Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

Comparison of catalytic properties and inhibition kinetics of two acetylcholinesterases from a lepidopteran insect

Acetylcholinesterase (AChE) is the primary target of organophosphate (OP) and carbamate (CB) insecticides. Many insect species have been shown to have two different AChE genes. The amino acid identity between the two lepidopteran AChEs is lower than 40%, and potential differences in enzymatic function have not been characterized. In this study, the cDNAs encoding two AChEs (Boma-AChE1 and Boma-AChE2) from Bombyx mandarina were sequenced, and the corresponding proteins were heterologously expressed to compare their enzymatic properties and interactions with insecticides in vitro. Both of these enzymes had high specific activities for acetylthiocholine iodide. Studies on substrate and inhibitor specificities confirmed that both enzymes belong to AChE. Insecticide inhibition assays indicated that Boma-AChE1 was more sensitive than Boma-AChE2 to eight of the nine insecticides tested. However, Boma-AChE2 was more sensitive than Boma-AChE1 to one of the OP insecticides, heptenophos. The results suggested that two AChEs from a lepidopteran insect have distinct catalytic properties and responses to different inhibitors.     

Reactivity In vitro toward substrate and inhibitors of acetylcholinesterase isozymes from electric eel electroplax and housefly brain

Five acetylcholinesterase isozymes from electric eel eletroplax (Electrophorus electricus) and four from housefly brain (Musca domestica L.), were separated by polyacrylamide gel electrophoresis combined with specific substrate staining and gel scanning procedures. The K_m values for acetylthiocholine varied over a 2.4-fold range for eel and a 4.2-fold range for housefly. The sensitivities to inhibition also varied: for eel, the range was 2.2-fold for malaoxon, 2.6-fold for Tetram and 2.1-fold for eserine. The corresponding values for housefly were 2.3, 1.5, and 1.9.     

Toxicological significance of acetylcholinesterase of the housefly thorax

Polyacrylamide gel electrophoresis of the 100,000g supernatant fraction of thoracic homogenates from the DDT/S housefly strain separated five bands having acetylcholinesterase activity. Ferguson plots indicated that the three bands of slowest mobility constituted members of the heterologous-size isomer family described for the head enzyme. The two faster migrating bands appeared as electrophoretic artifacts of juxtaposition with a 3.6 S nonacetylcholinesterase component; in the absence of juxtaposition, a single form was characterized with identical molecular properties to the 5.3 S fundamental unit of head acetylcholinesterase. The kinetic properties of isolated forms were consonant with the molecular data and no major differences in K_m values for acetylthiocholine were discovered between the thoracic and head enzymes. We suggest the preferential in vivo inhibition of thoracic acetylcholinesterase by organophosphate pesticides is unlikely the consequence of biochemical differences between the enzymes from the two regions.     

Change in activity of detoxifying enzymes in directionally selected population of tea mosquito bug (<Emphasis Type="Italic">Helopeltis theivora</Emphasis>) (Heteroptera: Miridae) by an organophosphate insecticide

The heteropteran bug, Helopeltis theivora is a polyphagous pest attacking the foliage crop tea and several other crops grown in the old world tropics. Application of synthetic insecticides, especially organophosphate was so far an effective and economic means of conventional management of the pest. Severe selection (bottlenecking) of H. theivora population by exposure to LC₈₀ dose of an organophosphate insecticide, monocrotophos resulted in 105 fold increase in tolerance level in the third generation. The total activity of general esterases (GE) and cytochrome P450 mono-oxygenases (CYP450) analogously increased in insecticide-selected F2 generation by 16.4 and 9.5 fold, respectively. Such enhanced enzyme activity could be related to the higher tolerance levels of the selected bug. Electropherogram of GE and CYP450 of the insecticide-selected generations indicated a change in isozyme profile with their elevated expressions. Induced isozymes in insecticide selected generations were only partially inhibited, when blocked by the insecticide. These findings imply that different groups (zones) of isozymes of the detoxifying enzymes are involved in imparting higher tolerance in insecticide-selected generations. Isozyme profiles of defence enzymes can be used as indices for identifying tolerance level in the field population of H. theivora, enabling tea planters to carry out rapid monitoring of the tolerance status of the pest populations, thereby providing a ready clue to choose an effective insecticide for pest management.     

Comparison of insecticide absorption and detoxification in larvae of the bollworm,Heliothis zea,and the tobacco budworm,H. virescens

Rates and nature of metabolism of several insecticides were compared in larvae of the bollworm, Heliothis zea (Boddie), and the tobacco budworm, H virescens (F.). Both species are resistant to organochlorine compounds. In addition, H. virescens is resistant to carbamate and organophosphate insecticides. All test insecticides were metabolized by larvae of both species under in vivo conditions. In most instances, metabolism was more rapid in H. virescens larvae.A series of in vivo and in vitro assays implicated high levels of NADPH-dependent oxidative enzymes as a major metabolic factor in resistance. Other work demonstrated that H. virescens larvae absorbed insecticides at a slower rate than did H. zea larvae. High resistance to organophosphates present in H. virescens may be due to interaction of factors for increased rate of detoxification and decreased rate of absorption.     

The relationship of esterases to organophosphorus insecticide tolerance in mosquitofish

The levels of peripheral acetylcholinesterase and carboxylesterases and their organophosphate sensitivities were studied in two populations of mosquitofish (Gambusia affinis). One population, highly resistant to organochlorine insecticides, demonstrated a low tolerance to parathion and methyl parathion. The organochlorine resistant fish possessed higher levels of both peripheral acetylcholinesterase and carboxylesterases. The sensitivities of these esterases to organophosphate inhibition were the same in both populations. The esterases were more sensitive to paraoxon than to methyl paraoxon. Carboxylesterases were far more sensitive to organophosphate inhibition than was acetylcholinesterase. Carboxylesterases, by their higher affinity for the organophosphates, may serve to protect acetylcholinesterase from inhibition.     

Integrated pest management approach for a new pest, Lacanobia subjuncta (Lepidoptera: Noctuidae), in Washington apple orchards

Bioassays of Lacanobia subjuncta (Grote and Robinson) larvae established baseline LC50 values and identified the potential of reduced-risk, organophosphate replacement and naturally derived insecticides (eg chloronicotinyls, spinosyns, oxadiazines, insect growth regulators, microbial insecticides and particle films) to control this pest. The toxicities of these products were compared with those of organophosphate, carbamate, chlorinated cyclodiene and synthetic pyrethroid insecticides used in the management of lepidopteran pests in Washington apple orchards. Field trials were conducted comparing candidate insecticides to conventional alternatives. Several new insecticides (eg spinosad, methoxyfenozide, indoxacarb and an aluminosilicate particle film) proved to be effective for the management of L subjuncta. We summarize the goals and challenges of developing an integrated pest management program for new and resurgent pests as insecticide tools continue to change, and propose a hypothesis for the sudden increase in pest status of L subjuncta based on organophosphate tolerances. The role of novel insecticides with unique modes of action in resistance management and the encouragement of biological control are also discussed.     

Role of oxidative stress in organophosphate insecticide toxicity - Short review

The objective of this paper is to present a short review of the state of knowledge regarding oxidative stress and its role in toxicity of organophosphate insecticides. The information has been obtained by searching the relevant literature using chemical abstracts, PubMed, scopus, medline and other data bases. The significance of the problem has been elucidated. Organophosphate insecticides (OP), apart from inhibition of cholinesterase and presence of cholinergic effects, oxidative stress and hyperglycemia has been reported by many authors as one of the adverse effects in poisoning by OP in both humans and animals. Oxidative stress induced by organophosphate leads to disturbances in the function of different organs and tissues. In subchronic or chronic OP exposition induction of oxidative stress has been reported, by many authors, as the main mechanism of its toxicity. Data were categorized according to animal studies (in vitro and in vivo) and clinical studies. On the basis of relevant literature it is concluded, that determination of oxidative stress parameters can be useful for monitoring people exposed to OP professionally. Supplementation with natural or synthetic antioxidant may be beneficial in OP poisoning, however the rat models of OP poisoning used in those studies do not completely reflect clinical situation. For this reason the clinical trials are needed to explore effectiveness of these antioxidants in protection against toxicity of OP.     

Insensitive acetylcholinesterase,high glutathione-S-transferase,and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly

The gene producing insensitive acetylcholinesterase in a tetrachlorvinphos-resistant strain of house fly was introduced into the genome of a susceptible strain. The resistance to a number of organophosphorus insecticides, which was high in the original strain (234- and more than 800-fold for paraoxon and tetrachlorvinphos, respectively), was much lower in the resulting strain, ranging from 53-fold for paraoxon to 3.9-fold for tetrachlorvinphos. Synergists further decreased these factors. The reduction in sensitivity of the acetylcholinesterase in vitro was about 20-fold for both inhibitors. The Michaelis parameters for hydrolysis of acetylcholine and acetylthiocholine of the mutant and normal acetylcholinesterase showed only minor differences. An increased rate of metabolism of the insecticides was found in the original strain. In vitro, glutathione-dependent detoxication was from 9- to 120-fold that of a susceptible strain, depending on the insecticide and the conditions used. The enzyme de-alkylated parathion and methylparaoxon, but there was no de-arylation of parathion, although it is important in other strains. In vitro, paraoxon and methylparaoxon were also hydrolysed, and this is not so in the susceptible strain. The high resistance present in the original strain seems to be due to joint action of at least three mechanisms.     

Dual role of esterases in insecticide resistance in the green rice leafhopper

The role of esterases as related to insecticide resistance was studied in an organophosphorus (OP)-resistant strain of the green rice leafhopper. As judged by p-nitrophenyl acetate hydrolysis, 21, 5, and 74% of the esterase activity was located in nuclei/mitochondria, microsomes, and the soluble fraction, respectively. All the fractions were active in hydrolyzing malathion, paraoxon, and fenvalerate. Hydrolysis of malathion and fenvalerate increased with time while that of paraoxon reached a plateau within 15 min. Since a considerable amount of p-nitrophenol was detected in the paraoxon reaction at 0°C and at zero time, the formation of p-nitrophenol may be due to phosphorylation of the esterases rather than phosphorotriesterase action. The results suggest a dual role for esterases in resistance mechanisms; a catalyst for hydrolysis of malathion and fenvalerate, and a binding protein for the oxygen analogs of other OP insecticides, both of which would protect the intrinsic target, acetylcholinesterase, from inhibition. Chromatofocusing of the soluble fraction resolved five esterase peaks, I–V. These esterases were active toward the three general substrates as well as for the three insecticides tested, except for Peak I in which the overall activity was too low. Thin-layer agar gel electrophoresis showed that the chromatofocusing peaks I–V corresponded to the electrophoretic bands E₁–E₅, some of which were previously shown to be associated with OP resistance. The dual role of these esterases may explain the cross-resistance between malathion and other OP insecticides as well as synergism between OP and carbamate insecticides.     

药剂对小菜蛾抗性及敏感品系乙酰胆碱酯酶抑制作用比较

采用浸叶法测定了云南通海、元谋和澜沧的小菜蛾plutella xylostella田间种群对常用杀虫剂的抗药性。结果表明,云南上述地区小菜蛾田间种群对各类杀虫剂均产生了不同程度的抗性。对有机磷类药剂的抗药性为1.74~31.1倍;对菊酯类药剂的抗药性为7.41~764倍;对阿维菌素类药剂则产生了 5.60~4.06×104倍的抗性。通过离体和活体试验测定了药剂对小菜蛾头部乙酰胆碱酯酶(AChE)的抑制作用。敌敌畏和灭多威对通海抗性品系AChE离体和活体内的抑制中浓度(I50)分别是敏感品系的209、26.5倍和2.21、2.16倍;敌敌畏对通海小菜蛾种群的离体和活体内抑制中时间(IT50)小于敏感品系,分别是敏感品系的0.32和0.17倍;而灭多威对通海小菜蛾种群的离体和活体内抑制中时间(IT50)则大于敏感品系,分别是敏感品系的1.37和1.74倍。     

Multiple forms of esterases in mouse,rat, and rabbit liver,and their role in hydrolysis of organophosphorus and pyrethroid insecticides

Six to seven esterases from mouse, rat, and rabbit liver microsomes were resolved by chromatofocusing in the pH range 7–4. Each esterase peak showed a different substrate specificity pattern with the substrates evaluated. Malathion and paraoxon hydrolysis always corresponded with p-nitrophenyl acetate and methylthiobutyrate hydrolysis, whereas the pattern of fenvalerate hydrolysis was more complicated. Phosphorotriester hydrolase activity was isolated, and was found to be more specific toward paraoxon than toward the other insecticides. Time-course studies of paraoxon hydrolysis indicated that the hydrolysis of paraoxon by carboxylesterase was an inhibitory reaction. This reaction and phosphorotriester hydrolase activity can serve as a detoxication reaction toward organophosphate insecticides.     

The effect of organophosphate insecticides on adrenal corticosterone formation

In vitro and in vivo experiments with Sprague-Dawley rats showed that the three organophosphate insecticides tested (see below) depressed endogenous corticosterone synthesis and blocked corticosteroidogenesis in response to ACTH and cAMP stimulation of a suspension of adrenal cells. Pregnenolone stimulation of adrenal cells was not inhibited at the insecticide concentrations which blocked the ACTH and cAMP stimulation of corticosteroidogenesis. It was concluded that the insecticides act beyond the site of action of ACTH and at or beyond the level of cAMP metabolism and prior to the metabolism of pregnenolone.Insecticides tested were: dichlorvos (O,O-dimethyl-O,2,2-dichlorovinyl phosphate), Dursban (phosphorothioic acid: O,O-diethyl O-3,5,6-trichloro-2-pyridyl ester), and Diazinon (phosphorothioic acid: O,O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl ester).     

Cross‐resistance and possible mechanisms of chlorpyrifos resistance in Laodelphax striatellus (Fallén)

BACKGROUND: Laodelphax striatellus (Fallén) is a major pest of cultivated rice and is commonly controlled in China with the organophosphate insecticides. To develop a better resistance management strategy, a chlorpyrifos‐resistant strain of L. striatellus was selected in the laboratory, and its cross‐resistance to other insecticides and possible mechanisms of the chlorpyrifos resistance were investigated. RESULTS: After 25 generations of selection with chlorpyrifos, the selected strain of L. striatellus developed 188‐fold resistance to chlorpyrifos in comparison with the susceptible strain, and showed 14‐ and 1.6‐fold cross‐resistance to dichlorvos and thiamethoxam respectively. There was no apparent cross‐resistance to abamectin. Chlorpyrifos was synergised by the inhibitor triphenyl phosphate; the carboxylesterase synergistic ratio was 3.8 for the selected strain, but only 0.92 for the susceptible strain. The carboxylesterase activity of the selected strain was approximately 4 times that of the susceptible strain, whereas there was no significant change in the activities of alkaline phosphatase, acid phosphatase, glutathione S‐transferase and cytochrome P450 monooxygenase between the strains. The Michaelis constant of acetylcholinesterase, maximum velocity of acetylcholinesterase and median inhibitory concentration of chlorpyrifos‐oxon on acetylcholinesterase were 1.7, 2.5 and 5 times higher respectively in the selected strain. CONCLUSION: The high cross‐resistance to the organophosphate dichlorvos in the chlorpyrifos‐resistant strain suggests that other non‐organophosphate insecticides would be necessary to counter resistance, should it arise in the field. Enhanced activities of carboxylesterase and the acetylcholinesterase insensitivity appear to be important mechanisms for chlorpyrifos resistance in L. striatellus. Copyright © 2010 Society of Chemical Industry     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

Laboratory efficacy screening of insecticides for control ofMaladera matrida Larvae

Maladera matrida Argaman (Coleoptera: Scarabaeidae) was first reported in the central coastal plain of Israel in the early 1980s and was subsequently described as a new species. By the late 1980s it had spread southward to the northwestern Negev desert region, where heavy infestations of this pest caused significant economic losses, especially to growers of peanuts and sweet potatoes. Larvae (white grubs) ofM. matrida feed on underground parts of plants. We used a laboratory soil-treatment assay for determining the relative toxicity of several insecticides to 3-week-old grubs. Insecticides, both registered and candidates for registration, were tested at the labeled rates for field crops. The residual activity of those insecticides that caused ≥60% mortality of grubs after the first week, was studied for two additional weeks, by replacing the grubs with new ones each week. The granular formulation of the organophosphate insecticides terbufos, chloropyrifos, ethoprop and isazofos induced a uniform high mortality to grubs throughout the study period. Chloropyrifos and ethoprop caused >90% mortality also at one tenth of the labeled rate. Also diazinon brought about >90% mortality after the first week, but subsequently lost its activity. Imidacloprid, bendiocarb, fenitrothion, acephate, methamidophos and carbaryl provided <50% mortality during the first week.     

Binding of organophosphate insecticides to lecithin: A proton magnetic resonance study

The interaction of four vinyl-type organophosphate insecticides with a phospholipid lecithin has been studied by proton magnetic resonance technique. The vinyl proton of organophosphate showed a low field chemical shift change when lecithin was added to a CCl₄ solution of the former. When organophosphate insecticide was added to a dilute solution of lecithin the choline protons peak also showed small chemical shift changes. These chemical shift changes suggest that the most probable binding site in the interaction involves a hydrogen bond formation between the vinyl proton and oxygen atom of phosphatidyl group of lecithin. Chemical shift data has been used to calculate the equilibrium constant of the interaction.     

Kinetic efficiency of mutant carboxylesterases implicated in organophosphate insecticide resistance

Resistance to organophosphorus (OP) insecticides in Lucilia cuprina arises from two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion. These mutations are not found naturally in the orthologous EST23 enzyme in Drosophila melanogaster. We have introduced the two mutations (G137D and W251L) into cloned genes encoding E3 and EST23 from susceptible L. cuprina and D. melanogaster and expressed them in vitro with the baculovirus system. The ability of the resultant enzymes to hydrolyse the phosphate ester of diethyl and dimethyl organophosphates was studied by a novel fluorometric assay, which also provided a sensitive titration technique for the molar amount of esterase regardless of its ability to hydrolyse the fluorogenic substrate used. Malathion carboxylesterase activity was also measured. The G137D mutation markedly enhanced (>30-fold) hydrolysis of both classes of phosphate ester by E3 but only had a similar effect on the hydrolysis of dimethyl organophosphate in EST23. Introduction of the W251L mutation into either gene enhanced dimethyl (23-30-fold) more than diethyl (6-10-fold) organophosphate hydrolysis and slightly improved (2-4-fold) malathion carboxylesterase activity, but only at high substrate concentration.     

Variability in resistance-related enzyme activities in field populations of the tarnished plant bug, Lygus lineolaris

Widespread use of Bt crops for control of lepidopterous pests has reduced insecticide use and provided the tarnished plant bug the opportunity to become a serious pest on mid-South cotton. Organophosphate insecticides have predominantly been used against plant bugs in recent years due to the reduced efficacy of other insecticides. In this study, a biochemical approach was developed to survey enzymatic levels associated with organophosphate resistance levels in field populations of the tarnished plant bug. Forty-three populations were collected from the delta areas of Arkansas, Louisiana, and Mississippi. Three esterase substrates and one substrate each of glutathione S-transferase (GST) and acetylcholinesterase (AChE) were used to determine corresponding detoxification enzyme activities in different populations. Compared to a laboratory susceptible colony, increases up to 5.29-fold for esterase, 1.96-fold for GST, and 1.97-fold for AChE activities were detected in the field populations. In addition to the survey of enzyme activities among the populations, we also examined the susceptibility of major detoxification enzymes to several inhibitors which could be used in formulations to synergize insecticide toxicity against the target pests. As much as 52-76% of esterase, 72-98% of GST, and 93% of AChE activities were inhibited in vitro. Revealing variable esterase and GST activities among field populations may lead to a better understanding of resistance mechanisms in the tarnished plant bug. This study also reports effective suppression of detoxification enzymes which may be useful in future insecticide resistance management program for the tarnished plant bug and other Heteropteran pests on Bt crops.