BIOCHEMICAL CHARACTERIZATION OF INSECTICIDE RESISTANCE IN INSECT PESTS OF VEGETABLES AND PREDATORY LADYBIRD BEETLES

Insecticide resistance and the underlying resistance mechanisms were studied in seven species of insect vegetable pests (aphids Aphis gossypii, Myzus persicae, Aphis cracciuora, Toxoptera citricidus and Lipaphis erysirni; diamond-back-moth Plutella xylostella; leafminer Liriomyza huidobrensis) and two predatory ladybird beetles (Coccinella sexrnaculatus and Thea cincta). Insects, collected from the Central Province, Sri Lanka were subjected to bioassays with malathion, chlorpyrifos, propoxur and permethrin by topical application and LC,, and LC,, values were obtained. M. persicae, P. xylostella and L. huidobrensis had high resistance to all the insecticides tested with the exception of M. persicae to propoxur. The aphid T. citricidus was the least tolerant pest to all four insecticides tested. Compared to the pest species, the ladybird beetle C. sexmaculatus had a moderate level of insecticide tolerance whereas T. cincta was more susceptible. DDT resistance, as determined by a discriminating concentration, was highest in P. xylostella and least in L. huidobrensis. Biochemical analysis of insect metabolic enzymes showed that elevation of esterases was the major resistance mechanism in aphids and P. xylostella. Highest esterase activity was present in M. persicae. Native polyacrylamide gel electrophoresis resolved elevated esterase isoenzymes in all species except in T. citricidus and T. cincta. Malathion carboxylesterase activity was found in P. xylostella, M. persicae, and L. huidobrensis. Higher glutathione S-transferase activities of P. xylostella and L. erysirni were correlated with their higher DDT resistance. Increased mono-oxygenase titres occurred in A. gossypii , M. persicae and T. citricidus. Insecticide inhibition of the organophosphate and carbamate target site acetylcholinesterases indicated that target site insensitivity is a major mechanism in L. huidobrensis.


INTRODUCTION
health sectors.Extensive use of insecticides has selected many insecticide resistant insect populations causing a severe problem in pest management programmes of the country.Although there are rich communities of insect predatory species that reduce pest populations, the use of insecticides affects both predators and pests.It is important to identify the insecticide cross-resistance spectra and the underlying resistance mechanisms of both pest and predatory insect populations to rationally select suitable insecticide groups which maximise pest control and minimise the effect on their natural enemies.We report here the resistance status of seven important vegetable insect pest species against organochlorines (DDT), organophosphates (malathion, chlorpyrifos), carbarnates (propoxur) and pyrethroids (permethrin).Two species of ladybird beetles were also used to determine the resistance status of predatory species in relation to their prey species.
Resistance to insecticides mainly occurs due to changes in insect metabolic enzymes or due to the development of insecticide insensitive target sites in the insect nervous system1.Increased metabolism is often caused by qualitative and/or quantitative changes of esterases, glutathione S-transferases and monooxygenases.Target site insensitivity is often caused by highly specific point mutations.Mutated target sites do not bind to insecticides but perform their normal physiological functions.Prevalence of these resistance mechanisms in the Insect pests are a serious ~r o b l e m in t r o ~i ~a l above mentioned insect populations were also agriculture.Extensive use of insecticides has been monitored.the major method of insect pest control in Sri Lanka for more than five decades.After DDT use METHODS AND MATERLALS was banned in 1977, malathion was used for house 'praying in insects: Five species of apterous adult aphids carbarnates were used (Homoptera: Aphididae) ie.Aphisgossypii, Myzus in 'yrethroids were persicad Aphis craccivora, Toxoptera citricidus introduced in mid 1990s into both agricult-ure and Torresponding Author and Lipaphis erysimi; adult diamondback moth Plutella xylostella (Lepidoptera: Yponomeutidae); and the adult ladybird beetles (Coleoptera: Coccinellidae) Coccinella sexmaculatus and Thea cincta were collected from agricultural crops in Kandy region.Adult leafminer Liriomyza huidobrensis (Diptera: Agromizidae) were collected from Nuwara Eliya area.Insects were directly used for bioassays or frozen a t -20°C.Frozen insects were used for biochemical assays within 2 wk.Chemicals a n d equipment: Chemicals were purchased from Sigma, U.K., unless otherwise stated.DDT (97.5% pure) and paraoxon (98% pure) were from Greyhound, UK.Malathion (97.5% pure) and chlorpyrifos (98% pure) were a gift from Cheminova, Denmark.Propoxur (98.5% pure) was from Bayer, Germany and permethrin (98% pure) was from Aventis, UK.The protein assay kit and mini-protean I1 electrophoresis kit were from Bio-Rad, LK.
Insect bioassays: Bioassays for malathion, chlorpyrifos, propoxur, permethrin and DDT were undertaken by topical application.Insecticide stock solutions were prepared in acetone.Dilutions were made with 70% ethanol.Final working solutions always had an acetone concentration of <0.01%.Insecticide solution (0.25 pl per 0.25 pg insect wet body weight) was applied to the thorax of each insect using a Burcard topical applicator and the mortalities were recorded after 24 h.2At least five concentrations of each insecticide (except DDT), aving mortalities between 0-100 % were used for each species.A minimum of 100 insects per concentration per species was tested.Controls were treated with solvent alone.Results were used only if the mortalities in the controls were <20%.Control mortalities were adjusted for using Abbott's for~nula.~Mortalities were plotted against insecticide concentrations to obtain logconcentration probit-mortality lines and LC,, and LC,,vaIues were calculated by regression analysis.DDT bioassays were carried out by applying a discriminating concentration (determined by preliminary experiments) of 1.4 pglmg wet body weight.
Biochemical assays with individual insects: Adult insects were individually subjected to esterase, glutathione S-transferase (GST), mono-oxygenase and protein assays using a kinetic microtitre plate reader (Bio-Tek, USA) a t 22 "C.At least 200 individuals from each population were tested.Each insect was homogenised in 150 p1 of distilled water and centrifuged at 10,000g for 2 min.Supernatants were tested individually for the enzyme activities in microtitre plates.
Esterase assay: Homogenate (10 p1) was mixed with 200 pl of 1 mMp-nitrophenyl acetate (pNPA) in 50 mM sodium phosphate buffer (pH 7.4).The increase in absorbance was monitored at 405 nm for 2 min.An extinction co-efficient of 6.53 mM-' (corrected for a path length of 0.6 cm) was used to convert the absorbance to Glutathion S-transferase assay: Homogenate (10 p1) was mixed with 200 pl of substrate solution [95 parts of 10.5 mM reduced glutathione in 100 mM phosphate buffer + 5 parts of 63 mM 1-chloro 2,4dinitrobenzene (CDNB) in methanol].The rate of reaction was measured a t 340 nm for 5 min.An extinction co-efficient of 5.76 (corrected for path length of 0.6 cm) was used to convert absorbance to m01es.~Mono-oxygenase assay: Homogenate (20 p1) was mixed with 80 pl of potassium phosphate buffer (pH 7.2) + 200 pl of 6.3 mM tetramethyl benzidine (TMBZ) working solution (0.01g TMBZ dissolved in 5 ml methanol and then in 15 ml of sodium acetate buffer pH 5.0) + 25 p1 of 3% H,O, solution in a microtitre plate well.After 2 h incubation at room temperature, the plate was read a t 630 nm as a n end point assay.7This assay does not measure mono-oxygenase activity but titrates the amount of bound haem in the insect homogenate.Since haem is present in the active site of monooxygenase and is the major haem component in insects, the amount of haem corresponds to the amount of mono-oxygenase present.By using a standard curve of pure cytochrome C, which contains one bound haem per molecule, an estimate of the amount of mono-oxygenases present was obtained and expressed as equivalent units of cytochrome P450.7 Protein assay: To obtain specific activities of the enzymes, protein concentrations of the homogenates were determined using a BIO-RAD protein determination kit, with bovine serum albumin as the standard p r ~t e i n .~ Homogenate (10 p1) was mixed with 300 pl of worlung solution (prepared according to the manufacturer's instructions).After incubation at 22°C for 5 min, the absorbance was read at 570 nm as an endpoint assay.
Malathion metabolism (malathion carboxylesterase assay): Batches of 5-80 individuals (equivalent to 10 mg wet weight) of each insect species were homogenized in 0.5 ml of 25 mM Tris-HC1 buffer (pH 7.5) and centrifuged at 13,000g for 5 min.Supernatant was incubated with 300 pM malathon for 2 h at 28OC.The mixture was then extracted twice with 0.   and the ladybird beetle T. cincta, had elevated elevated esterase bands would be below the limits esterase bands on native polyacrylamide gels of detection.12Elevated esterase bands were (Figure 1).The amount of protein per well in each completely inhibited by 0.1 mM paraoxon, partially electrophoresis experiment was set so that nun-inhibited by 0.1 mM propoxur and not inhbited by 0.1 mM permethnn except the slowest running band of M. persicae, which was not inhibited by any of the insecticides tested.Malathion carboxylesterase activity was found only in M. persicae, P. xylostella and L. huidobrensis, all of whch had h g h levels of resistance to malathion.Crude homogenates ofthese three species metabolised malathion into both its mono-and di-acid products after 2 hours incubation (Figure 2).Ladybird beetle species, which had a low level of tolerance to malathion did not have malathion carboxylesterase activities (results not shown).Higher DDT resistance, shown by P. xylostella and L. erysimi populations, was correlated with their elevated GST activity levels (Table 2).Involvement of mono-oxygenases in the insecticide resistance of these insect species was indirectly tested by quantifying bound haem.? A.
gossypii, M. persicae and A. craccivora had the highest mono-oxygenase estimates (Table 2).Lower mono-oxygenase content was estimated in L. huidobrensis and C. sexmaculatus.Bimolecular rate constants for the inhibition of AChEs with propoxur are a v e n in Table 3. AChEs are the target site of organophosphorous and carbamate insecticides.L. huidobrensis AChEs had the lowest inhibition rate suggesting a high frequency of insensitive AChE alleles in the leafminer population.In Sri Lanka, the organochlorines DDT and y-BHC were used extensively in agriculture until the mid 1970s.Organophosphates were introduced in the 1960s but the use of malathion and fenitrothion in agriculture was legally restricted because of their important role in malaria control.Carbamates were the major group of insecticides used in agriculture until the introduction of pyrethroids in the mid 1990s.The present study shows that both the agricultural insect pests and their natural enemies in Sri Lanka have evolved different insecticide tolerance patterns over the last five decades.The major resistance mechanism in the aphids and in the diamondback moth P. xylostella was elevated esterases.It has been reported that aphids and Culex mosquitoes confer organophosphate and carbamate resistance primarily through e s t e r a s e ~. ' ~~ 1 4 2 l5 The aphid E4 esterase also hydrolyses permethrin,16 while the mosquito esterases are unable to interact with this either as a substrate or as an i n h i b i t ~r .~, ~~ In the present study, the inhibition of elevated esterase bands by paraoxon and propoxur confirms that these esterases confer organophosphorous and carbamate insecticide resistance.The results also suggest that they are not reactive with permethrin.Extensive use of malathion in malaria control activities in Sri Lanka has contributed to the pressure for t h e selection of malathion carboxylesterase mechanism in several malaria vector population^.^Exposure of agricultural insects to malathion sprayings and the illegal use of malathion for agricultural purposes may have selected malathion carboxylesterase mechanism in some of the agricultural insects as well.Some of the tested populations had a very high DDT tolerance, despite 20 years without DDT use.GSTs are known as DDT hydrochlorinases because of their active role in DDT metabolism.17DDT tolerance seen in the tested populations was well correlated with their GST activity levels.Mean specific GST activities in some species were higher t h a n those of Sri Lankan malaria vectors Anopheles culicifacies and A. subpictus.18It is possible t h a t the DDT resistance has been maintained even after the cessation of DDT, through GSTs which act as a secondary source of organophosphate or pyrethroid r e s i s t a n ~e .~~~ 20 Increased metabolism by esterases and GSTs has been identified as a major mechanism in the storage pest of legumes, Callosobruchi~s r n a c ~l a t e s .~~ Mono-oxygenases can produce high levels of resistance to pyrethroids.However, the high pyrethroid resistance shown by P. xylostella with low mono-oxygenase estimates, may be due to the activity of other metabolic enzymes and/or altered pyrethroid target sites or a quantitative change in one or more mono-oxygenases which does not affect gross enzyme titres.
L. huidobrensis is a recently introduced insect pest in Sri Lanka.It caused a 95% loss of potato yield and a severe loss of beetroot and leek yields in NuwaraEliya area in 1997.Control of this pest was not easy due to its high resistance to the recommended insecticides.The present study shows that Nuwara Eliya L. huidobrensis population is highly resistant to organophosphates, carbamates and pyrethroids but susceptible to DDT.As it is evident from the present results, high resistance of this population to other groups of insecticides is probably due to the presence of insensitive insecticide target sites.

Table 2 .
High standard deviations indicate the heterogeneity among the individuals of each population.Increased esterase activities were observed in M. persicae, A. gossypii and L. erysimi.All species, except the aphid T. citricidus Bimolecular rate constant hi for AChE inhibition:Mass crude homogenates (100 mg wet weight) inRESULTSCalculated LC,, and LC,, values for malathion, chlorpyrifos, propoxur and permethrin for all the insect species are given in the Table1.Of the pest species, M .persicae,P.xylostella and L. huidobrensis had higher levels of resistance to all the insecticides tested, with the exception of M. persicae to propoxur.A. gossipi showed a high resistance to propoxur.The aphid T. citricidus was the least tolerant to all four insecticides.Although the ladybird beetle C. sexmaculatus had a moderate level of resistance to chlorpyrifos and high resistance to propoxur, its permethrin tolerance was low.Tolerance of T. cincta to all insecticides was very low.Heterogeneity of response to insecticide exposure is shown by the x2 values of the mortality curves and seen in M. persicae population for malathion and in A .craccivora and C. sexmaculatus populations for permethrin (Table1).Mortalities for DDT at the fixed dosage of 1.4 pglmg wet body weight were L. huidobrensis (92%), T. citricidus (63%), M. persicae (44%), A. gossypii (38%), A. craccivora (27%), C. sexmaculatus (20%), T. cincta (l7%), L. erysimi (16%) and P. xylostella (13%).

Table 2 : Mean values rt standard deviation of specific activities of esterases and glutathione S- transferases and estimates of mono-oxygenase content in different insect populations.
t glutathione S-transferase * equivalent units of cytochrome P450

Table 3 : Bimolecular rate constants (kis) for acetylcholinesterase-propoxur (1mM) inhibition reactions. Insect species ki (M-I min-' x lo4)
DISCUSSIONCompared to the pest specie, the predatory ladybird beetle C. sexmaculatus had a moderate level of insecticide tolerance whereas T. cincta was more susceptible.Similar levels of insecticide tolerance between aphids and ladybird beetles, have been reported previously as well.13 Brogdon W.G., McAllister J.C. & Vulule J.M.(1997).Heam peroxidase activity measured in single mosquitoes identifies individuals expressing an elevated oxidase for insecticide resistance.Journal o f American Mosquito Assoc~ation 13: 233.