A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH-Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5 kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.     

Cloning of a rice tau class GST isozyme and characterization of its substrate specificity

The GST cDNA was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU5 (GenBank Accession No. AF309377) sequence. The cDNA was composed of a 687-bp open reading frame encoding for 228 amino acids. The deduced amino acid sequence of this gene shared over 60% sequence identity with the sequences of the tau class ZmGSTU6 and ZmGSTU19. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTU5 formed a homo-dimer composed of 25 kDa subunit and its pI value was approximately 7.5. The OsGSTU5 displays high activities towards 1-chloro-2,4-dinitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. The activity of the OsGSTU5 was significantly inhibited by hematin and ethacrynic acid. The OsGSTU5 shows the highest activity towards chloro-s-triazine and acetanilide herbicides.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS-polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3-10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.     

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH₃-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I₅₀ value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.     

Activity of glutathione S-transferase toward some herbicides and its regulation by benoxacor in non-embryogenic callus and in vitro regenerated tissues of Zea mays

A procedure was developed to obtain non-embryogenic callus and regenerated lines from root segments of Zea mays grown in aseptic conditions. The activity of glutathione-S-transferases (GSTs), for non-embryogenic callus, was determined toward 1-chloro-2,4-dinitrobenzene (CDNB) and it was compared with that obtained for corn seedlings grown without hormones. For the callus masses, increases of specific activity toward CDNB and the kinetic parameter V_max were observed with respect to corn seedlings. The procedure permitted the regenerating of tissues from callus explants, therefore the GST(CDNB) activity and the effect of the safener benoxacor on its expression were investigated for the regenerated tissues grown in agarized substrate and in liquid medium. These explants showed a constitutive GST(CDNB) activity higher than corn seedlings and this activity was increased, for both tissues, in response to the presence of the safener benoxacor in the growing medium. The GST activity for the above tissues was also assayed toward benoxacor and terbuthylazine, metolachlor and fluorodifen herbicides. Measurable GST activity was found toward some of the above chemicals and it was found to be significantly enhanced in response to benoxacor treatment.     

Protective effects of Nigella sativa oil on propoxur-induced toxicity and oxidative stress in rat brain regions

Propoxur (PPr) is a widely used broad spectrum carbamate insecticide mainly used to control household pests. Because of the widespread use of pesticides for domestic and industrial applications, evaluation of their neurotoxic effects is of major concern to public health. The aim of the present study was to evaluate the possible protective effects of Nigella sativa oil (NSO), an antioxidant agent, against PPr-induced toxicity and oxidative stress in different brain regions of rats including cerebellum, cortex and hippocampus. In the present study, 32 male Sprague-Dawley rats were used and divided into four equal groups. Group 1 was allocated as the control group. Groups 2-4 were orally administered 1 ml/kg/bw/day NSO, 8.51 mg/kg/bw/day PPr or NSO plus PPr, respectively, for 30 days. Lipid peroxidation (LPO), protein carbonyl content (PCC) and acetylcholine esterase activity (AChE) were determined. Enzymatic antioxidant activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione-S-transferase (GST)] and non-enzymatic antioxidants [reduced glutathione (GSH)] were determined. PPr treatment significantly increased the levels of LPO, PCC and oxidized glutathione (GSSG) in brain regions. On the contrary, levels of GSH and the activities of SOD, CAT, GSH-Px, GST and AChE were significantly decreased. NSO treatment to PPr intoxicated rats restored such biochemical parameters to within control levels except GST activity, emphasizing its antioxidant role. We conclude that NSO significantly reduces PPr-induced toxicity and oxidative stress in rat brain regions via a free radicals scavenging mechanism.     

Biochemical and histological hepatic changes of Nile tilapia Oreochromis niloticus exposed to carbaryl

The purpose of this study was to evaluate biochemical and morphological responses induced by carbaryl in the liver of Nile tilapia (Oreochromis niloticus) exposed during 21 days to sublethal concentrations (0.25 and 0.5 mg L⁻¹), testing also recover for 14 days in clean water, after 14 days exposure. The activities of the following enzymes were measured: superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), and reduced (GSH) and oxidized glutathione (GSSG). Globally, our data showed that exposure to carbaryl decreased the SOD, CAT, GR, and GST activities, except for the SOD and GST activities after 14 days exposure to 0.25 mg L⁻¹. In contrast, after 14 days exposure the GR activity of the hepatic tissue from carbaryl-treated fish showed significant elevation in relation to the control. When fish were left to recover, a positive response was seen in the GSH and GSSG contents. The results of the recovery group suggest that the toxicity produced by carbaryl is reversible to some extent within 15 days. The liver histological analysis showed differences between fish concerning the cellular vacuolization degree (VD) of the hepatocytes. In fish exposed to carbaryl it was observed an increasing hepatocellular basophilia. No other histological alterations were observed when fish was exposed to carbaryl, except a few necrotic foci at day 7. The sections stained with PAS reaction showed that the vacuolization was always not due to glycogen deposits, thus suggesting lipid accumulation. The combined increased basophilia and glycogen depletion is a common, although non-specific, liver response to many toxicants. In short, this work shows a relation between histological and biochemical changes in liver and carbaryl exposure. The effects of carbaryl were observed at different concentrations.     

Allelochemical ethyl 2-methyl acetoacetate (EMA) induces oxidative damage and antioxidant responses in Phaeodactylum tricornutum

Ethyl 2-methyl acetoacetate (EMA) is a novel allelochemical exhibiting inhibitory effects on the growth of marine unicellular alga Phaeodactylum tricornutum (P. tricornutum). Oxidative damage and antioxidant responses in P. tricornutum were investigated to elucidate the mechanism involved in EMA inhibition on algal growth. The increase in reactive oxygen species (ROS) levels and malondialdehyde (MDA) contents following exposure to EMA suggested that alga was suffered from oxidative stress and severely damaged. The decrease in cell activity and cellular inclusions suggested that cell growth was greatly inhibited. The activities of the antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxide (GSH-PX) and glutathione S-transferase (GST) increased with the exposure concentration and decreased with the prolongation of exposure time. Cellular ascorbic acid (AsA) and reduced glutathione (GSH) systems were also involved in resisting oxidative stress of EMA by altering the composition of AsA and GSH pools. EMA exposure increased the contents of AsA, GSH, dehydroascorbate (DAsA) and glutathione (GSSG). However, the regeneration rate of AsA/DAsA did not change obviously between treatments and the control, while that of GSH/GSSG decreased significantly under 14 mmol/L EMA exposure on the 3rd day. These results showed that EMA-induced oxidative damage might be responsible for EMA inhibition on P. tricornutum growth and cellular antioxidant enzymes and non-enzymatic antioxidants were improved to counteract the oxidative stress.     

Biochemical effects of acute phoxim administration on antioxidant system and acetylcholinesterase in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of different concentrations of phoxim on acetylcholinesterase (AChE) and esterase (EST) activities, and antioxidant system after topical application to Oxya chinensis. The results showed that phoxim inhibited AChE activity, and did not cause significant changes in the EST activity and the levels of malondialdehyde (MDA) and reduced glutathione (GSH). After phoxim administration, superoxide (SOD) and catalase (CAT) activities showed a biphasic response with an initial increase followed by a decline in their activities. Glutathione reductase (GR) and glutathione peroxidase (GPx) activities were inhibited in comparison with the control. Glutathione S-transferase (GST) activity showed irregular changes. Its activity increased significantly at the concentrations of 0.06 and 0.12 μg/μL and decreased at the concentrations of 0.09 and 0.24 μg/μL compared with the control. Changes in SOD, CAT, GST, GPx, and GR activities indicated that phoxim caused oxidative damage in O. chinensis. However, no significant changes in MDA content suggested that these enzymes played important roles in scavenging the oxidative free radicals induced by phoxim in O. chinensis. The formation of oxygen free radicals might be a factor in the toxicity of phoxim.     

Effects of the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and liver biomarkers in the common carp (Cyprinus carpio)

The aim of the study was to evaluate the effect of subchronic exposure to the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). One year old fish were exposed for 28 days to LASSO MTX added to the tank water at concentrations of 240 and 2400 μg L⁻¹. The exposure did not affect fish biometric parameters. Glutathione-S-tranferase (GST) activity in liver (hepatopancreas) remained unchanged in exposed fish when compared to controls. However, significant induction of total cytochrome P 450 (CYP 450), ethoxyresorufin-O-deethylase (EROD) activity and elevated glutathione (GSH) in liver of exposed fish were detected.     

Effect of waterborne benomyl on the hematological and antioxidant parameters of the Nile tilapia, Oreochromis niloticus

The present study was conducted to determine the 96 h-LC₅₀ of benomyl to the Nile tilapia, Oreochromis niloticus and to investigate the biochemical or hematological indices of blood and the alterations in the antioxidant enzymes of this fish in response to sublethal concentrations of benomyl. Fish weighing 71.61 ± 12.05 g were used in this study; they were subjected to fasting for 4 weeks before treatment. An aqueous solution of benomyl (0, 0.5, 1, 2, 4, 8, and 16 mg L⁻¹) was administered for 96 h to determine the LC₅₀. The 96 h-LC₅₀ value of benomyl was 4.39 (3.23-5.60) mg L⁻¹ in the present study. For 5 weeks, the aqueous solution of benomyl (0, 100, 200, and 400 μg L⁻¹) was administered to investigate its effect on the hematological parameters and antioxidant enzymes. The predominant hematological findings in fish exposed to benomyl were as follows: no significant change in the Hb (g dL⁻¹) level, MCV (μm³), MCH (pg) and MCHC (%) as compared to the control. Benomyl exposure led to greater increases in the GPT, GOT (Karmen-unit), LDH (Wroblewski unit), total cholesterol, Fe, and Ca (mg dL⁻¹) values, whereas the levels of ALP (KA unit), total protein, triglyceride, albumin, and Mg (mg dL⁻¹) did not increase. Benomyl increased the in vivo HSI (%), GST (nmol min⁻¹ mg protein⁻¹), and SOD (U mg protein⁻¹) values in the fish livers in the test group, unlike those in the control group for 5 weeks. At concentrations higher than 100 μg L⁻¹, benomyl affected the GST and SOD levels of Nile tilapia in a dose- and time-dependent manner. The present findings suggest that the in vivo hepatotoxicity associated with benomyl may, in part, result from the hematological index, and antioxidants may provide limited protection against benomyl toxicity.     

Toxic effects of novel organophosphorus insecticide (RPR-V) on certain biochemical parameters of euryhaline fish, Oreochromis mossambicus

The euryhaline fish, Oreochromis mossambicus was exposed to sub-lethal concentration (0.017 mg L⁻¹) of a novel phosphorothionate, 2-butenoic acid-3-(diethoxy phosphinothionyl) ethyl ester (RPR-V) for 30 days and allowed to recover for 7 days. Important biomarker enzymes were assayed in plasma, brain, gill, liver, kidney, and muscle during exposure tenures of day-3, -7, -15, -30, and also at 7 days (withdrawal) after stopping treatment. Acetylcholinesterase (AChE) activities of brain, gill, and muscle were strongly inhibited by 67, 75, and 66%, respectively, on day-30. Exposure (time) dependent increases in alanine aminotransferase (ALAT), and aspartate aminotransferase (ASAT), acid phosphatase (AcP), and alkaline phosphatase (AkP), activities in plasma and kidney; AcP and AkP activities in gill were noticed. However, significant decrease in ALAT, ASAT, AcP, and AkP activities in liver was observed. The depletion of glycogen was observed in liver, brain, and gill tissues, an indication of typical stress related response of the fish with pesticide. A significant increase in lactate dehydrogenase (LDH) activity in gill and brain was observed and decreased in liver and muscle, indicating tissue damage and muscular harm. Depletion of glutathione (GSH) was observed in the above tissues, there by enhancing the lipid peroxidation resulting in cell damage. The induction in hepatic glutathione-S-transferase (GST) levels indicates the protection against the toxicity of xenobiotic-induced lipid peroxidation. There was a significant recovery in all the above biochemical parameters, in all the tissues of fish after a recovery period of 7 days. These results revealed that RPR-V affects the intermediary metabolism of O. mossambicus and the increase of biomarker enzymes in plasma, might be due to the necrosis of liver.     

Influence of three diets on susceptibility of selected insecticides and activities of detoxification esterases of Helicoverpa assulta (Lepidoptera: Noctuidae)

The oriental tobacco worm, Helicoverpa assulta Guenée, is one of the most destructive pests of tobacco and peppers in China. We determined the susceptibility of H. assulta reared on an artificial diet, chili pepper and tobacco to four insecticides (fenvalerate, phoxim, methomyl, indoxacarb) under laboratory conditions associated with the activities of acetylcholinesterase (AChE), carboxylesterase (CarE) and glutathione S-transferase (GST) in its larvae. H. assulta larvae that were fed with chili pepper were more susceptible to fenvalerate, indoxacarb, and phoxim than those that were fed with tobacco and the artificial diet, but not to methomyl. The larvae that were fed with chili pepper were 3.65-, 2.49-, 1.92- and 2.44-fold more susceptible to fenvalerate, phoxim, methomyl, and indoxacarb than those fed with tobacco, respectively. The AChE activities of H. assulta larvae that were fed with chili pepper and tobacco were 2.12 and 1.07 μmol mg⁻¹ 15 min⁻¹, respectively, almost 2-fold difference. The CarE activity of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 4.12, 7.40 and 7.12 μmol mg⁻¹ 30 min⁻¹, respectively. Similarly, the GST activities of H. assulta larvae that were fed with chili pepper, tobacco and the artificial diet was 52.02, 79.37 and 80.02 μmol mg⁻¹ min⁻¹, respectively. H. assulta larvae that were fed with chili pepper were more resistance to the tested insecticides. The low activities of AChE and the high activities of CarE and GST lead to H. assulta become more susceptible to the tested insecticides.     

A comparative study of rac- and S-metolachlor on some activities and metabolism of silkworm, Bombyx mori L.

The racemic product of metolachlor contains two R-enantiomers and two S-enantiomers. S-Metolachlor is a more effective herbicidal agent than rac-metolachlor; currently, it is widely used instead of rac-metolachlor. The comparative toxicity of rac- and S-metolachlor was assessed on the basis of their effects on some enzyme activities of the fifth instar of the silkworm, Bombyx mori L. These results suggest that rac-metolachlor and S-metolachlor have different effects on enzyme activities of fifth-instar silkworm larvae. Hemolymph lactate dehydrogenase and catalase activity levels in silkworms treated with rac-metolachlor were much lower than those in silkworms treated with S-metolachlor. Control hemolymph ACP activity was 44-73% higher than that of rac-metolachlor-treated hemolymph, but the difference was not large between S-metolachlor treatment and the control. Midgut alkaline phosphatase activity of silkworms treated with rac-metolachlor was 46% lower than control activity, and the effect of S-metolachlor on midgut alkaline phosphatase activity was minor. The difference between the two herbicides was apparent mainly during the first 5 days of the fifth instar, and then gradually disappeared. It is shown that compared with S-metolachlor, rac-metolachlor is more toxic to economically important silkworms because of its effects on some enzymes and on metabolism.     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

Effects of trichlorfon and sodium dodecyl sulphate on antioxidant defense system and acetylcholinesterase of Tilapia nilotica in vitro

The effects of organophosphorus insecticide trichlorfon, surfactant sodium dodecyl sulphate (SDS), and the mixture of trichlorfon and SDS on the antioxidant defense system and acetylcholinesterase (AChE) in Tilapia nilotica were assessed in vitro. Various concentrations of trichlorfon (0, 0.0001, 0.001, 0.01, 0.1 and 1 g/L) and SDS (0, 0.0625, 0.125, 0.25, 0.5, 1 g/L) were incubated with homogenate of liver and muscle, respectively, at 25 °C for 0, 30, 60 and 90 min. Two concentrations of mixture of trichlorfon and SDS (0.0001 g/L trichlorfon + 0.5 g/L SDS, 0.1 g/L trichlorfon + 0.5 g/L SDS) and 0.0001 g/L trichlorfon, 0.1 g/L trichlorfon, 0.5 g/L SDS and control, were incubated simultaneously with homogenate of liver and muscle, respectively, at 25 °C for 60 min. After incubation, the content of reduced-glutathione (GSH) and the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) in homogenate of liver were determined, and the activities of AChE in homogenate of muscle were also measured.Treatment with trichlorfon caused a significant concentration-dependent and time-related inhibition of AChE activity at all treatment concentrations and times since trichlorfon is a cholinesterase inhibitor. For the same trichlorfon treatment, an apparent decrease in GSH content was found in concentration of 0.01, 0.1, 1 g/L, whereas no significant alteration in antioxidant enzyme activity were found at all experiment concentrations and times, which might indicate that antioxidant enzymes have not involved in the metabolism of trichlorfon. The depletion of GSH might indicate that ROS could be involved in the toxic effects of trichlorfon. Exposure of SDS can inhibit activities of AChE, GST and CAT at concentrations of 0.5 and/or 1 g/L, which could be due to the denaturing process of SDS to the enzymes. For the mixture exposure of trichlorfon and SDS, the effect of the mixture of 0.0001 g/L trichlorfon and 0.5 g/L SDS on inhibition of AChE shows synergistic other than simple additive of trichlorfon and SDS. The combined effects of chemicals and detergents deserve to be particularly noted. It should be noted that the toxicity experiments were made in tissue homogenates instead of whole organisms. The responses against the toxic compounds will not be the same in both systems.     

Variation of acephate susceptibility and correlation with esterase and glutathione S-transferase activities in field populations of the tarnished plant bug, Lygus lineolaris

The tarnished plant bug (TPB) has increasingly become an economically important pest of cotton. Heavy dependence on insecticides, particularly organophosphates and pyrethroids, for TPB control facilitated resistance development to multiple classes of insecticides. To better understand resistance and explore ways to monitor resistance in field populations, this study examined acephate susceptibility and the activities of two major detoxification enzymes in nine field populations collected in the Delta region of Mississippi and Arkansas in 2010. Two Arkansas populations from Reed and Backgate had 3.5- and 4.3-fold resistance to acephate, as compared to a susceptible laboratory strain. Extensive planting of cotton and heavy chemical sprays is a major driving force for resistance development to acephate in Mid-south cotton growing areas. Reduced susceptibility to acephate was highly correlated with elevated esterase activities. The acephate-resistant populations from Backgate, Lula, and Reed consistently had higher (up to 5.3-fold) esterase activities than susceptible populations. Regression analysis of LC₅₀s with kinetic esterase activities revealed a significant polynomial quadratic relationship with R² up to 0.89. Glutathione S-transferase (GST) also had elevated activity in most populations, but the variations of GST activities were not significantly correlated with changes of acephate susceptibility. Finally, examination of esterase and GST inhibitors indicated that suppression rates (up to 70%) by two esterase inhibitors in 2010 were slightly lower than those detected in 2006, and ethacrynic acid (EA) inhibited GST effectively in both years. Two other GST inhibitors (sulfobromophthalein and diethyl maleate) displayed significantly lower suppression rates in 2010 than those detected in 2006, suggesting a potential genetic shift in pest populations and a necessity of continued monitoring for insecticide resistance with both bioassay and biochemical approaches. Results indicated that using major detoxification enzyme activities for resistance monitoring may provide insight into acephate resistance in field populations of TPB.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.