemical characterization of carboxytesterases associated with permethrin resistance in a near isogenic strain of Colorado Jpotato beetle. Pestic. Biochem. Physiol. 56: 208-219. OSAKABE, M.^^'rid SAKAGAMI, Y. 1993. Estirfiation of genetic variation in Japanese populations of the citrus red mite, Pandnychus citri (Me Gregor) (Acri: Tetranychidae) on the basis of esterase allele frequencies. Exp. Appl. Acarol. 17: 749-755. JOURNAL OF APPLIED ZOOLOGICAL RESEARCHES 7 1 OWUSU, O. E.; HORHKE, M. and HIRANO, C. 1996. Polyacrylamide gel electrophoresis assessments of esterases In cotton aphid (A'phidldae) resistance to dichlorvos. J. Econ. Entomol. 89(2): 302-306. RILEY, D.G.; TAN, W.J. and WOLFENBARGER, D. 2000. Activities of enzymes associated with inheritance of bifenthrin resistance In the whitetly, Bemisia argentifolii. Southwest. Entomol. 25: 201-211. SAHOO, B.; SAHOO, S.K. and SOMCHAUDHURY, A.K. 2003. Studies on the toxicity of newer molecules against tea red spider mite. Proc. Nat Sym. on Frontier Areas of Entomol.Res.lARI, N. Delhi, pp. 301. SUDDERUDDIN, K. I. 1973. An electrophoretic study of some hydrolases from an OP-susceptible and an OP- resistant strain of the green peach aphid, Myzus persicae. Comp. Biochem. Physiol. 44B : 923-929. SUDUKOV, Q.V.; ZIL-BERMINTS, I.V.; GOLOVKINA, L.S. and NOVOZHILOV, K.V. 1989. Problemy izbiratel’nosti Deistviya Insektltsidov I Akaritsidov I Ee Znachenie V, Zashchite Rastenii. 19: 64-69. VALLES, S.M. 1998. Toxicologlcal and biochemical studies with field populations ot German cockroach, Blattela germainica. Pestic. Biochem.Physiol. 62: 190-200. VAN ASPEREN, K. 1962. A study of house fly esterases by means of a sensitive colorimetric method. J. Insect. Physiol. 8: 401-416. YANG, X.; BUSCHMAN, L.L.; ZHU, K.Y. and MARGOLIES, D.C. 2002. Susceptibility and detoxifying enzyme activity In two spider mite species after selection with three Insecticides. J. Econ. Entomol. 95(2): 399-406. ZHAO, G.; ROSE, R.L.; HODGSON, E. and ROE, R.M. 1996. Biochemical mechanisms and diagnostic microassays for pyrethroid, carbamate, and organophosphate insecticide resistance / cross-resistance in the tobacco budworm, HeUothis virescens. Pestic. Biochem. P/iys/o/. 56: 183-195. ZHU, K.Y. and GAO, J-R. 1998. Kinetic properties and variability of esterases in organophosphate- susceptible and resistant greenbugs Schizaphis graminum (Aphididae). Pestic. Biochem. Physiol.62: 135-145. J. E nt Res. Soc. 8 (1 ): 27-36, 2006 ISSN: 1302-0250 Studies on Salivary and Midgut Enzymes of a Major Sucking Pest of Tea, Helopeltis theivora (Heteroptera: Miridae) from Darjeeling Plains, India Mayukh SARKER Ananda MUKHOPADHYAY University of North Bengal, Department of Zoology, Entomology Research Unit, Darjeeling, 734 013, INDIA, e-mail: [email protected] ABSTRACT Helopeltis theivora is a major pest of young leaves and buds of Camellia sinensis. To understand the feeding biology of this mirid bug, its digestive and oxidative enzymes from salivary glands and midgut were analysed. Three common hydrolytic enzymes, amylase, protease and lipase were detected both in its salivary gland and midgut homogenates. Catalase, peroxidase and polyphenol-oxidase were also detected from these homogenates. Catalase activity was higher in salivary gland than that of midgut, contrastingly, the activity of polyphenol-oxidase was greater in the midgut than in the salivary extract. Peroxidase activity was found to be similar. The presence of both hydrolysing and oxidoreductase enzymes in the salivary and midgut homogenates may be related to extra-oral digestion and defense, leading to tissue necrosis and phytotoxic effect in the tea leaves. Key words: H. theivora, tea, hydrolytic enzyme, catalase, polyphenol-oxidase, Darjeeling INTRODUCTION T ea, Camellia sinensis (L.) O. Kuntze is grown as a monoculture over contiguous areas of Daqeeling hills, plains and North-East India. The tea mosquito b u g , Helopeltis theivora Waterhouse injects watery saliva into plant tissues and causes severe injury to the growing shoots of tea bushes. Other than tea, the species also uses more than half a dozen alternate hosts, which include weeds, ornamental and fruit plants. The species has a wide distribution in S.E. Asia and China (Schuh, 1995). The chem ical composition of the saliva of heteropteran insects is crucial for effective feeding.'These insects rely heavily on saliva for extra-oral digestion 28 BARKER, M., MUKHOPADHYAY, A. (Cohen, 1998) and detoxification of defensive chemicals (M iles & Oertli, 1993). Sucking bugs deposit salivary secretion in or on plants when feeding, which significantly influences the physiology of the affected plant tissues. Some o f the secretions result in phytotoxemia (Gopalan, 1976). The ability of insects to use plant materials as food is indicated by the presence of specific digestive enzymes (Zeng & Cohen, 2000). In many heteropterans specific digestive enzymes for phytophagy include amylase and pectinase (Cohen, 1996). Proteolytic activity has also been detected in the salivary glands of m irid bugs such as Lygus rugulipennis (L au re m a et al., 1985) and Creontiades dilutus (Colebatch et a l, 2001). M irids use their digestive enzymes through the salivary canal to hquefy food into nutrient-rich slurry (M iles, 1972; Hori, 2000; Wheeler, 2001). The food slurry is ingested through the food canal and is passed into the alimentary canal where it is fiirther digested and absorbed (Cohen, 2000). Polyphenol-oxidase uses m olecular oxygen to catalyze two different types of reaction namely, the hydroxylation of monophenols to o-diphenols and the oxidation of polyphenols to quinones and further dark brown or black pigments, melanins (Robinson e ta l, 1991). Peroxidases use hydrogen peroxide to oxidize phenols and other aromatic derivatives (Deimann et al, 1991). Both these oxidoreductases have been identified in the salivary secretions of aphid species (M iles & Peng, 1989; Madhusudhan & M iles, 1993) and they were found to be involved in overcoming the plant defenses by neutralizing phenolics and their derivatives (M iles, 1969;Urbanska&Leszczynski, 1992). Heteropteran workers lament the lack of a thorough understanding of heteropteran feeding habits, especially that of M iridae (Wheeler, 2001). As the m irid, H. theivora causes serious damage to tea leaves resulting in the cessation o f growth, curling up and dying back, it was felt necessary to identify the hydrolyzing enzymes present in its salivary glands and midgut so that a better understanding o fthe feeding biology of the notorious pest is possible. Moreover it is of interest to know whether H. theivora possesses such enzymes as catalase, polyphenol-oxidase and peroxidase to metabolize tea phenolics and convert them into less toxic substances. MATERIALS AND METHODS Insect collection and maintenance Field populations of Helopeltisviexc collected from tea plantations of Daijeeling foothills, Terai and Dooars plains of West Bengal state, India. Studies on Salivary and Midgut Enzymes of a Major Sucking Pest 29 Specimens were reared in the laboratory on Tocklai clonal varieties (TV-1) for two generations at 25 ± 1 “ C; 85 ± 5 % R” with a photoperiod of (L; D) 12:12 in transparent containers (20 x 20 cm) with a regular supply of fresh tea twigs. Sample preparation Adults were used for enzyme extraction following a sUghtly modified method of Cohen (1993). The insects were placed for im m obilization at -20° C for 4 min and dissected in ice-cold phosphate buffer (pH 7.2) under a dissecting microscope. The salivary gland complex, including all lobes, accessory glands and tubules was exposed by gently pulling the head and protliorax away from the abdomen w itli fine forceps. Subsequently the midgut was removed by dissecting the body. The salivaiy glands of 10 insects were removed, placed in 1 ml of phosphate buffer, homogenized and centrifuged at 12, OOOX g for 10 min at 4° C. The supernatant was placed in a 1.5 ml centriflige tube and kept at 4“ C for use (w ithin 48 h). The midguts of the same 10 insects were homogenized and processed in the same way as the salivary glands. Protein concentrations of all enzyme samples were determined by Lowry’s metliod (1951) using bovine serum albumen as the standard. Five samples havingl 0 insects each were used for both the tissues. Each assay was repeated five times. Amylase assay Amylase activity in the salivary glands and midguts was determined after the method of Madhusudhan et al (1994) followed by the method of Sadasivam and Manickam (1996) using dinitrosalicylic acid reagent; and quantification of enzyme product was based on a standard curve prepared using various concentrations of maltose alone at 520 nm using UV-Vis spectrophotometer. The enzyme activity was expressed as |j.M / m in/ mg of protein. Protease assay Proteolytic activity was assayed after methods of Kunitz (1947) modified by Jayaraman (1981). 1% (w /v) casein was used as the substrate. 1 ml of casein prepared in 0.1 N NaOH was incubated with an equal volume of enzyme. After incubation for one hour, the reaction was terminated by the addition of 10% TCA and the acid-soluble peptides were quantified using the biuret reagent at 520 nm using UV-Vis spectrophotometer. The enzyme activity was expressed as |ig / mg of protein. 30 BARKER, M., MUKHOPADHYAY, A. Lipase assay Lipase activity was measured following the metliod of Sadasivam and Manickam (1996). The enzyme activity was calculated as m illiequivalent activity of free fatty acid/m in/g sample. Catalase assay 3 ml of Hydrogen peroxide and phosphate buffer was allowed to stabilize at 25 ± 2 ° C for 10 min. After the incubation period, 0.2 ml of salivary gland homogenate and midgut homogenate was added separately to the cuvette, and the change in absorbance was measured at 240 nm over a 30 sec period using a UV-Vis spectrophotometer. The protein concentration was measured as described above. Catalase activity was expressed as decrease in OD of hydi'ogen peroxide / min / mg ofprotein(Laurem aandVaris, 1991). Peroxidase assay Peroxidase activity was measured by monitoring the increase in absorbance by 0.1 and noting tlie time required in min. by using the slight modified method prescribed by Hampton (1963). The enzyme reaction was started by adding 3 ml of 0.1 M phosphate buffer (pH 7.0), 0.05 ml guiacol solution, 0.1 ml enzyme extract and 0.03 ml hydrogen peroxide in a cuvette. The activity of peroxidase was determined by increase in OD / min / mg of protein at 436 nm. Polyphenol-oxidase assay Polyphenol-oxidase activity was monitored following the m odified method of Hampton (1963) at 495 nm for 5 min after the start of the reaction. The reaction was initiated by adding 2.5 ml of phosphate buffer (pH 6.5) and 0.3 ml of catechol solution (O.OIM) and tlien 0.2 ml of enzyme extract. Enzyme activity was calculated as the increase in the absorbance/ m in / mg of protein RESULTS Enzyme studies The salivary gland and midgut of H. theivora were found to contain three common hydrolytic enzymes: amylase, proteaSe and lipase. Ashghtly higher quantity ofamylase was found in the midgut (0.0861 ±0.1 |iM /m in/m gofprotein)tlianthe salivary gland (0.0679 ± 0.1 / min / mg of protein). Sim ilarly, proteolytic enzyme Studies on Salivary and l^idgut Enzymes o f a Major Sucl activity was also higher in the midgut (19.06 ± 0.5 |^g / mg) than in the sahvary (17.54 ± 0.4 |j,g / mg) homogenate. The hpase activity could be detected but in a very low key both in the salivary (0.0314 ± 0.002 m illiequivalent (meq) of free fatty acid / m in/ g sample and the midgiit (0.0524 ± 0.004 m illiequivalent of free fatty acid / m in/ g sample) (Table 1). Among the oxidoreductase enzymes, catalase, peroxidase and polyphenol- oxidase were active both in salivary and midgut homogenate. Salivary gland homogenate initially showed more catalase activity as compared to that of the midgut (Fig. 1). W hile peroxidase activity was found to be sim ilar, polyphenol- oxidase activity was evidently more in the midgut than in the salivary gland homogenate (Fig. 2 and 3). Table 1. Digestive enzymes of the salivary gland (SG) and the inidgut (MG) homogenate of Helopeltis theivora (mean ± SD) Enzyme Enzyme Observed value per unit source SG 0.0679 ± 0.1 *(|J.M . mg protein min ') Amylase MG 0.0861 ± 0.1 “ (|J.M . mg protein '. min -I ')n SG 17.54 ± 0.4 |j,g / mg “ (Amount of protein, casein, utilized) Protease MG 19.06 ± 0.5 i-ig / mg ' (Amount of protein, casein, utilized) SG 0.0314 ± 0.002 “ * (Activity meq / min /g of sample) Lipase MG 0.0524 ± 0.004 (Activity meq / min /g of sample) Different letters in a column indicate significance difference of mean at p> 0.001 CONCLUSIONS AND DISCUSSION hi Hemiptera, the ability to use plant materials for food is very dependent on tlie presence of specific digestive enzymes. Digestive enzymes, advantageous for phytophagy, include amylases and proteases (Cohen, 1996; Gopalan, 1976). The plant feeding m irids usually have a high quantity of amylase in their salivary gland complex (Agusti & Cohen, 2000). Enhanced levels of amylase found in the salivary gland and midgut of H. theivora im plied an effective digestion of leaf starch both extraorally and after ingestion. 32 BARKER, M., MUKHOPADHYAY, A. Fig. 1. Catalase activity of the salivary gland (SG) and the midgut (MG) homogenate of H. theivora SG homogenate | MG homogenate Time ( Seconds) Fig. 2. Peroxidase activity of SG and MG of H. theivora. Studies on Salivary and Midgut Enzymes of a Major Sucking Pest 33 - SG honiogeiiate - MG honiogeiiate 0,03 0.028 c 0,026 'B 0,024 S 0,022 0,02 S. 0.018 S 0,016 " 0.014 •p 0,012 ^ 0,01 a 0,008 o 0,006 0,004 0,002 0 Time (Seconds) Fig. 3. Polyphenol-oxidase activity of SG and MG of H. theivora. The presence o f general protease activity both in the salivary gland and the midgut o f H. theivora indicates that this pest can w ell utilize the protein source of the tea leaf In an earlier study, protease was detected in Helopeltis corbisieri (K u m a r, 1970), and subsequently, the protease was found to be responsible for extra-intestinal digestion in Lygus disponsi (H o ri, 1970). W h ile in H theivora a very low activity of lipase was registered, the enzyme was found to be absent in the m irid, Lygiis sp. Lipase however is reported to occur in heteropterans at large (Nuorteva, 1954; B ro n s k ill er a/. 195 8; Feir & Beck, 1961). Lipase may be involved in extra-intestinal digestion and enlargement ofpore size in the protein-lipidlayer of the plasmamembrane of an individual cell (Branton, 1969). Hydrolytic salivary enzymes in minds may also be involved in the liberation of osm otically active substances into the intercellular space of the plant tissue causing an outflow of liquid that can be sucked back by the insect (M iles, 1987). The oxidoreductase enzymes of the salivary gland and midgut of H theivora appear to have a defensive role. O f these enzymes, catalase, peroxidase and polyphenol-oxidase are known to cause phytotoxaemia as w ell as detoxification of secondary metabolites of the plants. Catalase in the saliva has the ability to prevent the formation of harmful compounds (quinone), whereas the free amino acids present in the secretion interfere with plant defence chem icals to protect the digestive enzymes of saliva from denaturation (Laurema & Varis, 1991). The enzyme peroxidase is reported to 34 BARKER, M., MUKHOPADHYAY, A. degi-ade chlorophyll (M antile, 1980). Some aphids use salivary polyphenol-oxidase and peroxidase for detoxification of hannM plant phenolics (M iles & Peng, 1989). The polyphenol-oxidase and peroxidase in the saliva and midgut of H. theivora are possibly able to oxidize a wide range of phenolic compounds with a double efficiency as far as defence against tea plant phenolic compounds are concerned. The occurrence of salivary phenolase suggests that the enzyme, whether secreted alone or along w ith a substrate, may enable the insects to overcome the natural defences of host plants (M iles, 1972; Sengupta & M iles, 1975; U rb a n sk a etal. 1998; Peng & M iles, 1988). The presence of hydrolyzing enzymes in the salivary gland of H. theivora m ay be responsible for maceration and dissolution of plant tissue, facilitating peneti'ation of the stylets and rem oval of the cell contents. The amylase, protease and sm all quantity of lipase in the salivary glands and midgut may help extra-oral as w ell as lim iinal digestion at midgut level. The catalase, peroxidase and polyphenol-oxidase in the salivaiy glands and midgut homogenate were important since these were considered by M iles (1964) to counter host plant’s defence chem icals by speeding up the process of oxidation of phenolics to non-toxic end products. Hence the presence of these oxido-reductase enzymes in the salivary and midgut along with the basic hydrolyzing enzymes enable H. theivora to become one of the most destructive pests o f tea by depredating the young leaves and growing shoots of tea particulai'ly with a phytotoxic effect. REFERENCES Agusti, N., Cohen, A. C., 2000, Lygiis hespenis and L. lineolaris (Heiniptera, Miridae), phytophages, zoophages, or omnivores; evidence of feeding adaptations suggested by the salivary andmidgut digestive enzymes. Journal of Entomological Science, 35: 176-186. Branton, D., 1969, Membrane structure. Annual Review of Plant Physiology,20:209-23^. Bronskill, J. F., Salked, F. H., Friend, W. G., 1958, Anatomy, Histology and Secretions of salivai^ gland of the large milicweed bug, Oncopeltus fasciatus (Dallas) (Hemiptera, Lygaeidae). Canadian Journal of Zoology, 36: 961-968. Cohen, A. C., 1993, Organization of digestion and preliminaiy characterization of saiivaiy ti^psin- iike enzymes in a predaceous heteropteran, Zelus renardii. Journal of Insect Physiology, 39(10): 823-829. r Cohen, A. C., 1996, Plant feeding by predatory Heteroptera\ evolutionary and adaptational aspects of trophic switching. In: Alomar, O., Wiedenmann, R.N. (Eds.). Zoophagous Heteroptera: implications for life history and integrated pest management. Thomas Say Publications in Entomology, Entomological Society of America, Lanham, MD, 1-17. Studies on Salivary and l^idgut Enzymes of a Major SuMng Pest 3 5 Cohen, A. C., 1998, Solid-to-liquid feeding: the insect(s) stoiy on exti'a-oral digestion in predaceous A.nhi-opoda. American Entomologist, 44: 103-117. Cohen, A. C., 2000, How carnivorous bugs feed. In: Schaefer, C. W., Panizzi, A. R. (Eds.). The economic importance ofHeteroptera. CRC Press, Boca Raton, FL, 563-570. Colebatch, G. M., East, R, Cooper, R, 2001, Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutiis (Hemiptera, Miridae). bisect Biochemistry and Molecidar BioIog}>, 31:415-423. Deimann, W., Angernuiller, S., Steward, P. J., Fahinii, H. D., \ 99\, Peroxidases. In: Stoward, J. P., Pearse, A, G. E. (Eds.). Histochemistry Theoretical and Applied. Vol. 3. Churchill Livinstone, Edinburgh, 135-139. Feir, D., Beck, S. D., 1961, Salivary secretions o f Oncopeltns fasciatus (Hemiptera, Lygaeidae)- scienti fie note. Annals of Entomological Society of America, 54:316. Gopalan, M., 1976, Studies on salivary enzymes of Ragmiis iinportunitiis Distant (Hemiptera, Miridae). Current Science, 45 {5)\ 188-189. Hampton, R. E., 1963, Activity of some soluble oxidase in carrot slices infected with Thielaviopsis basicola. Phytopathologv, 53: 306-333. Hori, K., 1970, Some properties of proteases in the gut and in the salivary gland of Lygus disponsi Linnavuori (Hemiptera, Miridae). Research Bulletin of Obihiro University, 6:318-324. Hori, K., 2000, Possible causes of disease symptoms resulting from the feeding of phytophagous Heteroptera. In: Schaefer, C. W., Panizzi, A. R. (Eds.). The economic importance of Heteroptera. CRC Press, Boca Raton, FL, 11-35. Jayaraman, J., 1981, Laboratory Manual In Biochemistry, Wiley Eastern Limited, New Delhi, 132-133. Kumar, R., 1970, Occurrence of protease in the salivary glands of cocoa- capsids (Heteroptera, M iridae). Journal o f the New York Entomological Society, 78:198-200. Kunitz, M., 1947, Ci7Stalline soy bean trypsin inhibitor, general properties. Journal o f General W7>-5/o/og)',30:291-310. Laurema, S., Varis, A. L., 1991, Salivary aminoacids in (Heteroptera, Miridae). Insect Biochemistiy, 2 1: 759-765. Laurema, S., Varis, A. L., Miettinen, H., 1985, Studies on enzymes in the salivary glands of Lygus nigulipennis (Hemiptera, Miridae). Insect Biochemistry, 15:211 -224. L0W17,0.H ., Rosebrough,N. J., Farr, A. L., Randall, R. J., 1951, Protein measurement with Folin phenol reagent. Journal of Biological Chemistry, 193: 265-27 5. M adhusudhan, V. V., M iles, P. W., 1993, Detection o f enzymes secreted in the saliva of the spotted alfalfa aphid, Therioaphis trifolii (Monell) f. maculata (Hemiptera, Aphididae). In: Corey, S.A., Dali, D.J., Milne, W.M. (Eds.). Pest control and sustainable Agriculture. L31RO, Austialia, 333-334, Madhusudhan, V. V., Taylor, G. S., Miles, R W., 1994, The detection of salivary enzymes of phytophagous Hemiptera: a compilation of methods. Annals of Applied Biology, 124:405-412. Mantile, R, 1980, Catabolism of chlorophyll: Involvement of peroxidase? Zeitschrift fu er Pflanzenphysiologie, 99: 475-478. 36 BARKER, M., MUKHOPADHYAY, A. Miles, P. W., 1964, Studies on the salivary physiology of plant-bugs; oxidase activity in the salivary apparatus and saliva. Journal of Insect Physiology, 10; 121-129. Miles, P. W., 1969, Interaction of plant phenols and salivary phenolases in the relationship between plants and Hemiptera. Entomologia Experimentalis etApplicata, 12; 736-744. Miles, P. W., 1972, The saliva of Hemiptera. Advances in Insect Physiology, 9; 183-255. Miles, P. W., 1987, Feeding process of Aphidoidea in relation to effects on their food plants. In; Minks, A. K., Harrewijn, P (Eds.). Aphids; their biology. Natural Enemies and Control. Vol. 2A. Elsevier Science Publishers, Amsterdam, 321-339. Miles, P. W., Peng, Z., 1989, Studies on the salivary physiology of plant bugs: detoxification of phytochemicals by the salivary peroxidase. Journal of Insect Physiology, 35; 865-872. Miles, P. W., Oertli, J. J., 1993, The significance of antioxidants in the aphid-plant interaction; the redox hypothesis. Entomologia Experimentalis et Applicata, 67; 275-283. Nuorteva, P., 1954, Studies on the salivary enzymes of some bugs injuring wheat kernels. Annales Entomologici Fennici, 20; 102-124. Peng, Z., Miles, P. W., 1988, Studies on the salivary physiology of plant bugs; function of the catechol oxidase of the rose aphid. Journal of Insect Physiology, 34(11); 1027-1033. Robinson, J. M., Karnovsky, M. J., Stoward, P. J., Lewis, P. R., 1991, Oxidases. In; Steward, J. P., Pearse, A. G. E. (Eds.). Histochemistry Theoretical and Applied. Vol. 3. Churchill Livinstone, Edinburgh, 95-115. Sadasivam, S., Manickam, A., 1996, Biochemical Methods, 2"‘‘ edn. New Age International (P) Limited, New Delhi and TN AU, Coimbatore, 116-126. Schuh, R. T., 1995, Plant Bugs of the World (Insecta, Heteroptera, Miridae) Systematic Catalog, Distribution, Host list and Bibliography. The New York Entomological Society, N ew York, 511-516. Sengupta, G. C., Miles, P. W., 1975, Studies on the susceptibility of varieties of apple to the feeding of two strains of wooly aphis (Homoptera) in relation to the chemical content of the tissues ofthe host. Australian Journal of Agricultural Research, 26: 157-168. Urbanska, A., Leszczynski, B., 1992, Biochemical adaptations of cereal aphids to host-plants. In; Menken, S. B. J., Visser, H. J., Harrewijn, P., (Eds.). Proceedings of 8"' International Symposium on insect-Plant relationships. Kluwer Academic Publishers, Dordrecht, 277-279. Urbanska, A., Tjallingii, W. R, Dixon, A. F. G., Leszczynski, B., 1998, Phenol oxidising enzymes in the grain aphid’s saliva. Entomologia Experimentalis etApplicata, 86; 197-203. Wheeler, A. G. J., 2001, Biology of plant bugs (Hemiptera, Miridae): pests, predators, oppor tunists. Cornell University Press, Ithaca, NY, 507. Zeng, F., Cohen, A. C,, 2000, Comparison of a-amylase and protease activities of a zoophytophagous and two phytozoophagdus Heteroptera. Comparative Biochemistry and Physiology, 126A; 101-106. Received: September 01, 2005 Accepted: January 07, 2006 Two and a Bud 50:28-30, 2003 RESEARCH PAPER Expression of esterases in different tissues of tlie tea-pest, Helopeitis theivora exposed and uoexposed to synthetic pesticide sprays from Darjeeiing foothills and plains iiayukh Sarker and Ananda IVlukhopadhyay Entomology Research Unit, Department of Zoology, University of North Bengal, Darjeeling - 734 430, India ABSTRACT The general esterase zymograms were developed from salivary, midgut and whole body homogenates of Helopeitis theivora (Hemiptera: Miridae) sampled from discrete populations. Specimens were laboratory reared on TV-1 and TV-25 and collected from organic (synthetic pesticide free) and conventional (synthetic pesticide treated) tea plantations. They showed common two esterase bands in salivary gland homogenates with low staining intensity whereas in the midgut and whole body homogenates three prominent deeply stained esterase bands were apparent. The Rm values of these bands were 0.16, 0.20 and 0.31 respectively. Profile of general esterase bands of H. theivora when related to their host-plant variety did not show any host-clone based variation, however, in general, higher band intensities (stain) of the pesticide-exposed specimens (over unexposed ones) indicated a higher tolerance / resistance status due to formation of greater amount of esterases. INTRODUCTION homogenates was contemplated. The major objective of this study was to know the variation in the general esterase Tea, Camellia sinensis (L.) 0. Kuntze is grown as a profiles of salivary, midgut and whole body (without salivary monoculture over contiguous areas of Darjeeling hills and gland and gut) homogenates of H. theivora when reared in North-East India. Tea mosquito bug, Helopeitis theivora laboratoiy on TV-1 and TV-25 varieties. Esterase variation Waterhouse, is undisputedly the most destructive sucking was also compared between populations of H. theivora pest of young leaves and buds of tea. The chemical collected from organic (synthetic pesticide free) and composition of the saliva of hemipteran insects is crucial conventional (synthetic pesticide treated) tea plantations. for effective feeding, because these insects rely heavily on saliva for extra-oral digestion (Cohen, 1998) or detoxification MATERIALS AND IVIETHODS of defensive chemicals (Miles and Oertli, 1993). Varying degrees of susceptibility of insect pests when reared on Insect'collection and maintenance different plant species or varieties have been related to different levels of metabolizing enzymes, presumably Field populations of Helopeitis were collected from each induced by the plants (Yu, 1983; Ambrose and Regupathy, site of organic and conventional tea plantations of Darjeeling 1992). Many enzymes involved in detoxification pathways foothills, Terai and Dooars of West Bengal state, India. act on a broad array of substrates, including both naturally occurring plant allelochemicals and artificial pesticides Specimens vi/ere reared on Tocklai clonal varieties (TV-1 (Gordon, 1961). General esterases are common and TV-25) for two generations at 25 ± 1°C; 85 ± 5 % RH detoxification enzymes that are capable of degrading with a photoperiod of (L; D) 12:12 in transparent containers organophosphate and synthetic pyrethroid pesticides (2 0 X 2 0 cm) with a regular supply of fresh tea twigs. (Anspaugh etal., 1995; Valles, 1998). H. theivora is mainly controlled by organophosphate, carbamates and synthetic Enzyme extraction and Gel Electrophoresis pyrethroid pesticides. As the pest shows resurgence and recurrence despite pesticidal treatments, a study on the Adult males and females were used for enzyme extraction occurrence of general esterases in different tissue following the method of Cohen (1993) with a little modification. 28 The insects were placed for immobilization at -20°C for 4 min and dissected in ice-cold phosphate buffer (pH 7.2) under dissecting microscope. The salivary gland complex, 0.16- jincluding all lobes, accessory glands and tubules was 0 . 2 0 ' exposed by holding the abdomen with fine forceps and gently 0.31. pulling the head and prothorax away from the abdomen with another pair of fine forceps. Subsequently the midgut was removed by dissecting the body The pair of salivary glands of each individual (n = 1 0 ) (male and female) was place in 10)jl phosphate buffer. Volume of the buffer was adjusted to produce similar protein concentration in the homogenates for each sample. Homogenates were centrifuged at 12,000X g for 10 min at40°C.; the supernatant was placed in 1.5 ml centrifuge tube and preserved at -20°C. The midgut of the Fig. 1: Tissue specific PAGE profile of esterase isozymes of same insect was treated identically as the salivary glands. tine Helopeltis theivora 5 pi of supernatant was dispensed into each well of a vertical slab gel. Electrophoresis was earned out at constant current a) Salivary gland homogenate: (10mA) for about 1.5 hr on 8 % native polyacrylamide gel, TV-1 reared: Lane 1 c) Whole body homogenate: using Tris-glycine (pH 8.3) as running buffer (Davis, 1964). TV-25 reared: Lane 2 (Without salivary gland and midgut) b) Midgut homogenate: TV-1 reared: Lane 7 TV-1 reared: Lane 4 TV-25 reared: Lane 6,8 The gel was then stained for 60 min in dark at 37°C with TV-25 reared: Lane 3, 5 Fast Blue BB salt in 0.1 M phosphate buffer adding 0.03 M alpha napthyl acetate solution dissolved in acetone. infestans (Tavares ef a/,l 1998). |H. theivora when reared on Relative migration of esterase bands and zymogram were two different clonal varieties (TV-1 and TV-25) did not show determined by the Kodak digital science ID Image Analysis any major differences in the banding pattern, thus excluding iSoftware version 2.0.3. Relative mobility (Rm) was calculated any major influence of the host-variety on the variation of by: esterase isozymes in Helopeltis] in a similar case, lack of host influence was observed in the fall army worm where host- iDistance migrated by the specific bands (cm)/Dlstance ^3lant allelochemicals marginally affected its esterase activity migrated by the marker dye. (Yu ,1984). 1 : RESULTS AND DISCUSSION I n the present study the esterase zymograms were developed from salivary, midgut and whole body homogenates of three discrete populations of H, theivora i.e. i) specimens reared in laboratory on TV-1 and TV-25 clones ii) specimens collected from organic and iii) conventional plantations. All of them by and large showed a common banding pattern. Two faint a) esterase bands were detectable in salivary gland homogenate b) . Fig. 2; Z)TiK)grams of cslcra.st isozymes of midgul homogcnalc Hdopdtis fheivora with low-staining intensity, vis a vis in the midgut and collecicd from viirious sources wholebody homogenates three prominent esterase bands ii) Lane 1; Female specimens b) I .«nc 3: organic plaiitallon were apparent. These bands were slow moving, medium slow ! m e 2: M.'ilc spcciiiicn.s Lane 4; Pcsticidc treated planialioii Lane 5: l.^boratory reared specimens on moving, and fast moving with the Rm values of 0.16,0.20 and TV-1 tea done 0.31 respectively (Fig. la , b, c). Intensity of midgut esterase bands was higher in female specimens than in male (Fig. 2a). Direct measurement and quantification of detoxifying enzyme Esterase bands of female specimens exposed to pesticide lactivities in sucking insects is insufficient. So, in this maiden sprays, in conventionally managed tea plantations, showed a study on general esterase isozymes of the sucking bug, H. higher staining intensity than the ones either collected from theivora at different tissue levels, midgut esterase activity was organic plantations (free from synthetic pesticide sprays) or found to be higher in specimens from conventional plantations, reared in laboratory (Fig. 2b). possibly endovi/ing the bug with a greater insecticide tolerance {vis a vis resistance). Isozyme forms of Carboxylesterase occur in different tissues (Ahmad ntal. 1986). Tissue specific expression of esterases In resistant green bug, Schizaphis graminum, high levels of related to their functional roles has been recorded in Trialoma non-specific and intensely stained esterase bands were 29 obtained (Onoetal.,1994). Myzuspersicae has been reported Davis, B. J. (1964). Disc electrophoresis I! Method and to develop resistance to organophosphate, carbamate and application to human serum protein. Ann. B. T. Acad. synthetic pyrethroid insecticides through an increased amount Sci., 121:404-27. of esterases (Sawicki e ta i, 1978; Devonshire and Moores, Devonshire, A.L. and Moores, G.D. (1982). Acarboxyiesterase 1982). In the above case esterase-based resistance was with broad substrate specificity causes conferred either by gene amplification or enhanced gene organophosphorous, carbamate and pyrethroid expression. Amplification of esterase genes, often in resistance in peach-potato aphid, |Myzus persicae. combination with altered gene regulation results in the Pestic. Biochem. Physiol., 18:235-46. production of more esterases that hydrolyze insecticides (Field ef a/., 1998, 1999). Therefore, the high staining intensity of Field, L.M. and Devonshire, A.L. (1998). Evidence that the E4 esterase bands obsen/ed in the present zymograms could and FE4 esterase responsible for insecticide reasonably be due to greater production of esterases meant resistance in the aphid |Myzus persicae (Sulzer) are for hydrolyzing the insecticides to which the H. theivora was part of a gene family. Biochem. J. 330(1):169-73. exposed in the conventional tea plantations (Fig. 2b). In sheep blow fly, Lucilia cuprina, esterase band (E-3) was present Field, L.M., Blackman, R .L, Tyler, S.C. and Devonshire, A.L. both in susceptible and in resistant forms, but the esterase (1999). Relationship between amount of esterase and activity was higher in resistant forms due to amplification of gene copy number in insecticide-resistant Myzus esterase genes (Hughes and Raftos, 1985). Similar banding persicae (Sulzer). piochem. J., 339(3):737-42. patterns (Rm values) of esterases of H. theivora populations Gordon, H.T. (1961). 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